Patent Grant
11453906
The apparatus and methods disclosed herein relate generally to the high-throughput, automated execution of nucleic acid based assays, such as Polymerase Chain Reaction (PCR) in a microfluidic cartridge.
The medical diagnostics industry is a critical element of today's healthcare infrastructure. At present, however, in vitro 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 specialized equipment that is both expensive and only operable by trained clinicians. Such equipment may be found in only a few locations—often just one in any given urban area. This requires hospitals to send out samples for analyses to these locations, thereby incurring shipping costs and transportation delays, and possibly even sample loss or mishandling. Second, the equipment in question is typically not available “on-demand” but instead runs in batches, thereby delaying the processing time for many samples as they must wait for a machine to reach capacity before they can be run.
Understanding that diagnostic assays on biological samples may break down into several key steps, it is often desirable to automate one or more steps. For example, a biological sample, such as those obtained from a patient, can be used in nucleic acid amplification assays, in order to amplify a target nucleic acid (e.g., DNA, RNA, or the like) of interest. Once amplified, the presence of a target nucleic acid, or amplification product of a target nucleic acid (e.g., a target amplicon) reactor can be detected, wherein the presence of a target nucleic acid and/or target amplicon is used to identify and/or quantify the presence of a target (e.g., a target microorganism or the like). Often, nucleic acid amplification assays involve multiple steps, which can include nucleic acid extraction, nucleic acid amplification, and detection. It is desirable to automate certain steps of these processes.
There is a need for improved methods and devices for carrying out diagnostic assays on multiple biological samples in parallel. The embodiments described herein address this need and can advantageously be used in high throughput and multiplexed amplification and detection.
Certain embodiments disclosed herein contemplate a microfluidic cartridge having a plurality of sample lanes, each lane comprising a microfluidic network having, in fluid communication with one another, an inlet, an amplification chamber, a first amplification valve upstream of the amplification chamber and a second amplification valve downstream of the amplification chamber, a first amplification gate upstream of the amplification chamber and a second amplification gate downstream of the amplification chamber, a first channel leading from the inlet, via the first amplification valve and first amplification gate, to the amplification chamber, a plurality of detection chambers, a first detection valve upstream of the plurality of detection chambers and a second detection valve downstream of the plurality of detection chambers, and a second channel leading from the amplification chamber, via the second amplification gate and first detection valve, to the plurality of detection chambers.
Certain embodiments can further include a third channel in each of the microfluidic networks, the third channel leading from the amplification chamber, via the second amplification valve, to a first vent. Certain embodiments can further include a fourth channel in each of the microfluidic networks, the fourth channel leading from the plurality of detection chambers, via the second detection valve to a second vent.
Each of the plurality of sample lanes can be configured to amplify one or more polynucleotides independently of the other lanes and the amplification can be conducted by real-time amplification in the amplification chambers. Each of the inlets can be configured to accept a quantity of sample from a pipette tip and the quantity of each sample can be from 0.01-50 μl, e.g., 1-20 μl, such as 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 0.5 μl, 0.6 μl, 0.7 μl, 0.8 μl, 0.9 μl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, or more, or any amount in between. The inlets of the respective plurality of sample lanes can be spaced apart from one another to permit simultaneous loading from a multiple-pipette head dispenser. The amplification valves can include a temperature responsive substance that melts upon heating to seal a channel that communicates with the amplification chamber. The detection valves can comprise a temperature responsive substance that melts upon heating in order to seal a channel that communicates with the plurality of detection chambers. The amplification gates can include a temperature responsive substance that melts upon heating to open a channel that communicates with the amplification chamber. The detection gates can comprise a temperature responsive substance that melts upon heating in order to open a channel that communicates with the plurality of detection chambers. The amplification chamber can have a volume of 1-25, e.g., 5-10 μl. The plurality of detection chambers can each have a volume less than the volume of the corresponding amplification chamber, e.g., approximately 1 μl-5 μl. In some embodiments, the microfluidic cartridge of can have 12 sample lanes. In some embodiments, the microfluidic cartridge can include one or more detection windows disposed over the detection chambers.
Certain embodiments disclosed herein contemplate a diagnostic apparatus comprised of the microfluidic cartridge, and which further includes a plurality of separately controllable amplification heat sources thermally coupled to the amplification chambers. The apparatus can further include a plurality of detection heat sources thermally coupled to the detection chambers, wherein each amplification valve and each detection valve comprises a separately controllable valve heat source thermally coupled thereto, and each amplification gate comprises a separately controllable gate heat source thermally coupled thereto.
The apparatus can further include a multiple-pipette head dispenser, wherein the dispenser can be configured to introduce a plurality of samples into the plurality of sample lanes simultaneously. The dispenser can be configured to apply physical pressure to the microfluidic cartridge in order to bring the cartridge and the heaters in thermal communication with each other. The dispenser can be configured to introduce fluidic pressure into the plurality of sample lanes in order to move the plurality of samples.
In some embodiments, the detection heat source for each detection chamber can be individually and separately controllable. In other embodiments, the detection heat sources can also be separately controllable in pairs, in threes, fours, fives, sixes, sevens, eights, nines, tens, elevens, twelves, etc., wherein. In some embodiments, the detection heat sources themselves can also be separately controllable as a group of heat sources associated with the detection chambers of one (or more) sample lane(s).
The apparatus can further include an detector head having plurality of detector pairs, wherein each detector pair is comprised of an LED and a photodiode and each detector pair can be configured to detect the presence of one or more analyte(s), e.g., target nucleic acids of an amplified sample in the detection chambers. Two detector pairs of the detector head can be aligned collinearly and configured to detect the presence of one or more analyte(s) in two detection chambers of the plurality (e.g., six) detection chambers associated with one sample lane.
Certain embodiments of the disclosure include a method of amplifying a target nucleic acid in a sample including providing a microfluidic network, introducing the sample into the microfluidic network, the network comprising an amplification chamber and a plurality of detection chambers downstream of the amplification chamber, isolating the sample in the amplification chamber by closing amplification valves in the microfluidic network upstream and downstream of the amplification chamber, thermal cycling the amplification chamber under amplification conditions to create an amplified sample, wherein the amplified sample comprises a target amplicon when the sample comprises the target nucleic acid, opening an amplification gate upstream of the amplification chamber; and moving the amplified sample downstream to the plurality of detection chambers.
In certain embodiments, isolating the sample in the amplification chamber by closing amplification valves can include heating a temperature responsive substance to seal one or more channels that communicate with the amplification chamber and opening an amplification gate upstream of the amplification chamber can include heating a temperature responsive substance to remove the substance from an upstream channel in communication with the amplification chamber. Moving the amplified sample downstream can include applying fluidic pressure to the network and applying fluidic pressure to the network can include discharging fluid from a pipette tip into an inlet of the microfluidic network. The fluid discharged from the pipette tip may be a gas.
The method of amplifying a target nucleic acid can also include isolating the sample in the plurality of detection chambers by closing detection valves upstream and downstream of the plurality of detection chambers and contacting the amplified sample in one of more of the plurality of detection chambers with a detection probe, wherein the detection probe is specific for the target amplicon. The method can also include detecting the presence of the target amplicon in the sample in at least one of the detection chambers, which can be performed by contacting the amplified sample with a detection probe, wherein the detection probe is specific for the target amplicon. The detection probe can include a fluorescent moiety, and detecting the presence of the target amplicon in the amplified sample can include emitting light from a light source to activate the detection probe, and measuring the fluorescence of the detection probe with a photodiode.
In certain embodiments of the disclosure, the method can also include applying physical pressure to the microfluidic network in order to place the network and one or more heat sources in thermal communication. Applying physical pressure can include applying a force from a pipette head dispenser on the microfluidic network. The method of amplifying a target nucleic acid in a sample can be performed simultaneously on a plurality of networks, the plurality of networks comprising a microfluidic cartridge.
The method can further include amplifying a second target nucleic acid in the sample, wherein the amplified sample can include a second target amplicon when the sample comprises the second target nucleic acid. The method can further include detecting the presence of the target amplicon, the second target amplicon, or both in at least one of the detection chambers. Detecting the presence of the second target amplicon can include contacting the amplified sample with a second detection probe, wherein the second detection probe is specific for the second target amplicon. The second detection probe can be a second fluorescent moiety, and detecting the presence of the second target amplicon in the amplified sample can include emitting light from a light source to activate the second detection probe, and measuring the fluorescence of the second detection probe with a photodiode. The first fluorescent moiety and the second fluorescent moiety can be the same or the first fluorescent moiety and the second fluorescent moiety can be different. The detection probe and the second detection probe can be a capture nucleic acid and second capture nucleic acid, respectively, and the Tm of the detection probe and the second detection probe can be different. The detection probe can further include a quencher moiety. The second detection probe can include a second quencher moiety.
The thermal cycling can be an assay selected from the group consisting of: polymerase chain reaction, ligase chain reaction, nucleic acid sequence-based amplification, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification. The thermal cycling can be a multiplex polymerase chain reaction. The thermal cycling of each of the plurality of networks can be independently controlled.
Before the embodiments are further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the embodiments. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the embodiments, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the embodiments, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Definitions
In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
As used herein, the term “microfluidic” refers to volumes of less than 1 ml, preferably less than 0.9 ml, e.g., 0.8 ml, 0.7 ml, 0.6 ml, 0.5 ml, 0.4 ml, 0.3 ml, 0.2 ml, 0.1 ml, 90 μl, 80 μl, 70 μl, 60 μl, 50 μl, 40 μl, 30 μl, 20 μl, 10 μl, 5 μl, 4 μl, 3 μl, 2 μl, 1 μl, 0.9 μl, 0.8 μl, 0.7 μl, 0.6 μl, 0.5 μl, 0.4 μl, 0.3 μl, 0.2 μl, 0.1 μl, or less, e.g., nanoliter volumes in the range of 10-500 nanoliters, such as 100 nanoliters.
As used herein, the term “cartridge” refers to 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.
As used herein, the term “TRS”, “thermo-responsive substance” or “temperature responsive substance” refers to a substance that changes physical character upon heating, and that is relatively immobile at a first temperature and more mobile at a second temperature. A mass of TRS can be an essentially solid mass or an agglomeration of smaller particles. Examples of TRS's include, but are not limited to a eutectic alloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, and combinations thereof. A 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).
As used herein, the term “target nucleic acid” refers to a nucleic acid of interest. The term nucleic acid can refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
“Nucleic acid amplification assay” refers to any procedure that amplifies target nucleic acids from a template, including but not limited to polymerase chain reaction, e.g., multiplex PCR, ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) (Persing et al. (1993) Diagnostic Molecular Microbiology: Principles and Applications (American Society for Microbiology, Washington, D.C.).
Tm, or “melting temperature” refers to the temperature at which half of a given number of nucleic acid duplexes have separated into single strands—a phenomenon also referred to as “denaturing” or “de-annealing.” The melting temperature of a probe or primer—more precisely, the duplex formed by the probe or primer and a complementary single oligonucleotide strand—represents the single most widely used parameter to guide the design of probes and primers in assays involving hybridization. Many commercial software packages are available for this purpose, e.g., OLIGO™, VISUALOMP™, PRIMERSELECT™, ARRAY DESIGNER™, PRIMER3™, and others.
Diagnostic Apparatuses
Provided herein are diagnostic devices, or diagnostic apparatuses, that are configured to test whether an analyte of interest, e.g., a target nucleic acid, is present in a sample. More specifically, the diagnostic devices disclosed herein are configured for the amplification and/or detection of target nucleic acids in a sample, within a microfluidic cartridge.
In accordance with the embodiments disclosed herein, provided is a diagnostic device or diagnostic apparatus that comprises one or more of the following components: a microfluidic cartridge, a plurality of heat sources, a pipette-head dispenser, and an optical module. The components may be integral to the diagnostic device. Alternatively, the components may be removably incorporated into the device. For example, the microfluidic cartridge of the device can represent a removable/disposable component of the device as a whole, whereas the heat sources can be integral to the device.
The diagnostic devices disclosed herein are preferably configured to perform a plurality of nucleic acid amplification reactions in a plurality of microfluidic amplification chambers within a microfluidic cartridge, as well as to detect a plurality of target nucleic acids within a plurality of detection chambers within the microfluidic cartridge.
In some embodiments, the devices are configured to enable thermal cycling (e.g., cycles of heating and cooling) of amplification chambers and/or detection chambers within a microfluidic cartridge. Thermal cycling of the reaction chambers and detection chambers can be used to enable amplification of nucleic acids, e.g., target nucleic acids, as well as detection of nucleic acids, e.g., by observing the melting curves of amplified sample.
A PCR protocol may comprise guidelines for performing the successive annealing and denaturing of the polynucleotides in the reaction chamber prior to detection. Such guidelines, comprising a time profile for heating the chamber, may be referred to as a “protocol.” In certain embodiments, the apparatus may comprise an aperture plate which facilitates consistent heating and cooling of various reaction chambers, e.g., amplification chambers and/or detection chambers within a microfluidic cartridge by applying pressure to the cartridge.
Turning to the figures,
As seen in the embodiments shown in
In some embodiments, each specimen rack 24a, 24b can include multiple holders 26. The holders 26 can include receptacles for holding diagnostic reagents, such as nucleic acid amplification reagents. The racks 24 may also include specimen tubes (not shown) and mixing tubes (not shown) for preparing diagnostic-ready samples, such as amplification-ready samples. The apparatus 10 may prepare the desired reagents in the racks 24a, 24b using the dispenser 400. Further description of various fluid dispensers may be found in e.g., U.S. Patent Application Publication 2009-0130719 and U.S. Patent Application Publication 2009-0155123, incorporated herein by reference in their entirety. The prepared fluids may then be transferred to a microfluidic cartridge and be inserted into heater/optical modules 500a, 500b for processing and analysis.
In some embodiments, the reaction chambers within the microfluidic cartridge(s) (discussed below) can include one or more reagents, buffers, etc., used in the assays described herein, e.g., amplification and/or detection. For example, in some embodiments, the reaction chambers of the microfluidic cartridge can include, e.g., amplification primers, probes, nucleotides, enzymes such as polymerase, or the like. By way of example, in some embodiments, the reaction chambers can include lyophilized reagents, to which processed biological sample (e.g., a solution of extracted nucleic acids) can be added.
Also shown in
As will be described in more detail below, the diagnostic apparatus 10 may be configured to conduct real-time analysis on one or more samples. The sample to be tested may first be placed in a specimen tube (not shown) on the rack 24a or 24b. Reagents such as amplification reagents, (e.g., amplification primers, buffers, nucleotides, and the like) and/or detection reagents (e.g., probes and the like) may be located in the holders 26 on the rack 24a inside the diagnostic apparatus 10. The fluid dispenser 400 may mix and prepare the sample for diagnostic testing and may then deliver the prepared sample to the microfluidic cartridge 200 for thermal cycling and target nucleic acid detection in the heater/optical modules 500a, 500b. Alternatively, the fluid dispenser 400 may deliver nucleic acid samples to the reaction chambers of the microfluidic cartridge, wherein the reaction chambers, e.g., amplification chambers or detection, of the microfluidic cartridge already contain reagents for an amplification reaction or detection. Certain details and methods for processing polynucleotides may be found in e.g., U.S. Patent Application Publication 2009-0131650, U.S. Patent Application Publication 2010-0009351, U.S. Patent Application Publication 2009-0134069, all of which are incorporated herein by reference in their entirety.
As illustrated in
The cartridge 200 may include a label 204 attached to the top side of the cartridge 200. The label 204 may include a window portion 210 through which the detection chambers may be optically accessible by the detector head as discussed in further detail below, for the detection of target nucleic acids, e.g., target amplicons.
Microfluidic Cartridges
The term “cartridge,” as used herein, refers to a unit that may be disposable, or reusable in whole or in part, and that can be 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 heat, light, and the like, to) the cartridge. By microfluidic, as used herein, is meant that volumes of sample, and/or reagent, and/or amplified polynucleotide are from about 0.001 μl to about 999 μl, such as from 1-100 μl, or from 2-25 μl, as defined above. 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. Certain embodiments herein can also function with nanoliter volumes (in the range of 10-500 nanoliters, such as 100 nanoliters).
The cartridge 200 may comprise a plurality of sample lanes. The microfluidic cartridge 200 shown in
As described in more detail below with relation to
A microfluidic cartridge 200 may be constructed from a number of layers. Accordingly, one aspect of the present embodiments 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 amplification and detection on a sample in which the presence or absence of one or more polynucleotides is to be determined. In another embodiment, the microfluidic cartridge 200 can comprise a plurality of lanes, each including a reaction chamber, etched or molded in a single plane, such as in a molded plastic substrate, with each lane being closed by a cover layer, such as an adhesive plastic film layer. Embodiments with 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, or more lanes per cartridge are contemplated. For example, one suitable design is a single cartridge 200 having 12 sample lanes, each lane having an amplification chamber and six detection chambers.
In understanding the relative orientation of the cartridge 200, a sample lane 300 can be considered a column on the cartridge 200, running along a y-axis and multiple lanes 300a-l can be aligned side by side, in the direction of an x-axis. In certain embodiments disclosed herein, the x and y axes designated for the cartridge 200 may correspond to the x and y axes designated for the diagnostic apparatus 10 in
The spatial arrangement of the sample lane shown in
In the microfluidic network of the sample lane 300, the inlet 302 is the point at which a sample is input by the fluid dispenser 400 into the microfluidic network. The various components of the microfluidic network are spatially arranged in the sample lane 300 such that the inlet 302 can be considered the most upstream portion of the microfluidic network, meaning that in the progression of fluidic movement throughout the microfluidic network, all other components can be downstream of the inlet 302. The vent 368 and 376 are the most downstream components of the microfluidic network, meaning that in the progression of fluidic movement throughout the microfluidic network of the sample lane 300, the vents 368 and 376 are the components in the microfluidic network that are reached last as the fluid travels in the varying paths of the microfluidic network.
The inlet 302 can be manufactured to be frustoconical in shape with an appropriate conical angle so that industry-standard pipette tips (e.g., 2 μl, 10 μl, 20 μl, 200 μl, volumes, etc.) fit snugly, entering from the widest point of the inlet. Thus, in certain embodiments, an inlet comprises an inverted frustoconical structure of at least 1 mm height, and having a diameter at its widest point that accepts entry of a pipette tip, of from 1-5 mm. The apparatus herein may be adapted to suit other, later-arising, industry standards for pipette tips not otherwise described herein. Typically the volume of sample accepted via an inlet into a microfluidic network in a sample lane is from 0.1-20 μl, e.g., 5-10 μl. The fluid dispenser 400 can used to appropriately place the pipette tip in the inlet 302 of the sample lane 300. The inlet hole can be designed to fit a pipette tip snugly and to create a good seal around the pipette tip, within the cone of the inlet hole. Once the pipette tip lands within the cone, the conical shape guides the pipette and mechanically seals the combination to provide error free dispensing or withdrawal of fluid into the cartridge 200. However, the cone is designed such that the sealing is reversible to avoid pulling the cartridge 200 away from the recessed bay 524 in the receiving tray 520, when the pipette tips are lifted after completion of the dispensing operations.
As part of the first stage 206, the microfluidic network extends downstream from the inlet 302 along the first channel 360. The first channel 360 extends past, and is in fluid communication with, the amplification gate 310a and the amplification valve 330a. Downstream of the amplification gate 310a and the amplification valve 330a, the first channel can extend to, and be in fluid communication with, the amplification chamber 362. The second channel 364, which can be in fluid communication with the amplification chamber 362, can extend from the amplification chamber 362 through the second amplification gate 310b and through the first detection valve 370a, in the second stage 208 of the sample lane 300. In this manner, the second channel 364 extends from the first stage 206 of the microfluidic network of the sample lane 300 to the second stage 208. In the first stage 206, the third channel 366 is in fluid communication with the second channel 364 and can extend downstream from the second channel 364 at a point upstream of the second amplification gate 310b. The third channel 366 can be in fluid communication with the second amplification valve 330b and can extend from the second channel 364 to the first vent 368, and can be in fluid communication with the first vent 368.
In the second stage 208 of the microfluidic network, the second channel 364 can extend downstream through, and be in fluid communication with, the first detection valve 370a. Extending past the first detection valve 370a, the second channel 364 can split into two subchannels 365a and 365b, both of which are in fluid communication with the second channel 364. The subchannels 365a, 365b can each split again into additional subchannels 365a1-3 and 365b1-3, respectively. Subchannels 365a1-3 can extend through, and can be in fluid communication with, the detection chambers 372a-c, respectively. Likewise, subchannels 365b1-3 can extend through, and be in fluid communication with, the detection chambers 372d-f. Extending from detection chambers 372a-c, subchannels 365a1-3 can connect together again downstream of the detection chambers 372a-c as subchannel 367a. Extending from detection chambers 372d-f, subchannels 365b1-3 can connect together again downstream of the detection chambers 372d-f as subchannel 367b. Further downstream, subchannels 367a and 367b can connect together to form a fourth channel 374, which can extend to, and be in fluid communication with, the second vent 376. Along the fourth channel 374, upstream of the second vent 376, can be positioned a second detection valve 370b in fluid communication with the fourth channel 374. The vents 368 and 376 may be configured to drain excess sample from the microfluidic network and prevent a user from introducing any excess amount of liquid into the microfluidic cartridge 200.
The amplification chamber 362 can have a volume of about 5-10 μl, and particular embodiments of the amplification chamber 362 have a volume of about 8 μl. For amplification chambers of about 8 μl, the input volume of the sample can be approximately 10 μl. Sample input volumes may be greater than the volume of the amplification chamber 362 in order to account for sample volumes located in the channels and detection chambers 372a-f. Excess volume may also be drained from the microfluidic network through the vents 368 and 376.
In certain embodiments, the detection chambers 372a-f can each have a volume of about 1 μl. In some embodiments, the detection chambers 372a-f are of a volume equal to each other. In some embodiments, the detection chambers 372a-f may be of varying volumes. The detection chambers 372a-f may individually be of a volume less than the volume of the amplification chamber 362. In some embodiments, the detection chambers 372a-f may collectively be of a volume less than the volume of the amplification chamber 362.
In some embodiments, the inside walls of the channels in the amplification chamber 362 and detection chambers 372a-f can be made very smooth and polished to a shiny finish (for example, using a polish such as SPI A1, SPI A2, SPI A3, SPI B1, or SPI B2) during manufacture. The amplification chamber 362 and detection chambers 372a-f may be polished in order to minimize any microscopic air trapping in the surface of the chambers, which may cause bubbling during thermocycling. The presence of bubbles, particularly in the detection chambers 372a-f might cause a false reading. Furthermore, the amplification 362 and detection chambers 372a-f can be made shallow such that the temperature gradient across the depths of the chambers is minimized.
Turning now to
As shown in
As shown in
The TRS 345 can be deposited into the loading port 332 machined in the microfluidic substrate of the microfluidic cartridge 200. The loading port 332 of the microfluidic cartridge 200 can be dimensioned in such a way that a droplet of TRS can be accurately propelled to the bottom of the loading port 332 using, for example, compressed air. The microfluidic cartridge 200 can be maintained at a temperature above the melting point of the TRS thereby permitting the TRS to stay in a molten state immediately after it is dispensed. After the drop falls to the bottom of the loading port 332, the molten TRS 345 is drawn into the valve loading channel 334 by capillary action. The amount of TRS 345 that is dispensed into the loading port 332 can be approximately equal to the volume of the valve loading channel 334. The valve loading channel 334 can be structured so that even though the TRS 345 dispensed into the loading port 332 may vary between a minimum and a maximum dispensed amount, the TRS always fills up to, and stops at, the valve junction 336 because the junction 336 provides a higher cross section than that of the loading channel 334 and thus reduces the capillary forces. When the microfluidic cartridge 200 is cooled to a first temperature, the mass of immobile TRS is situated in the valve loading channel 334, not blocking the first channel 360 at the valve junction 336.
As shown in
A mass of TRS 345 can be deposited into the loading port 314 and passed into the gate loading channel 312. The loading port 314 and the gate loading channel 312 can be dimensioned in such a way that the deposited TRS 345 does not become immobilized in the gate loading channel 312, but extends into the gate junction 316. In some embodiments, the gate junction 316 can be narrower (e.g., approximately 150 μm wide and 100 μm deep) than the upstream 318 and downstream 320 portions of the first channel 360 with which it is in fluid communication. An upstream side 318 as well as a downstream side 320 of the gate junction 316 can be made wide (e.g., approximately 500 μm) and deep (e.g., approximately 500 μm) to help ensure the wax stops at the gate junction 316.
During operation of the diagnostic apparatus 10, a heater may be positioned external to the cartridge 200 and may be in thermal communication with the gate 310a. At the required time during operation of the diagnostic apparatus, the external heater may be used to melt the TRS 345 in gate 310a. The melted TRS 345 may be pushed downstream by upstream fluidic pressure and may move downstream along with the sample 350. The amount of TRS 345 melted and moved out of the gate junction 316 may be minimized for optimal gate 310 opening.
In various embodiments, the gate 310a can be configured to minimize the effective area or footprint of the gate within the network, such as by being bent as shown in
Fluid Dispensers
As mentioned above, the devices disclosed herein can include fluid dispensers. Turning to
In various embodiments, preparation of an amplification-ready sample in the diagnostic apparatus 10 can include one or more of the following steps: contacting a polynucleotide sample from the sample tube 32 with an amplification reagent mixture in one or more of the containers in the holder 26, the reagent mixture comprising a polymerase enzyme and a plurality of nucleotides (in some embodiments, the amplification reagent mixture can further include a positive control polynucleotide and a fluorogenic hybridization probe selective for at least a portion of the portion); in some embodiments, the amplification reagent mixture can be in the form of one or more lyophilized pellets, as stored in a receptacle on a holder, and the method can further include reconstituting the pellet with liquid to create an amplification reagent mixture solution. Various, such as one or more, of the liquid transfer operations associated with the foregoing steps can be accomplished by the automated fluid dispenser 400, having multiple pipette tips 402, under control of a microprocessor (not shown). As discussed below, in some embodiments, reagents, buffers, etc. used in the preparation of an amplification-ready sample are disposed within the microfluidic cartridge itself, e.g., within the amplification chamber. The dispenser can introduce the sample into the microfluidic network, where the sample is mixed with reagents, etc., within the network.
The fluid dispenser 400 can be configured to dispense a solution (e.g., of a prepared sample, amplification reagents, and probes, etc.) into the microfluidic cartridge 200. The fluid dispenser 400 can be configured to travel from a first set of positions above the rack 24 and holders 26 having various containers that hold reagents, etc., to a second set of positions above the cartridge 200, where pipette tips 402 can be inserted into the inlets 302 of the microfluidic cartridge 200. The cartridge 200 can be positioned in the receiving tray 520. The second set of positions is depicted schematically in
In some embodiments, the fluid dispenser 400 can be configured to accept or dispense, in a single operation, an amount of 1.0 ml of fluid or less, such as an amount of fluid in the range 10 nl-1 ml. Specifically, the fluid dispenser 400 can accept and dispense, in a single operation, an amount of about 5-15 μl of the sample. When transferring a sample containing extracted nucleic acid from a pipette tip 402 to an inlet 302 on the microfluidic cartridge 200, for example, using the fluid dispenser 400, a volume of air or other gas can also be introduced into the microfluidic network of the sample lanes 300 to provide propulsion or downstream movement to the sample. The volume of air or gas introduced into the microfluidic network can be between about 0.5 ml and about 5 ml, but depending on the volume of the pipette tip 402 and the volume of air or gas required for movement of the sample within the microfluidic network.
Heater/Optical Modules
After receiving the samples, the receiving tray 520 may be moved (e.g., mechanically or manually) on rails 522 to a position underneath the optical unit 502. In some embodiments, the receiving tray 520 may comprise an auto-loading device, which automatically aligns the cartridge once positioned beneath the optical module 502. In some embodiments, the recessed bay 524 of the receiving tray 520 may contain a heater substrate 600 which can be in thermal or physical communication with the cartridge 200 to activate certain cartridge components and conduct the thermal cycling necessary for nucleotide amplification and detection. In some embodiments, the receiving tray 520 may subsequently be raised to place the cartridge 200 in thermal and/or physical contact with the optical unit 502, such as in contact with an aperture plate 540 on the bottom side 506 of the optical unit 502.
As shown in
The device may comprise a plurality of detector pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more, such as the light source-photodetector pair 726. Each detector pair 726 can be comprised of a light source 726a, such as a light-emitting diode (LED), and a corresponding light detector 726b, such as a photodiode. The light source 726a may selectively emit light in an absorption band of a fluorescent moiety, and the light detector 726b may selectively detect light in an emission band of a fluorescent moiety.
In certain embodiments the light source 726a may comprise a bandpass-filtered diode that selectively emits light in the absorption band of the fluorescent probe. Light detector 726b may comprise a bandpass filtered photodiode that selectively detects light in the emission band of the fluorescent probe. In certain embodiments, a filter 726a1, such as a bandpass filter may be applied to the light source 726a's light. The light from the light source 726a passes through a filter before passing through the sample in the reaction chambers. In certain embodiments, the optical path-length for the light from the reaction chamber to the light detector 726b may be very small. The incident light from light source 726a generates fluorescence in the reaction chambers. Light from the reaction chamber then travels to the light detector 726b. Certain embodiments seek to mitigate any undesired light from entering the detector and thereby adversely affecting the light signal from the reaction chamber.
For the sake of explanation, a collection of cartridges or detector pairs along the length (x-axis) of the cartridge are referred to as a “row” and those along the width (y-axis) are referred to as a “column.” In some embodiments, two or more detector pairs, for example detector pairs 726 and 727 in
Each light source, such as, for example, light source 726a, may be configured to produce light of a wavelength specific to a specific fluorescent moiety contained in a reaction chamber, e.g. a detection chamber. Each light detector, such as for example 726b, may be configured to detect the light emitted from the fluorescent moieties associated with the light produced by the light emitter in the detector pair. The detector pairs may be configured to independently detect a plurality of fluorescent moieties having different fluorescent emission spectra, wherein in each reaction chamber (e.g., detection chamber), emission from each fluorescent moiety can be tracked and correlated to the presence or absence of one particular target polynucleotide. Although folded light paths can be used, one embodiment utilizes a detector and emitter pair where each is in direct optical contact with the reaction (detection) chamber, preferably simultaneously in such contact. Optionally, the photodetector and light source of a detector pair are aligned with the reaction chamber along lines that substantially intersect at an acute angle at the reaction chamber. The angle can be, for example, between about 5 and 70 degrees, preferably between about 8 and 60 degrees, more preferably between about 10 and 50 degrees.
In certain embodiments, the receiving tray 520 places the cartridge 200 in proximity to the heater substrate 600 or aperture layer 540, but does not mechanically couple and/or thereby place the layers in contact with one another. In this manner, the cartridge 200 may be thermally, but not mechanically, coupled to the heater substrate 600. In other embodiments, the receiving tray 520 places the heater substrate 600 in both mechanical and thermal contact with the cartridge 200 and the cartridge 200 in mechanical contact with the aperture layer 540. In various embodiments, the apparatus may include one or more force members (not shown) that are configured to apply pressure to the receiving tray 520 in order to thermally couple the heat sources in the heater substrate 600 to the microfluidic cartridge 200 positioned in the receiving tray 520. The application of pressure may be important to ensure consistent thermal contact between the heater substrate and the reaction chambers, gates, and valves, etc., in the microfluidic cartridge 200. When the receiving tray 520 is in a closed position, thereby being positioned under the aperture plate 540 of the optical unit 502, the force member, such as a motor assembly, positioned below the receiving tray 520 may begin traveling upwards towards the bottom side 506 of the optical unit 502, thereby bringing the receiving tray 520 closer to the optical unit 502. As the receiving tray 520 travels upwards towards the optical unit 502, the cartridge 200 may begin to come in contact with the bottom surface 506 of the aperture plate 540. The cartridge 200 may continue traveling upward until a sufficient pressure is received on the cartridge 200. The aperture plate 540 may be configured to apply an equal pressure across all points of the top of the cartridge 200 and thus, may press the cartridge 200 against the heater substrate 600 with uniform pressure. The aperture plate 540 may be selected to possess properties which facilitate this operation. For example, the material selection of the aperture plate 540 may provide very little deflection of the cartridge 200, when the cartridge 200 is pressed against the aperture plate 540.
The application of uniform pressure of the cartridge 200 against the heater substrate 600 may allow for uniform heating for each of the components of the cartridge 200 when desirable. Although uniform pressure and contact may be obtained between the heaters in the heater substrate 600 and the components (valves, gates, chambers, etc.) of the microfluidic networks in the cartridge 200, the heaters are not necessarily activated simultaneously. Thus, in certain embodiments, application of even pressure does not necessarily result in equal heating of different components of the cartridge 200. In some embodiments, both the activation of a specific heater in the heater substrate 600 along with the pressure applied by the aperture plate 540 to the cartridge 200 activate a particular component of cartridge 200.
In preferred embodiments, each heater in the heater substrate 600 can be a contact heater, such as a resistive heater (or network thereof), a radiator, a fluidic heat exchanger and a Peltier device, or the like. The contact heat source can be configured in the recessed bay 524 to be thermally coupled to one or more distinct locations of the microfluidic cartridge 200 received in the receiving tray 520, whereby the distinct locations are selectively heated (and/or cooled). The contact heat sources can each be configured in the heater substrate 600 to be independently thermally coupled to a different distinct location in a microfluidic cartridge 200 received in the receiving tray 520, whereby the distinct locations are independently heated. The contact heat sources can be configured to be in direct physical contact with distinct locations of a microfluidic cartridge 200 received in the receiving tray 520.
As illustrated in
The heaters in a heater lane 602 may be divided conceptually into first stage heaters 606 and second stage heaters 608, to correspond to the first stage 206 and second stage 208 of the microfluidic cartridge 200. Among the heaters in the first stage 606 may be a first amplification gate heater 610a, a second amplification gate heater 610b, a first amplification valve heater 630a, a second amplification valve heater 630b, and an amplification chamber heater 662. Included in the second stage heaters 608 may be a first detection valve heater 670a, a second detection valve heater 670b, and detection chamber heaters 672a-f.
When the microfluidic cartridge 200 is placed in the recessed bay 524 of the receiving tray 520, the various components (e.g., reaction chambers, valves, and gates) of the microfluidic networks in the sample lanes 300 of the cartridge 200 are aligned adjacent to, and above, the corresponding heaters in the heater substrate 600, and consequently can be in thermal contact with the corresponding heaters in the heater substrate 600. When the microfluidic cartridge 200 is placed in the recessed bay 524, the heaters of the heater substrate 600 may be in physical contact with the respective components. For example, the first amplification gate heater 610a can be aligned adjacent to the first amplification gate 310a; the second amplification gate heater 610b can be aligned adjacent to the second amplification gate 310b; the first amplification valve heater 630a can be aligned adjacent to the first amplification valve 330a; the second amplification valve heater 630b can be aligned adjacent to the second amplification valve 330b; and the amplification chamber heater 662 can be aligned adjacent to the amplification chamber 362. Similarly, the first detection valve heater 670a may be aligned adjacent to the first detection valve 370a; the second detection valve heater 670b may be aligned adjacent to the second detection valve 370b; and the detection chamber heaters 672a-f may be aligned adjacent to the respective detection chambers 372a-f.
In some embodiments, multiple heaters can be configured to simultaneously and uniformly activate to heat their respective corresponding cartridge components of the microfluidic network in the microfluidic cartridge 200. Each heater can be independently controlled by a processor and/or control circuitry used in conjunction with the apparatus 10 described herein. Generally, the heating of microfluidic components (gates, valves, chambers, etc.) in the microfluidic cartridge 200, is controlled by passing currents through suitably configured micro-fabricated heaters, as illustrated in
The heater substrate 600 may also include one or more heat sensors. In order to reduce the number of sensors or heaters required to control the heaters in a heater lane 602, the heaters may be used to sense temperature as well as provide heat, and thereby obviate the need to have a separate dedicated sensor for each heater. For example, the impedance and/or resistance of some materials change with the surrounding temperature. Accordingly, the resistance of the heater/sensors may be used as an indication of temperature when the sensors are not being actively heated.
In some embodiments, the heaters in the heater substrate 600 may be designed to have sufficient wattage to allow the heaters to be grouped in series or in parallel to reduce the number of electronically-controllable elements, thereby reducing the burden on the associated electronic circuitry. Heaters that are grouped together in this manner would be operated under synchronized and substantially simultaneous control.
In some embodiments, the first and second amplification valve heaters 630a, 630b can be grouped and configured to operate under synchronized control. In some embodiments, the first and second amplification gate heaters 610a, 610b can be grouped and configured to operate under synchronized control. In some embodiments, the first and second amplification valve heaters 630a, 630b and the amplification chamber heater 662 can be grouped and configured to operate under synchronized control. In some embodiments, the first and second detection valve heaters 670a, 670b can be grouped and configured to operate under synchronized control. In some embodiments, the detection chamber heaters 672a-f can be grouped and configured to operate under synchronized control. In some embodiments, the first and second detection valve heaters 670a, 670b and the detection chamber heaters 672a-f can be grouped and configured to operate under synchronized control. In some embodiments, the first stage heaters 606 can be grouped and configured to operate under synchronized control. In some embodiments, the second stage heaters 608 can be grouped and configured to operate under synchronized control.
In some embodiments, different combinations of the detection chamber heaters 672a-f can be grouped and configured to operate under synchronized control. For example, the detection chamber heaters on the same side of the second stage heaters 608 can be grouped and configured to operate under synchronized control. For example, in some embodiments, detection chamber heaters 672a-c can be grouped and configured to operate under synchronized control. For example, in some embodiments, detection chamber heaters 672d-f can be grouped and configured to operate under synchronized control.
In some embodiments, the detection chamber heaters on opposite sides of the second stage heaters can be grouped and configured to operate under synchronized control. For example, in some embodiments, the detection chamber heaters in a column, such as detection chamber heaters 672a and 672d, can be grouped and configured to operate under synchronized control. Similar grouping and configuring can be applied to heater groups including detection chamber heaters 672b and 672e and to heater groups including detection chamber heaters 672c and 672f The detection heaters 672a-f can be configured to operate individually and independently or they can be configured to operate in groups of two (pairs), three (thirds), four, five or six, etc., or the like.
In some embodiments, the heating in the heater substrate 600 can be controlled by periodically turning the current on and off to a respective heater 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 micro fabricated heater to a high voltage source (for example, 30V), which can be gated by the PWM signal. In some embodiments, the device includes 48 PWM signal generators. In some embodiments there will be two PWM signal generators associated with each reaction chamber. Operation of a PWM generator includes generating a signal with a chosen, programmable period (the end count) and 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 micro fabricated heater receives current and therefore a greater amount of heat produced.
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.
Methods of Detecting Target Nucleic Acids
In another aspect, provided herein are methods of detecting an analyte, e.g., a target nucleic acid from a sample, using the devices disclosed herein. The devices disclosed herein can advantageously be used to simultaneously analyze a single sample or multiple samples, for a plurality of target polynucleotides. Specifically, the “two-stage” design of the microfluidic cartridge enables for the testing and detection of many more targets than, for example, was previously possible in microfluidic cartridges that have a “one stage” design, where amplification and detection occur in the same chamber.
As used herein, the term “sample” can refer to a clinical specimen or sample from one or any number of sources, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration, peritoneal fluid, pleural fluid, effusions, ascites, and purulent secretions, lavage fluids, drained fluids, brush cytology specimens, biopsy tissue, explanted medical devices, infected catheters, pus, biofilms and semen) of virtually any organism, with mammalian samples, particularly human samples, and environmental samples (including, but not limited to, air, agricultural, water and soil samples) finding use in the invention. In addition, samples can be taken from food processing, which can include both input samples (e.g. grains, milk or animal carcasses), samples in intermediate steps of processing, as well as finished food ready for the consumer.
The microfluidic network in each sample lane 300 shown in
An amplification-ready sample can be prepared by mixing the sample nucleic acids (e.g., extracted sample nucleic acids) with amplification reagents. In some embodiments, the mixing is not performed within the microfluidic network of a microfluidic cartridge. For example, in some embodiments, an amplification-ready sample is prepared by mixing sample nucleic acids and amplification reagents within a container in a reagent holder 26. In other embodiments, the amplification-ready sample is prepared within the microfluidic network, e.g., the sample nucleic acids are mixed amplification reagents within a mixing chamber of the microfluidic network. Regardless of whether the mixture of sample nucleic acids and reagents occurs within the microfluidic network or outside of the microfluidic network, e.g., in a container in a reagent holder 26, the sample nucleic acids are introduced into the microfluidic network via an inlet port 302. Dispensing of the sample (whether amplification-ready or not) into the microfluidic network can be achieved either manually, or, for example, by an automated dispenser 400.
As discussed above, an amplification-ready sample is prepared by mixing nucleic acids from the sample to be analyzed with amplification reagents to create an amplification-ready sample. For example, an amplification-ready sample may include an amplification reagent mixture comprising a polymerase enzyme, a positive control nucleic acid, a fluorescent moiety specific for the positive control nucleic acids, and a plurality of nucleotides, and at least one fluorescent moiety that is selective for a target polynucleotide sequence.
Amplification
In some embodiments, the sample to be analyzed is dispensed within the microfluidic network via inlet port 302 and moved through to amplification chamber 362 as described elsewhere herein. The amplification-ready sample is isolated within the amplification chamber 362 by closing the amplification valves 330a, 330b in the microfluidic network upstream and downstream of the amplification chamber 362, respectively. In some embodiments, this is achieved by heating a TRS 345 to seal one or more channels (e.g., the first and second channels 360, 364 that communicate with the amplification chamber 362. Once isolated in the amplification chamber 362, the device may be activated to thermal cycle the amplification-ready sample within the amplification chamber 362 as described elsewhere herein, to generate an amplified sample. Specifically, as described elsewhere herein, the amplification chamber 362 of the microfluidic network is in thermal communication with, or thermally coupled to, an external heat source (e.g, the heater substrate 600). Thermal cycling of the amplification-ready sample creates an amplified sample, which, when target nucleic acids are present in the sample under analysis, includes amplicons of the target nucleic acids, i.e., target amplicons.
In some embodiments, the sample or specimen is contacted with a set of amplification primers under standard PCR conditions. For a review of PCR technology, including standard PCR conditions, applied to clinical microbiology, see DNA Methods in Clinical Microbiology, Singleton P., published by Dordrecht; Boston: Kluwer Academic, (2000) Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and “PCR Methods and Applications”, from 1991 to 1995 (Cold Spring Harbor Laboratory Press). Non-limiting examples of “PCR conditions” include the conditions disclosed in the references cited herein, such as, for example, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, with an annealing temperature of 72° C.; or 4 mM MgCl2, 100 mM Tris, pH 8.3, 10 mM KCl, 5 mM (NH4)2SO4, 0.15 mg BSA, 4% Trehalose, with an annealing temperature of 59° C., or 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, with an annealing temperature of 55° C., or the like.
In some embodiments, the amplification-ready sample is amplified within the amplification chamber 362 by the polymerase chain reaction (PCR). Generally, in PCR, a target polynucleotide sequence is amplified by reaction with at least one oligonucleotide primer or pair of oligonucleotide primers. In embodiments wherein the sample nucleic acids are amplified by PCR, the amplification ready sample minimally comprises template nucleic acid (except in the case of a negative control as described below) and oligonucleotide primers and/or probes in combination with suitable buffers, salts, and the like, and an appropriate concentration of a nucleic acid polymerase. As used herein, “nucleic acid polymerase” refers to an enzyme that catalyzes the polymerization of nucleoside triphosphates. Generally, the enzyme will initiate synthesis at the 3′-end of the primer annealed to the target sequence, and will proceed in the 5′-direction along the template until synthesis terminates. An appropriate concentration includes one that catalyzes this reaction in the presently described methods. Known DNA polymerases useful in the methods disclosed herein include, for example, E. coli DNA polymerase I, T7 DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase, Thermococcus litoralis DNA polymerase, Thermus aquaticus (Taq) DNA polymerase Pyrococcusfuriosus (Pfu) DNA polymerase, and the like.
In addition to the above components, the reaction mixture of the present methods includes primers, probes, and deoxyribonucleoside triphosphates (dNTPs). Usually the reaction mixture will further comprise four different types of dNTPs corresponding to the four naturally occurring nucleoside bases, i.e., dATP, dTTP, dCTP, and dGTP. In some of the embodiments disclosed herein, each dNTP will typically be present in an amount ranging from about 10 to 5000 μM, usually from about 20 to 1000 μM, about 100 to 800 μM, or about 300 to 600 μM.
As used herein, the terms “primer” and “probe” include, but are not limited to oligonucleotides or nucleic acids. The terms “primer” and “probe” encompass molecules that are analogs of nucleotides, as well as nucleotides. Nucleotides and polynucleotides, as used herein shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as NEUGENE™ polymers), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
In some embodiments, the “primers” or “probes” disclosed herein can contain locked nucleic acids (LNA). “Locked nucleic acids” (LNAs) are ribonucleotides which contain a methylene bridge which joins the 2′ oxygen of the ribose with the 4′ carbon (see
The terms nucleotide and polynucleotide include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′→P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA. The terms also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
It will be appreciated that, as used herein, the terms “nucleoside” and “nucleotide” will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with a halogen, an aliphatic group, or are functionalized as ethers, amines, or the like. Other modifications to nucleotides or polynucleotides involve rearranging, appending, substituting for, or otherwise altering functional groups on the purine or pyrimidine base which form hydrogen bonds to a respective complementary pyrimidine or purine. The resultant modified nucleotide or polynucleotide may form a base pair with other such modified nucleotidic units but not with A, T, C, G or U. For example, guanosine (2-amino-6-oxy-9-beta.-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-.beta.-D-ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-.beta.-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine. Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides may be prepared using the method described by Switzer et al. (1993), supra, and Mantsch et al. (1993) Biochem. 14:5593-5601, or by the method described U.S. Pat. No. 5,780,610 to Collins et al. The non-natural base pairs referred to as κ and π, may be synthesized by the method described in Piccirilli et al. (1990) Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo[4,3]-pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotidic units which form unique base pairs have been described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra, or will be apparent to those of ordinary skill in the art.
The primers and/or probes are preferably between 10 and 45 nucleotides in length. For example, the primers and or probes can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length. Primers and/or probes can be provided in any suitable form, included bound to a solid support, liquid, and lyophilized, for example. In some embodiments, the primers and/or probes include oligonucleotides that hybridize to a target nucleic acid sequence over the entire length of the oligonucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other. Where an oligonucleotide is referred to as “substantially complementary” with respect to a nucleic acid sequence herein, the two sequences can be fully complementary, or they may form mismatches upon hybridization, but retain the ability to hybridize under stringent conditions or standard PCR conditions as discussed below. As used herein, the term “standard PCR conditions” include, for example, any of the PCR conditions disclosed herein, or known in the art, as described in, for example, PCR 1: A Practical Approach, M. J. McPherson, P. Quirke, and G. R. Taylor, Ed., (c) 2001, Oxford University Press, Oxford, England, and PCR Protocols: Current Methods and Applications, B. White, Ed., (c) 1993, Humana Press, Totowa, N.J.
As used herein, the term “substantially complementary” refers to the complementarity between two nucleic acids, e.g., the complementary region of the capture probe and the target sequence, and/or between the linker sequence of the capture probe and the complementary region of the competitor nucleic acid. The complementarity need not be perfect; there may be any number of base pair mismatches that between the two nucleic acids. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to as “substantially complementary” herein, it is meant that the sequences are sufficiently complementary to the each other to hybridize under the selected reaction conditions. The relationship of nucleic acid complementarity and stringency of hybridization sufficient to achieve specificity is well known in the art and described further below in reference to sequence identity, melting temperature and hybridization conditions. Therefore, substantially complementary sequences can be used in any of the detection methods described herein. Such probes can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example discrimination between a target sequence and a non-target sequence. Accordingly, substantially complementary sequences can refer to sequences ranging in percent identity from 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 85, 80, 75 or less, or any number in between, compared to the reference sequence.
“Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.
“Moderately stringent conditions” may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The amplification-ready sample prepared in the first step of the methods of the embodiments disclosed herein further includes an aqueous buffer medium that includes a source of monovalent ions, a source of divalent cations, and a buffering agent. Any convenient source of monovalent ions, such as potassium chloride, potassium acetate, ammonium acetate, potassium glutamate, ammonium chloride, ammonium sulfate, and the like may be employed. The divalent cation may be magnesium, manganese, zinc, and the like, where the cation will typically be magnesium. Any convenient source of magnesium cation may be employed, including magnesium chloride, magnesium acetate, and the like. The amount of magnesium present in the buffer may range from 0.5 to 10 mM, and can range from about 1 to about 6 mM, or about 3 to about 5 mM. Representative buffering agents or salts that may be present in the buffer include Tris, Tricine, HEPES, MOPS, and the like, where the amount of buffering agent will typically range from about 5 to 150 mM, usually from about 10 to 100 mM, and more usually from about 20 to 50 mM, where in certain preferred embodiments the buffering agent will be present in an amount sufficient to provide a pH ranging from about 6.0 to 9.5, for example, about pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5. Other agents that may be present in the buffer medium include chelating agents, such as EDTA, EGTA, and the like. In some embodiments, the reaction mixture can include BSA, or the like.
In preparing the reaction mixture, the various constituent components may be combined in any convenient order. For example, the buffer may be combined with primer, polymerase, and then template nucleic acid, or all of the various constituent components may be combined at the same time to produce the reaction mixture.
Alternatively, commercially available premixed reagents can be utilized in the methods disclosed herein according to the manufacturer's instructions, or modified to improve reaction conditions (e.g., modification of buffer concentration, cation concentration, or dNTP concentration, as necessary), including, for example, TAQMAN® Universal PCR Master Mix (Applied Biosystems), OMNIMIX® or SMARTMIX® (Cepheid), IQ™ Supermix (Bio-Rad Laboratories), LIGHTCYCLER® FastStart (Roche Applied Science, Indianapolis, Ind.), or BRILLIANT® QPCR Master Mix (Stratagene, La Jolla, Calif.). In some embodiments, the reagents can be premixed and disposed within reaction chambers, e.g., amplification chambers and/or detection chambers, of the systems disclosed herein. In some embodiments, reagents can be lyophilized and predisposed within vessels in a reagent rack, or the like.
Following preparation of the reaction mixture, the reaction mixture can be subjected to primer extension reaction conditions (“conditions sufficient to provide polymerase-based nucleic acid amplification products”), i.e., conditions that permit for polymerase-mediated primer extension by addition of nucleotides to the end of the primer molecule using the template strand as a template. In many embodiments, the primer extension reaction conditions are amplification conditions, which conditions include a plurality of reaction cycles, where each reaction cycle comprises: (1) a denaturation step, (2) an annealing step, and (3) a polymerization step. In some embodiments, the reaction cycles do not include a specific amount of time allotted for annealing, but rather combine annealing and polymerization steps. The number of reaction cycles will vary depending on the application being performed, but will usually be at least 15, more usually at least 20, and may be as high as 60 or higher, where the number of different cycles will typically range from about 20 to 40. For methods where more than about 25, usually more than about 30 cycles are performed, it may be convenient or desirable to introduce additional polymerase into the reaction mixture such that conditions suitable for enzymatic primer extension are maintained.
The denaturation step comprises heating the reaction mixture to an elevated temperature and maintaining the mixture at the elevated temperature for a period of time sufficient for any double-stranded or hybridized nucleic acid present in the reaction mixture to dissociate. For denaturation, the temperature of the reaction mixture will usually be raised to, and maintained at, a temperature ranging from about 85 to 100° C., usually from about 90 to 98° C., and more usually from about 93 to 96° C., for a period of time ranging from about 3 to 120 sec, usually from about 3 sec.
Following denaturation, the reaction mixture can be subjected to conditions sufficient for primer annealing to template nucleic acid present in the mixture (if present), and for polymerization of nucleotides to the primer ends in a manner such that the primer is extended in a 5′ to 3′ direction using the nucleic acid to which it is hybridized as a template, i.e., conditions sufficient for enzymatic production of primer extension product. In this embodiment, the annealing and extension processes occur in the same step. The temperature to which the reaction mixture is lowered to achieve these conditions will usually be chosen to provide optimal efficiency and specificity, and will generally range from about 50 to 75° C., usually from about 55 to 70° C., and more usually from about 60 to 68° C., more particularly around 60° C. Annealing conditions will be maintained for a period of time ranging from about 15 sec to 30 min, usually from about 20 sec to 5 min, or about 30 sec to 1 minute, or about 30 seconds.
This step can optionally comprise one of each of an annealing step and an extension step with variation and optimization of the temperature and length of time for each step. In a two-step annealing and extension, the annealing step is allowed to proceed as above. Following annealing of primer to template nucleic acid, the reaction mixture will be further subjected to conditions sufficient to provide for polymerization of nucleotides to the primer ends as above. To achieve polymerization conditions, the temperature of the reaction mixture will typically be raised to or maintained at a temperature ranging from about 65 to 75° C., usually from about 67 to 73° C. and maintained for a period of time ranging from about 15 sec to 20 min, usually from about 30 sec to 5 min.
In some embodiments, the cycling can include a 15-minute initial denaturation at 95° C., which is performed only once, followed by a denaturation step at 95° C. for 1 second, and an annealing/elongation step at 60° C. for 25 seconds. This two-step cycle can be repeated multiple times, e.g., about 45 times. In some embodiments, a final elongation step can be added at 72° C. for 10 minutes.
In some embodiments, the cycling can include a 15-minute initial denaturation step at 95° C., is followed by multiple cycles (e.g., about 45 cycles) of: denaturation at 95° C. for 1 second, annealing at 60° C. for 9 seconds and elongation at 72° C. for 9 seconds. A final elongation step can be added of 72° C. for 10 minutes.
The person skilled in the art of nucleic acid amplification knows the existence of other rapid amplification procedures such as ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) (Persing et al. (1993) Diagnostic Molecular Microbiology: Principles and Applications (American Society for Microbiology, Washington, D.C.). The scope of the embodiments disclosed herein is not limited to the use of amplification by PCR, but rather includes the use of any rapid nucleic acid amplification methods or any other procedures described herein that can be readily carried out on the systems disclosed herein.
Detection
Following amplification, the amplified sample is then moved within the microfluidic network to one or more detection chamber 372a-f. In some embodiments, equal amounts of the amplified are moved to a plurality of detection chambers. For example, in some embodiments, equal volumes of the amplified sample are transferred into 2, 4, 6, 8, 10, 12, or more, detection chambers. Movement of the amplified sample within the microfluidic network is described elsewhere herein, and can comprise opening an amplification gate 310a upstream from the amplification chamber 362. In some embodiments, the amplification gate 310a comprises a TRS, and opening the gate comprises applying heat to the TRS. In some embodiments, heating a TRS within the amplification gate 310a removes the TRS from an upstream channel, e.g., the first channel 360, in communication with the amplification chamber 362.
Fluidic pressure, e.g., pressure from discharging fluid or gas into the network, can be applied to the network, in order to facilitate movement of the amplified sample within the microfluidic networks. For example, the application of a fluidic pressure can be achieved by discharging a fluid (e.g., a gas or liquid), from a pipette into an inlet of the microfluidic network that resides upstream from the sample, thereby forcing the sample downstream within the microfluidic network. Once the amplified sample is transferred into the detection chambers 372a-f, the amplified sample is isolated within the detection chambers 372a-f of the microfluidic network. In some embodiments, isolating the amplified sample within the detection chambers 372a-f can comprise closing detection valves 370a, 370b located upstream and downstream of the detection chambers 372a-f. In some embodiments, the detection valves 370a, 370b located upstream and downstream of the detection chamber(s) comprise a TRS, and are closed by heating the TRS to seal the upstream and downstream channels, respectively, that are in communication with the detection chambers 370a, 370b.
Once isolated inside the detection chambers 372a-f, the amplified sample can be contacted with, or mixed with a fluorescent moiety that is specific for the target nucleic acid and target amplicon, to create a detection mixture. In some embodiments, the fluorescent moiety can comprise an oligonucleotide probe operably coupled to a fluorescent moiety that is complementary to, or substantially complementary to, a nucleotide sequence within the target amplicon. In some embodiments, the term “specific for,” in reference to oligonucleotides and probes, refers to an oligonucleotide or probe that hybridizes exclusively or only to a cognate target sequence, under standard PCR/annealing conditions as described elsewhere herein.
In some embodiments, the microfluidic cartridges 200 can include oligonucleotide probes, e.g., in a lyophilized form, in the detection chambers 372a-f. In some embodiments, oligonucleotide probes can be added to the microfluidic network after the sample to be analyzed has been introduced into the microfluidic cartridge. In some embodiments, probes in the form of lyophilized beads can be placed in the detection chambers 372a-f of the cartridge 200 during the cartridge manufacturing process. In this embodiment, the lyophilized beads are larger in diameter than the diameters of the subchannels 365a1-365a3 and 365b1-365b3 leading into the detection chambers 372a-f and subchannels 367a, 367b leading out of the detection chambers 372a-f. In this manner, the lyophilized beads are blocked from leaving the detection chambers 372a-f after final manufacture of the cartridge 200 and remain in the detection chambers 372a-f during transportation and use.
In some embodiments, the probes are inserted into the detection chambers 372a-f during the manufacture of the cartridge 200 using a drying process. In this embodiment, an amount of probe in liquid solution is added to each of the detection chamber 372a-f; then the liquid is dried, evaporating the solvent and leaving the dried-on probe in each of the detection chambers 372a-f. Whether the probe used is a lyophilized bead or is dried-on, an excess amount of the probe can be placed in the detection chambers 372a-f to compensate for a portion that may dissolve in the sample 350 that passes through the detection chambers 372a-f and enters the subchannels 367a, 367b and the fourth channel 374.
The skilled artisan will appreciate that several probe technologies are useful in the embodiments described herein. By way of example, the embodiments disclosed herein can be used with TAQMAN™ probes, molecular beacon probes, SCORPION™ probes, Sunrise probes, and the like.
TaqMan™ assays are homogenous assays for detecting polynucleotides (see U.S. Pat. No. 5,723,591). In TAQMAN™ assays, two amplification primers flank a central TAQMAN™ probe oligonucleotide. The probe oligonucleotide contains a fluorophore and quencher. During the polymerization step of the amplification process, the 5′ nuclease activity of the polymerase cleaves the probe oligonucleotide, causing the fluorophore moiety to become physically separated from the quencher, which increases fluorescence emission. As more amplicon is created, the intensity of emission at the novel wavelength increases.
Molecular beacons are an alternative to TAQMAN™ probes for the detection of polynucleotides (see U.S. Pat. Nos. 6,277,607; 6,150,097; and 6,037,130). Molecular beacons are oligonucleotide hairpins which undergo a conformational change upon binding to a perfectly matched template. The conformational change of the oligonucleotide increases the physical distance between a fluorophore moiety and a quencher moiety present on the oligonucleotide. This increase in physical distance causes the effect of the quencher to be diminished, thereby increasing the signal derived from the fluorophore.
Another method of detection useful in the embodiments disclosed herein includes the “adjacent probes method.” In this method, PCR is used to amplify the target sequence in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence. One of the probes is labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair. Upon hybridization of the two probes with the target sequence, the donor fluorophore interacts with the acceptor fluorophore to generate a detectable signal. The sample is then excited with light at a wavelength absorbed by the donor fluorophore and the fluorescent emission from the fluorescence energy transfer pair is detected for the determination of that target amount. The “adjacent probes method” is disclosed, e.g., in U.S. Pat. No. 6,174,670B1.
Another method useful in the embodiments disclosed herein include the use of “sunrise primers.” Sunrise primers utilize a hairpin structure similar to molecular beacons, but in contrast to molecular beacons, are attached to a target binding sequence which serves as a primer. When the primer's complementary strand is synthesized, the hairpin structure is disrupted, thereby eliminating quenching. These primers detect amplified product and do not require the use of a polymerase with a 5′ exonuclease activity. Sunrise primers are described by Nazarenko et al. (Nucleic Acids Res. 25:2516-21 (1997) and in U.S. Pat. No. 5,866,336).
Yet another method useful in the embodiments disclosed herein include the use of SCORPION™ probes. SCORPION™ probes combine a primer with an added hairpin structure, similar to Sunrise primers. However, the hairpin structure of SCORPION™ probes is not opened by synthesis of the complementary strand, but by hybridization of part of the hairpin structure with a portion of the target which is downstream from the portion which hybridizes to the primer.
DzyNA-PCR is another method useful in the embodiments disclosed herein, that involves the use of a primer containing the antisense sequence of a DNAzyme, which is an oligonucleotide capable of cleaving specific RNA phosphodiester bonds. The primer binds to a target sequence and drives an amplification reaction producing an amplicon which contains the active DNAzyme. The active DNAzyme then cleaves a generic reporter substrate in the reaction mixture. The reporter substrate contains a fluorophore-quencher pair, and cleavage of the substrate produces a fluorescence signal which increases with the amplification of the target sequence. DzyNA-PCR is described in Todd et al., Clin. Chem. 46:625-30 (2000), and in U.S. Pat. No. 6,140,055.
Still other embodiments disclosed herein include the use of a “Q-PNA probe,” that is a quenchere-labeled peptide nucleic acid, in conjunction with a fluorophore-labeled oligonucleotide primer. The Q-PNA hybridizes to a tag sequence at the 5′ end of the primer. The use of Q-PNA probes is described in Fiandaca et al. Genome Research. 11:609-613 (2001).
Li et al. describes a double stranded probe having a quencher and fluorophore on opposite oligonucleotide strands. Li et al. Nucleic Acids Research. 30: 1-9. When not bound to the target, the strands hybridize to each other and the probe is quenched. However, when a target is present at least one strand hybridizes to the target resulting in a fluorescent signal.
Fluorescent Moieties
As discussed above, some embodiments, disclosed herein include contacting the an amplified sample with a fluorescent moiety, e.g., a fluourescent moiety that is operably coupled to an oligonucleotide that is specific for a polynucleotide sequence found within the target amplicon. Several fluorescent moieties useful in the embodiments disclosed herein are known in the art. By way of example, fluorophore labels and moieties useful in the embodiments and probes disclosed herein include, but are not limited to, dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, and the rhodamine family. Other families of dyes that can be used in the invention include, e.g., polyhalofluorescein-family dyes, hexachlorofluorescein-family dyes, coumarin-family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, chelated lanthanide-family dyes, the family of dyes available under the trade designation Alexa Fluor J, from Molecular Probes, and the family of dyes available under the trade designation Bodipy J, from Invitrogen (Carlsbad, Calif.). Dyes of the fluorescein family include, e.g., 6-carboxyfluorescein (FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED), 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxy-X-rhodamine (ROX), and 2′,4′,5′,7′-tetrachloro-5-carboxy-fluorescein (ZOE). Dyes of the carboxyrhodamine family include tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), Texas Red, R110, and R6G. Dyes of the cyanine family include Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7. Fluorophores are readily available commercially from, for instance, Perkin-Elmer (Foster City, Calif.), Molecular Probes, Inc. (Eugene, Oreg.), and Amersham GE Healthcare (Piscataway, N.J.).
As discussed above, in some embodiments, probes used in the methods disclosed herein can comprise a quencher. Quenchers may be fluorescent quenchers or non-fluorescent quenchers. Fluorescent quenchers include, but are not limited to, TAMRA, ROX, DABCYL, DABSYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds. Exemplary non-fluorescent quenchers that dissipate energy absorbed from a fluorophore include those available under the trade designation Black Hole™ from Biosearch Technologies, Inc. (Novato, Calif.), those available under the trade designation Eclipse™. Dark, from Epoch Biosciences (Bothell, Wash.), those available under the trade designation Qx1J, from Anaspec, Inc. (San Jose, Calif.), and those available under the trade designation Iowa Black™ from Integrated DNA Technologies (Coralville, Iowa).
Typically, a fluorophore and a quencher are used together, and may be on the same or different oligonucleotides. When paired together, a fluorophore and fluorescent quencher can be referred to as a donor fluorophore and acceptor fluorophore, respectively. A number of convenient fluorophore/quencher pairs are known in the art (see, for example, Glazer et al, Current Opinion in Biotechnology, 1997; 8:94-102; Tyagi et al., 1998, Nat. Biotechnol., 16:49-53) and are readily available commercially from, for instance, Molecular Probes (Junction City, Oreg.), and Applied Biosystems (Foster City, Calif.). Examples of donor fluorophores that can be used with various acceptor fluorophores include, but are not limited to, fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid, 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4-acetamido-4′-isothiocyanatostilbene-2-,2′-disulfonic acid derivatives. Acceptor fluorophores typically depend upon the donor fluorophore used. Examples of acceptor fluorophores include, but are not limited to, LC-Red 640, LC-Red 705, CyS, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorophores are readily available commercially from, for instance, Molecular Probes or Sigma Chemical Co. (St. Louis, Mo.).
In some embodiments, after the amplification reaction has been mixed with the probe(s), the detection reaction is subjected to a detection thermocycling reaction. The detection thermocycling can involve heating the detection reaction so as to denature the double-stranded polynucleotides, followed by continuously ramping the temperature up or down over a set period of time and analyzing melting and reannealing curves. The use of fluorescence melting curves to monitor hybridization has been described, e.g., in L. E. Morrison & L. M. Stols, Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution, 32 Biochemistry 3095-3104, 1993), U.S. Pat. No. 6,174,670, and in U.S. Pat. No. 6,140,054 the entire contents of which are each herein incorporated by reference.
In some embodiments, the detection comprises illuminating the detection reaction with a wavelength of light for eliciting fluorescence by the fluorescent moiety, and continuously monitoring, as a function of temperature, the fluorescence emitted. In some embodiments, more than one fluorescent moiety, e.g., 2, 3, 4, 5, 6, or more, wherein each fluorescent moiety is specific for a different nucleic acid sequence present in a target amplicon, can be mixed with the amplified sample in the detection chambers 372a-f. The use of more than one fluorescent moiety, wherein each fluorescent moiety is specific for a different nucleic acid sequence, enables the specific hybridization to (and thus detection of) more than one target nucleic acid sequence sequence, whether the different target nucleic acid sequences are present within a single amplicon, or whether the different target nucleic acid sequences are located within different amplicons, e.g., different amplicons generated by a multiplex PCR reaction in the amplification stage of the reaction.
In some embodiments, different oligonucleotide probes that are substantially complementary to different target nucleic acid sequences (within a single amplicon, or in different amplicons), and that are each operably coupled to a single fluorescent moiety can be used to detect multiple target sequences (e.g., different loci within a single amplicon or different amplicons) in a single detection reaction. For example, different oligonucleotides, preferably that have distinct Tm's can each be operably coupled to a single type of fluorescent moiety. Because each different oligonucleotide probe has a distinct Tm, the presence of the target nucleotide sequence within an amplicon(s) can be monitored by measuring the fluorescence emission as a function of temperature at a single emission wavelength. By way of example, in some embodiments, each detection chamber 372a-f can contain oligonucleotide probes that comprise two, three, four, or more different nucleic acid sequences, each having distinct Tm's, and each oligonucleotide being operably coupled to the same type of fluorescent moiety.
Alternatively, a single detection reaction can include a number of oligonucleotide different probes, each of which has a different oligonucleotide sequence, wherein each different oligonucleotide sequence is be operably coupled to different type of fluorescent moiety. This way, the each different probe within the detection reaction can be distinguished from other probes based on the distinguishable emission spectra of the fluorescent moieties. Preferably, the emission spectra of the different fluorescent moieties do not overlap, or do not have significant overlap, such that the different fluorescent moieties can be readily distinguished.
In some embodiments, the protocol used in the detection chambers 372a-f can include subjecting the detection reaction to an initial cycle to melt double stranded nucleic acids including any target amplicons, and lowering the temperature to allow the annealing of one or more oligonucleotide probes to anneal to complementary (or substantially complementary) sequences of the single stranded amplicon DNA, to form a duplex. By way of example, in some embodiments, the detection reaction can be heated in order to ensure complete denaturation of double stranded nucleic acids in the detection mixture, including denaturation of target amplicon sequences. By way of example only, the detection reaction can be heated to about 90-100° C. for a period of time, e.g., about 15 sec to about 1 min. Following denaturation, the temperature of the detection mixture is lowered to allow annealing of probe(s) to target sequence(s). By way of example only, the annealing step can include cooling the detection reaction to about 45° C. for a period of time, e.g., for about 15 seconds to a minute, or longer, or any period of time in between, e.g., 30 sec.
After the annealing step, the temperature of the detection mixture can be slowly raised within the detection chamber 372a-f, while continuously observing a fluorescence signal, in order to monitor (and record) the dissociation of, or melting of, the duplex formed by the probe and amplicon sequence. Specifically, the temperature of the detection mixture can incrementally ramped from the annealing temperature to a second temperature (e.g., the denaturation temperature). For example, in some embodiments, the detection reaction is ramped from the annealing temperature (or a first temperature that is lower than the Tm of any probe in the detection mixture), to the second temperature in increments of 0.05° C., 0.1° C., 0.2° C., 0.3° C., 0.4° C., 0.5° C., 0.6° C., 1.0° C., 1.5° C., 2.0° C., 2.5° C., 3.0° C., 3.5° C., 4.0° C., 4.5° C., 5.0° C., or greater, or any increment in between. Using the devices disclosed herein, the fluorescence signal emitted at each temperature increment is measured using the heater/detector module disclosed herein. In some embodiments, the ramping from annealing temperature to the second temperature (e.g., melting temperature), is referred to as a “melt protocol.” The change in fluorescence caused by the dissociation of a fluorescent probe from a cognate target sequence throughout the course of the melt protocol can be monitored over time/temperature during the melt protocol, and can be used to determine the presence and/or amount of a target sequence.
In some embodiments, the heating of the detection chambers 372a-f can be synchronized and staggered in order to achieve a desired detector cycle time. For example, in some embodiments, the total time for melt protocol/detection is less than 30 minutes, e.g., between about 8-27 minutes, 10-26 minutes, 15-25 minutes, 20-25 minutes, or the like. By way of example, in some embodiments, the melt protocol takes about 22-25 minutes. In some embodiments, each of the detection chambers 372a-f within a sample lane 300 is subjected to the same melt protocol. In some embodiments, some of the detection chambers 372a-f within a sample lane 300 are subjected to different melt protocols.
In some embodiments, more than one probe with a single species of fluorescent moiety can be used in the same detection chamber, wherein each probe comprises a different oligonucleotide sequence, and wherein each probe has a different Tm. For example, in some embodiments, two, three, four, or more, different probes with the same species of fluorescent moiety can be used in the same detection chamber, in order to detect two, three, four, or more, different target nucleic acid sequences.
Accordingly, in some embodiments, each lane 300 can be used to detect numerous target nucleic acid sequences. By way of example only, in some embodiments, 108 different target nucleic acid sequences can be detected in each of the lanes. Specifically, each detection chamber 372a-f can include multiple different probes, e.g., eighteen different probes, wherein the probes comprise eighteen different oligonucleotide sequences specific for eighteen different target nucleic acid sequences, and wherein the probes collectively comprise six different types of fluorescent moieties (i.e., three different oligonucleotide sequences having distinct Tm's can be coupled to a single type of fluorescent moiety). In this way, six different fluorescent moieties times three different oligonucleotide probes/each moiety times six different detection chambers, can be used to detect 108 different target nucleic acids.
Before introducing the sample 350 into the sample lane 300, the cartridge 200 can be positioned to receive the sample 350 from the fluid dispenser 400. Referring to
In some embodiments, prior to introduction of the sample 350 into the sample lane 300, the first and second amplification gates 310a, b of the sample lane 300 can be in a closed state, such that the TRS (e.g., wax) 345 is positioned in the first channel 360 to obstruct passage of the sample 350 through the gates 310a, 310b. In some embodiment, prior to introduction of the sample 350 into the sample lane 300, the first and second amplification valves 330a, 330b of the sample lane 300 can be in an open state, such that the TRS 345 is maintained in the wax loading channel 334, and therefore is not blocking passage of the sample 350 in the first channel 360 near the valves 330a, b. Thus, when the sample 350 is introduced into the microfluidic network, the sample 350 can bypass the closed amplification gate 310a (including the upstream 318 and downstream 320 sides of the amplification gate 310a) by following the route of the first channel 360 past the open first amplification valve 330a in the first channel 360. In this manner, the sample 350 can travel from the inlet 302, through the first channel 360, into the amplification chamber 362, thereby filling the amplification chamber 362 to capacity. In some embodiments the amplification chamber has a volumetric capacity of 8 μl. The volume of the sample 350 initially introduced into the sample 350 may be of a volume in excess of the volume of the amplification chamber 362. Any volume of the sample 350 in excess of the volume of the amplification chamber 362 can exit the amplification chamber 362 at a downstream side of the amplification chamber 362 and may enter the second channel 364.
The second channel 364 can extend from the amplification chamber 362 past the second amplification gate 310b into the second stage 208 of the microfluidic network of the sample lane 300. Because the second amplification gate 310b may be initially closed when the sample 350 is introduced into the sample lane 300, the sample 350 can be blocked by the second amplification gate 310b in the second channel 364 and diverted into the third channel 366. In the third channel 366, the sample can move past the second amplification valve 330b to the first vent 368. Any volume of the sample 350 in the third channel in excess of the volume of the third channel 366 can be drained and removed from the microfluidic network through the first vent 368.
In some embodiments, upon introduction of the sample 350 into the microfluidic network, the pipette tip 402 of the liquid dispenser 400 can be removed from the inlet 302 and the receiving tray 520 can be closed, thereby sliding the receiving tray 520 under the optical module 502. The force member (not shown) can then elevate the receiving tray 520 upward toward the bottom side 506 of the optical module 502 in order to press the cartridge 200 against the aperture plate 540 of the optical module 502. Pressing the cartridge 200 upward against the aperture plate 540 can cause the cartridge 200 to experience a physical pressure against the aperture plate 540. The physical pressure applied to the cartridge 200 by the aperture plate 540 can also cause the cartridge 200 to likewise apply a downward pressure against the heater substrate 600 in the receiving tray 520. The pressure applied to the cartridge 200 on its top side by the aperture plate 540 and on its bottom side by the heater substrate 600 can be applied in a substantially uniform manner throughout the cartridge 200 such that all portion of the cartridge 200 in the recessed bay 524 of the receiving tray 520 can experiencing the same degree of pressure.
With the cartridge 200 positioned in the recessed bay 524 of the receiving tray 520, the various heaters of the heater substrate 600 can be in physical and/or thermal contact with the corresponding components (e.g., valves and gates) of the sample lanes 300 in cartridge 200. After the sample 350 is introduced into the sample lane 300 through the inlet 302 and the sample 350 is positioned in the first stage 206 of the sample lane 300, as shown in
After completion of the amplification protocol in the amplification chamber 362, the amplified sample 350 can then be moved to the second stage 208 of the microfluidic network in the sample lane 300. Movement of the sample 350 from the first stage 206 into the detection chambers 372a-f of the second stage 208 is illustrated in
Prior to, and as a part of, advancing the sample 350 from the first stage 206 of the sample lane 300 to the second stage 208 of the sample lane 300, the receiving tray 520 can be opened in order to allow the fluid dispenser 400 to act on the cartridge 200. For each sample lane 300 being processed simultaneously in the cartridge 200, a pipette tip 402 of the fluid dispenser 400 can again be positioned in the inlet 302 of the sample lane 300. The actions of the fluid dispenser 400 on the cartridge 200 can be two-fold. The fluid dispenser 400 can be used to supply both physical pressure and air pressure to the cartridge 200.
When the receiving tray 520 is open and the cartridge 200 is no longer in a position under the aperture plate 540, the cartridge 200 is no longer receiving pressure from the aperture plate 540 to press it against the heater substrate 600. When receiving tray 520 is open and when the pipette tip 402 is inserted into the inlet 302 of a sample lane 300, the fluid dispenser can be activated to press the pipette tip 402 into the inlet 302 with additional pressure in order to press the cartridge 200 against the heater substrate 600. Thus, at this stage of the sample processing, the physical pressure between the cartridge 200 and the heater substrate 600 is provided by the physical pressure of the fluid dispenser 400 in pressing the pipette into the inlet 302.
When a pipette 402 is inserted into the inlet 302, the fluid dispenser 400 can introduce air or another gas into the microfluidic network of the sample lane 300 through the inlet 302, thus supplying an air pressure to the microfluidic network. As represented in
As the sample 350 is moved into the detection chambers 372a-f as described above, the sample 350 comes into contact with the probes that were initially placed in the detection chambers 372a-f prior to use or during manufacture of the cartridge 200. Upon entry of the sample into the detection chambers 372a-f, the probes are dissolved into the sample 350 solution and bind to the polynucleotides in the sample 350 as described herein. Because an excess portion of the sample 350 will pass through the detection chambers 372a-f into the subchannels 367a, 367b and fourth channel 374, an excess amount of the probe can also initially be placed into the detection chambers 372a-f to compensate for the portion that may lost as it is absorbed by and passes downstream with the excess portion of the sample 350.
It is noted that the first and second amplification valves 330a, 330b can remain closed when the first and second amplification gates 310a, 310b are opened. This is possible because when the first and second amplification gate heaters 610a, 610b are activated to heat up the TRS 645 in the gate junctions 316, the first and second amplification valve heaters 630a, 630b are not activated, thus leaving the TRS in the valve junctions 336 in a solidified state. Furthermore, the closed first and second amplification valves 330a, b are sufficiently stable to not open again when the air pressure is applied by the fluid dispenser 400, in part because the valve junctions 336 are wider than the gate junctions 316 and thus can hold a greater quantity of TRS 345, the greater quantity of TRS 345 being more difficult to dislodge from the valve junction 336. Thus, closing the initially-open first and second amplification valves 330a, 330b is the mechanism by which the sample 350 becomes isolated in the amplification chamber 362. Conversely, opening the initially-closed first and second amplification gates 310a, 310b (along with supplying the air pressure) is the mechanism by which the sample 350 is moved from the first stage 206 of the sample lane 300 to the second stage 208 of the sample lane 300.
As illustrated in
Following processing of the sample 350 in the amplification chamber 362 and deposition of the amplified samples in detection chambers 372a-f of the sample lane 300, the detector head 700 in the optical module 502 can conduct a detection procedure on the sample 350 in the detection chambers 372a-f to determine if specific analyte amplicons are present in the sample 350.
Referring again to
Because of the arrangement of the detection chambers 372a-f into rows and columns, each sample 350 in a sample lane 300 may undergo multiplexed amplicon detection. For example, each detection chamber 372a-f can be optically scanned by six different detector pairs, the detection chambers 372a-c being scanned by detector pairs in row R1 and the detection chambers 372d-f being scanned by detector pairs in row R2 of the detector head 700. In certain embodiments, the detector pairs in each column can be configured to emit and detect light of the same wavelength (color) as each other, but wherein the wavelength is different than the light emitted and detected by the detector pairs in the other columns. Each column of detector pairs may emit and detect a unique wavelength of light, and as such the detector 700 may emit and detect light of six different wavelengths. Accordingly, each detection chamber 372a-f in each of the sample lanes 300a-l may be optically scanned by six different wavelengths of light. Alternatively, the detector pairs individually in each column C1-C6 may emit and detect a unique wavelength of light. Thus, the detector may emit and detect light of 12 different wavelengths, a different length emitted and detected by each detector pair 722-733.
Each of the detection chambers 372a-f in each of the sample lanes 300a-l may be pre-loaded with fluorescence probes, each of which can be associated with a certain wavelength (color); each probe also specifically binds to (e.g., under stringent conditions), a particular sequence to be detected (e.g., a target amplicon, a positive control sequence, or the like). When the sample 350 is moved into the detection chambers 372a-f (as shown in
The detection data obtained from the detector head 700 can be recorded and associated with the samples 350 in the sample lanes 300a-l. Following completion of the detection procedure, the sample 350 may remain isolated in the detection chambers 372a-f in the second stage 208 of the sample lanes 300a-l. After the detection procedure is complete, the receiving tray 520 may be opened again and the cartridge 200 may be removed, in a manual or automated fashion, from the recessed bay 524 of the receiving tray 520. The cartridge 200 may be discarded.
The above description discloses several methods and systems of the embodiments disclosed herein. The embodiments disclosed herein are susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that the embodiments disclosed herein be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.
All references cited herein including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
The present application is a continuation of International Application No. PCT/US2012/063091, filed on Nov. 1, 2012, which claims priority to U.S. Provisional Application Ser. No. 61/556,032, filed Nov. 4, 2011. Each of the above applications is hereby incorporated by reference in its entirety.
The research leading to the present invention was supported, at least in part, by a grant under U01AI070428 from the National Institutes of Health. Accordingly, the Government may have certain rights in the invention.
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| Record of Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 (80 pages); Petitioner's Demonstratives for Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 (72 pages); Patent Owner's Demonstratives for Oral Hearing in IPR2019-00488 and IPR2019-00490 held Apr. 21, 2020 (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). |
| 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). |
| 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). |
| 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). |
| 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). |
| 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). |
| Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 10,625,262 (Paper 7 in IPR2021-00250) dated Jul. 15, 2021 (15 pages). |
| Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 10,632,466 (Paper 7 in IPR2021-00253) dated Jul. 15, 2021 (22 pages). |
| Decision Denying Institution of Inter Partes Review of U.S. Pat. No. 10,625,261 (Paper 7 in IPR2021-00251) dated Jul. 15, 2021 (24 pages). |
| Patent Owner's Response in Inter Partes Review of U.S. Pat. No. 8,709,787 and Exhibit List (Paper 29 in IPR 2020-01132) dated Jul. 15, 2021 (87 pages). |
| Decision Granting Institution of Inter Partes Review of U.S. Pat. No. 8,415,103 on Rehearing (Paper 23 in IPR2020-01133) dated Aug. 6, 2021 (20 pages). |
| Decision of U.S. Court of Appeal for the Federal Circuit Affirming Inter Partes Review Final Written Decisions Determining No Challenged Claims of U.S. Pat. Nos. 7,998,708 and 8,323,900 are Unpatentable (IPR2019-00488, IPR2019-00490, IPR2019-01493, and IPR2019-01494) dated Oct. 29, 2021 (12 pages). |
| Joint Motion to Terminate Inter Partes Review of U.S. Pat. No. 8,709,787 (Paper 37 in IPR 2020-01132) dated Nov. 15, 2021 (8 pages). |
| Joint Motion to Terminate Inter Partes Review of U.S. Pat. No. 8,415,103 (Paper 35 in IPR 2020-01133) dated Nov. 15, 2021 (8 pages). |
| First Amended and Supplemental Complaint filed by Becton, Dickinson and Company et al. on Jun. 25, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS, Infringement Action involving U.S. Pat. Nos. 7,998,708; 8,273,308; 8,323,900; 8,415,103; 8,703,069; 8,709,787; 10,494,663; 10,364,456; 10,443,088; 10,604,788; 10,625,261; 10,625,262; and U.S. Pat. No. 10,632,466 (55 pages). |
| Answer to First Amended and Supplemental Complaint filed by NeuModx Molecular, Inc. on Jul. 16, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (42 pages). |
| Defendant NeuModx's Initial Invalidity Contentions filed Sep. 30, 2020 in U.S. District Court, Delaware, Case #1:19-cv-01126-LPS (47 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-In-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). |
| Stipulation of Dismissal filed by Plaintiffs Becton, Dickinson and Company, Geneohm Sciences Canada, Inc. and HandyLab, Inc. and Defendants NeuMoDx Molecular, Inc., Qiagen GmbH, and Qiagen North American Holdings, Inc. on Nov. 12, 2021 in U.S. District Court, Delaware, Case # 1:19-cv-01226-LPS (3 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20140329301 A1 | Nov 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61556032 | Nov 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2012/063091 | Nov 2012 | US |
| Child | 14267232 | US |
