Microfluidic cartridges for enhanced amplification of polynucleotide-containing samples

Abstract

To provide: microfluidic cartridges that achieve improvement in both thermal uniformity and amplification efficiency and realize assays having an improved limit of detection (LOD) and an improved limit of quantification (LOQ); and methods and systems for amplifying polynucleotides contained in a plurality of samples.SOLUTION: The technology more specifically relates to a compressible pad applied to a microfluidic cartridge, where the microfluidic cartridge is configured to amplify nucleotides of interest, particularly from a plurality of biological samples in parallel, within microfluidic channels in the cartridge and permit detection of those nucleotides. Compressible pads of the present technology can be implemented in microfluidic cartridges having enhanced reaction chamber volumes, resulting in improved thermal uniformity and amplification efficiency in the cartridge.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 62 / 909,628, filed Oct. 2, 2019, which is hereby incorporated by reference in its entirety.

[0002] (Technical Field) The technology described herein generally relates to microfluidic cartridges. In one aspect, the technology more particularly relates to compressible pads applied to microfluidic cartridges, where the microfluidic cartridges are configured to receive and amplify target nucleotides. In another aspect, the technology relates to microfluidic cartridges having reaction chambers configured to receive and amplify a greater volume of fluid eluate from a processed sample. Embodiments of the cartridges described herein can amplify target nucleotides in parallel from multiple biological samples within microfluidic channels within the cartridge and enable detection of these nucleotides.

Background Art

[0003] The sensitivity of assays in molecular diagnostic tests is influenced by several factors. These factors include, among others, the extraction efficiency during specimen processing to obtain an amplification - ready sample, the amplification efficiency of the sample, and the thermal uniformity achieved within the reaction volume during the amplification process. Increasing the dimensions of the reaction volume contributes to improving the amplification efficiency, resulting in an improvement in the lower limit of detection (LOD) and the lower limit of quantification (LOQ). Thermal uniformity can be improved by improving the uniformity and distribution of heat transfer between the reaction volume and the heat source.

[0004] One implementation configuration of a current microfluidic cartridge has a reaction chamber with a reaction volume of about 4 μL. There are important advantages associated with cartridges that include a reaction chamber with such a small reaction volume. However, as the volume of the reaction chamber decreases, issues related to achieving the desired analysis sensitivity may arise. At the same time, as the volume of the reaction chamber increases to achieve improved amplification efficiency and overcome limitations in target delivery, issues related to achieving thermal uniformity may arise. Therefore, there is a need for a microfluidic cartridge that overcomes these issues, achieves both improved amplification efficiency and thermal uniformity, and results in an assay with improved lower limit of detection (LOD) and improved lower limit of quantification (LOQ).

[0005] The discussion regarding the background of the technology herein is included to explain the relevant context of the technology. This should not be construed as an admission that any of the matters referred to were published, known, or part of common general knowledge at the priority date of any of the claims.

[0006] Throughout the description and claims of this specification, the words "comprise", "comprising", "comprises" and their derivatives are not intended to exclude other additives, components, integers or steps.

Summary of the Invention

Means for Solving the Problems

[0007] The present technology includes methods and apparatuses for improving the pressure distribution throughout a microfluidic device, enhancing the thermal uniformity within the microfluidic device, and strengthening amplification parameters executed in the microfluidic device. Implementations of the technology of the present invention improve the characteristics of a microfluidic device that amplifies target nucleotides within a microfluidic channel. The present technology includes methods and apparatuses for improving the detection of these nucleotides.

[0008] The microfluidic device of the present technology can interact with a heating assembly that applies heat to a plurality of chambers within the microfluidic device where amplification occurs. The heating assembly can include an array of heaters configured to contact the microfluidic device. In some cases, the heating assembly is pressed against the microfluidic device and the array of heaters is disposed in thermal communication with the microfluidic device. In other cases, the microfluidic device is pressed against the heating assembly and the array of heaters is disposed in thermal communication with the microfluidic device. Embodiments of the microfluidic device according to the present technology can include a compressible pad that improves the distribution of pressure applied to the microfluidic device and enhances the uniformity of heat supplied to the microfluidic device. The compressible pad according to the present technology can enhance the uniformity of pressure applied to the microfluidic device, thereby reducing heat loss and improving the consistency and efficiency of amplification occurring in the plurality of chambers of the microfluidic device.

[0009] The microfluidic device of the present technology can also achieve an improvement in assay sensitivity by increasing the amplification chamber volume from about 4 μL to about 25 μL while still achieving optimal heat uniformity throughout the chamber during the amplification process. By increasing the amplification chamber of the present technology, a larger volume of eluate containing the DNA / RNA target analyte extracted from the sample can be received, thereby improving the assay sensitivity. The microfluidic device of the present technology can achieve a reaction chamber volume that is, in some cases, six times that of current microfluidic devices. When such a large volume reaction chamber of the present technology is combined with the improved pressure distribution and heat uniformity associated with the compressible pad of the present technology, the assay performance measured by the improvement in the lower limit of detection (LOD) and the lower limit of quantification (LOQ) is improved.

[0010] An implementation configuration of the improved microfluidic device includes a microfluidic cartridge. The microfluidic cartridge can include a first PCR reaction chamber. The microfluidic cartridge can include a second PCR reaction chamber. The microfluidic cartridge can include a first inlet in fluid communication with the first PCR reaction chamber. The microfluidic cartridge can include a second inlet in fluid communication with the second PCR reaction chamber. The microfluidic cartridge can include a compressible pad configured to increase compliance between the microfluidic cartridge and a heater.

[0011] In some embodiments, a microfluidic cartridge is provided that includes a second side opposite the first side. The microfluidic cartridge can include a first amplification chamber. The microfluidic cartridge can include a second amplification chamber. The microfluidic cartridge can include a first inlet disposed on the first side and in fluid communication with the first amplification chamber. The microfluidic cartridge can include a second inlet disposed on the first side and in fluid communication with the second amplification chamber. The microfluidic cartridge can include a compressible pad disposed on the first side. In some embodiments, the compressible pad is configured to provide more thorough and consistent heat transfer from a plurality of contact heat sources in contact with the second side of the microfluidic cartridge to the first amplification chamber and the second amplification chamber. In some embodiments, the compressible pad includes a first window over the first amplification chamber and a second window over the second amplification chamber. In some embodiments, the first window and the second window are configured such that light can be transmitted through the first side of the microfluidic cartridge to the first amplification chamber and the second amplification chamber, respectively.

[0012] In some embodiments, the first amplification chamber and the second amplification chamber have a volume of about 25 μL. In some embodiments, the first amplification chamber and the second amplification chamber have a width dimension of about 3.5 mm, a depth dimension of about 0.83 mm, and a length dimension of about 10 mm. In some embodiments, the microfluidic cartridge includes a label above the compressible pad. In some embodiments, the first amplification reaction chamber, the second amplification reaction chamber, the first inlet, and the second inlet are formed in a rigid substrate layer. In some embodiments, the second side of the microfluidic cartridge includes a flexible laminate layer below the first amplification chamber and the second amplification chamber. In some embodiments, the compressible pad includes a material having a compression force deflection of less than 30 psi. In some embodiments, the compressible pad includes a material having a compression force deflection of less than 20 psi. In some embodiments, the compressible pad improves the pressure distribution from components of the diagnostic test device. In some embodiments, the application of pressure to the compressible pad is configured to enhance the uniformity of heat application from a plurality of contact heat sources to the first amplification chamber and the second amplification chamber. In some embodiments, the compressible pad increases the uniformity of heat application to the first amplification chamber and the second amplification chamber. In some embodiments, the compressible pad enhances PCR amplification that depends on rapid temperature cycling.

[0013] In some embodiments, a method for amplification on a plurality of polynucleotide-containing samples is provided. The method can include introducing a plurality of samples into a microfluidic cartridge, the cartridge including a plurality of amplification chambers configured to enable thermal cycling of the plurality of samples independently of each other. The method can include moving the plurality of samples into respective ones of the plurality of amplification chambers. The method can include amplifying the polynucleotides included with the plurality of samples by applying continuous heating and cooling cycles to the amplification chambers. The method can include compressing a pad of the microfluidic cartridge during amplification. In some embodiments, the method can include applying pressure to a compressible pad to enhance contact between the microfluidic cartridge and a substrate including one or more heaters. In some embodiments, the method can include applying pressure to a compressible pad to enhance thermal uniformity. In some embodiments, the method can include applying pressure to a compressible pad to enhance amplification of the plurality of polynucleotide-containing samples.

[0014] In some embodiments, a system is provided. The system can include a microfluidic substrate. The microfluidic substrate can include a first PCR reaction chamber. The microfluidic substrate can include a second PCR reaction chamber. The microfluidic substrate can include a first inlet that is in fluid communication with the first PCR reaction chamber. The microfluidic substrate can include a second inlet that is in fluid communication with the second PCR reaction chamber. The microfluidic substrate can include a compressible pad. In some embodiments, the microfluidic cartridge is configured for use with an apparatus. The apparatus can include a bay configured to receive the microfluidic cartridge. The apparatus can include at least one heat source configured to be thermally coupled to the cartridge and apply a thermal cycle to perform PCR on one or more polynucleotide-containing samples within the cartridge. The apparatus can include a detector configured to detect the presence of one or more polynucleotides in one or more samples. The apparatus can include a processor coupled to the heat source and configured to control the heating of one or more regions of the microfluidic cartridge.

[0015] In some embodiments, the compressible pad is configured to improve the contact between the bay and the microfluidic cartridge. In some embodiments, the compressible pad is configured to improve the contact between at least one heat source and the cartridge. In some embodiments, the compressible pad is configured to be compressed by a detector disposed on the cartridge during detection. In some embodiments, the detector is configured to move downward to physically contact the cartridge in order to compress the compressible pad. In some embodiments, the cartridge is configured to move upward to physically contact the detector in order to compress the compressible pad. In some embodiments, the compressible pad is configured to be compressed by another component of the apparatus that applies pressure to the cartridge.

[0016] Details of one or more embodiments of the present technology are set forth in the accompanying drawings and the further description in this specification. Other features, objects, and advantages of the present technology will become apparent from this specification, the drawings, and the claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0018] The present technology relates to a microfluidic device configured to perform amplification of one or more polynucleotides by PCR or the like from one or more samples. Unless otherwise explicitly indicated, when the term PCR is used in this specification, it is intended to include any variant form of PCR, including but not limited to real-time and quantitative PCR, and any other form of polynucleotide amplification.

[0019] The microfluidic cartridge can be configured to receive thermal energy from one or more heating elements present in an external device with which the cartridge is in thermal communication. Exemplary such devices are further described herein, and additional embodiments of such devices are described in U.S. Patent Application No. 11 / 985,577, entitled "Microfluidic System for Amplifying and Detecting Polynucleotides in Parallel," filed Nov. 14, 2007, which is hereby incorporated by reference herein. This technology provides an apparatus for detecting polynucleotides in a sample, particularly a biological sample. More particularly, this technology relates to a microfluidic system that performs PCR on target nucleotides within a microfluidic channel and detects these nucleotides. The apparatus includes a microfluidic cartridge configured to receive a plurality of samples, and PCR can be performed individually on each sample, or on a group of samples, or on all of the plurality of samples simultaneously. U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed Nov. 14, 2007, is hereby incorporated by reference herein. U.S. Patent Application No. 11 / 940,310, entitled "Microfluidic Cartridge and Method of Using Same," filed Nov. 14, 2007, is hereby incorporated by reference herein. This technology provides a microfluidic substrate configured to perform PCR in parallel on a plurality of polynucleotide-containing samples. The substrate can be a single-layer substrate within the microfluidic cartridge. Also provided is a method of manufacturing a microfluidic cartridge that includes such a substrate. U.S. Patent Application No. 11 / 728,964, entitled "Integrated System for Processing Microfluidic Samples and Methods of Using Same," filed Mar. 26, 2007, is hereby incorporated by reference herein.The present technology provides an integrated device for processing polynucleotide-containing samples and providing diagnostic results therefor.

[0020] A cartridge means a unit that is disposable or can be reused in whole or in part and is configured to be used in combination with several other devices that are preferably and complementarily configured to receive the cartridge and operate (such as supplying energy) on the cartridge.

[0021] As used herein, microfluidics means that the volume of the sample and / or reagent and / or amplified polynucleotide is from about 0.1 μl to about 999 μl, for example, 1 to 100 μl, or 1 to 50 μl. In some embodiments, the volume is between 0 and 10 μl in smaller wells and between 10 and 30 μl in wider and deeper wells, as described herein. Similarly, when applied to a cartridge, the term microfluidics means that the various components and channels of the cartridge are configured to accept, and / or hold, and / or facilitate the passage of the microfluidic volume of the sample, reagent, or amplified polynucleotide. Certain embodiments herein can also function at nanoliter volumes (such as 100 nanoliters, in the range of 10 to 500 nanoliters).

[0022] One aspect of the present technology relates to a microfluidic cartridge having two or more sample lanes arranged such that analysis can be performed in parallel (e.g., simultaneously) in two or three or more lanes, with each lane being independently associated with a given sample (hereinafter referred to as a "sample lane").

[0023] A sample lane is an independently controllable set of elements capable of analyzing a sample according to the methods described herein and other methods known in the art. A sample lane includes, as further described herein, at least a sample inlet and a microfluidic network having one or more microfluidic components.

[0024] A cartridge can include a plurality of microfluidic networks, each network having various components, and each network is also configured to perform PCR on a sample for which the presence or absence of one or more polynucleotides is to be determined.

[0025] Embodiments of the present technology include a cartridge having a plurality of sample lanes (hereinafter referred to as a "multi-lane cartridge"). However, it will be understood that embodiments of the present technology can be implemented with a cartridge containing only one sample lane. A multi-lane cartridge is configured to receive a plurality of samples in series or in parallel, simultaneously or sequentially. In some embodiments, the multi-lane cartridge is configured to receive 24 samples, or any other suitable number of samples. In some cases, the multi-lane cartridge is configured to receive at least a first sample and a second sample, where the first sample and the second sample each contain one or more polynucleotides in a form suitable for amplification. The polynucleotides may be the same as or different from each other in different samples, and thus in different sample lanes of the cartridge. The cartridge can process each sample by increasing the concentration of the polynucleotide to be determined and / or by decreasing the concentration of an inhibitor with respect to the concentration of the polynucleotide to be determined.

[0026] The multi - lane cartridge includes at least a first sample lane having a first microfluidic network and a second sample lane having a second microfluidic network. Each of the first microfluidic network and the second microfluidic network includes the features described herein. The first microfluidic network is configured to amplify polynucleotides in a first sample, and the second microfluidic network is configured to amplify polynucleotides in a second sample.

[0027] In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to a sample mixture including PCR reagents and a neutralized polynucleotide sample under thermal cycling conditions suitable for creating PCR amplicons from the neutralized polynucleotide sample.

[0028] At least the external heat source can operate under the control of a computer processor configured to execute computer - readable instructions for operating one or more components of each sample lane independently of each other and for receiving signals from a detector that measures fluorescence from one or more chambers of a PCR reaction chamber.

[0029] Next, a non-limiting implementation configuration of the microfluidic cartridge according to the present technology will be described with reference to FIGS. 1A and 1B. FIG. 1A is a plan view of a microfluidic cartridge 100 having 24 independent sample lanes including sample lanes 102, 104, 106, 108. FIG. 1B is an enlarged view of a part of the cartridge 100 of FIG. 1A, illustrating reaction chambers 112, 114, 116, 118 of adjacent sample lanes 102, 104, 106, 108. The microfluidic network in each sample lane is typically configured to perform amplification such as by PCR on a PCR-compatible sample. The microfluidic network within each sample lane can receive and amplify a nucleic acid-containing sample extracted from a specimen using any suitable method. In an example of a cartridge that receives a PCR-compatible sample, the sample includes a mixture containing a polynucleotide sample neutralized with a PCR reagent and is suitable for being subjected to thermal cycling conditions to generate a PCR amplicon from the neutralized polynucleotide sample. In one example, the PCR-compatible sample is a PCR reagent mixture containing a polymerase enzyme, a positive control plasmid, a fluorescent hybridization probe selective for at least a part of the plasmid and a plurality of nucleotides, and at least one probe selective for a polynucleotide sequence. Exemplary probes are further described herein. In an embodiment of the present technology, the microfluidic network is configured to combine heat from an external heat source with a mixture containing a PCR reagent and a neutralized polynucleotide sample under thermal cycling conditions suitable for creating a PCR amplicon from the neutralized polynucleotide sample.

[0030] Next, another non-limiting implementation configuration of the microfluidic cartridge according to the present technology will be described with reference to FIGS. 2A and 2B. FIG. 2A is a plan view of a microfluidic cartridge 200 having 24 independent sample lanes including sample lanes 202, 204, 206, 208. FIG. 2B is an enlarged view of a part of the cartridge 200 of FIG. 2A illustrating reaction chambers 212, 214, 216, 218 of adjacent sample lanes 202, 204, 206, 208. Each of the sample lanes of the cartridge 200 includes a dedicated sample inlet configured to receive a sample. For example, sample lanes 202, 204, 206, 208 each include sample inlets 222, 224, 226, 228, and each sample inlet is configured to independently receive a sample. The cartridge 200 can be referred to as a multi-lane PCR cartridge having dedicated sample inlets. The sample inlet can be configured to receive a liquid transfer member (not shown) such as a syringe, pipette, or PCR tube containing a PCR-compatible sample. In an embodiment of the cartridge according to the present technology, one inlet operates in conjunction with one sample lane.

[0031] In the embodiment of FIG. 2A, each reaction chamber 212, 214, 216, 218 has at least one dimension that is larger than each reaction chamber 112, 114, 116, 118 of the embodiment of FIG. 1A. The reaction chambers 212, 214, 216, 218 can be considered wider, where the width dimension is measured along the X-axis of the microfluidic cartridge. The reaction chambers 212, 214, 216, 218 can be considered deeper, where the depth dimension is measured along the z-axis of the microfluidic cartridge. In some embodiments, the reaction chambers 212, 214, 216, 218 can be considered longer, and the length dimension is measured along the y-axis of the microfluidic cartridge. The length dimension and the width dimension can be arranged along perpendicular axes. In an exemplary embodiment, the reaction chambers 212, 214, 216, 218 are wider and deeper than the reaction chambers 112, 114, 116, 118. Each reaction chamber 212, 214, 216, 218 can have a larger volume than each reaction chamber 112, 114, 116, 118. As a result, each reaction chamber 212, 214, 216, 218 can hold a larger volume of fluid than each reaction chamber 112, 114, 116, 118.

[0032] In some embodiments, the cartridge 200 includes an increased thickness to accommodate the deeper reaction chambers of FIG. 2A, where the thickness dimension is measured along the z-axis of the microfluidic cartridge. The cartridge 200 can have a thickness of about 1.68 mm compared to the cartridge 100 that can have a thickness of about 1.24 mm. In some embodiments, the thicker cartridge may have inferior thermal performance characteristics compared to the thinner cartridge, including edge effect failures (outside the sample lane), reverse edge effect failures (inside the sample lane), and random failures. Embodiments of the compressible pad according to the present technology, as described herein, can improve the thermal conductivity and / or thermal coupling between the cartridge 200 and the heating device and reduce these failures.

[0033] The reaction chambers 212, 214, 216, 218 can have any shape. In an exemplary embodiment, the reaction chambers 212, 214, 216, 218 can have an oval shape. The edges of the reaction chambers 212, 214, 216, 218 can be rounded. Other shapes of the reaction chambers are also contemplated.

[0034] The chambers 212, 214, 216, 218 of the adjacent sample lanes 202, 204, 206, 208 are staggered with respect to each other. In some embodiments, all of the sample inlets are arranged along a single line 232 parallel to the X-axis of the microfluidic cartridge. The 24-lane cartridge has two banks 226, 228 of 12 PCR reaction chambers as shown in FIGS. 2A and 2B. Each network can include reaction chambers. In some embodiments, each network can include two valves on both sides of the reaction chamber. The valves are normally open initially and close the channel when actuated. The valves can include microvalves. In some embodiments, each network can include an outlet or vent. In some examples, the outlet or vent can allow the gas in the microfluidic network to avoid the microfluidic network when the sample moves from the inlet through the microfluidic network to the chamber. In some examples, the outlet or vent can allow the amplified sample to be removed from the microfluidic network.

[0035] In some embodiments, reaction chamber 212 in the first bank of reaction chamber 226 is aligned with reaction chamber 214 in the second bank of the PCR sample lanes. Reaction chambers 212, 214 can be aligned laterally with respect to a single line 232 of sample inlets. Adjacent networks can form a staggered reaction chamber, as shown in the illustrated embodiment. In some embodiments, the 24-lane cartridge has two banks of 12 reaction chambers 226, 228. One first bank of the 12 reaction chambers 226, 228 is closer to the inlet. The other bank of the 12 reaction chambers 226, 228 is further from the inlet. The first bank 226 of the 12 reaction chambers can be axially aligned along a first axis 256, and the second bank 228 of the 12 reaction chambers can be axially aligned along a second axis 258. The reaction chamber 212 of the first bank 226 of the 12 reaction chambers and the reaction chamber 214 of the second bank 228 of the reaction chambers can be aligned along a third axis 260. The third axis can be transverse or perpendicular to the first axis and / or the second axis. Other configurations are contemplated.

[0036] As an example, the reaction chambers 112, 114, 116, 118 can each be 4 microliter PCR reaction chambers. As an example, the reaction chambers 112, 114, 116, 118 can each be approximately 1.5 mm in width, approximately 0.30 mm (300 microns) in depth, and approximately 10 mm in length. The volume of the reaction chamber can be approximately 4 μl. These dimensions and layouts are exemplary, and it will be understood that departures from those shown do not conflict with equivalent ways of operating such cartridges. The microfluidic cartridge 100 can enable PCR to be performed in a concentrated reaction volume (about 4 μl) and enable rapid thermal cycling at about 20 seconds per cycle. As another example, typical dimensions of the reaction chamber are 150 μ in depth × 700 μ in width, and a typical volume is about 1.6 μl. The channels of the microfluidic network in the sample lane of the cartridge 100 can have cross-sectional dimensions of less than at least 1 millimeter. For example, the channels of such a network can have a width and / or depth of less than 1 mm (e.g., about 750 microns or less, about 500 microns or less, or about 250 microns or less).

[0037] In the implementation configuration of the present technology, the reaction chambers 212, 214, 216, 218 can have an increased width and / or an increased depth (however, the same or similar length) relative to the reaction chambers 112, 114, 116, 118 of the microfluidic cartridge 100. In the first embodiment, each of the reaction chambers 212, 214, 216, 218 has a width of about 3.5 mm, a depth of about 0.54 mm (540 microns), and a length of about 10 mm. The volume of the reaction chamber is about 16.8 μL. In the second embodiment, each of the reaction chambers 212, 214, 216, 218 has a width of about 2.5 mm, a depth of about 0.86 mm (860 microns), and a length of about 10 mm. The volume of the reaction chamber is about 18.6 μL. In some embodiments, each of the reaction chambers 212, 214, 216, 218 can be a PCR reaction chamber having a volume of about 25 microliters. In the third example shown in FIG. 2C, each of the reaction chambers 212, 214, 216, 218 has a width of about 3.5 mm, a depth of about 0.83 mm (830 microns), and a length of about 10 mm. The volume of the reaction chamber is about 25.2 μL. In the fourth embodiment, each of the reaction chambers 212, 214, 216, 218 has a width of about 2.5 mm, a depth of about 1.35 mm (1350 microns), and a length of about 10 mm. The volume of the reaction chamber is about 25.2 μL. In the context of the viral load assay test described in the following non-limiting examples, the third embodiment was determined to have shown optimal performance characteristics for an improved viral load assay test.

[0038] The reaction chambers of the above embodiments are summarized in the following table. TIFF2025102763000002.tif114164Table 1

[0039] Embodiments of the microfluidic cartridge described herein can include reaction chambers having different volumes. For example, in one non-limiting embodiment illustrated in FIG. 2D, the microfluidic cartridge 600 includes a reaction chamber 612 having a volume of about 4 μL and a reaction chamber 614 having a volume of about 16 μL. Embodiments of the microfluidic cartridge 600 are not limited to the specific arrangement of reaction chambers illustrated in FIG. 2D, and it will be understood that other arrangements and combinations of reaction chamber volumes are possible.

[0040] In some embodiments, the width of reaction chambers 212, 214, 216, 218 can be 1 to 4 mm (e.g., 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, between 1 mm and 2 mm, between 2 and 3 mm, between 3 and 4 mm, or a range between any two of the aforementioned values). In some embodiments, the depth of reaction chambers 212, 214, 216, 218 can be between 0 and 2 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 0.25 mm, 0.5 mm, 0.75 mm, 1 mm, 1.25 mm, 1.5 mm, 1.75 mm, between 0 and 0.5 mm, between 0 and 1 mm, between 1 and 1.5 mm, or a range between any two of the aforementioned values). In some embodiments, the length of reaction chambers 212, 214, 216, 218 can be between 8 mm and 12 mm (e.g., 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, between 9 mm and 11 mm, about 10 mm, or a range between any two of the aforementioned values). In some embodiments, the volume of reaction chambers 212, 214, 216, 218 can be between 10 μl and 30 μl (e.g., 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, 23 μl, 24 μl, 25 μl, 26 μl, 27 μl.28 μl, 29 μl, 30 μl, between 10 μl and 15 μl, between 15 μl and 20 μl, between 20 μl and 25 μl, between 25 μl and 30 μl, or a range between any two of the aforementioned values). These dimensions and layouts are exemplary, and it will be understood that departures from those shown do not conflict with equivalent ways of operating such cartridges. In some embodiments, each reaction chamber 112, 114, 116, 118 has a volume of 4 ml. In some embodiments, each reaction chamber 212, 214, 216, 218 has a volume of 25 ml, i.e., about 6 times the volume of reaction chambers 112, 114, 116, 118.

[0041] (Enhanced Microfluidic Cartridge with Larger Volume Reaction Chamber) The microfluidic cartridge 200 can be designed for nucleic acid amplification. As described herein, the microfluidic cartridge 200 has an increased volume PCR reaction chamber with a total volume of about 25.2 μl and can amplify a large volume of liquid eluate from the sample being processed. Specifically, embodiments of the microfluidic cartridge 200 can ensure that a larger proportion of the liquid eluate from the sample processing procedure is loaded into the PCR reaction chamber and can be amplified within the PCR reaction chamber. In some cases, there is a six-fold increase in the volume of the liquid eluate that can be amplified. The implementation configuration of the enhanced microfluidic cartridge of the present technology has a reaction chamber with a larger volume and thus can accommodate a larger input of liquid eluate. As a result, the enhanced microfluidic cartridge of the present technology enables a more consistent amplification process across samples and cartridges, reduces the variation in the amplification process across samples and cartridges, and improves the performance of the overall assay.

[0042] In an implementation configuration where the microfluidic cartridge 200 includes a plastic substrate layer, the geometric shape of each reaction chamber 212, 214, 216, 218 is formed within the plastic substrate layer on all sides except the side where each reaction chamber 212, 214, 216, 218 is sealed by a laminate layer as described herein. The sample nucleic acid and the PCR reagent mix can be loaded into the chamber through the inlet port and the microfluidic channels. Each reaction chamber 212, 214, 216, 218 can be sealed by a thermally activated wax valve that extends into the fluid path and cools on either side of the chamber. As described herein, heat is applied to each reaction chamber 212, 214, 216, 218 through the laminate layer on the bottom side of the cartridge 200 to perform the PCR reaction, and fluorescence changes are measured through an external optical system disposed to cover the chambers on the upper side of the cartridge 200.

[0043] The microfluidic cartridge 100 accommodates a reaction volume of approximately 4.2 μl for each of the reaction chambers 112, 114, 116, 118. In some embodiments, the microfluidic cartridge 200 achieves improved analytical sensitivity as compared to the microfluidic cartridge 100. In some embodiments, the larger PCR chamber volume of the microfluidic cartridge 200 overcomes the limitations of target delivery of the microfluidic cartridge 100. In some embodiments, the microfluidic cartridge 200 achieves improved sensitivity performance by increasing the PCR chamber volume to approximately 25.2 μl. In some embodiments, a larger volume of PCR chamber is desired because more DNA / RNA input from sample extraction can increase sensitivity. In some embodiments, a larger volume of PCR chamber provides better performance. In some embodiments, a larger volume of PCR chamber improves the lower limit of detection of amplification performed in the larger volume of PCR chamber. In some embodiments, a larger volume of PCR chamber improves the lower limit of quantification of amplification performed in the larger volume of PCR chamber. In some embodiments, a larger volume of PCR chamber improves PCR efficiency.

[0044] The sensitivity of the assay depends on multiple factors, including extraction efficiency, PCR efficiency, and thermal uniformity. In some embodiments, increasing the size of the chamber is one factor that improves the PCR efficiency that results in an improvement in the lower limit of detection and the lower limit of quantification. In some embodiments, the microfluidic cartridge 200 achieves a six-fold increase in volume compared to the microfluidic cartridge 100. Other configurations are contemplated (e.g., a two-fold increase in volume, a three-fold increase in volume, a four-fold increase in volume, a five-fold increase in volume, a six-fold increase in volume, a seven-fold increase in volume, an eight-fold increase in volume, or a range of any of the foregoing values of two or more). There is an upper limit to the amount by which the size of the chamber can be increased while achieving optimal temperature uniformity throughout the chamber during each cycle of the amplification protocol. This is particularly true for amplification protocols with specific optimized cycle times to achieve reliable PCR. In some embodiments, the microfluidic cartridge 200 can accommodate a larger eluate input from the sample processing procedures performed on the sample. In some embodiments, the microfluidic cartridge 200 can improve the lower limit of detection and the lower limit of quantification of the assay. In some embodiments, the microfluidic cartridge 200 can ensure that a larger proportion of the liquid eluate from sample processing can be loaded into the increased-size chamber. In some embodiments, the microfluidic cartridge 200 can facilitate more consistent PCR amplification. In some embodiments, the microfluidic cartridge 200 can reduce the variability of PCR amplification. In some embodiments, the microfluidic cartridge 200 can improve the overall performance of the assay performed on the sample.

[0045] The reaction chamber of a given sample lane has dimensions of length, width, and depth that allow PCR to amplify the polynucleotides present in the sample received within the reaction chamber. The top of each reaction chamber includes a window that allows for the detection of fluorescence from a fluorescent substance within the reaction chamber when a detector is positioned above the window. It should be understood that other configurations of the window can include, but are not limited to, a single window spanning across each PCR reactor over the width of the cartridge.

[0046] The sample inlets of adjacent sample lanes are spaced apart from each other to prevent any contamination of one sample inlet when introducing samples into adjacent sample inlets within the cartridge. In some embodiments, the sample inlets are configured to prevent the unintentional introduction of subsequent samples into a given sample lane after a sample has already been introduced into that sample lane. In some embodiments, the multi-sample cartridge is designed such that the distance between the centers of the sample inlets is 6 mm, which is the standard recognized in the industry. This means that, in a particular embodiment, the center-to-center distance between the inlet holes of the cartridge is 6 mm. The inlet holes can be manufactured in a conical shape with an appropriate conical angle such that industry-standard pipette tips (2 μl, 20 μl, 200 μl volume, etc.) fit snugly therein. The cartridges of the present specification can be adapted to other subsequent industry standards not otherwise described herein, as would be understood by one of ordinary skill in the art.

[0047] In some embodiments, the microfluidic cartridge includes first, second, and third layers that together define a plurality of microfluidic networks, each network having various components configured to perform PCR on a sample having one or more polynucleotides whose presence is to be determined. As described herein, the microfluidic cartridge can include a fourth layer designed to improve pressure distribution, enhance thermal uniformity, and strengthen PCR amplification. In some embodiments, the fourth layer is a compressible pad. Although four layers are described, the microfluidic cartridge can include fewer layers, and one or more of the layers can be combined into a single integrated layer. Although four layers are described, additional layers can be included, and one or more of the layers can be divided into two or more layers.

[0048] The cartridge includes one or more sample lanes, each sample lane being associated independently of a given sample for simultaneous processing, and each sample lane including a separately configured microfluidic network. The cartridge typically processes one or more samples by increasing the concentration (such as by amplification) of one or more polynucleotides to be determined present in each sample.

[0049] The cartridges herein include embodiments having three or more layers in their structure, as shown in Embodiment 300 of FIGS. 3A and 3B. Cartridge 300 includes a substrate 302, a laminate 304 (not visible in FIG. 3A), and a label 306. In cartridge 300, the microfluidic substrate 302 has an upper side 308 and a lower side 310 (not visible in FIG. 3A) on the opposite side of the substrate. Substrate 302 includes a plurality of microfluidic networks disposed in corresponding plurality of sample lanes 312. Cartridge 300 includes a plurality of cartridge lanes 330. In this non-limiting embodiment, cartridge 300 includes twelve cartridge lanes 330. In this non-limiting embodiment, each cartridge lane 330 corresponds to a region of cartridge 300 that includes two sample lanes 312. In this non-limiting embodiment, cartridge 300 includes twenty-four sample lanes 312 disposed in twelve parallel cartridge lanes 330. Cartridge 300 can include a laminate 304 attached to the lower side 310 of substrate 302 to seal various components (e.g., valves) of the microfluidic network. Laminate 304 can provide an effective heat transfer layer between a dedicated heating element (further described herein) and components within the microfluidic network. Cartridge 300 can include a label 306 attached to the upper side 308 of substrate 302. In some embodiments, each reaction chamber is formed within all of the microfluidic substrate layers except for one or more sides where each reaction chamber is sealed by one or more additional layers. In some embodiments, each reaction chamber is sealed by laminate 304. In some embodiments, each reaction chamber is sealed by laminate 304. In some embodiments, each reaction chamber is sealed by label 306.

[0050] The cartridge 300 can include a compressible pad 314. In some embodiments, the compressible pad 314 is disposed above the upper side 308 of the substrate 302. In some embodiments, the compressible pad 314 is disposed between the upper side 308 of the substrate 302 and the label 306. In one exemplary embodiment, the compressible pad 314 is disposed below the label 306. In another exemplary embodiment, the compressible pad 314 is disposed above the label 306. In embodiments where the compressible pad 314 is disposed above the label 306, the label 306 can cover and seal holes used in a manufacturing process for loading components such as valves of the microfluidic network with a thermoresponsive material. In such embodiments where the compressible pad 314 is disposed above the label 306, markings (detailed below) that would normally be included on the label 306 can be included on the compressible pad 314. In some embodiments, when the compressible pad 314 is disposed below the label 306, each reaction chamber is sealed by the compressible pad 314.

[0051] In some embodiments (not shown), the compressible pad 314 is disposed below the lower side 310 of the substrate 302. In some embodiments, the compressible pad 314 is disposed between the lower side 310 of the substrate 302 and the laminate 304. In some embodiments, the compressible pad 314 is above the laminate 304. In some embodiments, the compressible pad 314 is below the laminate 304. In such embodiments, the compressible pad can be formed of a thermally conductive material or can include thermally conductive properties.

[0052] Thus, embodiments of the microfluidic cartridge herein include embodiments in which the layers comprise a substrate 302, a laminate 304, and a label 306, and the compressible pad 314 is disposed adjacent to at least one of the layers. In some embodiments, the microfluidic cartridge consists essentially of four layers: a substrate, a laminate, a label, and a compressible pad. In some embodiments, the microfluidic cartridge comprises four layers: a substrate, a laminate, a label, and a compressible pad.

[0053] The microfluidic substrate layer 302 is typically injection molded from plastic, preferably zeonor plastic (cyclic olefin polymer), and includes a plurality of microfluidic networks (shown in FIGS. 1A and 2A). As described herein, in some embodiments, the substrate 302 comprises 24 reaction chambers containing materials for PCR amplification. Each microfluidic network includes reaction chambers and associated channels. In some embodiments, the microfluidic network includes one or more valves. The valves, when present, can be disposed on a first (e.g., lower) side (disposed toward the laminate). In some embodiments, the microfluidic network includes loading holes for loading wax or other thermally reactive materials into the valves. In some embodiments, the microfluidic network includes one or more vent channels. In some embodiments, the microfluidic network includes one or more liquid inlet holes on a second (e.g., upper) side (disposed toward the label layer). Typically, in a given cartridge, all of the microfluidic networks including the reaction chambers and inlet holes are defined together in a single substrate layer, namely substrate 302.

[0054] The substrate 302 can be formed from a material that improves the rigidity of the substrate (and thus the cartridge). The material from which the substrate 302 is formed can be rigid or non-deformable. Rigidity is advantageous because, as further described herein, it facilitates effective and uniform contact with the heating assembly. In some embodiments, the substrate 302 is impermeable to air or liquid, so that air or liquid can enter or exit the cartridge only through the inlet or various vents during operation of the cartridge. The material from which the substrate 302 is formed can be airtight with respect to air and other gases. Using an airtight material is also advantageous because it reduces the possibility that the concentrations of various types in liquid form will change during analysis. In some embodiments, the substrate 302 has low autofluorescence to facilitate the detection of polynucleotides during the amplification reaction performed in the microfluidic circuit defined herein. The use of a material with low autofluorescence is also important to ensure that background fluorescence does not interfere with the measurement of fluorescence from the analyte of interest.

[0055] The substrate 302 can have a region of reduced thickness to facilitate detection. In some embodiments, the region of reduced thickness can be located above each reaction chamber of each sample lane. In some embodiments, the region of reduced thickness can have an oval or elongated shape. The region of reduced thickness can have a surface area that is the same as or larger than the area of the corresponding reaction chamber.

[0056] The laminate layer 304 can be a heat-sealable laminate layer. The laminate layer 304 can typically have a thickness of about 100 to about 125 microns. The laminate layer 304 can be attached to the bottom surface of the microfluidic substrate 302 using, for example, thermal adhesion, pressure bonding, or a combination thereof. The laminate layer 304 can also be made from a material having an adhesive coating on only one side, and this side is the side that contacts the lower surface of the substrate 302. This layer 304 can be made from a single-coated tape having a layer of Adhesive 420 (registered trademark) manufactured by 3M (registered trademark). Exemplary tapes include single-sided forms of double-sided tapes having product numbers 9783, 9795, and 9795B and are available from 3M (registered trademark). The laminate layer is typically 50 - 200 μ thick, for example 125 μ thick. Other acceptable layers can be made from adhesive tapes that utilize microcapsule-based adhesives.

[0057] The label 306 can be made from polypropylene or other plastic having a pressure-sensitive adhesive. The label 306 can typically have a thickness of about 50 to 150 microns. In some embodiments, the label 306 can be configured to seal the wax filling hole of the valve of the substrate 302. In some embodiments, the label 306 can capture the air used for valve operation. In some embodiments, the label 306 can function as a location for operator markings. The label 306 can include identification characteristics such as the barcode number, lot number, and expiration date of the cartridge. In some embodiments, the label 306 has a space and a writable surface that allow the user to manually add identification annotations on the label. The label 306 can be a single molded layer, but it will be understood by those skilled in the art that the label 306 can be formed from two or more separate elements.

[0058] The label 306 can print various types of information, including but not limited to the manufacturer's logo, part number, and index number for each sample lane. In various embodiments, the label 306 can include a computer-readable or scannable portion that can include specific identification indicators such as a lot number, expiration date, or unique identifier. For example, the label 306 can include a barcode, radio frequency tag, or one or more computer-readable or optically scannable characters. The readable portion of the label 306 can be positioned to be readable by a sample identification verifier. The label 306 can include a cutout 318 from an edge or corner of the label 306.

[0059] In some embodiments, the microfluidic cartridge 300 further includes an alignment member 316 that ensures that the cartridge is received by a complementary diagnostic device in a single orientation, for example, in a receiving bay of the device. The alignment member 316 can be a cutout from an edge or corner of the cartridge (as shown in FIG. 3A), or can be a series of notches, wedge-shaped or curved cutouts, or other configurations of a shape that requires a specific orientation of the placement in the device.

[0060] In some embodiments, the microfluidic cartridge 300 has a size substantially the same as that of a 96-well plate conventionally used in the art. Advantageously, the cartridge can then be used with plate handlers used elsewhere in the art.

[0061] In some embodiments, the microfluidic cartridge 300 includes two or more positioning elements, i.e., fiducials, for use when filling the valve with a heat-responsive material. The positioning elements can be disposed on the substrate 302, typically on its upper surface. In some embodiments, the fiducials can be on the diagonal of the substrate, but are not limited to such a position.

[0062] As described herein, above each reaction chamber, there is a window 320 that enables optical detection such as fluorescence detection from a fluorescent substance in the reaction chamber, such as from a fluorescent hybridization probe, when the detector is located above the window 320. The plurality of windows 320 can be formed on the label 306. The number of windows 320 can correspond to the number of reaction chambers (e.g., 24 reaction chambers, 24 windows or 12 reaction chambers, 12 windows, etc., a 1:1 ratio). Other configurations are contemplated for the window 320, such as shape, position, and / or number. In the illustrated embodiment, the window 320 has an oval shape. The window 320 can have a surface area that is the same as or larger than the area of the corresponding reaction chamber.

[0063] Next, an embodiment of the compressible pad according to the present technology will be described. FIG. 4 is a diagram showing a non-limiting example of a compressible pad 314 according to the present technology. The cartridge 300 can include the compressible pad 314. The compressible pad 314 can be formed of a material with low deflection under compressive force, as described herein. The compressible pad 314 can be formed of a material that compresses easily, as described herein. The compressible pad 314 can be formed of a mechanically compliant material. For example, the mechanically flexible material of the compressible pad 314 can have a thickness of about 0.035 inches (about 0.9 mm). For example, other thicknesses such as about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, between 0 mm and 1 mm, between 0.5 mm and 1.5 mm, between 1 mm and 2 mm, between 1.5 mm and 2.5 mm, between 0 mm and 2 mm, between 0.5 mm and 2.5 mm, between 1 mm and 3 mm, between 1.5 mm and 3.5 mm, and others are also suitable.

[0064] In some embodiments, the compressible pad 314 is incorporated into a consumable, such as a microfluidic cartridge. In some embodiments, a compressible pad (not shown) is incorporated into a diagnostic device (e.g., into a detector that physically contacts the microfluidic cartridge during a detection procedure).

[0065] The compressible pad 314 can be a heat-sealable layer and can be attached to the microfluidic cartridge, for example, using a pressure-sensitive adhesive. The compressible pad 314 can be made compressible as described herein. The thickness of the compressible pad 314 can be from 0.1 to 2.5 mm when uncompressed, typically about 1.5 mm thick when uncompressed.

[0066] As described herein, the cartridge 300, particularly the substrate 302, can include an alignment member 316 that ensures that the cartridge is received by a complementary diagnostic device in a single orientation, for example, in a receiving bay of the device. The alignment member 316 can be a notch from the edge or corner of the cartridge (as shown in FIG. 3A), or can be a series of notches, wedge-shaped or curvilinear notches, or other configurations of a shape that requires a specific orientation of the placement in the device. The compressible pad 314 can include a notch 322 from the end or corner of the compressible pad 314. The notch 322 can correspond to the notch 318 of the label 306 shown in FIG. 3A.

[0067] As described herein, above each reaction chamber, there is a window 320 of the label 306 that enables optical detection in the reaction chamber when the detector is positioned above the window 320. The compressible pad 314 can include a plurality of windows 324. The number of windows 324 can correspond to the number of reaction chambers (e.g., 24 reaction chambers, 24 windows, or 12 reaction chambers, 12 windows, etc., a 1:1 ratio). Other configurations for the windows 324 are contemplated, such as shape, position, and / or number. In the illustrated embodiment, the windows 324 have an oval or elongated shape. The windows 324 can have a surface area that is the same as or larger than the area of the corresponding reaction chamber. The windows 324 can correspond to the windows 320 of the label 306 shown in FIG. 3 in terms of number and / or shape.

[0068] As described herein, the reaction chambers of adjacent sample lanes are arranged in a staggered pattern relative to each other. In some embodiments, all of the sample inlets are arranged along a single line parallel to the X-axis of the microfluidic cartridge 300. The reaction chambers in the first bank of sample lanes can be aligned with the reaction chambers in the second bank of sample lanes, and the reaction chambers can be aligned laterally with respect to the single line of sample inlets. In some embodiments, a 24-lane cartridge has two banks of 12 reaction chambers 326, 328. The first bank 326 of the 12 reaction chambers is proximate to an edge having an alignment member 316. The second bank 328 of the 12 reaction chambers is remote from the edge having the alignment member 316. The reaction chambers can form a grid. Other configurations are contemplated.

[0069] In some embodiments, a 24-lane cartridge has two banks of 12 windows formed from the windows 320 of the label 306 and the windows 324 of the compressible pad 314. The windows 320, 324 can form a grid. In some embodiments, the windows 320 of the label 306 and the windows 324 overlap each other to form pairs of windows through which light can pass. The surface area of each of the windows 320, 324 can be larger than the surface area of the corresponding reaction chamber. In the illustrated embodiment, each pair of windows 320, 324 encompasses the area surrounding one reaction chamber. In another embodiment (not shown), each pair of windows 320, 324 encompasses the area surrounding two or more reaction chambers. In yet another embodiment (not shown), each pair of windows 320, 324 encompasses the area surrounding a bank of reaction chambers. In this embodiment, the label 306 and the compressible pad 314 each include two windows, namely, a first window on the first bank 326 and a second window on the second bank 328. In a further embodiment (not shown), there is a single pair of windows 320, 324 that encompasses the area surrounding all 24 reaction chambers of the cartridge 300. In this embodiment, there is a single window across all of the reaction chambers of the cartridge 300.

[0070] In some embodiments, the compressible pad 314 can be a separate layer coupled to the label 306. The label 306 and / or the compressible pad 314 can include an adhesive surface to couple the components together. Other coupling methods are contemplated. FIG. 3A shows an embodiment in which the compressible pad 314 is adhered to the top of the PCR cartridge 300 and the white cartridge label 306 is adhered to the top of the compressible pad 314. The label 306 is partially removed to show the compressible pad 314 below the label 306.

[0071] In some embodiments, the compressible pad 314 and the label 306 can be combined into a single layer. In some embodiments, the label 306 can be omitted. In such embodiments, the compressible pad 314 can include an upper surface for displaying the barcode and manufacturing information as described above. In some embodiments, the compressible pad 314 is white or light-colored. In some embodiments, the label information can be printed directly on the compressible material, thereby eliminating the label 306. In some embodiments, omitting the label 306 can eliminate the possibility of delamination between the compressible pad 314 and the label 306.

[0072] In some embodiments, the compressible pad 314 is a completely separable compressible pad. In some embodiments, the compressible pad 314 is a separately or independently formed component. The compressible pad 314 can be disposed on the cartridge 300, such as on the upper surface 308 of the cartridge 300. In some embodiments, the compressible pad is applied on top of the label 306 of the cartridge 300 (this embodiment is not shown in FIG. 3A). In some embodiments, the compressible pad can be reusable after completion of PCR amplification, for example, by removing the compressible pad 314 from the first cartridge 300 and applying this same compressible pad 314 to the second cartridge 300. This embodiment, like other embodiments disclosed herein, shows an improvement in heat energy transfer to the reaction chamber. In some embodiments as described herein, the compressible pad 314 is integrated on or within the cartridge 300. For example, the compressible pad 314 is not intended to be reusable, and the disposal of the cartridge 300 after amplification of one or more samples also disposes of the compressible pad 314 integrated with the cartridge 300. The compressible pad 314 can be integrated into the configuration of the cartridge 300. When integrated, the compressible pad 314 can reduce the risk of peeling during use.

[0073] In some embodiments, the cartridge 300 is disposable. After PCR is performed on the sample and the presence or absence of the target polynucleotide is determined, the amplified sample remains on the cartridge and the cartridge is typically reused (if one or more sample lanes remain open) or discarded. If the user wishes to perform post-amplification analysis such as gel electrophoresis, the user can punch a hole through the stack 304 of the cartridge 300 and recover typically about 1.5 microliters of the PCR product. In one non-limiting embodiment, the user places an individual sample lane on a special narrow heating plate, maintains the temperature at which the wax in the valve of the sample lane melts, and then can aspirate the reacted sample from the inlet hole of the sample lane.

[0074] The microfluidic cartridge 300 can also be configured to be stackable, for example, for easy storage or transportation, or to be received by a loading device that holds a plurality of cartridges in proximity to each other without contacting each other. In various embodiments, during transportation and storage, the microfluidic cartridge can be further enclosed, for example, by a sealed pouch to reduce the effects of, for example, water vapor. The microfluidic cartridge can be sealed in the pouch with an inert gas. The microfluidic cartridge can be disposable, such as intended for single use. The microfluidic cartridge can be made disposable, for example, after one or more of its sample lanes have been used.

[0075] Next, a non-limiting example of a heating assembly according to the present technology will be described in detail. FIG. 5A is a diagram showing an exemplary heater module 400 of the receiving bay 402. The heater module 400 can include a concave surface that provides a platform for supporting a microfluidic cartridge in the receiving bay. In use, the cartridge 300 is typically thermally associated with an array of heat sources configured to apply heat to various components of the device (e.g., reaction chambers). Such an exemplary heater array including heat sources will be further described herein. Additional embodiments of the heater array are described in U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed on November 14, 2007, which is incorporated herein by reference.

[0076] FIG. 5B shows another exemplary heater module 700 of the receiving bay 702. In this non-limiting embodiment, the system includes two receiving bays 702 each configured to receive a microfluidic cartridge of the present technology. FIG. 5C shows an enlarged view of the heater module 700 of the left receiving bay 702. FIG. 5D shows an enlarged view of the heater module 700 of FIG. 5C with the microfluidic cartridge 200 received in the receiving bay 702.

[0077] The microfluidic substrate described herein is configured to receive heat from a contact heat source such as that found in a heater unit. The heater unit typically comprises a heater board or heater chip configured to supply heat to a specific region of the microfluidic substrate that, at a given time, includes, but is not limited to, one or more microfluidic components. For example, the heat source is configured such that a particular heating element is positioned adjacent to a particular component of the microfluidic network on the substrate. In certain embodiments, the device uniformly controls the heating of regions of the microfluidic network. In an exemplary embodiment, multiple heaters can be configured to simultaneously and uniformly heat regions such as a PCR reaction chamber of the microfluidic substrate.

[0078] The heater is located in a heater substrate layer directly beneath the microfluidic substrate. In a non-limiting example, the heater can be defined and etched by photolithography of a gold metal layer (typically about 3,000 Å thick). Layers of TiW of 400 Å are deposited on the top and bottom of the gold layer to function as adhesion layers. The substrate can be a glass, fused silica, or quartz wafer having a thickness of 0.4 mm, 0.5 mm, 0.7 mm, or 1 mm. A thin electrical insulation layer of silicon dioxide of 2 μm functions as an insulation layer on top of the metal layer. Additional thin electrically insulating layers such as parylene of 2 - 4 g / m can also be deposited on top of the silicon dioxide surface.

[0079] An exemplary set of heaters configured to periodically heat a PCR reaction chamber can be provided. It should be understood that heater configurations for actuating other regions of the microfluidic cartridge, such as other gates, valves, and actuators (if present in the cartridge), can be designed and deployed according to principles similar to those governing the heaters described herein.

[0080] Exemplary reaction chambers within a microfluidic substrate, typically chambers or channels having a certain volume, have long sides and short sides, each configured with an associated heating element. The reaction chamber may also sometimes be referred to herein as a PCR reactor, and the region of the cartridge in which the reaction chamber is located may sometimes be referred to as a zone. The heater substrate in this non-limiting example includes four heaters arranged along the sides of a given reaction chamber and configured to heat the given reaction chamber: namely, a long upper heater, a long lower heater, a short left heater, and a short right heater. The small gap between the long upper heater and the long lower heater results in a negligible temperature gradient (a difference of less than 1 °C between the two ends of the width of the reaction chamber at any point along the length of the reaction chamber), and thus a substantially uniform temperature throughout the reaction chamber. The heaters at the short edges of the reaction chamber provide heat to cancel out the gradient created by the two long heaters from the center to the edges of the reaction chamber.

[0081] One of ordinary skill in the art will understand that yet another configuration of one or more heaters positioned around the reaction chamber will be compatible with the methods and apparatuses described herein. For example, the "long" sides of the reaction zone can be configured to be heated by two or more heaters. Utilizing the low thermal conductivity of glass, or quartz, polyimide, FR4, ceramic, or fused silica substrates aids in the independent operation of various microfluidic components such as valves (if present in the cartridge) and the independent operation of various sample lanes, so that the particular orientation and configuration of the heaters are used to generate a uniform heating zone even on substrates with low thermal conductivity. One of ordinary skill in the art will further understand that the principles underlying the configuration of the heaters around the reaction zone are equally applicable to the placement of heaters adjacent to other components of the microfluidic cartridge such as actuators, valves, and gates (if present in the cartridge).

[0082] FIG. 38 shows a set of heater arrays of a heating device configured to apply heat to a microfluidic cartridge according to the present disclosure. For example, FIG. 38A illustrates a heater array configured to apply heat to a microfluidic cartridge including 24 sample lanes. FIG. 38B is an extended view of one array configured to apply heat to one reaction chamber of a 24-sample lane cartridge, including a heater and a temperature sensor through which current flows during operation.

[0083] In some embodiments, the heat source is controlled by a computer processor and operates according to a desired protocol. A processor configured to operate a microfluidic device is described, for example, in U.S. Patent Application No. 12 / 173,023, filed July 14, 2008, entitled "Integrated Apparatus for Performing Nucleic Acid Extraction and Diagnostic Testing on Multiple Biological Samples", which is hereby incorporated by reference. A processor, such as a microprocessor, is configured to control the functions of various components of the system as shown and thus communicate with each such component that requires control. It should be understood that many of these control functions can optionally be performed manually and are not under the control of the processor. Further, the order in which the various functions are described below is not limited to the order in which the processor executes instructions when the device is operating. Thus, the processor can be configured to receive data regarding a sample to be analyzed from a sample reader, which can be, for example, a barcode reader, an optical character reader, or an RFID scanner (radio frequency tag reader). Also, although a single processor is described as controlling all operations, it is understood that such operations may be distributed among multiple processors, if convenient.

[0084] The processor can be configured to receive user instructions from an input, and such instructions can include an instruction to start analyzing a sample and a selection of operating conditions. In various embodiments, the input can include, but is not limited to, one or more input devices such as a keyboard, a touch sensor surface, a microphone, a trackpad, a retina scanner, a holographic projection of an input device, and a mouse.

[0085] The processor can also be configured to communicate with a display, for example, to send information regarding the analysis to the display, thereby communicating it to the user of the system. Such information can include, but is not limited to, the current status of the device, the progress of the PCR thermal cycle, and a warning message in the event of a malfunction in either the system or the cartridge. Further, the processor can send one or more questions to be displayed on the display and prompt the user to enter a response thereto. Thus, in certain embodiments, the input and the display are integrated with each other.

[0086] Optionally, the processor can be further configured to send the analysis results to an output device such as a printer, a visual display, a display utilizing holographic projection, or a speaker, or a combination thereof.

[0087] The processor can further optionally be connected to a computer network via a communication interface such as a network interface. The communication interface can be one or more interfaces selected from the group consisting of a serial connection, a parallel connection, a wireless network connection, a USB connection, and a wired network connection. Thereby, when the system is properly addressed on the network, a remote user can access the processor and send instructions that can be stored in a memory (not shown) associated with the processor or in another computer-readable medium in communication with the processor, input data, or retrieve data. The interface can also thereby enable the extraction of data to a remote location such as a personal computer, a personal digital assistant, or a network storage device such as a computer server or a disk farm. The apparatus can further be configured so that a user can directly send analysis results by email to other parties such as medical providers, diagnostic facilities, or patients.

[0088] In addition, in various embodiments, the apparatus can further comprise a data storage medium configured to receive data from one or more of the processor, the input device, and the communication interface, and the data storage medium is one or more media selected from the group consisting of a hard disk drive, an optical disk drive, a flash card, and a CD-ROM.

[0089] The processor can further be configured to control various aspects of sample preparation and diagnostics, generally as follows and as described in more detail herein. The microfluidic cartridges 200, 300 are configured to operate with complementary racks (not shown). The racks are themselves configured to receive a plurality of biological samples in a form suitable for workup and diagnostic analysis, as well as a plurality of holders with various reagents, pipette tips and receptacles, as further described herein. The racks are configured such that during the workup of the samples, the samples are processed within their respective holders, the processing including being individually heated and cooled via a heater assembly. The heating function of the heater assembly can be controlled by the processor. The heater assembly operates in conjunction with a separator, such as a magnetic separator, that can be controlled by the processor to move in and out of proximity to one or more processing chambers associated with a holder in which particles, such as magnetic particles, are present.

[0090] A liquid dispenser (not shown) is similarly controllable by a processor and is configured to perform various suction and dispensing operations on each sample, liquid, and reagent in the holder to extract nucleic acids from the samples. The liquid dispenser can perform such operations simultaneously on a plurality of holders. The sample reader is configured to transmit identification indicators regarding the samples and, in some cases, the holders to the processor. In some embodiments, the sample reader is attached to the liquid dispenser, whereby the liquid dispenser can read indicators regarding samples located above it. In other embodiments, the sample reader is not attached to the liquid dispenser and is independently movable under the control of the processor. The liquid dispenser is also configured to take aliquots of the liquid containing the nucleic acids extracted from one or more samples and direct them to the receiving bays into which the microfluidic cartridges 200, 300 are received. The receiving bays communicate with a heater or a set of heaters, and the heaters can be controlled by the processor such that a specific region of the cartridge is heated at a specific time during analysis. The liquid dispenser is configured to take aliquots of the liquid containing the nucleic acids extracted from one or more samples and direct them to the respective inlets of the microfluidic cartridges. The cartridges are configured to amplify, such as by performing PCR on each nucleic acid. The processor is also configured to control a detector that receives diagnostic instructions from the cartridges. The diagnostic content can be transmitted to an output device and / or a display as described herein.

[0091] Suitable processors can be designed and manufactured according to design principles and semiconductor processing methods well-known in the art, respectively. In some embodiments, the apparatus includes a bay configured to selectively receive a microfluidic cartridge, and at least one heat source thermally coupled to the bay and coupled to a processor further described herein, the heat source being configured to heat individual sample lanes within the cartridge, and the processor being configured to control applying heat to the individual sample lanes individually, all at once, or in groups. In use, cartridges 200, 300 are typically thermally associated with an array of heat sources configured to operate components of the apparatus (e.g., valves, gates, and processing regions). In some embodiments, the heat source is operated by an operating system that operates the apparatus during use. The operating system includes a processor (e.g., a computer) configured to operate the heat source according to a desired protocol. In some embodiments, a temperature sensor is preferably configured to transmit information regarding the temperature in its vicinity to the processor at times when the heaters are not receiving the current by which they heat them. This can be achieved by appropriate control of the current cycle.

[0092] As described herein, applying pressure can facilitate contact between the microfluidic cartridge and the heat sources of the heater array. In some embodiments, the pressure can be about 1 psi. This pressure is sufficient to enhance the contact between the cartridge and the heat source to help achieve good thermal contact between the heat source and the heat-receivable portion of the cartridge. In some embodiments, the pressure can prevent expansion of the bottom laminate layer 304 such as can occur when the PCR channels are partially filled with liquid and the trapped air thermally expands during thermal cycling.

[0093] Each reaction chamber is heated through a series of cycles to perform amplification of nucleotides in the sample according to an amplification protocol. The inner wall of the channel in the PCR reactor is typically made very smooth during manufacturing and polished to a shiny finish. This is to minimize the minute amount of air trapped on the surface of the PCR channel that generates bubbles during the thermal cycle. The presence of bubbles, especially in the detection region of the PCR channel, can also cause incorrect or inaccurate readings when monitoring the progress of PCR.

[0094] Referring to FIG. 1A, the reaction chamber can have dimensions (such as a shallow depth) such that the temperature gradient across the depth of the channel is minimized. Referring to FIG. 2A, the reaction chamber is, for example, deeper and wider to accommodate a larger sample for PCR. In the exemplary embodiment of FIG. 2A, the wider and deeper well needs to ensure that the temperature gradient across the depth of the channel is minimized and increase the thermal contact between the cartridge and the heater substrate, thereby ensuring optimal thermal uniformity and enhancing PCR amplification. In some embodiments, the compressible pad 314 can enable the use of wider and deeper wells by improving the pressure distribution and thus increasing the contact between the microfluidic cartridge and the heater substrate.

[0095] In some embodiments, the region of the substrate 302 above the reaction chamber can be a thinned portion to reduce the heat mass and autofluorescence from the plastic within the substrate. Also, as described herein, the label 306 can include a window 320, and the compressible pad 314 can include a window 324 to enable visualization of the reaction chamber and transmission of light between the reaction chamber. The design of the cartridge 300 can enable an optical detector to more reliably monitor the progress of the reaction and detect fluorescence from a probe that binds to a certain amount of amplified nucleotides. In some embodiments, the region of the substrate 302 can be made of a material thinner than the remainder of the substrate 302 to reduce glare, autofluorescence, and excessive absorption of fluorescence.

[0096] As described herein, the microfluidic cartridge can be configured to be positioned in a complementary receiving bay within a device that includes a heater unit. Non-limiting examples of the heater unit are shown in FIGS. 5A and 5B - 5D. The heater unit is configured to supply heat at a specific time to a specific region of the cartridge that includes, but is not limited to, one or more reaction chambers. In certain embodiments, the device uniformly controls the heating of regions of the microfluidic network. In an exemplary embodiment, a plurality of heaters can be configured to simultaneously and uniformly heat a single region, such as a PCR reaction chamber of the microfluidic cartridge. In other embodiments, portions of different sample lanes are heated simultaneously and independently of each other.

[0097] The microfluidic cartridge 300 can have an alignment member 316 that mates with complementary features of the receiving bay. The alignment member 316 can be, for example, a notch on the edge of the cartridge 300, and the receiving bay can include complementary features for the alignment member 316. By selectively receiving the cartridge, the receiving bay can assist in positioning the cartridge so that the device can operate properly on the cartridge.

[0098] The receiving bay can also be configured so that the heat source of the device operating on the microfluidic cartridge 300 operates properly therein. For example, the contact heat source can be positioned in the receiving bay so that it can be thermally coupled to one or more separate locations on the microfluidic cartridge 300 that are selectively received in the bay. The microheaters within the heater module, as further described herein, are aligned with the corresponding heat-requiring microcomponents (valves, pumps, gates, reaction chambers, etc.). The microheaters arranged in a set to supply heat to a particular region of the cartridge 300 can be designed to be slightly larger than the microfluidic components that require heat, enabling the individual components to function effectively even when the cartridge is off-center from the heater set.

[0099] As further described elsewhere herein, the lower surface of the cartridge can have a layer of a mechanically compliant heat transfer laminate 304 that enables thermal contact between the microfluidic cartridge 300 and the heater substrate of the heater module. In some embodiments, a minimum pressure, such as 1 psi, can be utilized for the reliable operation of the reaction chambers present in the microfluidic cartridge, as described herein.

[0100] Referring back to FIG. 3, a PCR reaction chamber (e.g., a reaction chamber 150 μ deep × 700 μ wide) is shown in the substrate layer 302 of the cartridge 300. The laminate layer 304 of the cartridge (e.g., 125 μ thick) is directly beneath the PCR reaction chamber. In some embodiments, the region of the substrate 302 is made of a thinner material than the rest of the substrate 302, enabling the PCR reaction chamber to be more responsive to heating cycles (e.g., for rapid heating and cooling between temperatures appropriate for the denaturation and annealing steps). The heater is positioned in the heater module directly beneath the laminate layer 304 when the cartridge is received by the heater module.

[0101] In some embodiments, each reaction chamber is configured to have a long side and a short side. Each of the sides corresponds to a related heating element disposed on a heater substrate. Thus, the heater substrate includes four heaters disposed along the sides of the PCR reaction chamber and configured to heat the PCR reaction chamber: namely, a long upper heater, a long lower heater, a short left heater, and a short right heater. In some embodiments, the small gap between the long upper heater and the long lower heater results in a negligible temperature gradient (a difference of less than 1 °C between the two ends of the PCR channel at any point along the length of the PCR reaction chamber), and thus effectively provides a uniform temperature throughout the PCR reaction chamber. The heaters at the short edges of the PCR reactor supply heat to counteract the gradient generated by the two long heaters from the center of the reactor to the edges of the reactor. It will be understood by those skilled in the art that other configurations of one or more heaters located around the PCR reaction chamber are compatible with the methods and apparatuses described herein. For example, the "long" sides of the reaction chamber can be configured to be heated by two or more heaters.

[0102] The heat source can be, for example, a resistive heater or a network of resistive heaters. In some embodiments, at least one heat source can be a contact heat source selected from a resistive heater (or network thereof), a radiator, a fluid heat exchanger, and a Peltier element. The contact heat source can be configured to be thermally coupled to one or more different locations of a microfluidic cartridge received in the receiving bay at the receiving bay, whereby different locations are selectively heated. The contact heat source typically includes a plurality of contact heat sources, each configured to be independently thermally coupled to a different distinct location of a microfluidic cartridge received therein at the receiving bay, whereby distinct locations are independently heated. The contact heat source can be configured to physically and directly contact one or more distinct locations of a microfluidic cartridge received in the bay. In various embodiments, each contact heat source can heat a distinct location having a two-dimensional average diameter of from about 1 millimeter (mm) to about 15 mm (typically from about 1 mm to about 10 mm), or a surface area of from about 1 mm 2 to about 225 mm 2 (typically from about 1 mm 2 to about 100 mm 2 , or in some embodiments from about 5 mm 2 to about 50 mm 2 ). Various configurations of the heat source are further described in U.S. Patent Application No. 11 / 940,315, filed November 14, 2007, entitled "Heater Unit for Microfluidic Diagnostic System", which is incorporated herein by reference.

[0103] In some embodiments, the heater is a photolithographically defined and etched gold metal layer (typically about 3,000 Å thick). A 400 Å layer of TiW is deposited on top of and beneath the gold layer and can function as an adhesion layer. In some embodiments, the heater substrate is a glass, fused silica or quartz wafer having a thickness of 0.4 mm, 0.5 mm, 0.7 mm, or 1 mm. In some embodiments, a thin electrical insulation layer of silicon oxide of 2 μm functions as an insulation layer on top of the metal layer. In some embodiments, an additional thin electrically insulating layer such as parylene of 2-4 μm can also be deposited on top of the silicon oxide surface. In some embodiments, two long heaters and two short heaters extend in parallel and surround the regions corresponding to each PCR reaction chamber. An exemplary heater array is described in U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System", filed on November 14, 2007, the specification of which is hereby incorporated by reference in its entirety.

[0104] To create a uniform heating zone even on a substrate with poor thermal conductivity, specific orientations and arrangements of the heaters are used. The heater substrate can be formed of various materials including glass, quartz, polyimide, FR4, ceramic, fused silica substrates. The heater module is utilized to assist in the independent operation of various microfluidic components such as PCR reaction chambers and the independent operation of various sample lanes. A configuration for performing uniform heating for a single PCR reaction chamber can be applied to a multi-lane PCR cartridge in which multiple independent PCR reactions are performed. In other embodiments, as further described in U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System", filed on November 14, 2007, the heater can have an associated temperature sensor or can itself function as a sensor.

[0105] Generally, the heating of microfluidic components such as a PCR reaction chamber is controlled by passing an electric current through a properly configured and microfabricated heater. Under the control of a proper circuit, the sample lanes of a multi - lane cartridge can be controlled independently of each other. This means that not all heaters are heated simultaneously, and a given heater receives current only for a portion of the time it is needed for heating, resulting in higher energy efficiency of the device. Control systems and methods for controllably heating various heating elements are further described in U.S. Patent Application No. 11 / 940,315, titled "Heater Unit for Microfluidic Diagnostic System", filed on November 14, 2007.

[0106] An example of the thermal cycling performance in a PCR reaction chamber obtained with the configuration as described herein can include a protocol where the reaction mixture is heated to 92°C, held at this temperature for 1 second, then cooled to 62°C and set to remain there for 10 seconds. The illustrated cycle time is about 29 seconds, with 8 seconds required to heat from 62°C and stabilize at 92°C and 10 seconds required to cool from 92°C and stabilize at 62°C. To minimize the overall time required for effective PCR to produce a detectable amount of amplified material, it is important to minimize the time required for each cycle. Desirable cycle times are in the range of 15 - 30 seconds, such as 18 - 25 seconds, and 20 - 22 seconds. Generally, an average PCR cycle time of 25 seconds and a short cycle time of 20 seconds are typical with the technology described herein. In some non - limiting examples, using a reaction volume of less than a microliter (such as a few hundred nanoliters or less) allows the use of a related smaller PCR chamber and enables a short cycle time of 15 seconds.

[0107] Next, a non-limiting example of an optical detector suitable for use with the microfluidic cartridge of the present technology will be described. Referring to FIG. 6, one embodiment of an optical detector 500 is shown. As described above, the heater module 400 is disposed below the microfluidic cartridge 300. In some embodiments, a thermally conductive mechanically compliant layer such as the compressible pad 314 can be laid at the interface between the microfluidic cartridge 300 and the optical detector 500. Typically, the microfluidic cartridge 300 and the heater module 400 can be made flat at their respective interface surfaces, for example, flat within about 100 microns, more typically within about 25 microns. The compressible pad 314 can improve the thermal coupling between the microfluidic cartridge 300 and the heater module 400. The optical detector 500 can be disposed on the upper surface of the microfluidic cartridge 300.

[0108] In various embodiments, the apparatus can further include one or more force members configured to apply a force to at least a portion of the microfluidic cartridge 300 received in the receiving bay 402 that includes one or more heat sources. In the non-limiting embodiment shown in FIG. 6, the force member includes a lever assembly 502 associated with the optical detector 500. In some embodiments, the system is dependent on the pressure applied to the cartridge 300. The bottom surface of the optical detector 500 can be made flat (e.g., within 250 microns, typically within 100 microns, more typically within 25 microns), and the bottom surface can press the cartridge 300. The cartridge 300 can include the compressible pad 314. As a result, the optical detector 500 can compress the cartridge 300, thereby generating pressure and thus forming a thermal contact with the heater substrate of the underlying heater module 400, making the entire microfluidic cartridge 300 approximately uniform.

[0109] It will be understood that the present technology is not limited to an optical detector including the lever assembly 502. Other force members can be suitably implemented. In one embodiment, an automated platform including the optical detector 500 is lowered onto and pressed against the microfluidic cartridge 300, where the microfluidic cartridge 300 is received in a stationary receiving bay. The movement of the automated platform can be controlled by a processor of the diagnostic device. In another embodiment, an automated platform including the receiving bay 402 (and the microfluidic cartridge 300) is raised and pressed against the bottom surface of the optical detector 500, where the optical detector 500 remains stationary. The movement of the automated platform can be controlled by a processor of the diagnostic device.

[0110] Accordingly, an embodiment of the diagnostic apparatus according to the present technology is configured to apply a force to thermally couple at least one heat source to at least a part of the microfluidic cartridge 300. Applying the force is important for ensuring consistent thermal contact between the heater module 400 and the PCR reaction chamber within the microfluidic cartridge 300. In some embodiments, a lever assembly 502, a similar mechanical force member, or an automated platform can supply the force (e.g., 5 - 500 N, typically about 200 - 250 N) and generate a pressure (e.g., 2 psi) on the top or a part of the microfluidic cartridge 300. In embodiments where the optical detector 500 moves above the stationary receiving bay 402, the mechanical features of the optical detector 500 can press the microfluidic cartridge 300 after the optical detector 500 is in a predetermined position, so that the reaction chamber is in better thermal contact with the heater module 400. Thus, by positioning the optical detector 500, pressure can be applied to the cartridge 300. In embodiments where the receiving bay 402 moves below the stationary optical detector 500, the mechanical features of the receiving bay 402 can push up the microfluidic cartridge 300 after the receiving bay 402 is in a predetermined position, so that the reaction chamber is in better thermal contact with the heater module 400. Thus, by positioning the receiving bay 402, pressure can be applied to the cartridge 300.

[0111] Other configurations for applying pressure to the cartridge 300 to improve temperature uniformity and PCR efficiency are also contemplated, including applying pressure with another component of the diagnostic apparatus. In the illustrated embodiment, the pressure is applied to the upper surface of the cartridge 300 and the heater module 400 is disposed below the cartridge 300, but other configurations are also contemplated.

[0112] The optical detector 500 can include a light source that selectively emits light in the absorption band of the fluorescent dye and an optical detector that selectively detects light in the emission band of the fluorescent dye, where the fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. Alternatively, for example, the optical detector 500 can include a diode with a band-pass filter that selectively emits light in the absorption band of the fluorescent dye and a photodiode with a band-pass filter that selectively detects light in the emission band of the fluorescent dye. The optical detector 500 can be configured to independently detect a plurality of fluorescent dyes having different fluorescence emission spectra, and each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof. The optical detector 500 can be configured to independently detect a plurality of fluorescent dyes at a plurality of different positions in the microfluidic cartridge, and each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof in a different sample. The optical detector 500 can also be configured to detect the presence or absence of a target analyte in a sample within a PCR reaction chamber in a given sample lane and to condition the initiation of a thermal cycle upon a positive detection of the presence of the sample. In some embodiments, the cartridge and the device are configured such that the reading head of the optical detector 500 does not cover the sample inlet, thereby allowing the loading of a separate sample while another sample is undergoing a PCR thermal cycle. A further description of a suitably configured detector is described in U.S. Patent Application No. 11 / 940,321, entitled "Fluorescence Detector for Microfluidic Diagnostic System," filed on November 14, 2007, which is hereby incorporated by reference in its entirety. The present technology provides a fluorescence detector configured to detect light emitted from a probe characteristic of a polynucleotide. The polynucleotide is amplified within a microfluidic channel with which the detector is in optical communication. The detector is configured to detect trace amounts of polynucleotides such as those contained in the microchannel. The detector can also be multiplexed to be able to measure a plurality of polynucleotides simultaneously.

[0113] Although the various depictions herein describe a heater substrate disposed below the microfluidic cartridge and a detector disposed above the microfluidic cartridge, it will be understood that a reverse arrangement would function similarly. In such an embodiment, the heater is pressed down onto and into contact with the microfluidic substrate, and the detector is mounted below the substrate and arranged to emit light upwardly towards the microfluidic cartridge to collect light exiting the microfluidic cartridge downwardly towards the detector.

[0114] The compressible pad 314 can provide a number of advantages as described herein. The compressible pad 314 of the microfluidic cartridge 300 can be designed, for example, to improve the pressure distribution on the bottom surface of a detector on the top surface of the microfluidic cartridge and, thus, the distribution of pressure across the entire bottom surface of the microfluidic cartridge by the receiving bay. The compressible pad 314 of the microfluidic cartridge 300 can be designed, for example, to enhance thermal uniformity to improve the uniform contact between the cartridge and the heater module. The compressible pad 314 for the microfluidic cartridge 300 can be designed, for example, to enhance PCR amplification by facilitating the uniform application of heat to a wider and / or deeper reaction chamber.

[0115] As described above, in some embodiments, the compressible pad 314 is adhered to the top of the microfluidic cartridge 300. The cartridge 300 can include a substrate 302 made of a cycloolefin polymer (COP) as described herein. The cartridge 300 can include 24 microfluidic reaction chambers configured to contain molecular materials for PCR amplification. PCR amplification requires heating and cooling the fluid within each reaction chamber to a specific temperature within a given time. In use, the cartridge 300 is placed on top of a heater substrate. In some embodiments, the heater substrate is the surface with the heater below. In use, a compressive load is applied to firmly hold the cartridge 300 between the heater module 400 and the optical detector 500. The optical detector 500 can include a rigid surface including a rigid metal surface. The compressive load applied in embodiments of the present technology ensures physical contact between the cartridge 300 and the heater module 400, including optimally distributed physical contact between the cartridge 300 and the heater module 400. In some embodiments, heat is transferred from the heater to the fluid within the cartridge 300 via heat conduction or direct heater contact.

[0116] Due to surface roughness, mechanical variations, and / or inherent material irregularities, the rigid surfaces of the microfluidic cartridge 300 and the heater module 400 that are joined together may not provide sufficient flatness for optimal contact with each other. In some embodiments, the compressible pad 314 comprises a highly compressible material that is adhered onto the cartridge 300. The compressible pad 314 can improve the contact between the two rigid surfaces by introducing an element of compliance into another rigid body system. The compressibility of the material of the compressible pad 314 enables certain regions to be compressed by different amounts than other regions. This differential compression accommodates the inherent mechanical and material surface variations at the two surfaces and results in a much more uniform pressure distribution across the entire cartridge 300. The compliant pad 314 allows for a more uniform contact between the cartridge 300 and the heater module 400 and thus provides a more complete and consistent heat transfer to each of the microfluidic reaction chambers.

[0117] The compressible pad 314 enables a more uniform physical contact and pressure distribution between the cartridge 300 and the heater module 400. This is advantageous because a uniform pressure results in less heat loss and more heat can be directly transferred to the cartridge 300. This advantageously improves the uniformity of heating and directly impacts the achievement and consistency of PCR amplification in embodiments of the present technology.

[0118] The heater of the heater module 400 provides heat conduction to the fluid sample received in the cartridge 300. When the cartridge 300 without a compressible pad is compressed against the heater module 400, due to mechanical and material surface variations, as well as inherent flexures and curvatures on the surface of the heater and / or the microfluidic cartridge, there are regions with less contact than other regions. As a result, a non-uniform pressure distribution occurs across the cartridge 300. During use, regions with insufficient physical contact between the heater and the cartridge 300 will result in heat loss. Therefore, less heat is supplied to the reaction chamber with insufficient physical contact. As a result, delays and inconsistencies in PCR amplification occur. The physical contact inconsistencies can lead to significant variations in the performance of the entire assay.

[0119] Embodiments of the compressible pad 314 according to the present technology allow for some regions of the pad to be compressed more than other regions. This compressibility accommodates the inherent mechanical and material surface variations in the system and ensures that all regions of the cartridge 300 have a more uniform pressure distribution. The compressible pad 314 can improve the physical contact between the cartridge 300 and the heater within the heater module 400, reduce heat loss, and / or result in better PCR performance.

[0120] As described above, the compressible pad 314 can be incorporated directly into the label 306. The label 306 can include an upper surface for displaying barcodes, manufacturing information, and other types of information. The label 306 is made of a thin polyester face stock material, which does not have inherent compliance. In some embodiments, the compressible pad 314 is integrated directly into an existing label structure. There are various ways to achieve this. In a first non-limiting example, the label 306 can include an adhesive lower surface that can be bonded to the compressible pad 314 to form an integrated label-pad structure. In a second non-limiting example, the label information is applied directly onto the compressible material, and the polyester face stock of the label 306 is completely eliminated. In some embodiments, integrating the compliant material directly into an existing label can reduce peeling compared to other embodiments and can be easily introduced into existing manufacturing processes and supply chain systems. FIG. 3 shows an embodiment of the first non-limiting example described above, where the compressible pad 314 is adhered to the top of the PCR cartridge 300, and a white cartridge label 306 is attached on top of the compressible pad 314. The label 306 is peeled back to show the structure clearly.

[0121] In some embodiments, the compressible pad 314 is a completely separate compressible pad and does not form an integral part of the final manufactured microfluidic cartridge. The compressible pad 314 can be placed in contact with the cartridge 300, such as the upper surface 308 of the cartridge 300, in a reversible manner. In some embodiments, the compressible pad is applied on top of the label 306 of the cartridge 300 (this embodiment is not shown in FIG. 3A). In some embodiments, the compressible pad can be made reusable for application on another cartridge 300 after completion of PCR amplification. This embodiment, like other embodiments disclosed herein, shows improved heat energy transfer to the reaction chamber.

[0122] In some embodiments, a compressible pad is applied to the optical reader or its surface instead of the cartridge (embodiment not shown). One advantage of this embodiment is that the compressible pad is no longer part of the microfluidic cartridge. As described herein, in some embodiments, the microfluidic cartridge is disposable. In this embodiment, the compressible pad does not form part of the disposable microfluidic cartridge, but instead becomes a permanent part of the instrument (where each cartridge is used and discarded, so it is reused multiple times). The compressible pad in this embodiment can result in significant cost savings due to the reusability of the pad. In some embodiments, the optical detector 500 may be redesigned or modified to accommodate the reusable compressible pad. In some cases, the compressible pad in this example is replaced after a certain number of uses or after a certain period of time. By periodically replacing the compressible pad in this way, it can be ensured that the pad incorporated in the instrument has optimal compression characteristics.

[0123] As described herein, the thermal uniformity across the cartridge 300 depends on the physical contact between the cartridge and the surface of the heater module 400. In some embodiments, heat transfer to the cartridge 300 can depend on direct conduction. As described herein, there are surface irregularities, curvatures, and mechanical variations inherent to one or more of the heater substrate and the cartridge, and the two surfaces may not provide sufficient flatness for optimal contact with each other. Advantageously, embodiments of the technology include a compressible pad 314 incorporating a material with a very low compression force deflection. Compression force deflection is the amount of force required to compress a material a given distance. Materials with a low compression force deflection are more easily compressed. Embodiments of the compressible pad according to the present invention are highly compressible, so that different portions of the heater surface, the microfluidic cartridge, and the optical detector can be compressed by slightly different amounts depending on the timing at which these components contact other components. For example, the compressible pad 314 can be compressed by slightly different amounts depending on the timing and location at which different portions of the heater module 400 and / or the cartridge 300 first make contact between the surfaces. The compressible pad 314 can introduce a level of flexibility found in other rigid systems. The compressible pad 314 can adjust for any inherent variability throughout the system. Thus, the compressible pad 314 can improve the pressure distribution across the cartridge 300. The compressible pad 314 can thus help ensure that all 24 reaction chambers not only make sufficient contact with the heater of the heater module 400, but also make optimal contact with the heater of the heater module 400. This improved thermal uniformity can make PCR amplification more consistent, reduce variability, and improve the performance of the overall assay.

[0124] As described herein, there are two methods for determining the properties of materials: durometer testing and compression force deflection testing. These methods are useful for determining the relative hardness or firmness of materials. Durometer testing is effective for measuring the hardness of solid materials, for example, solid materials have a range of hardness. Compression force deflection (CFD) testing can be useful for the measurement of foams or spongy or other non-rigid materials. Both types of measurements are based on ASTM guidelines and methods, which are hereby incorporated by reference in their entirety.

[0125] Durometer testing utilizes a Shore hardness scale, such as Shore A. The Shore scale correlates with the testing apparatus utilized, particularly the configuration of the test indenter that contacts the material. The indenter applies a load to a small contact point on the material. In durometer testing, it is assumed that the surface of the material is of relatively uniform hardness with respect to the test contact point. Different Shore scales are generally used depending on the type of material, such as materials of different hardness. As an example, Shore A is generally useful for softer elastomeric materials and Shore D is generally useful for harder elastomeric materials.

[0126] In contrast, compression force deflection testing compresses the entire material sample, and the sample is typically about 10 cm. This method involves determining the stress at various levels of strain. This method can measure hardness or firmness at different compression levels. Compared to durometer testing, compression force deflection testing allows for a larger test sample, and a larger sample can facilitate more accurate measurement of the properties of the material.

[0127] In embodiments of the present technology, the inventors have found that durometer testing is typically less accurate than compression force deflection testing in determining the hardness of the compressible pad 314, and thus in evaluating the suitability of a particular compressible pad material to achieve improved PCR test results. The compressible pad 314 includes a compressible material as described herein. The hardness of these materials can depend on the level of compression. The hardness of these materials can depend on the area being tested and can vary from area to area being tested. As described herein, the indenter for durometer testing measures only a small point on the material and covers a small area of the entire surface of the material. This small point may not be representative of a larger sample, depending on the material of the compressible pad 314. In contrast, compression force deflection testing determines the average firmness for a larger sample size. Compression force deflection testing can determine the hardness of the material based on the compression level typical of the designed application. For example, compression force deflection testing can determine the hardness of the material based on the typical compression level of the testing device and, in some embodiments, the compression level of the optical detector 500 designed to compress the compressible pad 314. As explained herein, compression force deflection testing can be a more representative measurement of how the compressible pad 314 functions when applied to the microfluidic cartridge 300.

[0128] [Example 1] Although embodiments of the present technology have been generally described, further understanding can be obtained by referring to specific specific examples provided herein for illustrative purposes only and not intended to be limiting.

[0129] In this example, the identification of the material of the compressible pad 314 will be described. FIGS. 7A-7C show the results of an assay test of the target analyte without a compressible pad. FIGS. 8A-8D are diagrams showing the results of an assay test for the target analyte using a low durometer silicone compressible pad. FIGS. 9A-9D show the results of an assay test for the target analyte using a PORON® foam compressible pad.

[0130] As described herein, the microfluidic cartridges 100, 200, 300 can be used to perform amplification protocols on samples prepared to detect the presence or absence of many different types of analytes of interest. Embodiments of the automated molecular diagnostic test system described herein can prepare a specimen according to an analyte-specific assay to obtain a PCR-compatible sample, which is introduced into a microfluidic cartridge accepted within the system. One example of an analyte-specific test includes an assay test for a viral analyte of interest. This test relates to the detection of a viral analyte of interest using a molecular viral load assay. This assay is a real-time RT-PCR assay that quantifies the amount of the viral analyte of interest in the sample (the "viral load"). The assay can be performed on an automated molecular diagnostic test system according to the present technology as described above. The viral load is a numerical representation of the amount of virus in a given volume. This can be expressed as virus particles per milliliter. The higher the viral load, the more severe the viral infection is associated. By estimating the amount of live virus in a fluid specimen collected from a patient, the virus amount / mL can be calculated. For example, the viral load can be indicated by the number of RNA copies per milliliter of plasma. The assay can be used to track the viral load during antiretroviral therapy, whereby a caregiver can measure and evaluate changes in the amount of the viral analyte during treatment.

[0131] The assay of this example can use two RNA calibrator sequences, namely, Hi Cal and Lo Cal. The RNA calibrator sequence ("calibrator") is a synthetic RNA transcript of known sequence and quantity used to adjust the output of the assay measurement. This is in contrast to a "control", which is a standard sample that can be included in the assay to evaluate the validity of the test results (not to adjust the output of the test results). The calibrator is designed to bind to a molecule having a complementary base sequence and is also called a probe. This specific binding process is called hybridization. During sample preparation, a known amount of the calibrator is mixed with the patient specimen and the PCR reagents. The prepared specimen is amplified to detect and quantify the target nucleic acid (the viral analyte of interest) in the sample. The degree of hybridization between the calibrator and the corresponding probe is used to normalize the measured value of the target nucleic acid with the corresponding probe. The calibrator is designed to amplify with the same efficiency as the target nucleic acid and to react similarly to the factors of variation (such as variations in the device and matrix). In the following examples, the amounts (indicated by qCt measurements) of the Hi Cal calibrator, the viral analyte of interest, and the Lo Cal calibrator in the test sample were measured, and tests were performed to evaluate other characteristics (such as ymaxEP).

[0132] Quantification of the target nucleic acid in the sample depends on the relationship between fluorescence on a logarithmic scale and the number of amplification cycles. The number of cycles at which the fluorescence exceeds a given detection threshold may be called the cycle threshold (Ct). During amplification, the amount of the target nucleic acid doubles every cycle. Thus, for example, a sample with a cycle threshold 3 cycles earlier than other samples would contain 2 3 = 8 times more target nucleic acid. In the following assay tests, two parameters were examined. The first parameter is the qCt score indicating the first amplification cycle in which fluorescence was detected in a thermal cycling protocol including a plurality of amplification cycles. The second parameter is the ymaxEP score indicating the maximum fluorescence unit at the final stationary amplitude after a plurality of amplification cycles.

[0133] The optimal sample volume for the embodiments of the viral load assay test of the viral analyte described herein is in the range of about 25 μL (not about 4 μL). As described above, such a sample volume can be obtained using a wider and deeper reaction chamber in the thicker version of the microfluidic cartridge of the present technology (for a cartridge thickness of about 1.24 mm, the thickness of the cartridge with wider and deeper wells is about 1.68 mm). The thicker cartridge can correspond to an increased PCR reaction chamber that contains six times the volume as described above. However, thick cartridges implemented for viral load testing may have edge effect defects (outside the sample lane), reverse edge effect defects (inside the sample lane), and random defects. As described herein, when the compressible pad 314 was added to the top of the cartridge 300, the inventors of the present technology found that the results of the viral load assay test were significantly improved. The compressible pad 314 described herein can overcome the long-standing problem of incorporating pads into instruments or consumables (e.g., microfluidic cartridges). The compressible pad 314 can be considered a solution to the pressure distribution effect associated with microfluidic cartridges having increased thickness and increased volume wells. The following examples illustrate cartridge-based solutions, but in some embodiments, a compressible pad coupled to the optical detector 500 can include any of the features of the compressible pads described herein.

[0134] In this example, the viral load assay test included the cartridge 200 described herein, which had wells (e.g., PCR reaction chambers) that were wider and deeper than the cartridge 100 described herein. This study design used the Geometry C Prototype cartridge, in which each reaction chamber had a width dimension of approximately 3.5 mm, a depth dimension of approximately 0.83 mm, a length dimension of approximately 10 mm, and a volume of approximately 25.2 μL. The study design included a liquid master mix and cartridges manually filled by the tester. In each run, both the reaction chambers in the first bank and the reaction chambers in the second bank of the cartridge were tested. The test performed was PCR amplification. The tests were performed on two BD MAX™ devices (Becton, Dickinson and Company, Franklin Lakes, NJ). Two RNA calibrator sequences, Hi Cal and Lo Cal, were used for the viral load assay test.

[0135] Figures 7A-7C show the results of a viral load assay test without using the compressible pad according to the present technology. In this test, a cartridge 200 having wider and deeper wells was utilized without a compressible pad. FIG. 7A is a diagram showing the quantification of the target nucleic acid in the sample. This shows the relationship between the fluorescence amount and the number of amplification cycles on a logarithmic scale. The X-axis is the qCt score indicating the rate of change of fluorescence. The number of cycles is shown along the y-axis. During a thermal cycle such as PCR, the amount of the target nucleic acid doubles every cycle. Each colored line on the graph indicates a separate reaction chamber. As described herein, the microfluidic cartridge can include 24 sample lanes arranged in 12 cartridge lanes, and each cartridge lane corresponds to a region of a cartridge 300 including two sample lanes 312. Each cartridge lane can include a reaction chamber in the first bank of reaction chambers and a reaction chamber in the second bank of reaction chambers. The qCt scores of each reaction chamber within the same cartridge lane are assigned the same color in FIG. 7A. In each curve of FIG. 7A, there is a qCt score indicating the first amplification cycle at which fluorescence is detected. Each curve in FIG. 7A has a ymaxEP score indicating the maximum fluorescence unit at the final stationary amplitude after a plurality of cycles. This data is also shown in FIGS. 7B and 7C.

[0136] FIG. 7B is a diagram showing the individual qCt scores of each of the 24 reaction chambers of the cartridge 200. The upper graph shows the reaction chambers of the first bank of reaction chambers 226. The lower graph shows the reaction chambers of the second bank of reaction chambers 228. Twelve cartridge lanes are shown on the Y-axis. The qCt score indicating the first amplification cycle in which fluorescence was detected is shown on the X-axis. The qCt scores of Hi Cal are substantially constant among the cartridge lanes, and the qCt scores of Lo Cal are substantially constant among the cartridge lanes, but there is a large variation between cartridge lanes 5 and 10 in the first bank, indicating that the first amplification cycle in which fluorescence is detected varies greatly among the 24 detection chambers. Such variations in the reaction chambers are due to various factors such as surface variations, poor contact between the cartridge and the heater substrate, poor compression of the cartridge due to the application of force, and others. In particular, the reaction chambers in cartridge lanes 4-10 of the first bank have higher qCt scores for the target viral analyte than the reaction chambers in cartridge lanes 1-3 and 11-12 of the first bank. In particular, the reaction chambers in cartridge lanes 2, 3, 5, 10 of the second bank have higher qCt scores for the target viral analyte than the reaction chambers in cartridge lanes 1, 4, 6-9, 11-12 of the second bank.

[0137] Figure 7C is a diagram showing ymaxEP of each reaction chamber. The upper graph shows the reaction chambers included in the reaction chambers of the first bank. The lower graph shows the reaction chambers in the reaction chambers of the second bank. Twelve cartridge lanes are shown on the Y-axis. The ymaxEP score indicates the maximum fluorescence unit at the final stationary amplitude after multiple cycles. The ymaxEP scores of Hi Cal, Lo Cal, and the viral analyte sample vary depending on the reaction chamber. The final stationary amplitude is not consistent across the reaction chambers. This variation in the ymaxEP scores of the reaction chambers is due to various factors such as surface variation, poor contact between the cartridge and the heater substrate, and poor compression of the cartridge when force is applied. This variation indicates the inefficiency of the PCR reaction, such that the lanes of a certain cartridge do not reach the same maximum fluorescence. In particular, the reaction chambers in cartridge lanes 1, 2, 11, and 12 of the first bank have higher ymaxEP scores for the target viral analyte than the reaction chambers in cartridge lanes 3-10 of the first bank. In particular, the reaction chambers in cartridge lanes 1, 4, 6, 7, 8, 9, 11, and 12 of the second bank have higher ymaxEP scores for the target viral analyte compared to the reaction chambers in cartridge lanes 2, 3, 5, and 10 of the second bank. This baseline indicates the variations that may occur in the microfluidic cartridge 200 when the compressible pad of the present technology is not implemented. Overall, the amplification results in the reaction chambers are not consistent. For example, different reaction chambers have higher PCR efficiency than other chambers. In FIGS. 7A-7B, the data suggesting wide variations in both the qCt score and the ymaxEP score are not strongly clustered.

[0138] Figures 8A-8D are diagrams showing the results of a viral load assay test using a cartridge implementing a low durometer silicone compressible pad. The low durometer solid silicone used was BISCO® HT-6210 silicone by Rogers Corporation. Figure 8A shows an embodiment of a low durometer silicone compressible pad coupled to the top of the cartridge 200. Figure 8B is a diagram showing the quantification of the target nucleic acid in the sample. It is a diagram showing the relationship between fluorescence and the number of amplification cycles on a logarithmic scale. The x-axis is the qCt score indicating the rate of change of fluorescence. The y-axis is the number of cycles. In Figure 8B, it is suggested that the data is clustered more tightly in the initial amplification than in Figure 7A, and there is little variation in the qCt scores in each reaction chamber. In Figure 8B, it is suggested that the data is not tightly clustered as the amplitude becomes constant, and there is a large variation in the ymaxEP scores.

[0139] Figure 8C is a diagram showing the individual qCt scores of each reaction chamber. The upper graph shows the reaction chambers in the first bank of reaction chamber 226. The lower graph shows the reaction chambers of reaction chamber 228 in the second bank. The qCt scores of Hi Cal and Lo Cal are substantially constant between the lanes of the cartridge. However, for the viral analyte sample, there is variation in the qCt scores in cartridge lanes 5 and 7 of the first bank.

[0140] FIG. 8D is a diagram showing ymaxEP of each reaction chamber. The upper graph shows the reaction chambers in the reaction chambers of the first bank. The lower graph shows the reaction chambers included in the reaction chambers of the second bank. The ymaxEP scores of Lo Cal and the viral specimen sample vary in the reaction chambers of the first bank. The maximum fluorescence value of the final stationary amplitude after multiple cycles is not constant. In particular, the reaction chambers in cartridge lanes 1-3 and 9-12 of the first bank have higher ymaxEP scores for the viral analyte and Lo Cal than the reaction chambers in cartridge lanes 4-8 of the first bank. Therefore, the low durometer silicon compression pad results in an inconsistent PCR reaction as shown in the graph. In this example using a low durometer silicon compression pad, different reaction chambers can perform PCR more efficiently than other chambers.

[0141] Figures 9A-9D show the results of a viral load assay using a cartridge implementing a compressible pad made of PORON (registered trademark) foam. Figure 9A is a diagram showing an embodiment of a PORON (registered trademark) foam compressible pad coupled to the top of cartridge 200. The PORON (registered trademark) foam is a fine pitch open cell urethane foam by Rogers Corporation. The material used was PORON (registered trademark) cellular polyester urethane 4790-92. Figure 9B is a diagram explaining the quantification of the target nucleic acid in the sample. This shows the relationship between the amount of fluorescence and the number of amplification cycles on a logarithmic scale. The x-axis is the qCt score indicating the rate of change of fluorescence. The y-axis is the number of cycles. In Figure 9B, compared to Figures 7A and 8B, it is suggested that the data is more densely clustered for the initial amplification and there is less variation in the qCt scores of each reaction chamber. In Figure 9B, the data is more tightly clustered for the final amplification than in Figures 7A and 8B, suggesting less variation in the ymaxEP scores for each reaction chamber. In Figure 9B, from left to right, the first cluster of lines is related to Hi Cal, the second cluster of lines is related to Lo Cal, and the third cluster of lines is related to the viral analyte sample.

[0142] Figure 9C is a diagram showing the individual qCt scores of each reaction chamber. The upper graph shows the reaction chambers in the first bank of reaction chamber 226. The lower graph shows the reaction chambers of reaction chamber 228 in the second bank. The qCt scores of Hi Cal, Lo Cal, and the viral analyte sample are consistent across the entire cartridge lane. For the Hi Cal RNA calibrator sequence, the initial amplitude, in other words the first detection, occurred at approximately cycle 20. For the HiCal RNA calibrator sequence, the initial amplitude, that is, the first detection, occurred at approximately cycle 32. In the case of the Lo Cal RNA calibrator sequence, the initial amplitude, that is, the first detection, occurred at approximately cycle 36. These results are consistent across each cartridge lane. These results are consistent across each of the first bank and the second bank. These results are consistent for each of the 24 reaction chambers.

[0143] Figure 9D is a diagram showing the ymaxEP scores of each reaction chamber. The upper graph shows the reaction chambers included in the reaction chambers of the first bank. The lower graph shows the reaction chambers of the second bank. The ymaxEP scores of Hi Cal, Lo Cal, and the viral analyte sample are consistent across the entire cartridge lane (with relatively little variation). In the case of the Hi Cal RNA calibrator sequence, the maximum fluorescence unit at the final stationary amplitude after multiple cycles is approximately 2000. In the case of the Lo Cal RNA calibrator sequence, the maximum fluorescence unit at the final stationary amplitude after multiple repetitions is approximately 7000. In the case of the viral analyte sample sequence, the maximum fluorescence unit at the final stationary amplitude after multiple cycles is approximately 5500. These results are consistent for each cartridge lane. These results are consistent across each of the first bank and the second bank. These results are consistent for each of the 24 reaction chambers.

[0144] As outlined above, additional inspections were performed using compression pads of different materials. The additional inspections included a compressible pad formed of a graphite foil. The graphite foil was Laird's Tgon™ 820. Another inspection included a compression pad made of glass fiber coated with thermally conductive silicone. This material was ThermaCool® TF-1879. Further inspections included a compressible pad formed of a silicone sponge. The material was Rogers' BISCO® HT-800 silicone sponge. Yet another inspection included a compression pad with a thermally conductive coating applied to a thermally conductive silicone sponge. The material was ThermaCool® R-10404 silicone sponge. The inspection design was similar to that outlined above for the low durometer silicone compressible pads (Figs. 8A-8D) and PORON® foam compressible pads (Figs. 9A-9D).

[0145] In the evaluation of materials for use with the compressible pad 314, unexpected results were obtained for a selected group of materials. In some embodiments, the compressible pad includes a compressible material. In some embodiments, materials suitable for the compressible pad are selected based on compression force deflection (in this case, the amount of stress (measured in psi) required to deflect the material to 25% of its original height). Materials include those with a compression force deflection of less than 30 psi, less than 29 psi, less than 28 psi, less than 27 psi, less than 26 psi, less than 25 psi, less than 24 psi, less than 23 psi, less than 22 psi, less than 21 psi, less than 20 psi, less than 19 psi, less than 18 psi, less than 17 psi, less than 16 psi, less than 15 psi, less than 14 psi, less than 13 psi, less than 12 psi, less than 11 psi, less than 10 psi, less than 9 psi, less than 8 psi, less than 7 psi, less than 6 psi, less than 5 psi, less than 4 psi, less than 3 psi, less than 2 psi, less than 1 psi, and others. Materials can include those having a compression force deflection between 0 - 5 psi, 5 - 10 psi, 10 - 15 psi, 15 - 20 psi, 20 - 25 psi, 25 - 30 psi, 0 - 3 psi, 1 - 4 psi, 2 - 5 psi, 3 - 6 psi, 4 - 7 psi, 5 - 8 psi, 6 - 9 psi, 7 - 10 psi, 8 - 11 psi, 9 - 12 psi, 10 - 13 psi, 11 - 14 psi, 12 - 15 psi, 13 - 16 psi, 14 - 17 psi, 15 - 18 psi, 16 - 19 psi, 17 - 20 psi, 21 - 24 psi, 22 - 25 psi, 23 - 26 psi, 24 - 27 psi, 25 - 28 psi, 26 - 29 psi, 27 - 30 psi, etc. Materials can include those with a compression force deflection of 5 psi or less, 10 psi or less, 15 psi or less, 20 psi or less, 25 psi or less, 30 psi or less, and others. In some embodiments, the inspection method is 0.51 cm / min (0.2” / min) Strain Rate with Force Measured @ 25% Deflection. In some embodiments, it is in the range of 0.3 - 3.5 psi (2 - 24 kPa). In some embodiments, a typical value is 1.7 psi (12 kPa).

[0146] As described herein, both the durometer shore A measured at 25% and the compression force deflection are measurements of compressibility. In these measurements, the lower the numerical value, the easier it is to compress, and it is expected to lead to a better pressure distribution and better PCR performance. Unexpectedly, the inventors have discovered that durometer shore A is insufficient as an indicator of suitable materials (see FIGS. 7B-7D). Low durometer silicone typically has a hardness, durometer, shore "A" of 10, and PORON® foam typically has a hardness, durometer, shore "A" of less than 3. One skilled in the art would expect that both of these materials would be easily compressed and thus result in similar PCR performance. However, contrary to expectations, low durometer silicone showed significantly different performance from PORON® foam. Additional materials were also examined, but no correlation was found between durometer shore A and PCR performance.

[0147] Surprisingly, the compression force deflection measured at 25% was an excellent indicator of suitable materials. Low durometer silicone typically shows a compression force deflection of about 30 psi, and PORON® foam typically shows a compression force deflection of 2-5 psi. When additional materials were examined, a correlation was found between the amount of compression load deflection and PCR performance. In particular, materials with a compression force deflection measured at 25% deflection of less than 30 psi showed improved PCR performance. In some embodiments, materials having a compression force deflection of 0-20 psi had improved PCR performance.

[0148] As described herein, both the hardness, durometer, shore "A", and the compression force deflection measured at 25% determine the properties of the material. These methods are useful for determining the relative hardness or firmness of the material. One skilled in the art would expect that materials with a low hardness, durometer, shore "A" and a low compression load deflection measured at 25% would lead to better PCR performance. However, only the compression load deflection measured at 25% (rather than the hardness, durometer, shore "A") had a correlation indicating a material suitable for improving PCR performance.

[0149] In some embodiments, the compressible pad includes material properties as shown below. In some embodiments, the compressible pad includes a density according to ASTM D 3574-95, Test A. The density can be between 225 and 255 kg / m 3 3. In some embodiments, the compressible pad includes a thickness measured along the z-axis of the pad. The thickness can range from 0 to 5 mm, for example, 0 to 1 mm, 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, about 3 mm (0.12 inches) + / - 10%. In some embodiments, the compressible pad includes a hardness, durometer, Shore "O" according to ASTM D 2240-97, 2. It includes a compression set of 2 according to ASTM D 1667-90 Test D @ 23°C (73°F). In some embodiments, the compressible pad includes a compression set of 10 according to ASTM D 3574-95 Test D @ 70°C (158°F). In some embodiments, the compressible pad includes a resilience force due to a 4% vertical rebound according to ASTM D 2632-96.

[0150] In some embodiments, the compressible pad includes material properties as shown below. In some embodiments, the compressible pad includes a tensile strength of 120 psi (828 kPa) according to ASTM D412. In some embodiments, the compressible pad includes a thickness measured along the z-axis of the pad. The thickness can range from 0 to 5 mm, for example, 0 to 1 mm, 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, about 1 mm (0.035 inches) + / - 10%. In some embodiments, the compressible pad includes an elongation rate of 150% according to ASTM D412. In some embodiments, the compressible pad includes a hardness, durometer, Shore "A" of 13 according to ASTM D 2240. In some embodiments, the compressible pad includes a compression flexure of 18 psi (125 kPa) at 25% according to ASTM D1056. In some embodiments, the compressible pad includes a compression set of 15 according to ASTM D 1056. In some embodiments, the compressible pad includes 69 lbs / ft 3(1105 kg / m 3 ) including the density according to ASTM 297.

[0151] In some embodiments, the compressible pad includes material properties as shown below. In some embodiments, the compressible pad includes a thickness measured along the z-axis of the pad. The thickness can range from 0 to 5 mm, for example, 0 to 1 mm, 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, about 1 mm (0.032 inches) + / - 10%. In some embodiments, the compressible pad includes an elongation rate of 80% according to ASTM D412. In some embodiments, the compressible pad includes that the compression deflection at 25% according to ASTM D1056 is 9 psi (62 kPa). In some embodiments, the compressible pad includes that the compression set according to ASTM D1056 is less than 1 at 70 °C and less than 5 at 100 °C. In some embodiments, the compressible pad includes that the density according to ASTM1056 is 22 lbs / ft 3 (352 kg / m 3 ). In some embodiments, the compressible pad includes material properties including a range of any two values herein. In some embodiments, the compressible pad includes material properties including any value having ±50% of the values herein.

[0152] Embodiments of the compressible pads of the present technology are designed to improve heat transfer between a microfluidic cartridge and a heat source (e.g., an array of heat sources below the microfluidic cartridge). As described herein, virus load is a numerical representation of the amount of virus in a given volume. A microfluidic cartridge designed to determine virus load, such as via PCR, may require wider and deeper wells to increase the reaction volume and target detection. In these situations, heat transfer between a microfluidic cartridge with wider and deeper wells and the underlying heat source becomes extremely important. Contact can be improved by a more uniform pressure distribution so that each PCR reaction chamber optimally contacts the underlying heat source. By making the pressure distribution uniform, deterioration in the reproducibility of the thermal cycling protocol between sample lanes, cartridge lanes, or cartridges can be prevented. Also, a uniform pressure distribution can avoid hot spots or inefficiencies in heat transfer due to air conduction.

[0153] As described herein, the compressible pads of the present technology can be disposed on the upper or lower surface of the microfluidic cartridge. In some embodiments, the compressible pad on the bottom surface of the microfluidic cartridge can reduce heat conduction. In some embodiments, the compressible pad on the upper surface of the microfluidic cartridge may require a window or other cutout to enable optical detection. In some embodiments, the compressible pad is made as large as possible, for example, having the same spread as the surface area of the label as described herein. In some embodiments, the compressible pad can include at least 50% of the surface area of the surface of the cartridge (e.g., 50% of the upper surface of the cartridge), at least 60% of the surface, at least 70% of the surface, at least 80% of the surface, or at least 90% of the surface, among others.

[0154] Next, another non-limiting implementation configuration of the microfluidic cartridge according to the present technology will be described with reference to FIGS. 10 to 37. FIGS. 10 to 37 are diagrams of a microfluidic cartridge 200 including 24 independent sample lanes.

[0155] FIGS. 10 to 16 are diagrams showing a first embodiment of the microfluidic cartridge 200 without a compressible pad. FIG. 10 is a perspective view. FIG. 11 is a top view. FIG. 12 is a bottom view. FIG. 13 is a first side view. FIG. 14 is a second side view. FIG. 15 is a third side view. FIG. 16 is a fourth side view.

[0156] FIGS. 17 to 23 are diagrams showing a second embodiment of the microfluidic cartridge 200 provided with a compressible pad. FIG. 17A is a perspective view. FIG. 17B is an exploded perspective view. FIG. 18 is a top view. FIG. 19 is a bottom view. FIG. 20 is a first side view. FIG. 21 is a second side view. FIG. 22 is a third side view. FIG. 23 is a fourth side view.

[0157] FIGS. 24 to 30 are additional diagrams of the microfluidic cartridge of FIG. 10. FIG. 24 is a perspective view. FIG. 25 is a top view. FIG. 26 is a bottom view. FIG. 27 is a first side view. FIG. 28 is a second side view. FIG. 29 is a third side view. FIG. 30 is a fourth side view.

[0158] FIGS. 31 to 37 are additional diagrams of the microfluidic cartridge of FIG. 10. FIG. 31 is a perspective view. FIG. 32 is a top view. FIG. 33 is a bottom view. FIG. 34 is a first side view. FIG. 35 is a second side view. FIG. 36 is a third side view. FIG. 37 is a fourth side view. The dashed lines are used to illustrate features of the cartridge that do not form part of the claimed design.

[0159] The present disclosure relates to a molecular diagnostic testing apparatus, system, and method for determining the presence and / or amount of a target analyte in a sample. As used herein, "analyte" generally refers to a substance to be detected. For example, it can include antigenic substances, haptens, antibodies, and combinations thereof. Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes and those administered for illegal purposes), drug intermediates or by-products, bacteria, virus particles, and metabolites or antibodies of any of the above substances.

[0160] Specific examples of analytes include, but are not limited to, group B streptococcus, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginitis, bacterial vaginitis, Candida spp., Candida glabrata, Candida krusei, Salmonella spp., Shigella spp. / enteroinvasive Escherichia coli (EIEC), Campylobacter spp. (jejuni and coli) and Shiga toxin-producing E. coli (STEC, Shigella dysenteriae), Yersinia enterocolitica, enterotoxigenic E. coli (ETEC), Pseudomonas syringae, Vibrio spp. (Vibrio vulnificus / Vibrio parahaemolyticus), Giardia lamblia, Cryptosporidium spp. (C. parvum and C. hominis), Entamoeba histolytica, norovirus, rotavirus, adenovirus (40 / 41), sapovirus and human astrovirus, Cryptosporidium spp. (tcdB), MRSA, Staphylococcus aureus.Specific examples of analytes include, but are not limited to, ferritin, creatine kinase MB (CK-MB), human chorionic gonadotropin (hCG), digoxin, phenytoin, phenobarbital, carbamazepine, vancomycin, gentamicin, theophylline, valproic acid, quinidine, luteinizing hormone (LH); follicle-stimulating hormone (FSH); estradiol, progesterone; C-reactive protein (CRP); lipocalin; IgE antibody; cytokine; TNF-related apoptosis-inducing ligand (TRAIL); vitamin B2 microglobulin; interferon gamma-induced protein 10 (IP-10); interferon-induced GTP-binding protein (also called myxovirus (influenza virus) resistance 1, MX1, MxA, IFI-78K, IFI78, MX, MX dynamin-like GTPase 1); procalcitonin (PCT); glycated hemoglobin (Gly Hb); cortisol; digitoxin; N-acetyl procainamide (NAPA); procainamide; antibodies against rubella such as rubella IgG and rubella IgM; antibodies against toxoplasmosis such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylate; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies against hepatitis B core antigen such as anti-hepatitis B core antigen IgG and IgM (anti-HBC); human immunodeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1 and 2 (HTLV); hepatitis B e antigen (HBeAg); antibody against hepatitis B e antigen (Anti-HBe); influenza virus; thyroid-stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (T3); free triiodothyronine (F3); carcinoembryonic antigen (CEA); lipoprotein, cholesterol, and triglyceride; and alpha-fetoprotein (AFP).Illegally used drugs and controlled drugs include, but are not limited to, amphetamine; methamphetamine; barbiturates such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital; benzodiazepines such as librium and valium; cannabinoids such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and opium; fencyclidine; and propoxyphene, etc. For the purpose of the biological or environmental substance of interest, the analysis target can be further included.

[0161] The foregoing description is for the purpose of explaining various aspects of the present technology. The examples presented in this specification are not intended to limit the scope of the present technology. It will be apparent to those skilled in the art that many changes and modifications can be made to the present technology, which is currently fully described, without departing from the spirit or scope of the appended claims.

Claims

1. A microfluidic cartridge including a first side and a second side opposite the first side, a first amplification chamber, a second amplification chamber, a first inlet disposed on the first side and in fluid communication with the first amplification chamber, a second inlet disposed on the first side and in fluid communication with the second amplification chamber, a compressible pad disposed on the first side, comprising: the compressible pad is configured to provide more complete and consistent heat transfer from a plurality of contact heat sources in contact with the second side of the microfluidic cartridge to the first amplification chamber and the second amplification chamber, the compressible pad includes a first window above the first amplification chamber and a second window above the second amplification chamber, and the first window and the second window are each configured to allow light to pass through the first side of the microfluidic cartridge between the first amplification chamber and the second amplification chamber, a microfluidic cartridge.

2. The microfluidic cartridge according to claim 1, wherein the first amplification chamber and the second amplification chamber have a volume of about 25 μL.

3. The microfluidic cartridge according to claim 1, wherein the first amplification chamber and the second amplification chamber have a width dimension of about 3.5 mm, a depth dimension of about 0.83 mm, and a length dimension of about 10 mm.

4. The microfluidic cartridge according to claim 1, further comprising a label above the compressible pad.

5. The first amplification reaction chamber, the second amplification reaction chamber, the first inlet, and the second inlet are formed in a rigid substrate layer, and the second side of the microfluidic cartridge includes a flexible laminate layer below the first amplification chamber and the second amplification chamber. The microfluidic cartridge according to claim 1.

6. The microfluidic cartridge according to claim 1, wherein the compressible pad includes a material having a compressive force deflection of less than 30 psi.

7. The microfluidic cartridge according to claim 1, wherein the compressible pad includes a material having a compressive force deflection of less than 20 psi.

8. The microfluidic cartridge according to claim 1, wherein the compressible pad improves the pressure distribution from components of a diagnostic testing device.

9. The application of pressure to the compressible pad is configured to enhance the uniformity of heat application from the plurality of contact heat sources to the first amplification chamber and the second amplification chamber, the microfluidic cartridge according to claim 1.

10. The compressible pad enhances the uniformity of heat application to the first amplification chamber and the second amplification chamber, the microfluidic cartridge according to claim 1.

11. The compressible pad enhances PCR amplification that depends on rapid temperature cycling, the microfluidic cartridge according to claim 1.

12. A method of amplifying on a plurality of polynucleotide-containing samples, introducing a plurality of samples into a microfluidic cartridge, the cartridge including a plurality of amplification chambers configured to enable thermal cycling of the plurality of samples independently of each other, moving the plurality of samples to the respective plurality of amplification chambers, amplifying the polynucleotides contained in the plurality of samples by applying continuous heating and cooling cycles to the amplification chambers, compressing a pad of the microfluidic cartridge during amplification, comprising the method.

13. The method according to claim 12, further comprising applying pressure to the compressible pad to increase contact between the microfluidic cartridge and a substrate including one or more heaters.

14. The method according to claim 12, further comprising applying pressure to the compressible pad to enhance heat uniformity.

15. The method according to claim 12, further comprising applying pressure to the compressible pad to enhance amplification of the plurality of polynucleotide-containing samples.

16. A system comprising a microfluidic cartridge, the microfluidic cartridge comprising a first PCR reaction chamber, a second PCR reaction chamber, a first inlet in fluid communication with the first PCR reaction chamber, a second inlet in fluid communication with the second PCR reaction chamber, a compressible pad, and comprising the microfluidic cartridge is configured to be used with an apparatus, the apparatus comprising a bay configured to receive the microfluidic cartridge, At least one heat source that is thermally coupled to the cartridge and configured to apply a thermal cycle for performing PCR on one or more polynucleotide-containing samples within the microfluidic cartridge; A detector configured to detect the presence of one or more polynucleotides in the one or more samples; A processor coupled to the heat source and configured to control the heating of one or more regions of the microfluidic cartridge; comprising a system.

17. The system according to claim 16, wherein the compressible pad is configured to improve contact between the bay and the microfluidic cartridge.

18. The system according to claim 16, wherein the compressible pad is configured to improve contact between the at least one heat source and the microfluidic cartridge.

19. The system according to claim 16, wherein the compressible pad is configured to be compressed by a detector disposed above the microfluidic cartridge during detection.

20. The system according to claim 16, wherein the detector is configured to move downward to compress the compressible pad and physically contact the microfluidic cartridge.

21. The system according to claim 16, wherein the cartridge is configured to move upward to compress the compressible pad and physically contact the detector.

22. The system according to claim 16, wherein the compressible pad is configured to be compressed by another component of the apparatus that applies pressure to the microfluidic cartridge.

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