Microfluidic system for amplifying and detecting polynucleotides in parallel

Description

TECHNICAL FIELD

The technology described herein generally relates to systems for detecting polynucleotides in samples, particularly from biological samples. The technology more particularly relates to microfluidic systems that carry out PCR on nucleotides of interest within microfluidic channels, and detect those nucleotides.

BACKGROUND

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, 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 for analyses to these locations, thereby incurring shipping costs and transportation delays, and possibly even sample loss. Second, the equipment in question 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.

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. 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 PCR to amplify a vector of interest. Once amplified, the presence of a nucleotide of interest from 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.

There is a need for a method and apparatus of carrying out PCR and detection on prepared biological samples, and preferably with high throughput. In particular there is a need for an easy-to-use device that can deliver a diagnostic result on several samples in a short time.

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.

SUMMARY

The present technology addresses systems for detecting polynucleotides in samples, particularly from biological samples. In particular, the technology relates to microfluidic systems that carry out PCR on nucleotides of interest within microfluidic channels, and detect those nucleotides.

An apparatus, comprising: a receiving bay configured to receive a microfluidic cartridge; at least one heat source thermally coupled to the cartridge and configured to carry out PCR on a microdroplet of polynucleotide-containing sample, in the cartridge; a detector configured to detect presence of one or more polynucleotides in the sample; and a processor coupled to the detector and the heat source, configured to control heating of one or more regions of the microfluidic cartridge.

A method of carrying out PCR on a plurality of polynucleotide-containing samples, the method comprising: introducing the plurality of samples in to a microfluidic cartridge, wherein the cartridge has a plurality of PCR reaction chambers configured to permit thermal cycling of the plurality of samples independently of one another; moving the plurality of samples into the respective plurality of PCR reaction chambers; and amplifying polynucleotides contained with the plurality of samples, by application of successive heating and cooling cycles to the PCR reaction chambers.

The details of one or more embodiments of the technology are set forth in the accompanying drawings and further description herein. Other features, objects, and advantages of the technology will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary apparatus, a microfluidic cartridge, and a read head, as further described herein;

FIG. 2 shows an exemplary sample-preparation kit;

FIG. 3 shows a schematic diagram of an apparatus;

FIG. 4 shows a cross-section of a pipetting head and a cartridge in position in a microfluidic apparatus.

FIG. 5 shows introduction of a PCR-ready sample into a cartridge, situated in an instrument;

FIGS. 6A-6E show exemplary embodiments of an apparatus;

FIG. 7 shows an exploded view of an apparatus;

FIG. 8 shows a block diagram of control circuitry;

FIG. 9 shows a plan view of an exemplary multi-lane microfluidic cartridge;

FIG. 10A shows an exemplary multi-lane cartridge;

FIG. 10B shows a portion of an exemplary multi-lane cartridge;

FIGS. 11A-C show exploded view of an exemplary microfluidic cartridge;

FIG. 12 shows an exemplary highly-multiplexed microfluidic cartridge;

FIGS. 13-16 show various aspects of exemplary highly multiplexed microfluidic cartridges; and

FIGS. 17A-C show various aspects of a radially configured highly multiplexed microfluidic cartridge.

FIG. 18 shows an exemplary microfluidic network in a lane of a multi-lane cartridge;

FIGS. 19A-19D show exemplary microfluidic valves;

FIG. 20 shows an exemplary bubble vent;

FIG. 21 shows a cross-section of a microfluidic cartridge, when in contact with a heater substrate;

FIGS. 22A-22C shows various cut-away sections that can be used to improve cooling rates during PCR thermal cycling;

FIG. 23 shows a plot of temperature against time during a PCR process, as performed on a microfluidic cartridge as described herein;

FIG. 24 shows an assembly process for a cartridge as further described herein:

FIGS. 25A and 25B show exemplary deposition of wax droplets into microfluidic valves;

FIG. 26 shows an exemplary heater unit;

FIGS. 27A and 27B show a plan view of heater circuitry adjacent to a PCR reaction chamber;

FIG. 27C shows thermal images of heater circuitry in operation;

FIG. 28 shows an overlay of an array of heater elements on an exemplary multi-lane microfluidic cartridge, wherein various microfluidic networks are visible;

FIG. 29 shows a cross-sectional view of an exemplary detector;

FIG. 30 shows a perspective view of a detector in a read-head;

FIG. 31A, 31B shows a cutaway view of an exemplary detector in a read-head;

FIG. 32 shows an exterior view of an exemplary multiplexed read-head with an array of detectors therein;

FIG. 33 shows a cutaway view of an exemplary multiplexed read-head, as in FIG. 18;

FIG. 34 shows exemplary pre-amplifier circuitry for a fluorescence detector;

FIG. 35A shows effects of aperturing on fluorescence intensity; FIG. 35B shows a detector in cross section with an exemplary aperture;

FIG. 36 shows an exemplary layout for electronics and software components, as further described herein;

FIG. 37 shows an exemplary apparatus, a microfluidic cartridge, and a read head, as further described herein;

FIGS. 38-39 show positioning of a cartridge in an exemplary apparatus;

FIGS. 40 and 41 show removal of a heater unit from an exemplary apparatus;

FIGS. 42A and 42B show an exemplary heater unit and heater substrate;

FIGS. 43A and 43B show an exemplary apparatus having a detector mounted in a sliding lid;

FIGS. 44A-44C show a force member;

FIGS. 45A-45D show a force member associated with a detector;

FIG. 46 shows a block diagram of exemplary electronic circuitry in conjunction with a detector as described herein;

Additional figures are illustrated within the examples, and are further described therein.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Overview of Apparatus

The present technology relates to a system and related methods for amplifying, and carrying out diagnostic analyses on, polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) from biological samples. For example, the system and methods can 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. The nucleotides are typically provided to the system having been isolated or released from particles such as cells in the sample. The system includes a disposable microfluidic cartridge containing multiple sample lanes in parallel and a reusable instrument platform (a PCR analyzer apparatus) that can actuate on-cartridge operations, can detect (e.g., by fluorescence detection) and analyze the products of the PCR amplification in each of the lanes separately, in all simultaneously, or in groups simultaneously, and, optionally, can display the results on a graphical user interface.

A system, microfluidic cartridge, heater unit, detector, kit, methods, and associated computer program product, are now further described.

By cartridge is meant a unit that may be disposable, or reusable in whole or in part, and that is configured to be used in conjunction with some other apparatus that has been suitably and complementarily configured to receive and operate on (such as deliver energy to) the cartridge.

By microfluidic, as used herein, is meant that volumes of sample, and/or reagent, and/or amplified polynucleotide are from about 0.1 μl to about 999 μl, such as from 1-100 μl, or from 2-25 μl. Similarly, as applied to a cartridge, the term microfluidic means that various components and channels of the cartridge, as further described herein, are configured to accept, and/or retain, and/or facilitate passage of microfluidic volumes of sample, reagent, or amplified polynucleotide.

FIG. 1 shows a perspective view of an exemplary apparatus 100 consistent with those described herein, as well as various components thereof, such as exemplary cartridge 200 that contains multiple sample lanes, and exemplary read head 300 that contains detection apparatus for reading signals from cartridge 200. The apparatus 100 of FIG. 1 is able to carry out real-time PCR on a number of samples in cartridge 200 simultaneously. Preferably the number of samples is 12 samples, as illustrated with exemplary cartridge 200, though other numbers of samples such as 4, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and 48 are within the scope of the present description. In preferred operation of the apparatus, a PCR-ready solution containing the sample, and, optionally, one or more analyte-specific reagents (ASR's) is prepared, as further described elsewhere (see, e.g., U.S. patent application publication 2006-0166233, incorporated herein by reference), prior to introduction into cartridge 200. An exemplary kit for preparing a PCR-ready sample, for use with the system described herein, the kit comprising buffers, lysis pellets, and affinity pellets, is shown in FIG. 2.

System Overview

A schematic overview of a system 981 for carrying out analyses described herein is shown in FIG. 3. The geometric arrangement of the components of system 981 shown in FIG. 3, as well as their respective connectivities, is exemplary and not intended to be limiting.

A processor 980, such as a microprocessor, is configured to control functions of various components of the system as shown, and is thereby in communication with each such component. In particular, processor 980 is configured to receive data about a sample to be analyzed, e.g., from a sample reader 990, which may be a barcode reader, an optical character reader, or an RFID scanner (radio frequency tag reader). For example, the sample identifier can be a handheld bar code reader. Processor 980 can be configured to accept user instructions from an input 984, where such instructions may include instructions to start analyzing the sample, and choices of operating conditions.

Processor 980 can also be configured to communicate with an optional display 982, so that, for example, information including but not limited to the current status of the system, progress of PCR thermocycling, and any warning message in case of malfunction of either system or cartridge, as well as results of analysis, are transmitted to the display. Additionally, processor 980 may transmit one or more questions to be displayed on display 982 that prompt a user to provide input in response thereto. Thus, in certain embodiments, input 984 and display 982 are integrated with one another.

Processor 980 can be optionally further configured to transmit results of an analysis to an output device such as a printer, a visual display, or a speaker, or a combination thereof, the transmission being either directly through a directly dedicated printer cable, or wirelessly, or via a network connection.

Processor 980 is still further optionally connected via a communication interface such as a network interface to a computer network 988. The communication interface can be one or more interfaces selected from the group consisting of: a serial connection, a parallel connection, a wireless network connection and a wired network connection such as an ethernet, firewire, cable connection, or one using USB connectivity. Thereby, when the system is suitably addressed on the network, a remote user may access the processor and transmit instructions, input data, or retrieve data, such as may be stored in a memory (not shown) associated with the processor, or on some other computer-readable medium that is in communication with the processor. The computer network connection may also permit extraction of data to a remote location, such as a personal computer, personal digital assistant, or network storage device such as computer server or disk farm. The apparatus may further be configured to permit a user to e-mail results of an analysis directly to some other party, such as a healthcare provider, or a diagnostic facility, or a patient.

Although not shown in FIG. 3, in various embodiments, input 984 can include one or more input devices selected from the group consisting of: a keyboard, a touch-sensitive surface, a microphone, a track-pad, and a mouse. A suitable input device may further comprise a reader of formatted electronic media, such as, but not limited to, a flash memory card, memory stick, USB-stick, CD, or floppy diskette. An input device may further comprise a security feature such as a fingerprint reader, retinal scanner, magnetic strip reader, or bar-code reader, for ensuring that a user of the system is in fact authorized to do so, according to, for example, pre-loaded identifying characteristics of authorized users. An input device may additionally—and simultaneously—function as an output device for writing data in connection with sample analysis. For example, if an input device is a reader of formatted electronic media, it may also be a writer of such media. Data that may be written to such media by such a device includes, but is not limited to, environmental information, such as temperature or humidity, pertaining to an analysis, as well as a diagnostic result, and identifying data for the sample in question.

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, or one or more removable storage media such as a CD-R, CD-RW, USB-drive, and a flash card.

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 one or more samples 996 containing one or more polynucleotides in a form suitable for amplification and diagnostic analysis. The cartridge has dedicated regions within which amplification, such as by PCR, of the polynucleotides is carried out when the cartridge is situated in the apparatus.

The microfluidic cartridge is received by a receiving bay 992 configured to selectively receive the cartridge. For example, the receiving bay and the microfluidic cartridge can be complementary in shape so that the microfluidic cartridge is 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. The registration member can be, for example, a cut-out on an edge of the cartridge, such as a corner that is cut-off, or one or more notches that are made on one or more of the sides. 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 sources, detectors, force members, and the like) are positioned to properly operate on the microfluidic cartridge. In some embodiments, the apparatus can further include a sensor coupled to the processor, the sensor configured to sense whether the microfluidic cartridge is selectively received.

The receiving bay is in communication with a heater unit 998 that itself is controlled by processor 980 in such a way that specific regions of the cartridge, such as individual sample lanes, are independently and selectively heated at specific times during amplification and analysis. The processor can be configured to control application of heat to the individual sample lanes, separately, in all simultaneously, or in groups simultaneously.

The heat source can be, for example, a contact heat source such as a resistive heater or a network of resistive heaters, or a Peltier device, and the like. The contact heat source can be configured to be in direct physical contact with one or more distinct locations 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 mm²about 225 mm²(typically between about 1 mm²and about 100 mm², or in some embodiments between about 5 mm²and about 50 mm²).

In various embodiments, the heat source can be situated in an assembly that is removable from the apparatus, for example, to permit cleaning or to replace the heater configuration.

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 at 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 apparatus can further include one or more force members (not shown in FIG. 3) configured to apply force to thermally couple the at least one heat source at least a portion of a microfluidic cartridge received in the receiving bay.

In various embodiments, the one or more force members are 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 7 kilopascals, and still more preferably 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. The application of force is important to ensure consistent thermal contact between the heater wafer and the PCR reactor and microvalves in the microfluidic cartridge.

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 that ensures that the lid is securely closed before amplification reactions are applied to the samples in the cartridge.

The processor is also configured to receive signals from and control a detector 999 configured to detect a polynucleotide in a sample in one or more of the individual sample lanes, separately or simultaneously. The processor thereby provides an indication of a diagnosis from the cartridge 994. Diagnosis can be predicated on the presence or absence of a specific polynucleotide in a particular sample. The diagnosis can be transmitted to the output device 986 and/or the display 982, as described hereinabove.

The detector can be, for example, an optical detector that includes a light source that selectively emits light in an absorption band of a fluorescent dye, and a light detector that selectively detects light in an emission band of the fluorescent dye, wherein the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. Alternatively, 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 on a microfluidic cartridge, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof in a different sample.

A suitable processor 980 can be designed and manufactured according to, respectively, design principles and semiconductor processing methods known in the art.

The system in FIG. 3 is configured so that a cartridge with capacity to receive multiple samples can be acted upon by the system to analyze multiple samples—or subsets thereof—simultaneously, or to analyze the samples consecutively. It is also consistent that additional samples can be added to a cartridge, while previously added samples are being amplified and analyzed.

The system shown in outline in FIG. 3, as with other exemplary embodiments described herein, is advantageous at least because it does not require locations within the system suitably configured for storage of reagents. Neither does the system, or other exemplary embodiments herein, require inlet or outlet ports that are configured to receive reagents from, e.g., externally stored containers such as bottles, canisters, or reservoirs. Therefore, the system in FIG. 3 is self-contained and operates in conjunction with a microfluidic cartridge, wherein the cartridge has locations within it configured to receive mixtures of sample and PCR reagents.

The system of FIG. 3 may be configured to carry out operation in a single location, such as a laboratory setting, or may be portable so that it can accompany, e.g., a physician, or other healthcare professional, who may visit patients at different locations. The system is typically provided with a power-cord so that it can accept AC power from a mains supply or generator. An optional transformer (not shown) built into the system, or situated externally between a power socket and the system, transforms AC input power into a DC output for use by the system. The system may also be configured to operate by using one or more batteries and therefore is also typically equipped with a battery recharging system, and various warning devices that alert a user if battery power is becoming too low to reliably initiate or complete a diagnostic analysis.

The system of FIG. 3 may further be configured, for multiplexed cartridge analysis. In one such configuration, multiple instances of a system, as outlined in FIG. 3, are operated in conjunction with one another to accept and to process multiple cartridges, where each cartridge has been loaded with a different sample. Each component shown in FIG. 3 may therefore be present as many times as there are cartridges, though the various components may be configured in a common housing.

In still another configuration, a system is configured to accept and to process multiple cartridges, but one or more components in FIG. 3 is common to multiple cartridges. For example, a single device may be configured with multiple cartridge receiving bays, but a common processor and user interface suitably configured to permit concurrent, consecutive, or simultaneous, control of the various cartridges. In such an embodiment a single detector, for example, can scan across all of the multiple cartridges. It is further possible that such an embodiment, also utilizes a single sample reader, and a single output device.

In still another configuration, a system as shown in FIG. 3 is configured to accept a single cartridge, but wherein the single cartridge is configured to process more than 1, for example, 2, 3, 4, 5, or 6, samples in parallel, and independently of one another.

It is further consistent with the present technology that a cartridge can be tagged, e.g., with a molecular bar-code indicative of one or more of the samples, 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.

In various embodiments, the apparatus can further include an analysis port. The analysis port can be configured to allow an external sample analyzer 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 analyzer; for example, the analysis port can include a conduit in fluid communication with the microfluidic cartridge that direct a liquid sample to a chromatography apparatus, an optical spectrometer, a mass spectrometer, or the like.

Apparatus 100 may optionally comprise one or more stabilizing feet that cause the body of the device to be elevated above a surface on which system 100 is disposed, thereby permitting ventilation underneath system 100, and also providing a user with an improved ability to lift system 100. There may be 2, 3, 4, 5, or 6, or more feet, depending upon the size of system 100. Such feet are preferably made of rubber, or plastic, or metal, and in some embodiments may elevate the body of system 100 by from about 2 to about 10 mm above a surface on which it is situated. The stabilizing function can also be provided by one or more runners that run along one or more edges—or are inwardly displaced from one or more edges—of the underside of the apparatus. Such runners can also be used in conjunction with one or more feet. In another embodiment, a turntable situated on the underside permits the apparatus to be rotated in a horizontal or near-horizontal plane when positioned on, e.g., a benchtop, to facilitate access from a number of angles by a user.

FIG. 4 shows a schematic cross-sectional view of a part of an apparatus as described herein, showing input of sample into a cartridge 200 via a pipette 10 (such as a disposable pipette) and an inlet 202. Inlet 202 is preferably configured to receive a pipette or the bottom end of a PCR tube and thereby accept sample for analysis with minimum waste, and with minimum introduction of air. Cartridge 200 is disposed on top of and in contact with a heater substrate 400. Read head 300 is positioned above cartridge 200 and a cover for optics 310 restricts the amount of ambient light that can be detected by the read head.

FIG. 5 shows an example of 4-pipette head used for attaching disposable pipette tips, prior to dispensing PCR-ready sample into a cartridge.

Exemplary Systems

FIGS. 6A-6E show exterior perspective views of various configurations of an exemplary system, as further described herein. FIG. 6A shows a perspective view of a system 2000 for receiving microfluidic cartridge (not shown), and for causing and controlling various processing operations to be performed a sample introduced into the cartridge. The elements of system 2000 are not limited to those explicitly shown. For example, although not shown, system 2000 may be connected to a hand-held bar-code reader, as further described herein.

System 2000 comprises a housing 2002, which can-be made of metal, or a hardened plastic. The form of the housing shown in FIG. 6A embodies stylistic as well as functional features. Other embodiments of the technology may appear somewhat differently, in their arrangement of the components, as well as their overall appearance, in terms of smoothness of lines, and of exterior finish, and texture. System 2000 further comprises one or more stabilizing members 2004. Shown in FIG. 6A is a stabilizing foot, of which several are normally present, located at various regions of the underside of system 2000 so as to provide balance and support. For example, there may be three, four, five, six, or eight such stabilizing feet. The feet may be moulded into and made of the same material as housing 2002, or may be made of one or more separate materials and attached to the underside of system 2000. For example, the feet may comprise a rubber that makes it hard for system 2000 to slip on a surface on which it is situated, and also protects the surface from scratches. The stabilizing member of members may take other forms than feet, for example, rails, runners, or one or more pads.

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 FIG. 6A, also comprises a moveable lid 2010, having a handle 2008. The lid 2010 can slide back and forward. In FIG. 6A, the lid is in a forward position, whereby it is “closed”. In FIG. 6B, the lid is shown in a back position, wherein the lid is “open” and reveals a receiving bay 2014 that is configured to receive a microfluidic cartridge. Of course, as one of ordinary skill in the art would appreciate, the technology described herein is not limited to a lid that slides, or one that slides back and forward. Side to side movement is also possible, as is a configuration where the lid is “open” when positioned forward in the device. It is also possible that the lid is a hinged lid, or one that is totally removable.

Handle 2008 performs a role of permitting a user to move lid 2010 from 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 FIG. 6C, handle 2008 is shown in a depressed position, wherein force is thereby applied to lid 2014, and thus pressure is applied to a cartridge received in the receiving bay beneath the lid.

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 FIG. 6D, wherein a door 2012 is in an open position. Door 2012 is shown in a closed position in FIGS. 6A-C. The door is an optional component that permits a user to access a heater module 2020, and also a computer-readable medium input tray 2022. System 2000 can function without a door that covers heater module 2020 and medium input 2022, but such a door has convenience attached to it. Although the door 2012 is shown hinged at the bottom, it may also be hinged at one of its sides, or at its upper edge. Door 2012 may alternatively be a removable cover, instead of being hinged. Door 2012, may also be situated at the rear, or side of system 2000 for example, if access to the heater module and/or computer readable medium input is desired on a different face of the system. It is also consistent with the system herein that the heater module, and the computer readable medium input are accessed by separate doors on the same or different sides of the device, and wherein such separate doors may be independently hinged or removable.

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 FIG. 2D is an exemplary form of input 2022, a CD-Rom tray for accepting a CD, DVD, or mini-CD, or mini-DVD, in any of the commonly used readable, read-writable, and writable formats. Also consistent with the description herein is an input that can accept another form of medium, such as a floppy disc, flash memory such as memory stick, compact flash, smart data-card, or secure-data card, a pen-drive, portable USB-drive, zip-disk, and others. Such an input can also be configured to accept several different forms of media. Such an input 2022 is in communication with a processor (as described in connection with FIG. 3, though not shown in FIGS. 6A-E), that can read data from a computer-readable medium when properly inserted into the input.

FIG. 6E shows a plan view of a rear of system 2000. Shown are an air vent 2024, or letting surplus heat escape during an analysis. Typically, on the inside of system 2000, and by air vent 2024 and not shown in FIG. 6E, is a fan. Other ports shown in FIG. 6E are as follows: a power socket 2026 for accepting a power cord that will connect system 2000 to a supply of electricity; an ethernet connection 2028 for linking system 2000 to a computer network such as a local area network; an phone-jack connection 2032 for linking system 2000 to a communication network such as a telephone network; one or more USB ports 2030, for connecting system 2000 to one or more peripheral devices such as a printer, or a computer hard drive; an infra-red port for communicating with, e.g., a remote controller (not shown), to permit a user to control the system without using a touch-screen interface. For example, a user could remotely issue scheduling commands to system 2000 to cause it to start an analysis at a specific time in the future.

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 FIG. 6E are exemplary connections. It would be understood that various other features, including inputs, outputs, sockets, and connections, may be present on the rear face of system 2000, though not shown, or on other faces of system 2000.

An exploded view of an exemplary embodiment of the apparatus is shown in FIG. 7, particularly showing internal features of apparatus 2000. Apparatus 2000 can comprise a computer readable medium configured with hardware/firmware that can be employed to drive and monitor the operations on a cartridge used therewith, as well as software to interpret, communicate and store the results of a diagnostic test performed on a sample processed in the cartridge. Referring to FIG. 7, typical components of the apparatus 2000 are shown and include, for example, control electronics 2005, removable heater/sensor module 2020, detector 2009 such as a fluorescent detection module, display screen or optionally combined display and user interface 2006 (e.g., a medical grade touch sensitive liquid crystal display (LCD)). In some embodiments, lid 2010, detector 2009, and handle 2008 can be collectively referred to as slider module 2007. Additional components of apparatus 2000 may include one or more mechanical fixtures such as frame 2019 to hold the various modules (e.g., the heater/sensor module 2020, and/or the slider module 2007) in alignment, and for providing structural rigidity. Detector module 2009 can be placed in rails to facilitate opening and placement of cartridge 2060 in the apparatus 2000, and to facilitate alignment of the optics upon closing. Heater/sensor module 2020 can be also placed on rails for easy removal and insertion of the assembly.

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 FIG. 8, can include one or more functions in various embodiments, for example for, main control 900, multiplexing 902, display control 904, detector control 906, and the like. The main control function may serve as the hub of control electronics 840 in apparatus 2000 and can manage communication and control of the various electronic functions. The main control function can also support electrical and communications interface 908 with a user or an output device such as a printer 920, as well as optional diagnostic and safety functions. In conjunction with main control function 900, multiplexer function 902 can control sensor data 914 and output current 916 to help control heater/sensor module 2020. The display control function 904 can control output to and, if applicable, interpret input from touch screen LCD 846, which can thereby provide a graphical interface to the user in certain embodiments. The detector function 906 can be implemented in control electronics 840 using typical control and processing circuitry to collect, digitize, filter, and/or transmit the data from a detector 2009 such as one or more fluorescence detection modules.

Microfluidic Cartridge

The present technology comprises a microfluidic cartridge that is configured to carry out an amplification, such as by PCR, of one or more polynucleotides from one or more samples. It is to be understood that, unless specifically made clear to the contrary, where the term PCR is used herein, any variant of PCR including but not limited to real-time and quantitative, and any other form of polynucleotide amplification is intended to be encompassed. The microfluidic cartridge need not be self-contained and can be designed so that it receives thermal energy from one or more heating elements present in an external apparatus with which the cartridge is in thermal communication. An exemplary such apparatus is further described herein; additional embodiments of such a system are found in U.S. patent application Ser. No. 11/940,310, entitled “Microfluidic Cartridge and Method of Making Same”, and filed on even date herewith, the specification of which is incorporated herein by reference.

One aspect of the present technology relates to a microfluidic cartridge having two or more sample lanes arranged so that analyses can be carried out in two or more of the lanes in parallel, for example simultaneously, and wherein each lane is independently associated with a given sample.

A sample lane is an independently controllable set of elements by which a sample can be analyzed, according to methods described herein as well as others known in the art. A sample lane comprises at least a sample inlet, and a microfluidic network having one or more microfluidic components, as further described herein.

In various embodiments, a sample lane can include a sample inlet port or valve, and a microfluidic network that comprises, in fluidic communication one or more components selected from the group consisting of: at least one thermally actuated valve, a bubble removal vent, at least one thermally actuated pump, a gate, mixing channel, positioning element, microreactor, a downstream thermally actuated valve, and a PCR reaction chamber. The sample inlet valve can be configured to accept a sample at a pressure differential compared to ambient pressure of between about 70 and 100 kilopascals.

The cartridge can therefore include a plurality of microfluidic networks, each network having various components, and each network configured to carry out PCR on a sample in which the presence or absence of one or more polynucleotides is to be determined.

A multi-lane cartridge is configured to accept a number of samples in series or in parallel, simultaneously or consecutively, in particular embodiments 12 samples, wherein the samples include at least a first sample and a second sample, wherein the first sample and the second sample each contain one or more polynucleotides in a form suitable for amplification. The polynucleotides in question may be the same as, or different from one another, in different samples and hence in different lanes of the cartridge. The cartridge typically processes each sample by increasing the concentration of a polynucleotide to be determined and/or by reducing the concentration of inhibitors relative to the concentration of polynucleotide to be determined.

The multi-lane cartridge comprises at least a first sample lane having a first microfluidic network and a second lane having a second microfluidic network, wherein each of the first microfluidic network and the second microfluidic network is as elsewhere described herein, and wherein the first microfluidic network is configured to amplify polynucleotides in the first sample, and wherein the second microfluidic network is configured to amplify polynucleotides in the second sample.

In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to a sample mixture comprising PCR reagent and neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample.

At least the external heat source may operate under control of a computer processor, configured to execute computer readable instructions for operating one or more components of each sample lane, independently of one another, and for receiving signals from a detector that measures fluorescence from one or more of the PCR reaction chambers.

For example, FIG. 9 shows a plan view of a microfluidic cartridge 100 containing twelve independent sample lanes 101 capable of simultaneous or successive processing. The microfluidic network in each lane is typically configured to carry out amplification, such as by PCR, on a PCR-ready sample, such as one containing nucleic acid extracted from a sample using other methods as further described herein. A PCR-ready sample is thus typically a mixture comprising the PCR reagents and the neutralized polynucleotide sample, suitable for subjecting to thermal cycling conditions that create PCR amplicons from the neutralized polynucleotide sample. For example, a PCR-ready sample can include 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 is selective for a polynucleotide sequence. Exemplary probes are further described herein. Typically, the microfluidic network is configured to couple heat from an external heat source with the mixture comprising the PCR reagent 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 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 microfluidic cartridge can accommodate a negative control polynucleotide, wherein the microfluidic network can be configured to independently carry out PCR on each of a 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. Each lane of a multi-lane cartridge as described herein can perform two reactions when used in conjunction with two fluorescence detection systems per lane. A variety of combinations of reactions can be performed in the cartridge, such as two sample reactions in one lane, a positive control and a negative control in two other lanes; or a sample reaction and an internal control in one lane and a negative control in a separate lane.

FIG. 10A shows a perspective view of a portion of an exemplary microfluidic cartridge 200 according to the present technology. FIG. 10B shows a close-up view of a portion of the cartridge 200 of FIG. 10A illustrating various representative components. The cartridge 200 may be referred to as a multi-lane PCR cartridge with dedicated sample inlets 202. For example sample inlet 202 is configured to accept a liquid transfer member (not shown) such as a syringe, a pipette, or a PCR tube containing a PCR ready sample. More than one inlet 202 is shown in FIGS. 10A, 10B, wherein one inlet operates in conjunction with a single sample lane. Various components of microfluidic circuitry in each lane are also visible. For example, microvalves 204, and 206, and hydrophobic vents 208 for removing air bubbles, are parts of microfluidic circuitry in a given lane. Also shown is an ultrafast PCR reactor 210, which, as further described herein, is a microfluidic channel in a given sample lane that is long enough to permit PCR to amplify polynucleotides present in a sample. Above each PCR reactor 210 is a window 212 that permits detection of fluorescence from a fluorescent substance in PCR reactor 210 when a detector is situated above window 212. It is to be understood that other configurations of windows are possible including, but not limited to, a single window that straddles each PCR reactor across the width of cartridge 200.

In preferred embodiments, the multi-sample cartridge has a size substantially the same as that of a 96-well plate as is customarily used in the art. Advantageously, then, such a cartridge may be used with plate handlers used elsewhere in the art.

The sample inlets of adjacent lanes are reasonably spaced apart from one another to prevent any contamination of one sample inlet from another sample when a user introduces a sample into any one cartridge. In an embodiment, the sample inlets are configured so as to prevent subsequent inadvertent introduction of sample into a given lane after a sample has already been introduced into that lane. In certain embodiments, the multi-sample cartridge is designed so that a spacing between the centroids of sample inlets is 9 mm, which is an industry-recognized standard. This means that, in certain embodiments the center-to-center distance between inlet holes in the cartridge that accept samples from PCR tubes, as further described herein, is 9 mm. The inlet holes can be manufactured conical in shape with an appropriate conical angle so that industry-standard pipette tips (2 μl, 20 μl, 200 μl, volumes, etc.) fit snugly therein. The cartridge herein may be adapted to suit other, later-arising, industry standards not otherwise described herein, as would be understood by one of ordinary skill in the art.

In one embodiment, an exemplary microfluidic cartridge has 12 sample lanes. The inlet ports in this embodiment have a 6 mm spacing, so that, when used in conjunction with an automated sample loader having 4 heads, spaced equidistantly at 18 mm apart, the inlets can be loaded in three batches of four inlets: e.g., inlets 1, 4, 7, and 10 together, followed by 2, 5, 8, and 11, then finally 3, 6, 9, and 12, wherein the 12 inlets are numbered consecutively from one side of the cartridge to the other as shown.

A microfluidic cartridge as used herein may be constructed from a number of layers. Accordingly, one aspect of the present technology relates to a microfluidic cartridge that comprises a first, second, third, fourth, and fifth layers wherein one or more layers define a plurality of microfluidic networks, each network having various components configured to carry out PCR on a sample in which the presence or absence of one or more polynucleotides is to be determined. In various embodiments, one or more such layers are optional.

FIGS. 11A-C show various views of a layer structure of an exemplary microfluidic cartridge comprising a number of layers, as further described herein. FIG. 11A shows an exploded view; FIG. 11B shows a perspective view; and FIG. 11C shows a cross-sectional view of a sample lane in the exemplary cartridge. Referring to FIGS. 11A-C, an exemplary microfluidic cartridge 400 includes first 420, second 422, third 424, fourth 426, and fifth layers in two non-contiguous parts 428, 430 (as shown) that enclose a microfluidic network having various components configured to process multiple samples in parallel that include one or more polynucleotides to be determined.

Microfluidic cartridge 400 can be fabricated as desired. The cartridge can include a microfluidic substrate layer 424, typically injection molded out of a plastic, such as a zeonor plastic (cyclic olefin polymer), having a PCR channel and valve channels on a first side and vent channels and various inlet holes, including wax loading holes and liquid inlet holes, on a second side (disposed toward hydrophobic vent membrane 426). It is advantageous that all the microfluidic network defining structures, such as PCR reactors, valves, inlet holes, and air vents, are defined on the same single substrate 424. This attribute facilitates manufacture and assembly of the cartridge. Additionally, the material from which this substrate is formed is rigid or nondeformable, non-venting to air and other gases, and has a low autofluorescence to facilitate detection of polynucleotides during an amplification reaction performed in the microfluidic circuitry defined therein. Rigidity is advantageous because it facilitates effective and uniform contact with a heat unit as further described herein. Use of a non-venting material is also advantageous because it reduces the likelihood that the concentration of various species in liquid form will change during analysis. Use of a material having low auto-fluorescence is also important so that background fluorescence does not detract from measurement of fluorescence from the analyte of interest.

The cartridge can further include, disposed on top of the substrate 424, an oleophobic/hydrophobic vent membrane layer 426 of a porous material, such as 0.2 to 1.0 micron pore-size membrane of modified polytetrafluorethylene, the membrane being typically between about 25 and about 100 microns thick, and configured to cover the vent channels of microfluidic substrate 424, and attached thereto using, for example, heat bonding.

Typically, the microfluidic cartridge further includes a layer 428, 430 of polypropylene or other plastic label with pressure sensitive adhesive (typically between about 50 and 150 microns thick) configured to seal the wax loading holes of the valves in substrate 424, trap air used for valve actuation, and serve as a location for operator markings. In FIG. 4A, this layer is shown in two separate pieces, 428, 430, though it would be understood by one of ordinary skill in the art that a single piece layer would be appropriate.

In various embodiments, the label is 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. The label can be positioned such that it can be read by a sample identification verifier as further described herein.

The cartridge can further include a heat sealable laminate layer 422 (typically between about 100 and about 125 microns thick) attached to the bottom surface of the microfluidic substrate 424 using, for example, heat bonding. This layer serves to seal the PCR channels and vent channels in substrate 424. The cartridge can further include a thermal interface material layer 420 (typically about 125 microns thick), attached to the bottom of the heat sealable laminate layer using, for example, pressure sensitive adhesive. The layer 420 can be compressible and have a higher thermal conductivity than common plastics, thereby serving to transfer heat across the laminate more efficiently. Typically, however, layer 420 is not present.

The application of pressure to contact the cartridge to the heater of an instrument that receives the cartridge generally assists in achieving better thermal contact between the heater and the heat-receivable parts of the cartridge, and also prevents the bottom laminate structure from expanding, as would happen if the PCR channel was only partially filled with liquid and the air entrapped therein would be thermally expanded during thermocycling.

In use, cartridge 400 is typically thermally associated with an array of heat sources configured to operate the components (e.g., valves, gates, actuators, and processing region 410) of the device. Exemplary such heater arrays are further described herein. Additional embodiments of heater arrays are described in U.S. patent application Ser. No. 11/940,315, entitled “Heater Unit for Microfluidic Diagnostic System” and filed on even date herewith, the specification of which is incorporated herein by reference in its entirety. In some embodiments, the heat sources are controlled by a computer processor and actuated according to a desired protocol. Processors configured to operate microfluidic devices are described in, e.g., U.S. application Ser. No. 09/819,105, filed Mar. 28, 2001, which application is incorporated herein by reference.

In various embodiments, during transport and storage, the microfluidic cartridge can be further surrounded by a sealed pouch. The microfluidic cartridge can be sealed in the pouch with an inert gas. The microfluidic cartridge can be disposable for example after one or more of its sample lanes have been used.

Highly Multiplexed Embodiments

Embodiments of the cartridge described herein may be constructed that have high-density microfluidic circuitry on a single cartridge that thereby permit processing of multiple samples in parallel, or in sequence, on a single cartridge. Preferred numbers of such multiple samples include 20, 24, 36, 40, 48, 50, 60, 64, 72, 80, 84, 96, and 100, but it would be understood that still other numbers are consistent with the apparatus and cartridge herein, where deemed convenient and practical.

Accordingly, different configurations of lanes, sample inlets, and associated heater networks than those explicitly depicted in the FIGs and examples that can facilitate processing such numbers of samples on a single cartridge are within the scope of the instant disclosure. Similarly, alternative configurations of detectors and heating elements for use in conjunction with such a highly multiplexed cartridge are also within the scope of the description herein.

It is also to be understood that the microfluidic cartridges described herein are not to be limited to rectangular shapes, but can include cartridges having circular, elliptical, triangular, rhombohedral, square, and other shapes. Such shapes may also be adapted to include some irregularity, such as a cut-out, to facilitate placement in a complementary apparatus as further described herein.

In an exemplary embodiment, a highly multiplexed cartridge has 48 sample lanes, and permits independent control of each valve in each lane by suitably configured heater circuitry, with 2 banks of thermo cycling protocols per lane, as shown in FIG. 12. In the embodiment in FIG. 12, the heaters (shown superimposed on the lanes) are arranged in three arrays 502, 504, with 506, and 508. The heaters are themselves disposed within one or more substrates. Heater arrays 502, 508 in two separate glass regions only apply heat to valves in the microfluidic networks in each lane. Because of the low thermal conductivity of glass, the individual valves may be heated separately from one another. This permits samples to be loaded into the cartridge at different times, and passed to the PCR reaction chambers independently of one another. The PCR heaters 504,506 are mounted on a silicon substrate—and are not readily heated individually, but thereby permit batch processing of PCR samples, where multiple samples from different lanes are amplified by the same set of heating/cooling cycles. It is preferable for the PCR heaters to be arranged in 2 banks (the heater arrays 506 on the left and right 508 are not in electrical communication with one another), thereby permitting a separate degree of sample control.

FIG. 13 shows a representative 48-sample cartridge 600 compatible with the heater arrays of FIG. 12, and having a configuration of inlets 602 different to that depicted o other cartridges herein. The inlet configuration is exemplary and has been designed to maximize efficiency of space usage on the cartridge. The inlet configuration can be compatible with an automatic pipetting machine that has dispensing heads situated at a 9 mm spacing. For example, such a machine having 4 heads can load 4 inlets at once, in 12 discrete steps, for the cartridge of FIG. 13. Other configurations of inlets though not explicitly described or depicted are compatible with the technology described herein.

FIG. 14 shows, in close up, an exemplary spacing of valves 702, channels 704, and vents 796, in adjacent lanes 708 of a multi-sample microfluidic cartridge for example as shown in FIG. 13.

FIGS. 15 and 16 show close-ups of, respectively, heater arrays 804 compatible with, and inlets 902 on, the exemplary cartridge shown in FIG. 14.

FIGS. 17A and 17B show various views of an embodiment of a radially-configured highly-multiplexed cartridge, having a number of inlets 1002, microfluidic lanes 1004, valves 1005, and PCR reaction chambers 1006. FIG. 17C shows an array of heater elements 1008 compatible with the cartridge layout of FIG. 17A.

The various embodiments shown in FIGS. 12-17C are compatible with liquid dispensers, receiving bays, and detectors that are configured differently from the other specific examples described herein.

During the design and manufacture of highly multiplexed cartridges, photolithographic processing steps such as etching, hole drilling/photo-chemical drilling/sand-blasting/ion-milling processes should be optimized to give well defined holes and microchannel pattern. Proper distances between channels should be identified and maintained to obtain good bonding between the microchannel substate and the heat conducting substrate layer. In particular, it is desirable that minimal distances are maintained between pairs of adjacent microchannels to promote, reliable bonding of the laminate in between the channels.

The fabrication by injection molding of these complicated microfluidic structures having multiple channels and multiple inlet holes entails proper consideration of dimensional repeatability of these structures over multiple shots from the injection molding master pattern. Proper consideration is also attached to the placement of ejector pins to push out the structure from the mold without causing warp, bend or stretching of it. For example, impression of the ejector pins on the microfluidic substrate should not sink into the substrate thereby preventing planarity of the surface of the cartridge. The accurate placement of various inlet holes (such as sample inlet holes, valve inlet holes and vent holes) relative to adjacent microfluidic channels is also important because the presence of these holes can cause knit-lines to form that might cause unintended leak from a hole to a microchannel. Highly multiplexed microfluidic substrates may be fabricated in other materials such as glass, silicon.

The size of the substrate relative to the number of holes is also factor during fabrication because it is easy to make a substrate having just a simple microfluidic network with a few holes (maybe fewer than 10 holes) and a few microchannels, but making a substrate having over 24, or over 48, or over 72 holes, etc., is more difficult.

Microfluidic Networks

Particular components of exemplary microfluidic networks are further described herein.

Channels of a microfluidic network in a lane of cartridge typically have at least one sub-millimeter cross-sectional dimension. For example, channels of such a network 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).

FIG. 18 shows a plan view of a representative microfluidic circuit found in one lane of a multi-lane cartridge such as shown in FIGS. 10A and 10B. It would be understood by one skilled in the art that other configurations of microfluidic network would be consistent with the function of the cartridges and apparatus described herein. In operation of the cartridge, in sequence, sample is introduced through liquid inlet 202, optionally flows into a bubble removal vent channel 208 (which permits adventitious air bubbles introduced into the sample during entry, to escape), and continues along a channel 216. Typically, when using a robotic dispenser of liquid sample, the volume is dispensed accurately enough that formation of bubbles is not a significant problem, and the presence of vent channel 208 is not necessary. Thus, in certain embodiments, the bubble removal vent channel 208 is not present and sample flows directly into channel 216. Throughout the operation of cartridge 200, the fluid is manipulated as a microdroplet (not shown in the FIGs). Valves 204 and 206 are initially both open, so that a microdroplet of sample-containing fluid can be pumped into PCR reactor channel 210 from inlet hole 202 under influence of force from the sample injection operation. Upon initiating of processing, the detector present on top of the PCR reactor 210 checks for the presence of liquid in the PCR channel, and then valves 204 and 206 are closed to isolate the PCR reaction mix from the outside. In one embodiment, the checking of the presence of liquid in the PCR channel is by measuring the heat ramp rate, such as by one or more temperature sensors in the heating unit. A channel with liquid absent will heat up faster than one in which, e.g., a sample, is present.

Both valves 204 and 206 are closed prior to thermocycling to prevent or reduce any evaporation of liquid, bubble generation, or movement of fluid from the PCR reactor. End vent 214 is configured to prevent a user from introducing an excess amount of liquid into the microfluidic cartridge, as well as playing a role of containing any sample from spilling over to unintended parts of the cartridge. A user may input sample volumes as small as an amount to fill the region from the bubble removal vent (if present) to the middle of the microreactor, or up to valve 204 or beyond valve 204. The use of microvalves prevents both loss of liquid or vapor thereby enabling even a partially filled reactor to successfully complete a PCR thermocycling reaction.

The reactor 210 is a microfluidic channel that is heated through a series of cycles to carry out amplification of nucleotides in the sample, as further described herein, and according to amplification protocols known to those of ordinary skill in the art. The inside walls of the channel in the PCR reactor are typically made very smooth and polished to a shiny finish (for example, using a polish selected from SPI A1, SPI A2, SPI A3, SPI B1, or SPI B2) during manufacture. This is in order to minimize any microscopic quantities of air trapped in the surface of the PCR channel, which would causing bubbling during the thermocycling steps. The presence of bubbles especially in the detection region of the PCR channel could also cause a false or inaccurate reading while monitoring progress of the PCR. Additionally, the PCR channel can be made shallow such that the temperature gradient across the depth of the channel is minimized.

The region of the cartridge 212 above PCR reactor 210 is a thinned down section to reduce thermal mass and autofluorescence from plastic in the cartridge. It permits a detector to more reliably monitor progress of the reaction and also to detect fluorescence from a probe that binds to a quantity of amplified nucleotide. Exemplary probes are further described herein. The region 212 can be made of thinner material than the rest of the cartridge so as to permit the PCR channel to be more responsive to a heating cycle (for example, to rapidly heat and cool between temperatures appropriate for denaturing and annealing steps), and so as to reduce glare, autofluorescence, and undue absorption of fluorescence.

After PCR has been carried out on a sample, and presence or absence of a polynucleotide of interest has been determined, it is preferred that the amplified sample remains in the cartridge and that the cartridge is either used again (if one or more lanes remain unused), or disposed of Should a user wish to run a post amplification analysis, such as gel electrophoresis, the user may pierce a hole through the laminate of the cartridge, and recover an amount—typically about 1.5 microliter—of PCR product. The user may also place the individual PCR lane on a special narrow heated plate, maintained at a temperature to melt the wax in the valve, and then aspirate the reacted sample from the inlet hole of that PCR lane.

In various embodiments, the microfluidic network can optionally include at least one reservoir configured to contain waste.

Table 1 outlines typical volumes, pumping pressures, and operation times associated with various components of a microfluidic cartridge described herein.

TABLE 1

Pumping
Displacement
Time of

Operation
Pressure
Volume
Operation

Moving valve wax
~1-2 psi
<1 μl
5-15 seconds

plugs

Operation
Pump Used
Pump Design
Pump Actuation

Moving valve
Thermopneumatic
1 μl of
Heat trapped air to

wax plugs
pump
trapped air
~70-90 C.

Valves

A valve (sometimes referred to herein as a microvalve) is a component in communication with a channel, such that the valve 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 of the valve, 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 one embodiment, a valve can include a mass of a thermally responsive substance (TRS) that is relatively immobile at a first temperature and more mobile at a second temperature. The first and second temperatures are insufficiently high to damage materials, such as polymer layers of a microfluidic cartridge in which the valve is situated. A mass of TRS can be an essentially solid mass or an agglomeration of smaller particles that cooperate to obstruct the passage when the valve is closed. Examples of TRS's include a eutectic alloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, and combinations thereof. The TRS can also be a blend of variety of materials, such as an emulsion of thermoelastic polymer blended with air microbubbles (to enable higher thermal expansion, as well as reversible expansion and contraction), polymer blended with expancel material (offering higher thermal expansion), polymer blended with heat conducting microspheres (offering faster heat conduction and hence, faster melting profiles), or a polymer blended with magnetic microspheres (to permit magnetic actuation of the melted thermoresponsive material).

Generally, for such a valve, the second temperature is less than about 90° C. and the first temperature is less than the second temperature (e.g., about 70° C. or less). Typically, a chamber is in gaseous communication with the mass of TRS. The valve is in communication with a source of heat that can be selectively applied to the chamber of air and to the TRS. Upon heating gas (e.g., air) in the chamber and heating the mass of TRS to the second temperature, gas pressure within the chamber due to expansion of the volume of gas, forces the mass to move into the channel, thereby obstructing material from passing therealong.

An exemplary valve is shown in FIG. 19A. The valve of FIG. 19A has two chambers of air 1203, 1205 in contact with, respectively, each of two channels 1207, 1208 containing TRS. The air chambers also serve as loading ports for TRS during manufacture of the valve, as further described herein. In order to make the valve sealing very robust and reliable, the flow channel 1201 (along which, e.g., sample passes) at the valve junction is made narrow (typically 150 μm wide, and 150 μm deep or narrower), and the constricted portion of the flow channel is made at least 0.5 or 1 mm long such that the TRS seals up a long narrow channel thereby reducing any leakage through the walls of the channel. In the case of a bad seal, there may be leakage of fluid around walls of channel, past the TRS, when the valve is in the closed state. In order to minimize this, the flow channel is narrowed and elongated as much as possible. In order to accommodate such a length of channel on a cartridge where space may be at a premium, the flow channel can incorporate one or more curves 1209 as shown in FIG. 19A. The valve operates by heating air in the TRS-loading port, which forces the TRS forwards into the flow-channel in a manner so that it does not come back to its original position. In this way, both air and TRS are heated during operation.

In various other embodiments, a valve for use with a microfluidic network in a microfluidic cartridge herein can be a bent valve as shown in FIG. 19B. Such a configuration reduces the footprint of the valve and hence reduces cost per part for highly dense microfluidic cartridges. A single valve loading hole 1211 is positioned in the center, that serves as an inlet for thermally responsive substance. The leftmost vent 1213 can be configured to be an inlet for, e.g., sample, and the rightmost vent 1215 acts as an exit for, e.g., air. This configuration can be used as a prototype for testing such attributes as valve and channel geometry and materials.

In various other embodiments, a valve for use with a microfluidic network can include a curved valve as shown in FIG. 19C, in order to reduce the effective cross-section of the valve, thereby enabling manufacture of cheaper dense microfluidic devices. Such a valve can function with a single valve loading hole and air chamber 1221 instead of a pair as shown in FIG. 19A.

Gates

FIG. 19D shows an exemplary gate as may optionally be used in a microfluidic network herein. 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).

In various embodiments, a microfluidic network can include a narrow gate 380 as shown in FIG. 19D where a gate loading channel 382 used for loading wax from a wax loading hole 384 to a gate junction 386 can be narrower (e.g., approximately 150 μm wide and 100 microns deep). An upstream channel 388 as well as a downstream channel 390 of the gate junction 386 can be made wide (e.g., ˜500 μm) and deep (e.g., ˜500 μm) to help ensure the wax stops at the gate junction 386. The amount of gate material melted and moved out of the gate junction 386 may be minimized for optimal gate 380 opening. As an off-cartridge heater may be used to melt the thermally responsive substance in gate 380, a misalignment of the heater could cause the wax in the gate loading channel 382 to be melted as well. Therefore, narrowing the dimension of the loading channel may increase reliability of gate opening. In the case of excessive amounts of wax melted at the gate junction 386 and gate loading channel 382, the increased cross-sectional area of the downstream channel 390 adjacent to the gate junction 386 can prevent wax from clogging the downstream channel 390 during gate 380 opening. The dimensions of the upstream channel 388 at the gate junction 386 can be made similar to the downstream channel 390 to ensure correct wax loading during gate fabrication.

In various embodiments, the gate can be configured to minimize the effective area or footprint of the gate within the network and thus bent gate configurations, although not shown herein are consistent with the foregoing description.

Vents

In various embodiments, the microfluidic network can include at least one hydrophobic vent in addition to an end vent. A vent is a general outlet (hole) that may or may not be covered with a hydrophobic membrane. An exit hole is an example of a vent that need not be covered by a membrane.

A hydrophobic vent (e.g., a vent in FIG. 20) is a structure that permits gas to exit a channel while limiting (e.g., preventing) quantities of 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 GE Osmonics, Minnetonka, Minn.) that defines a wall of the channel. As described elsewhere herein, hydrophobic vents can be used to position a microdroplet of sample at a desired location within a microfluidic network.

The hydrophobic vents of the present technology are preferably constructed so that the amount of air that escapes through them is maximized while minimizing the volume of the channel below the vent surface. Accordingly, it is preferable that the vent is constructed so as to have a hydrophobic membrane 1303 of large surface area and a shallow cross section of the microchannel below the vent surface.

Hydrophobic vents are useful for bubble removal and 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 1305 (see FIG. 13). The length of the hydrophobic vent is typically 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 is 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 is typically about 75% or less (e.g., about 65% or less, about 60% or less) of the depth of the channel upstream 1301 and downstream (not shown) of the hydrophobic vent. For example, in some embodiments the channel depth within the hydrophobic vent is about 150 microns and the channel depth upstream and downstream of the hydrophobic vent is about 250 microns. Other dimensions are consistent with the description herein.

A width of the channel within the hydrophobic vent is typically 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 is about 400 microns, and the width of the channel upstream and downstream from the vent is about 250 microns. Other dimensions are consistent with the description herein.

The vent in FIG. 20 is shown in a linear configuration though it would be understood that it need not be so. A bent, kinked, curved, S-shaped, V-shaped, or U-shaped (as in item 208FIG. 11) vent is also consistent with the manner of construction and operation described herein.

Use of Cutaways in Cartridge and Substrate to Improve Rate of Cooling During PCR Cycling

During a PCR amplification of a nucleotide sample, a number of thermal cycles are carried out. For improved efficiency, the cooling between each application of heat is preferably as rapid as possible. Improved rate of cooling can be achieved with various modifications to the heating substrate and/or the cartridge, as shown in FIG. 21.

One way to achieve rapid cooling is to cutaway portions of the microfluidic cartridge substrate, as shown in FIG. 22A. The upper panel of FIG. 22A is a cross-section of an exemplary microfluidic cartridge taken along the dashed line A-A′ as marked on the lower panel of FIG. 22A. PCR reaction chamber 1601, and representative heaters 1603 are shown. Also shown are two cutaway portions, one of which labeled 1601, that are situated alongside the heaters that are positioned along the long side of the PCR reaction chamber. Cutaway portions such as 1601 reduce the thermal mass of the cartridge, and also permit air to circulate within the cutaway portions. Both of these aspects permit heat to be conducted away quickly from the immediate vicinity of the PCR reaction chamber. Other configurations of cutouts, such as in shape, position, and number, are consistent with the present technology.

Another way to achieve rapid cooling is to cutaway portions of the heater substrate, as shown in FIG. 22B. The lower panel of FIG. 22B is a cross-section of an exemplary microfluidic cartridge and heater substrate taken along the dashed line A-A′ as marked on the upper panel of FIG. 22B. PCR reaction chamber 901, and representative heaters 1003 are shown. Also shown are four cutaway portions, one of which labeled 1205, that are situated alongside the heaters that are situated along the long side of the PCR reaction chamber. Cutaway portions such as 1205 reduce the thermal mass of the heater substrate, and also permit air to circulate within the cutaway portions. Both of these aspects permit heat to be conducted away quickly from the immediate vicinity of the PCR reaction chamber. Four separate cutaway portions are shown in FIG. 22A so that control circuitry to the various heaters is not disrupted. Other configurations of cutouts, such as in shape, position, and number, are consistent with the present technology. These cutouts may be created by a method selected from: selective etching using wet etching processes, deep reactive ion etching, selective etching using CO₂laser or femtosecond laser (to prevent surface cracks or stress near the surface), selective mechanical drilling, selective ultrasonic drilling, or selective abrasive particle blasting. Care has to be taken to maintain mechanically integrity of the heater while reducing as much material as possible.

FIG. 22C shows a combination of cutouts and use of ambient air cooling to increase the cooling rate during the cooling stage of thermocycling. A substantial amount of cooling happens by convective loss from the bottom surface of the heater surface to ambient air. The driving force for this convective loss is the differential in temperatures between the glass surface and the air temperature. By decreasing the ambient air temperature by use of, for example, a peltier cooler, the rate of cooling can be increased. The convective heat loss may also be increased by keeping the air at a velocity higher than zero.

An example of thermal cycling performance in a PCR reaction chamber obtained with a configuration as described herein, is shown in FIG. 23 for a protocol that is set to heat up the reaction mixture to 92° C., and maintain the temperature for 1 second, then cool to 62° C., and stay for 10 seconds. The cycle time shown is about 29 seconds, with 8 seconds required to heat from 62° C. and stabilize at 92° C., and 10 seconds required to cool from 92° C., and stabilize at 62° C. To minimize the overall time required for a PCR effective to produce detectable quantities of amplified material, it is important to minimize the time required for each cycle. Cycle times in the range 15-30 s, such as 18-25 s, and 20-22 s, are desirable. In general, an average PCR cycle time of 25 seconds as well as cycle times as low as 20 seconds are typical with the technology described herein. Using reaction volumes less than a microliter (such as a few hundred nanoliters or less) permits use of an associated smaller PCR chamber, and enables cycle times as low as 15 seconds. An average cycle time of 25 seconds and as low as 20 seconds can be achieved by technology described herein, even without any forced cooling or implementing any thermal mass reductions described elsewhere herein.

Manufacturing Process for Cartridge

FIG. 24 shows a flow-chart 1800 for an embodiment of an assembly process for an exemplary cartridge as shown in FIG. 11A herein. It would be understood by one of ordinary skill in the art, both that various steps may be performed in a different order from the order set forth in FIG. 24, and additionally that any given step may be carried out by alternative methods to those described in the figure. It would also be understood that, where separate serial steps are illustrated for carrying out two or more functions, such functions may be performed synchronously and combined into single steps and remain consistent with the overall process described herein.

At 1802, a laminate layer is applied to a microfluidic substrate that has previously been engineered, for example by injection molding, to have a microfluidic network constructed in it; edges are trimmed from the laminate where they spill over the bounds of the substrate.

At 1804, wax is dispensed and loaded into the microvalves of the microfluidic network in the microfluidic substrate. An exemplary process for carrying this out is further described herein.

At 1806, the substrate is inspected to ensure that wax from step 1804 is loaded properly and that the laminate from step 1802 adheres properly to it. If a substrate does not satisfy either or both of these tests, it is usually discarded. If substrates repeatedly fail either or both of these tests, then the wax dispensing, or laminate application steps, as applicable, are reviewed.

At 1808, a hydrophobic vent membrane is applied to, and heat bonded to, the top of the microfluidic substrate covering at least the one or more vent holes, and on the opposite face of the substrate from the laminate. Edges of the membrane that are in excess of the boundary of the substrate are trimmed.

At 1810, the assembly is inspected to ensure that the hydrophobic vent membrane is bonded well to the microfluidic substrate without heat-clogging the microfluidic channels. If any of the channels is blocked, or if the bond between the membrane and the substrate is imperfect, the assembly is discarded, and, in the case of repeated discard events, the foregoing process step 1808 is reviewed.

At 1812, optionally, a thermally conductive pad layer is applied to the bottom laminate of the cartridge.

At 1814, two label strips are applied to the top of the microfluidic substrate, one to cover the valves, and a second to protect the vent membranes. It would be understood that a single label strip may be devised to fulfill both of these roles.

At 1816, additional labels are printed or applied to show identifying characteristics, such as a barcode #, lot # and expiry date on the cartridge. Preferably one or more of these labels has a space and a writable surface that permits a user to make an identifying annotation on the label, by hand.

Optionally, at 1818, to facilitate transport and delivery to a customer, assembled and labeled cartridges are stacked, and cartridges packed into groups, such as groups of 25, or groups of 10, or groups of 20, or groups of 48 or 50. Preferably the packaging is via an inert and/or moisture-free medium.

Wax Loading in Valves

In general, a valve as shown in, e.g., FIGS. 25A-C, is constructed by depositing a precisely controlled amount of a TRS (such as wax) into a loading inlet machined in the microfluidic substrate. FIGS. 25A and 25B show how a combination of controlled hot drop dispensing into a heated microchannel device of the right dimensions and geometry is used to accurately load wax into a microchannel of a microfluidic cartridge to form a valve. The top of FIG. 25A shows a plan view of a valve inlet 190 and loading channel 1902, connecting to a flow channel 1904. The lower portions of FIG. 25A show the progression of a dispensed wax droplet 1906 (having a volume of 75 nl±15 nl) through the inlet 1901 and into the loading channel 1902.

To accomplish those steps, a heated dispenser head can be accurately positioned over the inlet hole of the micro channel in the microfluidic device, and can dispense molten wax drops in volumes as small as 75 nanoliters with an accuracy of 20%. A suitable dispenser is also one that can deposit amounts smaller than 100 nl with a precision of +/−20%. The dispenser should also be capable of heating and maintaining the dispensing temperature of the TRS to be dispensed. For example, it may have a reservoir to hold the solution of TRS. It is also desirable that the dispense head can have freedom of movement at least in a horizontal (x-y) plane so that it can easily move to various locations of a microfluidic substrate and dispense volumes of TRS into valve inlets at such locations without having to be re-set, repositioned manually, or recalibrated in between each dispense operation.

The inlet hole of the microfluidic cartridge, or other microchannel device, is dimensioned in such a way that the droplet of 75 nl can be accurately propelled to the bottom of the inlet hole using, for example, compressed air, or in a manner similar to an inkjet printing method. The microfluidic cartridge is maintained at a temperature above the melting point of the wax thereby permitting the wax to stay in a molten state immediately after it is dispensed. After the drop falls to the bottom of the inlet hole 1901, the molten wax is drawn into the narrow channel by capillary action, as shown in the sequence of views in FIG. 25B. A shoulder between the inlet hole 1901 and the loading channel can facilitate motion of the TRS. The volume of the narrow section can be designed to be approximately equal to a maximum typical amount that is dispensed into the inlet hole. The narrow section can also be designed so that even though the wax dispensed may vary considerably between a minimum and a maximum shot size, the wax always fills up to, and stops at, the micro channel junction 1907 because the T-junction provides a higher cross section than that of the narrow section and thus reduces the capillary forces.

PCR Reagent Mixtures

In various embodiments, the sample for introduction into a lane of the microfluidic cartridge can include a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides.

In various embodiments, preparation of a PCR-ready sample for use with an apparatus and cartridge as described herein can include contacting a 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).

The PCR-ready sample can be prepared, for example, using the following steps: (1) collect sample in sample collection buffer, (2) transfer entire sample to lysis tube, mix, heat, and incubate for seven minutes, (3) place on magnetic rack, allow beads to separate, aspirate supernatant, (4) add 100 μl of Buffer 1, mix, place on magnetic rack, allow beads to separate, aspirate supernatant, (5) add 10 μl of Buffer 2, mix, place in high temperature heat block for 3 minutes, place on magnetic rack, allow beads to separate, transfer 5 μl supernatant, and (6) Add 5 μl of Buffer 3, transfer 1 to 10 μl of supernatant for PCR amplification and detection.

The PCR reagent mixture can be in the form of one or more lyophilized pellets and the steps by which the PCR-ready sample is prepared can involve reconstituting the PCR pellet by contacting it with liquid to create a PCR reagent mixture solution. In yet another embodiment, each of the PCR lanes may have dried down or lyophilized ASR reagents preloaded such that the user only needs to input prepared polynucleotide sample into the PCR. In another embodiment, the PCR lanes may have only the application-specific probes and primers pre-measured and pre-loaded, and the user inputs a sample mixed with the PCR reagents.

In various embodiments, the PCR-ready sample can include at least one probe that can be selective for a polynucleotide sequence, wherein the steps by which the PCR-ready sample is prepared involve contacting 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.

In various embodiments, the PCR-ready sample further includes a sample buffer.

In various embodiments, the PCR-ready sample includes at least one probe that is 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 various embodiments, the PCR reagent mixture can further include a polymerase enzyme, a positive control plasmid and a fluorogenic hybridization probe selective for at least a portion of the plasmid.

In various embodiments, the probe can be selective for a polynucleotide sequence that is 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, 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 is 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 is 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 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. coliO157: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 Stenotrophomonas 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 is characteristic of an organisms 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.

In various embodiments, a method of carrying out PCR on a sample can further include one or more of the following steps: 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 method for sampling a polynucleotide can include the steps of: placing a microfluidic cartridge containing a PCR-ready sample in a receiving bay of a suitably configured apparatus; carrying out PCR on the sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide in the sample, the PCR-ready sample 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 the at least one fluorogenic probe that is selective for a polynucleotide sequence, wherein the probe is selective for a polynucleotide sequence that is 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, the presence of the organism for which the one fluorogenic probe is selective is determined.

Carrying out PCR on a PCR-ready sample can additionally include: 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, a method of using the apparatus and cartridge described herein can further include one or more of the following steps: 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 that the sample was contaminated 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 that a PCR amplification has occurred if the plasmid probe is detected.

Kit

In various embodiments, the microfluidic cartridge as described herein can be provided in the form of a kit, wherein the kit can include a microfluidic cartridge, and a liquid transfer member (such as a syringe or a pipette). In various embodiments, the kit can further include instructions to employ the liquid transfer member to transfer a sample containing extracted nucleic acid from a sample container via a sample inlet to the microfluidic network on the microfluidic cartridge. In some embodiments, the microfluidic cartridge and the liquid transfer member can be sealed in a pouch with an inert gas.

Typically when transferring a sample from liquid dispenser, such as a pipette tip, to an inlet on the microfluidic cartridge, a volume of air is simultaneously introduced into the microfluidic network, the volume of air being between about 0.5 mL and about 5 mL. Presence of air in the microfluidic network, however, is not essential to operation of the cartridge described herein.

In various embodiments, the kit can further include at least one computer-readable label on the cartridge. The label can include, for example, a bar code, a radio frequency tag or one or more computer-readable characters. When used in conjunction with a similar computer-readable label on a sample container, such as a vial or a pouch, matching of diagnostic results with sample is thereby facilitated.

In some embodiments, a sample identifier of the apparatus described elsewhere herein is employed to read a label on the microfluidic cartridge and/or a label on the biological sample.

Heater Unit

An exemplary heater unit 2020 is shown in FIG. 26. The unit is configured to deliver localized heat to various selected regions of a cartridge received in a receiving bay 2014. Heater unit 2020 is configured to be disposed within a diagnostic apparatus during operation, as further described herein, and in certain embodiments is removable from that apparatus, for example to facilitate cleaning, or to permit reconfiguration of the heater circuitry. In various embodiments, heater unit 2020 can be specific to particular designs of microfluidic networks and microfluidic substrate layouts.

Shown in FIG. 26 is a heater unit having a recessed surface 2044 that provides a platform for supporting a microfluidic cartridge when in receiving bay 2014. In one embodiment, the cartridge rests directly on surface 2044. Surface 2044 is shown as recessed, in FIG. 2, but need not be so and, for example, may be raised or may be flush with the surrounding area of the heater unit. Surface 2044 is typically a layer of material that overlies a heater chip or board, or a heater substrate, that contains heater micro-circuitry configured to selectively and specifically heat regions of a microfluidic substrate, such as in a cartridge, in the receiving bay 2014.

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 FIG. 26 as an exemplary mechanical key 2045 is a diagonally cutout corner of area 2044 into which a complementarily cutoff corner of a microfluidic cartridge fits. Other registration members are consistent with the heater unit described herein, for example, a feature engineered on one or more edges of a cartridge including but not limited to: several, such as two or more, cut-out corners, one or more notches cut into one or more edges of the cartridge; or one or more protrusions fabricated into one or more edges of the cartridge. Alternative registration members include one or more lugs or bumps engineered into an underside of a cartridge, complementary to one or more recessed sockets or holes in surface 2044 (not shown in the embodiment of FIG. 26). Alternative registration members include one or more recessed sockets or holes engineered into an underside of a cartridge, complementary to one or more lugs or bumps on surface 2044. In general, the pattern of features is such that the cartridge possesses at least one element of asymmetry so that it can only be inserted in a single orientation into the receiving bay.

Also shown in FIG. 26 is a hand-grasp 2042 that facilitates removal and insertion of the heater unit into an apparatus by a user. Cutaway 2048 permits a user to easily remove a cartridge from receiving bay 2014 after a processing run where, e.g., a user's thumb or finger when grabbing the top of the cartridge, is afforded comfort space by cutaway 2048. Both cutaways 2042 and 2048 are shown as semicircular recesses in the embodiment of FIG. 26, but it would be understood that they are not so limited in shape. Thus, rectangular, square, triangular, half-oval, contoured, and other shaped recesses are also consistent with a heater unit as described herein.

In the embodiment of FIG. 26, which is designed to be compatible with an exemplary apparatus as further described herein, the front of the heater unit is at the left of the figure. At the rear of heater unit 2020 is an electrical connection 2050, such as an RS-232 connection, that permits electrical signals to be directed to heaters located at specific regions of area 2044 during sample processing and analysis, as further described herein. Thus, underneath area 2044 and not shown in FIG. 2 can be an array of heat sources, such as resistive heaters, that are configured to align with specified locations of a microfluidic cartridge properly inserted into the receiving bay. Surface 2044 is able to be cleaned periodically, for example with common cleaning agents (e.g., a 10% bleach solution), to ensure that any liquid spills that may occur during sample handling do not cause any short circuiting. Such cleaning can be carried out frequently when the heater unit is disposed in a diagnostic apparatus, and less frequently but more thoroughly when the unit is removed.

Other non-essential features of heater unit 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 unit 2020, to permit excess heat to escape, when heaters underneath receiving bay 2014, are in operation. The configuration of air vents in FIG. 26, as a linear array of square vents, is exemplary and it would be understood that other numbers and shapes thereof are consistent with routine fabrication and use of a heater unit. For example, although 5 square air vents are shown, other numbers such as 1, 2, 3, 4, 6, 8, or 10 air vents are possible, arranged on one side, or spread over two or more sides and/or faces of the heater unit. In further embodiments, air vents may be circular, rectangular, oval, triangular, polygonal, and having curved or squared vertices, or still other shapes, including irregular shapes. In further embodiments two or more vents need not be disposed in a line, parallel with one another and with an edge of the heater unit but may be disposed offset from one another.

Heater unit 2020 may further comprise one or more guiding members 2047 that facilitate inserting the heater unit into an apparatus as further described herein, for an embodiment in which heater unit 2020 is removable by a user. Heater unit 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 unit 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 2047 may perform one or more roles of ensuring that the heater unit is aligned correctly in the apparatus, and ensuring that the heater unit 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 FIG. 26 are on either side of receiving bay 2044 and stretch along a substantial fraction of the length of unit 2020, but such an arrangement of guiding members is exemplary. Other guiding members are consistent with use herein, and include but are not limited to other numbers of guiding members such as 1, 3, 4, 5, 6, or 8, and other positions thereof, including positioned in area 2051 of unit 2020, and need not stretch along as much of the length of unit 2020 as shown in FIG. 26, or may stretch along its entire length. Guiding members 2047 are shown having a non-constant thickness along their lengths. It is consistent herein that other guiding members may have essentially constant thickness along their lengths. At the end of the heater unit that is inserted into an apparatus, in the embodiment shown, the edges are beveled to facilitate proper placement.

Also shown in FIG. 26 is an optional region of fluorescent material, such as optically fluorescent material 2069, on area 2051 of heater unit 2020. The region of fluorescent material is configured to be detected by a detection system further described herein. The region 2069 is used for verifying the state of optics in the detection system prior to sample processing and analysis and therefore acts as a control, or a standard. For example, in one embodiment a lid of the apparatus in which the heater unit is disposed, when in an open position, permits ambient light to reach region 2069 and thereby cause the fluorescent material to emit a characteristic frequency or spectrum of light that can be measured by the detector for, e.g., standardization or calibration purposes. In another embodiment, instead of relying on ambient light to cause the fluorescent material to fluoresce, light source from the detection system itself, such as one or more LED's, is used to shine on region 2069. The region 2069 is therefore positioned to align with a position of a detector. Region 2069 is shown as rectangular, but may be configured in other shapes such as square, circular, elliptical, triangular, polygonal, and having curved or squared vertices. It is also to be understood that the region 2069 may be situated at other places on the heater unit 2020, according to convenience and in order to be complementary to the detection system deployed.

In particular and not shown in FIG. 26, heater/sensor unit 2020 can include, for example, a multiplexing function in a discrete multiplexing circuit board (MUX board), one or more heaters (e.g., a microheater), one or more temperature sensors (optionally combined together as a single heater/sensor unit with one or more respective microheaters, e.g., as photolithographically fabricated on fused silica substrates). The micro-heaters can provide thermal energy that can actuate various microfluidic components on a suitably positioned microfluidic cartridge. A sensor (e.g., as a resistive temperature detector (RTD)) can enable real time monitoring of the micro-heaters, for example through a feedback based mechanism to allow for rapid and accurate control of the temperature. One or more microheaters can be aligned with corresponding microfluidic components (e.g., valves, pumps, gates, reaction chambers) to be heated on a suitably positioned microfluidic cartridge. A microheater can be designed to be slightly bigger than the corresponding microfluidic component(s) on the microfluidic cartridge so that even though the cartridge may be slightly misaligned, such as off-centered, from the heater, the individual components can be heated effectively.

Heater Configurations to Ensure Uniform Heating of a Region

The microfluidic substrates described herein are configured to accept heat from a contact heat source, such as found in a heater unit described herein. The heater unit typically comprises a heater board or heater chip that is configured to deliver heat to specific regions of the microfluidic substrate, including but not limited to one or more microfluidic components, at specific times. For example, the heat source is configured so that particular heating elements are situated adjacent to specific components of the microfluidic network on the substrate. In certain embodiments, the apparatus uniformly controls the heating of a region of a microfluidic network. In an exemplary embodiment, multiple heaters can be configured to simultaneously and uniformly heat a region, such as the PCR reaction chamber, of the microfluidic substrate. The term heater unit, as used herein, may be used interchangeably to describe either the heater board or an item such as shown in FIG. 26.

Referring to FIGS. 27A and 27B, an exemplary set of heaters configured to heat, cyclically, PCR reaction chamber 1001 is shown. It is to be understood that heater configurations to actuate other regions of a microfluidic cartridge such as other gates, valves, and actuators, may be designed and deployed according to similar principles to those governing the heaters shown in FIGS. 27A and 27B.

Referring to FIGS. 27A and 27B, an exemplary PCR reaction chamber 1001 in a microfluidic substrate, typically a chamber or channel having a volume ˜1.6 μl, is configured with a long side and a short side, each with an associated heating element. A PCR reaction chamber may also be referred to as a PCR reactor, herein, and the region of a cartridge in which the reaction chamber is situated may be called a zone. The heater substrate therefore includes four heaters disposed along the sides of, and configured to heat, a given PCR reaction chamber, as shown in the exemplary embodiment of FIG. 27A: long top heater 1005, long bottom heater 1003, short left heater 1007, and short right heater 1009. The small gap between long top heater 1005 and long bottom heater 1003 results in a negligible temperature gradient (less than 1° C. difference across the width of the PCR channel at any point along the length of the PCR reaction chamber) and therefore an effectively uniform temperature throughout the PCR reaction chamber. The heaters on the short edges of the PCR reactor provide heat to counteract the gradient created by the two long heaters from the center of the reactor to the edge of the reactor.

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 chamber are consistent with the methods and apparatus described herein. For example, a ‘long’ side of the reaction chamber 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, polyimide, FR4, ceramic, 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 chamber are similarly applicable to the arrangement of heaters adjacent to other components of the microfluidic cartridge, such as actuators, valves, and gates.

Generally, the heating of microfluidic components, such as a PCR reaction chamber, is controlled by passing currents through suitably configured microfabricated heaters. Under control of suitable circuitry, the lanes of a multi-lane cartridge can then be controlled independently of one another. This can lead to a greater energy efficiency of the apparatus, because not all heaters are heating at the same time, and a given heater is receiving current for only that fraction of the time when it is required to heat. Control systems and methods of controllably heating various heating elements are further described in U.S. patent application Ser. No. 11/940,315, filed Nov. 14, 2007 and entitled “Heater Unit for Microfluidic Diagnostic System”.

In certain embodiments, each heater has an associated temperature sensor. In the embodiment of FIG. 27A, a single temperature sensor 1011 is used for both long heaters. A temperature sensor 1013 for short left heater, and a temperature sensor 1015 for short right heater are also shown. The temperature sensor in the middle of the reactor is used to provide feedback and control the amount of power supplied to the two long heaters, whereas each of the short heaters has a dedicated temperature sensor placed adjacent to it in order to control it. As further described herein, temperature sensors are preferably configured to transmit information about temperature in their vicinity to a processor in the apparatus at such times as the heaters are not receiving current that causes them to heat. This can be achieved with appropriate control of current cycles.

In order to reduce the number of sensor or heater elements required to control a PCR heater, the heaters may be used 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 four to just one, thereby reducing the burden on the associated electronic circuitry.

FIG. 27B shows expanded views of heaters and temperature sensors used in conjunction with a PCR reaction chamber of FIG. 27A. Temperature sensors 1001 and 1013 are designed to have a room temperature resistance of approximately 200-300 ohms. This value of resistance is determined by controlling the thickness of the metal layer deposited (e.g., a sandwich of 400 {acute over (Å)} TiW/3,000{acute over (Å)} Au/400 {acute over (Å)} TiW), and etching the winding metal line to have a width of approximately 10-25 μm and 20-40 mm length. The use of metal in this layer gives it a temperature coefficient of resistivity of the order of 0.5-20° C./ohms, preferably in the range of 1.5-3° C./ohms. Measuring the resistance at higher temperatures enables determination of the exact temperature of the location of these sensors.

The configuration for uniform heating, shown in FIG. 27A for a single PCR reaction chamber, can also be applied to a multi-lane PCR cartridge in which multiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and/or control circuitry used in conjunction with the apparatus described herein. FIG. 27C shows thermal images, from the top surface of a microfluidic cartridge when heated by heaters configured as in FIGS. 27A and 27B, when each heater in turn is activated, as follows: (A): Long Top only; (B) Long Bottom only; (C) Short Left only; (D) Short Right only; and (E) All Four Heaters on. Panel (F) shows a view of the reaction chamber and heaters on the same scale as the other image panels in FIG. 27C. Also shown in the figure is a temperature bar.

The configuration for uniform heating, shown in FIG. 27A for a single PCR reaction chamber, can be applied to a multi-lane PCR cartridge in which multiple independent PCR reactions occur. See, e.g., FIG. 28, which shows an array of heater elements suitable to heat a cartridge herein.

Heater Multiplexing (Under Software Control)

Another aspect of the heater unit described herein, relates to a control of heat within the system and its components. The method leads to a greater energy efficiency of the apparatus described herein, because not all heaters are heating at the same time, and a given heater is receiving current for only part of the time.

Generally, the heating of microfluidic components, such as a PCR reaction chamber, is controlled by passing currents through suitably configured microfabricated heaters. The heating can be further controlled by periodically turning the current on and off with varying pulse width modulation (PWM), wherein pulse width modulation refers to the on-time/off-time ratio for the current. The current can be supplied by connecting a microfabricated heater to a high voltage source (for example, 30 V), which can be gated by the PWM signal. In some embodiments, the device includes 48 PWM signal generators. Operation of a PWM generator includes generating a signal with a chosen, programmable, period (the end count) and a particular granularity. For instance, the signal can be 4000 μs (micro-seconds) with a granularity of 1 μs, in which case the PWM generator can maintain a counter beginning at zero and advancing in increments of 1 μs until it reaches 4000 μs, when it returns to zero. Thus, the amount of heat produced can be adjusted by adjusting the end count. A high end count corresponds to a greater length of time during which the microfabricated heater receives current and therefore a greater amount of heat produced. It would be understood that the granularity and signal width can take values other than those provided here without departing from the principles described herein.

Fluorescence Detection System, Including Lenses and Filters, and Multiple Parallel Detection for a Multi-Lane Cartridge

The detection system herein is configured to monitor fluorescence coming from one or more species involved in a biochemical reaction. The system can be, for example, an optical detector having a light source that selectively emits light in an absorption band of a fluorescent dye, and a light detector that selectively detects light in an emission band of the fluorescent dye, wherein the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof, as further described elsewhere herein. Alternatively, 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. 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. For example, the optical detector can be configured to independently detect a plurality of fluorescent dyes at a plurality of different locations of, for example, a microfluidic substrate, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. The detector further has potential for 2, 3 or 4 color detection and is controlled by software, preferably custom software, configured to sample information from the detector.

The detection system described herein is capable of detecting a fluorescence signal from nanoliter scale PCR reactions. Advantageously, the detector is formed from inexpensive components, having no moving parts. The detector can be configured to couple to a microfluidic cartridge as further described herein, and can also be part of a pressure application system, such as a sliding lid on an apparatus in which the detector is situated, that keeps the cartridge in place.

FIGS. 29-31B depict an embodiment of a highly sensitive fluorescence detection system that includes light emitting diodes (LED's), photodiodes, and filters/lenses for monitoring, in real-time, one or more fluorescent signals emanating from the microfluidic channel. The embodiment in FIGS. 29-31B displays a two-color detection system having a modular design that couples with a single microfluidic channel found, for example, in a microfluidic cartridge. It would be understood by one skilled in the art that the description herein could also be adapted to create a detector that just detects a single color of light. FIGS. 31A and 31B show elements of optical detector elements 1220 including light sources 1232 (for example, light emitting diodes), lenses 1234, light detectors 1236 (for example, photodiodes) and filters 1238. The detector comprises two LED's (blue and red, respectively) and two photodiodes. The two LED's are configured to transmit a beam of focused light on to a particular region of the cartridge. The two photo diodes are configured to receive light that is emitted from the region of the cartridge. One photodiode is configured to detect emitted red light, and the other photodiode is configured to detect emitted blue light. Thus, in this embodiment, two colors can be detected simultaneously from a single location. Such a detection system can be configured to receive light from multiple microfluidic channels by being mounted on an assembly that permits it to slide over and across the multiple channels. The filters can be, for example, bandpass filters, the filters at the light sources corresponding to the absorption band of one or more fluorogenic probes and the filters at the detectors corresponding to the emission band of the fluorogenic probes.

FIGS. 32 and 33 show an exemplary read-head comprising a multiplexed 2 color detection system that is configured to mate with a multi-lane microfluidic cartridge. FIG. 32 shows a view of the exterior of a multiplexed read-head. FIG. 33 is an exploded view that shows how various detectors are configured within an exemplary multiplexed read head, and in communication with an electronic circuit board.

Each of the detection systems multiplexed in the assembly of FIGS. 32 and 33 is similar in construction to the embodiment of FIGS. 29-31B. The module in FIGS. 32 and 33 is configured to detect fluorescence from each of 12 microfluidic channels, as found in, for example, the respective lanes of a 12-lane microfluidic cartridge. Such a module therefore comprises 24 independently controllable detectors, arranged as 12 pairs of identical detection elements. Each pair of elements is then capable of dual-color detection of a pre-determined set of fluorescent probes. It would be understood by one of ordinary skill in the art that other numbers of pairs of detectors are consistent with the apparatus described herein. For example, 4, 6, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and 48 pairs are also consistent and can be configured according to methods and criteria understood by one of ordinary skill in the art.

Detection Sensitivity, Time Constant and Gain

A typical circuit that includes a detector as described herein includes, in series, a preamplifier, a buffer/inverter, a filter, and a digitizer. Sensitivity is important so that high gain is very desirable. In one embodiment of the preamplifier, a very large, for example 100 GΩ, resistor is placed in parallel with the diode. Other values of a resistor would be consistent with the technology herein: such values typically fall in the range 0.5-100 GΩ, such as 1-50 GΩ, or 2-10 GΩ. An exemplary pre-amplifier circuit configured in this way is shown in FIG. 7. Symbols in the figure have their standard meanings in electronic circuit diagrams.

The FIG. 34 shows a current-to-voltage converter/pre-amplifier circuit suitable for use with the detection system. D11 is the photodetector that collects the fluorescent light coming from the microfluidic channel and converts it into an electric current. The accompanying circuitry is used to convert these fluorescent currents into voltages suitable for measurement and output as a final measure of the fluorescence.

A resistor-capacitor circuit in FIG. 34 contains capacitor C45 and resistor R25. Together, the values of capacitance of C45 and resistance of R25 are chosen so as to impact the time constant τ_c(equal to the product of R25 and C45) of the circuit as well as gain of the detection circuit. The higher the time constant, the more sluggish is the response of the system to incident light. It typically takes the duration of a few time constants for the photodetector to completely charge to its maximum current or to discharge to zero from its saturation value. It is important for the photo current to decay to zero between measurements, however. As the PCR systems described herein are intended to afford rapid detection measurements, the product R₂₅C₄₅should therefore be made as low as possible. However, the gain of the pre-amplifier whose circuitry is shown is directly proportional to the (fluorescent-activated) current generated in the photodetector and the resistance R₂₅. As the fluorescence signal from the microfluidic channel device is very faint (due to low liquid volume as well as small path lengths of excitation), it is thus important to maximize the value of R₂₅. In some embodiments, R₂₅is as high as 100 Giga-Ohms (for example, in the range 10-100 GΩ), effectively behaving as an open-circuit. With such values, the time-constant can take on a value of approximately 50-100 ms by using a low-value capacitor for C45. For example, the lowest possible available standard off-the-shelf capacitor has a value of 1 pF (1 picoFarad). A time-constant in the range 50-100 ms ensures that the photocurrent decays to zero in approximately 0.5 s (approx. 6 cycles) and therefore that approximately 2 samplings can be made per second. Other time constants are consistent with effective use of the technology herein, such as in the range 10 ms-1 s, or in the range 50 ms-500 ms, or in the range 100-200 ms. The actual time constant suitable for a given application will vary according to circumstance and choice of capacitor and resistor values. Additionally, the gain achieved by the pre-amplifier circuit herein may be in the range of 10⁷-5×10⁹, for example may be 1×10⁹.

As the resistance value for R25 is very high (˜100 GΩ), the manner of assembly of this resistor on the optics board is important for the overall efficiency of the circuit. Effective cleaning of the circuit during assembly and before use is important to achieve an optimal time-constant and gain for the optics circuit.

It is also important to test each photo-diode that is used, because many do not perform according to specification.

Sensitivity and Aperturing

The LED light passes through a filter before passing through the sample in the microfluidic channel (as described herein, typically 300μ deep). This is a very small optical path-length for the light in the sample. The generated fluorescence then also goes through a second filter, and into a photo-detector. Ultimately, then, the detector must be capable of detecting very little fluorescence. Various aspects of the detector configuration can improve sensitivity, however.

The illumination optics can be designed so that the excitation light falling on the PCR reactor is incident along an area that is similar to the shape of the reactor. As the reactor is typically long and narrow, the illumination spot should be long and narrow, i.e., extended, as well. The length of the spot can be adjusted by altering a number of factors, including: the diameter of the bore where the LED is placed (the tube that holds the filter and lens has an aperturing effect); the distance of the LED from the PCR reactor; and the use of proper lens at the right distance in between. As the width of the beam incident on the reactor is determined by the bore of the optical element (approximately 6 mm in diameter), it is typical to use an aperture (a slit having a width approximately equal to the width of the reactor, and a length equal to the length of the detection volume) to make an optimal illumination spot. A typical spot, then, is commensurate with the dimensions of a PCR reaction chamber, for example 1.5 mm wide by 7 mm long. FIG. 35A shows the illumination spot across 12 PCR reactors for the two different colors used. A typical aperture is made of anodized aluminum and has very low autofluoresence in the wavelengths of interest. FIG. 35B illustrates a cross-section of a detector, showing an exemplary location for an aperture 802.

The optimal spot size and intensity is importantly dependent on the ability to maintain the correct position of the LED's with respect to the center of the optical axis. Special alignment procedures and checks can be utilized to optimize this. The different illuminations can also be normalized with respect to each other by adjusting the power current through each of the LED's or adjusting the fluorescence collection time (the duration for which the photodetector is on before measuring the voltage) for each detection spot. It is also important to align the detectors with the axis of the micro-channels.

The aperturing is also important for successful fluorescence detection because as the cross-sectional area of the incident beam increases in size, so the background fluorescence increases, and the fluorescence attributable only to the molecules of interest (PCR probes) gets masked. Thus, as the beam area increases, the use of an aperture increases the proportion of collected fluorescence that originates only from the PCR reactor. Note that the aperture used in the detector herein not only helps collect fluorescence only from the reaction volume but it correspondingly adjusts the excitation light to mostly excite the reaction volume. The excitation and emission aperture is, of course, the same.

Based on a typical geometry of the optical exctiation and emission system and aperturing, show spot sizes as small as 0.5 mm by 0.5 mm and as long as 8 mm×1.5 mm have been found to be effective. By using a long detector (having an active area 6 mm by 1 mm) and proper lensing, the aperture design can extend the detection spot to as long as 15-20 mm, while maintaining a width of 1-2 mm using an aperture. Correspondingly, the background fluorescence decreases as the spot size is decreased, thereby increasing the detection sensitivity.

Use of Detection System to Measure/Detect Fluid in PCR Chamber

The fluorescence detector is sensitive enough to be able to collect fluorescence light from a PCR chamber of a microfluidic substrate. The detector can also be used to detect the presence of liquid in the chamber, a measurement that provides a determination of whether or not to carry out a PCR cycle for that chamber. For example, in a multi-sample cartridge, not all chambers will have been loaded with sample; for those that are not, it would be unnecessary to apply a heating protocol thereto. One way to determine presence or absence of a liquid is as follows. A background reading is taken prior to filling the chamber with liquid. Another reading is taken after microfluidic operations have been performed that should result in filling the PCR chamber with liquid. The presence of liquid alters the fluorescence reading from the chamber. A programmable threshold value can be used to tune an algorithm programmed into a processor that controls operation of the apparatus as further described herein (for example, the second reading has to exceed the first reading by 20%). If the two readings do not differ beyond the programmed margin, the liquid is deemed to not have entered the chamber, and a PCR cycle is not initiated for that chamber. Instead, a warning is issued to a user.

Exemplary Electronics and Software

The heater unit described herein can be controlled by various electronics circuitry, itself operating on receipt of computer-controlled instructions. FIG. 36 illustrates exemplary electronics architecture modules for operating a heater unit and diagnostic apparatus. It would be understood by one of ordinary skill in the art that other configurations of electronics components are consistent with operation of the apparatus as described herein. In the exemplary embodiment, the electronics architecture is distributed across two components of the apparatus: the Analyzer 2100 and a Heater unit 2102. The Analyzer apparatus as further described herein contains, for example, an Optical Detection Unit 2108, a Control Board 2114, a Backplane 2112, and a LCD Touchscreen 2110. The Control Board includes a Card Engine 2116 further described herein, and Compact Flash memory 2118, as well as other components. The Heater Assembly includes a Heater Board 2104 and a Heater Mux Board 2106, both further described herein.

In one embodiment, the Card Engine electronics module 2116 is a commercial, off the shelf “single board computer” containing a processor, memory, and flash memory for operating system storage.

The optional LCD+Touchscreen electronics module 2110 is a user interface, for example, driven through a touchscreen, such as a 640 pixel by 480 pixel 8 inch LCD and 5-wire touchscreen.

The Compact Flash electronics module 2118 is, for example, a 256 megabyte commercial, off the shelf, compact flash module for application and data storage. Other media are alternatively usable, such as USB-drive, smart media card, memory stick, and smart data-card having the same or other storage capacities.

The Backplane electronics module 2112 is a point of connection for the removable heater assembly 2102. Bare PC boards with two connectors are sufficient to provide the necessary level of connectivity.

The Control Board electronics module 2114 supports peripherals to the Card Engine electronics module 2116. In one embodiment, the peripherals include such devices as a USB host+slave or hub, a USB CDROM interface, serial ports, and ethernet ports. The Control Board 2114 can include a power monitor with a dedicated processor. The Control Board may also include a real time clock. The Control Board may further include a speaker. The Control Board 2114 also includes a CPLD to provide SPI access to all other modules and programming access to all other modules. The Control Board includes a programmable high voltage power supply. The Control Board includes a Serial-Deserializer interface to the LCD+Touchscreen electronics module 2110 and to the Optical Detection Unit electronics module 2108. The Control Board also includes module connectors.

In the exemplary embodiment, the optical detection unit electronics module 2108 contains a dedicated processor. The optical detection unit 2108 contains a serializer-deserializer interface. The optical detection unit 2108 contains LED drivers. The optical detection unit also contains high gain-low noise photodiode amplifiers. The optical detection unit can have power monitoring capability. The optical detection unit can also be remotely reprogrammable.

The Heater Board electronics module 2104 is preferably a glass heater board. The Heater Board has PCB with bonding pads for glass heater board and high density connectors.

In one embodiment, the heater mux (‘multiplex’) board electronics module 2106 has 24 high-speed ADC, 24 precision current sources, and 96 optically isolated current drivers for heating. The heater mux board has the ability to time-multiplex heating/measurement. The heater mux board has multiplexer banks to multiplex inputs to ADC, and to multiplex current source outputs. The heater mux board has a FPGA with a soft processor core and SDRAM. The heater mux board has a Power Monitor with a dedicated processor. The Heater Mux Board can be remotely reprogrammable.

In another embodiment, control electronics can be spread over four different circuit board assemblies. These include the MAIN board: Can serve as the hub of the Analyzer control electronics and manages communication and control of the other various electronic subassemblies. The main board can also serve as the electrical and communications interface with the external world. An external power supply (12V DC/10 A; UL certified) can be used to power the system. The unit can communicate via 5 USB ports, a serial port and an Ethernet port. Finally, the main board can incorporate several diagnostic/safety features to ensure safe and robust operation of the Analyzer.

MUX Board: Upon instruction from the main board, the MUX board can perform all the functions typically used for accurate temperature control of the heaters and can coordinate the collection of fluorescence data from the detector board.

LCD Board: Can contain the typical control elements to light up the LCD panel and interpret the signals from the touch sensitive screen. The LCD/touch screen combination can serve as a mode of interaction with the user via a Graphical User Interface.

Detector Board: Can house typical control and processing circuitry that can be employed to collect, digitize, filter, and transmit the data from the fluorescence detection modules.

Certain software can be executed in each electronics module. The Control Board Electronics Module executes, for example, Control Board Power Monitor software. The Card Engine electronics module executes an operating system, graphical user interface (GUI) software, an analyzer module, and an application program interface (api). The Optical Detection Unit electronics module executes an optics software module. The Heater Mux Board electronics module executes dedicated Heater Mux software, and Heater Mux Power Monitor software. Each of the separate instances of software can be modular and under a unified control of, for example, driver software.

The exemplary electronics can use Linux, UNIX, Windows, or MacOS, including any version thereof, as the operating system. The operating system is preferably loaded with drivers for USB, Ethernet, LCD, touchscreen, and removable media devices such as compact flash. Miscellaneous programs for configuring the Ethernet interface, managing USB connections, and updating via CD-ROM can also be included.

In the embodiment of FIG. 36, the analyzer module is the driver for specific hardware. The analyzer module provides access to the Heater Mux Module, the Optical Detection Unit, the Control Board Power Monitor, the Real Time Clock, the High Voltage Power Supply, and the LCD backlight. The analyzer module provides firmware programming access to the Control Board power monitor, the Optical Detection Unit, and the Heater Mux Module.

The API provides uniform access to the analyzer module driver. The API is responsible for error trapping, and interrupt handling. The API is typically programmed to be thread safe.

The GUI software can be based on a commercial, off-the-shelf PEG graphics library. The GUI can use the API to coordinate the self-test of optical detection unit and heater assembly. The GUI starts, stops, and monitors test progress. The GUI can also implement an algorithm to arrive on diagnosis from fluorescence data. The GUI provides access control to unit and in some embodiments has an HIS/LIS interface.

The Control Board Power Monitor software monitors power supplies, current and voltage, and signals error in case of a fault.

The Optics Software performs fluorescence detection which is precisely timed to turn on/off of LED with synchronous digitization of the photodetector outputs. The Optics Software can also monitor power supply voltages. The Optics Software can also have self test ability.

The Heater Mux Module software implements a “protocol player” which executes series of defined “steps” where each “step” can turn on sets of heaters to implement a desired microfluidic action. The Heater Mux Module software also has self test ability. The Heater Mux Module software contains a fuzzy logic temperature control algorithm.

The Heater Mux Power Monitor software monitors voltage and current levels. The Heater Mux Power Monitor software can participate in self-test, synchronous, monitoring of the current levels while turning on different heaters.

EXAMPLES

The following are exemplary aspects of various parts and functions of the system described herein.

Additional embodiments of a cartridge are found in U.S. patent application Ser. No. 11/940,310, entitled “Microfluidic Cartridge and Method of Making Same”, and filed on even date herewith, the specification of which is incorporated herein by reference.

Additional embodiments of heater units and arrays are described in U.S. patent application Ser. No. 11/940,315, entitled “Heater Unit for Microfluidic Diagnostic System” and filed on even date herewith, the specification of which is incorporated herein by reference in its entirety.

Further description of suitably configured detectors are described in U.S. patent application Ser. No. 11/940,321, filed on Nov. 14, 2007 and entitled “Fluorescence Detector for Microfluidic Diagnostic System”, incorporated herein by reference.

Example 1: Analyzer Having Removable Heater Unit

This non-limiting example describes pictorially, various embodiments of an apparatus, showing incorporation of a heater unit and a microfluidic cartridge operated on by the heater unit.

FIG. 37 shows an apparatus 1100 that includes a housing having a display output 1102, an openable lid 1104, and a bar code reader 1106. The cartridge is positioned in a single orientation in a receiving bay under the lid, FIG. 38. The lid of the apparatus can be closed to apply pressure to the cartridge, as shown in FIG. 39. The unit currently weighs about 20 lbs. and is approximately 10″ wide by 16″ deep by 13″ high.

FIGS. 40 and 41: The heating stage of the apparatus can be removable for cleaning, maintenance, or to replace a custom heating stage for a particular microfluidic cartridge. FIGS. 40 and 41 also show how a heater unit is insertable and removable from a front access door to an analyzer apparatus.

Example 2: Assembly of an Exemplary Heater Unit

FIG. 42A shows an exploded view of an exemplary heater unit. The unit has a top cover and a bottom cover that together enclose a Mux board (control board), a pressure support layer, and insulator film, and a microthermal circuit on a PCB. The last of these is the heat source that selectively heats regions of a microfluidic substrate placed in contact therewith through the top cover.

An exemplary heater substrate, FIG. 42B, consists of a photo-lithographically processed glass wafer bonded to a standard 0.100″ standard FR4 printed circuit board. The glass wafer is 0.5 mm thick and is cut into a rectangle the size of ˜3.5×4.25 inches. The glass substrate has numerous metal heaters and resistive temperature sensors photo-lithographically etched on the surface of the glass wafer. The substrate is aligned and bonded to the PCBoard using a compliant epoxy, ensuring flatness to within 2-3 mils over the surface of the wafer. The cured epoxy should withstand up to 120° C. for two hours minimum. Approximately 300-400 bond pads of the size of approximately 1 mm×0.25 mm, with exposed gold surfaces, are located along the two long edges of the wafer. These pads are wirebonded (ball-bonding) to corresponding pads on the PCB using 1.5 mil gold wires. Wire bonding is a threading process, standard in semiconductor FAB. Alternatively, a flip-chip method may be used, though such methods are more complicated and may warp the wafer because of thermal mismatch. Wire bonds should have good integrity and pass defined pull strength. The substrate is baked at 120° C. for two hours and then the wire bonds are encapsulated by a compliant epoxy that will protect the wirebonds but not damage the bonds even at 120° C. Encapsulant should not spill over predefined area around the wirebonds and should not be taller than a defined height. For example, instead of laying epoxy all over the substrate, lines (e.g., a hash pattern) of it are made so that epoxy cures and air escapes through side. Alternatively, a laminate fill (adhesive on both sides) can be used. Standard connectors are soldered to the PCB and then the unit is tested using a test set-up to ensure all heaters and sensors read the right resistance values.

Pictures of an exemplary Mux board and assembled heater unit are shown in FIGS. 27-29.

Example 3: Pulse Width Modulation for Heater Circuitry

In various embodiments, the operation of a PWM generator can also include a programmable start count in addition to the aforementioned end count and granularity. In such embodiments, multiple PWM generators can produce signals that can be selectively non-overlapping (e.g., by multiplexing the on-time of the various heaters) such that the current capacity of the high voltage power is not exceeded. Multiple heaters can be controlled by different PWM signal generators with varying start and end counts. The heaters can be divided into banks, whereby a bank defines a group of heaters of the same start count. For example, 36 PWM generators can be grouped into six different banks, each corresponding to a certain portion of the PWM cycle (500 ms for this example). The end count for each PWM generator can be selectively programmed such that not more than six heaters will be on at any given time. A portion of a PWM cycle can be selected as dead time (count 3000 to 4000 for this example) during which no heating takes place and sensitive temperature sensing circuits can use this time to sense the temperature. The table below represents a PWM cycle for the foregoing example:

Start Count
End Count
Max End count

Bank 1

PWM generator#1
0
150
500

PWM generator#2
0
220
500

. . .
. . .
. . .

PWM generator#6
0
376
500

Bank 2

PWM generator#7
500
704
1000

PWM generator#8
500
676
1000

. . .
. . .
. . .
. . .

PWM generator#12
500
780
1000

Bank 3

PWM generator#13
1000
1240
1500

PWM generator#14
1000
1101
1500

. . .
. . .
. . .
. . .

PWM generator#18
1000
1409
1500

Bank 4

PWM generator#19
1500
1679
2000

PWM generator#20
1500
1989
2000

. . .
. . .
. . .
. . .

PWM generator#24
1500
1502
2000

Bank 5

PWM generator#25
2000
2090
2500

PWM generator#26
2000
2499
2500

. . .
. . .
. . .
. . .

PWM generator#30
2000
2301
2500

Bank 6

PWM generator#31
2500
2569
3000

PWM generator#32
2500
2790
3000

. . .
. . .
. . .
. . .

PWM generator#36
2500
2678
3000

Example 4: Detector Integrated in Force Member

This non-limiting example describes pictorially, various embodiments of a detection system integrated into a force member, in an apparatus for carrying out diagnostics on microfluidic samples.

FIG. 43A: The lid of the apparatus can be closed, which can block ambient light from the sample bay, and place an optical detector contained in the lid into position with respect to the microfluidic cartridge.

FIG. 43B: The lid of the apparatus can be closed to apply pressure to the cartridge. Application of minimal pressure on the cartridge: after the slider compresses the cartridge, the slider can compress the compliant label of the cartridge. This can cause the bottom of the cartridge to be pressed down against the surface of the heater unit present in the heater module. Springs present in the slider can deliver, for example approximately 50 lb of pressure to generate a minimum pressure, for example 2 psi over the entire cartridge bottom.

Thermal interface: the cartridge bottom can have a layer of mechanically compliant heat transfer laminate that can enable thermal contact between the microfluidic substrate and the microheater substrate of the heater module. A minimal pressure of 1 psi can be employed for reliable operation of the thermal valves, gate and pumps present in the microfluidic cartridge.

Mechanicals and assembly: the Analyzer can have a simple mechanical frame to hold the various modules in alignment. The optics module can be placed in rails for easy opening and placement of cartridges in the Analyzer and error-free alignment of the optics upon closing. The heater/sensor module can be also placed on rails or similar guiding members for easy removal and insertion of the assembly.

Slider: the slider of the Analyzer can house the optical detection system as well as the mechanical assembly that can enables the optics jig to press down on the cartridge when the handle of the slider is turned down onto the analyzer. The optics jig can be suspended from the case of the slider at 4 points. Upon closing the slider and turning the handle of the analyzer down, 4 cams can turn to push down a plate that presses on 4 springs. On compression, the springs can deliver approximately 50 lb on the optical block. See FIGS. 44A-44C.

The bottom surface of the optics block can be made flat to within 100 microns, typically within 25 microns, and this flat surface can press upon the compliant (shore hardness approximately 50-70) label (approximately 1.5 mm thick under no compression) of the cartridge making the pressure more or less uniform over the cartridge. An optional lock-in mechanism can also be incorporated to prevent the slider from being accidentally knocked-off while in use.

FIG. 45A shows a side view of a lever assembly 1200, with lever 1210, gear unit 1212, and force member 1214. Assembly 1200 can be used to close the lid of the apparatus and (through force members 1214) apply force to a microfluidic cartridge 1216 in the receiving chamber 1217. One force member is visible in this cut away view, but any number, for example 4, can be used. The force members can be, for example, a manual spring loaded actuator as shown, an automatic mechanical actuator, a material with sufficient mechanical compliance and stiffness (e.g., a hard elastomeric plug), and the like. The force applied to the microfluidic cartridge 1216 can result in a pressure at the surface of the microfluidic cartridge 1216 of at least about 0.7 psi to about 7 psi (between about 5 and about 50 kilopascals), or in some embodiments about 2 psi (about 14 kilopascals.

FIG. 45B shows a side view of lever assembly 1200, with microfluidic cartridge 1216 in the receiving chamber 1217. A heat source 1219 (for example, a xenon bulb as shown) can function as a radiant heat source directed at a sample inlet reservoir 1218, where the heat can lyse cells in reservoir 1218. A thermally conductive, mechanically compliant layer 1222 can lie at an interface between microfluidic cartridge 1216 and thermal stage 1224. Typically, microfluidic cartridge 1216 and thermal stage 1224 can be planar at their respective interface surfaces, e.g., planar within about 100 microns, or more typically within about 25 microns. Layer 1222 can improve thermal coupling between microfluidic cartridge 1216 and thermal stage 1224. Optical detector elements 1220 can be directed at the top surface of microfluidic cartridge 1216.

FIGS. 45C and 45D show further cross-sectional views.

Example 6: Exemplary Optics Board

An exemplary optics board is shown schematically in FIG. 46, and is used to collect and amplify the fluorescent signature of a successful chemical reaction on a micro-fluidic cartridge, and control the intensity of LED's using pulse-width modulation (PWM) to illuminate the cartridge sample over up to four channels, each with two color options. Additionally, it receives instructions and sends results data back over an LVDS (low-voltage differential signaling) SPI (serial peripheral interface). In some embodiments there is a separate instance of this circuitry for each PCR channel that is monitored.

The power board systems include: a +12V input; and +3.3V, +3.6V, +5V, and −5V outputs, configured as follows: the +3.3V output contains a linear regulator, is used to power the LVDS interface, should maintain a +/−5% accuracy, and supply an output current of 0.35 A; the +3.6V output contains a linear regulator, is used to power the MSP430, should maintain a +/−5% accuracy, and supply an output current of 0.35 A; the +5V output contains a linear regulator, is used to power the plus rail for op-amps, should maintain a +/−5% accuracy, and supply an output current of 0.35 A; the −5V output receives its power from the +5V supply, has a mV reference, is used to power the minus rail for op-amps and for the photo-detector bias, should maintain a +/−1% voltage accuracy, and supply an output current of 6.25 mA+/−10%. Additionally, the power board has an 80 ohm source resistance, and the main board software can enable/disable the regulator outputs.

The main board interface uses a single channel of the LVDS standard to communicate between boards. This takes place using SPI signaling over the LVDS interface which is connected to the main SPI port of the control processor. The interface also contains a serial port for in-system programming.

The optical detection system of FIG. 46 comprises a control processor, LED drivers, and a photo-detection system. In the exemplary embodiment, the control processor is a TI MSP430F1611 consisting of a dual SPI (one for main board interface, and one for ADC interface) and extended SRAM for data storage. It has the functions of power monitoring, PWM LED control, and SPI linking to the ADC and main board. The LED drivers contain NPN transistor switches, are connected to the PWM outputs of the control processor, can sink 10 mA @ 12V per LED (80 mA total), and are single channel with 2 LEDs (one of each color) connected to each. The photo-detection system has two channels and consists of a photo-detector, high-sensitivity photo-diode detector, high gain current to voltage converter, unity gain voltage inverting amplifier, and an ADC. Additionally it contains a 16 channel Sigma-delta (only utilizing the first 8 channels) which is connected to the second SPI port of the control processor.

During assembly of the various components on to the PC board, such as may occur on a production line, there are the following considerations. The extremely high impedance of the photo-detection circuit means that a rigorous cleaning procedure must be employed. Such a procedure may include, for example: After surface mount components are installed, the boards are washed on a Weskleen and blow dried upon exiting conveyor. The belt speed can be set at 20-30. The boards are soaked in an alcohol bath for approximately 3 minutes, then their entire top and bottom surfaces are scrubbed using a clean, soft bristle brush. The boards are baked in a 105° F. (40° C.) oven for 30 minutes to dry out all components.

After all the components are installed: the soldered areas of the boards can be hand wash using deionized water and a soft bristle brush. The same soldered areas can be hand washed using alcohol and a soft bristle brush. The boards are allowed to air dry. Once the board is cleaned, the optical circuitry must be conformal coated to keep contaminates out.

The foregoing description is intended to illustrate various aspects of the present technology. It is not intended that the examples presented herein limit the scope of the present technology. The technology now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A method, comprising: providing a multi-lane microfluidic cartridge, each lane comprising an amplification region within which amplification of polynucleotides is carried out; andproviding an instrument, the instrument comprising a receiving bay configured to receive the multi-lane microfluidic cartridge, the instrument comprising one or more processors configured to control components of the instrument, the components of the instrument comprising: a plurality of separately controllable heat sources mounted in the instrument, a heat source of the plurality of separately controllable heat sources configured to direct heat to an amplification region of a lane of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay,a detector mounted in the instrument, the detector configured to detect the presence of an amplification product in the amplification region during amplification,an automatic pipetting machine mounted in the instrument, the automatic pipetting machine configured to load polynucleotide-containing samples into the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, wherein the automatic pipetting machine is configured to add a polynucleotide-containing sample to the multi-lane microfluidic cartridge after a different polynucleotide-containing sample has been amplified in the multi-lane microfluidic cartridge, wherein the instrument is configured to move the multi-lane microfluidic cartridge from a first location in an area inside the instrument to a second location in the area before amplification of polynucleotides is carried out.
2. The method of claim 1, wherein the multi-lane microfluidic cartridge comprises at least one reservoir configured to contain waste.
3. The method of claim 1, wherein the multi-lane microfluidic cartridge comprises a plurality of separate sample inlets aligned along a first axis, wherein the amplification regions of the multi-lane microfluidic cartridge are aligned along a second axis substantially parallel to the first axis, and wherein the detector does not cover the plurality of separate sample inlets of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
4. The method of claim 1, wherein the detector is configured to independently detect a plurality of fluorescent dyes in the amplification region of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, the plurality of fluorescent dyes having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.
5. The method of claim 1, wherein the instrument further comprises a touchscreen display and a second receiving bay configured to receive a second multi-lane microfluidic cartridge, and wherein the instrument is configured to: concurrently process a first polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a second polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay;consecutively process a third polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a fourth polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay; anddisplay results of analysis of the first, second, third, and fourth polynucleotide-containing samples on the touchscreen display.
6. The method of claim 1, wherein the amplified sample remains on the multi-lane microfluidic cartridge and the cartridge is disposed when all lanes are used.
7. The method of claim 1, wherein the instrument is configured to isolate polynucleotide-containing samples within the amplification regions of the multi-lane microfluidic cartridge after confirming that the polynucleotide-containing samples are present in the amplification regions.
8. The method of claim 1, further comprising providing a plurality of multi-lane microfluidic cartridges, wherein the instrument comprises multiple receiving bays configured to receive the plurality of multi-lane microfluidic cartridges, the one or more processors and a user interface configured to permit concurrent, consecutive, or simultaneous control of the plurality of multi-lane microfluidic cartridges when the plurality of multi-lane microfluidic cartridges are received in the multiple receiving bays.
9. The method of claim 1, wherein the detector is configured to move relative to the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
10. The method of claim 1, wherein the instrument comprises a mechanism configured to apply an external pressure to the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
11. A method, comprising: providing a multi-lane microfluidic cartridge, each lane comprising an amplification region within which amplification of polynucleotides is carried out; andproviding an instrument, the instrument comprising a receiving bay configured to receive the multi-lane microfluidic cartridge, the instrument further comprising: a plurality of separately controllable heat sources located in the instrument before the multi-lane microfluidic cartridge is received in the receiving bay, a heat source of the plurality of separately controllable heat sources configured to direct heat to a lane of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay,a detector configured to detect the presence of an amplification product in an amplification region of a lane of the multi-lane microfluidic cartridge during amplification, wherein the detector is configured to independently detect a plurality of fluorescent dyes in the amplification region of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, the plurality of fluorescent dyes having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof,an automatic pipetting machine configured to load polynucleotide-containing samples into the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, wherein after amplification has been carried out on a polynucleotide-containing sample, the amplified polynucleotide-containing sample is configured to remain in the multi-lane microfluidic cartridge while the multi-lane microfluidic cartridge receives another polynucleotide-containing sample in an unused lane of the multi-lane microfluidic cartridge from the automatic pipetting machine.
12. The method of claim 11, wherein the multi-lane microfluidic cartridge comprises a plurality of separate sample inlets aligned along a first axis, wherein the amplification regions of the multi-lane microfluidic cartridge are aligned along a second axis substantially parallel to the first axis, and wherein the detector does not cover the plurality of separate sample inlets of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
13. The method of claim 11, wherein the instrument is configured to isolate polynucleotide-containing samples within the amplification regions of the multi-lane microfluidic cartridge after confirming that the polynucleotide-containing samples are present in the amplification regions.
14. The method of claim 11, wherein the instrument further comprises a touchscreen display and a second receiving bay configured to receive a second multi-lane microfluidic cartridge, and wherein the instrument is configured to: concurrently process a first polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a second polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay;consecutively process a third polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a fourth polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay; anddisplay results of analysis of the first, second, third, and fourth polynucleotide-containing samples on the touchscreen display.
15. The method of claim 11, wherein the instrument comprises one or more processors configured to control the plurality of separately controllable heat sources, the detector, and the automatic pipetting machine.
16. The method of claim 11, wherein the instrument comprises a mechanism configured to apply an external pressure to the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
17. The method of claim 11, further comprising providing a plurality of multi-lane microfluidic cartridges, wherein the instrument comprises multiple receiving bays configured to receive the plurality of multi-lane microfluidic cartridges, the one or more processors and a user interface configured to permit concurrent, consecutive, or simultaneous control of the plurality of multi-lane microfluidic cartridges when the plurality of multi-lane microfluidic cartridges are received in the multiple receiving bays.
18. A method, comprising: providing a multi-lane microfluidic cartridge, each lane comprising an amplification region within which amplification of polynucleotides is carried out; andproviding an instrument, the instrument comprising a receiving bay configured to receive the multi-lane microfluidic cartridge, the instrument further comprising: a plurality of separately controllable heat sources configured at the receiving bay, a heat source of the plurality of separately controllable heat sources configured to direct heat to an amplification region of a lane of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, wherein the heat source is configured to cyclically heat in a series of heating phases to amplify polynucleotides in the amplification region, each heating phase comprising the heat source cycling between at least two temperatures,a detector configured to detect the presence of an amplification product in the amplification region during amplification,one or more processors configured to control the plurality of separately controllable heat sources, andan automatic pipetting machine configured to load polynucleotide-containing samples into the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, wherein the automatic pipetting machine is configured to add a polynucleotide-containing sample to the multi-lane microfluidic cartridge after a different polynucleotide-containing sample has been amplified in the multi-lane microfluidic cartridge, wherein the amplified polynucleotide-containing sample is configured to remain in the multi-lane microfluidic cartridge while the multi-lane microfluidic cartridge receives the added polynucleotide-containing sample in an unused lane of the multi-lane microfluidic cartridge from the automatic pipetting machine.
19. The method of claim 18, wherein the multi-lane microfluidic cartridge comprises a plurality of separate sample inlets aligned along a first axis, wherein the amplification regions of the multi-lane microfluidic cartridge are aligned along a second axis substantially parallel to the first axis, and wherein the detector does not cover the plurality of separate sample inlets of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
20. The method of claim 19, wherein the detector is configured to independently detect a plurality of fluorescent dyes in the amplification region of the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay, the plurality of fluorescent dyes having different fluorescent emission spectra, wherein each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.
21. The method of claim 20, wherein the instrument further comprises a touchscreen display and a second receiving bay configured to receive a second multi-lane microfluidic cartridge, and wherein the instrument is configured to: concurrently process a first polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a second polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay;consecutively process a third polynucleotide-containing sample in the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay and a fourth polynucleotide-containing sample in the second multi-lane microfluidic cartridge when the second multi-lane microfluidic cartridge is received in the second receiving bay; anddisplay results of analysis of the first, second, third, and fourth polynucleotide-containing samples on the touchscreen display.
22. The method of claim 18, wherein the microfluidic cartridge comprises at least one reservoir configured to contain waste.
23. The method of claim 18, wherein the instrument comprises a mechanism configured to apply an external pressure to the multi-lane microfluidic cartridge when the multi-lane microfluidic cartridge is received in the receiving bay.
24. The method of claim 18, wherein the instrument is configured to isolate polynucleotide-containing samples within the amplification regions of the multi-lane microfluidic cartridge after confirming that the polynucleotide-containing samples are present in the amplification regions.
25. The method of claim 18, wherein the one or more processors are configured to control the plurality of separately controllable heat sources, the detector, and the automatic pipetting machine.
26. The method of claim 25, further comprising providing a plurality of multi-lane microfluidic cartridges, wherein the instrument comprises multiple receiving bays configured to receive the plurality of multi-lane microfluidic cartridges, the one or more processors and a user interface configured to permit concurrent, consecutive, or simultaneous control of the plurality of multi-lane microfluidic cartridges when the plurality of multi-lane microfluidic cartridges are received in the multiple receiving bays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/796,239, filed Jul. 10, 2015, which is a continuation of U.S. patent application Ser. No. 13/692,929, filed Dec. 3, 2012 and issued as U.S. Pat. No. 9,080,207 on Jul. 14, 2015, which is a continuation of U.S. patent application Ser. No. 13/035,725, filed Feb. 25, 2011, issued as U.S. Pat. No. 8,323,900 on Dec. 4, 2012, which is a continuation of U.S. patent application Ser. No. 11/985,577, filed Nov. 14, 2007, issued as U.S. Pat. No. 7,998,708 on Aug. 16, 2011, which is a continuation-in-part of U.S. patent application Ser. No. 11/728,964, filed Mar. 26, 2007, issued as U.S. Pat. No. 9,040,288 on May 26, 2015, which claims the benefit of U.S. Provisional Patent Application No. 60/786,007, filed Mar. 24, 2006, and U.S. Provisional Patent Application No. 60/859,284, filed Nov. 14, 2006. U.S. patent application Ser. No. 11/985,577 claims the benefit of U.S. Provisional Patent Application No. 60/859,284, filed Nov. 14, 2006, and U.S. Provisional Patent Application No. 60/959,437, filed Jul. 13, 2007. The disclosures of U.S. patent application Ser. No. 13/692,929, U.S. patent application Ser. No. 13/035,725, U.S. patent application Ser. No. 11/985,577, U.S. patent application Ser. No. 11/728,964, U.S. Provisional Patent Application No. 60/859,284, and U.S. Provisional Patent Application No. 60/959,437 are considered part of the disclosure of this application, and are incorporated by reference herein in their entirety.

US Referenced Citations (1094)

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	Hat 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	Wiedenmann 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
5576218	Zurek et al.	Nov 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 et al.	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	Bachler 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
7705739	Northrup 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
7785868	Yuan et al.	Aug 2010	B2
D628305	Gorrec et al.	Nov 2010	S
7829025	Ganesan et al.	Nov 2010	B2
7858366	Northrup et al.	Dec 2010	B2
7867776	Kennedy et al.	Jan 2011	B2
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	Aug 2011	B2
8053214	Northrup	Nov 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	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
8592157	Petersen et al.	Nov 2013	B2
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	May 2015	B2
9051604	Handique	Jun 2015	B2
9080207	Handique	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	Oct 2020	B2
10821436	Handique et al.	Nov 2020	B2
10821446	Handique et al.	Nov 2020	B1
10822644	Steel et al.	Nov 2020	B2
10843188	Handique et al.	Nov 2020	B2
10844368	Duffy et al.	Nov 2020	B2
10857535	Handique et al.	Dec 2020	B2
10865437	Handique et al.	Dec 2020	B2
10875022	Williams et al.	Dec 2020	B2
10900066	Handique	Jan 2021	B2
10913061	Handique et al.	Feb 2021	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 et al.	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
20030088657	Eggers	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
20040076996	Kondo 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 et al.	Nov 2004	A1
20040240097	Evans	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
20070014695	Yue 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
20070178603	Takii et al.	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 et al.	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
20110060136	Matsunaga et al.	Mar 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
20150174579	Iten et al.	Jun 2015	A1
20150315631	Handique et al.	Nov 2015	A1
20160038942	Roberts	Feb 2016	A1
20170275702	Dahiya et al.	Sep 2017	A1
20180135102	Gubatayao et al.	May 2018	A1
20180333722	Handique	Nov 2018	A1
20190054467	Handique	Feb 2019	A1
20190054471	Williams et al.	Feb 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
20200139363	Handique et al.	May 2020	A1
20200156059	Handique et al.	May 2020	A1
20200156060	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
20210001334	Handique et al.	Jan 2021	A1
20210047676	Wu et al.	Feb 2021	A1
20210060565	Handique et al.	Mar 2021	A1
20210071234	Gubatayao et al.	Mar 2021	A1
20210087609	Handique et al.	Mar 2021	A1
20210121887	Handique et al.	Apr 2021	A1
20210123090	Handique et al.	Apr 2021	A1

Foreign Referenced Citations (249)

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
1934451	Mar 2007	CN
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
H03181853	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
H11-009258	Jan 1999	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 1998035013	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 2000066783	Nov 2000	WO
WO 2000073412	Dec 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 2002043864	Jun 2002	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-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

Non-Patent Literature Citations (444)

Entry
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).
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).
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.
Dittrich et al., “Single-molecule fluorescence detection in microfluidic channels—the Holy Grail in muTAS?”, Anal Bioanal Chem. (2005) 382(8):1771-1782.
EP Communication dated Aug. 9, 2006 for European Patent Application 02723636.3, filed Mar. 27, 2002.
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.
European Supplemental Search Report dated Aug. 5, 2010 for Application No. EP 08826342.1, filed Jul. 11, 2008.
European Supplemental Search Report dated Jul. 13, 2012 for Application No. EP 08843060.8, filed Jul. 14, 2008.
European Extended Search Report dated Feb. 16, 2017 for Application No. EP 16191793.5, filed Sep. 30, 2016.
European Extended Search Report dated May 11, 2017 for Application No. EP 16191787.7, filed Sep. 30, 2016.
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.
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 Preliminary Report on Patentability dated Jan. 19, 2010 for Application No. PCT/US2008/069897, filed Jul. 11, 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 and Written Opinion, dated Oct. 3, 2008, issued in International Application No. PCT/US2008/069897, filed Jul. 11, 2008.
International Search Report dated Jun. 17, 2009 for Application No. PCT/US2008/008640, filed Jul. 14, 2008.
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.
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.
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.
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 Common. (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.
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.
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.
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.
Ateya et al., “The good, the bad, and the tiny: a review of microflow cytometry”, Anal Bioanal Chern. (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.
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.
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 Microfabricated Devices, (2002) Ch. 8, pp. 183-206, CRC Press.
Bell M., “Integrated Microsystems in Clinical Chemistry”, in Integrated Microfabricated 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.
Burns et al., “Microfabricated Structures for Integrated DNA Analysis” Proc. Natl. Acad. Sci. USA (May 1996) 93: 5556-5561.
Cady et al., “Real-time PCR detection of Listeria monocytogenes using an integrated microfluidics platform”, Sensors Actuat B. (2005) 107:332-341.
Carlen et al., “Paraffin Actuated Surface Micromachined Valve,” in IEEE MEMS 2000 Conference, Miyazaki, Japan, (Jan. 2000) pp. 381-385.
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., “Design, fabrication, and packaging of a practical multianalyte-capable optical biosensor,” J Microlith Microfab Microsyst. (2006) 5(2):021105 in 8 pages.
Chang-Yen et al., “Spin-assembled nanofilms for gaseous oxygen sensing.” Sens Actuators B: Chemical (2007), 120(2):426-433.
Chaudhari et al., “Transient Liquid Crystal Thermometry of Microfabricated PCR Vessel Arrays”, J Microelectro Sys., (1998) 7(4):345-355.
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.
Chen et al., “Total nucleic acid analysis integrated on microfluidic devices,” Lab on a Chip. (2007) 7:1413-1423.
Cheng et al., “Biochip-Based Portable Laboratory”, Biochip Tech. (2001):269-289.
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 Polydimethylsiloxane Package for ‘Bio-Microfluidic System’”, IEEE Proceedings Electronic 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.
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.
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”, Electrophoresis. (2008) 29(16):3296-3305.
Dttrich 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 Microfabricated 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):2396-2402.
Edwards, “Silicon (Si),” in “Handbook of Optical Constants of Solids” (Ghosh & Palik eds., 1997) in 24 pages.
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.
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.
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.
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.
Hale et al., “Optical constants of Water in the 200-nm to 200-μm Wavelength Region”, Applied Optics, 12(3): 555-563 (1973).
Handal et al., “DNA mutation detection and analysis using miniaturized microfluidic systems”, Expert Rev Mol Diagn. (2006) 6(1):29-38.
Hansen et al., “Microfluidics in structural biology: smaller, faster . . . better”, Curr Opin Struct Biol. (2003) 13(5):538-544.
Harrison et al., “Capillary Electrophoresis and Sample Injection Systems Integrated on a Planar Glass Chip”, Anal. Chem., (1992) 64: 1926-1932.
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 (clEF): 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.
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.
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., “PCR Array on Chip—Thermal Characterization”, IEEE Sensors (2003) Conference Oct. 22-24, 2003; pp. 1045-1048.
Irawan et al., “Cross-Talk Problem on a Fluorescence Multi-Channel Microfluidic Chip System,” Biomed Micro. (2005) 7(3):205-211.
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.
Jiang et al., “Directing cell migration with asymmetric micropatterns” Proc. Natl. Acad. Sci. USA (2005) 102,975-978.
Kaigala et al., “An inexpensive and portable microchip-based platform for integrated RT-PCR and capillary electrophoresis”, 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.
Khandurina et al., “Bioanalysis in microfluidic devices,” J Chromatography A, (2002) 943:159-183.
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., “Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system” Sensors and Actuators B (2000) 63:138-146.
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 Chern. (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.
Liao et al., “Miniature RT-PCR system for diagnosis of RNA-based viruses,” Nucl Acids Res. (2005) 33(18):e156 in 7 pages.
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., “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.
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.
Malitson, “Interspecimen Comparison of the Refractive Index of Fused Silica,” J Optical Society of America, 55:1205-1209 (1965).
Manz et al., “Miniaturized Total Chemical Analysis Systems: a Novel Concept for Chemical Sensing,” Sensors and Actuators B1, (1990) 244-248.
Manz et al., “Design of an open-tubular column liquid chromatograph using silicon chip technology” Sensors and Actuators B (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.
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 System” (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.
Minco, “Conductive Heating Technologies for Medical Diagnostic Equipment,” (2006) in 13 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., “An integrated microfluidic for influenza and other genetic analyses,” Lab Chip, (2005), 5:1024-1032.
Palina et al., “Laser Assisted Boron Doping of Silicon Wafer Solar Cells Using Nanosecond and Picosecond Laser Pulses,” 2011 37th IEEE Photovoltaic Specialists Conference, p. 002193-002197, IEEE (2011).
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.
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.
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.
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.
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 gonorrhea,” 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 Transcription—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.
Rhee et al., “Drop Mixing in a Microchannel for Lab-on-a-Chip Applications” Langmuir (2008) 24 (2): 590-601.
Rodriguez et al., “Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis,” Electrophoresis, (2003), 24:172-178.
Rohsenow et al. [Eds.], Handbook of Heat Transfer, 3rd Edition McGraw-Hill Publishers (1998) Chapters 1 & 3; p. 108.
Roper et al., “Advances in Polymer Chain Reaction on Microfluidic Chips,” Anal. Chern., (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.
Sammarco et al., “Thermocapillary Pumping of Discrete Drops in Microfabricated Analysis Devices” AIChE Journal (1999) 45(2): 350-366.
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”, Anal 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, p. 4710521.
Schäferling et al., “Optical technologies for the read out and quality control of DNA and protein microarrays,” Anal Bioanal Chern, (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.
Shen et al., “A microchip-based PCR device using flexible printed circuit technology,” Sensors and Actuators B (2005), 105:251-258.
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., “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.
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), Chapter 10, pp. 97-129.
Squires et al., “Microfluidics: Fluid physics at the nanoliter scale”, Rev Modern Phys. (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., “Optimization of the performance of the polymerase chain reaction in silicon-based microstructures” Nucleic Acids Res. (1997) vol. 25, pp. 3164-3168.
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.
Terry et al., “A Gas Chromatographic Air Analyzer Fabricated on a Silicon Wafer” IEEE T Electron Dev (1979) 26:1880-1886.
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.
Velten et al., “Packaging of Bio-MEMS: Strategies, Technologies, and Applications,” IEEE Transactions on Advanced Packaging, (2005) 28(4):533-546.
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 Polymerase Chain Reaction Chip Utilizing Multiple Membrane-activated Micropumps,” (2006), MEMS 2006, Jan. 22-26, 2006, Istanbul, Turkey; 374-377.
Whitesides G.M., “The origins and the future of microfluidics” Nature (2006) 442(7101):368-373.
Woias P., “Micropumps—past, progress and future prospects” Sensors and 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.
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.
Wu et al., “Fabrication of Complex Three-dimensional Microchannel Systems in PDMS” J. Am. Chem. Soc. (2003) 125, 554-559.
Xiang et al., “Real Time PCR on Disposable PDMS Chip with a Miniaturized Thermal Cycler,” Giomedical 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-Eased 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 6, (2002), 82:75-81.
Zhang et al., “PCR Microfluidic Devices for DNA Amplification,” Biotechnology Advances, 24:243-284 (2006).
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 Gioarrays,” J Am Chem Soc., (2007), 129:9252-9253.
Zhang et al., “Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends,” Giotechnology Advances, (2007), 25:483-514.
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.
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.
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., “A Micromachined Integratable Thermal Reactor,” technical digest from International Electron Devices Meeting, IEEE, Washington, D.C., Dec. 2-5, 2001 (6 pages).
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.
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).
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).
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 pages).
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).
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).
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).
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).
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).
Petition for Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 2 in IPR2020-01137) dated Jun. 19, 2020 (86 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).
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).
Declaration of Mark A. Burns, Ph.D. (Exhibit N1101 in IPR2020-01132 and IPR2020-01133) dated Jun. 17, 2020 (253 pages).
Declaration of Mark A. Burns, Ph.D. (Exhibit N1201 in IPR2020-01136 and IPR2020-01137) dated Jun. 19, 2020 (205 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. Patent 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. dated Oct. 4, 2019 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (31 pages).
First Amended and Supplemental Complaint filed by Becton, Dickinson and Company et al. dated 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. dated Jul. 16, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (42 pages).
BDProbeTec™ ET Neisseria gonorrhoeae Amplified DNA Assay Package Insert, Jul. 2010 (13 pages).
BDProbeTec™ ET System Brochure, Aug. 2010 (9 pages).
Devarakonda et al., “The effect of PAMAM dendrimer generation size and surface functional group on the aqueous solubility of nifedipine”, Int J Pharma. 284(1-2): 133-140.
Gill et al., “Nucleic Acid Isothermal Amplification Technologies—A Review”, Nucleosides Nucleotides Nucleic Acids, (2008) 27(3): 224-243.
Northrup et al., “A MEMS-based Miniature DNA Analysis System.” Transducers '95—Eurosensors in Proc. 1995 (8th) IEEE Int. Conf. Solid-State Sens. Actuators, pp. 764-767.
Rush et al., “Dispersion by Pressure-Driven Flow in Serpentine Microfluidic Channels”, Ind Eng Chern Res., (2002) 41: 4652-4662.
Walker et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique”, Nucleic Acids Res. (1992) 20(7): 1691-1696.
Petition for Inter Partes Review of U.S. Pat. No. 10,625,262 (Paper 2 in IPR2021-00250) dated Nov. 25, 2020 (107 pages).
Petition for Inter Partes Review of U.S. Pat. No. 10,625,261 (Paper 2 in IPR2021-00251) dated Nov. 25, 2020 (117 pages).
Petition for Inter Partes Review of U.S. Pat. No. 10,632,466 (Paper 2 in IPR2021-00253) dated Nov. 25, 2020 (121 pages).
Declaration of Mark A. Burns, Ph.D. (Exhibit N1001 in IPR2021-00250, IPR2021-00251 and IPR2021-00253) dated Nov. 24, 2020 (311 pages).
Declaration of James L. Mullins, Ph.D. (Exhibit N1029 in IPR2021-00250, IPR2021-00251, and IPR2021-00253) dated Nov. 18, 2020 (54 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 14 in IPR2020-01091) dated Dec. 4, 2020 (21 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,703,069 (Paper 14 in IPR2020-01095) dated Dec. 4, 2020 (22 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,703,069 (Paper 15 in IPR2020-01100) dated Dec. 4, 2020 (19 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,273,308 (Paper 14 in IPR2020-01083) dated Jan. 7, 2021 (24 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 20 in IPR2020-01133) dated Jan. 20, 2021 (67 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 19 in IPR2020-01132) dated Jan. 20, 2021 (78 pages).
Declaration of M. Allen Northrup, Ph.D. in support of Patent Owner Preliminary Responses in IPR2020-01132 and IPR2020-01133 (Exhibit H2016) dated Jan. 20, 2021 (154 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 19 in IPR2020-01136) dated Jan. 20, 2021 (77 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 19 in IPR2020-01137) dated Jan. 20, 2021 (69 pages).
Declaration of M. Allen Northrup, Ph.D. in support of Patent Owner Preliminary Responses in IPR2020-01136 and IPR2020-01137 (Exhibit H2016) dated Jan. 20, 2021 (111 pages).
Opening Brief [Corrected] of Appellants Qiagen North American Holdings, Inc. and NeuMoDx Molecular Inc. in Appeals from the USPTO, PTAB, in Nos. IPR2019-00488, IPR2019-00490, IPR2019-01493 and IPR2019-01494 filed Jan. 22, 2021 in U.S. Court of Appeals for the Federal Circuit Case Nos. 20-2249, 20-2250, 20-2273 and 20-2276 (82 pages).
Decision Granting Institution of Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 20 in IPR2020-01132) dated Apr. 19, 2021 (33 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 21 in IPR2020-01133) dated Apr. 19, 2021 (24 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 20 in IPR2020-01136) dated Apr. 19, 2021 (19 pages).
Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 20 in IPR2020-01137) dated Apr. 19, 2021 (14 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 10,625,262 (Paper 6 in IPR2021-00250) dated Apr. 19, 2021 (71 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 10,625,261 (Paper 6 in IPR2021-00251) dated Apr. 19, 2021 (82 pages).
Patent Owner's Preliminary Response to Petition for Inter Partes Review of U.S. Pat. No. 10,632,466 (Paper 6 in IPR2021-00253) dated Apr. 19, 2021 (66 pages).
Declaration of James P. Landers, Ph.D. in support of Patent Owner Preliminary Responses in IPR2021-00250, IPR2021-00251, and IPR2021-00253 (Exhibit H2003) dated Apr. 19, 2021 (189 pages).
Defendant NeuModx's Joint Claim Construction Chart [Exhibit N1023] filed Oct. 21, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (25 pages).
Defendant NeuModx's Amended Answer, Affirmative Defenses, and Counterclaims to Plaintiffs' First Amended and Supplemental Complaint filed Dec. 11, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (43 pages).
Second Amended and Supplemental Complaint filed by Becton, Dickinson and Company et al. on Feb. 25, 2021 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (75 pages).
Defendant NeuMoDx's First Supplemental Invalidity Contentions filed Mar. 17, 2021 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (55 pages).
Defendant NeuModx's Answer, Affirmative Defenses, and Counterclaims to Plaintiffs' Second and Supplemental Complaint filed Mar. 18, 2021 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (67 pages).
Plaintiffs' Answer and/or Reply to Defendants' Counterclaims and Counterclaims-ln-Reply filed Apr. 22, 2021 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (127 pages).
Claim Construction (Markman) Order dated May 10, 2021 in in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (30 pages).
Brief for Appellee HandyLab, Inc. in Appeals from the USPTO, PTAB, in Nos. IPR2019-00488, IPR2019-00490, IPR2019-01493 and IPR2019-01494 filed May 24, 2021 in U.S. Court of Appeals for the Federal Circuit Case Nos. 20-2249, 20-2250, 20-2273 and 20-2276 (74 pages).
Reply Brief of Appellants Qiagen North American Holdings, Inc. and NeuMoDx Molecular, Inc. in Appeals from the USPTO, PTAB, in Nos. IPR2019-00488, IPR2019-00490, IPR2019-01493 and IPR2019-01494 filed Jun. 21, 2021 in U.S. Court of Appeals for the Federal Circuit Case Nos. 20-2249, 20-2250, 20-2273 and 20-2276 (44 pages).

Related Publications (1)

	Number	Date	Country
	20210010059 A1	Jan 2021	US

Provisional Applications (3)

Number	Date	Country
60959437	Jul 2007	US
60859284	Nov 2006	US
60786007	Mar 2006	US

Continuations (4)

	Number	Date	Country
Parent	14796239	Jul 2015	US
Child	16787977		US
Parent	13692929	Dec 2012	US
Child	14796239		US
Parent	13035725	Feb 2011	US
Child	13692929		US
Parent	11985577	Nov 2007	US
Child	13035725		US

Continuation in Parts (1)

	Number	Date	Country
Parent	11728964	Mar 2007	US
Child	11985577		US

Microfluidic system for amplifying and detecting polynucleotides in parallel

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

Field of Search

CPC

International Classifications

Disclaimer

Abstract