Analysis system with a microfluidic device, microfluidic device and related methods

Abstract

An analysis system having a housing with a chamber sized and configured to receive at least one microfluidic device. The system further 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 a sub-array selection module in communication with the controller. The sub-array selection module is configured to select a subgroup 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

[0001] Related Applications

[0002] 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.

[0003] Statement of Federal Support

[0004] This invention was made with government support under award number HR0011-12-2-0001 from the U.S. Department of Defense (DARPA). The government has certain rights in the invention.

[0005] Statement Regarding Electronic Submission of a Sequence Listing

[0006] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 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. The Sequence Listing is incorporated by reference herein in its entirety for its disclosure.

[0007] Copyright Retention

[0008] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner, The University of North Carolina at Chapel Hill, N.C., does not object to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyrights whatsoever. Technical Field

[0009] 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 array of encoded bead micro-wells. Background Art

[0010] 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. Probes are also sequence-specific, reducing false positives from primer-dimer formation or non-specific amplification. A typical method for 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

[0011] Embodiments of the invention provide methods for limiting photobleaching in encoded bead arrays, optionally while obtaining real-time PCR data.

[0012] Embodiments of the invention provide formulations of PCR master mixes and magnetic particle chemistries that are particularly suitable for encoded bead arrays.

[0013] Embodiments of the invention relate to sample analysis devices for assays, systems for analyzing signals from arrays of micro-wells of a fluid device, and assay methods.

[0014] 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 sub-array selection module in communication with the controller. The sub-array selection module is configured to select a subgroup of the 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.

[0015] 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 the bead retention segments of the micropores.

[0016] The microfluidic device may include a plurality of fluid channels, each fluid channel having multiple sets of micropores positioned along a length dimension associated with the direction between a sample input port and a second opposite port. The selected subgroup of the micropore sets 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 the aligned micropores from each of the plurality of fluid channels.

[0017] Before initial image acquisition and / or photoexcitation, the sub-array selection module identifies a subgroup of the micropore sets to define the positions of other micropore sets of the micropore sets of the microfluidic device.

[0018] The sub-array selection module may select different subgroups of the micropore sets 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 sets in the different fluid channels, and then direct the camera of the optical system to sequentially or simultaneously acquire images of the different subgroups of the micropore sets in the currently selected subgroup.

[0019] The sub-array 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 sets, and then direct the camera of the optical system to sequentially or simultaneously acquire images of the different subgroups of the micropore sets, optionally acquiring images of multiple pairs of adjacent micropore sets in the currently selected subgroup of micropores.

[0020] 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 corresponding to respective first and / or second wavelengths for beads of a first target encoding and beads of a second target encoding, for decoding bead sets held in the one or more sets of microwells of the at least one fluid channel of the microfluidic device.

[0021] The system can have a signal analysis module integrated in and / or coupled to the controller. The signal analysis module can be configured to obtain an analog signal for each microwell in a selected subgroup of the set of microwells. When the amplitude varies with the cycles of the assay, the analog signal can provide PCR data and determine the concentration of target molecules of a reaction associated with the defined bead type through the analog signal.

[0022] The signal analysis module can be further configured to obtain one or more digital PCR signals for the set of microwells.

[0023] When the amplitude varies with the number of PCR cycles of the input material in the one or more sets of microwells 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 / microwell. 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. For a given number of PCR cycles, a microwell reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or negative if the fluorescence signal intensity (Si) is always less than the threshold. Ct is the cycle number 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 where Bs is the background fluorescence signal, optionally measured as in a PCR negative reaction.

[0024] 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 can be obtained only for a single subgroup of the set of microwells in each fluid channel.

[0025] The analog signal can be the mean (optionally excluding outlier data), median, mode value, 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 microwells of other sets of microwells of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all sets of microwells of each fluid channel are imaged after different or each reaction cycle.

[0026] The optical system may have a camera with a field of view (FOV) that covers only a subgroup of the micro-wells in at least two, optionally all, adjacent fluid channels of the microfluidic device.

[0027] 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.

[0028] 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 micro-well sets 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 channels.

[0029] The system may further include a holder configured to hold a plurality of microfluidic devices in an aligned grid in the housing.

[0030] The holder may be formed of a heat-insulating material that provides a heat 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 outward.

[0031] The microfluidic device may further include a dye homogenizer, optionally the dye homogenizer includes oligonucleotides.

[0032] The dye homogenizer may include non-extendable oligonucleotides and / or partially double-stranded DNA.

[0033] The dye homogenizer may include partially double-stranded DNA containing biotin and / or a C1 - C20 hydrocarbon chain.

[0034] 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.

[0035] 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 micro-wells 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 micro-wells; and identifying a PCR reaction that is positive for a target substance and / or a molecular type associated with a bead type and / or a target molecule, at least in part based on the obtained signal intensity data.

[0036] 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 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.

[0037] The method may include changing, after each of a plurality of consecutive reaction steps of the assay (e.g., after each of the plurality of consecutive thermal cycles of the assay), the defined subgroup of the plurality of sets of micropores to a different defined subgroup, whereby after each reaction step some sets of micropores are not imaged.

[0038] After a plurality of consecutive reaction steps of the assay (e.g., after each of the plurality of consecutive thermal cycles of the assay), the defined subgroup may remain the same, whereby after each reaction step some sets of micropores are not imaged.

[0039] 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 row, second row, third row, and fourth row.

[0040] 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.

[0041] The method may further comprise: prior to the obtaining step, transmitting a photoexcitation signal only to a defined subgroup; digitally scanning a defined subgroup of the plurality of sets of microwells, after, before, or both before and after the transmitting and obtaining steps, 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 while the microwells are at an imaging temperature.

[0042] 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 (e.g., thermal cycling) of an assay, wherein consecutive subgroups of the one defined subgroup may be different from each other.

[0043] By continuously or concurrently obtaining images of different defined adjacent sets of microwells in different fluid channels of a defined subgroup of the set of microwells, a camera having 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.

[0044] Each of the set of microwells may have a microwell array having microwells in the range of 1,000 - 1,000,000.

[0045] 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, and optionally some or all of the microwells may be beadless or contain more than one bead.

[0046] The method may include electronically identifying the location of one or more sets of microwells at one or more locations within the microfluidic device prior to 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.

[0047] The method of claim 20, the method further comprising transmitting a photoexcitation signal only to a defined subgroup prior to the obtaining step, and selecting a filter to provide an encoded wavelength for the transmitting step prior to the transmitting.

[0048] The obtained signal intensity may include obtaining an analog signal that provides real-time PCR data when the amplitude varies with the number of PCR cycles of the input material in the microwell group, and the molecular concentration of the defined bead type is approximately equal to or greater than 1 molecule / microwell. 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, the microwell reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or the microwell reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold.

[0049] 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.

[0050] 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 microwell groups in one or more fluid channels.

[0051] The analog signal may be the mean, median, mode value, 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 microwells of other microwell groups of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all microwell groups of each fluid channel are imaged after different reaction steps.

[0052] The obtained signal intensity may be obtained by using a camera with a field of view (FOV) that covers only a subgroup of the microwell groups in at least two, optionally all adjacent fluid channels of the microfluidic device.

[0053] 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 microwell groups in each fluid channel, and wherein the analog signal contains the mean, median, mode value, 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 microwells of other microwell groups of the corresponding fluid channels of the microfluidic device, thus allowing single-cycle resolution even if not all microwell groups of each fluid channel are imaged after different reaction steps.

[0054] 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 into the fluid channel from the second port towards the first port; and then flowing a sealing oil into the fluid channel from the second port towards the first port, thereby sealing the sets of micropores and the fluid channel from each other.

[0055] The main mixture may optionally include a dye homogenizer. The dye homogenizer may optionally be or comprise 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)).

[0056] 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.

[0057] 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 comprises 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)).

[0058] 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).

[0059] 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.

[0060] 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.

[0061] At least some of the multiple fluid channels may be substantially parallel on the straight or arcuate length segments.

[0062] At least some of the multiple fluid channels may be arcuate substantially parallel channels and may provide the arcuate length segments.

[0063] At least some of the multiple fluid channels may be substantially parallel and extend radially between the outer peripheral portion of the device and the center of the device.

[0064] 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.

[0065] The first set of fluid channels and the second set of fluid channels may be circumferentially spaced apart.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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, define a subgroup of first adjacent micropores. The device may include a transparent substrate extending over the multiple sets of micropores of the fluid channels.

[0071] 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.

[0072] 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.

[0073] The fluid channels may extend radially across the device. The inlet port of the corresponding fluid channel may be located at an 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.

[0074] At least some of the fluid channels may be provided as concentric groups of fluid channels, each concentric group of fluid channels having an arcuate length segment.

[0075] The concentric groups of fluid channels having an arcuate length segment may be provided as concentric groups of a plurality of circumferentially spaced fluid channels.

[0076] The microfluidic device may further include a dye homogenizer.

[0077] The dye homogenizer may optionally include oligonucleotides.

[0078] The dye homogenizer may include oligonucleotides (e.g., non-extendable oligonucleotides and / or partially double-stranded DNA (e.g., partially double-stranded DNA containing biotin and / or a C1-C20 hydrocarbon chain)).

[0079] The dye homogenizer may be present in the main mixture present in the microfluidic device and / or the dye homogenizer may be attached to beads present in the microfluidic device.

[0080] Other embodiments relate to bead loading strategies and the use of isolated bead inputs and common reagent reservoirs / inputs as shown and / or described.

[0081] Other embodiments relate to fully integrated fluid 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 having pre-loaded reagents as shown and / or described.

[0082] Note that any one or more aspects or features described with respect to one embodiment can be incorporated into different embodiments, even though not specifically described with respect thereto. That is, all embodiments and / or features of any embodiment can 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, even though 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

[0083] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0084] The drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0085] Figure 1A is a schematic illustration of an exemplary fluid device according to an embodiment of the present invention.

[0086] Figure 1B is a schematic illustration of another exemplary fluid device according to an embodiment of the present invention.

[0087] Figure 2A is at Figure 1A a top perspective view of the fluid device shown in

[0088] Figure 2B is corresponding to Figure 2A a digital top perspective photographic view of a prototype of the fluid device.

[0089] FIG. 3A is an exemplary fluid device having non-cylindrical micro-wells according to an embodiment of the present invention.

[0090] FIG. 3B is an extremely magnified view of several micro-wells taken from a portion of the fluid device shown in FIG. 3A according to an embodiment of the present invention.

[0091] Figure 3CIs an example measurement signal generated by the non-cylindrical micro-wells of the fluid device shown in Figure 3A, illustrating a measurement signal separated from the bead background signal according to an embodiment of the present invention.

[0092] Figure 4A Is a graph of the raw fluorescence of negative and positive beads versus the number of cycles.

[0093] 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 the number of cycles.

[0094] Figures 5A - 5H Is a graph of the raw data versus the number of cycles obtained by combining real-time PCR data from different subgroups of micro-wells from different fluid channels during PCR according to an embodiment of the present invention.

[0095] Figure 6A -H is according to an embodiment of the present invention in Figures 5A - 5H Is a graph of the normalized data of the data shown in.

[0096] 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.

[0097] Figure 8 Is an example analysis system according to an embodiment of the present invention.

[0098] 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.

[0099] Figure 17 Is a flowchart of an example analysis method according to an embodiment of the present invention.

[0100] Figure 18 Is a data processing system according to an embodiment of the present invention.

[0101] Figure 19 Is a flowchart of the actions that can be performed after loading a fluid device with beads and then thermocycling according to an embodiment of the present invention.

[0102] 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.

[0103] Figure 21 Is a top view of a plurality of microchips held by an adiabatic holder according to an embodiment of the present invention.

[0104] Figure 22Box plots of the initial bead fluorescence of each bead in a 5-array from one channel of two 8-channel microchips, one microchip treated with a master mix containing 10X SYBR and the other microchip treated with a master mix containing 20X SYBR plus 5 μM non-extendable oligomers.

[0105] 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 is loaded with beads from the same sample according to an embodiment of the present invention.

[0106] Figure 24 Box plots of the encoded dye fluorescence signals from the mycoplasma group in a 12-plex respiration panel, associated with each bead in a 5-array of a microchip treated with SYBR or +NE oligomers and SYBR, according to an embodiment of the present invention.

[0107] Figure 25 Schematic diagram of an example of pdsDNA of a bead, according to an embodiment of the present invention. Detailed Description

[0108] 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.

[0109] Like numbers always represent like elements. In the drawings, for clarity, the thickness of some lines, layers, components, elements or features may be exaggerated. In the text and drawings, the abbreviations "FIG." and "Fig." of the word "Figure" may be used interchangeably.

[0110] 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 the 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".

[0111] 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.

[0112] 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 lack of a combination when interpreted in the alternative form ("or").

[0113] 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 composition 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.

[0114] 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 being equivalent to "comprising."

[0115] It will also be understood that as used herein, the terms "example," "exemplary," and their grammatical variations 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.

[0116] 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 may include any other ranges and / or individual values therein.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] For ease of description, spatial relative terms such as "below", "lower", "beneath", "above", "upper", 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 inverted, an element described as "below" or "beneath" another element or feature will be oriented "above" the other element or feature. Thus, the exemplary term "below" can encompass both an "above" and a "below" orientation. 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.

[0121] 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, the "first" element discussed below could also be referred to as the "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.

[0122] 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 micro-wells 20 in a coded micro-well array 120a (bead micro-well 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 the PCR, and a cycle of the PCR generally refers to a set of steps including denaturation of the template DNA, annealing of the primers to the single-stranded DNA template, and extension of the 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 recited herein.

[0123] In some embodiments, the designs and methods described herein can reduce the data collection time for large arrays (e.g., coded bead arrays, optionally including about 30,000 or more micro-wells), 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.

[0124] In some embodiments, the present invention may include, but is not limited to, substrates, devices, designs, solid supports (e.g., coded solid supports), steps, and / or methods as described in the following: U.S. Provisional Application No. 62 / 673,343 entitled "Compositions, Devices, and Methods for Improving a Surface Property of a Substrate", U.S. Provisional Application No. 62 / 736,525 entitled "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.

[0125] In some embodiments, the methods of the present invention include methods of delivering reagents and / or targets to reaction wells using beads (such as superparamagnetic beads), as described in U.S. Application Publication No. 2015 / 0211048 and International Application No. PCT / US2016 / 042913, the respective contents of which are incorporated herein by reference in their entirety.

[0126] 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. Optionally, the microchip can 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.

[0127] The term "major dimension" refers to the width and / or depth dimension of the fluid channel.

[0128] 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 within a size range of 10 millimeters or less, typically less than 900 microns and greater than 1 nm.

[0129] 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 further discussed below.

[0130] 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 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.

[0131] The term "circuit" refers to a fully hardware implementation or an implementation combining software and hardware. The term "module" refers to an implementation containing software and hardware or firmware.

[0132] The term "digital scan" and its derivatives refer to obtaining a digital image of part or all of a microfluidic device, typically through a camera.

[0133] 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 does not necessarily need to) be separated by a gap space 21 without microholes. Figure 1B Illustrates sets of microholes 20 that can be arranged continuously along the corresponding fluid channel 15. Figure 1B Also illustrates that adjacent ports of the input port 16 (the first port) can be aligned, rather than longitudinally offset (in the orientation shown) as in Figure 1A shown.

[0134] One or more sets of microholes 20 along the corresponding fluid channel 15 can 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 can be less than the length L of the footprint of the adjacent set of microholes 20.

[0135] As shown in Figure 1A and FIG. 2, at least one elongated fluid channel 15 includes eight adjacent fluid channels 15 1 、15 2 、15 3 、15 4 、15 5 、15 6 、15 7 and 15 8 However, a greater or lesser number of fluid channels 15 can be provided, and a greater or lesser number of sets of microholes 20 can be provided for all or any one of the fluid channels 15. For example, in some specific embodiments, the fluid device 10 can have between 2 - 2000 fluid channels 15, more typically 10 - 200 fluid channels 15.

[0136] As shown in Figure 1A each or some of the fluid channels 15 can be configured with multiple sets of microholes 20 to include a first set of microholes 20 1 、a second set of microholes 20 2 、a third set of microholes 20 3 、a fourth set of microholes 20 4 and a fifth set of microholes 205 However, more or fewer groups of micropores 20 can be provided. Figure 1B It is illustrated that the fluid channels 15 can each have three groups of micropores 20. In some embodiments, the fluid channels 15 of the fluid device 10 can have between 2 and 100 groups of micropores 20, more typically between 3 and 25 groups.

[0137] The interstitial space 21 between adjacent groups (micropore groups) of micropores 20 along the respective fluid channels 15 need not be provided by a physical gap or break in the continuous micropores 20, but can be determined 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 channels 15, as shown in Figure 1B .

[0138] Bubbles or constrictions for alignment beads can be provided in the fluid channels 15. Bubbles or constrictions can be provided between adjacent groups of micropores 20, which provide a corresponding array of micropores 120 (micropore array 120a) in the fluid channels 15 to control fluid movement / sealing or to facilitate or control bead loading.

[0139] Referring again to Figure 1A and FIG. 2, multiple groups of micropores 20 for the fluid channels 15 can be arranged in substantially parallel rows that are aligned and marked with position markers, such as alphanumeric markings for the rows, for R 1 -R 8 rows are shown marked with the letter markings A - E, and aligned parallel fluid channels 15, optionally marked in columns as C 1 -C 8 , 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 have markings in a human-readable form. More or fewer rows and more or fewer columns can be used. Adjacent channels 15n (shown as pairs 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 an interstitial space 19 provided by the substrate of the fluid device 10. Adjacent channels 15n can be separated by a distance that is smaller than the interstitial space 19 of other adjacent channels 15n. Although adjacent channels 15n are shown provided as pairs of channels 15p, for example, three, four, five or even more adjacent channels (including entire rows) can define an adjacent group of channels 15n.

[0140] The groups of micropores 20 can have a footprint with a rectangular outer perimeter 20r that encloses the corresponding group or array (micropore array 120a) of micropores 120 (single-bead micropores) (FIG. 3B). However, other geometries can be used.

[0141] Each fluid channel 15 may include a separate, dedicated input port 16 (fluid first port), which may be an input port for introducing a desired input material, optionally including a sample. Refer to Figure 2A 、 Figure 2B , the input port 16 (fluid port) may include a reservoir 16r having a through hole 16v passing through the covering substrate 10u to guide the input material from the reservoir 16r to the input port 16 of the fluid channel 15.

[0142] Each fluid channel 15 may merge into a second port 18 at an end opposite to the input port 16, and the second port 18 may be a main mixture / oil port. The second port 18 may also be connected to a reservoir 18r through a through hole 18v. The second port 18 may be in fluid communication with more than one fluid channel 15. The fluid device 10 may include a fluid manifold 18m in fluid communication with all or some of the fluid channels 15. Therefore, there may be a separate input port 16 for each fluid channel 15 and a smaller number of second ports 18 than the input ports 16, because some or all of the fluid channels 15 may merge into a common second port 18 via the manifold 18m or directly from the fluid channel extension 15e ( Figure 14 , Figure 15 ).

[0143] Although not shown, each fluid channel 15 may have its own separate second port 18. Different groups of fluid channels 15 may 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 the corresponding fluid channel 15 may be fluidly isolated from each other, and each fluid channel 15 may be fluidly isolated from other fluid channels 15.

[0144] As shown in Figure 1A 、 Figure 1B and FIG. 2, for the corresponding array 10a (channel array), the fluid channels 15 may be substantially parallel on a straight length segment including multiple groups of spaced micropores 20. The term "substantially" when referring to "parallel" means that the fluid channels 15 in the array 10a are parallel or nominally parallel on at least a part of the length segment (i.e., may vary by 10% or less at a slight angle from the adjacent centerlines C / L drawn through the corresponding 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 .

[0145] Referring to FIGS. 3A - 3C, each group or some groups of micropores 20 (micropore groups) may include a plurality of micropores 120 having a bead retention segment 120w and an assay signal segment 120s. The assay signal segment 120s may be in line with the bead retention segment 120w and parallel to and / or in line with the main surface of the top or covering substrate 10u of the fluid device 10 ( Figure 2A ). The assay signal segment 120s is in fluid communication with the bead retention segment 120w of the corresponding micropore 120. The assay signal segment 120s may generate a narrow - tailed assay signal 122, which allows separation of the bead background signal at the bead retention segment 120w (pore retention segment). In some embodiments, the assay signal segment 120s may 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 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 (pore). 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 micropore 120 (pore) may allow a high, typically single - bead - occupied loading of the reaction pore while increasing the corresponding reaction volume, potentially improving reaction efficiency.

[0146] Figures 9 - 11 and Figure 16 illustrate that the fluid device 10 may include multiple spaced - apart arrays 10a of fluid channels 1 、10a 2 、10a 3 、10a 4 (whereas Figure 11 also shows a fifth array 10a of fluid channels 15 5 ). Figure 16 Illustrate four circumferentially - extending arrays 10a of a group of 12 arrays 1 、10a 2 、10a 3 、10a 4 in three concentric groups.

[0147] Each array 10a of the fluid channels 15 may have a single common second port 18 for introducing a loading buffer, a sealing oil, and / or a master mixture.

[0148] The term "master mix" refers to a PCR master mix and can be added to the fluidic channel 15 to reach each set of microwells 20 before sealing the microwells 120 (wells) (Figure 3B) to each other. In some embodiments, an immiscible oil can be used as the sealing oil for sealing. The master mix of the present invention can be primer-free. That is, the PCR master mix of the present invention can exclude primers for some specific embodiments, such as the SiRCA platform.

[0149] As an alternative or in addition, the top substrate 10u( Figure 2A ) can comprise 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.

[0150] Figure 16 Illustrating that the fluidic channel 15 can be an arcuate fluidic channel 15a and circumferentially extends around at least a portion of the diameter of a circular geometry. Different arrays 10a of fluidic channels 1 -10a 4 can be circumferentially spaced apart and each array 10a can have a common single second port 18.

[0151] Figure 11 Showing a fluidic channel 15 extending towards the center, the center defining the location of the second port 18, wherein at least one inner leg 18i of the manifold 18m extends radially towards the second port, and the centerline C / L of the corresponding array A R extends radially across the fluidic device 10 towards the second port 18.

[0152] Figures 12 - 15 Showing the fluidic channel 15 configured as a radially inwardly extending channel 15r. The channel 15r extends radially inwards from the outer peripheral portion of the fluidic device 10, where the input port 16 (first port) is towards the center of the device to the second port 18. Figure 14 and Figure 15 Showing that the dimensions of the fluidic channel 15 decrease along its length towards the center of the fluidic device 10 to the second port 18, and when the microwell groups approach the second port 18, the size of the groups of microwells 20 decreases from the first group of microwells 20 1 to another group 20 2 、20 3 、20 4 、20. In this case, the groups of microwells 20 that are imaged simultaneously (collectively) can be arranged differently from other configurations (not parallel rectangles, etc.).

[0153] The array 10a (microchip array) can define the array positions of each set of micropores 20 of each fluid channel 15 corresponding to position addresses (e.g., row and column addresses). For example, a row and the associated 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 sets of micropores 20 as corner micropores 20c. In Figure 1A In the embodiment shown in FIGS. 1 and 2, the sets of micropores 20 at the array positions A1, A2, A7, A8, E1, E2, E7, and E8 are adjacent corner (sub)sets 20c of the sets of micropores 20. Additionally, more than two fluid channels 15 can be configured to provide adjacent sets of micropores to define a corner set 20c of micropores 20.

[0154] Note that in Figure 1A the fluid device 10 shown in FIGS. 1 and 2 has a rectangular outer perimeter 10p, whose length dimension is greater than the width dimension (“W”), optionally, the width dimension being between 15 - 30 mm wide. The length dimension can be 2 - 4 times or more the width dimension. The width dimension of the fluid channels 15 can correspond to the direction of the width dimension W of the fluid device 10. The fluid channels 15 are shown arranged extending along the length dimension of the fluid device 10. However, the fluid channels 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 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 ).

[0155] The corresponding sets 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 set of micropores 20 is above or below the adjacent first end of an adjacent and at least partially laterally aligned set of micropores in another fluid channel 15 (not shown).

[0156] Some sets or each set of micropores 120 (FIG. 3A) of the corresponding fluid channels 15 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.

[0157] 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), where the arrays can range from 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.

[0158] In exemplary embodiments, each fluid channel 15 can have between 2 - 10 groups (or more) of micropores 20 along its length, fluidically isolated after sealing. A group of micropores 20 can 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) can include fewer 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 houses a single bead (some may be unloaded while some may carry two beads, which may be undesirable in some applications).

[0159] In some embodiments, the fluid device 10 (microchip) can be configured to run assays with more than one bead in the micropores 120. See, for example, the exemplary 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.

[0160] The different groups of micropores 20 for the respective fluid channels 15 can have the same or different numbers of micropores 120 (FIG. 3B). The different groups of micropores 20 for each fluid channel 15 can have the same or different numbers of micropores 120 (FIG. 3B).

[0161] As in Figure 1AAs shown by the virtual box indicating a single field of view (FOV) 25 at positions A1, A2, the fluid device 10 may be configured such that a subgroup or subarray 10s of the array 10a (the entire micro-well array), shown as adjacent to the 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) to image different subgroups 10s of a group of micro-wells 20 (e.g., a single micro-well array 120a of multiple different fluid channels 15 (sample channels) covered by the FOV 25) at any one point in time. The adjacent group 15n may be arranged to have a subgroup or all (e.g., a portion or entire row) of the fluid channels 15 corresponding to the group of micro-wells 20.

[0162] 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 a group of micro-wells 20 of the fluid device 10( Figure 1A , Figure 1B ), which may be a subgroup of the group of micro-wells 20 and contains at least one group of micro-wells 20 from multiple adjacent sample channels of the fluid channels 15 (sample channels), but less than all the groups of micro-wells 20 (groups of micro-wells) of the fluid device 10 (microchip). For example, the defined wavelength range may be associated with light for assaying signals and / or encoding fluorophores.

[0163] As shown in Figure 1A , the defined subarray 10s may be associated with a single continuous position (e.g., an entire single row) of the array 10a of micro-wells of the fluid device 10. The defined subarray 10s may then be imaged or optically analyzed, typically by successively imaging the groups of micro-wells 20 in the corresponding adjacent channels 15n of the fluid channels 15 at a single position after a first assay cycle. That is, the assay comprises multiple 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 group of micro-wells 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 group of micro-wells 20 in the second row R2.

[0164] 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 polymers, such as silicone or other polymeric materials (e.g., PMMA, COC, COP, PDMS, PP, PE, PTFE, or Kapton (polyamide), and many other materials), and 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.

[0165] 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 being formed in one or more of the substrates 10u, 10b to have an open top surface and a closed bottom surface, and the sidewalls extending 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 the bottom can provide the groups of micropores 20, and a closed surface of the fluid channels 15 can be formed.

[0166] 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.

[0167] 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 a biological fluid, blood, serum, urine, dried blood, cell growth medium, lysed cells, a beverage, or food. The sample can also or alternatively include an environmental sample, such as water, air, or soil.

[0168] 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).

[0169] 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.

[0170] 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.

[0171] 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).

[0172] 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 in a reaction vessel (test tube, microwell, etc.) together with other reaction components.

[0173] As used herein, the term "magnetic" includes ferromagnetic, paramagnetic, and superparamagnetic properties.

[0174] Typically, 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.

[0175] 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 sets of microwells 20, each set containing an array of thousands of microwells 120 (microwell array 120a), typically with one bead occupying one bead retention segment 120w (bead well). The microwells 120 can have any suitable volume capacity. In some embodiments, the volume capacity of the microwells 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 microwells 120 is about 100 femtoliters.

[0176] Before sealing the pores from each other using immiscible oils, the 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 generated rapidly, without interference between primer sets. Signals from dsDNA intercalating dyes indicate target amplification, and the target ID is determined by the encoding of the beads. At low concentrations, single molecule counting (digital signal) allows precise analyte quantification. At higher concentrations, real-time PCR (analog signal), measured as the average of the fluorescence signals from all wells of the same reaction type, extends the quantification range above the digital signal saturation point.

[0177] 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 within 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 design variations. In a particular embodiment, it is preferred that one region has a bead retention segment 120w (pocket or container) (FIG. 3B) with a diameter of about 100% - about 150% of the bead diameter and a depth of about 50% - about 185% of the bead diameter, and has a fluidly connected region consisting of an assay signal segment 120s (narrow pocket or slit) (FIG. 3B) or other geometries into which a standard bead cannot physically fit. Both regions are fluidly 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 reaction wells while increasing their reaction volume, potentially improving reaction efficiency.

[0178] 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, such as up to about 1,000,000,000 reactions.

[0179] We have developed a 12-plex respiratory panel using synthetic targets. Excellent linearity was observed over the entire range from 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 immunological PCR with sub-pg / mL LOD.

[0180] Typically, digital assays do not require imaging of the assay signal after each amplification cycle. However, analog assays rely on 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). 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:

[0181]

[0182] Those skilled in the art will understand how to adjust for efficiencies less than 100%.

[0183] The resolution of concentration measurements (i.e., the precision with which concentration can be determined) depends on how frequently fluorescence is measured. Maximum resolution is obtained by imaging after each amplification cycle (i.e., approximately every doubling of amplicon concentration). However, frequent imaging results in photobleaching of the dye, which depends on the manner in which the dye is exposed 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.

[0184] 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 micropores 20 with associated micropore 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 the (shown in) can be assembled on a 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.

[0185] 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 with 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 through 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.

[0186] 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. Increasing the 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 the thermal cycling required to image a large number of arrays can be problematic because if given enough time, active polymerase tends to write dimers and non-specific products. This can lead to increased background signal and reduced assay specificity.

[0187] 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.

[0188] The properties of the encoded bead arrays can permit unique methods for mimicking PCR data collection and analysis. If the concentration of the target molecule is in the mimicking domain, the average number of molecules of each bead type will be approximately the same. Thus, the Ct of 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 typically approximate 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 a time and image many arrays in a short period of time without the need to translate long distances or through complex paths.

[0189] 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 a respective microwell 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 segments 120w (wells) from each group of microwells 20 across a row R 1 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 mimicking data without imaging the entire array 10a of different groups of microwells 20.

[0190] Figure 17A flowchart illustrating an exemplary method for identifying a target substance and / or target molecule. A fluid analysis device is provided having a first fluid channel with 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.

[0191] 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 other sets of micropores. The multiple sets of micropores along the first fluid channel are in fluidic communication only during loading and prior to the sealing step (block 602).

[0192] 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 sets of micropores are not imaged after each reaction step (block 612).

[0193] 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 sets of micropores are not imaged after each reaction step (block 614).

[0194] 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).

[0195] Optionally, a camera having a field of view (FOV) is used to obtain the signal intensity only from the defined subset, the field of view covering only the subset of the group of micropores in the corresponding fluid channel 15 (sample channel) and only covering 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 occupying 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).

[0196] Optionally, obtaining signal strength includes obtaining an analog signal. When the amplitude varies with the number of PCR cycles of the input material in the micropore array of the fluidic channel, the analog signal can provide real-time PCR data, and the concentration of target molecules 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 micropore reaction is identified as positive when the fluorescence signal is greater than the threshold, or a micropore reaction is identified as negative 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 to a reference reaction to determine the number of target molecules at the start of the PCR reaction.

[0197] Optionally, obtaining signal strength 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 array of the fluidic channel, and determining whether the PCR reaction is positive for the target molecule when Si >> Bs, 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 average, median, mode, 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 early PCR cycles) and then determining the average or median.

[0198] In some embodiments, reference Figure 19 and Figures 20A - 20D , loading the beads into the fluidic device 10 can be performed as follows. The fluidic 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 ), 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 in can be performed in the reverse order. After adding the bead slurry into 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 by a hard stop device 810 ( Figure 20C ) such that the beads do not enter the common manifold channel (which may allow bead mixing between samples) (block 740).

[0199] In each fluid channel 15, there are segmented groups, optionally physically spaced apart, of micro-holes 20 (block 704), shown as five groups of micro-holes 20 in Figure 1A which 1 -20 5 (each having 12.5k holes), for each material input or fluid channel 15, containing a total of thousands or millions of micro-holes 120, optionally about 62.5k micro-holes 120.

[0200] After the micro-holes 120 (FIG. 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-hole 16v, input port 16 ( Figure 20D ) by translating the magnet 800 towards the through-hole 16v (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 the 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).

[0201] Optionally, absorbent pads can be placed on the reservoir 16r and / or the through-hole 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 leaks out from the through-hole 16v and / or the reservoir 16r ( Figure 2A )(block 772).

[0202] Optionally, a second microfluidic channel, channel network, or reservoir can be used to store the waste effluent instead of allowing it to leak out from the through-hole. 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 the through-hole in a manner that prevents it from filling before the main mixture addition and / or sealing step using a wax or paraffin valve or a hydrophobic constriction. In some embodiments, after the beads are added, the through-hole 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.

[0203] 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.

[0204] The array 10a of groups of micro-wells 20 of the fluidic channels 15 is divided into sub-arrays, namely, A - E, herein referring to row and fluidic channel numbers (i.e., A1 is the group of micro-wells 20 at the upper left corner of the device 1 and E8 is the group of micro-wells 20 at the lower right corner of the device 5 ). A1 - A2 can be imaged by a single image acquisition.

[0205] Example data collection and processing method 1 (“Method 1”)

[0206] The 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 these angular 20c groups of micro-wells 20 in X, Y, and Z (focus height) 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 from 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.

[0207] 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, this 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.

[0208] 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 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 holes). 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 bank (F1 ,F 2 , Figure 8 ) is changed to the encoded wavelength, and a first set of encoded images is taken for each subgroup of the group of micro-wells 20. Subsequently, if necessary, a second set of encoded images is taken using a second filter set, and the process for decoding the bead group is repeated as needed.

[0209] 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 embedded dye signal for each well exceeds a set threshold. 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 that show strong signals early in the cycle can be considered non-specific amplification products or mis-identified beads.

[0210] 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 least one of the micro-wells 20 in the fluid channel 15 of the array 10a for each sample is imaged after each PCR cycle. 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 in each image. The average signal for each bead type can then 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 sub-arrays 1B, 1C, 1D, and 1E. At cycle 6, the data from the second image of sub-array 1A is used; at cycle 11, the data from the third image of sub-array 1A is used. This process is used for all eight fluid channels 15 (and each channel can have the same sample or different samples, i.e., eight samples) on each fluid device 10 (chip) to construct a real-time PCR curve.

[0211] 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 microwells 20) is not completely uniform. This can 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.

[0212] Example Data Collection and Processing Method 2 ("Method 2")

[0213] In some embodiments, data collection can be performed by imaging only a group of sub-arrays (a subgroup of the group of microwells 20) for all PCR cycles (in this non-limiting example, 30 cycles). In this method, using the position of the corner sub-arrays as described in Example Method 1 above, the camera can be aligned and focused. Then each group of microwells 20 is imaged at the imaging temperature before the PCR thermal cycle to obtain a baseline "pre-PCR" image.

[0214] After the first cycle of PCR, a group of microwells 20 in a row (a sub-array) is imaged. For example, different groups of microwells 20 in row C are imaged. The group of microwells 20 is imaged sequentially 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, for 30 PCR cycles, the same row will be imaged 30 times.

[0215] After the thermal cycle, a "post-PCR" image of the entire array is taken at the imaging temperature. The array is cooled (usually 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 cycle. The filter sets (F 1 , F 2 , 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 using a second filter set.

[0216] Similar to Example Method 1, the processing of imaging data can be divided into two domains: digital and real-time (analog). For this method, the digital PCR signal can be determined by comparing only the pre-PCR and post-PCR images to determine for each well whether the increase in the embedded dye signal exceeds a set threshold (Table 1). In some embodiments, the digital can be obtained separately from the post-PCR image.

[0217]

[0218] Table 1: Data was 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.

[0219] 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.

[0220] The analog signal processing of the imaged subarray is similar to traditional full-array imaging methods. The signal for 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 per bead, the average signal from several hundred beads should be sufficient to accurately and precisely determine the Ct.

[0221] The methods of the present invention (e.g., Example Methods 1 and 2 above) can have the advantage 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.

[0222] Advantages of collecting analog data across multiple sub - arrays (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 sub - array is only exposed to 20% of the illumination used for a standard collection protocol. 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.

[0223] Advantages of collecting analog data from a group of microwells 20 in a single row (i.e., a sub - array or subgroup of microwells as described with respect to Example Method 2) can include less reliance on the normalization of data for Ct determination, making the analysis more straightforward. Additionally, the pre - PCR image can be used to determine which row has the best bead loading for analog real - time PCR analysis. This can help improve problems that may occur if one or several sub - arrays are not loaded with enough beads for analog analysis (although this problem can be addressed by other means, such as tightly controlling array size, geometry, position, bead population size, and bead size).

[0224] 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, groups of microwells 20 in a single row in different fluid channels 15 can be imaged every cycle or every other cycle, combined with the 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 analog 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).

[0225] The fluid device 10 shown is merely exemplary 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 a favorable manner), and other designs may be used in the practice of the present invention. In some embodiments, the fluid device 10 of the present invention may 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 set of micro-wells 20. In some embodiments, the fluid device 10 of the present invention may be fabricated in silicon, glass, polymers (e.g., injection molded and / or thermally embossed plastics), and / or metal films. In some embodiments, the fluid device 10 of the present invention may be a composite structure. In some embodiments, the fluid device 10 of the present invention may contain only the bead / reaction well array and the array chamber, or may include other structures, such as sample handling microfluidic circuits and / or labeling or amplification reagents required for performing assays.

[0226] Electrical and / or mechanical circuits, actuators, and / or sensors may be integrated, attached, and / or assembled into and / or onto the fluid device 10, optionally for performing assays and / or reading out assays.

[0227] Polystyrene beads can absorb hydrophobic molecules as well as charged molecules from aqueous solutions. The long and narrow geometry of the micro-wells 120 (micro-well array) can result in the depletion of certain components (e.g., intercalating dyes) in the main mixture as the main mixture flows through the fluid channels 15. Beads in the first sub-array can absorb a greater amount of the component (e.g., intercalating dye) when they encounter the main mixture, while the last array can absorb less of the component 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, variations in the intercalating dye concentration 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 observed in some of the sub-arrays in Figures 5 and 6.

[0228] The dye uptake of the beads can be modified by including (e.g., adding) various reagents to effect more uniform dye (e.g., SYBR) uptake / distribution across the 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 the PCR and thus not associated with the intercalating dye), partially double-stranded DNA, ionic or non-ionic polymers such as dextran sulfate or polyamines, surfactants, detergents, phase transfer catalysts, dextrans, cyclodextrins, silicones, polysiloxanes, fluorocarbons, perfluorohydrocarbons, fluorosilicones, hydrocarbons, alcohols, hydrofluorocarbons, 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 intercalating dye are particularly well-suited for this purpose. The dye homogenizer can be miscible with the master mix. In some embodiments, the dye homogenizer is present in and / or added to the master mix. The dye homogenizer (e.g., partially double-stranded DNA) can be present in the master mix 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.

[0229] For example, oligonucleotides (oligomers) such as non-extendable oligomers (NE oligomers) 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'-attachment of biotin linked through 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 that can form dimers. As in Figure 22As shown, it can be seen that adding NE oligomers to the 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 the mycoplasma target sequence was compared between 5 subarrays in the channels of the chip, which had either SYBR alone (CV 23%) or SYBR plus NE oligomers (CV 4%) in the master mix. The reduced variation can lead to a more accurate determination of the average 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 melting point of the amplicon.

[0230] In some embodiments, the fluorescence signal from the encoded dye may be affected by the concentration of the intercalating dye, and it may become difficult to decode the array (e.g., assign beads to clusters during the encoding assay) when there are large differences in dye absorption among 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 non-extendable oligomers were added to the solution, the variation in the apparent encoded dye fluorescence signal was smaller (CV 5%).

[0231] As an alternative or in addition, differences in dye uptake during loading of the array with the master mixture can be addressed by other means. For example, in some embodiments, the 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. A partially double-stranded oligonucleotide (pdsDNA) can be used, which is optionally attached to the beads via biotin-TEG ligation chemistry available from IDT, since dsDNA associates more strongly with the intercalating dye than ssDNA. In some embodiments, prior to the assay, the 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 mixture containing all the reagents required for PCR (except 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 。

[0232] Figure 25 Showing the 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.

[0233] The melting point 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 since 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 modulate its uptake characteristics for dyes or other reagents.

[0234] 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.

[0235] In some embodiments, the fluid device 10 of the present invention may be fabricated 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.

[0236] 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.

[0237] 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 groups of microholes 20 is imaged at high resolution, or a lens system that allows 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.

[0238] 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.

[0239] 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 of the excitation energy used to record 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.

[0240] 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.

[0241] 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.

[0242] Figure 8 FIG. 4 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), the optical system (optical signal detector 220) including 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, a heat source 240 for applying heat to one or more fluid devices 10 during an assay cycle.

[0243] 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) F 1 、F 2 or even more filters, for example, by way of example, 2 - 100 filters, each or some capable of providing 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 sub - array selection module 250 to detect signals and / or image subgroups or sub - arrays 10s of an array 10a (microwell array) of a corresponding fluid device 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.

[0244] The system 200 can also include a signal analysis module 260. The signal analysis module 260 can analyze assay signal data from different groups of microwells.

[0245] System 200 can obtain an analog signal that defines a threshold cycle or cycle threshold "Ct", where 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.

[0246] Controller 210 can communicate with a sub - array selection module 250 (i.e., including computer program code) configured to select different groups of microwells during different assay cycles. The modules (sub - array selection module 250 and / or signal analysis module 260) can be located in whole or in part on the controller and / or the optical system (optical signal detector 220) or away 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.

[0247] Sub - array selection module 250 can be configured to identify a subgroup of (aligned) microwells in two, three, four, five or more adjacent fluid sample channels to provide position data. Optionally, the identified subgroup contains an adjacent group (i.e., corner group) of microwells 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 microwells 20 (microwell groups). As an alternative or in addition, alignment marks 27 (Figure 1) at one or more locations of the fluid device 10 can be used for this position data.

[0248] The modules (sub - array selection module 250 or signal analysis module 260) can be on system 200 (analysis system) or distributed among 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 a wired or wireless connection and can include various types of tangible non - transitory computer - readable media.

[0249] In use, cloud computing can be used to provide 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 traditional computing models, 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 function 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 where data is stored on multiple virtual servers rather than being hosted on one or more dedicated servers.

[0250] Controller 210 can communicate with server 410 or a computer via 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").

[0251] 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. System 1116 can be located on one machine or distributed across multiple machines. Processor 500 communicates with memory 536 via an address / data bus 548 and with input / output circuit 546 via an address / data bus 549. Input / output circuit 546 can be used to transfer information between a 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.

[0252] In particular, processor 500 (which can be incorporated into controller 210, Figure 8) can be a commercially available or customized 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 according to 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).

[0253] 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 according to 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.

[0254] The data 556 can include a (archived or stored) digital image data set 522. As further described in Figure 18As further described in, according to some embodiments of the present invention, the application program 554 includes a micro pore sub-group 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.

[0255] 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, 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, but may be provided by other arrangements and / or partitions of the functions between the data processing systems. For example, although Figure 18 is described as having various circuits and modules, one or more of these circuits or modules may be combined or separated without departing from the scope of the present invention.

[0256] For ease of development, computer program code for operating the data processing systems, 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 the 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 a wide area network (WAN), or can be connected to an external computer (e.g., via the Internet using an Internet service provider).

[0257] 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.

[0258] These computer program instructions can also be stored in a computer-readable memory that 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.

[0259] 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 boxes of the flowchart and / or block diagram.

[0260] The flowcharts and block diagrams in certain of the figures herein illustrate exemplary architectures, functions, and operations of possible implementations of the embodiments of the present invention. In this regard, each box in the 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 boxes may not occur in the order noted in the figures. For example, two boxes shown in succession may in fact be executed substantially concurrently, or the boxes may sometimes be executed in the reverse order, or two or more boxes may be combined, depending upon the functionality involved.

[0261] 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).

[0262] The foregoing describes the present invention, and should not be construed as limiting thereof. The present invention is defined by the appended claims, with equivalents of the claims being 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.

Claims

1. An analysis system, the system comprising: A housing, the housing comprising a chamber, the chamber being sized and configured to receive at least one microfluidic device; The at least one microfluidic device comprising a plurality of fluid channels, each fluid channel having a plurality of sets of micropores positioned along a length dimension associated with a direction between a sample input port and a second opposing port; An optical system coupled to the housing in optical communication with the at least one microfluidic device; wherein the optical system comprises a camera having a field of view that only covers a subarray of the plurality of sets of micropores in at least two, optionally all, adjacent fluid channels of the microfluidic device; A controller coupled to the optical system, wherein the controller comprises at least one processor; A heat source coupled to the optical system and thermally coupled to the at least one microfluidic device held in the housing; A subarray selection module comprising computer program code, in communication with the controller, wherein the subarray selection module is configured to select a subarray of the plurality of sets of micropores of at least one fluid channel of the microfluidic device for imaging by the optical system after a reaction step during an assay; And A signal analysis module integrated in and / or coupled to the controller, wherein the signal analysis module is configured to obtain a simulated PCR signal for each micropore in the selected subarray of the plurality of sets of micropores, and wherein the simulated PCR signal provides PCR data and the concentration of target molecules of a reaction associated with a defined bead type when the amplitude varies with the cycles of the assay, and wherein the signal analysis module is further configured to obtain one or more digital PCR signals for a part or all of the plurality of sets of micropores.

2. The system of claim 1, the system further comprising a magnet held in the housing adjacent to the 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 beads to guide the beads to travel along the fluid channel and into a bead retention segment of the plurality of sets of micropores.

3. The system of claim 1 or 2, wherein the selected subarray of the plurality of sets of micropores is associated with a single row of a first subarray of the plurality of sets of micropores of the plurality of fluid channels that is laterally aligned, wherein the single row of the first laterally aligned microarray of micropores is perpendicular to the plurality of fluid channels.

4. The system of claim 1 or 2, wherein prior to photoexcitation, the subarray selection module identifies a subarray of the plurality of sets of micropores to define the positions of other subarrays of the plurality of sets of micropores of the microfluidic device.

5. The system of claim 1 or 2, wherein the subarray selection module selects different subarrays of the plurality of sets of micropores of the microfluidic device at different reaction steps of the assay and directs the optical system to only excite the currently selected subarray of the plurality of sets of micropores and then directs the camera of the optical system to successively or concurrently acquire images of the different subarrays of the plurality of sets of micropores.

6. The system according to claim 1 or 2, wherein the sub-array selection module selects the same sub-array of multiple groups of microwells in at least some different consecutive reaction steps of the assay, and directs the optical system to transmit light only to the currently selected sub-array of the multiple groups of microwells, and then directs the camera of the optical system to sequentially or simultaneously acquire images of different sub-arrays of the multiple groups of microwells.

7. The system according to claim 1 or 2, 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 defines excitation light corresponding to a first wavelength and / or a second wavelength of beads encoded with a first target and beads encoded with a second target, for decoding bead sets held in one or more groups of microwells of at least one fluid channel of the microfluidic device.

8. The system according to claim 1 or 2, wherein when the amplitude varies with the number of PCR cycles of the input material in multiple groups of microwells of the fluid channel, the simulated PCR signal provides real-time PCR data, and the defined type of molecular concentration is equal to or greater than 1 molecule / microwell, and wherein the simulated PCR 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 microwell reaction is identified as positive when the fluorescence signal intensity (Si) is greater than the threshold, or a microwell reaction is identified as negative if the fluorescence signal intensity (Si) is always less than the threshold.

9. The system according to claim 8, wherein the Ct is the number of cycles at which Si >> Bs, where >> is at least 5 - 10% greater than Bs and / or 5 - 10 times the standard deviation of Bs, and wherein Bs is the background fluorescence signal.

10. The system according to claim 1 or 2, wherein the simulated PCR signal is obtained only for a single sub-array of the multiple groups of microwells in each fluid channel after every other or every one of the multiple different reaction steps of the assay, and / or wherein the simulated PCR signal is obtained for each sub-array of multiple groups of microwells in a row after every other or every one of the multiple different reaction steps of the assay.

11. The system according to claim 1 or 2, wherein the simulated PCR signal comprises the average value, median value, mode value or weighted value of Si.

12. The system according to claim 1 or 2, wherein the controller and / or the signal analysis module is configured to direct the optical system to acquire pre-PCR and post-PCR images and compare the signal intensities therebetween to determine positive and negative PCR reactions.

13. The system according to claim 1 or 2, wherein the controller and / or the signal analysis module is configured to direct the optical system to determine one or more concentrations of a target substance and / or one or more molecular concentrations in the original sample provided to one or more of the multiple fluid channels.

14. The system according to claim 1 or 2, the system further comprising a retainer configured to hold a plurality of microfluidic devices in an aligned grid within the housing.

15. The system according to claim 14, wherein the retainer comprises a thermally insulating material that provides a thermal barrier between adjacent microfluidic devices.

16. The system according to claim 15, wherein the retainer holds the plurality of microfluidic devices with the input ports of the fluid channels facing outward.

17. The system according to claim 1 or 2, wherein the plurality of fluid channels comprises a first fluid channel, a second fluid channel, a third fluid channel, and a fourth fluid channel.

18. The system according to claim 17, wherein the first, second, third, and fourth fluid channels comprise straight or arcuate segments that are substantially parallel to each other.

Images