Analysis system with microfluidic device, microfluidic

Abstract

The invention relates to an analysis system having a microfluidic device, a microfluidic device, and a related method. An analysis system has a housing having a chamber sized and configured to receive at least one microfluidic device. The system also includes 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 held in the housing; and the sub-array selection module is communicated with the controller. The subarray selection module is configured to select a subset of the set of micropores of at least one fluid channel of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during an assay.

Description

This application is a divisional application of the patent application for "Analysis Systems with Microfluidic Devices, Microfluidic Devices, and Related Methods" with the filing date of November 5, 2019, the application number of 201980036615.0 (PCT / US2019 / 059773).

[0001] Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 62 / 760,604, filed on November 13, 2018, and U.S. Provisional Patent Application Serial No. 62 / 825,523, filed on March 28, 2019, the contents of which are incorporated herein by reference as if fully set forth herein.

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

[0003] Statement Regarding Electronic Submission of a Sequence Listing A sequence listing in ASCII text format, submitted under 37 C.F.R. § 1.821, titled 5470-860WO_ST25.txt, 817 bytes in size, generated on October 29, 2019, and submitted via EFS-Web, is provided in lieu of a paper copy. This sequence listing is incorporated by reference herein in its entirety.

[0004] Copyright Retention Part of the disclosure of this patent document contains copyrighted material. The copyright owner, The University of North Carolina at Chapel Hill, N.C., does not oppose the reproduction by anyone of the patent document or the patent disclosure as it appears in the patent files or records of the U.S. Patent and Trademark Office, but reserves all copyrights in other respects whatsoever. Technical Field

[0005] The present invention relates to systems, fluidic devices (e.g., high-throughput microfluidic devices), and methods of using them, e.g., adapted to obtain polymerase chain reaction (PCR) data from an encoded bead microwell array. Background Art

[0006] Polymerase chain reaction (PCR) is a highly sensitive method for amplifying fragments of genomic DNA (gDNA) or complementary DNA (cDNA). PCR has many applications, such as detecting trace nucleic acids to determine the presence of pathogenic organisms, gene expression, genotyping, genetic engineering or modification, and forensic applications. PCR amplification provides outstanding target identification and quantification over a wide range of analyte concentrations. However, simultaneous and quantitative analysis of many analytes by PCR has proven to be extremely challenging. Detection based on intercalating dye fluorescence can only determine the total dsDNA concentration, and thus it is not possible to analyze multiple templates simultaneously in a single reaction vessel using this detection method. Fluorescent probe technologies (i.e., TaqMan, molecular beacons, or other chemistries) can be used for low-level multiplexing of reactions because different-colored fluorescent probes can be used as signaling reporters to amplify each target. The probes are also sequence-specific, reducing false positives from primer-dimer formation or non-specific amplification. The typical method of multiplexing with conventional microtiter plates or microfluidic real-time PCR (rt-PCR) is to use a small number of reaction wells, each containing three different-colored probes. However, designing multiplex primer and probe sets is generally considered challenging because they require an additional level of careful design and optimization to ensure compatibility with each other. Although multiplexing using at least six dyes has been demonstrated, multiplexing by this method is ultimately limited by spectral overlap between the instrument and the dyes, limited to approximately four-color detection, with one color typically reserved for an internal standard dye. SUMMARY OF THE INVENTION

[0007] Embodiments of the present invention provide methods for limiting photobleaching in encoded bead arrays, optionally while obtaining real-time PCR data.

[0008] Embodiments of the present invention provide formulations of PCR master mixes and magnetic particle chemistries that are particularly suitable for encoded bead arrays.

[0009] Embodiments of the present invention relate to sample analysis devices for assays, systems for analyzing signals from arrays of micro-wells of a fluid device, and assay methods.

[0010] Embodiments of the present invention relate to an analysis system. The system includes: a housing that contains a chamber sized and configured to receive at least one microfluidic device; an optical system coupled to the housing that is 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 held in the housing; and a subarray selection module in communication with the controller. The subarray selection module is configured to select a subgroup of a micropore group of at least one fluid channel 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 may include at least one magnet held in the housing adjacent to the at least one microfluidic device. The magnet may be configured to translate along at least one fluid channel during a bead loading operation to magnetically couple to beads to guide the beads to travel along the fluid channel and into a bead retention segment of the micropores.

[0012] The microfluidic device may include a plurality of fluid channels, each fluid channel having multiple groups of micropores positioned along a length dimension associated with a direction between a sample input port and a second opposing port. The selected subgroup of the micropore groups may be associated with a single row of a first subgroup of laterally aligned micropores of the plurality of fluid channels, optionally associated with a subgroup of aligned micropores from each of the plurality of fluid channels.

[0013] Before initial image acquisition and / or photoexcitation, the subarray selection module identifies the subgroup of the micropore groups to define the positions of other micropore groups of the micropore groups of the microfluidic device.

[0014] The subarray selection module may select different subgroups of the micropore groups of the microfluidic device at different reaction steps of the assay (e.g., different thermal cycles of the assay), and direct the optical system to only excite the currently selected subgroup of the micropore groups in the different fluid channels, and then direct the camera of the optical system to sequentially or concurrently acquire images of the different subgroups of the micropore groups in the currently selected subgroup.

[0015] The subarray selection module may select the same subgroup of micropores at at least some different consecutive reaction steps of the assay (e.g., different thermal cycles of the assay), and direct the optical system to transmit light only to the currently selected subgroup of the micropore groups, and then direct the camera of the optical system to sequentially or concurrently acquire images of the different subgroups of the micropore groups, optionally acquiring images of multiple pairs of adjacent micropore groups in the currently selected subgroup of micropores.

[0016] The optical system can have at least a first filter and / or a second filter (optionally more than two, e.g., between 3 and 100), the at least first filter and / or second filter defining excitation light of a corresponding first wavelength and / or second wavelength for beads of a first target encoding and beads of a second target encoding, for decoding a set of bead groups held in the one or more sets of micropores of the at least one fluid channel of the microfluidic device.

[0017] The system can have a signal analysis module integrated in the controller and / or coupled to the controller. The signal analysis module can be configured to obtain an analog signal for each micropore in a selected subgroup of the set of micropores. When the amplitude varies with the cycles of the assay, the analog signal can provide PCR data and determine the concentration of the target molecule of the reaction associated with the defined bead type through the analog signal.

[0018] The signal analysis module can be further configured to obtain one or more digital PCR signals for the set of micropores.

[0019] When the amplitude varies with the number of PCR cycles of the input material in the one or more sets of micropores of the fluid channel, the analog signal can provide real-time PCR data, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / micropore. The analog signal can define a threshold cycle or cycle threshold Ct and the number / concentration of target molecules of the target substance and / or molecular type, and for a given number of PCR cycles, a micropore reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or as negative if the fluorescence signal intensity (Si) is always less than the threshold. Ct is the number of cycles where Si >> Bs, where >> means at least 5 - 10% greater than Bs, optionally twice Bs, and / or 5 - 10 times the standard deviation of Bs, and where Bs is the background fluorescence signal, optionally measured in a PCR negative reaction.

[0020] After every other or every one of a plurality of different reaction steps (e.g., different thermal cycles of the assay), the analog signal can be obtained only for a single subgroup of the set of micropores in each fluid channel.

[0021] The analog signal can be the average value of Si (optionally excluding outlier data), median, mode value, or weighted value as the analog signal corresponding to each defined type of bead, and is provided as an estimate of the real-time PCR curve of similar reactions in the micropores of other sets of micropores of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all sets of micropores of each fluid channel are imaged after different or each reaction cycle.

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

[0023] In some specific embodiments, the defined FOV may optionally cover between 2 - 10 or more adjacent fluid channels and / or between 10 - 50% of the total number of fluid channels of the microfluidic device.

[0024] The controller and / or the signal analysis module may be configured to direct the optical system to obtain pre - PCR and post - PCR images and compare the signal intensities between them, optionally together with simulated data obtained using a selected subgroup of the micropore sets at different reaction steps of the assay, to determine positive and negative PCR reactions and optionally to determine one or more concentrations of the target substance and / or one or more molecular concentrations in the original sample provided to the fluid channels.

[0025] The system may further include a holder configured to hold a plurality of microfluidic devices in an aligned grid in the housing.

[0026] The holder may be formed of a thermally insulating material that provides a thermal barrier between adjacent microfluidic devices. Optionally, the holder may hold the plurality of microfluidic devices with the (sample / bead) input ports of the fluid channels facing outwards.

[0027] The microfluidic device may further include a dye homogenizer, optionally the dye homogenizer comprises an oligonucleotide.

[0028] The dye homogenizer may include non - extendable oligonucleotides and / or partially double - stranded DNA.

[0029] The dye homogenizer may include partially double - stranded DNA containing biotin and / or a C1 - C20 hydrocarbon chain.

[0030] The dye homogenizer may be present in the master mixture present in the microfluidic device and / or the dye homogenizer may be attached to the beads present in the microfluidic device.

[0031] Other embodiments relate to methods of analyzing a sample, such as for identifying a target substance and / or a target molecule. The method includes: providing a fluid analysis device having a first fluid channel with multiple sets of micropores positioned along the first fluid channel; obtaining signal intensity data (optionally in response to a transmitted light excitation signal) only from a defined subgroup of the multiple sets of micropores; and identifying a PCR reaction that is positive for a target substance and / or molecular type associated with a bead type and / or a target molecule, at least in part based on the obtained signal intensity data.

[0032] The method may include loading with a bead slurry (pretreated with one or more samples), and then sealing the plurality of sets of micropores along the first fluid channel before the obtaining step such that after sealing, each set of micropores is fluidically isolated from the other sets of micropores. The plurality of sets of micropores along the first fluid channel may be in fluid communication only during the loading and before the sealing step.

[0033] The method may include, after each of a plurality of successive reaction steps of the assay (e.g., after each of the plurality of successive thermal cycles of the assay), changing the defined subgroup of the plurality of sets of micropores to a different defined subgroup, whereby after each reaction step some of the sets of micropores are not imaged.

[0034] After a plurality of successive reaction steps of the assay (e.g., after each of the plurality of successive thermal cycles of the assay), the defined subgroup may remain the same, whereby after each reaction step some of the sets of micropores are not imaged.

[0035] The fluid analysis device may include a plurality of fluid channels having corresponding micropores (optionally, between 2 and 100), including but not limited to: a second fluid channel having a plurality of sets of micropores spaced along the second fluid channel; a third fluid channel having a plurality of sets of micropores spaced along the third fluid channel; and a fourth second fluid channel having a plurality of sets of micropores spaced along the fourth fluid channel. The first set of micropores of the plurality of sets of micropores in each of the first, second, third, and fourth fluid channels may be aligned in a first row. The second set of micropores of the plurality of sets of micropores in each of the first, second, third, and fourth fluid channels may be aligned in a second row. The third set of micropores of the plurality of sets of micropores in each of the first, second, third, and fourth fluid channels may be aligned in a third row. The fourth set of micropores of the plurality of sets of micropores in each of the first, second, third, and fourth fluid channels may be aligned in a fourth row. The defined subgroup may be a single (partial or complete) row among the first, second, third, and fourth rows.

[0036] The first, second, third, and fourth fluid channels may have straight or arcuate segments that are substantially parallel to each other and provide the first, second, third, and fourth sets of micropores.

[0037] The method may further comprise: prior to the obtaining step, transmitting a photoexcitation signal only to a defined subgroup; digital scanning, after, before, or both before and after the transmitting and obtaining steps, the defined subgroup of the plurality of sets of microwells to obtain an image of the set of microwells for identifying positive and negative PCR reactions associated with digital PCR; and electronically identifying microwells in the set of microwells that are positive for one or more target analyte molecules when the microwells are at an imaging temperature.

[0038] The exciting and obtaining may be performed to scan only one defined subgroup of the set of microwells after each of a plurality of consecutive reaction steps (such as thermal cycling) of an assay, wherein consecutive subgroups of the one defined subgroup may be different from each other.

[0039] By continuously or simultaneously obtaining images of different defined adjacent sets of microwells in different fluid channels of the defined subgroup of the set of microwells, a camera with a field of view (FOV) may be used to obtain the signal intensity only from the defined subgroup, the field of view having coverage of only one set of microwells or only a subgroup of the set of microwells in all of the fluid channels or a subgroup of adjacent fluid channels.

[0040] Each of the set of microwells may have a microwell array having microwells in the range of 1,000 - 1,000,000.

[0041] The fluid analysis device may have a plurality of spaced-apart fluid channels, each fluid channel having the set of microwells containing the microwell array. During an assay, the fluid channels may be fluidically isolated. At least some of the microwells of the microwell array may contain a single bead, optionally some or all of the microwells may be beadless or contain more than one bead.

[0042] The method may include electronically identifying the location of one or more sets of microwells at one or more locations in the microfluidic device before the transmitting and obtaining steps, and defining the location of other sets of microwells of the set of microwells based on at least a portion of the identified location.

[0043] The method of claim 20, the method further comprising transmitting a photoexcitation signal only to the defined subgroup before the obtaining step, and selecting a filter to provide an encoded wavelength for the transmitting step before the transmitting.

[0044] The obtained signal intensity may include obtaining an analog signal that provides real-time PCR data as the amplitude varies with the number of PCR cycles of the input material in the micro-well group, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / micro-well. The analog signal may define a threshold cycle or cycle threshold Ct and the number / concentration of target molecules of the target substance and / or molecular type, and for a given number of PCR cycles, a micro-well reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or the micro-well reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold.

[0045] Ct may be the number of cycles where Si >> Bs, where >> means at least 5 - 10% greater than Bs, optionally twice Bs, and / or 5 - 10 times the standard deviation of Bs, and where Bs is the background fluorescence signal, optionally measured in a PCR negative reaction.

[0046] After every other or every one of a plurality of different reaction steps of the assay (e.g., different thermal cycles of the assay), the analog signal may be obtained for only a single subgroup of the micro-well group in one or more fluid channels.

[0047] The analog signal may be the average, median, mode, or weighted value of Si (optionally discarding outlier data) as the analog signal corresponding to each defined type of bead, and may be provided as an estimate of the real-time PCR curve of similar reactions in the micro-wells of other micro-well groups of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all micro-well groups of each fluid channel are imaged after different reaction steps.

[0048] The obtained signal intensity may be obtained by using a camera with a field of view (FOV) that covers only a subgroup of the micro-well groups in at least two, optionally all adjacent fluid channels of the microfluidic device.

[0049] After a plurality of different reaction steps of the assay (e.g., different thermal cycles of the assay), the analog signal may be obtained for only a single group of the micro-well group in each fluid channel, and wherein the analog signal contains the average, median, mode, or weighted value of Si as the analog signal corresponding to each defined type of bead, and is provided as an estimate of the real-time PCR curve of similar reactions in the micro-wells of other micro-well groups of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all micro-well groups of each fluid channel are imaged after different reaction steps.

[0050] The fluid analysis device having the first fluid channel may include a plurality of additional fluid channels, the first fluid channel having the plurality of sets of micropores spaced along the first fluid channel, each additional fluid channel having a corresponding plurality of sets of micropores spaced along its respective length. The fluid analysis device may have a separate material input port for each of the first fluid channel and the plurality of additional fluid channels, and at least some of the fluid channels share a common opposite second port. The method may further include, prior to the obtaining step: fluidly loading a bead slurry pre-exposed to a respective sample for analysis into the respective input port; magnetically guiding the bead slurry along the fluid channel into different sets of micropores; flowing a main fluid mixture containing a dye from the second port towards the first port into the fluid channel; and then flowing a sealing oil from the second port towards the first port into the fluid channel, thereby sealing the micropore sets and the fluid channel from each other.

[0051] The main mixture may optionally include a dye homogenizer. The dye homogenizer may optionally be or include an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)).

[0052] The method may include placing a magnet adjacent to the fluid analysis chip and translating the magnet towards the second port before the main fluid mixture and the sealing oil flow.

[0053] The fluid analysis device (optionally the first fluid channel and / or the plurality of sets of micropores) may include a dye homogenizer, optionally wherein the dye homogenizer includes an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)).

[0054] The dye homogenizer may be present in the main mixture present in the fluid analysis device (e.g., present in the first fluid channel and / or the plurality of sets of micropores) and / or the dye homogenizer may be attached to the beads present in the fluid analysis device (e.g., present in the first fluid channel and / or the plurality of sets of micropores).

[0055] Still other embodiments relate to a microfluidic device. The device includes a plurality of fluid channels. Each of the plurality of fluid channels has a length dimension corresponding to the direction between a first port and an opposite second port, wherein at least a portion of the length dimension is configured as a straight or arcuate length segment. Each of the fluid channels includes a plurality of sets of micropores positioned along the straight or arcuate length segment of the length dimension.

[0056] The multiple sets of micropores in the multiple fluid channels may be arranged in rows, columns, or both rows and columns. The rows or columns correspond to the straight or arcuate length segments.

[0057] At least some of the multiple fluid channels may be substantially parallel on the straight or arcuate length segments.

[0058] At least some of the multiple fluid channels may be substantially parallel arcuate channels and may provide the arcuate length segments.

[0059] At least some of the multiple fluid channels may be substantially parallel and radially extend between the outer peripheral portion and the center of the device.

[0060] The multiple fluid channels may have a first set of fluid channels and a second set of fluid channels spaced apart from the first set. The first set of fluid channels may terminate at a first main mixture port as the second port, and the second set of fluid channels may terminate at a second main mixture port as the second port.

[0061] The first set of fluid channels and the second set of fluid channels may be circumferentially spaced apart.

[0062] The multiple fluid channels may include at least two fluid channels defining a first adjacent group and at least two channels defining a second adjacent group adjacent to the first adjacent group, each channel having a spatially aligned set of micropores.

[0063] The device may include a first interstitial space between each of the first fluid channels and the second fluid channels of the first adjacent group and the second adjacent group. The device may further include a second interstitial space between the first adjacent group and the second adjacent group, and the second interstitial space may have a lateral extent greater than that of the first interstitial space.

[0064] The multiple sets of micropores of each of the multiple fluid channels may have a common configuration. The multiple sets of micropores of each of the fluid channels may be aligned with each other in rows and / or columns. The multiple fluid channels may keep the respective input materials fluidly isolated from each other.

[0065] At least multiple sets of the micropores for the respective fluid channels may each include a quantity of micropores in the range of 1,000 - 1,000,000. The micropores of the set of micropores may be sized and configured to hold and retain a single bead, thereby allowing 1,000 - 1,000,000,000 reactions in the microfluidic device.

[0066] A group of first and second sets of micropores from at least a first fluid channel and a second fluid channel, optionally paired first and second sets of micropores, defines a subgroup of first sets of adjacent micropores. The device may include a transparent substrate extending over the sets of micropores of the fluid channels.

[0067] Each fluid channel of the plurality of fluid channels may have a separate first port at a first end as the first port, and alternating first ports of the plurality of first ports may be located at a first longitudinal position on the device, and the alternating other first ports may be located at a second longitudinal position on the device, the second longitudinal position being spaced from the first longitudinal position in the length dimension.

[0068] The first port may be a material input port and may be located at the first end of the corresponding fluid channel. The device may further include a fluid manifold that connects opposite second ends of at least some of the fluid channels to the second port.

[0069] The fluid channels may extend radially across the device. The inlet port of the corresponding fluid channel may be located at the outer peripheral portion of the device. The second port may be a single second port located at the center of the device connected to each of the fluid channels.

[0070] At least some of the fluid channels may be provided as concentric groups of fluid channels, each of the concentric groups of fluid channels having the arcuate length segment.

[0071] The concentric groups of fluid channels having the arcuate length segment may be provided as concentric groups of a plurality of circumferentially spaced fluid channels.

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

[0073] The dye homogenizer may optionally include oligonucleotides.

[0074] The dye homogenizer may include oligonucleotides. (For example, non-extendable oligonucleotides and / or partially double-stranded DNA (e.g., partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)).

[0075] The dye homogenizer may be present in the main mixture present in the microfluidic device and / or the dye homogenizer is attached to the beads present in the microfluidic device.

[0076] Other embodiments relate to bead loading strategies and the use of isolated bead inputs and common reagent reservoirs / inputs as shown and / or described.

[0077] Other embodiments relate to fully integrated fluidic analysis chips having raw (biological) sample input, sample preparation and processing, followed by loading of beads (pre-saturated with the corresponding sample) into a micro-well array, associated kits and microfluidic chips with pre-loaded reagents as shown and / or described.

[0078] Note that any one or more aspects or features described with respect to one embodiment may be incorporated into different embodiments, although not specifically described with respect thereto. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to change any originally filed claim or to file any new claim accordingly, including the right to amend any originally filed claim to depend on and / or incorporate any feature of any other claim, although not originally claimed in that manner. These and other objects and / or aspects of the invention will be explained in detail in the specification set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0080] The drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0081] Figure 1A is a schematic illustration of an exemplary fluidic device according to an embodiment of the invention.

[0082] Figure 1B is a schematic illustration of another exemplary fluidic device according to an embodiment of the invention.

[0083] Figure 2A is in Figure 1A a top perspective view of the fluidic device shown in

[0084] Figure 2B is corresponding to Figure 2A a digital top perspective photographic view of a prototype of the fluidic device.

[0085] FIG. 3A is an exemplary fluidic device having non-cylindrical micro-wells according to an embodiment of the invention.

[0086] FIG. 3B is a highly magnified view of several micro-wells taken from a portion of the fluidic device shown in FIG. 3A according to an embodiment of the invention.

[0087] Figure 3CIs an example measurement signal generated by the non-cylindrical micro-wells of the fluid device shown in FIG. 3A, illustrating a measurement signal separated from the bead background signal according to an embodiment of the present invention.

[0088] Figure 4A Is a graph of the raw fluorescence of negative and positive beads versus cycles.

[0089] Figure 4B Is a graph of the fluorescence of positive and negative slits of the micro-wells according to an embodiment of the present invention versus cycles.

[0090] Figures 5A - 5H Is a graph of the raw data versus cycles obtained by combining real-time PCR data of different subgroups of micro-wells from different fluid channels during PCR according to an embodiment of the present invention.

[0091] Figure 6A -H is according to an embodiment of the present invention in Figures 5A - 5H Graph of the normalized data of the data shown.

[0092] Figures 7A - 7H Is a graph of real-time PCR data collected from a set of bead wells in different fluid channels according to an embodiment of the present invention.

[0093] Figure 8 Is an example analysis system according to an embodiment of the present invention.

[0094] Figures 9 - 16 Is a schematic top view of different example devices with different fluid channel configurations according to an embodiment of the present invention.

[0095] Figure 17 Is a flowchart of an example analysis method according to an embodiment of the present invention.

[0096] Figure 18 Is a data processing system according to an embodiment of the present invention.

[0097] Figure 19 Is a flowchart of the operations that can be performed, after loading, on a fluid device loaded with beads and then thermally cycled according to an embodiment of the present invention.

[0098] Figures 20A - 20D Is an enlarged side perspective view of an example loading operation of a microchip using a magnet according to an embodiment of the present invention.

[0099] Figure 21 Is a top view of a plurality of microchips held by an adiabatic holder according to an embodiment of the present invention.

[0100] Figure 22Box plots of the initial bead fluorescence of each bead in the 5-arrays of one channel from two 8-channel microchips, where one microchip was treated with a master mix containing 10X SYBR and the other microchip was treated with a master mix containing 20X SYBR plus 5 μM non-extendable oligomers, according to an embodiment of the present invention.

[0101] Figure 23 Graph of the average molecules / bead (digital positive signal) of mycoplasma within a 12-plex respiration panel assay (although related to the digital positive signal, it can be a combination of digital and analog). Each point represents the result from one of the 5-arrays in a single channel of an 8-channel microchip, where each array was loaded with beads from the same sample according to an embodiment of the present invention.

[0102] Figure 24 Box plots of the encoded dye fluorescence signals from the mycoplasma groups in the 12-plex respiration panel, associated with each bead in the 5-arrays of the microchips treated with SYBR or +NE oligomers and SYBR, according to an embodiment of the present invention.

[0103] Figure 25 Schematic diagram of an example of pdsDNA of a bead, according to an embodiment of the present invention. Detailed Description

[0104] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. However, the present invention may 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.

[0105] The same numbers always represent the same elements. In the drawings, for clarity, the thickness of certain lines, layers, components, elements, or features may be exaggerated. The abbreviations "FIG." and "Fig." of the word "Figure" may be used interchangeably in the text and the drawings.

[0106] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit 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. It will be further understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "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" shall be interpreted to include X and Y. As used herein, the phrase "between about X and Y" means "between about X and about Y". As used herein, the phrase "from about X to Y" means "from about X to about Y".

[0107] 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. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict in terms, the present specification shall control.

[0108] In addition, as used herein, "and / or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative form ("or").

[0109] Unless the context otherwise indicates, the various features of the invention described herein are specifically intended to be used in any combination. In addition, the invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. By way of illustration, if the specification states that a composite comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be omitted and dispensed with.

[0110] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) shall be interpreted to include the recited material or steps "and those that do not materially affect one or more of the basic and novel characteristics of the claimed invention." See, re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, the term "consisting essentially of" as used herein shall not be interpreted as equivalent to "comprising."

[0111] It will also be understood that, as used herein, the terms "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting examples and / or alternative embodiments discussed herein and are not intended to indicate a preference for one or more of the embodiments discussed herein as compared to one or more other embodiments.

[0112] When referring to measurable values (such as amounts or concentrations, etc.), the term "about" as used herein means including the specified value and 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 including X and variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. The ranges of measurable values provided herein can include any other ranges and / or individual values therein.

[0113] As used herein, the terms "increase," "increase," "increased," "increasing," "enhance," and like terms indicate that a specified parameter is elevated by 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 in the text.

[0114] As used herein, the terms "decrease," "decrease," "decreased," "decreasing," "inhibit," and like terms mean that a specified parameter is decreased by 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 in the text.

[0115] It should be understood that when an element is referred to as being “on,” “attached to,” “connected to,” “coupled with,” “in contact with,” or the like another element, it can be directly on, attached to, connected to, coupled with, or in contact with the other element, or intervening elements may also be present. In contrast, when an element is referred to as, for example, “directly on,” “directly attached to,” “directly connected to,” “directly coupled with,” or “directly in contact with” another element, there are no intervening elements. Those skilled in the art will also understand that a structure or feature described as being “adjacent” to another feature may have portions that overlap with or are located beneath the adjacent feature.

[0116] For ease of description, spatial relative terms such as “below,” “beneath,” “under,” “above,” “on,” etc. may be used herein to describe the relationship of one element or feature to another element or feature(s) as illustrated in the figures. It should be understood that the spatial relative terms are intended to encompass different 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 is turned over, an element described as “below” or “beneath” another element or feature will then be oriented “above” the other element or feature. Thus, the exemplary term “below” can encompass both an orientation of “above” and “below.” The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatial relative descriptors used herein are to be interpreted accordingly. Similarly, unless specifically stated otherwise, terms such as “upward,” “downward,” “vertical,” “horizontal,” etc. are for purposes of explanation only.

[0117] It will be understood that although terms such as “first,” “second,” etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below may also be referred to as a “second” element without departing from the teachings of the present invention. Unless specifically stated otherwise, the order of operations (or steps) is not limited to the order presented in the claims or the figures.

[0118] 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 can be used to obtain real-time and digital polymerase chain reaction (PCR) data from a set of microwells 20 in an encoded microwell array 120a (bead microwell array) (FIG. 3B) and / or different fluid channels 15. In some embodiments, the fluidic device 10, method, and / or analytical system of the present invention can be used for applications such as non-PCR reactions, loop-mediated isothermal amplification (LAMP), and / or enzyme reactions for protein assays. 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 the amplicon concentration is collected after each cycle of PCR, and a cycle of PCR generally refers to a set of steps including denaturation of template DNA, annealing of primers to single-stranded DNA templates, and extension of primers by polymerase. See Arya et al., Expert Rev. Mol. Diagn. 5(2), (2005), pp. 209-219. 0.1586 / 14737159.5.2.2, the content of which is incorporated herein by reference as if fully set forth herein.

[0119] In some embodiments, the designs and methods described herein can reduce the data collection time for large arrays (e.g., encoded bead arrays, optionally including about 30,000 or more microwells), while also reducing the effects of photobleaching. The methods described herein can be applicable to other applications where varying signals are collected at various time points.

[0120] In some embodiments, the present invention may include, but is not limited to, substrates, devices, designs, solid supports (e.g., encoded solid supports), steps, and / or methods as described in the following: U.S. Provisional Application No. 62 / 673,343 titled "Compositions, Devices, and Methods for Improving a Surface Property of a Substrate", U.S. Provisional Application No. 62 / 736,525 titled "Compounds, Compositions, and Methods for Improving Assays", and / or as described in U.S. Patent No. 9,617,589, U.S. Application Publication No. 2015 / 0211048, International Publication No. WO 2017 / 112025, International Application No. PCT / US2016 / 042913, International Application No. PCT / US2016 / 043463, and / or International Application No. PCT / US2016 / 055407, the respective contents of which are incorporated herein by reference in their entirety.

[0121] In some embodiments, the methods of the present invention include methods of delivering reagents and / or targets to reaction wells using beads (e.g., superparamagnetic beads), 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 entirety.

[0122] The terms "microchip" and "microfluidic chip" are used interchangeably and refer to a substantially planar, thin device. The microfluidic chip can be rigid, semi-rigid, or flexible. The term "thin" refers to a thickness dimension of 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. The width and length of the microchip are typically less than about 6 inches, more typically between about 1 inch and 6 inches. However, in some embodiments, the microchip can be longer, such as having a length and / or width of 1 foot or longer. The microchip can have a perimeter that is polygonal, rectangular, circular, or other desired shape. The microchip can optionally have a width dimension that is less than the length dimension. In some embodiments, the diameter or width dimension of the microfluidic chip can be about 2.13 inches (54 mm) and / or the diameter or length dimension can be about 3.4 inches (85.5 mm). The microchip can include fluid channels of millimeter, micron, and / or nanometer dimensions.

[0123] The term "major dimension" refers to the width and / or depth dimension of the fluid channel.

[0124] The terms "micron-sized" and "microfluidic" with respect to fluid channels refer to fluid flow channels having a width and / or depth of millimeter, sub-millimeter, or smaller dimensions (e.g., the term includes millimeter, micron, and nanometer-sized channels, including segments or chambers provided by the channels). The channel can have at least one segment having a width and / or depth in the size range of 10 mm or less, typically less than 900 microns and greater than 1 nm.

[0125] The term "microwell" refers to a reaction well that is sized and configured to have a small reaction volume of about 100 microliters or less, and in some embodiments, can optionally accommodate a single bead, as will be further discussed below.

[0126] The term "bead" refers to a solid-phase component, such as a particle, granule, or microsphere, typically a magnetic or superparamagnetic microsphere, which can be one or more materials that are porous, surface-porous, or non-porous, such as polymers, photoresists, plastics, glass, silica, metals, or semi-metal oxides (including but not limited to alumina, titanium oxide, zirconium oxide, or other oxides), quantum dots, metal particles, etc., which are suitable for reaction wells.

[0127] The term "circuit" refers to a fully hardware implementation or an implementation combining software and hardware. The term "module" refers to an implementation that includes software and hardware or firmware.

[0128] The term "digital scanning" and its derivatives refer to obtaining a digital image of part or all of a microfluidic device, typically via a camera.

[0129] Now refer to Figure 1A 、 Figure 1B and FIG. 2, which shows an exemplary fluid device 10. The fluid device 10 includes at least one elongated fluid channel 15 having multiple sets of spaced-apart microholes 20 along the length of the corresponding elongated fluid channel 15. Each set of adjacent microholes 20 along the corresponding fluid channel 15 may (but is not necessarily required to) be separated by a gap space 21 without microholes. Figure 1B Illustrates sets of microholes 20 that may be arranged continuously along the corresponding fluid channel 15. Figure 1B Also illustrates that adjacent ports of the input port 16 (the first port) may be aligned, rather than longitudinally offset (in the orientation shown) as in Figure 1A .

[0130] One or more sets of microholes 20 along the corresponding fluid channel 15 may have different geometric footprints. In the case of using sets of spaced-apart microholes 20 as shown in Figure 1A , the length of the gap space 21 may be less than the length L of the footprint of adjacent sets of microholes 20.

[0131] As shown in Figure 1A and FIG. 2, at least one elongated fluid channel 15 includes eight adjacent fluid channels 151, 152, 153, 154, 155, 156, 157, and 158. However, a greater or lesser number of fluid channels 15 may be provided, and a greater or lesser number of sets of microholes 20 may be provided for all or any one of the fluid channels 15. For example, in some specific embodiments, the fluid device 10 may have between 2 and 2000 fluid channels 15, more typically 10 to 200 fluid channels 15.

[0132] As shown in Figure 1A , each or some of the fluid channels 15 may be configured with multiple sets of microholes 20 to include a first set of microholes 201, a second set of microholes 202, a third set of microholes 203, a fourth set of microholes 204, and a fifth set of microholes 205. However, a greater or lesser number of sets of microholes 20 may be provided. Figure 1B Illustrates that the fluid channels 15 may each have three sets of microholes 20. In some embodiments, the fluid channels 15 of the fluid device 10 may have between 2 and 100 sets of microholes 20, more typically 3 to 25 sets.

[0133] The interstitial space 21 between groups (micropore groups) of adjacent micropores 20 along a respective fluid channel 15 need not be provided by a physical gap or break in the continuous micropores 20, but can be defined by the field of view of the imaging system and / or the illumination window of the radiation source. The groups of micropores 20 can be provided as a continuous array of micropores 120 along the length of the respective fluid channel 15, as shown in Figure 1B as shown.

[0134] Bubbles or constrictions for alignment beads can be provided in the fluid channel 15. Bubbles or constrictions can be provided between adjacent groups of micropores 20, which provide a respective array (micropore array 120a) of micropores 120 in the fluid channel 15 to control fluid movement / sealing or to facilitate or control bead loading.

[0135] Referring again to Figure 1A and FIG. 2, multiple groups of micropores 20 for the fluid channels 15 can be arranged in aligned substantially parallel rows, marked with position markers, such as alphanumeric markers for the rows, shown as letter markers A - E for rows R1 - R8, and aligned parallel fluid channels 15, optionally marked in columns as C1 - C8, which define an array 10a of micropores of the fluid device 10. However, other formats can be used, and the fluid device 10 need not be marked in a human - readable form. More or fewer rows and more or fewer columns can be used. Adjacent channels 15n (shown as pair 15p of fluid channels 15) can be closely spaced and separated from another adjacent group of adjacent channels 15n (i.e., another pair of channels 15p of fluid channels 15) by the interstitial space 19 provided by the substrate of the fluid device 10. Adjacent channels 15n can be separated by a distance smaller than the interstitial space 19 of other adjacent channels 15n. Although adjacent channels 15n are shown as provided in pairs of channels 15p, for example, three, four, five or even more adjacent channels (including an entire row) can define an adjacent group of channels 15n.

[0136] The groups of micropores 20 can have a footprint with a rectangular outer perimeter 20r that encloses a respective group or array (micropore array 120a) of micropores 120 (single - bead micropores) (FIG. 3B). However, other geometries can be used.

[0137] Each fluid channel 15 can include a separate, dedicated input port 16 (fluid first port), which can be an input port for introducing a desired input material, optionally including a sample. Referring to Figure 2A 、 Figure 2B , the input port 16 (fluid port) can include a reservoir 16r having a through - hole 16v through the overlying substrate 10u to direct the input material from the reservoir 16r to the input port 16 of the fluid channel 15.

[0138] Each fluid channel 15 can merge at one end opposite the input port 16 into a second port 18, which can be the main mixture / oil port. The second port 18 can also be connected to a reservoir 18r through a through-hole 18v. The second port 18 can be in fluid communication with more than one fluid channel 15. The fluid device 10 can include a fluid manifold 18m in fluid communication with all or some of the fluid channels 15. Thus, there can be a separate input port 16 for each fluid channel 15 and a smaller number of second ports 18 than input ports 16, because some or all of the fluid channels 15 can merge into a common second port 18 via the manifold 18m or directly from a fluid channel extension 15e( Figure 14 , Figure 15 ).

[0139] Although not shown, each fluid channel 15 can have its own separate second port 18. Different groups of fluid channels 15 can be in fluid communication with corresponding different second ports 18 (main mixture / oil ports)( Figure 16 ). As will be further discussed below, once bead-loaded and sealed, each group of micropores 20 in a corresponding fluid channel 15 can be fluidly isolated from each other, and each fluid channel 15 can be fluidly isolated from other fluid channels 15.

[0140] As shown in Figure 1A , Figure 1B and FIG. 2, for a corresponding array 10a (channel array), the fluid channels 15 can be substantially parallel over a straight length segment including multiple groups of spaced-apart micropores 20. The term "substantially" when referring to "parallel" means that the fluid channels 15 in the array 10a are parallel or nominally parallel over at least a portion of the length segment (i.e., can vary at an angle of 10% or less slightly from adjacent centerlines C / L drawn through the respective longitudinal extension centers of the fluid channels 15): shown as straight length segments in FIGS. 1, 2, Figure 9 , Figures 10 - 15 and shown as arcuate length segments in Figure 16 .

[0141] Referring to FIGS. 3A-3C, each group or some groups of micropores 20 (micropore groups) can include multiple micropores 120 having a bead retention segment 120w and an assay signal segment 120s. The assay signal segment 120s can be in line with the bead retention segment 120w and parallel to and / or with the top or covering substrate 10u of the fluid device 10( Figure 2A) The main surface is straight. The assay signal segment 120s is in fluid communication with the bead retention segment 120w of the corresponding micro-well 120. The assay signal segment 120s can generate a narrow-tailed assay signal 122, which allows separation of the bead background signal at the bead retention segment 120w (well retention segment). In some embodiments, the assay signal segment 120s can have a geometry into which beads cannot physically enter. The bead retention segment 120w and the assay signal segment 120s are fluidly connected such that reagents and / or analytes released from the beads held in the bead retention segment 120w can diffuse or otherwise mix in the entire common solution volume of the bead retention segment 120w (well). By spatially separating the beads from the assay signal segment 120s (detection region), the contribution of bead fluorescence to the signal can be reduced or eliminated, improving the signal-to-noise ratio. The geometry of the micro-well 120 (well) can allow a high, typically single-bead occupancy loading of the reaction well while increasing the corresponding reaction volume, potentially improving reaction efficiency.

[0142] Figures 9 - 11 and Figure 16 illustrate that the fluid device 10 can include multiple spaced-apart arrays 10a1, 10a2, 10a3, 10a4 of fluid channels (whereas Figure 11 also shows a fifth array 10a5 of fluid channels 15). Figure 16 Illustrate three concentric groups of four circumferentially extending arrays 10a1, 10a2, 10a3, 10a4 of 12 array groups.

[0143] Each array 10a of the fluid channels 15 can have a single common second port 18 for introducing a loading buffer, a sealing oil, and / or a master mixture.

[0144] The term "master mixture" refers to a PCR master mixture and can be added to the fluid channels 15 before sealing the micro-wells 120 (wells) (Figure 3B) from each other to reach each group of micro-wells 20. In some embodiments, an immiscible oil can be used as the sealing oil for sealing. The master mixture of the present invention can be primer-free. That is, the PCR master mixture of the present invention can exclude primers for some specific embodiments, such as the SiRCA platform.

[0145] As an alternative or in addition, the top substrate 10u ( Figure 2A ) can include a flexible substrate, such as silicone (e.g., polydimethylsiloxane (PDMS)), and sealing can be achieved by pressing the flexible substrate flat onto the array 10a.

[0146] Figure 16It is illustrated that the fluid channel 15 can be an arcuate fluid channel 15a and circumferentially extends around at least a portion of the diameter of a circular geometry. Different arrays 10a1 - 10a4 of fluid channels can be circumferentially spaced apart, and each array 10a can have a common single second port 18.

[0147] Figure 11 Illustrated is a fluid channel 15 extending towards a center that defines the location of the second port 18, wherein at least one inner leg 18i of a manifold 18m extends radially towards the second port, and the centerline C / L of a corresponding array A R extends radially across the fluid device 10 towards the second port 18.

[0148] Figures 12 - 15 Illustrated is a fluid channel 15 configured as a radially inwardly extending channel 15r. The channel 15r extends radially inwards from an outer peripheral portion of the fluid device 10, where an input port 16 (first port) is towards the center of the device to the second port 18. Figure 14 and Figure 15 Illustrated is that the size of the fluid channel 15 decreases along its length towards the center of the fluid device 10 to the second port 18, and when the group of micropores approaches the second port 18, the size of the group of micropores 20 decreases from a first group of micropores 201 to other groups 202, 203, 204. In this case, the groups of micropores 20 that are imaged simultaneously (collectively) can be arranged differently from other configurations (not parallel rectangles, etc.).

[0149] An array 10a (microchip array) can define the array position of each group of micropores 20 of each fluid channel 15 corresponding to position addresses (e.g., row and column addresses). For example, a row and the relevant positions on that row, such as 1A (or A1), 2A (or A2), 7E (or E7), and 8E (or E8). The array 10a can be configured to provide some groups of micropores 20 as corner micropores 20c. In Figure 1A the embodiment shown in and Figure 2, the groups of micropores 20 at the array positions A1, A2, A7, A8, E1, E2, E7, and E8 are adjacent corner (sub) - groups 20c of the groups of micropores 20. Additionally, more than two fluid channels 15 can be configured to provide adjacent groups of micropores to define a corner group 20c of micropores 20.

[0150] Note that in Figure 1AThe fluid device 10 shown in and FIG. 2 has a rectangular outer perimeter 10p, the length dimension of which is greater than the width dimension (“W”), optionally between 15 - 30 mm wide. The length dimension can be 2 - 4 times or more the width dimension. The width dimension of the fluid channel 15 can correspond to the direction of the width dimension W of the fluid device 10. The fluid channel 15 is shown arranged to extend along the length dimension of the fluid device 10. However, the fluid channel 15 can alternatively be oriented to extend across at least a portion of the width dimension of the fluid device 10. In some embodiments, some of the fluid channels 15 can extend along the length dimension and some along the width dimension( Figure 9 , Figure 10 ). In some embodiments, the fluid channels 15 extend in a radial direction( Figures 11 - 14 ). In some embodiments, the fluid channels 15 extend circumferentially( Figure 16 ).

[0151] The respective groups of micropores 20 in each fluid channel 15 can be aligned to have the same longitudinal and lateral extent and position, as shown in Figure 1A , for example, or can be staggered, where the first end of a group of micropores 20 is above or below the adjacent first end of an adjacent and at least partially laterally aligned group of micropores in another fluid channel 15 (not shown).

[0152] Some groups of the respective fluid channels 15 or the micropores 120 (FIG. 3A) of each group of micropores 20 can be provided as a dense array of micropores having between 1,000 - 1,000,000 or more micropores 120, more typically 1,000 - 100,000 or 1,000 - 50,000 micropores 120.

[0153] In some embodiments, the fluid channels 15 of the fluid device 10 (microfluidic device) can analyze a corresponding sample with groups of micropores 20 each providing an array of micropores 120 (micropore array 120a), the array being in the range of 1,000 - 1,000,000 or greater, optionally 10,000 - 200,000. The fluid device 10 (microfluidic device) can include a plurality of fluid channels 15, typically between 10 - 100, providing up to 20,000,000 cumulative micropores 120 in the fluid device 10.

[0154] In an example embodiment, each fluid channel 15 may have micropores 20 along its length that are between 2 - 10 groups (or more), fluidically isolated after sealing. A group of micropores 20 may have any suitable number of micropores 120. In some embodiments, each group of micropores 20 has the same number of micropores 120. In some embodiments, one or more groups of micropores 20 (in some embodiments, each group) may contain less than 12,000 micropores 120 (e.g., 1,000, 2,000, 5,000, 8,000, 10,000, or 12,000 micropores 120). In some embodiments, one or more groups of micropores 20 (in some embodiments, each group) have at least 1,000 micropores 120 (e.g., 1,000, 10,000 or more, e.g., 12,000 - 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 micropores 120). Each micropore 120 (FIG. 3B) typically accommodates a single bead (some may be unloaded, while some may carry two beads, which may be undesirable in some applications).

[0155] In some embodiments, the fluid device 10 (microchip) may be configured to run assays with more than one bead in the micropores 120. See, for example, the example multi - bead assays 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.

[0156] The different groups of micropores 20 for the respective fluid channels 15 may have the same or different numbers of micropores 120 (FIG. 3B). The different groups of micropores 20 for each fluid channel 15 may have the same or different numbers of micropores 120 (FIG. 3B).

[0157] As shown by the virtual boxes indicating a single field of view (FOV) 25 at positions A1, A2 in Figure 1A , the fluid device 10 may be configured such that a subgroup or sub - array 10s of the array 10a (the entire micropore array) (shown as adjacent to group 15n, optionally a pair 15p of fluid channels 15) occupies the entire single FOV of an optical signal detector (220, Figure 8 )(e.g., a camera). This allows the optical signal detector 220 ( Figure 8)(e.g., at least one camera) image different subgroups 10s of the set of microwells 20 (e.g., a single microwell array 120a of a plurality of different fluid channels 15 (sample channels) covered by the FOV 25) at any given point in time. Adjacent sets 15n may be arranged as a subgroup or all (e.g., a partial or entire row) of the fluid channels 15 of the corresponding set of microwells 20.

[0158] As will be further discussed below, the light excitation source 225 ( Figure 8 ) may be configured to transmit excitation light having a defined wavelength range to a defined subarray 10s of the array 10a (the entire array) of the set of microwells 20 of the fluid device 10 ( Figure 1A , Figure 1B ) which may be a subgroup of the set of microwells 20 and contains at least one set of microwells 20 from a plurality of adjacent sample channels of the fluid channel 15 (sample channel), but less than all of the set of microwells 20 (microwell group) of the fluid device 10 (microchip). For example, the defined wavelength range may be associated with light for assaying signals and / or encoding fluorophores.

[0159] As shown in Figure 1A , the defined subarray 10s may be associated with a single contiguous location (e.g., an entire single row) of the array 10a of microwells of the fluid device 10. The defined subarray 10s may then be imaged or optically analyzed, typically by successively imaging the set of microwells 20 in the corresponding adjacent channels 15n of the fluid channel 15 at a single location after a first assay cycle. That is, the assay comprises a plurality of assay cycles associated with thermal cycling. The defined subarray 10s that are excited and imaged may change after each assay cycle (i.e., at the end of the first assay cycle). In some embodiments, at the end of the first assay cycle, the system 200 (analysis system) ( Figure 8 ) only excites and images a subgroup 10s of the array 10a associated with the set of microwells 20 in the first row R1, while at the end of the second assay cycle, the system 200 (analysis system) only excites and images the set of microwells 20 in the second row R2.

[0160] The fluid device 10 may comprise an upper substrate 10u and a lower substrate 10b attached together ( Figure 2A , Figure 2B)。The upper substrate 10u and the lower substrate 10b can be the same or different. Either or both of the substrates 10u, 10b can be rigid and include, for example, glass, quartz, silicon, or a suitable metal. The covering substrate 10u can be visually transmissive and is typically transparent. Either or both of the substrates 10u, 10b can be a polymer, such as silicone or other polymeric materials (such as PMMA, COC, COP, PDMS, PP, PE, PTFE, or Kapton (polyamide), and many other materials), and it can provide one or more arrays 10a of micropores. In some embodiments, either or both of the substrates 10u, 10b can include silicon (e.g., can be a silicon wafer) and / or can be functionalized (e.g., silanized) with a hydrophobic compound (such as an alkylsilane and / or an alkylthiol). In some embodiments, either or both of the substrates 10u, 10b include silicon silanized with an alkylsilane.

[0161] The fluid channels 15 having spaced-apart groups of a plurality of micropores 20 can have sidewalls and a bottom plate, wherein each group of micropores 20 provides a corresponding micropore array 120a, the bottom plate is formed in one or more substrates 10u, 10b to have an open top surface and a closed bottom surface, and the sidewalls extend therebetween. One or more spacers, top substrates, membranes, or covers can be used. The top substrate, membrane, or cover can seal, cover, or otherwise enclose the upper surface of one or more fluid channels and / or the array of reaction pores. In some embodiments, the fluid channels 15 can be etched into the top substrate 10u, and a group of micropores 20 can be provided at the bottom, and a closed surface of the fluid channels 15 can be formed.

[0162] The analyte in the material input can be any analyte of interest, including, for example, various mixtures, including synthetic and biological macromolecules, nanoparticles, small molecules, DNA, nucleic acids / polynucleic acids, peptides, proteins, etc. The analyte can be one or more analyte molecules.

[0163] The material input can contain a sample or an analyte of a sample and can include one or more polar metabolites, such as amino acids or charged molecules, molecules, peptides, and proteins. The sample and / or analyte can also or alternatively include molecules extracted from biological fluids, blood, serum, urine, dried blood, cell growth media, lysed cells, beverages, or foods. The sample can also or alternatively include environmental samples, such as water, air, or soil.

[0164] 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, the oligonucleotide can be from about 15 nucleotides to about 50 nucleotides, or from about 20 nucleotides to about 25 nucleotides, which can be used, for example, as primers for polymerase chain reaction (PCR) amplification assays and / or as probes in hybridization assays or in microarrays. The oligonucleotides of the present invention can be natural or synthetic, such as DNA, RNA, PNA, LNA, modified backbones, etc., or any combination thereof, as is well known in the art. The oligonucleotides of the present invention can be single-stranded, double-stranded or partially double-stranded. In some embodiments, the oligonucleotide is non-extendable (e.g., by PCR).

[0165] Probes and primers (including those for amplification and / or detection) are oligonucleotides of any suitable length (including naturally occurring oligonucleotides such as DNA as well as synthetic and / or modified oligonucleotides), but typically range in length from 5, 6 or 8 nucleotides, up to 40, 50 or 60 nucleotides, or longer. Such probes and / or primers can be immobilized on or conjugated to a solid support, such as beads, chips, pins or microtiter plate wells, and / or conjugated to or labeled with a detectable group, such as a fluorescent compound, a chemiluminescent compound, a radioactive element or an enzyme.

[0166] The polymerase chain reaction (PCR) can be performed according to known techniques. See, for example, U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. Typically, PCR involves, first, treating a nucleic acid sample under hybridization conditions with an oligonucleotide primer (e.g., in the presence of a thermostable DNA polymerase) for each strand of a specific sequence to be detected, so that an extension product of each primer complementary to each nucleic acid strand is synthesized, wherein the primer is sufficiently complementary to each strand of a specific sequence to hybridize therewith, so that an extension product is synthesized from each primer, which, when separated from its complement, can be used as a template for synthesizing extension products of other primers, and then treating the sample under denaturing conditions, separating the primer extension products from their templates if one or more sequences to be detected are present. These steps are repeated cyclically until the desired degree of amplification is obtained. The detection of the sequence of amplification can be carried out as follows: by adding an oligonucleotide probe (for example, an oligonucleotide probe of the present invention) that can hybridize with the reaction product to the reaction product, the probe carries a detectable label, and then detects the label according to known techniques, or by directly observing on a gel. The sequence of amplification can also be detected by adding an intercalating dye to the reaction mixture and monitoring the fluorescence signal intensity, which is proportional to the total mass of the double-stranded DNA. Other non-intercalating dyes can also be used, but higher fluorescence signals are produced in the presence of dsDNA. Although described about PCR reactions according to embodiments of the present invention, it should be understood that other nucleic acid amplification methods can be used, such as reverse transcription PCR (RT-PCR), including isothermal amplification techniques such as rolling circle amplification or loop-mediated isothermal amplification (LAMP), and nucleic acid sorting methods can also be used. In addition, other methods can be used, such as enzyme amplification reactions, such as enzyme-linked immunosorbent assay (ELISA). In such a case, micropore temperature control can be used to control amplification reactions to optimize the imaging of micropore arrays, and some regions can only use images (digital signals) before and after the complete reaction for imaging, while other regions or subarrays can be imaged during the reaction to obtain analog signals.

[0167] DNA amplification techniques (such as aforementioned) can include the use of probes, a pair or a group of three, four, five or six or more probes, or two pairs of probes, its specific binding contains the DNA of interested polymorphism or mutation, but under identical hybridization conditions, not so strongly combined with the DNA that does not contain interested polymorphism, and it can be used as one or more primers for amplifying DNA or its part in an amplified reaction. In this article, such probes are sometimes referred to as amplification probes or primers. In some embodiments, two or more probes can be used, for example, in a competitive method, for detecting single nucleotide polymorphisms (SNPs) and / or other mutations and / or rare alleles (such as insertion and deletion).

[0168] The term "reagent" refers to any substance or compound, including primers, nucleic acid templates, and amplification enzymes, that is added to a system to cause a chemical reaction or to observe whether a reaction occurs. Amplification reagents or reagents refer to those reagents (deoxyribonucleotide triphosphates, buffers, etc.) that are commonly used for amplification in addition to primers, nucleic acid templates, and amplification enzymes. Typically, the amplification reagents are placed and contained, together with other reaction components, in a reaction vessel (test tube, micro-well, etc.).

[0169] The term "magnetic" as used herein includes ferromagnetic, paramagnetic, and superparamagnetic properties.

[0170] Generally, an oligonucleotide probe for detecting DNA containing a polymorphism or mutation of interest is an oligonucleotide probe that binds to the DNA encoding the mutation or polymorphism under the same hybridization conditions but does not bind well to DNA that does not contain the mutation or polymorphism. The oligonucleotide probe is labeled with a suitable detectable group, such as those described below. In this document, such a probe is sometimes referred to as a detection probe or primer.

[0171] Embodiments of the present invention can be used for singleplex reactions in a compact array (SiRCA), which is a platform for sensitive, highly multiplexed nucleic acid (NA) and protein quantification. SiRCA combines sample preparation with digital PCR precision in a large-scale parallel, highly multiplexed format. NA-SiRCA is a magnetic bead-based variant of PCR, where the assay panel can be made up of sets of microbeads uniquely encoded with fluorescent dyes. Before combining the sets, each set can be functionalized with different primer pairs specific for a particular target NA. During the assay, the primers on the beads can hybridize to the NA targets, capturing, concentrating, and purifying them from the sample matrix. The beads can be washed and loaded into a set of micro-wells 20, each set containing an array of thousands of micro-wells 120 (micro-well array 120a), typically with one bead occupying one bead retention segment 120w (bead well). The micro-wells 120 can have any suitable volume capacity. In some embodiments, the volume capacity of the micro-wells 120 ranges from about 10 microliters or less, such as 10 femtoliters to less than about 10 microliters, or about 10, 50, or 100 femtoliters to about 200, 500, or 1,000 femtoliters. In some embodiments, the volume capacity of the micro-wells 120 is about 100 femtoliters.

[0172] Before sealing the pores from each other using immiscible oil, a PCR master mix (containing all reagents except primers) is added. After heating, the beads release primer sets and captured targets, forming singleplex PCRs in each microwell 120. Thousands of spatially multiplexed PCRs are rapidly generated with no interference between primer sets. Signals from a dsDNA intercalating dye indicate target amplification, and the target ID is determined by the encoding of the beads. At low concentrations, single molecule counting (digital signal) allows for precise analyte quantification. At higher concentrations, real-time PCR (analog signal), measured as the average fluorescence signal from all wells of the same reaction type, extends the quantification range above the digital signal saturation point.

[0173] The use of non-cylindrical microwell geometries can improve detection in bead array-based technologies. These well geometries are fabricated such that one region of the well is optimized for magnetic loading and bead retention, while another region of the well is for signal detection. After loading the beads into the bead region of the well, a small volume of reagent fluid is isolated in the well using a method such as immiscible fluid sealing. An example single array chip with inserts showing the microwells 120 is shown in FIG. 3A. Other embodiments may include variations in the design. In a particular embodiment, it is preferred that one region has a bead retention segment 120w (pocket or container) (FIG. 3B) that is about 100% - about 150% of the bead diameter in diameter and about 50% - about 185% of the bead diameter in depth, and has a fluid-connected region consisting of a measurement signal segment 120s (narrow pocket or slit) (FIG. 3B) or other geometries into which a standard bead cannot physically fit. Both regions are fluid-connected such that reagents or analytes released from the beads can diffuse or otherwise mix throughout the common solution volume. By spatially separating the beads from the detection region, the contribution of bead background fluorescence to the signal can be reduced or eliminated, improving the signal-to-noise ratio ( Figure 4B ). Additionally, these geometries allow for high single occupancy loading of the reaction wells while increasing their reaction volume, potentially improving reaction efficiency.

[0174] NA-SiRCA can be performed using beads in a microtube format and / or a microwell array 120a on a fluid device 10 (chip), allowing for about 35,000 or even more reactions, e.g., up to about 1,000,000,000 reactions.

[0175] We have developed a 12-plex respiratory panel using synthetic targets. Excellent linearity was observed over the entire range of 10 copies / μL to 10,000,000 copies / μL using a combination of digital and analog signals. Digital quantification at low concentrations (50 - 150 copies / μL) showed high precision with low variance, allowing discrimination between small copy number changes. We have shown that RNA can be assayed with reverse transcriptase. Additional assays under development include multiplex protein assays (cytokines) using immuno-PCR with sub-pg / mL LOD.

[0176] Typically, digital assays do not require imaging of the assay signal after each amplification cycle. However, analog assays rely on the detection of the threshold cycle (Ct) to determine how many NA molecules initially bound to the beads. Ct is typically determined as the first cycle in which the signal is significantly higher than the background signal (5 - 10% or more higher). The difference in Ct can be used to determine the initial concentration of the sample. Assuming 100% amplification efficiency in the PCR reaction, the Ct (C tS ) from the target sample can be compared to a reference reaction (C tR ) with a known initial concentration to determine the concentration in the target sample: Those skilled in the art will understand how to adjust for efficiencies less than 100%.

[0177] The resolution of concentration measurements (i.e., the precision with which the concentration can be determined) depends on how frequently the fluorescence is measured. Maximum resolution is obtained by imaging after each amplification cycle (i.e., approximately every doubling of the amplicon concentration). However, frequent imaging results in photobleaching of the dye, which depends on the exposure of the dye to the excitation source. This is particularly significant for small volume PCR reactions used in SiRCA. Figure 4A Traces of real-time PCR plots are shown where the effect of photobleaching is evident by the negative slope of the baseline signal.

[0178] The microfluidic device of the present invention can optionally provide one or more (e.g., 1, 2, 3, 4 or more) samples to be tested in a high-throughput application and / or manner. The microfluidic device of the present invention can have a single substrate that includes one or more arrays 10a having multiple sets of microwells 20 with associated microwell arrays 120a thereon (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, optionally on a single array 10a or multiple arrays 10a (the latter in Figures 9 - 11 and Figure 16Two or more fluid channels 15 provided in (shown in) the substrate can be assembled on the substrate to form a fluid device 10 (aggregated multi-sample device) for high-throughput applications. In some embodiments, one or more sets of microwells 20 in a plurality of fluid channels 15 in one or more arrays 10a of the fluid device 10 (microfluidic device) can be thermocycled simultaneously.

[0179] Digital PCR can be accomplished in such formats because the array can be imaged once before PCR and once after PCR to obtain digital PCR data. In some embodiments, the array (e.g., an array including about 40,000 wells) is imaged by a sensor having a pixel density high enough such that light from one well is collected by at least one pixel. In some embodiments, each microwell 120 of the devices of the present invention can be imaged by about 4 - about 20 pixels, or about 4 - about 100, 150, or 200 pixels. In some embodiments, the systems, devices, and / or methods of the present invention provide one or more sets of aligned or partially aligned microwells 20 in two or more adjacent fluid channels 15 for imaging with a single sensor without custom optics and / or precise alignment.

[0180] An alternative to custom optics is to use a precision positioning stage to move the camera and / or the microfluidic chip horizontally, vertically, and / or rotationally such that the array can be imaged in a sequential manner after the extension step of each PCR cycle. This method allows imaging of many arrays at the cost of increased assay time. For fluid channels 15 having sets providing 30,000 - 40,000 or more microwells 20, e.g., 62.5k microwells 120 per fluid channel 15 (sample channel), translation and imaging typically add about 1 second to about 10 seconds per cycle for each array added, although high-performance hardware can reduce this time at significant expense. The increased imaging time effectively increases the extension time of the PCR cycle. Although the PCR reaction rate is cycle-dependent rather than time-dependent, the long delays in thermal cycling required for imaging a large number of arrays can be problematic because active polymerase tends to write dimers and non-specific products if given enough time. This can lead to increased background signal and reduced assay specificity.

[0181] Some embodiments of the present invention provide solutions to these imaging and / or data collection problems, e.g., by taking advantage of the unique features of the encoded microwell arrays 120a (bead microwell arrays) (FIG. 3B) according to embodiments of the present invention.

[0182] The properties of the encoded bead arrays can permit unique methods of mimicking PCR data collection and analysis. If the concentration of the target molecules is in the analog domain, the average number of molecules per bead type will be approximately the same. Thus, the Ct for each bead group will be independent of the position of the beads in the array, and different regions of the array can be imaged at different cycles of PCR while still obtaining single-cycle resolution. That is, after each cycle of PCR, it is not necessary to image the entire array, only a representative portion of the array. In some embodiments, instead of using a typical approximately square array, an elongated array can be used such that the arrays from each sample can be positioned in close proximity. This can permit the camera to image two or more sample arrays at once and image many arrays in a short period of time without the need to translate long distances or through complex paths.

[0183] One implementation of the method can be illustrated by the following example of the layout of the fluidic device 10 shown in Figure 1A . In this design, the fluidic channels 15 are grouped (shown as pairs 15p) such that a subgroup of the array 10s of groups of microwells 20, each having corresponding microwells 120 (FIG. 3B) from two or more fluidic channels 15 (optionally having two or more different samples), can be imaged by the camera in a single frame (the field of view of the camera is represented by the frame boxes 25 at 1A, 2A in Figure 1A , Figure 1B ). In this example, a representative portion of the bead retention fragments 120w (wells) from each group of microwells 20 across a row R1 can be imaged in only four acquisitions across the width of the device (successive acquisitions A1 (also interchangeably referred to as "1A"), A2 (also interchangeably referred to as "2A"), then A3 (also interchangeably referred to as "3A"), A4 (also interchangeably referred to as "4A"), then A5 (also interchangeably referred to as "5A"), A6 (also interchangeably referred to as "1A"), then A7 (also interchangeably referred to as "7A"), A8 (also interchangeably referred to as "8A")), with only short lateral translations between the frames. Thus, in some embodiments, the method of the present invention includes collecting analog data without imaging the entire array 10a of different groups of microwells 20.

[0184] Figure 17A flowchart illustrating an exemplary method for identifying a target substance and / or target molecule. A fluid analysis device having a first fluid channel is provided, the first fluid channel having multiple sets of micropores spaced along the first fluid channel (block 600). An optical excitation signal is (optionally) transmitted only to a defined subset of the multiple sets of micropores (block 610). In response to transmitting the optical excitation signal, signal intensity data is obtained only from the defined subset of the multiple sets of micropores (optionally) (block 620). The bead type that is positive for the target substance and / or target molecule is 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, an assay (such as a protein assay) can produce a chemiluminescent signal that can be detected and / or imaged without optical excitation.

[0185] The method may optionally include loading and then sealing the multiple sets of micropores along the first fluid channel prior to the transmission step such that after sealing, each set of micropores is fluidically isolated from the other micropore sets. The multiple sets of micropores along the first fluid channel are in fluidic communication only during loading, prior to the sealing step (block 602).

[0186] Optionally, the method may include changing the defined subset of the multiple sets of micropores to a different defined subset after each of a plurality of consecutive reaction steps of the assay (e.g., after each of a plurality of consecutive thermal cycles of the assay), whereby some micropore sets are not imaged after each reaction step (block 612).

[0187] Optionally, after a plurality of consecutive reaction steps of the assay (e.g., after each of a plurality of consecutive thermal cycles of the assay), the defined subset remains the same, whereby some micropore sets are not imaged after each reaction step (block 614).

[0188] Optionally, the method includes digitally scanning the defined subset of the multiple sets of micropores after, before, or before and after the transmission and acquisition steps; and electronically identifying the pores that are positive for one or more target analyte molecules when the array of pores is at an imaging temperature (block 624).

[0189] Optionally, obtaining the signal intensity only from the defined subset using a camera having a field of view (FOV) that covers only a subset of the group of micropores 20 in the corresponding fluid channel 15 (sample channel) and only a subset of one or more adjacent, neighboring fluid channels 15, by way of example, 2 - 10 or more adjacent fluid channels in the first, second, third, and fourth or more fluid channels, by continuously obtaining images of the subset that occupies a single physical location of the fluid device 10 (microchip), e.g., continuously obtaining data from different individual rows of the first, second, third, and fourth or more corresponding rows (block 622).

[0190] Optionally, obtaining the signal intensity includes obtaining an analog signal. When the amplitude varies with the number of PCR cycles of the input material in the micropore set of the fluid channel, the analog signal can provide real-time PCR data, and the concentration of the target molecule of one or more of the defined bead types is approximately equal to or greater than 1 molecule / micropore, and wherein the analog signal defines a threshold cycle or cycle threshold Ct, for a given number of PCR cycles, a positive micropore reaction is identified when the fluorescence signal is greater than the threshold, or a negative micropore reaction is identified if the fluorescence signal is always less than the threshold, where Ct is the number of cycles where Si >> Bs, where >> is at least 5-10% greater than Bs, optionally twice Bs, and / or 5-10 times the standard deviation of Bs, and wherein Si is the fluorescence signal intensity and Bs is the background fluorescence signal (box 626), as measured in a PCR negative reaction. The Ct can be compared with a reference reaction to determine the number of target molecules at the start of the PCR reaction.

[0191] Optionally, obtaining the signal intensity includes obtaining an analog signal, which can provide real-time PCR data when the amplitude varies with the measured number of PCR cycles of the input material in the micropore set of the fluid channel, and determining whether the PCR reaction is positive for the target molecule when Si >> Bs, which is at least 10% greater than Bs, optionally twice Bs, and / or 5-10 times the standard deviation of Bs. Si can be calculated as the mean, median, mode value, or weighted value of the analog signals corresponding to each defined type of bead. In some embodiments, Si can be calculated by first excluding outliers (usually in the early PCR cycles) and then determining the mean or median.

[0192] In some embodiments, reference Figure 19 and Figures 20A - 20D , loading the beads into the fluid device 10 can be carried out as follows. The fluid device 10 is wetted with a loading buffer and positioned on a holder 900 ( Figure 21 ) coupled to or cooperating with an array of linear magnets or magnets 800 (box 700). The through holes 16v of the input port 16 can optionally be offset in position in two adjacent rows to allow bead loading with a minimum risk of cross-channel contamination (box 702). When the magnet 800 is held below the odd-numbered channels ( Figure 20A ), a bead slurry from the material input can be pipetted into the through holes 16v (box 710). Then the magnet 800 is moved so that it stays below the even-numbered through holes ( Figure 20B ), and the beads are pulled into the odd-numbered channels (box 720). Of course, in Figure 2A and Figure 20BThe steps shown can be carried out in the reverse order. After adding the bead slurry to the even-numbered through-holes, the beads are pulled into the micro-holes (array chamber) using the magnet 800( Figure 20C )(block 730). By sliding the magnet under each group of micro-holes 20 (array) along the fluid channel 15 towards the manifold channel and / or the second port 18 (main mixing port), the group of micro-holes (array segment) is loaded with beads, and optionally the movement is controlled with a hard stop device 810( Figure 20C ) such that the beads do not enter the common manifold channel (which could allow bead mixing between samples) (block 740).

[0193] In each fluid channel 15, there are segmented groups, optionally physically spaced apart, of micro-holes 20 (block 704), shown in Figure 1A as five groups of micro-holes 201 - 205 (each having 12.5k holes), containing a total of thousands or millions of micro-holes 120 for each material input or fluid channel 15, optionally approximately 62.5k micro-holes 120.

[0194] After the micro-holes 120 (Figure 3B) of each group of micro-holes 20 in the fluid channel 15 are loaded with beads, the remaining beads are magnetically dragged back towards the through-holes 16v, input port 16( Figure 20D ) by translating the magnet 800 (block 750). The main mixture flows under pressure applied to the common second port 18 (reservoir) until it fills the fluid channel 15 (block 760). Then sealing oil is pumped under pressure until the sealing oil seals the group of micro-holes 20 and displaces the excess main mixture from the fluid channel 15 (block 770).

[0195] Optionally, absorbent pads can be placed on the reservoir 16r and / or the through-holes 16v to collect the aqueous solution when the aqueous solution is displaced from the fluid device 10, or a partial vacuum can be used to remove the liquid when the liquid oozes out from the through-holes 16v and / or the reservoir 16r( Figure 2A )(block 772).

[0196] Optionally, a second microfluidic channel, channel network, or reservoir can be used to store the waste effluent instead of allowing it to ooze out from the through-holes. Such a waste reservoir, whether microfluidic or as a separate, optionally attached, reservoir or group of reservoirs, can be fluidly connected to the channel and / or through-holes in a way that prevents it from filling before the addition of the main mixture and / or the sealing step, using a wax or paraffin valve or a hydrophobic constriction. In some embodiments, after the beads are added, the through-holes can be sealed with a membrane such that the membrane blocks the aqueous flow and / or the oil flow, optionally allowing air to escape through the membrane. In some embodiments, the membrane can cover other through-holes or ports and act as a path for air to escape from the microfluidic channel, chamber, or through-hole.

[0197] The fluid device 10 is then placed on the thermal cycling microscope stage (frame 780) of a test system 200h( Figure 8 ) for thermal cycling, PCR, and imaging.

[0198] An array 10a of groups of micro-wells 20 in the fluidic channels 15 is divided into sub-arrays, namely, A - E, which herein refer to rows and fluidic channel numbers (i.e., A1 is the group of micro-wells 201 at the upper left corner of the device, and E8 is the group of micro-wells 205 at the lower right corner of the device). A1 - A2 can be imaged by a single image acquisition.

[0199] Example data collection and processing method 1 (“Method 1”) A camera (electronic signal detector 222, Figure 8 ) can be initially aligned and focused on sub-groups of groups of micro-wells 20 at positions A1 - A2, A7 - A8, E7 - E8, and then E1 - E2. The positions of the corner 20c groups of these X, Y, and Z (focus height) micro-wells 20 can be used to calculate the positions of all internal groups of micro-wells 20. Focus can be collected while the array 10a is maintained at the imaging temperature (usually an extended temperature of about 60 °C to about 72 °C) to eliminate dimensional changes due to thermal expansion. Then, each group of micro-wells 20 in each fluidic channel 15 is imaged at the imaging temperature before the PCR thermal cycling to obtain a baseline “before PCR” image.

[0200] After the first cycle of PCR, sub-groups of groups of micro-wells 20 in the first row (A1 - A2, A3 - A4, A5 - A6, and A7 - A8) are sequentially imaged in a total of four frames. After the second cycle of PCR, the second row (B1 - B2, B3 - B4, B5 - B6, and B7 - B8) is imaged. After the E row is imaged after the 5th PCR cycle, the series is repeated, and the A row is imaged after the 6th PCR cycle. If the 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 position of the next row while a denaturation step is performed before the next annealing / extension / imaging step.

[0201] After completion of the thermal cycling, a “post-PCR” image of the entire array 10a is taken at the imaging temperature. The array 10a is cooled (usually cooled to about 20 °C - about 25 °C), and another set of post-PCR images is taken (to clarify problem areas of the chip where, due to failure of the sealing technique, the aqueous master mix may connect two or more wells). If desired, the array 10a can be imaged at a series of temperatures to determine the melting point and / or range of the amplicons detected after the PCR cycles. The filter sets (F1, F2, Figure 8) is changed to the encoding wavelength, and a first set of encoded images is taken for each subgroup of the group of micro-wells 20, and subsequently, if desired, a second set of encoded images is taken using a second filter set, and the process is repeated as needed for decoding the bead groups.

[0202] The processing of the imaging data can be divided into two domains: digital and analog. The digital PCR signal can be determined by comparing the pre-PCR and post-PCR images to determine whether the increase in the signal of the intercalating dye exceeds a set threshold for each well. In some embodiments, only the post-PCR image may be needed to determine digital positive or negative. Alternatively, the pre-PCR and post-PCR images can be used, optionally together with six thermal cycle images, to determine whether the fluorescence signal changes during the imaging process in a manner consistent with the PCR amplification of the correct amplicon. For example, at low concentrations of the target, where some portions of the PCR reaction population of the target are negative, an increase in signal is expected in later cycles, and outliers showing strong signals early in the cycle can be considered non-specific amplification products or mis-identified beads.

[0203] At high concentrations of the target where most or all of the PCR reactions are positive, real-time analog signals are used. Although the signal from each group of micro-wells 20 in the array 10a has a resolution of five cycles (since each sub-array is imaged only once every five PCR cycles), at each PCR cycle, at least one of the micro-wells 20 of the fluid channel 15 of the array 10a for each sample is imaged. Since the target molecules should be equally distributed over a given bead group, the data from the images can be combined to reconstruct a Ct curve with single-cycle resolution, even though the same wells are not imaged after each cycle. This is achieved by averaging the signal intensities from each bead group for each sample for each image. Then the average signal for each bead type can be plotted as a real-time PCR curve, as shown in Figures 5A - 5H as shown in Figure 5A As can be seen, the real-time signal for cycle 1 comes from the first image of the group of micro-wells 20 at position 1A, while the data for cycles 2, 3, 4, and 5 come from the first images of the sub-arrays 1B, 1C, 1D, and 1E. At cycle 6, the data from the second image of the sub-array 1A is used; at cycle 11, the data from the third image of the sub-array 1A is used. This process is used for all eight fluid channels 15 on each fluid device 10 (chip) (and each channel can have the same sample or different samples, i.e., eight samples) to construct a real-time PCR curve.

[0204] As shown in Figures 5A - 5HAs can be seen, for a given fluid channel 15 and / or sample, the fluorescence intensity baseline across the sub-arrays (groups of micro-wells 20) is not completely uniform. This may be due to several factors, including the effective concentration of the embedded dye, the focus of the image, and / or the effects of different levels of silanization of the device. If they are normalized by dividing the PCR image signal by the pre-PCR image signal ( Figures 6A - 6H ), the baseline is more linear and the calling of the cycle threshold can be more reproducible. The irregularity of the maximum PCR signal will have a low impact on the quantification of the target. A protocol that helps control the dye concentration and thus the fluorescence signal intensity across all arrays will be discussed later.

[0205] Example data collection and processing method 2 ("Method 2") In some embodiments, data collection can be performed by imaging only a group of sub-arrays (a subgroup of the group of micro-wells 20) for all PCR cycles (in this non-limiting example, 30 cycles). In this method, using the positions of the corner sub-arrays as described in Example Method 1 above, the camera can be aligned and focused. Then each group of micro-wells 20 is imaged at the imaging temperature before the PCR thermal cycling to obtain a baseline "pre-PCR" image.

[0206] After the first cycle of PCR, a group of micro-wells 20 in a row (a sub-array) is imaged, for example, different groups of micro-wells 20 in row C are imaged. The group of micro-wells 20 is imaged successively in a total of four boxes at positions C1-C2, C3-C4, C5-C6, and C7-C8. After the second cycle of PCR, the same row is imaged again. This process continues for 30 PCR cycles, and in this example, as seen in FIGS. 7A-7H, the same row will be imaged 30 times for 30 PCR cycles.

[0207] After the thermal cycling, a "post-PCR" image of the entire array is taken at the imaging temperature. The array is cooled (usually cooled to about 20°C to about 25°C) and another set of post-PCR images is taken. If desired, the array can be imaged at a series of temperatures to determine the melting point or range of the amplicons detected after the PCR cycles. The filter sets (F1, F2, Figure 8 ) are changed, and a first set of encoded images is taken for each sub-array, followed by a second set of encoded images taken using the second filter set.

[0208] Similar to Example Method 1, the processing of the imaging data can be divided into two domains: digital and real-time (analog). For this method, the digital PCR signal can be determined only by comparing the pre-PCR and post-PCR images to determine whether the increase in the embedded dye signal for each well exceeds a set threshold (Table 1). In some embodiments, the digital can be obtained separately from the post-PCR image.

[0209] 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 a synthetic DNA sequence of FluA incorporated into different sample channels. The values in the table represent the percentage positive of the corresponding encoded bead sets.

[0210] The set of microwells 20 that are not imaged during PCR will have very little photobleaching, while the imaged subarray may show significant photobleaching. In some embodiments, this may require different thresholds to distinguish digital positives from digital negatives.

[0211] Analog signal processing of the imaged subarray is similar to traditional full-array imaging methods. The signal of each bead cluster is averaged and plotted against the number of PCR cycles ( Figures 7A - 7H ). For analog signals where the target molecule is more than 1 copy / bead, the average signal from several hundred beads should be sufficient to accurately and precisely determine the Ct.

[0212] The methods of the present invention (e.g., the above-described example methods 1 and 2) can have advantages over typical data collection schemes where all wells in the array are imaged at each PCR cycle. In some embodiments, the methods of the present invention reduce data collection time while producing single-cycle real-time PCR resolution for analog quantification. The compact, narrow design of the well array can allow imaging of fluid-separated arrays from multiple samples in a single image.

[0213] Advantages of collecting analog data across multiple subarrays (e.g., as described with respect to example method 1) can include reduced photobleaching. For example, example method 1 can provide significantly reduced photobleaching because each subarray is only exposed to 20% of the illumination used for the standard collection scheme. Data collected over several cycles, even at lower cycle resolution, may be more reliable for identifying false-positive beads for digital quantification because only pre-PCR and post-PCR images are compared. Additionally, a lower degree of photobleaching can allow for sufficient signal at lower concentrations of intercalating dyes (e.g., SYBR Green I) than those required for imaging all 30 cycles. Since intercalating dyes can inhibit PCR, for some targets, lower concentrations can improve PCR efficiency or melting point determination.

[0214] Advantages of collecting simulated data (i.e., sub-arrays or subgroups of microwells as described for Example Method 2) from a single row of microwells 20 can include less reliance on normalization of data used for Ct determination, making the analysis more straightforward. Additionally, pre-PCR images can be used to determine which row has the best bead loading for simulated real-time PCR analysis. This can help improve problems that may occur if one or several sub-arrays are not loaded with sufficient beads for simulated analysis (although this problem can be addressed by other means, such as tightly controlling array size, geometry, position, bead population size, and bead size).

[0215] Example Method 1 and Example Method 2 are merely illustrative examples of the device designs and data collection methods of the present invention, and various combinations and / or modifications can be used. For example, a single row of microwells 20 groups in different fluid channels 15 can be imaged every cycle or every other cycle, in combination with imaging of other sub-arrays at various different intervals. Although some combinations may require more acquisition time, in some embodiments, the improved quality of digital or simulated data may be worth such a trade-off. In some embodiments, all or most groups of microwells 20 can be imaged during some PCR cycles (and typically not every and / or not every PCR cycle).

[0216] The illustrated fluid device 10 is merely a demonstration of a design concept (i.e., dividing the bead / reaction well array into a series of sub-arrays, separating and positioning them such that they can be imaged in an advantageous manner), and other designs can be used in the practice of the present invention. In some embodiments, the fluid device 10 of the present invention can include one or more arrays 10a of fluid channels 15 for more than eight fluid channels 15, such as 9 - 1,000 fluid channels 15, including 16, 32, 64, 96, 384, etc. or more fluid channels 15, each fluid device 10 having a corresponding group of microwells 20. In some embodiments, the fluid device 10 of the present invention can be fabricated in silicon, glass, polymer (e.g., injection molded and / or thermally embossed plastic), and / or metal film. In some embodiments, the fluid device 10 of the present invention can be a composite structure. In some embodiments, the fluid device 10 of the present invention can contain only the bead / reaction well array and the array chamber, or can include other structures, such as sample handling microfluidic circuits and / or labeling or amplification reagents required for performing assays.

[0217] Electrical and / or mechanical circuits, actuators, and / or sensors can be integrated, attached, and / or assembled into and / or onto the fluid device 10, optionally for performing assays and / or reading out assays.

[0218] Polystyrene beads can absorb hydrophobic molecules as well as charged molecules from aqueous solutions. The long and narrow geometry of the micropores 120 (micropore arrays) can result in the depletion of certain components (such as the embedded dye) in the main mixture as the main mixture flows through the fluid channel 15. The beads in the first subarray can absorb a larger amount of components (such as the embedded dye) when encountering the main mixture, while the last array can absorb less components from the partially depleted solution. When the component is a dye, this can result in a gradient of dye concentration across the chip and thus, when comparing the arrays in the channels, can result in an initial bead fluorescence (Figure 20). These differences in dye absorption can lead to several complexities in the assay, as too much dye may inhibit PCR, while too little dye results in a weak amplification signal. Additionally, changes in the concentration of the embedded dye may cause an undesired change in the apparent fluorescence intensity of the encoded dye, which may make decoding the array more difficult. This effect may be one of the reasons for the lower intensity signals in some of the subarrays observed in Figures 5 and 6.

[0219] The dye absorption of the beads can be modified by including (e.g., adding) various reagents to cause more uniform dye (e.g., SYBR) absorption / distribution across the entire array. These reagents (also referred to herein as "one or more dye homogenizers") can include, but are not limited to, oligonucleotides of various lengths, such as single-stranded DNA, RNA, double-stranded DNA (optionally having a low melting temperature such that the reagent is double-stranded during addition to the channel and sealing of the wells, but single-stranded during the imaging step of PCR and thus not associated with the embedded dye), partially double-stranded DNA, ionic or non-ionic polymers such as dextran sulfate or polyamines, surfactants, detergents, phase transfer catalysts, dextran, cyclodextrin, silicone, polysiloxane, fluorocarbon, polyfluorohydrocarbon, fluorosiloxane, hydrocarbon, alcohol, hydrofluorocarbon, 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 fluorescence signal when associated with the embedded dye are particularly well-suited for this purpose. The dye homogenizer can be miscible with the main mixture. In some embodiments, the dye homogenizer is present in and / or added to the main mixture. The dye homogenizer (e.g., partially double-stranded DNA) can be present in the main mixture at a concentration in the range of about 100 nM to about 100 μM or any range and / or value therein (e.g., about 1, 5, or 10 μM to about 15, 25, 50, or 100 μM). In some embodiments, the dye homogenizer can be attached (e.g., covalently and / or non-covalently) to a portion of the bead. In some embodiments, the bead can contain an amount of the dye homogenizer in the range of about 300 pM, 1 nM, or 3 nM to about 300 μM or 3 mM.

[0220] For example, an oligonucleotide (oligomer), such as a non-extendable oligomer (NE oligomer) optionally containing biotin (e.g., biotin at the 3' end), can alter (e.g., reduce) dye (e.g., SYBR) binding during addition of the master mix. In some embodiments, the dye homogenizer can comprise an ssDNA molecule having a 3'-attached biotin linked via a hexacarbon chain, but other modifications and / or linkers (e.g., C1-C20 hydrocarbon chains, such as C1-C20 alkyl branched or unbranched) can also function. In some embodiments, the non-extendable oligomer can be used as a dye homogenizer because it is less likely to negatively impact PCR than additional primers capable of forming dimers. As shown in Figure 22 , it can be seen that addition of an NE oligomer to a master mix containing SYBR Green I reduces the variation in average initial bead fluorescence (CV 0.7%) compared to SYBR alone (CV 4.8%). Since variations in SYBR absorption (and thus the concentration of SYBR in the PCR reaction) can modify target amplification and fluorescence signals, variations in SYBR absorption between arrays result in variations in the number of positive reactions (i.e., digital PCR signals). In Figure 23 , the average number of target molecules per bead of a mycoplasma target sequence was compared between 5 subarrays in the channels of a chip that had either SYBR alone (CV 23%) or SYBR plus an NE oligomer (CV 4%) in the master mix. The reduced variation can lead to a more accurate determination of the average number of molecules per bead and thus a more accurate determination of the concentration of mycoplasma. Additionally, controlling the concentration of the intercalating dye across the entire array can potentially result in smaller variations in the melting temperature of dsDNA and thus enable a more accurate determination of the amplicon melting point.

[0221] In some embodiments, the fluorescence signal from the encoded dye can be affected by the concentration of the intercalating dye, and it can become difficult to decode the array (e.g., assign beads to clusters during the encoding assay) when there are large differences in dye absorption by the beads. In Figure 24 , samples with SYBR alone in the master mix showed a displacement of the fluorescence signal observed in the encoded dye fluorescence channels between different subarrays (CV 14%). When a non-extendable oligomer was added to the solution, the variation in the apparent encoded dye fluorescence signal was smaller (CV 5%).

[0222] As an alternative or in addition, differences in dye uptake during loading of the array with the master mix can be addressed by other means. For example, in some embodiments, beads can be soaked in a buffer containing an intercalating dye prior to loading the beads into the microwell array. The beads can be soaked before, during, and / or after incubation with the sample. As an alternative or in addition, the surface of the beads can be functionalized to alter dye uptake. For example, when more primers bind to the surface of the beads, the absorbance of the dye increases compared to when fewer primers are bound. Partially double-stranded oligonucleotides (pdsDNA) can be used, which are optionally linked to the beads via biotin-TEG ligation chemistry available from IDT, because dsDNA associates more strongly with the intercalating dye than ssDNA. In some embodiments, prior to the assay, beads with pdsDNA oligomers are soaked in a buffer containing an intercalating dye. The beads are then incubated with the sample in hybridization buffer, washed, and loaded into the microwell array. In some embodiments, prior to sealing the reaction wells from each other, a master mix containing all of the reagents required for PCR (except for primers and intercalating dye) and optionally including a dye homogenizer can be introduced into the channels of the microwell array, optionally with an immiscible sealing oil. Examples of pdsDNA are shown in Figure 25 .

[0223] Figure 25 Showing 5'-biotin TEG ligation from IDT (top). Examples of partially double-stranded DNA primers are shown (bottom). The DNA primers shown have a first strand with the sequence SEQ ID NO:1; and a second strand with the sequence SEQ ID NO:2.

[0224] The melting temperature of the pdsDNA that can be used to alter dye uptake can be optimized such that under storage and / or hybridization conditions, the pdsDNA can remain partially double-stranded; however, during PCR, the pdsDNA can dissociate to produce ssDNA primers with low background fluorescence because its association with the intercalating dye is not as strong. In some embodiments, the linker attaching the primer to the biotin functional group (e.g., a carbon chain, optionally a C1-C20 hydrocarbon) can be modified to modulate its hydrophobicity and / or ionic charge and thus its uptake characteristics for dyes or other reagents.

[0225] In some embodiments, the fluidic device 10 of the present invention can be fabricated using a holder 900 that holds a plurality of fluidic devices 10 (microchips), optionally configured in a grid 900g, as in Figure 21As shown. The retainer 900 may include an outer periphery 910 enclosing the closed grid 900g, and may define a lateral adiabatic barrier region 920 and a longitudinal adiabatic barrier region 930 between the sides and ends of adjacent fluid devices 10. The second ports 18 (main mixture ports) may be present facing each other across the barrier segments (longitudinal adiabatic barrier regions 930). The input ports 16 may be present facing the outer periphery 910 of the retainer 900. In some embodiments, the retainer 900 is made of an adiabatic material, and the fluid device 10 (chip) may include a thermally conductive substrate. Each of the smaller fluid devices 10 (microarray chips) in the grid 900g may be thermally cycled independently. The thermal cycling and / or imaging of the fluid devices 10 (microarray chips) in the grid 900g may be synchronized by the system 200 (testing system) such that each fluid device 10 (microarray chip) is imaged in a defined sequence.

[0226] In some embodiments, the fluid device 10 of the present invention may be manufactured from a substrate (such as plastic) that is an insulator, and optionally, different regions, areas, or locations of the same substrate may be thermally cycled and / or imaged in a defined sequence.

[0227] The fluid device 10 of the present invention may be used with commercially available automated sample preparation (such as pipetting robots and / or bead washing robots, such as Tecan, Hamilton, Beckman, or other automated systems), or it may be operated using a customized and dedicated reader device or platform. The reader used in the present invention may include a large format design suitable for high-volume in vitro testing, a benchtop format suitable for clinical laboratories or physician offices, a handheld format optimized for production sites or consumer or forensic testing, or a format incorporated into other systems.

[0228] The optical system (optical signal detector 220) that may be used ( Figure 8 ) includes but is not limited to dual-light or multi-light imaging systems, in which the entire array portion (or most of it) of the fluid device 10 may be imaged at low resolution, and a smaller portion of a selected group of the group of microholes 20 is imaged at high resolution, or a lens system that allows for rapid changes in the field of view, such as a zoom lens. Alternatively, other detection systems may be used, including electrochemical, absorption, and / or chemiluminescence detection.

[0229] The devices and / or methods of the present invention can be used in immunological PCR, reverse transcriptase PCR, singleplex reactions of proteins in compact arrays (protein-SiRCA), and many other variants of PCR or nucleic acid amplification. Different means for detecting amplicons can be used, such as molecular beacons or hydrolysis probes (e.g., TaqMan probes), or any such means known to those skilled in the art. Decoding images for determining the identity of each bead can be acquired before, during, and / or after the thermal cycling images.

[0230] According to some embodiments, a method for obtaining high-resolution real-time PCR data can be performed while reducing the effect of photobleaching on the assay signal in singleplex reactions in a compact array (SiRCA). The fluidic device 10 of the present invention can comprise two or more fluid channels 15, each channel having a set of microwells 20, optionally for one or more samples, and the fluidic device 10 (array) can use sub-composite imaging of the sets of microwells 20. Subgroups of different sets of microwells 20 can be imaged sequentially, where one segment is imaged after each amplification cycle. For each image, the average signal of each type of reaction for each sample can be calculated. By plotting the average signal from each reaction type for each amplification cycle, the cycle threshold for each reaction type can be determined with single-cycle resolution without the time required to image the entire array after each PCR cycle. For example, a fluidic device 10 that is 14 mm long × 1.25 mm wide with fluid channels 15 can be divided into five (row) segments A, B, C, D, and E. After cycle 1 of PCR, segment A is imaged. After PCR cycle 2, segment B is imaged, and so on. After 30 PCR cycles, each row / segment can be imaged six times. Although signals from each bead type can be / will be obtained for each PCR cycle, each row / segment (and thus each reaction) will only receive 1 / 5 dose of the excitation energy for recording fluorescence data. Thus, the method reduces the effect of photobleaching while maintaining signal-cycle real-time PCR resolution. Additionally, by narrowing the sets of microwells 20 in the corresponding fluid channels 15 (sample fluid channels) to one dimension, two or more fluid channels 15 having corresponding sets of microwells 20 can be placed close enough together such that they can be imaged quickly (e.g., one frame can contain more than one array), facilitating the ability to collect real-time PCR data for multiple samples with single-cycle resolution with reasonable imaging times.

[0231] The methods of the present invention can be applied to any array of encoded reactions such that the identity of the reactions can be determined, typically in real time.

[0232] In some embodiments, the devices of the present invention can be used to replace any multiplexed PCR or immunoassay panel. Applications of the embodiments of the present invention include but are not limited to biomedical or biological research, diagnosing diseases (including infectious diseases and / or oncology) through nucleic acid or protein biomarkers, veterinary applications, forensic analysis or genotyping, environmental monitoring, counterfeit detection, and / or biopharmaceutical production or quality control applications.

[0233] Figure 8 is a schematic illustration of an example system 200 (analysis system). The system 200 can include at least one controller 210 (generally including at least one processor) in communication with an optical system (optical signal detector 220), and the optical system (optical signal detector 220) includes an electronic signal detector 222, such as an optical detector including a camera or other imaging device or other signal detection device. The system 200 can also include a housing 200h and a heat source 240 for applying heat to one or more fluid devices 10 during an assay cycle.

[0234] The optical system (optical signal detector 220) can optionally include an excitation source 225 and one or more filters, which can include different filters (or filter sets) F1, F2 or even more filters, for example, by way of example, 2 - 100 filters, each or some of which can provide different encoded wavelengths for exciting beads held in one or more sets of microwells 20. The optical system (optical signal detector 220) can include a subarray selection module 250 to detect signals and / or image subgroups or subarrays 10s of an array 10a (microwell array) of corresponding fluid devices 10 (optionally in response to directing the optical system to selectively detect only one or more subgroups). Imaging and / or excitation can be performed continuously or in parallel for the defined subgroups. In some embodiments, light excitation is not required. For example, in some embodiments, an assay (such as a protein assay) can produce a chemiluminescent signal, which can be detected and / or imaged without light excitation.

[0235] The system 200 can also include a signal analysis module 260. The signal analysis module 260 can analyze assay signal data from different sets of microwells.

[0236] System 200 can obtain an analog signal defining a threshold cycle or cycle threshold "Ct", and when the fluorescence signal is greater than the threshold, the threshold cycle or cycle threshold "Ct" identifies the PCR reaction of the target substance and / or molecular type as positive, or when the fluorescence signal does not rise above the threshold, the PCR reaction of the target substance and / or molecular type is identified as negative. That is, Ct is the cycle where Si >> Bs, where >> is at least 5 - 10% greater than Bs, optionally twice Bs, and where Si is the fluorescence signal intensity and Bs is the background fluorescence signal. The Ct of an unknown concentration sample can be compared with the Ct of a known concentration sample to calculate the initial target concentration.

[0237] Controller 210 can communicate with a sub - array selection module 250 (i.e., including computer program code) configured to select different groups of micro - wells during different assay cycles. The modules (sub - array selection module 250 and / or signal analysis module 260) can be all or partially on the controller and / or the optical system (optical signal detector 220) or remote from the controller and / or the optical system. System 200 (analysis system) can include at least one processor (i.e., digital signal processor) and can include a transceiver 214.

[0238] Sub - array selection module 250 can be configured to identify a subgroup of (aligned) micro - wells in two, three, four, five or more adjacent fluid sample channels to provide position data. Optionally, the identified subgroup includes an adjacent group (i.e., corner group) of micro - wells in two or more adjacent fluid channels. The module (sub - array selection module 250) can use information having known spacing dimensions and array configuration to define the positions of other groups of micro - wells 20 (micro - well groups). As an alternative or in addition, alignment marks 27 (Figure 1) at one or more positions of the fluid device 10 can be used for this position data.

[0239] The modules (sub - array selection module 250 or signal analysis module 260) can be on system 200 (analysis system) or distributed in one or more servers 300. Server 300 can be embodied as an independent server or can be included as part of other computing infrastructure. Server 300 can be embodied as one or more enterprise, application, personal, pervasive and / or embedded computer systems, which can be independent or interconnected via public and / or private, real and / or virtual, wired and / or wireless networks including the Internet, and can include various types of tangible, non - transitory computer - readable media. Server 300 can also communicate with the network via wired or wireless connections and can include various types of tangible non - transitory computer - readable media.

[0240] In use, cloud computing can be used to provide the server 300, which includes providing computing resources on demand via a computer network. The resources can be embodied as various infrastructure services (e.g., computing, storage, etc.) as well as applications, databases, file services, mail, etc. In a traditional computing model, both data and software are typically fully contained on the user's computer; in cloud computing, the user's computer can contain little software or data (possibly an operating system and / or a web browser) and can merely be used as a display terminal for processes occurring on an external computer network. Cloud computing services (or an aggregation of multiple cloud resources) are typically referred to as the "cloud". Cloud storage can include a model of networked computer data storage in which data is stored on multiple virtual servers rather than being hosted on one or more dedicated servers.

[0241] The controller 210 can communicate with the server 410 or a computer via the transceiver 214 and / or a computer network or a cellular network. For a computer network, this can include one or more of a local area network (LAN), a wide area network (WAN), and can include a private intranet and / or the public Internet (also known as the World Wide Web or the "Web" or the "Internet").

[0242] As described in Figure 18 As illustrated, embodiments of the present invention can be configured as a data processing system 1116, which can include one or more processors 500, a memory 536, and an input / output circuit 546. One or more processors 500 can be part of an image processing circuit 500c. The data processing system can be incorporated into, for example, one or more of a personal computer, a database, a workstation W, a server, a router, etc. The system 1116 can be located on one machine or distributed across multiple machines. The processor 500 communicates with the memory 536 via an address / data bus 548 and with the input / output circuit 546 via an address / data bus 549. The input / output circuit 546 can be used to transfer information between the memory (memory and / or storage medium) 536 and another computer system or a network using, for example, an Internet Protocol (IP) connection. These components can be conventional components, such as those used in many conventional data processing systems, which can be configured to operate as described herein.

[0243] In particular, the processor 500 (which can be incorporated into the controller 210, Figure 8) can be a commercially available or custom microprocessor, microcontroller, digital signal processor, etc. The memory 536 can include any memory device and / or storage medium that contains software and data for implementing the functional circuits or modules used in accordance with the embodiments of the present invention. The memory 536 can 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, the memory 536 can be a content-addressable memory (CAM).

[0244] As further described in Figure 18 , the memory (and / or storage medium) 536 can include several types of software and data used in a data processing system: an operating system 552; application programs 554; input / output device drivers 558; and data 556. As will be understood by those skilled in the art, the operating system 552 can be any operating system suitable for use with a data processing system, such as or an operating system, or 95, Windows98, Windows2000, or WindowsXP operating systems, Unix, or Linux TM , IBM, OS / 2, AIX, and zOS are trademarks of International Business Machines Corporation in the United States, other countries, or both, while Linux is a trademark of Linus Torvalds in the United States, other countries, or both. Microsoft and Windows are trademarks of Microsoft Corporation in the United States, other countries, or both. The input / output device drivers 558 typically include software routines accessed by the application programs 554 through the operating system 552 to communicate with devices (such as the input / output circuit 546 and certain memory 536 components). The application programs 554 describe programs that implement the various features of the circuits and modules in accordance with some embodiments of the present invention. Finally, the data 556 represents the static and dynamic data used by the application programs 554, the operating system 552, the input / output device drivers 558, and other software programs that may be located in the memory 536.

[0245] The data 556 can include a (archived or stored) digital image data set 522. As further described in Figure 18As further described herein, in some embodiments of the present invention, the application program 554 includes a micropore subgroup selection module (sub-array selection module 250) and a signal analysis module 260. The application program 554 may be located in 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.

[0246] Although the present invention is described with reference to Figure 18 the application program 554 and the modules (sub-array selection module 250 and signal analysis module 260) in Figure 18 it, those skilled in the art will understand that other configurations also fall within the scope of the present invention. For example, these circuits and modules may also be incorporated into the operating system 552 or other such logical partitions of the data processing system, rather than as the application program 554. In addition, although the application program or modules (sub-array selection module 250, signal analysis module 260) are described in a single data processing system, those skilled in the art will understand that such functions may be distributed across one or more data processing systems, for example, in the type of client / server arrangement described above. Therefore, the present invention should not be construed as limited to the Figure 18 configuration described in

[0247] For ease of development, computer program code for operating a data processing system, method steps or actions, modules or circuits (or portions thereof) discussed herein can be written in a high-level programming language (Python, Java, AJAX (Asynchronous JavaScript), C, and / or C++). Additionally, computer program code for operating exemplary embodiments can also be written in other programming languages, such as but not limited to interpreted languages. Some modules or routines can be written in assembly language or even microcode to enhance performance and / or memory usage. Some modules or routines can also be written in scripting languages, including open-source scripting languages. However, the embodiments are not limited to a particular programming language. As described above, the functionality of any or all program modules can also be implemented using discrete hardware components, one or more application-specific integrated circuits (ASICs), or programmed digital signal processors or microcontrollers. The program code can execute entirely on one computer (e.g., a workstation), partially on one computer, execute as a stand-alone software package, partially on the workstation computer, and partially on another local and / or remote computer, or execute entirely on another local or remote computer. In the latter case, the other local or remote computer can be connected to the user's computer via a local area network (LAN) or wide area network (WAN), or can be connected to an external computer (e.g., via the Internet using an Internet service provider).

[0248] The present invention will be 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, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions / acts specified in one or more blocks of the flowchart and / or block diagram.

[0249] These computer program instructions can also be stored in a computer-readable memory, which can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means for implementing the functions / acts specified in one or more blocks of the flowchart and / or block diagram.

[0250] Computer program instructions can also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process such that the instructions executed on the computer or other programmable apparatus provide steps for implementing some or all of the functions / acts specified in one or more blocks of a flowchart and / or block diagram.

[0251] The flowcharts and block diagrams in some of the figures herein illustrate the exemplary architectures, functions, and operations of possible implementations of the embodiments of the present invention. In this regard, each block in a flowchart or block diagram represents a module, segment, or portion of code that contains one or more executable instructions for implementing the specified one or more logical functions. 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, 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, depending upon the functionality involved.

[0252] In particular, the controller 210( Figure 8 ) and / or the processor 500( Figure 18 ) may include any commercially available or custom microprocessor, microcontroller, digital signal processor, etc. The memory may include any storage device and / or storage medium that contains software and data for implementing the functional circuits or modules used in accordance with the embodiments of the present invention. The 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, the memory may be a content addressable memory (CAM).

[0253] The foregoing describes the present invention, and should not be construed as limiting thereof. The present invention is defined by the appended claims, and equivalents thereof are included therein. All publications, patent applications, patents, patent publications, and other references cited herein are hereby incorporated by reference in their entirety for the teachings relevant to the sentences and / or paragraphs in which the references are presented. This application relates to the following embodiments: 1. An analysis system, the system comprising: A housing that includes 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 retained in the housing; and A sub-array selection module, which is in communication with the controller, wherein the sub-array selection module is configured to select a subgroup of a micropore group of at least one fluid channel of the microfluidic device for imaging via the optical system after a reaction step (e.g., one thermal cycle) during an assay. 2. The system of embodiment 1, the system further comprising a magnet held in the housing adjacent to at least one microfluidic device, the magnet being configured to translate along at least one fluid channel during a bead loading operation to magnetically couple to the beads to guide the beads to travel along the fluid channel and into the bead retention segments of the micropores. 3. The system of embodiment 1, wherein the microfluidic device comprises a plurality of fluid channels, each fluid channel having multiple groups of micropores, the multiple groups of micropores being positioned along a length dimension associated with the direction between a sample input port and a second opposite port, and wherein the selected subgroup of the micropore groups is associated with a single row of a first subgroup of laterally aligned micropores of the multiple fluid channels, optionally associated with a subgroup of aligned micropores from each of the multiple fluid channels. 4. The system of embodiment 1, wherein prior to the light excitation, the sub-array selection module identifies a subgroup of the micropore groups to define the positions of other micropore groups of the micropore groups of the microfluidic device. 5. The system of embodiment 1, wherein the sub-array selection module selects different subgroups of the micropore groups of the microfluidic device at different reaction steps (e.g., different thermal cycles of the assay), and guides the optical system to only excite the currently selected subgroup of the micropore groups in the different fluid channels, and then guides the camera of the optical system to continuously or concurrently acquire images of the different subgroups of the micropore groups in the currently selected subgroup. 6. The system of embodiment 1, wherein the sub-array selection module selects the same subgroup of micropores at at least some different consecutive reaction steps (e.g., different thermal cycles of the assay), and guides the optical system to transmit light only to the currently selected subgroup of the micropore groups, and then guides the camera of the optical system to continuously or concurrently acquire images of the different subgroups of the micropore groups, optionally acquiring images of multiple pairs of adjacent micropore groups in the currently selected subgroup of micropores. 7. The system of embodiment 1, wherein the optical system comprises at least a first filter and / or a second filter, the at least first filter and / or second filter defining excitation light of a first wavelength and / or a second wavelength corresponding to beads with a first target encoding and beads with a second target encoding for decoding bead groups held in the one or more groups of micropores of at least one fluid channel of the microfluidic device. 8. The system of embodiment 1, the system further comprising a signal analysis module integrated in and / or coupled to the controller, wherein the signal analysis module is configured to obtain an analog signal for each micropore in a selected subgroup of the micropore group, and wherein the analog signal provides PCR data when the amplitude varies with the cycles of the assay, and the concentration of the target molecule of the reaction associated with the defined bead type is determined by the analog signal. 9. The system of embodiment 8, wherein the signal analysis module is further configured to obtain one or more digital PCR signals of the micropore group. 10. The system of embodiment 8, wherein when the amplitude varies with the number of PCR cycles of the input material in the one or more sets of micropores of the fluid channel, the analog signal provides real-time PCR data, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / micropore, and wherein the analog signal defines the threshold cycle or cycle threshold Ct and the number / concentration of target molecules of the target substance and / or molecular type, and for a given number of PCR cycles, a micropore reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or a micropore reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold. 11. The system of embodiment 10, wherein the Ct is the number of cycles at which Si >> Bs, where >> means at least 5 - 10% greater than Bs, optionally twice Bs, and / or 5 - 10 times the standard deviation of Bs, and wherein Bs is the background fluorescence signal, optionally measured in a PCR negative reaction. 12. The system of embodiment 8, wherein the analog signal is obtained only for a single subgroup of the micropore group in each fluid channel after every other or every one of a plurality of different reaction steps (e.g., different thermal cycles of the assay). 13. The system of embodiment 8, wherein the analog signal comprises the average value of Si (optionally excluding outlier data), median value, mode value or weighted value as the analog signal corresponding to each defined type of bead, and is provided as an estimate of the real-time PCR curve of the micropores in other micropore groups of the corresponding fluid channels of the microfluidic device, thereby allowing single-cycle resolution even if not all micropore groups of each fluid channel are imaged after different or each reaction cycle. 14. The system of embodiment 1, wherein the optical system comprises a camera having a field of view (FOV) that only covers a subgroup of the micropores of the microfluidic device, wherein the subgroup is located in at least two, optionally all (e.g., entire rows) adjacent fluid channels of the microfluidic device. 15. The system according to embodiment 1, wherein the controller and / or the signal analysis module are configured to direct the optical system to obtain pre-PCR and post-PCR images and compare the signal intensities therebetween, optionally together with simulated data obtained using a selected subgroup of the microwell groups at different reaction steps of the assay, to determine positive and negative PCR reactions and optionally to determine one or more concentrations and / or one or more molecular concentrations of the target substance in the original sample provided to the fluid channel. 16. The system according to embodiment 1, the system further comprising a holder configured to hold a plurality of microfluidic devices in an aligned grid in the housing. 17. The system according to embodiment 1, wherein the holder comprises a heat insulating material providing a thermal barrier between adjacent microfluidic devices, optionally wherein the holder holds the plurality of microfluidic devices with the input ports of the fluid channels facing outwards. 18. The system according to embodiment 1, wherein the microfluidic device further comprises a dye homogenizer, optionally wherein the dye homogenizer comprises oligonucleotides, optionally non-extendable oligonucleotides and / or partially double-stranded DNA (e.g., partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain). 19. The system according to embodiment 18, wherein the dye homogenizer is present in the master mixture present in the microfluidic device and / or wherein the dye homogenizer is attached to the beads present in the microfluidic device. 20. A method for identifying a target substance and / or a target molecule, the method comprising: providing a fluid analysis device having a first fluid channel with a plurality of groups of microwells positioned along the first fluid channel; obtaining signal intensity data only from a defined subgroup of the plurality of groups of microwells; and identifying a PCR reaction positive for a target substance and / or molecular type associated with a bead type and / or a target molecule, at least in part based on the obtained signal intensity data. 21. The method according to embodiment 20, the method further comprising loading and then sealing the plurality of groups of microwells along the first fluid channel prior to the obtaining step such that after sealing, each group of microwells is fluidically isolated from the other groups of microwells, wherein the plurality of groups of microwells along the first fluid channel are in fluid communication only during the loading and prior to the sealing step. 22. The method according to embodiment 20, the method further comprising, after each of a plurality of successive reaction steps being assayed (e.g., after each of the plurality of successive thermal cycles being assayed), changing the defined subgroup of the plurality of sets of microwells to a different defined subgroup, whereby after each reaction step some of the microwell sets are not imaged. 23. The method according to embodiment 20, wherein for a plurality of successive reaction steps being assayed (e.g., after each of the plurality of successive thermal cycles being assayed), the defined subgroup remains the same, whereby after each reaction step some of the microwell sets are not imaged. 24. The method according to embodiment 20, wherein the fluid analysis device further comprises more than one plurality of fluid channels, including but not limited to: A second fluid channel having a plurality of sets of microwells spaced along the second fluid channel; A third fluid channel having a plurality of sets of microwells spaced along the third fluid channel; and A fourth second fluid channel having a plurality of sets of microwells spaced along the fourth fluid channel, wherein a first set of microwells of the plurality of sets of microwells in each of the first, second, third, and fourth fluid channels is aligned in a first row, wherein a second set of microwells of the plurality of sets of microwells in each of the first, second, third, and fourth fluid channels is aligned in a second row, wherein a third set of microwells of the plurality of sets of microwells in each of the first, second, third, and fourth fluid channels is aligned in a third row, wherein a fourth set of microwells of the plurality of sets of microwells in each of the first, second, third, and fourth fluid channels is aligned in a fourth row, and wherein the defined subgroup is a single row of the first row, second row, third row, and fourth row. 25. The method according to embodiment 24, wherein the first, second, third, and fourth fluid channels comprise straight or arcuate segments that are substantially parallel to each other and provide the first, second, third, and fourth sets of microwells. 26. The method according to embodiment 20, the method further comprising: Before the obtaining step, transmitting a light excitation signal only to the defined subgroup, and obtaining the signal in response to the transmission of the light excitation signal; After, before, or both before and after the transmitting and obtaining steps, digitally scanning the defined subgroup of the plurality of sets of microwells to obtain an image of the microwell sets for identifying positive and negative PCR reactions associated with digital PCR; and electronically identifying microwells in the microwell sets that are positive for one or more target analyte molecules when the microwells are at an imaging temperature. 27. The method according to embodiment 20, wherein the excitation and acquisition are performed to image only a sub-constituent defined by one of the micro-well groups after each of a plurality of consecutive reaction steps (e.g., thermal cycling) of the assay, wherein only consecutive sub-groups of one defined sub-group are different from each other. 28. The method according to embodiment 20, wherein the signal intensity is obtained only from the defined sub-group using at least one camera having a field of view (FOV) by obtaining images of different defined micro-well groups either sequentially or in parallel, the field of view covering only one set of micro-wells or only a sub-group of the micro-well group in at least two adjacent fluid channels of the fluid channel. 29. The method according to embodiment 20, wherein each of the micro-well groups comprises a micro-well array within the range of 1,000 - 1,000,000 corresponding micro-wells, wherein the fluid analysis device comprises a plurality of spaced-apart fluid channels, each fluid channel having a micro-well group containing the micro-well array, wherein the fluid channels are fluidically isolated, and wherein at least some of the micro-wells of the micro-well array contain a single bead, optionally wherein some or all of the micro-wells are bead-free or contain more than one bead. 30. The method according to embodiment 20, the method further comprising electronically identifying the positions of one or more sets of micro-wells at one or more positions within the microfluidic device before the transfer and acquisition steps, and defining the positions of other micro-well groups of the micro-well group based on at least a portion of the identified positions. 31. The method according to embodiment 20, the method further comprising transmitting a photoexcitation signal only to the defined sub-group before the acquisition step, and selecting a filter to provide an encoding wavelength for the transmission step before the transmission. 32. The method according to embodiment 20, further, the obtaining the signal intensity comprises obtaining an analog signal, which can provide real-time PCR data when the amplitude varies with the number of PCR cycles of the input material in the micro-well group, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / micro-well, and wherein the analog signal defines a threshold cycle or cycle threshold Ct and the number / concentration of target molecules of the target substance and / or molecular type, and for a given number of PCR cycles, a micro-well reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or a micro-well reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold. 33. The method according to embodiment 32, wherein the Ct is the number of cycles at which Si >> Bs, where >> is at least 5 - 10% greater than Bs, optionally twice Bs, and / or 5 - 10 times the standard deviation of Bs, and wherein Bs is the background fluorescence signal, optionally as measured in a PCR negative reaction. 34. The method according to embodiment 32, wherein after every other or every one of a plurality of different reaction steps of the assay (e.g., different thermal cycles of the assay), the simulated signal is obtained for only a single subgroup of the micropore groups in one or more fluid channels. 35. The method according to embodiment 32, wherein the simulated signal comprises an average value, median value, mode value or weighted value of Si (optionally discarding outlier data) as the simulated signal corresponding to each defined type of bead, and is provided as an estimate of the real-time PCR curve of a similar reaction in the micropores of other micropore groups of the corresponding fluid channels of the microfluidic device, thereby allowing single-cycle resolution even if not all micropore groups of each fluid channel are imaged after different reaction steps. 36. The method according to embodiment 20, wherein the obtained signal intensity is obtained by using a camera having a field of view (FOV) that only covers a subgroup of the micropore groups of the microfluidic device and is located in at least two, optionally all, adjacent fluid channels of the microfluidic device. 37. The method according to embodiment 32, wherein after a plurality of different reaction steps of the assay (e.g., different thermal cycles of the assay), the simulated signal is obtained for only a single group of the micropore groups in each fluid channel, and wherein the simulated signal comprises an average value, median value, mode value or weighted value of Si as the simulated signal corresponding to each defined type of bead, and is provided as an estimate of the real-time PCR curve of a similar reaction in the micropores of other micropore groups of the corresponding fluid channels of the microfluidic device, thereby allowing single-cycle resolution even if not all micropore groups of each fluid channel are imaged after different reaction steps. 38. The method according to embodiment 20, wherein the fluid analysis device having the first fluid channel comprises a plurality of additional fluid channels, the first fluid channel having the plurality of groups of micropores spaced along the first fluid channel, each of the additional fluid channels comprising a corresponding plurality of groups of micropores spaced along its respective length, and wherein the fluid analysis device further comprises separate material input ports for each of the first fluid channel and the plurality of additional fluid channels, and at least some of the fluid channels share a common opposite second port, and the method further comprises, before the obtaining step: Loading a bead slurry fluid pre-exposed to a corresponding sample for analysis into the corresponding input port; Magnetically guiding the bead slurry along the fluid channels into different groups of micropores; Cause a fluid main mixture containing a dye to flow from the second port towards the first port into the fluid channel, where the main mixture optionally contains a dye homogenizing agent, optionally where the dye homogenizing agent is or contains an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)); and then cause a sealing oil to flow from the second port towards the first port into the fluid channel, thereby sealing the micropore group and the fluid channel from each other. 39. The method according to embodiment 38, the method further comprising placing a magnet adjacent to the fluid analysis chip and translating the magnet towards the second port before the fluid main mixture and the sealing oil flow. 40. The method according to embodiment 20, where the fluid analysis device (optionally the first fluid channel and / or the multiple groups of micropores) contains a dye homogenizing agent, optionally where the dye homogenizing agent contains an oligonucleotide (e.g., a non-extendable oligonucleotide and / or a partially double-stranded DNA (e.g., a partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)). 41. The system according to embodiment 40, where the dye homogenizing agent is present in the main mixture present in the fluid analysis device (e.g., present in the first fluid channel and / or the multiple groups of micropores) and / or where the dye homogenizing agent is attached to the beads present in the fluid analysis device (e.g., present in the first fluid channel and / or the multiple groups of micropores). 42. A microfluidic device for analysis, comprising: A plurality of fluid channels, where each of the plurality of fluid channels has a length dimension corresponding to the direction between a first port and an opposite second port, where at least a portion of the length dimension is configured as a straight or arcuate length segment, and where each of the fluid channels contains multiple groups of micropores positioned along the straight or arcuate length segment of the length dimension. 43. The microfluidic device according to embodiment 42, where the multiple groups of micropores in the plurality of fluid channels are arranged in rows, columns, or rows and columns, and where the row or the column corresponds to the straight or arcuate length segment. 44. The microfluidic device according to embodiment 42, where at least some of the plurality of fluid channels are substantially parallel on the straight or arcuate length segment. 45. The microfluidic device according to embodiment 42, where at least some of the plurality of fluid channels are arcuate substantially parallel channels and contain the arcuate length segment. 46. The microfluidic device according to embodiment 42, wherein at least some of the plurality of fluid channels are substantially parallel and extend radially between the outer peripheral portion of the device and the center of the device. 47. The microfluidic device according to embodiment 42, wherein the plurality of fluid channels includes a first set of fluid channels and a second set of fluid channels spaced apart from the first set, wherein the first set of fluid channels terminates at a first main mixture port as the second port, and the second set of fluid channels terminates at a second main mixture port as the second port. 48. The microfluidic device according to embodiment 42, wherein the first set of fluid channels and the second set of fluid channels are circumferentially spaced apart. 49. The microfluidic device according to embodiment 42, wherein the plurality of fluid channels includes at least two fluid channels defining a first adjacent group and at least two channels defining a second adjacent group adjacent to the first adjacent group, each channel having a spatially aligned group of micropores. 50. The microfluidic device according to embodiment 49, wherein the device further includes a first interstitial space between each of the first and second fluid channels of the first adjacent group and the second adjacent group, wherein the device further includes a second interstitial space between the first adjacent group and the second adjacent group, and wherein the second interstitial space has a greater lateral extent than the first interstitial space. 51. The device according to embodiment 42, wherein the plurality of sets of micropores of each of the plurality of fluid channels has a common configuration, wherein the plurality of sets of micropores of each of the fluid channels are aligned with each other in rows and / or columns, and wherein the plurality of fluid channels keep the respective input materials fluidly isolated from each other. 52. The microfluidic device according to embodiment 42, wherein at least the plurality of sets of micropores for the respective fluid channels each includes a quantity of micropores in the range of 1,000 - 1,000,000, and wherein the micropores of the group of micropores are sized and configured to hold and retain a single bead, thereby allowing 1,000 - 1,000,000,000 reactions in the microfluidic device. 53. The microfluidic device according to embodiment 42, wherein a group of a first set of micropores and a second set of micropores from at least a first fluid channel and a second fluid channel, optionally a paired first set of micropores and a second set of micropores, defines a subgroup of a first set of adjacent micropores, and wherein the device includes a transparent substrate extending over the plurality of sets of micropores of the fluid channels. The microfluidic device according to embodiment 42, wherein each fluid channel of the plurality of fluid channels comprises a separate first port at a first end as the first port, and wherein alternating first ports of the plurality of first ports are located at a first longitudinal position on the device, and the alternating other first ports are located at a second longitudinal position on the device, the second longitudinal position being spaced from the first longitudinal position in the length dimension. The microfluidic device according to embodiment 42, wherein the first port is a material input port and is located at the first end of the corresponding fluid channel, and wherein the device further comprises a fluid manifold that connects the opposite second ends of at least some of the fluid channels to the second port. The microfluidic device according to embodiment 42, wherein the fluid channels extend radially across the device, wherein the inlet port of the corresponding fluid channel is located at the outer peripheral portion of the device, and wherein the second port is a single second port located at the center of the device connected to each of the fluid channels. The microfluidic device according to embodiment 42, wherein at least some of the fluid channels are provided as concentric groups of fluid channels, each concentric group of fluid channels having an arcuate length segment. The microfluidic device according to embodiment 42, wherein the concentric group of fluid channels having an arcuate length segment is provided as a plurality of circumferentially spaced concentric groups of fluid channels. The microfluidic device according to embodiment 42, wherein the microfluidic device further comprises a dye homogenizer, optionally wherein the dye homogenizer comprises oligonucleotides (e.g., non-extendable oligonucleotides and / or partially double-stranded DNA (e.g., partially double-stranded DNA comprising biotin and / or a C1-C20 hydrocarbon chain)). The microfluidic device according to embodiment 59, wherein the dye homogenizer is present in the main mixture present in the microfluidic device and / or wherein the dye homogenizer is attached to the beads present in the microfluidic device.

Claims

1. An analysis system, the system comprising: A housing, the housing including a chamber sized and configured to receive at least one microfluidic device; An optical system coupled to the housing that is 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 retained in the housing; And A sub-array selection module in communication with the controller, wherein the sub-array selection module is configured to select a subgroup of the micro-pore groups of at least one fluid channel of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during an assay.

2. The system of claim 1, the system further comprising a magnet retained in the housing adjacent to the at least one microfluidic device, the magnet configured to translate along at least one fluid channel during a bead loading operation to magnetically couple to beads to guide the beads to travel along the fluid channel and into the bead retention segment of the micro-pores.

3. The system of claim 1, wherein the microfluidic device includes a plurality of fluid channels, each fluid channel having multiple groups of micro-pores positioned along a length dimension associated with the direction between a sample input port and a second opposing port, and wherein the selected subgroup of the micro-pore groups is associated with a single row of a first subgroup of laterally aligned micro-pores of the plurality of fluid channels, optionally associated with a subgroup of the aligned micro-pores of each of the plurality of fluid channels.

4. The system of claim 1, wherein prior to the light excitation, the sub-array selection module identifies the subgroup of the micro-pore groups to define the positions of the other micro-pore groups of the micro-pore groups of the microfluidic device.

5. The system of claim 1, wherein the sub-array selection module selects different subgroups of the micro-pore groups of the microfluidic device at different reaction steps of the assay (e.g., different thermal cycles of the assay), and directs the optical system to only excite the currently selected subgroup of the micro-pore groups in the different fluid channels, and then directs the camera of the optical system to sequentially or simultaneously acquire images of the different subgroups of the micro-pore groups in the currently selected subgroup.

6. The system of claim 1, wherein the sub-array selection module selects the same subgroup of micro-pores at at least some different consecutive reaction steps of the assay (e.g., different thermal cycles of the assay), and directs the optical system to transmit light only to the currently selected subgroup of the micro-pore groups, and then directs the camera of the optical system to sequentially or simultaneously acquire images of the different subgroups of the micro-pore groups, optionally acquiring images of multiple pairs of adjacent micro-pore groups in the currently selected subgroup of micro-pores.

7. The system of claim 1, wherein the optical system comprises at least a first filter and / or a second filter, and the at least first filter and / or second filter define an excitation light of a first wavelength and / or a second wavelength corresponding to beads of a first target encoding and beads of a second target encoding for decoding a bead set held in the one or more sets of micro-wells of the microfluidic device.

8. The system of claim 1, the system further comprising a signal analysis module integrated in the controller and / or coupled to the controller, wherein the signal analysis module is configured to obtain an analog signal of each micro-well in a selected subgroup of the set of micro-wells, and wherein when the amplitude varies with the cycles of the assay, the analog signal provides PCR data and determines the concentration of target molecules of a reaction associated with a defined bead type through the analog signal.

9. The system of claim 8, wherein the signal analysis module is further configured to obtain one or more digital PCR signals of the set of micro-wells.

10. The system of claim 8, wherein when the amplitude varies with the number of PCR cycles of the input material in the one or more sets of micro-wells of the fluid channel, the analog signal provides real-time PCR data, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / micro-well, and wherein the analog signal defines a threshold cycle or cycle threshold Ct and the number / concentration of target molecules of the target substance and / or molecular type, and for a given number of PCR cycles, a micro-well reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or a micro-well reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold.

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