Microfluidic cartridge for enhancing amplification of polynucleotide-containing samples - Patent Application 20070123633

Abstract

The present technology generally relates to microfluidic cartridges. More specifically, the present technology relates to compressible pads applied to microfluidic cartridges, which are configured to amplify nucleotides of interest from multiple biological samples in parallel within microfluidic channels within the cartridge and enable detection of these nucleotides. The compressible pads of the present technology can be implemented in microfluidic cartridges with improved reaction chamber volumes, resulting in improved thermal uniformity and amplification efficiency within the cartridge. Assays using the microfluidic cartridges of the present technology can advantageously improve the limit of detection (LOD) and limit of quantitation (LOQ).

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 909,628, filed October 2, 2019, which is 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 a compressible pad applied to a microfluidic cartridge, the microfluidic cartridge configured to receive and amplify nucleotides of interest. In another aspect, the technology relates to a microfluidic cartridge having a reaction chamber configured to receive and amplify a larger volume of fluid eluate from a processed sample. Cartridge embodiments described herein can amplify nucleotides of interest from multiple biological samples in parallel within microfluidic channels within the cartridge, allowing for the detection of these nucleotides. [Background technology]

[0003] The sensitivity of an assay in molecular diagnostic testing depends on several factors. These factors include, among others, the efficiency of extraction during specimen processing to obtain an amplification-ready sample, the efficiency of sample amplification, and the thermal uniformity achieved within the reaction volume during the amplification process. Increasing the size of the reaction volume contributes to increased amplification efficiency, leading to improved limits of detection (LOD) and higher limits 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 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 reaction chambers with such small reaction volumes. However, as the reaction chamber volume decreases, challenges associated with achieving the desired analytical sensitivity may arise. At the same time, as the reaction chamber volume increases to achieve improved amplification efficiency and overcome target delivery limitations, challenges associated with achieving thermal uniformity may arise. Thus, there is a need for a microfluidic cartridge that overcomes these challenges and achieves both improved amplification efficiency and thermal uniformity to result in assays with improved limits of detection (LOD) and improved limits of quantification (LOQ).

[0005] The discussion of the background of the present technology herein is included to describe the relevant state of the art and is not to be construed as an admission that any of the subject matter referred to was published, well known, or part of the common general knowledge as of the priority date of any of the claims.

[0006] Throughout the description and claims of this specification, the word "comprise" and its derivatives such as "comprising" and "comprises" are not intended to exclude other additives, components, integers or steps. Summary of the Invention [Means for solving the problem]

[0007] The technology includes methods and apparatus for improving pressure distribution across a microfluidic device, increasing thermal uniformity within the microfluidic device, and enhancing amplification parameters performed in the microfluidic device. Implementations of the technology of the present invention improve the characteristics of microfluidic devices that amplify nucleotides of interest within microfluidic channels. The technology includes methods and apparatus for improving detection of these nucleotides.

[0008] The microfluidic device of the present technology can interact with a heating assembly that applies heat to multiple chambers in 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 presses against the microfluidic device, placing the array of heaters in thermal communication with the microfluidic device. In other cases, the microfluidic device presses against the heating assembly, placing the array of heaters in thermal communication with the microfluidic device. An embodiment of a microfluidic device according to the present technology can include a compressible pad that improves the distribution of pressure applied to the microfluidic device and increases the uniformity of heat delivered to the microfluidic device. The compressible pad according to the present technology can increase the uniformity of pressure applied to the microfluidic device, thereby reducing heat loss and improving the consistency and efficiency of amplification occurring in multiple chambers of the microfluidic device.

[0009] The microfluidic devices of the present technology are also capable of achieving improved assay sensitivity by increasing the amplification chamber volume from about 4 μL to about 25 μL while still achieving optimal thermal uniformity throughout the chamber during the amplification process. The increased amplification chamber of the present technology can receive a larger volume of eluate containing DNA / RNA target analytes extracted from the specimen, thereby improving assay sensitivity. The microfluidic devices of the present technology achieve in some cases six times the reaction chamber volume compared to current microfluidic devices. The larger volume reaction chamber of the present technology, combined with the improved pressure distribution and thermal uniformity associated with the compressible pad of the present technology, improves assay performance as measured by improved limit of detection (LOD) and limit of quantification (LOQ).

[0010] An implementation 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 first side and an opposing second 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 and second amplification chambers. 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 and second windows are configured to allow light to transmit through a first side of the microfluidic cartridge to the first and second amplification chambers, respectively.

[0012] In some embodiments, the first and second amplification chambers have a volume of about 25 μL. In some embodiments, the first and second amplification chambers 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, a second side of the microfluidic cartridge includes a flexible laminate layer below the first and second amplification chambers. In some embodiments, the compressible pad includes a material having a compressive force deflection of less than 30 psi. In some embodiments, the compressible pad includes a material having a compressive force deflection of less than 20 psi. In some embodiments, the compressible pad improves pressure distribution from components of the diagnostic testing device. In some embodiments, the application of pressure to the compressible pad is configured to increase uniformity of application of heat from the multiple contact heat sources to the first and second amplification chambers. In some embodiments, the compressible pad increases uniformity of application of heat to the first and second amplification chambers. In some embodiments, the compressible pad enhances PCR amplification that relies on rapid temperature cycling.

[0013] In some embodiments, a method is provided for amplifying on a plurality of polynucleotide-containing samples. The method can include introducing a plurality of samples into a microfluidic cartridge, the cartridge including a plurality of amplification chambers configured to allow thermal cycling of the plurality of samples independently of one another. The method can include moving the plurality of samples to respective plurality of amplification chambers. The method can include amplifying polynucleotides included with the plurality of samples by applying successive 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 in fluid communication with the first PCR reaction chamber. The microfluidic substrate can include a second inlet in fluid communication with the second PCR reaction chamber. The microfluidic substrate can include a compressible pad. In some embodiments, a microfluidic cartridge is configured for use with the device. The device can include a bay configured to receive the microfluidic cartridge. The device can include at least one heat source thermally coupled to the cartridge and configured to apply a thermal cycle to perform PCR on one or more polynucleotide-containing samples in the cartridge. The device can include a detector configured to detect the presence of one or more polynucleotides in the one or more samples. The device can include a processor coupled to the heat source and configured to control heating of one or more regions of the microfluidic cartridge.

[0015] In some embodiments, the compressible pad is configured to improve contact between the bay and the microfluidic cartridge. In some embodiments, the compressible pad is configured to improve 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 downwardly into physical contact with the cartridge to compress the compressible pad. In some embodiments, the cartridge is configured to move upwardly into physical contact with the detector to compress the compressible pad. In some embodiments, the compressible pad is configured to be compressed by another component of the device that applies pressure to the cartridge.

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

[0017] [Figure 1A] FIG. 1 is a plan view of an example of a multi-lane microfluidic cartridge. [Figure 1B] FIG. 1B is an enlarged view of a portion of the cartridge of FIG. 1A showing the reaction chamber. [Figure 2A] FIG. 13 is a plan view of another example of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 2B] FIG. 2B is an enlarged view of a portion of the cartridge of FIG. 2A showing the reaction chamber. [Figure 2C] FIG. 2B shows an example of a reaction chamber of the cartridge of FIG. 2A. [Figure 2D] FIG. 13 shows yet another example of a multi-lane microfluidic cartridge with reaction chambers of various volumes. [Figure 3A] 13 shows a cutaway layer diagram of a further embodiment of a cartridge including a compressible pad. [Figure 3B] FIG. 3B is an exploded perspective view of the cartridge of FIG. 3A. [Figure 4] FIG. 3B shows a compressible pad of the cartridge of FIG. 3A. [Figure 5A] FIG. 1 illustrates an example of a heater module in a receiving bay. [Figure 5B] FIG. 1 shows an example of a system having two receiving bays. [Figure 5C] FIG. 1 shows an example of a system having two receiving bays. [Figure 5D] FIG. 1 shows an example of a system having two receiving bays. [Figure 6] FIG. 2 shows an optical detector. [Figure 7A] FIG. 13 shows the results of an assay test for an analyte of interest without a compressible pad. [Figure 7B] FIG. 13 shows the results of an assay test for an analyte of interest without a compressible pad. [Figure 7C] FIG. 13 shows the results of an assay test for an analyte of interest without a compressible pad. [Figure 8A] FIG. 1 shows the results of assay testing of analytes of interest using low durometer silicone compressible pads. [Figure 8B] FIG. 1 shows the results of assay testing of analytes of interest using low durometer silicone compressible pads. [Figure 8C] FIG. 1 shows the results of assay testing of analytes of interest using low durometer silicone compressible pads. [Figure 8D] FIG. 1 shows the results of assay testing of analytes of interest using low durometer silicone compressible pads. [Figure 9A] 1 shows the results of an analyte assay test using a PORON® foam compressible pad. [Figure 9B] 1 shows the results of an analyte assay test using a PORON® foam compressible pad. [Figure 9C] 1 shows the results of an analyte assay test using a PORON® foam compressible pad. [Figure 9D] 1 shows the results of an analyte assay test using a PORON® foam compressible pad. [Figure 10] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 11] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 12] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 13] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 14] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 15] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 16] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 17] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 18] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 19] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 20] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 21] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 22] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 23] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 24] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 25] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 26] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 27]FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 28] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 29] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 30] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 31] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 32] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 33] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 34] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 35] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Diagram 36] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 37] FIG. 13 is a diagram of yet another embodiment of a multi-lane cartridge with reaction chambers having enhanced features. [Figure 38A] FIG. 1 illustrates an example embodiment of a heater array and heater element microstructure of a heating device configured to apply heat to a microfluidic cartridge. [Figure 38B] FIG. 1 illustrates an example embodiment of a heater array and heater element microstructure of a heating device configured to apply heat to a microfluidic cartridge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present technology relates to microfluidic devices configured to perform amplification, such as by PCR, of one or more polynucleotides from one or more samples. Unless expressly indicated otherwise, when the term PCR is used herein, it is intended to encompass any variant of PCR, including but not limited to real-time and quantification, 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 described further herein, and additional embodiments of such devices are described in U.S. Patent Application No. 11 / 985,577, filed Nov. 14, 2007, entitled "Microfluidic System for Amplifying and Detecting Polynucleotides in Parallel," which is incorporated herein by reference. The present technology provides devices for detecting polynucleotides in samples, particularly biological samples. The present technology more particularly relates to microfluidic systems that perform PCR on nucleotides of interest in microfluidic channels and detect these nucleotides. The present device includes a microfluidic cartridge configured to receive multiple samples, and can perform PCR on each sample individually, on a group of multiple samples, or on all of the multiple samples simultaneously. U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed November 14, 2007, is incorporated herein by reference. U.S. Patent Application No. 11 / 940,310, entitled "Microfluidic Cartridge and Method of Using Same," filed November 14, 2007, is incorporated herein by reference. The present technology provides a microfluidic substrate configured to perform PCR in parallel on multiple polynucleotide-containing samples. The substrate can be a single layer substrate within a microfluidic cartridge. Also provided is a method of manufacturing a microfluidic cartridge including such a substrate. U.S. Patent Application No. 11 / 728,964, entitled "Integrated System for Processing Microfluidic Samples and Methods of Using Same," filed March 26, 2007, is incorporated herein by reference.The present technology provides an integrated device for processing polynucleotide-containing samples and providing diagnostic results thereon.

[0020] By cartridge, we mean a unit that may be disposable or reusable in whole or in part and that is configured for use in conjunction with some other device that is suitably and complementarily configured to receive the cartridge and operate on it (such as providing energy).

[0021] Microfluidic as used herein means that the volume of sample and / or reagents and / or amplified polynucleotide is between about 0.1 μl and about 999 μl, e.g., 1-100 μl, or 1-50 μl. In some embodiments, the volume is between 0-10 μl for smaller wells and between 10-30 μl for wider and deeper wells, as described herein. Similarly, when applied to a cartridge, the term microfluidic 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 sample, reagent, or amplified polynucleotide, as further described herein. Certain embodiments herein can also function with nanoliter volumes (in the range of 10-500 nanoliters, such as 100 nanoliters).

[0022] One aspect of the present technology relates to a microfluidic cartridge having two or more sample lanes arranged such that analyses can be performed in parallel (e.g., simultaneously) in the two or more lanes, each lane 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 as well as other methods known in the art. A sample lane includes at least a sample inlet and a microfluidic network having one or more microfluidic components, as further described herein.

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

[0025] Embodiments of the present technology include cartridges having multiple sample lanes (hereinafter referred to as "multi-lane cartridges"). However, it will be understood that embodiments of the present technology can be implemented in cartridges that include as few as one sample lane. The multi-lane cartridge is configured to accept multiple samples in series or parallel, simultaneously or sequentially. In some embodiments, the multi-lane cartridge is configured to accept 24 samples, or any other suitable number of samples. In some cases, the multi-lane cartridge is configured to accept at least a first sample and a second sample, where the first sample and the second sample each include one or more polynucleotides in a form suitable for amplification. The polynucleotides may be the same 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 decreasing the concentration of an inhibitor relative 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 including features described herein, the first microfluidic network configured to amplify polynucleotides in a first sample and the second microfluidic network 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 the neutralized polynucleotide sample under thermocycling conditions suitable for generating 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 one another, and for receiving signals from a detector that measures fluorescence from one or more of the PCR reaction chambers.

[0029] A non-limiting implementation of a microfluidic cartridge according to the present technology will now be described with reference to Figures 1A and 1B. Figure 1A is a plan view of a microfluidic cartridge 100 having 24 separate sample lanes, including sample lanes 102, 104, 106, and 108. Figure 1B is an enlarged view of a portion of the cartridge 100 of Figure 1A illustrating the reaction chambers 112, 114, 116, and 118 of adjacent sample lanes 102, 104, 106, and 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 in 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 receiving a PCR-compatible sample, the sample includes a mixture including PCR reagents and a neutralized polynucleotide sample, suitable for subjecting the neutralized polynucleotide sample to thermal cycling conditions to generate PCR amplicons. In one example, the PCR-ready sample is a PCR reagent mixture that includes a polymerase enzyme, a positive control plasmid, a fluorescent hybridization probe selective for at least a portion of the plasmid and a plurality of nucleotides, and at least one probe selective for a polynucleotide sequence. Exemplary probes are described further herein. In an embodiment of the present technology, the microfluidic network is configured to couple heat from an external heat source to a mixture that includes the PCR reagents and the neutralized polynucleotide sample under thermocycling conditions suitable for generating PCR amplicons from the neutralized polynucleotide sample.

[0030] Another non-limiting implementation of a microfluidic cartridge according to the present technology will now be described with reference to Figures 2A and 2B. Figure 2A is a plan view of a microfluidic cartridge 200 having 24 independent sample lanes, including sample lanes 202, 204, 206, and 208. Figure 2B is an enlarged view of a portion of the cartridge 200 of Figure 2A illustrating the reaction chambers 212, 214, 216, and 218 of adjacent sample lanes 202, 204, 206, and 208. Each sample lane of the cartridge 200 includes a dedicated sample inlet configured to receive a sample. For example, the sample lanes 202, 204, 206, and 208 include sample inlets 222, 224, 226, and 228, respectively, each sample inlet configured to receive a sample independently. The cartridge 200 can be referred to as a multi-lane PCR cartridge with a dedicated sample inlet. The sample inlets can be configured to receive a liquid transfer member (not shown), such as a syringe, a pipette, or a PCR tube containing a PCR-compatible sample. In an embodiment of a 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 greater 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, where the length dimension is measured along the y-axis of the microfluidic cartridge. The length and width dimensions can be disposed along the vertical axis. 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 chamber 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, which can have a thickness of about 1.24 mm. In some embodiments, the thicker cartridge may have poorer thermal performance characteristics than the thinner cartridge, including edge effect failures (outside the sample lane), inverse edge effect failures (inside the sample lane), and random failures. Compressible pad embodiments according to the present technology, as described herein, can improve thermal conductivity and / or thermal coupling between the cartridge 200 and the heating device, reducing 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 reaction chambers are also contemplated.

[0034] The chambers 212, 214, 216, 218 of adjacent sample lanes 202, 204, 206, 208 are staggered with respect to each other. In some embodiments, the sample inlets are all 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 Figures 2A and 2B. Each network can include a reaction chamber. In some embodiments, each network can include two valves on either side of the reaction chamber. The valves are initially normally open and close the channels 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 gases in the microfluidic network to avoid the microfluidic network as the sample moves through the microfluidic network from the inlet 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, the reaction chambers 212 in the first bank of reaction chambers 226 are aligned with the reaction chambers 214 in the second bank of PCR sample lanes. The reaction chambers 212, 214 can be aligned laterally with respect to a single line 232 of sample inlets. The adjacent network can form staggered reaction chambers as shown in the illustrated embodiment. In some embodiments, a 24-lane cartridge has two banks of 12 reaction chambers 226, 228. One first bank of 12 reaction chambers 226, 228 is closer to the inlet. The other bank of 12 reaction chambers 226, 228 is further away from the inlet. The first bank 226 of 12 reaction chambers can be aligned axially along a first axis 256, and the second bank 228 of 12 reaction chambers can be aligned axially along a second axis 258. The reaction chambers 212 of the first bank of twelve reaction chambers 226 and the reaction chambers 214 of the second bank of reaction chambers 228 may be aligned along a third axis 260. The third axis may 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 a 4 microliter PCR reaction chamber. As an example, the reaction chambers 112, 114, 116, 118 can each be about 1.5 mm wide, about 0.30 mm (300 microns) deep, and about 10 mm long. The volume of the reaction chambers can be about 4 μl. It will be understood that these dimensions and layouts are exemplary, and that deviations from those shown are not inconsistent with equivalent methods of operation of such cartridges. The microfluidic cartridge 100 can allow PCR to be performed in concentrated reaction volumes (about 4 μl) and can allow rapid thermal cycling at about 20 seconds per cycle. As another example, typical dimensions of the reaction chambers are 150 μm deep by 700 μm wide, with a typical volume of about 1.6 μl. The channels of the microfluidic network in the sample lanes of the cartridge 100 can have cross-sectional dimensions of at least one millimeter or less. For example, the channels of such a network can have a width and / or depth of less than 1 mm (eg, about 750 microns or less, about 500 microns or less, or about 250 microns or less).

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

[0038] The reaction chambers of the above examples are summarized in the table below. TIFF0007645250000001.tif114164Table 1

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

[0040] In some embodiments, the width of the reaction chambers 212, 214, 216, 218 can be 1-4 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 foregoing values). In some embodiments, the depth of the 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 of any two of the foregoing values). In some embodiments, the length of the 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 foregoing values). In some embodiments, the reaction chambers 212, 214, 216, 218 can have a volume 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 foregoing values). It will be understood that these dimensions and layouts are exemplary and that deviations from those shown are not inconsistent with equivalent methods of operation of 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., approximately six 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, allowing for the amplification of large volumes of liquid eluate from the specimen being processed. In particular, embodiments of the microfluidic cartridge 200 can ensure that a greater percentage of the liquid eluate from the sample processing procedure can be loaded into the PCR reaction chamber and amplified therein. In some cases, there is a six-fold increase in the volume of liquid eluate that can be amplified. Implementations of the enhanced microfluidic cartridge of the present technology have a larger volume reaction chamber and can accommodate a larger liquid eluate input. As a result, the enhanced microfluidic cartridge of the present technology allows for a more consistent amplification process across samples and cartridges, reducing the variability of the amplification process across samples and cartridges, and improving the overall assay performance.

[0042] In implementations in which the microfluidic cartridge 200 includes a plastic substrate layer, the geometry of each reaction chamber 212, 214, 216, 218 is formed in the plastic substrate layer on all sides except one side where each reaction chamber 212, 214, 216, 218 is sealed by a laminate layer as described herein. Sample nucleic acid and PCR reagent mix can be loaded into the chambers through an inlet port and a microfluidic channel. Each reaction chamber 212, 214, 216, 218 can be sealed by a heat-activated wax valve that spans the fluid path and cools on either side of the chamber. 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 the fluorescence change is measured through external optics positioned over the chamber on the top side of the cartridge 200, as described herein.

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

[0044] The sensitivity of the assay depends on several factors, including extraction efficiency, PCR efficiency, and thermal uniformity. In some embodiments, increasing the chamber size contributes to improving PCR efficiency resulting in improved limits of detection and quantification. In some embodiments, the microfluidic cartridge 200 achieves a 6-fold increase in volume compared to the microfluidic cartridge 100. Other configurations are contemplated (e.g., 2-fold increase in volume, 3-fold increase in volume, 4-fold increase in volume, 5-fold increase in volume, 6-fold increase in volume, 7-fold increase in volume, 8-fold increase in volume, or a range of any of the above values, 2 or 3 or more). There is an upper limit to how much the chamber size can be increased while still achieving optimal temperature uniformity across the chamber during each cycle of the amplification protocol. This is especially true for amplification protocols that have 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 procedure performed on the analyte. In some embodiments, the microfluidic cartridge 200 can improve the limits of detection and quantification of the assay. In some embodiments, the microfluidic cartridge 200 can ensure that a greater proportion of 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 variability in PCR amplification. In some embodiments, the microfluidic cartridge 200 can improve the overall performance of an assay performed on a sample.

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

[0046] The sample inlets of adjacent sample lanes are spaced apart from one another to prevent any contamination of one sample inlet when introducing a sample into an adjacent sample inlet in the cartridge. In some embodiments, the sample inlets are configured to prevent the unintentional introduction of a subsequent sample 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 to have an industry-accepted standard of 6 mm center-to-center spacing of the sample inlets. This means that in certain embodiments, the cartridge has a 6 mm center-to-center distance between the inlet holes. The inlet holes can be manufactured in a conical shape with an appropriate cone angle so that industry standard pipette tips (2 μl, 20 μl, 200 μl capacity, etc.) fit snugly therein. The cartridges herein can be adapted to other subsequently emerging industry standards not otherwise described herein, as would be understood by one of skill in the art.

[0047] In some embodiments, the microfluidic cartridge includes a first, second, and third layer 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, increase thermal uniformity, and enhance 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 layers can be combined into a single integrated layer. Although four layers are described, additional layers can be included and one or more layers can be separated into two or more layers.

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

[0049] Cartridges herein include embodiments having three or more layers in their structure, as shown in embodiment 300 in Figures 3A and 3B. Cartridge 300 includes a substrate 302, a laminate 304 (not visible in Figure 3A), and a label 306. In cartridge 300, microfluidic substrate 302 has an upper side 308 and a lower side 310 (not visible in Figure 3A) on the opposite side of the substrate. Substrate 302 includes a plurality of microfluidic networks arranged in a corresponding plurality of sample lanes 312. Cartridge 300 includes a plurality of cartridge lanes 330. In this non-limiting embodiment, cartridge 300 includes 12 cartridge lanes 330. In this non-limiting embodiment, each cartridge lane 330 corresponds to an area of ​​cartridge 300 that includes two sample lanes 312. In this non-limiting embodiment, cartridge 300 includes 24 sample lanes 312 arranged in 12 parallel cartridge lanes 330. The cartridge 300 may include a laminate 304 attached to the underside 310 of the substrate 302 to seal various components (e.g., valves) of the microfluidic network. The laminate 304 may provide an effective heat transfer layer between a dedicated heating element (described further herein) and components in the microfluidic network. The cartridge 300 may include a label 306 attached to the topside 308 of the substrate 302. In some embodiments, each reaction chamber is formed in 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 the laminate 304. In some embodiments, each reaction chamber is sealed by the laminate 304. In some embodiments, each reaction chamber is sealed by the label 306.

[0050] The cartridge 300 can include a compressible pad 314. In some embodiments, the compressible pad 314 is disposed above the top side 308 of the base 302. In some embodiments, the compressible pad 314 is disposed between the top side 308 of the base 302 and the label 306. In an 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 the manufacturing process to load components, such as valves, of the microfluidic network with a thermally responsive material. In such embodiments where the compressible pad 314 is disposed above the label 306, the compressible pad 314 can include markings (described in more detail below) that would normally be included on the label 306. 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 underside 310 of the base 302. In some embodiments, the compressible pad 314 is disposed between the underside 310 of the base 302 and the stack 304. In some embodiments, the compressible pad 314 is above the stack 304. In some embodiments, the compressible pad 314 is below the stack 304. In such embodiments, the compressible pad may be formed of a thermally conductive material or may include thermally conductive properties.

[0052] Thus, embodiments of microfluidic cartridges herein include those that are comprised of layers including a substrate 302, a laminate 304, and a label 306, with a compressible pad 314 disposed adjacent to at least one of the layers. In some embodiments, a microfluidic cartridge is comprised of essentially four layers: a substrate, a laminate, a label, and a compressible pad. In some embodiments, a 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 (as 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 a reaction chamber and associated channels. In some embodiments, the microfluidic network includes one or more valves. The valves, if present, can be located on the first (e.g., bottom) side (located 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 the second (e.g., top) side (located toward the label layer). Typically, in a given cartridge, all of the microfluidic network including the reaction chambers and inlet holes are together defined in a single substrate layer, substrate 302 .

[0054] The substrate 302 may be formed from a material that enhances the rigidity of the substrate (and thus the cartridge). The material from which the substrate 302 is formed may be rigid or non-deformable. Rigidity is advantageous because it facilitates effective and uniform contact with a heating assembly, as further described herein. In some embodiments, the substrate 302 is impermeable to air or liquid, such that entry and exit of air or liquid during operation of the cartridge is only possible through inlets or various vents. The material from which the substrate 302 is formed may be non-permeable to air and other gases. The use of non-permeable materials is also advantageous because it reduces the likelihood that the concentrations of various species in liquid form will change during an analysis. In some embodiments, the substrate 302 has low autofluorescence to facilitate detection of polynucleotides during amplification reactions performed in the microfluidic circuits defined herein. The use of materials with low autofluorescence is also important to ensure that background fluorescence does not corrupt the measurement of fluorescence from the analyte of interest.

[0055] The substrate 302 can have regions of reduced thickness to facilitate detection. In some embodiments, a region of reduced thickness can be present above each reaction chamber in 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 be about 100 to about 125 microns thick. The laminate layer 304 can be attached to the bottom surface of the microfluidic substrate 302 using, for example, thermal bonding, pressure bonding, or a combination thereof. The laminate layer 304 can also be made from a material that has an adhesive coating on only one side, the side that contacts the bottom surface of the substrate 302. The layer 304 can be made from a single coated tape having a layer of Adhesive 420® from 3M® Corporation. Exemplary tapes include single sided forms of double sided tape having product numbers 9783, 9795, and 9795B, available from 3M®. The laminate layer is typically 50 to 200 μ thick, for example 125 μ thick. Another acceptable layer can be made from an adhesive tape that utilizes a microcapsule based adhesive.

[0057] The label 306 can be made from polypropylene or other plastic with a pressure sensitive adhesive. The label 306 can typically be about 50-150 microns thick. In some embodiments, the label 306 can be configured to seal a wax-loaded hole of a valve in the substrate 302. In some embodiments, the label 306 can trap air used for valve actuation. In some embodiments, the label 306 can serve as a location for operator marking. The label 306 can include identifying characteristics such as a cartridge bar code number, a lot number, and an expiration date. In some embodiments, the label 306 has spaces and a writable surface that allow a user to manually make identifying annotations on the label. The label 306 can be a single molded layer, although one skilled in the art will appreciate that the label 306 can be formed of two or more separate elements.

[0058] The label 306 can be printed with various types of information, including, but not limited to, a manufacturer's logo, a part number, and an index number for each of the sample lanes. In various embodiments, the label 306 includes a computer readable or scannable portion that can include a specific identification indicia, such as a lot number, an expiration date, or a unique identifier. For example, the label 306 can include a bar code, a radio frequency tag, or one or more computer readable or optically scannable characters. The readable portion of the label 306 can be positioned so that it can be read by a sample identification verifier. The label 306 can include a notch 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 it can be a series of notches, wedge or curved cutouts, or other configurations whose shape requires a unique orientation for 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, or fiducials, for use in loading the valves with thermally responsive material. The positioning elements can be located on the substrate 302, typically on its top surface. In some embodiments, the fiducials can be located diagonally across the substrate, but are not limited to such a location.

[0062] As described herein, above each reaction chamber is a window 320 that allows for optical detection, such as detection of fluorescence from a fluorescent material in the reaction chamber, such as from a fluorescent hybridization probe, when a detector is positioned above the window 320. A plurality of windows 320 can be formed in the label 306. The number of windows 320 can correspond to the number of reaction chambers (e.g., 1:1, such as 24 reaction chambers, 24 windows or 12 reaction chambers, 12 windows). Other configurations, such as shapes, positions, and / or numbers of windows 320, are contemplated. 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 a compressible pad according to the present technology will be described. FIG. 4 illustrates a non-limiting example of a compressible pad 314 according to the present technology. The cartridge 300 can include a compressible pad 314. The compressible pad 314 can be formed of a material that has a low deflection of a 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). Other thicknesses are also suitable, such as, for example, 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, etc.

[0064] In some embodiments, the compressible pad 314 is incorporated into a consumable product, such as a microfluidic cartridge. In some embodiments, the compressible pad (not shown) is incorporated into a diagnostic instrument (e.g., into a detector that is in physical contact with 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 using, for example, a pressure sensitive adhesive. The compressible pad 314 can be compressible as described herein. The compressible pad 314 can have a thickness of 0.1-2.5 mm uncompressed, typically about 1.5 mm thick uncompressed.

[0066] As described herein, the cartridge 300, and particularly the base 302, can include alignment members 316 that ensure 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 members 316 can be cut out 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 require a unique orientation for placement in the device. The compressible pad 314 can include cutouts 322 from an end or corner of the compressible pad 314. The cutouts 322 can correspond to the cutouts 318 of the label 306 shown in FIG. 3A.

[0067] As described herein, above each reaction chamber is a window 320 in the label 306 that allows optical detection at the reaction chamber when a detector is positioned above the window 320. The compressible pad 314 can include multiple windows 324. The number of windows 324 can correspond to the number of reaction chambers (e.g., 1:1, such as 24 reaction chambers, 24 windows, or 12 reaction chambers, 12 windows, etc.). Other configurations are contemplated for the windows 324, such as shapes, positions, and / or numbers. 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 in number and / or shape to the windows 320 in the label 306 shown in FIG. 3.

[0068] As described herein, the reaction chambers of adjacent sample lanes are staggered relative to each other. In some embodiments, the sample inlets are all arranged along a single line parallel to the X-axis of the microfluidic cartridge 300. The reaction chambers in a first bank of sample lanes can be aligned with the reaction chambers in a second bank of sample lanes, and the reaction chambers can be aligned laterally 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 12 reaction chambers is close to the edge with the alignment members 316. The second bank 328 of 12 reaction chambers is far from the edge with the alignment members 316. The reaction chambers can form a grid. Other configurations are contemplated.

[0069] In some embodiments, the 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 and 324 of the label 306 overlap each other to form a pair of windows that allow light to pass therethrough. The surface area of ​​each of the windows 320, 324 can be greater than the surface area of ​​the corresponding reaction chamber. In the illustrated embodiment, each pair of windows 320, 324 encompasses the area around one reaction chamber. In another embodiment (not shown), each pair of windows 320, 324 encompasses the area around two or more reaction chambers. In yet another embodiment (not shown), each pair of windows 320, 324 encompasses the area around a bank of reaction chambers. In this embodiment, the label 306 and the compressible pad 314 each include two windows, 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 perimeter of 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 bonded to the label 306. The label 306 and / or the compressible pad 314 can include an adhesive surface to bond the components together. Other bonding methods are contemplated. Figure 3A shows an embodiment in which the compressible pad 314 is bonded to the top of the PCR cartridge 300 and a white cartridge label 306 is bonded to the top of the compressible pad 314. The label 306 has been partially removed to show the compressible pad 314 underneath 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 a top surface for displaying a bar code 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 onto 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 separate or independently formed component. The compressible pad 314 can be disposed on the cartridge 300, such as on the top 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 a first cartridge 300 and applying this same compressible pad 314 to a second cartridge 300. This embodiment, as well as other embodiments disclosed herein, shows improved thermal 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 discarding the cartridge 300 after amplification of one or more samples also discards the compressible pad 314 that is integrated with the cartridge 300. The compressible pad 314 may be integrated into the construction of the cartridge 300. When integrated, the compressible pad 314 may reduce the risk of detachment 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 polynucleotide of interest 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 a post-amplification analysis, such as gel electrophoresis, the user can drill a hole through the laminate 304 of the cartridge 300 and retrieve the PCR product, typically in the amount of about 1.5 microliters. In one non-limiting embodiment, the user can place each individual sample lane on a specialized narrow heating plate, maintained at a temperature that melts the wax in the valve of that sample lane, and then aspirate the reacted sample through the inlet hole of that sample lane.

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

[0075] A non-limiting example of a heating assembly according to the present technology will now be described in detail. FIG. 5A illustrates an exemplary heater module 400 in a 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). Exemplary such heater arrays including heat sources are further described herein. Additional embodiments of heater arrays are described in U.S. Patent Application No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed November 14, 2007, the specification of which is incorporated herein by reference.

[0076] 5B illustrates another exemplary heater module 700 in a 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 illustrates a close-up view of the heater module 700 in the left receiving bay 702. FIG. 5D illustrates a close-up view of the heater module 700 of FIG. 5C with a microfluidic cartridge 200 received in the receiving bay 702.

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

[0078] The heater is located in a heater substrate layer directly beneath the microfluidic substrate. In a non-limiting example, the heater can be photolithographically defined and etched from a gold metal layer (typically about 3,000 Å thick). A 400 Å layer of TiW is deposited on top and bottom of the gold layer to act as an adhesion layer. 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 electrically insulating layer of 2 μm silicon oxide acts as an insulating layer on top of the metal layer. An additional thin electrically insulating layer such as 2-4 g / m parylene can also be deposited on top of the silicon oxide surface.

[0079] An exemplary set of heaters configured to cyclically heat the PCR reaction chamber can be provided, it being 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 similar principles as those governing the heaters described herein.

[0080] An exemplary reaction chamber in a microfluidic substrate, typically a chamber or channel having a volume, is configured with a long side and a short side, each with an associated heating element. The reaction chamber may also be referred to herein as a PCR reactor, and the area of ​​the cartridge in which the reaction chamber is located may 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: a long top heater, a long bottom heater, a short left heater, and a short right heater. The small gap between the long top heater and the long bottom heater results in a negligible temperature gradient (less than 1°C difference across the width of the reaction chamber at any point along the length of the reaction chamber), thus resulting in a virtually uniform temperature throughout the reaction chamber. The heaters at the short edges of the reaction chamber provide heat to counteract the gradient created by the two long heaters from the center to the edges of the reaction chamber.

[0081] Those skilled in the art will appreciate that further configurations of one or more heaters located around the reaction chamber are compatible with the methods and apparatus described herein. For example, the "long" side of the reaction zone can be configured to be heated by two or more heaters. Since the low thermal conductivity of glass, or quartz, polyimide, FR4, ceramic, or fused silica substrates is utilized to aid in the independent operation of various microfluidic components such as valves (if present in the cartridge) and the independent operation of various sample lanes, specific orientations and configurations of heaters are used to generate uniform zones of heating even on substrates with low thermal conductivity. Those skilled in the art will further appreciate that the principles underlying the configuration of heaters around the reaction zone can be similarly applied 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]

[00136] Figure 38 illustrates a set of heater arrays of a heating device configured to apply heat to a microfluidic cartridge according to the present disclosure. For example, Figure 38A illustrates a heater array configured to apply heat to a microfluidic cartridge including 24 sample lanes. Figure 38B is a blown up view of one array configured to apply heat to one reaction chamber of the 24 sample lane cartridge, including a heater and a temperature sensor that applies electrical current during operation.

[0083] In some embodiments, the heat source is controlled by a computer processor and operates according to a desired protocol. Processors configured to operate the microfluidic device are described, for example, in U.S. Patent Application Serial No. 12 / 173,023, entitled "Integrated Apparatus for Performing Nucleic Acid Extraction and Diagnostic Testing on Multiple Biological Samples," filed July 14, 2008, which is incorporated herein by reference. A processor, such as a microprocessor, is configured to control the functions of the various components of the system as shown, and is in communication with each such component that requires control. It should be understood that many of such control functions may optionally be performed manually and are not under the control of the processor. Furthermore, 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 may be configured to receive data regarding the sample to be analyzed from a sample reader, which may 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 will be appreciated that such operations may be distributed among several processors as convenient.

[0084] The processor can be configured to accept user instructions from an input, which can include instructions to initiate analysis of a sample and selection of operating conditions. In various embodiments, the input can include one or more input devices, such as, but not limited to, a keyboard, a touch sensitive surface, a microphone, a track pad, a retina scanner, a holographic projection of an input device, and a mouse.

[0085] The processor may also be configured to communicate with the display, for example to send information about the analysis to the display for communication to a user of the system. Such information may include, but is not limited to, the current status of the instrument, the progress of a PCR thermal cycle, and warning messages in the event of a malfunction of either the system or the cartridge. Additionally, the processor may send one or more questions that are displayed on the display, prompting the user to enter input in response thereto. Thus, in certain embodiments, the input and the display are integrated with each other.

[0086] The processor may optionally be further configured to transmit the analysis results to an output device such as a printer, a visual display, a display using holographic projection, or a speaker, or any combination thereof.

[0087] The processor may further optionally be connected to a computer network via a communications interface, such as a network interface. The communications interface may 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. This allows a remote user to access the processor, when the system is properly addressed on the network, to send instructions, input data, or retrieve data that may be stored in a memory (not shown) associated with the processor or other computer-readable medium in communication with the processor. The interface may also allow 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 disk farm. The device may further be configured to allow a user to email analysis results directly to another party, such as a healthcare provider, a diagnostic facility, or a patient.

[0088] Additionally, in various embodiments, the device may further include a data storage medium configured to accept data from one or more of the processor, the input device, and the communication interface, the data storage medium being one or more media selected from the group consisting of a hard disk drive, an optical disk drive, a flash card, and a CD-ROM.

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

[0090] The liquid dispenser (not shown), also controllable by the processor, is configured to perform various aspirating and dispensing operations on each sample, liquid, and reagent in the holder to extract nucleic acid from the sample. The liquid dispenser can perform such operations on multiple holders simultaneously. The sample reader is configured to transmit identification indicia for the samples and possibly the holder to the processor. In some embodiments, the sample reader is attached to the liquid dispenser, such that the liquid dispenser can read indicia for the sample above which it is positioned. In other embodiments, the sample reader is not attached to the liquid dispenser, but is independently movable under the control of the processor. The liquid dispenser is also configured to take aliquots of liquid containing extracted nucleic acid from one or more samples and direct these to a receiving bay where the microfluidic cartridges 200, 300 are received. The receiving bay is in communication with a heater or set of heaters, which can be controlled by the processor such that specific regions of the cartridge are heated at specific times during analysis. The liquid dispenser is configured to take aliquots of liquid containing extracted nucleic acid from one or more samples and direct them to the inlets of each of the microfluidic cartridges. The cartridge is configured to amplify, such as by performing PCR, each nucleic acid. The processor is also configured to control a detector that receives diagnostic instructions from the cartridge. The diagnostics can be transmitted to an output device and / or display, as described herein.

[0091] Suitable processors may be designed and manufactured according to design principles and semiconductor processing methods well known in the art. In some embodiments, the device 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 as further described herein, the heat source configured to heat individual sample lanes in the cartridge, and the processor configured to control the application of heat to the individual sample lanes individually, all at the same time, or groups at the same time. In use, the cartridge 200, 300 is typically thermally associated with an array of heat sources configured to operate components of the device (e.g., valves, gates, and processing regions). In some embodiments, the heat sources are operated by an operating system that operates the device during use. The operating system includes a processor (e.g., a computer) configured to operate the heat sources according to a desired protocol. In some embodiments, the temperature sensors are preferably configured to transmit information regarding the temperature in their vicinity to the processor at times such that the heaters are not receiving current to heat them. This can be achieved by appropriate control of the current cycles.

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

[0093] Each reaction chamber is heated through a series of cycles to carry out the amplification of nucleotides in the sample according to an amplification protocol. The inner walls of the channels in the PCR reactor are typically made very smooth during manufacture and polished to a glossy finish. This is to minimize minute amounts of air being trapped on the surface of the PCR channel which would cause bubbles during thermal cycling. The presence of air bubbles, especially in the detection region of the PCR channel, can also cause erroneous or inaccurate readings when monitoring the progress of the PCR.

[0094] With reference to FIG. 1A, the reaction chamber can have dimensions (e.g., shallow depth) such that temperature gradients across the channel depth are minimized. With reference to FIG. 2A, the reaction chamber is deeper and wider to accommodate larger samples, for example, for PCR. In the exemplary embodiment of FIG. 2A, wider and deeper wells are required to ensure that temperature gradients across the channel depth are minimized and to increase thermal contact between the cartridge and the heater base, which can ensure optimal thermal uniformity and enhance PCR amplification. In some embodiments, compressible pads 314 can enable the use of wider and deeper wells by improving pressure distribution and thus increasing contact between the microfluidic cartridge and the heater base.

[0095] In some embodiments, the area of ​​the substrate 302 above the reaction chambers can be thinned to reduce thermal mass and autofluorescence from the plastic in 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 allow visualization of and transmission of light to and from the reaction chambers. The design of the cartridge 300 can allow an optical detector to more reliably monitor the progress of the reaction and also detect fluorescence from probes that bind to an amount of amplified nucleotides. In some embodiments, the area of ​​the substrate 302 can be made of a thinner material than the remainder of the substrate 302 to reduce glare, autofluorescence, and excessive absorption of fluorescent light.

[0096] As described herein, a microfluidic cartridge can be configured to be positioned in a complementary receiving bay in a device that includes a heater unit. Non-limiting examples of heater units are shown in FIG. 5A and FIG. 5B-5D. The heater unit is configured to provide heat to specific regions of the cartridge at specific times, including but 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, multiple 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 one another.

[0097] The microfluidic cartridge 300 can have an alignment member 316 that fits into a complementary feature of the receiving bay. The alignment member 316 can be, for example, a notch on an edge of the cartridge 300, and the receiving bay can include a complementary feature to the alignment member 316. By selectively receiving the cartridge, the receiving bay can help position the cartridge so that a device can properly operate on the cartridge.

[0098] The receiving bay can also be configured so that a heat source of the device operating on the microfluidic cartridge 300 can operate appropriately therein. For example, a contact heat source can be positioned in the receiving bay so that it can be thermally coupled to one or more distinct locations on the microfluidic cartridge 300 that is selectively received in the bay. Micro-heaters in a heater module as described further herein are aligned with corresponding heat-demanding micro-components (valves, pumps, gates, reaction chambers, etc.). Micro-heaters arranged in sets to provide heat to specific regions of the cartridge 300 can be designed to be slightly larger than the microfluidic components requiring heat, allowing the individual components to function effectively even if the cartridge is off-center from the heater set.

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

[0100] Referring back to FIG. 3, a PCR reaction chamber (e.g., a 150μ deep by 700μ wide reaction chamber) is shown in the substrate layer 302 of the cartridge 300. The laminate layer 304 of the cartridge (e.g., 125μ thick) is directly beneath the PCR reaction chamber. In some embodiments, an area of ​​the substrate 302 may be made of a thinner material than the remainder of the substrate 302 to make the PCR reaction chamber more responsive to heating cycles (e.g., for rapid heating and cooling between temperatures appropriate for the denaturation and annealing steps). A heater is located 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 side corresponds to an associated heating element disposed on the heater base. The heater base thus includes four heaters disposed along the sides of the PCR reaction chamber and configured to heat the PCR reaction chamber: a long top heater, a long bottom heater, a short left heater, and a short right heater. In some embodiments, the small gap between the long top heater and the long bottom heater results in a negligible temperature gradient (less than 1° C. difference between both ends of the PCR channel at any point along the length of the PCR reaction chamber), thus resulting in an effectively uniform temperature throughout the PCR reaction chamber. The heaters at the short edges of the PCR reactor provide heat to offset the gradient created by the two long heaters from the center of the reactor to the edges of the reactor. Those skilled in the art will appreciate that other configurations of one or more heaters located around the PCR reaction chamber are compatible with the methods and apparatus 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 in the receiving bay to be thermally coupled to one or more different locations of a microfluidic cartridge received in the receiving bay, thereby selectively heating the different locations. The contact heat source typically includes a plurality of contact heat sources, each configured in the receiving bay to be independently thermally coupled to a different separate location of a microfluidic cartridge received therein, thereby independently heating the separate locations. The contact heat source can be configured to be in direct physical contact with one or more separate locations of a microfluidic cartridge received in the bay. In various embodiments, each contact heat source is a separate location having an average diameter in two dimensions of about 1 millimeter (mm) to about 15 mm (typically about 1 mm to about 10 mm), or a separate location having an average diameter in two dimensions of about 1 mm. 2 ~Approx. 225mm 2 (Typically about 1 mm 2 ~ approx. 100mm 2 , or in some embodiments, about 5 mm 2 ~ approx. 50mm 2 The heat source may be configured to heat discrete locations having a surface area of ​​100 mm or less. Various configurations of heat sources are further described in U.S. patent application Ser. No. 11 / 940,315, filed Nov. 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 can be deposited on top and bottom of the gold layer to act as an adhesion layer. In some embodiments, the heater substrate is a glass, fused silica or quartz wafer with a thickness of 0.4 mm, 0.5 mm, 0.7 mm, or 1 mm. In some embodiments, a thin electrically insulating layer of 2 μm silicon oxide acts as an insulating layer on top of the metal layer. In some embodiments, an additional thin electrically insulating layer such as 2-4 μm parylene can also be deposited on top of the silicon oxide surface. In some embodiments, two long heaters and two short heaters run in parallel, surrounding the areas corresponding to each PCR reaction chamber. An exemplary heater array is described in U.S. patent application Ser. No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed Nov. 14, 2007, the specification of which is incorporated herein by reference in its entirety.

[0104] A specific heater orientation and placement is used to create a uniform heating zone even on substrates with poor thermal conductivity. The heater substrate can be made of a variety of materials, including glass, quartz, polyimide, FR4, ceramic, and fused silica substrates. The heater module is utilized to facilitate independent operation of various microfluidic components, such as PCR reaction chambers, and independent operation of various sample lanes. The configuration that provides uniform heating to 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, the heater can have an associated temperature sensor or may function as a sensor itself, as further described in U.S. patent application Ser. No. 11 / 940,315, entitled "Heater Unit for Microfluidic Diagnostic System," filed Nov. 14, 2007.

[0105] Generally, heating of microfluidic components such as PCR reaction chambers is controlled by passing electrical current through appropriately configured microfabricated heaters. Under the control of appropriate circuitry, the sample lanes of a multi-lane cartridge can be controlled independently of each other. This makes the device more energy efficient, since not all heaters are heated at the same time, and a given heater only receives electrical current for a portion of the time it needs to be heated. Control systems and methods for controllably heating various heating elements are further described in U.S. patent application Ser. No. 11 / 940,315, filed Nov. 14, 2007, entitled "Heater Unit for Microfluidic Diagnostic System."

[0106] An example of thermal cycling performance in a PCR reaction chamber configured as described herein may include a protocol set to heat the reaction mixture to 92°C, hold this temperature for 1 second, then cool to 62°C and dwell for 10 seconds. The cycle time shown is approximately 29 seconds, requiring 8 seconds to heat from 62°C and stabilize at 92°C, and 10 seconds to cool from 92°C and stabilize at 62°C. It is important to minimize the time required for each cycle to minimize the overall time required for effective PCR to generate detectable amounts of amplified material. Cycle times of 15-30 seconds, such as 18-25 seconds, and 20-22 seconds, are desirable. In general, average PCR cycle times of 25 seconds, and short cycle times of 20 seconds are typical for the technology described herein. In some non-limiting examples, the use of reaction volumes of less than a microliter (such as hundreds of nanoliters or less) allows the use of associated smaller PCR chambers, allowing cycle times as short as 15 seconds.

[0107] A non-limiting example of an optical detector suitable for use with the microfluidic cartridge of the present technology will now 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 a compressible pad 314, can be laid down 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 flat at their respective interface surfaces, for example, within about 100 microns, more typically within about 25 microns. The compressible pad 314 can improve thermal coupling between the microfluidic cartridge 300 and the heater module 400. The optical detector 500 can be disposed on the top 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 containing the one or more heat sources. In a 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 relies on a pressure being applied to the cartridge 300. The bottom surface of the optical detector 500 can be flat (e.g., within 250 microns, typically within 100 microns, more typically within 25 microns) and the bottom surface can press against the cartridge 300. The cartridge 300 can include a compressible pad 314. As a result, the optical detector 500 can compress the cartridge 300, thereby creating a pressure and thus forming a thermal contact with the underlying heater base of the heater module 400, approximately uniform throughout the microfluidic cartridge 300.

[0109] It will be appreciated that the present technique is not limited to an optical detector including a lever assembly 502. Other force members may be suitably implemented. In one embodiment, an automation platform including the optical detector 500 is lowered and pressed onto the microfluidic cartridge 300, where the microfluidic cartridge 300 is received in a receiving bay that remains stationary. The movement of the automation platform may be controlled by a processor of the diagnostic device. In another embodiment, an automation 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 automation platform may be controlled by a processor of the diagnostic device.

[0110] Thus, embodiments of diagnostic devices according to the present technology are configured to apply a force to thermally couple at least one heat source to at least a portion of the microfluidic cartridge 300. The application of force is important to ensure consistent thermal contact between the heater module 400 and the PCR reaction chamber in the microfluidic cartridge 300. In some embodiments, a lever assembly 502, similar mechanical force member, or automated platform can provide a force (e.g., 5-500 N, typically about 200-250 N) to generate a pressure (e.g., 2 psi) on top of or a portion of the microfluidic cartridge 300. In embodiments in which the optical detector 500 moves over the stationary receiving bay 402, the mechanical features of the optical detector 500 can press against the microfluidic cartridge 300 after the optical detector 500 is in place, bringing the reaction chamber into better thermal contact with the heater module 400. Positioning of the optical detector 500 in this manner can apply pressure to the cartridge 300. In embodiments where the receiving bay 402 moves beneath the stationary optical detector 500, the mechanical features of the receiving bay 402 may be forced up against the microfluidic cartridge 300 after the receiving bay 402 is in place, bringing the reaction chamber into better thermal contact with the heater module 400. Positioning the receiving bay 402 in this manner may allow pressure to 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 device. In the illustrated embodiment, pressure is applied to the top surface of the cartridge 300 and the heater module 400 is located below the cartridge 300, although other configurations are contemplated.

[0112] The optical detector 500 can include a light source that selectively emits light in an absorption band of a fluorescent dye, and an optical detector that selectively detects light in an emission band of a fluorescent dye, where the fluorescent dye corresponds to a fluorescent polynucleotide probe or fragment thereof. Alternatively, for example, the optical detector 500 can include a bandpass filtered diode that selectively emits light in an absorption band of a fluorescent dye, and a bandpass filtered photodiode that selectively detects light in an emission band of a fluorescent dye. The optical detector 500 can be configured to independently detect multiple fluorescent dyes having different fluorescence emission spectra, each fluorescent dye corresponding to a fluorescent polynucleotide probe or fragment thereof. The optical detector 500 can be configured to independently detect multiple fluorescent dyes at multiple different locations of the microfluidic cartridge, each fluorescent dye corresponding to a fluorescent polynucleotide probe or fragment thereof in a different sample. The optical detector 500 can also be configured to detect the presence or absence of an analyte of interest in a sample in a PCR reaction chamber of a given sample lane and condition the initiation of thermal cycling upon positive detection of the presence of the sample. In some embodiments, the cartridge and device are configured such that the read head of the optical detector 500 does not cover the sample inlet, thereby allowing loading of a separate sample while another sample is undergoing PCR thermal cycling. Further description of a suitably configured detector is provided in U.S. Patent Application No. 11 / 940,321, entitled "Fluorescence Detector for Microfluidic Diagnostic System," filed November 14, 2007, which is incorporated herein by reference in its entirety. The present technology provides a fluorometric detector configured to detect light emitted in response to a probe characteristic of a polynucleotide. The polynucleotide is amplified in a microfluidic channel with which the detector is in optical communication. The detector is configured to detect minute amounts of the polynucleotide, such as those contained in the microchannel. The detector may also be multiplexed to allow multiple polynucleotides to be measured simultaneously.

[0113] While 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 an inverted configuration would work as well. In such an embodiment, the heater would be pressed down onto and in contact with the microfluidic substrate, and the detector would be mounted below the substrate and positioned to emit light upwards towards the microfluidic cartridge and collect light exiting the microfluidic cartridge downwards 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 of the bottom surface of the detector on the top surface of the microfluidic cartridge and thus the pressure distribution across the bottom surface of the microfluidic cartridge by the receiving bay. The compressible pad 314 of the microfluidic cartridge 300 can be designed to enhance thermal uniformity, for example, to improve uniform contact between the cartridge and the heater module. The compressible pad 314 for the microfluidic cartridge 300 can be designed to enhance PCR amplification, for example, by promoting uniform application of heat to a wider and / or deeper reaction chamber.

[0115] As mentioned 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 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 in each reaction chamber to a specific temperature within a given time. In use, the cartridge 300 is placed on a heater substrate. In some embodiments, the heater substrate is a surface with an underlying heater. In use, a compressive load is applied to hold the cartridge 300 securely 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 thermal 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 brought together may not be flat enough to provide optimal contact with one another. In some embodiments, the compressible pad 314 comprises a highly compressible material that is applied on top of the cartridge 300. The compressible pad 314 can improve the contact between the two rigid surfaces by introducing an element of compliance into an otherwise rigid system. The compressibility of the material of the compressible pad 314 allows some areas to compress a different amount than other areas. This differential compression accommodates the inherent mechanical and material surface variations in the two surfaces, resulting in a much more uniform pressure distribution across the cartridge 300. The compliant pad 314 allows for more uniform contact between the cartridge 300 and the heater module 400, thus providing more complete and consistent heat transfer to each of the microfluidic reaction chambers.

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

[0118] The heater of the heater module 400 provides thermal conduction to the fluid sample received in the cartridge 300. When the cartridge 300 without the compressible pad is compressed against the heater module 400, there are areas that have less contact than other areas due to mechanical and material surface variations and inherent bending and curvature in the heater surface and / or the surface of the microfluidic cartridge. This results in an uneven pressure distribution across the cartridge 300. In use, areas with poor physical contact between the heater and the cartridge 300 will experience heat loss. Thus, less heat is delivered to reaction chambers with poor physical contact. This results in delays and inconsistencies in PCR amplification. Inconsistencies in physical contact can result in significant variations in overall assay performance.

[0119] An embodiment of the compressible pad 314 according to the present technology allows some areas of the pad to be more compressed than others. This compressibility accommodates inherent mechanical and material surface variations in the system and ensures that all areas of the cartridge 300 have a more even pressure distribution. The compressible pad 314 can improve physical contact between the cartridge 300 and the heater in the heater module 400, reducing heat loss and / or resulting in better PCR performance.

[0120] As mentioned above, the compressible pad 314 can be directly integrated into the label 306. The label 306 can include a top surface for displaying bar codes and manufacturing information as well as other types of information. The label 306 is made of a thin polyester facestock material, which has no inherent compliance. In some embodiments, the compressible pad 314 is directly integrated 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 underside that can bond 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 facestock of the label 306 is completely eliminated. In some embodiments, integrating the compliant material directly into an existing label can reduce delamination 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 glued to the top of the PCR cartridge 300 and a white cartridge label 306 is attached over the compressible pad 314. The label 306 has been removed to allow a clear view of the structure.

[0121] In some embodiments, the compressible pad 314 is an entirely separate compressible pad and does not form an integral part of the final manufactured microfluidic cartridge. The compressible pad 314 can be reversibly placed in contact with the cartridge 300, such as on the top surface 308 of the cartridge 300. In some embodiments, the compressible pad is applied over the label 306 of the cartridge 300 (this embodiment is not shown in FIG. 3A). In some embodiments, the compressible pad can be reusable for application onto another cartridge 300 after completion of PCR amplification. This embodiment, as well as other embodiments disclosed herein, exhibits improved thermal 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 it is reused multiple times as each cartridge is used and discarded). The compressible pad in this embodiment can generate significant cost savings due to the reusability of the pad. In some embodiments, the optical detector 500 may be redesigned or modified to accommodate a reusable compressible pad. In some cases, the compressible pad in this example is replaced after a certain number of uses or after a certain time. Periodically replacing the compressible pad in this way can ensure that the pad incorporated in the instrument has optimal compression characteristics.

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

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

[0125] Durometer testing utilizes a shore harness scale, e.g., Shore A. The Shore scale correlates to the configuration of the test equipment utilized, specifically the test indenter that contacts the material. The indenter applies a load to a small contact point on the material. Durometer testing assumes that the surface of the material is of relatively uniform hardness relative to the test contact point. Different Shore scales are typically used for different types of materials, such as materials of different hardness. As an example, Shore A is generally useful for softer elastomeric materials, while Shore D is generally useful for harder elastomeric materials.

[0126] In contrast, the compression force deflection test compresses the entire material sample, which is usually around 10 cm. The method involves determining the amount of stress at various levels of strain. The method can measure hardness or hardness at different compression levels. Compared to the durometer test, the compression force deflection test allows for a larger test sample, and a larger sample can make it easier to measure the material's properties more accurately.

[0127] In an embodiment of the present technology, the inventors have found that durometer testing is typically less accurate than compressive force deflection testing to determine the hardness of the compressible pad 314, and thus to evaluate 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 compression level. The hardness of these materials can depend on the test area and can vary from test area to test area. As described herein, the durometer test indenter measures only a small point on the material, covering 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, compressive force deflection testing determines an average hardness for a larger sample size. Compressive force deflection testing can determine the hardness of a material based on a compression level typical for the designed application. For example, compressive force deflection testing can determine the hardness of a material based on a typical compression level of the test device and, in some embodiments, the compression level of the optical detector 500 designed to compress the compressible pad 314. As described herein, the compressive force deflection test may be a more representative measurement of how the compressible pad 314 performs when applied to the microfluidic cartridge 300 .

[0128] [Example 1] Having generally described embodiments of the present technology, a further understanding can be obtained by reference to specific illustrative examples, which are provided herein for purposes of illustration only and are not intended to be limiting.

[0129] This example describes the identification of the material of the compressible pad 314. Figures 7A-7C show the results of an assay test for an analyte of interest without a compressible pad. Figures 8A-8D show the results of an assay test for an analyte of interest with a low durometer silicone compressible pad. Figures 9A-9D show the results of an assay test for an analyte of interest with 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. The embodiments of the automated molecular diagnostic testing system described herein can prepare specimens according to analyte-specific assays to obtain PCR-ready samples, which are introduced into the microfluidic cartridges that are received within the system. An example analyte-specific test includes an assay test for a viral analyte of interest. The test is for detection of the viral analyte of interest using a molecular viral load assay. The assay is a real-time RT-PCR assay that quantifies the amount of the viral analyte of interest in the sample ("viral load"). The assay can be performed in an automated molecular diagnostic testing system according to the present technology, as described above. Viral load is a numerical representation of the amount of virus in a given volume. It can be expressed as viral particles per mL. Higher viral loads are associated with more severe viral infections. The amount of live virus in a fluid sample taken from a patient can be estimated to calculate viral load / mL. For example, viral load can be expressed as RNA copies per ml of plasma. The assay can be used to track viral load during antiretroviral therapy, allowing caregivers to measure and evaluate changes in the amount of viral analytes during treatment.

[0131] The assay of this embodiment can use two RNA calibrator sequences, Hi Cal and Lo Cal. An RNA calibrator sequence ("calibrator") is a synthetic RNA transcript of known sequence and quantity used to calibrate the output of the assay measurement. This contrasts with a "control", which is a standard sample that can be included in an assay to evaluate the validity of the test result (not to calibrate the output of the test result). A calibrator is designed to bind to a molecule with a complementary base sequence, also called a probe. This specific binding process is called hybridization. During sample preparation, a known amount of calibrator is mixed with the patient specimen and PCR reagents. The prepared specimen is amplified to detect and quantify the target nucleic acid (viral analyte of interest) in the sample. The degree of hybridization between the calibrator and the corresponding probe is used to normalize the measurement 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 respond similarly to variables (such as instrument and matrix variations). In the examples below, tests were performed to measure the amounts of Hi Cal calibrator, viral analyte of interest, and Lo Cal calibrator in test samples (as indicated by qCt measurements) and to assess other properties (e.g., ymaxEP).

[0132] Quantification of target nucleic acid in a sample depends on the relationship between fluorescence and amplification cycle number on a logarithmic scale. The cycle number at which fluorescence crosses a given detection threshold is sometimes called the cycle threshold (Ct). During amplification, the amount of target nucleic acid doubles with every cycle. Thus, for example, a sample with a cycle threshold 3 cycles earlier than another sample will have a 2 3 = 8 times more target nucleic acid. In the following assay test, two parameters were tested. The first parameter is the qCt score, which indicates the first amplification cycle at which fluorescence was detected in a thermal cycling protocol that includes multiple amplification cycles. The second parameter is the ymaxEP score, which indicates the maximum fluorescence units at the final resting amplitude after multiple amplification cycles.

[0133] The optimal sample volume for the embodiment of the viral load assay test for the viral analyte of interest described herein is in the range of about 25 μL (rather than about 4 μL). As described above, such a sample volume can be obtained using a wider and deeper reaction chamber in a thicker version of the microfluidic cartridge of the present technology (about 1.24 mm cartridge thickness vs. about 1.68 mm thickness for the cartridge with wider and deeper wells). The thicker cartridge can accommodate an increased PCR reaction chamber, including six times the volume, as described above. However, the thicker cartridge implemented for viral load testing can experience edge effect defects (outside the sample lane), reverse edge effect defects (inside the sample lane), and random defects. When a compressible pad 314 was added to the top of the cartridge 300 as described herein, 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 long-standing challenges of incorporating pads into instruments or consumables (e.g., microfluidic cartridges). The compressible pad 314 can be considered a solution to the pressure distribution effects associated with microfluidic cartridges having wells of increased thickness and increased volume. While the following example describes a cartridge-based solution, in some embodiments the compressible pad coupled to the optical detector 500 can include any of the compressible pad features described herein.

[0134] In this example, the viral load assay test includes a cartridge 200 as described herein, which has wider and deeper wells (e.g., PCR reaction chambers) than the cartridge 100 as described herein. The study design used a Geometry C Prototype cartridge, where each reaction chamber has a width dimension of about 3.5 mm, a depth dimension of about 0.83 mm, a length dimension of about 10 mm, and a volume of about 25.2 μL. The study design included a liquid master mix and a cartridge that was manually filled by a 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 a PCR amplification. The test was performed on two BD MAX™ instruments (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] 7A-7C show the results of a viral load assay test without the use of a compressible pad according to the present technology. In this test, a cartridge 200 with wider and deeper wells was utilized without a compressible pad. FIG. 7A illustrates the quantification of target nucleic acid in a sample. This shows the amount of fluorescence versus the number of amplification cycles on a logarithmic scale. The x-axis is the qCt score, which indicates the rate of change of fluorescence. The number of cycles is shown along the y-axis. During thermal cycling, such as PCR, the amount of target nucleic acid doubles with each cycle. Each colored line on the graph represents a separate reaction chamber. As described herein, a microfluidic cartridge can include 24 sample lanes arranged in 12 cartridge lanes, with each cartridge lane corresponding to an area of ​​the cartridge 300 that includes two sample lanes 312. Each cartridge lane can include a reaction chamber in a first bank of reaction chambers and a reaction chamber in a second bank of reaction chambers. The qCt scores of each reaction chamber in the same cartridge lane are assigned the same color in FIG. 7A. For each curve in Figure 7A, there is a qCt score indicating the first amplification cycle in which fluorescence is detected. For each curve in Figure 7A, there is a ymaxEP score indicating the maximum fluorescence units at the final resting amplitude after multiple cycles. This data is also shown in Figures 7B and 7C.

[0136] FIG. 7B shows the individual qCt scores for each of the 24 reaction chambers of cartridge 200. The top graph shows the reaction chambers of the first bank of reaction chambers 226. The bottom graph shows the reaction chambers of the second bank of reaction chambers 228. The 12 cartridge lanes are shown on the y-axis. The qCt scores, which indicate the first amplification cycle at which fluorescence is detected, are shown on the x-axis. While the qCt scores for Hi Cal are fairly consistent between cartridge lanes and the qCt scores for Lo Cal are fairly consistent between cartridge lanes, there is a large variation between cartridge lanes 5 and 10 of the first bank, indicating that the first amplification cycle at which fluorescence is detected varies significantly between the 24 detection chambers. This variation in reaction chambers can be attributed to a variety of factors, including surface variations, poor contact between the cartridge and 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 viral analyte of interest 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, and 10 of the second bank have higher qCt scores for the viral analyte of interest than the reaction chambers in cartridge lanes 1, 4, 6-9, and 11-12 of the second bank.

[0137] FIG. 7C shows the ymaxEP for each reaction chamber. The top graph shows the reaction chambers in the first bank of reaction chambers. The bottom graph shows the reaction chambers in the second bank of reaction chambers. The 12 cartridge lanes are shown on the Y-axis. The ymaxEP score indicates the maximum fluorescence units at the final resting amplitude after multiple cycles. The ymaxEP score for Hi Cal, the ymaxEP score for Lo Cal, and the ymaxEP score for the viral analyte sample vary from reaction chamber to reaction chamber. The final resting amplitude is not consistent across reaction chambers. This variability in reaction chamber ymaxEP scores is due to a variety of factors including surface variability, poor contact between the cartridge and heater substrate, and poor compression of the cartridge under applied force. This variability indicates inefficiencies in the PCR reaction, causing lanes of a cartridge to not meet 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 viral analyte of interest 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 viral analyte of interest compared to the reaction chambers in cartridge lanes 2, 3, 5, and 10 of the second bank. This baseline is indicative of the variability that may occur in the microfluidic cartridge 200 if 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 Figures 7A-7B, the data suggesting wide variation in both qCt scores and ymaxEP scores are not tightly clustered.

[0138] 8A-8D show 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. FIG. 8A shows an embodiment of a low durometer silicone compressible pad bonded to the top of a cartridge 200. FIG. 8B shows quantification of target nucleic acid in a sample. A logarithmic scale plot of fluorescence versus amplification cycle number. The x-axis is qCt score, which indicates the rate of change in fluorescence. The y-axis is cycle number. In FIG. 8B, the data is more tightly clustered in the initial amplification than in FIG. 7A, suggesting less variability in the qCt scores in each reaction chamber. In FIG. 8B, the data is less tightly clustered as the amplitude becomes more constant, suggesting more variability in the ymaxEP scores.

[0139] 8C illustrates the individual qCt scores for each reaction chamber. The top graph shows the reaction chambers in the first bank of reaction chambers 226. The bottom graph shows the reaction chambers in the second bank of reaction chambers 228. The qCt scores for Hi Cal and Lo Cal are fairly consistent between cartridge lanes. However, for the viral analyte sample, there is variability in the qCt scores in cartridge lanes 5 and 7 of the first bank.

[0140] FIG. 8D shows the ymaxEP of each reaction chamber. The top graph shows the reaction chambers in the first bank of reaction chambers. The bottom graph shows the reaction chambers in the second bank of reaction chambers. The Lo Cal ymaxEP scores and the ymaxEP scores of the viral analyte samples vary among the reaction chambers in the first bank. The maximum fluorescence values ​​of the final resting amplitude after multiple cycles are not consistent. In particular, the reaction chambers in cartridge lanes 1-3 and 9-12 in the first bank have higher ymaxEP scores for the viral analyte and Lo Cal than the reaction chambers in cartridge lanes 4-8 in the first bank. Thus, the low duro silicone compressible pad produces inconsistent PCR reactions as shown in the graph. In this example using the low duro silicone compressible pad, different reaction chambers can perform PCR more efficiently than other chambers.

[0141] 9A-9D show results of a viral load assay test using a cartridge incorporating a compressible pad made of PORON® foam. FIG. 9A shows an embodiment of a PORON® foam compressible pad bonded to the top of a cartridge 200. The PORON® foam is a fine pitch open cell urethane foam from Rogers Corporation. The material used was PORON® Cellular Polyester Urethane 4790-92. FIG. 9B illustrates the quantification of target nucleic acid in a 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, which indicates the rate of change in fluorescence. The y-axis is the number of cycles. In FIG. 9B, the data is more tightly clustered for the initial amplification compared to FIG. 7A and FIG. 8B, suggesting less variability in the qCt scores for each reaction chamber. In FIG. 9B, the data is more tightly clustered for the final amplification compared to FIG. 7A and FIG. 8B, suggesting less variability in the ymaxEP scores for each reaction chamber. In FIG. 9B, from left to right, the first cluster of lines relates to Hi Cal, the second cluster of lines relates to Lo Cal, and the third cluster of lines relates to viral analyte samples.

[0142] FIG. 9C illustrates the individual qCt scores for each reaction chamber. The top graph shows the reaction chambers in the first bank of reaction chambers 226. The bottom graph shows the reaction chambers in the second bank of reaction chambers 228. The qCt scores for the Hi Cal, Lo Cal, and viral analyte samples are consistent across the cartridge lanes. For the Hi Cal RNA calibrator sequence, the initial amplitude or first detection occurred at about cycle 20. For the HiCal RNA calibrator sequence, the initial amplitude or first detection occurred at about cycle 32. For the Lo Cal RNA calibrator sequence, the initial amplitude or first detection occurred at about cycle 36. These results are consistent across each cartridge lane. These results are consistent across each bank of the first and second banks. These results are consistent for each reaction chamber out of the 24 reaction chambers.

[0143] FIG. 9D shows the ymaxEP scores for each reaction chamber. The top graph shows the reaction chambers in the first bank of reaction chambers. The bottom graph is the reaction chambers in the second bank. The ymaxEP scores for the Hi Cal, Lo Cal, and viral analyte samples are consistent (relatively low variability) across cartridge lanes. For the Hi Cal RNA calibrator sequence, the maximum fluorescence units at the final resting amplitude after multiple cycles is about 2000. For the Lo Cal RNA calibrator sequence, the maximum fluorescence units at the final resting amplitude after multiple cycles is about 7000. For the viral analyte sample sequence, the maximum fluorescence units at the final resting amplitude after multiple cycles is about 5500. These results are consistent for each cartridge lane. These results are consistent for each bank of the first and second banks. These results are consistent for each reaction chamber of the 24 reaction chambers.

[0144] Additional tests were conducted using compression pads of different materials as outlined above. Additional tests included compressible pads made of graphite foil. The graphite foil was Tgon™ 820 from Laird. Another test included a compression pad made of fiberglass coated with thermally conductive silicone. This material was TF-1879 from ThermaCool®. A further test included a compressible pad made of silicone sponge. The material was BISCO® HT-800 silicone sponge from Rogers. Yet another test included a compression pad made of thermally conductive silicone sponge with a thermally conductive coating. The material was R-10404 silicone sponge from ThermaCool®. The test design was similar to that outlined above for the low durometer silicone compressible pads (FIGS. 8A-8D) and the PORON® foam compressible pads (FIGS. 9A-9D).

[0145] In evaluating materials for use with the compressible pad 314, unexpected results were obtained for a selected group of materials. In some embodiments, the compressible pad comprises a compressible material. In some embodiments, materials suitable for the compressible pad are selected based on the compressive force deflection (in this case the amount of stress (measured in psi) to deflect the material to 25% of its original height). Materials include those with compressive force deflection 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, etc. The material can include materials having a compressive 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 materials with compressive 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, etc. In some embodiments, the test method is 0.51 cm / min (0.2" / min) Strain Rate with Force Measured @ 25% Deflection. In some embodiments, the range is 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 durometer Shore A and compression force deflection measured at 25% are measures of compressibility. In these measurements, lower numbers indicate easier compression, which is expected to translate into better pressure distribution and better PCR performance. Unexpectedly, the inventors have discovered that durometer Shore A is a poor indicator of a suitable material (see Figures 7B-7D). Low durometer silicones typically have a hardness, durometer, Shore "A" of 10, and PORON® foams typically have a hardness, durometer, Shore "A" of less than 3. One skilled in the art would expect that both of these materials would compress easily and thus provide similar PCR performance. However, contrary to expectations, low durometer silicones performed significantly differently than PORON® foams. Additional materials were also tested, but no correlation was found between durometer Shore A and PCR performance.

[0147] Surprisingly, the compression deflection measured at 25% was a good indicator of a suitable material. Low durometer silicones typically exhibit a compression deflection of about 30 psi, and PORON® foams typically exhibit a compression deflection of 2-5 psi. Additional materials were also tested and found to correlate with the amount of compression load deflection and PCR performance. In particular, materials with a compression deflection of less than 30 psi measured at 25% deflection exhibited improved PCR performance. In some embodiments, materials having a compression deflection of 0-20 psi had improved PCR performance.

[0148] As described herein, both hardness, durometer, Shore "A", and compressive force deflection measured at 25% determine the properties of a material. These methods are useful for determining the relative hardness or stiffness of a material. One skilled in the art would expect that a material with lower hardness, durometer, Shore "A" and lower compressive load deflection measured at 25% would lead to better PCR performance. However, only the compressive load deflection measured at 25% (not hardness, durometer, Shore "A") correlated to indicate a material suitable for improving PCR performance.

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

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

[0151] In some embodiments, the compressible pad comprises material properties as set forth below: In some embodiments, the compressible pad comprises a thickness measured along the z-axis of the pad. The thickness can range from 0-5 mm, e.g., 0-1 mm, 1-2 mm, 2-3 mm, 3-4 mm, 4-5 mm, about 1 mm (0.032 inches) + / - 10%. In some embodiments, the compressible pad comprises an elongation per ASTM D412 of 80%. In some embodiments, the compressible pad comprises a compression deflection at 25% per ASTM D1056 of 9 psi (62 kPa). In some embodiments, the compressible pad comprises a compression set per ASTM D1056 of less than 1 at 70°C and less than 5 at 100°C. In some embodiments, the compressible pad comprises a density per ASTM 1056 of 22 lbs / ft 3 (352kg / m 3 In some embodiments, the compressible pad comprises a material property that includes a range of any two values ​​herein. In some embodiments, the compressible pad comprises a material property that includes any value having ±50% of the values ​​herein.

[0152] The compressible pad embodiment of the present technology is designed to improve heat transfer between a microfluidic cartridge and an associated heat source (e.g., an array of heat sources below a microfluidic cartridge). As described herein, viral load is a numerical representation of the amount of virus in a given volume. Microfluidic cartridges designed to determine viral load, such as via PCR, may require wider and deeper wells to increase larger reaction volumes and target detection. In these situations, heat transfer between a microfluidic cartridge with wider and deeper wells and the underlying heat source becomes critical. Contact can be improved by a more uniform pressure distribution so that each PCR reaction chamber is in optimal contact with the underlying heat source. Uniform pressure distribution can prevent poor reproducibility of thermal cycling protocols across sample lanes, cartridge lanes, or cartridges. Uniform pressure distribution can also avoid inefficiencies in heat transfer due to hot spots or air conduction.

[0153] As described herein, the compressible pad of the present technology can be placed on the top or bottom surface of the microfluidic cartridge. In some embodiments, a compressible pad on the bottom surface of the microfluidic cartridge can reduce thermal conduction. In some embodiments, a compressible pad on the top surface of the microfluidic cartridge may require a window or other cutout to allow for optical detection. In some embodiments, the compressible pad is made as large as possible, e.g., coextensive with 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 top 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, etc.

[0154] Another non-limiting implementation of a microfluidic cartridge according to the present technology will now be described with reference to Figures 10 to 37. Figures 10 to 37 are diagrams of a microfluidic cartridge 200 that includes 24 independent sample lanes.

[0155] Figures 10-16 show a first embodiment of a microfluidic cartridge 200 without compressible pads. Figure 10 is a perspective view. Figure 11 is a top view. Figure 12 is a bottom view. Figure 13 is a first side view. Figure 14 is a second side view. Figure 15 is a third side view. Figure 16 is a fourth side view.

[0156] Figures 17-23 show a second embodiment of a microfluidic cartridge 200 with compressible pads. Figure 17A is a see-through view. Figure 17B is an exploded perspective view. Figure 18 is a top view. Figure 19 is a bottom view. Figure 20 is a first side view. Figure 21 is a second side view. Figure 22 is a third side view. Figure 23 is a fourth side view.

[0157] Figures 24-30 are additional views of the microfluidic cartridge of Figure 10. Figure 24 is a perspective view. Figure 25 is a top view. Figure 26 is a bottom view. Figure 27 is a first side view. Figure 28 is a second side view. Figure 29 is a third side view. Figure 30 is a fourth side view.

[0158] Figures 31-37 are additional views of the microfluidic cartridge of Figure 10. Figure 31 is a perspective view. Figure 32 is a top view. Figure 33 is a bottom view. Figure 34 is a first side view. Figure 35 is a second side view. Figure 36 is a third side view. Figure 37 is a fourth side view. 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 molecular diagnostic testing devices, systems, and methods for determining the presence and / or amount of an analyte of interest in a sample. As used herein, "analyte" generally refers to the substance being detected. For example, it may 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 therapeutic and illicit administration), 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 streptococcosis, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginitis, bacterial vaginosis, Candida group, Candida glabrata, Candida krusei, Salmonella spp., Shigella spp. / enteroinvasive E. coli (EIEC), Campylobacter spp. (jejuni and coli) and Shiga toxin producing bacteria (STEC, Shigella dysenteriae), Yersinia enterocolitica, enterotoxigenic E. coli (ETEC), Plesiomonas shigelloides, 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, and Staphylococcus aureus.Additional examples of analytes include, but are not limited to, ferritin, creatinine 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 antibodies; cytokines; TNF-related apoptosis-inducing ligand (TRAIL); vitamin B2 microglobulin; interferon gamma-inducible protein 10 (IP-10); interferon-inducible GTP-binding protein (also known as 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 to rubella, such as rubella IgG and rubella IgM;Antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM);Testosterone;Salicylates;Acetaminophen;Hepatitis B surface antigen (HBsAg);Antibodies to 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 to hepatitis B e antigen (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (T3); free triiodothyronine (F3); carcinoembryonic antigen (CEA); lipoproteins, cholesterol, and triglycerides; and alpha-fetoprotein (AFP).Drugs of abuse and controlled substances include, but are not limited to, amphetamines, methamphetamines, 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, opium, phencyclidine, and propoxyphene. Analytes can also be included for purposes of biological or environmental substances of interest.

[0161] The above description is intended to illustrate various aspects of the present technology. The examples presented herein are not intended to limit the scope of the technology. Although the present technology has now been fully described, it will be apparent to those skilled in the art that many changes and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. 1. A microfluidic cartridge comprising a first side and an opposing second side, a first amplification chamber; a second amplification chamber; and 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 adhered to the first side; Equipped with 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 including a first window above the first amplification chamber and a second window above the second amplification chamber, the first window and the second window configured to allow light transmission through the first side of the microfluidic cartridge between the first amplification chamber and the second amplification chamber, respectively. Microfluidic cartridge.

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

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

4. The microfluidic cartridge of claim 1 , wherein the microfluidic cartridge comprises a label above the compressible pad.

5. 10. The microfluidic cartridge of claim 1, wherein the first amplification chamber, the second amplification chamber, the first inlet, and the second inlet are formed in a rigid base layer, and the second side of the microfluidic cartridge includes a flexible laminate layer below the first amplification chamber and the second amplification chamber.

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

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

8. The microfluidic cartridge of claim 1 , wherein the compressible pad improves pressure distribution from a component of a diagnostic testing instrument.

9. 2. The microfluidic cartridge of claim 1, wherein application of pressure to the compressible pad is configured to increase uniformity of application of heat from the multiple contact heat sources to the first amplification chamber and the second amplification chamber.

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

11. The microfluidic cartridge of claim 1 , wherein the compressible pad enhances PCR amplification that relies on rapid temperature cycling.

12. 1. A method of amplifying on a plurality of polynucleotide-containing samples, comprising: introducing a plurality of samples into a microfluidic cartridge, the microfluidic cartridge including a plurality of amplification chambers configured to allow thermal cycling of the plurality of samples independently of one another, the microfluidic cartridge comprising a compressible pad adhered to a first side of the microfluidic cartridge; transferring the plurality of samples to the respective plurality of amplification chambers; amplifying polynucleotides contained in the plurality of samples by applying successive heating and cooling cycles to the amplification chambers by one or more heaters in contact with a second side of the microfluidic cartridge opposite the first side; compressing the compressible pad of the microfluidic cartridge during amplification; A method comprising:

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

14. The method of claim 12 further comprising applying pressure to the compressible pad to enhance thermal uniformity.

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

16. 1. A system comprising a microfluidic cartridge, The microfluidic cartridge comprises: a first side and an opposing second side; a first PCR reaction chamber; a second PCR reaction chamber; and 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 adhered to the first side; Equipped with the microfluidic cartridge is configured for use with an apparatus; The apparatus comprises: a bay configured to receive the microfluidic cartridge; at least one heat source configured to be thermally coupled to the second side of the microfluidic cartridge and configured to apply thermal cycles to one or more polynucleotide-containing samples in the microfluidic cartridge to perform PCR; 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 heating of one or more regions of the microfluidic cartridge; Including, system.

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

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

20. 17. The system of claim 16, wherein the detector is configured to move downward into physical contact with the microfluidic cartridge to compress the compressible pad.

21. 17. The system of claim 16, wherein the microfluidic cartridge is configured to move upward to compress the compressible pad into physical contact with the detector.

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

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