Analytical systems equipped with microfluidic devices, and related methods for microfluidic devices.

Abstract

The present invention provides an analytical system comprising a housing having a chamber of a size and configuration that accommodates at least one microfluidic device. [Solution] The system includes an optical system coupled to a housing in optical communication with at least one microfluidic device, a controller coupled to the optical system, a heat source coupled to the optical system and thermally coupled to at least one microfluidic device fixed to the housing, and a subarray selection module in communication with the controller. The subarray selection module is configured to select a subset of microwell sets of at least one fluid passage of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during inspection.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefits and priority of U.S. Provisional Patent Application No. 62 / 760,604, filed on 13 November 2018, and U.S. Provisional Patent Application No. 62 / 825,523, filed on 28 March 2019, and the contents of both applications are incorporated into this application by reference as if they were fully described.

[0002] (Indication of federal government support) This invention was made with government support under authorization number HR0011-12-2-0001 granted by the U.S. Department of Defense (DARPA). The U.S. Government has certain rights to this invention.

[0003] (Description regarding electronic filing of sequence listings) A sequence listing in ASCII text format, filed pursuant to §1.821 of the Patent Act Enforcement Regulations, named 5470-860WO_ST25.txt, with a size of 817 bytes, created on October 29, 2019, and submitted via EFS-Web, is presented in lieu of a paper copy. This sequence listing is incorporated by reference into the specification for its disclosure.

[0004] (Copyright reservation) A portion of the disclosure in this patent document contains materials that are protected by copyright. The copyright holder, the University of North Carolina of Chapel Hill, North Carolina, as recorded in the U.S. Patent and Trademark Office patent files or records, will not object to any reproduction of the patent document or patent disclosure by any party, but reserves all other rights.

[0005] The present invention relates to a system, a fluid device (e.g., a high-capacity microfluidic device) suitable for, for example, acquiring polymerase chain reaction (PCR) data from a coded bead microwell array, and a method for using the same. [Background technology]

[0006] Polymerase chain reaction (PCR) is a highly sensitive method for amplifying segments of genomic DNA (gDNA) or complementary DNA (cDNA). PCR has many applications, such as detecting trace amounts of nucleic acids to determine the presence of pathogenic organisms, gene expression, genotyping, genetic manipulation or modification, and forensic applications. PCR amplification allows for significant target identification and quantification over a wide range of analyte concentrations. However, simultaneous quantitative analysis of many analytes by PCR has proven extremely difficult. Detection based on insertion dye fluorescence can only determine the overall concentration of dsDNA, and therefore simultaneous analysis of multiple templates in a single reactor using this detection method is not possible. Since each target is amplified using different colored fluorescent probes as signaling reporters, fluorescent probe techniques (i.e., TaqMan, molecular beacons, or other chemical structures) can be used for low-level multiplexing of the reaction. The probes are also sequence-specific, reducing false positives due to primer dimer formation or non-specific amplification. Conventional multiplexing methods using either microtiter plates or microfluidic real-time PCR (rt-PCR) typically involve a small number of reaction wells, each containing three different color probes. However, designing multiplexing primer and probe sets is generally considered challenging, requiring additional levels of careful design and optimization to ensure compatibility. While multiplexing with at least six dyes has been demonstrated, this method ultimately limits the detection of approximately four colors due to spectral overlap between instruments and dyes, and typically involves providing one color as an inner reference dye. [Overview of the Initiative] [Means for solving the problem]

[0007] Embodiments of the present invention provide a method for limiting photobleaching in an encoded bead array while optionally acquiring real-time PCR data.

[0008] Embodiments of the present invention provide formulations of PCR master mixes and chemical structures of magnetic particles that are particularly suitable for use with coded bead arrays.

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

[0010] Embodiments of the present invention relate to an analytical system. The system includes a housing encompassing a chamber having a size and configuration for receiving at least one microfluidic device; an optical system coupled to the housing in optical communication with at least one microfluidic device; a controller coupled to the optical system; a heat source coupled to the optical system and thermally coupled to at least one microfluidic device fixed to the housing; and a subarray selection module in communication with the controller. The subarray selection module is configured to select a subset of microwell sets of at least one fluid passage of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during the examination.

[0011] The system may include at least one magnet fixed to the housing adjacent to at least one microfluidic device. The magnet may be configured to move in parallel along at least one fluid passage during the bead loading process, magnetically coupling to the bead, and guiding the bead to move along the fluid passage to the bead-holding segment of the microwell.

[0012] A microfluidic device comprises multiple fluid passages, each comprising multiple microwell sets arranged along a length dimension associated with the direction between a sample introduction port and a second port opposite it. The selected subset of microwell sets may be a first microwell subset of transversely aligned fluid passages, or optionally a single transverse row of one subset of aligned microwells from each of the fluid passages.

[0013] Prior to initial image acquisition and / or photoexcitation, the subarray selection module identifies a subset of microwellsets to define the locations of other microwellsets in the microfluidic device.

[0014] The subarray selection module selects different subsets of microwellsets of a microfluidic device at different reaction steps of the inspection (e.g., different thermal cycles of the inspection), instructs the optical system to excite only the currently selected subset of microwellsets in different fluid passages, and instructs the optical system's camera to sequentially or in parallel capture images of different subsets of microwellsets in the currently selected subset.

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

[0016] The optical system may have at least first and / or second filters (optionally more than two, such as 3 to 100) that define the first and / or second excitation light wavelengths corresponding to the first and second target coded beads in order to decode the bead sets held in one or more microwell sets in at least one fluid passage of the microfluidic device.

[0017] The system may have a signal analysis module integrated with and / or coupled to the controller. The signal analysis module may be configured to acquire analog signals for each microwell of a selected subset of the microwell set. The analog signals can provide PCR data as amplitude changes over the test cycle, and the concentration of the target molecule for a given bead type and associated reaction is determined by the analog signals.

[0018] The signal analysis module may also have a configuration to acquire digital PCR signals from microwell sets.

[0019] The analog signal provides real-time PCR data as the amplitude change of the input substance in one or more microwell sets of fluid passages with respect to the number of PCR cycles, where the molecular concentration of a given bead type is approximately 1 molecule, equal to 1 molecule, or greater than 1 molecule per microwell. The analog signal can define a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescence signal intensity (Si) is above a threshold and the number of target molecules / target molecular concentration for the target species and / or molecular type, or identifies a negative reaction when the fluorescence signal intensity (Si) is always below a threshold for a given number of PCR cycles. Ct is the number of cycles where Si >> Bs, where >> is at least 5–10%, optionally greater than 2 times, and / or 5–10 times the standard deviation of Bs, where Bs is optionally the background fluorescence signal measured in a PCR negative reaction.

[0020] An analog signal can be acquired only for a single subset of the microwell sets of each fluid passage, every other or after each of a plurality of different reaction steps of the assay (e.g., different thermal cycles of the assay).

[0021] The analog signal is the mean value of Si (optionally excluding outlier data), median, mode, or weighted value as an analog signal corresponding to each defined bead type, and is provided as an estimated value of the real-time PCR curve for a similar reaction in the microwells of other microwell sets of the corresponding fluid passage of the microfluidic device, enabling single-cycle resolution even if not all microwell sets of each fluid passage are imaged after different reaction cycles or after each reaction cycle.

[0022] The optical system can have a camera with a field of view (FOV) that covers only at least two, optionally all, adjacent fluid passages of the microfluidic device and microwell subsets.

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

[0024] The controller and / or signal analysis module instructs the optical system to acquire pre- and post-PCR images and, optionally, compares the signal intensities in between with the analog data acquired using a selected subset of microwell sets of different reaction steps of the assay to determine positive and negative PCR reactions and optionally determine the concentration of the target species and / or molecular concentration of the original sample provided to the fluid passage.

[0025] The system can further include a holder configured to fix a plurality of microfluidic devices to an alignment grid in the housing.

[0026] The holder is formed of an insulating material that provides a thermal barrier between adjacent microfluidic devices, and optionally the holder can secure multiple microfluidic devices with the (sample / bead) input ports facing outward into the fluid passage.

[0027] The microfluidic device further includes a dye homogenizer, which optionally includes oligonucleotides.

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

[0029] The dye homogenizer may contain biotin and / or partially double-stranded DNA comprising C1-C20 hydrocarbon chains.

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

[0031] Other embodiments relate to methods for analyzing a sample, such as for identifying a target species and / or target molecule. This method includes preparing a fluid analysis device comprising a first fluid passage containing a plurality of microwell sets arranged on the first fluid passage; acquiring signal intensity data from only a specified subset of the plurality of microwell sets (optionally receiving a photoexcitation signal); and identifying PCR reactions that are positive for a bead type and / or a target species and / or molecular type associated with the target molecule, based at least in part on the acquired signal intensity data.

[0032] This method may include loading (a bead slurry pretreated with a sample) and then sealing a plurality of microwell sets on a first fluid passage before the acquisition step such that each microwell set is fluidly isolated from the others after sealing. The plurality of microwell sets on the first fluid passage may be in fluid communication only during loading, which precedes the sealing step.

[0033] This method may include changing a predetermined subset of microwellsets to a different predetermined subset after each of the multiple sequential reaction steps of the test (for example, after each of the multiple sequential thermal cycles of the test), thereby ensuring that some microwellsets are not imaged after each reaction step.

[0034] Because the specified subset remains the same across multiple consecutive reactions of the test (for example, after each of multiple consecutive thermal cycles of the test), some microwell sets are not imaged after each reaction step.

[0035] The fluid analysis device may include a number of fluid passages, each having between 2 and 100 microwells, including, but not limited to, a second fluid passage having a plurality of spaced-apart microwell sets in the second fluid passage, a third fluid passage having a plurality of spaced-apart microwell sets in the third fluid passage, and a fourth second fluid passage having a plurality of spaced-apart microwell sets in the fourth fluid passage. The first microwell sets of each of the first, second, third, and fourth fluid passages may be aligned as a first row. The second microwell sets of each of the first, second, third, and fourth fluid passages may be aligned as a second row. The third microwell sets of each of the first, second, third, and fourth fluid passages may be aligned as a third row. The fourth microwell sets of each of the first, second, third, and fourth fluid passages may be aligned as a fourth row. A specified subset may be a single row (part or all of) from the first, second, third, or fourth row.

[0036] The first, second, third, and fourth fluid passages may have linear or arcuate segments that are substantially parallel to each other and form the first, second, third, and fourth microwellsets.

[0037] This method may further include transmitting a photoexcitation signal to only a specified subset before the acquisition step, digitally scanning a specified subset of multiple microwell sets before, after, or before / after the transmission and acquisition steps to acquire images of the microwell sets for identifying positive and negative PCR reactions associated with digital PCR, and electronically identifying microwells in a microwell set that are positive for one or more target analyte molecules while the microwells are at imaging temperature.

[0038] The excitation and acquisition steps are performed such that only one specified subset of the microwell set is scanned after each of the multiple sequential reaction steps of the test (e.g., thermal cycling), and each sequential specified subset may be different from the others.

[0039] Acquiring signal intensity from only a specified subset can be done using a camera with a field of view (FOV) that covers only one microwellset or only a subset of microwellsets in all or a subset of adjacent fluid passages within a fluid passage, by sequentially or in parallel acquiring images of different specified adjacent microwellsets in different fluid passages within a specified subset of microwellsets.

[0040] Each microwell set may have a microwell array containing microwells ranging from 1,000 to 1,000,000.

[0041] A fluid analysis device may have multiple spaced fluid passages, each comprising a set of macrowells including a microwell array. The fluid passages may be in a fluid-isolated state during analysis. At least some of the microwells in the microwell array may contain a single bead, and optionally some or all of the microwells may contain no beads or one or more beads.

[0042] This method may include electronically identifying one or more microwellset locations at one or more positions in a microfluidic device prior to the transmission and acquisition steps, and defining the locations of other microwellsets based on at least a portion of the identified locations.

[0043] The method of claim 20 further comprises transmitting the photoexcitation signal to only a specified subset before the acquisition step, and selecting a filter that provides a coded wavelength for the transmission step before the transmission.

[0044] The acquisition of signal intensity involves obtaining an analog signal that can provide real-time PCR data as amplitude change versus number of PCR cycles for the material introduced into the microwell set, where the molecular concentration of a given bead type is approximately 1 molecule per microwell, equal to 1 molecule, or greater than 1 molecule. The analog signal can define a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescence signal intensity (Si) is above a threshold and the target number of molecules / target molecular concentration of the target species and / or molecular type, or that identifies it as negative when the fluorescence signal intensity (Si) is always below a threshold for a given number of PCR cycles.

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

[0046] Analog signals may be acquired for only a single subset of microwellsets of one or more fluid passages after every other or each of the multiple different reaction steps of the test (e.g., different thermal cycles of the test).

[0047] The analog signal is the mean, median, mode, or weighted value of Si (optionally with outlier data removed) as an analog signal corresponding to each specified bead type, and can be provided as an estimate of a real-type PCR curve for similar reactions in the microwells of other microwell sets in the fluid passage of the microfluidic device, thereby enabling a single cycle resolution even if all microwell subsets of each fluid passage are not imaged after different reaction steps.

[0048] Signal intensity can be acquired by using cameras with a field of view (FOV) that covers at least two microfluidic devices, and optionally only a subset of the microwellsets of all adjacent fluid passages.

[0049] After multiple different reaction steps of the test (e.g., different thermal cycles of the test), analog signals are acquired for only a single set of microwell sets in each fluid passage. The analog signals include the mean, median, mode, or weighted values ​​of Si as analog signals corresponding to each specified bead type, and are provided as estimates of real-time PCR curves for similar reactions of microwells in other microwell sets in the fluid passage of the microfluidic device. This enables single-cycle resolution even if all microwell sets in each fluid passage are not imaged after different reaction steps.

[0050] A fluid analysis device comprising a first fluid passage containing a plurality of spaced-apart microwell sets along the first fluid passage may include a plurality of additional fluid passages, each containing the plurality of spaced-apart microwell sets along the length thereof. The fluid analysis device has separate substance input ports for the first fluid passage and each of the plurality of additional fluid passages, and at least some fluid passages share a common opposite second port. The method further comprises, prior to the acquisition step, fluidly loading a bead slurry pre-exposed to the sample into the input port for analysis, magnetically guiding the bead slurry to flow into different microwell sets on the fluid passage, introducing a fluid master mix containing a dye from the second port through the fluid passage to the first port, and sealing the microwell sets and the fluid passage from each other by flowing a sealing oil from the second port through the fluid passage to the first port.

[0051] The master mix may optionally contain a dye homogenizer. The dye homogenizer may optionally be or may contain oligonucleotides (e.g., non-expandable oligonucleotides and / or partially double-stranded DNA (e.g., biotin and / or partially double-stranded DNA containing C1-C20 hydrocarbon chains)).

[0052] This method may include placing a magnet adjacent to the fluid analysis tip and moving the magnet parallel to a second port before the fluid master mix and sealed oil flow.

[0053] The fluid analysis device (optionally a first fluid passage and / or multiple microwell sets) includes a dye homogenizer, which optionally includes oligonucleotides (e.g., non-expandable oligonucleotides and / or partially double-stranded DNA (e.g., biotin and / or partially double-stranded DNA containing C1-C20 hydrocarbon chains)).

[0054] The dye homogenizer may be present in the master mix of the fluid analysis device (e.g., in the first fluid passage and / or in multiple microwell sets), and / or the dye homogenizer may be attached to beads present in the fluid analysis device (e.g., in the first fluid passage and / or in multiple microwell sets).

[0055] Another embodiment relates to a microfluidic device. The device includes a plurality of fluid passages. Each of the plurality of fluid passages has a length dimension corresponding to the direction between a first port and a second port opposite to it, and at least a portion of the length dimension is configured as a linear or arc-shaped length segment. Each of the fluid passages includes a plurality of microwell sets arranged along the linear or arc-shaped length segment of the length dimension.

[0056] Multiple microwellsets of multiple fluid passages may be arranged in horizontal rows, vertical rows, or both horizontal and vertical rows. The horizontal or vertical rows correspond to linear or arc-shaped length segments.

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

[0058] At least some of the fluid passages are arc-shaped and substantially parallel, which can provide arc-shaped length segments.

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

[0060] Multiple fluid passages may have a first fluid passage set and a second fluid passage set spaced apart from the first set. The first fluid passage set may terminate at a first master mix port as a second port, and the second fluid passage set may terminate at a second master mix port as a second port.

[0061] The first and second fluid passage sets can be spaced apart in the circumferential direction.

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

[0063] The device may include a first gap space between the first and second fluid passages of the first and second neighborhood sets, respectively. The device may further include a second gap space between the first and second neighborhood sets, the second gap space having a larger lateral range than the first gap space.

[0064] Multiple microwell sets in each of multiple fluid passages may have a common configuration. Multiple microwell sets in each of the fluid passages may be aligned with each other in rows and / or columns. Multiple fluid passages may hold the input material in a state of fluid isolation from one another.

[0065] Each of at least several microwell sets for the fluid passage may contain a quantity of microwells ranging from 1,000 to 1,000,000. The microwells of the microwell set, having a size and configuration that fixes and holds a single bead, enable 100 to 1 billion reactions in the microfluidic device.

[0066] At least a set of first and second microwell sets from the first and second fluid passages, optionally a pair, constitutes the first set, comprising adjacent microwell subsets. The device may include a transparent substrate extending across multiple microwell sets of the fluid passage.

[0067] Each of the multiple fluid passages has a first port at its first end, which serves as a first port, and one of the alternating first ports may be located at a first longitudinal position of the device, while the other alternating first port may be located at a second longitudinal position of the device, spaced lengthwise from the first longitudinal position.

[0068] The first port is a material input port and may be located at the first end of the fluid passage. The device further includes a fluid manifold connecting the opposite second ends of at least several fluid passages to the second port.

[0069] The fluid passages may extend radially within the device. The inlet ports of these fluid passages may be located on the outer periphery of the device. The second ports may be a single second port located at the center of the device, connected to each of the fluid passages.

[0070] At least some of the fluid passages may be provided as a concentric set of fluid passages, each having an arc-shaped length segment.

[0071] A set of concentric fluid passages having arc-shaped length segments can be provided as a plurality of concentric fluid passage sets spaced apart in the circumferential direction.

[0072] Microfluidic devices may also contain dye homogenizers.

[0073] The dye homogenizer may optionally contain oligonucleotides.

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

[0075] The dye homogenizer may be present in the master mix of the microfluidic device, and / or the dye homogenizer may be attached to the beads present in the microfluidic device.

[0076] Other embodiments relate to bead loading strategies and the use of a reagent reservoir / injection unit common to the isolated bead injection unit as illustrated and / or described.

[0077] Other embodiments relate to a fully integrated fluid analysis chip in which a live (biological) sample is introduced, the sample is prepared and processed, and then beads (pre-saturated with the sample) are loaded into a microwell array; an associated kit; and a microfluidic chip including pre-loaded reagents as illustrated and / or described.

[0078] It should be noted that one or more aspects or features described in relation to one embodiment may be incorporated into other embodiments, even if not described as explicitly as in this embodiment. That is, all embodiments and / or features of any embodiment may be combined in some way and / or in some combination. The applicant reserves the right to amend the claims of the original application or to file new claims as appropriate, including the right to amend the claims of the original application to include features dependent on and / or incorporated from other claims, even if not claimed in the original application. The above and other objects and / or aspects of the present invention are described in detail in the following specification. [Brief explanation of the drawing]

[0079] The patent or application file must include at least one color drawing. A copy of this patent or patent application publication, along with the color drawing, will be provided by the Patent Office upon request and payment of the necessary fees. The attached drawings, which are incorporated into the specification and form part of it, illustrate embodiments of the invention and, together with the description, serve to explain the principle of the invention. [Figure 1A] This is a schematic diagram of an example of a fluid device according to an embodiment of the present invention. [Figure 1B] This is a schematic diagram of another fluid device example according to an embodiment of the present invention. [Figure 2A] Figure 1A is a top perspective view of the fluid device shown. [Figure 2B] This is a top-view digital photograph of a prototype of the fluid device corresponding to Figure 2A. [Figure 3A] This is an example of a fluid device including a non-cylindrical microwell according to an embodiment of the present invention. [Figure 3B]This is an ultra-magnified view of a small microwell in a portion of the fluid device shown in Figure 3A, according to an embodiment of the present invention. [Figure 3C] This is an example of an inspection signal generated by a non-cylindrical microwell in the fluid device shown in Figure 3A, according to an embodiment of the present invention, and shows an inspection signal separate from the bead background signal. [Figure 4A] This is a raw graph of the fluorescence pair cycles for negative and positive beads. [Figure 4B] This is a graph of fluorescence pair cycles for the positive and negative slits of a microwell according to an embodiment of the present invention. [Figure 5A] ~ [Figure 5H] This is a graph of raw data during a cycle, obtained by combining real-time PCR data from different microwell subsets with different fluid pathways during PCR according to an embodiment of the present invention. [Figure 6A] ~ [Figure 6H] These are graphs of normalized data shown in Figures 5A to 5H according to embodiments of the present invention. [Figure 7A] ~ [Figure 7H] This is a graph of real-time PCR data collected from a single row of bead well sets with different fluid passages according to an embodiment of the present invention. [Figure 8] This is an example of an analysis system according to an embodiment of the present invention. [Figure 9] ~ [Figure 16] This is a schematic top view of different device examples having fluid passages with different configurations according to embodiments of the present invention. [Figure 17] This is a flowchart illustrating an example of an analysis method according to an embodiment of the present invention. [Figure 18] This is a data processing system according to an embodiment of the present invention. [Figure 19] This is a flowchart of the operations that may be performed for loading beads into a fluid device and for thermal cycling after loading, according to embodiments of the present invention. [Figure 20A] ~ [Figure 20D] This is an enlarged side perspective view of an example of microchip loading using a magnet according to an embodiment of the present invention. [Figure 21] This is a top view of a plurality of microchips held by an insulating holder according to an embodiment of the present invention. [Figure 22] This is a box plot of the initial bead fluorescence of each bead in a five-array consisting of one lane with two eight-passage microchips according to an embodiment of the present invention, where one microchip is treated with a master mix containing 10X SYBR and the other microchip is treated with a master mix containing 20X SYBR and 5 μM of non-expandable oligo. [Figure 23] This is a graph of the average number of molecules per bead (digital positive signal) for Mycoplasma in a 12-plex respiratory panel test (related to the digital positive signal, but a combination of digital and analog may also be used). Each point represents a result from one of five arrays in a single lane of an 8-passage microchip according to an embodiment of the present invention, with beads from the same sample loaded into each array. [Figure 24] This is a box plot of encoded dye fluorescence signals from a mycoplasma population in a 12-plex respiratory panel associated with each bead in a five-bead array of microchips treated with SYBR or +NE Oligo and SYBR, according to an embodiment of the present invention. [Figure 25] This is a schematic example of pdsDNA in beads according to an embodiment of the present invention. [Modes for carrying out the invention]

[0080] The present invention will now be described in more detail below with reference to the accompanying drawings illustrating embodiments of the invention. However, the present invention can be embodied in many different ways and should not be construed as being limited to the embodiments presented herein. Rather, these embodiments are provided so that this disclosure is thorough and complete and fully conveys the scope of the invention to those skilled in the art.

[0081] Similar numbers refer to the same elements throughout. In the figures, the thickness of a line, layer, component, element, or feature may be exaggerated for clarity. The abbreviations "FIG." and "Fig." for "Figure" may be used interchangeably in the text and figures.

[0082] The terms used herein are intended solely to describe specific embodiments and are not intended to limit the invention. Where used herein, the singular forms “a,” “an,” and “the” are also intended to include the plural forms unless the context explicitly states otherwise. Furthermore, “comprises” and / or “comprising,” where used herein, indicate the presence of the described features, steps, operations, elements, and / or components, but not to mention that they do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. Where used herein, “and / or” includes any and all combinations of one or more related list items. Where used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted as including X and Y. Where used herein, “between about X and Y” means “between about X and about Y.” When used in this context, phrases like "from about X to Y" mean "from about X to about Y".

[0083] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Furthermore, terms as defined in commonly used dictionaries should be interpreted in a way consistent with their meaning in the context of this application and related art, and should not be interpreted in an ideal or overly formal sense unless explicitly defined to do so. The terminology used herein to describe the invention is intended solely to describe specific embodiments and is not intended to limit the invention. All publications, patent applications, patents, and other prior art referenced herein are incorporated in their entirety by reference. In the event of any inconsistency in terminology, this specification shall prevail.

[0084] Furthermore, when used here, “and / or” refers to and includes any or all possible combinations of one or more related list items, as well as the absence of any combination when interpreted as an alternative ("or").

[0085] Unless the context indicates otherwise, the various features of the invention described herein are expressly intended to be used in any combination. Furthermore, in some embodiments of the invention, features or combinations of features presented herein may be excluded or omitted. For illustrative purposes, where the specification states that a composite includes components A, B, and C, it is expressly intended that any one of A, B, C, or any combination thereof may be omitted or abandoned.

[0086] When used herein, the transitional phrase “consisting essentially of” (and its grammatical variation) shall be interpreted as including the substance or step described in the claimed invention that “does not substantially affect the basic and novel nature.” See re Herz case: 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis added). Also see MPEP (Patent Examination Manual) §2111.03. Therefore, when used herein, the phrase “consisting essentially of” should not be interpreted as equivalent to “comprising.”

[0087] When used herein, the words “example,” “exemplary,” and their grammatical variations are intended to refer to the non-restrictive examples and / or variant embodiments described herein, and are not intended to indicate that one or more embodiments described herein are preferable to one or more other embodiments.

[0088] When referring to measurable values ​​such as quantity or concentration, the word "about" used herein means that, along with the specified value, it includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, when X is a measurable value, "about X" means that, along with X, it includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. The ranges presented herein for measurable values ​​may include other values ​​and / or individual values ​​within them.

[0089] When used here, words similar to “increase,” “increases,” “increased,” “increasing,” and “enhance” refer to an increase of at least approximately 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%, and 500% or more of the specified parameter, unless explicitly stated otherwise in the text.

[0090] When used herein, “reduce,” “reduces,” “reduced,” “reduction,” “inhibit,” and similar terms refer to a reduction of at least approximately 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, and 100% of the specified parameter, unless otherwise explicitly stated in the text.

[0091] When an element is described as being “on,” “attached,” “connected,” “coupled,” or “contacting” another element, it may be directly on, attached to, connected to, coupled to, or in contact with the other element, or an intervening element may also exist. In contrast, when an element is described as, for example, “directly on,” “directly attached,” “directly connected,” “directly coupled,” or “directly contacting” another element, no intervening element exists. A reference to a structure or feature “adjacent” to another feature will also be recognized by those skilled in the art, which may have a portion above or below the adjacent feature.

[0092] Spatial relative terms such as “under,” “below,” “lower,” “over,” “upper,” and others may be used to facilitate descriptions of the relationship between one element or feature shown in a diagram and another element or feature. It will be understood that spatial relative terms are intended to include various orientations of the device during use or operation, in addition to the orientation depicted in the diagram. For example, if the device in the diagram were inverted, an element described as “under” or “beneath” (directly below) another element or feature would have an “over” (above) orientation of that other element or feature. Thus, the exemplary term “under” (below) may include both “over” (above) and “under” (below) orientations. The device may have other orientations (rotated 90 degrees or other orientations), and the spatial relative descriptors used here will be interpreted accordingly. Similarly, “upwardly,” “downwardly,” “vertical,” “horizontal,” and others are used here for descriptive purposes only unless otherwise specified.

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

[0094] In general, embodiments of the present invention relate to analytical systems, fluid devices, and methods for using them. In some embodiments, the fluid device may include a high-capacity fluid device. The fluid device 10 of the present invention may be used to acquire real-time and digital polymerase chain reaction (PCR) data from a coded bead microwell array 120a (Figure 3B) and / or a set or subset of microwells 20 in different fluid passages 15. In some embodiments, the fluid device 10, method, and / or analytical system of the present invention may be used for applications such as non-PCR reactions, loop-mediated isothermal amplification (LAMP), and / or enzymatic reactions for protein analysis. As is well known to those skilled in the art, real-time PCR is defined as a polymerase chain reaction in which signals related to amplicon concentration are collected after each cycle of PCR, and a PCR cycle generally refers to a complete set of steps including denaturation of template DNA, annealing of primers to a single-stranded DNA template, and expansion of primers by polymerase. Please refer to the Expert Rev. Mol. Diagn. 5(2), (2005), pp. 209-219, 0.1586 / 14737159.5.2.2 by Arya et al., whose contents are incorporated into this application by reference as if they were fully described therein.

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

[0096] In some embodiments, the present invention includes, but is not limited to, substrates, devices, designs, solid supports (e.g., coded solid supports), steps, and / or methods described in U.S. Provisional Application No. 62 / 673,343, entitled “Compositions, devices, and methods for improving the surface properties of a substrate,” U.S. Provisional Application No. 62 / 736,525, entitled “Compounds, compositions, and methods for improving inspection,” and / or U.S. Patent No. 9,617,589, U.S. Publication No. 2015 / 0211048, International Publication No. WO2017 / 112025, International Application No. PCT / US2016 / 042913, International Application No. PCT / US2016 / 043463, and / or International Application No. PCT / US2016 / 055407, the contents of each thereof being incorporated in whole by reference.

[0097] In some embodiments, the methods of the present invention include methods for delivering reagents and / or targets to reaction wells using beads (e.g., superparamagnetic beads), as described, for example, in U.S. Patent Publication No. 2015 / 0211048 and International Application No. PCT / US2016 / 042913, the contents of each of which are incorporated in whole by reference in this application.

[0098] The terms “microchip” and “microfluidic chip” are used interchangeably and refer to substantially planar, thin devices. Microfluidic chips 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. Microchips are generally smaller than about 6 inches and more commonly have a width and length between about 1 inch and 6 inches. However, in some embodiments, microchips may be larger in length and / or width, such as more than 1 foot. Microchips may have an outer periphery that is polygonal, rectangular, circular, or other desired shape. Microchips may optionally have a width dimension smaller than their length dimension. In some embodiments, microfluidic chips may have a diameter or width dimension of about 2.13 inches (54 mm) and / or a diameter or width dimension of about 3.4 inches (85.5 mm). Microchips may include millimeter-sized, micro-sized, and / or nano-sized fluid passages.

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

[0100] The terms "micro-size" and "microfluidic" in relation to fluid passages refer to fluid channels having widths and / or depths of millimeters, sub-millimeters, or smaller (for example, the term includes millimeter, micrometer, and nanometer-sized passages that include segments or chambers provided within the passage). A passage may have at least segments having widths and / or depths in the size range of 10 millimeters or less, generally less than 900 microns and greater than 1 nm.

[0101] The term "microwell" refers to a reaction well having a small reaction volume of approximately 100 microliters or less, and in some embodiments, it may optionally hold a single bead, as described below.

[0102] The term "beads" refers to solid-state components such as polymers, photoresists, plastics, glass, silicon dioxide, metals or metalloid oxides (including, but not limited to, aluminum oxide, titanium oxide, zirconium oxide, or other oxides), quantum dots, metal particles, and other materials that may be porous, surface-porous, or non-porous, generally such as magnetic or superparamagnetic spheres.

[0103] The term "circuit" refers to a complete hardware embodiment or an embodiment combining software and hardware. The term "module" refers to an embodiment that includes software and hardware or firmware.

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

[0105] Now, referring to Figures 1A, 1B, and 2, an example of the fluid device 10 is shown. The fluid device 10 comprises at least one elongated fluid passage 15 having a plurality of spaced-apart microwell sets 20 located along the length of the elongated fluid passage 15. Each neighboring microwell set 20 on the fluid passage 15 may, but does not necessarily, be separated by a gap space 21 that does not contain microwells. Figure 1B illustrates the microwell sets 20 arranged to be continuous on the fluid passage 15. Figure 1B also illustrates that the neighboring ports of the first port 16 may be aligned in ways other than the longitudinal offset (in the orientation of the figure) shown in Figure 1A.

[0106] One or more microwell sets 20 on the fluid passage 15 may have different geometric installation ranges. When spaced-out microwells 20 as shown in Figure 1A are used, the gap space 21 may have a length shorter than the length L of the installation range of the neighboring microwell sets 20.

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

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

[0109] The gaps 21 between adjacent microwell sets 20 on the fluid passage 15 do not need to be provided by physical gaps or interruptions in the continuous microwells 20, but can be determined by the field of view of the imaging system and / or the illumination window of the illumination source. As shown in Figure 1B, the microwell sets 20 may be provided as a continuous array of microwells 20 along the length of the fluid passage 15.

[0110] The fluid passage 15 may be provided with bubbles or constrictions for aligning the beads. Bubbles or constrictions between neighboring sets of microwells 20 that form the array 120a of the microwells 120 in the fluid passage 15 may be provided for either controlling fluid movement / sealing or facilitating or controlling bead loading.

[0111] Referring again to Figures 1A and 12, the multiple microwell sets 20 of the fluid passage 15 can be arranged as substantially parallel aligned rows with part markings such as letter markings A to E for row R1 to R8, and as optionally vertical aligned parallel passages 15 marked C1 to C8 defining the microwell array 10a of device 10. However, other marking formats may be used, and device 10 does not require a human-readable marking format. More or fewer rows and more or fewer vertical rows may be used. Neighboring passages 15n, shown as pairs 15p of passage 15, are located slightly apart and separated from other adjacent sets of neighboring passages 15n, i.e., other pairs 15p of passages 15 separated by gap spaces 19 provided by the substrate of device 10. Neighboring passages 15n may be separated by a smaller distance than the gap spaces 19 between other adjacent neighboring passages 15n. Although the neighboring passage 15n is shown as being provided as a passage pair 15p, for example, three, four, five, or more adjacent passages, including the entire row, can define a neighboring passage set 15n.

[0112] The microwell set 20 may have an installation area with a rectangular outer perimeter 20r that surrounds the set or array 120a of single-bead microwells 120 (Figure 3B). However, other geometric shapes may be used.

[0113] Each fluid passage 15 contains an independent, dedicated first fluid port 16, which may optionally be an input port for introducing a desired input substance containing a sample. Referring to Figures 2A and 2B, the fluid port 16 may contain a reservoir 16r having vias 16v of a cover substrate 10u that guide the input substance from the reservoir 16r to the input port 16 of the fluid passage 15.

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

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

[0116] As shown in Figures 1A, 1B, and 2, for the passage array 10a, the fluid passages 15 may be substantially parallel over linear length segments containing multiple spaced microwellsets 20. The term "substantially" when referring to "parallel" means that the fluid passages 15 of the array 10a are parallel or nominally parallel (i.e., they may deviate by an angle of less than 10% from the adjacent centerline C / L passing through the longitudinal extension center of the fluid passage 15) over at least a portion of the length segments shown as linear length segments in Figures 1, 2, 9, 10-15 and as arcuate length segments in Figure 16).

[0117] Referring to FIGS. 3A - 3C, each or some of the microwell sets 20 may include a plurality of microwells 120 including a bead - holding segment 120w and an inspection - signal segment 120s. The inspection - signal segment 120s is juxtaposed with the bead - holding segment 120w and may be parallel and / or in series with respect to the upper portion of the device 10 or the primary surface of the cover substrate 10u (FIG. 2A). The inspection - signal segment 120s is in fluid communication with the bead - holding segment 120w of the respective microwell 120. The inspection - signal segment 120s generates a tapered inspection signal 122 that enables the separation of the bead background signal in the well - holding segment 120w. In some embodiments, the inspection - signal segment 120s may have a geometry that physically prevents beads from entering. The segments 120w, 120s are fluid - connected such that the sample and / or analyte released from the beads held in segment 120w diffuses or otherwise mixes throughout the common solution volume of the well 120w. By spatially separating the beads from the detection region 120s, the influence of bead fluorescence on the signal is reduced or eliminated, improving the signal - to - noise ratio. The geometry of the well 120 generally allows for a high loading into the reaction well occupied by a single bead while increasing the reaction volume, potentially improving reaction efficiency.

[0118] FIGS. 9 - 11, 16 illustrate that the device 10 may include a plurality of spaced - apart fluid - passage arrays 10 a5 , 10 a2 , 10 a3 [[ID=!0]], 10 a4 (and FIG. 11 also shows the fifth array 10 a5 of fluid passages 15). FIG. 16 illustrates three concentric sets by four arrays 10 a1 , 10 a2 , 10 a3 , 10 a4 that circumferentially extend for twelve array sets.

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

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

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

[0122] Figure 16 illustrates that the fluid passage 15 is an arc-shaped fluid passage 15a, extending circumferentially to at least a portion of the diameter of the circular geometric configuration. Different fluid passage arrays 10 a1 ~10 a4 The elements are spaced apart in the circumferential direction, and each array 10a may have a common single second port 18.

[0123] Figure 11 shows a fluid passage 15 extending toward the center defining the position of the second port 18, and at least the inner leg 18i of the manifold 18m extends radially toward the second port, and the array A R The centerline C / L extends radially in the device 10 toward the second port 18.

[0124] Figures 12-15 show a fluid passage 15 configured as a passage 15r extending radially inward. The passage 15r extends radially inward from the outer periphery of the device 10, which has the first port 16, toward the center of the device toward the second port 18. Figures 14 and 15 show the fluid passage 15 decreasing in size along its length toward the center of the device 10 toward the second port 18, and the microwell set 20 decreases in size from the first microwell set 201 toward another set 202, 203, 204 as it approaches the second port 18. In this case, the microwell subsets 20 that are imaged simultaneously (together) may be arranged differently from other configurations (such as parallel rectangles).

[0125] The microchip array 10a can define array regions for each microwell set 20 of each fluid passage 15 corresponding to positional addresses such as row and column addresses. For example, row and column-related positions such as 1A (or A1), 2A (or A2), 7E (or E7), and 8E (or E8). The array 10a may be configured to provide several microwell sets 20 as corner microwells 20c. In the embodiments shown in Figures 1A and 2, the microwell sets 20 of array regions A1, A2, A7, A8, E1, E2, E7, E8 are near-corner (sub)sets 20c of the microwell set 20. Again, two or more fluid passages 15 may be configured to provide adjacent microwell sets that define corner sets 20c of the microwells 20.

[0126] The device 10 shown in Figures 1A and 12 has a rectangular outer perimeter 10p with a length dimension greater than its width dimension ("W"), and the width dimension is optionally between 15 and 30 mm. The length dimension can be 2 to 4 times the width dimension. The width dimension of the fluid passage 15 may correspond to the direction of the width dimension W of the device 10. The passage 15 is shown as being arranged to extend along the length dimension of the device 10. However, the fluid passage 15 may alternatively be oriented to extend along at least a portion of the width dimension of the device 10. In some embodiments, some fluid passages 15 may extend along the length dimension and some along the width dimension (Figures 9, 10). In some embodiments, the fluid passage 15 extends radially (Figures 11-14). In some embodiments, the fluid passage 15 extends circumferentially (Figure 16).

[0127] The microwellsets 20 in each passage 15 may be aligned to have the same longitudinal and lateral range and position, for example, as shown in Figure 1A, or they may be arranged alternately, with the first end of a microwellset 20 above or below the adjacent first end of an adjacent microwellset in another fluid passage 15 (not shown) that is aligned at least partially laterally.

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

[0129] In some embodiments, the passages 15 of the microfluidic device 10 can analyze the sample in a microwell set 20, each having an array 120a of microwells 120, which can range from 1,000 to 1,000,000 or more, and optionally from 10,000 to 200,000. The microfluidic device 10 generally includes multiple fluid passages 15, typically between 10 and 100, and the device 10 is provided with a total of up to 20 million microwells 120.

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

[0131] In some embodiments, the microchip 10 may be configured to perform inspections involving one or more beads in the microwell 120. See, for example, the examples of multi-bead inspections described in Patent No. 9,617,589 and PCT / US2016 / 042913 (and also US2019 / 0054470), which are incorporated herein by reference as sufficiently described herein.

[0132] Each of the different microwell sets 20 in the passage 15 may have the same or different number of microwells 120 (Figure 3B).

[0133] As shown by the virtual frames representing a single field of view (FOV) 25 in parts A1 and A2 in Figure 1A, the device 10 may be configured such that a subset or subarray 10s of the entire microwell array 10a, shown as a neighbor set 15n of a passage 15, or optionally as a pair 15p, covers the entire single FOV of a camera-like optical signal detector (220, Figure 8). In this way, at least one camera-like optical signal detector 220 (Figure 8) can image different subsets 10s of a microwell set 20, such as a single microwell array 120a of multiple different sample passages 15 covered by the FOV 25, at any given time point. The neighbor set 15n may be arranged as a subset including the microwell set 20 (e.g., part or all of a row) or as all of the passages 15.

[0134] As will be described later, the photoexcitation source 225 (Figure 8) may be configured to transmit excitation light of a specified wavelength range to a specified subarray 10s of the entire array 10a of the microwellsets 20 of the device 10 (Figures 1A, 1B), which is a subset of the microwellsets 20 and includes at least one of the microwellsets 20 of the multiple adjacent passages of the sample passage 15, but not all of the microwellsets 20 of the microchip 10. The specified wavelength range may be related, for example, to light and / or coded fluorophores for the inspection signal.

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

[0136] Device 10 may include upper and lower substrates 10u, 10b (Figures 2A, 2B) that are attached together. The upper substrate 10u may be the same as or different from the lower substrate 10b. Either or both of the substrates 10u, 10b are rigid and may include, for example, glass, quartz, or a suitable metal. The cover substrate 10u may be visually translucent, generally transparent. Either or both of the substrates 10u, 10b are polymers such as silicone or polymeric materials (PMMA, COC, COP, PDMS, PP, PE, PTFE, Kapton (polyamide), and many others) and form one or more microwell arrays 10a. In some embodiments, either or both of the substrates 10u, 10b contain silicon (e.g., a silicon wafer) and / or may be functionalized with hydrophobic compounds such as alkylsilanes and / or alkylthiols (e.g., silane treatment). In some embodiments, either or both of the substrates 10u, 10b contain silicon treated with alkylsilane.

[0137] A fluid passage 15 comprising a plurality of spaced-apart microwell sets 20, each of which forms the microwell array 120a, has a side wall and a floor formed on one or more substrates 10u, 10b such that the side wall extends between the open top surface and the closed bottom surface. One or more spacers, upper substrates, membranes, or covers may be used. The upper substrates, membranes, or covers can seal, cover, or otherwise close the top surface of the fluid passage and / or the array of reaction wells. In some embodiments, the passage 15 can be etched into the upper substrate 10u, with the bottom becoming a microwell set 20 and forming the closed surface of the passage 15.

[0138] The analyte in the material input section may be any target analyte, including, for example, a mixture of various materials including synthetic and biopolymers, nanoparticles, small molecules, DNA, nucleic acids / polynucleic acids, peptides, proteins, and others. The analyte may consist of one or more analyte molecules.

[0139] The input material includes the sample or analyte of the sample and may contain one or more polar metabolites such as amino acids, charged molecules, molecules, peptides, and proteins. The sample and / or analyte may also, or alternatively, contain molecules extracted from biological fluids, blood, serum, urine, dried blood, cell growth media, lysed cells, beverages, or food. The sample may also, or alternatively, contain environmental samples such as water, air, or soil.

[0140] The term “oligonucleotide” refers to a nucleic acid sequence of at least about 5 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, 500 nucleotides). In some embodiments, for example, an oligonucleotide may be about 15 to about 50 nucleotides, or about 20 to about 25 nucleotides, which can be used, for example, as a primer for polymerase chain reaction (PCR) amplification testing and / or as a probe or microarray for cross-breeding testing. The oligonucleotides of the present invention may be natural or synthetic, for example, DNA, RNA, PNA, LNA, denatured backbone, etc., or any combination known in the art. The oligonucleotides of the present invention may be single-stranded, double-stranded, or partially double-stranded. In some embodiments, the oligonucleotides are non-expandable (e.g., by PCR).

[0141] Probes and primers, including those for amplification and / or detection, are oligonucleotides of appropriate length (DNA and naturally occurring oligonucleotides such as synthetic and / or denatured oligonucleotides), but can generally range in length from 5, 6, or 8 nucleotides to 40, 50, or 60 nucleotides or longer. Such probes or primers may be immobilized or bound to a solid support such as beads, tips, pins, or microtiter plate wells, and / or bound to or labeled with detectable bases such as fluorescent compounds, chemiluminescent compounds, radioactive elements, or enzymes.

[0142] Polymerase chain reaction (PCR) can be performed according to well-known techniques. See, for example, U.S. Patents 4,683,195, 4,683,202, 4,800,159, and 4,965,188. Generally, PCR involves first treating a nucleic acid sample with one oligonucleotide primer for each strand of specific sequences to be detected under hybridization conditions (e.g., in the presence of a thermostable DNA polymerase) so that an expanded product of each primer complementary to each nucleic acid strand is synthesized, provided that the primers are sufficiently complementary to each strand of specific sequences to be hybridized so that the expanded product synthesized from each primer serves as a template for the synthesis of the expanded product of the other primer when separated from its complement, and then treating the sample under denaturation conditions to separate the primer expanded products from their templates, if one or more sequences to be detected are present. These steps are repeated periodically until the desired degree of amplification is obtained. The amplified sequence can be detected by adding a detectable labeled oligonucleotide probe (e.g., the oligonucleotide probe of the present invention) that is capable of cross-reacting with the reaction product to the reaction product and then detecting the label according to known techniques, or by direct visualization on a gel. The amplified sequence can also be detected by adding an insertion dye to the reaction mixture and monitoring the fluorescence signal intensity proportional to the total mass of double-stranded DNA. Other non-insertive dyes that produce a high fluorescence signal in the presence of dsDNA can also be used. Although embodiments of the present invention have been described in relation to PCR reactions, it should be understood that other nucleic acid amplification methods, such as reverse transcription PCR (RT-PCR) including isothermal amplification techniques such as rolling circle amplification or loop-mediated isothermal amplification (LAMP), and nucleic acid sequencing methods can also be used. Additionally, other processes, such as enzyme amplification reactions like enzyme-linked immunosorbent assays (ELISA), can be used. In such cases, microwell temperature control is used to control the amplification reaction and optimize imaging of the microwell array, with some areas being imaged only before and after use of the complete reaction image (digital signal), while other areas or subarrays may be imaged during the reaction to acquire analog signals.

[0143] The DNA amplification techniques described above require the use of two probe pairs, which are specifically bound to DNA containing the target polymorphism or mutant, but not strongly bound to DNA that does not contain the target polymorphism under the same cross-conforming conditions, and which function as one or more primers for the amplification of the DNA or a portion thereof in the amplification reaction. Such probes are sometimes referred to here as amplification probes or primers. In some embodiments, two or more probes may be used, for example, in a free-competition process for the detection of single nucleotide polymorphisms (SNPs) and / or other mutants and / or rare alleles such as indels.

[0144] The term "reagent" refers to a substance or compound, including primers, nucleic acid templates, and amplification enzymes, that are added to a system to cause a chemical reaction or to confirm whether a reaction is occurring. One or more amplification reagents refer to these reagents (deoxyribonucleotide triphosphate, buffer, etc.) used for amplification, excluding primers, nucleic acid templates, and amplification enzymes. Generally, amplification reagents, along with other reaction components, are placed and contained in a reaction vessel (test tube, microwell, etc.).

[0145] The term "magnetism" as used here includes ferromagnetic, paramagnetic, and superparamagnetic properties.

[0146] Generally, oligonucleotide probes used to detect DNA containing a target polymorphism or mutant are oligonucleotide probes that bind to the DNA encoding this mutant or polymorphism, but do not bind to DNA that does not contain the mutant or polymorphism under the same cross conditions. Oligonucleotide probes are labeled with appropriate detectable bases, such as those listed below. Such probes are sometimes referred to here as detection probes or primers.

[0147] Embodiments of the invention can be used for singleplex reactions (SiRCA) in a compact array, a platform for highly sensitive, highly multiplex nucleic acids (NAs), and protein quantification. SiRCA combines sample preparation in a massively parallel, highly multiplex format with the precision of digital PCR. NA-SiRCA is a PCR modification based on magnetic beads, in which a test panel is fabricated from sets of microbeads uniquely encoded with fluorescent dyes. Each set is functionalized with different primer pairs for specific target NAs before the sets are combined. During testing, the primers on the beads are cross-conjugated with NA targets, which can then be captured, concentrated, and purified from the sample matrix. The beads are washed and loaded into a microwell set 20, each containing an array 120a with thousands of microwells 120, where typically one bead occupies one beadwell 120w. The microwells 120 can have a suitable volume. In some embodiments, the microwell 120 has a volume ranging from 10 femtoliters to less than about 10 microliters, or from about 10, 50, 100 femtoliters to about 200, 500, 1,000 femtoliters, or less than about 10 microliters. In some embodiments, the microwell 120 has a volume of about 100 femtoliters.

[0148] The PCR master mix (containing all reagents except primers) is added before sealing the wells together using immiscible oil. Upon heating, the beads release the primer set and captured target, forming singleplex PCRs in each microwell. Thousands of spatially multiplex PCRs are rapidly generated without interference between primer sets. The signal from the dsDNA insertion dye indicates target amplification, and the target ID is determined from the bead encoding. At low concentrations, precise analyte quantification is possible by single-molecule counting (digital signal). At high 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.

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

[0150] NA-SiRCA is performed using beads containing a microtube format and / or microwell array 120a on tip 10, enabling over 35,000 reactions, including approximately 1 billion reactions.

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

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

number

[0153] The resolution of concentration measurement (i.e., the accuracy of concentration determination) depends on the frequency with which fluorescence is measured. Maximum resolution is achieved by imaging after each amplification cycle (i.e., each image is approximately equal to twice the amplicon concentration). However, frequent imaging results in photobleaching of the dye, dependent on the dye's exposure to the excitation source. This is particularly important for the small-volume PCR reactions used in SiRCA. Figure 4A shows a trace of a real-time PCR plot where the effect of photobleaching is evident from the negative slope of the baseline signal.

[0154] The microfluidic device of the present invention can provide one or more (e.g., 1, 2, 3, 4 or more) samples to be tested in optionally high-volume applications and / or methods. The microfluidic device of the present invention may have a single substrate comprising one or more arrays 10a having multiple microwell sets 20, including associated microwell arrays 120a (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In some embodiments, two or more fluid passages 15, optionally provided as a single array 10a or multiple arrays 10a (the latter illustrated in Figures 9-11, 16), are incorporated into the substrate to form an intensive multi-sample device 10 for high-volume applications. In some embodiments, one or more microwell sets 20 in multiple fluid passages 15 of one or more arrays 10a of the microfluidic device 10 undergo thermal cycling simultaneously.

[0155] Digital PCR of this form can be achieved because the array is imaged once before PCR and once after PCR to acquire digital PCR data. In some embodiments, the array (e.g., an array containing about 40,000 wells) is imaged by a sensor with a sufficiently high pixel density so that light from one well is collected by at least one pixel. In some embodiments, each microwell 120 of the device of the present invention may be imaged with about 4 to about 20 pixels, or about 4 to about 100, 150, or 200 pixels. In some embodiments, the system, device, and / or method of the present invention provides one or more aligned or partially aligned microwell sets 20 in two or more adjacent fluid passages 15 imaged using a single sensor without custom optical elements and / or high-precision alignment.

[0156] An alternative to custom optical elements is the use of a high-precision positioning stage that moves either a camera and / or a microfluidic chip horizontally, vertically, and / or rotates so that arrays can be imaged sequentially after the expansion step of each PCR cycle. This approach allows imaging of many arrays at the expense of longer test times. For a passage 15 with 30,000 to 40,000 or more microwell sets 20, such as 62.5k microwells 120 per sample passage 15, translation and imaging can generally add about 1 to 10 seconds to each cycle of each additional array, although high-performance hardware can reduce this time, although it is more expensive. The increased imaging time effectively increases the expansion time of the PCR cycle. While the PCR reaction rate is cycle-dependent and not time-dependent, active polymerases tend to do so if given enough time to write dimers and nonspecific products, so long delays in the thermal cycles required to image a large number of arrays are problematic. This leads to increased background signal and decreased test specificity.

[0157] Some embodiments of the present invention provide solutions to these imaging and / or data acquisition problems, for example, by utilizing unique features in the coded microbead microwell array 120a (Figure 3B) according to an embodiment of the present invention.

[0158] The properties of coded bead arrays enable unique methods for analog PCR data acquisition and analysis. When the concentration of the target molecule is in the analog region, the average number of molecules for each bead type is approximately the same. Therefore, the Ct of each bead set is independent of the bead position in the array, and different areas of the array are imaged in different PCR cycles, yet single-cycle resolution is still achieved. That is, it is not necessary to image the entire array after each PCR cycle; only a representative portion of the array needs to be imaged. In some embodiments, instead of using a common, nearly rectangular array, a long array may be used so that the arrays from each sample are positioned in high proximity. Thus, the camera can image two or more sample arrays simultaneously, and many arrays in a short time, without the need for translation along complex paths over long distances.

[0159] Using the device 10 layout shown in Figure 1A, one example of this method can be illustrated by the following example. In this design, the fluid passages 15 are grouped into sets (shown as pairs 15p) so that a subset of an array 10s of microwell sets 20, each having a well 120 (Figure 3B) from two or more fluid passages 15 (optionally two or more different samples), is imaged by a camera in a single frame (in Figures 1A and 1B, the camera's field of view is indicated by frame boxes 25 1A and 2A). In this example, a representative portion of the wells 120w from each microwellset 20 in a single row R1 is imaged in just four acquisitions across the width of the device (sequential acquisitions of A1 (also interchangeably referred to as "1A"), A2 (also interchangeably referred to as "2A"), and A3 (also interchangeably referred to as "3A"), A4 (also interchangeably referred to as "4A"), and A5 (also interchangeably referred to as "5A"), A6 (also interchangeably referred to as "1A"), and A7 (also interchangeably referred to as "7A"), and A8 (also interchangeably referred to as "8A")), with only short lateral translations between frames. Thus, in some embodiments, the method of the present invention includes acquiring analog data without imaging the entire array 10a of different microwellsets 20.

[0160] Figure 17 is a flowchart illustrating an example of a method for identifying a target species and / or target molecule. A fluid analysis device is prepared, comprising a first fluid passage containing a plurality of spaced-apart microwellsets on the first fluid passage (block 600). A photoexcitation signal is (optionally) transmitted to only a specified subset of the plurality of microwellsets (block 610). (Optionally) signal intensity data is acquired from only a specified subset of the plurality of microwellsets in response to the transmission of the photoexcitation signal (block 620). Based at least in part on the signal intensity data, bead types that are positive for the target species and / or target molecule are identified (block 630). However, in some embodiments, photoexcitation is not required to acquire a signal. For example, in some embodiments, the test (e.g., protein testing) can generate a chemiluminescent signal that can be detected and / or imaged without photoexcitation.

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

[0162] Optionally, this method may include changing a predetermined subset of microwellsets to a different predetermined subset after each of the multiple sequential reaction steps of the test (for example, after each of the multiple sequential thermal cycles of the test), thereby ensuring that some microwellsets are not imaged after each reaction step (block 612).

[0163] Optionally, a defined subset remains the same across multiple consecutive reaction steps of the test (e.g., after each of multiple consecutive thermal cycles of the test), and therefore some microwell sets are not imaged after each reaction step (block 614).

[0164] Optionally, this method includes digitally scanning a specified subset of a set of microwells after, before, or before / after the transmission and acquisition steps, and electronically identifying wells that are positive for one or more target analyte molecules while the array of wells is at imaging temperature (block 624).

[0165] Optionally, by sequentially acquiring images of a subset occupying a single physical part of the microchip 10, for example, by sequentially acquiring data from a different single row among the first, second, third, fourth, or more such rows, signal intensity can be acquired from only a subset of the microwell set 20 of the sample passage 15 and only a subset of one or more neighboring fluid passages 15, for example, using a camera with a field of view (FOV) that covers two to ten or more neighboring fluid passages among the first, second, third, fourth, or more fluid passages (block 622).

[0166] Optionally, acquiring signal intensity may include acquiring an analog signal. The analog signal can provide real-time PCR data as amplitude change of input substance in a microwell set of fluid passages versus PCR cycle number, where the concentration of one or more specified bead-type target molecules is approximately 1 molecule per microwell, equal to 1 molecule, or greater than 1 molecule, and the analog signal defines a threshold cycle or cycle threshold Ct that identifies a microwell reaction as positive when the fluorescence signal is greater than the threshold, or negative when the fluorescence signal is always less than the threshold for a given number of PCR cycles, where Ct is the number of cycles Si >> Bs, where >> is at least 5-10% greater than Bs, optionally twice as great, and / or 5-10 times the standard deviation of Bs, where Si is the fluorescence signal intensity and Bs is the background fluorescence signal measured in a PCR negative reaction (block 626). Ct may be compared to a reference reaction to determine the number of molecules at the beginning of the PCR reaction.

[0167] Optionally, obtaining signal intensity involves providing real-time PCR data as amplitude changes over the number of PCR cycles for testing the material introduced into the microwellset of the fluid passage, and obtaining an analog signal that can determine whether the PCR reaction is positive for the target molecule when Si >> Bs is greater than at least 10% of Bs, optionally twice as great, and / or 5 to 10 times the standard deviation of Bs. Si can be calculated as the mean, median, mode, and weighted values ​​of the analog signals corresponding to each specified bead type. In some embodiments, Si can be calculated by excluding outliers (typically in the initial PCR cycles) and then determining the mean or median.

[0168] In some embodiments, referring to Figures 19 and 20A-20D, the loading of beads into the device 10 may be carried out as follows: The device 10 is moistened with loading buffer and placed in a holder 900 (Figure 21) coupled to or cooperating with a linear magnet or an array of magnets 800 (block 700). To enable bead loading with minimal risk of inter-pathway contamination, the vias 16v of the input port 16 may optionally be positionally offset in two neighboring adjacent rows (block 702). With the magnets 800 fixed below the odd-numbered passages (Figure 20A), the bead slurry from the material input section is pipetted into the vias 16v (block 710). The magnets 800 then move to a position below the even-numbered vias (Figure 20B), attracting the beads to the odd-numbered passages (block 720). Needless to say, the steps shown in Figures 2A and 20B may be carried out in reverse order. After adding the bead slurry to the even-numbered vias, magnets 800 are used to draw the beads into the microwells (array chambers) (Figure 20C) (block 730). By sliding the magnets below each array set 20 along the fluid passage 15 to the manifold passage and / or master mix port 18, the microwell sets (array segments) are loaded with beads, and the operation is optionally controlled by a rigid stop device 810 (Figure 20C) so that beads do not enter the common manifold passage (which allows for bead mixing between samples) (block 740).

[0169] Each passage 15 is provided with a total of several thousand or several million microwells 120 for each material input section or fluid passage 15, and optionally, five microwell sets 201 to 205 (each with a well of 12.5k) containing approximately 62.5k microwells 120, as shown in Figure 1A, which are optionally physically separated segments of microwells 20 (block 704).

[0170] After beads are loaded into the microwells 120 (Figure 3B) of each microwell set 20 in the fluid passage 15, the remaining beads are magnetically pulled back towards via 16v by moving magnet 800 parallel to via 16v, the input port (Figure 20D) (block 750). The master mix flows under pressure applied to the common reservoir 18 until it fills the passage 15 (block 760). Then, it is pumped under pressure until sealing oil seals the microwell set 20 and the excess master mix is ​​drained from the passage 15 (block 770).

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

[0172] Optionally, a second microfluidic passage, passage network, or reservoir may be used to collect waste fluid instead of allowing it to leak out of the via. The microfluidic and such a waste reservoir, either as an optional additional reservoir or reservoir set, may be fluidly connected to the passage and / or via by a wax or paraffin valve or hydrophobic constriction in such a manner as to prevent filling prior to the master mix addition and / or sealing step. In some embodiments, the via may be sealed with a membrane after bead addition so that the membrane blocks the flow of water and / or oil and optionally allows the release of air from the membrane. In some embodiments, the membrane may cover other vias or ports to act as a pathway for air to be released from the microfluidic passage, chamber, or via.

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

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

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

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

[0177] After the thermal cycling is complete, “post-PCR” images are taken for the entire array 10a at the imaging temperature. Array 10a is cooled (typically from about 20°C to about 25°C), and another set of post-PCR images is taken (to highlight the problem area on the tip where the aqueous master mix connects two or more wells due to a defect in the sealing technique). If desired, array 10a is imaged at a series of temperatures to determine the melting point and / or range of the amplicons detected after PCR cycling. Filter sets (F1, F2, Figure 8) are swapped to match the coding wavelength, and the first coded image set is acquired for each subset of the microwell set 20, followed by a second coded image set using the second filter set if necessary, and repeated as needed for decoding the bead set.

[0178] The processing of imaging data can be divided into two areas: digital and analog. The digital PCR signal can be determined by comparing pre-PCR and post-PCR images to determine whether the increase in the insertion dye signal exceeds a set threshold in each well. In some embodiments, only the post-PCR image is required to determine digital positivity or negativity. Alternatively, the pre-PCR and post-PCR images may be used, optionally along with six thermal cycle images, to determine whether the fluorescence signal changed during the imaging process in a manner consistent with the PCR amplification of the accurate amplicon. For example, in low-concentration targets where some portion of the target's PCR reaction population is negative, an increase in signal is expected in later cycles, and outliers showing a strong signal early in the cycling cycle may be considered nonspecific amplification products or beads of specific misidentification.

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

[0180] As seen in Figures 5A-5H, the fluorescence intensity baseline is not perfectly uniform across a given fluid passage 15 and / or sample subarray (a subset of microwells 20). This is due to several factors, including the effective concentration of the insertion dye, the focus of the image, and / or the effect of different levels of silane treatment on the device. If the PCR image signal is normalized by splitting with the pre-PCR image signal (Figures 6A-6H), the baseline may be more linear and the cycle threshold call may be more repeating. Irregularity in the maximum PCR signal should have a weak impact on target quantification. Protocols to help control the dye concentration and thus the fluorescence signal intensity across all arrays will be discussed below.

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

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

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

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

[0185] Microwell sets 20 (i.e., subarrays) that were not imaged during PCR may exhibit very slight photobleaching, while imaged subarrays may show significant photobleaching. Thus, in some embodiments, different thresholds are required to distinguish digitally positive from digitally negative.

[0186] The analog signal processing of the imaged subarrays is similar to that of conventional whole-array imaging methods. The signals are averaged across each bead population and displayed relative to the number of PCR cycles (Figures 7A-7H). For analog signals where the target molecule has more than one copy per bead, an average signal from several hundred beads should be sufficient for accurate and precise Ct determination.

[0187] For example, methods of the present invention, such as Method Examples 1 and Method 2 described above, may have advantages over common data acquisition protocols in which all wells of an array are imaged after each PCR cycle. In some embodiments, methods of the present invention reduce data acquisition time while generating real-time PCR resolution for a single cycle for analog quantification. Small, narrow well arrays can image fluid separation arrays from numerous samples in a single image.

[0188] The advantages of collecting analog data across multiple subarrays (as described in Example Method 1) include reduced photobleaching. For example, since each subarray is exposed to only 20% of the irradiation used in a standard acquisition protocol, a significant reduction in photobleaching is achieved in Example Method 1. Even with low cycle resolution, data collected over several cycles is more reliable for identifying false-positive beads for digital quantification than comparing only pre-PCR and post-PCR images. Additionally, with less photobleaching, a proper signal can be obtained with lower concentrations of insertion dye (e.g., SYBR Green I) than is required to image all 30 cycles. Since insertion dyes can inhibit PCR, lower concentrations may improve PCR efficiency or melting point determination for some targets.

[0189] The advantages of collecting analog data from a single row of microwells in a microwell set 20 (i.e., a subarray or subset of microwells as described in Method Example 2) may include reducing the reliance on data normalization for Ct determination, thus simplifying the analysis. Additionally, pre-PCR images can be used to determine which row has the optimal bead loading for analog real-time PCR analysis. This can help mitigate problems that may arise if one or a few subarrays are not loaded with enough beads for analog analysis (although this problem can be addressed by other means such as strict control over array size, geometry, site, bead population size, and bead size).

[0190] Examples 1 and 2 of the method are merely illustrative examples of the device design and data acquisition method of the present invention, and various combinations and / or modifications may be used. For example, a single row of microwellsets 20 with different fluid passages 15 may be imaged cycle by cycle or every other cycle in combination with imaging of other subarrays at various different intervals. Some combinations require more supplement time, but in some embodiments, the improvement in the quality of digital or analog data may be worth such a trade-off. In some embodiments, all or most of the microwellsets 20 may be imaged in several PCR cycles (generally not in every and / or each PCR cycle).

[0191] The device 10 in the figure is merely an example of the design concept (i.e., the division of the bead / reaction well array into a series of subarrays with spacing and arrangements that allow for imaged in an advantageous manner), and other designs may be used in the practice of the invention. In some embodiments, the device 10 of the present invention may include one or more arrays of fluid passages 15, such as 9 to 1000 fluid passages 15, with each device 10 containing 16, 32, 64, 96, 384 or more fluid passages 15, including the microwell set 20, for a total of 8 or more fluid passages 15. In some embodiments, the device 10 of the present invention may be made of silicon, glass, or polymer (e.g., plastics by injection molding and / or hot embossing) and / or metal film. In some embodiments, the device 10 of the present invention may be a composite structure. In some embodiments, the device 10 of the present invention may contain only the bead / reaction well array and array chamber, or may include other structures such as sample processing microfluidic circuits and / or labeling or amplification reagents necessary for performing the test.

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

[0193] Polystyrene beads can absorb both hydrophobic and charged molecules from aqueous solutions. The long, narrow geometric shape of the microarray 20 results in a reduction of certain components, such as insertion dyes, from the master mix as they flow through the passage 15. The beads of the first subarray, which merge with the master mix, absorb a large amount of components (e.g., insertion dye), while the final array absorbs less from the partially reduced solution. When the component is a dye, this can result in a gradient in the dye concentration at the tip, and therefore in the initial bead fluorescence, compared to the array in the passage (Figure 20). Too much dye can inhibit PCR, and too little dye can result in a weak amplification signal, so these differences in dye absorption introduce some complexity to the test. Additionally, variations in insertion dye concentration cause undesirable variations in the apparent fluorescence intensity of the encoding dye, which can make decoding the array more difficult. This effect may be one of the causes of the low-intensity signals in some subarrays observed in Figures 5 and 6.

[0194] Bead-mediated dye absorption can be regulated by including (e.g., adding) various reagents that produce more uniform dye absorption / distribution (e.g., SYBR) across the array. These reagents (also called "dye homogenizers") may include, but are not limited to, single-stranded DNA, RNA, double-stranded DNA (optionally, oligonucleotides of various lengths such as single-stranded DNA, RNA, double-stranded DNA (which is double-stranded during addition to the pathway and sealing of the wells but single-stranded during the PCR imaging step and has a low melting temperature so as not to be associated with the insertion dye), partially double-stranded DNA, ionic or nonionic polymers such as dextran sulfate or polyamines, surfactants, detergents, phase transfer catalysts, dextran, cyclodextran, silicones, polysilicones, fluorocarbons, polyfluorocarbons, fluorosilicones, hydrocarbons, alcohols, hydrofluorocarbons, and biomolecules such as peptides, proteins, lipids, carbohydrates, glycans, complex molecules, nucleic acids, and / or modified nucleic acids. Reagents and / or compounds with known PCR compatibility and low fluorescence signals associated with insertion dyes are particularly well suited for this purpose. Dye homogenizers may be miscible with the master mix. In some embodiments, a dye homogenizer is present in and / or added to the master mix. The dye homogenizer (e.g., partially double-stranded DNA) may be present in the master mix at concentrations ranging from about 100 nM to about 100 μM, or any of the ranges and / or values ​​within that range (e.g., about 1, 5, 10 μM to about 15, 25, 50, 100 μM). In some embodiments, the dye homogenizer may be attached to a portion of the beads (e.g., by covalent and / or non-covalent bonding). In some embodiments, the beads may contain an amount of dye homogenizer ranging from about 300 pM, 1 nM, or 3 nM to about 300 μM or 3 mM.

[0195] For example, oligonucleotides (oligonucleotides) such as non-expandable oligonucleotides (NE oligonucleotides) that optionally contain biotin (e.g., biotin at the 3' end) can modulate (e.g., reduce) dye (e.g., SYBR) binding during master mix addition. In some embodiments, the dye homogenizer may contain an ssDNA molecule with a 3' attachment site of biotin linked by a 6-carbon chain, but other modification sites and / or binding sites (e.g., C1-C20 hydrocarbon chains such as C1-C20 alkyl branched or unbranched chains) may also function. In some embodiments, non-expandable oligonucleotides may be used as dye homogenizers because they are less likely to negatively impact PCR than additional primers that can form dimers. As shown in Figure 22, the addition of NE oligonucleotides to a master mix containing SYBR Green I reduces the variability of the average initial bead fluorescence (CV 0.7%) compared to SYBR alone (CV 4.8%). Changes in SYBR absorption (and therefore SYBR concentration in the PCR reaction) can modulate target amplification and fluorescence signals; therefore, 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 for mycoplasma target sequences was compared between SYBR (CV 23%) alone and SYBR plus NE oligo (CV 4%) in the master mix across five subarrays in the chip's pathway. The reduction in variation results in a more accurate determination of the average molecule per bead and, therefore, a more accurate determination of mycoplasma concentration. Additionally, controlling the insertion dye concentration across the array leads to a reduction in dsDNA melting temperature variation, thus enabling a more accurate determination of the amplicon melting point.

[0196] In some embodiments, the fluorescence signal from the coding dye is affected by the concentration of the insertion dye, and when there are large differences in dye absorption by the beads, decoding the array can become difficult (e.g., clustering the beads during coding analysis). In Figure 24, a sample containing only SYBR in the master mix shows a shift in the fluorescence signal observed in the coding dye fluorescence pathway (CV 14%) between different subarrays. The apparent dispersion of the coding dye fluorescence signal was less when a non-expandable oligo was added to the solution (CV 5%).

[0197] Alternatively or additionally, differences in dye absorption during loading of the master mix into the array may be addressed by other means. For example, in some embodiments, beads may be immersed in a buffer containing the insertion dye before loading the beads into the microwell array. Beads may be immersed before, during, and / or after incubation with the sample. Alternatively or additionally, the surface of the beads may be functionalized to modulate dye absorption. For example, binding more primers to the surface of the beads increases dye absorption compared to when fewer primers are bound. Since dsDNA binds more strongly to the insertion dye than ssDNA, partially double-stranded oligonucleotides (pdsDNA) optionally linked to the beads via biotin-TEG binding chemical structures available from IDT may be used. In some embodiments, beads containing pdsDNA oligos are immersed in a buffer containing the insertion dye before testing. The beads are then incubated with the sample in a cross-buffer buffer, washed, and loaded into the microwell array. In some embodiments, a master mix containing all the reagents necessary for PCR except primers and insertion dyes, and optionally including a dye homogenizer, may be introduced into the pathways of a microwell array before the reaction wells are optionally sealed together with miscible sealing oil. An example of pdsDNA is shown in Figure 25.

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

[0199] The melting point of pdsDNA, which can be used to adjust dye absorption, can be optimized so that the pdsDNA remains partially double-stranded under storage and / or cross-pollination conditions. However, during PCR, the pdsDNA dissociates, resulting in ssDNA primers with low background fluorescence because they do not strongly bind to the inserted dye. In some embodiments, the binding site (e.g., a carbon chain, optionally a C1-C20 hydrocarbon) to which the primer attaches to the biotin functional group can be adjusted to harmonize its hydrophobicity and / or ionic charge with respect to the absorption properties of the dye or other reagents.

[0200] In some embodiments, the device 10 of the present invention may be fabricated using a holder 900 that secures multiple microchips 10 or devices, optionally configured as a grid 900g as shown in Figure 21. The holder 900 encompasses an outer periphery 910 surrounding the grid 900g and can define transverse and longitudinal thermal insulation barrier regions 920, 930 between adjacent devices 10 on both sides and edges. Master mix ports 18 are positioned opposite each other across the barrier segment 930. Input ports 16 are positioned opposite the outer periphery 910 of the holder 900. In some embodiments, the holder 900 may be fabricated from a thermal insulation material, and the chips 10 may encompass a thermally conductive substrate. Each of the small microarray chips / devices 10 in the grid 900g may undergo thermal cycling independently. Thermal cycling and / or imaging of the microarray chips / devices 10 in the grid 900g may be synchronized by a test system 200 so that each microarray chip / device 10 is imaged in a specified sequence.

[0201] In some embodiments, the device 10 of the present invention is manufactured from a substrate which is an insulating material such as plastic, and optionally different zones, areas, or parts of the same substrate are subjected to thermal cycling and / or imaging in a predetermined sequence.

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

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

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

[0205] According to several embodiments, a method for obtaining high-resolution real-time PCR can be performed using a singleplex reaction (SiRCA) in a compact array while mitigating the effect of photobleaching on the test signal. The device 10 of the present invention comprises two or more fluid passages 15, each having a microwell set 20 for optionally one or more samples, and the array 10 can be imaged using a subset of the microwell sets 20. Different subsets of microwell sets 20 can be imaged in a sequence in which one segment is imaged after each amplification cycle. For each image, the average signal for each reaction type can be calculated for each sample. By displaying the average signal from each reaction type for each amplification cycle, the cycle threshold can be determined for each reaction type at single-cycle resolution without requiring time to image the entire array after every PCR cycle. For example, a fluid device 10 with a length of 14 mm and a width of 1.25 mm, including the passages 15, can be divided into five (row) segments A, B, C, D, and E. After PCR cycle 1, segment A is imaged. Segment B is imaged after PCR cycle 2, and so on. Each row / segment may be imaged 6 times after 30 PCR cycles. Signals from each bead type may be acquired for every PCR cycle, but each row / segment (and therefore each reaction) receives only 1 / 5 of the excitation energy dose used to record fluorescence data. Thus, this approach reduces the effects of photobleaching while maintaining signal cycle real-time PCR resolution. Additionally, by narrowing the microwell set 20 of the sample fluid passage 15 to one dimension, two or more passages 15 containing the subset of the microwells 20 are placed close enough to be imaged at high speed (e.g., one frame contains one or more arrays), facilitating the ability to acquire real-time PCR data for a large number of samples at single-cycle resolution within a reasonable imaging time.

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

[0207] In some embodiments, the device of the present invention may be used as a substitute for multiplex PCR or immunoassay panels. Applications of embodiments of the present invention include, but are not limited to, biomedical or biological research, diagnosis of diseases (including infectious diseases and / or tumors) using nucleic acid or protein biomarkers, veterinary applications, forensic analysis or genotyping, environmental monitoring, counterfeit detection, and / or biopharmaceutical production and quality control applications.

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

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

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

[0211] System 200 can acquire an analog signal that defines a threshold cycle or cycle threshold "Ct" which identifies a PCR reaction for a target species and / or molecular type as positive when the fluorescence signal is greater than the threshold, or negative when the fluorescence signal does not rise above the threshold. That is, Ct is a Si >> Bs cycle, where >> is at least 5-10% and optionally twice as large as Bs, where Si is the fluorescence signal intensity and Bs is the background fluorescence signal. The initial target concentration can be calculated by comparing the Ct of an unknown concentration of sample with the Ct of a known concentration of sample.

[0212] The controller 210 is in communication with (i.e., includes computer program code for) a subarray selection module 250 that has a configuration for selecting different microwell sets during different examination cycles. Modules 250 and / or 260 are mounted whole or partially, or are located remotely from the controller and / or optical system 220. The analysis system 200 includes at least one processor (i.e., a digital signal processor) and may include a transceiver 214.

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

[0214] Modules 250 and 260 may be installed in the analysis system 200 or distributed within one or more servers 300. Servers 300 may be implemented as standalone servers or housed as part of other computing infrastructure. Servers 300 may be implemented as one or more enterprise, application, personal, popular, and / or embedded computer systems, either standalone or interconnected by public and / or private, real and / or virtual, wired and / or wireless networks including the internet, and may include various types of tangible, non-transient, computer-readable media. Servers 300 may also communicate with networks via wired or wireless connections and may include various types of tangible, non-transient, computer-readable media.

[0215] Server 300 may be provided using cloud computing, which involves providing computing resources on demand via a computer network. Resources can be embodied as applications, databases, file services, email, etc., along with various infrastructure services (e.g., computing, storage, etc.). In traditional computing models, both data and software are generally housed entirely on the user's computer. In cloud computing, the user's computer may house mostly software or data (perhaps an operating system and / or a web browser) and function only as a display terminal for processes occurring on a network of external computers. Cloud computing services (or a collection of many cloud resources) are generally referred to as the "cloud." Cloud storage includes a model of network-connected computer data storage, where data is stored on a number of virtual servers rather than on one or more dedicated servers.

[0216] The controller 210 can communicate with the server 410 or computer via the transceiver 214 and / or a computer network or cellular network. Regarding computer networks, this includes one or more local area networks (LANs) and wide area networks (WANs), and may include private intranets and / or the public internet (also known as the World Wide Web, "Web," or "Internet").

[0217] As illustrated in Figure 18, embodiments of the present invention have a configuration as a data processing system 1116 which may include (one or more) processors 500, memory 536, and input / output circuitry 546. One or more processors 500 may be part of image processing circuitry 500c. The data processing system may be incorporated into one or more of, for example, a personal computer, database, workstation W, server, router, or the like. System 1116 may reside in a single machine or be distributed across multiple machines. The processors 500 communicate with memory 536 via address / data bus 548 and with input / output circuitry 546 via address / data bus 549. The input / output circuitry 546 may be used to transmit information between memory (memory and / or storage medium) 537 and another computer system or network, for example, using an Internet Protocol (IP) connection. These components may be conventional components, such as those used in many conventional data processing systems having a configuration that functions as described herein.

[0218] In particular, the processor 500 (which may be incorporated into the controller 210 in Figure 8) may be a commercially available or custom microprocessor, microcontroller, digital signal processor, or the like. The memory 536 may include a memory device and / or storage medium that houses software and data used to run a functional circuit or module used in embodiments of the present invention. The memory 536 includes, 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 may be a content-addressable memory (CAM).

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

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

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

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

[0223] The present invention is described with partial reference to flowcharts and / or block diagrams relating to methods, apparatus (systems) and computer program products according to embodiments of the present invention. It will be understood that each block in the flowcharts and / or block diagrams, and combinations of blocks in the flowcharts and / or block diagrams, can be executed by computer program instructions. These computer program instructions may be provided to the processor of a general-purpose computer, a dedicated computer, or another programmable data processing device that produces machines, such that the instructions, executed via the processor of the computer or other programmable data processing device, provide means for performing the functions / operations specified in one or more blocks of the flowcharts and / or block diagrams.

[0224] Computer program instructions that can instruct a computer or other programmable data processing device to function in a particular manner can also be stored in computer-readable memory. Thus, products are produced that include instruction means to perform functions / operations specified in one or more blocks of a flowchart and / or block diagram, based on instructions stored in computer-readable memory.

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

[0226] The flowcharts and block diagrams in certain figures of this application illustrate the exemplary architecture, function, and operation of possible executions of embodiments of the present invention. In this regard, each block in the flowchart or block diagram represents a module, segment, or code portion containing one or more executable instructions for performing a specified logic function. It should also be noted that in some alternative executions, the functions described in the blocks may be performed in an order other than that shown in the figures. For example, depending on the required function, two consecutively shown blocks may actually be executed substantially simultaneously, blocks may sometimes be executed in reverse order, or two or more blocks may be combined.

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

[0228] The foregoing is an example of the present invention and should not be construed as an limitation thereof. The present invention is defined by the following claims, and equivalents of these claims are included in this application. All publications, patent applications, patents, patent gazettes, and other prior art referenced herein are incorporated by reference in their entirety with respect to teachings relating to the sentences and / or paragraphs in which the references are presented. [Explanation of Symbols]

[0229] 10 Fluid Devices 10a array 10b Lower base material 10p outer edge 10s subarray 10u upper base material 15,151,152,153,154,155,156,157,158 fluid passage 15 n Nearby passage 16 Input Ports 16r Reservoir 16V via 18 Master Mix / Oil Port 18m Fluid Manifold 18r Reservoir 18V Via 19 Interstitial space 20, 201, 202, 203, 204, 205 microwells 20c Corner Microwells 20r outer circumference 21 Interstitial space 25 Frame Box 27. Alignment Markers 120 microwells 120a microwell array 120s Test signal element 120W bead-holding segment 122 Tapered Inspection Signal 200 Analysis Systems 200h Test System 210 Controller 214 transceivers 220 Optical signal detectors 222 Cameras 225 Photoexcitation source 240 Heat source 250 Subarray Selector Module 260 Signal Analysis Modules 300 servers 548,549 Address / Data Bus 800 magnets 810 Hard Stop Device 900 Holder 900g grid 910 Outer periphery 920,930 barrier segments 1116 Data Processing System C1, C2, C3, C4, C5, C6, C7, C8 (vertical column) Rows R1, R2, R3, R4, R5

Claims

1. It is an analysis system, A housing comprising a chamber having a size and configuration for receiving at least one microfluidic device, An optical system coupled to the housing in a state of optical communication with at least one microfluidic device, A controller coupled to the optical system, A heat source coupled to the optical system and thermally coupled to at least one microfluidic device fixed to the housing, A subarray selection module in communication with the controller, having a configuration to select a subset of microwells of at least one fluid passage of the microfluidic device for imaging by the optical system after a reaction step (e.g., one thermal cycle) during inspection, An analytical system that encompasses all aspects.

2. The system of claim 1, further comprising a magnet fixed to the housing adjacent to at least one microfluidic device, wherein the magnet is configured to move in parallel along at least one fluid passage and magnetically couple to a bead during bead loading, and to guide the bead to move along the fluid passage to a bead-holding segment of the microwell.

3. The system of claim 1, wherein the microfluidic device comprises a plurality of fluid passages, each having a plurality of microwell sets arranged along a length dimension relating to the direction between a sample input port and a second port opposite to it, and the selectable subset of the microwell sets is associated with a single transverse row of laterally aligned first microwell subsets from the plurality of fluid passages, and optionally one aligned microwell subset from each of the plurality of fluid passages.

4. The system according to claim 1, wherein, prior to photoexcitation, the subarray selection module identifies a subset of the microwellsets that define the locations of other microwellsets in the microfluidic device.

5. The system of claim 1, wherein the subarray selection module selects different subsets of the microwellsets of the microfluidic device in different reaction steps of the inspection (e.g., different thermal cycles of the inspection), instructs the optical system to excite only the currently selected subset of the microwellsets of the different fluid passages, and instructs the camera of the optical system to sequentially or in parallel capture images of the different subsets of the microwellsets of the currently selected subset.

6. The system of claim 1, wherein the subarray selection module selects the same microwell subset in at least several different sequential reaction steps of the inspection (e.g., different thermal cycles of the inspection), instructs the optical system to transmit light only to the currently selected subset of the microwell set, and instructs the camera of the optical system to sequentially or in parallel capture images of different subsets of the microwell set, optionally pairs of adjacent microwell sets from the simultaneously selected microwell subsets.

7. The system according to claim 1, wherein the optical system comprises at least a first and / or second filter that defines first and / or second wavelengths of excitation light corresponding to first and second target coded beads for decoding a set of beads held in one or more of the microwell sets of at least one fluid passage of the microfluidic device.

8. The system of claim 1, further comprising a signal analysis module integrated with and / or coupled to the controller, wherein the signal analysis module is configured to acquire analog signals for each microwell of the selected subset of the microwell set, the analog signals providing PCR data as amplitude changes of the test cycle, and the concentration of a target molecule for a given bead type and associated reaction is determined by the analog signals.

9. The system according to claim 8, wherein the signal analysis module further has a configuration to acquire the digital PCR signal of the microwellset.

10. The system of claim 8, wherein the analog signal provides real-time PCR data as an amplitude change over the number of PCR cycles for one or more input substances in the microwell set of the fluid passage, the molecular concentration of a specified bead type is about one molecule, equal to one molecule, or greater than one molecule per microwell, and the analog signal defines a threshold cycle or cycle threshold Ct which identifies a microwell reaction as positive when the fluorescence signal intensity (Si) is greater than a threshold and the number of target molecules / target molecular concentration for a target species and / or molecular type, or identifies a negative reaction when the fluorescence signal intensity (Si) is always lower than a threshold for a given number of PCR cycles.

11. The system of claim 10, wherein the Ct is the number of cycles of Si >> Bs, where >> is at least 5 to 10%, optionally twice, and / or 5 to 10 times the standard deviation of Bs, and Bs is optionally a background fluorescence signal measured by a PCR negative reaction.

12. The system of claim 8, wherein the analog signal is acquired for only a single subset of the microwellsets of each fluid passage after every other or each of the multiple different reaction steps of the test (e.g., different thermal cycles of the test).

13. The system of claim 8, wherein the analog signal comprises the mean value (optionally excluding outlier data), median, mode, and weighted value of Si as the analog signal corresponding to each specified bead type, and is provided as an estimate of a real-time PCR curve for similar reactions in the microwells of other microwell sets for the fluid passage of the microfluidic device, thereby enabling single-cycle resolution even if all microwell sets of each fluid passage are not imaged after different reaction cycles or each reaction cycle.

14. The system of claim 1, wherein the optical system includes a camera having a field of view (FOV) that covers only a subset of microwells of the microfluidic device, and the subset is located in at least two, or optionally all (e.g., entire rows) of adjacent fluid passages of the microfluidic device.

15. The system of claim 1, wherein the controller and / or the signal analysis module optionally instructs the optical system to acquire pre and post-PCR images and compare the signal intensity between them, along with analog data acquired using the selected subset of microwells at different reaction steps of the test, to determine positive and negative PCR reactions, and optionally determine the concentration of the target species and / or molecular concentration in the source sample provided to the fluid passage.

16. The system of claim 1, further comprising a holder having a configuration for fixing a plurality of microfluidic devices to an alignment grid of the housing.

17. The system according to claim 1, wherein the holder includes an insulating material that provides a thermal barrier between adjacent microfluidic devices, and optionally the holder fixes the plurality of microfluidic devices with the input ports to the fluid passage facing outward.

18. The system of claim 1, wherein the microfluidic device further comprises a dye homogenizer, optionally comprising an oligonucleotide, optionally a non-expandable oligonucleotide and / or partially double-stranded DNA (e.g., biotin and / or a partially double-stranded standard DNA comprising a C1-120 hydrocarbon chain).

19. The system according to claim 18, wherein the dye homogenizer is present in the master mix present in the microfluidic device, and the dye homogenizer adheres to the beads present in the microfluidic device.

20. A method for identifying a target species and / or target molecule, A fluid analysis device is provided with a first fluid passage that includes a plurality of microwell sets arranged on the first fluid passage, The method involves obtaining signal intensity data from only a specified subset of the aforementioned multiple microwell sets, Based on at least a portion of the acquired signal intensity data, identify PCR reactions that are positive for the bead type and / or target species and / or molecular type associated with the target molecule, A method of including.

21. The method of claim 20, further comprising loading and sealing the plurality of microwell sets on the first fluid passage prior to the acquisition step such that each microwell set is fluid-isolated from one another after sealing, wherein the plurality of microwell sets on the first fluid passage are fluid-communicated only during the loading that precedes the sealing step.

22. The method of claim 20, further comprising changing the predetermined subset of the plurality of microwell sets to a different predetermined subset after each of the plurality of sequential reaction steps of the examination (for example, after each of the plurality of sequential thermal cycles of the examination), thereby ensuring that some microwell subsets are not imaged after each reaction step.

23. The method of claim 20, wherein the specified subset remains the same across multiple consecutive reaction steps of the test (for example, after each of the multiple consecutive thermal cycles of the test), so that some microwell sets are not imaged after each reaction step.

24. The aforementioned fluid analysis device further, The second fluid passage includes a plurality of spaced-apart microwell sets, A third fluid passage comprising a plurality of spaced-apart microwell sets, A fourth second fluid passage including a plurality of spaced-apart microwell sets on the fourth fluid passage, It includes a number of fluid passages that include one or more of these, not exclusively, The first microwell set of each of the plurality of microwell sets in the first, second, third, and fourth fluid passages is aligned as the first row, the second microwell set of each of the plurality of microwell sets in the first, second, third, and fourth fluid passages is aligned as the second row, the third microwell set of each of the plurality of microwell sets in the first, second, third, and fourth fluid passages is aligned as the third row, and the fourth microwell set of each of the plurality of microwell sets in the first, second, third, and fourth fluid passages is aligned as the fourth row, The aforementioned subset is a single row from the first, second, third, and fourth rows. The method of claim 20.

25. The method of claim 24, wherein the first, second, third, and fourth fluid passages include linear or arcuate segments that are substantially parallel to each other and form the first, second, third, and fourth microwellsets.

26. moreover, Prior to the acquisition step, the photoexcitation signal is transmitted only to the specified subset, and the signal is acquired upon receiving the transmitted photoexcitation signal. After, before, or before / after the transmission and acquisition step, the predetermined subset of the plurality of microwell sets is digitally scanned to acquire images of the microwell sets for identifying positive and negative PCR reactions associated with the digital PCR. Electronically identifying the microwells of the microwell set that are positive for one or more target analyte molecules while the microwells are at imaging temperature, The method of claim 20, which includes the method of claim 20.

27. The method of claim 20, wherein the excitation and acquisition are performed after each of a plurality of sequential reaction steps of the examination (e.g., thermal cycling) to image only one specified subset of the microwell set, and the sequential sets of the single specified subset are different from each other.

28. The method of claim 20, wherein the signal intensity is obtained from only the specified subset by using at least one camera having a field of view (FOV) that covers only the microwellset of at least one of two adjacent passages in the fluid passage or only a subset of the microwellset, by successively or in parallel acquiring images of different specified microwellsets.

29. The method of claim 20, wherein each of the microwell sets comprises a microwell array of 1,000 and 1,000,000 microwells, and the fluid analysis device comprises a plurality of spaced fluid passages, each comprising the microwell set containing the microwell array, wherein the fluid passages are in a fluid isolation state, and at least some of the microwells of the microwell array contain a single bead, and optionally some or all of the microwells contain no beads or contain one or more beads.

30. The method of claim 20, comprising: electronically identifying a portion of one or more microwellsets located at one or more locations in the microfluidic device prior to the transmission and acquisition steps; and defining the locations of other microwellsets based at least partially on the portion of the identified portion.

31. The method of claim 20 further comprises transmitting the photoexcitation signal to only the specified subset before the acquisition step, and selecting a filter that provides the encoded wavelength to the transmission step before the transmission.

32. The method of claim 20, further comprising obtaining an analog signal that can provide real-time PCR data as amplitude change versus number of PCR cycles for the substance introduced into the microwell set, wherein the molecular concentration of a given bead type is about one molecule, equal to one molecule, or greater than one molecule per microwell, and the analog signal specifies a microwell reaction that is positive when the fluorescence signal intensity (Si) is higher than a threshold, and the number of target molecules / target molecular concentration for a target species and / or molecular type, and defines a threshold cycle or cycle threshold Ct that is negative when the fluorescence signal intensity (Si) is always lower than the threshold for a given number of PCR cycles.

33. The method of claim 32, wherein Ct is the number of cycles of Si >> Bs, where >> is at least 5 to 10%, optionally twice as large as Bs, and / or 5 to 10 times the standard deviation of Bs, and Bs is optionally a background fluorescence signal measured by a PCR negative reaction.

34. The method of claim 32, wherein the analog signal is acquired for only a single subset of the microwellset in one or more fluid passages after every other or after each of a plurality of different reaction steps of the test (e.g., different thermal cycles of the test).

35. The method of claim 32, wherein the analog signal includes the mean, median, mode, or weighted value of Si (optionally discarded for outlier data) as the analog signal corresponding to each specified bead type, and is provided for the fluid passage of the microfluidic device as an estimate of a real-time PCR curve for similar reactions in the microwells of other microwell sets, thereby enabling single-cycle resolution even if not all microwell sets of each fluid passage are imaged after different reaction steps.

36. The method of claim 20, wherein the signal intensity is acquired by using a camera having a field of view (FOV) that covers only a subset of the microwellset of the microfluidic device and that is present in at least two, optionally all, adjacent fluid passages of the microfluidic device.

37. The method of claim 32, wherein the analog signal is acquired for only a single set of the microwell sets of each fluid passage after a plurality of different reaction steps of the test (e.g., different thermal cycles of the test), and the analog signal comprises the mean, median, mode, or weighted value of Si as the analog signal corresponding to each specified bead type, and is provided for the fluid passage of the microfluidic device as an estimate of a real-time PCR curve for similar reactions of microwells in other microwell sets, thereby enabling single-cycle resolution even if all microwell sets of each fluid passage are not imaged after different reaction steps.

38. The fluid analysis device comprising the first fluid passage including the plurality of microwell sets spaced apart on the first fluid passage comprises a plurality of additional fluid passages, each encompassing the plurality of microwell sets spaced apart over the length thereof, and the fluid analysis device further comprises separate material input ports for each of the first fluid passage and the plurality of additional fluid passages, and at least some of the fluid passages share opposite common second ports. Furthermore, before the acquisition step, The bead slurry, which has been pre-exposed on the sample for analysis, is loaded into the input port by fluid. The bead slurry is magnetically guided to flow into different microwell sets on the fluid passage, A fluid master mix containing a dye is flowed from the second port through the fluid passage to the first port, wherein the master mix optionally contains a dye homogenizer, and optionally the dye homogenizer is or contains oligonucleotides (e.g., non-expandable oligonucleotides and / or partially double-stranded DNA (e.g., biotin and / or partially double-stranded DNA containing C1-C20 hydrocarbon chains)), and By flowing sealing oil from the second port through the fluid passage to the first port, the microwell set and the fluid passage are sealed from each other. A method that includes The method of claim 20.

39. The method of claim 38, further comprising placing a magnet adjacent to the fluid analysis chip and moving the magnet parallel to the second port before flowing the fluid master mix and sealing oil.

40. The method of claim 20, wherein the fluid analysis device (optionally the first fluid passage and / or the plurality of microwell sets) comprises a dye homogenizer, and optionally the dye homogenizer comprises oligonucleotides (e.g., non-expandable oligonucleotides and / or partially double-stranded DNA (e.g., biotin and / or partially double-stranded DNA comprising C1-C20 hydrocarbon chains)).

41. The system of claim 40, wherein the dye homogenizer is present in a master mix present in the fluid analyzer (for example, present in the first fluid passage and / or the plurality of microwell sets), and / or the dye homogenizer is attached to beads present in the fluid analyzer (for example, present in the first fluid passage and / or the plurality of microwell sets).

42. A microfluidic device for analysis, A plurality of fluid passages, each having a length dimension corresponding to the direction between a first port and a second port opposite to it, wherein at least a portion of the length dimension is configured as a linear or arc-shaped length segment, and each fluid passage encloses a plurality of microwell sets arranged in the linear or arc-shaped length segment of the length dimension. A microfluidic device for analysis that encompasses [the relevant information].

43. The microfluidic device according to claim 42, wherein the plurality of microwellsets of the plurality of fluid passages are arranged in horizontal rows, vertical rows, or horizontal and vertical rows, and the horizontal rows or vertical rows correspond to the linear or arc-shaped length segments.

44. The microfluidic device according to claim 42, wherein at least some of the plurality of fluid passages are substantially parallel in the linear or arc-shaped length segments.

45. The microfluidic device of claim 42, wherein at least some of the plurality of fluid passages are arc-shaped and substantially parallel passages that encompass the arc-shaped length segments.

46. The microfluidic device according to claim 42, wherein at least some of the plurality of fluid passages are substantially parallel and extend radially between the outer periphery of the device and the center of the device.

47. The microfluidic device according to claim 42, wherein the plurality of fluid passages include a first fluid passage set and a second fluid passage set spaced apart from the first set, the first fluid passage set terminates at a first master mix port as the second port, and the second fluid passage set terminates at a second master mix port as the second port.

48. The microfluidic device according to claim 42, wherein the first and second fluid passage sets are spaced apart in the circumferential direction.

49. The microfluidic device according to claim 42, wherein the plurality of fluid passages each comprises a spatially aligned set of microwells, each including at least two fluid passages defining a first neighborhood set and at least two passages defining a second neighborhood set adjacent to the first neighborhood set.

50. A microfluidic device according to claim 49, further comprising a first gap space between the first and second fluid passages of the first and second neighborhood sets, and further comprising a second gap space between the first and second neighborhood sets, wherein the second gap space has a larger lateral range than the first gap space.

51. The device according to claim 42, wherein each of the plurality of microwell sets in the plurality of fluid passages has a common configuration, and each of the plurality of microwell sets in the fluid passages is aligned with one another in horizontal and / or vertical rows, and the plurality of fluid passages hold the input substance in a state of fluid isolation from one another.

52. The microfluidic device according to claim 42, wherein each of at least a plurality of microwell sets for the fluid passage comprises a quantity in the range of 1,000 to 1,000,000 microwells, and the microwells of the microwell set have a size and configuration that fixes and holds a single bead, thereby enabling 100 to 1 billion reactions in the microfluidic device.

53. The microfluidic device of claim 42, comprising a transparent substrate extending across the plurality of microwell sets of the fluid passage, defining a first set of first and second microwell sets from at least first and second fluid passages, optionally paired by adjacent microwell subsets.

54. The microfluidic device according to claim 42, wherein each of the plurality of fluid passages includes a separate first port at a first end as the first port, one of the alternating first ports is located at a first longitudinal position of the device, and the other alternating first port is located at a second longitudinal position of the device spaced apart from the first longitudinal position by the length dimension.

55. The microfluidic device according to claim 42, wherein the first port is a material input port located at the first end of the fluid passage, and the device further comprises a fluid manifold connecting at least some of the opposite second ends of the fluid passage to the second port.

56. The microfluidic device according to claim 42, wherein the fluid passages extend radially in the device, the input ports of the fluid passages are located on the outer periphery of the device, and the second ports are a single second port located at the center of the device to which each of the fluid passages is connected.

57. The microfluidic device according to claim 42, wherein at least some of the fluid passages are provided as a set of concentric fluid passages, each having one of the arc-shaped length segments.

58. The microfluidic device according to claim 42, wherein the concentric fluid passage set having the arc-shaped length segment is provided as a plurality of concentric fluid passage sets spaced apart in the circumferential direction.

59. The microfluidic device according to claim 42, wherein the microfluidic device further comprises a dye homogenizer, optionally comprising an oligonucleotide (e.g., non-expandable oligonucleotide and / or partially double-stranded DNA (e.g., biotin and / or partially double-stranded DNA comprising a C1-C20 hydrocarbon chain)).

60. The microfluidic device according to claim 59, wherein the dye homogenizer is present in a master mix present in the microfluidic device, and / or the dye homogenizer is attached to beads present in the microfluidic device.

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