Analytical system including a microfluidic device, a microfluidic device and related methods

Abstract

To provide analysis systems with a housing having a chamber sized and configured to receive at least one microfluidic device.SOLUTION: A system includes: an optic system coupled to a housing in optical communication with at least one microfluidic device 10; a controller coupled to the optic system; a heat source coupled to the optic system and thermally coupled to the at least one microfluidic device held in the housing; and a sub-array 10s selection module in communication with the controller. The sub-array selection module is configured to select a sub-set of sets of microwells of at least one fluid channel of the microfluidic device for imaging by the optic system after a reaction step (e.g., one thermocycle) during an assay.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 760,604, filed November 13, 2018, and U.S. Provisional Patent Application No. 62 / 825,523, filed March 28, 2019, the contents of both of which are incorporated by reference as if fully set forth herein.

[0002] (Statement of Federal Government Support) This invention was made with government support under Grant No. HR0011-12-2-0001 awarded by the U.S. Department of Defense (DARPA). The U.S. Government has certain rights in this invention.

[0003] (Statement regarding electronic filing of sequence listings) A Sequence Listing in ASCII text format, filed under 37 CFR § 1.821, entitled 5470-860WO_ST25.txt, 817 bytes in size, created on October 29, 2019, and submitted via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference for its disclosure.

[0004] (Copyright reserved) A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner, The University of North Carolina at Chapel Hill, North Carolina, has no objection to the reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0005] The present invention relates to systems, fluidic devices (eg, high-throughput microfluidic devices), and methods of use thereof that are suitable for obtaining, for example, polymerase chain reaction (PCR) data from coded bead microwell arrays. [Background technology]

[0006] Polymerase chain reaction (PCR) is a highly sensitive method for amplifying segments of genomic DNA (gDNA) or complementary DNA (cDNA). PCR has many applications, including the detection of trace amounts of nucleic acids to determine the presence of pathogenic organisms, gene expression, genotyping, genetic manipulation or modification, and forensic science. PCR amplification provides excellent target specificity and quantification across a wide range of analyte concentrations. However, simultaneous quantitative analysis of many analytes by PCR has proven extremely challenging. Detection based on intercalating dye fluorescence can only determine the overall concentration of dsDNA; therefore, simultaneous analysis of multiple templates in a single reaction vessel using this detection method is not possible. Fluorescent probe technology (i.e., TaqMan, molecular beacons, or other chemistries) can be used for low-level multiplexing of reactions, as each target is amplified using a differently colored fluorescent probe as a signaling reporter. The probes are also sequence-specific, reducing false positives due to primer-dimer formation or nonspecific amplification. A common multiplexing method, either using traditional microtiter plates or microfluidic real-time PCR (rt-PCR), uses a small number of reaction wells, each containing three different color probes. However, designing multiplexed primer and probe sets is generally considered challenging, requiring careful design and an additional level of optimization to ensure compatibility. While multiplexing with at least six dyes has been demonstrated, multiplexing with this method is ultimately limited to detection of approximately four colors due to instrumentation and spectral overlap between the dyes, typically with one color for the internal reference dye. Summary of the Invention [Means for solving the problem]

[0007] Embodiments of the present invention provide methods to limit photobleaching on coded bead arrays, optionally during acquisition of real-time PCR data.

[0008] Embodiments of the present invention provide PCR master mix and magnetic particle chemistry formulations that are particularly suitable for use with coded bead arrays.

[0009] The present invention relates to a sample analysis device for testing, a system for analyzing signals from an array of microwells in a fluidic device, and a testing method.

[0010] An embodiment of the present invention relates to an analytical system including a housing containing a chamber sized and configured to receive at least one microfluidic device, an optical system coupled to the housing in optical communication with the at least one microfluidic device, a controller coupled to the optical system, a heat source coupled to the optical system and thermally coupled to the at least one microfluidic device secured to the housing, and a subarray selection module in communication with the controller, the subarray selection module configured to select a subset of a set of microwells in at least one fluid path of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during an assay.

[0011] The system can include at least one magnet secured to the housing adjacent to the at least one microfluidic device, the magnet configured to translate along the at least one fluid path during a bead loading operation to magnetically couple to the beads and guide the beads along the fluid path to a bead-retaining segment of the microwell.

[0012] The microfluidic device includes a plurality of fluid passages, each comprising a plurality of sets of microwells arranged along a length dimension associated with a direction between a sample entry port and an opposing second port, and the selected subset of the sets of microwells may be associated with a first subset of microwells in a horizontal array of the plurality of fluid passages, optionally a single row with one subset of aligned microwells from each fluid passage of the plurality of fluid passages.

[0013] Prior to initial image capture and / or photoexcitation, a subarray selection module identifies a subset of the set of microwells to define the location of other sets of microwells in the microfluidic device.

[0014] The subarray selection module selects different subsets of the microwell sets of the microfluidic device for different reaction steps of the test (e.g., different thermal cycles of the test), instructs the optical system to excite only the currently selected subset of the microwell sets of different fluid paths, and instructs the cameras of the optical system to capture images of the different subsets of the microwell sets in the currently selected subset, either sequentially or in parallel.

[0015] The subarray selection module selects the same microwell subset for at least some different sequential reaction steps of the test (e.g., different thermal cycles of the test), instructs the optical system to transmit light only to the currently selected subset of microwell sets, and instructs the camera of the optical system to capture paired images of different subsets of microwell sets, optionally adjacent microwell sets, of the currently selected microwell subset, either sequentially or in parallel.

[0016] The optical system can include at least first and / or second filters (optionally more than two, such as 3-100) that define respective first and / or second excitation light wavelengths corresponding to the first and second target-encoded beads for decoding sets of beads held in one or more sets of microwells in at least one fluid passage of the microfluidic device.

[0017] The system can include a signal analysis module integrated with and / or coupled to the controller. The signal analysis module can be configured to acquire an analog signal for each microwell of a selected subset of the set of microwells. The analog signal can provide PCR data as an amplitude change with cycles of testing, and the concentration of the target molecule for a reaction associated with a defined bead type can be determined from the analog signal.

[0018] The signal analysis module may further be configured to acquire digital PCR signals for the set of microwells.

[0019] The analog signal provides real-time PCR data as amplitude changes versus PCR cycle number for input material in one or more sets of microwells in the fluidic pathway, where the molecular concentration of a given bead type is about, equal to, or greater than one molecule per microwell. The analog signal can define a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescent signal intensity (Si) is higher than the threshold and the target molecule number / target molecule concentration for the target species and / or molecule type, or negative when the fluorescent signal intensity (Si) is consistently lower than the threshold for a given number of PCR cycles. Ct is the cycle number where Si >> Bs, where >> is at least 5-10% greater than Bs, optionally 2-fold greater, and / or 5-10 times the standard deviation of Bs, and Bs is optionally the background fluorescent signal measured in negative PCR reactions.

[0020] Analog signals may be acquired for only a single subset of the microwell sets in each fluid path after every other or each of several different reaction steps of the test (eg, different thermal cycles of the test).

[0021] The analog signal is the mean (optionally excluding outlier data), median, mode, or weighted value of the Si as analog signal corresponding to each defined bead type and serves as an estimate of real-time PCR curves for similar reactions in microwells of other sets of microwells in that fluid channel of the microfluidic device, thereby enabling single cycle resolution without imaging every set of microwells in each fluid channel after different reaction cycles or each reaction cycle.

[0022] The optical system may include a camera with a field of view (FOV) that covers only a subset of the microwells of at least two, and optionally all, adjacent fluid channels of the microfluidic device.

[0023] In some particular embodiments, the defined FOV covers between 2 and 10 or more adjacent fluid paths, and / or optionally between 10 and 15% of the total number of fluid paths in the microfluidic device.

[0024] The controller and / or signal analysis module may be configured to instruct the optical system to acquire pre- and post-PCR images, optionally along with analog data acquired using selected subsets of the microwell sets for different reaction steps of the test, compare signal intensities between them, determine positive and negative PCR reactions, and optionally determine the target species concentration and / or molecular concentration of the original sample provided to the fluid path.

[0025] The system may further include a holder configured to secure the plurality of microfluidic devices to the alignment grid of the housing.

[0026] The holder is formed of a thermal insulating material that provides a thermal barrier between adjacent microfluidic devices, and optionally the holder can hold multiple microfluidic devices with their (sample / bead) input ports to the fluid pathways facing outwards.

[0027] The microfluidic device further comprises a dye-uniformity agent, and optionally the dye-uniformity agent includes an oligonucleotide.

[0028] The dye-uniformizing agent may comprise a non-extending oligonucleotide and / or partially double-stranded DNA.

[0029] The dye-uniformizing agent may contain biotin and / or partially double-stranded DNA that includes a C1-C20 hydrocarbon chain.

[0030] The dye uniformity agent can be present in a master mix present in the microfluidic device and / or the dye uniformity agent can be attached to beads present in the microfluidic device.

[0031] Another embodiment relates to a method of analyzing a sample, such as to identify a target species and / or target molecule, comprising providing a fluid analysis device with a first fluid path including a plurality of microwell sets disposed on the first fluid path, acquiring signal intensity data from only a defined subset of the plurality of microwell sets (optionally in response to transmission of an optical excitation signal), and identifying PCR reactions that are positive for a target species and / or molecule type associated with the bead type and / or target molecule based at least in part on the acquired signal intensity data.

[0032] The method may include loading (with a sample-pretreated bead slurry) and then sealing a plurality of microwell sets on a first fluid path prior to the acquiring step such that after sealing, each microwell set is fluidically isolated from one another. The plurality of microwell sets on the first fluid path may be in fluid communication only during loading prior to the sealing step.

[0033] The method may include changing a defined subset of the plurality of microwell sets to a different defined subset after each of a plurality of sequential reaction steps of the test (e.g., after each of a plurality of sequential thermal cycles of the test) so that some microwell sets are not imaged after each reaction step.

[0034] The defined subset remains the same across multiple successive reactions of the assay (eg, after each of multiple successive thermal cycles of the assay), such that some sets of microwells are not imaged after each reaction step.

[0035] The fluid analysis device may include multiple fluid paths, including, without limitation, a second fluid path including a plurality of microwell sets spaced apart along the second fluid path, a third fluid path including a plurality of microwell sets spaced apart along the third fluid path, and a fourth fluid path including a plurality of microwell sets spaced apart along the fourth fluid path, with any number of microwells between 2 and 100 of such microwells. A first microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths may be arranged as a first row. A second microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths may be arranged as a second row. A third microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths may be arranged as a third row. A fourth microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths may be arranged as a fourth row. The defined subset may be (part or all of) a single row of the first, second, third, or fourth row.

[0036] The first, second, third, and fourth fluid paths may have straight or arcuate segments that are substantially parallel to one another and define the first, second, third, and fourth sets of microwells.

[0037] The method may further include transmitting an optical excitation signal to only the defined subset prior to the acquiring step; digitally scanning the defined subset of the plurality of microwell sets after, before, or both before and after the transmitting and acquiring steps to acquire images of the microwell sets to identify positive and negative PCR reactions associated with digital PCR; and electronically identifying microwells of the microwell set that are positive for one or more target analyte molecules while the microwells are at the imaging temperature.

[0038] The excitation and acquisition steps are performed to scan only one defined subset of the set of microwells after each of multiple successive reaction steps (eg, thermal cycles) of the assay, and each successive defined subset may be different from each other.

[0039] Acquisition of signal intensities from only a defined subset can be performed using a camera with a field of view (FOV) that covers only one microwell set or only a subset of microwell sets in all or a subset of adjacent fluid paths of the fluid paths by sequentially or in parallel acquiring images of different defined adjacent microwell sets in different fluid paths of the defined subset of microwell sets.

[0040] Each of the microwell sets can have a microwell array containing between 1,000 and 1 million microwells.

[0041] The fluid analysis device can have a plurality of spaced apart fluid passages, each containing a set of macrowells comprising a microwell array. The fluid passages can be in fluid isolation during testing. At least some of the microwells in the microwell array can contain a single bead, and optionally some or all of the microwells can contain no beads, one or more beads.

[0042] The method may include electronically identifying the locations of one or more sets of microwells located at one or more locations on the microfluidic device prior to the transmitting and acquiring steps, and defining the locations of other sets of microwells based at least in part on the identified locations.

[0043] The method of claim 20 further includes transmitting an optical excitation signal to only the defined subset prior to the acquiring step, and selecting a filter prior to the transmitting step that provides a coding wavelength for the transmitting step.

[0044] Obtaining signal intensities includes obtaining analog signals that can provide real-time PCR data as amplitude change versus PCR cycle number for input materials of a set of microwells, where the molecule concentration of a given bead type is about, equal to, or greater than 1 molecule per microwell. The analog signals can define a threshold cycle or cycle threshold Ct that identifies a microwell reaction and target molecule number / target molecule concentration of a target species and / or molecule type as positive when the fluorescent signal intensity (Si) is higher than a threshold, or negative when the fluorescent signal intensity (Si) is consistently lower than the threshold for a given number of PCR cycles.

[0045] Ct is the cycle number where Si>>Bs, where >> is at least 5-10% greater than Bs, optionally 2-fold greater, and / or 5-10 times the standard deviation of Bs, and Bs is optionally the background fluorescence signal measured in negative PCR reactions.

[0046] Analog signals may be acquired for only a single subset of the microwell sets of one or more fluid pathways after every other or each of several different reaction steps of the test (eg, different thermal cycles of the test).

[0047] The analog signal is the mean, median, mode, or weighted value of the analog signal Si (optionally with outlier data removed) corresponding to each defined bead type and can serve as an estimate of real-time PCR curves for similar reactions in microwells of other sets of microwells in that fluid path of the microfluidic device, thereby allowing single cycle resolution without having to image all microwell subsets in each fluid path after different reaction steps.

[0048] Acquisition of signal intensity can be performed using a camera with a field of view (FOV) that covers only a subset of the microwell sets of at least two, and optionally all, adjacent fluid channels of the microfluidic device.

[0049] Analog signals are obtained for only a single set of microwell sets in each fluid path after multiple different reaction steps of the test (e.g., different thermal cycles of the test), and the analog signals comprise the mean, median, mode, or weighted value of Si as the analog signal corresponding to each defined bead type and serve as estimates of real-time PCR curves for similar reactions in microwells of other microwell sets in that fluid path of the microfluidic device, thereby allowing single cycle resolution without imaging all microwell sets in each fluid path after different reaction steps.

[0050] A fluid analysis device having a first fluid passage containing a plurality of microwell sets spaced apart along the length of the first fluid passage can include a plurality of additional fluid passages, each containing a plurality of microwell sets, spaced apart along the length of the first fluid passage. The fluid analysis device has a separate material input port for each of the first fluid passage and the plurality of additional fluid passages, with at least some of the fluid passages sharing a common, opposite second port. The method further includes, prior to the acquiring step, fluidically loading a bead slurry pre-exposed to the sample of interest for analysis into the input port, magnetically directing the bead slurry to flow into different microwell sets on the fluid passage, flowing a fluidic master mix containing a dye from the second port through the fluid passage to the first port, and sealing the microwell sets and the fluid passage from one another by flowing sealing oil from the second port through the fluid passage to the first port.

[0051] The master mix may optionally include a dye-uniformity agent, which may optionally be or include an oligonucleotide (e.g., a non-extending oligonucleotide and / or partially double-stranded DNA (e.g., partially double-stranded DNA including biotin and / or a C1-C20 hydrocarbon chain).

[0052] The method can include placing a magnet adjacent to the fluidic analysis chip and translating the magnet to the second port prior to flow of the fluidic master mix and sealing oil.

[0053] The fluid analysis device (optionally the first fluid passageway and / or the plurality of microwell sets) comprises a dye-uniform agent, and optionally the dye-uniform agent comprises an oligonucleotide (e.g., a non-extending oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain)).

[0054] The dye uniformity agent may be present in a master mix present in the fluid analysis device (e.g., present in the first fluid path and / or the plurality of microwell sets), and / or the dye uniformity agent may be attached to beads present in the fluid analysis device (e.g., present in the first fluid path and / or the plurality of microwell sets).

[0055] Yet another embodiment relates to a microfluidic device. The device includes a plurality of fluid passages. Each of the plurality of fluid passages has a length dimension corresponding to a direction between a first port and an opposing second port, at least a portion of the length dimension being configured as a linear or arcuate length segment. Each of the fluid passages includes a plurality of microwell sets arranged along the linear or arcuate length segment of the length dimension.

[0056] The plurality of microwell sets of the plurality of fluid passages may be arranged in rows, columns, or rows and columns, where a row or column corresponds to a linear or arcuate length segment.

[0057] At least some of the plurality of fluid passages may be substantially parallel over linear or arcuate length segments.

[0058] At least some of the plurality of fluid passageways may be arcuate, substantially parallel passageways providing arcuate length segments.

[0059] At least some of the plurality of fluid passageways may be substantially parallel and extend radially between the outer periphery of the device and the center of the device.

[0060] The plurality of fluid passageways can include a first set of fluid passageways and a second set of fluid passageways spaced apart from the first set, the first set of fluid passageways terminating in a first master mix port as the second port, and the second set of fluid passageways terminating in a second master mix port as the second port.

[0061] The first and second sets of fluid passages may be circumferentially spaced apart.

[0062] The plurality of fluid passages includes at least two fluid passages defining a first neighborhood set and at least two passages defining a second neighborhood set adjacent to the first neighborhood set, each containing a spatially aligned set of microwells.

[0063] The device can include a first gap space between each of the first and second fluid passages of the first and second proximal sets, and the device can further include a second gap space between the first and second proximal sets, the second gap space having a greater lateral extent than the first gap space.

[0064] The plurality of microwell sets in each of the plurality of fluid paths can have a common configuration. The plurality of microwell sets in each of the plurality of fluid paths can be aligned with one another in rows and / or columns. The plurality of fluid paths can maintain input materials of interest in fluid isolation from one another.

[0065] Each of the at least a plurality of microwell sets for the fluid pathway can include a quantity ranging from 1,000 to 1,000,000 microwells, each of which is sized and configured to immobilize and hold a single bead, thereby enabling 100 to 1 billion reactions in the microfluidic device.

[0066] A set, optionally a pair, of first and second sets of microwells from at least the first and second fluid paths define a first set of adjacent microwell subsets. The device may include a transparent substrate extending across the plurality of microwell sets of the fluid paths.

[0067] Each fluid passage of the plurality of fluid passages may have another first port at a first end as the first port, with one of the alternating first ports being at a first vertical position of the device and the other of the alternating first ports being at a second vertical position of the device spaced apart in the length dimension from the first vertical position.

[0068] The first port may be a substance input port and may be located at a first end of the fluid passageway. The device further includes a fluid manifold connecting opposite second ends of at least some of the fluid passageways with the second port.

[0069] The fluid passages can extend radially through the device, the inlet ports for the fluid passages can be located at the periphery of the device, and the second port can be a single second port located at the center of the device connected to each of the fluid passages.

[0070] At least some of the fluid passages may be provided as concentric sets of fluid passages each having an arcuate length segment.

[0071] The concentric fluid passage sets having arcuate length segments may be provided as a plurality of circumferentially spaced concentric fluid passage sets.

[0072] The microfluidic device may further include a dye homogenizing agent.

[0073] The dye-uniformizing agent may optionally include an oligonucleotide.

[0074] The dye-uniformizing agent can include oligonucleotides (eg, non-extending oligonucleotides and / or partially double-stranded DNA (eg, partially double-stranded DNA including biotin and / or a C1-C20 hydrocarbon chain)).

[0075] The dye uniformity agent can be present in a master mix present in the microfluidic device and / or the dye uniformity agent can be attached to beads present in the microfluidic device.

[0076] Other embodiments relate to bead loading strategies and the use of a common reagent reservoir / input with the segregated bead inputs shown and / or described.

[0077] Other embodiments relate to fully integrated fluid analysis chips for live (biological) sample input, sample preparation and processing, followed by loading of beads (pre-saturated with the sample) into the microwell array, and associated kits and microfluidic chips including pre-loaded reagents as shown and / or described.

[0078] It should be noted that one or more aspects or features described with respect to one embodiment may be incorporated into a different embodiment, even if not as specifically described in that embodiment. That is, all and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to modify the claims of the original application or to submit new claims as appropriate, including the right to amend the claims of the original application to depend on and / or incorporate features of other claims even if not claimed in the original application. These and other objects and / or aspects of the present invention are described in detail in the specification below. [Brief explanation of the drawings]

[0079] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. [Figure 1A] 1 is a schematic diagram of an example fluidic device according to an embodiment of the present invention. [Figure 1B] 1 is a schematic diagram of another example fluidic device according to an embodiment of the present invention. [Figure 2A] FIG. 1B is a top perspective view of the fluidic device shown in FIG. 1A. [Figure 2B] FIG. 2B is a digital photograph of a top perspective view of a prototype of a fluidic device corresponding to FIG. 2A. [Figure 3A] 1 is an example of a fluidic device including non-cylindrical microwells according to an embodiment of the present invention. [Figure 3B]3B is a highly enlarged view of a few microwells from a portion of the fluidic device shown in FIG. 3A, according to an embodiment of the present invention. [Figure 3C] 3B is an example test signal generated by a non-cylindrical microwell of the fluidic device shown in FIG. 3A, showing the test signal separate from the bead background signal, according to an embodiment of the present invention. [Figure 4A] 1 is a raw graph of fluorescence versus cycle for negative and positive beads. [Figure 4B] 10 is a graph of fluorescence versus cycle for positive and negative slits of a microwell according to an embodiment of the present invention. [Figure 5A] ~ [Figure 5H] 10 is a graph of raw data during a cycle obtained by combining real-time PCR data from different microwell subsets of different fluid paths during PCR according to an embodiment of the present invention. [Figure 6A] ~ [Figure 6H] 5A-5H are graphs of normalized data for the data shown in FIGS. 5A-5H in accordance with an embodiment of the present invention. [Figure 7A] ~ [Figure 7H] 10 is a graph of real-time PCR data collected from one row with different fluid path bead well sets according to an embodiment of the present invention. [Figure 8] 1 is an example of an analysis system according to an embodiment of the present invention. [Figure 9] ~ [Figure 16] 1A-1D are schematic top view illustrations of different example devices having fluid passageways of different configurations according to embodiments of the present invention. [Figure 17] 1 is a flowchart of an example analysis method according to an embodiment of the present invention. [Figure 18] 1 is a data processing system according to an embodiment of the present invention. [Figure 19] 1 is a flowchart of operations that may be performed for loading beads into a fluidic device and post-loading thermal cycling, according to an embodiment of the present invention. [Figure 20A] ~ [Figure 20D] 1 is an enlarged side perspective view of an example of a microchip loading operation using a magnet, according to an embodiment of the present invention. FIG. [Figure 21] FIG. 1 is a top view of multiple microchips held by an insulating holder according to an embodiment of the present invention. [Figure 22] 1 is a box plot of the initial bead fluorescence for each bead in five arrays consisting of one lane of two eight-channel microchips, one microchip treated with a master mix containing 10X SYBR and the other microchip treated with a master mix containing 20X SYBR and 5 μM non-extending oligo, according to an embodiment of the invention. [Figure 23] 1 is a graph of the average molecules per bead (digital positive signal) for Mycoplasma in a 12-plex respiratory panel test (related to the digital positive signal, but could be a combination of digital and analog). Each point represents a result from one of five arrays in a single lane of an 8-channel microchip, with beads from the same sample loaded into each array, according to an embodiment of the invention. [Figure 24] 10 is a box plot of coded dye fluorescent signals from the mycoplasma populations of a 12-plex respiratory panel associated with each bead of five arrays of a microchip treated with SYBR or +NE Oligo and SYBR, according to an embodiment of the invention. [Figure 25] 1 is a schematic of an example of pdsDNA on beads according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0081] Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. The abbreviations "FIG." and "Fig." may be used interchangeably in the text and figures to represent the word "Figure."

[0082] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, "comprises" and / or "comprising," when used herein, specify the presence of stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, "between about X and Y" means "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y".

[0083] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Furthermore, terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined as such. The terminology used to describe the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict of terminology, the present specification will control.

[0084] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted as alternatives ("or").

[0085] Unless the context dictates otherwise, it is expressly intended that the various features of the invention described herein be used in any combination. It is also contemplated that in some embodiments of the invention, any feature or combination of features presented herein may be excluded or omitted. For purposes of illustration, if the specification describes a composite comprising components A, B, and C, it is expressly intended that any or any combination of A, B, and C may be omitted and discarded.

[0086] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) shall be construed to include substances or steps recited in the claimed invention "that do not materially affect the basic and novel characteristics." See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original). See also MPEP § 2111.03. Therefore, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising."

[0087] As used herein, the words "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example and / or alternative embodiments described herein and are not intended to indicate preference for one or more embodiments described herein over one or more other embodiments.

[0088] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to encompass the specified value, as well as variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, "about X," where X is a measurable value, means to encompass X, as well as variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​may include other values ​​and / or individual values ​​therein.

[0089] As used herein, the terms "increase," "increases," "increased," "increasing," "enhance," and similar terms refer to an increase in the specified parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more, unless expressly stated otherwise within the context.

[0090] As used herein, "reduce," "reduces," "reduced," "reduction," "inhibit," and similar terms refer to a reduction in a specified parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%, unless expressly stated otherwise within the context.

[0091] When an element is referred to as being "on," "attached," "connected," "coupled," "contacting," etc., another element, it means that the element is directly on, attached to, connected to, coupled with, or in contact with the other element, or intervening elements may be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached," "directly connected," "directly coupled," or "directly contacting" another element, there are no intervening elements present. Those skilled in the art will also recognize that references to structures or features located "adjacent" to another feature can have portions above or below the adjacent feature.

[0092] Spatially relative terms such as "under," "below," "lower," "over," "upper," and the like may be used to facilitate the description of the relationship of one element or feature to another element or feature shown in the figures. It will be understood that spatially relative terms are intended to encompass various orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were inverted, an element described as "under" or "beneath" another element or feature would have an orientation "over" that other element or feature. Thus, the exemplary term "under" can encompass both an "over" and "under" orientation. The device may have other orientations (such as being rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein should be interpreted accordingly. Similarly, terms such as "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless otherwise specified.

[0093] Although terms such as "first" and "second" are used herein to describe various elements, it will be understood that these elements should not be limited to these terms. These terms may be used only to distinguish one feature from another. Thus, a "first" element below may be referred to as a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless otherwise specified.

[0094] Generally, embodiments of the present invention relate to analytical systems, fluidic devices, and methods of using the same. In some embodiments, the fluidic device may include a high-throughput fluidic device. The fluidic device 10 of the present invention may be used to acquire real-time and digital polymerase chain reaction (PCR) data from a coded bead microwell array 120a ( FIG. 3B ) and / or a set or subset of microwells 20 in different fluidic pathways 15. In some embodiments, the fluidic device 10, methods, and / or analytical systems of the present invention may be used in applications such as non-PCR reactions, loop-mediated isothermal amplification (LAMP), and / or enzymatic reactions for protein testing. As is well known to those skilled in the art, real-time PCR is defined as a polymerase chain reaction in which a signal related to amplicon concentration is collected after each cycle of PCR, and a cycle of PCR generally refers to a complete set of steps including denaturation of template DNA, annealing of primers to single-stranded DNA templates, and extension of the primers by a polymerase. See Arya et al., Expert Rev. Mol. Diagn., 5(2), (2005) pp. 209-219, 0.1586 / 14737159.5.2.2, the contents of which are incorporated herein by reference as if fully set forth.

[0095] In some embodiments, the designs and methods described herein can reduce data collection times for large arrays (e.g., coded bead arrays optionally containing about 30,000 or more microwells) while also mitigating the effects of photobleaching. The methods described herein are also applicable to other applications in which change signals are collected at various time points.

[0096] In some embodiments, the present invention includes, but is not limited to, substrates, devices, designs, solid supports (e.g., coded solid supports), steps, and / or methods described in U.S. Provisional Application No. 62 / 673,343, entitled "Compositions, Devices, and Methods for Improving Surface Properties of Substrates," U.S. Provisional Application No. 62 / 736,525, entitled "Compounds, Compositions, and Methods for Improving Testing," and / or described in U.S. Patent No. 9,617,589, U.S. Application Publication No. 2015 / 0211048, International Publication No. WO2017 / 112025, International Application No. PCT / US2016 / 042913, International Application No. PCT / US2016 / 043463, and / or International Application No. PCT / US2016 / 055407, the contents of each of which are incorporated herein by reference in their entirety.

[0097] In some embodiments, the methods of the invention include methods of using beads (e.g., superparamagnetic beads) to deliver reagents and / or targets to reaction wells, such as those described in U.S. Application Publication No. 2015 / 0211048 and International Application No. PCT / US2016 / 042913, the contents of each of which are incorporated herein by reference in their entireties.

[0098] The terms "microchip" and "microfluidic chip" are used interchangeably and refer to a substantially planar, thin device. Microfluidic chips can be rigid, semi-rigid, or flexible. The term "thin" refers to a thickness dimension that is 10 mm or less, such as between 10 mm and 0.1 mm, and can be about 3 mm, about 2.5 mm, about 2 mm, about 1.5 mm, about 1 mm, or about 0.5 mm. Microchips typically have widths and lengths smaller than about 6 inches, more typically between about 1 inch and 6 inches. However, in some embodiments, microchips can be larger in length and / or width, such as 1 foot or more. Microchips can have a perimeter that is polygonal, rectangular, circular, or any other desired shape. Microchips can optionally have a width dimension that is smaller than their length dimension. In some embodiments, microfluidic chips can have a diameter or width dimension that is about 2.13 inches (54 mm) and / or a diameter or width dimension that is about 3.4 inches (85.5 mm). Microchips can include millimeter-sized, micro-sized, and / or nano-sized fluid passages.

[0099] The term "primary dimension" refers to the width and / or depth dimension of a fluid passageway.

[0100] The terms "microsized" and "microfluidic" with respect to fluid passageways refer to fluid flow paths having widths and / or depths of millimeter, submillimeter, or smaller sizes (e.g., the terms include millimeter-, micrometer-, and nanometer-sized passageways, including segments or chambers provided in the passageways). A passageway may have at least a segment having a width and / or depth of 10 millimeters or less, typically in the size range of less than 900 microns and greater than 1 nm.

[0101] The term "microwell" refers to a reaction well sized and configured to have a small reaction volume of about 100 microliters or less, and in some embodiments, can optionally hold a single bead, as further described below.

[0102] The term "bead" refers to a solid phase member such as a particle, granule, or microsphere, typically a magnetic or superparamagnetic microsphere, which may be a porous, superficially porous, or non-porous material such as a polymer, photoresist, plastic, glass, silicon dioxide, metal or semi-metal oxide (including, but not limited to, aluminum oxide, titanium oxide, zirconium oxide, or other oxides), quantum dots, metal particles, or the like, suitable for use in a reaction well.

[0103] The term "circuit" refers to an entirely hardware embodiment or an embodiment combining software and hardware. The term "module" refers to an embodiment including software and hardware or firmware.

[0104] The term "digital scanning" and its derivatives generally refer to acquiring a digital image of part or all of a microfluidic device via a camera.

[0105] 1A, 1B, and 2, an example of a fluidic device 10 is shown. The fluidic device 10 includes at least one elongated fluid passage 15 with a plurality of spaced-apart microwell sets 20 located along the length of the elongated fluid passage 15. Adjacent microwell sets 20 on the fluid passage 15 can be, but need not be, separated by interstitial spaces 21 that do not contain microwells. FIG. 1B illustrates the microwell sets 20 arranged contiguously on the fluid passage 15. FIG. 1B also illustrates that adjacent ports of a first port 16 can be aligned other than vertically offset (in the orientation of the figure) as shown in FIG. 1A.

[0106] One or more microwell sets 20 on a given fluid pathway 15 may have different geometric footprints. When spaced microwells 20 are used, such as those shown in FIG. 1A, the interstitial space 21 may have a length that is less than the footprint length L of adjacent microwell sets 20.

[0107] 1A and 2, the at least one elongated fluid passage 15 includes eight adjacent fluid passages 151, 152, 153, 154, 155, 156, 157, and 158. However, more or fewer passages 15 may be provided, and more or fewer sets of microwells 20 may be provided for all or any one of the fluid passages 15. For example, in some particular embodiments, the fluidic device 10 may have between 2 and 2000 fluid passages 15, more typically between 10 and 200 fluid passages 15.

[0108] As shown in FIG. 1A, each or some of the fluid paths 15 can have multiple microwell sets 20, including a first microwell set 201, a second microwell set 202, a third microwell set 203, a fourth microwell set 204, and a fifth microwell set 205. However, more or fewer microwell sets 20 can be provided. FIG. 1B shows that each of the fluid paths 15 can have three microwell sets 20. In some embodiments, the fluid paths 15 of the fluidic device 10 can have between 2 and 100 microwell sets 20, more typically between 3 and 25 microwell sets 20.

[0109] The gaps 21 between adjacent microwell sets 20 on a given fluid path 15 need not be provided by physical gaps or interruptions in consecutive microwells 20, but may be defined by the field of view of an imaging system and / or the illumination window of an illumination source. As shown in FIG. 1B, the microwell sets 20 may be provided as a continuous array of microwells 20 spanning the length of the given fluid path 15.

[0110] Bubbles or constrictions for aligning beads may be provided in the fluid path 15. Bubbles or constrictions between adjacent sets of microwells 20 in a given array 120a of microwells 120 in the fluid path 15 may be provided to either control fluid movement / containment and facilitate or control bead loading.

[0111] 1A and 2, the plurality of microwell sets 20 of the fluid passageways 15 may be arranged as substantially parallel aligned rows labeled with location designations, such as alphanumeric designations for the rows, as shown by letter designations A-E for rows R1-R8, and optionally as aligned parallel passageways 15 labeled C1-C8, defining the microwell array 10a of the device 10. However, other designation formats may be used, and the device 10 does not require human-readable designations. More or fewer rows and more or fewer columns may be used. Neighboring passageways 15n, shown as pairs 15p of passageways 15, are spaced slightly apart and separated from other adjacent sets of neighboring passageways 15n, i.e., other pairs 15p of passageways 15, separated by interstitial spaces 19 provided by the substrate of the device 10. Neighboring passageways 15n may be separated by a distance less than the interstitial spaces 19 of other adjacent neighboring passageways 15n. Although the adjacent passages 15n are shown as being arranged as passage pairs 15p, for example, three, four, five, or more adjacent passages, including an entire row, may define a adjacent passage set 15n.

[0112] A microwell set 20 may have a footprint with a rectangular perimeter 20r that surrounds that set or array 120a of single bead microwells 120 (FIG. 3B), although other geometries may be used.

[0113] Each fluid passage 15 includes a separate, dedicated fluid first port 16, which may be an input port for introducing a desired input substance, optionally including a sample. Referring to Figures 2A and 2B, the fluid port 16 may include a reservoir 16r with a via 16v in the cover substrate 10u that directs the input substance from the reservoir 16r to the input port 16 of the fluid passage 15.

[0114] Each fluid passage 15 may merge at the end opposite the input port 16 into a second port 18, which may be a master mix / oil port 18. The second port 18 may also be connected to a reservoir 18r through a via 18v. The second port 18 is in fluid communication with one or more fluid passages 15. The device 10 may include a fluid manifold 18m in fluid communication with all or some of the fluid passages 15. Thus, some or all of the fluid passages 15 may merge into a common second port 18 through the manifold 18m or directly from a fluid passage extension 15e (FIGS. 14 and 15), so that a separate input port 16 for each fluid passage 15 and fewer second ports 18 than input ports 16 may be provided.

[0115] Although not shown, each fluid passage 15 may have its own independent second port 18. Different sets of fluid passages 15 may be in fluid communication with different corresponding master mix / oil ports 18 (FIG. 16). As described further below, when beads are loaded and sealed, each microwell set 20 of a corresponding fluid passage 15 is in fluid isolation from one another, and each fluid passage 15 is in fluid isolation from the other fluid passages 15.

[0116] 1A, 1B, and 2, for a given channel array 10a, the fluid channels 15 can be substantially parallel along linear length segments that include a plurality of spaced-apart microwell sets 20. The term "substantially" when referring to "parallel" means that the fluid channels 15 of that array 10a are parallel or nominally parallel (i.e., can vary in angle by no more than 10% from an adjacent centerline C / L passing through a given longitudinal center of the fluid channels 15) along at least a portion of the length segments, which are shown as linear length segments in FIGS. 1, 2, 9, 10-15 and as arcuate length segments in FIG. 16.

[0117] 3A-3C, each or some of the microwell sets 20 may include a plurality of microwells 120, each including a bead-retaining segment 120w and a test signal segment 120s. The test signal segment 120s is juxtaposed with the bead-retaining segment 120w and may be parallel and / or in-line with respect to the primary surface of the top or cover substrate 10u (FIG. 2A) of the device 10. The test signal segment 120s is in fluid communication with the bead-retaining segment 120w of the corresponding microwell 120. The test signal segment 120s may be beads The retention segment 120w generates a tapered test signal 122 that allows separation of the bead background signal. In some embodiments, the test signal segment 120s can have a geometry that is physically impenetrable to beads. The segments 120w, 120s are fluidly connected so that sample and / or analyte released from beads retained in segment 120w diffuse or otherwise mix throughout the common solution volume of the well 120w. By spatially separating the beads from the detection region 120s, the contribution of bead fluorescence to the signal is reduced or eliminated, improving the signal-to-noise ratio. The geometry of the well 120 allows for high loading of reaction wells, typically occupied by a single bead, while potentially increasing the reaction volume in question, improving reaction efficiency.

[0118] 9-11 and 16 show that the device 10 includes a plurality of spaced apart fluid passage arrays 10 a1 ,10 a2 ,10 a3 ,10 a4 11 illustrates that the fifth array 10 of fluid passages 15 may include a5 (Also shown) Figure 16 shows the circumferential extension of four arrays 10 for a set of 12 arrays. a1 ,10 a2 ,10 a3 ,10 a4 Illustrates three concentric sets of

[0119] Each array 10a of fluid passages 10 may have a single common second port 18 for introducing loading buffer, sealing oil, and / or master mix.

[0120] The term "master mix" refers to a PCR master mix that is added to fluid pathway 15 to reach each microwell set 20 before sealing wells 120 (FIG. 3B) together, which in some embodiments may be sealed using an immiscible oil as the sealing oil. Master mixes of the present invention may not contain primers; that is, PCR master mixes of the present invention may exclude primers for some specific embodiments, such as the SiRCA platform.

[0121] Alternatively or additionally, the upper substrate 10u (FIG. 2A) may comprise a flexible substrate such as silicone (e.g., polydimethylsiloxane (PDMS)), and sealing may be achieved by pressing the flexible substrate flat against the array 10a.

[0122] 16 illustrates that the fluid passages 15 are arcuate fluid passages 15a that extend circumferentially across at least a portion of the diameter of a circular geometry. a1 ~10 a4 are circumferentially spaced apart, and each array 10a may have a single second port 18 in common.

[0123] FIG. 11 shows the fluid passages 15 extending toward the center that define the location of the second port 18, and at least the inner leg 18i of the manifold 18m extends radially toward the second port to form the array A. R The centerline C / L extends radially through the device 10 toward the second port 18.

[0124] 12-15 show fluid passageways 15 configured as radially inwardly extending passageways 15r. Passageways 15r extend radially inward from the outer periphery of device 10, including first port 16, toward the center of the device to second port 18. FIGS. 14 and 15 show fluid passageways 15 that decrease in size along their length toward the center of device 10 to second port 18, with microwell sets 20 decreasing in size from a first set 201 to another set 202, 203, 204 as they approach second port 18. In this case, the microwell subsets 20 that are imaged simultaneously may be arranged in other configurations (e.g., other than a parallel rectangle).

[0125] The microchip array 10a may define an array site for each microwell set 20 in each fluid pathway 15 that corresponds to a positional address, such as a row and column address. For example, a row and its associated location, such as 1A (or A1), 2A (or A2), 7E (or E7), and 8E (or E8). The array 10a may be configured to provide some microwell sets 20 as corner microwells 20c. In the embodiment shown in FIGS. 1A and 2, the microwell sets 20 in array sites A1, A2, A7, A8, E1, E2, E7, and E8 are adjacent corner (sub)sets 20c of microwell sets 20. Again, two or more fluid pathways 15 may be configured to provide adjacent microwell sets that define corner sets 20c of microwells 20.

[0126] The device 10 shown in FIGS. 1A and 2 has a rectangular periphery 10p with a length dimension greater than a width dimension (“W”), optionally between 15 and 30 mm wide. The length dimension can be 2× to 4× the width dimension. The width dimension of the fluid passages 15 can correspond to the width dimension W of the device 10. The passages 15 are shown as extending along the length dimension of the device 10. However, the fluid passages 15 can alternatively be oriented to extend across at least a portion of the width dimension of the device 10. In some embodiments, some fluid passages 15 can extend along the length dimension and some along the width dimension (FIGS. 9 and 10). In some embodiments, the fluid passages 15 extend radially (FIGS. 11-14). In some embodiments, the fluid passages 15 extend circumferentially (FIG. 16).

[0127] The microwell sets 20 in each passage 15 may be aligned to have the same vertical and horizontal extents and positions, for example as shown in FIG. 1A, or may be staggered so that the first ends of the microwell sets 20 are located above or below the adjacent first ends of adjacent and at least partially horizontally aligned microwell sets of another fluid passage 15 (not shown).

[0128] Some or each microwell 120 (FIG. 3A) of a microwell set 20 of a given fluid pathway 15 may be provided as a dense array of 1,000 to 1,000,000 or more microwells 120, or more typically 1,000 to 100,000 or 1,000 to 50,000 microwells 120.

[0129] In some embodiments, the channels 15 of the microfluidic device 10 can analyze samples of interest in microwell sets 20, each comprising an array 120a of microwells 120, which can range from 1,000 to 1,000,000 or more, optionally ranging from 10,000 to 200,000. A microfluidic device 10 typically includes a plurality of fluid channels 15, ranging from 10 to 100, for a total of up to 20 million microwells 120 in the device 10.

[0130] In example embodiments, each passageway 15 may have 2-10 (or more) microwell sets 20 along its length in fluid isolation after sealing. A microwell set 20 may have any suitable number of microwells 120. In some embodiments, each microwell set 20 has the same number of microwells 120. In some embodiments, one or more microwell sets 20 (and in some embodiments, each set) may contain fewer than 12,000 microwells 120 (e.g., 1,000, 2,000, 5,000, 8,000, 10,000, or 12,000 microwells 120). In some embodiments, one or more microwell sets 20 (and in some embodiments, each set) have at least 1,000 microwells 120 (e.g., 1,000, 10,000 or more, e.g., 12,000 to 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,000,000,000 or more microwells 120). Each microwell 120 (FIG. 3B) typically holds a single bead (some may be unloaded, and some may have two loaded beads, which may be undesirable in some applications).

[0131] In some embodiments, microchip 10 can be configured to perform tests involving one or more beads in microwells 120. See, for example, examples of multi-bead tests described in 9,617,589 and PCT / US2016 / 042913 (also US2019 / 0054470), the contents of which are incorporated herein by reference as if fully set forth herein.

[0132] The different microwell sets 20 of a given passage 15 may have the same or different numbers of microwells 120 (FIG. 3B). The different microwell sets 20 of each passage 15 may have the same or different numbers of microwells 120 (FIG. 3B).

[0133] As shown by the virtual frame representing a single field of view (FOV) 25 at locations A1 and A2 in Figure 1A, device 10 may be configured such that a neighboring set 15n of passages 15, optionally a subset or sub-array 10s of the entire microwell array 10a, shown as a pair 15p, covers the entire single FOV of an optical signal detector (220, Figure 8), such as a camera. This allows at least one optical signal detector 220 (Figure 8), such as a camera, to image different subsets 10s of microwell sets 20, such as a single microwell array 120a, of multiple different sample passages 15 covered by the FOV 25 at any one time. The neighboring set 15n may be arranged as a subset or all passages 15 that include that microwell set 20 (e.g., part or an entire row).

[0134] As further described below, optical excitation source 225 (FIG. 8) can be configured to transmit excitation light in a defined wavelength range to a defined subarray 10s of the entire array 10a of microwell sets 20 of device 10 (FIGS. 1A, 1B), which is a subset of microwell sets 20 and includes at least one microwell set 20 in a plurality of adjacent passages of sample passage 15, but not all microwell sets 20 of microchip 10. The defined wavelength range can be associated with light and / or encoding fluorophores for, for example, a test signal.

[0135] As shown in FIG. 1A , a given subarray 10s may be associated with a single contiguous location, such as an entire single row of the entire microwell array 10a of the device 10. The given subarray 10s may be imaged or optically analyzed by sequentially imaging subsets of the microwell sets 20 in corresponding adjacent passages 15n of passages 15 at a single location, typically after a first inspection cycle. That is, the inspection may involve multiple inspection cycles associated with thermal cycling. The given subarray 10s that is excited and imaged may change after each inspection cycle, i.e., at the end of the first inspection cycle. In some embodiments, the analysis system 200 ( FIG. 8 ) only excites and images the subset 10s of the array 10a associated with the subset 20 of microwells in the first row R1 at the end of the first inspection cycle, and at the end of the second inspection cycle, the analysis system 200 only excites and images the subset 20 of microwells in the second row R2.

[0136] The device 10 may include upper and lower substrates 10u, 10b (FIGS. 2A and 2B) that are attached together. The upper substrate 10u may be the same as or different from the lower substrate 10b. Either or both of the substrates 10u, 10b may be rigid and may comprise, for example, glass, quartz, or a suitable metal. The cover substrate 10u may be optically transparent, typically transparent. Either or both of the substrates 10u, 10b may be a polymer, such as silicone or a polymeric material (PMMA, COC, COP, PDMS, PP, PE, PTFE, Kapton (polyamide), and many others), and may comprise one or more microwell arrays 10a. In some embodiments, either or both of the substrates 10u, 10b may comprise silicon (e.g., silicon wafers) and / or may be functionalized (e.g., silanized) with hydrophobic compounds, such as alkylsilanes and / or alkylthiols. In some embodiments, either or both of the substrates 10u, 10b may comprise silicon that has been silanized with alkylsilanes.

[0137] A fluid passageway 15 comprising a plurality of spaced-apart microwell sets 20, with each microwell set 20 becoming a corresponding microwell array 120a, has sidewalls and a floor formed in one or more substrates 10u, 10b such that the sidewalls extend therebetween, with an open top surface and a closed bottom surface. One or more spacers, top substrates, membranes, or covers may be used. The top substrates, membranes, or covers may seal, cover, or otherwise close the top surface of the fluid passageway and / or the array of reaction wells. In some embodiments, the passageway 15 is etched into the top substrate 10u, with the bottom becoming a microwell set 20 and forming the closed surface of the passageway 15.

[0138] The analyte at the substance input can be any analyte of interest, including various mixtures containing, for example, synthetic and biological polymers, nanoparticles, small molecules, DNA, nucleic acids / polynucleic acids, peptides, proteins, etc. The analyte can be one or more analyte molecules.

[0139] Input materials include samples or sample analytes, and may include one or more polar metabolites such as amino acids, charged molecules, molecules, peptides, and proteins. Samples and / or analytes may also, or alternatively, include molecules extracted from biological fluids, blood, serum, urine, dried blood, cell growth medium, lysed cells, beverages, or food. Samples may also, or alternatively, include environmental samples such as water, air, and soil.

[0140] The term "oligonucleotide" refers to a nucleic acid sequence of at least about 5 nucleotides to about 500 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 nucleotides). In some embodiments, for example, an oligonucleotide can be about 15 to about 50 nucleotides, or about 20 to about 25 nucleotides, which can be used, for example, as a primer in a polymerase chain reaction (PCR) amplification assay and / or as a probe in a hybridization assay or microarray. Oligonucleotides of the invention can be natural or synthetic, e.g., DNA, RNA, PNA, LNA, modified backbones, etc., or any combination known in the art. Oligonucleotides of the invention can be single-stranded, double-stranded, or partially double-stranded. In some embodiments, the oligonucleotides are non-extendable (e.g., by PCR).

[0141] Probes and primers, including those for either amplification and / or detection, can be oligonucleotides of any suitable length (DNA and naturally occurring oligonucleotides, such as synthetic and / or modified oligonucleotides), but can generally range from 5, 6, or 8 nucleotides in length to 40, 50, 60 or more nucleotides in length. Such probes or primers can be immobilized or attached to a solid support, such as a bead, chip, pin, or microtiter plate well, and / or can be conjugated to or labeled with a detectable base, such as a fluorescent compound, a chemiluminescent compound, a radioactive element, or an enzyme.

[0142] Polymerase chain reaction (PCR) can be performed according to well-known techniques. See, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188. Generally, PCR involves first treating a nucleic acid sample (e.g., in the presence of a thermostable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridization conditions to synthesize extension products of each primer complementary to each nucleic acid strand, where the primers are sufficiently complementary to each strand of the hybridized specific sequence that the extension products synthesized from each primer, when separated from their complements, serve as templates for the synthesis of extension products of the other primer; and then treating the sample under denaturing conditions to separate the primer extension products from their templates, if present, for the sequence or sequences to be detected. These steps are repeated cyclically until the desired degree of amplification is achieved. Detection of amplified sequences can be achieved by adding a detectably labeled oligonucleotide probe (e.g., an oligonucleotide probe of the present invention) capable of hybridizing to the reaction product to the reaction product and then detecting the label according to well-known techniques, or by direct visualization on a gel. Amplified sequences can also be detected by adding an intercalating dye to the reaction mixture and monitoring the fluorescent signal intensity, which is proportional to the total mass of double-stranded DNA. Other dyes that are non-intercalating but produce a high fluorescent signal in the presence of dsDNA can also be used. While embodiments in accordance with the present invention have been described with reference to PCR reactions, it should be understood that other nucleic acid amplification methods, such as reverse transcription PCR (RT-PCR), including isothermal amplification techniques such as rolling circle amplification or loop-mediated isothermal amplification (LAMP), can be used, as can nucleic acid sequencing methods. Additionally, other processes, such as enzyme amplification reactions like enzyme-linked immunosorbent assays (ELISAs), can be used. In such cases, microwell temperature control can be used to control the amplification reaction and optimize imaging of the microwell array, with some areas imaged only before and after the complete reaction (digital signal), while other areas or subarrays can be imaged during the reaction to obtain analog signals.

[0143] Such DNA amplification techniques require the use of a single probe, a pair or set of three, four, five, six, or more probes, or a pair of two probes that specifically bind to DNA containing the polymorphism or mutation of interest but do not bind strongly to DNA not containing the polymorphism of interest under the same hybridization conditions, and that function as one or more primers for amplifying the DNA or a portion thereof in an amplification reaction. Such probes are sometimes referred to herein as amplification probes or primers. In some embodiments, more than one probe may be used, such as in a competitive process for the detection of single nucleotide polymorphisms (SNPs) and / or other mutations and / or rare alleles such as indels.

[0144] The term "reagent" refers to substances or compounds, including primers, nucleic acid templates, and amplification enzymes, that are added to a system to initiate a chemical reaction or to determine whether a reaction is occurring. Amplification reagent(s) generally refers to those reagents (deoxyribonucleotide triphosphates, buffers, etc.) used in amplification, excluding primers, nucleic acid templates, and amplification enzymes. Amplification reagents, along with other reaction components, are typically placed and contained in a reaction vessel (test tube, microwell, etc.).

[0145] As used herein, the term "magnetic" includes ferromagnetic, paramagnetic, and superparamagnetic properties.

[0146] Generally, the oligonucleotide probes used to detect DNA containing a polymorphism or mutation of interest are those that bind to DNA encoding the mutation or polymorphism but do not bind to DNA that does not contain the mutation or polymorphism under the same hybridization conditions. The oligonucleotide probes are labeled with an appropriate detectable base, such as those listed below. Such probes are sometimes referred to herein as detection probes or primers.

[0147] Embodiments of the invention can be used for singleplex reactions in compact arrays (SiRCA), a platform for highly sensitive and highly multiplexed nucleic acid (NA) and protein quantification. SiRCA combines sample preparation with digital PCR precision in a massively parallel, highly multiplexed format. NA-SiRCA is a magnetic bead-based PCR variant in which test panels are fabricated from sets of microbeads uniquely coded with fluorescent dyes. Each set is functionalized with a different primer pair for a specific target NA before the sets are combined. During testing, the bead primers hybridize to the NA target, allowing them to be captured, enriched, and purified from the sample matrix. The beads are washed and loaded into microwell sets 20, each containing an array 120a containing thousands of microwells 120, with one bead typically occupying one bead well 120w. The microwells 120 can have any suitable volume. In some embodiments, the microwells 120 have a volume ranging from about 10 femtoliters to less than about 10 microliters, such as from about 10 femtoliters to less than about 10 microliters, or from about 10, 50, 100 femtoliters to about 200, 500, 1,000 femtoliters, hi some embodiments, the microwells 120 have a volume of about 100 femtoliters.

[0148] A PCR master mix (containing all reagents except primers) is added before sealing the wells together using immiscible oil. Upon heating, the beads release the primer set and captured target, forming a singleplex PCR in each microwell 120. Thousands of spatially multiplexed PCRs are rapidly generated without interference between primer sets. Signal from the dsDNA intercalating dye indicates target amplification, and target identity is determined from the bead coding. At low concentrations, single-molecule counting (digital signal) enables precise analyte quantification. At high concentrations, real-time PCR (analog signal), measured as the average of the fluorescent signal from all wells of the same reaction type, extends the quantification range above the digital signal saturation point.

[0149] The use of non-cylindrical microwell geometries can improve detection in microbead array-based technologies. These well geometries are designed so that one area of ​​the well is optimized for magnetic loading and bead retention, while another area of ​​the well is used for signal detection. After loading the beads into the bead region of the well, a small volume of reagent fluid is isolated within the well using methods such as immiscible fluid sealing. An example of a single array chip is shown in FIG. 3A, including an inset showing the microwells 120. Other embodiments may include design variations. In certain embodiments, one area includes a pocket or reservoir 120w (FIG. 3B) with a diameter of about 100 to about 150% of the bead diameter and a depth of about 50 to about 185% of the bead diameter, and a fluidic connection region consisting of a narrow pocket or slit 120s (FIG. 3B) or other geometry into which standard beads do not physically fit. Both regions are fluidically connected so that reagents or analytes released from the beads diffuse or otherwise mix within a common solution volume. By spatially separating the beads from the detection region, the contribution of bead background fluorescence to the signal is reduced or eliminated, improving the signal-to-noise ratio (Figure 4B). Additionally, these geometries allow for highly single-occupancy loading of the reaction wells while increasing their reaction volume, potentially improving reaction efficiency.

[0150] NA-SiRCA is performed using a microtube format and / or bead containing microwell array 120a on chip 10, allowing for more than about 35,000 reactions, such as up to about 1 billion reactions.

[0151] We developed a 12-plex respiratory panel using synthetic targets. Excellent linearity was observed using digital and analog synthetic signals across the entire range from 10 copies / μL to 10,000,000 copies / μL. Digital quantification of low concentrations (50-150 copies / μL) was displayed with high accuracy and low variability, allowing discrimination between subtle copy number variations. We demonstrated that RNA can be tested using reverse transcription. Additional tests in development include multiplex protein tests (cytokines) using immuno-PCR with low pg / mL LODs.

[0152] Generally, digital testing does not require imaging of the test signal after each amplification cycle. However, analog testing relies on detection of the threshold cycle (Ct) to determine how many NA molecules are initially bound to the beads. Ct is generally determined as the first cycle in which the signal is significantly above (5-10% or more) the background signal. The difference in Ct can be used to determine the initial concentration of the sample. 100% amplification efficiency of a PCR reaction is achieved by

number

[0153] The resolution of concentration measurements (i.e., the precision with which concentration is determined) depends on how frequently fluorescence is measured. Imaging after every amplification cycle (i.e., each approximately equal to twice the amplicon concentration) provides the greatest resolution. However, frequent imaging results in photobleaching of the dye in a manner that depends on the dye's exposure to the excitation source. This is particularly important for small PCR reactions used in SiRCA. Figure 4A shows a trace of a real-time PCR plot in which the effect of photobleaching is evident by the negative slope of the baseline signal.

[0154] Microfluidic devices of the invention can accommodate one or more (e.g., 1, 2, 3, 4, or more) samples to be tested, optionally in high-throughput applications and / or procedures. Microfluidic devices of the invention can have a single substrate including one or more arrays 10a with multiple microwell sets 20 including associated microwell arrays 120a (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more). In some embodiments, two or more fluid pathways 15, optionally provided as a single array 10a or multiple arrays 10a (the latter illustrated in Figures 9-11 and 16), are incorporated into the substrate to form an integrated multi-sample device 10 for high-throughput applications. In some embodiments, one or more microwell sets 20 in multiple fluid pathways 15 of one or more arrays 10a of the microfluidic device 10 are simultaneously thermal cycled.

[0155] This form of digital PCR can be achieved because the array is imaged once before PCR and once after PCR to obtain digital PCR data. In some embodiments, the array (e.g., an array containing about 40,000 wells) is imaged with a sensor having a pixel density high enough that light from one well is collected by at least one pixel. In some embodiments, each microwell 120 in a device of the invention can be imaged with about 4 to about 20 pixels, or about 4 to about 100, 150, or 200 pixels. In some embodiments, the systems, devices, and / or methods of the invention result in one or more aligned or partially aligned microwell sets 20 in two or more adjacent fluid paths 15 being imaged using a single sensor, without custom optics and / or precision alignment.

[0156] An alternative to custom optics is the use of a precision positioning stage that translates either the camera and / or the microfluidic chip horizontally, vertically, and / or rotationally so that arrays can be imaged sequentially after the extension step of each PCR cycle. This approach allows for imaging of many arrays at the expense of longer test times. For channels 15 with as many as 30,000 to 40,000 or more microwell sets 20, such as 62.5k microwells 120 per sample channel 15, translation and imaging typically adds about 1 to 10 seconds to each cycle for each additional array, although costly high-performance hardware can reduce this time. Increasing the imaging time effectively increases the extension time of the PCR cycle. While PCR reaction kinetics is cycle-dependent, not time-dependent, the long thermal cycle delays required to image many arrays are problematic because active polymerases tend to write dimers and nonspecific products if given sufficient time to do so. This leads to increased background signal and reduced test specificity.

[0157] Some embodiments of the present invention provide solutions to these imaging and / or data collection problems, such as by utilizing unique features of coded microbead microwell array 120a (FIG. 3B) according to embodiments of the present invention.

[0158] The properties of coded bead arrays enable a unique approach to analog PCR data collection and analysis. When the concentration of target molecules is in the analog region, the average number of molecules of each bead type is approximately the same. Therefore, the Ct of each bead set is independent of the bead position on the array, and different areas of the array can be imaged at different PCR cycles while still achieving single-cycle resolution. That is, it is not necessary to image the entire array after each PCR cycle; only a representative portion of the array needs to be imaged. In some embodiments, instead of using a typical roughly rectangular array, a long array can be used so that the arrays from each sample are positioned in close proximity. In this way, the camera can image two or more sample arrays at once, and many arrays in a short time, without having to translate along complex paths over long distances.

[0159] Using the layout of device 10 shown in FIG. 1A, one implementation of this method can be illustrated by the following example. In this design, fluid paths 15 are grouped into sets (shown as pairs 15p) such that a subset of an array 10s of microwell sets 20, each comprising relevant wells 120 (FIG. 3B) from two or more fluid paths 15 (optionally two or more different samples), is imaged by the camera in a single frame (in FIGS. 1A and 1B, the camera's field of view is marked by frame boxes 25 labeled 1A and 2A). In this example, a representative portion of wells 120w from each microwell set 20 in one row R1 are imaged across the width of the device in only four captures: A1 (also interchangeably referred to as "1A"), A2 (also interchangeably referred to as "2A"), and A3 (also interchangeably referred to as "3A"), A4 (also interchangeably referred to as "4A"), and A5 (also interchangeably referred to as "5A"), A6 (also interchangeably referred to as "6A"), and A7 (also interchangeably referred to as "7A"). 6A "), then A7 (also referred to interchangeably as "7A"), and A8 (also referred to interchangeably as "8A"), with only a short lateral translation between frames. Thus, in some embodiments, methods of the present invention involve collecting analog data without imaging the entire array 10a of different microwell sets 20.

[0160] 17 is a flowchart illustrating an example method for identifying a target species and / or target molecule. A fluid analysis device is provided having a first fluid path including a plurality of microwell sets spaced apart along the first fluid path (Block 600). An optical excitation signal is (optionally) transmitted to only a predetermined subset of the plurality of microwell sets (Block 610). Signal intensity data is (optionally) obtained from only the predetermined subset of the plurality of microwell sets in response to the optical excitation signal (Block 620). Bead types that are positive for the target species and / or target molecule are identified based at least in part on the signal intensity data (Block 630). However, in some embodiments, optical excitation is not required to obtain a signal. For example, in some embodiments, a test (e.g., a protein test) can generate a chemiluminescent signal that can be detected and / or imaged without optical excitation.

[0161] The method may optionally include loading and sealing a plurality of microwell sets on the first fluid path prior to the transferring step, such that each microwell set is fluidically isolated from one another after sealing. The plurality of microwell sets on the first fluid path are in fluid communication only during loading prior to the sealing step (block 602).

[0162] Optionally, the method may include changing a defined subset of the plurality of microwell sets to a different defined subset after each of a plurality of sequential reaction steps of the test (e.g., after each of a plurality of sequential thermal cycles of the test) so that some microwell sets are not imaged after each reaction step (block 612).

[0163] Optionally, the defined subset remains the same across multiple successive reaction steps of the test (e.g., after each of multiple successive thermal cycles of the test), such that some sets of microwells are not imaged after each reaction step (block 614).

[0164] Optionally, the method includes digitally scanning a defined subset of the plurality of sets of microwells after, before, or after the transmitting and acquiring steps, and electronically identifying wells that are positive for one or more target analyte molecules while the array of wells is at the imaging temperature (block 624).

[0165] Optionally, acquisition of signal intensities from only a defined subset can be performed by sequentially acquiring images of the subset occupying a single physical portion of the microchip 10, for example, by sequentially acquiring data from different single rows among the first, second, third, fourth or more rows, using a camera having a field of view (FOV) covering only a subset of the microwell set 20 of the sample passage 15 in question and only a subset of one or more nearby adjacent fluid passages 15, for example, 2 to 10 or more adjacent fluid passages among the first, second, third, fourth or more fluid passages (block 622).

[0166] Optionally, acquiring signal intensities includes acquiring analog signals. The analog signals can provide real-time PCR data as amplitude change of input material in a set of microwells in the fluid path versus PCR cycle number, where the concentration of target molecules of one or more defined bead types is about, equal to, or greater than one molecule per microwell. The analog signals define a threshold cycle or cycle threshold Ct that identifies microwell reactions as positive when the fluorescent signal is greater than the threshold or negative when the fluorescent signal is consistently less than the threshold for a given number of PCR cycles, where Ct is the cycle number where Si >> Bs, where >> is at least 5-10% greater than Bs, optionally 2-fold greater, and / or 5-10 times the standard deviation of Bs, Si is the fluorescent signal intensity, and Bs is the background fluorescent signal measured in negative PCR reactions (block 626). Ct can be compared to a reference reaction to determine the number of molecules at the beginning of the PCR reaction.

[0167] Optionally, obtaining signal intensities includes providing real-time PCR data as amplitude changes with PCR cycle number of input substance tests in a set of microwells in the fluid pathway, and obtaining an analog signal that can determine whether a PCR reaction is positive for the target molecule when Si>>Bs is at least 10% greater than Bs, optionally 2-fold greater, and / or 5-10 times the standard deviation of Bs. Si can be calculated as the mean, median, mode, or weighted value of the analog signals corresponding to each defined bead type. In some embodiments, Si can be calculated by determining the mean or median after removing outliers (typically in early PCR cycles).

[0168] In some embodiments, referring to Figures 19 and 20A-20D, loading of beads into device 10 can be performed as follows: Device 10 is wetted with loading buffer and placed in a holder 900 (Figure 21) coupled to or cooperating with a linear magnet or array of magnets 800 (block 700). To allow for bead loading with minimal risk of inter-passage contamination, vias 16v of input port 16 can optionally be positionally offset in two adjacent rows (block 702). With magnets 800 fixed under odd-numbered passages (Figure 20A), bead slurry from the material input is pipetted into vias 16v (block 710). Magnets 800 are then moved so that they are positioned under even-numbered vias (Figure 20B), attracting beads into odd-numbered passages (block 720). Of course, Figures 20A The steps shown in Figure 20B can be performed in reverse order. After adding the bead slurry to the even vias, a magnet 800 is used to attract the beads to the microwells (array chambers) (Figure 20C) (block 730). Beads are loaded into the microwell sets (array segments) by sliding the magnet under each array set 20 along the fluid path 15 into the manifold passages and / or master mix port 18, with the movement optionally controlled by a rigid stop device 810 (Figure 20C) so that the beads do not enter the common manifold passage (which allows for mixing of beads between samples) (block 740).

[0169] Each passage 15 is provided with an optionally physically spaced segmented set of microwells 20, shown in FIG. 1A as five microwell sets 201-205 (each with 12.5k wells) containing a total of thousands or millions of microwells 120, optionally approximately 62.5k microwells 120, for each material input or fluid passage 15 (block 704).

[0170] After the beads are loaded into the microwells 120 (FIG. 3B) of each microwell set 20 in the fluidic pathway 15, the remaining beads are magnetically drawn back toward the vias 16v by translating the magnet 800 toward the via 16v, the input port (FIG. 20D) (block 750). The master mix flows under pressure applied to the common reservoir 18 until it fills the pathway 15 (block 760). It is then pumped under pressure until the sealing oil seals the microwell sets 20 and expels excess master mix from the pathway 15 (block 770).

[0171] Optionally, an absorbent pad may be placed over the reservoir 16r and / or via 16v to collect the aqueous solution as it is discharged from the device 10, or a partial vacuum may be used to remove the liquid as it leaks from the via 16v and / or reservoir 16r (FIG. 2A) (block 772).

[0172] Optionally, a second microfluidic channel, channel network, or reservoir can be used to accumulate waste fluid instead of allowing it to leak through the via. Such waste reservoirs, either as microfluidic and optionally as a separate reservoir or set of reservoirs, can be fluidly connected to the channels and / or vias by wax or paraffin valves or hydrophobic constrictions in a manner that prevents filling before the master mix addition and / or sealing steps. In some embodiments, vias can be sealed with a membrane after bead addition, such that the membrane prevents the flow of water and / or oil and optionally allows air to escape through the membrane. In some embodiments, the membrane can cover other vias or ports to act as a path for air to escape from the microfluidic channel, chamber, or via.

[0173] The device 10 is then placed on the thermal cycling microscope stage of inspection system 200h (FIG. 8) for thermal cycling, PCR, and imaging (block 780).

[0174] The array 10a of microwell sets 20 of a fluid path 15 is divided into subarrays, A through E, referred to here by row and fluid path number (i.e., A1 is the subset of microwells 201 in the upper left corner of the device, E8 is the subset of microwells 205 found in the lower right corner of the device, etc.), and A1-A2 can be imaged in a single image capture.

[0175] Data collection and processing method example 1 ("Method 1") The camera (222, FIG. 8) is aligned and focused on subsets of microwell sets 20, first at positions A1-A2, A7-A8, E7-E8, and then E1-E2. The locations of these corners 20c of the subsets of microwells 20 in X, Y, and Z (focus height) can be used to calculate the positions of all the inner microwell sets 20. Focusing is performed while the array 10a is held at the imaging temperature (typically an expanded temperature of about 60°C to about 72°C) to eliminate dimensional changes due to thermal expansion. Every microwell set 20 in each passage is then imaged at the imaging temperature prior to PCR thermal cycling to obtain a baseline "pre-PCR" image.

[0176] After the first PCR cycle, a subset of the microwell sets 20 in the first row (A1-A2, A3-A4, A5-A6, A7-A8) are imaged sequentially for a total of four frames. After the second PCR cycle, the second row is imaged (B1-B2, B3-B4, B5-B6, B7-B8). After imaging row E after the fifth PCR cycle, the sequence is repeated, and row A is imaged after the sixth PCR cycle. If this process continues for 30 PCR cycles, each row will be imaged six times. To reduce the time required for stage translation, the stage will move to the site of the next row while a denaturation step is performed before the next annealing / extension / imaging step is performed.

[0177] After thermal cycling is complete, a "post-PCR" image is taken of the entire array 10a at the imaging temperature. The array 10a is cooled (typically to about 20°C to about 25°C), and another set of post-PCR images is taken (to identify problem areas on the chip where the aqueous master mix has contacted two or more wells due to a defective sealing technique). If desired, the array 10a is imaged at a series of temperatures to determine the melting point and / or range of amplicons detected after PCR cycling. Filter sets (F1, F2, Figure 8) are swapped to match the encoding wavelengths, and a first set of encoded images is acquired for each subset of the microwell sets 20, followed by a second set of encoded images using a second filter set, if necessary, and repeated as needed to decode the bead sets.

[0178] Processing of imaging data can be divided into two areas: digital and analog. Digital PCR signals can be determined by comparing pre- and post-PCR images to determine whether the increase in intercalating dye signal exceeds a set threshold for each well. In some embodiments, only the post-PCR image is required to determine a digital positive or negative result. Alternatively, the pre- and post-PCR images, optionally along with six thermal cycle images, can be used to determine whether the fluorescent signal changed during the imaging process in a manner consistent with PCR amplification of the correct amplicon. For example, for low-concentration targets where some portion of the target PCR reaction population is negative, an increase in signal in later cycles is expected, and outliers showing strong signals early in cycling can be considered nonspecific amplification products or misidentified beads.

[0179] For high-concentration targets where most or all PCR reactions are positive, real-time analog signals are used. Signals from each subset of microwells 20 in array 10a have a five-cycle resolution (each subarray is imaged once every five PCR cycles), with at least one subset of microwells 20 in passage 15 of array 10a for each sample imaged after each PCR cycle. Because target molecules should be equally distributed across a given bead population, it is possible to combine data from images to reconstruct a single-cycle resolution Ct curve, even if the same wells are not imaged after every cycle. This is achieved by averaging the signal intensities from each bead set for each sample in each image. The average signal for each bead type can then be displayed as a real-time PCR curve, as shown in Figures 5A-5H. As seen in Figure 5A, the real-time signal used for cycle 1 is obtained from the first image of microwell 20 at position 1A, while data for cycles 2, 3, 4, and 5 are obtained from the first images of subarrays 1B, 1C, 1D, and 1E. In cycle 6, data from the second image of subarray 1A is used, and in cycle 11, data from the third image of subarray 1A is used. This process is used for all eight fluidic channels 15 of each chip 10 (each channel having the same or a different sample, i.e., eight samples) to construct a real-time PCR curve.

[0180] As can be seen in Figures 5A-5H, the fluorescence intensity baseline is not completely uniform for a given fluidic pathway 15 and / or sample subarray (subset of microwells 20). This is due to several factors, including the effective concentration of the intercalating dye, the focus of the image, and / or the effect of different levels of silanization on the device. If the PCR image signal is normalized by dividing by the pre-PCR image signal (Figures 6A-6H), the baseline will be more linear and the cycle threshold call may be more repeatable. Irregularities in the maximum PCR signal should have a weaker effect on target quantification. We now discuss protocols that help control the dye concentration and therefore the intensity of the fluorescent signal across all arrays.

[0181] Data collection and processing method example 2 ("Method 2") In some embodiments, data collection may be performed by imaging only a set of subarrays (a subset of microwell sets 20) for every PCR cycle, in this non-limiting example, 30 cycles. In this approach, the camera is aligned and focused using the corner subarray sites as described in Example Method 1 above. Every microwell set 20 (i.e., every subarray) is then imaged at the imaging temperature prior to PCR thermal cycling to obtain a baseline "pre-PCR" image.

[0182] After the first PCR cycle, a different microwell set 20 in one row of the microwell set 20 (subarray), e.g., row C, is imaged. Microwell sets 20 in positions C1-C2, C3-C4, C5-C6, and C7-C8 are imaged sequentially for a total of four frames. After the second PCR cycle, the same row is imaged again. This process continues for 30 PCR cycles; in this example, the same row would be imaged 30 times for 30 PCR cycles, as seen in Figures 7A-7H.

[0183] After thermal cycling, a "post-PCR" image is acquired for the entire array at the imaging temperature. The array is cooled (typically to about 20°C to about 25°C) and another set of post-PCR images is acquired. If desired, the array can be imaged at a range of temperatures to determine the melting point or range of amplicons detected after PCR cycling. The filter sets (F1, F2, Figure 8) are swapped and a first set of coded images is acquired for each subarray, followed by a second set of coded images using the second filter set.

[0184] As with Example Method 1, the processing of image data can be divided into two areas: digital and real-time (analog). For this method, the digital PCR signal can be determined simply by comparing the pre-PCR and post-PCR images to determine whether the increase in intercalating dye signal exceeds a set threshold for each well (Table 1). In some embodiments, the digital signal can be obtained from the post-PCR signal alone. [Table 1] Table 1: Data were collected using a combination of real-time and digital processing as described in Method 2. Specifically, row C was collected as a real-time trace, while the other regions were analyzed digitally. Each sample contained 5,000 copies / µL of rhinovirus and influenza A synthetic DNA sequences spiked into different sample passages. Values ​​in the table represent percent positives for the corresponding coded bead set.

[0185] Microwell sets 20 (i.e., subarrays) that were not imaged during PCR may have very little photobleaching, whereas imaged subarrays may exhibit significant photobleaching. Thus, in some embodiments, different thresholds are required to distinguish digital positives from digital negatives.

[0186] Analog signal processing of imaged subarrays is similar to traditional whole-array imaging methods. Signals are averaged for each bead population and plotted against PCR cycle number (Figures 7A-7H). For analog signals with more than one copy of the target molecule per bead, the average signal from several hundred beads should be sufficient for accurate and precise Ct determination.

[0187] Methods of the invention, such as those described above in Example Methods 1 and 2, can have advantages over typical data collection protocols in which every well of an array is imaged every PCR cycle. In some embodiments, methods of the invention reduce data collection time while generating single-cycle real-time PCR resolution for analog quantification. The compact, narrow well array design allows for imaging of fluid separation arrays from multiple samples in a single image.

[0188] Advantages of collecting analog data across multiple subarrays (as described with respect to Example Method 1) can include reduced photobleaching. For example, significant photobleaching reduction occurs in Example Method 1 because each subarray is exposed to only 20% of the irradiance used in standard collection protocols. Data collected over several cycles, even at low cycle resolution, is more reliable for identifying false-positive beads for digital quantification than comparison of only pre- and post-PCR images. Additionally, the reduced degree of photobleaching allows for adequate signal to be obtained with lower concentrations of intercalating dye (e.g., SYBR Green I) than are required to image all 30 cycles. Because intercalating dyes can inhibit PCR, lower concentrations may improve determination of PCR efficiency or melting temperature for some targets.

[0189] Advantages of collecting analog data from a single row of microwell set 20 (i.e., a subarray or subset of microwells as described with respect to Example Method 2) may include less reliance on data normalization for Ct determination, making analysis simpler. Additionally, pre-PCR images may be used to determine which rows have optimal bead loading for analog real-time PCR analysis. This may help ameliorate problems that can arise when one or a few subarrays do not have enough beads loaded for analog analysis (although this problem may be addressed by other means, such as strict control over array size, geometry, location, bead population size, and bead size).

[0190] Example methods 1 and 2 are merely illustrative examples of device designs and data collection methods of the present invention, and various combinations and / or adjustments may be used. For example, single rows of microwell sets 20 in different fluidic pathways 15 may be imaged every cycle or every other cycle in combination with imaging of other subarrays at various different intervals. Some combinations require more acquisition time, but in some embodiments, the improved quality of the digital or analog data may be worth such a trade-off. In some embodiments, all or most of the microwell sets 20 may be imaged in several PCR cycles (generally not every and / or every PCR cycle).

[0191] The illustrated device 10 is merely illustrative of a design concept (i.e., division of a bead / reaction well array into a series of subarrays spaced and positioned so that they can be imaged in an advantageous manner), and other designs may be used in the practice of the invention. In some embodiments, a device 10 of the invention may include one or more arrays 10a of fluid passages 15, such as 8 or more fluid passages 15, such as 9-1000 fluid passages 15 per device 10, including 16, 32, 64, 96, 384, etc., fluid passages 15 containing microwell sets 20 of interest. In some embodiments, a device 10 of the invention may be fabricated from silicon, glass, or polymers (e.g., injection-molded and / or hot-embossed plastic) and / or metal films. In some embodiments, a device 10 of the invention may be a composite structure. In some embodiments, a device 10 of the invention may contain only a bead / reaction well array and an array chamber, or may include other structures, such as sample processing microfluidic circuitry and / or labeling or amplification reagents, necessary to perform a test.

[0192] Optionally, electrical and / or mechanical circuits, actuators, and / or sensors may be integrated, mounted, and / or incorporated into and / or on device 10 for the performance and / or reading of tests.

[0193] Polystyrene beads can absorb both hydrophobic and charged molecules from aqueous solutions. The long, thin geometry of the microarray 20 results in the depletion of certain components, such as intercalating dyes, from the master mix as it flows through the channel 15. The beads in the first subarrays that meet the master mix absorb a large amount of the component (e.g., intercalating dye), while the final arrays absorb less from the partially depleted solution. When the component is a dye, this can result in a gradient in dye concentration and therefore initial bead fluorescence at the chip compared to the channel array (Figure 20). These differences in dye absorption introduce some complications into testing, as too much dye can inhibit PCR and too little dye results in a weak amplification signal. Additionally, variations in intercalating dye concentration can cause undesirable variations in the apparent fluorescence intensity of the coding dye, which can make array interpretation more difficult. This effect may be one of the reasons for the low signal intensity in some subarrays observed in Figures 5 and 6.

[0194] Dye absorption by the beads can be tailored by including (e.g., adding) various reagents that result in more uniform dye (e.g., SYBR) absorption / distribution across the array. These reagents (also called "dye uniformity agents") can include, but are not limited to, single-stranded DNA, RNA, double-stranded DNA (optionally with a low melting temperature so that the reagent is double-stranded during addition to the channel and sealing of the well, but single-stranded during the imaging step of PCR and does not associate with the intercalating dye), oligonucleotides of various lengths such as partially double-stranded DNA, ionic or nonionic polymers such as dextran sulfate or polyamines, surfactants, detergents, phase transfer catalysts, dextran, cyclodextran, silicone, polysilicone, fluorocarbon, polyfluorocarbon, fluorosilicone, hydrocarbon, alcohol, hydrofluorocarbon, and biomolecules such as peptides, proteins, lipids, carbohydrates, glycans, complex molecules, nucleic acids, and / or modified nucleic acids. Reagents and / or compounds with known PCR compatibility and low fluorescent signals associated with intercalating dyes are particularly suitable for this purpose. The dye uniformity agent can be miscible with the master mix. In some embodiments, a dye-uniformity agent is present in and / or added to the master mix. The dye-uniformity agent (e.g., partially double-stranded DNA) may be present in the master mix at a concentration ranging from about 100 nM to about 100 μM, or any range and / or value therein (e.g., from about 1, 5, 10 μM to about 15, 25, 50, 100 μM). In some embodiments, the dye-uniformity agent may be attached (e.g., by covalent and / or non-covalent attachment) to a portion of a bead. In some embodiments, the bead may include an amount of dye-uniformity agent ranging from about 300 pM, 1 nM, or 3 nM to about 300 μM or 3 mM.

[0195] For example, oligonucleotides (oligos), such as non-extendable oligos (NE oligos), optionally containing biotin (e.g., at the 3' end), can modulate (e.g., reduce) dye (e.g., SYBR) binding during master mix addition. In some embodiments, the dye-uniformity agent can include ssDNA molecules containing a 3' attachment of biotin linked by a six-carbon chain, although other modifications and / or linkages (e.g., C1-C20 hydrocarbon chains, such as C1-C20 alkyl branched or unbranched chains) can also function. In some embodiments, non-extendable oligos can be used as dye-uniformity agents because they are less likely to negatively impact PCR than additional primers capable of dimer formation. As shown in Figure 22, adding NE oligos to a master mix containing SYBR Green I reduces the variability in average initial bead fluorescence (CV 0.7%) compared to SYBR alone (CV 4.8%). Because changes in SYBR absorption (and thus SYBR concentration in PCR reactions) can modulate target amplification and fluorescent signal, variability in SYBR absorption between arrays results in variability in the number of positive reactions (i.e., digital PCR signals). In Figure 23, the average number of target molecules per bead for the Mycoplasma target sequence was compared across five subarrays of a chip channel using either SYBR alone (CV 23%) or SYBR plus NE oligos (CV 4%) in the master mix. This reduced variability results in more accurate determination of the average molecules per bead and, therefore, more accurate determination of Mycoplasma concentration. Additionally, controlling intercalating dye concentration across the array leads to reduced variability in dsDNA melting temperatures, thereby enabling more accurate determination of amplicon melting points.

[0196] In some embodiments, the fluorescent signal from the coding dye is affected by the concentration of the intercalating dye, and large differences in dye absorption by the beads can make arrays difficult to read (e.g., causing beads to cluster during coding analysis). In Figure 24, a sample containing only SYBR in the master mix shows a shift in fluorescent signal observed in the coding dye fluorescence path between different subarrays (CV 14%). The variance in the apparent coding dye fluorescent signal was less when non-extending oligos were added to the solution (CV 5%).

[0197] Alternatively or additionally, differences in dye absorption during loading of the master mix onto the array may be addressed by other means. For example, in some embodiments, beads may be soaked in a buffer containing an intercalating dye before loading them onto the microwell array. The beads may be soaked before, during, and / or after incubation with the sample. Alternatively or additionally, the surface of the beads may be functionalized to tailor dye absorption. For example, more primers bound to the surface of the beads increases dye absorption compared to fewer primers. Because dsDNA binds intercalating dyes more strongly than ssDNA, partially double-stranded oligonucleotides (pdsDNA) may be used, optionally linked to beads via biotin-TEG conjugation chemistry available from IDT. In some embodiments, beads containing pdsDNA oligos are soaked in an intercalating dye-containing buffer before testing. The beads are then incubated with the sample in hybridization buffer, washed, and loaded onto the microwell array. In some embodiments, a master mix containing all the reagents necessary for PCR except for primers and intercalating dyes, and optionally including a dye homogenizing agent, can be introduced into the channels of the microwell array, optionally before sealing the reaction wells together with miscible sealing oil. An example of pdsDNA is shown in Figure 25.

[0198] Figure 25 shows 5' biotin TEG conjugation from IDT (top). An example of a partially double-stranded DNA primer is shown (bottom). The DNA primer shown has a first strand with the sequence of SEQ ID NO:1 and a second strand with the sequence of SEQ ID NO:2.

[0199] The melting temperature of the pdsDNA, which can be used to adjust the dye absorption, can be optimized so that the pdsDNA remains partially double-stranded under storage and / or hybridization conditions. However, during PCR, the pdsDNA dissociates, resulting in ssDNA primers that are not strongly associated with the intercalating dye and therefore have low background fluorescence. In some embodiments, the linker (e.g., a carbon chain, optionally a C1-C20 hydrocarbon) attaching the primer to the biotin functionality can be adjusted to match its hydrophobicity and / or ionic charge and, therefore, absorption properties with respect to dyes or other reagents.

[0200] In some embodiments, the device 10 of the present invention can be fabricated using a holder 900 for securing multiple microchips 10 or devices, optionally configured as a grid 900g as shown in FIG. 21 . The holder 900 can include a perimeter 910 that surrounds the grid 900g, defining horizontal and vertical insulating barrier regions 920, 930 between the sides and ends of adjacent devices 10. Master mix ports 18 are located opposite each other across the barrier segment 930. Input ports 16 are located opposite the perimeter 910 of the holder 900. In some embodiments, the holder 900 is fabricated from an insulating material, and the chips 10 can include a thermally conductive substrate. Each of the miniature microarray chips / devices 10 of the grid 900g can be independently thermally cycled. Thermal cycling and / or imaging of the microarray chips / devices 10 of the grid 900g can be synchronized by the test system 200 so that each microarray chip / device 10 is imaged in a predetermined sequence.

[0201] In some embodiments, the device 10 of the present invention is fabricated from a substrate that is an insulating material, such as plastic, and optionally different zones, areas, or regions of the same substrate are thermally cycled and / or imaged in a defined sequence.

[0202] The device 10 of the present invention can be used with commercially available automated sample preparation methods (e.g., pipetting robots and / or bead washing robots such as Tecan, Hamilton, Beckman, or other automated systems) or can be operated using customized dedicated reader equipment or platforms. Readers for use with the present invention can include large-scale designs suitable for high-volume in vitro testing, benchtop versions suitable for clinical laboratories or clinics, portable versions ideal for production floor, consumer, or forensic testing, or versions integrated into other systems.

[0203] Optical systems 220 (FIG. 8) that may be utilized include, but are not limited to, dual or multi-optical imaging systems in which the entire array portion (or most of it) of device 10 is imaged at low resolution and a small portion of a selected subset of microwells 20 is imaged at high resolution, or lens systems that allow rapid field of view changes, such as a zoom lens. Alternatively, other detection systems may be used, including electrochemical, absorption, and / or chemiluminescence detection.

[0204] The devices and / or methods of the present invention can be used for immuno-PCR, reverse transcription-PCR, protein singleplex reactions in compact arrays (protein-SiRCA), as well as many other variations of PCR or nucleic acid amplification. Different means of detecting amplicons can be used, such as molecular beacons or hydrolysis probes (e.g., TaqMan probes), or any such means known in the art. The decoding images used to determine the properties of each bead can be acquired before, during, and / or after the thermal cycling images.

[0205] According to some embodiments, a method for obtaining high-resolution real-time PCR can be performed in singleplex reactions in a compact array (SiRCA) while mitigating the effects of photobleaching on test signals. The device 10 of the present invention includes two or more fluidic channels 15, each with a microwell set 20, optionally for one or more samples. The array 10 can be imaged using a subset of the microwell sets 20. Different subsets of the microwell sets 20 can be imaged in sequence, with one segment imaged after each amplification cycle. For each image, the average signal for each reaction type can be calculated for each sample. By displaying the average signal from each reaction type for each amplification cycle, cycle thresholds can be determined for each reaction type at single-cycle resolution without the need to image the entire array after every PCR cycle. For example, a 14 mm long, 1.25 mm wide fluidic device 10 containing the channels 15 can be divided into five (row) segments A, B, C, D, and E. After cycle 1 of PCR, segment A is imaged. Segment B is imaged after cycle 2 of PCR, and so on. Each row / segment can be imaged six times after 30 PCR cycles. While signal from each bead type can be acquired for every PCR cycle, each row / segment (and therefore each reaction) receives only 1 / 5 of the dose of excitation energy used to record the fluorescence data. This approach therefore mitigates the effects of photobleaching while maintaining single-cycle real-time PCR resolution. Additionally, by narrowing the microwell set 20 of a given sample fluid passage 15 to one dimension, two or more passages 15 containing a given subset of microwells 20 can be placed in sufficient proximity to be imaged at high speed (e.g., one frame contains more than one array), facilitating the ability to collect real-time PCR data at single-cycle resolution for multiple samples in a reasonable imaging time.

[0206] The methods of the present invention can be applied to any reaction array that is coded so that the nature of the reaction can be determined, generally in real time.

[0207] In some embodiments, the devices of the present invention may be used in place of multiplex PCR or immunoassay panels. Applications of embodiments of the present invention include, but are not limited to, biomedical or biological research, disease diagnosis (including infectious disease and / or oncology) by nucleic acid or protein biomarkers, veterinary applications, forensic analysis or genotyping, environmental monitoring, counterfeit product detection, and / or biopharmaceutical production and quality control applications.

[0208] 8 is a schematic diagram of an example analysis system 200. System 200 may include at least one controller (typically including at least one processor) in communication with an optical system 220 that includes an electronic signal detector 222, such as an optical detector that includes a camera or other imaging or signal detection device. System 200 may also include a housing 200h and a heat source 240 for applying heat to one or more fluidic devices 10 during an inspection cycle.

[0209] The optical system 220 may optionally include an excitation source 225 and one or more filters, including different filters (or filter sets) F1, F2, or more, e.g., 2-100 filters, each or some of which may provide a different coded wavelength for exciting beads held in one or more microwell sets 20. The optical system 220 may include a subarray selection module 250 that detects signals and / or images subsets or subarrays 10s of the microwell array 10a of the fluidic device 10 (optionally, selectively, upon directing the optical system to only one or more subsets). Imaging and / or excitation may be performed sequentially or in parallel for defined subsets. In some embodiments, optical excitation is not required. For example, in some embodiments, a test (e.g., a protein test) may emit a chemiluminescent signal that can be detected and / or imaged without optical excitation.

[0210] The system 200 may also include a signal analysis module 260. The signal analysis module 260 can analyze test signal data from different sets of microwells.

[0211] System 200 can acquire an analog signal that defines a threshold cycle or cycle threshold "Ct" that identifies a PCR reaction for a target species and / or molecule type as positive when the fluorescent signal is greater than the threshold, or negative when the fluorescent signal does not rise above the threshold. That is, Ct is the cycle where Si>>Bs, where >> is at least 5-10%, and optionally 2-fold, greater than Bs, where Si is the fluorescent signal intensity, and Bs is the background fluorescent signal. The Ct of a sample of unknown concentration can be compared to the Ct of a sample of known concentration to calculate the initial target concentration.

[0212] The controller 210 is in communication with (i.e., contains computer program code for) a subarray selection module 250 configured to select different sets of microwells during different test cycles. Modules 250 and / or 260 may be wholly or partially on-board or remote from the controller and / or optical system 220. The analysis system 200 includes at least one processor (i.e., a digital signal processor) and may include a transceiver 214.

[0213] Subarray selection module 250 is configured to identify a subset of (aligned) microwells of two, three, four, five, or more adjacent fluid sample paths to provide site data, and optionally, the identified subset includes adjacent sets of microwells (i.e., corner sets) of two or more adjacent fluid paths. Module 250 uses this information, along with known spacing dimensions and array configuration, to define the sites of other microwell sets 20. Alignment features 27 (FIG. 1) of one or more sites on device 10 can alternatively or additionally be used for this site data.

[0214] Modules 250 and 260 may be integrated into analysis system 200 or distributed within one or more servers 300. Server 300 may be embodied as a standalone server or may be housed as part of another computing infrastructure. Server 300 may be embodied as one or more enterprise, application, personal, pervasive, and / or embedded computer systems that may be standalone or interconnected by public and / or private, real and / or virtual, wired and / or wireless networks, including the Internet, and may include various types of tangible, non-transitory, computer-readable media. Server 300 may also communicate with networks via wired or wireless connections and may include various types of tangible, non-transitory, computer-readable media.

[0215] In use, server 300 may be provided using cloud computing, which involves the provision of computing resources on demand over a computer network. The resources may be embodied as applications, databases, file services, email, etc., along with various infrastructure services (e.g., computation, storage, etc.). In traditional computing models, both data and software are typically contained entirely on the user's computer. In cloud computing, the user's computer may contain mostly software or data (perhaps an operating system and / or web browser) and serve only as a display terminal for processes occurring on a network of external computers. A cloud computing service (or a collection of multiple cloud resources) may be generally referred to as a "cloud." Cloud storage includes a model of networked computer data storage in which data is stored on multiple virtual servers rather than on one or more dedicated servers.

[0216] The controller 210 can communicate with the server 410 or computer via the transceiver 214 and / or a computer or cellular network, which may include one or more of a local area network (LAN), a wide area network (WAN), and may include a private intranet and / or the public Internet (also known as the World Wide Web or "Web" or "Internet").

[0217] As shown in FIG. 18, an embodiment of the present invention is configured as a data processing system 1116, which may include one or more processors 500, memory 536, and input / output circuitry 546. One or more processors 500 may be part of image processing circuitry 500c. The data processing system may be incorporated into one or more of, for example, a personal computer, a database, a workstation W, a server, a router, etc. The system 1116 may reside on one machine or be distributed among multiple machines. The processor 500 communicates with the memory 536 via an address / data bus 548 and with the input / output circuitry 546 via an address / data bus 549. The input / output circuitry 546 may be used to transfer information between the memory (memory and / or storage medium) 537 and another computer system or network, for example, using an Internet Protocol (IP) connection. These components may be conventional components, such as those used in many conventional data processing systems configured to function as described herein.

[0218] In particular, processor 500 (which may be incorporated into controller 210 of FIG. 8) may be a commercially available or custom microprocessor, microcontroller, digital signal processor, etc. Memory 536 may include memory devices and / or storage media containing software and data used to implement functional circuits or modules used in accordance with embodiments of the present invention. Memory 536 may include, but is not limited to, the following types of devices: ROM, PROM, EPROM, EEPROM, flash memory, SRAM, DRAM, and magnetic disks. In some embodiments of the present invention, memory 536 may be a content-addressable memory (CAM).

[0219] 18, memory (and / or storage media) 536 may include several categories of software and data used by the data processing system, operating system 552, application programs 554, input / output device drivers 558, and data 556. As will be appreciated by those skilled in the art, operating system 552 may be any operating system suitable for use with a data processing system, such as the IBM®, OS / 2®, AIX®, zOS® operating systems, Microsoft® Windows® 95, Windows 98, Windows 2000, or Windows XP operating systems, and Unix or Linux®, IBM, OS / 2, AIX, and zOS are trademarks of IBM (International Business Machines Corporation) in the United States and / or other countries, while Linux is a trademark of Linus Torvalds in the United States and / or other countries. Microsoft and Windows are trademarks of Microsoft Corporation in the United States and / or other countries. I / O device drivers 558 generally include software routines accessed by application programs 554 through operating system 552 to communicate with devices such as I / O circuitry 546 and certain memory 536 components. Application programs 554 are examples of programs that implement various features of circuits and modules according to some embodiments of the present invention. Finally, data 556 represents static or dynamic data used by application programs 554, operating system 552, I / O device drivers 558, and other software programs that may reside in memory 536.

[0220] The data 556 may include (archived or saved) digital image datasets 522. As further illustrated in Figure 18, according to some embodiments of the present invention, the application programs 554 include a microwell subset selection module 250 and a signal analysis module 260. The application programs 554 may be located on a local server (or processor) and / or database or a remote server (or processor) and / or database, or a combination of local and remote databases and / or servers.

[0221] Although the present invention has been illustrated with reference to application program 554 and modules 250 and 260 in FIG. 18, those skilled in the art will recognize that other configurations are within the scope of the present invention. For example, rather than application program 554, these circuits and modules may be incorporated into operating system 552 or other such logic of a data processing system. Furthermore, while application program or modules 250 and 260 are illustrated in a single data processing system, those skilled in the art will recognize that such functionality may be distributed across one or more data processing systems, for example, in a client / server configuration of the type described above. Thus, the present invention should not be construed as limited to the configuration illustrated in FIG. 18, but may be provided by other configurations and / or functionalities among data processing systems. For example, although FIG. 18 is illustrated as having various circuits and modules, one or more of these circuits or modules may be combined or separated without departing from the scope of the present invention.

[0222] Computer program code for carrying out the operation of the data processing systems, method steps or acts, modules, or circuits (or portions thereof) described herein may be written in a high-level programming language such as Python, Java, AJAX (Asynchronous JavaScript), C, and / or C++ for development convenience. In addition, computer program code for carrying out the operation of exemplary embodiments may be written in other programming languages, such as, but not limited to, interpreted languages. Some modules or routines may also be written in assembly language or microcode to improve performance and / or memory usage. Some modules or routines may also be written in scripting languages, including open-source scripting languages. However, embodiments are not limited to a particular programming language. As noted above, the functionality of any or all of the program modules may be implemented using discrete hardware components, one or more application-specific integrated circuits (ASICs), or programmed digital signal processors or microprocessors. The program code may be executed entirely on one computer (e.g., a workstation), partially on one computer as a separate software package, partially on the workstation computer and partially on another computer, local and / or remote, or entirely on another local or remote computer. In the latter scenario, other local or remote computers may be connected to the user's computer through a local area network (LAN) or wide area network (WAN), or a connection to external computers may be provided (e.g., through the Internet using an Internet Service Provider).

[0223] The present invention has been described in part with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, executed by the processor of the computer or other programmable data processing apparatus, provide means for performing the function(s) / act(s) specified in one or more of the blocks in the flowchart illustrations and / or block diagrams.

[0224] Computer program instructions that can instruct a computer or other programmable data processing apparatus to function in a particular manner may also be stored in a computer-readable memory, such that the instructions stored in the computer-readable memory produce an article of manufacture that includes instruction means for performing the functions / acts specified in one or more blocks of the flowcharts and / or block diagrams.

[0225] Computer program instructions are loaded into a computer or other programmable data processing apparatus such that the instructions, executed by the computer or other programmable apparatus, provide steps for performing some or all of the functions / operations specified in one or more blocks of the flowcharts and / or block diagrams, and a computer-implemented process is provided by a series of operational steps performed by the computer or other programmable apparatus.

[0226] The flowcharts and block diagrams in certain figures herein illustrate the exemplary architecture, functionality, and operation of possible implementations of embodiments of the present invention. In this regard, each block in the flowcharts or block diagrams represents a module, segment, or code portion containing one or more executable instructions for performing the specified logical function(s). It should also be noted that in some alternative implementations, the functions noted in the blocks may occur out of the order noted in the figures. For example, depending on the functionality required, two blocks shown in succession may in fact be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, or two or more blocks may be combined.

[0227] In particular, controller 210 (FIG. 8) and / or processor 500 (FIG. 18) may include commercially available or custom microprocessors, microcontrollers, digital signal processors, etc. Memory may include memory devices and / or storage media containing software and data used to implement functional circuits or modules used in accordance with embodiments of the present invention. Memory may include, but is not limited to, the following types of devices: ROM, PROM, EPROM, EEPROM, flash memory, SRAM, DRAM, and magnetic disks. In some embodiments of the present invention, memory may be content-addressable memory (CAM).

[0228] The foregoing is illustrative of the invention and should not be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included herein. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for any teachings relevant to the sentence and / or paragraph in which the reference is presented. [Appendix 1] 1. An analytical system comprising: a housing including a chamber sized and configured to receive at least one microfluidic device; an optical system coupled to the housing in optical communication with the at least one microfluidic device; a controller coupled to the optical system; a heat source coupled to the optical system and thermally coupled to the at least one microfluidic device secured to the housing; a subarray selection module in communication with the controller configured to select a subset of a set of microwells in at least one fluid path of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) under test; An analytical system that encompasses: [Appendix 2] 2. The system of claim 1, further comprising a magnet fixed to the housing adjacent to at least one microfluidic device, the magnet configured to translate on at least one fluid path to magnetically couple to beads during a bead loading operation and guide the beads to move on the fluid path to the bead-retaining segment of the microwell. [Appendix 3] 2. The system of claim 1, wherein the microfluidic device includes a plurality of fluid passages, each having a plurality of microwell sets arranged along a length dimension associated with a direction between a sample input port and an opposite second port, and wherein the selected subset of microwell sets is associated with a single row of laterally aligned first microwell subsets of the plurality of fluid passages, optionally one aligned microwell subset for each passage of the plurality of fluid passages. [Appendix 4] 2. The system of claim 1, wherein prior to optical excitation, the subarray selection module identifies a subset of the set of microwells that defines the locations of other sets of microwells on the microfluidic device. [Appendix 5] 2. The system of claim 1, wherein the subarray selection module selects different subsets of the microwell set of the microfluidic device for different reaction steps of the test (e.g., different thermal cycles of the test), instructs the optical system to excite only a currently selected subset of the microwell set of the different fluid paths, and instructs a camera of the optical system to capture images of the different subsets of the microwell set of the currently selected subset sequentially or in parallel. [Appendix 6] 2. The system of claim 1, wherein the subarray selection module selects the same microwell subset for at least some different sequential reaction steps of the test (e.g., different thermal cycles of the test), instructs the optical system to transmit light only to a currently selected subset of the microwell set, and instructs a camera of the optical system to capture images of different subsets of the microwell set, optionally pairs of adjacent microwell sets of the simultaneously selected microwell subsets, in sequential or parallel fashion. [Appendix 7] 2. The system of claim 1, wherein the optical system includes at least first and / or second filters that define first and / or second wavelengths of excitation light corresponding to first and second target-encoded beads for decoding bead sets held in one or more of the microwell sets in at least one fluid passage of the microfluidic device. [Appendix 8] 2. The system of claim 1, further comprising a signal analysis module integrated with and / or coupled to the controller, the signal analysis module configured to acquire an analog signal for each microwell of the selected subset of the microwell set, the analog signal providing PCR data as amplitude changes over the test cycle, and the concentration of a target molecule for a reaction associated with a specified bead type being determined from the analog signal. [Appendix 9] 9. The system of claim 8, wherein the signal analysis module is further configured to acquire digital PCR signals for the set of microwells. [Appendix 10] 9. The system of claim 8, wherein the analog signal provides real-time PCR data as amplitude changes in PCR cycle number for input materials in one or more of the sets of microwells in the fluid pathway, wherein the molecule concentration of a defined bead type is about, equal to, or greater than 1 molecule per microwell, and the analog signal defines a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescent signal intensity (Si) is greater than a threshold and the target molecule number / target molecule concentration for a target species and / or molecule type, or negative when the fluorescent signal intensity (Si) is consistently less than the threshold for a given number of PCR cycles. [Appendix 11] 11. The system of claim 10, wherein Ct is the cycle number where Si>>Bs, where >> is at least 5-10% greater than Bs, optionally 2-fold greater, and / or 5-10-fold greater than the standard deviation of Bs, and Bs is optionally the background fluorescence signal measured in a negative PCR reaction. [Appendix 12] 9. The system of claim 8, wherein the analog signal is acquired for only a single subset of the microwell sets in each fluid path after every other or each of a plurality of different reaction steps of the test (e.g., different thermal cycles of the test). [Appendix 13] 9. The system of claim 8, wherein the analog signal comprises a mean (optionally excluding outlier data), median, mode, or weighted value of the analog signal Si corresponding to each defined bead type and is provided as an estimate of real-time PCR curves for similar reactions in microwells of other microwell sets for that fluid path of the microfluidic device, thereby enabling single cycle resolution without imaging all microwell sets of each fluid path after different reaction cycles or each reaction cycle. [Appendix 14] 2. The system of claim 1, wherein the optical system includes a camera having a field of view (FOV) that covers only a subset of the microwells of the microfluidic device, the subsets being located in at least two, and optionally all (e.g., entire rows) of adjacent fluid passages of the microfluidic device. [Appendix 15] The system of Appendix 1, wherein the controller and / or the signal analysis module is optionally configured to instruct the optical system to acquire pre- and post-PCR images and compare signal intensities therebetween, along with analog data acquired using the selected subset of microwell sets at different reaction steps of the test, to determine positive and negative PCR reactions, and optionally to determine the concentration of target species and / or molecule concentration in the original sample provided to the fluid path. [Appendix 16] 10. The system of claim 1, further comprising a holder configured to secure a plurality of microfluidic devices to an alignment grid of the housing. [Appendix 17] 2. The system of claim 1, wherein the holder includes an insulator that provides a thermal barrier between adjacent microfluidic devices, and optionally, the holder secures the plurality of microfluidic devices with input ports to the fluid passages facing outward. [Appendix 18] 2. The system of claim 1, wherein the microfluidic device further comprises a dye-uniformity agent, and optionally, the dye-uniformity agent comprises an oligonucleotide, optionally a non-extendable oligonucleotide and / or partially double-stranded DNA (e.g., partially double-stranded standard DNA comprising biotin and / or a C1-120 hydrocarbon chain). [Appendix 19] 20. The system of claim 18, wherein the dye uniformity agent is present in a master mix present in the microfluidic device, and the dye uniformity agent is attached to beads present in the microfluidic device. [Appendix 20] 1. A method for identifying a target species and / or a target molecule, comprising: providing a first fluid passage in a fluid analysis device, the first fluid passage including a plurality of microwell sets disposed on the first fluid passage; acquiring signal intensity data from only a defined subset of said plurality of microwell sets; identifying PCR reactions that are positive for a target species and / or molecule type associated with the bead type and / or target molecule based at least in part on the signal intensity data obtained; A method that encompasses [Appendix 21] 21. The method of claim 20, further comprising loading and sealing the plurality of microwell sets on the first fluid path prior to the acquiring step such that after sealing, each microwell set is fluidically isolated from one another, and wherein the plurality of microwell sets on the first fluid path are in fluid communication only during the loading prior to the sealing step. [Appendix 22] 21. The method of claim 20, further comprising changing the defined subset of the plurality of microwell sets to a different defined subset after each of a plurality of sequential reaction steps of the test (e.g., after each of a plurality of sequential thermal cycles of the test), so that some microwell subsets are not imaged after each reaction step. [Appendix 23] 21. The method of claim 20, wherein the defined subset remains the same across multiple successive reaction steps of the test (e.g., after each of multiple successive thermal cycles of the test), such that some sets of microwells are not imaged after each reaction step. [Appendix 24] The fluid analysis device further comprises: a second fluid path including a plurality of sets of microwells spaced apart on the second fluid path; a third fluid path including a plurality of microwell sets spaced apart on the third fluid path; a fourth second fluid path including a plurality of sets of microwells spaced apart on the fourth fluid path;

[0023] The invention further comprises a plurality of fluid passages, including, but not limited to, one or more of: a first microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a first row, a second microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a second row, a third microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a third row, and a fourth microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a fourth row; the defined subset is a single row among the first, second, third, and fourth rows; Appendix 20 method. [Appendix 25] 25. The method of claim 24, wherein the first, second, third, and fourth fluid paths comprise linear or arcuate segments that are substantially parallel to one another and define the first, second, third, and fourth microwell sets. [Appendix 26] moreover, transmitting an optical excitation signal to only the predetermined subset before the acquiring step, and acquiring the signal in response to the transmission of the optical excitation signal; digitally scanning the defined subset of the plurality of microwell sets after, before, or both before and after the transmitting and acquiring steps to acquire images of the microwell sets to identify positive and negative PCR reactions associated with digital PCR; electronically identifying microwells of the set of microwells that are positive for one or more target analyte molecules while the microwells are at an imaging temperature; The method of claim 20, [Appendix 27] 21. The method of claim 20, wherein the excitation and acquisition are performed to image only one defined subset of the set of microwells after each of multiple sequential reaction steps (e.g., thermal cycles) of the test, and each of the consecutive only one defined subsets is different from each other. [Appendix 28] The method of claim 20, wherein the acquisition of signal intensities from only the defined subsets is performed using at least one camera having a field of view (FOV) covering only one microwell set or only a subset of the microwell sets in at least two adjacent ones of the fluid paths by sequentially or parallelly acquiring images of different defined microwell sets. [Appendix 29] 21. The method of claim 20, wherein each of the microwell sets comprises a microwell array of between 1,000 and 1 million of said microwells, and the fluid analysis device comprises a plurality of spaced apart fluid passageways each comprising the microwell set comprising the microwell array, the fluid passageways being in fluid isolation, and at least some of the microwells of the microwell array containing a single bead, and optionally some or all of the microwells containing no beads or one or more beads. [Appendix 30] 21. The method of claim 20, comprising electronically identifying the locations of one or more microwell sets located at one or more locations on the microfluidic device prior to the transmitting and acquiring steps, and defining the locations of other microwell sets based at least in part on the locations of the identified locations. [Appendix 31] 21. The method of claim 20, further comprising transmitting an optical excitation signal to only the defined subset prior to the acquiring step, and selecting, prior to the transmitting, a filter that provides a coding wavelength for the transmitting step. [Appendix 32] 21. The method of claim 20, further comprising acquiring an analog signal capable of providing real-time PCR data as change in amplitude versus PCR cycle number for input material of the set of microwells, wherein the molecule concentration of a defined bead type is about, equal to, or greater than 1 molecule per microwell, and wherein the analog signal defines a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescent signal intensity (Si) is higher than a threshold and the number of target molecules / concentration of target molecules for a target species and / or molecule type, and negative when the fluorescent signal intensity (Si) is consistently lower than the threshold for a given number of PCR cycles. [Appendix 33] 33. The method of claim 32, wherein Ct is the cycle number where Si>>Bs, where >> is at least 5-10%, optionally 2-fold greater than Bs, and / or 5-10 times the standard deviation of Bs, and Bs is optionally the background fluorescence signal measured in a negative PCR reaction. [Appendix 34] 33. The method of claim 32, wherein the analog signal is acquired for only a single subset of the microwell set in one or more fluid paths after every other or each of a plurality of different reaction steps of the test (e.g., different thermal cycles of the test). [Appendix 35] 33. The method of claim 32, wherein the analog signal comprises a mean, median, mode, or weighted value of Si (optionally discarding outlier data) as the analog signal corresponding to each defined bead type and serves as an estimate of real-time PCR curves for similar reactions in microwells of other microwell sets for that fluid path of the microfluidic device, thereby enabling single cycle resolution without having to image all of the microwell sets of each fluid path after different reaction steps. [Appendix 36] 21. The method of claim 20, wherein acquiring the signal intensity is performed by using a camera having a field of view (FOV) covering only a subset of the set of microwells of the microfluidic device and covering at least two, and optionally all, adjacent fluid paths of the microfluidic device. [Appendix 37] 33. The method of claim 32, wherein the analog signal is obtained for only a single set of the microwell sets in each fluid path after multiple different reaction steps of the test (e.g., different thermal cycles of the test), and the analog signal comprises a mean, median, mode, or weighted value of the analog signal Si corresponding to each defined bead type and serves as an estimate of real-time PCR curves for similar reactions in microwells in other microwell sets for that fluid path of the microfluidic device, thereby enabling single cycle resolution without imaging all microwell sets in each fluid path after different reaction steps. [Appendix 38] the fluid analysis device comprising the first fluid passage including the plurality of microwell sets spaced apart on the first fluid passage, further comprising a plurality of additional fluid passages each including the plurality of microwell sets spaced apart along the length, the fluid analysis device further comprising a separate material input port for each of the first fluid passage and the plurality of additional fluid passages, at least some of the fluid passages sharing an opposite common second port; Furthermore, before the obtaining step, fluidly loading the input port with a bead slurry pre-exposed to the sample for analysis; magnetically directing the bead slurry to flow into different sets of microwells on the fluid path; flowing a fluid master mix comprising a dye from the second port through the fluid passageway to the first port, the master mix optionally comprising a dye uniformity agent, and optionally the dye uniformity agent being or comprising an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain)); and sealing the microwell set and the fluid passage from each other by flowing sealing oil from the second port through the fluid passage to the first port; The method includes: Appendix 20 method. [Appendix 39] 39. The method of claim 38, further comprising placing a magnet adjacent to the fluid analysis chip and translating the magnet to the second port before flowing the fluid master mix and sealing oil. [Appendix 40] 21. The method of claim 20, wherein the fluid analysis device (optionally the first fluid path and / or the plurality of microwell sets) comprises a dye-uniform agent, and optionally the dye-uniform agent comprises an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain)). [Appendix 41] The system of claim 40, wherein the dye uniformity agent is present in a master mix present in the fluid analysis device (e.g., present in the first fluid path and / or the plurality of microwell sets) and / or the dye uniformity agent is attached to beads present in the fluid analysis device (e.g., present in the first fluid path and / or the plurality of microwell sets). [Appendix 42] A microfluidic device for analysis, comprising: a plurality of fluid passageways, each having a length dimension corresponding to a direction between a first port and an opposite second port, at least a portion of said length dimension being configured as a linear or arcuate length segment, each fluid passageway containing a plurality of microwell sets disposed in said linear or arcuate length segment of said length dimension; A microfluidic device for analysis comprising: [Appendix 43] 43. The microfluidic device of claim 42, wherein the plurality of microwell sets of the plurality of fluid paths are arranged in rows, columns, or rows and columns, the rows or columns corresponding to the linear or arcuate length segments. [Appendix 44] 43. The microfluidic device of claim 42, wherein at least some of the plurality of fluid passages are substantially parallel in the linear or arcuate length segments. [Appendix 45] 43. The microfluidic device of claim 42, wherein at least some of the plurality of fluid passageways are arcuate, substantially parallel passageways that comprise the arcuate length segments. [Appendix 46] 43. The microfluidic device of claim 42, wherein at least some of the plurality of fluid passages are substantially parallel and extend radially between a periphery of the device and a center of the device. [Appendix 47] 43. The microfluidic device of claim 42, wherein the plurality of fluid passages includes a first set of fluid passages and a second set of fluid passages spaced apart from the first set, the first set of fluid passages terminating in a first master mix port as the second port, and the second set of fluid passages terminating in a second master mix port as the second port. [Appendix 48] 43. The microfluidic device of claim 42, wherein the first and second fluid passage sets are circumferentially spaced apart. [Appendix 49] 43. The microfluidic device of claim 42, wherein the plurality of fluid passages includes at least two fluid passages defining a first proximal set and at least two passages defining a second proximal set adjacent the first proximal set, each containing a spatially aligned set of microwells. [Appendix 50] 50. The microfluidic device of claim 49, further comprising a first gap space between each of the first and second fluid passages of the first proximal set and the second proximal set, and a second gap space between the first proximal set and the second proximal set, the second gap space having a larger lateral extent than the first gap space. [Appendix 51] 43. The device of claim 42, wherein the plurality of microwell sets in each of the plurality of fluid paths have a common configuration, the plurality of microwell sets in each of the fluid paths are aligned with one another in rows and / or columns, and the plurality of fluid paths maintain the input substances in fluid isolation from one another. [Appendix 52] 43. The microfluidic device of claim 42, wherein each of the at least a plurality of microwell sets for the fluid pathway comprises a quantity ranging from 1,000 to 1,000,000 microwells, and the microwells of the microwell sets are sized and configured to immobilize and retain a single bead, thereby enabling 100 to 1 billion reactions in the microfluidic device. [Appendix 53] 43. The microfluidic device of claim 42, wherein the device includes a transparent substrate extending across at least first and second sets of microwells from first and second fluid paths, optionally defining a first set of adjacent microwell subsets, and the plurality of microwell sets in the fluid paths. [Appendix 54] 43. The microfluidic device of claim 42, wherein each fluid passage of the plurality of fluid passages includes a separate first port at a first end as the first port, one of the alternating first ports being at a first vertical position of the device and the other of the alternating first ports being at a second vertical position of the device spaced apart from the first vertical position in the length dimension. [Appendix 55] 43. The microfluidic device of claim 42, wherein the first port is a material input port located at a first end of the fluid passage, the device further including a fluid manifold connecting opposite second ends of at least some of the fluid passages to the second port. [Appendix 56] 43. The microfluidic device of claim 42, wherein the fluid passages extend radially in the device, the input ports for the fluid passages are located at the periphery of the device, and the second port is a single second port located at the center of the device connected to each of the fluid passages. [Appendix 57] 43. The microfluidic device of claim 42, wherein at least some of the fluid passages are provided as concentric fluid passage sets, each having the arcuate length segment. [Appendix 58] 43. The microfluidic device of claim 42, wherein the concentric fluid passage sets having arcuate length segments are provided as a plurality of circumferentially spaced concentric fluid passage sets. [Appendix 59] 43. The microfluidic device of claim 42, further comprising a dye-uniformizing agent, and optionally, the dye-uniformizing agent comprises an oligonucleotide (e.g., a non-extending oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain)). [Appendix 60] 60. The microfluidic device of claim 59, wherein the dye uniformity agent is present in a master mix present in the microfluidic device and / or the dye uniformity agent is attached to beads present in the microfluidic device. [Explanation of symbols]

[0229] 10 Fluidic Devices 10a Array 10b Lower base material 10p outer periphery 10s sub-array 10u upper base material 15,151,152,153,154,155,156,157,158 fluid passage 15 n Nearby passage 16 Input port 16r reservoir 16v via 18 Master Mix / Oil Port 18m fluid manifold 18r reservoir 18v via 19 Interstitial space 20, 201, 202, 203, 204, 205 microwells 20c Corner microwell 20r outer periphery 21 Interstitial space 25 Frame Box 27 Alignment Mark 120 microwells 120a Microwell Array 120s Inspection Signal Elements 120w Bead Retaining Segment 122 Tapered Inspection Signal 200 Analysis System 200h Test System 210 Controller 214 Transceiver 220 Optical Signal Detector 222 Camera 225 Photoexcitation source 240 Heat source 250 Subarray Selector Module 260 Signal Analysis Module 300 servers 548,549 Address / Data Bus 800 magnets 810 Hard Stop Device 900 holder 900g grid 910 Outer periphery 920,930 Barrier Segments 1116 Data Processing System C1,C2,C3,C4,C5,C6,C7,C8 column R1,R2,R3,R4,R5 row

Claims

1. A method for identifying target species and / or target molecules in a microfluidic device configured to perform PCR reactions in a plurality of sets of microwells, comprising: exciting a defined subarray of the plurality of microwell sets of the microfluidic device, the microfluidic device comprising at least a first fluidic channel having some of the plurality of microwell sets disposed along the first fluidic channel; acquiring signal intensity data from only the defined subarray of the plurality of microwell sets, wherein acquiring the signal intensity data comprises acquiring an analog signal capable of providing real-time PCR data as amplitude change versus PCR cycle number for an input material of the plurality of microwell sets, wherein the molecule concentration of the defined type is about, equal to, or greater than 1 molecule per microwell, and wherein the analog signal defines a threshold cycle or cycle threshold Ct that identifies a microwell reaction and number of target molecules / concentration of a target species and / or molecule type as positive if the fluorescent signal intensity (Si) is higher than a threshold, or negative if the fluorescent signal intensity (Si) is consistently lower than the threshold for a given number of PCR cycles; scanning some or all of the plurality of microwell sets after, before, or both before and after the PCR reactions to obtain images of the plurality of microwell sets to identify positive and negative PCR reactions associated with digital PCR; electronically identifying microwells of the plurality of sets of microwells that are positive for one or more target analyte molecules; A method comprising:

2. The method of claim 1, further comprising the steps of fluidly loading each sample with a pre-exposed bead slurry for analysis, and sealing the plurality of microwell sets along the first fluid channel prior to the acquiring step such that after sealing, each microwell set of the plurality of microwell sets is fluidly isolated from one another, wherein the plurality of microwell sets along the first fluid channel are in fluid communication only during the loading prior to the sealing step.

3. 10. The method of claim 1, further comprising changing the defined subarray of the plurality of microwell sets to a different defined subarray after each of a plurality of successive reaction steps of an assay, wherein some microwells of the plurality of microwell sets are not imaged after each of the reaction steps.

4. The fluid analysis device, a second fluid path including a plurality of microwell sets disposed along the second fluid path; a third fluid path including a plurality of microwell sets disposed along the third fluid path; a fourth fluid path including a plurality of microwell sets disposed along the fourth fluid path; a first microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a first row, a second microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a second row, a third microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a third row, and a fourth microwell set of the plurality of microwell sets in each of the first, second, third, and fourth fluid paths is arranged as a fourth row; The method of claim 1.

5. The method of claim 4, wherein the first, second, third and fourth fluid passages comprise straight or arc-shaped segments that are substantially parallel to one another.

6. The method of claim 1, wherein the microfluidic device further comprises a second fluid passage adjacent to the first fluid passage.

7. The method of claim 1, wherein the microfluidic device is configured to have a plurality of spaced-apart fluid flow paths, the first fluid passage is one of the plurality of spaced-apart fluid flow paths, each of the plurality of spaced-apart fluid flow paths includes a plurality of microwell sets, and the fluid flow paths are in a fluid isolation state.

8. The method of claim 1, further comprising, prior to the exciting and acquiring steps, electronically identifying the positions of one or more microwell sets of the plurality of microwell sets located at one or more locations on the microfluidic device, and identifying the positions of other of the microwell sets based at least in part on the identified positions.

9. The method of claim 1, further comprising the step of selecting a filter that provides a coded wavelength for the excitation step before the acquiring step and before the excitation step.

10. The method described in claim 1, wherein Ct is the cycle number of Si>>Bs, where >> is at least 5 to 10% greater than Bs and / or 5 to 10 times the standard deviation of Bs, and Bs is the background fluorescence signal.

11. The method of claim 1, wherein the microfluidic device comprises a plurality of fluid passages, the first fluid passage being one of the plurality of fluid passages, the plurality of fluid passages being arranged with their respective microwells aligned in different columns, and the analog signal being acquired for each specified subarray of the plurality of microwell sets, which changes after every other or each of a plurality of different reaction steps of an assay.

12. The method of claim 1, wherein the analog signal comprises a mean, median, mode, or weighted value of Si.

13. The method of claim 1, wherein the acquired signal intensity data is acquired using a camera having a field of view (FOV) that covers only a specified subarray of the plurality of microwell sets of the microfluidic device at any one time.

14. The microfluidic device comprising the first fluid passage including the plurality of microwell sets spaced along the first fluid passage, further comprising a plurality of additional fluid passages, each of the plurality of additional fluid passages including a respective one of the plurality of microwell sets spaced along its respective length, the microfluidic device further comprising a separate material input port for the first fluid passage and each of the plurality of additional fluid passages, at least some of the fluid passages sharing an opposite common port; The method may further comprise, before the obtaining step: fluidly loading a bead slurry into the input port that has been pre-exposed to each sample for analysis; magnetically directing the bead slurry to flow into different sets of the plurality of microwell sets along the fluid pathway; flowing a fluidic master mix including a dye from the opposite port through the fluid passageway to the material input port; sealing the microwell and the fluid passage from each other by flowing sealing oil from the opposite port through the fluid passage to the substance input port; Including, The method of claim 1.

15. The method of claim 14, further comprising the steps of placing a magnet adjacent to the microfluidic device and moving the magnet to the opposite port before flowing the fluid master mix and sealing oil.

16. The method described in claim 4, wherein the defined subarray of the plurality of microwell sets is a single one of the first, second, third and fourth columns.

17. The method of claim 1, wherein the defined subarray of the plurality of microwell sets includes microwells in the range of 1,000 to 1,000,000 individual microwells.

18. The method described in claim 1, wherein at least some of the microwells in each set of the plurality of microwell sets contain a single bead.

19. The method described in claim 10, wherein >> is twice as large as Bs.

20. The method described in claim 10, wherein Bs is the background fluorescence signal measured in a negative PCR reaction.

21. The method described in claim 7, wherein the defined subarrays of the plurality of microwell sets are present in at least two adjacent fluid passages of a plurality of spaced apart fluid passages.

22. The method of claim 11, wherein each of the defined subarrays of the plurality of microwell sets includes two sets of microwells.

Images