WO2021144396A1

WO2021144396A1 - Microfluidic device and method for automated split-pool synthesis

Info

Publication number: WO2021144396A1
Application number: PCT/EP2021/050754
Authority: WO
Inventors: Sedide OZTURK; Juraj Topolancik; Jaeyoung Yang
Original assignee: F. Hoffmann-La Roche Ag; Roche Diagnostics Gmbh; Roche Sequencing Solutions, Inc.
Priority date: 2020-01-17
Filing date: 2021-01-15
Publication date: 2021-07-22
Also published as: EP4090464A1

Abstract

Described herein are systems and methods for randomly dividing a population of particles into two or more sub-populations, reacting each formed sub-population of particles with a different reagent, pooling the reacted sub-populations of particles back together.

Description

MICROFLUIDIC DEVICE AND METHOD FOR AUTOMATED SPLIT-

POOL SYNTHESIS

BACKGROUND OF THE DISCLOSURE

[0001] Driven by recent advances in flow cytometry and RNA/DNA sequencing, significant progress has been achieved in single-cell characterization. Microfluidic systems including cell sorters and micro-well reactors have been employed to separate tens of thousands of cells into isolated compartments where their mRNAs are reverse transcribed and amplified for sequencing. Significant enhancement in throughput, however, is needed to enable analysis of millions of individual cells, which is necessary to identify all different cell types and to fully understand their function and their response to changes in the microenvironment. Future improvements in cell manipulation and isolation of cells in separate compartments will likely come with a considerable increase in complexity and the cost of instrumentation.

[0002] An alternative approach to single cell analysis that does not require partitioning and confinement involves employing molecular tags to identify reads from individual cells, such as through ensemble processing in conventional microwell plates while retaining single-cell resolution. Unique barcodes are assigned to each cell by split-pool barcoding (SPBC) or quantum barcoding (QBC). This can be done, for example, by labeling each cell's mRNAs during reverse transcription or by labeling cell-specific antibodies with specific DNA oligonucleotides. In each split-pool cycle, fixed cells or nuclei are randomly distributed into N wells containing specific barcodes as shown in FIG. I A.

[0003] After barcodes are appended through ligation, the unattached barcodes are washed away and the cells or nuclei are pooled together. The process can be repeated multiple times by redistributing the cells or nuclei into the same or another set of the wells. This is repeated a sufficient number of times to reach high probability that each cell or nucleus in the final pool holds a unique barcode. For example, if "m" number of cells or nuclei are started with and are then split them into "N" wells, and if the process is repeated "X" times, then the "m" different number of cells or nuclei will be eventually sharing "N^x" unique barcodes.

[0004] Since the number of unique tags grows exponentially with the number of barcoding rounds, significant throughput enhancement can be achieved simply by adding a few additional cycles. It is believed, however, that using larger micro-well plates does not reduce the number of needed QBC cycles dramatically, but it does add a significant number of pipetting steps (see FIGS. IB and 1C). For these processes, the optimal size of a micro-well plate used in conventional QBC is determined by: (1) the efficiency of ligation and/or reverse transcription reactions, (2) the losses of cellular material during pipetting between different micro-wells and various washing and rinsing steps needed to remove the unbound tags and other reagents, and, ultimately, (3) by the cost of sequencing which limits the length of the barcodes and hence the number of allowed QBC cycles.

[0005] While QBC can certainly be performed in micro-well plates by liquid handling robots to achieve ultra-high-throughput analysis of single cells, the non parallel nature of liquid manipulation makes conventional automated platforms not particularly ideal for QBC protocol. For example, a QBC process performed in four micro-wells requires eight sequential pipetting steps as shown schematically in FIG. ID. Depending on the micro-well plate size, it is believed that a full QBC process could require between 200 and 18,500 pipetting steps to move cells around the microarray with a single pipette tip moving at any one time. It will be appreciated that each of these pipetting steps potentially contributes to a loss of material and genetic information.

[0006] Microfluidic systems are of significant value for acquiring and analyzing chemical and biological information using very small volumes of liquid. Microfluidic systems can be broadly defined as systems leveraging micrometer scale channels, to manipulate and process low volume fluid samples (Whitesides, 2006). Use of microfluidic systems can increase the response time of reactions, minimize sample volume, and lower reagent and consumables consumption. When volatile or hazardous materials are used or generated, performing reactions in microfluidic volumes also enhances safety and reduces disposal quantities. Such devices are used, for example, in medical diagnostics, genomic analysis, DNA forensics, and "lab-on- a-chip" chemical analyzers, and they can be fabricated using common microfabrication techniques such as photolithography.

BRIEF SUMMARY OF THE DISCLOSURE

[0007] Applicant has developed a system and method capable of randomly dividing a population of particles (e.g. cells or cellular components) into two or more reaction channels, independently reacting each population of divided particles with a separate reagent in the two or more reaction channels and pooling the divided populations of particles back together simultaneously, repeatedly, and without considerable material losses. In some embodiments, the microfluidic devices and methods of the present disclosure are adapted for performing any number of chemical reactions and/or chemical synthesis. In some embodiments, the microfluidic devices and methods of the present disclosure are adapted for split-pool synthesis, split-pool barcoding, and/or quantum barcoding.

[0008] In some embodiments, the microfluidic devices and methods of the present disclosure are suitable for use in labeling particles, e.g. cells and/or cellular components, with a statistically unique barcode, where the statistically unique barcode is iteratively generated after repeated split-pool synthesis cycles. In some embodiments, the statistically unique barcodes include concatenated nucleic acid sequences. In some embodiments, the microfluidic devices of the present disclosure facilitate the implementation of the quantum barcoding protocol described in the U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0009] In a first aspect of the present disclosure is a microfluidic chip including: a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each of the at least two reaction channels are in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the particle transfer conduit comprises at least two levels of binary branching. In some embodiments, the particle transfer conduit comprises at least three of binary branching. In some embodiments, the particle transfer conduit comprises at least four of binary branching. In some embodiments, the reaction array comprises at least two reaction channels. In some embodiments, the reaction array comprises at least four reaction channels. In some embodiments, the reaction array comprises at least eight reaction channels. In some embodiments, the pooling chamber includes at least two pooling channels. In some embodiments, the pooling chamber includes at least four pooling channels. In some embodiments, the at least two reaction channels each comprise a plurality of hydrodynamic trapping elements. In some embodiments, the plurality of hydrodynamic trapping elements are configured to capture particles during a first fluid flow through the at least two reaction channels in a first direction; and wherein the plurality of hydrodynamic trapping elements are configured to release the captured particles during a second fluid flow through the at least two reaction channels in a second direction. In some embodiments, the first and second fluid flows are in opposite directions.

[0010] In some embodiments, the hydrodynamic trapping elements comprise at least two trapping members. In some embodiments, the hydrodynamic trapping elements comprise at least three trapping members. In some embodiments, one of the at least three trapping members is offset longitudinally relative to another two of the at least three trapping members. In some embodiments, the at least three trapping members have a symmetrical cross-sectional shape. In some embodiments, the at least three trapping members have a asymmetrical cross-sectional shape. In some embodiments, the at least three trapping members have a shape selected from the group consisting of a triangular shape, a diamond shape, and a teardrop shape.

[0011] In some embodiments, the hydrodynamic trapping elements are arranged in substantially parallel rows within each of the reaction channels. In some embodiments, a lateral offset distance between two adjacent substantially parallel rows of hydrodynamic trapping elements varies from between about 0.5 pm to about 3 pm. In some embodiments, the lateral offset distance varies by about 2 pm. In some embodiments, a lateral offset distance between the hydrodynamic trapping elements within each of the reaction channels decreases between a first row of hydrodynamic trapping elements and a last row of the hydrodynamic trapping elements.

[0012] In some embodiments, the lateral offset distance decreases a predetermined amount for every predetermined number of substantially parallel rows of hydrodynamic trapping elements. In some embodiments, the lateral offset distance decreases until the lateral offset distance is about equal to a gap spacing between two trapping members of the hydrodynamic trapping elements.

[0013] In some embodiments, the at least two reaction channels each comprise two or more hydrodynamic trapping zones, wherein each of the two or more hydrodynamic trapping zones comprise substantially parallel rows of hydrodynamic trapping elements, wherein each of the two or more hydrodynamic trapping zones are separated by a free flow zone. In some embodiments, a lateral offset distance between two adjacent substantially parallel rows of hydrodynamic trapping elements within each hydrodynamic trapping zone are different. In some embodiments, the at least two reaction channels have a tapered shape.

[0014] In some embodiments, the microfluidic chip further includes a fluid introduction conduit, wherein the fluid introduction conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the fluid introduction conduit.

[0015] In some embodiments, the microfluidic chip is adapted for split-pool barcoding or quantum barcoding. In some embodiments, the microfluidic chip is adapted to facilitate the flow of one or more fluids (e.g. buffers) and/or reagents (e.g. assayable polymer subunits, nucleotides, oligonucleotide, antibodies, etc.) to facilitate split-pool barcoding or quantum barcoding. In some embodiments, the microfluidic chip facilitates implementation of the quantum barcoding protocol described in the U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety. [0016] In a second aspect of the present disclosure is a system including a microfluidic chip, a fluidics module, and a control system, wherein the microfluidic chip includes a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the fluidics module comprises at least one pump. In some embodiments, the fluidics module comprises at least two pumps. In some embodiments, the system further includes at least two reagent reservoirs, wherein each of the at least two reagent reservoirs is in fluidic communication with only one of the at least two reaction channels. In some embodiments, the microfluidic system is adapted for split-pool barcoding or quantum barcoding. In some embodiments, the microfluidic system is adapted to facilitate the flow of one or more fluids (e.g. buffers) and/or reagents (e.g. assayable polymer subunits, nucleotides, oligonucleotides, antibodies, etc.) to facilitate split-pool barcoding or quantum barcoding. In some embodiments, the microfluidic device facilitates implementation of the quantum barcoding protocol described in the U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0017] In a third aspect of the present disclosure is a population of uniquely labeled particles prepared using a microfluidic system including a microfluidic chip, a fluidics module, and a control system, wherein the microfluidic chip includes a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the fluidics module comprises at least one pump. In some embodiments, the microfluidic system further includes at least two reagent reservoirs, wherein each reagent reservoir of the at least two reagent reservoirs is in fluidic communication with only one of the at least two reaction channels. [0018] In some embodiments, each uniquely labeled particle includes a different tag, label, or barcode. In some embodiments, the barcode includes a concatemeric nucleic acid sequence. In some embodiments, the uniquely labeled particles include a series of assayable polymer subunits. In some embodiments, each uniquely labeled particle includes a different cell originating barcode. In some embodiments, each uniquely labeled particle comprises a barcode having the structure of any of those described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0019] In a fourth aspect of the present disclosure is a method of functionalizing particles with one or more reagents (e.g. assayable polymer subunits, nucleotides, oligonucleotides, etc.) including: flowing a population of particles in a fluid (e.g. a buffer) through a particle splitting conduit to provide two or more sub populations of particles, wherein each of the two or more sub-populations of particles comprises a random distribution of particles from the population of particles; flowing each sub-population of particles in the fluid through a different reaction channel towards a plurality of hydrodynamic trapping elements so as to independently capture each sub-population of particles within one of the different reaction channels; flowing a different reagent through each different reaction channel so as to react each captured sub-population of particles with a different reagent; and flowing each of the sub-populations of reacted particles from the different reaction channels to a pooling chamber to form a pool of reacted particles.

[0020] In some embodiments, the reacted particles are randomly distributed within the pooling chamber. In some embodiments, the reacted particles are captured within a plurality hydrodynamic trapping elements within the pooling chamber. In some embodiments, a first fluid flow to effectuate the independent capture of each sub-population of particles in the one of the different reaction channels is in a first direction; and wherein a second fluid flow to effectuate a release of each captured sub-population of particles from each different reaction channel is in a second direction. In some embodiments, the method further comprises flowing a wash fluid through each of the reaction channels prior to flowing each of the sub-populations of reacted particles from the different reaction channels. In some embodiments, the method further comprises sequentially repeating each of the aforementioned steps.

[0021] In some embodiments, the population of particles includes cells. In some embodiments, the population of particles includes cellular components. In some embodiments, the different reagents comprise oligonucleotide sequences. In some embodiments, the different reagents comprise assayable polymer subunits. In some embodiments, the different reagents include any of those described in the U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0022] In some embodiments, the hydrodynamic trapping elements comprise at least three trapping members, wherein one of the at least three trapping members is offset longitudinally relative to another two of the at least three trapping members. In some embodiments, the hydrodynamic trapping elements are arranged in substantially parallel rows within the different reaction channels. In some embodiments, a lateral offset distance between two adjacent parallel rows of hydrodynamic trapping elements varies from between about 0.5 pm to about 3 pm. In some embodiments, a lateral offset distance between the hydrodynamic trapping elements decreases between a first row of hydrodynamic trapping elements and a last row of the hydrodynamic trapping elements. In some embodiments, the lateral offset distance decreases a predetermined amount for every predetermined number of substantially parallel rows of hydrodynamic trapping elements. In some embodiments, the lateral offset distance decreases until the lateral offset distance is about equal to a gap spacing between two trapping members of the hydrodynamic trapping elements.

[0023] In some embodiments, each of the different reaction channels comprise two or more hydrodynamic trapping zones including the substantially parallel rows of hydrodynamic trapping elements, wherein each of the two or more hydrodynamic trapping zones are separated by a free flow zone. In some embodiments, a lateral offset distance between two adjacent rows of the substantially parallel rows of hydrodynamic trapping elements within each hydrodynamic trapping zone are different. [0024] In a fifth aspect of the present disclosure is a population of uniquely labeled particles prepared according to a process including: flowing a population of particles in a fluid in a first direction through a particle splitting conduit, wherein the particle splitting conduit comprises one or more levels of binary branching and wherein each of the one or more levels of binary branching of the particle splitting conduit is in fluidic communication with a reaction channel, wherein the flowing of the population of particles through the particle splitting conduit randomly divides the population of particles into two or more sub-populations of particles; flowing each sub-population of particles in the fluid through a different reaction channel toward a plurality of hydrodynamic trapping elements so as to independently capture each sub-population of particles within one of the different reaction channels; flowing a different reagent (e.g. assayable polymer subunits, nucleotides, oligonucleotides, etc.) through each different reaction channel so as to react each captured sub population of particles with the different reagent; and flowing each of the sub populations of reacted particles in a fluid from the different reaction channels through the particle splitting conduit and to a pooling chamber to form a pool of reacted particles; and repeating the aforementioned steps a pre-determined number of times.

[0025] In some embodiments, the pooling chamber comprises two or more pooling channels. In some embodiments, the pooling chamber comprises a plurality of hydrodynamic trapping elements. In some embodiments, the population is further prepared by flowing a wash fluid through each of the reaction channels prior to flowing each of the sub-populations of reacted particles from the different reaction channels. In some embodiments, the population of particles comprises cells. In some embodiments, the labels are barcodes including unique concatemeric nucleotide sequences. In some embodiments, the different reagents comprise oligonucleotides.

[0026] In some embodiments, the hydrodynamic trapping elements including three trapping members, and wherein the hydrodynamic trapping elements are arranged in substantially parallel rows within each different reaction channel. In some embodiments, the population is further prepared by imaging the reaction channels after flowing each of the sub-populations of reacted particles from the different reaction channels. In some embodiments, the population is further prepared by imaging the pooling chamber after flowing the population of particles through the particle splitting conduit.

[0027] In a sixth aspect of the present disclosure is a use of the microfluidic chip to carry out split-pool synthesis, wherein the microfluidic chip includes: a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit.

[0028] In a seventh aspect of the present disclosure is a use of the microfluidic chip to carry out split-pool barcoding and/or quantum barcoding, wherein the microfluidic chip includes: a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit.

[0029] In an eighth aspect of the present disclosure is a substrate including a pooling chamber, a reaction array including at least two reaction channels, and a particle splitting conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle splitting conduit comprises one or more levels of binary branching, and wherein each reaction channel is in fluidic communication with a branch of the one or more levels of binary branching of the particle splitting conduit; and wherein each of the at least two reaction channels are fluidically coupled with one branch of a fluid splitting conduit having one or more levels of binary branching. In some embodiments, the at least two reaction channels and the pooling chamber comprise a plurality of hydrodynamic trapping elements. In some embodiments, the plurality of hydrodynamic trapping elements are configured to capture particles during a first fluid flow through the at least two reaction channels in a first direction; and wherein the plurality of hydrodynamic trapping elements are configured to release the captured particles during a second fluid flow through the at least two reaction channels in a second direction.

[0030] In a ninth aspect of the present disclosure is a kit including a microfluidic chip and one or more buffers, wherein the microfluidic chip includes: a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the kit further includes one or more reagents. In some embodiments, the reagents comprise oligonucleotides.

[0031] In a tenth aspect of the present disclosure is a kit including a microfluidic chip and a sequencing device, wherein the microfluidic chip includes: a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the kit further includes one or more reagents. [0032] In an eleventh aspect of the present disclosure is an apparatus including a sequencing device coupled to a microfluidic system, the microfluidic system including a microfluidic chip, a fluidics module, and a control system, wherein the microfluidic chip includes a pooling chamber, a reaction array including at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit. In some embodiments, the fluidics module comprises at least one pump. In some embodiments, the system further includes at least two reagent reservoirs, wherein each reagent reservoir of the at least two reagent reservoirs is in fluidic communication with only one of the at least two reaction channels.

BRIEF DESCRIPTION OF THE FIGURES [0033] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0034] FIG. 1A is a schematic representation of split-pool barcoding. The number of unique barcodes grows exponentially with the number of barcoding rounds.

[0035] FIG. IB illustrates the calculated numbers of split-pool cycles.

[0036] FIG. 1C illustrates the number of pipetting steps needed to avoid barcode collisions for various cell populations and micro-well plate sizes.

[0037] FIG. ID depicts a conventional fluidic manipulation in a QBC protocol.

[0038] FIG. 2 depicts a system including a microfluidic device and a controller in accordance with one embodiment of the present disclosure.

[0039] FIG. 3 depicts a microfluidic device including a pooling chamber and a reaction chamber, where the reaction chamber includes a plurality of reaction channels in accordance with one embodiment of the present disclosure.

[0040] FIG. 4A depicts a microfluidic device including a pooling chamber and a reaction chamber, where the reaction chamber includes a plurality of reaction channels and where the pooling chamber includes a plurality of pooling channels in accordance with one embodiment of the present disclosure. [0041] FIG. 4B depicts a microfluidic device including a pooling chamber and a reaction chamber, where the reaction chamber includes a plurality of reaction channels and where the pooling chamber includes a plurality of pooling channels in accordance with one embodiment of the present disclosure. [0042] FIG 5A depicts a plurality of reaction channels in accordance with one embodiment of the present disclosure.

[0043] FIG. 5B depicts a plurality of reaction channels in accordance with one embodiment of the present disclosure. [0044] FIG. 5C depicts a plurality of reaction channels in accordance with one embodiment of the present disclosure.

[0045] FIGS. 6A, 6B, and 6C illustrate hydrodynamic trapping elements, where each hydrodynamic trapping element is comprised of three hydrodynamic trapping members in accordance with one embodiment of the present disclosure. [0046] FIGS. 7A and 7B depict teardrop-shaped hydrodynamic trapping elements arranged into clusters of hydrodynamic trapping elements in accordance with one embodiment of the present disclosure.

[0047] FIG. 7C illustrates a reaction channel including alternating hydrodynamic trapping zones and free flow zones in accordance with one embodiment of the present disclosure.

[0048] FIGS. 8A - 8E illustrate the operation of the microfluidic device of

FIG. 4 in accordance with one embodiment of the present disclosure.

[0049] FIG. 9 provides a flow chart illustrating a method of collecting populations of reacted particles in accordance with one embodiment of the present disclosure.

[0050] FIG. 10 provides a flow chart illustrating a method of collecting populations of reacted particles by flowing fluids through a microfluidic device in accordance with one embodiment of the present disclosure. DETAILED DESCRIPTION

[0051] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0052] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0053] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0054] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0055] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, for example, the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (for example "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0056] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0057] As used herein, the terms "Cell Origination Barcode" and "COB" each refer to a unique code that can be associated to a specific cell of origin. In some embodiments, upon binding of the COB to a common linker moiety (e.g. common linker oligo) associated with an ESB, the COB code identifies the cells of origin of the target molecule to which the UBA/ESB complex is bound. Thus, in some embodiments, the COBs of the disclosure comprise two main portions: (i) a sequence specific for a common linker moiety (e.g. common linker oligo) associated with a UBA/ESB probe; and (ii) an unique code that can be associated to a specific cell of origin. In some embodiments, COBs are modular structures. In some embodiments, the COB comprises a plurality of different assayable polymer subunits (APS). In some embodiments, the COBs comprise a plurality of APSs attached in linear combination. In some embodiments, a COB is a molecular entity containing certain basic elements: (i) a plurality of APSs including label attachment regions attached in linear combination to form a backbone, and (ii) complementary polynucleotide sequences, including a label, which are complementary and are attached to the label attachment regions of the backbone. The term "label attachment region" includes a region of defined polynucleotide sequence within a given backbone that may serve as an individual attachment point for a detectable molecule. In some embodiments, the COBs comprise a plurality of different APSs attached in linear combination, wherein the APSs comprise small molecules of deterministic weight. In some embodiments, the COB comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more unique APSs attached in a linear combination. In some embodiments, the COB comprises 4 or more APSs attached in linear combination. UBAs, ESB, and COBs are further described herein and in United States Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0058] As used herein, the terms "couple" or "coupling" refer to the joining, bonding (e.g. covalent bonding), or linking of one molecule or atom to another molecule or atom.

[0059] As used herein, the term "cell," refers to a prokaryotic cell or a eukaryotic cell. The cell may be an adherent or a non-adherent cell, such as an adherent prokaryotic cell, adherent eukaryotic cell, non-adherent prokaryotic cell, or non-adherent eukaryotic cell. A cell may be a yeast cell, a bacterial cell, an algae cell, a fungal cell, or any combination thereof. A cell may be a mammalian cell. A cell may be a primary cell obtained from a subject. A cell may be a cell line or an immortalized cell. A cell may be obtained from a mammal, such as a human or a rodent. A cell may be a cancer or tumor cell. A cell may be an epithelial cell. A cell may be a red blood cell or a white blood cell. A cell may be an immune cell such as a T cell, a B cell, a natural killer (NK) cell, a macrophage, a dendritic cell, or others. A cell may be a neuronal cell, a glial cell, an astrocyte, a neuronal support cell, a Schwann cell, or others. A cell may be an endothelial cell. A cell may be a fibroblast or a keratinocyte. A cell may be a pericyte, hepatocyte, a stem cell, a progenitor cell, or others. A cell may be a circulating cancer or tumor cell or a metastatic cell. A cell may be a marker specific cell such as a CD8+ T cell or a CD4+ T cell. A cell may be a neuron. A neuron may be a central neuron, a peripheral neuron, a sensory neuron, an interneuron, a intraneuron, a motor neuron, a multipolar neuron, a bipolar neuron, or a pseudo-unipolar neuron. A cell may be a neuron supporting cell, such as a Schwann cell. A cell may be one of the cells of a blood-brain barrier system. A cell may be a cell line, such as a neuronal cell line. A cell may be a primary cell, such as cells obtained from a brain of a subject. A cell may be a population of cells that may be isolated from a subject, such as a tissue biopsy, a cytology specimen, a blood sample, a fine needle aspirate (FNA) sample, or any combination thereof. A cell may be obtained from a bodily fluid such as urine, milk, sweat, lymph, blood, sputum, amniotic fluid, aqueous humor, vitreous humor, bile, cerebrospinal fluid, chyle, chyme, exudates, endolymph, perilymph, gastric acid, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, serous fluid, smegma, sputum, tears, vomit, or other bodily fluid. A cell may comprise cancerous cells, non- cancerous cells, tumor cells, non-tumor cells, healthy cells, or any combination thereof.

[0060] As used herein, the term "channel" refers to an enclosed passage within a microfluidic chip through which a fluid can flow. The channel can have one or more openings for introduction of a fluid. Each channel may include a coating, e.g. a hydrophilic or hydrophobic coating.

[0061] As used herein, the terms "Epitope Specific Barcode" or "ESB" refer to unique codes that can be associated to a specific target molecule. ESBs are molecules or assemblies that are designed to bind with at least one EGBA (defined herein) or part of an EGBA; and can, under appropriate conditions, form a molecular complex including the ESB, the EGBA and the target molecule. ESBs can comprise at least one identity identification portion that allow them to bind to or interact with at least one UBA; typically in a sequence-specific, a confirmation-specific manner, or both; for example but not limited to UBA-antibody binding, aptamer-target binding, and the like. In some embodiments, the ESB are attached, directly or indirectly, to the UBA. In other embodiments, the ESBs bind to the UBAs in a cell or sample, e.g., as part of the assay procedure. UBAs and ESB are further described herein and in United States Patent No. 10, 144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0062] As used herein, the term "fluid" refers to any liquid or liquid composition, including water, solvents, buffers, solutions (e.g. polar solvents, non polar solvents), and/or mixtures. The fluid may be aqueous or non-aqueous. Non limiting examples of fluids include washing solutions, rinsing solutions, acidic solutions, alkaline solutions, transfer solutions, and hydrocarbons (e.g., alkanes, isoalkanes and aromatic compounds such as xylene). In some embodiments, washing solutions include a surfactant to facilitate spreading of the washing liquids over the specimen-bearing surfaces of the slides. In some embodiments, acid solutions include deionized water, an acid (e.g., acetic acid), and a solvent. In some embodiments, alkaline solutions include deionized water, a base, and a solvent. In some embodiments, transfer solutions include one or more glycol ethers, such as one or more propylene-based glycol ethers (e.g., propylene glycol ethers, di(propylene glycol) ethers, and tri (propylene glycol) ethers, ethylene-based glycol ethers (e.g., ethylene glycol ethers, di(ethylene glycol) ethers, and tri(ethylene glycol) ethers), and functional analogs thereof. Non-liming examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine- N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N- morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N- morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine(Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxy ethyl- 1 - piperazineethanesulfonic acid (HEPES), 2-

{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In some embodiments, the unmasking agent is water. In other embodiments, the buffer may be comprised of tris(hydroxymethyl)methylamine (TRIS), 2-(N- morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine(Bicine), N -tris(hydroxymethyl)methylglycine (Tricine), 4-2-hy droxy ethyl- 1- piperazineethanesulfonic acid (HEPES), 2-

{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof. Additional wash solutions, transfer solutions, acid solutions, and alkaline solutions are described in United States Patent Application Publication No. 2016/0282374, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0063] As used herein, the term "hydrodynamic trapping element" refers to a particle trap in which the force of a fluid in motion plays a role in capturing the particle within the hydrodynamic trapping element and/or retaining the particle trapped in its position within the hydrodynamic trapping element.

[0064] As used herein, the term "label" refers to a detectable moiety that may be atoms or molecules, or a collection of atoms or molecules. A label may provide a chemical, optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature which may be detected.

[0065] As used herein, "microfluidic" refers to a system or device having one or more fluidic channels, conduits, or chambers that are generally fabricated at the millimeter to nanometer scale. As such, a "microfluidic device," as used herein, refers to any device that allows for the precise control and manipulation of fluids that are geometrically constrained to structures in which at least one dimension (width, length, height) may be less than 1 mm. In some embodiments, the microfluidic device includes a microfluidic chip including one or more channels and/or conduits.

[0066] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotide or nucleoside monomer units wherein the oligomer optionally includes non-nucleotide monomer units, and/or other chemical groups attached at internal and/or external positions of the oligomer. The oligomer can be natural or synthetic and can include naturally-occurring oligonucleotides, or oligomers that include nucleosides with non-naturally-occurring (or modified) bases, sugar moieties, phosphodiester-analog linkages, and/or alternative monomer unit chiralities and isomeric structures (e.g., 5'- to 2'-linkage, L-nucleosides, a-anomer nucleosides, b- anomer nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNA)).

[0067] As used herein, "particles" include natural and/or synthetic chemicals or biological entities. Examples of particles include cells, components of cells, nuclei, organelles, etc.

[0068] As used herein, the term "plurality" refers to two or more, for example, 3 or more, 4 or more, 5 or more, etc.

[0069] As used herein, a "reaction" between two reactive groups (such as between a reagent and a particle each including a different reactive group) may mean that a covalent linkage is formed between two reactive groups or two reactive functional groups; or may mean that the two reactive groups or two reactive functional groups associate with each other, interact with each other, hybridize to each other, hydrogen bond with each other, etc. In some embodiments, a "reaction" between two reactive groups includes binding events. [0070] As used herein, the term "reagent" refers to solutions or suspensions including one or more agents capable of covalently or non-covalently reacting with, coupling with, interacting with, or hybridizing to another entity. Non-limiting examples of such agents include specific-binding entities, antibodies (primary antibodies, secondary antibodies, or antibody conjugates), nucleic acid probes, oligonucleotide sequences, detection probes, chemical moieties bearing a reactive functional group or a protected functional group, enzymes, solutions or suspensions of dye or stain molecules.

[0071] As used herein, the term "sequence" when used in reference to a nucleic acid, refers to the order of nucleotides (or bases). In cases, where different species of nucleotides are present, the sequence includes an identification of the species of nucleotide (or base) at respective positions in of the nucleic acid or oligonucleotide.

[0072] As used herein, the terms "sequencing" or "DNA sequencing" refer to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modem automated DNA sequencing instruments.

[0073] As used herein, the phrase "split-pool synthesis" refers to one non limiting example of a combinatorial synthesis process in which a reaction mixture is divided into several different aliquots prior to performing a reaction, and wherein each aliquot receives a different chemical entity to be reacted with, coupled with, etc., e.g. a monomer, an oligomer, an assayable polymer subunit, etc. Following the coupling reaction, the aliquots are combined (pooled), mixed, and divided (split) into a new set of aliquots prior to performing the next round of coupling. In general, the approach may be used for a variety of coupling reactions and conjugation chemistries including, but not limited to, amino acid (or short peptide) coupling reactions to produce longer peptides of fully or partially random amino acid sequences, the coupling of deoxyribonucleotides (or short DNA oligonucleotides) to produce longer DNA oligonucleotides of fully or partially random base sequences, or the coupling of ribonucleotides (or short RNA oligonucleotides) to produce longer RNA oligonucleotides of fully or partially random base sequences, ligation reactions, polymerase chain reactions, click-chemistry coupling reactions, etc. Any of a variety of chemical monomers, e.g., amino acids, small molecules, short peptides, short oligonucleotides, etc., may thus be utilized. In some embodiments, a split-pool synthesis is adapted for split-pool barcoding and/or quantum barcoding, where particles are iteratively reacted with agents, such as monomeric agents, for the generation of statistically unique barcodes.

[0074] As used herein, the term "substrate" refers to an organic or inorganic sheet, tube, sphere, container, pad, film or slide. In some embodiments, the substrate is flat but may take on alternative surface configurations. For example, the substrate may include raised or depressed regions, such as microfluidic channels, chambers, conduits, apertures, ports, etc. For example, the substrate may be functionalized glass, Si, Ge, GaAs, GaP, S1O2, S1N4, modified silicon, nitrocellulose and nylon membranes, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations thereof. Other suitable substrate materials are be readily apparent to those of skill in the art and include polymers and copolymers, substrates for lithography, etc. The surface of the substrate may also contain reactive groups, which could be carboxyl, amino, hydroxyl, thiol, or the like. In some embodiments, the surface of the substrate is optically transparent and will have surface Si — OH functionalities, such as are those found on silica surfaces. In some embodiments, the substrate includes one or more coatings, e.g. hydrophilic coatings or hydrophobic coatings.

[0075] As used herein, the term "substantially" means the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. In some embodiments, "substantially" means within about 5%. In some embodiments, "substantially" means within about 10%. In some embodiments, "substantially" means within about 15%. In some embodiments, "substantially" means within about 20%.

[0076] As used herein, the terms "unique binding agent" or "UBAs" refer to molecules or assemblies that are designed to bind with at least one target molecule, at least one target molecule surrogate, or both; and can, under appropriate conditions, form a molecular complex including the UB A and the target molecule. Examples of target molecules include, but are not limited to, proteins, nucleic acids, lipids, carbohydrates, ions, small molecules, organic monomers, and drugs. In some embodiments, the UBAs that bind to a target protein or a target mRNA. The terms "protein," "polypeptide," "peptide," and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acids of any length. In some embodiments, the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids or synthetic amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. In some embodiments, "UBAs" include at least one reaction portion that facilitates their binding to or interaction with at least one target molecule, at least one part of at least one target molecule, at least one target molecule surrogate, at least part of a target molecule surrogate, or combinations thereof; typically in a sequence-specific manner, a confirmation-specific manner, or both (e.g. antigen-antibody binding, aptamer-target binding, and the like). In some embodiments, the UBAs comprise an identity portion or at least part of an identity portion, for example, an ESB, a COB, an ESB and/or a linker oligo. In certain embodiments, the UBAs comprise a capture region. In some embodiments, the capture region is used for the isolation of the UBA and/or immobilization of the UBA into a surface. In some embodiments, the capture region can be an affinity tag, a bead, a slide, an array, a microdroplet, an enclosure in a microfluidic device or any other suitable capture region in the art. In some embodiments, the capture region is the ESB, for example the ESB can be a detectable bead such as a bead with a unique spectral signature (e.g. a bead that has been internally dyed with red and infrared fluorophores). Capture regions can define reaction volumes in which manipulation of compositions of the disclosure can take place. UBAs, ESB, and COBs are further described herein and in United States Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0077] OVERVIEW

[0078] Applicant has developed a microfluidic device configured to (i) randomly divide a population of particles into multiple sub-populations where, in some embodiments, each sub-population of particles is transferred to one of a plurality of reaction channels; (ii) permit each of the sub-population of particles to be separately reacted with a different reagent, such as in one of the plurality of reaction channels, to provide different reacted sub-populations of particles; and (iii) pool the different reacted sub-populations of particles together (such as simultaneously and/or randomly) to a separate compartment (e.g. a compartment different than any of the reaction populations). In some embodiments, the particles are cells or components of cells. [0079] In some embodiments, the microfluidic device is configured to repeat the aforementioned process a predetermined number of times and to do so without any considerable loss of material, contamination from an outside environment, and/or damage to the particles themselves. In some embodiments, repeated cycling of the process allows each of the particles to be randomly reacted with a different reagent each time the process is repeated, e.g. each time the process is repeated a different oligonucleotide sequence or assayable polymer subunits may be appended to the particle so as to provide a population of particles each having a statistically unique concatemeric nucleotide sequence or a statistically unique sequence of assayable polymer subunits. In some embodiments, the process is repeated until each of the particles in the population includes a moiety which is statistically different, e.g. a different concatemeric nucleotide sequence. These and other embodiments will be described herein.

[0080] In some embodiments, the disclosed microfluidic devices, methods, and/or kits facilitate the detection and quantification of individual target molecules in biological samples. In some embodiments, the microfluidic devices and methods described herein enable detection and quantification of one or more target molecules in individual cells or sub-cellular units (including macromolecular complexes) present in the sample, where the sample comprises a large population of cells or a mixture of multiple sub-cellular units of macromolecular complexes.

[0081] In some embodiments, the microfluidic device facilitates implementation of the quantum barcoding (QBC) protocol described in the U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety. Briefly, the QBC protocol comprises the use of unique binding agents (UBA) to bind each of the target molecules, the use of epitope-specific barcodes (ESB) optionally attached to and identifying the UBAs, and assembling cell-originating barcodes (COB) on the UBAs (and optionally ESBs) such that each of the variety of target molecules present in the cell is labeled with the same unique barcode particular to that cell. The method of assembling cell-originating barcodes (COB) involves a split-pool synthesis step, such as described herein. As described above, using the most basic calculation, if a different sub-code is present in each container or well, after M rounds of splitting the population of particles into N wells, N^M different codes will be assembled from sub-codes. For example, 3 rounds of splitting into 96-well plates, can generate about 106 unique barcodes, enough to individually label each particle in a typical volume of a sample.

[0082] MICROFLUIDIC DEVICE

[0083] In one aspect of the present disclosure are microfluidic devices including a microfluidic chip.

[0084] In some embodiments, the microfluidic devices 100 may be utilized to facilitate any number of chemical reactions and also may be used for chemical synthesis. In some embodiments, the microfluidic devices 100 may be utilized in labeling particles. In some embodiments, the microfluidic devices 100 of the present disclosure are configured to facilitate split-pool synthesis, e.g. split-pool barcoding and/or quantum barcoding. In some embodiments, the microfluidic devices 100 of the present disclosure facilitate the quantum barcoding process described herein and set forth in U.S. Patent No. 10,144,950, the disclosure of which is incorporated by reference herein in its entirety.

[0085] With reference to FIG. 2, in one aspect of the present disclosure is a microfluidic device 100 including a fluidics module 402, a control system 401, and a microfluidic chip 400. In some embodiments, the microfluidic device 100 further includes one or more reservoirs 403 for storing fluids, reagents, and/or particles (e.g. reagent reservoirs, particle collection vessels, particle storage vessels, and/or waste collection vessels. In some embodiments, the microfluidic devices are communicatively coupled to one or more sensors (temperature sensors and/or fluid flow rate sensors) and/or imaging modules which are configured to provide feedback to the control system.

[0086] In some embodiments, the microfluidic chip 400 includes a pooling chamber 110 and a reaction array 120 (see FIGS. 2, 3, and 4). In some embodiments, the microfluidic chip 400 further includes one or more conduits for flowing fluids, reagents, and/or particles (e.g. particle splitting conduits, fluid splitting conduits, particle transfer conduits). In some embodiments, the fluidic module includes one or more pumps (e.g. fluid withdrawal pumps, fluid infusion pumps, particle withdrawal pumps). In some embodiments, the one or more pumps are in fluidic communication with the components of the microfluidic chip 400 so as to allow fluids, reagents, and/or particles to be flowed in one or more directions between the various components of the microfluidic device, and/or to and/or from one or more reservoirs. In some embodiments, the fluidic module includes one or more internal or external valves, such as valves disposed in any of the conduits. Each of these components are described in more detail herein.

[0087] Microfluidic Chip

[0088] In some embodiments, the microfluidic device 100 includes a microfluidic chip 400. With reference to FIGS. 3, 4A, and 4B, in some embodiments, the microfluidic chip 400 includes a pooling chamber 110 and reaction array 120 in fluidic communication with each other such that fluids and/or particles may be transferred from the pooling chamber 110 to the reaction array 120 or from the reaction array 120 to the pooling chamber 110. In some embodiments, the pooling chamber 110 and the reaction array 120 are fluidically connected via a particle transfer conduit 130.

[0089] Reaction Array

[0090] In some embodiments, the reaction array 120 includes a plurality of reaction channels 121. In some embodiments, the reaction array 120 may include two reaction channels 121. In some embodiments, the reaction array 120 includes between 2 and 64 reaction channels 121. In other embodiments, the reaction array 120 includes between 4 and 32 reaction channels 121. In other embodiments, the reaction array 120 may include four reaction channels 121. In other embodiments, the reaction array 120 may include 8 reaction channels 121. In yet other embodiments, the reaction array 120 may include 16 reaction channels 121. In further embodiments, the reaction array 120 may include 32 reaction channels 121.

[0091] In some embodiments, the reaction channels 121 of the reaction array

120 serve as reusable chambers for the reaction of particles with one or more introduced reagents. In some embodiments, the reaction array 120 is configured to (i) permit an inward flow of fluid, reagents, and/or particles, such that fluid and/or particles may be received and at least temporarily stored within the reaction channels 121; (ii) permit a reaction to occur between the received particles and one or more introduced reagents; and/or (iii) permit an outward flow of fluids and reacted particles. [0092] Given that the reaction array 120 includes a plurality of reaction channels 121, the flow of fluid and/or particles through the reaction array 120 (either an inward flow or an outward flow) is divided among the plurality of reaction channels 121. In some embodiments, the flow rate through each of the reaction channels 121 may be substantially the same. In other embodiments, the flow rate through each of the reaction channels 121 may be different. In some embodiments, the flow rate through each of the reactions channels 121 is monitored, e.g. using one or more flow rate sensors. In some embodiments, each reaction channel of the plurality of reaction channels are arranged substantially parallel to one another and within the same plane as depicted in FIG. 5A. In other embodiments, the reaction channels are stacked on top of each other as depicted in FIG. 5B.

[0093] In some embodiments, the lengths of the reaction channels 121 may be the same. In other embodiments, the lengths of the reaction channels 121 may be of varying lengths, e.g. to equalize pressure and/or retain fluid flow rates which are substantially the same between the various reaction channels. In some embodiments, the reaction channels 121 are rectangular. In some embodiments, the reaction channels 121 are tubular. In other embodiments, the reaction channels 121 taper from a first end to a second end (see, e.g. FIGS. 5A and 5C). In some embodiments, the reaction channels are linear (see, e.g., FIGS. 4A and 4B). In other embodiments, the reaction channels 121 are serpentine (not depicted). In some embodiments, the reaction channels all have the same shapes. In other embodiments, at least one of the reaction channels 121 has a different shape.

[0094] The reaction channels 121 may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns. In some embodiments, the dimensions of the reaction channels may be selected such that fluid is able to freely flow. In other embodiments, the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel. By way of example only, assuming the volume of a cell is about 1,000 cubic micrometers and one would want to trap about 1,000,000 cells in a trapping array (such as any of those disclosed herein) with an about 30% fill factor (e.g. a percentage of channel volume occupied by trapping pillars), in some embodiments the channels would need to be aboutlOmi crons deep, about 11.5mm wide, and about 11.5mm long.

[0095] In some embodiments, each of the reaction channels 121 includes a plurality of hydrodynamic trapping elements 200 (described further herein). In some embodiments, the total number of hydrodynamic trapping elements 200 in each of the reaction channels 121 in the aggregate at least approximates the number of particles introduced into the microfluidic device 100 or anticipated to be introduced into the microfluidic device 100. In other embodiments, the total number of hydrodynamic trapping elements 200 in each of the reaction channels 121 in the aggregate is at least 10% greater than the number of particles introduced into the microfluidic device. In yet other embodiments, the total number of hydrodynamic trapping elements 200 in each of the reaction channels 121 in the aggregate is at least 15% greater than the number of particles introduced into the microfluidic device. In further embodiments, the total number of hydrodynamic trapping elements 200 in each of the reaction channels 121 in the aggregate is at least 20% greater than the number of particles introduced into the microfluidic device.

[0096] In some embodiments, the total number of hydrodynamic trapping elements in each of the reaction channels 121 in the aggregate is at least 10,000. In some embodiments, the total number of hydrodynamic trapping elements in each of the reaction channels 121 in the aggregate is at least 100,000. In other embodiments, the total number of hydrodynamic trapping elements in each of the reaction channels 121 in the aggregate is at least 1,000,000. In yet other embodiments, the total number of hydrodynamic trapping elements in each of the reaction channels 121 in the aggregate is at least 10,000,000.

[0097] In some embodiments, each reaction channel 121 is in fluidic communication with a branch of a particle spilling conduit 160 and also in fluidic communication with a branch of a fluid splitting conduit 165, which are each described further herein. In some embodiments, each of the reaction channels are in fluidic communication with a reagent reservoir 152. In some embodiments, each reaction channel includes a first aperture and a second aperture, wherein the first and second apertures are located on opposite longitudinal ends of the reaction channel. In some embodiments, the first aperture of each reaction channel 121 is in fluidic communication with a branch of a particle splitting conduit 160 and at least one reagent reservoir 152; while the second aperture of each reaction channel 121 is in fluidic communication with a branch of the fluid splitting conduit 160. In some embodiments, each of the first and second apertures of each of the reaction channels 121 may optionally include a valve having one or more ports, e.g. flow inward or outward may be restricted at one or both apertures via a valve.

[0098] Pooling Chamber

[0099] In some embodiments, the microfluidic device 100 includes a pooling chamber 110 which serves as a storage vessel for fluid and/or particles. In some embodiments, the pooling chamber 110 is configured to receive fluids and/or particles such as during an inward fluid flow into the pooling chamber. In some embodiments, the fluids may be received from a fluid reservoir 151 in fluidic communication with the pooling chamber 110, such as via a fluid introduction conduit 170. In some embodiments, fluid and/or particles may be received into the pooling chamber 110 from the reaction array 120 via the particle transfer conduit 130. In some embodiments, the pooling chamber 110 is further configured to facilitate the outward flow of fluids and/or particles, such that the fluid and/or particles may be transferred to the reaction array 120, such as via the transfer conduit 130. Alternatively, fluids and/or particles may be transferred outward from the pooling chamber 110 to a particle collection vessel in communication with pump 141.

[0100] In some embodiments, the pooling chamber includes a series of pooling channels 111. In some embodiments, the pooling chamber 110 includes at least 2 pooling channels 111. In other embodiments, the pooling chamber 110 includes 4 pooling channels 111 (see, e.g., FIG. 4A). In other embodiments, the pooling chamber 110 includes 6 pooling channels 111. In other embodiments, the pooling chamber 110 includes 8 pooling channels 111 (see, e.g., FIG. 4B). In other embodiments, the pooling chamber includes 12 pooling channels. In yet other embodiments, the number of pooling channels 111 is equal to the number of reaction channels 121 (see, e.g., FIG. 4B). In some embodiments, the pooling chamber includes between 2 and 64 pooling channels.

[0101] In other embodiments, the number of pooling channels 111 differs from the number of reaction channels 121 (see, e.g., FIG. 4A). In further embodiments, the pooling chamber includes 0 pooling channels (see, e.g., FIG. 3). In some embodiments, the flow rate through each of the pooling channels 111 may be substantially the same. In other embodiments, the flow rate through each of the pooling channels 11 lmay be different.

[0102] In some embodiments, the lengths of the pooling channels 111 may be the same length. In other embodiments, the lengths of the pooling channels 111 may be of varying lengths, e.g. to equalize pressure and/or retain fluid flow rates which are substantially the same between the various pooling channels. In some embodiments, the pooling channels 111 are rectangular. In some embodiments, the pooling channels 111 are tubular. In other embodiments, the pooling channels 111 taper from a first end to a second end. In some embodiments, the pooling channels 111 are linear. In other embodiments, the pooling channels are serpentine. In some embodiments, the pooling channels 111 all have the same shapes. In other embodiments, at least one of the pooling channels 111 has a different shape.

[0103] In some embodiments, the size of the pooling chamber approximates the size of all of the reaction chambers. In other embodiments, the size of the pooling chamber is larger than the aggregate size of all reaction channels. In yet other embodiments, the aggregate size of all pooling channels is greater than the aggregate size of all reaction channels.

[0104] As with the reaction array 120 and the reaction channels 121 included therein, the pooling chamber 110 and/or the pooling channels 111 include a plurality of hydrodynamic trapping elements 200. In those embodiments where the pooling chamber 110 does not include pooling channels, the pooling chamber itself includes the plurality of hydrodynamic trapping elements. In those embodiments where the pooling chamber 110 includes two or more pooling channels 111, each of the pooling channels 111 includes a plurality of hydrodynamic trapping elements. In some embodiments, the total number of hydrodynamic trapping elements 200 in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate at least approximates the number of particles introduced into the microfluidic device. In some embodiments, the total number of hydrodynamic trapping elements in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate is at least 1,000. In some embodiments, the total number of hydrodynamic trapping elements in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate is at least 10,000. In other embodiments, the total number of hydrodynamic trapping elements in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate is at least 100,000. In yet other embodiments, the total number of hydrodynamic trapping elements in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate is at least 1,000,000. In further embodiments, the total number of hydrodynamic trapping elements in the pooling chamber 110 or in each of the pooling channels 111 in the aggregate is at least 10,000,000.

[0105] As noted above, the pooling chamber 110 is in fluidic communication with a particle transfer conduit 130 and a fluid introduction conduit 170. In those embodiments where the pooling chamber 110 includes two or more pooling channels 111, each of the pooling channels 111 is in fluidic communication with a particle splitting conduit 161 and a fluid splitting conduit 166 (see, e.g., FIGS. 4A AND 4B. In some embodiments, each of the pooling channels includes a first aperture and a second aperture, wherein the first and second apertures are located on opposite ends of the pooling chambers. In some embodiments, the first aperture of each pooling channel 111 is in fluidic communication with a branch of a particle splitting conduit 161; while the second aperture of each pooling channel 111 is in fluidic communication with a branch of the fluid splitting conduit 166. In some embodiments, each of the first and second apertures of each of the pooling channels 111 may optionally include a valve having one or more ports. [0106] Hydrodynamic Trapping Elements

[0107] In some embodiments, the plurality of hydrodynamic trapping elements 200 are each adapted to trap particles introduced during a first fluid flow through the reaction channels 121, pooling chamber 110, or pooling channels 111 in a first direction. In some embodiments, the plurality of hydrodynamic trapping elements 200 are further adapted to release the trapped particles during a second fluid flow through the reaction channels 121, pooling chamber 110, or pooling channels 111 in a second direction, such as where the first and second fluid flow directions are opposite each other. With this in mind, the plurality of hydrodynamic trapping elements 200 may have any size and shape provided that they facilitate the trapping of a particle during fluid flow in the first direction and facilitate the release of the trapped particle during fluid flow in the second, opposite direction.

[0108] In some embodiments, each of the hydrodynamic trapping elements

200 are capable of trapping a single particle. In some embodiments, no more than two particles may occupy any individual hydrodynamic trapping element 200 at one time. In some embodiments, a volumetric fluid flow through an unoccupied hydrodynamic trapping element 200 may be substantially higher than a volumetric fluid flow through an occupied hydrodynamic trapping element, such that no more than one particle may occupy any individual hydrodynamic trapping element 200 at one time.

[0109] In some embodiments, an imaging device may be positioned to monitor the trapping of particles within the trapping elements. In this manner, feedback from the monitoring using the imaging device may be used to alter fluid flow rates or pressures within the microfluidic chip such that particle trapping may be optimized (e.g. to prevent clogging of particles near an aperture of a reaction channel, or to prevent cell lysis). In some embodiments, an imaging device may be used to determine if all of the particles have been flowed out from the reaction channels 121 of the reaction array 120 Likewise, in other embodiments, and imaging device may be used to determine if all of the particles have been flowed out from the pooling chamber 110 (or any pooling channels 111 therein). If the pooling chamber and/or reaction array include any remaining particles, additional fluid may be flowed and/or the flow rate may be altered to ensure the transfer of those particles from one chamber to another (or to a collection vessel).

[0110] In some embodiments, the hydrodynamic trapping elements 200 include one or more trapping members 201, separated by a gap "G," for capturing a particle from a hydrodynamic fluid flow through the reaction channel, the pooling chamber, or the pooling channel. In some embodiments, the hydrodynamic trapping elements 200 include one or more trapping members 201 having a shape which facilitates the trapping of particles having various sizes, e.g. varying diameters. In some embodiments, the one or more trapping members have a symmetrical cross- sectional shape which helps to maintain a hydrodynamic flow within the reaction channels, pooling chamber, or pooling channels and helps to minimize hydrodynamic drag. In some embodiments, each of the trapping members may have a triangular shape profile in cross-section (see FIG. 6B). In some embodiments, each of the trapping members may have a diamond shape profile in cross-section (see FIG. 6C). In some embodiments, each of the trapping members may have a round shape profile in cross-section. In some embodiments, each of the trapping members may have an ovate shape profile in cross-section. In some embodiments, each of the trapping members may have an oblong shape profile in cross-section. In some embodiments, each of the trapping members may have a teardrop shape profile in cross-section (FIGS. 7A - 7B).

[0111] In some embodiments, each hydrodynamic trapping element 200 includes three trapping members 201 (see FIG. 6 A). In some embodiments, each of the trapping members 201 are arranged parallel to each other, but where a middle of the three trapping members 201 is offset longitudinally relative to the other two trapping members, such as depicted in FIGS. 6 A - 6C and 7 A - 7B. In some embodiments, a gap "G" between first and third trapping members may be varied. In some embodiments, the gap "G" may range from about 1000 nm to about 2000 nm. In other embodiments, the gap "G" may range from about 2,000 nm to about 5,000 nm. In yet other embodiments, the gap "G" may range from about 5,000 nm to about 10,000 nm. [0112] With reference to FIG. 6, in some embodiments, the longitudinal offset "H" between first and third and second and third trapping members 201 may be varied. In some embodiments, the longitudinal offset "H" may range from about 1,000 nm to about 2,000 nm. In other embodiments, the longitudinal offset "H" may range from about 2,000 nm to about 5,000 nm. In yet other embodiments, the longitudinal offset "H" may range from about 5,000 nm to about 10,000nm.

[0113] In some embodiments, the hydrodynamic trapping elements 200 are arranged in parallel rows within each reaction channel 121, pooling chamber 110, or pooling channel 111. In some embodiments, the hydrodynamic trapping elements 200 in a first row are offset laterally from those in a second row such as by a lateral offset distance "D" (see, e.g., FIGS. 6, 7A, and 7B). In some embodiments, the lateral offset distance "D" may range from about 5,000 nm to about 10,000 nm. In other embodiments, the lateral offset distance "D" may range from about 10,000 nm to about 20,000 nm. In yet other embodiments, the lateral offset distance "D" may range from about 20,000 nm to about 50,000 nm. In some embodiments, each individual row of hydrodynamic trapping elements is spaced apart by a distance "R" (see FIG. 6). In some embodiment, the spacing between rows "R" may range from about 5,000 nm to about 10,000 nm. In other embodiments, the spacing between rows "R" may range from about 10,000 nm to about 20,000 nm. In yet other embodiments, the spacing between rows "R" may range from about 20,000 nm to about 50,000 nm.

[0114] In some embodiments, by varying at least one of the gap "G," the longitudinal offset "H," the lateral offset "D," and/or the row distance "R," the particle trapping ability and/or hydrodynamic flow through the reaction channels, pooling chamber, or pooling channels may be altered. In addition, the dimensions and/or relative shape of the trapping members themselves may be varied (e.g. a height of the trapping member, a width of the trapping member, a geometric angle, or a curvature) to further alter particle trapping ability and/or the hydrodynamic fluid flow through the reaction channels, the pooling chamber, or pooling channels.

[0115] In some embodiments, at least the parameters "G" and/or "D" are varied to (i) maximize the trapping efficiency of a population of introduced particles having various sizes and/or shapes, (ii) to avoid particle loss from the reaction channels 121, the pooling chamber 110, or pooling channels 111; and/or to avoid clogging of particles near or within an aperture of any of the reaction channels 121, the pooling chamber 110, or the pooling channels 111. In some embodiments, the parameter "D" is varied between successive rows of hydrodynamic trapping elements, or successively between a predetermined number of rows of hydrodynamic trapping elements. For example, the parameter "D" may vary between any two adjacent rows of hydrodynamic trapping elements, either in the reaction channels, pooling chamber, and or pooling channels. In some embodiments, the parameter "D" may be iteratively varied until the parameter "D" roughly equals the parameter "G," i.e. until the lateral offset distance "D" is roughly equal to the gap "G" (see, e.g., FIG. 7B).

[0116] In some embodiments, the distance "D" may be iteratively reduced by a predetermined amount of an original distance "D" (e.g. by about 2pm) after a predetermined number of rows of hydrodynamic trapping elements. For example, rows 1 - 10 of a reaction channel, pooling chamber, or pooling channel may each include hydrodynamic trapping elements offset laterally by a distance of 2 about 4 pm; rows 11 - 20 may each include hydrodynamic trapping elements offset laterally by a distance of about 22 pm; rows 21 - 30 may each include hydrodynamic trapping elements offset laterally by a distance of about 20 pm; rows 31 -40 may each include hydrodynamic trapping elements offset laterally by a distance of about 18 pm; rows 41 - 50 may each include hydrodynamic trapping elements offset laterally by a distance of about 16 pm; rows 51 - 60 may each include hydrodynamic trapping elements offset laterally by a distance of about 14 pm; rows 61 - 70 may each include hydrodynamic trapping elements where the distance of about 12 pm; and rows 71 through 80 may each include hydrodynamic trapping elements where the distance D is about equal to the distance G.

[0117] Alternatively, in some embodiments, the distance "D" may be iteratively reduced by a predetermined percentage of an original distance "D" (e.g. by about 10% of an original distance "D") after a predetermined number of rows of hydrodynamic trapping elements. For example, rows 1 - 10 of a reaction channel, pooling chamber, or pooling channel may each include hydrodynamic trapping elements offset laterally by a distance "D," rows 11 - 20 may each include hydrodynamic trapping elements offset laterally by a distance ("D" - [20% * "D"]); rows 21 - 30 may each include hydrodynamic trapping elements offset laterally by a distance ("D" - [40% * "D"]); rows 31 - 40 may each include hydrodynamic trapping elements offset laterally by a distance ("D" - [60% * "D"]); rows 41 - 50 may each include hydrodynamic trapping elements offset laterally by a distance ("D" - [80% * "D"]); rows 51 - 60 may each include hydrodynamic trapping elements offset laterally by a distance ("D" - [90% * "D"]); and rows 61 - 70 may each include hydrodynamic trapping elements where the distance "D" is about equal the value "G."

[0118] In some embodiments, the reaction arrays 121, pooling chamber 110, or pooling channels 111 include one or more hydrodynamic trapping zones (210) (areas including a plurality of hydrodynamic trapping elements) and one or more free-flow zones (220) (areas free of hydrodynamic trapping elements) (see FIG. 7C). In some embodiments, the fluid flow in free-flow zones and in the hydrodynamic trapping zones and is laminar or streamlined and is therefore not generally conducive to trapping particles without optimizing the geometry of trap arrays. In some embodiments, an example of approaches to trap geometry optimization can be found in Biomicrofluidicics. 2013 Jan; 7(1): 014112, the disclosure of which is hereby incorporated by reference herein in its entirety. In some embodiments, efficient trapping occurs at zone boundaries (e.g. the transition from free-flow to trapping arrays) where the sudden appearance of obstacles locally changes flow characteristics from laminar to turbulent and the trapping is most efficient. Cascading free-flow zones and trapping zones introduces multiple zone boundaries and helps increase the trapping efficiency, i.e. maximize the occupancy of traps in the trapping arrays.

[0119] In some embodiments, each hydrodynamic trapping zone 210 is separated from another hydrodynamic trapping zone 210 by a free flow zone 220. In some embodiments, the free flow zones 220 are the same size as a hydrodynamic zone 210. In other embodiments, the free flow zones 220 are smaller than the hydrodynamic trapping zones 210, e.g. about 25% smaller than the hydrodynamic trapping zones 210, about 50% smaller than the hydrodynamic trapping zones 210, about 75% smaller than the hydrodynamic trapping zones 210, or about 90% smaller than the hydrodynamic trapping zones 210. In yet other embodiments, the free flow zones 220 are larger than the hydrodynamic trapping zones 210, e.g. about 25% larger than the hydrodynamic trapping zones 210, about 50% larger than the hydrodynamic trapping zones 210, about 75% larger than the hydrodynamic trapping zones 210, or about 90% larger than the hydrodynamic trapping zones 210. In some embodiments, each of the hydrodynamic trapping zones 210 are the same length, while the length of the free flow zones 220 is varied. In other embodiments, each of the free flow zones 220 are about the same length, while the length of the hydrodynamic trapping zones 210 is varied.

[0120] In some embodiments, each hydrodynamic trapping zone 210 comprises parallel rows of hydrodynamic trapping elements, where each row of hydrodynamic trapping elements are the same and spaced the same distance "D" from each other. For example, for a first hydrodynamic trapping zone, each of the first hydrodynamic trapping elements may be laterally offset by the same distance "Dl," regardless of the number of rows within the first hydrodynamic trapping zone. For a second hydrodynamic trapping zone, each of the second hydrodynamic trapping elements may be laterally offset by the same distance "D2," regardless of the number of rows within the second hydrodynamic trapping zone, where D2 is less than Dl. Likewise, for a third hydrodynamic trapping zone, each of the third hydrodynamic trapping elements may be laterally offset by the same distance "D3," regardless of the number of rows within the third hydrodynamic trapping, and where D3 is less than D2. In some embodiments, the lateral offset distance "D" may be iteratively reduced in each hydrodynamic trapping zone until the lateral offset distance "D" about equals the parameter "G." Following the above example further, for a fourth hydrodynamic trapping zone, each of the fourth hydrodynamic trapping elements may be laterally offset by the same distance "D4," regardless of the number of rows within the fourth hydrodynamic trapping, and where D4 is less about equal to "G." In addition, the parameter "D," the parameters "G," "H," and "R" may likewise be varied from zone to zone. [0121] As noted herein, the reaction channels may taper from a first end to a second end (see, e.g., FIGS. 5A and 5C). In some embodiments, the reaction channel widens from top to bottom such that each subsequent hydrodynamic trapping zone and each subsequent free flow zone becomes progressively wider while the distance "D" decreases. In some embodiments, the taper helps to ensure that the flow rate through the trapping array is about constant and the particles do not accelerate with the flow. In some embodiments, increasing the density of trapping arrays by reducing the distance between trapping elements also reduces the effective channel cross sections, making the fluid with particle flow faster, thereby increasing the risk that the fast moving particles will get wedged firmly in the trapping element, making it difficult to remove it during pooling step. In some embodiments, this effect can be eliminated by gradually widening the channel width as the distance between traps decreases to keep the effective flow cross-section about constant.

[0122] In some embodiments, the reaction channels 121, pooling chamber 110, and pooling channels 111 include stacks of hydrodynamic trapping elements.

For example, rather than all hydrodynamic trapping elements be arranged within one plane within a channel or chamber, layers of hydrodynamic elements may be positioned on top of each other (e.g. along a z-axis of the channel or chamber). In this manner, the quantity of hydrodynamic trapping elements within any channel or chamber may be increased.

[0123] Conduits

[0124] The microfluidic device of the present disclosure includes a plurality of conduits, including particle transfer conduits, fluid splitting conduits, particle splitting conduits, and fluid introduction conduits, as described further herein. In some embodiments, the conduits facilitate the transfer of fluids, reagents, and/or particles from one component of the microfluidic device to another. In general, the conduits of the present disclosure are in communication with at least one of a reservoir, a pump, a reaction channel, a pooling channel, or a pooling chamber. [0125] Particle Transfer Conduit

[0126] As noted herein, fluids and/or particles may be transferred from the pooling chamber 110 to the reaction array 120 and vice versa through a particle transfer conduit 130 in fluidic communication with both the pooling chamber 110 and the reaction array 120. In some embodiments, the particle transfer conduit 130 is in further fluidic communication with a particle storage vessel 150 and/or a pump 141. In some embodiments, the particle transfer conduit 130 is in fluidic communication with at least one particle splitting conduit 160 (described herein). In other embodiments, the particle transfer conduit 130 is in fluidic communication with two particle splitting conduits 160 and 161. Examples of particle transfer conduits and their arrangement relative to other components are illustrated in FIGS. 3, 4 A, and 4B.

[0127] In some embodiments, the particle transfer conduit 130 includes one or more valves which enable the flow of fluid and/or particles to be regulated between the pooling chamber 110 and reaction array 120, or any other components in fluidic communication therewith, e.g. pumps, reservoirs, or vessels fluidically coupled thereof. In some embodiments, the particle transfer conduit 130 includes a single 3-port valve 182, such that depending on the arrangement and movement of the valve (i) fluid and/or particles may be received into the particle transfer conduit 130 from the particle storage vessel 150; (ii) fluid and/or particles may be withdrawn from the particle transfer conduit 130 via pump 141 for collection and/or transfer to a collection vessel (not depicted); and/or (iii) fluid and/or or particles may be transferred between the pooling chamber 110 and the reaction array 120 (or any particle splitting conduits 160 and 161 disposed therebetween).

[0128] Particle Splitting Conduit

[0129] As noted above, the particle transfer conduit 130 is in fluidic communication with at least one particle splitting conduit 160 such that each of the individual reaction channels 121 of the reaction array 120 may be in fluidic communication with the particle transfer conduit 130 and/or any upstream components (particle storage vessels, particle collection vessels, pumps, etc.). In some embodiments, the particle transfer conduit 130 is in fluidic communication with two particle splitting conduits, e.g. a first particle splitting conduit 160 in fluidic communication with each of the reaction channels 121 of the reaction array 120 and a second particle splitting conduit 161 in fluidic communication with each of the pooling channels 111 of the pooling chamber 110.

[0130] In some embodiments, the particle splitting conduit 160 includes one or more binary splitting branches which serve to randomly divide the fluid flow from the particle transfer conduit 130 to each of the reaction channels 121. Similarly, the particle splitting conduit 161 includes one or more binary splitting branches which serve to randomly divide the fluid flow from the particle transfer conduit 130 to each of the pooling channels 111. In addition, the one or more binary splitting branches of the particle splitting conduits 160 and/or 161 serve to randomly divide the number of particles transferred to each reaction channel 121 (and/or each pooling channel 11) via the hydrodynamic fluid flow into each reaction channel 121 (and/or each pooling channel 111) such that the number of particles received by each reaction channel 121 (and/or each pooling channel 111) is substantially the same. In some embodiments, the one or more binary splitting branches serve to randomly divide the quantity of particles substantially equally at each level of branching. For example, assuming that a pool of 1000 particles are introduced into the particle transfer conduit 130, at the first branching, about 500 particles are transferred to each branch. Then, those about 500 particles are further randomly divided such that about 250 particles are transferred to each subsequent branch. This process is then repeated for each branching level of the particle splitting conduit 160 or 161 until the particles enter one of the reaction channels 121 and/or pooling channels 111.

[0131] As noted above, the particle splitting conduit 160 and/or 161 includes one or more binary splitting branches. In some embodiments, the particle splitting conduit 160 and/or 161 includes two binary levels of branching (e.g. a primary branch which is split into two secondary branches). In other embodiments, the particle splitting conduit 160 and/or 161 includes three binary levels of branching (e.g. a primary branch which is split into two secondary branches, and where each of the two second branches are split into two tertiary branches). In yet other embodiments, the particle splitting conduit 160 and/or 161 includes four binary levels of branching (e.g. a primary branch which is split into two secondary branches, and where each of the two second branches are split into two tertiary branches, and where each of the formed tertiary branches are further split to two quaternary branches). In further embodiments, the particle splitting conduit 160 and/or 161 includes five or more binary levels of branching.

[0132] In some embodiments, the number of branches and the number of binary branching levels of the particle splitting conduit 160 and/or 161 varies with the number and/or arrangement of reaction channels 121 within the reaction array 120 and/or with the number and/or arrangement of pooling channels 111 within the pooling chamber 110. For example, a particle splitting conduit 161 may include a primary branch and two secondary branches, as depicted in FIG. 4A. In other embodiments, and as depicted in FIG. 4 A, a particle transfer conduit 160 may include a primary branch, two secondary branches, and four tertiary branches.

[0133] The particle splitting conduits may optionally include one or more valves. The particle splitting conduits themselves may have any size and/or shape, e.g. rectangular, tubular, etc.

[0134] Fluid Splitting Conduits

[0135] In some embodiments, and as noted above, each of the reaction channels and each of the pooling channels (if present) are in communication with a fluid splitting conduit. In some embodiments, the fluid splitting conduit is adapted to divide the amount of fluid being infused or withdrawn by one or more pumps and/or fluid reservoirs in communication therewith and to equally distribute the divided fluid among the reaction channels 121 and pooling channels 111 (if present). In some embodiments, the fluid splitting conduit serves to transfer fluid to and from one or more pumps in fluid communication thereto, thereby facilitating inward and outward fluid flows from each of the reaction channels 121.

[0136] With reference to FIGS. 2 - 4, in some embodiments, each of the reaction channels 121 of the reaction array 120 is in fluidic communication with one or more pumps 142 and/or 143 via a fluid splitting conduit 165. Likewise, and with references to FIGS. 3 and 4, in some embodiments, each of the pooling channels 111 of the pooling chamber 110 is in fluidic communication with a fluid reservoir via a fluid splitting conduit 166. As with the particle splitting conduit 160 and/or 161, in some embodiments the fluid splitting conduit 165 and/or 166 includes one or more binary splitting branches. In some embodiments, the fluid splitting conduit 165 and/or 166 includes two binary levels of branching, three binary levels of branching, four binary levels of branching, five binary levels of branching, etc. In some embodiments, the number of branches and the number of binary branching levels of the fluid splitting conduit 165 and/or 166 varies with the number and/or arrangement of the reaction channels within the reaction array and/or with the arrangement of the pooling channels within the pooling chamber. In some embodiments, the fluid splitting conduit 165 and/or 166 includes one or more valves, e.g. one or more valves within a primary branch or one or more valves in one or more branches.

[0137] Microfluidic Chip Fabrication

[0138] The microfluidic chips of the present disclosure may be fabricated according to any method known to those of ordinary skilled in the art. Suitable methods of fabrication include lithography, 3D printing, laser etching, and embossing.

[0139] A microfluidic chip may be fabricated of any material suitable for forming a channel and/or conduit. Non-limiting examples of materials include polymers (e.g., polyethylene, polystyrene, polymethylmethacrylate, polycarbonate, poly(dimethylsiloxane), PTFE, PET, and a cyclo-olefin copolymer), glass, quartz, and silicon. The material forming the microfluidic chip and any associated components (e.g., a cover) may be hard or flexible. Those of ordinary skill in the art can readily select suitable material(s) based upon e.g., its rigidity, its inertness to (e.g., freedom from degradation by) a fluid to be passed through it, its robustness at a temperature at which a particular device is to be used, its transparency/opacity to light (e.g., in the ultraviolet and visible regions), and/or the method used to fabricate features in the material. For instance, for injection molded or other extruded articles, the material used may include a thermoplastic (e.g., polypropylene, polycarbonate, acrylonitrile-butadiene-styrene, nylon 6), an elastomer (e.g., polyisoprene, isobutene-isoprene, nitrile, neoprene, ethylene-propylene, hypalon, silicone), a thermoset (e.g., epoxy, unsaturated polyesters, phenolics), or combinations thereof.

[0140] The microfluidic chips disclosed herein are typically constructed by single and multilayer soft lithography (MLSL) techniques and/or sacrificial-layer encapsulation methods. The MLSL techniques are particularly useful in some embodiments for producing microfluidic devices which comprise both the control channel and the flow channel. In general, the MLSL technique involves casting a series of elastomeric layers on a micro-machined mold, removing the layers from the mold and then fusing the layers together. In the sacrificial-layer encapsulation approach, patterns of photoresist are deposited wherever a channel is desired. The use of these techniques to fabricate elements of microfluidic devices is described, for example, by Unger et al. (2000) Science 288:113-116; by Chou, et al. (2000) “Integrated Elastomer Fluidic Lab-on-a-chip-Surface Patterning and DNA Diagnostics, in Proceedings of the Solid State Actuator and Sensor Workshop, Hilton Head, S.C.; in PCT Publication WO 01/01025; and in U.S. patent application Ser. No. 09/679,432, filed Oct. 3, 2000, all of which are incorporated herein by reference in their entireties.

[0141] MLSL takes advantage of well-established photolithography techniques and advances in microelectronic fabrication technology. The first step in MLSL is to draw a design using computer drafting software, which is then printed on high-resolution masks. Silicon wafers covered in photoresist are exposed to ultraviolet light, which is filtered out in certain regions by the mask. Depending on whether the photoresist is negative or positive, either areas exposed (negative) or not (positive) will crosslink and the resist will polymerize. The unpolymerized resist is soluble in a developer solution and is subsequently washed away. By combining different photoresists and spin coating at different speeds, wafers can be patterned with a variety of different shapes and heights. The wafers are then used as molds to transfer the patterns to polydimethylsiloxane (PDMS). In MSL, stacking different layers of PDMS cast from different molds on top of each other is used to create channels in overlapping "flow" and "control" layers. The two (or more) layers are bound together by mixing a potting prepolymer component and a hardener component at complementary stoichiometric ratios to achieve vulcanization. In order to create a simple microfluidic chip, a "thick" layer is cast from the mold containing the flow layer, and the "thin" layer is cast from the mold containing the control layer. After partial vulcanization of both layers, the flow layer is peeled off the mold, and manually aligned to the control layer. These layers are allowed to bond, and then this double slab is peeled from the control mold, and then holes for inlets and outlets are punched and the double slab is bonded to a blank layer of PDMS. After allowing more time to bond, the completed device is mounted on glass slides. [0142] Reservoirs and Vessels

[0143] The microfluidic device 100 may be fluidically coupled to any number of reagent reservoirs, particle storage vessels, particle collection vessels, fluid reservoirs, waste collection reservoirs, etc. Each of the reservoirs may be fluidically coupled the microfluidic device 100 via a conduit. In some embodiments, each of the reservoirs include a valve such that the flow of fluids from the reservoir may be controlled. In some embodiments, the volume of a fluid reservoir ranges from between about 10pL to about lmL . In some embodiments, the volume of a fluid reservoir ranges from between about lmL to about lOmL. In some embodiments, the volume of a particle loading reservoir ranges from between about 10pL to about lmL. In some embodiments, the volume of a particle loading reservoir ranges from between about 100 pL to about lmL. In some embodiments, the volume of a particle collection reservoir ranges from between about 10pL to about lmL. In some embodiments, the volume of a particle collection reservoir ranges from between about lmL to about lOmL. [0144] In some embodiments, the microfluidic device includes a separate reagent reservoir 152 for each different reagent. In some embodiments, the number of reagent reservoirs 152 are equal to the number of reaction channels 121. In some embodiments, each different reagent reservoir 152 is in fluidic communication with a different reaction channel 121 via a separate reagent conduit 172. In some embodiments, each reagent conduit includes a valve 183, e.g. a 2-way valve, such that reagent may be withdrawn from a reagent reservoir 152 and flowed to a reaction channel 121 via the reagent conduit 172. In some embodiments, the volume of a reagent reservoir ranges from between about 10pL to about lOOpL. In some embodiments, the volume of a reagent reservoir ranges from between about 100 pL to about lmL. [0145] Fluidics Module

[0146] Fluid Introduction Conduit. Pumping Conduit and Reagent Conduits

[0147] The microfluidic device 100 may include any number of conduits to facilitate the transfer of fluids, reagents, and/or particles between any of the components of the microfluidic device. With reference to FIGS. 3, 4 A, and 4B, in some embodiments, the microfluidic device 100 includes a fluid introduction conduit

170, a pumping conduit 171, and a plurality of reagent conduits 172. In some embodiments, the fluid introduction conduit 170 facilitates the transfer of fluid from one or more fluid reservoirs 151 into the microfluidic device 100. In some embodiments, the fluid introduction conduit 170 is in direct fluidic communication with the pooling chamber 110 (see, e.g., FIG. 3). In embodiments where the pooling chamber 110 includes a plurality of pooling channels 111, the fluid introduction conduit 170 is indirectly in fluidic communication with the pooling chamber 110 via a fluid splitting conduit 166 (see, e.g., FIG. 4 A). In some embodiments, the fluid splitting conduit 166 may be configured similarly to fluid splitting conduit 165. In some embodiments, the fluid introduction 170 conduit includes one or more valves 181 depending on the number of fluid reservoirs 151 in communication with the microfluidic system 100. In some embodiments, the fluid introduction conduit 170 is in communication with a single fluid reservoir 151 and includes a single valve 181 (e.g. a 1-port valve). [0148] In some embodiments, the pumping conduit 171 facilitates the transfer of fluid between the reaction channels 121 and one or more pumps 142 and/or 143. In some embodiments, the pumping conduit 171 includes one or more valves 180 depending on the number of pumps 142 and/or 143 used to infuse or withdraw fluids from the microfluidic system 100. [0149] In some embodiments, the plurality of reagent conduits 172 facilitate the transfer of fluid between the reaction channels 121 and the one or more reagent reservoirs 152. In some embodiments, a separate reagent reservoir 152 and a separate reagent conduit 172 are in communication with each individual reaction channel 121 of the reaction array 120. In this manner, reagents from a single reagent reservoir may be transferred via a reagent conduit to one of the reaction channels such that the particles within that reaction channel may be reacted with just that reagent. In some embodiments, each of the reagent conduits 172 includes a valve, e.g. a 2-way valve, such that reagents may be flowed into the reaction channels 121.

[0150] Pumps

[0151] In some embodiments, the microfluidic device 100 is in fluidic communication with one or more pumps. In some embodiments, the microfluidic device is in fluidic communication with two pumps. In other embodiments, the microfluidic device is in fluidic communication with three pumps. In yet other embodiments, the microfluidic device is in fluidic communication with four or more pumps.

[0152] In some embodiments, the one or more pumps facilitate the movement of fluid, reagents, and/or particles within the chambers, channels, and/or conduits of the microfluidic device. Any pump may be utilized within the microfluidic device of the present disclosure provided that the pump selected allows for control of the volume loaded into or discharged from the microfluidic device. In some embodiments, the one or more pumps are pressure pumps. In other embodiments, the one or more pumps are piezo-electric pumps. In some embodiments, the one or more pumps are peristaltic pumps. In some embodiments, the one or more pumps are syringe pumps. In some embodiments, the one or more pumps are volumetric pumps.

[0153] In some embodiments, the one or more pumps of the present disclosure have a volume ranging from between about lmL to about lOmL. In other embodiments, the one or more pumps of the present disclosure have a volume ranging from between about lOmL to about lOOmL. In some embodiments, the one or more pumps of the present disclosure may deliver a flow rate of between about 1 pL/minute to about 1 OpL/minute. In other embodiments, the one or more pumps of the present disclosure may deliver a flow rate of between about 1 OpL/minute to about lOOpL/minute.

[0154] In some embodiments, the one or more pumps are syringe pumps. In some embodiments, the one or more syringe pumps have a volume of between about lmL to about lOmL and can deliver a flow rate of between about 1 pL/minute to about lOOpL/minute. Suitable syringe pumps are available from Chemyx (Stafford, TX), KD Scientific (Holliston, MA).

[0155] In some embodiments, each of the one or more pumps of the microfluidic device 100 are provided for a single purpose, e.g. infusing fluids, withdrawing fluids, or withdrawing particles. In other embodiments, any single pump may be used for multiple purposes. For example, one pump may facilitate both infusion and withdrawal of fluid.

[0156] In some embodiments, the microfluidic device is in communication with one or more of a "fluid injection pump," a "fluid withdrawal pump," and/or a particle withdrawal pump." A "fluid infusion pump," as used herein, refers to any device through which fluid may be introduced into a microfluidic device, including into any of the chambers, channels, or conduits of the microfluidic devices of the present disclosure. As such, a fluid infusion pump 142 can be used to deliver any fluid to any chamber, channel, and/or conduit and/or any particles included within the fluid may be moved from one component of the microfluidic device 100 to another through the actions of the fluid injection pump 142

[0157] A "fluid withdrawal pump," as used herein, refers to any device through which fluid may be removed from a microfluidic device, including from any of the chambers, channels, or conduits of the microfluidic devices of the present disclosure, or from any one or more of fluid reservoirs and/or reagent reservoirs in fluidic communication therewith. As such, a fluid withdrawal pump 143 can be used to remove any fluid or reagent from any chamber, channel, conduit and/or reservoir; and any particles included within the fluid may be moved from one component of the microfluidic device 100 to another through the actions of the fluid withdrawal pump 143.

[0158] A "particle withdrawal pump," as used herein, refers to any device through which fluid may be removed from a microfluidic device, including from any of the chambers, channels, or conduits of the microfluidic devices 100 of the present disclosure, or from any one or more of fluid reservoirs and/or reagent reservoirs in fluidic communication therewith. As such, a fluid withdrawal pump 141 can be used to remove any fluid or reagent from any chamber, channel, conduit and/or reservoir; and any particles included within the fluid may be moved from one component of the microfluidic device 100 to another through the actions of the fluid withdrawal pump 141.

[0159] In some embodiments, the one or more pumps are micropumps. In some embodiments, the micropumps are mechanical pumps (e.g. diaphragm micropumps and peristaltic micropumps). In some embodiments, the micropumps are non-mechanical pumps (e.g. valveless micropumps, capillary pumps, and chemically powered pumps). Devices are known for through pumping of small fluid quantities. For example, U.S. Pat. Nos. 5,094,594, 5,730,187 and 6,033,628 disclose devices which can pump fluid volumes in the nanoliter or picoliter range, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0160] Other pumps suitable for use with microfluidic devices are disclosed in U.S. Patent No. 10, 208,739; and in U.S. Publication Nos. 2015/0050172 and 2017/0167481, the disclosures of which are each hereby incorporated by reference herein in their entireties.

[0161] Valves

[0162] The microfluidic device 100 of the present disclosure may include one or more valves, such as valves positioned internal or external to the microfluidic chip. In some embodiments, the valves may be disposed within any conduit of the microfluidic device 100, with any portion of a conduit of the microfluidic device 100, or at a junction of any two conduits of the microfluidic device 100. In some embodiments, each of the valves of the microfluidic device 100 includes one or more ports, e.g. 1-port, 2-ports, or 3 -ports. Any type of valve may be utilized provided that the valve allows the flow of fluid, reagents, and/or particles throughout the microfluidic device 100 to be regulated, e.g. starting/ stopping fluid flow, controlling the quantities of fluid flow, etc. In some embodiments, the valves are controlled based on signals from a control system, e.g. the control system may command a valve to actuate to a first position, to a second position, or a third position such that fluid, reagent, and/or particle flow may be regulated.

[0163] Non-limiting examples of suitable microfluidic valves are described in U.S. Patent No. 10,197,188; in U.S. Patent Publication Nos. 2008/0236668 and 2006/0180779; and in PCT Publication No. WO/2018/104516, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, the microfluidic valves may be internal to the microfluidic chip.

[0164] Control System and Other Modules

[0165] The presently disclosed microfluidic devices are communicatively coupled to a control system 401. In some embodiments, the system may further include one or more pressure sensors, temperature sensors, flow rate sensors, and/or imaging modules. In some embodiments, the sensors may be coupled to the control system to permit feedback control of the microfluidic system.

[0166] In some embodiments, the systems of the present disclosure a control system 401 is used to send instructions to the various pumps and/or valves so as to regulate a fluid flow (e.g. direction of a fluid and/or reagent flow, a volume of fluid flow, or a flow rate) of any fluids and/or reagents passing through the microfluidic chip. In some embodiments, the control system 401 is configured to send instructions to actuate one or more valves to open or close, including one or more valves disposed in a conduit a or channel. In some embodiments, the control system is configured to send instructions to regulate the operation of one or more pumps in fluidic communication with the microfluidic chip, such as to cause the pump to infuse or withdraw fluids, reagents, and/or particles from the microfluidic chip 400. In some embodiments, the control module 401 may direct a first fluid flow in a first path, such that the flow may be ON when populating a plurality of hydrodynamic trapping elements, and the flow may be turned OFF during a transferring of particles in a second fluid flow in a second path.

[0167] In some embodiments, the control system is configured to receive data from an imaging module or sensor (e.g. a flow rate sensor, a temperature sensor, a pressure sensor, a chemical analyzer), process the received data, and regulate fluid a fluid flow, a temperature, a pressure, etc. based on the received and processed data. In some embodiments, feedback control involves the detection of one or more events or processes occurring in the present microfluidic systems. In some embodiments, detection may involve, for example, determination of at least one characteristic of a fluid, a component within a fluid, interaction between components within regions of the microfluidic chip, or a condition within a region of the microfluidic device (e.g., temperature, pressure, particle distribution, particle aggregation, etc.). By way of example, the control system 401, in some embodiments, is configured to execute a series of instructions to control or operate one or more system components to perform one or more operations, e.g. preprogrammed operations or routines, or to receive feedback from one or more sensor communicatively coupled to the system and command the one or more system components to operate (or cease to operate) depending on the sensor feedback received. In some embodiments, the one or more preprogrammed operations or routines can be performed by one or more programmable processors executing one or more computer programs to perform action, including by operating on received sensor feedback data or imaging data and commanding system components based on that received feedback.

[0168] The control system 401, in some embodiments, includes one or more memories and a programmable processor. To store information, the control system 401 can include, without limitation, one or more storage elements, such as volatile memory, non-volatile memory, read-only memory (ROM), random access memory (RAM), or the like. In some embodiments, the control system 201 is a stand-alone computer, which is external to the system. The storage and/or memory device can be one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some instances, the device is volatile memory and requires power to maintain stored information. In other instances, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In still other instances, the non-volatile memory comprises flash memory. The non-volatile memory can comprise dynamic random-access memory (DRAM). The non-volatile memory can comprise ferroelectric random access memory (FRAM). The non-volatile memory can comprise phase-change random access memory (PRAM). The device can be a storage device including, by way of non limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage.

[0169] In some embodiments, the control system 201 is a networked computer which enables control of the system remotely. The term "programmed processor" encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable microprocessor, a computer, a system on a chip, or multiple ones, or combinations, of the foregoing. The apparatus can include special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). The apparatus also can include, in addition to hardware, code that creates an execution environment for the computer program in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, a cross platform runtime environment, a virtual machine, or a combination of one or more of them. The apparatus and execution environment can realize various different computing model infrastructures, such as web services, distributed computing and grid computing infrastructures.

[0170] The system may comprise an imaging module. In some embodiments, the imaging module includes a microscope and a light source. In some embodiments, the imaging module includes a camera. The imaging module may permit a user to visualize one or more hydrodynamic traps, channels, conduits, or combinations thereof. In some embodiments, the imaging module may measure one or more parameters and deliver the measured one or more parameters to the control module (e.g. presence or absence of cells; presence or absence of one or more reagents; presence or absence of one or more labels). In that regard, the imaging module permits feedback control of the system of the present disclosure. For example, in the instance that particles are detected in an area beyond the last row of hydrodynamic trapping elements in any one reaction channel, the control system may send signals to one or more pumps and/or valves to alter a fluid flow rate, a fluid flow direction, etc.

[0171] In some embodiments, the microfluidic chip, reagent reservoirs, fluid reservoirs, and/or any conduits may be in communication with one or more heating and/or cooling modules. Suitable heating and/or cooling modules include heating blocks, Peltier devices, and/or thermoelectric modules. Suitable Peltier devices include any of those described within U.S. Pat. Nos. 4,685,081, 5,028,988, 5,040,381, and 5,079,618, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, the control system may be in communication with the one or more heating and/or cooling elements and command the heating and/or cooling elements to active and heat and/or cool the microfluidic chip, reagent reservoirs, fluid reservoirs, and/or conduits to a pre determined temperature for a pre-determined amount of time. For example, a control module may direct a supply of heat from at least one heating element to the microfluidic chip such that a predetermined temperature is reached and/or maintained. The pre-determined temperature may be input to the control system by a user or may be provided within pre-programmed instructions or routines.

[0172] In some embodiments, the system may further include one or more chemical analyzers. In some embodiments, the one or more chemical analyzers may be used to detect cellular components, reagents, byproducts, etc. within a collected waste stream.

[0173] In some embodiments, the microfluidic chip or any of the individual processing conduits may be in communication with one or more mixing modules. In some embodiments, the one or more mixing modules an acoustic wave generator, such as a transducer. In some embodiments, the transducer is a mechanical transducer. In other embodiments, the transducer is a piezoelectric transducer. In some embodiments, the transducer is composed of a piezoelectric wafer that generates a mechanical vibration. In some embodiments, a surface transducer is used to distribute or mix a fluid volume on-slide. Suitable devices and methods for contactless mixing and/or agitation are described in PCT Publication No. WO/2018/215844, the disclosure of which is hereby incorporated by reference.

[0174] In some embodiments, the system may be further coupled to a sequencing device for "next generation sequencing." The term "next generation sequencing" refers to sequencing technologies having high-throughput sequencing as compared to traditional Sanger- and capillary electrophoresis-based approaches, wherein the sequencing process is performed in parallel, for example producing thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (anywhere from 25 - 500 bp) but many hundreds of thousands or millions of reads in a relatively short time. The term "next- generation sequencing" refers to the so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms currently employed by Illumina, Life Technologies, and Roche etc. Next-generation sequencing methods may also include nanopore sequencing methods or electronic-detection based methods such as Ion Torrent technology commercialized by Life Technologies.

[0175] METHODS

[0176] The present disclosure is also directed to methods of using any of the microfluidic devices and/or microfluidic chips described herein for split-pool synthesis, e.g. split-pool barcoding and/or quantum barcoding. In some embodiments, the microfluidic devices 100 may be used in any method involving a split-pool step to label one or more particles or targets associated with particles present in a mixture of many like particles. In some embodiments, the particle may be a cell or a sub-cellular macromolecular entity.

[0177] In some embodiments, any of the microfluidic devices and/or microfluidic chips described herein may be configured to carry out any of the methods described in U.S. Patent No. 10,144,950 (including with any of the fluids and/or reagents there described), the disclosure of which is hereby incorporated by reference herein in its entirety. In some embodiments, any of the microfluidic devices and/or microfluidic chips described herein may be configured and/or operated to provide any of the uniquely labeled particles (e.g. cells) described in U.S. Patent No. 10,144950, e.g. a population of particles (e.g. cells) each uniquely labeled with a different series of assayable polymer subunits.

[0178] The present disclosure also provides methods of (i) randomly dividing a population of particles into multiple sub-populations, (ii) reacting each of the sub-populations with a different reagent, and then (iii) simultaneously pooling the reacted sub-populations back together. In some embodiments, these steps are repeated sequentially. In some embodiments, the sequential process may be repeated at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 12 times, at least 16 times, at least 20 times, at least 24 times, at least 28 times, at least 32 times, at least 36 times, at least 40 times, at least 44 times, at least 48 times, at least 56 times, at least 64 times, etc. Given that the process of dividing the particles into multiple sub-populations is random, each of the particles may be uniquely reacted over the course of the sequential and repetitive processing to provide a particle that includes a statistically unique chemical moiety, e.g. a statistically unique barcode, label, tag, nucleotide sequence, sequence of assayable polymer subunits, etc.

[0179] This concept is demonstrated by following two particles derived from a population of particles through a microfluidic device. Assume that particles 1 and 2 are divided after passing through a particle splitting conduit, where particle 1 passes into reaction channel 3 and particle passes into reaction channel 7. Trapped particles 1 and 2 may be reacted with reagents "A" and "B," respectively to provide PI -A and P2-B. These particles may then be pooled and again randomly split. Assume for a second cycle that particle 1 is passed into reaction channel 7 and particle 2 is passed into reaction channel 8. Trapped particles 1 and 2 may be reacted with reagents "B" and "C," respectively to provide Pl-A-B and P2-B-C. These particles may then be pooled and again randomly split. Assume for a third cycle that particle 1 is passed into reaction channel 1 and particle 2 is also passed into reaction channel 1. Trapped particles 1 and 2 may be reacted with reagents "D" and "D," respectively to provide Pl-A-B-D and P2-B-C-D. These particles may then be pooled and again randomly split. Assume for a fourth cycle that particle 1 is passed into reaction channel 1 and particle 2 is also passed into reaction channel 8. Trapped particles 1 and 2 may be reacted with reagents "D" and "C," respectively to provide Pl-A-B-D-D and P2-B-C-D-C. From this example, it is clear that as the particles are randomly split from the population and reacted, they may be modified such that they are distinguishable from each other.

[0180] While the methods described herein utilize, in some embodiments, the microfluidic devices and systems described herein, any type of microfluidic device and system capable of (i) randomly dividing a population particles, (ii) facilitating the independent reaction of the sub-populations of particles with a different reagent; and (iii) pooling the sub-populations of particles together, may be utilized. In some embodiments, the method is provided within a closed system thereby minimizing contamination.

[0181] In accordance with the foregoing, FIG. 9 depicts a method of retrieving a population of particles to be processed ("retrieving"), randomly dividing the retrieved population of particles into two or more sub-populations ("dividing"), reacting each formed sub-population of particles with a different reagent ("reacting"), pooling the reacted sub-populations of particles back together ("pooling"), and then collecting the reacted particles ("collecting"). Additional steps may be included within the method, such as steps of washing the reacted sub populations and or a step of imaging the trapped particles before and/or after reaction. In some embodiments, the processed depicted by FIG. 9 is performed using a microfluidic device, including any one of the microfluidic devices 100 of the present disclosure.

[0182] In some embodiments, a population of particles is first retrieved (step

310) and/or provided to a loading vessel in fluidic communication with a microfluidic device. In some embodiments, the population of particles includes cells and/or nuclei (or any combination thereof). In some embodiments, the particles have been pre-treated with one or more reagents to facilitate further reaction, coupling and/or hybridization of one or more moieties subsequently introduced reagents. In some embodiments, the particles have been pre-treated in accordance with the methods described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety. [0183] Subsequently, in some embodiments, the received population of particles are retrieved from the loading vessel and randomly divided into two or more sub-populations of particles (step 311). In some embodiments, each of the two or more sub-populations of particles are each flowed to (and housed within) a separate reaction channel in a microfluidic device such that each sub-population within each different reaction channel may be independently reacted with a different reagent. For example, all particles in a first sub-population may be reacted with a first reagent; while all particles in a second sub-population may be reacted with a second reagent.

[0184] In some embodiments, the random dividing of the population of received particles (step 311) includes passing the population of particles through a particle splitting conduit having a plurality of branches. In some embodiments, the passing of the population of received particles through the particle splitting conduit having the plurality of branches includes flowing the particles in a fluid (e.g. a buffer or other non-reactive liquid) through the particle splitting conduit. In some embodiments, as the population of received particles passes through each branch of the particle splitting conduit having the plurality of branches, the population of received particles is divided into two sub-populations of particles. In some embodiments, the particle splitting conduit includes two binary levels of branching, three levels of binary branching, four levels or binary branching, or five or more binary levels of branching. For example, if the population of received particles is passed through a particle splitting conduit having a single binary branch, the population of received particles will be divided into two sub-populations of particles.

[0185] In some embodiments, each of the particles of each of the two or more sub-populations of particles are temporarily and reversibly trapped within hydrodynamic traps provided within each reaction channel. In some embodiments, each of the particles of each of the two or more sub-populations of particles are trapped within the hydrodynamic traps by flowing a fluid (e.g. a buffer) in a first direction through the reaction channels, namely a direction of fluid flow into the separate reaction channels. In some embodiments, the particles remain reversibly trapped while the fluid flow is maintained in this direction. In some embodiments, each of the particles reversibly trapped within the hydrodynamic trapping elements are maintained in communication with one or more fluids and/or reagents introduced into the reaction channel.

[0186] Once the population of retrieved particles is randomly divided and flowed into each of the separate reaction channels, each sub-population of particles housed in each separate reaction channel is reacted with a different reagent (step 312). For example, the particles within a first sub-population in a first reaction channel may be reacted with a first reagent (e.g. a first oligonucleotide); while the particles within a second sub-population of in second reaction channel may be reacted with a second reagent (e.g. a second oligonucleotide). In some embodiments, each of the separate reaction channels housing each sub-population of particles is in communication with a different reagent reservoir. In some embodiments, each different reagent with each different reagent reservoir is independently transferred into one of the separate reaction channels, such as through a discrete reagent conduit. In some embodiments, the particles in each sub-population are allowed time to react (or incubate) with each of the introduced reagents, e.g. a time period ranging from between about one minute to about 60 minutes, from about one minute to about 30 minutes, or from about one minute to about 15 minutes. Suitable fluids and reagents (e.g. assayable polymer subunits) are further described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0187] In some embodiments, the reagent flowed to each of the reaction channels is at room temperature. In other embodiments, the reagent flowed to each of the reaction channels is at an elevated temperature, e.g. a temperature ranging from between about 25°C to about 100°C, a temperature ranging from between about 25°C to about 85°C, or a temperature ranging from between about 25°C to about 70°C. In yet other embodiments, the reagent flowed to each of the reaction channels is at a sub-ambient temperature, e.g. a temperature ranging from between about -5°C to about 15°C, a temperature ranging from between about 0°C to about 15°C. a temperature ranging from between about 5°C to about 10°C. In some embodiments, the reagent is heated or cooled within the reagent reservoir to a pre-determined temperature prior to being transferred to each separate compartment within the microfluidic device. For example, a thermoelectric heating and/or cooling element may be coupled to the reagent reservoir to effectuate cooling. In some embodiments, the temperature of the reagent transferred into each separate compartment is heated or cooled as it is transferred, e.g. each reagent conduit is in thermal communication with a heating element, such as a printed heated element.

[0188] In some embodiments, the reacted sub-populations are each optionally washed one or more times with a fluid, e.g. a wash fluid or a buffer, so as to remove any impurities and/or unreacted reagents. In some embodiments, the reacted sub-populations are washed once. In other embodiments, the reacted sub populations are washed twice (e.g. with the same or different fluids). In yet other embodiments, the reacted sub-populations are washed three or more times (e.g. with the same or different fluids). In some embodiments, the sub-populations of washed by flowing a fluid in a directed that maintains each particle trapped within a hydrodynamic trapping element.

[0189] Following the reaction of each sub-population of particles within a different reagent, each of the sub-populations of particles are pooled together (step 313). In some embodiments, the pooling together of the different sub-populations of particles comprises transferring each of the sub-populations from each of the separate reaction channels to a pooling chamber, e.g. a pooling chamber including 0 pooling channels, a pooling chamber including at least 2 pooling channels, a pooling chamber including at least 4 pooling channels, a pooling chamber including at least 8 pooling channels, etc. In some embodiments, the particles are randomly pooled together as they are flowed out of the separate reactions channels and through the particle splitting conduit toward the pooling chamber. In some embodiments, the transfer includes releasing the trapped particles from the hydrodynamic traps within each separate reaction channel. In some embodiments, the release of the trapped particles comprises flowing a fluid (e.g. a buffer) through the separate reaction channels in a direction opposite the fluid flow direction utilized to trap the particles within the hydrodynamic trapping elements.

[0190] In some embodiments, the flow of fluid and/or particles from each of the reaction channels 121 is monitored using an imaging device. In some embodiments, the flow of fluid and/or particles is monitored in real-time. In some embodiments, an imaging device may be used to determine if substantially all of the particles have been flowed out from the reaction channels 121 of the reaction array 120. If the reaction channels include any remaining particles, additional fluid may be flowed and/or the flow rate may be altered to ensure the transfer of those particles from the reaction channels. For example, the flow rate can be increased to exert larger hydrodynamic force on the remaining particles.

[0191] In some embodiments, the steps of dividing (step 311), reacting, reagent (step 312), optional washing, and pooling (step 313) are be repeated (step 314) a pre-determined number of times, e.g. two or more times, three or more times, four or more times, five or more times, 10 or more times, 15 or more times, 20 or more times, 40 or more times, 50 or more times, etc. For example, the pooled population of particles may be flowed from the pooling chamber and back through a particle splitting conduit where different sub-populations of particles are again randomly divided into different reaction channels and differentially reacted. Suitable fluids and reagents and methods for such differential reaction are further described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety. In some embodiments, an imaging device (e.g. a camera or microscope) may be used to determine if all of the particles have been flowed out from the pooling chamber 110 and back to the reaction channels. If the pooling chamber includes any remaining particles, additional fluid may be flowed and/or the flow rate may be altered to ensure the transfer of those particles from the pooling chamber to the reaction channels.

[0192] Once a pre-determined number of rounds (step 314) of processing have been performed, the pooled population of reacted particles is collected from the pooling chamber. The collected population of reacted particles may then be used in downstream operations. For example, each particle after having been processed may include a unique chemical moiety that may be detected such that each particle may be uniquely identified. By way of sample, each particle may include a unique concatemeric barcode sequence that may be sequenced, e.g. with next-generation sequencing, thereby facilitating single particle identification. [0193] Methods of flowing fluids, reagents, and/or particles through a microfluidic device, such as any one of the microfluidic devices of the present disclosure, are depicted in FIG. 10. In some embodiments, a population of particles is first retrieved (step 320) and/or provided to a loading vessel in fluidic communication with a microfluidic device. In some embodiments, the population of particles includes cells and/or nuclei (or any combination thereof). In some embodiments, the particles have been pre-treated with one or more reagents to facilitate further reaction, coupling, and/or hybridization of one or more moieties introduced reagents.

[0194] In some embodiments, the population of retrieved particles provided within the loading vessel is flowed from the loading vessel and through a particle splitting conduit in fluidic communication therewith to randomly divide the provided population of particles into two or more sub-populations of particles (step 321). In some embodiments, the population of particles is flowed through one or more binary splitting branches of a particle splitting conduit and into one of a plurality of reaction channels, where each binary splitting branch of the particle splitting conduit is in fluidic communication with one reaction channel. In some embodiments, the retrieved population of particles is randomly divided two times, four times, eight times, sixteen times, etc., depending on the levels of binary splitting branches within the particle splitting conduit. In some embodiments, each reaction channel is provided with one of the sub-populations of particles. In some embodiments, the particles are flowed in a buffer solution.

[0195] In some embodiments, the retrieved particles are flowed through the particle splitting conduit by withdrawing fluid from the microfluidic device through one or more pumps fluidically coupled to the microfluidic device, e.g. one or more fluid withdrawal pumps fluidically coupled to the microfluidic device. In some embodiments, the fluid is withdrawn by flowing fluid into and through a plurality of reaction channels in communication with the one or more binary splitting branches of the particle splitting conduit. In some embodiments, the inward flow of the fluid through the plurality of reaction compartments and from the particle splitting conduit facilitates the reversible trapping of particles within hydrodynamic trapping elements disposed within the reaction channels. As discussed herein, an outward flow of the fluid from the plurality of reaction compartments and toward the particle splitting conduit releases the trapped particles from the hydrodynamic trapping elements. In some embodiments, the flow of fluid and/or particles into each of the reaction channels 121 is monitored using an imaging device. In some embodiments, the flow of fluid and/or particles is monitored in real-time.

[0196] After the retrieved population of particles is randomly divided into sub-populations and provided within the separate reaction channels, the different sub-populations of particles within the different reaction channel are each independently reacted with different reagents (e.g. any of the reagents described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety). In some embodiments, the reactions are facilitated by flowing a different reagent (or even a mixture including components necessary to carry out a specific chemical reaction) into and through each of the different reaction channels (step 322). In some embodiments, each different reagent is maintained in a different reagent reservoir, where each reagent reservoir is coupled to one of the reaction channels via a reagent conduit. In some embodiments, a reagent is flowed inward into the reaction channels from each of the reagent reservoirs by withdrawing fluid and/or reagent from the microfluidic device through one or more pumps fluidically coupled to the microfluidic device, e.g. one or more fluid withdrawal pumps fluidically coupled to the microfluidic device. Suitable fluids and reagents (e.g. assayable polymer units) are further described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0197] In some embodiments, the particles in each sub-population are allowed time to react (or incubate) with each of the introduced reagents, e.g. a time period ranging from between about one minute to about 60 minutes, from about one minute to about 30 minutes, or from about one minute to about 15 minutes. In some embodiments, the reagent provided to each of the reaction channels is at room temperature. In other embodiments, the reagent provided to each of the reaction channels is at an elevated temperature, e.g. a temperature ranging from between about 25°C to about 100°C, a temperature ranging from between about 25°C to about 85°C, or a temperature ranging from between about 25°C to about 70°C. In yet other embodiments, the reagent flowed to each of the reaction channels is at a sub-ambient temperature, e.g. a temperature ranging from between about -5°C to about 15°C, a temperature ranging from between about 0°C to about 15°C. a temperature ranging from between about 5°C to about 10°C. In some embodiments, the reagent is heated or cooled within the reagent reservoir to a pre-determined temperature prior to being transferred to each separate compartment within the microfluidic device.

[0198] In some embodiments, one or more wash fluids and/or buffers are optionally flowed through each of the reaction channels to remove impurities and/or to remove unreacted reagent (step 323). In some embodiments, step 323 may be repeated one or more times, e.g. two times, three times, four times, or five or more times. In some embodiments, each of the repeated washes may be conducted with the same or different fluid. In some embodiments, the wash fluid or buffer is flowed inward into and through the reaction channels from one or more fluid reservoirs in communication with the microfluidic device by withdrawing fluid and/or reagent from the microfluidic device through one or more pumps fluidically coupled to the microfluidic device, e.g. one or more fluid withdrawal pumps fluidically coupled to the microfluidic device.

[0199] Following the flowing of one or more wash fluids and/or buffers into and through the reaction channels, a fluid is flowed through each of the reaction channels to transfer each of the reacted sub-populations of particles from the reaction channels and to a pooling chamber, allowing for the different sub-populations of particles may be pooled together (step 324). In some embodiments, the pooling of particles is randomly, such that the pooled particles are randomly distributed within the pooling chamber and/or within the pooling channels. For example, particles from four different reaction channels are randomly mixed during the pooling processes. In some embodiments, the flow of fluid through the reaction channels in the pooling step is in a direction opposite the flow of fluid used to introduce the particles into the reaction channels. In this manner, the flow of fluid for pooling facilitates the release of the particles trapped within the hydrodynamic trapping elements. In some embodiments, the fluid is flowed inward into the reaction channels and towards the particle splitting conduit by infusing the fluid into the reaction arrays using one or more pumps fluidically coupled to the microfluidic device, e.g. one or more fluid infusion pumps fluidically coupled to the microfluidic device. In some embodiments, the particles flow out of the reaction channels, through the one or more levels of binary splitting branches of the particle splitting conduit, and into the pooling chamber (and/or into one or more pooling channels disposed therein). As the particles are passed through at least the particle splitting conduit and toward the pooling chamber, the particles are randomly distributed.

[0200] In some embodiments, the steps of flowing a population of particles through a particle splitting conduit (step 321), flowing a different reagent through each of the separate reaction channels (step 322), flowing a washing fluid or buffer through each of the reaction channels (step 323), and flowing a buffer through each of the reaction channels to transfer the sub-populations to a pooling chamber (step 324) may be repeated (step 325) a pre-determined number of times, e.g. two or more times, three or more times, four or more times, five or more times, etc. For example, the pooled population of particles may be flowed from the pooling chamber (e.g. out of the pooling chamber) and back through a particle splitting conduit where the particle population is again randomly divided and where different sub-populations of particles are again provided into different reaction channels for reaction with different reagents.

[0201] Once all of the desired reactions have been run, the population of reacted particles are collected from the pooling chamber by flowing a fluid outward from the pooling chamber and into a collection vessel in communication with the microfluidic device (step 326). In some embodiments, the fluid is flowed outward from the pooling chamber by withdrawing fluid from the pooling chamber and into a collection vessel using one or more pumps fluidically coupled to the microfluidic device, e.g. one or more particle withdrawal pumps fluidically coupled to the microfluidic device. The population of reacted particles may then be used in downstream operations. For example, each particle after having been processed may include a unique chemical moiety that may be detected such that each particle may be uniquely identified. By way of sample, each particle, e.g. a cell or a nucleus, may include a unique concatemeric barcode sequence that may be sequenced thereby facilitating single particle identification.

[0202] FIGS. 8A - 8E illustrate a method of sequentially randomly dividing a population of particles into sub-populations (FIG. 8A), reacting each of the randomly divided sub-populations of particles with a different reagent (FIG. 8B), washing the reacted sub-populations of particles to remove excess reagent, impurities, or byproducts (FIG. 8C), pooling the reacted sub-populations of particles (FIG. 8D), and collecting the reacted population of particles (FIG. 8E). In some embodiments, the particles are cells or cell components. Each of the sequential steps are described herein. Of course, the steps illustrated in FIGS. 8 A - 8D may be repeated any pre-determined number of times such that the particles may be reacted a pre-determined number of times, e.g. 2 or more times, 4 or more times, 6 or more times, 10 or more times, 20 or more times, 40 or more times, 50 or more times, or 60 or more times. In some embodiments, the method facilitates the labeling of each cell or cell component with a statistically unique barcode (see, for example, the methods described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety).

[0203] In some embodiments, the particles are randomly divided and reacted a pre-determined number of times such that each particle may include a statistically uniquely identifiable chemical moiety, e.g. a concatemeric molecular barcode. While the steps described below utilize a microfluidic device having a particular configuration, any microfluidic device may be utilized to facilitate the reaction of the introduced particles with any series of reagents. Indeed, while FIGS. 8A through 8E depict the use of a specific microfluidic device, any microfluidic device may be adapted to carry out the steps of the method described in relation to FIGS. 8A to 8E.

[0204] FIG. 8A illustrates a first step of combinatorial reaction process, namely randomly dividing an introduced population of particles into a plurality of sub-populations ("particle dividing" step). With regard to the microfluidic device 100 depicted in FIG. 8 A, valve 180 and valve 182 are opened to permit fluid and/or particles to flow from the particle storage vessel 150, through a particle transfer conduit 130, and into a particle splitting conduit 160. By flowing the fluid and/or particles through the particle splitting conduit 160, the fluid and/or particles are randomly divided into a plurality of sub-populations. In some embodiments, the particle splitting conduit 160 includes one or more levels of binary splitting branches, such that when the flow of particles passes through a binary splitting branch, the flow of particles is divided in half. This process of dividing the flow of particles in half is repeated for each level of binary splitting branches included within the particle splitting conduit 160.

[0205] In some embodiments, the particles within each of the sub populations of particles become reversibly trapped within hydrodynamic trapping elements 200 provided within a plurality of reaction channels 121 as the fluid is flowed (i) out of the binary splitting branches of the particle splitting conduit 160, (ii) into and through the individual reaction channels 121, and (iii) out of the reaction channels 121 and through a fluid splitting conduit 165 (see the arrows in FIG. 8A illustrating the direction of fluid and/or particle flow). In some embodiments, the flow of fluid and/or particles in the particles dividing step is effectuated using a fluid withdrawal pump 143 in fluidic communication with the microfluidic device. In some embodiments, the fluid withdrawal pump 143 is in further communication with a waste collection vessel. During the particles dividing step, each of valves 183 remain closed such that no reagent is flowed from any of the reagent reservoirs 152 and through the reagent conduits 172 into the reaction channels. Likewise, valve 181 remains closed such that no additional fluid is received into the microfluidic device from a fluid reservoir 151.

[0206] Next, and with reference to FIG. 8B, valve 182 is closed and each of the vales 183 are opened. In some embodiments, each of the valves 183 are opened simultaneously. In this way, reagents are permitted to flow from the reagent reservoirs and into reaction channels 121. In some embodiments, as fluid and/or reagents are withdrawn from the microfluidic device using the fluid withdrawal pump 143, reagent is flowed from each of the reagent reservoirs 152 and into one of the reaction channels 121 (see the arrows in FIG. 8B illustrating the direction of fluid and/or reagent flow). In some embodiments, the particles trapped within the hydrodynamic trapping elements 200 within the reaction channels 121 are reacted with the reagents flowed from the reagent reservoirs, thereby providing reacted particles. In some embodiments, the particles are cells and/or nuclei, and the reaction is a ligation reaction. In some embodiments, the different reagents in each different reservoir includes a different assayable polymer submit.

[0207] Subsequently, and with reference to FIG. 8C, a wash fluid or buffer is flowed into and through the reaction channels to remove any residual reagent, impurities, and/or byproducts from the reaction channels 121. In some embodiments, a wash fluid and/or buffer is flowed through each of the reaction channels once. In other embodiments, a wash fluid and/or buffer is flowed through each of the reaction channels twice. In yet other embodiments, a wash fluid and/or buffer is flowed through each of the reaction channels three or more times. In some embodiments, the process of removing residual reagent, impurities, and/or byproducts is repeated using the same or different wash fluids and/or buffers. In some embodiments, valves 183 are each closed and valves 181 and 182 are opened to permit fluid of a fluid from a fluid reservoir 151 in communication with the microfluidic device. In some embodiments, fluid is flowed from the fluid reservoir 151, through the reaction channels 121, and into a waste collection vessel. In some embodiments, the flow fluid in this step is maintained through the use of a fluid withdrawal pump in fluidic communication with the microfluidic device (see the arrows in FIG. 8C illustrating the direction of fluid flow).

[0208] Next, the reacted particles in each of the reaction channels 121 are transferred from the reaction channels 121 and into a pooling chamber 110. In some embodiments, the transfer of the reacted particles from the reaction channels 121 and into the pooling chamber 100 is effectuated by reversing the fluid flow through the microfluidic device (compare the arrows indicative of fluid flow in FIGS. 8D and 8C). In some embodiments, valve 180 is configured to allow buffer to be infused into the microfluidic device using a fluid infusion pump 142 in fluidic communication with the microfluidic device. In some embodiments, the particles are then pushed from the reaction channels 121, through the particle splitting conduit 160, and into the pooling chamber 110. In some embodiments, the particle splitting conduit 160 facilitates the random distribution of the particles as they are transferred from the reaction channels to the pooling chamber 110. In some embodiments, the pooling chamber 110 may include two or more pooling channels 111 in fluidic communication with a particle splitting conduit 161 such that the particles are randomly divided into the two or more pooling channels 111. [0209] The processes depicted in FIGS. 8A through 8D may be repeated a pre-determined number of times, e.g. two or more times, four or more times, 6 or more times, 8 or more times, 12 or more times, 16 or more times, 20 or more times, 24 or more times, 36 or more times, etc. As depicted in FIG. 8E, once the pre determined number of reactions has been performed, the reacted particles may be collected. In some embodiments, valves 180, 181, and 182, are configured such that fluid and/or particles is permitted to flow from a fluid reservoir 151, through the pooling chamber 110, and into a particle collection vessel. In some embodiments, fluid flow is effectuated using a particle withdrawal pump 141 in fluidic communication with the microfluidic device (see the direction of the arrows in FIG. 8E).

[0210] In some embodiments, the particles introduced into the microfluidic chip are pre-sorted. For example, a received sample of particles may be sorted into a first population of particles and into a second population of particles, where the first and second populations of particles have different average diameters. [0211] In embodiments where the particles in a sample are cells, the cells may be sorted prior to any of the split-pool synthesis methods described herein. In some embodiments, tumor cells and normal cells may be pre-sorted prior to introduction to any microfluidic chip. In some embodiments, it is believed that normal cells have a size ranging from between about 4 pm to about 12 pm depending, of course, on the type of cell or the tissue in which the cell originated, and whether the tissue from which the cell originated was preserved, e.g. formalin-fixed a paraffin embedded. In some embodiments, normal cells isolated from formalin-fixed tissues have a size which ranges from between about 5 pm to about 12 pm. In yet other embodiments, normal cells from fixed tissue have a size which is less than 12 pm. [0212] In some embodiments, it is believed that tumor cells have a size ranging from between about 9 gm to about 100 gm depending, of course, on the type of cell or the tissue in which the cell originated, and whether the tissue from which the cell originated was preserved, e.g. formalin-fixed a paraffin embedded. In some embodiments, tumor cells isolated from fixed tissue have a size which ranges from between about 9 gm to about 20 gm. In other embodiments, tumor cells isolated from fixed tissue have a size which ranges from between about 9 gm to about 50 gm. In other embodiments, tumor isolated from fixed tissue cells have a size which ranges from between about 12 gm to about 25 gm. In yet other embodiments, tumors cells isolated from fixed tissue have a size which is greater than 12 gm.

[0213] In embodiments where the particles in a sample are cell nuclei, the nuclei may be sorted prior to any of the split-pool synthesis methods described herein. In some embodiments, tumor nuclei and normal nuclei may be pre-sorted prior to introduction to any microfluidic chip. In some embodiments, it is believed that normal nuclei isolated from fixed tissue have a size ranging from between about

4.5 gm to about 9 gm depending, of course, on the type of cell or the tissue in which the nuclei originated, and whether the tissue from which the nuclei originated was preserved, e.g. formalin-fixed a paraffin embedded. In other embodiments, normal nuclei have a size which ranges from between about 5 gm to about 8.5 gm. In yet other embodiments, normal cells have a size which is less than 8.5 gm. It is anticipated that normal nuclei isolated from fresh tissue may have a size range that is similar or slightly larger than those isolated from fixed tissue.

[0214] It is believed that tumor nuclei isolated from fixed tissue have a size ranging from between about 7.5 gm to about 20 gm depending, of course, on the type of cell or the tissue in which the nuclei originated, and whether the tissue from which the nuclei originated was preserved, e.g. formalin-fixed a paraffin embedded. In other embodiments, tumor nuclei have a size which ranges from between about

8.5 gm to about 20 gm. In other embodiments, tumor nuclei have a size which ranges from between about 9 gm to about 18 gm. In other embodiments, tumor nuclei have a size which ranges from between about 9.5 gm to about 15 gm. In yet other embodiments, tumors cells have a size which is greater than about 8.5 gm. [0215] Sorting of particles, including the sorting of cells and/or cell nuclei, may be accomplished using any upstream sorting device or process. Examples of suitable upstream sorting devices include deterministic lateral displacement devices, hydrophoretic filtration devices, hydrodynamic filtration devices, microfluidic devices utilizing inertial focusing in curved channels, and microfluidic devices utilizing inertial focusing in straight channels. Additional devices and methods of sorting particles, including cells and/or nuclei, are described in PCT Application No. PCT/EP2018/058809, the disclosure of which is hereby incorporated by reference herein in its entirety. [0216] The microfluidic devices of the present disclosure may be used to implement any of the processes described in the following references: U.S. Patent No. 10,144,950; A. M. Klein, L. Mazutis, I. Akartuna, N. Tallapragada, A. Veres, V. Li, et al. "Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells," Cell, Vol. 161, 1187-201, 20151; C. Trapnell, "Defining Cell Types and States with Single-Cell Genomics," Genome Res., Vol 25, 1491-1498, 2015; V. Svensson, K. N. Natarajan, L.-H. Ly, R. J. Miragaia, C. Labalette, I. C. Macaulay, et al. "Power Analysis of Single-Cell RNA-Sequencing Experiments," Nat. Methods, Vol. 14, 381-387, 2017; E. Z. Macosko, A. Basu, R. Satija, J. Nemesh, K. Shekhar, M. Goldman, et al. "Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets", Cell, Vol. 161, 1202-1214, 2015; A. B. Rosenberg, C. M. Roco, R. A. Muscat, A. Kuchina, P. Sample, Z. Yao, L. T. Graybuck, D. J. Peeler, S. Mukheijee, W. Chen, S. H. Pun, D. L. Sellers, B. Tasic, G. Seelig, "Single-Cell Profiling of the Developing Mouse Brain and Spinal Cord with Split-Pool Barcoding", Science, Vol. 360, 176-182, 2018; T. M. Gierahn, M. H. Wadsworth II, T. K. Hughes, B. D. Bryson, A. Butler, R. Satija, S. Fortune, J. C. Love, and A. K. Shalek, "Seq-Well: Portable, Low-Cost RNA Sequencing of Single Cells at High Throughput", Nat. Methods, Vol. 14, 395-398, 2017.

[0217] Additional Barcoding Embodiments

[0218] In some embodiments, the microfluidic devices 100 and/or microfluidic chips of the present disclosure facilitate a quantum barcoding process and, more specifically, facilitate one or more split-pool steps of a quantum barcoding process. In some embodiments, the microfluidic devices 100 and/or microfluidic child’s facilitate the assembly of a cell-originating barcode (COB) a particle, such as on a cell or a component of a cell, to which a unique binding agent (UB A) has bound. In some embodiments, the microfluidic devices 100 and/or microfluidic chips are configured to automate the split-pool synthesis process described herein. In some embodiments, the microfluidic devices 100 and/or microfluidic chips are adapted for pooling and splitting cell populations two or more times, such as described herein, to achieve the step-wise assembly of the code (COB). In some embodiments, the microfluidic devices 100 and/or microfluidic chips are configured to achieve suitable reaction conditions for any enzymatic and non-enzymatic steps of barcode assembly to occur, such as any of those processes described in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein. In some embodiments, the microfluidic devices 100 and/or microfluidic chips may be configured to provide a supply of buffers (such as from any of the reservoirs or vessels described herein) and may be adapted to achieve temperatures suitable for the enzymatic and non-enzymatic steps of assembling barcodes (COBs) from assayable polymeric subunits to occur. Suitable heating and/or cooling elements are described herein.

[0219] In some embodiments, the microfluidic devices 100 and/or microfluidic chips facilitate the quantum barcoding workflow. Briefly, the workflow involves contacting one or more specific agents, for example unique binding agents, with each particle in a population of particles (e.g. to each cell within a population of cells). By way of example only, a UBA can be an antibody and an entity can be a cell. In some embodiments, the process further includes the step of assembling a unique barcode characteristic of each particle (described as a cell-originating barcode (COB) herein) upon each specific agent bound to the entity. By way of example only, each of the one or more types of antibodies bound to the same cell will carry the same barcode characteristic of the cell. In some embodiments, the COBs are assembled from assayable polymer subunits (APSs) in the course of the QBC workflow as described herein and as set forth in U.S. Patent No. 10,144,950.

[0220] In some embodiments, unique binding agents (UBAs) bind to target molecules and serve as a site of assembly of barcodes using the microfluidic devices 100 and/or microfluidic chips of the present disclosure. Binding of the UBA to the target molecule may occur external to the microfluidic devices 100 and/or microfluidic chips described herein. In some embodiments, the microfluidic devices 100 and/or microfluidic chips comprises an optional upstream reaction chamber where UBA-target binding is to occur (e.g. an upstream chamber or vessel in fluidic communication with the microfluidic device 100 and/or microfluidic chip). In embodiments where the microfluidic device 100 includes a UBA-target binding chamber, the chamber is configured to supply a suitable buffer and further adapted to supply temperature and mechanical conditions (e.g., agitation) for the binding to occur. Any of the heating and/or cooling elements and/or transducers described here in may be utilized for this purpose.

[0221] In some embodiments, and as noted above, UBAs are molecules or molecular assemblies that bind at least one target molecule. Non-limiting examples of target molecules includes proteins, nucleic acids, lipids, carbohydrates, and drugs including large and small molecule drugs. Accordingly, and in some embodiments, a UBA may be an antibody, including IgA, IgG, IgM and components or fragments of antibodies that bind specifically to the target molecule. In some embodiments, the UBA is an aptamer. Aptamers include nucleic acid aptamers (i.e., single-stranded DNA molecules or single-stranded RNA molecules) and peptide aptamers. In some embodiments, aptamers bind target molecules in a highly specific, conformation- dependent manner, typically with very high affinity, although aptamers with lower binding affinity can be selected if desired. Aptamers can be designed and optimized using the SELEX process, see Gold, J. Biol. Chem., 270(23): 13581 84 (1995); S. Jayasena, Clin. Chem., 45:1628-50 (1999). In some embodiments, the UBA is a peptoid. Peptoids are short sequences of N-substituted glycines synthetic peptides that bind proteins. In some embodiments, small size peptoids improve diffusion and kinetics of the methods described herein. Any suitable method known in the art to generate peptoids can be used, see e.g., Simon et ah, PNAS 15: 89(20): 9367 — 9371 (1992), incorporated herein by reference. In some embodiments, the UBA is a nucleic acid (modified or unmodified DNA or RNA) complementary or at least partially complementary to the target nucleic acid (also DNA or RNA). [0222] In some embodiments, the microfluidic devices 100 and/or microfluidic chips of the present disclosure are adapted to facilitate the detection of multiple target molecules. In some embodiments, the present disclosure provides a UBA population for use in a multiplexed assay. Each UBA in the population is specific for a target molecule and two or more target molecules are detected. In some embodiments, two or more target molecules are detected and the target molecules are of the same kind, e.g., two or more protein targets. In other embodiments, two or more target molecules are detected and the target molecules are of different kinds, e.g., a protein target and a nucleic acid target (DNA or RNA). In each instance, multiple target molecules (of the same or different kinds) present in the cell will become associated with the cell-originating barcode (COB). Using the COB, the targets will be associated with the cell of origin as described herein.

[0223] In some embodiments, the UBAs include an identity portion termed an Epitope-Specific Barcode (ESB) that identifies the UBA. For example, specific nucleic acid UBAs (probes) can be identified by their sequence or a portion thereof and do not require a separate ESB. A non-nucleic acid UBA, e.g., an antibody UBA, or a peptide and some nucleic acid UBAs, e.g., an aptamer or a random nucleic acid UBA may comprise an Epitope-Specific Barcode (ESB) that enables identifying the UBA by nucleic acid sequencing. ESB can be a nucleic acid, e.g., an oligonucleotide. Each ESB comprises a unique code that can be associated to a specific target molecule. ESB can be conjugated to the protein UBA and can be made a 5’-part or a 3’-part of a nucleic acid UBA. In certain embodiments, the ESBs comprise common linker moiety, for example, a linker oligo to which a cell originating barcode (COB) can be assembled as described in the next section. Through attachment to the COB, the ESB can be read together with the COB.

[0224] In some embodiments, binding of the ESB to the UBA may occur outside of the microfluidic device 100 described herein. In some embodiments, binding of the ESB to the UBA may occur prior to exposing the UBA to the target. In some embodiments, the microfluidic device 100 includes an optional upstream reaction chamber where UBA-ESB binding is to occur. In such embodiments, the microfluidic device 100 includes an UBA-ESB binding chamber adapted to supply a suitable buffer and further configured to provide suitable temperature and mechanical conditions (e.g., agitation) for the binding to occur. Any of the heating and/or cooling elements and/or transducers described here in may be utilized for this purpose.

[0225] In some embodiments, the microfluidic devices 100 are configured to automate assembly of a cell origination barcode (COB). In some embodiments, each COB includes a unique code that can be associated with a specific entity of origin, e.g., a cell (or another macromolecular entity). In some embodiments, the COBs are modular structures including a plurality of different assayable polymer subunits (APS). In some embodiments, the APSs are attached in a linear combination to form a COB. In some embodiments, APSs and COBs include nucleic acids which can be sequenced with or without a prior amplification step. In some embodiments, detection of the COB sequence allows for the detection of the presence of the target molecule in the mixture (qualitative analysis). By way of example, when using fluorescent labels, a COB having a unique identity or unique spectral signature is associated with a UB A that recognizes a specific target molecule or a portion thereof. In some embodiments, detection of the COB signal, such as the spectral code of a fluorescently labeled COB allows detection of the presence of the target molecule in the mixture (qualitative analysis). Other examples of qualitative and quantitative detection of COBs are described in detail in U.S. Patent No. 10,144,950, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0226] In some embodiments, a COB may be assembled by stepwise addition of assayable polymer subunits (APSs) including, e.g., oligonucleotides. Any of the methods described herein of repeatedly and sequentially splitting, reacting, and pooling may be utilized to assembly any COB from any different APSs. In some embodiments, the COB can be attached to the UBA via a common linker (CL) to which the first APS is annealed or ligated. The assembly of COBs and their optional attachment to common linkers is described in detail in U.S. Patent No. 10,144,950. [0227] In some embodiments, the assembly of cell originating barcodes

(COBs) from assayable polymer subunits (APSs) involves a process of split-pool. In some embodiments, the microfluidic devices 100 are configured to pool and split the cells into reaction channels. In some embodiments, the microfluidic devices 100 are configured to introduce the sub-code subunits (APSs) to the cells in the "split" step. In this process, the sample is split into multiple reaction channels, and a different APS is flowed into each of the reaction channels. After binding of the APS to the growing COB, the split sample is pooled back together. In the next round, the sample is split again into multiple reaction channels and a different APS is flowed into each other reaction channels. In some embodiments, the microfluidic devices 100 are configured to provide conditions facilitating the subunit (APS) attachment to occur. Any of the methods described herein of repeatedly and sequentially splitting, reacting, and pooling may be utilized to assembly any COB from any different APSs. [0228] Methods of annealing and ligating APSs together to form a COB are described in U.S. Patent No. 10,144,950, the disclosure of which is incorporated by reference herein in its entirety. For example, each APS can be designed to selectively hybridize to an annealing region of an APS added during the previous round. Alternatively, APSs can anneal to an annealing primer added during each round and optionally be ligated together. In yet another alternative, all APSs can serially anneal to a single linker including multiple binding regions for APS specific to each round of annealing. In some embodiments, APSs are linked via CLICK chemistry, e.g., CLICK chemistry linkage of oligonucleotides, see, e.g. El-Sagheer et al. (PNAS, 108:28, 11338-11343, 2011). Many other variations of APS structure and methods of connecting APSs are described in in U.S. Patent No. 10,144,950.

[0229] An alternative method of assembling a COB from a series of APSs is described in a U.S. Application Serial No. 16/250,974, filed on January 17, 2019, the disclosure of which is hereby incorporated by reference herein in its entirety. Briefly, the UBA can comprise an anchor oligonucleotide to which a linker is annealed. APSs may then be annealed to the linker but instead of ligation, each APS is copied by extending the extendable end of the linker by a DNA polymerase. The assembled COB then comprises a copy of the series of annealed APSs. The APSs themselves may be optionally dissociated from the growing COB.

[0230] Each APS in a given round can comprise a unique sub-code sequence that is different from the rest of the APSs in that round. The sub-code may comprise a unique nucleotide sequence (code). Each assembled COB may comprise an additional barcode characteristic of the COB or characteristic of the sample. [0231] Some embodiments of the present disclosure relate to the assembly of COBs on the UBA molecules (e.g., antibody molecules) bound to targets on the surface of cells. COBs can, for example, be assembled associated with UBAs targeting cell surface components such as peptide epitopes of cell surface proteins. In other embodiments, UBAs are delivered into cells or into cellular compartments where targets are present, e.g., intracellular proteins, mRNA or DNA targets. In such embodiments, COBs are assembled associated with UBAs inside the cell. Cells may be fixed to facilitate one or both of UBA binding and COB assembly inside the cell. Many cell permeabilization methods are known in the art and can be used for this purpose.

[0232] In some embodiments, the quantum barcoding (QBC) procedure is performed on bodies that are not cells, including organelles and peptide assemblies or other macromolecular assemblies where a target molecule may be present. For example, the QBC procedure may be performed on MHC-antigen and MHC-ag-ab complexes.

Claims

Patent Claims

1. A microfluidic chip comprising: a pooling chamber, a reaction array comprising at least two reaction channels, and a particle transfer conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle transfer conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the particle transfer conduit.

2. The microfluidic chip of claim 1, wherein the particle transfer conduit comprises at least two levels of binary branching.

3. The microfluidic chip of claim 1, wherein the particle transfer conduit comprises at least three of binary branching.

4. The microfluidic chip of claim 1, wherein the pooling chamber includes at least two pooling channels.

5. The microfluidic chip of claim 1, wherein the at least two reaction channels each comprise a plurality of hydrodynamic trapping elements.

6. The microfluidic chip of claim 5, wherein the plurality of hydrodynamic trapping elements are configured to capture particles during a first fluid flow through the at least two reaction channels in a first direction; and wherein the plurality of hydrodynamic trapping elements are configured to release the captured particles during a second fluid flow through the at least two reaction channels in a second direction.

7. The microfluidic chip of claim 5, wherein the hydrodynamic trapping elements comprise at least two trapping members.

8. The microfluidic chip of claim 5, wherein the hydrodynamic trapping elements comprise at least three trapping members, wherein one of the at least three trapping members is offset longitudinally relative to another two of the at least three trapping members.

9. The microfluidic chip of claim 5, wherein the plurality of hydrodynamic trapping elements are arranged in substantially parallel rows within each of the reaction channels.

10. The microfluidic chip of claim 1, wherein the at least two reaction channels each comprise two or more hydrodynamic trapping zones, wherein each hydrodynamic trapping zone comprises substantially parallel rows of hydrodynamic trapping elements, wherein each of the two or more hydrodynamic trapping zones are separated by a free flow zone.

11. The microfluidic chip of claim 1, wherein the at least two reaction channels have a tapered shape.

12. The microfluidic chip of claim 1, further comprising a fluid introduction conduit, wherein the fluid introduction conduit terminates in one or more binary branches, and wherein each reaction channel is in fluidic communication with a branch of the one or more binary branches of the fluid introduction conduit.

13. A system comprising the microfluidic chip of any one of claims 1 - 12, wherein the system further comprises a fluidics module and a control system.

14. A population of uniquely labeled particles prepared using the system of claim 13.

15. A method of functionalizing particles with one or more reagents comprising:

(a) flowing a population of particles in a fluid through a particle splitting conduit to provide two or more sub-populations of particles, wherein each of the two or more sub-populations of particles comprises a random distribution of particles from the population of particles;

(b) flowing each sub-population of particles in the fluid through a different reaction channel towards a plurality of hydrodynamic trapping elements so as to independently capture each sub population of particles within one of the different reaction channels;

(c) flowing a different reagent through each different reaction channel so as to react each captured sub-population of particles with a different reagent; and

(d) flowing each of the sub-populations of reacted particles from the different reaction channels to a pooling chamber to form a pool of reacted particles.

16. A population of uniquely labeled particles prepared according to a process comprising:

(a) flowing a population of particles in a fluid in a first direction through a particle splitting conduit, wherein the particle splitting conduit comprises one or more levels of binary branching and wherein each of the one or more levels of binary branching of the particle splitting conduit is in fluidic communication with a reaction channel, wherein the flowing of the population of particles through the particle splitting conduit randomly divides the population of particles into two or more sub-populations of particles;

(b) flowing each sub-population of particles in the fluid through a different reaction channel toward a plurality of hydrodynamic trapping elements so as to independently capture each sub population of particles within one of the different reaction channels;

(d) flowing each of the sub-populations of reacted particles in a fluid from the different reaction channels through the particle splitting conduit and to a pooling chamber to form a pool of reacted particles; and

(e) repeating steps (a) through (e) a pre-determined number of times.

17. A use of the microfluidic chip of any of one claims 1 - 12 in split-pool synthesis, split-pool barcoding and/or quantum barcoding.

18. A substrate comprising a pooling chamber, a reaction array comprising at least two reaction channels, and a particle splitting conduit fluidically coupling the pooling chamber to the reaction array, wherein the particle splitting conduit comprises one or more levels of binary branching, and wherein each reaction channel is in fluidic communication with a branch of the one or more levels of binary branching of the particle splitting conduit; and wherein each of the at least two reaction channels are fluidically coupled with one branch of a fluid splitting conduit having one or more levels of binary branching.

19. A kit comprising the microfluidic chip of any one of claims 1 - 12 and a component selected from a group consisting of buffers, reagents, a chip for sequencing, or a chip for conducting digital droplet polymerase chain reaction.