Patent Grant
10913061
The technology described herein relates to an integrated apparatus for processing polynucleotide-containing samples and carrying out diagnostic tests on the same. More specifically, the technology relates to an apparatus for obtaining a diagnostic result on a biological sample using a microfluidic cartridge that receives the sample, in conjunction with a bench-top system. Methods of using the technology are also described herein.
The medical diagnostics industry is a critical element of today's healthcare infrastructure. At present, however, diagnostic analyses no matter how routine have become a bottleneck in patient care. There are several reasons for this. First, there are usually several steps in a diagnostic analysis between collecting the sample, and obtaining a diagnostic result, that require different levels of skill by operators, and different levels of complexity of equipment. For example, a biological sample, once extracted from a patient, must be put in a form suitable for a processing regime that typically involves using polymerase chain reaction (PCR) to amplify a nucleotide of interest. Once amplified, the presence of a nucleotide of interest in the sample needs to be determined unambiguously. Sample preparation is a process that is susceptible to automation but is also relatively routinely carried out in almost any location. By contrast, steps such as PCR and nucleotide detection have customarily only been within the compass of specially trained individuals having access to specialist equipment. Second, many diagnostic analyses can only be done with highly specialist equipment that is both expensive and only operable by trained clinicians. Such equipment is found in only a few locations—often just one in any given urban area. This means that most hospitals are required to send out samples to these locations for analysis, thereby incurring shipping costs and transportation delays, and possibly even sample loss, or mix-up. Third, some specialist equipment is typically not available ‘on-demand’ but instead runs in batches, thereby delaying the processing time for many samples because they must wait for a machine to fill up before they can be run.
The analysis of a biological sample to accomplish a particular diagnosis typically includes detecting one or more polynucleotides present in the sample. One example of detection is qualitative detection, which relates, for example, to the determination of the presence of the polynucleotide and/or the determination of information related to, for example, the type, size, presence or absence of mutations, and/or the sequence of the polynucleotide. Another example of detection is quantitative detection, which relates, for example, to the determination of the amount of polynucleotide present. Detection may therefore generally include both qualitative and quantitative aspects. Detecting polynucleotides qualitatively often involves establishing the presence of extremely small quantities in a sample. In order to improve sensitivity, therefore, the amount of polynucleotide in question is often amplified. For example, some detection methods include polynucleotide amplification by polymerase chain reaction (PCR) or a related amplification technique. Such techniques use a cocktail of ingredients, including one or more of an enzyme, a probe, and a labeling agent. Therefore, detection of polynucleotides can require use of a variety of different reagents, many of which require sensitive handling to maintain their integrity, both during use, and over time.
Understanding that sample flow breaks down into several key steps, it would be desirable to consider ways to automate as many of these as possible, and, desirably, to facilitate accomplishing as many as possible with a single machine that can be made available, on demand, to many users. There is therefore need for a method and apparatus of carrying out steps of sample preparation, PCR, and detection on biological samples in such a way that as few separate steps as possible are carried out.
The discussion of the background to the technology herein is included to explain the context of the technology. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as at the priority date of any of the claims.
Throughout the description and claims of the specification the word “comprise” and variations thereof, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.
An apparatus, comprising: a receiving bay configured to receive an insertable microfluidic cartridge; at least one heat source thermally coupled to the cartridge and configured to apply heat to one or more selected regions of the cartridge at one or more selected times, in order to: create a micro-droplet of a polynucleotide-containing biological sample held on the cartridge; cause the micro-droplet to move between one or more positions on the microfluidic cartridge; lyse cells, where present in the biological sample, thereby releasing polynucleotides from the cells; prepare one or more of the polynucleotides for amplification; and amplify one or more of the polynucleotides; a detector configured to detect presence of the one or more amplified polynucleotides; and a processor coupled to the detector and the at least one heat source, wherein the processor is configured to control applying heat to the one or more selected regions of the microfluidic cartridge at one or more selected times.
The system herein further comprises an integrated system, comprising an apparatus and a complementary cartridge, wherein together the apparatus and cartridge process a sample that has been injected into the cartridge, and provide a diagnostic result on the sample.
The receiving bay of the apparatus can be configured to selectively receive the microfluidic cartridge, as further described herein and exemplified by the accompanying drawings. For example, the receiving bay and the microfluidic cartridge can be complementary in shape so that the microfluidic cartridge can be selectively received in, e.g., a single orientation. The microfluidic cartridge can have a registration member that fits into a complementary feature of the receiving bay. By selectively receiving the cartridge, the receiving bay can help a user to place the cartridge so that the apparatus can properly operate on the cartridge. The receiving bay can also be configured so that various components of the apparatus that can operate on the microfluidic cartridge (heat pumps, peltier coolers, heat-removing electronic elements, detectors, force members, and the like) can be positioned to properly operate on the microfluidic cartridge. For example, a contact heat source can be situated in the receiving bay such that it can be thermally coupled to one or more distinct locations of a microfluidic cartridge that can be selectively received in the receiving bay.
The heat pump can be, for example, a heat source such as a resistor, a reversible heat pump such as a liquid-filled heat transfer circuit or a thermoelectric element, a radiative heat source such as a xenon lamp, and the like. The heat pump may be used not only to provide heat to the microfluidic elements but also to remove heat from microfluidic elements such as to reduce activity of certain reagents, freeze liquid in a microchannel to change its phase from liquid to solid, reduce the pressure of an air chamber to create a partial vacuum, etc.)
In various embodiments of the apparatus: the apparatus can further include a registration member that is complementary to the microfluidic cartridge, whereby the receiving bay receives the microfluidic cartridge in a single orientation; the apparatus can further include a sensor coupled to a processor, the sensor configured to sense whether the microfluidic cartridge can be selectively received.
The processor can be programmable to operate the detector to detect a polynucleotide or a probe thereof in a microfluidic cartridge located in the receiving bay.
The detector can be, for example, an optical detector. For example, the detector can include a light source that emits light in an absorption band of a fluorescent dye and a light detector that detects light in an emission band of the fluorescent dye, wherein the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. For example, the optical detector can include a bandpass-filtered diode that selectively emits light in the absorption band of the fluorescent dye and a bandpass filtered photodiode that selectively detects light in the emission band of the fluorescent dye; or for example, the optical detector can be configured to independently detect a plurality of fluorescent dyes having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof; or for example, the optical detector can be configured to independently detect a plurality of fluorescent dyes at a plurality of different locations in the cartridge, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.
The processor can be, for example, programmable to operate the at least one heat pump.
In various embodiments, the at least one heat pump can be a contact heat source selected from a resistive heater, a radiator, a fluidic heat exchanger and a Peltier device. The contact heat source can be configured at the receiving bay to be thermally coupled to a distinct location in a microfluidic cartridge received in the receiving bay, whereby the distinct location can be selectively heated. At least one additional contact heat source can be included, wherein the contact heat sources can be each configured at the receiving bay to be independently thermally coupled to a different distinct location in a microfluidic cartridge received in the receiving bay, whereby the distinct locations can be independently heated. The contact heat source can be configured to be in direct physical contact with a distinct location of a microfluidic cartridge received in the receiving bay. In various embodiments, each contact source heater can be configured to heat a distinct location having an average diameter in 2 dimensions from about 1 millimeter (mm) to about 15 mm (typically about 1 mm to about 10 mm), or a distinct location having a surface area of between about 1 mm2 about 225 mm2 (typically between about 1 mm2 and about 100 mm2, or in some embodiments between about 5 mm2 and about 50 mm2).
In various embodiments, the apparatus can include a compliant layer at the contact heat source, configured to thermally couple the contact heat source with at least a portion of a microfluidic cartridge received in the receiving bay. The compliant layer can have a thickness of between about 0.05 and about 2 millimeters, and a Shore hardness of between about 25 and about 100.
In various embodiments, at least one heat pump can be a radiative heat source configured to direct heat to a distinct location of a microfluidic cartridge received in the receiving bay.
In various embodiments, the one or more force members configured to apply force to at least a portion of a microfluidic cartridge received in the receiving bay.
In various embodiments, the one or more force members can be configured to apply force to thermally couple the at least one heat pump to at least a portion of the microfluidic cartridge. The one or more force members can be configured to operate a mechanical member at the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir, a valve or a pump.
In various embodiments, the one or more force members can be configured to apply force to a plurality of locations in the microfluidic cartridge. The force applied by the one or more force members can result in an average pressure at an interface between a portion of the receiving bay and a portion of the microfluidic cartridge of between about 5 kilopascals and about 50 kilopascals, for example, the average pressure can be at least about 14 kilopascals. At least one force member can be manually operated. At least one force member can be mechanically coupled to a lid at the receiving bay, whereby operation of the lid operates the force member.
In various embodiments, the apparatus can further include a lid at the receiving bay, the lid being operable to at least partially exclude ambient light from the receiving bay. The lid can be, for example, a sliding lid. The lid can include the optical detector. A major face of the lid at the optical detector or at the receiving bay can vary from planarity by less than about 100 micrometers, for example, less than about 25 micrometers. The lid can be configured to be removable from the apparatus. The lid can include a latching member.
In various embodiments, the apparatus can further include at least one input device coupled to the processor.
In various embodiments, the apparatus can further include a heating stage configured to be removable from the apparatus wherein at least one heat pump can be located in the heating stage.
In various embodiments, the cartridge can further include an analysis port. The analysis port can be configured to allow an external sample system to analyze a sample in the microfluidic cartridge; for example, the analysis port can be a hole or window in the apparatus which can accept an optical detection probe that can analyze a sample in situ in the microfluidic cartridge.
In some embodiments, the analysis port can be configured to direct a sample from the microfluidic cartridge to an external sample system; for example, the analysis port can include a conduit in fluid communication with the microfluidic cartridge that directs a liquid sample to a chromatography apparatus, an optical spectrometer, a mass spectrometer, or the like.
In some embodiments, the apparatus can include a receiving bay configured to receive a microfluidic cartridge in a single orientation; at least one radiative heat source thermally coupled to the receiving bay; at least two contact heat sources configured in the receiving bay to be thermally coupled to distinct locations, whereby the distinct locations can be selectively heated; one or more force members configured to apply force to at least a portion of the microfluidic cartridge received in the receiving bay, wherein at least one of the one or more force members can be configured to apply force to thermally couple the contact heat sources to the distinct locations, and at least one of the one or more force members can be configured to operate a mechanical member at the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir; a lid at the receiving bay, the lid being operable to at least partially exclude ambient light from the receiving bay, the lid comprising an optical detector configured to independently detect one or more fluorescent dyes, optionally having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof; at least one input device selected from the group consisting of a keyboard, a touch-sensitive surface, a microphone, and a mouse, at least one data storage medium selected from the group consisting of a hard disk drive, an optical disk drive, a communication interface selectede from the group consisting of: a serial connection, a parallel connection, a wireless network connection, and a wired network connection, a sample identifier selected from an optical character reader, a bar code reader, and a radio frequency tag reader; at least one output selected from a display, a printer, a speaker; and a processor coupled to the detector, the sensor, the heat sources, the input, and the output.
A microfluidic cartridge can include a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor. In some embodiment, the microfluidic cartridge also includes a registration member.
In various embodiments of the microfluidic cartridge, the microfluidic cartridge can further include a sample inlet valve in fluid communication with the microfluidic network. The sample inlet valve can be configured to accept a sample at a pressure differential compared to ambient pressure of between about 20 kilopascals and 200 kilopascals, for example between about 70 kilopascals and 110 kilopascals.
In various embodiments, the microfluidic network can include a filter in fluid communication with the sample inlet valve, the filter being configured to separate at least one component from a sample mixture introduced at the sample inlet.
In various embodiments, the microfluidic network can include at least one thermally actuated pump in fluid communication with the microfluidic network. The thermally actuated pump can include a thermoexpansive material selected from a gas, a liquid vaporizable at a temperature between 25° C. and 100° C. at 1 atmosphere, and an expancel polymer.
In various embodiments, the microfluidic network can include at least one thermally actuated valve in fluid communication with the microfluidic network. The thermally actuated valve can include a material having a solid to liquid phase transition at a temperature between 25° C. and 100° C. at 1 atmosphere.
In various embodiments, the microfluidic network can include at least one sealed reservoir containing a reagent, a buffer or a solvent. The sealed reservoir can be, for example, a self-piercing blister pack configured to bring the reagent, the buffer or the solvent into fluid communication with the microfluidic network.
In various embodiments, the microfluidic network can include at least at least one hydrophobic vent.
In various embodiments, the microfluidic network can include at least one reservoir configured to receive and to contain waste such as fluids and/or particulate matter such as cellular debris.
In various embodiments, the retention member can include a polyalkylene imine or a polycationic polyamide, for example, polyethylene imine, poly-L-lysine or poly-D-lysine. The retention member can be in the form of one or more particles. The retention member can be removable from the microfluidic cartridge.
In various embodiments, the microfluidic network can include a lysis reagent. The lysis reagent can include one or more lyophilized pellets of surfactant, wherein the microfluidic network can be configured to contact the lyophilized pellet of surfactant with a liquid to create a lysis reagent solution. The microfluidic network can be configured to contact a sample with the lysis reagent to produce a lysed sample.
In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to the sample to produce the lysed sample. For example, the microfluidic network can be configured to contact the retention member and the lysed sample to create a polynucleotide-loaded retention member.
In various embodiments, the microfluidic cartridge can further include a filter configured to separate the polynucleotide-loaded retention member from liquid.
In various embodiments, the microfluidic cartridge can further include a reservoir containing a wash buffer, wherein the microfluidic network can be configured to contact the polynucleotide-loaded retention member with the wash buffer, for example, the wash buffer can have a pH of at least about 10.
In various embodiments, the microfluidic cartridge can include a reservoir containing a release buffer, wherein the microfluidic cartridge can be configured to contact the polynucleotide-loaded retention member with the release buffer to create a released polynucleotide sample.
In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to the polynucleotide-loaded retention member to create the released polynucleotide sample.
In various embodiments, the microfluidic cartridge can include a reservoir containing a neutralization buffer, wherein the microfluidic network can be configured to contact the released polynucleotide sample with the neutralization buffer to create a neutralized polynucleotide sample.
In various embodiments, the microfluidic cartridge can include a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides. The PCR reagent mixture can be in the form of one or more lyophilized pellets, and the microfluidic network can be configured to contact the PCR pellet with liquid to create a PCR reagent mixture solution.
In various embodiments, the microfluidic network can be configured to couple heat from an external heat source with the PCR reagent mixture and the neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample.
In various embodiments, the PCR reagent mixture can further include a positive control plasmid and a fluorogenic hybridization probe selective for at least a portion of the plasmid.
In various embodiments, the microfluidic cartridge can include a negative control polynucleotide, wherein the microfluidic network can be configured to independently contact each of the neutralized polynucleotide sample and the negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons of the neutralized polynucleotide sample and PCR amplicons of the negative control polynucleotide.
In various embodiments, the microfluidic cartridge can include at least one probe that can be selective for a polynucleotide sequence, wherein the microfluidic cartridge can be configured to contact the neutralized polynucleotide sample or a PCR amplicon thereof with the probe. The probe can be a fluorogenic hybridization probe. The fluorogenic hybridization probe can include a polynucleotide sequence coupled to a fluorescent reporter dye and a fluorescence quencher dye. The PCR reagent mixture can further include a positive control plasmid and a plasmid fluorogenic hybridization probe selective for at least a portion of the plasmid and the microfluidic cartridge can be configured to allow independent optical detection of the fluorogenic hybridization probe and the plasmid fluorogenic hybridization probe.
In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism, for example any organism that employs deoxyribonucleic acid or ribonucleic acid polynucleotides. Thus, the probe can be selective for any organism. Suitable organisms include mammals (including humans), birds, reptiles, amphibians, fish, domesticated animals, farmed animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for components of organisms that employ their own polynucleotides, for example mitochondria. In some embodiments, the probe can be selective for microorganisms, for example, organisms used in food production (for example, yeasts employed in fermented products, molds or bacteria employed in cheeses, and the like) or pathogens (e.g., of humans, domesticated or wild mammals, domesticated or wild birds, and the like). In some embodiments, the probe can be selective for organisms selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.
In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of Group B Streptococcus.
In various embodiments, the microfluidic cartridge can be configured to allow optical detection of the fluorogenic hybridization probe.
In various embodiments, the microfluidic cartridge can further include a computer-readable label. For example, the label can include a bar code, a radio frequency tag or one or more computer-readable characters. The label can be formed of a mechanically compliant material. For example, the mechanically compliant material of the label can have a thickness of between about 0.05 and about 2 millimeters and a Shore hardness of between about 25 and about 100.
In various embodiments, the microfluidic cartridge can be further surrounded by a sealed pouch, during handling and storage, and prior to being inserted into the chamber. The microfluidic cartridge can be sealed in the pouch with an inert gas. The sealed pouch may also contain a packet of dessicant. The microfluidic cartridge can bedisposable.
In various embodiments, the microfluidic cartridge can contain one or more sample lanes. For example, a sample lane can include a thermally actuated pump, a thermally actuated valve, a sample inlet valve, a filter, and at least one reservoir. The lanes can be independent of each other, or can be partially dependent, for example, the lanes can share one or more reagents such as the lysis reagent.
In some embodiments, the microfluidic cartridge can include a registration member; and a microfluidic network. The microfluidic network includes, in fluidic communication: at least one thermally actuated pump; at least one thermally actuated valve; a sample inlet valve configured to accept a sample at a pressure differential compared to ambient pressure of between about 70 kilopascals and 110 kilopascals; a retention member selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor, the retention member being in the form of a plurality of particles formed of a polyalkylene imine or a polycationic polyamide; a filter configured to separate the polynucleotide-loaded retention member from liquid; a plurality of reservoirs, at least said one said reservoir being a sealed, self-piercing blister pack reservoir. The plurality of reservoirs can contain among them: a lysis reagent, the microfluidic network being configured to contact a sample introduced at the sample inlet with the lysis reagent and the retention member to create a polynucleotide-loaded retention member; a reservoir containing a wash buffer, the microfluidic network being configured to contact the polynucleotide-loaded retention member with the wash buffer; a reservoir containing a release buffer, the microfluidic network being configured to contact the polynucleotide-loaded retention member with the release buffer to create a released polynucleotide sample; a neutralization buffer, the microfluidic network being configured to contact the released polynucleotide sample with the neutralization buffer to create a neutralized polynucleotide sample; a PCR reagent mixture comprising a polymerase enzyme, a positive control plasmid, a fluorogenic hybridization probe selective for at least a portion of the plasmid and a plurality of nucleotides; and at least one probe that can be selective for a polynucleotide sequence, wherein the microfluidic network can be configured to contact the neutralized polynucleotide sample or a PCR amplicon thereof with the probe. Further, the microfluidic network can be configured to couple heat from an external heat source with the PCR reagent mixture and the neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample.
In various embodiments, a polynucleotide analysis system can include both the the microfluidic cartridge and the apparatus, as further described herein.
In various embodiments, a polynucleotide sample kit can include a microfluidic cartridge comprising a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor a sample container; and a liquid transfer member such as a syringe.
In various embodiments, the polynucleotide sample kit can further include instructions to employ the liquid transfer member to transfer a sample from the sample container to the microfluidic network.
In various embodiments, the polynucleotide sample kit can further include instructions to employ the liquid transfer member to direct a sample from the sample container, and a volume of air into the microfluidic network, the volume of air being between about 0.5 mL and about 5 mL.
In various embodiments, the polynucleotide sample kit can further include a filter, and, for example, instructions to employ the liquid transfer member to direct a sample from the sample container through the filter into the microfluidic network.
In various embodiments, the polynucleotide sample kit can further include at least one computer-readable label on the sample container. The label can include, for example, a bar code, a radio frequency tag or one or more computer-readable characters. The microfluidic cartridge can be sealed in a pouch with an inert gas.
In various embodiments, the polynucleotide sample kit can further include a sampling member; a transfer container; and instructions to contact the sampling member to a biological sample and to place the sampling member in the transfer container.
In various embodiments, the polynucleotide sample kit can further include a sample buffer, and, for example, instructions to contact the sampling member and the sample buffer.
In various embodiments, the polynucleotide sample kit can further include at least one probe that can be selective for a polynucleotide sequence, e.g., the polynucleotide sequence that is characteristic of a pathogen selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.
In some embodiments, the polynucleotide sample kit can include a microfluidic cartridge comprising a microfluidic network, a retention member in fluid communication with the microfluidic network, and a flurogenic probe, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor, the flurogenic probe being selective for a polynucleotide sequence that can be characteristic of a pathogen selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses; a sample container; a liquid transfer member; a sampling member; a transfer container; a sample buffer; and instructions. The instructions can include instructions to: employ the liquid transfer member to transfer a sample from the sample container to the microfluidic network; employ the liquid transfer member to direct a sample from the sample container and a volume of air into the microfluidic network, the volume of air being between about 0.5 mL and about 5 mL; employ the liquid transfer member to direct a sample from the sample container through a filter into the microfluidic network; and contact the sampling member to a biological sample and to place the sampling member in the transfer container.
A method for sampling a polynucleotide can include the steps of contacting the retention member at the microfluidic cartridge with a biological sample, the biological sample comprising at least one polynucleotide, thereby producing a polynucleotide-loaded retention member in the microfluidic cartridge; separating at least a portion of the biological sample from the polynucleotide-loaded retention member; and releasing at least a portion of a polynucleotide from the polynucleotide-loaded retention member, thereby creating a released polynucleotide sample.
In various embodiments, the method can further include one or more of the following steps: placing the microfluidic cartridge in the receiving bay of the apparatus; operating the force member in the apparatus to apply pressure at an interface between a portion of the receiving bay and a portion of the microfluidic cartridge (e.g., creating a pressure between about 5 kilopascals and about 50 kilopascals, or in some embodiments, at least about 14 kilopascals); employing the force member to apply force to a mechanical member in the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir, a valve or a pump, to release at least one reagent, buffer, or solvent from a reservoir in the microfluidic chip; and/or closing the lid to operate the force member, wherein the force member can be mechanically coupled to a lid at the receiving bay.
In some embodiments, the method can further include employing a sample identifier to read a label on the microfluidic cartridge or a label on the biological sample.
In some embodiments, the method can further include introducing a crude biological sample into the microfluidic cartridge and separating the biological sample from the crude biological sample in the microfluidic cartridge, e.g., using a filter in the cartridge, or the biological sample can be separated from a crude biological sample prior to introducing the biological sample into the microfluidic cartridge.
In some embodiments, the method can further include lysing the biological sample, for example, using heat, a lysis reagent, and the like. In some embodiments, wherein the microfluidic cartridge comprises one or more lyophilized pellets of lysis reagent, the method can further include reconstituting the lyophilized pellet of surfactant with liquid to create a lysis reagent solution.
In various embodiments, the method can further include one or more of the following: heating the biological sample in the microfluidic cartridge; pressurizing the biological sample in the microfluidic cartridge at a pressure differential compared to ambient pressure of between about 20 kilopascals and 200 kilopascals, or in some embodiments between about 70 kilopascals and 110 kilopascals.
In some embodiments, the portion of the biological sample separated from the polynucleotide-loaded retention member can include at least one polymerase chain reaction inhibitor selected from the group consisting of hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide. In some embodiments, the method can further include separating the polynucleotide-loaded retention member from substantially all of the polymerase chain reaction inhibitors in the biological sample.
In various embodiments, the method can further include one or more of the following: directing a fluid in the microfluidic cartridge by operating a thermally actuated pump or a thermally actuated valve; contacting the polynucleotide-loaded retention member with a wash buffer; heating the polynucleotide-loaded retention member to a temperature of at least about 50° C. (in some embodiments, the temperature can be 100° C. or less); heating the polynucleotide-loaded retention member for less than about 10 minutes; contacting the polynucleotide-loaded retention member with a release buffer to create a released polynucleotide sample (for example, in some embodiments, the the release buffer can have a volume of less than about 50 microliters, the release buffer can include a detergent, and/or the release buffer can have a pH of at least about 10); and/or contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample.
In various embodiments, the method can further include one or more of the following: contacting the neutralized polynucleotide sample with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides (in some embodiments, the PCR reagent mixture can further include a positive control plasmid and a fluorogenic hybridization probe selective for at least a portion of the plasmid); in some embodiments, the PCR reagent mixture can be in the form of one or more lyophilized pellets, and the method can further include reconstituting the PCR pellet with liquid to create a PCR reagent mixture solution; heating the PCR reagent mixture and the neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample; contacting the neutralized polynucleotide sample or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; independently contacting each of the neutralized polynucleotide sample and a negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons of the neutralized polynucleotide sample and PCR amplicons of the negative control polynucleotide; and/or contacting the neutralized polynucleotide sample or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence.
In various embodiments, the method can further include one or more of the following: determining the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the neutralized polynucleotide sample or a PCR amplicon thereof; determining a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof; and/or in some embodiments, wherein the PCR reagent mixture further comprises a positive control plasmid and a plasmid probe selective for at least a portion of the plasmid, the method further including determining a PCR reaction has occurred if the plasmid probe is detected.
In various embodiments, the method does not comprise centrifugation of the polynucleotide-loaded retention member.
In some embodiments, the method for sampling a polynucleotide can include: placing a microfluidic cartridge in the receiving bay of an apparatus; operating a force member in the apparatus to apply pressure at an interface between a portion of the receiving bay and a portion of the microfluidic cartridge, the force operating to release at least one reagent, buffer, or solvent from a reservoir in the microfluidic cartridge; lysing a biological sample in the microfluidic cartridge to create a lysed biological sample; contacting a retention member at a microfluidic cartridge with the lysed biological sample, the biological sample comprising at least one polynucleotide, thereby producing a polynucleotide-loaded retention member in the microfluidic cartridge, wherein the retention member is in the form of a plurality of particles of a polyalkylene imine or a polycationic polyamide; contacting the polynucleotide-loaded retention member with a wash buffer; heating the polynucleotide-loaded retention member to a temperature of at least about 50° C. for less than about 10 minutes; contacting the polynucleotide-loaded retention member with a release buffer to create a released polynucleotide sample. contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample; contacting the neutralized polynucleotide sample with a PCR reagent mixture under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample, the PCR reagent mixture comprising a polymerase enzyme, a positive control plasmid a fluorogenic hybridization probe selective for at least a portion of the plasmid, and a plurality of nucleotides. contacting the neutralized polynucleotide sample or a PCR amplicon thereof with at least one fluorogenic probe that can be selective for a polynucleotide sequence, wherein the probe can be selective for a polynucleotide sequence that can be characteristic of an organism selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses; and detecting the fluorogenic probe and determining the presence of the organism for which the one fluorogenic probe can be selective.
In various embodiments, a computer program product includes computer readable instructions thereon for operating the apparatus.
In some embodiments, a computer program product includes computer readable instructions thereon for causing the system to create a released polynucleotide sample from a biological sample. The computer readable instructions can include instructions for contacting the retention member with the biological sample under conditions suitable for producing a polynucleotide-loaded retention member; separating at least a portion of the biological sample from the polynucleotide-loaded retention member; and releasing at least a portion of a polynucleotide from the polynucleotide-loaded retention member, thereby creating a released polynucleotide sample.
In various embodiments, the computer program product can include one or more instructions to cause the system to: output an indicator of the placement of the microfluidic cartridge in the receiving bay; read a sample label or a microfluidic cartridge label; output directions for a user to input a sample identifier; output directions for a user to load a sample transfer member with the biological sample; output directions for a user to apply a filter to the sample transfer member; output directions for a user to introduce the biological sample into the microfluidic cartridge; output directions for a user to cause the biological sample to contact a lysis reagent in the microfluidic cartridge; output directions for a user to place the microfluidic cartridge in the receiving bay; output directions for a user to operate a force member in the apparatus to apply pressure at an interface between a portion of the receiving bay and a portion of the microfluidic cartridge; output directions for a user to close the lid to operate the force member; and/or output directions for a user to pressurize the biological sample in the microfluidic cartridge by injecting the biological sample with a volume of air between about 0.5 mL and about 5 mL.
In various embodiments, the computer program product can include one or more instructions to cause the system to: lyse the biological sample; lyse the biological sample with a lysis reagent; reconstitute a lyophilized pellet of surfactant with liquid to create a lysis reagent solution; heat the biological sample; separate the polynucleotide-loaded retention member from at least a portion of the biological sample; separate the polynucleotide-loaded retention member from substantially all of the polymerase chain reaction inhibitors in the biological sample; direct a fluid in the microfluidic cartridge by operating a thermally actuated pump or a thermally actuated valve; contact the polynucleotide-loaded retention member with a wash buffer; heat the polynucleotide-loaded retention member to a temperature of at least about 50° C. (in some embodiments, the temperature can be about 100° C. or less); heat the polynucleotide-loaded retention member for less than about 10 minutes; contact the polynucleotide-loaded retention member with a release buffer to create a released polynucleotide sample; and/or contact the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample.
In various embodiments, the computer program product can include one or more instructions to cause the system to: contact the neutralized polynucleotide sample with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides; heat the PCR reagent mixture and the neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample; contact the neutralized polynucleotide sample or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; independently contact each of the neutralized polynucleotide sample and a negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons of the neutralized polynucleotide sample and PCR amplicons of the negative control polynucleotide; contact the neutralized polynucleotide sample or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; output a determination of the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the neutralized polynucleotide sample or a PCR amplicon thereof; and/or output a determination of a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof.
In various embodiments, the computer program product can include one or more instructions to cause the system to automatically conduct one or more of the steps of the method.
In various embodiments, wherein the microfluidic network comprises two or more sample lanes each including a thermally actuated pump, a thermally actuated valve, a sample inlet valve, a filter, and at least one reservoir, wherein the computer readable instructions can be configured to independently operate each said lane in the system.
In some embodiments, the computer program product includes computer readable instructions thereon for causing a system to create a released polynucleotide sample from a biological sample. The system can include a microfluidic cartridge comprising a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor; and an apparatus comprising a receiving bay configured to selectively receive the microfluidic cartridge; at least one heat pump configured to be thermally coupled to the microfluidic cartridge in the receiving bay; a detector; and a programmable processor coupled to the detector and the heat pump. The computer readable instructions can include instructions for: lysing a biological sample by contacting the biological sample with a lysis reagent and heating to produce a lysed sample; contacting the retention member with the biological sample and/or the lysed sample to produce a polynucleotide-loaded retention member; separating at least a portion of the biological sample from the polynucleotide-loaded retention member; contacting the polynucleotide-loaded retention member with a wash buffer; contacting the polynucleotide-loaded retention member with a release buffer and or heat to release at least a portion of a polynucleotide from the polynucleotide-loaded retention member, thereby creating a released polynucleotide sample; contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample; independently contacting each of the neutralized polynucleotide sample and a negative control polynucleotide with a PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons, the PCR reagent mixture comprising a polymerase enzyme, a plurality of nucleotides, a positive control plasmid and a plasmid probe selective for at least a portion of the plasmid; determining a PCR reaction has occurred if the plasmid probe is detected; contacting the neutralized polynucleotide sample or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence; determining the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the neutralized polynucleotide sample or a PCR amplicon thereof; and determining a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof.
The details of one or more embodiments of the technology are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the technology will be apparent from the description and drawings, and from the claims. Like reference symbols in the various drawings indicate like elements.
A system, microfluidic cartridge, kit, methods, and computer program product, are now further described
Analysis of biological samples often includes determining whether one or more polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) can be present in the sample. For example, one may analyze a sample to determine whether a polynucleotide indicative of the presence of a particular pathogen (such as a bacterium or a virus) can be present. The polynucleotide may be a sample of genomic DNA, or may be a sample of mitochondrial DNA. Typically, biological samples can be complex mixtures. For example, a sample may be provided as a blood sample, a tissue sample (e.g., a swab of, for example, nasal, buccal, anal, or vaginal tissue), a biopsy aspirate, a lysate, as fungi, or as bacteria. Polynucleotides to be determined may be contained within particles (e.g., cells (e.g., white blood cells and/or red blood cells), tissue fragments, bacteria (e.g., gram positive bacteria and/or gram negative bacteria), fungi, spores). One or more liquids (e.g., water, a buffer, blood, blood plasma, saliva, urine, spinal fluid, or organic solvent) can typically be part of the sample and/or can be added to the sample during a processing step.
Methods for analyzing biological samples include providing a biological sample (e.g., a swab), releasing polynucleotides from particles (e.g., cells such as bacteria) of the sample, amplifying one or more of the released polynucleotides (e.g., by polymerase chain reaction (PCR)), and determining the presence (or absence) of the amplified polynucleotide(s) (e.g., by fluorescence detection). Biological samples, however, typically include inhibitors (e.g., mucousal compounds, hemoglobin, faecal compounds, and DNA binding proteins) that can inhibit determining the presence of polynucleotides in the sample. For example, such inhibitors can reduce the amplification efficiency of polynucleotides by PCR and other enzymatic techniques for determining the presence of polynucleotides. If the concentration of inhibitors is not reduced relative to the polynucleotides to be determined, the analysis can produce false negative results. The methods and related systems herein for processing biological samples (e.g., samples having one or more polynucleotides to be determined) are typically able to reduce the concentration of inhibitors relative to the concentration of polynucleotides to be determined by methods further described herein.
Various aspects of a system and a microfluidic cartridge are described herein. Additional disclosures of various components thereof may be found in U.S. application Ser. No. 11/580,267, and in provisional application Ser. No. 60/859,284, the specifications of which are hereby incorporated by reference.
System Overview
A schematic overview of a system 981 for carrying out analyses described herein is shown in
Although not shown in
Additionally, in various embodiments, the apparatus can further comprise a data storage medium configured to receive data from one or more of the processor, an input device, and a communication interface, the data storage medium being one or more media selected from the group consisting of: a hard disk drive, an optical disk drive, a flash card, and a CD-Rom.
Processor 980 is further configured to control various aspects of sample diagnosis, as follows in overview, and as further described in detail herein. The system is configured to operate in conjunction with a complementary cartridge 994, such as a microfluidic cartridge. The cartridge is itself configured, as further described herein, to receive a biological sample 996 in a form suitable for work-up and diagnostic analysis. The cartridge is received by a receiving bay 992 in the system. The receiving bay is in communication with a heater 998 that itself is controlled by processor 980 in such a way that specific regions of the cartridge are heated at specific times during sample work-up and analysis. The processor is also configured to control a detector 999 that receives an indication of a diagnosis from the cartridge 994. The diagnosis can be transmitted to the output device 986 and/or the display 982, as described hereinabove.
A suitable processor 980 can be designed and manufactured according to, respectively, design principles and semiconductor processing methods known in the art.
The system shown in outline in
The system of
The system of
In still another configuration, a system is configured to accept and to process multiple cartridges, but one or more components in
In still another configuration, a system as shown in
It is further consistent with the present technology that a cartridge can be tagged, e.g., with a molecular bar-code indicative of the sample, to facilitate sample tracking, and to minimize risk of sample mix-up. Methods for such tagging are described elsewhere, e.g., in U.S. patent application publication Ser. No. 10/360,854, incorporated herein by reference.
Exemplary Systems
System 2000 comprises a housing 2002, which can be made of metal, or a hardened plastic. The form of the housing shown in
System 2000 further comprises a display 2006, which may be a liquid crystal display, such as active matrix, an OLED, or some other suitable form. It may present images and other information in color or in black and white. Display 2006 may also be a touch-sensitive display and therefore may be configured to accept input from a user in response to various displayed prompts. Display 2006 may have an anti-reflective coating on it to reduce glare and reflections from overhead lights in an laboratory setting. Display 2006 may also be illuminated from, e.g., a back-light, to facilitate easier viewing in a dark laboratory.
System 2000, as shown in
Handle 2008 performs a role of permitting a user to move lid 2010 form one position to another, and also performs a role of causing pressure to be forced down on the lid, when in a closed position, so that pressure can be applied to a cartridge in the receiving bay 2014. In
In one embodiment, the handle and lid assembly are also fitted with a mechanical sensor that does not permit the handle to be depressed when there is no cartridge in the receiving bay. In another embodiment, the handle and lid assembly are fitted with a mechanical latch that does not permit the handle to be raised when an analysis is in progress.
A further configuration of system 2000 is shown in
Heater module 2020 is preferably removable, and is further described hereinbelow.
Computer readable medium input 2022 may accept one or more of a variety of media. Shown in
Features shown on the rear of system 2000 may be arranged in any different manner, depending upon an internal configuration of various components. Additionally, features shown as being on the rear of system 2000, may be optionally presented on another face of system 2000, depending on design preference. Shown in
An exploded view of an exemplary embodiment of the apparatus is shown in
Embodiments of apparatus 2000 also include software (e.g., for interfacing with users, conducting analysis and/or analyzing test results), firmware (e.g., for controlling the hardware during tests on the cartridge 812), and one or more peripheral communication interfaces shown collectively as 2031 for peripherals (e.g., communication ports such as USB/Serial/Ethernet to connect to storage such as compact disc or hard disk, to connect input devices such as a bar code reader and/or a keyboard, to connect to other computers or storage via a network, and the like).
Control electronics 840, shown schematically in the block diagram in
In various embodiments, fluorescent detection module 2009 can be a miniaturized, highly sensitive fluorescence detection system which can, for example, be capable of real-time analysis of a fluorescent signal emanating from a suitably positioned microfluidic cartridge, as shown in
In various embodiments, slider module 2007 of the apparatus 2000 can house the detection module 2009 (e.g., optical detection system) as well as mechanical assembly/optics jig 856 to press down on microfluidic cartridge 2020 when the handle 2008 of the slider module 2007 is pressed down.
Removable Heater Module
An exemplary removable heater module 2020 is shown in
Area 2044 is configured to accept a microfluidic cartridge in a single orientation. Therefore area 2044 can be equipped with a registration member such as a mechanical key that prevents a user from placing a cartridge into receiving bay 2014 in the wrong configuration. Shown in
Also shown in
In the embodiment of
Other non-essential features of heater module 2020 are as follows. One or more air vents 2052 can be situated on one or more sides (such as front, rear, or flanking) or faces (such as top or bottom) of heater module 2020, to permit excess heat to escape, when heaters underneath receiving bay 2014, are in operation. The configuration of air vents in
Heater module 2020 may further comprise one or more guiding members 2047 that facilitate inserting the heater module into an apparatus as further described herein for an embodiment in which heater module 2020 is removable by a user. Heater module is advantageously removable because it permits system 2000 to be easily reconfigured for a different type of analysis, such as employing a different cartridge with a different registration member and/or microfluidic network, in conjunction with the same or a different sequence of processing operations. In other embodiments, heater module 2020 is designed to be fixed and only removable, e.g., for cleaning, replacement, or maintenance, by the manufacturer or an authorized maintenance agent, and not routinely by the user. Guiding members may perform one or more roles of ensuring that the heater module is aligned correctly in the apparatus, and ensuring that the heater module makes a tight fit and does not significantly move during processing and analysis of a sample, or during transport of the apparatus. Guiding members shown in the embodiment of
Adjacent receiving bay 2014 is a non-contact heating element 2046, such as lamp, set into a recessed area. 2053. Recessed area 2053 may also be configured with a reflector, or a reflective coating, so that as much as thermal and optical energy from non-contact heating element 2046 as possible is directed outwards towards receiving bay 2014. Element 2046 is a heat lamp in certain embodiments. Element 2046 is configured to receive electrical energy and thereby heat up from the effects of electrical resistance. Element 2046 provides a way of heating a raised region of a cartridge received in receiving bay 2014. The raised region of the cartridge (see, e.g.,
Also shown in
Heater module 2020 also comprises an array of heaters, situated beneath area 2044 and not shown in
In particular and not shown in
Non-contact heater 2046 can also serve as a radiation heat source to heat one section of a suitably positioned microfluidic cartridge. For example, a 20 W Xenon lamp may be used as the non-contact heating element 2046. In various embodiments, heater/sensor module 2020 can be specific to particular cartridge designs and can be easily replaceable through the front panel of the apparatus 800. Heater/sensor module 2020 can be configured to permit cleaning of heating surface 2044 with common cleaning agents (e.g., a 10% bleach solution).
Referring to
It would be understood by one of ordinary skill in the art that still other configurations of one or more heater(s) situated about a PCR reaction zone are consistent with the methods and apparatus described herein. For example, a ‘long’ side of the reaction zone can be configured to be heated by two or more heaters. Specific orientations and configurations of heaters are used to create uniform zones of heating even on substrates having poor thermal conductivity because the poor thermal conductivity of glass, or quartz, or fused silica substrates is utilized to help in the independent operation of various microfluidic components such as valves and independent operation of the various PCR lanes. It would be further understood by one of ordinary skill in the art, that the principles underlying the configuration of heaters around a PCR reaction zone are similarly applicable to the arrangement of heaters adjacent to other components of the microfluidic cartridge, such as actuators, valves, and gates.
In certain embodiments, each heater has an associated temperature sensor. In the embodiment of
In order to reduce the number of sensor or heater elements required to control a PCR heater, we may use the heaters to sense as well as heat, and thereby obviate the need to have a separate dedicated sensor for each heater. In another embodiment, each of the four heaters may be designed to have an appropriate wattage, and connect the four heaters in series or in parallel to reduce the number of electronically-controllable elements from 4 to just 1, thereby reducing the burden on the associated electronic circuitry.
The configuration for uniform heating, shown in
Each heater can be independently controlled by a processor and/or control circuitry used in conjunction with the apparatus described herein.
Microfluidic Cartridge
Other than tower 2064, cartridge 2060 is substantially planar such that it can be easily handled by an operator and can be easily matched to a complementary receiving bay of an apparatus such as shown in
Cartridge 2060 further comprises a port 2068 through which a detector can receive a signal directly or indirectly from one or more polynucleotides in the sample, during processing or amplification, in order to provide a user with a diagnostic result on the sample.
Cartridge 2060 can further comprise a registration member such as a mechanical key, complementary to a corresponding registration member in the receiving bay. Shown in
The integrated system, as described herein, comprises an apparatus configured to receive a microfluidic cartridge, and a microfluidic cartridge. It is consistent with the system described herein that a number of different configurations of microfluidic cartridge, and purposes thereof, are compatible with suitably configured apparati. Thus, for example, although benefits are described wherein a single cartridge is capable of accepting a collected biological sample, working up the sample, including lysing cells to liberate and collect polynucleotides contained therein, applying pre-amplification preparatory steps to the polynucleotides, amplifying the polynucleotides, and causing the amplified polynucleotides to be detected, it is also consistent with the descriptions herein that other microfluidic cartridges can be used. Such other cartridges can be configured to carry out fewer, such as one or more, of the aforementioned steps, and, correspondingly the apparatus for use therewith is configured to cause fewer such steps to be effectuated. It is to be understood therefore, that when presenting various exemplary configurations of microfluidic cartridge herein, the various components thereof can be used interchangeably (e.g., an exemplary valve described in connection with one cartridge can also be used in a network described in connection with another cartridge) both without modification, and with suitable adjustments or modifications of geometry or size, as appropriate.
Aspects of Microfluidic Cartridges
Accordingly, the technology herein also comprises a microfluidic cartridge having attributes, as follows. Thus the technology includes a microfluidic cartridge that is configured to process one or more polynucleotides, e.g., to concentrate the polynucleotide(s) and/or to separate the polynucleotide(s) from inhibitor compounds, (e.g., hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide) that might inhibit detection and/or amplification of the polynucleotides.
The microfluidic cartridge can be configured to contact the polynucleotides and a relatively immobilized compound that preferentially associates with (e.g., retains) the polynucleotides as opposed to inhibitors. An exemplary compound is a poly-cationic polyamide (e.g., poly-L-lysine and/or poly-D-lysine), or polyethyleneimine (PEI), which may be bound to a surface (e.g., surfaces of one or more particles). The compound retains the polynucleotides so that the polynucleotides and inhibitors may be separated, such as by washing the surface to which the compound and associated polynucleotides are bound. Upon separation, the association between the polynucleotide and compound may be disrupted to release (e.g., separate) the polynucleotides from the compound and surface.
In some embodiments, the surface (e.g., surfaces of one or more particles) can be modified with a poly-cationic substance such as a polyamide or PEI, which may be covalently bound to the surface. The poly-cationic polyamide may include at least one of poly-L-lysine and poly-D-lysine. In some embodiments, the poly-cationic polyamide (e.g., the at least one of the poly-L-lysine and the poly-D-lysine) has an average molecular weight of at least about 7500 Da. The poly-cationic polyamide (e.g., the at least one of the poly-L-lysine and the poly-D-lysine) may have an average molecular weight of less than about 35,000 Da (e.g., an average molecular weight of less than about 30,000 Da (e.g., an average molecular weight of about 25,000 Da)). The poly-cationic polyamide (e.g., the at least one of the poly-L-lysine and the poly-D-lysine) may have a median molecular weight of at least about 15,000 Da. The poly-cationic polyamide (e.g., the at least one of the poly-L-lysine and the poly-D-lysine) may have a median molecular weight of less than about 25,000 Da (e.g., a median molecular weight of less than about 20,000 Da (e.g., a median molecular weight of about 20,000 Da). If the polycationic material is PEI, its molecular weight is preferably in the range 600-800 Daltons.
In other embodiments, the microfluidic cartridge includes a surface having a poly-cationic polyamide or PEI bound thereto and a sample introduction passage in communication with the surface for contacting the surface with a fluidic sample.
In some embodiments, the apparatus includes a heat source configured to heat an aqueous liquid in contact with the surface to at least about 65° C.
In some embodiments, the cartridge includes a reservoir of liquid having a pH of at least about 10 (e.g., about 10.5 or more). The cartridge can be configured to contact the surface with the liquid (e.g., by actuating a pressure source to move the liquid).
Another aspect of the microfluidic cartridge relates to a retention member, e.g., a plurality of particles such as beads, comprising bound PEI, or poly-lysine, e.g., poly-L-lysine, and related methods and systems. An exemplary method for processing a sample includes contacting a retention member with a mixture that includes providing a mixture including a liquid and an amount of polynucleotide. The retention member may be configured to preferentially retain polynucleotides as compared to polymerase chain reaction inhibitors. Substantially all of the liquid in the mixture can be removed from the retention member. The polynucleotides can be released from the retention member. The polynucleotide may have a size of less than about 7.5 Mbp.
The liquid may be a first liquid, and removing substantially all of the liquid from the retention member may include contacting the retention member with a second liquid.
Contacting the retention member with a second liquid can include actuating a thermally actuated pressure source to apply a pressure to the second liquid. Contacting the retention member with a second liquid can include opening a thermally actuated valve to place the second liquid in fluid communication with the retention member.
The second liquid may have a volume of less than about 50 microliters, and may include a detergent (e.g., SDS).
The retention member may include a surface having a compound configured to bind polynucleotides preferentially to polymerase chain reaction inhibitors (such inhibitors including, for example, hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide).
The surface may include a poly-lysine (e.g., poly-L-lysine and/or poly-D-lysine) or PEI.
Releasing polynucleotides from the retention member may include heating the retention member to a temperature of at least about 50° C. (e.g., at about 65° C.). The temperature may be insufficient to boil the liquid in the presence of the retention member during heating. The temperature may be 100° C. or less (e.g., less than 100° C., about 97° C. or less). The temperature may be maintained for less than about 10 minutes (e.g., for less than about 5 minutes, for less than about 3 minutes). The releasing may be performed without centrifugation of the retention member.
In certain embodiments, PCR inhibitors can be rapidly removed from clinical samples to create a PCR-ready sample. Methods herein therefore may comprise the preparation of a polynucleotide-containing sample that can be substantially free of inhibitors. Such samples may be prepared from, e.g., crude lysates resulting from thermal, chemical, ultrasonic, mechanical, electrostatic, and other lysing techniques. The samples may be prepared without centrifugation. The samples may be prepared using other microfluidic devices or on a larger scale.
The retention member may be used to prepare polynucleotide samples for further processing, such as amplification by polymerase chain reaction. In certain embodiments, more than 90% of a polynucleotide present in a sample may be bound to the retention member, released, and recovered.
In certain embodiments, a polynucleotide may be bound to the retention member, released, and recovered, in less than about 10 minutes (e.g., less than about 7½ minutes, less than about 5 minutes, or less than about 3 minutes).
A polynucleotide may be bound to a retention member, released, and recovered without subjecting the polynucleotide, retention member, and/or inhibitors to centrifugation.
Separating the polynucleotides and inhibitors generally excludes subjecting the polynucleotides, inhibitors, processing region, and/or retention member to sedimentation (e.g., centrifugation).
In various embodiments, the microfluidic cartridge can include a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides. The PCR reagent mixture can be in the form of one or more lyophilized pellets, and the microfluidic network can be configured to contact the PCR pellet with liquid to create a PCR reagent mixture solution.
In various embodiments, the microfluidic cartridge can be configured to couple heat from an external heat source with the PCR reagent mixture and the neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample.
In various embodiments, the PCR reagent mixture can further include a positive control plasmid and a fluorogenic hybridization probe selective for at least a portion of the plasmid.
In various embodiments, the microfluidic cartridge can include a negative control polynucleotide, wherein the microfluidic network can be configured to independently contact each of the neutralized polynucleotide sample and the negative control polynucleotide with the PCR reagent mixture under thermal cycling conditions suitable for independently creating PCR amplicons of the neutralized polynucleotide sample and PCR amplicons of the negative control polynucleotide.
In various embodiments, the microfluidic cartridge can include at least one probe that can be selective for a polynucleotide sequence, wherein the microfluidic cartridge can be configured to contact the neutralized polynucleotide sample or a PCR amplicon thereof with the probe. The probe can be a fluorogenic hybridization probe. The fluorogenic hybridization probe can include a polynucleotide sequence coupled to a fluorescent reporter dye and a fluorescence quencher dye. The PCR reagent mixture can further include a positive control plasmid and a plasmid fluorogenic hybridization probe selective for at least a portion of the plasmid and the microfluidic cartridge can be configured to allow independent optical detection of the fluorogenic hybridization probe and the plasmid fluorogenic hybridization probe.
In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism, for example any organism that employs deoxyribonucleic acid or ribonucleic acid polynucleotides. Thus, the probe can be selective for any organism. Suitable organisms include mammals (including humans), birds, reptiles, amphibians, fish, domesticated animals, farmed animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for components of organisms that employ their own polynucleotides, for example mitochondria. In some embodiments, the probe can be selective for microorganisms, for example, organisms used in food production (for example, yeasts employed in fermented products, molds or bacteria employed in cheeses, and the like) or pathogens (e.g., of humans, domesticated or wild mammals, domesticated or wild birds, and the like). In some embodiments, the probe can be selective for organisms selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.
In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism selected from the group consisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Staphylococcus; Streptococcus (e.g., α, β or γ-hemolytic, Group A, B, C, D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, and E. faecium (formerly S. faecalis, S. durans, S. faecium); nonenterococcal group D streptococci, e.g., S. bovis and S. equines; Streptococci viridans, e.g., S. mutans, S. sanguis, S. salivarius, S. mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus; S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides, N. gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E. rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanovii and L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis, B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella, e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii; Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E. coli, E. coli O157:H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g., S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii), S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S. heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S. saint-paul; Shigella, e.g., subgroups: A, B, C, and D, such as S. flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis, P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia (P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y. pestis, Y. enterocolitica; Haemophilus, e.g., H. influenzae, H. parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus, B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis; Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei, Burkholderia mallei, Burkholderia cepacia and Stenotrophomorias maltophilia; Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori (Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus, V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and the nonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C. difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides, e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaninogenica; genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum, T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspecies pallidum; genus Borrelia, e.g., B. burgdorferi; genus Leptospira; Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus; Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. avium M. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, the M. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M. asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M. malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma, e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae, M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M. genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T. vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H. capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides, e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides, e.g., P. brasiliensis; Penicillium, e.g., P. marneffei; Sporothrix, e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus; diseases caused by Bipolaris, Cladophialophora, Cladosporium, Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis, Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T. beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P. falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa of the genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L. major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis; Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria or Acanthamoeba; Entamoeba histolytica; Giardia lamblia; genus Cryptosporidium, e.g., C. parvum; Isospora belli; Cyclospora cayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostoma duodenale or Necator americanus; Strongyloides stercoralis Toxocara, e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella, e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea; Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocerca volvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, e.g., S. japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium; Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis; Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothrium latum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E. granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses, coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3, and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2; varicella-zoster virus; human T-lymphotrophic virus (type I and type II); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis (alphaviruses e.g., Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis); Viral hemorrhagic fevers (filoviruses, e.g., Ebola, Marburg, and arenaviruses, e.g., Lassa, Machupo); Smallpox (variola); retroviruses e.g., human immunodeficiency viruses 1 and 2; human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, and 35.
In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism selected from the group consisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Acinetobacter Baumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcus faecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus, methecillin-resistant Staphylococcus aureus (MRSA), Streptococcus viridans, Listeria monocytogenes, Enterococcus spp., Streptococcus Group B, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus epidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilus influenzae, Neisseria gonorrhoeee, Moraxella catarrahlis, Salmonella sps., Chlamydia trachomatis, Peptostreptococcus productus, Peptostreptococcus anaerobius, Lactobacillus fermentum, Eubacterium lentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacter spp., Salmonella spp., smallpox (variola major), Yersina Pestis, Herpes Simplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II).
In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of Group B Streptococcus.
The technology herein also comprises a microfluidic cartridge having a component for inhibiting motion of fluid. The component comprises a channel, a first mass of a thermally responsive substance (TRS) disposed on a first side of the channel, a second mass of a TRS disposed on a second side of the channel opposite the first side of the channel, a gas pressure source associated with the first mass of the TRS. Actuation of the gas pressure source drives the first mass of the TRS into the second mass of the TRS and obstructs the channel.
The microfluidic cartridge can include a second gas pressure source associated with the second mass of the TRS. Actuation of the second gas pressure source drives the second mass of TRS into the first mass of TRS. At least one (e.g., both) of the first and second masses of TRS may be a wax.
Another aspect of the microfluidic cartridge includes a component for obstructing a channel of a microfluidic cartridge. A mass of a TRS can be heated and driven across the channel (e.g., by gas pressure) into a second mass of TRS. The second mass of TRS may also be driven (e.g., by gas pressure) toward the first mass of TRS.
Another aspect of the microfluidic cartridge is an actuator. The actuator includes a channel, a chamber connected to the channel, at least one reservoir of encapsulated liquid disposed in the chamber, and a gas surrounding the reservoir within the chamber. Heating the chamber expands the reservoir of encapsulated liquid and pressurizes the gas. Typically the liquid has a boiling point of about 90° C. or less. The liquid may be a hydrocarbon having about 10 carbon atoms or fewer. The liquid may be encapsulated by a polymer.
An actuator may include multiple reservoirs of encapsulated liquid disposed in the chamber. The multiple reservoirs may be dispersed within a solid (e.g., a wax). The multiple reservoirs may be disposed within a flexible enclosure (e.g., a flexible sack).
Another aspect of the microfluidic cartridge includes pressurizing a gas within a chamber of the device to create a gas pressure sufficient to move a liquid within a channel of the microfluidic device. Pressurizing the gas typically expands at least one reservoir of encapsulated liquid disposed within the chamber. Expanding the at least one reservoir can include heating the chamber. Pressurizing the gas can include expanding multiple reservoirs of encapsulated liquid.
Another aspect of a microfluidic cartridge for use herein includes combining (e.g., mixing) first and second liquid volumes. The device includes a mass of a temperature responsive substance (TRS) that separates first and second channels of the device. The device can be configured to move a first liquid along the first channel so that a portion (e.g., a medial portion) of the first liquid can be adjacent the TRS, and to move a second liquid along the second channel so that a portion (e.g., a medial portion) of second liquid can be adjacent the TRS. A heat source can be actuated to move the TRS (e.g., by melting, dispersing, fragmenting). The medial portions of the first and second liquids typically combine without being separated by a gas interface. Typically, only a subset of the first liquid and a subset of the second liquid can be combined. The liquids mix upon being moved along a mixing channel. The liquids when combined should be moved at least two droplet lengths to get good mixing by interlayering and transverse diffusion (perpendicular to the length of the microchannel) without having to rely on longitudinal diffusion alone for mixing (see, also, e.g., “Mathematical modeling of drop mixing in a slit-type microchannel”, K Handique, et al., J. Micromech. Microeng., 11 548-554, (2001), incorporated herein by reference). By moving the combined drop by a drop length, the liquid in the middle of the receding drop is caused to move to the front of the leading drop and then towards the channel wall. At the receding end of the drop, liquid moves from the wall towards the center of the drop. This motion of the drop results in interlayering between the two liquids. Further interlayering can be achieved by repeating the method additional times, such as over further drop lengths.
The microfluidic cartridge further includes a lyophilized reagent particle. In some embodiments, the lyophilized particles include multiple smaller particles each having a plurality of ligands that preferentially associate with polynucleotides as compared to PCR inhibitors. The lyophilized particles can also (or alternatively) include lysing reagents (e.g., enzymes) configured to lyse cells to release polynucleotides. The lyophilized particles can also (or alternatively) include enzymes (e.g., proteases) that degrade proteins.
Cells can be lysed by combining a solution of the cells, e.g., in a microfluidic droplet, with the lyophilized particles thereby reconstituting the particles. The reconstituted lysing reagents lyse the cells. The polynucleotides associate with ligands of the smaller particles. During lysis, the solution may be heated (e.g., radiatively using a lamp such as a heat lamp, or by a contact heat source.
In some embodiments, lyophilized particles include reagents (e.g., primers, control plasmids, polymerase enzymes) for performing PCR.
Another aspect of the microfluidic cartridge includes a liquid reservoir capable of holding a liquid (e.g., a solvent, a buffer, a reagent, or combination thereof). In general, the reservoir can have one or more of the following features, as further described in international application publication no. WO2006/079082.
The reservoir can include a wall that can be manipulated (e.g., pressed or depressed) to decrease a volume within the reservoir. For example, the reservoir can include a piercing member (e.g., a needle-like or otherwise pointed or sharp member) that ruptures another portion of the reservoir (e.g., a portion of the wall) to release liquid. The piercing member can be internal to the reservoir such that the piercing member ruptures the wall from an inner surface of the reservoir (e.g., wall) outwards.
In general, the wall resists passage of liquid or vapor therethrough. In some embodiments, the wall lacks stretchiness. The wall may be flexible. The wall may be, e.g., a metallic layer, e.g., a foil layer, a polymer, or a laminate including a combination thereof. The wall may be formed by vacuum formation (e.g., applying a vacuum and heat to a layer of material to draw the layer against a molding surface). The molding surface may be concave such that the wall can be provided with a generally convex surface.
Exemplary liquids held by the reservoir include water and aqueous solutions including one or more salts (e.g., magnesium chloride, sodium chloride, Tris buffer, or combination thereof). The reservoir can retain the liquid (e.g., without substantial evaporation thereof) for a period of time (e.g., at least 6 months or at least a year). In some embodiments, less than 10% (e.g., less than about 5%) by weight of the liquid evaporates over a year.
The piercing member may be an integral part of a wall of the reservoir. For example, the reservoir can include a wall having an internal projection, which may be in contact with liquid in the reservoir. The reservoir also includes a second wall opposite the piercing member. During actuation, the piercing member can be driven through the second wall (e.g., from the inside out) to release liquid.
In some embodiments, a maximum amount of liquid retained by a reservoir can be less than about 1 ml. For example, a reservoir may hold about 500 microliters or less (e.g., 300 microliters or less). Generally, a reservoir holds at least about 25 microliters (e.g., at least about 50 microliters). The reservoir can introduce within about 10% of the intended amount of liquid (e.g., 50±5 μl).
The reservoir can deliver a predetermined amount of liquid that can be substantially air-free (e.g., substantially gas-free). Upon introduction of the liquid, the substantially air and/or gas free liquid produces few or no bubbles large enough to obstruct movement of the liquid within the microfluidic device. Use of a piercing member internal to the reservoir can enhance an ability of the reservoir to deliver substantially air and/or gas free liquids.
In some embodiments, the reservoir can be actuated to release liquid by pressing (e.g., by one's finger or thumb or by mechanical pressure actuation). The pressure may be applied directly to a wall of the reservoir or to a plunger having a piercing member. In various embodiments, minimal pressure can be required to actuate the reservoir. An automated system can be used to actuate (e.g., press upon) a plurality of reservoirs simultaneously or in sequence.
Actuation of the reservoir may include driving a piercing member through a wall of the reservoir. In some embodiments, the reservoir does not include a piercing member. Instead, internal pressure generated within the reservoir ruptures a wall of the reservoir allowing liquid to enter the microfluidic device.
Upon actuating a reservoir to introduce liquid into the microfluidic device, liquid generally does not withdraw back into the reservoir. For example, upon actuation, the volume of the reservoir may decrease to some minimum but generally does not increase so as to withdraw liquid back into the reservoir. For example, the reservoir may stay collapsed upon actuation. In such embodiments, the flexible wall may be flexible but lack hysteresis, elasticity, or stretchiness. Alternatively or in combination, the reservoir may draw in air from a vent without withdrawing any of the liquid.
The reservoir preserves the reactivity and composition of reagents therein (e.g., the chemicals within the reservoir may exhibit little or no change in reactivity over 6 months or a year).
The flexible wall of the reservoir can limit or prevent leaching of chemicals therethrough. The reservoir can be assembled independently of a microfluidic cartridge and then secured to the microfluidic cartridge.
Exemplary Microfluidic Cartridge for Processing Polynucleotides
Referring to
Fabrication of Cartridge
Microfluidic cartridge 200 can be fabricated as desired. Typically, layers 205, 207, and 209 can be formed of a polymeric material. Components of network 201 can typically be formed by molding (e.g., by injection molding) layers 207, 209. Layer 205 can typically be a flexible polymeric material (e.g., a laminate) that can be secured (e.g., adhesively and/or thermally) to layer 207 to seal components of network 201. Layers 207 and 209 may be secured to one another using adhesive. Other methods of cartridge fabrication suitable for application herein can be found described in U.S. provisional patent application Ser. No. 60/859,284, filed Nov. 14, 2006, and incorporated herein by reference in its entirety.
Microfluidic Network
An exemplary arrangement of components of network 201 is as follows, as further described in U.S. Patent Application Publication No. 2006/0166233, incorporated herein by reference.
Network 201 includes an inlet 202 by which sample material can be introduced to the network and an output 236 by which a processed sample can be removed (e.g., expelled by or extracted from) network 201. A channel 204 extends between inlet 202 and a junction 255. A valve 206 can be positioned along channel 204. A reservoir channel 240 extends between junction 255 and an actuator 244. Gates 242 and 246 can be positioned along channel 240. A channel 257 extends between junction 255 and a junction 259. A valve 208 can be positioned along channel 257. A reservoir channel 246 extends between junction 259 and an actuator 248. Gates 250 and 252 can be positioned along channel 246. A channel 261 extends between junction 259 and a junction 263. A valve 210 and a hydrophobic vent 212 can be positioned along channel 261. A channel 256 extends between junction 263 and an actuator 254. A gate 258 can be positioned along channel 256.
A channel 214 extends between junction 263 and a processing chamber 220, which has an inlet 265 and an outlet 267. A channel 228 extends between processing chamber outlet 267 and a waste reservoir 232. A valve 234 can be positioned along channel 228. A channel 230 extends between processing chamber outlet 267 and output 236.
Particular components of microfluidic network 201 are further described as follows.
Processing Chamber
Referring also to
A filter 219 prevents particles 218 from passing downstream of processing region 220. A channel 287 connects filter 219 with outlet 267. Filter 219 has a surface area within processing region 220 that can be larger than the cross-sectional area of inlet 265. For example, in some embodiments, the ratio of the surface area of filter 219 within processing chamber 220 to the cross-sectional area of inlet 265 (which cross-sectional area is typically about the same as the cross-sectional area of channel 214) can be at least about 5 (e.g., at least about 10, at least about 20, at least about 30). In some embodiments, the surface area of filter 219 within processing region 220 can be at least about 1 mm2 (e.g., at least about 2 mm2, at least about 3 mm2). In some embodiments, the cross-sectional area of inlet 265 and/or channel 214 can be about 0.25 mm2 or less (e.g., about 0.2 mm2 or less, about 0.15 mm2 or less, about 0.1 mm2 or less). The larger surface area presented by filter 219 to material flowing through processing chamber 220 helps prevent clogging of the processing region while avoiding significant increases in the void volume (described hereinbelow) of the processing region.
Particles 218 can be modified with at least one ligand that retains polynucleotides (e.g., preferentially as compared to inhibitors). Typically, the ligands retain polynucleotides from liquids having a pH about 9.5 or less (e.g., about 9.0 or less, about 8.75 or less, about 8.5 or less). As a sample solution moves through processing chamber 220, polynucleotides can be retained while the liquid and other solution components (e.g., inhibitors) can be less retained (e.g., not retained) and exit the processing region. In general, the ligands release polynucleotides when the pH can be about 10 or greater (e.g., about 10.5 or greater, about 11.0 or greater). Consequently, polynucleotides can be released from the ligand modified particles into the surrounding liquid.
Exemplary ligands on particles 218 include, for example, polyamides (e.g., poly-cationic polyamides such as poly-L-lysine, poly-D-lysine, poly-DL-ornithine) and PEI. Other ligands include, for example, intercalators, poly-intercalators, minor groove binders polyamines (e.g., spermidine), homopolymers and copolymers comprising a plurality of amino acids, and combinations thereof. In some embodiments, the ligands have an average molecular weight of at least about 5,000 Da (e.g., at least about 7,500 Da, of at least about 15,000 Da). In some embodiments, the ligands have an average molecular weight of about 50,000 Da or less (e.g., about 35,000, or less, about 27,500 Da or less). In some embodiments, the ligand can be a poly-lysine ligand attached to the particle surface by an amide bond.
In certain embodiments, the ligands on the particles 218 can be resistant to enzymatic degradation, such as degradation by protease enzymes (e.g., mixtures of endo- and exo-proteases such as pronase) that cleave peptide bonds. Exemplary protease resistant ligands include, for example, poly-D-lysine and other ligands that can be enantiomers of ligands susceptible to enzymatic attack.
Particles 218 can typically be formed of a material to which the ligands can be associated. Exemplary materials from which particles 218 can be formed include polymeric materials that can be modified to attach a ligand. Typical polymeric materials provide or can be modified to provide carboxylic groups and/or amino groups available to attach ligands. Exemplary polymeric materials include, for example, polystyrene, latex polymers (e.g., polycarboxylate coated latex), polyacrylamide, polyethylene oxide, and derivatives thereof. Polymeric materials that can used to form particles 218 are described in U.S. Pat. No. 6,235,313 to Mathiowitz et al., which patent is incorporated herein by reference. Other materials include glass, silica, agarose, and amino-propyl-tri-ethoxy-silane (APES) modified materials.
Exemplary particles that can be modified with suitable ligands include carboxylate particles (e.g., carboxylate modified magnetic beads (Sera-Mag Magnetic Carboxylate modified beads, Part #3008050250, Seradyn) and Polybead carboxylate modified microspheres available from Polyscience, catalog no. 09850). In some embodiments, the ligands include poly-D-lysine and the beads comprise a polymer (e.g., polycarboxylate coated latex). In other embodiments, the ligands include PEI.
In general, the ratio of mass of particles to the mass of polynucleotides retained by the particles can be no more than about 25 or more (e.g., no more than about 20, no more than about 10). For example, in some embodiments, about 1 gram of particles retains about 100 milligrams of polynucleotides.
Typically, the total volume of processing chamber 220 (including particles 218) between inlet 265 and filter 219 can be about 15 microliters or less (e.g., about 10 microliters or less, about 5 microliters or less, about 2.5 microliters or less, about 2 microliters or less). In an exemplary embodiment, the total volume of processing region 220 can be about 2.3 microliters. In some embodiments, particles 218 occupy at least about 10 percent (e.g., at least about 15 percent) of the total volume of processing region 220. In some embodiments, particles 218 occupy about 75 percent or less (e.g., about 50 percent or less, about 35 percent or less) of the total volume of processing chamber 220.
In some embodiments, the volume of processing chamber 220 that can be free to be occupied by liquid (e.g., the void volume of processing chamber 220 including interstices between particles 218) can be about equal to the total volume minus the volume occupied by the particles. Typically, the void volume of processing region 220 can be about 10 microliters or less (e.g., about 7.5 microliters or less, about 5 microliters or less, about 2.5 microliters or less, about 2 microliters or less). In some embodiments, the void volume can be about 50 nanoliters or more (e.g., about 100 nanoliters or more, about 250 nanoliters or more). For example, in an exemplary embodiment, the total volume of processing chamber 220 can be about 2.3 microliters, the volume occupied by particles can be about 0.3 microliters, and the volume free to be occupied by liquid (void volume) can be about 2 microliters.
Particles 218 typically have an average diameter of about 20 microns or less (e.g., about 15 microns or less, about 10 microns or less). In some embodiments, particles 218 have an average diameter of at least about 4 microns (e.g., at least about 6 microns, at least about 8 microns).
In some embodiments, a volume of channel 287 between filter 219 and outlet 267 can be substantially smaller than the void volume of processing chamber 220. For example, in some embodiments, the volume of channel 287 between filter 219 and outlet 267 can be about 35% or less (e.g., about 25% or less, about 20% or less) of the void volume. In an exemplary embodiment, the volume of channel 287 between filter 219 and outlet 267 can be about 500 nanoliters.
The particle density can typically be at least about 108 particles per milliliter (e.g., about 109 particles per milliliter). For example, a processing region with a total volume of about 1 microliter may include about 103 beads.
Filter 219 typically has pores with a diameter smaller than the diameter of particles 218. In an exemplary embodiment, filter 219 has pores having an average width of about 8 microns, where particles 218 have an average diameter of about 10 microns.
In some embodiments, at least some (e.g., all) of the particles can be magnetic. In alternative embodiments, few (e.g., none) of the particles are magnetic.
In some embodiments, at least some (e.g., all) the particles can be solid. In some embodiments, at least some (e.g., all) the particles can be porous (e.g., the particles may have channels extending at least partially within them).
Further components that may be found in microfluidic network 201 are as follows.
Channels
Channels of microfluidic network 201 typically have at least one sub-millimeter cross-sectional dimension. For example, channels of network 201 may have a width and/or a depth of about 1 mm or less (e.g., about 750 microns or less, about 500 microns, or less, about 250 microns or less).
Valves
A valve can be a component that has a normally open state allowing material to pass along a channel from a position on one side of the valve (e.g., upstream of the valve) to a position on the other side of the valve (e.g., downstream of the valve). Upon actuation, the valve transitions to a closed state that prevents material from passing along the channel from one side of the valve to the other. For example, in
A mass of TRS can be an essentially solid mass or an agglomeration of smaller particles that cooperate to obstruct the passage. Examples of TRS's include a eutectic alloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, and combinations thereof. The first and second temperatures can be insufficiently high to damage materials, such as polymer layers of cartridge 200. Generally, the second temperature can be less than about 90° C. and the first temperature can be less than the second temperature (e.g., about 70° C. or less).
A gate can be a component that can have a closed state that does not allow material to pass along a channel from a position on one side of the gate to another side of the gate, and an open state that does allow material to pass along a channel from a position on one side of the gate to another side of the gate. Actuation of an open gate can transition the gate to a closed state in which material is not permitted to pass from one side of the gate (e.g., upstream of the gate) to the other side of the gate (e.g., downstream of the gate). Upon actuation, a closed gate can transition to an open state in which material is permitted to pass from one side of the gate (e.g., upstream of the gate) to the other side of the gate (e.g., downstream of the gate). For example, gate 242 in
In various embodiments, a microfluidic network 201 can include a narrow gate 380 as shown in
In various embodiments, the gate can be configured to minimize the effective area or footprint of the gate within the network, such as bent gate 392 as shown in
In the microfluidic cartridge of
The portion of channel 247 between gates 250 and 252 form a fluid reservoir 281 configured like reservoir 279 to hold a liquid (e.g., a solution) with limited or no evaporation. During operation of cartridge 200, the liquid of reservoir 281 can typically be used as a release liquid into which polynucleotides that had been retained by particles 218 can be released. An exemplary release liquid can be a hydroxide solution (e.g., a NaOH solution) having a concentration of, for example, between about 2 mM hydroxide (e.g., about 2 mM NaOH) and about 500 mM hydroxide (e.g., about 500 mM NaOH). In some embodiments, liquid in reservoir 281 can be a hydroxide solution having a concentration of about 25 mM or less (e.g., a hydroxide concentration of about 15 mM).
Reservoirs 279, 281 typically each independently hold at least about 0.375 microliters of liquid (e.g., at least about 0.750 microliters, at least about 1.25 microliters, at least about 2.5 microliters). In some embodiments, reservoirs 279, 281 each independently hold about 7.5 microliters or less of liquid (e.g., about 5 microliters or less, about 4 microliters or less, about 3 microliters or less).
Actuators
An actuator can be a component that provides a gas pressure that can move material (e.g., sample material and/or reagent material) between one location in a network e.g., network 201, and another location. For example, referring to
In one embodiment, shown in
When the TEM can be heated (e.g., to a temperature of at least about 50° C. (e.g., to at least about 75° C., or at least about 90° C.)), the liquid vaporizes and increases the volume of each sealed reservoir and of mass 273. Carrier 277 softens allowing mass 273 to expand. Typically, the TEM can be heated to a temperature of less than about 150° C. (e.g., about 125° C. or less, about 110° C. or less, about 100° C. or less) during actuation. In some embodiments, the volume of the TEM expands by at least about 5 times (e.g., at least about 10 times, at least about 20 times, at least about 30 times).
Vents
A hydrophobic vent (e.g., vent 212) can be a structure that permits gas to exit a channel while limiting (e.g., preventing) liquid from exiting the channel. Typically, hydrophobic vents include a layer of porous hydrophobic material (e.g., a porous filter such as a porous hydrophobic membrane from Osmonics) that defines a wall of the channel. As described hereinbelow, hydrophobic vents can be used to position a microdroplet of sample at a desired location within network 201.
The hydrophobic vents of the present technology are preferably constructed so that the amount of air that escapes through them can be maximized while minimizing the volume of the channel below the vent surface. Accordingly, it is preferable that the vent can be constructed so as to have a hydrophobic membrane of large surface area and a shallow cross section of the microchannel below the vent surface.
Hydrophobic vents typically have a length of at least about 2.5 mm (e.g., at least about 5 mm, at least about 7.5 mm) along a channel. The length of the hydrophobic vent can typically be at least about 5 times (e.g., at least about 10 times, at least about 20 times) larger than a depth of the channel within the hydrophobic vent. For example, in some embodiments, the channel depth within the hydrophobic vent can be about 300 microns or less (e.g., about 250 microns or less, about 200 microns or less, about 150 microns or less).
The depth of the channel within the hydrophobic vent can typically be about 75% or less (e.g., about 65% or less, about 60% or less) of the depth of the channel upstream and downstream of the hydrophobic vent. For example, in some embodiments the channel depth within the hydrophobic vent can be about 150 microns and the channel depth upstream and downstream of the hydrophobic vent can be about 250 microns.
A width of the channel within the hydrophobic vent can typically be at least about 25% wider (e.g., at least about 50% wider) than a width of the channel upstream from the vent and downstream from the vent. For example, in an exemplary embodiment, the width of the channel within the hydrophobic vent can be about 400 microns and the width of the channel upstream and downstream from the vent can be about 250 microns.
In use, cartridge 200 can typically be thermally associated with an array of heat sources configured to operate various components (e.g., valves, gates, actuators, and processing region 220) of the cartridge. In some embodiments, the heat sources can be controlled by a processor in a system such as that further described herein, which operates to receive and to monitor the cartridge during use. The processor (e.g., a microprocessor) is configured to actuate the heat sources individually and at different times, according to a desired protocol. Processors configured to operate microfluidic cartridges, suitable for use or for modification for use herein, are described in U.S. application Ser. No. 09/819,105, filed Mar. 28, 2001 (now U.S. Pat. No. 7,010,391), which patent is incorporated herein by reference. In other embodiments, the heat sources can be integral with the cartridge itself.
Cartridge 200 may be operated as follows. Valves of network 201 can be fabricated in an open state. Gates of network 201 can be fabricated in a closed state. A fluid sample, such as a biological sample as further described herein, comprising polynucleotides can be introduced to network 201 via inlet 202. For example, sample can be introduced with a syringe having a Luer fitting. The syringe provides pressure to initially move the sample within network 201. Sample passes along channels 204, 257, 261, and 214 to inlet 265 of processing region 220. The sample passes through processing region 220, exits via outlet 267, and passes along channel 228 to waste chamber 232. When the trailing edge (e.g., the upstream liquid-gas interface) of the sample reaches hydrophobic vent 212, pressure provided by the introduction device (e.g., the syringe) can be released from network 201 stopping further motion of the sample.
Typically, the amount of sample introduced can be about 500 microliters or less (e.g., about 250 microliters or less, about 100 microliters or less, about 50 microliters or less, about 25 microliters or less, about 10 microliters or less). In some embodiments, the amount of sample can be about 2 microliters or less (e.g., about 0.5 microliters or less).
Polynucleotides entering processing region 220 pass through interstices between the particles 218. Polynucleotides of the sample contact retention member 216 and can be preferentially retained as compared to liquid of the sample, and certain other sample components (e.g., inhibitors). Typically, retention member 220 retains at least about 50% of polynucleotides (e.g., at least about 75%, at least about 85%, at least about 90%) of the polynucleotides present in the sample that entered processing region 220. Liquid of the sample and inhibitors present in the sample exit the processing region 220 via outlet 267 and enter waste chamber 232. Processing region 220 can typically be at a temperature of about 50° C. or less (e.g., 30° C. or less) during introduction of the sample.
Processing continues by washing retention member 216 with liquid of reservoir 279 to separate remaining inhibitors from polynucleotides retained by retention member 216. To wash retention member 216, valve 206 can be closed and gates 242, 246 of first reservoir 240 can be opened. Actuator 244 can be actuated to move wash liquid within reservoir 279 along channels 257, 261, and 214, through processing region 220, and into waste reservoir 232. The wash liquid moves sample that may have remained within channels 204, 257, 261, and 214 through the processing region and into waste chamber 232. Once the trailing edge of the wash liquid reaches vent 212, the gas pressure generated by actuator 244 can be vented and further motion of the liquid can be stopped.
The volume of wash liquid moved by actuator 244 through processing region 220 can typically be at least about 2 times the void volume of processing region 220 (e.g., at least about 3 times the void volume) and can be about 10 times the void volume or less (e.g., about 5 times the void volume or less). Processing region can typically be at a temperature of about 50° C. or less (e.g., 30° C. or less) during washing. Exemplary wash fluids include liquids described with respect to reservoirs 279 and 281, herein.
Processing continues by releasing polynucleotides from retention member 216. Typically, wash liquid from reservoir 279 can be replaced with release liquid (e.g., an hydroxide solution) from reservoir 281 before releasing the polynucleotides. Valve 208 can be closed and gates 250, 252 can be opened. Actuator 248 can be actuated, thereby moving release liquid within reservoir 281 along channels 261, 214 and into processing region 220 and in contact with retention member 216. When the trailing edge of release liquid from reservoir 281 reaches hydrophobic vent 212, pressure generated by actuator 248 can be vented stopping the further motion of the liquid. The volume of liquid moved by actuator 248 through processing region 220 can typically be at least about equal to the void volume of the processing region 220 (e.g., at least about 2 times the void volume) and can be about 10 times the void volume or less (e.g., about 5 times the void volume or less).
Once retention member 216 with retained polynucleotides has been contacted with liquid from reservoir 281, a releasing step can typically be performed. Typically, the releasing includes heating release liquid present within processing region 216. Generally, the liquid can be heated to a temperature insufficient to boil liquid in the presence of the retention member. In some embodiments, the temperature can be 100° C. or less (e.g., less than 100° C., about 97° C. or less). In some embodiments, the temperature can be about 65° C. or more (e.g., about 75° C. or more, about 80° C. or more, about 90° C. or more). In some embodiments, the temperature is maintained for about 1 minute or more (e.g., about 2 minutes or more, about 5 minutes or more, about 10 minutes or more). In some embodiments, the temperature can be maintained for about 30 minutes (e.g., about 15 minutes or less, about 10 minutes or less, about 5 minutes or less). In an exemplary embodiment, processing region 220 can be heated to between about 65 and 90° C. (e.g., to about 70° C.) for between about 1 and 7 minutes (e.g., for about 2 minutes). Such temperatures and times vary according to the sample and can be chosen accordingly by one of ordinary skill in the art.
The polynucleotides can be released into the liquid present in the processing region 220 (e.g., the polynucleotides can typically be released into an amount of release liquid having a volume about the same as the void volume of the processing region 220). Typically, the polynucleotides can be released into about 10 microliters or less (e.g., about 5 microliters or less, about 2.5 microliters or less) of liquid.
In certain embodiments, the ratio of the volume of original sample moved through the processing region 220 to the volume of liquid into which the polynucleotides can be released can be at least about 10 (e.g., at least about 50, at least about 100, at least about 250, at least about 500, at least about 1000). In some embodiments, polynucleotides from a sample having a volume of about 2 ml can be retained within the processing region, and released into about 4 microliters or less (e.g., about 3 microliters or less, about 2 microliters or less, about 1 microliter or less) of liquid.
The liquid into which the polynucleotides can be released typically includes at least about 50% (e.g., at least about 75%, at least about 85%, at least about 90%) of the polynucleotides present in the sample that entered processing region 220. The concentration of polynucleotides present in the release liquid may be higher than in the original sample because the volume of release liquid can typically be less than the volume of the original liquid sample moved through the processing region. For example the concentration of polynucleotides in the release liquid may be at least about 10 times greater (e.g., at least about 25 times greater, at least about 100 times greater) than the concentration of polynucleotides in the sample introduced to cartridge 200. The concentration of inhibitors present in the liquid into which the polynucleotides can be released can generally be less than concentration of inhibitors in the original fluid sample by an amount sufficient to increase the amplification efficiency for the polynucleotides.
The time interval between introducing the polynucleotide containing sample to processing region 220 and releasing the polynucleotides into the release liquid can typically be about 15 minutes or less (e.g., about 10 minutes or less, about 5 minutes or less).
Liquid including the released polynucleotides may be removed from the processing region 220 as follows. Valves 210 and 234 can be closed. Gates 238 and 258 can be opened. Actuator 254 can be actuated to generate pressure that moves liquid and polynucleotides from processing region 220, into channel 230, and toward outlet 236. The liquid with polynucleotides can be removed using, for example, a syringe or automated sampling device. Depending upon the liquid in contact with retention member 216 during polynucleotide release, the solution with released polynucleotide may be neutralized with an amount of buffer (e.g., an equal volume of 25-50 mM Tris-HCl buffer pH 8.0).
While releasing the polynucleotides has been described as including a heating step, the polynucleotides may be released without heating. For example, in some embodiments, the liquid of reservoir 281 has an ionic strength, pH, surfactant concentration, composition, or combination thereof that releases the polynucleotides from the retention member at ambient temperature, without need for additional heating.
While the polynucleotides have been described as being released into a single volume of liquid present within processing region 220, other configurations can be used. For example, polynucleotides may be released with the concomitant (stepwise or continuous) introduction of fluid into and/or through processing region 220. In such embodiments, the polynucleotides may be released into liquid having a volume of about 10 times or less (e.g., about 7.5 times or less, about 5 times or less, about 2.5 times or less, about 2 times or less) than the void volume of the processing region 220.
While reservoirs 279, 281 have been described as holding liquids between first and second gates, other configurations can be used. For example, liquid for each reservoir may be held within a pouch (e.g., a blister pack, as further described herein) isolated from network 201 by a generally impermeable membrane. The pouch can be configured so that a user can rupture the membrane driving liquid into reservoirs 279, 281 where actuators 244, 248 can move the liquid during use.
While processing regions have been described as having microliter scale dimensions, other dimensions can be used. For example, processing regions with surfaces (e.g., particles) configured to preferentially retain polynucleotides as opposed to inhibitors may have large volumes (e.g., many tens of microliters or more, at least about 1 milliliter or more). In some embodiments, the processing region has a bench-top scale.
While processing region 220 has been described as having a retention member formed of multiple surface-modified particles, other configurations can be used. For example, in some embodiments, processing region 220 includes a retention member configured as a porous member (e.g., a filter, a porous membrane, or a gel matrix) having multiple openings (e.g., pores and/or channels) through which polynucleotides pass. Surfaces of the porous member can be modified to preferentially retain polynucleotides. Filter membranes available from, for example, Osmonics, can be formed of polymers that may be surface-modified and used to retain polynucleotides within processing region 220. In some embodiments, processing region 220 includes a retention member configured as a plurality of surfaces (e.g., walls or baffles) through which a sample passes. The walls or baffles can be modified to preferentially retain polynucleotides.
While processing region 220 has been described as a component of a microfluidic network, other configurations can be used. For example, in some embodiments, the retention member can be removed from a processing region for processing elsewhere. For example, the retention member may be contacted with a mixture comprising polynucleotides and inhibitors in one location and then moved to another location at which the polynucleotides can be removed from the retention member.
While reservoirs 275 have been shown as dispersed within a carrier, other configurations may be used. For example, reservoirs 275 can be encased within a flexible enclosure (e.g., a membrane, for example, an enclosure such as a sack). In some embodiments, reservoirs can be loose within chamber 272. In such embodiments, actuator 244 may include a porous member having pores too small to permit passage of reservoirs 275 but large enough to permit gas to exit chamber 272.
Exemplary Microfluidic Cartridge Having a Lysing Chamber
Further microfluidic cartridge with various components are described in U.S. provisional application No. 60/553,553 filed Mar. 17, 2004 by Parunak, et al., and patent application publication no. 2005-0084424, which applications are incorporated herein by reference.
While microfluidic cartridges have been described that are configured to receive polynucleotides already released from cells, microfluidic cartridges for use herein can also be configured to release polynucleotides from cells (e.g., by lysing the cells). For example, referring to
Network 304 can be substantially defined between layers L2 and L3 but extends in part between all three layers L1-L3. Microfluidic network 304 includes various microfluidic components as further described herein, including channels Ci, valves Vi, double valves V′i, gates Gi, mixing gates MGi, vents Hi, gas actuators (e.g., pumps) Pi, a first processing region B1, a second processing region B2, detection zones Di, air vents AVi, and waste zones Wi.
Components of network 304 can typically be thermally actuated. As seen in
Further components of exemplary microfluidic cartridge 300 are as follows.
Air Vents
Air vents AVi can allow gas (e.g., air) displaced by the movement of liquids within network 304 to be vented so that pressure buildup does not inhibit desired movement of the liquids. For example, air vent AV2 permits liquid to move along channel C14 and into channel C16 by venting gas downstream of the liquid through vent AV2.
Valves
Valves Vi can have a normally open state allowing material to pass along a channel from a position on one side of the valve (e.g., upstream of the valve) to a position on the other side of the valve (e.g., downstream of the valve). The valves Vi can have a similar structure to valves of microfluidic cartridge 200, as further described herein.
As seen in
The TRS masses 314, 316 and chambers 318, 320 of double valve Vi′ can be in thermal contact with a corresponding heat source HV11′ of heat source network 312. Actuating heat source HV11′ causes TRS masses 314, 316 to transition to a more mobile second state (e.g., a partially melted state) and increases the pressure of gas within chambers 318, 320. The gas pressure drives TRS masses 314, 316 across channel C11 and closes valve HV11′ (
Returning to
As seen in
Actuators
Actuators Pi can provide a gas pressure to move material (e.g., sample material and/or reagent material) between one location of network 304 and another location. Actuators Pi can be similar in form to actuators of cartridge 200. For example, each actuator Pi includes a chamber with a mass 273 of TEM that can be heated to pressurize gas within the chamber. Each actuator Pi includes a corresponding gate Gi (e.g., gate G2 of actuator P1) that prevents liquid from entering the chamber of the actuator. The gate can typically be actuated (e.g., opened) to allow pressure created in the chamber of the actuator to enter the microfluidic network.
Waste Chambers
Waste chambers Wi can receive waste (e.g., overflow) liquid resulting from the manipulation (e.g., movement and/or mixing) of liquids within network 304. Typically, each waste chamber Wi has an associated air vent that allows gas displaced by liquid entering the chamber to be vented.
Processing Regions
First processing region B1 of network 304 can be a component that allows polynucleotides to be concentrated and/or separated from inhibitors of a sample. Processing region B1 can be configured and operated as processing region 220 of cartridge 200. In some embodiments, first processing region B1 includes a retention member (e.g., multiple particles (e.g., microspheres or beads), a porous member, multiple walls) having at least one surface modified with one or more ligands as described for processing region 220. For example, the ligand can include one or more polyamides (e.g., poly-cationic polyamides such as poly-L-lysine, poly-D-lysine, poly-DL-ornithine), or polyethyleneimine. In some embodiments, particles of the retention member can be disposed in lysing chamber 302 and can be moved into processing region B1 along with sample material.
Second processing region B2 can be a component that allows material (e.g., sample material) to be combined with compounds (e.g., reagents) for determining the presence of one or more polynucleotides. In some embodiments, the compounds include one or more PCR reagents (e.g., primers, control plasmids, and polymerase enzymes).
Lyophilized Particles
In some embodiments, the compounds for determining the presence of one or more polynucleotides can be stored within a processing region such as B2 as one or more lyophilized particles (e.g., pellets). The particles generally have a room temperature (e.g., about 20° C.) shelf-life of at least about 6 months (e.g., at least about 12 months). Liquid entering the second processing region B2 dissolves (e.g., reconstitutes) the lyophilized compounds.
Typically, the lyophilized particle(s) of processing region B2 have an average volume of about-5 microliters or less (e.g., about 4 microliters or less, about 3 microliters or less, about 2 microliters or less). In some embodiments, the lyophilized particle(s) of processing region B2 have an average diameter of about 4 mm or less (e.g., about 3 mm or less, about 2 mm or less) In an exemplary embodiment the lyophilized particle(s) have an average volume of about 2 microliters and an average diameter of about 1.35 mm. In other embodiments, the lyophilized particles may have a diameter of about 5 mm or less (e.g., about 2.5 mm or less, about 1.75 mm or less).
Lyophilized particles for determining the presence of one or more polynucleotides typically include multiple compounds. In some embodiments, the lyophilized particles include one or more compounds used in a reaction for determining the presence of a polynucleotide and/or for increasing the concentration of the polynucleotide. For example, lypophilized particles can include one or more enzymes for amplifying a polynucleotide, as by PCR.
Exemplary lyophilized particles include exemplary reagents for the amplification of polynucleotides associated with group B streptococcus (GBS) bacteria. In some embodiments, the lyophilized particles include one or more of a cryoprotectant, one or more salts, one or more primers (e.g., GBS Primer F and/or GBS Primer R), one or more probes (e.g., GBS Probe—FAM), one or more internal control plasmids, one or more specificity controls (e.g., Streptococcus pneumoniae DNA as a control for PCR of GBS), one or more PCR reagents (e.g., dNTPs and/or dUTPs), one or more blocking or bulking agents (e.g., non-specific proteins (e.g., bovine serum albumin (BSA), RNAseA, or gelatin), and a polymerase (e.g., glycerol-free Taq Polymerase). Of course, other components (e.g., other primers and/or specificity controls) can be used for amplification of other polynucleotides.
Cryoprotectants generally help increase the stability of the lypophilized particles and help prevent damage to other compounds of the particles (e.g., by preventing denaturation of enzymes during preparation and/or storage of the particles). In some embodiments, the cryoprotectant includes one or more sugars (e.g., one or more dissacharides (e.g., trehalose, melizitose, raffinose)) and/or one or more poly-alcohols (e.g., mannitol, sorbitol).
Lyophilized particles can be prepared as desired. A method for making lyophilized particles includes forming a solution of reagents of the particle and a cryoprotectant (e.g., a sugar or poly-alcohol). Typically, compounds of the lyophilized particles can be combined with a solvent (e.g., water) to make a solution, which can be then placed (e.g., dropwise, in discrete aliquots (e.g., drops) such as by pipette) onto a chilled hydrophobic surface (e.g., a diamond film or a polytetrafluorethylene surface). In general, the temperature of the surface can be reduced to near the temperature of liquid nitrogen (e.g., about −150° F. or less, about −200° F. or less, about −275° F. or less), such as by use of a cooling bath of a cryogenic agent directly underneath. The solution can be dispensed without contacting the cryogenic agent. The solution freezes as discrete particles. The frozen particles can be subjected to a vacuum, typically while still frozen, for a pressure and time sufficient to remove the solvent (e.g., by sublimation) from the pellets. Such methods are further described in international patent application publication no. WO 2006/119280, incorporated herein by reference.
In general, the concentrations of the compounds in the solution from which the particles are made can be higher than when reconstituted in the microfluidic cartridge. Typically, the ratio of the solution concentration to the reconstituted concentration can be at least about 3 (e.g., at least about 4.5). In some embodiments, the ratio can be about 6.
An exemplary solution for preparing lyophilized pellets for use in the amplification of polynucleotides indicative of the presence of GBS can be made by combining a cryoprotecant (e.g., 120 mg of trehalose as dry powder), a buffer solution (e.g., 48 microliters of a solution of IM Tris at pH 8.4, 2.5M KCl, and 200 mM MgCl2), a first primer (e.g., 1.92 microliters of 500 micromolar GBS Primer F (Invitrogen)), a second primer (e.g., 1.92 microliters of 500 micromolar GBS Primer R (Invitrogen)), a probe (e.g., 1.92 microliters of 250 micromolar GBS Probe—FAM (IDT/Biosearch Technologies)), a control probe (e.g., 1.92 microliters of 250 micromolar Cal Orange 560 (Biosearch Technologies)), a template plasmid (e.g., 0.6 microliters of a solution of 105 copies plasmid per microliter), a specificity control (e.g., 1.2 microliters of a solution of 10 nanograms per microliter (e.g., about 5,000,000 copies per microliter) Streptococcus pneumoniae DNA (ATCC)), PCR reagents (e.g., 4.8 microliters of a 100 millimolar solution of dNTPs (Epicenter) and 4.microliters of a 20 millimolar solution of dUTPs (Epicenter)), a bulking agent (e.g., 24 microliters of a 50 milligram per milliliter solution of BSA (Invitrogen)), a polymerase (e.g., 60 microliters of a 5 U per microliter solution of glycerol-free Taq Polymerase (Invitrogen/Eppendorf)) and a solvent (e.g., water) to make about 400 microliters of solution. About 200 aliquots of about 2 microliters each of this solution can be frozen and desolvated as described above to make 200 pellets. When reconstituted, the 200 particles make a PCR reagent solution having a total volume of about 2.4 milliliters.
Reagent Reservoirs
As seen in
A portion of enclosure 329 can be formed as an actuation mechanism (e.g., a piercing member 331) oriented toward the lower wall 333 of each enclosure. When cartridge 300 can be used, reagent reservoirs Ri can be actuated by depressing piercing member 331 to puncture wall 333. Piercing member 331 can be depressed by a user (e.g., with a thumb) or by the operating system used to operate cartridge 300.
Wall 333 can typically be formed of a material having a low vapor transmission rate (e.g., Aclar, a metallized (e.g. aluminum) laminate, a plastic, or a foil laminate) that can be ruptured or pierced. Reservoir 330 holds an amount of liquid suited for cartridge 300. For example, the reservoir may hold up to about 200 microliters. The piercing member 331 may account for a portion (e.g., up to about 25%) of that volume. The material of the laminate inside the blister that may touch corrosive reagent such as basic sodium hydroxide should not corrode even after six to twelve months of exposure.
In general, reservoirs Ri can be formed and filled as desired. For example, the upper wall of the enclosure can be sealed to the lower wall 333 (e.g., by adhesive and/or thermal sealing). Liquid can be introduced into the reservoir by, for example, an opening at the lower end of the piercing member 331. After filling, the opening can be sealed (e.g., by heat sealing through the localized application of heat or by the application of a sealing material (e.g., capping material 341)).
When wall 333 can be punctured, fluid from the reservoir enters network 333. For example, as seen in
In the configuration shown, reagent reservoir R1 typically holds a release liquid (e.g., a hydroxide solution as described above for cartridge 200) for releasing polynucleotides retained within processing region B1. Reagent reservoir R2 typically holds a wash liquid (e.g., a buffer solution as described above for cartridge 200) for removing un-retained compounds (e.g., inhibitors) from processing region B1 prior to releasing the polynucleotides. Reagent reservoir R3 typically holds a neutralization buffer (e.g., 25-50 mM Tris-HCl buffer at pH 8.0). Reagent reservoir R4 typically holds deionized water.
While reservoirs have been shown as having a piercing member formed of a wall of the reservoir, other configurations are possible. For example, in some embodiments, the reservoir includes a needle-like piercing member that extends through an upper wall of the reservoir into the sealed space toward a lower wall of the reservoir. The upper wall of the reservoir may be sealed at the needle-like piercing member (e.g., with an adhesive, an epoxy). In use, the upper wall can be depressed driving the piercing member through the lower wall forcing liquid in the sealed space to enter a microfluidic network.
While reservoirs have been described as including an actuation mechanism (e.g., a piercing member), other configurations are possible. For example, in some embodiments, a lower wall of the sealed space of the reservoir includes a weakened portion that overlies an opening to a microfluidic network. The lower wall material (e.g., laminate, polymer film, or foil) that overlies the opening can be thick enough to prevent loss of the liquid within the sealed space but thin enough to rupture upon the application of pressure to the liquid therein. Typically, the material overlying the opening can be thinner than the adjacent material. Alternatively, or in addition, the weakened material can be formed by leaving this material relatively unsupported as compared to the surrounding material of the lower wall.
While reservoirs have been described as having a sealed spaced formed in part by a wall of the sealed space, other configurations are possible. For example, referring to
Referring to
Referring to
While the reservoirs have been described as having a sealed space that may be stationary with respect to a piercing member, other configurations are possible. For example,
Referring to
While reservoirs have been described as having a piercing member that can be secured with respect to some portion of the reservoir, other configurations are possible. For example, referring to
As another example,
As yet another example,
While reservoirs have been described as having an enclosed space that can be fixed or otherwise integral with a portion of the reservoir, other configurations are possible. For example, referring to
While reservoirs have been described as generally overlying an inlet to a microfluidic network, other configurations are possible. For example, referring to
A still further embodiment of a reservoir with a piercing member is shown in
Yet another embodiment of a reservoir with a piercing member is shown in
It is to be understood that the dimensions of the reservoir, piercing element, shell and moulding shown in
Furthermore, the materials of the various embodiments can also be chosen so that the cartridge has a shelf-life of about a year. By this it is meant that the thickness of the various materials can be such that they resist loss, through means such as diffusion, of 10% of the liquid volume contained therein over a desired shelf-life period.
Preferably the volume of the reservoir can be around 150 μl before a shell is depressed. Upon depression of a shell, the volume can preferably be deformed to around half its original volume.
It is to be noted that completely filling the blister pack with a liquid reagent—with no remaining space for an air bubble results in a blister that requires application of significantly greater force than is preferable. Accordingly, the blister(s) are typically filled to about 80-95% of their volume, thereby reserving about 5-20%, typically 10-15% of the volume for air. Thus, in one embodiment, a blister that has a total volume of 200 μl is filled with 170 μl of liquid.
Lysing Chamber
Exemplary lysing chamber 302, as shown in
Waste chamber 308 includes a waste portion W6 by which liquid can enter chamber 308 from network 304 and a vent 310 by which gas displaced by liquid entering chamber 308 can exit.
Lysing Reagent Particles
Lyophilized reagent particles LP of lysing chamber 302 include one or more compounds (e.g., reagents) configured to release polynucleotides from cells (e.g., by lysing the cells). For example, particles LP can include one or more enzymes configured to reduce (e.g., denature) proteins (e.g., proteinases, proteases (e.g., pronase), trypsin, proteinase K, phage lytic enzymes (e.g., PlyGBS)), lysozymes (e.g., a modified lysozyme such as ReadyLyse), cell specific enzymes (e.g., mutanolysin for lysing group B streptococci)).
In some embodiments, particles LP alternatively or additionally include components for retaining polynucleotides as compared to inhibitors. For example, particles LP can include multiple particles 218 surface modified with ligands as described above for processing chamber of cartridge 200. Particles LP can include enzymes that reduce polynucleotides that might compete with a polynucleotide to be determined for binding sites on the surface modified particles. For example, to reduce RNA that might compete with DNA to be determined, particles LP may include an enzyme such as an RNAase (e.g., RNAseA ISC BioExpress (Amresco)).
In an exemplary embodiment, particles LP include a cryoprotecant, particles modified with ligands configured to retain polynucleotides as compared to inhibitors, and one or more enzymes.
Typically, particles LP have an average volume of about 35 microliters or less (e.g., about 27.5 microliters or less, about 25 microliters or less, about 20 microliters or less). In some embodiments, the particles LP have an average diameter of about 8 mm or less (e.g., about 5 mm or less, about 4 mm or less) In an exemplary embodiment the lyophilized particle(s) have an average volume of about 20 microliters and an average diameter of about 3.5 mm.
Particles LP can be prepared as desired. Typically, the particles can be prepared using a cryoprotectant and chilled hydrophobic surface as described hereinabove for other reagent particles. For example, a solution for preparing particles LP can be prepared by combining a cryoprotectant (e.g., 6 grams of trehalose), a plurality of particles modified with ligands (e.g., about 2 milliliters of a suspension of carboxylate modified particles with poly-D-lysine ligands), a protease (e.g., 400 milligrams of pronase), an RNAase (e.g., 30 milligrams of RNAseA (activity of 120 U per milligram), an enzyme that digests peptidoglycan (e.g., ReadyLyse (e.g., 160 microliters of a 30,000 U per microliter solution of ReadyLyse)), a cell specific enzyme (e.g., mutanolysin (e.g., 200 microliters of a 50 U per microliter solution of mutanolysin), and a solvent (e.g., water) to make about 20 milliters. About 1,000 aliquots of about 20 microliters each of this solution can be frozen and desolvated as described above to make 1,000 pellets. When reconstituted, the pellets can typically be used to make a total of about 200 milliliters of solution.
Exemplary Operation of Microfluidic Cartridge
In use, various components of cartridge 300 can be operated as follows. Valves Vi and Vi′ of network 304 can be configured in the open state. Gates Gi and mixing gates MGi of network 304 can be configured in the closed state. Reagent ports R1-R4 can be depressed, e.g., by application of mechanical force, to introduce liquid reagents into network 304, as described hereinabove. A sample can be introduced to lysing chamber 302 via port SP1 and combined with lyophilized particles LP within primary lysing chamber 306. Typically, the sample includes a combination of particles (e.g., cells) and a buffer solution. For example, an exemplary sample includes about 2 parts whole blood to 3 about parts buffer solution (e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA, and 1% SDS). Another exemplary sample includes group B streptococci and a buffer solution (e.g., a solution of 20 mM Tris at pH 8.0, 1 mM EDTA, and 1% Triton X-100).
In general, the volume of sample introduced can be smaller than the total volume of primary lysing chamber 306. For example, the volume of sample may be about 50% or less (e.g., about 35% or less, about 30% or less) of the total volume of chamber 306. A typical sample has a volume of about 3 milliliters or less (e.g., about 1.5 milliliters or less). A volume of gas (e.g., air) can generally be introduced to primary chamber 306 along with the sample. Typically, the volume of gas introduced can be about 50% or less (e.g., about 35% or less, about 30% or less) of the total volume of chamber 306. The volume of sample and gas combine to pressurize the gas already present within chamber 306. Valve 307 of port SP1 prevents gas from exiting chamber 306. Because gates G3, G4, G8, and G10 can be in the closed state, the pressurized sample can be prevented from entering network 304 via port SP2.
The sample dissolves particles LP in chamber 306. Reconstituted lysing reagents (e.g., ReadyLyse, mutanolysin) begin to lyse cells of the sample releasing polynucleotides. Other reagents (e.g., protease enzymes such as pronase) begin to reduce or denature inhibitors (e.g., proteins) within the sample. Polynucleotides from the sample begin to associate with (e.g., bind to) ligands of particles 218 released from particles LP. Typically, the sample within chamber 306 can be heated (e.g., to at least about 50° C., to at least about 60° C.) for a period of time (e.g., for about 15 minutes or less, about 10 minutes or less, about 7 minutes or less) while lysing occurs. In some embodiments, optical energy can be used at least in part to heat contents of lysing chamber 306. For example, the operating system used to operate cartridge 300 can include a light source 399 (e.g., a lamp primarily emitting light in the infrared) disposed in thermal and/or optical contact with chamber 306. Such a light source can be that shown in connection with heater module 2020,
Continuing with the operation of cartridge 300, G2 can be actuated (e.g., opened) providing a path between port SP2 of primary lysing chamber 306 and port W6 of lysing waste chamber 308. The path extends along channel C9, channel C8, through processing region B1, and channel C11. Pressure within chamber 306 drives the lysed sample material (containing lysate, polynucleotides bound to particles 218, and other sample components) along the pathway. Particles 218 (with polynucleotides) can be retained within processing region B1 (e.g., by a filter) while the liquid and other components of the sample flow into waste chamber 308. After a period of time (e.g., between about 2 and about 5 minutes), the pressure in lysing chamber 306 can be vented by opening gate G1 to create a second pathway between ports SP2 and W6. Double valves V1′ and V8′ can be closed to isolate lysing chamber 302 from network 304.
Operation of cartridge 300 continues by actuating pump P1 and opening gates G2,G3 and G9. Pump P1 drives wash liquid in channel C2 downstream of junction J1 through processing region B1 and into waste chamber W5. The wash liquid removes inhibitors and other compounds not retained by particles 218 from processing region B1. When the trailing edge of the wash liquid (e.g., the upstream interface) passes hydrophobic vent H14, the pressure from actuator P1 vents from network 304, stopping further motion of the liquid. Double valves V2′ and V9′ can be closed.
Operation continues by actuating pump P2 and opening gates G6, G4 and G8 to move release liquid from reagent reservoir R1 into processing region B1 and into contact with particles 218. Air vent AV1 vents pressure ahead of the moving release liquid. Hydrophobic vent H6 vents pressure behind the trailing edge of the release liquid stopping further motion of the release liquid. Double valves V6′ and V10′ can be closed.
Operation continues by heating processing region B1 (e.g., by heating particles 218) to release the polynucleotides from particles 218. The particles can be heated as described above for cartridge 200. Typically, the release liquid includes about 15 mM hydroxide (e.g., NaOH solution) and the particles can be heated to about 70° C. for about 2 minutes to release the polynucleotides from the particles 218.
Operation continues by actuating pump P3 and opening gates G5 and G10 to move release liquid from process region B1 downstream. Air vent AV2 vents gas pressure downstream of the release liquid allowing the liquid to move into channel C16. Hydrophobic vent H8 vents pressure from upstream of the release liquid stopping further movement. Double valve V11′ and valve V14 can be closed.
Referring to
Before actuating mixing gate MG11, the release liquid at junction J4 and the neutralization buffer at a junction J6 between channels C13 and C12 can be separated by mass 324 of TRS (e.g., the liquids are not typically spaced apart by a volume of gas). To combine the release liquid and neutralization buffer, pump P4 and gates G12, G13, and MG11 can be actuated. Pump P4 drives the volume of neutralization liquid between junctions J5 and J6 and the volume of release liquid between junctions J4 and J3 into mixing channel C15 (
The volume of neutralization buffer combined with the release liquid can be determined by the channel dimensions between junction J5 and J6. Typically, the volume of combined neutralization liquid can be about the same as the volume of combined release liquid. In some embodiments, the volume of liquid positioned between junctions J5 and J6 can be less than about 5 microliters (e.g., about 4 microliters or less, about 2.5 microliters or less). In an exemplary embodiment the volume of release liquid between junctions J5 and J6 can be about 2.25 microliters (e.g., the total volume of release liquid and neutralization buffer can be about 4 microliters).
Returning to
Continuing with operation of cartridge 300, actuator P5 and gates G14, G15 and G17 can be actuated to dissolve the lyophilized PCR particle present in second processing region B2 in water from reagent reservoir R4. Hydrophobic vent H10 vents pressure from actuator P5 upstream of the water stopping further motion. Dissolution of a PCR-reagent pellet typically occurs in about 2 minutes or less (e.g., in about 1 minute or less). Valve V17 can be closed.
Continuing with operation of cartridge 300, actuator P6 and gate G16 can be actuated to drive the dissolved compounds of the lyophilized particle from processing region B2 into channel C31, where the dissolved reagents mix to form a homogenous dissolved lyophilized particle solution. Actuator P6 moves the solution into channels C35 and C33 (vented downstream by air vent AV5). Hydrophobic vent H9 vents pressure generated by actuator P6 upstream of the solution stopping further motion. Valves V18, V19, V20′, and V22′ can be closed.
Continuing with operation of cartridge 300, actuator P7 and gates G18, MG20 and G22 can be actuated to combine (e.g., mix) a portion of neutralized release liquid in channel 32 between gate MG20 and gate G22 and a portion of the dissolved lyophilized particle solution in channel C35 between gate G18 and MG20. The combined liquids travel along a mixing channel C37 and into detection region D2. An air vent AV3 vents gas pressure downstream of the combined liquids. When the upstream interface of the combined liquids passes hydrophobic vent H13, the pressure from actuator P7 can be vented and the combined liquids can be positioned within detection region D2.
Actuator P8 and gates MG2, G23, and G19 can be actuated to combine a portion of water from reagent reservoir R4 between MG2 and gate G23 with a second portion of the dissolved lyophilized particle solution in channel C33 between gate G19 and MG2. The combined liquids travel along a mixing channel C41 and into detection region D1. An air vent AV4 vents gas pressure downstream of the combined liquids. When the upstream interface of the combined liquids passes hydrophobic vent H12, the pressure from actuator P8 can be vented and the combined liquids can be positioned within detection region D1.
Continuing with operation of cartridge 300, double valves V26′ and V27′ can be closed to isolate detection region D1 from network 304 and double valves V24′ and V25′ can be closed to isolate detection region D2 from network 304. The contents of each detection region (neutralized release liquid with sample polynucleotides in detection region D2 with PCR reagents from dissolved lyophilized particle solution and deionized water with PCR reagents from dissolved lyophilized particle solution in detection region D1) can be subjected to heating and cooling steps to amplify polynucleotides (if present in detection region D2). The double valves of each detection region prevent evaporation of the detection region contents during heating. The amplified polynucleotides can typically be detected using fluorescence detection. Thus, typically above one or both of detection regions D1, D2, is a window (as in, e.g.,
While cartridges for carrying out various stages of processing samples have been shown and described herein as having a generally planar configuration, other configurations can be used and are consistent with an integrated system as described herein. For example, a cartridge having a generally tube-like or vial-like configuration is described in U.S. patent application publication no. 2006-0166233, incorporated herein by reference.
The following Examples are illustrative and are not intended to be limiting.
This non-limiting example describes various exemplary embodiments of an apparatus, system, microfluidic cartridge, kit, methods, and computer program product, as shown in
For example,
Apparatus 800 can be used with a sample kit 810, shown in
Microfluidic cartridge 812, as depicted in
Referring to
In preparation for testing a sample, pipette tip 820 from kit 810 may be attached to the syringe 822, and a sample may be drawn from sample container 814 into syringe 822. Referring to
Referring to
As shown in
This non-limiting example describes various embodiments of an apparatus, system, microfluidic cartridge, kit, methods, and computer program product, in particular, various aspects of using apparatus 800 as described in Example 1 relating to exemplary aspects of the method and the computer program product.
Referring to
A user may then remove the microfluidic cartridge 812 and the sample container 814 from the sealed pouch 824. The software and apparatus 800 may be configured to request that the bar code 813 on the microfluidic cartridge 812 and/or the bar code 815 on the sample container 814 be scanned in using the bar code reader 806, as in
Various embodiments of the software and apparatus 800 may be configured to allow a user to perform one or more optional functions (e.g., adjustments to apparatus 800 or the software) including, but not limited to: modifying user settings, modifying logout settings, setting the system clock, modifying display settings, modifying QC requirements, setting report preferences, configuring an attached printer, configuring a network connection, sending data via a network connection, selecting or adapting data analysis protocols, or the like.
In various embodiments, the software can include a user interface and device firmware. The user interface software can allow for aspects of interaction with the user including, but not limited to, entering patient/sample information, monitoring test progress, error warnings, printing test results, uploading of results to databases, updating software, and the like. The device firmware can operate apparatus 800 during the analytical tests and can have a generic portion that can be test independent and a portion specific to the test being performed. The test specific portion (“protocol”) can specify the microfluidic operations and their order to accomplish the test.
This non-limiting example describes various embodiments of the claimed apparatus, system, microfluidic cartridge, kit, methods, and computer program product. In one embodiment, the apparatus 800, shown in
Cell lysis can be achieved by methods known to the art, for example, heat and/or chemical activation. In some embodiments, after a 1.0+/−0.2 mL sample can be injected into the lysis reservoir 830 via the sample inlet 826, the apparatus 800 may cause the sample to be mixed with lysis reagents from the wet reagent storage 838 and heated (e.g., for 7 minutes) in the lysis reservoir 830. Using this protocol, greater than 90% lysis efficiency has been achieved for GBS and other bacterial cells. The lysis reagents may also incorporate a cationic polyamide modified polycarbonate—polystyrene latex bead based DNA affinity matrix (e.g., retention member 821) to capture the negatively charged DNA which may be released during the lytic process. The affinity beads can bind to negatively charged DNA with very high affinity while potential PCR inhibitors can either fail to bind or can be removed during subsequent wash steps.
In one exemplary embodiment, the apparatus 800 may also automate the capture and cleaning of DNA from crude sample lysate (e.g., GBS sample lysate) to generate “PCR-ready” DNA. The contents of the lysis reservoir 830 (e.g., 1.0+/−0.2 mL sample and reagents) may be transferred to the DNA processing chamber 840. Affinity beads with bound DNA from the input sample can be trapped using an in-line bead column (e.g., a filter with specific pore size) and an on-cartridge pump can be used to wash the affinity beads to remove non-specifically bound moieties as well as soluble inhibitors by performing a buffer exchange. The bound DNA may be released by known methods, for example, by heating the affinity beads (e.g., to 80° C.) and/or by using a release buffer. The intact DNA can be recovered with this single step release in very small volume (3-4 μl) thereby achieving a significant concentration of the original target DNA. Other methods known to the art can be employed by the system to achieve cell lysis and DNA capture, wash, and release.
In the example described here, the basis for the real-time PCR assay used is the TaqMan® assay, the schematic operation of as depicted in
This process can typically occur, for example, in every thermal cycle and should not interfere with the exponential accumulation of product. The increase in fluorescence signal can typically be detected if the target sequence can be complementary to the probe and can be amplified during PCR. TaqMan® assay can offer a two-fold stringency (the primer typically binds and the probe typically binds to the target sequence) and hence detection of any nonspecific amplification can be reduced or eliminated.
Real-time PCR primers and probe sets for GBS (Streptococcus agalactiae) have been designed & tested using clinical samples. The PCR reagents may include a pair of hybridization primers specific to the portion of the cfb gene between positions 328 and 451 encoding the CAMP factor (Christie, Atkins and Munch-Petersen, see, e.g., Boll Ist Sieroter Milan, (1955 July-August); 34(7-8):441-52). The CAMP factor is a diffusible extracellular protein and is produced by the majority of GBS. The gene encoding CAMP factor, cfb gene (GenBank access number: X72754), can be present in GBS isolate and has been used for the development of a PCR based identification of GBS (Danbing K., et al., (2000), Clinical Chemistry, 46, 324-331). Further, a specific TaqMan® style fluorogenic probe has also been designed & tested, in one example, to recognize the sequence amplified between the primers allowing real-time detection by using fluorescence measurements.
In order to evaluate the DNA clean-up process and monitor the performance of our primers at run-time, positive internal control plasmids (e.g., as depicted in
In an exemplary embodiment, a robust system for carrying out rapid thermo-cycling using a microfluidic volume was designed, developed, and implemented in a microfluidic format. The microfluidic volumes that can be accommodated range from about 0.01 μl to about 10 μl, wherein the principal limitation on the lower limit is sensitivity of detection. Exemplary volumes are in the range 0.5-4.5 μl. Still other exemplary volumes are 2 μl.
In various embodiments, any number (e.g., 0, 1, 2, or all) of the reagents for performing the PCR can be incorporated on the cartridge in a lyophilized format. At the time of use, the lyophilized PCR reagents can be reconstituted using, for example, deionized water, which may be stored on the microfluidic cartridge 812 in a blister format (e.g., in a self-pierceable reservoir 828). The reconstituted PCR reagents can be aliquoted into, e.g., two parts. In various embodiments, PCR ready DNA (output of the sample preparation module) can be mixed one aliquot and sent to the first PCR channel for real-time PCR (sample PCR). DI water containing non-target DNA can be mixed with the second aliquot of the PCR reagents and can be sent to the other PCR chamber (Negative PCR) to serve as the negative control.
In various embodiments, a microfluidic system (e.g., microfluidic cartridge 812) can include components such as micropumps for moving/mixing liquid drops, microreactors for performing thermally initiated biochemical reactions, and microvalves or microgates to enable control of the liquid pumping operations as well as to isolate regions of the cartridge such as the PCR chambers during thermocycling.
In some embodiments, a liquid drop handling system can be used to produce liquid-sample injection and motion based on thermally actuated pumps (e.g. thermo-pneumatic pumping) that may be operated electronically without the use of mechanical valves. For example, by heating air trapped inside chambers that can be connected to the main channel, significant air pressure can be generated for thermo-pneumatic pumping. Increasing the temperature of the air can cause the pressure inside the chamber to rise until the pressure can be high enough to split off a drop (meter an aliquot) and move it to the desired location. This technique can be implemented as an on-cartridge actuation mechanism and may use, for example, molded chambers, channels and heaters. Typically, this can avoid mechanical moving parts and can facilitate fabrication.
In some embodiments, thermally expansive materials such as gas, readily vaporizable liquid (e.g., vaporizable between 25° C. and 100° C. at 1 atmosphere), and/or a thermally-expanding polymer (e.g., Expancel) may be introduced in thermally actuated pumps (e.g., thermopneumatic air chambers), which may minimize the size of the pumps to generate differential pressures greater than 5 psi. These materials can expand, e.g., by over 100% when a threshold temperature can be reached, causing it to partially or completely fill up the thermopneumatic chamber causing further compression of the air.
For example,
An exemplary clinical sample input into the mirofluidic cartridge 812 can have a volume of approximately 1 milliliter. After enzymatic/thermal lysis of the cells, the released DNA can be bound to affinity-microbeads. These microbeads can be, for example, on the order of 10 microns in size. In various embodiments, a total amount of beads in the range of a few million can be used per microfluidic cartridge 812 for DNA concentration. In some cases, a minimum pressure of 10 psi (e.g., 10 psi, 11 psi, or 15 psi) may be used to concentrate the beads against an inline-filter area of a few square millimeters (pore size of 8 microns) in a few minutes (e.g. 3 minutes). This pressure can be generated, for example, by injecting extra air (e.g., 1-3 mL) into the bulk lysis chamber of the microfluidic cartridge 812. In some embodiments, a one-way duckbill valve at the luer inlet can be used to minimize or prevent air pressure from escaping through the inlet.
Reagents which can be employed for sample preparation and PCR reactions can be pumped into the microfluidic network by depressing the reagent blister domes by the slider of the instrument during use of the instrument.
In exemplary embodiments, the enzymes typically employed for cell lysis, DNA capture, and for performing real-time PCR can be lyophilized into pellets and stored at different locations of the cartridge. The contact of air can be minimized by storing the pellets in the microfluidic cartridge 812, for example, in a nitrogen purged chamber or a in a channel structure sealed on either ends of the microchannel by thermal gates. Buffers typically employed for sample preparation, reagent hydration and PCR can be stored in hermetically sealed reagent blisters (e.g., the self-pierceable reservoirs 828). Materials used for making the reagent blister can have high moisture vapor barrier and can minimize the loss of liquid during storage of the cartridge over a year. Waste generated from the clinical sample as well as the various wash buffers can be stored on-board the cartridge in chambers and microchannels (e.g., the waste reservoir 832, which can typically be leak-resistant).
A plurality of steps that can be used for the accurate, real-time PCR based diagnosis of pathogens (e.g., Group B Streptococcus (GBS) colonization in prenatal women) was integrated into a single, microfluidic technology based, disposable cartridge, as shown in
In various embodiments, samples (e.g., GBS samples) can be introduced through the sample inlet 826, which may have a luer fitting for accommodating a syringe. A pre-filter (e.g., attached to the syringe) can be used to remove at least a portion of crude impurities from the sample and, in some embodiments, the sample (e.g., 1 mL) can be lysed in the lysis chamber using heat and/or lytic enzymes. Enzymes such as pronase, protienaseK, and RNAaseA can be used (e.g., during the lysis step) to remove the inhibitory proteinaceous matter and competing RNA molecules. Referring to
In some embodiments, the waste chamber is equipped with an anti-foaming agent, such as simeticone. Vigorous bubble formation can occur in the waste chamber because liquid enters it at high speed and, upon mixing with air in the waste chamber, foams. It is undesirable if the foam overflows from the waste chamber. Presence of a de-foaming agent can mitigate this phenomenon. The defoaming agent can be present in powder, tablet, pellet, or particle form.
In some embodiments, the beads can be subject to washing to remove unbound and non-specifically bound matter and the cleaned DNA can be released into a small volume (˜3 μl) compartment and concentrated (e.g., by a factor of about 300). In various embodiments, the concentrated DNA can then be mixed with the appropriate PCR reagents and/or be sent to a PCR channel for real-time PCR. An Internal Control plasmid (along with its cognate Probe) may also be included in the first PCR channel, which can act as a positive control. In a second PCR channel (if present), DI water containing non-target DNA and the internal control can be mixed along with the PCR reagents, to act as a negative control.
A user may introduce a sample in the bulk lysis sample through luer-duckbill valve (e.g., the sample inlet 826), shake gently to dissolve lysis reagent pellets, and introduce an excess amount of air (e.g., 0.25-0.75 ml) into the lysis chamber to over-pressurize the lysis chamber. The absolute pressure (P) generated in the lysis chamber having a chamber volume, Vchamber, is related to the amount of liquid sample injected, Vsample, and the volume of extra air injected, Vextra air, by the formula:
The microfluidic cartridge 812 then may be placed in the apparatus 800 and the slider module 848 closed. On closing, the slider module 848 may press reagent blisters (e.g., self-piercing reservoirs 828), causing them to burst and release reagents (e.g., wash buffer, release buffer, neutralization buffer and water) into the channels with reagents.
In various embodiments, the apparatus 800 may perform any or all of the following steps. In referring to
Referring to
Referring to
Referring to
Referring to
In various embodiments, referring to
Referring to
This non-limiting example shows CAD views of various exemplary embodiments of the apparatus, system, microfluidic cartridge, kit, methods and computer program product, as further described herein.
Carboxylate surface magnetic beads (Sera-Mag Magnetic Carboxylate modified, Part #3008050250, Seradyn) at a concentration of about 1011 mL−1 were activated for 30 minutes using N-hydroxylsuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in a pH 6.1 500 mM 2-(N-Morpholinio)-ethanesulfonic acid (MES) buffer solution. Activated beads were incubated with 3,000 Da or 300,000 Da average molecular weight poly-L-lysine (PLL). After 2 washes to remove unbound PLL, beads were ready for use.
Referring to
In use, sample introduced via inlet 310′ passed along channel and through processing region 320′. Excess sample material passed along channel 308′ and exited device 300′ via outlet 316′. Polynucleotides were preferentially retained by the beads as compared to inhibitors. Once sample had been introduced, additional liquids, e.g., a wash liquid and/or a liquid for use in releasing the retained polynucleotides were introduced via inlet 326′.
Retention of polynucleotides by the poly-L-lysine modified beads of device 300′ was demonstrated by preparing respective devices comprising processing regions having a volume of about 1 μL including about 1000 beads. The beads were modified with poly-L-lysine of between about 15,000 and 30,000 Da. Each processing region was filled with a liquid comprising herring sperm DNA (about 20 uL of sample with a concentration of about 20 mg/mL) thereby placing the beads and liquid in contact. After the liquid and beads had been in contact for 10 minutes, the liquid was removed from each processing region and subjected to quantitative real-time PCR to determine the amount of herring sperm DNA present in the liquid.
Two controls were performed. First, an otherwise identical processing region was packed with un-modified beads, i.e., beads that were identical with the poly-L-lysine beads except for the activation and poly-L-lysine incubation steps. The liquid comprising herring sperm DNA was contacted with these beads, allowed to stand for 10 minutes, removed, and subjected to quantitative real-time PCR. Second, the liquid comprising the herring sperm DNA (“the unprocessed liquid”) was subjected to quantitative real-time PCR.
Referring to
Devices having processing regions were packed with 3,000 Da poly-L-lysine modified beads. Liquid comprising polynucleotides obtained from group B streptococci (GBS) was contacted with the beads and incubated for 10 minutes as above for the herring sperm DNA. This liquid had been obtained by subjecting about 10,000 GBS bacteria in 10 μl of 20 mM Tris pH 8, 1 mM EDTA, 1% Triton X-100 buffer to thermal lysing at 97° C. for 3 min.
After 10 minutes, the liquid in contact with the beads was removed by flowing about 10 μl of wash solution (Tris-EDTA pH 8.0 with 1% Triton X 100) through the processing region. Subsequently, about 1 μl of 5 mM NaOH solution was added to the processing region. This process left the packed processing region filled with the NaOH solution in contact with the beads. The solution in contact with the beads was heated to 95° C. After 5 minutes of heating at 95° C., the solution in contact with the beads was removed by eluting the processing region with a volume of solution equal to three times the void volume of the processing region.
Referring to
As seen in
Buccal cells from the lining of the cheeks provide a source of human genetic material (DNA) that may be used for single nucleotide polymorphism (SNP) detection. A sample comprising buccal cells was subjected to thermal lysing to release DNA from within the cells. Device 300 was used to separate the DNA from concomitant inhibitors as described above. A cleaned-up sample corresponding to aliquot E2 of
Referring to
Blood acts as a sample matrix in variety of diagnostic tests including detection of infectious disease agents, cancer markers and other genetic markers. Hemoglobin present in blood samples is a documented potent inhibitor of PCR. Two 5 ml blood samples were lysed in 20 mM Tris pH 8, 1 mM EDTA, 1% SDS buffer and introduced to respective devices 300, which were operated as described above to prepare two clean-up samples. A third 5 ml blood sample was lysed and prepared using a commercial DNA extraction method Puregene, Gentra-Systems, MN. The respective cleaned-up samples and sample subjected to the commercial extraction method were used for a Allelic discrimination analysis (CYP2D6*4 reagents, Applied Biosystems, CA). Each sample contained an amount of DNA corresponding to about 1 ml of blood.
Referring to
The preparation of polynucleotide samples for further processing often includes subjecting the samples to protease treatment in which a protease cleaves peptide bonds of proteins in the sample. An exemplary protease is pronase, a mixture of endo- and exo-proteases. Pronase cleaves most peptide bonds. Certain ligands, such as poly-L-lysine can be susceptible to rupture by pronase and other proteases. Thus, samples are generally not subjected to protease treatment in the presence of the retention member if the ligands bound thereto are susceptible to the proteases.
Poly-D-lysine, the dextro enantiomer of poly-lysine resists cleavage by pronase and other proteases. The ability of a retention member comprising bound poly-D-lysine to retain DNA even when subjected to a protease treatment was studied.
Eight (8) samples were prepared. A first group of 4 samples contained 1000 GBS cells in 10 μl buffer. A second group of 4 samples contained 100 GBS cells in 10 μl buffer. Each of the 8 samples was heated to 97° C. for 3 min to lyse the GBS cells. Four (4) sample sets were created from the heated samples. Each sample set contained 1 sample from each of the first and second groups. The samples of each sample sets were treated as follows.
Referring to
The samples of sample set 2 were subjected to pronase incubation to prepare respective protein cleaved samples, which were then heated to inactivate the proteases. The protein-cleaved, heated samples were contacted with respective retention members each comprising a set of poly-D-lysine modified beads. After 5 minutes, the respective sets of beads were washed with 5 microliters of a 5 mM NaOH solution to separate inhibitors and products of protein cleavage from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated to 80 (eighty) ° C. for 2 minutes to release the DNA. The solutions with released DNA were neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and shown in
The samples of sample set 3 were subjected to pronase incubation to prepare respective protein cleaved samples. The proteases were not deactivated either thermally or chemically. The protein-cleaved samples were contacted with respective retention members each comprising a set of poly-L-lysine modified beads. After 5 minutes, the respective sets of beads were washed with 5 microliters of a 5 mM NaOH solution to separate inhibitors and products of protein cleavage from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated to 80 (eighty) ° C. for 2 minutes to release the DNA. The solutions with released polynucleotides were each neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and shown in
The samples of sample set 4 were subjected to pronase incubation to prepare respective protein cleaved samples. The proteases were not deactivated either thermally or chemically. The protein-cleaved samples were contacted with respective retention members each comprising a set of poly-D-lysine modified beads. After 5 minutes, the respective sets of beads were washed with 5 microliters of a 5 mM NaOH solution to separate inhibitors and products of protein cleavage from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated to 80 (eighty) ° C. for 2 minutes to release the DNA. The solutions with released polynucleotides were each neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and shown in
As seen in
This non-limiting example describes, in the form of an operator's manual, various embodiments of the claimed apparatus, microfluidic cartridge, kit, methods, and computer program product, in particular directed to a single cartridge system including a microfluidic PCR assay for the qualitative detection of microorganisms, such as Group B Streptococcus (GBS). Further descriptions pertinent to GBS analysis are presented in Example 14.
Sample Preparation Kit, which May Include
Positive Control Swab specimen containing Limit of Detection sample of, e.g., GBS bacteria in dehydrated form;
System with Barcode reader, both of which as further described herein, fitted with, e.g., a 115V or 220V power cord.
Additional Optional Equipment:
The description in this example, is suitable to test samples for presence of GBS, further details of which are provided in Example 14.
Explanation of Test Application
The apparatus and materials can be used to test for a variety of pathogens and microorganisms, as further described herein. One example is to test for GBS, as further described in Example 14. The tests can be performed in the near-patient setting by clinicians who are not extensively trained in laboratory procedures. A QC routine can be built into the User Interface of the system in order provide continued Quality Assurance for the GBS test. The test can also be performed in a central hospital “stat” laboratory, provided that the sample testing occurs within the time-frame required by the physician requesting the test.
Exemplary Warnings & Precautions
Other warnings and precautions specific to GBS are presented in Example 14.
Exemplary Specimen Collection
Cartridge should typically be packaged in biohazard protective materials as described by the International Standards.
Exemplary Cleaning Procedure for System
A solution of 10% bleach (0.5% sodium hypochlorite), followed by a clean water rinse can be used to disinfect as well as reduced potential DNA contamination.
DNA contamination can typically be accomplished by cleaning with bleach or materials suitable for eliminating DNA contamination. Chemicon™ Nucleic acid removers can also be used to be used after cleaning with ordinary disinfectants.
Typically, alcohol or ordinary sanitary wipes will not reduce DNA contamination of the instrument.
Exemplary Reagent Lot Verification
Purpose—Assessment of cartridge and sample kit lots and verification of total system performance.
Recommendation: Upon receipt of a new lot of cartridges or sample kits, a quality control set can be run to confirm, for example, whether the reagent set includes (1) external control and (1) negative external control.
Exemplary Quality Control Routine
The external positive control serves to monitor and calibrate the sensitivity of the specimen preparation steps and the assay, and can be used to minimize the risk of false negative results. If the test fails, it may invalidate the results from that batch of cartridges, and should be reported to the supplier.
Purpose—Verification of total system performance including assessment of sample handling technique. Test can be performed at a User selected preset interval.
Recommendation: When test is performed, run a QC set including (1) Positive external control and (1) Negative external control, If QC test fails to yield expected results, contact manufacturer.
Exemplary Quality Control Routine Directions
The System may perform a System initiation test when it is powered up. As the Start Up Routine of each Patient Test Sample or QC Sample, the System may run a self-test to determine if, for example, the electronics, optical system, heaters and temperature sensors are functioning as intended.
Exemplary Internal Controls (On-Cartridge)
Reagents which can be used in the assay may be included on the cartridge to reduce the potential for user handling errors and contamination. Two types of on-cartridge positive and negative controls strategies can be incorporated within each microfluidic cartridge to monitor individual PCR assay performance. Two examples are:
1) Positive Internal Control Plasmid (ICP):
An internal control plasmid can provide a control for integrity of the PCR assay reagents as well as being an indicator for presence of PCR inhibitory substances in the specimen, i.e. it can be a control for false negative results. During thermocycling, amplification of this region may produce a distinct fluorogenic signal in both PCR lanes. Failure to amplify the internal control sequence, in the absence of a positive sample, can be indicative of either a failure of the reagent mix or presence of PCR inhibitors in the specimen. This can invalidate the results of the test as indicated by an error code on the instrument. “IND” can be displayed to report an indeterminate result.
2) Negative Internal Control PCR Lane:
A parallel lane can be used to run a second PCR with reagents from the same mix without any sample. This can provide a control against false positive results due to contamination of the reagents. A failed result in the negative control lane can invalidate the result and can be indicated by an error code on the instrument. “IND” can be displayed to report an indeterminate result.
Exemplary Real Time PCR for GBS DNA Detection
In various embodiments, the test can use real-time Polymerase Chain Reaction (PCR) for the amplification of a cfb gene sequence of GBS recovered from clinical samples and fluorogenic target-specific hybridization for the detection of the amplified DNA. The cfb gene encodes the CAMP factor, a diffusible extracellular protein which is typically present in GBS isolates. The Group B Streptococcus (GBS) detection test can be an integrated, raw-sample-to-result type of nucleic acid amplification assay. A TaqMan fluorogenic probe may be used to detect the PCR amplicons. Reagents used in the assay may be included on the cartridge to reduce potential for user handling errors and contamination.
Exemplary Potential Precautions
In some embodiments, the test system may not be qualified for identifying targets such as GBS DNA, in specimens other than vaginal and/or rectal specimens. Urine and Blood specimens may not be qualified.
In some embodiments, a patient undergoing antibiotic treatment may not be able to obtain a correct diagnosis with this or other diagnostic tests.
In some embodiments, the test may not yield a GBS culture suitable for direct identification of the bacteria by a microbiologist. In some embodiments, the test may not provide susceptibility results that are needed to recommend a treatment for penicillin-allergic persons.
Exemplary Potential Interfering Substances
In some embodiments, urine and vaginal secretions if present in very large quantities, may interfere with the test.
Typically, contaminants such as blood, meconium, and amniotic fluid contamination of the samples is not likely to interfere with the test.
Typically, drug interference (such as present in vaginal and rectal secretions), other than antibiotics, are not known at this time to interfere with PCR.
Exemplary Performance Characteristics and Interpretation
The flow chart in
Exemplary Cross Reacting Substances
Specificity of the primers and probes can be tested with real-time PCR (Taqman assay) using genomic DNAs isolated from the following organisms: nine GBS serotypes (serotype la, lb, lc, II, III, IV, V, VI and VII; American Type Culture Collection and National Center for Streptococcus, Canada); 10 clinical GBS isolates; 60 clinical samples; a wide variety of gram-positive and gram-negative bacterial strains as well as two yeast strains and RSV type I and 2.
Exemplary Microorganisms
Pseudomones aeruginosa
Proteus mirabilis
Kiebsiella oxytoca
Kiebsiefla pneurnoniae
Escherichia cot (clinical isolate 1)
Escherichia coli (clinical isolate 2)
Acinetobacter baumannd
Serra. marcescens
Entembacter aerugenes
Enterococcus Maclean
Staphylococcus aureus (clinical isolate 1)
Staphylococcus aureus (clinical isolate 2)
Streptococcus pyogenes
Streptococcus viridans
Listena monocytogenes
Enterococcus sps.
Cendida glabrata
Candida albicans
Streptococcus Group C
Streptococcus Group G
Streptococcus Group F
Enterococcus feecalis
Streptococcus pneumoniae
Staphylococcus epiderrnidis (C−)
Gardenerella vaginalis
Micrococcus spa
Haemophilus influenza°
Neisseria gonorrhoeae
Moraxella catarrahlis
Salmonella sps.
Chlamytha trechomatis
Peptostreptococcus product.
Peptostreptococcus anaerobe.
Lactobacillus lennentum
Eubacterium lentum
Exemplary Troubleshooting Chart
This non-limiting example describes, in the form of user instructions, various embodiments of the claimed apparatus, microfluidic cartridge, kit, methods, and computer program product, in particular directed to a microfluidic cartridge for use in a microfluidic PCR assays including the qualitative detection of microorganisms such as Group B Streptococcus.
Presence of Group B Streptococci (GBS) remains a leading cause of serious neonatal infection despite great progress in prevention since the 1990's with sepsis, pneumonia, and meningitis affecting the baby after birth. The GBS test system can be used for the rapid, qualitative detection of Group B streptococcus (GBS) DNA in vaginal/rectal samples.
Exemplary Use and Indications for Use
In various embodiments, the GBS test system can be used for the rapid, qualitative detection of microorganisms in clinical samples, such as Group B streptococcus (GBS) DNA in vaginal/rectal samples.
Typical indications for use of the GBS test include, for example, a rapid screening test in the prenatal care regimen for the maternity patient to determine the need for antibiotic treatment during labor, as described by the CDC guidelines (Centers for Disease Control and Prevention. Prevention of Perinatal Group B Streptococcal Disease: Revised Guideline from CDC. Morbidity and Mortality Weekly Report, Aug. 16, 2002; 51 (No. RR-11); 1-24). The test can provide rapid results at the point of care or in a central laboratory with rapid turnaround service during the intrapartum and prepartum phase of the maternity patient. The test can also be used to detect GBS DNA in vaginal or rectal samples of any subject suspected of GBS infection. See also Mark A. Burns, Brian N. Johnson, Sundaresh N. Brahmasandra, Kalyan Handique, James R. Webster, Madhavi Krishnan, Timothy S. Sammarco, Piu M. Man, Darren Jones, Dylan Heldsinger, Carlos H. Mastrangelo, David T. Burke “An Integrated Nanoliter DNA Analysis Device” Science, Vol. 282, 16 Oct. 1998.
Explanation of Test Application
The Center for Disease Control and Prevention recommends universal prenatal screening for vaginal/rectal GBS colonization of all women at 35-37 weeks gestation in order to determine the need for prophylactic antibiotics during labor and delivery. The current CDC recommendation is the culture-based test method (Standard Culture Method), from which results are typically available in 48-72 hours, compared to about 30 minutes for the present test. The test can utilize automated sample preparation and real-time PCR to identify the cfb gene in the GBS genome which is an established identification sequence that encodes the CAMP factor. The CAMP factor is an extra cellular protein typically present in GBS isolates. The CAMP factor can be used for the presumptive identification of GBS bacteria in clinical samples by the culture method. This test can be performed in the near-patient setting by clinicians who are not extensively trained in laboratory procedures. A QC routine can be built into the User Interface in order provide continued Quality Assurance for the GBS test. The test can also be performed in a central hospital “stat” laboratory provided that the sample testing occurs within the time-frame required by the maternity department.
Possible Contraindications
The GBS test may be contraindicated for persons with allergy to polyester contained in the specimen collection swab.
Exemplary Warnings & Precautions
Warnings herein may be additional to those of general application shown in Example 13, herein.
When test is complete, results may be clearly displayed. Results can be printed or stored as determined by lab procedures.
Exemplary Disposal of Materials
Cartridge and collection kit should be treated as biohazard.
Exemplary Recommended Lab Quality Control Routine
Exemplary Test Verification
On a weekly basis, (1) positive external control and (1) negative external control can be run. A QC set to confirm system total system performance. This procedure is also recommended when training new users.
Exemplary Quality Control Routine Directions:
May run samples as directed on display screens. If a QC test fails to yield expected results, manufacturer can be contacted.
The external positive control can include a lyophilized aliquot of Streptococcus agalactiae (GBS) cells that are reconstituted at run-time with sample collection buffer. The number of GBS cells in the external positive control may be approximately equal to the Minimum Detectable Limit (MDL) of the test.
Exemplary quality control tests also include an System Self-test QC, as described in Example 14.
Exemplary Internal Controls (On-Cartridge)
Typical reagents for the assay may be included on the cartridge to reduce the potential for user handling errors and contamination. Two types of on-cartridge positive and negative controls strategies may be incorporated within each microfluidic cartridge to monitor individual PCR assay performance.
An exemplary positive internal control plasmid for GBS is a double stranded circular DNA molecule containing a 96 bp region comprised of a unique 39 bp artificial DNA sequence flanked by the forward and reverse sequences from the cfb gene can be included in the lyophilized master mix along with a second distinct fluorogenic probe specific to this unique sequence. The Failure to amplify the internal control sequence, in the absence of a positive GBS sample, can be indicative of either a failure of the reagent mix or presence of PCR inhibitors in the specimen.
Exemplary Real Time PCR for GBS DNA Detection
The test can utilize real-time Polymerase Chain Reaction (PCR) for the amplification of a cfb gene sequence of GBS recovered from clinical samples. A fluorogenic target-specific Tacman® probe can be used for the detection of the amplified DNA. The cfb gene encodes the CAMP factor, a diffusible extracellular protein which is typically present in GBS isolates. The Group B Streptococcus (GBS) detection test may be an integrated, raw-sample-to-result type of nucleic acid amplification assay. Typical reagents for the assay can be included on the cartridge to reduce potential for user handling errors and cross contamination.
Exemplary Possible Test Limitations
In some embodiments, the test system may not be qualified for identifying GBS DNA in specimens other than vaginal/rectal specimens. For example, urine and blood specimens may not be qualified in some embodiments.
In some embodiments, a patient undergoing antibiotic treatment may not be able to obtain a correct GBS diagnosis.
In some embodiments, the test may not yield a GBS culture suitable for direct identification of the bacteria by a microbiologist.
Exemplary Performance Characteristics and Interpretation
10-30% of pregnant women are colonized with GBS. The GBS colonization cut-off is determined to be approximately 1000 copies of DNA/sample determined by amplification of the cfb gene sequence. The user is referred to the flow-chart of
Exemplary Possible Interfering Substances
In some embodiments, urine or vaginal secretions, or mucus, if present in large quantities, may interfere with test.
In some embodiments, blood, meconium, or amniotic fluid contamination of the samples is typically not likely to interfere with the test.
In some embodiments, drug interference (present in vaginal and rectal secretions), other than antibiotics, are typically not known at this time to interfere with PCR.
Exemplary Results Interpretation and Expected Values
10-30% of pregnant women can be colonized with GBS. The GBS colonization cut-off can be determined to be approximately 1000 copies of DNA/sample determined by amplification of the cfb gene sequence.
PCR reactions can be interpreted as positive or negative for GBS and Internal control. A logical algorithm may be used to determine if sample is Positive or Negative or Indeterminate.
Exemplary Storage and Stability Information
Each reference cited herein is incorporated by reference in its entirety, including: U.S. Pat. Nos. 6,057,149, 6,048,734, 6,130,098, 6,271,021, 6,911,183, CA2,294,819, 6,575,188, 6,692,700, 6,852,287, and Canadian patent application no. CA 2,294,819.
A number of embodiments of the technology have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the technology. Accordingly, other embodiments are within the scope of the following claims.
This application is a continuation of U.S. application Ser. No. 14/719,692, filed May 22, 2015, which is a continuation of U.S. application Ser. No. 11/728,964, filed Mar. 26, 2007 and issued as U.S. Pat. No. 9,040,288 on May 26, 2015, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60/786,007, filed Mar. 24, 2006, and 60/859,284, filed Nov. 14, 2006. The disclosures of all of the above-referenced prior applications, publications, and patents are considered part of the disclosure of this application, and are incorporated by reference herein in their entirety. This application is also related to, and incorporates herein by reference the specifications of U.S. Design application Ser. Nos. 29/257,028, 29/257,029, and 29/257,030, all of which filed on Mar. 27, 2006, and also U.S. patent application Ser. No. 11/580,267, filed Oct. 11, 2006, also incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| D189404 | Nicolle | Dec 1960 | S |
| 3050239 | Williams | Aug 1962 | A |
| 3905772 | Hartnett et al. | Sep 1975 | A |
| 3985649 | Eddelman | Oct 1976 | A |
| 4018089 | Dzula et al. | Apr 1977 | A |
| 4018652 | Lanham et al. | Apr 1977 | A |
| 4038192 | Serur | Jul 1977 | A |
| 4055395 | Honkawa et al. | Oct 1977 | A |
| D249706 | Adamski | Sep 1978 | S |
| 4139005 | Dickey | Feb 1979 | A |
| D252157 | Kronish et al. | Jun 1979 | S |
| D252341 | Thomas | Jul 1979 | S |
| D254687 | Fadler et al. | Apr 1980 | S |
| 4212744 | Oota | Jul 1980 | A |
| D261033 | Armbruster | Sep 1981 | S |
| D261173 | Armbruster | Oct 1981 | S |
| 4301412 | Hill et al. | Nov 1981 | A |
| 4439526 | Columbus | Mar 1984 | A |
| 4457329 | Werley et al. | Jul 1984 | A |
| 4466740 | Kano et al. | Aug 1984 | A |
| 4472357 | Levy et al. | Sep 1984 | A |
| 4504582 | Swann | Mar 1985 | A |
| 4522786 | Ebersole | Jun 1985 | A |
| D279817 | Chen et al. | Jul 1985 | S |
| D282208 | Lowry | Jan 1986 | S |
| 4599315 | Terasaki et al. | Jul 1986 | A |
| 4612873 | Eberle | Sep 1986 | A |
| 4612959 | Costello | Sep 1986 | A |
| D288478 | Carlson et al. | Feb 1987 | S |
| 4647432 | Wakatake | Mar 1987 | A |
| 4654127 | Baker et al. | Mar 1987 | A |
| 4673657 | Christian | Jun 1987 | A |
| 4678752 | Thorne et al. | Jul 1987 | A |
| 4683195 | Mullis et al. | Jul 1987 | A |
| 4683202 | Mullis | Jul 1987 | A |
| 4698302 | Whitehead et al. | Oct 1987 | A |
| D292735 | Lovborg | Nov 1987 | S |
| 4720374 | Ramachandran | Jan 1988 | A |
| 4724207 | Hou et al. | Feb 1988 | A |
| 4795698 | Owen et al. | Jan 1989 | A |
| 4798693 | Mase et al. | Jan 1989 | A |
| 4800022 | Leonard | Jan 1989 | A |
| 4827944 | Nugent | May 1989 | A |
| 4841786 | Schulz | Jun 1989 | A |
| D302294 | Hillman | Jul 1989 | S |
| 4855110 | Marker et al. | Aug 1989 | A |
| 4871779 | Killat et al. | Oct 1989 | A |
| 4889818 | Gelfand et al. | Dec 1989 | A |
| 4895650 | Wang | Jan 1990 | A |
| 4902624 | Columbus et al. | Feb 1990 | A |
| 4914710 | Ward et al. | Apr 1990 | A |
| 4919829 | Gates et al. | Apr 1990 | A |
| 4921809 | Schiff et al. | May 1990 | A |
| 4935342 | Seligson et al. | Jun 1990 | A |
| 4946562 | Guruswamy | Aug 1990 | A |
| 4948561 | Hinckley et al. | Aug 1990 | A |
| 4949742 | Rando et al. | Aug 1990 | A |
| D310413 | Bigler et al. | Sep 1990 | S |
| 4963498 | Hillman | Oct 1990 | A |
| 4965188 | Mullis et al. | Oct 1990 | A |
| 4967950 | Legg et al. | Nov 1990 | A |
| D312692 | Bradley | Dec 1990 | S |
| 4978502 | Dole et al. | Dec 1990 | A |
| 4978622 | Mishell et al. | Dec 1990 | A |
| 4989626 | Takagi et al. | Feb 1991 | A |
| 4994373 | Stavrianopoulos et al. | Feb 1991 | A |
| 4997772 | Sutton et al. | Mar 1991 | A |
| 5001417 | Pumphrey et al. | Mar 1991 | A |
| 5004583 | Guruswamy et al. | Apr 1991 | A |
| 5048554 | Kremer | Sep 1991 | A |
| 5053199 | Keiser et al. | Oct 1991 | A |
| 5060823 | Perlman | Oct 1991 | A |
| 5061336 | Soane | Oct 1991 | A |
| 5064618 | Baker et al. | Nov 1991 | A |
| 5071531 | Soane | Dec 1991 | A |
| 5089233 | DeVaney, Jr. et al. | Feb 1992 | A |
| 5091328 | Miller | Feb 1992 | A |
| D324426 | Fan et al. | Mar 1992 | S |
| 5096669 | Lauks et al. | Mar 1992 | A |
| D325638 | Sloat et al. | Apr 1992 | S |
| 5126002 | Iwata et al. | Jun 1992 | A |
| 5126022 | Soane et al. | Jun 1992 | A |
| D328135 | Fan et al. | Jul 1992 | S |
| D328794 | Frenkel et al. | Aug 1992 | S |
| 5135627 | Soane | Aug 1992 | A |
| 5135872 | Pouletty et al. | Aug 1992 | A |
| 5147606 | Charlton et al. | Sep 1992 | A |
| 5147777 | Sutton et al. | Sep 1992 | A |
| 5155166 | Danielson et al. | Oct 1992 | A |
| 5169512 | Wedenmann et al. | Dec 1992 | A |
| 5173269 | Mon et al. | Dec 1992 | A |
| D333522 | Gianino | Feb 1993 | S |
| 5186339 | Heissler | Feb 1993 | A |
| 5192507 | Taylor et al. | Mar 1993 | A |
| 5208163 | Charlton et al. | May 1993 | A |
| 5217694 | Gibler et al. | Jun 1993 | A |
| 5223226 | Wittmer et al. | Jun 1993 | A |
| 5229297 | Schnipelsky et al. | Jul 1993 | A |
| 5231015 | Cummins et al. | Jul 1993 | A |
| D338275 | Fischer et al. | Aug 1993 | S |
| 5234809 | Boom et al. | Aug 1993 | A |
| 5250263 | Manz | Oct 1993 | A |
| 5252743 | Barrett et al. | Oct 1993 | A |
| 5256376 | Callan et al. | Oct 1993 | A |
| 5273716 | Northrup et al. | Dec 1993 | A |
| 5275787 | Yuguchi et al. | Jan 1994 | A |
| 5282950 | Dietze et al. | Feb 1994 | A |
| 5296375 | Kricka et al. | Mar 1994 | A |
| 5304477 | Nagoh et al. | Apr 1994 | A |
| 5304487 | Wilding et al. | Apr 1994 | A |
| D347478 | Pinkney | May 1994 | S |
| 5311896 | Kaartinen et al. | May 1994 | A |
| 5311996 | Duffy et al. | May 1994 | A |
| 5316727 | Suzuki et al. | May 1994 | A |
| 5327038 | Culp | Jul 1994 | A |
| 5334499 | Burdick et al. | Aug 1994 | A |
| 5338671 | Scalice et al. | Aug 1994 | A |
| 5339486 | Persic, Jr. | Aug 1994 | A |
| D351475 | Gerber | Oct 1994 | S |
| D351913 | Hieb et al. | Oct 1994 | S |
| 5364591 | Green et al. | Nov 1994 | A |
| 5372946 | Cusak et al. | Dec 1994 | A |
| 5374395 | Robinson | Dec 1994 | A |
| 5384499 | Pedersen et al. | Jan 1995 | A |
| 5389339 | Petschek et al. | Feb 1995 | A |
| D356232 | Armstrong et al. | Mar 1995 | S |
| 5397709 | Berndt | Mar 1995 | A |
| 5401465 | Smethers et al. | Mar 1995 | A |
| 5411708 | Moscetta et al. | May 1995 | A |
| 5414245 | Hackleman | May 1995 | A |
| 5415839 | Zaun et al. | May 1995 | A |
| 5416000 | Allen et al. | May 1995 | A |
| 5422271 | Chen et al. | Jun 1995 | A |
| 5422284 | Lau | Jun 1995 | A |
| 5427946 | Kricka et al. | Jun 1995 | A |
| 5443791 | Cathcart et al. | Aug 1995 | A |
| 5466574 | Liberti et al. | Nov 1995 | A |
| 5474796 | Brennan | Dec 1995 | A |
| 5475487 | Mariella, Jr. et al. | Dec 1995 | A |
| D366116 | Biskupski | Jan 1996 | S |
| 5486335 | Wilding et al. | Jan 1996 | A |
| 5494639 | Grzegorzewski | Feb 1996 | A |
| 5498392 | Wilding et al. | Mar 1996 | A |
| 5503803 | Brown | Apr 1996 | A |
| 5516410 | Schneider et al. | May 1996 | A |
| 5519635 | Miyake et al. | May 1996 | A |
| 5529677 | Schneider et al. | Jun 1996 | A |
| 5559432 | Logue | Sep 1996 | A |
| 5565171 | Dovichi et al. | Oct 1996 | A |
| 5569364 | Hooper et al. | Oct 1996 | A |
| 5578270 | Reichler et al. | Nov 1996 | A |
| 5578818 | Kain et al. | Nov 1996 | A |
| 5579928 | Anukwuem | Dec 1996 | A |
| 5580523 | Bard | Dec 1996 | A |
| 5582884 | Ball et al. | Dec 1996 | A |
| 5582988 | Backus et al. | Dec 1996 | A |
| 5585069 | Zanucchi et al. | Dec 1996 | A |
| 5585089 | Queen et al. | Dec 1996 | A |
| 5585242 | Bouma et al. | Dec 1996 | A |
| 5587128 | Wilding et al. | Dec 1996 | A |
| 5589136 | Northrup et al. | Dec 1996 | A |
| 5593838 | Zanzucchi et al. | Jan 1997 | A |
| 5595708 | Berndt | Jan 1997 | A |
| 5599432 | Manz et al. | Feb 1997 | A |
| 5599503 | Manz et al. | Feb 1997 | A |
| 5599667 | Arnold, Jr. et al. | Feb 1997 | A |
| 5601727 | Bormann et al. | Feb 1997 | A |
| 5603351 | Cherukuri et al. | Feb 1997 | A |
| 5605662 | Heller et al. | Feb 1997 | A |
| 5609910 | Hackleman | Mar 1997 | A |
| D378782 | LaBarbera et al. | Apr 1997 | S |
| 5628890 | Carter et al. | May 1997 | A |
| 5630920 | Friese et al. | May 1997 | A |
| 5631337 | Sassi et al. | May 1997 | A |
| 5632876 | Zanzucchi et al. | May 1997 | A |
| 5632957 | Heller et al. | May 1997 | A |
| 5635358 | Wilding et al. | Jun 1997 | A |
| 5637469 | Wilding et al. | Jun 1997 | A |
| 5639423 | Northrup et al. | Jun 1997 | A |
| 5639428 | Cottingham | Jun 1997 | A |
| 5643738 | Zanzucchi et al. | Jul 1997 | A |
| 5645801 | Bouma et al. | Jul 1997 | A |
| 5646039 | Northrup et al. | Jul 1997 | A |
| 5646049 | Tayi | Jul 1997 | A |
| 5647994 | Tuunanen et al. | Jul 1997 | A |
| 5651839 | Rauf | Jul 1997 | A |
| 5652141 | Henco et al. | Jul 1997 | A |
| 5652149 | Mileaf et al. | Jul 1997 | A |
| D382346 | Buhler et al. | Aug 1997 | S |
| D382647 | Staples et al. | Aug 1997 | S |
| 5654141 | Mariani et al. | Aug 1997 | A |
| 5658515 | Lee et al. | Aug 1997 | A |
| 5667976 | Van Ness et al. | Sep 1997 | A |
| 5671303 | Shieh et al. | Sep 1997 | A |
| 5674394 | Whitmore | Oct 1997 | A |
| 5674742 | Northrup et al. | Oct 1997 | A |
| 5681484 | Zanzucchi et al. | Oct 1997 | A |
| 5681529 | Taguchi et al. | Oct 1997 | A |
| 5683657 | Mian | Nov 1997 | A |
| 5683659 | Hovatter | Nov 1997 | A |
| 5699157 | Parce et al. | Dec 1997 | A |
| 5700637 | Southern | Dec 1997 | A |
| 5705813 | Apffel et al. | Jan 1998 | A |
| 5721136 | Finney et al. | Feb 1998 | A |
| 5725831 | Reichler et al. | Mar 1998 | A |
| 5726026 | Wilding et al. | Mar 1998 | A |
| 5726404 | Brody | Mar 1998 | A |
| 5726944 | Pelley et al. | Mar 1998 | A |
| 5731212 | Gavin et al. | Mar 1998 | A |
| 5744366 | Kricka et al. | Apr 1998 | A |
| 5746978 | Bienhaus et al. | May 1998 | A |
| 5747666 | Willis | May 1998 | A |
| 5750015 | Soane et al. | May 1998 | A |
| 5755942 | Zanzucchi et al. | May 1998 | A |
| 5762874 | Seaton et al. | Jun 1998 | A |
| 5763262 | Wong et al. | Jun 1998 | A |
| 5770029 | Nelson et al. | Jun 1998 | A |
| 5770388 | Vorpahl | Jun 1998 | A |
| 5772966 | Maracas et al. | Jun 1998 | A |
| 5779868 | Parce et al. | Jul 1998 | A |
| 5783148 | Cottingham et al. | Jul 1998 | A |
| 5787032 | Heller et al. | Jul 1998 | A |
| 5788814 | Sun et al. | Aug 1998 | A |
| 5800600 | Lima-Marques et al. | Sep 1998 | A |
| 5800690 | Chow et al. | Sep 1998 | A |
| 5804436 | Okun et al. | Sep 1998 | A |
| D399959 | Prokop et al. | Oct 1998 | S |
| 5819749 | Lee et al. | Oct 1998 | A |
| 5827481 | Bente et al. | Oct 1998 | A |
| 5842106 | Thaler et al. | Nov 1998 | A |
| 5842787 | Kopf-Sill et al. | Dec 1998 | A |
| 5846396 | Zanzucchi et al. | Dec 1998 | A |
| 5846493 | Bankier et al. | Dec 1998 | A |
| 5849208 | Hayes et al. | Dec 1998 | A |
| 5849486 | Heller et al. | Dec 1998 | A |
| 5849489 | Heller | Dec 1998 | A |
| 5849598 | Wilson et al. | Dec 1998 | A |
| 5852495 | Parce | Dec 1998 | A |
| 5856174 | Lipshutz et al. | Jan 1999 | A |
| 5858187 | Ramsey et al. | Jan 1999 | A |
| 5858188 | Soane et al. | Jan 1999 | A |
| 5863502 | Southgate et al. | Jan 1999 | A |
| 5863708 | Zanzucchi et al. | Jan 1999 | A |
| 5863801 | Southgate et al. | Jan 1999 | A |
| 5866345 | Wilding et al. | Feb 1999 | A |
| 5869004 | Parce et al. | Feb 1999 | A |
| 5869244 | Martin et al. | Feb 1999 | A |
| 5872010 | Karger et al. | Feb 1999 | A |
| 5872623 | Stabile et al. | Feb 1999 | A |
| 5874046 | Megerle | Feb 1999 | A |
| 5876675 | Kennedy | Mar 1999 | A |
| 5880071 | Parce et al. | Mar 1999 | A |
| 5882465 | McReynolds | Mar 1999 | A |
| 5883211 | Sassi et al. | Mar 1999 | A |
| 5885432 | Hooper et al. | Mar 1999 | A |
| 5885470 | Parce et al. | Mar 1999 | A |
| 5895762 | Greenfield et al. | Apr 1999 | A |
| 5900130 | Benvegnu et al. | May 1999 | A |
| 5911737 | Lee et al. | Jun 1999 | A |
| 5912124 | Kumar | Jun 1999 | A |
| 5912134 | Shartle | Jun 1999 | A |
| 5914229 | Loewy | Jun 1999 | A |
| 5916522 | Boyd et al. | Jun 1999 | A |
| 5916776 | Kumar | Jun 1999 | A |
| 5919646 | Okun et al. | Jul 1999 | A |
| 5919711 | Boyd et al. | Jul 1999 | A |
| 5922591 | Anderson et al. | Jul 1999 | A |
| 5927547 | Papen et al. | Jul 1999 | A |
| 5928161 | Krulevitch et al. | Jul 1999 | A |
| 5928880 | Wilding et al. | Jul 1999 | A |
| 5929208 | Heller et al. | Jul 1999 | A |
| D413391 | Lapeus et al. | Aug 1999 | S |
| 5932799 | Moles | Aug 1999 | A |
| 5935401 | Amigo | Aug 1999 | A |
| 5939291 | Loewy et al. | Aug 1999 | A |
| 5939312 | Baier et al. | Aug 1999 | A |
| 5942443 | Parce et al. | Aug 1999 | A |
| 5944717 | Lee et al. | Aug 1999 | A |
| D413677 | Dumitrescu et al. | Sep 1999 | S |
| D414271 | Mendoza | Sep 1999 | S |
| 5948227 | Dubrow | Sep 1999 | A |
| 5948363 | Gaillard | Sep 1999 | A |
| 5948673 | Cottingham | Sep 1999 | A |
| 5955028 | Chow | Sep 1999 | A |
| 5955029 | Wilding et al. | Sep 1999 | A |
| 5957579 | Kopf-Sill et al. | Sep 1999 | A |
| 5958203 | Parce et al. | Sep 1999 | A |
| 5958349 | Petersen et al. | Sep 1999 | A |
| 5958694 | Nikiforov | Sep 1999 | A |
| 5959221 | Boyd et al. | Sep 1999 | A |
| 5959291 | Jensen | Sep 1999 | A |
| 5935522 | Swerdlow et al. | Oct 1999 | A |
| 5964995 | Nikiforov et al. | Oct 1999 | A |
| 5964997 | McBride | Oct 1999 | A |
| 5965001 | Chow et al. | Oct 1999 | A |
| 5965410 | Chow et al. | Oct 1999 | A |
| 5965886 | Sauer et al. | Oct 1999 | A |
| 5968745 | Thorp et al. | Oct 1999 | A |
| 5972187 | Parce et al. | Oct 1999 | A |
| 5973138 | Collis | Oct 1999 | A |
| D417009 | Boyd | Nov 1999 | S |
| 5976336 | Dubrow et al. | Nov 1999 | A |
| 5980704 | Cherukuri et al. | Nov 1999 | A |
| 5980719 | Cherukuri et al. | Nov 1999 | A |
| 5981735 | Thatcher et al. | Nov 1999 | A |
| 5985651 | Hunicke-Smith | Nov 1999 | A |
| 5989402 | Chow et al. | Nov 1999 | A |
| 5992820 | Fare et al. | Nov 1999 | A |
| 5993611 | Moroney, III et al. | Nov 1999 | A |
| 5993750 | Ghosh et al. | Nov 1999 | A |
| 5997708 | Craig | Dec 1999 | A |
| 6001229 | Ramsey | Dec 1999 | A |
| 6001231 | Kopf-Sill | Dec 1999 | A |
| 6001307 | Naka et al. | Dec 1999 | A |
| 6004450 | Northrup et al. | Dec 1999 | A |
| 6004515 | Parce et al. | Dec 1999 | A |
| 6007690 | Nelson et al. | Dec 1999 | A |
| 6010607 | Ramsey | Jan 2000 | A |
| 6010608 | Ramsey | Jan 2000 | A |
| 6010627 | Hood, III | Jan 2000 | A |
| 6012902 | Parce | Jan 2000 | A |
| D420747 | Dumitrescu et al. | Feb 2000 | S |
| D421130 | Cohen et al. | Feb 2000 | S |
| 6024920 | Cunanan | Feb 2000 | A |
| D421653 | Purcell | Mar 2000 | S |
| 6033546 | Ramsey | Mar 2000 | A |
| 6033880 | Haff et al. | Mar 2000 | A |
| 6043080 | Lipshutz et al. | Mar 2000 | A |
| 6043880 | Andrews et al. | Mar 2000 | A |
| 6046056 | Parce et al. | Apr 2000 | A |
| 6048734 | Burns et al. | Apr 2000 | A |
| 6054034 | Soane et al. | Apr 2000 | A |
| 6054277 | Furcht et al. | Apr 2000 | A |
| 6056860 | Amigo et al. | May 2000 | A |
| 6057149 | Burns et al. | May 2000 | A |
| 6062261 | Jacobson et al. | May 2000 | A |
| 6063341 | Fassbind et al. | May 2000 | A |
| 6063589 | Kellogg et al. | May 2000 | A |
| 6068751 | Neukermans | May 2000 | A |
| 6068752 | Dubrow et al. | May 2000 | A |
| 6071478 | Chow | Jun 2000 | A |
| 6074725 | Kennedy | Jun 2000 | A |
| 6074827 | Nelson et al. | Jun 2000 | A |
| D428497 | Lapeus et al. | Jul 2000 | S |
| 6086740 | Kennedy | Jul 2000 | A |
| 6096509 | Okun et al. | Aug 2000 | A |
| 6100541 | Nagle et al. | Aug 2000 | A |
| 6102897 | Lang | Aug 2000 | A |
| 6103537 | Ullman et al. | Aug 2000 | A |
| 6106685 | McBride et al. | Aug 2000 | A |
| 6110343 | Ramsey et al. | Aug 2000 | A |
| 6117398 | Bienhaus et al. | Sep 2000 | A |
| 6123205 | Dumitrescu et al. | Sep 2000 | A |
| 6123798 | Gandhi et al. | Sep 2000 | A |
| 6130098 | Handique et al. | Oct 2000 | A |
| 6132580 | Mathies et al. | Oct 2000 | A |
| 6132684 | Marino | Oct 2000 | A |
| 6133436 | Koster et al. | Oct 2000 | A |
| D433759 | Mathis et al. | Nov 2000 | S |
| 6143250 | Tajima | Nov 2000 | A |
| 6143547 | Hsu | Nov 2000 | A |
| 6149787 | Chow et al. | Nov 2000 | A |
| 6149872 | Mack et al. | Nov 2000 | A |
| 6156199 | Zuk, Jr. | Dec 2000 | A |
| 6158269 | Dorenkott et al. | Dec 2000 | A |
| 6167910 | Chow | Jan 2001 | B1 |
| 6168948 | Anderson et al. | Jan 2001 | B1 |
| 6171850 | Nagle et al. | Jan 2001 | B1 |
| 6174675 | Chow et al. | Jan 2001 | B1 |
| 6180950 | Olsen | Jan 2001 | B1 |
| D438311 | Yamanishi et al. | Feb 2001 | S |
| 6190619 | Kilcoin et al. | Feb 2001 | B1 |
| 6194563 | Cruickshank | Feb 2001 | B1 |
| D438632 | Miller | Mar 2001 | S |
| D438633 | Miller | Mar 2001 | S |
| D439673 | Brophy et al. | Mar 2001 | S |
| 6197595 | Anderson et al. | Mar 2001 | B1 |
| 6211989 | Wulf et al. | Apr 2001 | B1 |
| 6213151 | Jacobson et al. | Apr 2001 | B1 |
| 6221600 | MacLeod et al. | Apr 2001 | B1 |
| 6228635 | Armstrong et al. | May 2001 | B1 |
| 6232072 | Fisher | May 2001 | B1 |
| 6235175 | Dubrow et al. | May 2001 | B1 |
| 6235313 | Mathiowitz et al. | May 2001 | B1 |
| 6235471 | Knapp et al. | May 2001 | B1 |
| 6236456 | Giebeler et al. | May 2001 | B1 |
| 6236581 | Foss et al. | May 2001 | B1 |
| 6238626 | Higuchi et al. | May 2001 | B1 |
| 6251343 | Dubrow et al. | Jun 2001 | B1 |
| 6254826 | Acosta et al. | Jul 2001 | B1 |
| 6259635 | Khouri et al. | Jul 2001 | B1 |
| 6261431 | Mathies et al. | Jul 2001 | B1 |
| 6267858 | Parce et al. | Jul 2001 | B1 |
| D446306 | Ochi et al. | Aug 2001 | S |
| 6271021 | Burns et al. | Aug 2001 | B1 |
| 6274089 | Chow et al. | Aug 2001 | B1 |
| 6280967 | Ransom et al. | Aug 2001 | B1 |
| 6281008 | Komai et al. | Aug 2001 | B1 |
| 6284113 | Bjornson et al. | Sep 2001 | B1 |
| 6284470 | Bitner et al. | Sep 2001 | B1 |
| 6287254 | Dodds | Sep 2001 | B1 |
| 6287774 | Nikiforov | Sep 2001 | B1 |
| 6291248 | Haj-Ahmad | Sep 2001 | B1 |
| 6294063 | Becker et al. | Sep 2001 | B1 |
| 6300124 | Blumenfeld et al. | Oct 2001 | B1 |
| 6302134 | Kellogg et al. | Oct 2001 | B1 |
| 6302304 | Spencer | Oct 2001 | B1 |
| 6303343 | Kopf-sill | Oct 2001 | B1 |
| 6306273 | Wainright et al. | Oct 2001 | B1 |
| 6306590 | Mehta et al. | Oct 2001 | B1 |
| 6310199 | Smith et al. | Oct 2001 | B1 |
| 6316774 | Giebeler et al. | Nov 2001 | B1 |
| 6319469 | Mian et al. | Nov 2001 | B1 |
| 6319474 | Krulevitch et al. | Nov 2001 | B1 |
| 6322683 | Wolk et al. | Nov 2001 | B1 |
| 6326083 | Yang et al. | Dec 2001 | B1 |
| 6326147 | Oldham et al. | Dec 2001 | B1 |
| 6326211 | Anderson et al. | Dec 2001 | B1 |
| 6334980 | Hayes et al. | Jan 2002 | B1 |
| 6337435 | Chu et al. | Jan 2002 | B1 |
| 6353475 | Jensen et al. | Mar 2002 | B1 |
| 6358387 | Kopf-sill et al. | Mar 2002 | B1 |
| 6366924 | Parce | Apr 2002 | B1 |
| 6368561 | Rutishauser et al. | Apr 2002 | B1 |
| 6368871 | Christel et al. | Apr 2002 | B1 |
| 6370206 | Schenk | Apr 2002 | B1 |
| 6375185 | Lin | Apr 2002 | B1 |
| 6375901 | Robotti et al. | Apr 2002 | B1 |
| 6379884 | Wada et al. | Apr 2002 | B2 |
| 6379929 | Burns et al. | Apr 2002 | B1 |
| 6379974 | Parce et al. | Apr 2002 | B1 |
| 6382254 | Yang et al. | May 2002 | B1 |
| 6391541 | Petersen et al. | May 2002 | B1 |
| 6391623 | Besemer et al. | May 2002 | B1 |
| 6395161 | Schneider et al. | May 2002 | B1 |
| 6398956 | Coville et al. | Jun 2002 | B1 |
| 6399025 | Chow | Jun 2002 | B1 |
| 6399389 | Parce et al. | Jun 2002 | B1 |
| 6399952 | Maher et al. | Jun 2002 | B1 |
| 6401552 | Elkins | Jun 2002 | B1 |
| 6403338 | Knapp et al. | Jun 2002 | B1 |
| 6408878 | Unger et al. | Jun 2002 | B2 |
| 6413401 | Chow et al. | Jul 2002 | B1 |
| 6416642 | Alajoki et al. | Jul 2002 | B1 |
| 6420143 | Kopf-sill | Jul 2002 | B1 |
| 6425972 | McReynolds | Jul 2002 | B1 |
| D461906 | Pham | Aug 2002 | S |
| 6428987 | Franzen | Aug 2002 | B2 |
| 6430512 | Gallagher | Aug 2002 | B1 |
| 6432366 | Ruediger et al. | Aug 2002 | B2 |
| 6440725 | Pourahmadi et al. | Aug 2002 | B1 |
| D463031 | Slomski et al. | Sep 2002 | S |
| 6444461 | Knapp et al. | Sep 2002 | B1 |
| 6447661 | Chow et al. | Sep 2002 | B1 |
| 6447727 | Parce et al. | Sep 2002 | B1 |
| 6448047 | Dattagupta et al. | Sep 2002 | B2 |
| 6448064 | Vo-Dinh et al. | Sep 2002 | B1 |
| 6453928 | Kaplan et al. | Sep 2002 | B1 |
| 6458259 | Parce et al. | Oct 2002 | B1 |
| 6461570 | Ishihara et al. | Oct 2002 | B2 |
| 6465257 | Parce et al. | Oct 2002 | B1 |
| 6468761 | Yang et al. | Oct 2002 | B2 |
| 6472141 | Nikiforov | Oct 2002 | B2 |
| D466219 | Wynschenk et al. | Nov 2002 | S |
| 6475364 | Dubrow et al. | Nov 2002 | B1 |
| D467348 | McMichael et al. | Dec 2002 | S |
| D467349 | Niedbala et al. | Dec 2002 | S |
| 6488897 | Dubrow et al. | Dec 2002 | B2 |
| 6495104 | Unno et al. | Dec 2002 | B1 |
| 6498497 | Chow et al. | Dec 2002 | B1 |
| 6500323 | Chow et al. | Dec 2002 | B1 |
| 6500390 | Boulton et al. | Dec 2002 | B1 |
| D468437 | McMenamy et al. | Jan 2003 | S |
| 6506609 | Wada et al. | Jan 2003 | B1 |
| 6509186 | Zou | Jan 2003 | B1 |
| 6509193 | Tajima | Jan 2003 | B1 |
| 6511853 | Kopf-sill et al. | Jan 2003 | B1 |
| D470595 | Crisanti et al. | Feb 2003 | S |
| 6515753 | Maher | Feb 2003 | B2 |
| 6517783 | Horner et al. | Feb 2003 | B2 |
| 6520197 | Deshmukh et al. | Feb 2003 | B2 |
| 6521181 | Northrup et al. | Feb 2003 | B1 |
| 6521188 | Webster | Feb 2003 | B1 |
| 6524456 | Ramsey et al. | Feb 2003 | B1 |
| 6524532 | Northrup | Feb 2003 | B1 |
| 6524790 | Kopf-sill et al. | Feb 2003 | B1 |
| D472324 | Rumore et al. | Mar 2003 | S |
| 6534295 | Tai et al. | Mar 2003 | B2 |
| 6537432 | Schneider et al. | Mar 2003 | B1 |
| 6537771 | Farinas et al. | Mar 2003 | B1 |
| 6540896 | Manz et al. | Apr 2003 | B1 |
| 6544734 | Briscoe et al. | Apr 2003 | B1 |
| 6547942 | Parce et al. | Apr 2003 | B1 |
| 6555389 | Ullman et al. | Apr 2003 | B1 |
| 6556923 | Gallagher et al. | Apr 2003 | B2 |
| D474279 | Mayer et al. | May 2003 | S |
| D474280 | Niedbala et al. | May 2003 | S |
| 6558916 | Veerapandian et al. | May 2003 | B2 |
| 6558945 | Kao | May 2003 | B1 |
| 6565815 | Chang et al. | May 2003 | B1 |
| 6569607 | McReynolds | May 2003 | B2 |
| 6572830 | Burdon et al. | Jun 2003 | B1 |
| 6575188 | Parunak | Jun 2003 | B2 |
| 6576459 | Miles et al. | Jun 2003 | B2 |
| 6579453 | Bächler et al. | Jun 2003 | B1 |
| 6589729 | Chan et al. | Jul 2003 | B2 |
| 6592821 | Wada et al. | Jul 2003 | B1 |
| 6597450 | Andrews et al. | Jul 2003 | B1 |
| 6602474 | Tajima | Aug 2003 | B1 |
| 6613211 | Mccormick et al. | Sep 2003 | B1 |
| 6613512 | Kopf-sill et al. | Sep 2003 | B1 |
| 6613580 | Chow et al. | Sep 2003 | B1 |
| 6613581 | Wada et al. | Sep 2003 | B1 |
| 6614030 | Maher et al. | Sep 2003 | B2 |
| 6620625 | Wolk et al. | Sep 2003 | B2 |
| 6623860 | Hu et al. | Sep 2003 | B2 |
| 6627406 | Singh et al. | Sep 2003 | B1 |
| D480814 | Lafferty et al. | Oct 2003 | S |
| 6632655 | Mehta et al. | Oct 2003 | B1 |
| 6633785 | Kasahara et al. | Oct 2003 | B1 |
| D482796 | Oyama et al. | Nov 2003 | S |
| 6640981 | Lafond et al. | Nov 2003 | B2 |
| 6649358 | Parce et al. | Nov 2003 | B1 |
| 6664104 | Pourahmadi et al. | Dec 2003 | B2 |
| 6669831 | Chow et al. | Dec 2003 | B2 |
| 6670133 | Knapp et al. | Dec 2003 | B2 |
| 6670153 | Stern | Dec 2003 | B2 |
| D484989 | Gebrian | Jan 2004 | S |
| 6672458 | Hansen et al. | Jan 2004 | B2 |
| 6681616 | Spaid et al. | Jan 2004 | B2 |
| 6681788 | Parce et al. | Jan 2004 | B2 |
| 6685813 | Williams et al. | Feb 2004 | B2 |
| 6692700 | Handique | Feb 2004 | B2 |
| 6695009 | Chien et al. | Feb 2004 | B2 |
| 6699713 | Benett et al. | Mar 2004 | B2 |
| 6706519 | Kellogg et al. | Mar 2004 | B1 |
| 6720148 | Nikiforov | Apr 2004 | B1 |
| 6730206 | Ricco et al. | May 2004 | B2 |
| 6733645 | Chow | May 2004 | B1 |
| 6734401 | Bedingham et al. | May 2004 | B2 |
| 6737026 | Bergh et al. | May 2004 | B1 |
| 6740518 | Duong et al. | May 2004 | B1 |
| D491272 | Alden et al. | Jun 2004 | S |
| D491273 | Biegler et al. | Jun 2004 | S |
| D491276 | Langille | Jun 2004 | S |
| 6750661 | Brooks et al. | Jun 2004 | B2 |
| 6752966 | Chazan | Jun 2004 | B1 |
| 6756019 | Dubrow et al. | Jun 2004 | B1 |
| 6762049 | Zou et al. | Jul 2004 | B2 |
| 6764859 | Kreuwel et al. | Jul 2004 | B1 |
| 6766817 | da Silva | Jul 2004 | B2 |
| 6773567 | Wolk | Aug 2004 | B1 |
| 6777184 | Nikiforov et al. | Aug 2004 | B2 |
| 6783962 | Olander et al. | Aug 2004 | B1 |
| D495805 | Lea et al. | Sep 2004 | S |
| 6787015 | Lackritz et al. | Sep 2004 | B2 |
| 6787016 | Tan et al. | Sep 2004 | B2 |
| 6787111 | Roach et al. | Sep 2004 | B2 |
| 6790328 | Jacobson et al. | Sep 2004 | B2 |
| 6790330 | Gascoyne et al. | Sep 2004 | B2 |
| 6811668 | Berndt et al. | Nov 2004 | B1 |
| 6818113 | Williams et al. | Nov 2004 | B2 |
| 6819027 | Saraf | Nov 2004 | B2 |
| 6824663 | Boone | Nov 2004 | B1 |
| D499813 | Wu | Dec 2004 | S |
| D500142 | Crisanti et al. | Dec 2004 | S |
| D500363 | Fanning et al. | Dec 2004 | S |
| 6827831 | Chow et al. | Dec 2004 | B1 |
| 6827906 | Bjornson et al. | Dec 2004 | B1 |
| 6838156 | Neyer et al. | Jan 2005 | B1 |
| 6838680 | Maher et al. | Jan 2005 | B2 |
| 6852287 | Ganesan | Feb 2005 | B2 |
| 6858185 | Kopf-sill et al. | Feb 2005 | B1 |
| 6859698 | Schmeisser | Feb 2005 | B2 |
| 6861035 | Pham et al. | Mar 2005 | B2 |
| 6878540 | Pourahmadi et al. | Apr 2005 | B2 |
| 6878755 | Singh et al. | Apr 2005 | B2 |
| 6884628 | Hubbell et al. | Apr 2005 | B2 |
| 6887693 | McMillan et al. | May 2005 | B2 |
| 6893879 | Petersen et al. | May 2005 | B2 |
| 6900889 | Bjornson et al. | May 2005 | B2 |
| 6905583 | Wainright et al. | Jun 2005 | B2 |
| 6905612 | Dorian et al. | Jun 2005 | B2 |
| 6906797 | Kao et al. | Jun 2005 | B1 |
| 6908594 | Schaevitz et al. | Jun 2005 | B1 |
| 6911183 | Handique et al. | Jun 2005 | B1 |
| 6914137 | Baker | Jul 2005 | B2 |
| 6915679 | Chien et al. | Jul 2005 | B2 |
| 6918404 | Dias da Silva | Jul 2005 | B2 |
| D508999 | Fanning et al. | Aug 2005 | S |
| 6939451 | Zhao et al. | Sep 2005 | B2 |
| 6940598 | Christel et al. | Sep 2005 | B2 |
| 6942771 | Kayyem | Sep 2005 | B1 |
| 6951632 | Unger et al. | Oct 2005 | B2 |
| 6958392 | Fomovskaia et al. | Oct 2005 | B2 |
| D512155 | Matsumoto | Nov 2005 | S |
| 6964747 | Banerjee et al. | Nov 2005 | B2 |
| 6977163 | Mehta | Dec 2005 | B1 |
| 6979424 | Northrup et al. | Dec 2005 | B2 |
| 6984516 | Briscoe et al. | Jan 2006 | B2 |
| D515707 | Sinohara et al. | Feb 2006 | S |
| D516221 | Wohlstadter et al. | Feb 2006 | S |
| 7001853 | Brown et al. | Feb 2006 | B1 |
| 7004184 | Handique et al. | Feb 2006 | B2 |
| D517554 | Yanagisawa et al. | Mar 2006 | S |
| 7010391 | Handique et al. | Mar 2006 | B2 |
| 7023007 | Gallagher | Apr 2006 | B2 |
| 7024281 | Unno | Apr 2006 | B1 |
| 7036667 | Greenstein et al. | May 2006 | B2 |
| 7037416 | Parce et al. | May 2006 | B2 |
| 7038472 | Chien | May 2006 | B1 |
| 7039527 | Tripathi et al. | May 2006 | B2 |
| 7040144 | Spaid et al. | May 2006 | B2 |
| 7049558 | Baer et al. | May 2006 | B2 |
| D523153 | Akashi et al. | Jun 2006 | S |
| 7055695 | Greenstein et al. | Jun 2006 | B2 |
| 7060171 | Nikiforov et al. | Jun 2006 | B1 |
| 7066586 | da Silva | Jun 2006 | B2 |
| 7069952 | McReynolds et al. | Jul 2006 | B1 |
| 7072036 | Jones et al. | Jul 2006 | B2 |
| 7099778 | Chien | Aug 2006 | B2 |
| D528215 | Malmsater | Sep 2006 | S |
| 7101467 | Spaid | Sep 2006 | B2 |
| 7105304 | Nikiforov et al. | Sep 2006 | B1 |
| D531321 | Godfrey et al. | Oct 2006 | S |
| 7118892 | Ammann et al. | Oct 2006 | B2 |
| 7118910 | Unger et al. | Oct 2006 | B2 |
| 7122799 | Hsieh et al. | Oct 2006 | B2 |
| 7135144 | Christel et al. | Nov 2006 | B2 |
| 7138032 | Gandhi et al. | Nov 2006 | B2 |
| D534280 | Gomm et al. | Dec 2006 | S |
| 7150814 | Parce et al. | Dec 2006 | B1 |
| 7150999 | Shuck | Dec 2006 | B1 |
| D535403 | Isozaki et al. | Jan 2007 | S |
| 7160423 | Chien et al. | Jan 2007 | B2 |
| 7161356 | Chien | Jan 2007 | B1 |
| 7169277 | Ausserer et al. | Jan 2007 | B2 |
| 7169601 | Northrup et al. | Jan 2007 | B1 |
| 7169618 | Skold | Jan 2007 | B2 |
| D537951 | Okamoto et al. | Mar 2007 | S |
| D538436 | Patadia et al. | Mar 2007 | S |
| 7188001 | Young et al. | Mar 2007 | B2 |
| 7192557 | Wu et al. | Mar 2007 | B2 |
| 7195986 | Bousse et al. | Mar 2007 | B1 |
| 7205154 | Corson | Apr 2007 | B2 |
| 7208125 | Dong | Apr 2007 | B1 |
| 7235406 | Woudenberg et al. | Jun 2007 | B1 |
| 7247274 | Chow | Jul 2007 | B1 |
| D548841 | Brownell et al. | Aug 2007 | S |
| D549827 | Maeno et al. | Aug 2007 | S |
| 7252928 | Hafeman et al. | Aug 2007 | B1 |
| 7255833 | Chang et al. | Aug 2007 | B2 |
| 7270786 | Parunak et al. | Sep 2007 | B2 |
| D554069 | Bolotin et al. | Oct 2007 | S |
| D554070 | Bolotin et al. | Oct 2007 | S |
| 7276208 | Sevigny et al. | Oct 2007 | B2 |
| 7276330 | Chow et al. | Oct 2007 | B2 |
| 7288228 | Lefebvre | Oct 2007 | B2 |
| 7297313 | Northrup et al. | Nov 2007 | B1 |
| D556914 | Okamoto et al. | Dec 2007 | S |
| 7303727 | Dubrow et al. | Dec 2007 | B1 |
| D559995 | Handique et al. | Jan 2008 | S |
| 7315376 | Bickmore et al. | Jan 2008 | B2 |
| 7323140 | Handique et al. | Jan 2008 | B2 |
| 7332130 | Handique | Feb 2008 | B2 |
| 7338760 | Gong et al. | Mar 2008 | B2 |
| D566291 | Parunak et al. | Apr 2008 | S |
| 7351377 | Chazan et al. | Apr 2008 | B2 |
| D569526 | Duffy et al. | May 2008 | S |
| 7374949 | Kuriger | May 2008 | B2 |
| 7390460 | Osawa et al. | Jun 2008 | B2 |
| 7419784 | Dubrow et al. | Sep 2008 | B2 |
| 7422669 | Jacobson et al. | Sep 2008 | B2 |
| 7440684 | Spaid et al. | Oct 2008 | B2 |
| 7476313 | Siddiqi | Jan 2009 | B2 |
| 7480042 | Phillips et al. | Jan 2009 | B1 |
| 7494577 | Williams et al. | Feb 2009 | B2 |
| 7494770 | Wilding et al. | Feb 2009 | B2 |
| 7514046 | Kechagia et al. | Apr 2009 | B2 |
| 7518726 | Rulison et al. | Apr 2009 | B2 |
| 7521186 | Burd Mehta | Apr 2009 | B2 |
| 7527769 | Bunch et al. | May 2009 | B2 |
| D595423 | Johansson et al. | Jun 2009 | S |
| 7553671 | Sinclair et al. | Jun 2009 | B2 |
| D596312 | Giraud et al. | Jul 2009 | S |
| D598566 | Allaer | Aug 2009 | S |
| 7578976 | Northrup et al. | Aug 2009 | B1 |
| D599234 | Ito | Sep 2009 | S |
| 7595197 | Brasseur | Sep 2009 | B2 |
| 7604938 | Takahashi et al. | Oct 2009 | B2 |
| 7622296 | Joseph et al. | Nov 2009 | B2 |
| 7628902 | Knowlton et al. | Dec 2009 | B2 |
| 7633606 | Northrup et al. | Dec 2009 | B2 |
| 7635588 | King et al. | Dec 2009 | B2 |
| 7645581 | Knapp et al. | Jan 2010 | B2 |
| 7670559 | Chien et al. | Mar 2010 | B2 |
| 7674431 | Ganesan | Mar 2010 | B2 |
| 7689022 | Weiner et al. | Mar 2010 | B2 |
| 7704735 | Facer et al. | Apr 2010 | B2 |
| 7723123 | Murphy et al. | May 2010 | B1 |
| D618820 | Wilson et al. | Jun 2010 | S |
| 7727371 | Kennedy et al. | Jun 2010 | B2 |
| 7727477 | Boronkay et al. | Jun 2010 | B2 |
| 7744817 | Bui | Jun 2010 | B2 |
| D621060 | Handique | Aug 2010 | S |
| D628305 | Gorrec et al. | Nov 2010 | S |
| 7829025 | Ganesan et al. | Nov 2010 | B2 |
| 7867776 | Kennedy et al. | Jan 2011 | B2 |
| D632799 | Canner et al. | Feb 2011 | S |
| 7892819 | Wilding et al. | Feb 2011 | B2 |
| D637737 | Wilson et al. | May 2011 | S |
| 7955864 | Cox et al. | Jun 2011 | B2 |
| 7987022 | Handique et al. | Jul 2011 | B2 |
| 7998708 | Handique et al. | Aug 2011 | B2 |
| 8071056 | Burns et al. | Dec 2011 | B2 |
| 8088616 | Handique | Jan 2012 | B2 |
| 8105783 | Handique | Jan 2012 | B2 |
| 8110158 | Handique | Feb 2012 | B2 |
| 8133671 | Williams et al. | Mar 2012 | B2 |
| 8182763 | Duffy et al. | May 2012 | B2 |
| 8246919 | Herchenbach et al. | Aug 2012 | B2 |
| 8273308 | Handique et al. | Sep 2012 | B2 |
| D669597 | Cavada et al. | Oct 2012 | S |
| 8287820 | Williams et al. | Oct 2012 | B2 |
| 8323584 | Ganesan | Dec 2012 | B2 |
| 8323900 | Handique et al. | Dec 2012 | B2 |
| 8324372 | Brahmasandra et al. | Dec 2012 | B2 |
| 8415103 | Handique | Apr 2013 | B2 |
| 8420015 | Ganesan et al. | Apr 2013 | B2 |
| 8440149 | Handique | May 2013 | B2 |
| 8470586 | Wu et al. | Jun 2013 | B2 |
| 8473104 | Handique et al. | Jun 2013 | B2 |
| D686749 | Trump | Jul 2013 | S |
| D687567 | Jungheim et al. | Aug 2013 | S |
| D692162 | Lentz et al. | Oct 2013 | S |
| 8679831 | Handique et al. | Mar 2014 | B2 |
| D702854 | Nakahana et al. | Apr 2014 | S |
| 8685341 | Ganesan | Apr 2014 | B2 |
| 8703069 | Handique et al. | Apr 2014 | B2 |
| 8709787 | Handique | Apr 2014 | B2 |
| 8710211 | Brahmasandra et al. | Apr 2014 | B2 |
| 8734733 | Handique | May 2014 | B2 |
| D710024 | Guo | Jul 2014 | S |
| 8765076 | Handique et al. | Jul 2014 | B2 |
| 8765454 | Zhou et al. | Jul 2014 | B2 |
| 8768517 | Handique et al. | Jul 2014 | B2 |
| 8852862 | Wu et al. | Oct 2014 | B2 |
| 8883490 | Handique et al. | Nov 2014 | B2 |
| 8894947 | Ganesan et al. | Nov 2014 | B2 |
| 8895311 | Handique et al. | Nov 2014 | B1 |
| D729404 | Teich et al. | May 2015 | S |
| 9028773 | Ganesan | May 2015 | B2 |
| 9040288 | Handique et al. | May 2015 | B2 |
| 9051604 | Handique | Jun 2015 | B2 |
| 9080207 | Handique et al. | Jul 2015 | B2 |
| D742027 | Lentz et al. | Oct 2015 | S |
| 9186677 | Williams et al. | Nov 2015 | B2 |
| 9217143 | Brahmasandra et al. | Dec 2015 | B2 |
| 9222954 | Lentz et al. | Dec 2015 | B2 |
| 9234236 | Thomas et al. | Jan 2016 | B2 |
| 9238223 | Handique | Jan 2016 | B2 |
| 9259734 | Williams et al. | Feb 2016 | B2 |
| 9259735 | Handique et al. | Feb 2016 | B2 |
| 9347586 | Williams et al. | May 2016 | B2 |
| 9480983 | Lentz et al. | Nov 2016 | B2 |
| 9528142 | Handique | Dec 2016 | B2 |
| 9618139 | Handique | Apr 2017 | B2 |
| D787087 | Duffy et al. | Jun 2017 | S |
| 9670528 | Handique et al. | Jun 2017 | B2 |
| 9677121 | Ganesan et al. | Jun 2017 | B2 |
| 9701957 | Wilson et al. | Jul 2017 | B2 |
| 9745623 | Steel | Aug 2017 | B2 |
| 9765389 | Gubatayao et al. | Sep 2017 | B2 |
| 9789481 | Petersen et al. | Oct 2017 | B2 |
| 9802199 | Handique et al. | Oct 2017 | B2 |
| 9815057 | Handique | Nov 2017 | B2 |
| 9958466 | Dalbert et al. | May 2018 | B2 |
| 10065185 | Handique | Sep 2018 | B2 |
| 10071376 | Williams et al. | Sep 2018 | B2 |
| 10076754 | Lentz et al. | Sep 2018 | B2 |
| 10100302 | Brahmasandra et al. | Oct 2018 | B2 |
| 10139012 | Handique | Nov 2018 | B2 |
| 10179910 | Duffy et al. | Jan 2019 | B2 |
| 10234474 | Williams et al. | Mar 2019 | B2 |
| 10351901 | Ganesan et al. | Jul 2019 | B2 |
| 10364456 | Wu et al. | Jul 2019 | B2 |
| 10443088 | Wu et al. | Oct 2019 | B1 |
| 10494663 | Wu et al. | Dec 2019 | B1 |
| 10571935 | Handique et al. | Feb 2020 | B2 |
| 10590410 | Brahmasandra et al. | Mar 2020 | B2 |
| 10604788 | Wu et al. | Mar 2020 | B2 |
| 10619191 | Ganesan et al. | Apr 2020 | B2 |
| 10625261 | Williams et al. | Apr 2020 | B2 |
| 10625262 | Williams et al. | Apr 2020 | B2 |
| 10632466 | Williams et al. | Apr 2020 | B1 |
| 10695764 | Handique et al. | Jun 2020 | B2 |
| 10710069 | Handique et al. | Jul 2020 | B2 |
| 10717085 | Williams et al. | Jul 2020 | B2 |
| 10731201 | Handique et al. | Aug 2020 | B2 |
| 10781482 | Gubatayao et al. | Sep 2020 | B2 |
| 10799862 | Handique et al. | Oct 2020 | B2 |
| 20010005489 | Roach et al. | Jun 2001 | A1 |
| 20010012492 | Acosta et al. | Aug 2001 | A1 |
| 20010016358 | Osawa et al. | Aug 2001 | A1 |
| 20010018513 | Baker | Aug 2001 | A1 |
| 20010021355 | Baugh et al. | Sep 2001 | A1 |
| 20010023848 | Gjerde et al. | Sep 2001 | A1 |
| 20010038450 | McCaffrey et al. | Nov 2001 | A1 |
| 20010045358 | Kopf-Sill et al. | Nov 2001 | A1 |
| 20010046702 | Schembri | Nov 2001 | A1 |
| 20010048899 | Marouiss et al. | Dec 2001 | A1 |
| 20010051340 | Singh et al. | Dec 2001 | A1 |
| 20010055765 | O'Keefe et al. | Dec 2001 | A1 |
| 20020001848 | Bedingham et al. | Jan 2002 | A1 |
| 20020008053 | Hansen et al. | Jan 2002 | A1 |
| 20020009015 | Laugharn, Jr. et al. | Jan 2002 | A1 |
| 20020014443 | Hansen et al. | Feb 2002 | A1 |
| 20020015667 | Chow | Feb 2002 | A1 |
| 20020021983 | Comte et al. | Feb 2002 | A1 |
| 20020022261 | Anderson | Feb 2002 | A1 |
| 20020037499 | Quake et al. | Mar 2002 | A1 |
| 20020039783 | McMillan et al. | Apr 2002 | A1 |
| 20020047003 | Bedingham et al. | Apr 2002 | A1 |
| 20020053399 | Soane et al. | May 2002 | A1 |
| 20020054835 | Robotti et al. | May 2002 | A1 |
| 20020055167 | Pourahmadi et al. | May 2002 | A1 |
| 20020058332 | Quake et al. | May 2002 | A1 |
| 20020060156 | Mathies et al. | May 2002 | A1 |
| 20020068357 | Mathies et al. | Jun 2002 | A1 |
| 20020068821 | Gundling | Jun 2002 | A1 |
| 20020086443 | Bamdad | Jul 2002 | A1 |
| 20020090320 | Burow et al. | Jul 2002 | A1 |
| 20020092767 | Bjornson et al. | Jul 2002 | A1 |
| 20020094303 | Yamamoto et al. | Jul 2002 | A1 |
| 20020131903 | Ingenhoven et al. | Sep 2002 | A1 |
| 20020141903 | Parunak et al. | Oct 2002 | A1 |
| 20020143297 | Francavilla et al. | Oct 2002 | A1 |
| 20020155010 | Karp et al. | Oct 2002 | A1 |
| 20020155477 | Ito | Oct 2002 | A1 |
| 20020169518 | Luoma et al. | Nov 2002 | A1 |
| 20020173032 | Zou et al. | Nov 2002 | A1 |
| 20020176804 | Strand et al. | Nov 2002 | A1 |
| 20020187557 | Hobbs et al. | Dec 2002 | A1 |
| 20020192808 | Gambini et al. | Dec 2002 | A1 |
| 20030008308 | Enzelberger et al. | Jan 2003 | A1 |
| 20030008320 | Baker | Jan 2003 | A1 |
| 20030019522 | Parunak | Jan 2003 | A1 |
| 20030022392 | Hudak | Jan 2003 | A1 |
| 20030036067 | Schwartz | Feb 2003 | A1 |
| 20030049833 | Chen et al. | Mar 2003 | A1 |
| 20030059823 | Matsunaga et al. | Mar 2003 | A1 |
| 20030064507 | Gallagher et al. | Apr 2003 | A1 |
| 20030072683 | Stewart et al. | Apr 2003 | A1 |
| 20030073106 | Johansen et al. | Apr 2003 | A1 |
| 20030083686 | Freeman et al. | May 2003 | A1 |
| 20030087300 | Knapp et al. | May 2003 | A1 |
| 20030096310 | Hansen et al. | May 2003 | A1 |
| 20030099954 | Miltenyi et al. | May 2003 | A1 |
| 20030124611 | Schwartz | Jul 2003 | A1 |
| 20030127327 | Kurnik | Jul 2003 | A1 |
| 20030134333 | Dehlinger et al. | Jul 2003 | A1 |
| 20030136679 | Bohn et al. | Jul 2003 | A1 |
| 20030156991 | Halas et al. | Aug 2003 | A1 |
| 20030180192 | Seippel | Sep 2003 | A1 |
| 20030186295 | Colin et al. | Oct 2003 | A1 |
| 20030190608 | Blackburn et al. | Oct 2003 | A1 |
| 20030199081 | Wilding et al. | Oct 2003 | A1 |
| 20030211517 | Carulli et al. | Nov 2003 | A1 |
| 20040014202 | King et al. | Jan 2004 | A1 |
| 20040014238 | Krug et al. | Jan 2004 | A1 |
| 20040018116 | Desmond et al. | Jan 2004 | A1 |
| 20040018119 | Massaro | Jan 2004 | A1 |
| 20040022689 | Wulf et al. | Feb 2004 | A1 |
| 20040029258 | Heaney et al. | Feb 2004 | A1 |
| 20040029260 | Hansen et al. | Feb 2004 | A1 |
| 20040037739 | McNeely et al. | Feb 2004 | A1 |
| 20040043479 | Briscoe et al. | Mar 2004 | A1 |
| 20040053290 | Terbrueggen et al. | Mar 2004 | A1 |
| 20040063217 | Webster et al. | Apr 2004 | A1 |
| 20040065655 | Brown | Apr 2004 | A1 |
| 20040072278 | Chou et al. | Apr 2004 | A1 |
| 20040072375 | Gjerde et al. | Apr 2004 | A1 |
| 20040086427 | Childers et al. | May 2004 | A1 |
| 20040086956 | Bachur | May 2004 | A1 |
| 20040132059 | Scurati et al. | Jul 2004 | A1 |
| 20040141887 | Mainquist et al. | Jul 2004 | A1 |
| 20040151629 | Pease et al. | Aug 2004 | A1 |
| 20040157220 | Kurnool et al. | Aug 2004 | A1 |
| 20040161788 | Chen et al. | Aug 2004 | A1 |
| 20040171515 | Hamers et al. | Sep 2004 | A1 |
| 20040189311 | Glezer et al. | Sep 2004 | A1 |
| 20040197810 | Takenaka et al. | Oct 2004 | A1 |
| 20040200909 | McMillan et al. | Oct 2004 | A1 |
| 20040209331 | Ririe | Oct 2004 | A1 |
| 20040209354 | Mathies et al. | Oct 2004 | A1 |
| 20040224317 | Kordunsky et al. | Nov 2004 | A1 |
| 20040235154 | Oh | Nov 2004 | A1 |
| 20040240097 | Evans | Dec 2004 | A1 |
| 20040259237 | Kellogg | Dec 2004 | A1 |
| 20050009174 | Nikiforov et al. | Jan 2005 | A1 |
| 20050013737 | Chow et al. | Jan 2005 | A1 |
| 20050019902 | Mathies et al. | Jan 2005 | A1 |
| 20050037471 | Liu et al. | Feb 2005 | A1 |
| 20050041525 | Pugia et al. | Feb 2005 | A1 |
| 20050042639 | Knapp et al. | Feb 2005 | A1 |
| 20050048540 | Inami et al. | Mar 2005 | A1 |
| 20050058574 | Bysouth et al. | Mar 2005 | A1 |
| 20050058577 | Micklash et al. | Mar 2005 | A1 |
| 20050064535 | Favuzzi et al. | Mar 2005 | A1 |
| 20050069898 | Moon et al. | Mar 2005 | A1 |
| 20050084424 | Ganesan et al. | Apr 2005 | A1 |
| 20050106066 | Saltsman et al. | May 2005 | A1 |
| 20050112754 | Yoon et al. | May 2005 | A1 |
| 20050121324 | Park et al. | Jun 2005 | A1 |
| 20050129580 | Swinehart et al. | Jun 2005 | A1 |
| 20050130198 | Ammann et al. | Jun 2005 | A1 |
| 20050133370 | Park et al. | Jun 2005 | A1 |
| 20050135655 | Kopf-sill et al. | Jun 2005 | A1 |
| 20050142036 | Kim et al. | Jun 2005 | A1 |
| 20050158781 | Woudenberg et al. | Jul 2005 | A1 |
| 20050170362 | Wada et al. | Aug 2005 | A1 |
| 20050186585 | Juncosa et al. | Aug 2005 | A1 |
| 20050196321 | Huang | Sep 2005 | A1 |
| 20050202470 | Sundberg et al. | Sep 2005 | A1 |
| 20050202489 | Cho et al. | Sep 2005 | A1 |
| 20050202504 | Anderson et al. | Sep 2005 | A1 |
| 20050205788 | Itoh | Sep 2005 | A1 |
| 20050208676 | Kahatt | Sep 2005 | A1 |
| 20050214172 | Burgisser | Sep 2005 | A1 |
| 20050220675 | Reed et al. | Oct 2005 | A1 |
| 20050227269 | Lloyd et al. | Oct 2005 | A1 |
| 20050233370 | Ammann et al. | Oct 2005 | A1 |
| 20050238545 | Parce et al. | Oct 2005 | A1 |
| 20050239127 | Ammann et al. | Oct 2005 | A1 |
| 20050266489 | Ammann et al. | Dec 2005 | A1 |
| 20050276728 | Muller-Cohn et al. | Dec 2005 | A1 |
| 20060002817 | Bohm et al. | Jan 2006 | A1 |
| 20060003373 | Ammann et al. | Jan 2006 | A1 |
| 20060041058 | Yin et al. | Feb 2006 | A1 |
| 20060057039 | Morse et al. | Mar 2006 | A1 |
| 20060057629 | Kim | Mar 2006 | A1 |
| 20060058519 | Deggerdal et al. | Mar 2006 | A1 |
| 20060062696 | Chow et al. | Mar 2006 | A1 |
| 20060081539 | Safar et al. | Apr 2006 | A1 |
| 20060094004 | Nakajima et al. | May 2006 | A1 |
| 20060094108 | Yoder et al. | May 2006 | A1 |
| 20060113190 | Kurnik | Jun 2006 | A1 |
| 20060133965 | Tajima et al. | Jun 2006 | A1 |
| 20060134790 | Tanaka et al. | Jun 2006 | A1 |
| 20060148063 | Fauzzi et al. | Jul 2006 | A1 |
| 20060154341 | Chen | Jul 2006 | A1 |
| 20060165558 | Witty et al. | Jul 2006 | A1 |
| 20060165559 | Greenstein et al. | Jul 2006 | A1 |
| 20060177376 | Tomalia et al. | Aug 2006 | A1 |
| 20060177855 | Utermohlen et al. | Aug 2006 | A1 |
| 20060183216 | Handique | Aug 2006 | A1 |
| 20060201887 | Siddiqi | Sep 2006 | A1 |
| 20060205085 | Handique | Sep 2006 | A1 |
| 20060207944 | Siddiqi | Sep 2006 | A1 |
| 20060210435 | Alavie et al. | Sep 2006 | A1 |
| 20060223169 | Bedingham et al. | Oct 2006 | A1 |
| 20060228734 | Vann et al. | Oct 2006 | A1 |
| 20060246493 | Jensen et al. | Nov 2006 | A1 |
| 20060246533 | Fathollahi et al. | Nov 2006 | A1 |
| 20060269641 | Atwood et al. | Nov 2006 | A1 |
| 20060269961 | Fukushima et al. | Nov 2006 | A1 |
| 20070004028 | Lair et al. | Jan 2007 | A1 |
| 20070009386 | Padmanabhan et al. | Jan 2007 | A1 |
| 20070020699 | Carpenter et al. | Jan 2007 | A1 |
| 20070020764 | Miller | Jan 2007 | A1 |
| 20070026421 | Sundberg et al. | Feb 2007 | A1 |
| 20070042441 | Masters et al. | Feb 2007 | A1 |
| 20070048188 | Bigus | Mar 2007 | A1 |
| 20070054413 | Aviles et al. | Mar 2007 | A1 |
| 20070077643 | Nakamura et al. | Apr 2007 | A1 |
| 20070077648 | Okamoto et al. | Apr 2007 | A1 |
| 20070092901 | Ligler et al. | Apr 2007 | A1 |
| 20070098600 | Kayyem et al. | May 2007 | A1 |
| 20070099200 | Chow et al. | May 2007 | A1 |
| 20070104617 | Coulling et al. | May 2007 | A1 |
| 20070116613 | Elsener | May 2007 | A1 |
| 20070154895 | Spaid et al. | Jul 2007 | A1 |
| 20070177147 | Parce | Aug 2007 | A1 |
| 20070178607 | Prober et al. | Aug 2007 | A1 |
| 20070184463 | Molho et al. | Aug 2007 | A1 |
| 20070184547 | Handique et al. | Aug 2007 | A1 |
| 20070196237 | Neuzil et al. | Aug 2007 | A1 |
| 20070196238 | Kennedy et al. | Aug 2007 | A1 |
| 20070199821 | Chow | Aug 2007 | A1 |
| 20070215554 | Kreuwel et al. | Sep 2007 | A1 |
| 20070218459 | Miller et al. | Sep 2007 | A1 |
| 20070231213 | Prabhu et al. | Oct 2007 | A1 |
| 20070243626 | Windeyer et al. | Oct 2007 | A1 |
| 20070248958 | Jovanovich | Oct 2007 | A1 |
| 20070261479 | Spaid et al. | Nov 2007 | A1 |
| 20070269861 | Williams et al. | Nov 2007 | A1 |
| 20070292941 | Handique et al. | Dec 2007 | A1 |
| 20080000774 | Park et al. | Jan 2008 | A1 |
| 20080003649 | Maltezos et al. | Jan 2008 | A1 |
| 20080017306 | Liu et al. | Jan 2008 | A1 |
| 20080056948 | Dale et al. | Mar 2008 | A1 |
| 20080069729 | McNeely | Mar 2008 | A1 |
| 20080090244 | Knapp et al. | Apr 2008 | A1 |
| 20080095673 | Xu | Apr 2008 | A1 |
| 20080118987 | Eastwood et al. | May 2008 | A1 |
| 20080124723 | Dale et al. | May 2008 | A1 |
| 20080176230 | Owen et al. | Jul 2008 | A1 |
| 20080192254 | Kim et al. | Aug 2008 | A1 |
| 20080226502 | Jonsmann et al. | Sep 2008 | A1 |
| 20080240898 | Manz et al. | Oct 2008 | A1 |
| 20080247914 | Edens et al. | Oct 2008 | A1 |
| 20080257882 | Turner | Oct 2008 | A1 |
| 20080280285 | Chen et al. | Nov 2008 | A1 |
| 20080308500 | Brassard | Dec 2008 | A1 |
| 20090047180 | Kawahara | Feb 2009 | A1 |
| 20090066339 | Glezer et al. | Mar 2009 | A1 |
| 20090136385 | Handique et al. | May 2009 | A1 |
| 20090148933 | Battrell et al. | Jun 2009 | A1 |
| 20090189089 | Bedingham et al. | Jul 2009 | A1 |
| 20090223925 | Morse et al. | Sep 2009 | A1 |
| 20090325164 | Vossenaar et al. | Dec 2009 | A1 |
| 20090325276 | Battrell et al. | Dec 2009 | A1 |
| 20100009351 | Brahmasandra et al. | Jan 2010 | A1 |
| 20100120129 | Amshey et al. | May 2010 | A1 |
| 20100233763 | Shigeura et al. | Sep 2010 | A1 |
| 20100284864 | Holenstein et al. | Nov 2010 | A1 |
| 20110008825 | Ingber et al. | Jan 2011 | A1 |
| 20110027151 | Handique et al. | Feb 2011 | A1 |
| 20110097493 | Kerr et al. | Apr 2011 | A1 |
| 20110127292 | Sarofim et al. | Jun 2011 | A1 |
| 20110158865 | Miller et al. | Jun 2011 | A1 |
| 20110287447 | Norderhaug | Nov 2011 | A1 |
| 20110300033 | Battisti | Dec 2011 | A1 |
| 20120122231 | Tajima | May 2012 | A1 |
| 20120160826 | Handique | Jun 2012 | A1 |
| 20120171678 | Maltezos et al. | Jul 2012 | A1 |
| 20120258463 | Duffy et al. | Oct 2012 | A1 |
| 20130183769 | Tajima | Jul 2013 | A1 |
| 20130217013 | Steel et al. | Aug 2013 | A1 |
| 20130315800 | Yin et al. | Nov 2013 | A1 |
| 20140030798 | Wu et al. | Jan 2014 | A1 |
| 20140227710 | Handique et al. | Aug 2014 | A1 |
| 20140329301 | Handique et al. | Nov 2014 | A1 |
| 20150045234 | Stone et al. | Feb 2015 | A1 |
| 20150064702 | Handique et al. | Mar 2015 | A1 |
| 20150142186 | Handique et al. | May 2015 | A1 |
| 20150174579 | Iten et al. | Jun 2015 | A1 |
| 20150315631 | Handique et al. | Nov 2015 | A1 |
| 20150328638 | Handique et al. | Nov 2015 | A1 |
| 20160038942 | Roberts | Feb 2016 | A1 |
| 20180112252 | Handique | Apr 2018 | A1 |
| 20180135102 | Gubatayao et al. | May 2018 | A1 |
| 20180154364 | Handique et al. | Jun 2018 | A1 |
| 20180333722 | Handique | Nov 2018 | A1 |
| 20190054467 | Handique | Feb 2019 | A1 |
| 20190054471 | Williams et al. | Feb 2019 | A1 |
| 20190106692 | Brahmasandra et al. | Apr 2019 | A1 |
| 20190144849 | Duffy et al. | May 2019 | A1 |
| 20190145546 | Handique | May 2019 | A1 |
| 20190151854 | Baum et al. | May 2019 | A1 |
| 20190154719 | LaChance et al. | May 2019 | A1 |
| 20190284606 | Wu et al. | Sep 2019 | A1 |
| 20190324050 | Williams et al. | Oct 2019 | A1 |
| 20200010872 | Ganesan et al. | Jan 2020 | A1 |
| 20200139363 | Handique et al. | May 2020 | A1 |
| 20200156059 | Handique et al. | May 2020 | A1 |
| 20200164363 | Handique et al. | May 2020 | A1 |
| 20200215536 | Handique et al. | Jul 2020 | A1 |
| 20200216831 | Brahmasandra et al. | Jul 2020 | A1 |
| 20200291388 | Brahmasandra et al. | Sep 2020 | A1 |
| 20200324293 | Handique et al. | Oct 2020 | A1 |
| 20200325523 | Brahmasandra et al. | Oct 2020 | A1 |
| 20200325524 | Handique et al. | Oct 2020 | A1 |
| Number | Date | Country |
|---|---|---|
| 1357102 | Mar 2002 | AU |
| 3557502 | Jul 2002 | AU |
| 4437602 | Jul 2002 | AU |
| 4437702 | Jul 2002 | AU |
| 764319 | Aug 2003 | AU |
| 2574107 | Sep 1998 | CA |
| 2294819 | Jan 1999 | CA |
| 1312287 | Apr 2007 | CN |
| 1942590 | Apr 2007 | CN |
| 1968754 | May 2007 | CN |
| 101466848 | Jun 2009 | CN |
| 101522909 | Sep 2009 | CN |
| 103540518 | Jan 2014 | CN |
| 19755479 | Jun 1999 | DE |
| 19929734 | Dec 1999 | DE |
| 19833293 | Jan 2000 | DE |
| 0136126 | Apr 1985 | EP |
| 0365828 | May 1990 | EP |
| 0483620 | May 1992 | EP |
| 0402994 | Nov 1994 | EP |
| 0393744 | Jan 1995 | EP |
| 0688602 | Dec 1995 | EP |
| 0707077 | Apr 1996 | EP |
| 0698046 | Mar 1997 | EP |
| 0766256 | Apr 1997 | EP |
| 0772494 | May 1997 | EP |
| 0810030 | Dec 1997 | EP |
| 1059458 | Dec 2000 | EP |
| 1064090 | Jan 2001 | EP |
| 1077086 | Feb 2001 | EP |
| 1346772 | Sep 2003 | EP |
| 1541237 | Jun 2005 | EP |
| 1574586 | Sep 2005 | EP |
| 1621890 | Feb 2006 | EP |
| 1745153 | Jan 2007 | EP |
| 1780290 | May 2007 | EP |
| 1792656 | Jun 2007 | EP |
| 2372367 | Oct 2011 | EP |
| 2672301 | Aug 1992 | FR |
| 2795426 | Dec 2000 | FR |
| 2453432 | Apr 2009 | GB |
| S50-100881 | Aug 1975 | JP |
| 58212921 | Dec 1983 | JP |
| S62-119460 | May 1987 | JP |
| H01-502319 | Aug 1989 | JP |
| H 03181853 | Aug 1991 | JP |
| 04-053555 | May 1992 | JP |
| 06-064156 | Sep 1994 | JP |
| 07-020010 | Jan 1995 | JP |
| H07-290706 | Nov 1995 | JP |
| H08-122336 | May 1996 | JP |
| H08-173194 | Jul 1996 | JP |
| H08-211071 | Aug 1996 | JP |
| H08-285859 | Nov 1996 | JP |
| H08-337116 | Dec 1996 | JP |
| H09-304385 | Nov 1997 | JP |
| H09-325151 | Dec 1997 | JP |
| 2001-502790 | Jan 1998 | JP |
| H01-219669 | Sep 1998 | JP |
| H10-327515 | Dec 1998 | JP |
| H11-501504 | Feb 1999 | JP |
| H11-503315 | Mar 1999 | JP |
| 2000-514928 | Apr 1999 | JP |
| H11-156231 | Jun 1999 | JP |
| H11-316226 | Nov 1999 | JP |
| H11-515106 | Dec 1999 | JP |
| 2000-180455 | Jun 2000 | JP |
| 2000-266760 | Sep 2000 | JP |
| 2000-275255 | Oct 2000 | JP |
| 2001-502319 | Feb 2001 | JP |
| 2001-204462 | Jul 2001 | JP |
| 2001-509437 | Jul 2001 | JP |
| 3191150 | Jul 2001 | JP |
| 2001-515216 | Sep 2001 | JP |
| 2001-523812 | Nov 2001 | JP |
| 2001-527220 | Dec 2001 | JP |
| 2002-503331 | Jan 2002 | JP |
| 2002-085961 | Mar 2002 | JP |
| 2002-517735 | Jun 2002 | JP |
| 2002-215241 | Jul 2002 | JP |
| 2002-540382 | Nov 2002 | JP |
| 2002-544476 | Dec 2002 | JP |
| 2003-500674 | Jan 2003 | JP |
| 2003-047839 | Feb 2003 | JP |
| 2003-047840 | Feb 2003 | JP |
| 2003-516125 | May 2003 | JP |
| 2003-164279 | Jun 2003 | JP |
| 2003-185584 | Jul 2003 | JP |
| 2003-299485 | Oct 2003 | JP |
| 2003-329693 | Nov 2003 | JP |
| 2003-329696 | Nov 2003 | JP |
| 2003-532382 | Nov 2003 | JP |
| 2004-003989 | Jan 2004 | JP |
| 2004-506179 | Feb 2004 | JP |
| 2004-150797 | May 2004 | JP |
| 2004-283728 | Oct 2004 | JP |
| 2004-531360 | Oct 2004 | JP |
| 2004-533838 | Nov 2004 | JP |
| 2004-534157 | Nov 2004 | JP |
| 2004-361421 | Dec 2004 | JP |
| 2004-536291 | Dec 2004 | JP |
| 2004-536689 | Dec 2004 | JP |
| 2005-009870 | Jan 2005 | JP |
| 2005-010179 | Jan 2005 | JP |
| 2005-511264 | Apr 2005 | JP |
| 2005-514718 | May 2005 | JP |
| 2005-518825 | Jun 2005 | JP |
| 2005-176613 | Jul 2005 | JP |
| 2005-192439 | Jul 2005 | JP |
| 2005-192554 | Jul 2005 | JP |
| 2005-519751 | Jul 2005 | JP |
| 2005-204661 | Aug 2005 | JP |
| 2005-525816 | Sep 2005 | JP |
| 2005-291954 | Oct 2005 | JP |
| 2005-532043 | Oct 2005 | JP |
| 2005-323519 | Nov 2005 | JP |
| 2005-533652 | Nov 2005 | JP |
| 2005-535904 | Nov 2005 | JP |
| 2006-021156 | Jan 2006 | JP |
| 2006-055837 | Mar 2006 | JP |
| 2006-094866 | Apr 2006 | JP |
| 2006-145458 | Jun 2006 | JP |
| 2006-167569 | Jun 2006 | JP |
| 2006-284409 | Oct 2006 | JP |
| 2007-024742 | Feb 2007 | JP |
| 2007-074960 | Mar 2007 | JP |
| 2007-097477 | Apr 2007 | JP |
| 2007-101364 | Apr 2007 | JP |
| 2007-510518 | Apr 2007 | JP |
| 2007-514405 | Jun 2007 | JP |
| 2007-178328 | Jul 2007 | JP |
| 2007-535933 | Dec 2007 | JP |
| 2009-515140 | Apr 2009 | JP |
| 2009-542207 | Dec 2009 | JP |
| 3193848 | Oct 2014 | JP |
| 1020060044489 | May 2006 | KR |
| 2418633 | May 2011 | RU |
| WO 1988006633 | Sep 1988 | WO |
| WO 1990012350 | Oct 1990 | WO |
| WO 1992005443 | Apr 1992 | WO |
| WO 1994005414 | Mar 1994 | WO |
| WO 1994011103 | May 1994 | WO |
| WO 1995033846 | Dec 1994 | WO |
| WO 1996000228 | Jan 1996 | WO |
| WO 1996004547 | Feb 1996 | WO |
| WO 1996018731 | Jun 1996 | WO |
| WO 1996039547 | Dec 1996 | WO |
| WO 1997005492 | Feb 1997 | WO |
| WO 1997016835 | May 1997 | WO |
| WO 1997021090 | Jun 1997 | WO |
| WO 1997022825 | Jun 1997 | WO |
| WO 1997027324 | Jul 1997 | WO |
| WO 1998000231 | Jan 1998 | WO |
| WO 1998007019 | Feb 1998 | WO |
| WO 1998022625 | May 1998 | WO |
| WO 199835013 | Aug 1998 | WO |
| WO 1998038487 | Sep 1998 | WO |
| WO 1998049548 | Nov 1998 | WO |
| WO 1998050147 | Nov 1998 | WO |
| WO 1998053311 | Nov 1998 | WO |
| WO 1999001688 | Jan 1999 | WO |
| WO 1999009042 | Feb 1999 | WO |
| WO 1999012016 | Mar 1999 | WO |
| WO 1999017093 | Apr 1999 | WO |
| WO 1999029703 | Jun 1999 | WO |
| WO 1999033559 | Jul 1999 | WO |
| WO 1999060397 | Nov 1999 | WO |
| WO 2000022436 | Apr 2000 | WO |
| WO 2000075623 | Dec 2000 | WO |
| WO 2000078455 | Dec 2000 | WO |
| WO 2001005510 | Jan 2001 | WO |
| WO 2001014931 | Mar 2001 | WO |
| WO 2001027614 | Apr 2001 | WO |
| WO 2001028684 | Apr 2001 | WO |
| WO 2001030995 | May 2001 | WO |
| WO 2001041931 | Jun 2001 | WO |
| WO 2001046474 | Jun 2001 | WO |
| WO 2001054813 | Aug 2001 | WO |
| WO 2001089681 | Nov 2001 | WO |
| WO 2001089705 | Nov 2001 | WO |
| WO 2001092569 | Dec 2001 | WO |
| WO 2002048164 | Jun 2002 | WO |
| WO 2002052002 | Jul 2002 | WO |
| WO 2002072264 | Sep 2002 | WO |
| WO 2002078845 | Oct 2002 | WO |
| WO 2002086454 | Oct 2002 | WO |
| WO 2002094185 | Nov 2002 | WO |
| WO 2003007677 | Jan 2003 | WO |
| WO 2003012325 | Feb 2003 | WO |
| WO 2003012406 | Feb 2003 | WO |
| WO 2003048295 | Jun 2003 | WO |
| WO 2003055605 | Jul 2003 | WO |
| WO 2003076661 | Sep 2003 | WO |
| WO 2003078065 | Sep 2003 | WO |
| WO 2003080868 | Oct 2003 | WO |
| WO 2003087410 | Oct 2003 | WO |
| WO 2004007081 | Jan 2004 | WO |
| WO 2004010760 | Feb 2004 | WO |
| WO 2004048545 | Jun 2004 | WO |
| WO 2004055522 | Jul 2004 | WO |
| WO 2004056485 | Jul 2004 | WO |
| WO 2004074848 | Sep 2004 | WO |
| WO 2004094986 | Nov 2004 | WO |
| WO 2005008255 | Jan 2005 | WO |
| WO 2005011867 | Feb 2005 | WO |
| WO 2005030984 | Apr 2005 | WO |
| WO 2005072353 | Aug 2005 | WO |
| WO 2005094981 | Oct 2005 | WO |
| WO 2005107947 | Nov 2005 | WO |
| WO 2005108571 | Nov 2005 | WO |
| WO 2005108620 | Nov 2005 | WO |
| WO 2005116202 | Dec 2005 | WO |
| WO 2005118867 | Dec 2005 | WO |
| WO 2005120710 | Dec 2005 | WO |
| WO 2006010584 | Feb 2006 | WO |
| WO 2006032044 | Mar 2006 | WO |
| WO 2006035800 | Apr 2006 | WO |
| WO 2006043642 | Apr 2006 | WO |
| WO 2006066001 | Jun 2006 | WO |
| WO 2006079082 | Jul 2006 | WO |
| WO 2006081995 | Aug 2006 | WO |
| WO 2006113198 | Oct 2006 | WO |
| WO 2006118420 | Nov 2006 | WO |
| WO 2006119280 | Nov 2006 | WO |
| WO 2007044917 | Apr 2007 | WO |
| WO 2007050327 | May 2007 | WO |
| WO 2007064117 | Jun 2007 | WO |
| WO 2007075919 | Jul 2007 | WO |
| WO 2007091530 | Aug 2007 | WO |
| WO 2007112114 | Oct 2007 | WO |
| WO 2007120240 | Oct 2007 | WO |
| WO 2007120241 | Oct 2007 | WO |
| WO 2008005321 | Jan 2008 | WO |
| WO 2008030914 | Mar 2008 | WO |
| WO 2008060604 | May 2008 | WO |
| WO 2008149282 | Dec 2008 | WO |
| WO 2009012185 | Jan 2009 | WO |
| WO 2009054870 | Apr 2009 | WO |
| WO 2010118541 | Oct 2010 | WO |
| WO 2010130310 | Nov 2010 | WO |
| WO 2010140680 | Dec 2010 | WO |
| WO 2011009073 | Jan 2011 | WO |
| WO 2011101467 | Aug 2011 | WO |
| Entry |
|---|
| Allemand et al., “pH-Dependent Specific Binding and Combing of DNA”, Biophys J. (1997) 73(4): 2064-2070. |
| Bollet, C. et al., “A simple method for the isolation of chromosomal DNA from Gram positive or acid-fast bacteria”, Nucleic Acids Research, vol. 19, No. 8 (1991), p. 1955. |
| Brahmasandra et al., On-chip DNA detection in microfabricated separation systems, SPIE Conference on Microfluidic Devices and Systems, 1998, vol. 3515, pp. 242-251, Santa Clara, CA. |
| Breadmore, M.C. et al., “Microchip-Based Purification of DNA from Biological Samples”, Anal. Chem., vol. 75 (2003), pp. 1880-1886. |
| Brody, et al., Diffusion-Based Extraction in a Microfabricated Device, Sensors and Actuators Elsevier, 1997, vol. A58, No. 1, pp. 13-18. |
| Broyles et al., “Sample Filtration, Concentration, and Separation Integrated on Microfluidic Devices” Analytical Chemistry (American Chemical Society), (2003) 75(11): 2761-2767. |
| Burns et al., “An Integrated Nanoliter DNA Analysis Device”, Science 282:484-487 (1998). |
| Carlen et al., “Paraffin Actuated Surface Micromachined Valve,” in IEEE MEMS 2000 Conference, Miyazaki, Japan, (Jan. 2000) pp. 381-385. |
| Chung, Y. et al., “Microfluidic chip for high efficiency DNA extraction”, Miniaturisation for Chemistry, Biology & Bioengineering, vol. 4, No. 2 (Apr. 2004), pp. 141-147. |
| Cooley et al., “Applications of Ink-Jet Printing Technology to BioMEMS and Microfluidic Systems”, Proceedings, SPIE Conference on Microfluids and BioMEMS, (Oct. 2001), 12 pages. |
| Edwards, “Silicon (Si),” in “Handbook of Optical Constants of Solids” (Ghosh & Palik eds., 1997) in 24 pages. |
| Goldmeyer et al., “Identification of Staphylococcus aureus and Determination of Methicillin Resistance Directly from Positive Blood Cultures by Isothermal Amplification and a Disposable Detection Device”, J Clin Microbiol. (Apr. 2008) 46(4): 1534-1536. |
| Hale et al., “Optical constants of Water in the 200-nm to 200-μm Wavelength Region”, Applied Optics, 12(3): 555-563 (1973). |
| Handique et al, “Microfluidic flow control using selective hydrophobic patterning”, SPIE, (1997) 3224: 185-194. |
| Handique et al., “On-Chip Thermopneumatic Pressure for Discrete Drop Pumping”, Anal. Chem., (2001) 73(8):1831-1838. |
| Handique et al., “Nanoliter-volume discrete drop injection and pumping in microfabricated chemical analysis systems”, Solid-State Sensor and Actuator Workshop (Hilton Head, South Carolina, Jun. 8-11, 1998), pp. 346-349. |
| Handique et al., “Mathematical Modeling of Drop Mixing in a Slit-Type Microchannel”, J. Micromech. Microeng., 11:548-554 (2001). |
| Handique et al., “Nanoliter Liquid Metering in Microchannels Using Hydrophobic Patterns”, Anal. Chem., 72(17):4100-4109 (2000). |
| Harding et al., “DNA isolation using Methidium-Spermine-Sepharose”, Meth Enzymol. (1992) 216: 29-39. |
| Harding et al., “Rapid isolation of DNA from complex biological samples using a novel capture reagent—methidium-spermine-sepharose”, Nucl Acids Res. (1989) 17(17): 6947-6958. |
| He, et al., Microfabricated Filters for Microfluidic Analytical Systems, Analytical Chemistry, American Chemical Society, 1999, vol. 71, No. 7, pp. 1464-1468. |
| Ibrahim, et al., Real-Time Microchip PCR for Detecting Single-Base Differences in Viral and Human DNA, Analytical Chemistry, American Chemical Society, 1998, 70(9): 2013-2017. |
| International Preliminary Report on Patentability and Written Opinion dated Jan. 19, 2010 for Application No. PCT/US2008/008640, filed Jul. 14, 2008. |
| International Search Report and Written Opinion dated Apr. 4, 2008 for PCT/US2007/007513, filed Mar. 26, 2007. |
| International Search Report and Written Opinion dated Jan. 5, 2009 for PCT/US2007/024022, filed Nov. 14, 2007. |
| International Search Report dated Jun. 17, 2009 for Application No. PCT/US2008/008640, filed Jul. 14, 2008. |
| Khandurina et al., Microfabricated Porous Membrane Structure for Sample Concentration and Electrophoretic Analysis, Analytical Chemistry American Chemical Society, 1999, 71(9): 1815-1819. |
| Kim et al., “Electrohydrodynamic Generation and Delivery of Monodisperse Picoliter Droplets Using a Poly(dimethylsiloxane) Microchip”, Anal Chem. (2006) 78: 8011-8019. |
| Kopp et al., Chemical Amplification: Continuous-Flow PCR on a Chip, www.sciencemag.org, 1998, vol. 280, pp. 1046-1048. |
| Kuo et al., “Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis”, J Biotech. (2007) 129: 383-390. |
| Kutter et al., Solid Phase Extraction on Microfluidic Devices, J. Microcolumn Separations, John Wiley & Sons, Inc., 2000, 12(2): 93-97. |
| Labchem; Sodium Hydroxide, 0,5N (0.5M); Safety Data Sheet, 2015; 8 pages. |
| Lagally et al., Single-Molecule DNA Amplification and Analysis in an Integrated Microfluidic Device, Analytical Chemistry, American Chemical Society, 2001, 73(3): 565-570. |
| Livache et al., “Polypyrrole DNA chip on a Silicon Device: Example of Hepatitis C Virus Genotyping”, Analytical Biochemistry, (1998) 255: 188-194. |
| Malitson, “Interspecimen Comparison of the Refractive Index of Fused Silica,” J Optical Society of America, 55:1205-1209 (1965). |
| Mastrangelo et al., Microfabricated Devices for Genetic Diagnostics. Proceedings of the IEEE (1998) 86(8):1769-1787. |
| Mascini et al., “DNA electrochemical biosensors”, Fresenius J. Anal. Chem., 369: 15-22, (2001). |
| Meyers, R.A., Molecular Biology and Biotechnology: A Comprehensive Desk Reference; VCH Publishers, Inc. New York, NY; (1995) pp. 418-419. |
| Nakagawa et al., Fabrication of amino silane-coated microchip for DNA extraction from whole blood, J of Biotechnology, Mar. 2, 2005, 116: 105-111. |
| Northrup et al., A Miniature Analytical Instrument for Nucleic Acids Based on Micromachined Silicon Reaction Chambers, Analytical Chemistry, American Chemical Society, 1998, 70(5): 918-922. |
| Oh K.W. et al., “A Review of Microvalves”, J Micromech Microeng. (2006) 16:R13-R39. |
| Oleschuk et al., Trapping of Bead-Based Reagents within Microfluidic Systems: On-Chip Solid-Phase Extraction and Electrochromatography, Analytical Chemistry, American Chemical Society, 2000, 72(3): 585-590. |
| Pal et al., “Phase Change Microvalve for Integrated Devices”, Anal Chem. (2004) 76: 3740-3748. |
| Palina et al., “Laser Assisted Boron Doping of Silicon Wafer Solar Cells Using Nanosecond and Picosecond Laser Pulses,” 2011 37th IEEE Photovoltaic Specialists Conference, pp. 002193-002197, IEEE (2011). |
| Paulson et al., “Optical dispersion control in surfactant-free DNA thin films by vitamin B2 doping,” Nature, Scientific Reports 8:9358 (2018) published at www.nature.com/scientificreports, Jun. 19, 2018. |
| Plambeck et al., “Electrochemical Studies of Antitumor Antibiotics”, J. Electrochem Soc.: Electrochemical Science and Technology (1984), 131(11): 2556-2563. |
| Roche et al. “Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells” Faseb J (2005) 19: 1341-1343. |
| Ross et al., Analysis of DNA Fragments from Conventional and Microfabricated PCR Devices Using Delayed Extraction MALDI-TOF Mass Spectrometry, Analytical Chemistry, American Chemical Society, 1998, 70(10): 2067-2073. |
| Sanchez et al., “Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis”, PNAS (2004) 101(7): 1933-1938. |
| Sarma, K.S., “Liquid Crystal Displays”, Chapter 32 in Electrical Measurement, Signal Processing, Displays, Jul. 15, 2003, ISBN: 978-0-8493-1733-0, Retrieved from the Internet: URL: http://http://197.14.51.10:81/pmb/ELECTRONIQUE/Electrical Measurement Signal Processing and Displays/Book/1733ch32.pdf; 21 pages. |
| Shoffner et al., Chip PCR.I. Surface Passivation of Microfabricated Silicon-Glass Chips for PCR, Nucleic Acids Research, Oxford University Press, (1996) 24(2): 375-379. |
| Smith, K. et al., “Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples”, Journal of Clinical Microbiology, vol. 41, No. 6 (Jun. 2003), pp. 2440-2443. |
| Tanaka et al., “Improved Method of DNA Extraction from Seeds Using Amine-Dendrimer Modified Magnetic Particles”, Proceedings of the 74th Annual Meeting of the Electrochemical Society of Japan; Abstract #2E09 on p. 149, Mar. 29, 2007; Faculty of Engineering, Science University of Tokyo; 4 pages. |
| Wang, “Survey and Summary, from DNA Biosensors to Gene Chips”, Nucleic Acids Research, 28(16):3011-3016, (2000). |
| Waters et al., Microchip Device for Cell Lysis, Multiplex PCR Amplification, and Electrophoretic Sizing, Analytical Chemistry, American Chemical Society, 1998, 70(1): 158-162. |
| Weigl, et al., Microfluidic Diffusion-Based Separation and Detection, www.sciencemag.org, 1999, vol. 283, pp. 346-347. |
| Wu et al., “Polycationic dendrimers interact with RNA molecules: polyamine dendrimers inhibit the catalytic activity of Candida ribozymes”, Chem Commun. (2005) 3: 313-315. |
| Yoza et al., “Fully Automated DNA Extraction from Blood Using Magnetic Particles Modified with a Hyperbranched Polyamidoamine Dendrimer”, J Biosci Bioeng, 2003, 95(1): 21-26. |
| Yoza et al., DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer, J Biotechnol., Mar. 20, 2003, 101(3): 219-228. |
| Zhang et al., “PCR Microfluidic Devices for DNA Amplification,” Biotechnology Advances, 24:243-284 (2006). |
| Zhou et al., “Cooperative binding and self-assembling behavior of cationic low molecular-weight dendrons with RNA molecules”, Org Biomol Chem. (2006) 4(3): 581-585. |
| Zhou et al., “PAMAM dendrimers for efficient siRNA delivery and potent gene silencing”, Chem Comm.(Camb.) (2006) 22: 2362-2364. |
| Zou et al., “A Micromachined Integratable Thermal Reactor,” technical digest from International Electron Devices Meeting, IEEE, Washington, D.C., Dec. 2-5, 2001 (6 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 7,998,708 (Paper 1 in IPR2019-00488) dated Dec. 20, 2018 (94 pages). |
| Declaration of Bruce K. Gale, Ph.D. (Exhibit 1001 in IPR2019-00488 and IPR2019-00490) dated Dec. 20, 2018 (235 pages). |
| Patent Owner Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 7,998,708 and Exhibit List (Papers 5 and 6 in IPR2019-00488) dated Apr. 18, 2019 (79 pages). |
| Decision instituting Inter Partes Review of U.S. Pat. No. 7,998,708 (Paper 8 in IPR2019-00488) dated Jul. 16, 2019 (20 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,323,900 (Paper 1 in IPR2019□00490) dated Dec. 20, 2018 (85 pages). |
| Declaration of Michael G. Mauk, Ph.D. in Support of Patent Owner Preliminary Responses in IPR2019-00488 and IPR2019-00490 dated Apr. 18, 2019 (43 pages). |
| Patent Owner Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,323,900 and Exhibit List (Papers 5 and 6 in IPR2019-00490) dated Apr. 18, 2019 (73 pages). |
| Decision instituting Inter Partes Review of U.S. Pat. No. 8,323,900 (Paper 8 in IPR2019-00490) dated Jul. 16, 2019 (23 pages). |
| Becker H., “Hype, hope and hubris: the quest for the killer application in microfluidics”, Lab on a Chip, The Royal Society of Chemistry (2009) 9:2119-2122. |
| Becker H., “Collective Wisdom”, Lab on a Chip, The Royal Society of Chemistry (2010) 10:1351-1354. |
| Chaudhari et al., “Transient Liquid Crystal Thermometry of Microfabricated PCR Vessel Arrays”, J Microelectro Sys., (1998) 7(4):345-355. |
| Chang-Yen et al., “Design, fabrication, and packaging of a practical multianalyte-capable optical biosensor,” J Microlith Microfab Microsyst. (2006) 5(2):021105 in 8 pages. |
| Chen et al., “Total nucleic acid analysis integrated on microfluidic devices,” Lab on a Chip. (2007) 7:1413-1423. |
| Cui et al., “Design and Experiment of Silicon PCR Chips,” Proc. SPIE 4755, Design, Test, Integration, and Packaging of MEMS/MOEMS 2002, (Apr. 19, 2002) pp. 71-76. |
| Grunenwald H., “Optimization of Polymerase Chain Reactions,” in Methods in Molecular Biology, PCR Protocols., Second Edition by Bartlett et al. [Eds.] Humana Press (2003) vol. 226, pp. 89-99. |
| Handal et al., “DNA mutation detection and analysis using miniaturized microfluidic systems”, Expert Rev Mol Diagn. (2006) 6(1):29-38. |
| Irawan et al., “Cross-Talk Problem on a Fluorescence Multi-Channel Microfluidic Chip System,” Biomed Micro. (2005) 7(3):205-211. |
| Khandurina et al., “Bioanalysis in microfluidic devices,” J Chromatography A, (2002) 943:159-183. |
| Liao et al., “Miniature RT-PCR system for diagnosis of RNA-based viruses,” Nucl Acids Res. (2005) 33(18):e156 in 7 pages. |
| Lin et al., “Thermal Uniformity of 12-in Silicon Wafer During Rapid Thermal Processing by Inverse Heat Transfer Method,” IEEE Transactions on Semiconductor Manufacturing, (2000) 13(4):448-456. |
| Manz et al., “Miniaturized Total Chemical Analysis Systems: a Novel Concept for Chemical Sensing,” Sensors and Actuators B1, (1990) 244-248. |
| Minco, “Conductive Heating Technologies for Medical Diagnostic Equipment,” (2006) in 13 pages. |
| Picard et al., Laboratory Detection of Group B Streptococcus for Prevention of Perinatal Disease, Eur. J. Clin. Microbiol. Infect. Dis., Jul. 16, 2004, 23: 665-671. |
| Rohsenow et al. [Eds.], Handbook of Heat Transfer, 3rd Edition McGraw-Hill Publishers (1998) Chapters 1 & 3; pp. 108 (in two parts). |
| Shen et al., “A microchip-based PCR device using flexible printed circuit technology,” Sensors and Actuators B (2005), 105:251-258. |
| Spitzack et al., “Polymerase Chain Reaction in Miniaturized Systems: Big Progress in Little Devices”, in Methods in Molecular Biology—Microfluidic Techniques, Minteer S.D. [Ed.] Humana Press (2006), pp. 97-129. |
| Squires et al., “Microfluidics: Fluid physics at the nanoliter scale”, Rev Modern Phys. (2005) 77:977-1026. |
| Velten et al., “Packaging of Bio-MEMS: Strategies, Technologies, and Applications,” IEEE Transactions on Advanced Packaging, (2005) 28(4):533-546. |
| Zhang et al., “Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends,” Nucl Acids Res., (2007) 35(13):4223-4237. |
| Patent Owner's Response in Inter Partes Review of U.S. Pat. No. 8,323,900 and Exhibit List (Paper 25 in IPR2019-00490) dated Oct. 16, 2019 (80 pages). |
| Patent Owner's Response in Inter Partes Review of U.S. Pat. No. 7,998,708 and Exhibit List (Paper 25 in IPR 2019-00488) dated Oct. 16, 2019 (93 pages). |
| Transcript of Deposition of Bruce K. Gale, Ph.D., in Support of Patent Owner's Responses (Exhibit 2012 in IPR2019-00488 and IPR2019-00490), taken Sep. 24, 2019 (124 pages). |
| Declaration of M. Allen Northrup, Ph.D. in Support of Patent Owner's Responses (Exhibit 2036 in IPR2019-00488 and IPR2019-00490) dated Oct. 16, 2019 (365 pages). |
| Complaint filed by Becton, Dickinson et al., v. NeuModx Molecular, Inc. on Jun. 18, 2019 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS, Infringement Action involving U.S. Pat. Nos. 7,998,708; 8,273,308; 8,323,900; 8,415,103; 8,703,069; and 8,709,787 (29 pages). |
| Answer to Complaint filed by NeuModx Molecular, Inc. on Aug. 9, 2019 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (24 pages). |
| Amended Answer to Complaint filed by NeuModx Molecular, Inc. on Oct. 4, 2019 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (31 pages). |
| Altet et al., [Eds.] “Thermal Transfer and Thermal Coupling in IC's”, Thermal Testing of Integrated Circuits; Chapter 2 (2002) Springer Science pp. 23-51. |
| Ateya et al., “The good, the bad, and the tiny: a review of microflow cytometry”, Anal Bioanal Chem. (2008) 391(5):1485-1498. |
| Auroux et al., “Miniaturised nucleic acid analysis”, Lab Chip. (2004) 4(6):534-546. |
| Baechi et al., “High-density microvalve arrays for sample processing in PCR chips”, Biomed Microdevices. (2001) 3(3):183-190. |
| Baker M., “Clever PCR: more genotyping, smaller volumes.” Nature Methods (May 2010) 70(5):351-356. |
| Becker H. “Fabrication of Polymer Microfluidic Devices”, in Biochip Technology (2001), Chapter 4, pp. 63-96. |
| Becker H., “Microfluidic Devices Fabricated by Polymer Hot Embossing,” in Integrated Microfabricated Biodevices: Advanced Technologies for Genomics, Drug Discovery, Bioanalysis, and Clinical Diagnostics (2002), Chapter 13, 32 pages. |
| Becker H., “Microfluidics: A Technology Coming of Age”, Med Device Technol. (2008) 19(3):21-24. |
| Becker et al., “Portable CE system with contactless conductivity detection in an injection molded polymer chip for on-site food analysis”, SPIE Proceedings MOEMS-MEMS 2008 Micro and Nanofabrication (2008) vol. 6886 in 8 pages. |
| Belgrader et al., “Rapid PCR for Identity Testing Using a Battery-Powered Miniature Thermal Cycler”, J Forensic Sci. (1998) 43(2):315-319. |
| Belgrader et al., “A minisonicator to rapidly disrupt bacterial spores for DNA analysis.”, Anal Chem. (1999) 71(19):4232-4236. |
| Belgrader et al., “Real-time PCR Analysis on Nucleic Acids Purified from Plasma Using a Silicon Chip”, Micro Total Analysis Systems 2000 (pp. 525-528). Springer, Dordrecht. |
| Belgrader et al., “A microfluidic cartridge to prepare spores for PCR analysis”, Biosens Bioelectron. (2000) 14(10-11):849-852. |
| Belgrader et al., “A Battery-Powered Notebook Thermal Cycler for Rapid Multiplex Real-Time PCR Analysis”, Anal Chem. (2001) 73(2):286-289. |
| Belgrader et al., “Rapid and Automated Cartridge-based Extraction of Leukocytes from Whole Blood for Microsatellite DNA Analysis by Capillary Electrophoresis”, Clin Chem. (2001) 47(10):1917-1933. |
| Belgrader et al., “A Rapid, Flow-through, DNA Extraction Module for Integration into Microfluidic Systems”, Micro Total Analysis Systems (2002) pp. 697-699). Springer, Dordrecht. |
| Belgrader et al., “Development of a Battery-Powered Portable Instrumentation for Rapid PCR Analysis”, in Integrated Microfabicated Devices, (2002) Ch. 8, pp. 183-206, CRC Press. |
| Bell M., “Integrated Microsystems in Clinical Chemistry”, in Integrated Microfabicated Devices, (2002) Ch. 16, pp. 415-435, CRC Press. |
| Berthier et al., “Managing evaporation for more robust microscale assays Part 1. Volume loss in high throughput assays”, Lab Chip (2008) 8(6):852-859. |
| Berthier et al., “Managing evaporation for more robust microscale assays Part 2. Characterization of convection and diffusion for cell biology”, Lab Chip (2008) 8(6):860-864. |
| Berthier et al., “Microdrops,” in Microfluidics for Biotechnology (2006), Chapter 2, pp. 51-88. |
| Biomerieux Press Release: “bioMérieux—2018 Financial Results,” dated Feb. 27, 2019, accessed at www.biomerieux.com, pp. 13. |
| Blanchard et al., “Micro structure mechanical failure characterization using rotating Couette flow in a small gap”, J Micromech Microengin. (2005) 15(4):792-801. |
| Blanchard et al., “Single-disk and double-disk viscous micropumps”, Sensors and Actuators A (2005) 122:149-158. |
| Blanchard et al., “Performance and Development of a Miniature Rotary Shaft Pump”, J Fluids Eng. (2005) 127(4):752-760. |
| Blanchard et al., “Single-disk and double-disk viscous micropump”, ASME 2004 Inter'l Mechanical Engineering Congress & Exposition, Nov. 13-20, 2004, Anaheim, CA, IMECE2004-61705:411-417. |
| Blanchard et al., “Miniature Single-Disk Viscous Pump (Single-DVP), Performance Characterization”, J Fluids Eng. (2006) 128(3):602-610. |
| Brahmasandra et al., “Microfabricated Devices for Integrated DNA Analysis”, in Biochip Technology by Cheng et al., [Eds.] (2001) pp. 229-250. |
| Bu et al., “Design and theoretical evaluation of a novel microfluidic device to be used for PCR”, J Micromech Microengin. (2003) 13(4):S125-S130. |
| Cady et al., “Real-time PCR detection of Listeria monocytogenes using an integrated microfluidics platform”, Sensors Actuat B. (2005) 107:332-341. |
| Carles et al., “Polymerase Chain Reaction on Microchips” in Methods in Molecular Biology—Microfluidic Techniques, Reviews & Protocols by Minteer S.D. [Ed.] Humana Press (2006), vol. 321; Chapter 11, pp. 131-140. |
| Chang-Yen et al., “A novel integrated optical dissolved oxygen sensor for cell culture and micro total analysis systems”, IEEE Technical Digest MEMS International Conference Jan. 24, 2002, 4 pages. |
| Chang-Yen et al., “A PDMS microfluidic spotter for fabrication of lipid microarrays”, IEEE 3rd EMBS Special Topic Conference May 12-15, 2005; 2 pages. |
| Chang-Yen et al., “Design and fabrication of a multianalyte-capable optical biosensor using a multiphysics approach”, IEEE 3rd EMBS Special Topic Conference May 12-15, 2005; 2 pages. |
| Chang-Yen et al., “A Novel PDMS Microfluidic Spotter for Fabrication of Protein Chips and Microarrays”, IEEE J of Microelectromech Sys. (2006) 15(5): 1145-1151. |
| Chang-Yen et al., “Spin-assembled nanofilms for gaseous oxygen sensing.” Sens Actuators B: Chemical (2007), 120(2):426-433. |
| Chen P-C., “Accelerating micro-scale PCR (polymerase chain reactor) for modular lab-on-a-chip system”, LSU Master's Theses—Digital Commons, (2006) 111 pages. |
| Cheng et al., “Biochip-Based Portable Laboratory”, Biochip Tech. (2001):296-289. |
| Cho et al., “A facility for characterizing the steady-state and dynamic thermal performance of microelectromechanical system thermal switches”, Rev Sci Instrum. (2008) 79(3):034901-1 to -8. |
| Chong et al., “Disposable Polydimethylsioxane Package for ‘Bio-Microfluidic System’”, IEEE Proceedings Electonic Components and Technology (2005); 5 pages. |
| Chou et al., “A miniaturized cyclic PCR device—modeling and experiments”, Microelec Eng. (2002) 61-62:921-925. |
| Christel et al., “Nucleic Acid Concentration and PCR for Diagnostic Applications”, in Micro Total Analysis Systems. (1998) D.J. Harrison et al. [Eds.] pp. 277-280. |
| Christel et al., “Rapid, Automated Nucleic Acid Probe Assays Using Silicon Microstructures for Nucleic Acid Concentration”, J Biomech Eng. (1999) 121(1):22-27. |
| Christensen et al., “Characterization of interconnects used in PDMS microfluidic systems”, J Micromech Microeng. (2005) 15:928 in 8 pages. |
| Crews et al, “Rapid Prototyping of a Continuous-Flow PCR Microchip”, Proceedings of the AiChE Annual Meeting(Nov. 15, 2006) (335a) 3 pages. |
| Crews et al., Thermal gradient PCR in a continuous-flow microchip. In Microfluidics, BioMEMS, and Medical Microsystems V; Jan. 2007; vol. 6465, p. 646504; 12 pages. |
| Crews et al., “Continuous-flow thermal gradient PCR”, Biomed Microdevices. (2008) 10(2):187-195. |
| Cui et al., “Electrothermal modeling of silicon PCR chips”, In MEMS Design, Fabrication, Characterization, and Packaging, (Apr. 2001) (vol. 4407, pp. 275-280. |
| Danaher Press Release: “Danaher to Acquire Cepheid for $53.00 per share, or approximately $4 Billion,” dated Sep. 6, 2016, accessed at www.danaher.com, pp. 3. |
| Demchenko A.P., “The problem of self-calibration of fluorescence signal in microscale sensor systems”, Lab Chip. (2005) 5(11):1210-1223. |
| Dineva et al., “Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings”, Analyst. (2007) 132(12):1193-1199. |
| Dishinger et al., “Multiplexed Detection and Applications for Separations on Parallel Microchips”, Electophoresis. (2008) 29(16):3296-3305. |
| Dittrich et al., “Single-molecule fluorescence detection in microfluidic channels—the Holy Grail in muTAS?”, Anal Bioanal Chem. (2005) 382(8):1771-1782. |
| Dittrich et al., “Lab-on-a-chip: microfluidics in drug discovery”, Nat Rev Drug Discov. (2006) 5(3):210-208. |
| Dunnington et al., “Approaches to Miniaturized High-Throughput Screening of Chemical Libraries”, in Integrated Microfabicated Devices, (2002) Ch. 15, pp. 371-414, CRC Press. |
| Eddings et al., “A PDMS-based gas permeation pump for on-chip fluid handling in microfluidic devices”, J Micromech Microengin. (2006) 16(11). |
| Edwards et al., “Micro Scale Purification Systems for Biological Sample Preparation”, Biomed Microdevices (2001) 3(3):211-218. |
| Edwards et al., “A microfabricated thermal field-flow fractionation system”, Anal Chem. (2002) 74(6):1211-1216. |
| Ehrlich et al., “Microfluidic devices for DNA analysis”, Trends Biotechnol. (1999) 17(8):315-319. |
| El-Ali et al., “Simulation and experimental validation of a SU-8 based PCR thermocycler chip with integrated heaters and temperature sensor”, Sens Actuators A: Physical (2004) 110(1-3):3-10. |
| Erickson et al., “Joule heating and heat transfer in poly(dimethylsiloxane) microfluidic systems”, Lab Chip (2003) 3(3):141-149. |
| Erickson et al., “Integrated Microfluidic Devices”, Analytica Chim Acta. (2004) 507:11-26. |
| Erill et al., “Development of a CMOS-compatible PCR chip: comparison of design and system strategies”, J Micromech Microengin. (2004) 14(11):1-11. |
| Fair R.B., Digital microfluidics: is a true lab-on-a-chip possible? Microfluidics Nanofluid. (2007) 3:245-281. |
| Fan et al., “Integrated Plastic Microfluidic Devices for Bacterial Detection”, in Integrated Biochips for DNA Analysis by Liu et al. [Eds], (2007) Chapter 6, pp. 78-89. |
| Fiorini et al., “Disposable microfluidic devices: fabrication, function, and application”, Biotechniques (2005) 38(3):429-446. |
| Frazier et al., “Integrated micromachined components for biological analysis systems”, J Micromech. (2000) 1(1):67-83. |
| Gale et al., “Micromachined electrical field-flow fractionation (mu-EFFF) system”, IEEE Trans Biomed Eng. (1998) 45(12):1459-1469. |
| Gale et al., “Geometric scaling effects in electrical field flow fractionation. 1. Theoretical analysis”, Anal Chem. (2001) 73(10):2345-2352. |
| Gale et al., “BioMEMS Education at Louisiana Tech University”, Biomed Microdevices, (2002) 4:223-230. |
| Gale et al., “Geometric scaling effects in electrical field flow fractionation. 2. Experimental results”, Anal Chem. (2002) 74(5):1024-1030. |
| Gale et al., “Cyclical electrical field flow fractionation”, Electrophoresis. (2005) 26(9):1623-1632. |
| Gale et al., “Low-Cost MEMS Technologies”, Elsevier B.V. (2008), Chapter 1.12; pp. 342-372. |
| Garst et al., “Fabrication of Multilayered Microfluidic 3D Polymer Packages”, IEEE Proceedings Electronic Components & Tech, Conference May/Jun. 2005, pp. 603-610. |
| Gärtner et al., “Methods and instruments for continuous-flow PCR on a chip”, Proc. SPIE 6465, Microfluidics, BioMEMS, and Medical Microsystems V, (2007) 646502; 8 pages. |
| Giordano et al., “Toward an Integrated Electrophoretic Microdevice for Clinical Diagnostics”, in Integrated Microfabricated Biodevices: Advanced Technologies for Genomics, Drug Discovery, Bioanalysis, and Clinical Diagnostics (2002) Chapter 1; pp. 1-34. |
| Graff et al., “Nanoparticle Separations Using Miniaturized Field-flow Fractionation Systems”, Proc. Nanotechnology Conference and Trade Show (NSTI) (2005); pp. 8-12. |
| Greer et al., “Comparison of glass etching to xurography prototyping of microfluidic channels for DNA melting analysis”, J Micromech Microengin. (2007) 17(12):2407-2413. |
| Guijt et al., “Chemical and physical processes for integrated temperature control in microfluidic devices”, Lab Chip. (2003) 3(1):1-4. |
| Gulliksen A., “Microchips for Isothermal Amplification of RNA”, Doctoral Thesis (2007); Department of Mol. Biosciences—University of Oslo; 94 pages. |
| Guttenberg et al., “Planar chip device for PCR and hybridization with surface acoustic wave pump”, Lab Chip. (2005) 5(3):308-317. |
| Haeberle et al., “Microfluidic platforms for lab-on-a-chip applications”, Lab Chip. (2007) 7(9):1094-1110. |
| Hansen et al., “Microfluidics in structural biology: smaller, faster . . . better”, Curr Opin Struct Biol. (2003) 13(5):538-544. |
| Heid et al., “Genome Methods—Real Time Quantitative PCR”, Genome Res. (1996) 6(10):986-994. |
| Henry C.S. [Ed], “Microchip Capillary electrophoresis”, Methods in Molecular Biology, Humana Press 339 (2006) Parts I-IV in 250 pages. |
| Herr et al., “Investigation of a miniaturized capillary isoelectric focusing (cIEF) system using a full-field detection approach”, Solid State Sensor and Actuator Workshop, Hilton Head Island (2000), pp. 4-8. |
| Herr et al., “Miniaturized Isoelectric Focusing (μIEF) As a Component of a Multi-Dimensional Microfluidic System”, Micro Total Analysis Systems (2001) pp. 51-53. |
| Herr et al., Miniaturized Capillary Isoelectric Focusing (cIEF): Towards a Portable High-Speed Separation Method. In Micro Total Analysis Systems (2000) Springer, Dordrecht; pp. 367-370. |
| Holland et al., “Point-of-care molecular diagnostic systems—past, present and future”, Curr Opin Microbiol. (2005) 8(5):504-509. |
| Hong et al., “Integrated nanoliter systems”, Nat Biotechnol. (2003) 21(10):1179-1183. |
| Hong et al., “Molecular biology on a microfluidic chip”, J Phys.: Condens Matter (2006) 18(18):S691-S701. |
| Hong et al., “Integrated Nucleic Acid Analysis in Parallel Matrix Architecture”, in Integrated Biochips for DNA Analysis by Liu et al. [Eds], (2007) Chapter 8, pp. 107-116. |
| Horsman et al., “Forensic DNA Analysis on Microfluidic Devices: A Review”, J Forensic Sci. (2007) 52(4):784-799. |
| Hsieh et al., “Enhancement of thermal uniformity for a microthermal cycler and its application for polymerase chain reaction”, Sens Actuators B: Chemical. (2008) 130(2):848-856. |
| Huang et al., “Temperature Uniformity and DNA Amplification Efficiency in Micromachined Glass PCR Chip”, TechConnect Briefs; Tech Proc. of the 2005 NSTI Nanotechnology Conference and Trade Show. (2005) vol. 1:452-455. |
| Huebner et al., “Microdroplets: A sea of applications?”, Lab Chip. (2008) 8(8):1244-1254. |
| Iordanov et al., “PCT Array on Chip—Thermal Characterization”, IEEE Sensors (2003) Conference Oct. 22-24, 2003; pp. 1045-1048. |
| Ji et al., “DNA Purification Silicon Chip”, Sensors and Actuators A: Physical (2007) 139(1-2):139-144. |
| Jia et al., “A low-cost, disposable card for rapid polymerase chain reaction”, Colloids Surfaces B: Biointerfaces (2007) 58:52-60. |
| Kaigala et al., “An inexpensive and portable microchip-based platform for integrated RT-PCR and capillary electophoresis”, The Analyst (2008) 133(3):331-338. |
| Kajiyama et al., “Genotyping on a Thermal Gradient DNA Chip”, Genome Res. (2003) 13(3):467-475. |
| Kang et al., “Simulation and Optimization of a Flow-Through Micro PCR Chip”, NSTI—Nanotech (2006) vol. 2, pp. 585-588. |
| Kantak et al.,“Microfluidic platelet function analyzer for shear-induced platelet activation studies”, 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Med and Biol. (May 2002) 5 pages. |
| Kantak et al., “Microfabricated cyclical electrical field flow fractionation”, 7th International Conference on Miniaturized Chomical and Biochem Analysis Sys. (2003) pp. 1199-1202. |
| Kantak et al., “Platelet function analyzer: Shear activation of platelets in microchannels”, Biomedical Microdevices (2003) 5(3):207-215. |
| Kantak et al., “Characterization of a microscale cyclical electrical field flow fractionation system”, Lab Chip. (2006) 6(5):645-654. |
| Kantak et al., “Effect of carrier ionic strength in microscale cyclical electrical field-flow fractionation”, Anal Chem. (2006) 78(8):2557-2564. |
| Kantak et al., “Improved theory of cyclical electrical field flow fractions”, Electrophoresis (2006) 27(14):2833-2843. |
| Karunasiri et al.,“Extraction of thermal parameters of microbolometer infrared detectors using electrical measurement”, SPIE's Inter'l Symposium on Optical Science, Engineering, and Instrumentation; Proceedings (1998) vol. 3436, Infrared Technology and Applications XXIV; (1998) 8 pages. |
| Kelly et al., “Microfluidic Systems for Integrated, High-Throughput DNA Analysis,” Analytical Chemistry, (2005), 97A-102A, Mar. 1, 2005, in 7 pages. |
| Kim et al., “Reduction of Microfluidic End Effects in Micro-Field Flow Fractionation Channels”, Proc. MicroTAS 2003, pp. 5-9. |
| Kim et al., “Multi-DNA extraction chip based on an aluminum oxide membrane integrated into a PDMS microfluidic structure”, 3rd IEEE/EMBS Special Topic Conference on Microtechnology in Med and Biol. (May 2005). |
| Kim et al., “Geometric optimization of a thin film ITO heater to generate a uniform temperature distribution”, (2006), Tokyo, Japan; pp. 293-295; Abstract. |
| Kim et al., “Micro-Raman thermometry for measuring the temperature distribution inside the microchannel of a polymerase chain reaction chip”, J Micromech Microeng. (2006) 16(3):526-530. |
| Kim et al., “Patterning of a Nanoporous Membrane for Multi-sample DNA Extraction”, J Micromech Microeng. (2006) 16:33-39. |
| Kim et al., “Performance evaluation of thermal cyclers for PCR in a rapid cycling condition”, Biotechniques. (2008) 44(4):495-505. |
| Kim et al., “Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AIO(x) membrane”, Lab Chip. (2008) 8(9):1516-1523. |
| Kogi et al., “Microinjection-microspectroscopy of single oil droplets in water: an application to liquid/liquid extraction under solution-flow conditions”, Anal Chim Acta. (2000) 418(2):129-135. |
| Kopf-Sill et al., “Creating a Lab-on-a-Chip with Microfluidic Technologies”, in Integrated Microfabricated Biodevices: Advanced Technologies for Genomics, Drug Discovery, Bioanalysis, and Clinical Diagnostics (2002) Chapter 2; pp. 35-54. |
| Kricka L.J., “Microchips, Bioelectronic Chips, and Gene Chips—Microanalyzers for the Next Century”, in Biochip Technology by Cheng et al. [Eds]; (2006) Chapter 1, pp. 1-16. |
| Krishnan et al., “Polymerase chain reaction in high surface-to-volume ratio SiO2 microstructures”, Anal Chem. (2004) 76(22):6588-6593. |
| Kuswandi et al., “Optical sensing systems for microfluidic devices: a review”, Anal Chim Acta. (2007) 601(2):141-155. |
| Lagally et al., “Genetic Analysis Using Portable PCR-CE Microsystem”, Proceedings 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems (2003) pp. 1283-1286. |
| Lagally et al., “Integrated portable genetic analysis microsystem for pathogen/infectious disease detection”, Anal Chem. (2004) 76(11):3152-3170. |
| Lauerman L.H., “Advances in PCR technology”, Anim Health Res Rev. (2004) 5(2):247-248. |
| Lawyer et al., “High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquaticus DNA Polymerase and a Truncated Form Deficient in 5′to 3′Exonuclease Activity.” Genome research (1993) 2(4):275-287. |
| Lee et al., “Submicroliter-volume PCR chip with fast thermal response and very power consumption”, 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems, (2003) pp. 187-190. |
| Lee et al., “Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption”, Lab Chip. (2004) 4(4):401-407. |
| Lewin et al., “Use of Real-Time PCR and Molecular Beacons to Detect Virus Replication in Human Immunodeficiency Virus Type 1-infected Individuals on Prolonged Effective Antiretroviral Therapy”. J Virol. (1999) 73(7), 6099-6103. |
| Li et al., “Effect of high-aspect-ratio microstructures on cell growth and attachment”, 1st Annual Inter'l IEEE-EMBS Special Topic Conference on Microtechnologies in Med and Biol. Proceedings Cat. No. 00EX451; (Oct. 2000) Poster 66, pp. 531-536. |
| Li PCH., “Micromachining Methods et al.” in Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, CRC Press (2005), Chapter 2-3 to 2-5; pp. 10-49. |
| Li PCH., “Microfluidic Flow” in Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, CRC Press (2005), Chapter 3, pp. 55-99. |
| Li PCH., “Detection Methods” in Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, CRC Press (2005), Chapter 7, pp. 187-249. |
| Li PCH., “Applications to Nucleic Acids Analysis” in Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, CRC Press (2005), Chapter 9; pp. 293-325. |
| Li et al., “A Continuous-Flow Polymerase Chain Reaction Microchip With Regional Velocity Control”, J Microelectromech Syst. (2006) 15(1):223-236. |
| Lien et al., “Integrated reverse transcription polymerase chain reaction systems for virus detection”, Biosens Bioelectron. (2007) 22(8):1739-1748. |
| Lien et al., “Microfluidic Systems Integrated with a Sample Pretreatment Device for Fast Nucleic-Acid Amplification”, J Microelectro Sys. (2008) 17(2):288-301. |
| Lifesciences et al., “Microfluidics in commercial applications; an industry perspective.” Lab Chip (2006) 6:1118-1121. |
| Lin et al., “Simulation and experimental validation of micro polymerase chain reaction chips”, Sens Actuators B: Chemical. (2000) 71(1-2):127-133. |
| Linder et al., “Microfluidics at the Crossroad with Point-of-care Diagnostics”, Analyst (2007) 132:1186-1192. |
| Liu et al., “Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing”, Anal Chem. (2007) 79(5):1881-1889. |
| Liu et al. [Eds], Integrated Biochips for DNA Analysis—Biotechnology Intelligence Unit; Springer/Landes Bioscience (2007) ISBN:978-0-387-76758-1; 216 pages. |
| Locascio et al., “ANYL 67 Award Address—Microfluidics as a tool to enable research and discovery in the life sciences”, Abstract; The 236th ACS National Meeting (Aug. 2008); 2 pages. |
| Mahjoob et al., “Rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification”, Inter'l J Heat Mass Transfer. (2008) 51(9-10):2109-2122. |
| Marcus et al., “Parallel picoliter rt-PCR assays using microfluidics”, Anal Chem. (2006) 78(3):956-958. |
| Mariella R.P. Jr., “Microtechnology”, Thrust Area Report FY 96 UCRL-ID-125472; Lawrence Livermore National Lab., CA (Feb. 1997) Chapter 3 in 44 pages. |
| Mariella R., “Sample preparation: the weak link in microfluidics-based biodetection”, Biomed Microdevices. (2008) 10(6):777-784. |
| McMillan et al., “Application of advanced microfluidics and rapid PCR to analysis of microbial targets”, In Proceedings of the 8th international symposium on microbial ecology (1999), in 13 pages. |
| Melin et al., “Microfluidic large-scale integration: the evolution of design rules for biological automation”, Annu Rev Biophys Biomol Struct. (2007) 36:213-231. |
| Merugu et al., “High Throughput Separations Using a Microfabricated Serial Electric Split Ssystem” (2003), Proceedings of μTAS 2003, 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems, Oct. 5-9, 2003, Squaw Valley, California; 1191-1194, in 3 pages. |
| Miao et al., “Low cost micro-PCR array and micro-fluidic integration on single silicon chip”, Int'l J Comput Eng Science (2003) 4(2):231-234. |
| Miao et al., “Flip-Chip packaged micro-plate for low cost thermal multiplexing”, Int'l J Comput Eng Science. (2003) 4(2):235-238. |
| Micheletti et al., “Microscale Bioprocess Optimisation”, Curr Opin Biotech. (2006) 17:611-618. |
| MicroTAS 2005., “Micro Total Analysis Systems”, Proceedings 9th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences; Presentations/Posters/Articles for Conference; Boston, MA in Oct. 10-12, 2005 in 1667 pages. |
| MicroTAS 2007., “Micro Total Analysis Systems”, Proceedings 11th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences; Presentations/Posters/Articles for Conference; Paris, France in Oct. 7-11, 2007 in 1948 pages. |
| MicroTAS 2007., “Micro Total Analysis Systems”, Advance Program for the Proceedings 11th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences; Presentations/Posters/Articles for Conference; Paris, France in Oct. 7-11, 2007 in 42 pages. |
| Mitchell et al., “Modeling and validation of a molded polycarbonate continuous-flow polymerase chain reaction device,” Microfluidics, BioMEMS, and Medical Microsystems, Proc. SPIE (2003) 4982:83-98. |
| Myers et al., “Innovations in optical microfluidic technologies for point-of-care diagnostics”, Lab Chip (2008) 8:2015-2031. |
| Namasivayam et al., “Advances in on-chip photodetection for applications in miniaturized genetic analysis systems”, J Micromech Microeng. (2004) 14:81-90. |
| Narayanan et al., “A microfabricated electrical SPLITT system,” Lab Chip, (2006) 6:105-114. |
| Neuzil et al., “Disposable real-time microPCR device: lab-on-a-chip at a low cost, ” Mol. Biosyst., (2006) 2:292-298. |
| Neuzil et al., “Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes,” Nucleic Acids Research, (2006) 34(11)e77, in 9 pages. |
| Nguyen et al. [Eds], “Microfluidics for Internal Flow Control: Microfluidics” in Fundamentals and Applications of Microfluidics; 2nd Edition (2006) Introduction Chapter 1, pp. 1-9. |
| Nguyen et al. [Eds], “Microfluidics for Internal Flow Control: Microvalves” in Fundamentals and Applications of Microfluidics; (2006) 2nd Edition, Chapter 6, pp. 211-254. |
| Nguyen et al. [Eds], “Microfluidics for Internal Flow Control: Micropumps” in Fundamentals and Applications of Microfluidics; (2006) 2nd Edition, Chapter 7, pp. 255-309. |
| Nguyen et al. [Eds], “Microfluidics for Life Sciences and Chemistry: Microdispensers” in Fundamentals and Applications of Microfluidics; (2006) , Chapter 11, pp. 395-418. |
| Nguyen et al. [Eds], “Microfluidics for Life Sciences and Chemistry: Microreactors” in Fundamentals and Applications of Microfluidics; (2006) 2nd Edition, Chapter 13, pp. 443-477. |
| Ning et al., “Microfabrication Processes for Silicon and Glass Chips”, in Biochip Technology, CRC-Press (2006) Chapter 2, pp. 17-38. |
| Northrup et al., “A MEMs-based Miniature DNA Analysis System,” Lawrence Livermore National Laboratory, (1995), submitted to Transducers '95, Stockholm, Sweden, Jun. 25-29, 1995, in 7 pages. |
| Northrup et al., “Advantages Afforded by Miniaturization and Integration of DNA Analysis Instrumentation,” Microreaction Technology, (1998) 278-288. |
| Northrup et al., “A New Generation of PCR Instruments and Nucleic Acid Concentration Systems,” in PCR Applications: Protocols for Functional Genomics, (1999), Chapter 8, pp. 105-125. |
| Northrup, “Microfluidics, A few good tricks,” Nature materials (2004), 3:282-283. |
| Northrup et al., “Microfluidics-based integrated airborne pathogen detection systems,” Abstract, Proceedings of the SPIE, (2006), vol. 6398, Abstract in 2 pages. |
| Oh et al., “World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays,” Lab Chip, (2005), 5:845-850. |
| Ohno et al., “Microfluidics: Applications for analytical purposes in chemistry and biochemistry,” Electrophoresis (2008), 29:4443-4453. |
| Pal et al., “Phase Change Microvalve for Integrated Devices,” Anal. Chem. (2004), 76(13):3740-3748, Jul. 1, 2004, in 9 pages. |
| Pal et al., “An integrated microfluidic for influenza and other genetic analyses,” Lab Chip, (2005), 5:1024-1032, in 9 pages. |
| Pamme, “Continuous flow separations in microfluidic devices,” Lab Chip, (2007), 7:1644-1659. |
| Pang et al., “A novel single-chip fabrication technique for three-dimensional MEMS structures,” Institute of Microelectronics, Tsinghua University, Beijing, P.R. China, (1998), IEEE, 936-938. |
| Pang et al., “The Study of Single-Chip Integrated Microfluidic System,” Tsinghua University, Beijing, P.R. China, (1998), IEEE, 895-898. |
| Papautsky et al., “Effects of rectangular microchannel aspect ratio on laminar friction constant”, in Microfluidic Devices and Systems II (1999) 3877:147-158. |
| Petersen, Kurt E., “Silicon as a Mechanical Material.” Proceedings of the IEEE, (May 1982) 70(5):420-457. |
| Petersen et al., “Toward Next Generation Clinical Diagnostic Instruments: Scaling and New Processing Paradigms,” Biomedical Microdevices (1998) 1(1):71-79. |
| Poser et al., “Chip elements for fast thermocycling,” Sensors and Actuators A, (1997), 62:672-675. |
| Pourahmadi et al., “Toward a Rapid, Integrated, and Fully Automated DNA Diagnostic Assay for Chlamydia trachomatis and Neisseria gonorrhoeae,” Clinical Chemistry, (2000), 46(9):1511-1513. |
| Pourahmadi et al., “Versatile, Adaptable and Programmable Microfluidic Platforms for DNA Diagnostics and Drug Discovery Assays,” Micro Total Analysis Systems, (2000), 243-248. |
| Raisi et al., “Microchip isoelectric focusing using a miniature scanning detection system,” Electrophoresis, (2001), 22:2291-2295. |
| Raja et al., “Technology for Automated, Rapid, and Quantitative PCR or Reverse Transcriptin-PCR Clinical Testing,” Clinical Chemistry, (2005), 51(5):882-890. |
| Reyes et al., “Micro Total Analysis Systems. 1. Introduction, Theory, and Technology”, Anal Chem (2002) 74:2623-2636. |
| Rodriguez et al., “Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis,” Electrophoresis, (2003), 24:172-178. |
| Roper et al., “Advances in Polymer Chain Reaction on Microfluidic Chips,” Anal. Chem., (2005), 77:3887-3894. |
| Ross et al., “Scanning Temperature Gradient Focusing for Simultaneous Concentration and Separation of Complex Samples,” Micro Total Analysis Systems 2005, vol. 2, (2005), Proceedings of μTAS 2005, Ninth International Conference on Miniaturized Systems for Chemistry and Life Sciences, Oct. 9-13, 2005, Boston, Massachusetts; 1022-1024. |
| Ross et al., “Simple Device for Multiplexed Electrophoretic Separations Using Gradient Elution Moving Boundary Electrophoresis with Channel Current Detection,” Anal. Chem., (2008), 80(24):9467-9474. |
| Sadler et al., “Thermal Management of BioMEMS: Temperature Control for Ceramic-Based PCR and DNA Detection Devices,” IEEE Transactions on Components and Packaging Technologies, (2003) 26(2):309-316. |
| Sant et al., “An Integrated Optical Detector for Microfabricated Electrical Field Flow Fractionation System,” Proceedings of μTAS 2003, 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems, Oct. 5-9, 2003, Squaw Valley, California; pp. 1259-1262. |
| Sant et al., “Geometric scaling effects on instrumental plate height in field flow fractionation”, J Chromatography A (2006) 1104:282-290. |
| Sant H.J., “Reduction of End Effect-Induced Zone Broadening in Field-Flow Fractionation Channels”, Anl Chem. (2006) 78:7978-7985. |
| Sant et al., “Microscale Field-Flow Fractionation: Theory and Practice”, in Microfluidic Technologies for Miniaturized Analysis Systems. (2007) Chapter 12, pp. 4710521. |
| Schäferling et al., “Optical technologies for the read out and quality control of DNA and protein microarrays,” Anal Bioanal Chem, (2006), 385: 500-517. |
| Serpengüzel et al., “Microdroplet identification and size measurement in sprays with lasing images”, Optics express (2002) 10(20):1118-1132. |
| Shackman et al., “Gradient Elution Moving Boundary Electrophoresis for High-Throughput Multiplexed Microfluidic Devices,” Anal. Chem. (2007), 79(2), 565-571. |
| Shackman et al., “Temperature gradient focusing for microchannel separations,” Anal Bioanal Chem, (2007), 387:155-158. |
| Shadpour et al., “Multichannel Microchip Electrophoresis Device Fabricated in Polycarbonate with an Integrated Contact Conductivity Sensor Array,” Anal Chem., (2007), 79(3), 870-878. |
| Sia et al., “Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies,” Electrophoresis, (2003), 24:3563-3576. |
| Sigurdson M., “AC Electrokinetic Enhancement for Assay Enhancement”, ProQuest LLC (2008) Doctoral Thesis UMI Microform 3319791 in 24 pages. |
| Singh et al., “PCR thermal management in an integrated Lab on Chip,” Journal of Physics: Conference Series, (2006), 34:222-227. |
| Situma et al., “Merging microfluidics with microarray-based bioassays”, Biomol Engin. (2006) 23:213-231. |
| Smith et al., “(576d) Micropatterned fluid lipid bilayers created using a continuous flow microspotter for multi-analyte assays,” (2007), Biosensors II, 2007 AIChE Annual Meeting, Nov. 8,2007, Abstract in 2 pages. |
| Sommer et al., “Introduction to Microfluidics”, in Microfluidics for Biological Applications by Tian et al. [Eds] (2008) Chapter 1, pp. 1-34. |
| Squires et al., “Microfluidics: Fluid physics at the nanoliter scale,” Reviews of Modern Physics, (2005), 77(3):977-1026. |
| Sundberg et al., “Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis,” Biomed Microdevices, (2007), 9:159-166, in 8 pages. |
| Tabeling, P. [Ed.], “Physics at the micrometric scale,” in Introduction to Microfluidics (2005) Chapter 1, pp. 24-69. |
| Tabeling, P. [Ed.], “Hydrodynamics of Microfluidic Systems”, in Introduction to Microfluidics; (2005) Chapter 2, pp. 70-129. |
| Tabeling, P. [Ed.], Introduction to Microfluidics; (2005) Chapters 5-7, pp. 216-297. |
| Taylor et al., Fully Automated Sample Preparation for Pathogen Detection Performed in a Microfluidic Cassette, in Micro Total Analysis Systems, Springer (2001), pp. 670-672. |
| Taylor et al., “Lysing Bacterial Spores by Sonication through a Flexible Interface in a Microfluidic System,” Anal. Chem., (2001), 73(3):492-496. |
| Taylor et al., “Microfluidic Bioanalysis Cartridge with Interchangeable Microchannel Separation Components,” (2001), The 11th International Conference on Solid-State Sensors and Actuators, Jun. 10-14, 2001, Munich, Germany; 1214-1247. |
| Taylor et al., “Disrupting Bacterial Spores and Cells using Ultrasound Applied through a Solid Interface,” (2002), 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine & Biology, May 2-4, 2002, Madison, Wisconsin; 551-555. |
| Thorsen et al., “Microfluidic Large-scale integration,” Science, (2002), 298:580-584. |
| Toriello et al., “Multichannel Reverse Transcription-Polymerase Chain Reaction Microdevice for Rapid Gene Expression and Biomarker Analysis,” Anal. Chem., (2006) 78(23):7997-8003. |
| Ugaz et al., “Microfabricated electrophoresis systems for DNA sequencing and genotyping applications,” Phil. Trans. R. Soc. Lond. A, (2004), 362:1105-1129. |
| Ugaz et al., “PCR in Integrated Microfluidic Systems”, in Integrated Biochips for DNA Analysis by Liu et al. [Eds]; (2007) Chapter 7, pp. 90-106. |
| Ullman et al., “Luminescent oxygen channeling assay (LOCI™): sensitive, broadly applicable homogeneous immunoassay method”. Clin Chem. (1996) 42(9), 1518-1526. |
| Vinet et al., “Microarrays and microfluidic devices: miniaturized systems for biological analysis,” Microelectronic Engineering, (2002), 61-62:41-47. |
| Wang et al., “From biochips to laboratory-on-a-chip system”, in Genomic Signal Processing and Statistics by Dougherty et al. [Eds]; (2005) Chapter 5, pp. 163-200. |
| Wang et al., “A disposable microfluidic cassette for DNA amplification and detection”, Lab on a Chip (2006) 6(1):46-53. |
| Wang et al., “Micromachined Flow-through Polimerase Chain Reaction Chip Utilizing Multiple Membrane-activated Micropumps,” (2006), MEMS 2006, Jan. 22-26, 2006, Istanbul, Turkey; 374-377. |
| Woolley a.T., “Integrating Sample Processing and Detection with Microchip Capillary Electrophoresis of DNA”, in Integrated Biochips for DNA Analysis by Liu et al. [Eds]; (2007) Chapter 5, pp. 68-77. |
| Xiang et al., “Real Time PCR on Disposable PDMS Chip with a Miniaturized Thermal Cycler,” Biomedical Microdevices, (2005), 7(4):273-279. |
| Xuan, “Joule heating in electrokinetic flow,” Electrophoresis, (2008), 298:33-43. |
| Yang et al., “High sensitivity PCR assay in plastic micro reactors,” Lab Chip, (2002), 2:179-187. |
| Yang et al., “An independent, temperature controllable-microelectrode array,” Anal. Chem., (2004), 76(5):1537-1543. |
| Yang et al., “Cost-effective thermal isolation techniques for use on microfabricated DNA amplification and analysis devices,” J Micromech Microeng, (2005), 15:221-230. |
| Yobas et al., Microfluidic Chips for Viral RNA Extraction & Detection, (2005), 2005 IEEE, 49-52. |
| Yobas et al., “Nucleic Acid Extraction, Amplification, and Detection on Si-Based Microfluidic Platforms,” IEEE Journal of Solid-State Circuits, (2007), 42(8):1803-1813. |
| Yoon et al., “Precise temperature control and rapid thermal cycling in a micromachined DNA polymer chain reaction chip,” J. Micromech. Microeng., (2002), 12:813-823. |
| Zhang et al, “Temperature analysis of continuous-flow micro-PCR based on FEA,” Sensors and Actuators B, (2002), 82:75-81. |
| Zhang et al, “Continuous□Flow PCR Microfluidics for Rapid DNA Amplification Using Thin Film Heater with Low Thermal Mass,” Analytical Letters, (2007), 40:1672-1685, in 15 pages. |
| Zhang et al, “Direct Adsorption and Detection of Proteins, Including Ferritin, onto Microlens Array Patterned Bioarrays,” J Am Chem Soc., (2007), 129:9252-9253. |
| Zhang et al, “Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trens,” Biotechnology Advances, (2007), 25:483-514. |
| Zhao et al, “Heat properties of an integrated micro PCR vessel,” Proceedings of SPIE, (2001), International Conference on Sensor Technology, 4414:31-34. |
| Zou et al., “Micro-assembled multi-chamber thermal cycler for low-cost reaction chip thermal multiplexing,” Sensors and Actuators A, (2002), 102:114-121. |
| Zou et al., “Miniaturized Independently Controllable Multichamber Thermal Cycler,” IEEE Sensors Journal, (2003), 3(6):774-780. |
| Petitioner's Reply to Patent Owner's Response to Petition in Inter Partes Review of U.S. Pat. No. 7,998,708 and Exhibit List (Paper 32 in IPR 2019-00488) dated Jan. 31, 2020 (34 pages). |
| Petitioner's Reply to Patent Owner's Response to Petition in Inter Partes Review of U.S. Pat. No. 8,323,900 and Exhibit List (Paper 32 in IPR 2019-00490) dated Jan. 31, 2020 (35 pages). |
| Second Declaration of Bruce K. Gale, Ph.D. (Exhibit 1026 in IPR2019-00488 and IPR2019-00490) dated Jan. 31, 2020 (91 pages). |
| Transcript of Deposition of M. Allen Northrup, Ph.D., (Exhibit 1027 in IPR2019-00488 and IPR2019-00490), taken Dec. 19, 2019 (109 pages). |
| Patent Owner's Sur-Reply in Inter Partes Review of U.S. Pat. No. 8,323,900 (Paper 42 in IPR2019-00490) dated Mar. 12, 2020 (39 pags). |
| Patent Owner's Sur-Reply in Inter Partes Review of U.S. Pat. No. 7,998,708 (Paper 43 in IPR 2019-00488) dated Mar. 12, 2020 (41 pages). |
| Transcript of Second Deposition of Bruce K. Gale, Ph.D., (Exhibit 2068 in IPR2019-00488 and IPR2019-00490), taken Feb. 19, 2020 (352 pages). |
| Anderson et al., “Microfluidic biochemical analysis system” Proc. 1997 IEEE Int. Conf. Solid-State Sens. Actuat., 1997, pp. 477-480. |
| Anderson et al., “Advances in Integrated Genetic Analysis” Micro Total Analysis Systems '98 Conference Proceedings, D. Kluwer Academic Publishers, 1998, in 6 pages. |
| Anderson et al., “A Miniature Integrated Device for Automated Multistep Genetic Assays” Nucleic Acids Research (2000) 28(12), i-vi. |
| Hsueh et al., “A microfabricated, electrochemiluminescence cell for the detection of amplified DNA” Proc. 1995 IEEE Int. Conf. Solid-State Sens. Actuators, 1995, pp. 768-771. |
| Hsueh et al., “DNA quantification with an electrochemiluminescence microcell” Proc. 1997 IEEE Int. Conf. Solid-State Sens. Actuators, 1997, pp. 175-178. |
| Jiang et al., “Directing cell migration with asymmetric micropatterns” Proc. Natl. Acad. Sci. USA. (2005) 102, 975-978. |
| Lagally et al., “Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system” Sens Actuator B—Chem (2000) 63:138-146. |
| Manz et al., “Design of an open-tubular column liquid chromatograph using silicon chip technology” Sensors and Actuators B: Chemical (1990) 1:249-255. |
| Manz et al., “Planar chips technology for miniaturization and integration of separation techniques into monitoring systems: Capillary electrophoresis on a chip” Journal of Chromatography A (1992) 593:253-258. |
| Rhee et al., “Drop Mixing in a Microchannel for Lab-on-a-Chip Applications” Langmuir, (2008) 24 (2): 590-601. |
| Sammarco et al., “Thermocapillary Pumping of Discrete Drops in Microfabricated Analysis Devices” AIChE Journal, (1999) 45(2): 350-366. |
| Taylor et al., “Optimization of the performance of the polymerase chain reaction in silicon-based microstructures” Nucleic Acids Res., 1997, vol. 25, pp. 3164-3168. |
| Terry et al., “A Gas Chromatographic Air Analyzer Fabricated on a Silicon Wafer” IEEE T Electron Dev (1979) 26:1880-1886. |
| Whitesides G.M., “The origins and the future of microfluidics” Nature (2006) 442(7101):368-373. |
| Woias P., “Micropumps—past, progress and future prospects” Sens. Actuators, B. (2005) 105, 28-38. |
| Woolley et al., “Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device” Anal. Chem., 1996, vol. 68, pp. 4081-4086. |
| Wu et al., “Fabrication of Complex Three-dimensional Microchannel Systems in PDMS” J. Am. Chem. Soc. (2003) 125, 554-559. |
| Record of Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 in 80 pages; Petitioner's Demonstratives for Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 in 72 pages; Patent Owner's Demonstratives for Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 in 88 pages; Patent Owner's Objections to Petitioner's Oral Hearing Demonstratives in IPR2019-00488 and IPR2019-00490 dated Apr. 16, 2020 (4 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 2 in IPR2020-01083) dated Jun. 12, 2020 (104 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 2 in IPR2020-01091) dated Jun. 12, 2020 (105 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,803,069 (Paper 2 in IPR2020-01095) dated Jun. 12, 2020 (84 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,803,069 (Paper 3 in IPR2020-01100) dated Jun. 12, 2020 (83 pages). |
| Declaration of Mark A. Burns, Ph.D. (Exhibit N1001 in IPR2020-01083, IPR2020-01091, IPR2020-01095 and IPR2020-01100) dated Jun. 12, 2020 (378 pages). |
| Burns et al., “Microfabricated Structures for Integrated DNA Analysis” Proc. Natl. Acad. Sci. USA (May 1996) 93: 5556-5561. |
| Harrison et al., “Capillary Electrophoresis and Sample Injection Systems Integrated on a Planar Glass Chip”, Anal. Chem., (1992) 64: 1926-1932. |
| Petition for Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 2 in IPR2020-01132) dated Jun. 18, 2020 (96 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 2 in IPR2020-01133) dated Jun. 18, 2020 (96 pages). |
| Declaration of Mark A. Burns, Ph.D. (Exhibit N1101 in IPR2020-01132 and IPR2020-01133) dated Jun. 17, 2020 (253 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 2 in IPR2020-01136) dated Jun. 19, 2020 (85 pages). |
| Petition for Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 2 in IPR2020-01137) dated Jun. 19, 2020 (86 pages). |
| Declaration of Mark A. Burns, Ph.D. (Exhibit N1210 in IPR2020-01136 and IPR2020-01137) dated Jun. 19, 2020 (205 pages). |
| Zhang et al., “Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device,” Analytical Biochemistry, (2009) 387:102-112. |
| Judgment/Final Written Decision Determining No Challenged Claims Unpatentable in Inter Partes Review of U.S. Pat. No. 7,998,708 (Paper No. 52 in IPR2019-00488) dated Jul. 14, 2020 (43 pages). |
| Judgment/Final Written Decision Determining No Challenged Claims Unpatentable in Inter Partes Review of U.S. Pat. No. 8,323,900 (Paper No. 51 in IPR2019-00490) dated Jul. 14, 2020 (43 pages). |
| First Amended and Supplemental Complaint filed by Becton, Dickinson and Company et al. on Jun. 25, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (55 pages). |
| Answer to Amended and Supplemental Complaint filed by NeuModx Molecular, Inc. on Jul. 16, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (42 pages). |
| U.S. File History of U.S. Appl. No. 60/491,264, filed Jul. 31, 2003 (50 pages). |
| U.S. File History of U.S. Appl. No. 60/491,269, filed Jul. 31, 2003 (59 pages). |
| U.S. File History of U.S. Appl. No. 60/491,539, filed Aug. 1, 2003 (55 pages). |
| U.S. File History of U.S. Appl. No. 60/553,553, filed Mar. 17, 2004 (59 pages). |
| U.S. File History of U.S. Appl. No. 60/726,066, filed Oct. 11, 2005 (68 pages). |
| U.S. File History of U.S. Appl. No. 60/786,007, filed Mar. 24, 2006 (247 pages). |
| U.S. File History of U.S. Appl. No. 60/859,284, filed Nov. 14, 2006 (121 pages). |
| Petitioner's Notice of Appeal in Inter Partes Review of U.S. Pat. No. 7,998,708 (Paper No. 54 in IPR2019-00488) dated Sep. 9, 2020 (48 pages). |
| Petitioner's Notice of Appeal in Inter Partes Review of U.S. Pat. No. 8,323,900 (Paper No. 53 in IPR2019-00490) dated Sep. 9, 2020 (48 pages). |
| Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,703,069 (Paper 13 in IPR2020-01095) dated Sep. 17, 2020 (77 pages). |
| Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 13 in IPR2020-01091) dated Sep. 17, 2020 (70 pages). |
| Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,703,069 (Paper 14 in IPR2020-01100) dated Sep. 17, 2020 (59 pages). |
| Declaration of M. Allen Northrup, Ph.D. in Support of Patent Owner Preliminary Responses in IPR2020-01091, IPR2020-01095 and IPR2020-01100 (Exhibit H2003) dated Sep. 16, 2020 (137 pages). |
| Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 13 in IPR2020-01083) dated Oct. 22, 2020 (88 pages). |
| Declaration of M. Allen Northrup, Ph.D. in support of Patent Owner Preliminary Responses in IPR2020-01083, IPR2020-01091, IPR2020-01095 and IPR2020-01100 (Exhibit H2003) dated Oct. 21, 2020 (171 pages). |
| Defendant NeuModx's Initial Invalidity Contentions filed Sep. 30, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (47 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20200156060 A1 | May 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60786007 | Mar 2006 | US | |
| 60859284 | Nov 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14719692 | May 2015 | US |
| Child | 16730763 | US | |
| Parent | 11728964 | Mar 2007 | US |
| Child | 14719692 | US |
