Systems and methods for identifying biomolecule interactions

Abstract

Provided herein are methods and devices for a microfluidic platform aimed at the high-throughput discovery of allosteric drugs. An aspect provided herein is a microfluidic system comprising: one or more elongated reaction chambers; a plurality of reservoir chambers, wherein each reservoir chamber of the plurality of reservoir chambers is located on a same side of the elongated reaction chamber wherein each reservoir chamber of the plurality is connected in an angular orientation; one or more members positioned adjacent to the elongated reaction chamber and configured to move orthogonally to a surface of the reaction chamber; and a set of channels fluidically connecting at least two of the plurality of reservoir chambers to the reaction chamber. Another aspect provided herein is a method for operating an imaging system, comprising: providing a substrate comprising the microfluidic device, wherein the microfluidic device includes a plurality of channels, at least one channel comprising: one or more reaction chambers; one or more reservoir chambers fluidically coupled to the one or more reaction chambers; and one or more members positioned adjacent to the one or more reaction chambers and configured to move along an axis orthogonal to the one or more reaction chambers; and providing a transillumination subsystem comprising a light source and a detector, wherein the light source is configured to provide light to the microfluidic device, and wherein the detector is configured to detect at least a portion of the light.

Description

Attorney Docket No. 69414-702601SYSTEMS AND METHODS FOR IDENTIFYING BIOMOLECULE INTERACTIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 728,515 filed on December 5, 2024, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Active-site targeting drugs often fail during development due to issues like poor cell permeability or off-target toxicity, particularly for certain classes of enzymes, such as protein phosphatases. Despite their potential for therapeutic intervention in diseases like cancer and diabetes, protein phosphatases, for example, are often considered “undruggable” due to their highly charged and similar active sites. Current FDA-approved drugs for these targets are limited.SUMMARY

[0003] The present disclosure provides systems, devices, platforms, methods, and techniques for high-throughput therapeutic candidate screening. In some embodiments, the systems, devices, platforms, methods, and techniques are configured to identify various binding sites, encompassing both active and allosteric sites, discover binding molecules, and assess the functional effects of these molecules on their respective targets within a microfluidic environment. The instant disclosure provides devices and methods for a microfluidic platform for high-throughput therapeutic enzymology, identifying various binding sites, encompassing both active and allosteric sites, discovering binding molecules, and assessing the desired functional effects.

[0004] In one aspect disclosed herein is a method for identifying a candidate binding partner of a target biomolecule, the method comprising: (a) providing a device, wherein the device comprises a reaction chamber; (b) providing the target biomolecule and one or more candidate binding partners of the target biomolecule; (c) mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device; (d) subsequent to (c), tagging the target biomolecule, the candidate binding partners, or both with a barcode in the device; and (e) identifying the barcode, thereby identifying the target biomolecule, the candidate binding partner, additional information about an interaction between the target biomolecule and the one or more candidate binding partners, or any combination thereof.Attorney Docket No. 69414-702601

[0005] In some embodiments, the reaction chamber is amongst a plurality of reaction chambers, wherein the device comprises the plurality of reaction chambers. In some embodiments, the one or more candidate binding partners comprise an additional barcode that encodes a unique identifier corresponding to the candidate binding partner. In some embodiments, the one or more candidate binding partners comprises a small molecule. In some embodiments, the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule. In some embodiments, the one or more candidate binding partners interact with one or more binding sites of the target biomolecule. In some embodiments, the method further comprises identifying one or more allosteric binding sites of the target biomolecule. In some embodiments, the method further comprises inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site. In some embodiments, the method further comprises trapping a bound interaction between the target biomolecule and the one or more candidate binding partners, washing away unbound compounds, then eluting a solution from the reaction zone, thereby collecting the bound interaction. In some embodiments, the reaction chamber comprises micro-chambers having a volume configured for screening of a plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule, wherein the volume comprises between 0.5 nL and 50 nL.

[0006] In some embodiments, the method further comprises introducing single- or multimutant target biomolecules, including unnatural or extant allelic variants, to the target biomolecule, and assessing the effect of the introduced variants on identification of binding partners. In some embodiments, the method further comprises performing scanning mutagenesis on the target biomolecule to assess the effects on binding partner identification. In some embodiments, the method further comprises performing mutagenesis on the target biomolecule to localize binding sites of binding partners. In some embodiments, the method further comprises performing mutagenesis to introduce multiple mutations in the target biomolecule and assessing effects of the multiple mutations on binding partner identification. In some embodiments, the method further comprises performing a plurality of screens with a plurality of target biomolecules to identify a plurality of sets of binding partners of the plurality of target biomolecules, wherein the plurality of target biomolecules comprises theAttorney Docket No. 69414-702601 target biomolecule. In some embodiments, the plurality of target biomolecules comprises one or more variants of the target biomolecule.

[0007] In some embodiments, performing the plurality of screens comprises identifying variation amongst the plurality of sets of binding partners. In some embodiments, the method further comprises quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor. In some embodiments, the method further comprises administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, a target in vitro expression plasmid, a protein-identifying barcode, or any combination thereof.

[0008] In some embodiments, the method includes the expression and immobilization of the target biomolecule in the device. In some embodiments, the device further comprises an array configured to allow programming via microarray prints. In some embodiments, the method further comprises, prior to (b), disposing one or more nucleic acid molecules onto a surface adjacent to the device, wherein the one or more nucleic acid molecules encode the target biomolecule, the candidate binding partner, the barcode or any combination thereof. In some embodiments, the one or more nucleic acid molecules comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes the target biomolecule, wherein the second nucleic acid molecule encode the barcode, and wherein the barcode corresponds to the target biomolecule. In some embodiments, the first nucleic acid molecule is provided in a first chamber of the device, and wherein the second nucleic acid molecule is provided in a second chamber of the device, and wherein the first chamber of the device and the second chamber of the device are fluidically coupled to the reaction chamber. In some embodiments, the device further comprises an array configured to introduce a DNA barcode encoding the target biomolecule via microarray prints, wherein the printed DNA barcode is in a reservoir chamber adjacent to the DNA encoding of the target biomolecule and connected to a same reaction chamber. In some embodiments, the reservoir chamber is an elongated reservoir chamber. In some embodiments, the reaction chamber is an elongated reaction chamber. In some embodiments, the method includes expressing the target biomolecule or multiple proteins off the device, followed by immobilization of the target biomolecule or proteins on the device. In some embodiments, the method involves flowing on DNA-tagged small molecules onto the device. In some embodiments, the method involves flowing on DNA-tagged protein molecules onto the device. In some embodiments, the method further comprises attaching in situ barcodes to molecules on the device. In someAttorney Docket No. 69414-702601 embodiments, the method further comprises the elution of in situ barcoded small molecules. In some embodiments, the method further comprises the elution of in situ barcoded macro molecules, including DNA, RNA, and protein. In some embodiments, protein interactors are introduced by a microarray print. In some embodiments, the method further comprises using spatial barcoding to map interactors to their respective target biomolecule. In some embodiments, the method further comprises using temporal barcoding to tag reaction products based on the order of generation. In some embodiments, the method further comprises sequentially flowing a first library of interactors onto the reaction zone, thereby causing at least one interactor of the first library of interactors to bind, flowing a first barcode to label bound interactors, eluting the first library of interactors, subsequently flowing a second library of interactors onto the reaction zone, flowing a second barcode to label bound interactors, and eluting the second library of interactors, wherein each barcode corresponds to a specific library of interactors. In some embodiments, the method further comprises employing temporal barcoding to tag interactors based on the order of introduction to the chip. In some embodiments, barcoding is performed on-chip.

[0009] In another aspect disclosed herein is a microfluidic system comprising: (a) one or more reaction chambers comprising a reaction surface; (b) a plurality of reservoir chambers fluidically connected to the reaction chamber via a set of channels; (c) one or more members positioned adjacent to the reaction chamber and configured to move along an axis orthogonal to the reaction surface; and (d) a hydraulic control module configured to actuate the one or more members.

[0010] In some embodiments, each reservoir chamber of the plurality of reservoir chambers is located on a same side of the reaction chamber.

[0011] In some embodiments, at least one reaction chamber is an elongated reaction chamber. In some embodiments, at least one reservoir chamber of the plurality is an elongated chamber. In some embodiments, the set of channels fluidically connects at least two reservoir chambers of the plurality to the reaction chamber. In some embodiments, the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface. In some embodiments, the orthogonal movement of the member is configured to control fluidic mixing between at least one reservoir chamber of the plurality of reservoir chambers and one or more reaction chambers. In some embodiments, each of the plurality of reservoir chambers is oriented at an angle relative to at least one surface of a reaction chamber of the one or more reaction chambers. In some embodiments, the plurality of reservoir chambers has a denser packing efficiency compared to aAttorney Docket No. 69414-702601 microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, the denser packing efficiency is at least 1.75-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, a volume of at least one reaction chamber is at least 4.71-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, a member area of the reaction chamber is at least 2 to 10-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, at least one reservoir chamber of the plurality of reservoir chambers are reversibly separated from the one or more reaction chambers, or made contiguous with the one or more reaction chambers by a plurality of neck valves. In some embodiments, the microfluidic system further comprises micro-chambers with volumes between 0.5 nL and 50 nL, wherein the micro-chambers are configured to house incubations and reactions.

[0012] In some embodiments, the system is configured to simultaneously express and purify a plurality of proteins, including enzymes or variants thereof, wherein the plurality of proteins comprises from at least one to at least 1,000 proteins or variants thereof. In some embodiments, the system is configured to quantitatively assay all or a subset of proteins or protein variants simultaneously. In some embodiments, a second reservoir chamber is configured to introduce a unique molecular barcode for multiplexing across multiple protein variants, while maintaining isolation between chambers. In some embodiments, the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area. In some embodiments, the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8. In some embodiments, the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6. In some embodiments, the application of a stretch value of 600 microns to the elongatedAttorney Docket No. 69414-702601 reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4. In some embodiments, the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3. In some embodiments, the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 1200 chambers per chip. In some embodiments, the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 900 chambers per chip. In some embodiments, the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 700 chambers per chip. In some embodiments, the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 600 chambers per chip.

[0013] In another aspect disclosed herein is a method of identifying one or more binding partners of one or more target biomolecules, the method comprising: (a) providing a device, wherein the device comprises a plurality of reaction chambers on a surface, wherein the plurality of reaction chambers comprises one or more sets of reaction chambers; (b) providing a target biomolecule of the one or more target biomolecules and a plurality of candidate binding partners of the target biomolecule to a set of reaction chambers, wherein the surface is configured to display the one or more target biomolecules over at least 3% of the surface; and (c) identifying one or more binding partners of the target biomolecule from a subset of the plurality of candidate binding partners bound to the target biomolecule.

[0014] In some embodiments, the method further comprises (i) mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device, and (ii) generating one or more complexes comprising the target molecule and the subset of the plurality of candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 10,000 unique candidate binding partners. In some embodiments, the method does not comprise tagging the one or more complexes with a barcode in the device. In some embodiments, the plurality of candidate binding partners is configured to interact with two or more binding sites on the target biomolecule. In some embodiments, the one or more target biomolecules comprises one or more variants of the target biomolecule. In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during (b). In some embodiments, the method further comprises providing a second target biomolecule of the one or more target biomolecules and theAttorney Docket No. 69414-702601 plurality of candidate binding partners of the second target biomolecule to the additional set of reaction chambers.

[0015] In some embodiments, the method further comprises identifying one or more binding partners of the second target biomolecule from the plurality of candidate binding partners.

[0016] In some embodiments, the method further comprises identifying a difference between the one or more binding partners of the target biomolecule and the one or more binding partners of the second target biomolecule. In some embodiments, the method further comprises identifying one or more binding sites of the one or more binding partners. In some embodiments, the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing the target biomolecule and the second target biomolecule.

[0017] In some embodiments, the plurality of reaction chambers comprises at least 4200 reaction chambers. In some embodiments, the plurality of reaction chambers covers at least 30% of the surface. In some embodiments, the plurality of reaction chambers displays at least 0.3 pmol of protein. In some embodiments, (b) does not comprise providing the target biomolecule from a reservoir chamber on the surface adjacent to a reaction chamber of the set of reaction chambers.

[0018] In some embodiments, the method further comprises eluting the subset of the plurality of candidate binding partners from the one or more complexes. In some embodiments, the method further comprises providing the subset of the plurality of candidate binding partners to a set of reaction chambers on a different surface. In some embodiments, the method further comprises obtaining an unbound subset of the plurality of candidate binding partners, wherein the unbound subset of the plurality of candidate binding partners is not bound to the target biomolecule. In some embodiments, the method further comprises providing the unbound subset of the plurality of candidate binding partners to the set of reaction chambers. In some embodiments, the method further comprises expressing the one or more target biomolecules external to the device.

[0019] In some embodiments, the plurality of candidate binding partners comprises tags. In some embodiments, the tags comprise nucleic acids. In some embodiments, the plurality of candidate binding partners comprises small molecules. In some embodiments, the plurality of candidate binding partners comprises proteins. In some embodiments, the plurality of candidate binding partners comprises nucleic acids. In some embodiments, the target biomolecule comprises DNA, RNA, a protein, or a combination thereof.Attorney Docket No. 69414-702601

[0020] In some embodiments, the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule. In some embodiments, the method further comprises inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site. In some embodiments, the plurality of reaction chambers comprises reaction chambers having a volume between 0.5 nL and 50 nL.

[0021] In some embodiments, the method further comprises quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor. In some embodiments, the method further comprises administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, or any combination thereof.

[0022] In another aspect disclosed herein is a microfluidic system comprising: (a) a plurality of reaction chambers on a surface, wherein the surface is configured to display one or more target biomolecules over at least 3% of the surface; (b) one or more members positioned adjacent to one or more reaction chambers of the plurality of reaction chambers configured to move along an axis orthogonal to the surface; and (c) a hydraulic control module configured to actuate the one or more members.

[0023] In some embodiments, the plurality of reaction chambers covers at least 30% of the surface. In some embodiments, the plurality of reaction chambers comprises at least 4200 reaction chambers. In some embodiments, the plurality of reaction chambers displays at least 0.3 pmol of protein. In some embodiments, the plurality of reaction chambers comprises a set of reaction chambers, wherein reaction chambers of the set of reaction chambers are fluidly connected. In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are configured to not be fluidly connected to the set of reaction chambers at any point during use of the microfluidic system. In some embodiments, the plurality of reaction chambers is not connected to a reservoir chamber adjacent to a reaction chamber of the plurality of reaction chambers on the surface.

[0024] In some embodiments, the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface. In some embodiments, the plurality of reaction chambers comprises one or more reaction chambers having a volume between 0.5 nL and 50 nL. In some embodiments, the microfluidic system is configured toAttorney Docket No. 69414-702601 quantitatively assay the one or more target biomolecules, or a subset thereof, simultaneously. In some embodiments, at least one reaction chamber of the plurality of reaction chambers is an elongated reaction chamber. In some embodiments, the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area. In some embodiments, the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8. In some embodiments, the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6. In some embodiments, the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4. In some embodiments, the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3. In some embodiments, the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 6000 chambers per chip. In some embodiments, the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 4500 chambers per chip. In some embodiments, the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 3500 chambers per chip. In some embodiments, the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 3000 chambers per chip. In some embodiments, the surface has an area greater than 1100 mm2. In some embodiments, the surface has a dimension of 25 mm by 43 mm.

[0025] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.Attorney Docket No. 69414-702601BRIEF DESCRIPTION OF THE DRAWINGS

[0026] A better understanding of the features and advantages of the present subject matter will be obtained by reference to the following detailed description that sets forth illustrative embodiments and the accompanying drawings of which:

[0027] FIG. 1A illustrates an example of affinity selection processes.

[0028] FIG. IB illustrates an example of tandem forward and reverse screening workflows.

[0029] FIG. 1C illustrates an example of how mutational scanning identifies sites to screen.

[0030] FIG. 2A illustrates an example of valve states.

[0031] FIG. 2B illustrates an example of flow for a given valve state.

[0032] FIG. 3 illustrates an example of in situ barcoding.

[0033] FIG. 4 illustrates an example of the relative abundance of recovered vs loaded simulated DNA-encoded Libraries (DELs).

[0034] FIG. 5 illustrates an example of the process of target expression, in situ barcode ligation, interaction elution and sequencing, and target identification using barcodes.

[0035] FIG. 6 illustrates an example of how temporal barcoding reactions tag products based on the order of generation.

[0036] FIG. 7 illustrates examples of chip architectures.

[0037] FIG. 8A illustrates an example of a stretched chamber configuration.

[0038] FIG. 8B illustrates an example of the relationship between the stretch value and the number of chambers, as well as the fold change in chamber volume compared to a nonstretched configuration.

[0039] FIG. 8C illustrates an example of the relationship between the stretch value, the number of chambers and the change in button valve area compared to a non-stretched configuration.

[0040] FIG. 8D illustrates an example of the relationship between the stretch value, the number of chambers, and the total internal volume of the device.

[0041] FIG. 9A illustrates an example of an imaging setup.

[0042] FIG. 9B illustrates examples of fluidic control components.

[0043] FIG. 9C illustrates an example of a combined imaging and fluidic setup.

[0044] FIG. 10A illustrates an example of on-chip expression of two protein phosphatases.

[0045] FIG. 10B illustrates an example of on-chip protein expression using various miniprep kits and print buffer composition.

[0046] FIG. 10C illustrates an example of protein expression using printed plasmid on-chip.Attorney Docket No. 69414-702601

[0047] FIG. 11A illustrates an example of two human protein phosphatases (SHP2 and PTP1B).

[0048] FIG. 11B illustrates an example of reproducible, on-chip activity assays for two human protein phosphatases (SHP2 and PTP1B), measuring turnover number (£cat) across different experiments using the systems described herein.

[0049] FIG. 11C illustrates an example of reproducible, on-chip activity assays for two human protein phosphatases (SHP2 and PTP1B), measuring Michaelis constant (KM) across different experiments using the systems described herein.

[0050] FIG. 12 illustrates an example of an on-chip binding assay for identifying interactors (e.g., candidate binding partners), including the workflow and data analysis steps.

[0051] FIG. 13A illustrates an example of a device configuration for multiplexed DNA- Encoded Library (DEL) screening.

[0052] FIG. 13B illustrates an example of chamber-specific in situ barcoding of interactions.

[0053] FIG. 14 illustrates an example of a hydraulics valve control workflow.

[0054] FIG. 15A & FIG. 15B show examples of solenoid arrays operating with pressurized water.

[0055] FIG. 16 illustrates an example of an integrated optics and hydraulics setup.

[0056] FIG. 17A - FIG. 17C illustrate examples of an integrated optics and hydraulics setup with a controlling system.

[0057] FIG. 18A illustrates an example of a trans-illumination setup implemented in a macroscopy imaging system.

[0058] FIG. 18B illustrates the configuration and functionality of a trans-illumination setup with an emphasis on excitation and emission pathways for imaging in a macroscopy system.

[0059] FIG. 19 illustrates an example of light collimation and expansion, as well as filters for excitation and emission.

[0060] FIG. 20A illustrates an example of a device with chambers for plasmid DNA and barcode DNA pairs.

[0061] FIG. 20B illustrates example data of enrichment factors for tool compounds.

[0062] FIG. 20C illustrates example data of enrichment factors for a tool compound.

[0063] FIG. 21A illustrates an example of a device with chambers for plasmid DNA and barcode DNA pairs.

[0064] FIG. 21B illustrates example data of relative enrichment factors for tool compounds.

[0065] FIG. 22A illustrates an example of a device with chambers organized into distinct blocks and a schematic of the chambers in a block.Attorney Docket No. 69414-702601

[0066] FIG. 22B illustrates example data describing binding of members in a library pool.

[0067] FIG. 22C illustrates example data describing binding of members in a library pool compared to a control.

[0068] FIG. 23A illustrates an example of a device with chambers organized into distinct blocks.

[0069] FIG. 23B illustrates an example schematic of screening multiple targets across different blocks against a library.INCORPORATION BY REFERENCE

[0070] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.DETAILED DESCRIPTION

[0071] While preferred embodiments of the present subject matter have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present subject matter. It should be understood that various alternatives to the embodiments of the present subject matter described herein may be employed in practicing the present subject matter.

[0072] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0073] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.Attorney Docket No. 69414-702601

[0074] When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The terms “about” and “approximately,” as used herein, when preceding a numerical value indicates the value plus or minus a range of 10%. For example, about 10 may be reasonably understood to convey 9, 10, or 11, or a range of numerical values spanning from 9 to 11. Whenever “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, the term “about” or “approximately” applies to each of the numerical values in that series of numerical values.

[0075] As used herein, the terms "elongated" or "stretched" generally refer to a similar modification to the shape of a chamber, and these terms are used interchangeably throughout this application. Both terms, "elongated" or "stretched", denote that the chamber is altered or extended along one axis relative to its original shape, thereby leading to a structural expansion. A schematic representation of an "elongated" or a "stretched" shape can be found in FIG. 8A, which depicts a chamber in its stretched or elongated state compared to its original form.

[0076] As used herein, the terms "interactor" or "binding partner" generally imply a similar function and are used interchangeably. Both terms, "interactor" or "binding partner", denote molecules that interact or associate in some way with a biomolecule like a protein, leading to a biochemical interaction or connection.

[0077] As used herein, the term "on-chip" refers to activities taking place directly on or within a microfluidic device, such as a microfluidic chip. This is an in situ approach, implying that operations are performed within the same system where they function or where the results are needed. As used herein, the terms "system", "device", “microfluidic device” “microchip” or “chip” generally imply a similar function and are used interchangeably.

[0078] The present disclosure provides systems, devices, methods, and techniques configured for high-throughput screening, imaging, and the analysis of molecular interactions. Provided herein are systems and methods configured to provide numerous benefits, including identifying the binding partners of a target biomolecule. In some cases, the system comprises a device with a plurality of reaction chambers in which the biomolecule like a protein and potential binding partners are mechanically entrapped for assessment. In some instances, the candidate binding partner (e.g., interactor) is identified through a barcode (e.g., such as a DNA barcode). In some instances, barcodes that are present on the interactor prior to introduction to the device or introduced in situ may attach to the candidate binding partners prior to providing the candidate binding partners to a microchannel. In some embodiments,Attorney Docket No. 69414-702601 the barcodes that are present on the interactor prior to the introduction in the device can be introduced by means other than ligation. For example, barcode DNA can be covalently attached to the interactor through a variety of chemical reactions, such as with an NHS ester and a reactive amine. Polymerase Chain Reaction (PCR) is one such method, where barcodes are integrated into the primers used, and during amplification, they become part of the resultant DNA strands. Random Priming is another method, which involves attaching barcodes to random hexamer primers used to copy and barcode the total RNA or DNA content of a sample. The CRISPR / Cas9 gene-editing technology can be utilized to insert barcodes directly into genomic DNA. Transposon-Mediated Barcoding uses transposons, a type of genetic element, to insert barcodes randomly into DNA. Lastly, barcodes can be inserted via Cloning, where they are incorporated into a plasmid or other vector, which is then integrated into the genome.

[0079] In some embodiments, the devices provided comprise a two-layer microfluidic device configured for high throughput microfluidic enzyme kinetics such that users may express, purify and assay, for example, up to 2000 enzyme variants (e.g., mutants, homologs, fusion constructs, etc.) in two days. In some embodiments, the system is equally applicable and efficient for lower throughput, even with fewer than 1000 variants. In some embodiments, the microfluidic device comprises a number of chambers (for example, about 2000) that contain volume for massive parallelization (e.g., nanoliters, microliters, etc.). In some embodiments, the flow paths between and within these chambers (in the flow layer of the device) may be reversibly closed via a series of on-chip, push-down valves that are actuated by pressurizing fluidic paths directly above the flow channels (in the control layer of the device). The microfluidic device may be aligned and bonded to a glass slide that contains a printed grid containing a number of unique DNA spots corresponding to the number of chambers, encoding different enzyme variants (e.g., one variant per spot). For example, this alignment may enclose each DNA spot within an expression chamber (e.g., “reservoir” or “reservoir chamber”) and spatially links its sequence identity to its location on the chip. In some embodiments, all variants may be then expressed simultaneously by flowing cell-free in vitro transcription-translation (IVTT) mix into the expression chambers, which yields a single variant per chamber. For example, a deflectable member (called a button valve) may selectively mask parts of the chamber from reagents flowing through the device to selectively pattern specific regions of the device with an anti-GFP antibody. In further examples, utilizing the antibody patch under each member, expressed variants (configured to have aAttorney Docket No. 69414-702601GFP tag) may be then affinity-purified to program each reaction chamber with an enzyme variant.

[0080] In some embodiments, the microfluidic devices provided herein comprise about 100 microchambers to about 5,000 microchambers. In some embodiments, the microfluidic devices provided herein comprise about 100 microchambers to about 250 microchambers, about 100 microchambers to about 500 microchambers, about 100 microchambers to about 750 microchambers, about 100 microchambers to about 1,000 microchambers, about 100 microchambers to about 1,250 microchambers, about 100 microchambers to about 1,500 microchambers, about 100 microchambers to about 1,750 microchambers, about 100 microchambers to about 2,000 microchambers, about 100 microchambers to about 2,500 microchambers, about 100 microchambers to about 3,000 microchambers, about 100 microchambers to about 5,000 microchambers, about 250 microchambers to about 500 microchambers, about 250 microchambers to about 750 microchambers, about 250 microchambers to about 1,000 microchambers, about 250 microchambers to about 1,250 microchambers, about 250 microchambers to about 1,500 microchambers, about 250 microchambers to about 1,750 microchambers, about 250 microchambers to about 2,000 microchambers, about 250 microchambers to about 2,500 microchambers, about 250 microchambers to about 3,000 microchambers, about 250 microchambers to about 5,000 microchambers, about 500 microchambers to about 750 microchambers, about 500 microchambers to about 1,000 microchambers, about 500 microchambers to about 1,250 microchambers, about 500 microchambers to about 1,500 microchambers, about 500 microchambers to about 1,750 microchambers, about 500 microchambers to about 2,000 microchambers, about 500 microchambers to about 2,500 microchambers, about 500 microchambers to about 3,000 microchambers, about 500 microchambers to about 5,000 microchambers, about 750 microchambers to about 1,000 microchambers, about 750 microchambers to about 1,250 microchambers, about 750 microchambers to about 1,500 microchambers, about 750 microchambers to about 1,750 microchambers, about 750 microchambers to about 2,000 microchambers, about 750 microchambers to about 2,500 microchambers, about 750 microchambers to about 3,000 microchambers, about 750 microchambers to about 5,000 microchambers, about 1,000 microchambers to about 1,250 microchambers, about 1,000 microchambers to about 1,500 microchambers, about 1,000 microchambers to about 1,750 microchambers, about 1,000 microchambers to about 2,000 microchambers, about 1,000 microchambers to about 2,500 microchambers, about 1,000 microchambers to about 3,000 microchambers, about 1,000 microchambers to about 5,000Attorney Docket No. 69414-702601 microchambers, about 1,250 microchambers to about 1,500 microchambers, about 1,250 microchambers to about 1,750 microchambers, about 1,250 microchambers to about 2,000 microchambers, about 1,250 microchambers to about 2,500 microchambers, about 1,250 microchambers to about 3,000 microchambers, about 1,250 microchambers to about 5,000 microchambers, about 1,500 microchambers to about 1,750 microchambers, about 1,500 microchambers to about 2,000 microchambers, about 1,500 microchambers to about 2,500 microchambers, about 1,500 microchambers to about 3,000 microchambers, about 1,500 microchambers to about 5,000 microchambers, about 1,750 microchambers to about 2,000 microchambers, about 1,750 microchambers to about 2,500 microchambers, about 1,750 microchambers to about 3,000 microchambers, about 1,750 microchambers to about 5,000 microchambers, about 2,000 microchambers to about 2,500 microchambers, about 2,000 microchambers to about 3,000 microchambers, about 2,000 microchambers to about 5,000 microchambers, about 2,500 microchambers to about 3,000 microchambers, about 2,500 microchambers to about 5,000 microchambers, or about 3,000 microchambers to about 5,000 microchambers. In some embodiments, the microfluidic devices provided herein comprise about 100 microchambers, about 250 microchambers, about 500 microchambers, about 750 microchambers, about 1,000 microchambers, about 1,250 microchambers, about 1,500 microchambers, about 1,750 microchambers, about 2,000 microchambers, about 2,500 microchambers, about 3,000 microchambers, or about 5,000 microchambers. In some embodiments, the microfluidic devices provided herein comprise at least about 100 microchambers, about 250 microchambers, about 500 microchambers, about 750 microchambers, about 1,000 microchambers, about 1,250 microchambers, about 1,500 microchambers, about 1,750 microchambers, about 2,000 microchambers, about 2,500 microchambers, or about 3,000 microchambers. In some embodiments, the microfluidic devices provided herein comprise at most about 250 microchambers, about 500 microchambers, about 750 microchambers, about 1,000 microchambers, about 1,250 microchambers, about 1,500 microchambers, about 1,750 microchambers, about 2,000 microchambers, about 2,500 microchambers, about 3,000 microchambers, or about 5,000 microchambers.

[0081] In some embodiments, the microfluidic device may be aligned and bonded to a glass slide that contains a printed grid. This grid contains a number of unique DNA spots, each corresponding to the number of microchambers. Each spot may encode a different enzyme variant (e.g., one variant per spot) or multiple printed spots may encode the same enzyme variant. In some cases, the systems accommodate single or multi-mutant proteins, permittingAttorney Docket No. 69414-702601 an assessment of how these variations impact the identification of binding partners. In some instances, the method includes small molecules as candidate binding partners (e.g., interactors). Additional applications of the systems and methods provided include predicting compounds that may bind to various binding sites, encompassing both active and allosteric sites, using bioinformatics or other computational models. The systems and methods may be versatile and adaptable for multiple forward and reverse screens, and for other applications such as analyzing specific interactions between DNA sequences and transcription factor binding.

[0082] In some embodiments, the system may include microfluidic devices comprising an elongated chamber (e.g., “an elongated reaction chamber”), multiple elongated or nonelongated reservoir chambers, and connecting channels. In some embodiments, the microfluidic device’s configuration facilitates spatial and temporal barcoding for the identification and mapping of interactors to a protein variant of interest. The microfluidic device’s configuration may allow for high-throughput workflow, making the microfluidic device capable of handling large scale applications.

[0083] In some embodiments, a reaction chamber of the microfluidic device comprises a member or a structural component positioned adjacent to the reaction chamber and designed to move along an axis orthogonal to a reaction surface within the reaction chamber. The design of the member includes, but is not limited to, valves, buttons, or similar mechanisms configured to regulate flow, isolate the reaction zone, or manage interactions within the reaction chamber. In some embodiments, the member comprises a hydraulically actuated system for movement, a pneumatically or hydraulically actuated module using air or gas pressure, or a mechanically actuated mechanism, such as a spring, to return the member to its original position after actuation. Furthermore, the member enables applications such as isolating reaction zones, dynamically regulating reagent delivery, facilitating surface-based interactions, and providing precise control over the experimental environment in microfluidic or biochemical systems.

[0084] In some embodiments, the members described herein move orthogonally to a reaction surface within or adjacent to a reaction chamber. In some embodiments, the member comprises a member area comprising a specified region within or adjacent to a reaction chamber that houses a portion of the member configured to interact with the reaction surface. In some embodiments, the member area is configured to control access to the reaction chamber, regulate fluid flow, or isolate a corresponding portion of the reaction surface. The member situated within the member area can move along an axis orthogonal to the reactionAttorney Docket No. 69414-702601 surface, allowing for precise control of interactions between the reaction surface and member area. In some embodiments, the member area comprises a hydraulically controlled module for actuating the member with fluid power, a pneumatically controlled module for dictating member movement with air or gas pressure, or a mechanically controlled module that utilizes mechanisms like springs to return the member to its original position post-actuation. In some embodiments, the member area includes systems for fluidic regulation, isolation mechanisms to prevent cross-contamination, and configurations permitting sequential reagent delivery or sample collection. In some embodiments, the member area enables controlled interactions within the reaction chamber, facilitating high-precision reagent mixing, or dynamically managing fluid flow in microfluidic or biochemical workflows.

[0085] FIG. 2A illustrates an example of valve or member states. In some embodiments, the microfluidic device comprises a member configured to motion on an axis orthogonal to a surface within a microchamber of the microfluidic device to generate a valve state. In some embodiments, the valve states described in FIGs. 2A & 2B are example valve states of the microfluidic systems described herein. The figures depict representations of states that include elements denoting chambers, including reservoir chambers 1 and 2 and a reaction chamber. The figure also shows various valves such as neck valve 1 and 2, sandwich valve, and member (depicted as “button valve”), which may be in different states at different steps. These valves are shown in gray when closed (e.g., preventing fluid flow) and in a light color when open (e.g., permissive of fluid flow). The control and operation of these valves may be done using fluid that is pressurized by air or water. FIG. 2B illustrates an example of flow for a given valve state. In some embodiments, the microfluidic device may comprise fluid flow for a given valve state. The top-down view and front-on view show the direction of fluid through the reaction chambers or reservoir chambers in the respective steps. The control of the valves allowing, causing, or restricting the fluid flow within or between these chambers may be achieved using fluid that is pressurized by air or water.

[0086] In some embodiments, the methods of utilizing the systems and microfluidic devices described herein comprise providing the microfluidic device. In some embodiments, the microfluidic device comprises a plurality of reaction chambers. In some embodiments, the method further comprises providing a target biomolecule (or variant of interest thereof) and one or more candidate binding partners of the target biomolecule within the microfluidic device. In some embodiments, the method further comprises mechanically trapping the target biomolecule and one or more candidate binding partners of the target biomolecule between a surface of the microfluidic device, which is referred to as the chip itself, and a secondAttorney Docket No. 69414-702601 surface, which can include a surface like a glass slide or similar material, within the reaction chamber of the microfluidic device. In some embodiments, the method comprises tagging the one or more candidate binding partners, the target biomolecule, or both, with a proteinidentifying barcode subsequent to the mechanical trapping. In some embodiments, the method comprises tagging the one or more candidate binding partners, the target biomolecule, or both, in the device with a protein-identifying barcode subsequent to the mechanical trapping. In some embodiments, the method comprises identifying the proteinidentifying barcode on the one or more candidate binding partners of the target biomolecule, the target biomolecule, or both, to identify the protein that the one or more candidate binding partners interact with (or a specific binding site on the protein). In some embodiments, the one or more candidate binding partners comprise a small molecule. In some embodiments, the first surface or the second surface comprises a member configured to move on an orthogonal axis to a surface of a reaction zone. In some embodiments, one or more candidate binding partners are selected to interact with various binding sites, encompassing both active and allosteric sites of the target biomolecule. In some embodiments, the one or more candidate binding partners comprises a plurality of candidate binding partners that are randomly selected. In some embodiments, the one or more candidate binding partners comprises a plurality of candidate binding partners that are designed for testing against the target biomolecule of binding. In some embodiments, the one or more candidate binding partners comprises a plurality of candidate binding partners that are commercially available as a library. In some embodiments, one or more candidate binding partners are selected to interact with an allosteric binding site of the target biomolecule. In some embodiments, the method comprises identifying one or more allosteric binding sites of the target biomolecule. In some embodiments, an allosteric binding site of the protein may be inputted into a bioinformatics or other computational model to predict a compound or multiple compounds that bind to the allosteric binding site. In some embodiments, these compounds comprise biologies (e.g., proteins, antibodies, etc.) or small molecules.In some embodiments, the method comprises eluting a solution containing the target biomolecule (or variants thereof), one or more candidate binding partners, or both, from the reaction chamber after mechanically trapping at least a subset of the target biomolecule (or variants thereof), one or more candidate binding partners, or both, thereby removing one or more unbound binding compounds. In some embodiments, the method comprises rinsing a solution through the reaction chamber, thereby removing one or more unbound binding compounds. In some embodiments, the method comprises performing multiple forward andAttorney Docket No. 69414-702601 reverse screens simultaneously. In some embodiments, the reaction chamber comprises micro chambers (or “micro-chambers”) having a volume suitable for performing multiple screenings. In some embodiments, screenings on a plurality of proteins or variants can be performed simultaneously. In some embodiments, the method may be employed for screening of a plurality of proteins or variants thereof, the plurality ranging from at least one to at least 1,000 proteins or variants thereof. In some embodiments, the method comprises introducing single- or multi-mutant proteins having structural variants or extant allelic variants, to the target biomolecule, to evaluate additional binding sites for binding partners. In some embodiments, the method comprises performing scanning mutagenesis on the target biomolecule to assess the effects on binding site identification. In some embodiments, the method comprises performing mutagenesis on the target biomolecule to generate a library of mutant proteins known to exist naturally in the human population, to assess the effects on binding partner identification. In some embodiments, the method comprises performing mutagenesis to introduce multiple mutations in the target biomolecule to assess the effects on binding partner identification. In some embodiments, the method comprises performing a plurality of screens with a plurality of target biomolecules to identify a plurality of sets of binding partners of the plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule. In some embodiments, the plurality of target biomolecules comprises target biomolecules with different sequences, structures, or modifications (e.g., post-translational modifications or unnatural amino acids). In some embodiments, the plurality of target biomolecules comprises one or more variants of the target biomolecule. In some embodiments, the plurality of target biomolecules comprises at least 17 unique target biomolecules. In some embodiments, the one or more variants comprises a point mutation, an insertion, a deletion, or a combination thereof relative to the target biomolecule. In some embodiments, the one or more variants comprises a different post-translational modification (PTM), more PTM or fewer PTM relative to the target biomolecule. In some embodiments, performing the plurality of screens comprises identifying variation amongst the plurality of sets of binding partners. In some embodiments, the plurality of target biomolecules comprises multiple protein homologs to assess the specificity of binding partner interactions with the target biomolecule. In some embodiments, the method may be utilized for the identification of DNA sequence motifs for transcription factor binding. In some embodiments, the method may be utilized for analyzing interactions between DNA sequences and transcription factor binding. In some embodiments, the methodAttorney Docket No. 69414-702601 comprises counting bound sequences to deduce affinities or relative affinities for each sequence.

[0087] In some embodiments, the method comprises analyzing small molecule interactions with target biomolecules. In some embodiments, the method comprises analyzing antibody interactions with target biomolecules. In some embodiments, the method comprises analyzing the presence of certain proteins or peptides using mRNA display libraries. In some embodiments, the method comprises analyzing the presence of certain proteins or peptides using cDNA display libraries.

[0088] In some embodiments, the method further comprises preparing a chip for expression. In some embodiments, the method further comprises preparing a surface of the device for coupling biomolecules (e.g., DNA, RNA, peptides / proteins, etc.). The preparation can include treating or modifying the device surface in such a way that it promotes or allows the coupling of these biological molecules. In some embodiments, the DNA can be printed onto the device surface or onto a glass substrate in an initial step. In some embodiments, the device comprises a glass substate. Then it can be followed by the preparation of a target in vitro expression plasmid. In some embodiments, the method further comprises administering components that may include reagents such as biotinylated albumin (e.g., biotinylated Bovine Serum Albumin), a neutravidin, a biotinylated antibody (e.g., a biotinylated anti-green fluorescent protein antibody), and target in vitro expression plasmid, to a surface of the microfluidic device or reaction chamber.

[0089] In some embodiments, the method comprises expression and immobilization of target biomolecules on the microfluidic device. In some embodiments, the method comprises expression of the target biomolecule, or multiple proteins, off the microfluidic device, then immobilization of the protein or proteins on the microfluidic device. In some embodiments, the method comprises flowing DNA-tagged small molecules into the microfluidic device. In some embodiments, the method comprises flowing DNA-tagged peptide or protein molecules into the microfluidic device. In some embodiments, the method comprises flowing DNA- tagged antibodies into the microfluidic device. In some embodiments, the method comprises flowing RNA-tagged peptide or protein molecules into the microfluidic device.

[0090] In some embodiments, the method comprises attaching barcodes in situ (e.g., on the microfluidic device). In some embodiments, the method further comprises the elution of in situ barcoded molecules. In some embodiments, the method further comprises the elution of in situ barcoded interactors or proteins of interest. In some embodiments, protein interactors may be introduced to all reaction chambers from a common inlet. In some embodiments, theAttorney Docket No. 69414-702601 method comprises spatial barcoding that maps interactors to the target biomolecule. In some embodiments, the target biomolecule, in addition to the interactor (e.g., candidate binding partner), comprises a uniquely identifying barcode. In some embodiments, both the barcode ligated to the target biomolecule, and the barcode ligated to the interactor are determined to identify a binding site on the target biomolecule. In some embodiments, knowledge of the spatial position of the target biomolecule and the identity of the barcode ligated to the interactor determined after the binding reaction are used to identify a binding site on the target biomolecule.

[0091] In some embodiments, the method comprises temporal barcoding to tag reaction products based on the order of generation. In some embodiments, the method comprises temporal barcoding to tag reagents or reactants based on the order in which they were flowed onto the chip. In some embodiments, the method comprises temporal barcoding to tag binding partners that interact with the target biomolecule based on the order in which they were flowed onto the chip. In some embodiments, barcoding may be performed on-chip (in situ barcoding).

[0092] In some embodiments, interactors comprise small molecules. In some embodiments, the target biomolecule comprises unnatural amino acids. In some embodiments, the target biomolecule comprises post-translation modifications comprising additional chemical entities like phosphate, sugars, and ubiquitin protein.

[0093] In some embodiments, the systems described herein are configured to permit screening of a diverse range of library types against each other. This broad scope of screening capability extends to biomolecules such as peptides, proteins, DNA, and RNA. Not only limited to naturally occurring biomolecules, the systems described herein may also facilitate the screening of potentially bioactive synthetic molecules or macromolecules. Small molecules can also be included in the screening process. A large number of interactions can be screened in a relatively short amount of time, significantly improving the efficiency of current discovery processes. Moreover, the systems described herein may not be limited to studying interactions involving just two molecules. The systems described herein can be configured to analyze more complex interactions involving multiple molecules or molecular complexes, further increasing its versatility.

[0094] In some embodiments, the systems described herein may be utilized for screening libraries composed of derivatized biomolecules. In some embodiments, the derivatized biomolecules comprise post-translationally modified amino acids, unnatural amino acid incorporation, and synthetic post-translational modifications. In some embodiments, theAttorney Docket No. 69414-702601 primary targets for the screens comprise proteins or their variants. However, the platform is not limited to proteins and could be adapted for screening against other types of targets. In some embodiments, these targets are expressed off-chip or, in other scenarios, expressed in situ on the platform (e.g., within a chamber fluidically coupled to a reaction chamber of the microfluidic devices described herein). In some embodiments, the system comprises various classes of interactor tags. In some embodiments, the types of interactor tags comprise DNA, which is commonly used as a barcode, such as ligating barcodes onto DNA-encoded libraries (DELs). The systems described herein may not be limited to the use of DNA interactor tags. The systems described herein may also facilitate the use of other types of tags, such as RNA, peptides, and small molecules. Additionally, non-biological macromolecules, including synthetic polymers, may be used as interactor tags. In some embodiments, the identity of the interactor may be encoded by a tag embedded in an element of the interactor from the start, such as a DNA tag conjugated to the small molecule that contains a barcode uniquely identifying that small molecule. In some cases, the tag may be sequenced using nextgeneration short-read sequencing or nanopore sequencing. In other instances, hybridization or binding of specifically recognizing probes to the tag can occur, which may involve the use of microarrays or other multiplexed immunoassays. Additionally, the systems described herein can perform charge-to-mass or other biophysical assays such as chromatography and mass- spectrometry. The depth of interactor identity information recovery may vary on the systems described herein. In some embodiments, it may be qualitative, identifying whether an interactor is present or not. In other cases, it may be semi-quantitative, recovering the interactor affinity or abundance in eluate rank order. Moreover, it may be quantitative, recovering actual or scaled interactor counts. In some instances, measurements of absolute counts can yield relative or rank order binding affinities.

[0095] The systems described herein can accommodate a diversity of approaches, such as variations on how to introduce targets. Targets may be prepared off-chip and flowed on-chip, or prepared on-chip from plasmids, or prepared on-chip from linear amplicons. In other instances, interactors, which are typically prepared off-chip, may be flowed on-chip. Interactors may also be printed on-chip.

[0096] In some embodiments, the microfluidic system comprises an elongated reaction chamber, and a plurality of reservoir chambers, which may also be elongated. In some embodiments, each reservoir chamber of the plurality of reservoir chambers is located on the same side of the elongated reaction chamber. In some embodiments, the microfluidic system comprises a set of channels connecting at least two of the plurality of reservoir chambers toAttorney Docket No. 69414-702601 the reaction chamber. In some embodiments, a member disposed adjacent to the elongated reaction chamber is configured to move orthogonal to a surface of the elongated reaction chamber. In some embodiments, each reservoir chamber of the plurality of reservoir chambers is coupled to the elongated reaction chamber in an angular orientation (e.g., 90° angle to the elongated reaction chamber). In some embodiments, the plurality of reservoir chambers comprises a denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, the denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber may be about 1 to 11 fold.

[0097] In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 1 fold to about 11 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 1 fold to about 2 fold, about 1 fold to about 3 fold, about 1 fold to about 4 fold, about 1 fold to about 5 fold, about 1 fold to about 6 fold, about 1 fold to about 7 fold, about 1 fold to about 8 fold, about 1 fold to about 9 fold, about 1 fold to about 10 fold, about 1 fold to about 11 fold, about 2 fold to about 3 fold, about 2 fold to about 4 fold, about 2 fold to about 5 fold, about 2 fold to about 6 fold, about 2 fold to about 7 fold, about 2 fold to about 8 fold, about 2 fold to about 9 fold, about 2 fold to about 10 fold, about 2 fold to about 11 fold, about 3 fold to about 4 fold, about 3 fold to about 5 fold, about 3 fold to about 6 fold, about 3 fold to about 7 fold, about 3 fold to about 8 fold, about 3 fold to about 9 fold, about 3 fold to about 10 fold, about 3 fold to about 11 fold, about 4 fold to about 5 fold, about 4 fold to about 6 fold, about 4 fold to about 7 fold, about 4 fold to about 8 fold, about 4 fold to about 9 fold, about 4 fold to about 10 fold, about 4 fold to about 11 fold, about 5 fold to about 6 fold, about 5 fold to about 7 fold, about 5 fold to about 8 fold, about 5 fold to about 9 fold, about 5 fold to about 10 fold, about 5 fold to about 11 fold, about 6 fold to about 7 fold, about 6 fold to about 8 fold, about 6 fold to about 9 fold, about 6 fold to aboutAttorney Docket No. 69414-70260110 fold, about 6 fold to about 11 fold, about 7 fold to about 8 fold, about 7 fold to about 9 fold, about 7 fold to about 10 fold, about 7 fold to about 11 fold, about 8 fold to about 9 fold, about 8 fold to about 10 fold, about 8 fold to about 11 fold, about 9 fold to about 10 fold, about 9 fold to about 11 fold, or about 10 fold to about 11 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 1 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, or about 11 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 6.9 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 7 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by about 10 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at least about 1 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at least about 6.9 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on oppositeAttorney Docket No. 69414-702601 sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at least about 7 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at least about 10 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at most about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, or about 11 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at most about 6.9 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at most about 7 fold. In some embodiments, the elongated reaction chamber comprises a surface area greater than the reaction chamber of a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber by at most about 10 fold.

[0098] In some embodiments, the elongated reaction chamber comprises at least a 6 fold greater member surface area compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, the elongated reaction chamber comprises a 6.9- fold greater member area compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber. In some embodiments, the member is configured to move on an axisAttorney Docket No. 69414-702601 orthogonal to the surface of the elongated reaction chamber. In some embodiments, the reservoir chambers of the plurality of reservoirs are reversibly separated or made contiguous with the reaction chamber by two neck valves. In some embodiments, one or more reservoir chambers of the plurality of reservoir chambers is an elongated reservoir chamber.

[0099] In some embodiments, the microfluidic system further comprises micro-chambers with volumes of between about 0.1 nL to about 10 nL configured to perform incubations and reactions. In some embodiments, the microfluidic system comprises micro-chambers with volumes of between about 0.1 nL to 0.2 nL, about 0.2 nL to 0.3 nL, about 0.3 nL to 0.4 nL, about 0.4 nL to 0.5 nL, about 0.5 nL to 0.6 nL, about 0.6 nL to 0.7 nL, about 0.7 nL to 0.8 nL, about 0.8 nL to 0.9 nL, and about 0.9 nL to 1 nL, about 1 nL to 1.4 nL, about 1.4 nL to1.8 nL, about 1.8 nL to 2.2 nL, about 2.2 nL to 2.6 nL, about 2.6 nL to 3 nL, about 3 nL to3.4 nL, about 3.4 nL to 3.8 nL, about 3.8 nL to 4.2 nL, about 4.2 nL to 4.6 nL, about 4.6 nL to 5 nL, about 5 nL to 5.4 nL, about 5.4 nL to 5.8 nL, about 5.8 nL to 6.2 nL, about 6.2 nL to 6.6 nL, about 6.6 nL to 7 nL, about 7 nL to 7.4 nL, about 7.4 nL to 7.8 nL, about 7.8 nL to8.2 nL, about 8.2 nL to 8.6 nL, about 8.6 nL to 9 nL, about 9 nL to 9.4 nL, about 9.4 nL to9.8 nL, and about 9.8 nL to 10 nL. In some embodiments, the microfluidic system comprises micro-chambers with volumes of between about 0.6 nL and about 2.8 nL configured to perform incubations and reactions.

[0100] In some embodiments, the system comprises an array that allows for programming and / or control via microarray prints. In some embodiments, the system comprises a two-layer polydimethylsiloxane structure on top of a glass layer. In some embodiments, the system comprises integrated push-down, “Quake style” valves. Quake style valves may utilize additional channels, often perpendicular to the targeted microfluidic channels. The additional channels share a thin, common member with the targeted channels. When fluid at sufficient pressure is applied through the additional channels, the shared member is deflected and obstructs the flow of fluid (e.g., interaction between the member area and reaction surface as described herein). In some embodiments, the internal volume range of the device comprises about 0.1 pL to about 0.2 pL. In some embodiments, the range comprises about 0.2 pL to about 0.3 pL. In some instances, the range may vary from about 0.3 pL to about 0.4 pL, from about 0.4 pL to about 0.5 pL, or from about 0.5 pL to about 0.6 pL. Further, the internal volume range may span from about 0.6 pL to about 0.7 pL, from about 0.7 pL to about 0.8 pL, or from about 0.8 pL to about 0.9 pL. In some cases, the range comprises about 0.9 pL to about 1 pL. In additional embodiments, the internal volume range comprises about 1 pL to about 2 pL, from about 2 pL to about 3 pL, from about 3 pL to about 4 pL, or from about 4Attorney Docket No. 69414-702601 pL to about 5 pL. In other instances, the range may be from about 5 pL to about 6 pL, from about 6 pL to about 7 gL, from about 7 gL to about 8 gL, from about 8 gL to about 9 gL, or from about 9 pL to about 10 gL. In some embodiments, the internal volume range comprises about 10 pL to about 20 pL, from about 20 pL to about 30 pL, from about 30 pL to about 40 pL, or from about 40 pL to about 50 pL. Additionally, the range comprises about 50 pL to about 60 pL, from about 60 pL to about 70 pL, from about 70 pL to about 80 pL, from about 80 pL to about 90 pL, or from about 90 pL to about 100 pL.

[0101] In some embodiments, the system simultaneously expresses and purifies a plurality of proteins, including enzymes, or variants thereof, the plurality ranging from at least one to at least 1,000 proteins or variants thereof. In some embodiments, the system simultaneously expresses and purifies a plurality of proteins, including enzymes, or variants thereof, the plurality comprising more than 1,000 proteins or variants thereof. In some embodiments, the system assays all, or a subset of, proteins or protein variants at once. In some embodiments, when members are open, the fluid flows through all reaction chambers in the device. In some embodiments, when the members are closed, the fluid flows within all the reaction chambers and around the volume partitioned off by the member without disturbing variants and their interactors immobilized between the member area and the reaction surface of a microfluidic device.

[0102] In some embodiments, when sandwich valves are closed, individual chambers become their own unique environments, thereby permitting high-throughput workflow. In some embodiments, the introduction of one or more reservoir chambers allows for multiplexing multiple protein variants by introducing a unique molecular barcode in situ while the reaction chambers are isolated from each other. In some embodiments, the barcode is a DNA barcode. In some embodiments, the DNA barcode is printed in a reservoir chamber connected to a reaction chamber. In some embodiments, another reservoir chamber comprising DNA encoding a protein is adjacent to the reservoir chamber comprising the DNA barcode and is connected to the same reaction chamber. In some embodiments, the printed DNA barcode is configured to uniquely identify the protein variant.

[0103] In some embodiments, the reaction chamber may accommodate variable stretch values leading to corresponding chamber volume and member area fold changes. In some embodiments, the reaction chamber may be designed to accommodate variable stretch values ranging from about 0 to about 1000 microns, leading to corresponding changes in a number of chambers ranging from about 100 to about 2000 and a chamber volume fold-change over the non-stretched design from about 0.1 to about 10. In some embodiments, the stretch valueAttorney Docket No. 69414-702601 may range from about 0 to about 10 microns, from about 10 microns to about 20 microns, from about 20 microns to about 30 microns, from about 30 microns to about 40 microns, from about 40 microns to about 50 microns, from about 50 microns to about 60 microns, from about 60 microns to about 70 microns, from about 70 microns to about 80 microns, from about 80 microns to about 90 microns, from about 90 microns to about 100 microns, from about 100 microns to about 200 microns, from about 200 microns to about 300 microns, from about 300 microns to about 400 microns, from about 400 microns to about 500 microns, from about 500 microns to about 600 microns, from about 600 microns to about 700 microns, from about 700 microns to about 800 microns, from about 800 microns to about 900 microns, and from about 900 microns to about 1000 microns.

[0104] In some embodiments, a number of chambers comprises about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 1000 to about 1100, from about 1100 to about 1200, from about 1200 to about 1300, from about 1300 to about 1400, from about 1400 to about 1500, from about 1500 to about 1600, from about 1600 to about 1700, from about 1700 to about 1800, from about 1800 to about 1900, and from about 1900 to about 2000.

[0105] In some embodiments, the chamber volume fold-change over the non-stretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10.

[0106] In some embodiments, the button valve area fold-change over the non-stretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10.

[0107] In some embodiments, the total internal volume of the device comprises about 0.1 to about 0.2 (*0.6 pL), from about 0.2 to about 0.3 (*0.6 pL), from about 0.3 to about 0.4 (*0.6 pL), from about 0.4 to about 0.5 (*0.6 pL), from about 0.5 to about 0.6 (*0.6 pL), from about 0.6 to about 0.7 (*0.6 pL), from about 0.7 to about 0.8 (*0.6 pL), from about 0.8 to about 0.9 (*0.6 pL), from about 0.9 to about 1.0 (*0.6 pL), continuing in this manner with a step size of 0.1 up to about 9.9 to about 10 (*0.6 pL).Attorney Docket No. 69414-702601

[0108] In some embodiments, stretching a reaction chamber by a specified stretch value increases the internal volume and surface area of the microfluidic device (e.g., see FIG. 8A- 8D). In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2 and a member area fold change of about 3 over the non-stretched design. In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2.1 and a member area fold change of about 2.8 over the non-stretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3 and a member area fold change of about 5 over the non-stretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3.25 and a member area fold change of about 4.6 over the non-stretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4 and a member area fold change of about 7 over the non-stretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4.4 and a member area fold change of about 6.45 over the non-stretched design. In some embodiments, a stretch value of 800 microns generates a chamber volume fold change of about 5 and a member area fold change of about 8 over the non-stretched design. In some embodiments, a stretch value of 800 microns generates a chamber volume fold change of about 5.5 and a member area fold change of about 8.28 over the non-stretched design.

[0109] In some embodiments, a stretch value of 200 microns generates a configuration of approximately 1200 chambers per chip. In some embodiments, a stretch value of 400 microns generates a configuration of approximately 900 chambers per chip. In some embodiments, a stretch value of 600 microns generates a configuration of approximately 700 chambers per chip. In some embodiments, a stretch value of 800 microns may lead to a configuration of approximately 600 chambers per chip.

[0110] FIG. 8A provides an example of a schematic of a chamber within a microfluidic device exhibiting a stretched configuration. The microfluidics device can comprise chambers with a stretched configuration. In some cases, the chamber has a width of 225 microns. FIG. 8B provides a non-limiting example of a plot comparing the stretch value in microns against the number of chambers and the chamber volume fold change over a non-stretched configuration. It may be inferred that a greater stretch value may lead to a smaller number of chambers but an increased volume fold change over a non-stretched configuration. FIG. 8C provides a non-limiting example of a plot comparing stretch value in microns versus the number of chambers and the member area fold change over a non-stretched configuration. In some cases, a greater stretch value leads to a smaller number of chambers but an increasedAttorney Docket No. 69414-702601 member area fold changes over a non-stretched configuration. FIG. 8D provides a nonlimiting example of a plot comparing the stretch value in microns versus the number of chambers sets and the total internal volume of the device. As illustrated in FIG. 8D, in some cases, a greater stretch value may result in a smaller number of chambers sets but an increased total internal volume of the device.

[0111] FIG. 7 illustrates examples of chip architecture. The top-down view of the flow layer of the microfluidic device shows reservoir chambers with the same volume not stretched, including reservoir chamber 1 and 2 connected to one reaction chamber. Alternatively, the illustrated view shows reservoir chambers with different volumes, one or both stretched. The top-down view of the control layer of the microfluidic device displays member, sandwich valve, neck valve 1 and neck valve 2.

[0112] FIG. 13A illustrates an example of a device configuration for multiplex DNA- Encoded Library (DEL) screening. In the example, more than one protein (e.g., enzyme variants) can be displayed on the device, each bound onto separate areas (e.g., reaction chambers) of the device. Barcodes can be introduced from a reservoir chamber adjacent the area for bound protein to tag a fraction of the DEL bound to the protein displayed in the area.

[0113] In another aspect disclosed herein are methods for identifying one or more binding partners of one or more target biomolecules. In some embodiments, the method comprises (a) providing a device. The device can be referred to as a microfluidic device or as a chip. The device can comprise a plurality of reaction chamber on a surface. The plurality of reaction chambers can comprise one or more sets of reaction chambers. In some embodiments, the method comprises (b) providing a target biomolecule of the one or more target biomolecules and a plurality of candidate binding partners of the target biomolecule to a set of reaction chambers. In some embodiments, the method comprises providing a target biomolecule of the one or more target biomolecules to a set of reaction chambers. In some embodiments, the method comprises providing a plurality of candidate binding partners of the target biomolecules to the set of reaction chambers. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.3%, at least about 6.5%, at least about 6.7%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 10.5%, at least about 11%, at least about 11.5%, at least about 12%, at least about 12.5%, at least about 13%, at least about 13.5%, at least about 14%, at least about 14.5%, at least about 15%, at least about 15.5%, atAttorney Docket No. 69414-702601 least about 16%, at least about 16.5%, at least about 17%, at least about 17.5%, at least about 18%, at least about 18.3%, at least about 18.5%, at least about 18.6%, at least about 18.7%, at least about 19%, at least about 19.5%, at least about 20%, at least about 20.5%, at least about 21%, at least about 21.5%, at least about 22%, at least about 22.5%, at least about 23%, at least about 24%, or at least about 25% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 3% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 6% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 6.3% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 18% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 18.6% of the surface. The surface, as referred to above, is an active surface that is a portion of the larger surface (e.g. a glass slide). The active surface, as referred to herein, refers to a portion of the larger surface comprising the plurality of reaction chambers. The active surface can be substantially covered by the plurality of reaction chambers. The active surface can have a boundary that is set by the outer sides of the plurality of reaction chambers. An example of the active surface is illustrated in the left panel of FIG. 22A, wherein the active surface is the portion of the glass slide covered by the grid formed by the plurality of reaction chambers and the active surface’s boundary can be set by the outermost sides of the outermost chambers in the grid. In some embodiments, the method comprises (c) identifying one or more binding partners of the target biomolecule. In some embodiments, the one or more binding partners is identified from a subset of the plurality of candidate binding partners. In some embodiments, the subset of the plurality of candidate binding partners are bound to the target biomolecule.

[0114] In some embodiments, the method comprises mechanically trapping the target biomolecule and the one or more candidate binding partners. In some embodiments, the trapping occurs between a first surface and a second surface of the device. In some cases, the first surface and second surface can be surfaces within a reaction chamber of the set of reaction chambers. In some embodiments, the first surface and the second surface comprise the surface the reaction chamber is on. In some embodiments, the first surface and the second surface comprise a surface like a glass slide or similar material. In some embodiments, the method comprises generating one or more complexes comprising the target molecule and the subset of the plurality of candidate binding partners. In some embodiments, the secondAttorney Docket No. 69414-702601 surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule. In some embodiments, the member is a button valve. In some embodiments, the method further comprises administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin (e.g., biotinylated Bovine Serum Albumin), a neutravidin, a biotinylated antibody (e.g., a biotinylated anti-green fluorescent protein antibody), a biotinylated nanobody, or any combination thereof.

[0115] In some embodiments, the plurality of candidate binding partners comprises at least 10, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 100,000, at least about 500,000, at least about 1 million, at least about 5 million, at least about 10 million, at least about 50 million, at least about 100 million, at least about 500 million, at least about 1 billion, at least about 5 billion, at least about 10 billion, or at least about 50 billion unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 1,000 unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 10,000 unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 1 million unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 10 million unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 100 million unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 1 billion unique candidate binding partners. In some embodiments, the plurality of candidate binding partners comprises at least 10 billion unique candidate binding partners.

[0116] In some embodiments, the method comprises providing the one or more target biomolecules to the plurality of reaction chambers. In some embodiments, the one or more target biomolecules comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least48, at least 49, at least 50, at least 51, or at least 52, at least 60, at least 70, at least 80, at least 90, or at least 100 unique target biomolecules. In some embodiments, the one or more targetAttorney Docket No. 69414-702601 biomolecules comprises at least 8 unique target biomolecules. In some embodiments, the one or more target biomolecules comprises at least 16 unique target biomolecules. In some embodiments, the one or more target biomolecules comprises at least 32 unique target biomolecules. In some embodiments, the method comprises providing different unique target biomolecule to different sets of reaction chambers in the plurality of reaction chambers. In some embodiments, the method comprises providing each unique target biomolecule to different sets of reaction chambers in the plurality of reaction chambers. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 93 million-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 186 million-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 372 million-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 744 million-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 1.488 billion-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 1.5 billion-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 2.976 billion-fold greater than a number of unique target biomolecules in the one or more target biomolecules. In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners at least about 3 billion-fold greater than a number of unique target biomolecules in the one or more target biomolecules.

[0117] In some embodiments, a number of unique candidate binding partners in the plurality of candidate binding partners is less than a number of unique target biomolecules in the one or more target biomolecules. For example, the number of unique candidate binding partners can be less than the number of unique target biomolecules by about 8 fold, about 16 fold, or about 32 fold.Attorney Docket No. 69414-702601

[0118] In some embodiments, the method does not comprise tagging the one or more complexes with a barcode (e.g., nucleic acid barcode) in the device. In some embodiments, the method does not comprise introducing a barcode from a reservoir chamber on the surface of the device into a reaction chamber of the plurality of reaction chambers.

[0119] In some embodiments, the set of reaction chambers from the one or more sets of reaction chambers comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more reaction chambers. In some embodiments, the set of reaction chambers from the one or more sets of reaction chambers comprises 2 or more reaction chambers. In some embodiments, reaction chambers of the set of reaction chambers are fluidly connected during (b). In some embodiments, reaction chambers of the set of reaction chambers are fluidly connected during providing of the target biomolecule. In some embodiments, reaction chambers of the set of reaction chambers are fluidly connected during providing of the plurality of candidate binding partners. In some embodiments, the reaction chambers of the set of reaction chambers are fluidly connected in series. In some embodiments, the reaction chambers of the set of reaction chambers are fluidly connected in parallel.

[0120] In some embodiments, the plurality of candidate binding partners is configured to interact with two or more binding sites on the target biomolecule. In some embodiments, the plurality of candidate binding partners is randomly selected. In some embodiments, the plurality of candidate binding partners is designed for testing against the target biomolecule of binding. In some embodiments, the plurality of candidate binding partners is commercially available as a library. In some embodiments, different candidate binding partners of the plurality of candidate binding partners interact with different binding sites on the target biomolecule. In some embodiments, the plurality of target biomolecules comprises target biomolecules with different sequences, structures, or modifications (e.g., post- translational modifications or unnatural amino acids). In some embodiments, the one or more target biomolecules comprises one or more variants of the target biomolecule. In some embodiments, the one or more variants comprises a point mutation, an insertion, a deletion, or a combination thereof relative to the target biomolecule. In some embodiments, the one or more variants comprises a different post-translational modification (PTM), more PTM or fewer PTM relative to the target biomolecule. In some embodiments, the one or more variants can bind to different candidate binding partners of the plurality of candidate binding partnersAttorney Docket No. 69414-702601 than the target biomolecule. In some embodiments, the one or more variants and the target biomolecule can bind to the same candidate binding partners with different affinities.

[0121] In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during providing of the target biomolecule (b). In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during providing of the target biomolecule. In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during providing of the plurality of candidate binding partners. In some embodiments, the plurality of reaction chambers comprises at least 3 sets, at least 4 sets, at least 5 sets, at least 6 sets, at least 7 sets, at least 8 sets, at least 9 sets, at least 10 sets, at least 11 sets, at least 12 sets, at least 13 sets, at least 14 sets, at least 15 sets, at least 16 sets, at least 17 sets, at least 18 sets, at least 19 sets, at least 20 sets, at least 21 sets, at least 22 sets, at least 23 sets, at least 24 sets, at least 25 sets, at least 26 sets, at least 27 sets, at least 28 sets, at least 29 sets, at least 30 sets, at least 31 sets, at least 32 sets, at least 40 sets, at least 50 sets, at least 60 sets, at least 70 sets, at least 80 sets, at least 90 sets, or at least 100 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 8 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 16 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 32 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises as many sets of reaction chambers as there are unique target biomolecules in the one or more target biomolecules. In some embodiments, the one or more target biomolecules comprises the target biomolecule and one or more variants thereof and the method comprises providing each unique target biomolecule into different sets in the plurality of reaction chambers.

[0122] In some embodiments, the method comprises providing a second target biomolecule of the one or more target biomolecules and the plurality of candidate binding partners of the second target biomolecule to the additional set of reaction chambers. In some embodiments, the second target biomolecule is a variant of the target biomolecule. In some embodiments, the method comprises identifying one or more binding partners of the second target biomolecule from the plurality of candidate binding partners. In some embodiments, the method comprises comprising identifying a difference between the one or more bindingAttorney Docket No. 69414-702601 partners of the target biomolecule and the one or more binding partners of the second target biomolecule. In some embodiments, the method comprises identifying one or more binding sites of the one or more binding partners of the one or more target biomolecules (e.g., the target biomolecule, the second target biomolecule, or variants thereof). In some embodiments, identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing the target biomolecule and the second target biomolecule. In some embodiments, identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing a sequence of the target biomolecule and a sequence the second target biomolecule. In some embodiments, the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing a structure of the target biomolecule and a structure the second target biomolecule.

[0123] In some embodiments, reaction chambers of one set of reaction chambers are not fluidly connected to reaction chambers of a different set amongst the sets of reaction chambers during (b). In some embodiments, reaction chambers of one set of reaction chambers are not fluidly connected to reaction chambers of a different set amongst the sets of reaction chambers during providing of the target biomolecule. In some embodiments, reaction chambers of one set of reaction chambers are not fluidly connected to reaction chambers of a different set amongst the sets of reaction chambers during providing of the second target biomolecule or a variant of the target biomolecule. In some embodiments, reaction chambers of one set of reaction chambers are not fluidly connected to reaction chambers of a different set amongst the sets of reaction chambers during providing of the plurality of candidate binding partners.

[0124] In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 500 nM, at least about 1 pM, at least about 5 pM, at least about 10 pM, at least about 15 pM, at least about 20 pM, at least about 25 pM, at least about 30 pM, at least about 35 pM, at least about 40 pM, at least about 45 pM, or at least about 50 pM. In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 1 nM. In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 10 nM. In someAttorney Docket No. 69414-702601 embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 20 nM. In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 1 pM. In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 10 pM. In some embodiments, the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least about 25 pM. In some embodiments, a binding partner of the one or more binding partners of the target biomolecule has a binding affinity to no more than about 100 nM, about 1 pM, or about 10 pM. In some embodiments, the plurality of reaction chambers comprises at least about 4000, at least about 4100, at least about 4200, at least about 4300, at least about 4400, at least about 4500, at least about 4600, at least about 4700, at least about 4800, at least about 4900, at least about 5000, at least about 5100, at least about 5200, at least about 5300, at least about 5400, at least about 5500, at least about 5600, at least about 5700, at least about 5800, at least about 5900, or at least about 6000 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4096 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4100 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4192 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4200 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4992 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 5000 reaction chambers. In some embodiments, the plurality of reaction chambers covers at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the surface. In some embodiments, the plurality of reaction chambers covers at least about 30% of the surface. In some embodiments, the plurality of reaction chambers covers at least about 60% of the surface. In some embodiments, the plurality of reaction chambers displays at least about 0.3 pmol, at least about 1 pmol, at least about 2 pmol, at least about 3 pmol, at least about 4 pmol, at least about 5 pmol, at least about 6 pmol, at least about 7 pmol, at least about 8 pmol, at least about 9 pmol, at least about 10 pmol, at least about 11 pmol, at least about 12 pmol, at least about 13 pmol, at least about 14 pmol, at least about 15 pmol, at least about 16 pmol, at least about 17 pmol, at least about 18 pmol, at least about 19 pmol, at least about 20 pmol, or at least about 25 pmol of the one or more target biomolecules (e.g.,Attorney Docket No. 69414-702601 protein). In some embodiments, the plurality of reaction chambers displays at least about 0.3 pmol of the protein. In some embodiments, the plurality of reaction chambers displays at least about 3 pmol of the protein. In some embodiments, the plurality of reaction chambers displays at least about 10 pmol of the protein. In some embodiments, providing the target biomolecule does not comprise providing the target biomolecule from a reservoir chamber on the surface adjacent to a reaction chamber of the set of reaction chambers.

[0125] In some embodiments, the method comprises eluting the subset of the plurality of candidate binding partners from the one or more complexes. In some embodiments, eluting comprises providing a protease (e.g., Proteinase K) to the one or more complexes. In some embodiments, the method comprises providing the subset of the plurality of candidate binding partners to a set of reaction chambers on a different surface. In some embodiments, the method comprises providing the target biomolecule and the subset of the plurality of candidate binding partners to a set of reaction chambers on a different surface. In some embodiments, the different surface can be on a different device. In some embodiments, another subset of the subset of the plurality of candidate binding partners can bind to the target biomolecule and eluted. The eluted subset can be identified or reanalyzed in reaction chambers of another surface of another device.

[0126] In some embodiments, the method comprises obtaining an unbound subset of the plurality of candidate binding partners, wherein the unbound subset of the plurality of candidate binding partners is not bound to the target biomolecule. In some embodiments, the method comprises providing the unbound subset of the plurality of candidate binding partners to the set of reaction chambers. Providing the unbound subset of the plurality of candidate binding partners to the set of reaction chambers containing the target biomolecule can provide another opportunity for members of the unbound subset to bind to the target biomolecule. After providing the unbound subset to the set of reaction chambers, the resulting unbound subset can be provided again to the set of reaction chambers. Reintroduction of the unbound subset to the set of reaction chambers can increase the sensitivity of identifying binding partners of the target molecule.

[0127] In some embodiments, the method further comprises expressing the one or more target biomolecules external to the device. In some embodiments, the expressing is not on the surface of the device. In some embodiments, the expressing comprises cell free expression. In some embodiments, the expressing comprises in vitro transcription and translation.

[0128] In some embodiments, the plurality of candidate binding partners comprises tags. For example, the plurality of candidate binding partners can be a DNA-encoded library (DEL). InAttorney Docket No. 69414-702601 some embodiments, the tags are unique for each of the unique candidate binding partners. In some embodiments, identifying the one or more binding partners of the target biomolecule comprises identifying the tags from the subset of the plurality of candidate binding partners. In some embodiments, the tags comprise nucleic acids (e.g., DNA and / or RNA). In some embodiments, the tags can comprise single stranded DNA. In some embodiments, the tags can comprise double stranded DNA. In some embodiments, identifying the tags comprises sequencing the tags from the subset of the plurality of candidate binding partners. For example, identifying the one or more binding partners of the target biomolecule can comprise eluting the subset of the plurality of candidate binding partners bound to the target molecules in the one or more complexes and identifying the tags on the subset of the plurality of candidate binding partners by sequencing the tags. The sequence of the tags can be unique to each unique candidate binding partner in the plurality of candidate binding partners.Sequencing the tags from the subset of the plurality of candidate binding partners can identify each unique candidate binding partner of the subset as a binding partner of the target biomolecule.

[0129] In some embodiments, the plurality of candidate binding partners comprises small molecules. In some embodiments, the plurality of candidate binding partners comprises proteins. In some embodiments, the plurality of candidate binding partners comprises nucleic acids (e.g., DNA and / or RNA).

[0130] In some embodiments, providing the target biomolecule and the plurality of candidate binding partners can be through a common inlet. In some embodiments, providing the target biomolecule and the plurality of candidate binding partners can be through fluidically separate inlets.

[0131] In some embodiments, the target biomolecule can be a DNA, RNA, a protein, or a combination thereof. In some embodiments, the target biomolecule is a protein. In some embodiments, the target molecule can be an enzyme. In some embodiments, the one or more target biomolecules can comprise the target biomolecule and variants thereof.

[0132] In some embodiments, the method further comprises inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site. In some embodiments, the method is configured to perform multiple forward and reverse screens simultaneously.Attorney Docket No. 69414-702601

[0133] In some embodiments, the plurality of reaction chambers comprises reaction chambers having a volume between 0.5 nL and 50 nL. In some embodiments, the plurality of reaction chambers comprises micro-chambers as described elsewhere herein.

[0134] In some embodiments, the method comprises quantifying bound interactors (e.g., the one or more binding partners) to determine binding affinities or relative binding affinities for at least 2 interactors. In some embodiments, the method comprises quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor.

[0135] In some embodiments, the method is used for analyzing interactions between DNA sequences and transcription factors. For example, the one or more target biomolecules can comprise one or more transcription factors and the plurality of candidate binding partners can comprise different DNA sequences. In some embodiments, the method is employed for analyzing small molecule interactions with the one or more target biomolecules. In some embodiments, the method comprises analyzing antibody interactions with target biomolecules. For example, the plurality of candidate binding partners can comprise different antibodies. In some embodiments, the method is employed for analyzing interactions of peptides or proteins with the one or more target biomolecules using mRNA display libraries. In some embodiments, the method is employed for analyzing interactions of peptides or proteins with the one or more target biomolecules using cDNA display libraries.

[0136] In some embodiments, the target biomolecule is engineered to contain one or more unnatural amino acids. In some embodiments, the target biomolecule is post-translationally modified with one or more chemical entities. In some embodiments, the one or more chemical entities is selected from the group consisting of phosphate, sugars, and ubiquitin protein.

[0137] In another aspect disclosed herein are microfluidic systems comprising a plurality of reaction chambers on a surface. In some embodiments, the surface is configured to display one or more target biomolecules. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.3%, at least about 6.5%, at least about 6.7%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 10.5%, at least about 11%, at least about 11.5%, at least about 12%, at least about 12.5%, at least about 13%, at least about 13.5%, at least about 14%, at least about 14.5%, at least about 15%, at least about 15.5%, at least about 16%, at least about 16.5%, at least about 17%, at least about 17.5%, at least aboutAttorney Docket No. 69414-70260118%, at least about 18.3%, at least about 18.5%, at least about 18.6%, at least about 18.7%, at least about 19%, at least about 19.5%, at least about 20%, at least about 20.5%, at least about 21%, at least about 21.5%, at least about 22%, at least about 22.5%, at least about 23%, at least about 24%, or at least about 25% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 3% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 6% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 6.3% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 18% of the surface. In some embodiments, the surface is configured to display the one or more target biomolecules over at least about 18.6% of the surface. The surface, as referred to above, is an active surface that is a portion of the larger surface (e.g. a glass slide). The active surface, as referred to herein, refers to a portion of the larger surface comprising the plurality of reaction chambers. The active surface can be substantially covered by the plurality of reaction chambers. The active surface can have a boundary that is set by the outer sides of the plurality of reaction chambers. An example of the active surface is illustrated in the left panel of FIG. 22A, wherein the active surface is the portion of the glass slide covered by the grid formed by the plurality of reaction chambers and the active surface’s boundary can be set by the outermost sides of the outermost chambers in the grid. In some embodiments, the microfluidic system comprises one or more members positioned adjacent to one or more reaction chambers of the plurality of reaction chambers. In some embodiments, the one or more members is configured to move along an axis orthogonal to the surface. In some embodiments, the microfluidic device comprises a hydraulic control module configured to actuate the one or more members.

[0138] In some embodiments, the plurality of reaction chambers covers at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the surface. In some embodiments, the plurality of reaction chambers covers at least about 30% of the surface. In some embodiments, the plurality of reaction chambers covers at least about 60% of the surface. In some embodiments, the plurality of reaction chambers comprises at least about 4000, at least about 4100, at least about 4200, at least about 4300, at least about 4400, at least about 4500, at least about 4600, at least about 4700, at least about 4800, at least about 4900, at least about 5000, at least about 5100, at least about 5200, at least about 5300, atAttorney Docket No. 69414-702601 least about 5400, at least about 5500, at least about 5600, at least about 5700, at least about 5800, at least about 5900, or at least about 6000 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4096 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4100 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4192 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4200 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 4992 reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least about 5000 reaction chambers. In some embodiments, the plurality of reaction chambers displays at least about 0.3 pmol, at least about 1 pmol, at least about 2 pmol, at least about 3 pmol, at least about 4 pmol, at least about 5 pmol, at least about 6 pmol, at least about 7 pmol, at least about 8 pmol, at least about 9 pmol, at least about 10 pmol, at least about 11 pmol, at least about 12 pmol, at least about 13 pmol, at least about 14 pmol, at least about 15 pmol, at least about 16 pmol, at least about 17 pmol, at least about 18 pmol, at least about 19 pmol, at least about 20 pmol, or at least about25 pmol of the one or more target biomolecules (e.g., protein). In some embodiments, the plurality of reaction chambers displays at least about 0.3 pmol of the protein. In some embodiments, the plurality of reaction chambers displays at least about 3 pmol of the protein. In some embodiments, the plurality of reaction chambers displays at least about 10 pmol of the protein.

[0139] In some embodiments, the plurality of reaction chambers comprises a set of reaction chambers, wherein reaction chambers of the set of reaction chambers are fluidly connected. In some embodiments, the reaction chambers of the set of reaction chambers are fluidly connected in series. In some embodiments, the reaction chambers of the set of reaction chambers are fluidly connected in parallel.

[0140] In some embodiments, the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are configured to not be fluidly connected to the set of reaction chambers at a point (or any point) during use of the microfluidic system. In some embodiments, the use of the microfluidic system is described the methods disclosed elsewhere herein. In some embodiments, the plurality of reaction chambers comprises at least 3 sets, at least 4 sets, at least 5 sets, at least 6 sets, at least 7 sets, at least 8 sets, at least 9 sets, at least 10 sets, at least 11 sets, at least 12 sets, at least 13 sets, at least 14 sets, at least 15 sets, at least 16 sets, at least 17 sets, at least 18 sets, at least 19 sets, at least 20 sets, at least 21 sets, at least 22 sets, at least 23 sets, at least 24 sets, at least 25 sets, atAttorney Docket No. 69414-702601 least 26 sets, at least 27 sets, at least 28 sets, at least 29 sets, at least 30 sets, at least 31 sets, at least 32 sets, at least 40 sets, at least 50 sets, at least 60 sets, at least 70 sets, at least 80 sets, at least 90 sets, or at least 100 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 8 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 16 sets of reaction chambers. In some embodiments, the plurality of reaction chambers comprises at least 32 sets of reaction chambers.

[0141] In some embodiments, the plurality of reaction chambers is not connected to a reservoir chamber adjacent to a reaction chamber of the plurality of reaction chambers on the surface. In some embodiments, the surface does not comprise a reservoir chamber adjacent to a reaction chamber of the plurality of reaction chambers.

[0142] In some embodiments, the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface. In some embodiments, the reaction surface is a surface in the reaction chamber. In some embodiments, the hydraulic control module is a hydraulic control module described elsewhere herein.

[0143] In some embodiments, the plurality of reaction chambers comprises reaction chambers having a volume between 0.5 nL and 50 nL. In some embodiments, the plurality of reaction chambers comprises micro-chambers as described elsewhere herein.

[0144] In some embodiments, the microfluidic system comprises a two-layer elastomer structure positioned on top of a glass layer. In some embodiment, the two-layer elastomer structure layered on top of the glass layer forms one or more cavities between the layers. In some embodiments, the elastomer structure comprises polydimethylsiloxane (PDMS). In some embodiments, the one or more cavities form the plurality of reaction chambers.

[0145] In some embodiments, the microfluidic device further comprises integrated pushdown valves configured in a Quake-style arrangement, or a pneumatically or hydraulically actuated elastomeric valve arrangement for controlling fluid flow. In some embodiments, valves configured in a Quake-style arrangement are Quake style valves described elsewhere herein.

[0146] In some embodiments, the microfluidic system is configured to quantitatively assay the one or more target biomolecules, or a subset thereof, simultaneously. In some embodiments, when the one or more members are open, fluid flows through the full volume of all reaction chambers in the plurality of reaction chambers. In some embodiments, at least one reaction chamber of the plurality of reaction chambers is an elongated reaction chamber. In some embodiments, the elongated reaction chamber comprises variable stretch valuesAttorney Docket No. 69414-702601(e.g., stretch values described elsewhere herein), resulting in proportional fold changes to chamber volume and member area. In some embodiments, the variable stretch values results in proportional fold changes to chamber volume and member area. In some embodiments, the chamber volume fold-change over the non-stretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10. In some embodiments, the member area fold-change over the nonstretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10. In some embodiments, the total internal volume of the device comprises about 0.1 to about 0.2 (*0.6 pL), from about 0.2 to about 0.3 (*0.6 pL), from about 0.3 to about 0.4 (*0.6 pL), from about 0.4 to about 0.5 (*0.6 pL), from about 0.5 to about 0.6 (*0.6 pL), from about 0.6 to about 0.7 (*0.6 pL), from about 0.7 to about 0.8 (*0.6 pL), from about 0.8 to about 0.9 (*0.6 pL), from about 0.9 to about 1.0 (*0.6 pL), continuing in this manner with a step size of 0.1 up to about 9.9 to about 10 (*0.6 pL). In some embodiments, stretching a reaction chamber by a specified stretch value increases the internal volume and surface area of the microfluidic device (e.g., see FIG. 8A-8D). In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2 and a member area fold change of about 3 over the nonstretched design. In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2.1 and a member area fold change of about 2.8 over the nonstretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3 and a member area fold change of about 5 over the nonstretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3.2 and a member area fold change of about 4.6 over the nonstretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4 and a member area fold change of about 7 over the nonstretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4.3 and a member area fold change of about 6.4 over the nonstretched design. In some embodiments, a stretch value of 800 microns generates a chamber volume fold change of about 5 and a member area fold change of about 8 over the nonstretched design. In some embodiments, a stretch value of 800 microns generates a chamberAttorney Docket No. 69414-702601 volume fold change of about 5.5 and a member area fold change of about 8.3 over the nonstretched design. In some embodiments, a stretch value of 200 microns generates a configuration of approximately 1200 chambers per chip. In some embodiments, a stretch value of 200 microns generates a configuration of approximately 6000 chambers per chip. In some embodiments, a stretch value of 400 microns generates a configuration of approximately 900 chambers per chip. In some embodiments, a stretch value of 400 microns generates a configuration of approximately 4500 chambers per chip. In some embodiments, a stretch value of 600 microns generates a configuration of approximately 700 chambers per chip. In some embodiments, a stretch value of 600 microns generates a configuration of approximately 3500 chambers per chip. In some embodiments, a stretch value of 800 microns may lead to a configuration of approximately 3000 chambers per chip.

[0147] In some embodiments, the surface has an area greater than about 1065 mm2. In some embodiments, the surface has an area greater than about 1100 mm2. In some embodiments, the surface has an area of about 1065 mm2. In some embodiments, the surface has an area of about 1100 mm2. In some embodiments, the surface has an area of about 1300 mm2. In some embodiments, the surface has an area of about 1325 mm2. In some embodiments, the surface has an area of about 1400 mm2. In some embodiments, the surface has a dimension of about 25.5 mm by 43.5 mm. In some embodiments, the surface has a dimension of about 25 mm by 43 mm. In some embodiments, the surface has a dimension of about 26 mm by 44 mm. In some embodiments, the surface has a dimension of about 28.8 mm by 46 mm. In some embodiments, the surface has a dimension of about 28 mm by 46 mm. In some embodiments, the surface has a dimension of about 29 mm by 46 mm.Hydraulic Control Systems

[0148] In some embodiments, the systems and microfluidic devices described herein are powered by a hydraulic system. In some embodiments, the hydraulic system is configured to control the valves on the microchip. In some embodiments, the hydraulic system comprises a hydraulic pump. In some embodiments, the hydraulic pump is the primary source of power in the hydraulic system and creates a flow of hydraulic fluid configured to pressurize the system. In some embodiments, the hydraulic pump controls valves within the microfluidic devices of the system, the control valves managing the flow and direction of the hydraulic fluid, controlling its distribution to various components of the system. In some embodiments, the hydraulic fluid comprises a medium like oil or water through which power is transmitted in the hydraulic system by compressing and changing position to allow force to be transferred through the system. In some embodiments, the system comprises an actuator, and the actuatorAttorney Docket No. 69414-702601 may be a hydraulic cylinder or a hydraulic motor, which converts the fluid power into mechanical power. In some embodiments, the system comprises a reservoir. In some embodiments, the reservoir holds the hydraulic fluid, supplies it to the pump, and helps dissipate heat from the fluid and hydraulic lines. In some embodiments, the system comprises pipes or tubes that carry the hydraulic fluid from one part of the system to another, and a pressure regulator. In some embodiments, the pressure regulator or relief valve controls and maintains the hydraulic pressure in the system to prevent the pressure from exceeding the specified limits. In some embodiments, the system comprises filters which are used to remove contaminants from the hydraulic fluid, keeping the system functioning smoothly and extending its lifespan.

[0149] In some embodiments, the hydraulic microfluidic control system comprises a common reservoir feeding solenoid valves controlling the flow of solution through the microfluidic device. In some embodiments, at least one solenoid valve is filled with liquid. In some embodiments, all solenoid valves are filled with liquid. In some embodiments, the system is configured to extend to an arbitrary number of valves. In some embodiments, the hydraulic control module actuates the member by creating a pressure differential between the reaction chamber and the reservoir chambers. In some instances, the hydraulic control module may cause the member to move in response to the member actuation, altering the flow of fluids in the channels.

[0150] In some embodiments, a microfluidic system is provided, where the hydraulic control module employs a set of pistons connected to a movable member. By adjusting the positions of the pistons, the member may be actuated, offering a mechanical means to control the reaction within the chamber. In some embodiments, the system comprises at least one hydraulic control module. In some cases, at least one hydraulic control module comprises at least one pump. In some instances, at least one pump comprises a pulsating hydraulic pressure pump. In some cases, at least one hydraulic control module comprises at least one motor-driven syringe. In some instances, at least one motor-driven syringe comprises a plunger for displacement control. For example, at least one hydraulic control module may be configured to actuate a movable member to facilitate the mixing of reagents within a reaction chamber. In further examples, the actuation of the movable member is configured to control the dynamics and conditions within the reaction chamber. As an example, the system may utilize rhythmic movement of the member to enhance reagent mixing and optimize reaction conditions.Attorney Docket No. 69414-702601

[0151] In some embodiments, the device integrates a hydraulic control system and a fluidbased system. This system may operate in conjunction with trans fluorescence illumination imaging. All valves on-chip may be controlled using hydraulic pressurization which may include members, sandwich valves, a neck valve for each one of a plurality of reservoir chambers, all the valves for flow inputs, and the outlet valve. In some embodiments, an air compressor may generate up to 115 psi of pressure. A neck valve refers to a specially designed valve structure that often resembles a narrow "neck" or constriction. Its primary function is to allow for precise control over fluid flow. By opening and closing the valve depending on applied pressure or other stimuli, it acts as a gate to regulate the movement of tiny volumes of liquid.

[0152] In some embodiments, an air compressor may generate up to about 1 psi of pressure to about 110 psi of pressure. In some embodiments, an air compressor may generate up to about 1 psi of pressure to about 10 psi of pressure, about 1 psi of pressure to about 20 psi of pressure, about 1 psi of pressure to about 30 psi of pressure, about 1 psi of pressure to about 40 psi of pressure, about 1 psi of pressure to about 50 psi of pressure, about 1 psi of pressure to about 60 psi of pressure, about 1 psi of pressure to about 70 psi of pressure, about 1 psi of pressure to about 80 psi of pressure, about 1 psi of pressure to about 90 psi of pressure, about 1 psi of pressure to about 100 psi of pressure, about 1 psi of pressure to about 110 psi of pressure, about 10 psi of pressure to about 20 psi of pressure, about 10 psi of pressure to about 30 psi of pressure, about 10 psi of pressure to about 40 psi of pressure, about 10 psi of pressure to about 50 psi of pressure, about 10 psi of pressure to about 60 psi of pressure, about 10 psi of pressure to about 70 psi of pressure, about 10 psi of pressure to about 80 psi of pressure, about 10 psi of pressure to about 90 psi of pressure, about 10 psi of pressure to about 100 psi of pressure, about 10 psi of pressure to about 110 psi of pressure, about 20 psi of pressure to about 30 psi of pressure, about 20 psi of pressure to about 40 psi of pressure, about 20 psi of pressure to about 50 psi of pressure, about 20 psi of pressure to about 60 psi of pressure, about 20 psi of pressure to about 70 psi of pressure, about 20 psi of pressure to about 80 psi of pressure, about 20 psi of pressure to about 90 psi of pressure, about 20 psi of pressure to about 100 psi of pressure, about 20 psi of pressure to about 110 psi of pressure, about 30 psi of pressure to about 40 psi of pressure, about 30 psi of pressure to about 50 psi of pressure, about 30 psi of pressure to about 60 psi of pressure, about 30 psi of pressure to about 70 psi of pressure, about 30 psi of pressure to about 80 psi of pressure, about 30 psi of pressure to about 90 psi of pressure, about 30 psi of pressure to about 100 psi of pressure, about 30 psi of pressure to about 110 psi of pressure, about 40 psi of pressure to about 50 psiAttorney Docket No. 69414-702601 of pressure, about 40 psi of pressure to about 60 psi of pressure, about 40 psi of pressure to about 70 psi of pressure, about 40 psi of pressure to about 80 psi of pressure, about 40 psi of pressure to about 90 psi of pressure, about 40 psi of pressure to about 100 psi of pressure, about 40 psi of pressure to about 110 psi of pressure, about 50 psi of pressure to about 60 psi of pressure, about 50 psi of pressure to about 70 psi of pressure, about 50 psi of pressure to about 80 psi of pressure, about 50 psi of pressure to about 90 psi of pressure, about 50 psi of pressure to about 100 psi of pressure, about 50 psi of pressure to about 110 psi of pressure, about 60 psi of pressure to about 70 psi of pressure, about 60 psi of pressure to about 80 psi of pressure, about 60 psi of pressure to about 90 psi of pressure, about 60 psi of pressure to about 100 psi of pressure, about 60 psi of pressure to about 110 psi of pressure, about 70 psi of pressure to about 80 psi of pressure, about 70 psi of pressure to about 90 psi of pressure, about 70 psi of pressure to about 100 psi of pressure, about 70 psi of pressure to about 110 psi of pressure, about 80 psi of pressure to about 90 psi of pressure, about 80 psi of pressure to about 100 psi of pressure, about 80 psi of pressure to about 110 psi of pressure, about 90 psi of pressure to about 100 psi of pressure, about 90 psi of pressure to about 110 psi of pressure, or about 100 psi of pressure to about 110 psi of pressure. In some embodiments, an air compressor may generate up to about 1 psi of pressure, about 10 psi of pressure, about 20 psi of pressure, about 30 psi of pressure, about 40 psi of pressure, about 50 psi of pressure, about 60 psi of pressure, about 70 psi of pressure, about 80 psi of pressure, about 90 psi of pressure, about 100 psi of pressure, or about 110 psi of pressure. In some embodiments, an air compressor may generate up to at least about 1 psi of pressure, about 10 psi of pressure, about 20 psi of pressure, about 30 psi of pressure, about 40 psi of pressure, about 50 psi of pressure, about 60 psi of pressure, about 70 psi of pressure, about 80 psi of pressure, about 90 psi of pressure, or about 100 psi of pressure. In some embodiments, an air compressor may generate up to at most about 10 psi of pressure, about 20 psi of pressure, about 30 psi of pressure, about 40 psi of pressure, about 50 psi of pressure, about 60 psi of pressure, about 70 psi of pressure, about 80 psi of pressure, about 90 psi of pressure, about 100 psi of pressure, or about 110 psi of pressure.

[0153] This pressure may feed into two digital pressure regulators, one for controlling on- chip valve pressure and one for controlling flow pressure. Each of these pressure controllers may have 4 channels, which allows the control of 4 microfluidic devices independently and in parallel. Each channel of the pressure regulator may pressurize a “common reservoir” filled with water. The pressurized water from the “common reservoir” may feed a series of liquid-filled solenoids that may be individually opened or closed. Each solenoid mayAttorney Docket No. 69414-702601 pressurize a control valve on-chip. The pressures may be digitally adjusted through a digital pressure controller from 0 to 100 psi or higher. In some embodiments, up to 4 independent devices may be run in parallel, as the pressure for each common reservoir may be individually adjusted. Each common reservoir may pressurize a series of at least 18 solenoid valves that are filled with liquid. In some embodiments, when there is no hydraulic pressure, the valves are open, and when there is hydraulic pressure, the valves may be closed. In some embodiments, all on-chip valves are normally open and will only close when pressurized. In some embodiments, on-chip valves are normally closed and will only open when pressurized. In some embodiments, each bank of solenoids may control at least 18 individual valves. This number may be arbitrarily increased or decreased.

[0154] FIG. 14 illustrates an example of a hydraulics valve control workflow. The system may comprise an air compressor, contributing to the generation of pressurized air for system operation, and a digital pressure regulator for control valves that may optimize functioning by modulating air pressure. In some cases, the system may include up to four pressurized liquid reservoirs, or more. In some instances, the pressurized liquid reservoirs may serve as storage for the system's liquid. Additionally, solenoid banks, each equipped with 18 solenoids, may serve as electronic components associated with controlling the liquid flow in the system. The microfluidic device, which may operate up to four devices in parallel, is depicted as the central component of the system. In some instances, the system may feature 18 valves for valve control to regulate the flow of liquid through the system. The system may also incorporate a pressurized water and air supply to potentially assist fluid flow and control valve operation. A digital pressure regulator for fluid flow may be included to manage the pressure within multiple fluid flow lines. For example, this may demonstrate a configuration of the hydraulic valve control system within microfluidic devices.

[0155] In some embodiments, the system may comprise one or more hydraulic valves. In some embodiments, the system may comprise an air compressor, digital pressure regulator for control valves or a digital pressure regulator for fluid flow, pressurized liquid reservoirs, solenoid banks and microfluidic device. Valves and fluid flow may be controlled via pressurized water, pressurized air, or a combination thereof.

[0156] FIG. 15A - FIG. 15B show examples of solenoid arrays operating with pressurized water.Imaging Systems

[0157] In some embodiments, an imaging system for imaging the systems and microfluidic devices described herein comprise a substrate that includes the microfluidic device. In someAttorney Docket No. 69414-702601 embodiments, the imaging system comprises a field comprising a plurality of channels within the microfluidic device. In some embodiments, the imaging system comprises a light source configured to provide light to a sample. In some embodiments, the light source and the sample are separated by the microfluidic device. Additionally, a detector may be positioned on a side of the substrate opposite of the side of the substrate where the light source is located. In some embodiments, a channel of the plurality of channels comprises a reaction chamber, one or more reservoir chambers fluidically coupled to the reaction chamber, and a member disposed adjacent to the reaction chamber and configured to move on an axis orthogonal to the reaction chamber.

[0158] In some embodiments, the imaging system includes a macroscope with a macro lens system of approximately equal to or less than lx magnification. In some embodiments, the system comprises an integrated Fly’s eye homogenizer for achieving near-uniform illumination.

[0159] In some embodiments, the imaging system comprises a stacked arrangement of emission filters to enhance the signal-to-noise ratio. In some embodiments, the imaging system comprises an emission filter placement within the infinity space between lenses for improved optical performance.

[0160] In some embodiments, the imaging system comprises an integrated hydraulic microfluidic control system. In some embodiments, the imaging system is configured for trans-illumination rather than epi-illumination. In some embodiments, the imaging system may be configured for side-on illumination rather than epi-illumination. In some embodiments, the imaging system further comprises a system for analyzing biochemical events (e.g., such as a kinetic rate) involving surface immobilized fluorophores. In some embodiments, the imaging system further comprises a system for analyzing the fast kinetics of biochemical events involving solution-phase fluorophores. In some embodiments, the imaging system further comprises a system for analyzing biochemical events (e.g., such as the kinetic rate) using luminescence techniques.

[0161] FIG. 9A illustrates an example of an imaging system described herein. As displayed in FIG. 9A, the imaging system comprises a collimator, an excitation filter wheel, a microfluidic device, an emission filter wheel, a camera, a motorized XY stage, a Z-axis linear actuator, and two macro lenses. FIG. 9C depicts an example rendering of the combined imaging and fluidic setup, which may comprise a digital flow pressure regulator, a digital control pressure regulator, liquid-filled solenoid arrays, common liquid reservoirs, a custom enclosure, a microfluidic device, and a fluorescence macroscope.Attorney Docket No. 69414-702601

[0162] FIG. 18A illustrates an example of a trans-illumination setup implemented in a macroscopy imaging system. The system utilizes a macro lens configuration with a magnification of <lx to capture a full view of the sample. The trans-illumination subsystem comprises a light source positioned to provide uniform illumination through the sample, permitting high-quality imaging across a variety of microfluidic device configurations. The detector is configured to receive the transmitted light, capturing detailed optical signals from the full sample. The setup emphasizes uniform illumination and imaging, suitable for analyzing large-scale microfluidic devices with precision. The illustrated approach allows for the observation of the entire device in a single field of view, ensuring comprehensive analysis and accurate data collection.

[0163] In some embodiments, the system comprises at least one substrate. In some cases, the at least one substrate comprises at least one microfluidic device. In some instances, the at least one microfluidic device comprises a plurality of channels. In some cases, the at least one channel comprises at least one reaction chamber. In some instances, the at least one reaction chamber comprises one or more reservoir chambers fluidically coupled to the reaction chamber. For example, the at least one reaction chamber may be configured to facilitate chemical or biochemical reactions within the microfluidic device. In further examples, the reaction process is configured to permit precise fluid control and reaction monitoring.

[0164] In some embodiments, the system comprises at least one substrate. In some cases, the at least one substrate comprises at least one microfluidic device. In some instances, the at least one microfluidic device comprises a plurality of channels on a surface. In some cases, the at least one channel comprises at least one reaction chamber. In some instances, the at least one reaction chamber is an elongated reaction chamber. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.3%, at least about 6.5%, at least about 6.7%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 10.5%, at least about 11%, at least about 11.5%, at least about 12%, at least about 12.5%, at least about 13%, at least about 13.5%, at least about 14%, at least about 14.5%, at least about 15%, at least about 15.5%, at least about 16%, at least about 16.5%, at least about 17%, at least about 17.5%, at least about 18%, at least about 18.3%, at least about 18.5%, at least about 18.6%, at least about 18.7%, at least about 19%, at least about 19.5%, at least about 20%, at least about 20.5%, at least about 21%, at least about 21.5%, at least about 22%, atAttorney Docket No. 69414-702601 least about 22.5%, at least about 23%, at least about 24%, or at least about 25% of the surface. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 3% of the surface. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 6% of the surface. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 6.3% of the surface. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 18% of the surface. In some embodiments, the surface is configured to display one or more target biomolecules over at least about 18.6% of the surface. The surface, as referred to above, is an active surface that is a portion of the larger surface (e.g. a glass slide). The active surface, as referred to herein, refers to a portion of the larger surface comprising the plurality of reaction chambers. The active surface can be substantially covered by the plurality of reaction chambers. The active surface can have a boundary that is set by the outer sides of the plurality of reaction chambers. An example of the active surface is illustrated in the left panel of FIG. 22A, wherein the active surface is the portion of the glass slide covered by the grid formed by the plurality of reaction chambers and the active surface’s boundary can be set by the outermost sides of the outermost chambers in the grid. In some embodiments, the one or more target biomolecules comprises one or more target biomolecules described elsewhere herein. In some cases, the plurality of channels comprises one or more members positioned adjacent to the one or more reaction chambers and configured to move along an axis orthogonal to the one or more reaction chambers. In some embodiments, elongated reaction chamber comprises variable stretch values (e.g., stretch values described elsewhere herein), resulting in proportional fold changes to chamber volume and member area. In some embodiments, the chamber volume fold-change over the non-stretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10. In some embodiments, the member area fold-change over the nonstretched design comprises about 0.1 to about 0.2, from about 0.2 to about 0.3, from about 0.3 to about 0.4, from about 0.4 to about 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, from about 0.8 to about 0.9, from about 0.9 to about 1.0, continuing in this manner with a 0.1 step size up to about 9.9 to about 10. In some embodiments, the total internal volume of the device comprises about 0.1 to about 0.2 (*0.6 pL), from about 0.2 to about 0.3 (*0.6 pL), from about 0.3 to about 0.4 (*0.6 pL), from aboutAttorney Docket No. 69414-7026010.4 to about 0.5 (*0.6 pL), from about 0.5 to about 0.6 (*0.6 pL), from about 0.6 to about 0.7 (*0.6 pL), from about 0.7 to about 0.8 (*0.6 pL), from about 0.8 to about 0.9 (*0.6 pL), from about 0.9 to about 1.0 (*0.6 pL), continuing in this manner with a step size of 0.1 up to about 9.9 to about 10 (*0.6 pL). In some embodiments, stretching a reaction chamber by a specified stretch value increases the internal volume and surface area of the microfluidic device (e.g., see FIG. 8A-8D). In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2 and a member area fold change of about 3 over the nonstretched design. In some embodiments, a stretch value of 200 microns generates a chamber volume fold change of about 2.1 and a member area fold change of about 2.8 over the nonstretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3 and a member area fold change of about 5 over the nonstretched design. In some embodiments, a stretch value of 400 microns generates a chamber volume fold change of about 3.2 and a member area fold change of about 4.6 over the nonstretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4 and a member area fold change of about 7 over the nonstretched design. In some embodiments, a stretch value of 600 microns generates a chamber volume fold change of about 4.3 and a member area fold change of about 6.45 over the nonstretched design. In some embodiments, a stretch value of 800 microns generates a chamber volume fold change of about 5 and a member area fold change of about 8 over the nonstretched design. In some embodiments, a stretch value of 800 microns generates a chamber volume fold change of about 5.5 and a member area fold change of about 8.3 over the nonstretched design. In some embodiments, a stretch value of 200 microns generates a configuration of approximately 1200 chambers per chip. In some embodiments, a stretch value of 200 microns generates a configuration of approximately 6000 chambers per chip. In some embodiments, a stretch value of 400 microns generates a configuration of approximately 900 chambers per chip. In some embodiments, a stretch value of 400 microns generates a configuration of approximately 4500 chambers per chip. In some embodiments, a stretch value of 600 microns generates a configuration of approximately 700 chambers per chip. In some embodiments, a stretch value of 600 microns generates a configuration of approximately 3500 chambers per chip. In some embodiments, a stretch value of 800 microns may lead to a configuration of approximately 3000 chambers per chip. In some embodiments, the surface has an area greater than about 1065 mm2. In some embodiments, the surface has an area greater than about 1100 mm2. In some embodiments, the surface has an area of about 1065 mm2. In some embodiments, the surface has an area of about 1100 mm2. In someAttorney Docket No. 69414-702601 embodiments, the surface has an area of about 1300 mm2. In some embodiments, the surface has an area of about 1325 mm2. In some embodiments, the surface has an area of about 1400 mm2. In some embodiments, the surface has a dimension of about 25.5 mm by 43.5 mm. In some embodiments, the surface has a dimension of about 25 mm by 43 mm. In some embodiments, the surface has a dimension of about 26 mm by 44 mm. In some embodiments, the surface has a dimension of about 28.8 mm by 46 mm. In some embodiments, the surface has a dimension of about 28 mm by 46 mm. In some embodiments, the surface has a dimension of about 29 mm by 46 mm. In some embodiments, a number of chambers comprises about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, from about 900 to about 1000, from about 1000 to about 1100, from about 1100 to about 1200, from about 1200 to about 1300, from about 1300 to about 1400, from about 1400 to about 1500, from about 1500 to about 1600, from about 1600 to about 1700, from about 1700 to about 1800, from about 1800 to about 1900, from about 1900 to about 2000, from about 2000 to about 2100, from about 2100 to about 2200, from about 2200 to about 2300, from about 2300 to about 2400, from about 2400 to about 2500, from about 2500 to about 2600, from about 2600 to about 2700, from about 2700 to about 2800, from about 2800 to about 2900, from about 2900 to about 3000, from about 3000 to about 3100, from about 3100 to about 3200, from about 3200 to about 3300, from about 3300 to about 3400, from about 3400 to about 3500, from about 3500 to about 3600, from about 3600 to about 3700, from about 3700 to about 3800, from about 3800 to about 3900, from about 3900 to about 4000, from about 4000 to about 4100, from about 4100 to about 4200, from about 4200 to about 4300, from about 4300 to about 4400, from about 4400 to about 4500, from about 4500 to about 4600, from about 4600 to about 4700, from about 4700 to about 4800, from about 4800 to about 4900, from about 4900 to about 5000, from about 5000 to about 5100, from about 5100 to about 5200, from about 5200 to about 5300, from about 5300 to about 5400, from about 5400 to about 5500, from about 5500 to about 5600, from about 5600 to about 5700, from about 5700 to about 5800, from about 5800 to about 5900, from about 5900 to about 6000, from about 6000 to about 6100, from about 6100 to about 6200, from about 6200 to about 6300, from about 6300 to about 6400, from about 6400 to about 6500, from about 6500 to about 6600, from about 6600 to about 6700, from about 6700 to about 6800, from about 6800 to about 6900, or from about 6900 to about 7000.Attorney Docket No. 69414-702601

[0165] In some embodiments, the system comprises at least one transillumination subsystem. In some cases, the at least one transillumination subsystem comprises at least one light source. In some instances, the at least one light source is configured to illuminate the microfluidic device for imaging purposes. In some cases, the at least one transillumination subsystem further comprises at least one detector. In some instances, the at least one detector is configured to detect light transmitted through the microfluidic device. For example, the at least one transillumination subsystem may be configured to provide uniform illumination and high-quality optical signal detection for imaging. In further examples, the detection process is configured to enhance image clarity and permit real-time or near real-time monitoring of biochemical events.

[0166] As an example, the system may also incorporate a macroscope configured to capture images with <lx magnification. In some instances, the macroscope includes a tandem macro lens system to ensure high-resolution imaging of the full sample area. Additionally, the system may include emission filters within the optical path to improve the signal-to-noise ratio for fluorescence-based imaging. For instance, the macroscope may be configured to analyze kinetics of biochemical events involving fluorophores, either surface-immobilized or in solution. In further examples, the system may be adapted for both trans-illumination and side-on illumination, providing versatile imaging options for various experimental setups.

[0167] FIG. 18B illustrates the configuration and functionality of a trans-illumination setup with an emphasis on excitation and emission pathways for imaging in a macroscopy system. This figure highlights the interaction of excitation and emission light within the sample, demonstrating how the trans-illumination process facilitates optical signal detection. The excitation light is directed downward through the sample, permitting the excitation of fluorescent or optically active elements within the microfluidic device. The emitted light, corresponding to the detected signal is captured by the optical setup positioned below the sample. This demonstrates the dual role of the trans-illumination system: delivering precise excitation light and detecting the emitted signal with high fidelity. This configuration adds functionality by showcasing how trans-illumination can support fluorescence imaging or other light-based analyses, ensuring effective excitation and efficient signal capture. It complements the macroscope system shown in FIG. 18A by incorporating fluorescence capabilities, broadening the system's applications in biochemical, biological, and microfluidic analyses.

[0168] FIG. 19 illustrates an example of light collimation and expansion, as well as filters for excitation and emission. In some embodiments, the microfluidic device imaging system mayAttorney Docket No. 69414-702601 display an example set of components for light collimation and expansion, in addition to filter wheels for excitation and emission. These components may include a liquid light guide adapter for channeling light into the system, retainer rings for securing optical components, a fly’s eye homogenizer for achieving uniform illumination, a collimator for directing light, a thread adapter for component integration, and a lens tube for housing the optical pathway. Additionally, the figure illustrates a filter wheel that houses multiple 2-inch filters, including a 4 mm spacer, a custom 3D printed filter cell, and two stacked 2-inch filters for emission control. This configuration facilitates precise wavelength selection and uniform light distribution, ensuring accurate and reliable imaging in fluorescence or other wavelengthsensitive applications.

[0169] In some embodiments, a method for operating an imaging system is provided. In some cases, the method comprises illuminating a specimen with uniform light from a Fly’s eye homogenizer. In some instances, the illumination involves using the Fly’s eye homogenizer to ensure even distribution of light across the specimen.

[0170] In some embodiments, the method comprises detecting the transmitted light from the specimen. In some cases, the detection involves using a macroscope. In some instances, the macroscope comprises a macro lens system to capture the transmitted light.

[0171] For example, the method may include creating an overview image of the specimen. In some cases, this involves capturing light from the Fly’s eye homogenizer as processed through the macroscope. In further examples, the method may include capturing light from a shallow region centered on the specimen plane. In some instances, this is achieved by the macroscope focusing on the desired plane.

[0172] As an example, the method may also involve analyzing fast kinetics, such as those of biochemical events involving surface-immobilized fluorophores. This process may provide detailed insights into biochemical interactions occurring on the specimen surface.

[0173] In some embodiments, the method may further include analyzing the fast kinetics of biochemical events involving solution-phase fluorophores. In some embodiments, the method may further include analyzing kinetics of enzyme reactions. In some cases, the enzyme is surface immobilized.

[0174] In some embodiments, the method may further involve analyzing kinetics of enzyme reactions. In some cases, the enzyme is in solution. In some embodiments, the method may further include controlling fluidic movements by a hydraulic microfluidic control system comprising a common reservoir feeding solenoid valves filled with liquid.Attorney Docket No. 69414-702601

[0175] FIG. 17A - FIG. 17C represent examples of an integrated optics and hydraulics setup with a controlling system. As shown, the system may comprise water reservoirs, hydraulic system control, connectors, tubing, and fixtures.

[0176] In some embodiments, the system comprises at least one high-throughput microfluidic device. In some cases, at least one high-throughput microfluidic device comprises an integrated fluidic control and optical setup. This setup, in some instances, includes a compact structure that allows for an increased capacity in terms of the number of addressable control lines and flow lines. For example, at least one high-throughput microfluidic device may be used to perform enzyme activity assays for surface scanning mutagenesis libraries to elucidate allostery. In further examples, the high-throughput microfluidic device is configured to facilitate highly simultaneous screenings of small molecules against various targets. In further examples, incorporation of fewer components into the setup enhances the robustness and scalability of the device, thus presenting it as a promising tool for efficient drug discovery processes.

[0177] In some embodiments, the system comprises at least one microfluidic device control system. In some cases, at least one microfluidic device control system includes a liquid reservoir located downstream of the solenoids. In some instances, the solenoid operates while filled with pressurized air. In some cases, the microfluidic device control system may utilize one liquid reservoir for each solenoid valve. For instance, the system accommodates a 1 : 1 ratio of liquid reservoirs to on-chip control valves and has the capability to handle 18 or more valves.

[0178] In some embodiments, the system comprises at least one integrated optics and hydraulics setup. In some cases, at least one integrated optics and hydraulics setup comprises at least one digital flow pressure regulator. In some instances, at least one digital flow pressure regulator is configured to modulate pressure for fluid flow. In some cases, at least one integrated optics and hydraulics setup comprises at least one digital control pressure regulator. In some instances, at least one digital control pressure regulator is configured to regulate pressure for control valves.

[0179] For example, at least one integrated optics and hydraulics setup may include arrays of liquid-filled solenoids, which may be filled with liquid for optimal functioning. In further examples, the system may include at least one common liquid reservoir configured to serve as shared storage for the system’s liquid. As an example, at least one integrated optics and hydraulics setup may also include a custom enclosure configured to provide specialized housing for the system.Attorney Docket No. 69414-702601

[0180] In some embodiments, the system comprises at least one microfluidic device. In some cases, at least one microfluidic device is positioned within the integrated optics and hydraulics setup, where the intended operations may take place. In further examples, the system may include at least one fluorescence macroscope configured to offer visual inspection or recording of the system’s activity.

[0181] FIG. 16 illustrates an example of an integrated optics and hydraulics setup. This setup may comprise a digital flow pressure regulator, a digital control pressure regulator, liquid- filled solenoid arrays, common liquid reservoirs, a custom enclosure, a microfluidic device, and a fluorescence macroscope. In some embodiments, the system comprises at least one integrated setup for controlling multiple devices. In some cases, at least one integrated setup comprises at least one microfluidic control system. In some instances, at least one microfluidic control system comprises a configuration for controlling up to four devices simultaneously. In some cases, at least one integrated setup comprises at least one computational module. In some instances, at least one computational module is configured to manage and analyze data from high-throughput enzymology experiments.Applications of the Systems and Microfluidic Devices

[0182] FIG. 1A provides an example of an affinity selection process that can be performed onto many proteins simultaneously. Affinity selections performed onto many proteins at once refers to a process in which the binding strength (affinity) of different molecules to various proteins is tested simultaneously in a high-throughput manner. Rather than evaluating each potential drug-target interaction one at a time, this high-throughput approach allows for the rapid identification of promising molecules that show high affinity to their target biomolecules. As shown in the FIG. 1A, the process comprises three steps. The first step, binding, depicts three example proteins bonding from protein 1 to protein 3, with each protein showcasing different sites that interact with members of a library of potential interactors. The second step, washing, involves removing individual library members that do not interact with the target, leaving only the interacting members. The final step, elution and identification, leads to the identification of each specific interactor and protein site, which may include an allosteric or an active-site pocket. FIG. IB illustrates an example of forward and reverse screening workflows in tandem. In some embodiments, forward screens identify all target binders, while reverse screens localize binders to specific regions or mutations in the target biomolecule, permitting comprehensive interaction analysis. In some embodiments, applications of the microfluidics devices described herein include a forward screen that may identify all target binders (such as an example of a wild type of protein equipped with bindingAttorney Docket No. 69414-702601 pocket 1 and binding pocket 2), which interactors may engage with. Applications of the microfluidics device may also include reverse screens that may localize binders (such as a protein with mutation 1 interacting with the first interactor but not interacting with a second interactor, and mutation 2 interacting with the second interactor but not the first interactor). FIG. 1C provides an example of how mutational scanning identifies sites to screen. Using the microfluidic device described herein to assay enzyme reaction kinetics for each mutant may identify which mutations have a significant effect on enzyme activity (e.g., such as that shown in FIG. 1C). The figure illustrates the results of mutational scanning to identify critical sites within a protein structure. On the left side of the FIG. 1C, a three-dimensional model of a wild-type (WT) protein is shown, with its sequence labeled as“MPEEKSAVT. . PENFR.” Several mutants (Mut 1, Mut 2, Mut 3, . . . , Mut N) are listed, each featuring specific amino acid substitutions (e.g., “MAEEKSAVT. . .PENFR” for Mut 1), culminating in a final mutant (Mut N) with a substitution at the end of the sequence. The right side of the FIG. 1C, displays a 3D model highlighting regions with significant effects upon mutation marked in a darker color.

[0183] FIG. 3 illustrates an example process of interactor selection with in situ barcoding. In vitro expression plasmids and corresponding in situ barcodes may be printed on a glass slide prior to aligning a microfluidic device onto the glass slide, which results in the barcodes and expression plasmids being partitioned in separate reservoir chambers. After the microfluidic device is aligned, as depicted in the FIG. 3, the selection process may start with preparing the chip for expression where components like biotinylated BSA, neutravidin, or biotinylated antibody are introduced into the reaction chamber, with the neck valves being closed to separate the reservoir chambers from the reaction chamber. In vitro expression components are then flowed onto the chip and one set of neck valves corresponding to the reservoir chamber containing the in vitro expression plasmid is opened, allowing for protein to be expressed from the plasmid, migrate to the reaction chamber, and immobilize to a surface in the reaction chamber. Next, an interactor library is flowed into the chip and members of the library are allowed to interact with the immobilized target biomolecule. The next step involves opening the second set of neck valves corresponding to the reservoir chamber containing the in situ barcodes to resuspend the in situ barcodes and using DNA ligase to attach those barcodes onto the members of the interactor library that are bound to the immobilized target biomolecule in the reaction chamber. Finally, in situ barcoded interactors are eluted from the reaction chamber using reagents such as proteinase K or other solutions to reverse the interaction between the interactor and the target biomolecule or cause theAttorney Docket No. 69414-702601 interactor-target pair to become unbound from the reaction chamber surface or cleave one or more surface-immobilized components to allow the interactor and one or more associated molecular components to become unbound. The barcodes from the target biomolecules, interactors, or both, can be processed (e.g., with high throughput sequencing) to identify specific interactions between the target biomolecule and the interactors.

[0184] The systems and devices described herein are particularly adapted for the applications described herein. The methods described herein are particularly efficient when applied to the systems and devices described herein. FIG. 4 illustrates an example of the relative abundance of recovered vs loaded simulated DNA-encoded Libraries (DELs). The x-axis represents the abundance of simulated DEL members loaded into the chip, counted as the relative proportion of unique molecular identifiers (UMIs) present for each unique simulated DEL member. The y-axis represents the abundance of simulated DEL members recovered after performing in situ barcoding and eluting from the chip, counted as the relative proportion of unique molecular identifiers (UMIs) present for each unique simulated DEL member. Data points show relative UMI counts for different in situ barcodes ligated onto simulated DEL members on-chip (barcode 2, barcode 3, barcode 4) and off-chip control, and a dashed line showing the expected results. According to R squared values shown in the figure, there is mostly a linear relationship between the loaded and recovered simulated DEL members, demonstrating that the system is quantitative across multiple orders of magnitude and that in situ barcoding does not systematically affect recovery of flowed on simulated interactor members.

[0185] Similar to FIG. 3, FIG. 5 illustrates an example assay utilizing the systems and methods described herein. Specifically, FIG. 5 describes the processes of target expression, interaction, in situ barcode ligation, elution, sequencing, and target identification using barcodes. In some embodiments, positive control interactors with known affinity for a target biomolecule demonstrate higher binding to the target compared to negative control proteins (e.g., showcasing the system's specificity and reliability). The microfluidic device may contain multiple targets in reservoir chambers, with corresponding barcodes in one or more fluidically coupled reservoir chambers. The target biomolecule may be expressed from the reservoir chambers and immobilized in the reaction chamber. A library of potential small molecule interactors ligated to DNA barcodes may be flowed on to bind to the target biomolecules. The barcodes from the reservoir chamber may then be ligated onto the target biomolecule, bound members of the interactor library, or both, which may be subsequently eluted. The target biomolecule barcodes, the bound interactor barcodes, or both, may then beAttorney Docket No. 69414-702601 sequenced to identify the interactors themselves and the protein that they interacted with on the chip (or a specific binding site on the protein). This may then be used to identify which interactors are bound to which protein. Positive control interactors that have known affinity for a given target biomolecule may display more binding to the target biomolecule than to a negative control protein for which it does not have significant affinity. FIG. 5 shows an example system of two reaction chambers. A first reaction chamber is connected to a reservoir chamber containing a barcode 1 and another reservoir chamber containing a target plasmid. A second reaction chamber is connected to a reservoir chamber containing a barcode 2 and another reservoir chamber containing a negative control plasmid. The process includes expressing targets and flowing on the interactor library. Then the barcode 1 and barcode 2 are introduced into the first and second reaction chamber respectively. The process includes ligating barcodes onto the bound interactors in situ and eluting bound and barcoded interactors. The interactors are then sequenced to determine the identity of the interactors and the protein that they interacted with (e.g., target or negative control).

[0186] FIG. 6 illustrates an example of how temporal barcoding tags reaction products based on the order of generation. As shown in FIG. 6, at Timepoint 1, barcode 1 may be introduced into the reaction chamber when the neck valves to the reservoir chambers are closed. At Timepoint 2, barcode 2 may be introduced into the reaction chamber and neck valves to the reservoir chambers may also be closed.

[0187] FIG. 12 illustrates an example of an on-chip binding assay for discovering allosteric inhibitors, including the workflow and data analysis steps. Two clinical candidate allosteric inhibitors are used to demonstrate the workflow, though the system is adaptable for broader applications. As shown in the example of FIG. 12, two clinical candidate allosteric inhibitors of SHP2 may be spiked in a DEL library, which may interact with SHP2 and eGFP. The bound fraction may be collected and sequenced to determine binders and their rank order affinities. Both clinical candidates may be successfully recovered, with their fraction of unique molecular identifiers reflecting their relative reported IC50s. The graph titled as the "Resolve rank order affinities” illustrates the analysis of bound interactors using in situ barcodes to determine target identity by comparing the binding of an interactor library to the target versus a negative control. The scatterplot maps the target bound fraction (e.g., SHP2 binding) (%) on the x-axis against the negative control bound fraction (e.g., eGFP binding) (%) on the y-axis, with each data point representing an individual interactor from the library. The diagonal dashed line serves as a threshold for specificity, indicating equal binding to the target and negative control. Most interactor library members are clustered near the origin,Attorney Docket No. 69414-702601 showing low binding to both the target and the negative control, which suggests non-specific or weak interactions. Notably, two positive controls (Novartis TNO-155 tool compound as a Positive Control 1 and Revolution Medicines RMC -4550 tool compound as a Positive Control 2) are highlighted as outliers, displaying significantly higher binding to the target compared to the negative control. These positive controls validate the accuracy of the assay, confirming the ability of the method to identify specific interactors among the libraries.

[0188] FIG. 13A-B illustrates an example of chamber-specific in situ barcoding of interactions. A device configuration may include an extra reservoir chamber containing printed barcodes and additional valving. In some cases, the device configuration permits in situ barcoding of the bound fraction with single-stranded or double-stranded DNA to link the identity of interactors to their target.

[0189] For example, at least one integrated setup may be configured to permit prospective discovery of allosteric drug candidates by screening protein / small-molecule interactions. In further examples, the screening process is configured to identify and validate therapeutic interventions for traditionally “undruggable” targets.

[0190] In some embodiments, the system addresses challenges in drug discovery for targeting enzyme active sites. In some cases, active-site targeting drug candidates often fail during development due to poor cell permeability or off-target toxicity caused by a highly charged or conserved active site. In some instances, the system is configured to permit the identification of allosteric modulators, which bind outside the active site and offer an improved therapeutic approach for diseases such as cancer, diabetes, and autoimmune disorders.

[0191] For example, the system may provide a microfluidic platform capable of high- throughput screening for protein / small-molecule interactions. In further examples, this platform may dramatically enhance the ability to discover therapeutic candidates for diseases refractory to traditional drug discovery efforts. The platform may lay the foundation for a scalable experimental and computational system for therapeutic enzymology.

[0192] In some embodiments, the system comprises at least one microfluidic discovery engine. In some cases, at least one microfluidic discovery engine comprises at least one functional assessment module. In some instances, at least one functional assessment module is configured to measure the biophysical properties of enzyme mutants, such as activity and stability. In some cases, at least one microfluidic discovery engine comprises at least one surface scanning mutagenesis platform. In some instances, at least one surface scanning mutagenesis platform is configured to identify candidate allosteric sites on the surface of an enzyme.Attorney Docket No. 69414-702601

[0193] For example, at least one microfluidic discovery engine may be configured to process and analyze more than 2000 mutants at high throughput. In further examples, the system may leverage surface scanning mutagenesis to elicit candidate allosteric sites on a therapeutically valuable, under-drugged human enzyme. As an example, this system may support an improved, high-throughput screening assay to identify site- and target-specific allosteric binders.

[0194] In some cases, the system addresses the need for identifying candidate allosteric sites, discovering molecules that bind specifically to these sites, and determining if these binders induce the desired functional effects. In some instances, site-directed mutagenesis may be used to localize binding sites, where the mutational site is the binding site if binding is ablated.

[0195] For example, at least one microfluidic discovery engine may offer a scalable, low-cost solution for accurately measuring multi-parameter functional readouts of enzyme mutants. In further examples, this platform may establish a foundation for high-throughput therapeutic enzymology, permitting the prospective discovery of allosteric drugs with site- and targetspecific efficacy. In some embodiments, the systems described herein can increase throughput and accuracy of mutational studies while reducing the cost.

[0196] In some cases, all variants may be assayed simultaneously by flowing fluorogenic substrates into the device, isolating the reaction chambers via valving, and measuring enzyme activity through time-lapse fluorescence imaging. In some instances, as the variants are surface immobilized the products of a reaction may be washed out and variants may be reassayed to measure a broad range of biophysical properties including Michaelis-Menten kinetics. In some cases, High-Throughput Microfluidic Enzyme Kinetics (HT-MEK) conducted on the systems and devices described herein may permit biophysical measurements with 1000-fold miniaturization and efficiency (e.g., by simultaneously screening a plurality of variants) compared to classical enzymology.

[0197] In some embodiments, the systems described herein comprise at least one therapeutic discovery platform. In some cases, at least one therapeutic platform may comprise at least one enzymology engine. In some instances, at least one enzymology engine may be configured to rapidly assay more than 2000 human enzymes. In some cases, at least one therapeutic platform comprises at least one surface scanning mutant library. In some instances, at least one surface scanning mutant library may be configured to discover allosteric surface sites on under drugged human protein phosphatases of high therapeutic value.Attorney Docket No. 69414-702601

[0198] For example, at least one therapeutic platform may be configured to perform high- throughput enzymology assays at a fraction of the cost of existing high-throughput microfluidic enzymology platforms (e.g., HT-MEK). In further examples, the system may establish proof-of-concept for an improved on-chip binding assay configured to identify allosteric small molecules that are both site- and target-specific. As an example, this approach may address limitations of current methods, such as complexity, imaging speed constraints, and unsuitability for industrial use.

[0199] In some embodiments, the systems described herein may be configured to overcome the limitations of current HT-MEK platforms. In some cases, the systems described herein aim to provide a reliable, scalable, and cost-effective solution for analyzing biophysical processes and performing high-throughput screening assays for hit discovery. In some instances, the system may be configured to identify allosteric sites and discover small molecules with therapeutic potential, permitting new drug discovery approaches for challenging targets.

[0200] For example, the systems described herein may integrate rapid enzymology assays with mutant library screening to discover and validate allosteric binding sites. In further examples, the system may establish a robust, scalable tool for therapeutic development, permitting site- and target-specific discovery of allosteric small molecules. This innovative approach aims to advance therapeutic enzymology by addressing the speed, complexity, and cost challenges of current platforms. In some embodiments, the system comprises at least one imaging and control setup. In some cases, at least one imaging and control setup comprises at least one optical imaging module. In some instances, at least one optical imaging module is configured to provide sensitive, large field-of-view (FOV) fluorescence imaging at a fraction of the cost of existing setups. In some cases, at least one imaging and control setup may comprise at least one fluidic control system. In some instances, at least one fluidic control system may be configured to reduce experimental complexity while retaining full functionality to improve the success rate of experiments.

[0201] For example, at least one imaging and control setup may be configured to permit the quantification of fast biophysical reactions while scaling cost-effectively for multiple experimental setups. In further examples, the imaging module and fluidic control system may work in tandem to enhance experimental throughput and reliability. As an example, this setup may integrate seamlessly with computational frameworks to analyze results.

[0202] In some embodiments, the system may comprise at least one computational framework. In some cases, at least one computational framework may comprise modules forAttorney Docket No. 69414-702601 analyzing results from site-directed mutagenesis experiments. In some instances, the computational framework may be configured to rediscover previously known allosteric sites and / or may identify improved allosteric sites for therapeutically valuable protein phosphatases.

[0203] For example, the system may include capabilities for simultaneous screening of small molecule libraries against enzymes of interest. In further examples, the system may integrate sequencing technologies and computational analysis to resolve rank order affinities of binders. As an example, the system may screen large commercial DNA-encoded chemical libraries (DELs) to identify both known and improved allosteric binders.

[0204] In some embodiments, the system may comprise at least one therapeutic platform. In some cases, at least one therapeutic platform comprises at least one experimental setup. In some instances, at least one experimental setup comprises optics and fluidics capable of automated multi-channel fluorescence imaging of an entire microfluidic device. In some cases, at least one experimental setup comprises at least one imaging module configured to provide high sensitivity while being robust, cost-effective, and scalable. In some instances, at least one fluidic component is configured to support microfluidic assays at reduced reaction volumes.

[0205] For example, the at least one therapeutic platform may be configured to benchmark performance against current setups, improving at least two performance metrics such as field- of-view (FOV), sensitivity, cost, or robustness while maintaining comparable performance in others. In further examples, this setup may streamline high-throughput assays. As an example, the platform may scale reaction volumes down from 100 pL to 1 nL while retaining the sensitivity configured to resolve rank order affinities of binders.

[0206] In some embodiments, the system comprises at least one target assessment module. In some cases, at least one target assessment module comprises tools for expressing full-length enzyme targets in sufficient quantities. In some instances, this module is configured to assay targets at high throughput while maintaining exceptional accuracy to detect subtle functional effects.

[0207] For example, at least one target assessment module may facilitate rediscovery of previously known allosteric sites on protein phosphatases of interest. In further examples, the module may perform a comprehensive surface scanning study on targets that demonstrate the highest accuracy in recapitulating known allosteric sites.

[0208] In some embodiments, the system comprises at least one high-throughput binding assay module. In some cases, at least one binding assay module comprises tools for screeningAttorney Docket No. 69414-702601DNA-encoded libraries (DELs) containing millions of interactions. In some instances, at least one binding assay module may use an improved device configuration or on-chip molecular biology to encode target identities spatially or with DNA tags.

[0209] For example, the binding assay module may be configured to rediscover previously known allosteric binders spiked into DEL libraries and identify new binders with site-specific and target-specific binding properties. In further examples, the system may establish proof- of-concept for high-throughput on-chip assays (e.g., assays capable of testing multiple targets and / or target variants simultaneously) capable of transformative therapeutic discovery, demonstrating scalability and cost-effectiveness while resolving complex binding interactions.

[0210] For example, at least one therapeutic discovery platform may be configured to systematically and scientifically probe the feasibility of allosteric drug discovery for underdrugged human enzymes. In further examples, the proof-of-concept module may be configured to detect subtle allosteric effects or identify suitable binders emerging from screening efforts. As an example, this platform may also address market risks by demonstrating the discovery of improved chemical matter, such as hits and leads, alongside a robust and scalable discovery process within limited capital and time constraints.

[0211] For example, the development system may include modules for screening underdrugged human enzymes to identify potential binders. In further examples, these modules may refine experimental conditions to maximize the detection of subtle functional effects and ensure reliable output for downstream applications. This stepwise and methodical approach aims to minimize risks while advancing the feasibility of the platform for transformative therapeutic discoveries.EXAMPLES

[0212] The following illustrative examples are representative of embodiments of the applications, systems, and methods described herein and are not meant to be limiting in any way.Example 1: Optimizing On-Chip Protein Expression for Allosteric Site Discovery in Therapeutically Relevant Phosphatases

[0213] In this case, systems and methods for high-throughput screening were provided to effectively optimize on-chip protein expression of human protein phosphatases. Specifically, the system, resulted in enhanced expression levels of SHP2 (residues 1-525) and PTP1B (residues 1-403).Attorney Docket No. 69414-702601

[0214] Initially, human protein phosphatases were provided as the enzymes of interest, including SHP2 and PTP1B. SHP2 and PTP1B are two examples of undrugged protein phosphatases associated with various diseases, including solid tumors, diabetes, and autoimmune disorders.

[0215] Next, an initial optimization of on-chip in vitro expression of SHP2 and PTP1B was performed using an NEB PURExpress kit. The assay was quantified using a GFP tag fused to the enzyme of interest (FIG. 10A).

[0216] The optimization process involved systematically testing various buffer compositions (i.e., different concentrations of BSA, trehalose, and NaCl in DI water) to determine the optimal conditions for printing plasmid DNA on glass slides. A buffer composition of 10 mg / ml BSA, 12 mg / ml trehalose, and 10 mM NaCl was selected. This buffer consistently yielded the highest expression levels and facilitated visualization of the printed spots and provided precise alignment with the microfluidic device.

[0217] Following the optimization of buffer conditions, an additional study evaluated the effect of different DNA purification methods on expression levels. The Zymo miniprep kit produced an order of magnitude more DNA than the previously used Qiagen kit, further enhancing expression when combined with the optimized print buffer. FIG. 10B illustrates an example of the effect of a miniprep kit and print buffer on protein expression. In this case, the protein was a SHP2 1-525 protein. The figure illustrates the effect of using an optimized miniprep kit and print buffer on the expression of the target enzyme SHP2 (residues 1-525). The vertical axis represents the on-chip expression of the target enzyme in relative fluorescence units (RFU) after subtracting background values, plotted on a logarithmic scale. As shown in FIG. 10B, three conditions were evaluated: the previously reported protocol, the optimized print buffer alone, and the combination of the optimized print buffer with the Zymo miniprep kit. The results demonstrate a stepwise improvement in expression levels, with the optimized print buffer alone surpassing the previous protocol and the combination of the optimized print buffer and Zymo miniprep kit yielding the highest expression levels.

[0218] Next, the effect of printed plasmid DNA concentration for on-chip protein expression was studied. As illustrated in FIG. 10C, protein yield did not exhibit a direct correlation with printed DNA concentration, emphasizing the necessity for individual optimization for each enzyme. Data was obtained to compare the expression levels of SHP2 (residues 1-525) and PTP1B (residues 1-403) across DNA concentrations ranging from 25 to 400 ng / pl. SHP2 consistently showed higher expression levels than PTP1B across all concentrations. Notably,Attorney Docket No. 69414-702601 the difference in expression levels between the two enzymes was more pronounced at lower DNA concentrations, the difference being reduced as DNA concentration increased.Example 2: On-Chip Activity Assays for Human Protein Phosphatases Using a Fluorogenic Substrate

[0219] In this case, systems and methods for high-throughput screening were provided that accurately perform on-chip activity assays of human protein phosphatases. Specifically, the initial activity assays for Wild Type (WT) and selected mutants of PTP1B (residues 1-321) and SHP2 (residues 1-525) exhibited a high level of reproducibility in measuring both kcat and K\\ across multiple experiments.

[0220] Initially, human protein phosphatases were provided as the enzymes of interest, including SHP2 and PTP1B. SHP2 and PTP1B are two examples of undrugged protein phosphatases associated with various diseases, including solid tumors, diabetes, and autoimmune disorders.

[0221] In this case, on-chip activity assays were conducted using a fluorogenic substrate DiFMUP, which generates a fluorescent signal upon enzymatic activity. Here, the system was configured to test WT and selected mutants of PTP1B (residues 1-321) and SHP2 (residues 1-525) to determine their catalytic turnover rates (£cat) and Michaelis-Menten constants KM across two independent experiments.

[0222] FIG. HA illustrates structural models of PTP1B and SHP2. FIG. 11B and FIG. 11C illustrate the reproducibility of kcaland KM measurements, respectively, for these enzymes.

[0223] As shown in FIG. 11B, kcat values for both PTP1B and SHP2 variants displayed a high level of reproducibility, with data points clustering tightly along the unity line between experiments. The x-axis represents kcalvalues measured in Experiment 1, while the y-axis represents kcalvalues measured in Experiment 2. The close agreement between the two axes underscores the consistency of the measurements across replicates. SHP2 variants (green) and PTP1B variants (blue) showed minimal deviation across experiments, underscoring the reliability of the system. The data was obtained over a range of kcalvalues from approximately 0.01 to 100 s-1. This demonstrated the ability of the system to measure both low and high catalytic turnover rates. Here, most variants fall within the 2-fold agreement boundaries, demonstrating the ability of the system to consistently reproduce accurate kinetic parameter measurements across diverse enzyme variants. The deviation from the unity line shows reproducibility of the system.

[0224] As shown in FIG. 11C, KM values for both PTP1B and SHP2 variants exhibited a high level of reproducibility, with data points clustering tightly along the unity line betweenAttorney Docket No. 69414-702601 experiments. The x-axis represents KM values measured in Experiment 1, while the y-axis represents K values measured in Experiment 2. The close alignment of data points along the unity line illustrates the consistency of the measurements across replicates. SHP2 variants (green) and PTP1B variants (blue) displayed minimal deviation across experiments, underscoring the reliability of the system.

[0225] The data covered a broad dynamic range of KM values, spanning approximately 1 to 1000 pM, demonstrating the system's ability to accurately measure both high- and low- affinity substrate binding. Variants that deviated significantly from the unity line, including those with larger error bars, either represent outliers or reflect unique structural or allosteric influences affecting substrate binding. Here, most variants, however, fell within the 2-fold agreement boundaries, further validating the system's reproducibility and robustness in measuring binding parameters across diverse enzyme variants. These findings confirm that the high-throughput system provides a reliable and precise platform for quantifying both substrate binding (KM) and enzymatic turnover (£cat) across a wide range of protein phosphatase variants.Example 3: Benchmarking and Surface Scanning for Allosteric Site Discovery in Human Protein Phosphatases

[0226] In this case, a system is provided that rapidly discovers allosteric sites in human protein phosphatases.

[0227] Here, the system is configured to validate on-chip measurements of biophysical constants, including kcat, KM, and £cat / KM, for target biomolecules, like human protein phosphatases (such as SHP2 and PTP1B). This validation ensures the accuracy and reliability of the on-chip system, providing its application in systematic surface scanning to identify target allosteric sites.

[0228] First, the system measures biophysical constants, including kcat, KM, and kcat / KM, for WT and selected mutants of SHP2 and PTP1B (as examples) on-chip. These measurements are benchmarked, such as against off-chip data generated using alternative methods to establish a baseline of accuracy.

[0229] If discrepancies are observed between on-chip and off-chip measurements, the system implements systematic optimizations. These optimizations include exploring different designs, linking strategies, and positions for the protein tag used for quantification and purification of the expressed enzyme (with data currently shown for a C-terminus eGFP tag). Additionally, truncated versions of SHP2 and PTP1B are assessed to identify constructs with enhanced stability and activity.Attorney Docket No. 69414-702601

[0230] To ensure properly folded and active enzymes, cell-free expression conditions are optimized by systematically adjusting parameters such as temperature, reaction time, and additives like disulfide bond enhancers. In this case, the system is compatible with existing commercial assays, which can be adapted for on-chip use. Alternatively, different on-chip fluorogenic assays are introduced to further enhance measurement precision and sensitivity.

[0231] Next, mutations are introduced to generate a surface scanning library for the system (SHP2 or PTP1B). Each surface position is mutated to at least two residues with distinct biophysical properties, such as valine and glycine, to systematically evaluate their impact on enzyme function and binding (e.g., allosteric binding). Measurements of biophysical constants (£cat, KM, and kcalI KM are performed on-chip for each mutant, and functional effects are analyzed to determine if they result from gross misfolding, equilibrium unfolding, or the presence of long-lived misfolded proteins. This process investigates whether specific mutations influence the enzyme's structure or function and elucidates potential allosteric effects.

[0232] Next, the surface scan focuses on re-discovering known allosteric sites for SHP2 and PTP1B, validating the ability of the system to identify functionally relevant regions. This information is then used to establish the computational framework necessary to determine whether a surface site is allosteric. The full surface scan is executed only for the enzyme system (SHP2 or PTP1B) with comparable on-chip measurements, off-chip data, and literature values, ensuring reliable and accurate results.

[0233] If on-chip measurements fail to align with off-chip data for either system, additional optimizations are applied to refine the platform. If known allosteric sites are not recapitulated for the first system studied, alternative systems or modified scanning approaches are explored. This experimental plan provides a systematic approach to optimize on-chip measurements and discover alternative allosteric sites in human protein phosphatases, advancing their therapeutic potential.Example 4: High-throughput small molecule screening for Allosteric Inhibitors of SHP2

[0234] In this case, a system was provided having a high-throughput sequencing-based binding assay capable of screening DNA-encoded chemical libraries (DELs) against protein targets and resolving their rank order affinities. Here, the capacity of the system to identify allosteric inhibitors of SHP2 from a large chemical library and measure their relative binding affinities is demonstrated.

[0235] The experiment began with the preparation of a 370,000-species DEL library. Here, two allosteric inhibitors of SHP2, Novartis TNO-155 (IC50 = 11 nM) and RevolutionAttorney Docket No. 69414-702601Medicines RMC-4550 (IC50 = 0.58 nM), were spiked into the library at equal concentrations to other library members. Next, as illustrated in FIG. 12 (i), the DEL library was flowed onto a microfluidic chip, where SHP2 was immobilized on the left block and eGFP was immobilized on the right block as a control. After incubation, the library was allowed to interact with the immobilized proteins. The bound fractions for SHP2 and eGFP were trapped using button valves, and unbound fractions were washed off-chip. Next, as illustrated in FIG. 12 (ii), the bound fractions were then eluted separately for SHP2 and eGFP and collected for further analysis.

[0236] Next-generation sequencing was then used to analyze the collected fractions, providing molecular counting to identify binders and determine their rank order affinities as illustrated in FIG. 12 (iii). The sequencing analysis effectively identified both clinical candidates, TNO-155 and RMC-4550, as specific binders to SHP2, with no notable binding to the control protein, eGFP. This demonstrates the high specificity of the assay for its target protein, SHP2. The fraction of unique molecular identifiers (UMIs) for each compound accurately reflected their reported IC50 values. RMC-4550 (IC50 = 0.58 nM) exhibited a higher UMI fraction than TNO-155 (IC50 = 11 nM), consistent with their binding affinities. This result demonstrates the ability of the system to resolve rank order affinities for binders within the library (FIG. 12 (iv)). In FIG. 12 (iv), the scatter plot illustrates SHP2 binding percentages for the recovered molecules compared to eGFP binding percentages, with clear separation of the clinical candidates from non-specific binders.

[0237] This system provides a sequencing-based microfluidic assay for high-throughput screening of DEL libraries. The successful recovery of both clinical candidates from a 370,000-species library and the accurate reflection of their rank order affinities confirms the utility of the system in identifying target-specific binders and assessing their relative affinities. This system provides a robust and scalable tool for drug discovery efforts targeting allosteric sites.Example 5: High-throughput small molecule screening for Allosteric Inhibitors of SHP2

[0238] In this case, a system is provided that multiplexes DNA-encoded library (DEL) screening assay to the discovery of allosteric binders that are both site-specific and targetspecific. This system screens hundreds to thousands of targets simultaneously, including wild-type (WT) proteins, surface mutants, and counter-screen targets such as paralogs or homologs. The system localizes binding sites to candidate allosteric regions and ensures specificity to the target of interest while excluding undesirable off-target binders.Attorney Docket No. 69414-702601

[0239] Here, the system includes a microfluidic device. The microfluidics device has two side chambers connected to a central reaction chamber (FIG. 13A). The first side chamber contains plasmid DNA encoding the enzyme variant of interest, while the second side chamber contains printed barcodes used to tag the bound fraction for each target. These barcodes link the identity of the binders to the specific target they interact with. The microfluidic device also includes button valves, neck valves, and sandwich valves that ensure precise fluid handling and chamber-specific interactions. The microfluidic device can simultaneously screen multiple targets and associate target identity with binders in an accurate and efficient manner.

[0240] The microfluidics device is configured for chamber-specific tagging to further enhance the precision of associating binders with their respective targets. As illustrated in FIG. 13B, here, chamber-specific tagging is performed in situ by integrating DNA barcodes specific to each target into the bound fraction. DNA polymerase-based or ligase-based methods are systematically evaluated for barcode integration with DNA-encoded library members. Fluorophore-conjugated DNA and PCR amplification are used to validate the fidelity and efficiency of barcode incorporation, ensuring robust association of target-specific information with binders. As illustrated in FIG. 13B, the tagging workflow includes binding, trapping the interaction, washing, tagging with a barcode, and eluting the bound fraction, providing a streamlined process for encoding target specificity into the bound molecules.

[0241] Next, in this case, a large library of DNA-tagged small molecules, such as DNA- encoded chemical libraries (DELs), is screened against protein targets of interest to resolve the rank order affinities of binders. The allosteric inhibitors of the protein phosphatase SHP2 are tagged and spiked into a commercial DEL library comprising, for example, 370,000 species of the library at equal concentrations to the other library members. The screen includes WT SHP2 as well as several surface mutants.

[0242] Next, unique molecular identifiers (UMIs) for each tool compound are measured across WT and mutant variants to assess binding ablation caused by mutations at the binding site. Here, the system detects site-specific interactions and quantifies changes in binding due to target mutations.

[0243] In this case, the system performs DEL screening with 100- to 1000-fold greater throughput than alternative methods. The ability of the system to screen across hundreds to thousands of targets simultaneously and to associate binders with specific targets and binding sites unlocks the discovery of allosteric small molecules that are both site-specific and target specific.Attorney Docket No. 69414-702601Example 6: Epifluorescent microfluidic imaging and control setup

[0244] In this case, a system was developed with an integrated fluidic control and optical setup to perform and analyze high-throughput biochemical assays. While the system demonstrated precise fluorescence measurements and robust fluidic control, it was limited in scalability, field of view (FOV), and temporal resolution.

[0245] The system incorporated improved integration and packaging to fit the setup inside a custom dark enclosure. The system included an enclosure containing flow pressure regulators, control pressure regulators, solenoid valve arrays, liquid reservoirs, and microfluidic devices. The system utilized an inverted epi-fluorescence microscope (e.g., such as a Nikon Ti2) for fluorescence quantification on the microfluidic device. However, the microscope's limited FOV (~25 mm2) necessitated rastering (-16-25 tiled images) to capture the full chip area (-500 mm2), which increased imaging time to approximately one minute. This significantly reduced temporal resolution, hindering the ability to quantify fast biophysical reactions. Additionally, the high cost of the microscope (> $100k) and its excessive features for most microfluidic applications constrained the scalability of the system.

[0246] The system included a fluidic control system that provided on-chip valve actuation and fluid manipulation, with 48 solenoid valves organized into six banks, configured for pressures up to 45 psi, and 30 flow lines controlled via three banks capable of pressures up to 5 psi. The configuration of the fluidic control system provided independent control of flow and pressure for complex experiments. However, the fluidic control system’s reliance on hundreds of pneumatic and hydraulic connections introduced risks of failure and leakage, which limited its robustness for industrial applications and making scaling up cost prohibitive.Example 7: Proposed Development of a Scalable, Integrated Fluidic Control and Fluorescence Imaging System

[0247] In this case, a system is provided that has an integrated fluidic control and fluorescence imaging setup. Here, the system is configured to quantify fast biophysical reactions, such as enzymatic or binding / unbinding reactions, while achieving significant improvements in field of view (FOV), temporal resolution, and cost-efficiency. The provided system is a scalable industrial discovery tool configured for high-throughput biochemical assays testing

[0248] The system incorporates a tandem-lens epifluorescence macroscope and is configured to achieve a 10-fold increase in FOV and temporal resolution while reducing costs by five-Attorney Docket No. 69414-702601 fold compared to the current state-of-the-art Nikon Ti2 inverted microscope (FIG. 16). The macroscope is configured with a trans-fluorescence illumination scheme, placing the excitation filter above the object and the emission filter below in the nearly infinity-corrected space between the two lenses. By eliminating the need for a dichroic mirror, the lenses are positioned closer together, reducing image clipping and vignetting while providing larger FOVs. The system also supports fully automated XYZ movement and multi-channel fluorescence imaging, compatible with fluorophores such as GFP, DAPI, and Cy5.

[0249] The system includes a fluidic control configuration to improve scalability and robustness while reducing complexity. This system includes 4 reservoirs (e.g., as opposed to alternative systems that require 48), utilizing a common pressurized reservoir upstream of water-compatible solenoids. The system configuration enhances scalability and robustness while reducing complexity. The system includes 54 controllable valves (e.g., an increase from 48 in alternative systems) and 40 flow lines (e.g., an increase from 30 in alternative systems), as illustrated in FIG. 15. The system is configured to maintain constant reservoir pressurization, minimizing pressure cycles, enhancing reliability, and supporting digitized pressure control for more complex microfluidic operations. Additionally, digital pressure regulators provide precise fluid management, on-chip fault detection, and remediation.

[0250] The system integrates the tandem-lens macroscope, water-compatible solenoid arrays, and common liquid reservoirs into a compact, custom-built enclosure (FIG. 16). This streamlined configuration reduces the number of components, lowering costs and enhancing scalability for industrial research applications. The system includes an enhanced field of view (FOV), sensitivity across fluorescent channels (e.g., GFP, DAPI, Cy5), and a cost-effective setup, validated through comparisons with the Nikon Ti2 microscope equipped with lx, 2x, and 4x objectives. It is designed to accurately measure fast biophysical reactions, such as enzymatic and binding / unbinding reactions with half-lives of tens of seconds — processes that are challenging to quantify using current setups — thanks to its improved temporal resolution.

[0251] The system provides a scalable, robust, and low-cost platform for high-throughput biochemical assays. By offering improved FOV, reducing components, and enhancing reliability, it significantly advances the functionality of microfluidic research tools, providing fast, sensitive, and scalable discovery efforts.Example 8: Single Chamber Selectivity

[0252] To determine the number of reaction chambers needed to observe selectivity, a slide was printed with plasmid encoding a target biomolecule (“Target”), plasmid encoding EGFP, or buffer alone without plasmid. Target was printed in 364 spots, EGFP was printed in 268Attorney Docket No. 69414-702601 spots, and buffer was printed in 40 spots. Each printed plasmid was then subsequently encoded with a printed barcode, which is a 3’ phosphorylated and partially double-stranded custom sequence. There were 9 barcodes associated with Target plasmid, which were printed in 1, 2, 4, 8, 16, 32, 64, 109, or 128 spots, respectively. There were 9 barcodes associated with the EGFP plasmid, which were printed in 1, 2, 4, 8, 13, 16, 32, 64, or 128 spots, respectively. There were 6 barcodes associated with the buffer, which were printed in 1, 2, 4, 8, 9, or 16 spots, respectively. After printing, chips were aligned on the print such that each plasmid and barcode pair were encapsulated in reservoirs adjacent to the same reaction chamber (FIG. 20A). In this manner, each barcode was associated with a distinct target (Target, EGFP, or buffer) and with a distinct number of chambers, enabling demultiplexing of both the target and number of chambers from a single barcode (e.g., BC31 represents 16 chambers of Target, while BC18 represents 32 chambers of EGFP).

[0253] Standard patterning of the chip was performed by blocking with bovine serum albumin (BSA) across the whole chip excluding the button valves, then sequentially flowing biotinylated BSA, neutravidin, and biotinylated anti-GFP nanobody with the button valves open. Printed plasmids (Target, EGFP) were expressed in situ by flowing in vitro transcription and translation (IVTT) mix into the chip, then opening the neck valve to the printed target template. Once the reaction chamber and the reservoir with the printed template or buffer only were saturated with IVTT mixture, the sandwich valves were closed and the button valves were opened to isolate individual reaction chambers from each other while allowing the expressed target to diffuse to the button and interact with the anti-GFP nanobody. After expression was complete, unbound protein and remaining IVTT was washed out and the neck valve to the printed target template was closed.

[0254] Next, the binding assay with a pool of four tool compounds was performed. The pool comprised an equimolar mixture of four tool compounds that included two known strong binders of Target (S5 and S4, respective unconjugated IC50s of 0.58nM and 1 InM) and two known binders of other targets that were not expected to have any affinity for Target or EGFP (S6 and S9). Each tool compound was covalently conjugated to a unique DNA tag that encoded the identity of the molecule. The pool was flowed onto the chip to all reaction chambers, and the button valves were opened to expose the bound target to the pool of potential binders. After the tool compound was flowed onto the chip for 30 minutes, the button valves were closed to trap any tool compounds interacting with the target, then the chip was rinsed to remove any unbound tool compound.Attorney Docket No. 69414-702601

[0255] After the selection assay was complete and nonbinders were removed, a solution containing T4 ligase was flowed onto the chip. The neck valves separating the printed barcode chambers were depressurized to allow the barcode to be solubilized in the T4 solution, then the neck valves were closed again to isolate individual barcodes. After 10 minutes of solubilization, sandwich valves were closed to isolate individual reaction chambers, then the neck valves were reopened to allow the barcode to diffuse into the reaction chamber. After 20 minutes, the button valves were opened to allow the barcode and T4 ligase to interact with any molecules of tool compound present underneath the button. Ligation proceeded for 30 minutes to ensure any tool compound was ligated with the corresponding barcode. After this point, the DNA tag on each tool compound now encoded the identity of the molecule, the identity of the target it bound to, and the number of reaction chambers that barcode was associated with. Finally, the target and any bound tool compounds were eluted by flowing Proteinase K through the chip with the button valves open, and the whole pool was collected.

[0256] After elution, sequencing libraries were prepared by amplifying the DNA tags with custom primers - one primer bound to a common sequence upstream of the variable region that encodes the identity of the molecule, and the other primer bound to a common sequence in the printed barcode downstream of the variable region. Both primers additionally contained common sequences used for Readl and Read2 in Illumina sequencing. After this first PCR, the reaction was cleaned with Ampure beads to remove primers and contaminating protein. A second PCR was performed with primers that bound the Readl and Read2 region of the first set of primers and also contained unique indices as well as the P5 and P7 handles necessary to bind the flow cell of an Illumina sequencing cartridge. Libraries were quantified with TapeStation and qPCR, then sequenced with an Illumina NextSeq 2000.

[0257] After sequencing, reads were decoded to determine the identity of the bound molecule and the printed barcode. To analyze the relative strength of binding, the enrichment factor for each tool compound, which is defined as the relative abundance of a given tool compound in the final readout divided by the relative abundance of the given tool compound in the initial pool, was determined. The enrichment factor of each tool compound for each barcode was calculated. Calculations indicated that S5 was most highly enriched for Target (SHP2 WT), followed by S4, and there was very little of either negative control (S6 or S9) (FIG. 20B). This pattern of selectivity was conserved across all barcodes, which are denoted by individual points in the plot. While the negative control conditions (EGFP and buffer) also had a slight enrichment for S5, it was much less than the enrichment for Target and the enrichment for theAttorney Docket No. 69414-702601 negative control compounds was much higher. Because each individual barcode corresponded to a set number of chambers, robustness of the selection was able to be compared across different groups of chambers. Results demonstrated whether the selection was performed in a single chamber or up to 128 chambers, the selectivity for S5 to Target (SHP2) was essentially identical, confirming that selectivity can be achieved in as little as one ~lnL reaction chamber (FIG. 20C) despite the fact that typical (classical) off-chip DEL selections are performed in >100pL volumes.Example 9: Multiplexed Affinity Selection

[0258] To determine whether selectivity of DEL members binding to particular protein target variants could be sensitively observed, a slide microarray printed with plasmid encoding WT Target, 10 plasmids with different mutations, and five different negative controls plasmids and buffer (EGFP only, buffer only, 2x antitargets, and lx nontarget) was prepared. Each variant template or buffer was printed in 45 chambers, respectively. Each printed plasmid was then subsequently encoded with a printed barcode, which is a 3’ phosphorylated and partially double-stranded custom sequence. There were 4 barcodes associated with every unique plasmid type, with each barcode printed in 11 or 13 chambers. After printing, chips were aligned on the print such that each plasmid and barcode pair were encapsulated in reservoirs adjacent to the same reaction chamber (FIG. 21 A).

[0259] Patterning of the chip and target expression was performed as described in Example 8. Next, the binding assay with a pool of four tool compounds was performed, which comprised ligating barcodes in situ and eluting bound compounds as described in Example 8. Sequencing libraries were prepared as described in Example 8.

[0260] After sequencing and decoding reads, the enrichment factor of each tool compound was calculated as described in Example 8. The enrichment factor for each tool compound for each barcode was then normalized to one of the negative control compounds by dividing the enrichment factor for a given tool compound by the enrichment factor for the negative control compound. This provides the relative enrichment factor, which is a metric of how much more enriched a given tool compound is compared to the negative control. Results demonstrated that the WT Target had the highest relative enrichment factor for the strongest known binder, some mutants had intermediate relative enrichment factors, and others fully ablated binding and reduced the relative enrichment factor to the same level as the negative control targets (FIG. 21B). The binding was very consistent across chambers. Error bars in FIG. 21B represent standard error of the mean for the relative enrichment factor across the four barcode replicates for each target.Attorney Docket No. 69414-702601

[0261] Together, these data show that the assay is capable of discriminating changes in binding of tool compound conjugates due to a single point mutation, and that variations in binding strength can be sensitively and quantitatively determined from a single selection for a large number of variants.Example 10: Parallelized Affinity Selection

[0262] A selection of a 3 billion (3B) member DEL on a panel of 8 targets was performed. There were two replicates of WT Target, one truncated Target, three Target mutants, one antitarget, and one negative control (EGFP). A parallelized assay was used to perform this selection. Briefly, a parallelized chip with 4192 chambers organized into 8 distinct blocks (FIG. 22A) was patterned by blocking with BSA across the whole chip excluding the button valves, then sequentially flowing biotinylated BSA, neutravidin, and biotinylated anti-GFP nanobody with the button valves open. Targets were expressed in standard PCR tube strips using in vitro transcription and translation mixture, then each target was flowed into an individual block from separate inlets. Button valves were opened to allow binding to the anti- GFP nanobody, then buttons were closed to protect bound protein while unbound protein was rinsed off the chip.

[0263] A single tube (aliquot) of DNA encoded library comprising 3B individual molecules with 1 million (IM) copies of each molecule was split into 8 separate sub-aliquots. Each subaliquot was then loaded into an individual block of the parallelized chip and flowed onto the chip with button valves open to allow for molecules to interact with the bound protein. The unbound flowthrough was collected and reflowed on the same block twice, for a total of three rounds of flow for each target. After the final flow, buttons were closed to protect bound DEL members, and the rest of the chip was rinsed. Finally, bound protein / DEL complexes were eluted by flowing Proteinase K with the buttons open. The eluted fraction for each target was collected in a separate tube, heat inactivated, then saved for the future.

[0264] Two more rounds of selection were performed as described above, except that the input to the chip in each case was the eluted fraction from the prior selection round rather than a naive DEL (of the original input composition) as was the case in the first round. After each round of selection, qPCR was performed to quantify the output and selection was stopped after three rounds when each condition reached approximately 20 million (20M) total DEL members.

[0265] After selection was completed, libraries were kept in separate tubes for two rounds of PCR as described in Example 8 to amplify the library and add necessary sequences for Illumina sequencing. During the second round of PCR, each library was barcoded with aAttorney Docket No. 69414-702601 distinct index to allow for pooled sequencing. Libraries were pooled and sequenced on a NextSeq 2000. Sequences were then processed for each library to decode the identity of the DEL member, and unique molecular identifiers present in the DEL were used to determine counts of bound members for each condition. After decoding, binding was first visualized with a 3D plot. Each DEL member is composed of three separate building blocks, also referred to as cycles, so a particular fully synthesized DEL member can be visualized by plotting the point on three axes where each axis represents the identity of one of the three building blocks. Each building block has a set of potential structures, so each unique member of the DEL is synthesized by combining a unique set of three building blocks. In FIG. 22B, each point represents an individual DEL member, and the size and color of each point represent the number of copies of that member observed binding to a particular target. The plot is filtered to only show DEL features that were determined to be significantly enriched over background. In this visualization, plane features represent many individual DEL members that all have one building block in common, while line features represent individual DEL members that have two building blocks in common. When comparing Target against EGFP, FIG. 22C demonstrates that different features were enriched between the two conditions, indicating that there were building blocks that have differential binding affinity for Target compared to EGFP.

[0266] To examine the differences across multiple targets, individual single building blocks across the full DEL were analyzed for their enrichment in each target. A significance score was calculated for each building block by normalizing the observed counts to its pre-selection abundance. Significance scores were then ranked separately for each target. Figure X shows the top 25 most significant building blocks for WT Target. As shown by looking across the variants, different features have different selectivity. For example, Feature 1 is a target specific binder that is highly enriched across all target variants but substantially less enriched in the antitarget and negative control, whereas Feature 2 is a general non-specific binder that is highly enriched across all conditions. Most interesting are features such as Feature 7, which is highly enriched in WT target and the three mutants, but is almost completely lost in the negative controls and the truncated target, suggesting that this particular building block interacts strongly with a region of the target that is absent in the truncated mutant. Together, these data demonstrate that this assay can effectively perform selection assays using highly diverse and large DELs using a fraction of the quantity of DEL typically required for such selections using traditional DEL selection methods, and can provide structure-affinity relationship information from a single primary selection.Attorney Docket No. 69414-702601Example 11: Parallelized Selection Schematic

[0267] FIGs. 23A-B illustrates a parallelized selection workflow using devices with no reservoir chambers. Distinct sets or blocks of reaction chambers can be fluidically separated from each other, enabling flow of distinct reagents to each block. Target protein can be expressed, then flowed onto the chip and bound under the button in each block. Next, an aliquot of a library of potential binders can be flowed onto each block and allowed to interact with the bound target. Finally, the bound interactors can be eluted from the device and separately collected. If necessary, the eluent from one selection assay can be used as the input for a subsequent selection assay for as many iterations as necessary to specifically enrich for target binders.

[0268] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EMBODIMENTS1. A method for identifying a candidate binding partner of a target biomolecule, the method comprising: a. providing a device, wherein the device comprises a reaction chamber; b. providing the target biomolecule and one or more candidate binding partners of the target biomolecule;Attorney Docket No. 69414-702601 c. mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device; d. subsequent to (c), tagging the target biomolecule, the candidate binding partners, or both with a barcode in the device; and e. identifying the barcode, thereby identifying the target biomolecule, the candidate binding partner, additional information about an interaction between the target biomolecule and the one or more candidate binding partners, or any combination thereof.2. The method of embodiment 1, wherein the target biomolecule comprises DNA, RNA or a protein.3. The method of embodiment 1, wherein the target biomolecule comprises any combination of DNA, RNA or a protein.4. The method of embodiment 1, wherein the reaction chamber is amongst a plurality of reaction chambers, wherein the device comprises the plurality of reaction chambers.5. The method of embodiment 1, wherein the one or more candidate binding partners comprise an additional barcode that encodes a unique identifier corresponding to the candidate binding partner.6. The method of embodiment 1, wherein the one or more candidate binding partners comprises a small molecule.7. The method of embodiment 1, wherein the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule.8. The method of embodiment 7, wherein the member is a button valve.9. The method of embodiment 1, wherein the one or more candidate binding partners interact with one or more binding sites of the target biomolecule.10. The method of embodiment 1, wherein the method further comprises identifying one or more allosteric binding sites of the target biomolecule.11. The method of embodiment 1, wherein the method further comprises inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site.Attorney Docket No. 69414-70260112. The method of embodiment 1, wherein the method further comprises trapping a bound interaction between the target biomolecule and the one or more candidate binding partners, washing away unbound compounds, then eluting a solution from the reaction zone, thereby collecting the bound interaction.13. The method of embodiment 1, wherein the method is configured to perform multiple forward and reverse screens simultaneously.14. The method of embodiment 1, wherein the reaction chamber comprises microchambers having a volume configured for screening of a plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule, wherein the volume comprises between 0.5 nL and 50 nL.15. The method of embodiment 1, wherein the method is employed for screening of up to 1,000 target biomolecules, or at least 1,000 target biomolecules, or more, or variants thereof.16. The method of embodiment 1, further comprising introducing single- or multimutant target biomolecules, including unnatural or extant allelic variants, to the target biomolecule, and assessing the effect of the introduced variants on identification of binding partners.17. The method of embodiment 1, further comprising performing scanning mutagenesis on the target biomolecule to assess the effects on binding partner identification.18. The method of embodiment 1, further comprising performing mutagenesis on the target biomolecule to localize binding sites of binding partners.19. The method of embodiment 1, further comprising performing mutagenesis to introduce multiple mutations in the target biomolecule and assessing effects of the multiple mutations on binding partner identification.20. The method of embodiment 1, further comprising performing a plurality of screens with a plurality of target biomolecules to identify a plurality of sets of binding partners of the plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule.21. The method of embodiment 20, wherein the plurality of target biomolecules comprises at least 17 unique target biomolecules.22. The method of embodiment 20 or embodiment 21, wherein the plurality of target biomolecules comprises one or more variants of the target biomolecule.Attorney Docket No. 69414-70260123. The method of embodiment 22, wherein the one or more variants comprises a point mutation, an insertion, a deletion, or a combination thereof relative to the target biomolecule.24. The method of any one of embodiments 20-23, wherein performing the plurality of screens comprises identifying variation amongst the plurality of sets of binding partners.25. The method of embodiment 1, further comprising quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor.26. The method of embodiment 1, wherein the method is used for analyzing interactions between DNA sequences and transcription factors.27. The method of embodiment 1, wherein the method is employed for analyzing small molecule interactions with target biomolecules.28. The method of embodiment 1, wherein the method is employed for analyzing antibody interactions with target biomolecules.29. The method of embodiment 1, wherein the method is employed for analyzing interactions of peptides or proteins with target biomolecules using mRNA display libraries.30. The method of embodiment 1, wherein the method is employed for analyzing interactions of peptides or proteins with target biomolecules using cDNA display libraries.31. The method of embodiment 1, further comprising administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, a target in vitro expression plasmid, a protein-identifying barcode, or any combination thereof.32. The method of embodiment 1, wherein the method includes the expression and immobilization of the target biomolecule in the device.33. The method of embodiment 1, wherein the device further comprises an array configured to allow programming via microarray prints.34. The method of embodiment 1, further comprising, prior to (b), disposing one or more nucleic acid molecules onto a surface adjacent to the device, wherein the one or more nucleic acid molecules encode the target biomolecule, the candidate binding partner, the barcode or any combination thereof.35. The method of embodiment 34, wherein the one or more nucleic acid molecules comprises a first nucleic acid molecule and a second nucleic acid molecule, whereinAttorney Docket No. 69414-702601 the first nucleic acid molecule encodes the target biomolecule, wherein the second nucleic acid molecule encode the barcode, and wherein the barcode corresponds to the target biomolecule.36. The method of embodiment 35, wherein the first nucleic acid molecule is provided in a first chamber of the device, and wherein the second nucleic acid molecule is provided in a second chamber of the device, and wherein the first chamber of the device and the second chamber of the device are fluidically coupled to the reaction chamber.37. The method of embodiment 1, wherein the device further comprises an array configured to introduce a DNA barcode encoding the target biomolecule via microarray prints, wherein the printed DNA barcode is in a reservoir chamber adjacent to the DNA encoding of the target biomolecule and connected to a same reaction chamber.38. The method of embodiment 37, wherein the reservoir chamber is an elongated reservoir chamber.39. The method of embodiment 1, wherein the reaction chamber is an elongated reaction chamber.40. The method of embodiment 1, wherein the method includes expressing the target biomolecule or multiple proteins off the device, followed by immobilization of the target biomolecule or proteins on the device.41. The method of embodiment 1, wherein the method involves flowing on DNA-tagged small molecules onto the device.42. The method of embodiment 1, wherein the method involves flowing on DNA-tagged protein molecules onto the device.43. The method of embodiment 1, wherein the method involves flowing on RNA-tagged protein molecules onto the device.44. The method of embodiment 1, further comprising attaching in situ barcodes to molecules on the device.45. The method of embodiment 1, further comprising the elution of in situ barcoded small molecules.46. The method of embodiment 1, further comprising the elution of in situ barcoded macro molecules, including DNA, RNA, and protein.47. The method of embodiment 1, wherein protein interactors are introduced to the reaction chamber from a common inlet.Attorney Docket No. 69414-70260148. The method of embodiment 1, wherein protein interactors are introduced by a microarray print.49. The method of embodiment 1, further comprising using spatial barcoding to map interactors to their respective target biomolecule.50. The method of embodiment 1, further comprising using temporal barcoding to tag reaction products based on the order of generation.51. The method of embodiment 1, further comprising sequentially flowing a first library of interactors onto the reaction zone, thereby causing at least one interactor of the first library of interactors to bind, flowing a first barcode to label bound interactors, eluting the first library of interactors, subsequently flowing a second library of interactors onto the reaction zone, flowing a second barcode to label bound interactors, and eluting the second library of interactors, wherein each barcode corresponds to a specific library of interactors.52. The method of embodiment 1, further comprising employing temporal barcoding to tag interactors based on the order of introduction to the chip.53. The method of embodiment 1, wherein barcoding is performed on-chip.54. The method of embodiment 1, further comprising one or more interactors, wherein the one or more interactors include small molecules.55. The method of embodiment 1, wherein the target biomolecule is engineered to contain one or more unnatural amino acids.56. The method of embodiment 1, wherein the target biomolecule is post-translationally modified with one or more chemical entities.57. The method of embodiment 56, wherein the one or more chemical entities is selected from the group consisting of phosphate, sugars, and ubiquitin protein.58. A microfluidic system comprising: a. one or more reaction chambers comprising a reaction surface; b. a plurality of reservoir chambers fluidically connected to the reaction chamber via a set of channels; c. one or more members positioned adjacent to the reaction chamber and configured to move along an axis orthogonal to the reaction surface; and d. a hydraulic control module configured to actuate the one or more members.59. The microfluidic system of embodiment 58, wherein each reservoir chamber of the plurality of reservoir chambers is located on a same side of the reaction chamber.Attorney Docket No. 69414-70260160. The microfluidic system of embodiment 58, wherein at least one reaction chamber is an elongated reaction chamber.61. The microfluidic system of embodiment 58, wherein at least one reservoir chamber of the plurality is an elongated chamber.62. The microfluidic system of embodiment 58, wherein the set of channels fluidically connects at least two reservoir chambers of the plurality to the reaction chamber.63. The microfluidic system of embodiment 58, wherein the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface.64. The microfluidic system of embodiment 63, wherein the orthogonal movement of the member is configured to control fluidic mixing between at least one reservoir chamber of the plurality of reservoir chambers and one or more reaction chambers.65. The microfluidic system of embodiment 58, wherein each of the plurality of reservoir chambers is oriented at an angle relative to at least one surface of a reaction chamber of the one or more reaction chambers.66. The microfluidic system of embodiment 58, wherein the plurality of reservoir chambers has a denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.67. The microfluidic system of embodiment 58, wherein the denser packing efficiency is at least 1.75-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.68. The microfluidic system of embodiment 58, wherein a volume of at least one reaction chamber is at least 4.71-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.69. The microfluidic system of embodiment 68, wherein a member area of the reaction chamber is at least 2 to 10-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on oppositeAttorney Docket No. 69414-702601 sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.70. The microfluidic system of embodiment 58, wherein at least one reservoir chamber of the plurality of reservoir chambers are reversibly separated from the one or more reaction chambers, or made contiguous with the one or more reaction chambers by a plurality of neck valves.71. The microfluidic system of embodiment 58, further comprising micro-chambers with volumes between 0.5 nL and 50 nL, wherein the micro-chambers are configured to house incubations and reactions.72. The microfluidic system of embodiment 58, further comprising an array configured to allow programming via microarray prints.73. The microfluidic system of embodiment 58, further comprising a two-layer elastomer structure positioned on top of a glass layer.74. The microfluidic system of embodiment 58, further comprising integrated pushdown valves configured in a Quake-style arrangement or a pneumatically or hydraulically actuated elastomeric valve arrangement for controlling fluid flow.75. The microfluidic system of embodiment 58, wherein the system is configured to simultaneously express and purify a plurality of proteins, including enzymes or variants thereof, wherein the plurality of proteins comprises from at least one to at least 1,000 proteins or variants thereof.76. The microfluidic system of embodiment 58, wherein the system is configured to quantitatively assay all or a subset of proteins or protein variants simultaneously.77. The microfluidic system of embodiment 58, wherein, when the members are open, fluid flows through the full volume of all reaction chambers in the device.78. The microfluidic system of embodiment 58, wherein, when the members are closed, fluid flows around the volume partitioned off by the member without disturbing purified variants or their interactors.79. The microfluidic system of embodiment 58, further comprising one or more sandwich valves, wherein, when the one or more sandwich valves are closed, the elongated reaction chamber and / or one or more of the pluralities of reservoir chambers are isolated as unique environments separated from other reaction chambers, thereby permitting high-throughput workflows.Attorney Docket No. 69414-70260180. The microfluidic system of embodiment 58, wherein a second reservoir chamber is configured to introduce a unique molecular barcode for multiplexing across multiple protein variants, while maintaining isolation between chambers.81. The microfluidic system of embodiment 60, wherein the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area.82. The microfluidic system of embodiment 81, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.83. The microfluidic system of embodiment 81, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.84. The microfluidic system of embodiment 81, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.85. The microfluidic system of embodiment 81, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.86. The microfluidic system of embodiment 81, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 1200 chambers per chip.87. The microfluidic system of embodiment 81, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 900 chambers per chip.88. The microfluidic system of embodiment 81, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 700 chambers per chip.89. The microfluidic system of embodiment 81, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 600 chambers per chip.90. An imaging system for imaging a microfluidic device, the system comprising: a. a substrate comprising the microfluidic device, wherein the microfluidic device comprises a plurality of channels, wherein at least one channel of the plurality of channels comprises:Attorney Docket No. 69414-702601 i. one or more reaction chambers; ii. one or more reservoir chambers fluidically coupled to the reaction chamber; and iii. one or more members disposed adjacent to the reaction chamber and configured to move along an axis orthogonal to the reaction chamber; b. a transillumination subsystem comprising a light source and a detector, wherein the light source is configured to provide light to the substrate, wherein the substrate is configured to provide the light from the light source to the detector, and wherein the detector is configured to detect at least a portion of the light emitted from the substrate.91. The imaging system of embodiment 90, wherein at least one reservoir chamber of the one or more reservoir chambers is an elongated reservoir chamber.92. An imaging system for imaging a microfluidic device, the system comprising: a. a substrate comprising the microfluidic device, wherein the microfluidic device comprises a plurality of channels on a surface, wherein at least one channel of the plurality of channels comprises one or more reaction chambers, wherein at least one of the one or more reaction chambers is an elongated reaction chamber; wherein the surface is configured to display one or more target biomolecules over at least 3% of the surface and; b. a transillumination subsystem comprising a light source and a detector, wherein the light source is configured to provide light to the substrate, wherein the substrate is configured to provide the light from the light source to the detector, and wherein the detector is configured to detect at least a portion of the light emitted from the substrate.93. The imaging system of embodiment 92, wherein the surface is configured to display the one or more target biomolecules over at least 6% of the surface.94. The imaging system of embodiment 92 or embodiment 93, wherein the plurality of channels comprises one or more members positioned adjacent to the one or more reaction chambers and configured to move along an axis orthogonal to the one or more reaction chambers, and wherein the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area.Attorney Docket No. 69414-70260195. The imaging system of embodiment 94, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.96. The imaging system of embodiment 94, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.97. The imaging system of embodiment 94, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.98. The imaging system of embodiment 94, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.99. The imaging system of embodiment 94, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 1200 chambers per chip.100. The imaging system of embodiment 94, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 900 chambers per chip.101. The imaging system of embodiment 94, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 700 chambers per chip.102. The imaging system of embodiment 94, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 600 chambers per chip.103. The imaging system of embodiment 94, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 6000 chambers per chip.104. The imaging system of embodiment 94, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 4500 chambers per chip.105. The imaging system of embodiment 94, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 3500 chambers per chip.Attorney Docket No. 69414-702601106. The imaging system of embodiment 94, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 3000 chambers per chip.107. The imaging system of any one of embodiments 90-106, wherein the detector is configured to detect at least a portion of the light emitted from the substrate and at least a portion of the light emitted from the light source.108. The imaging system of any one of embodiments 90-107, wherein the detector is configured to detect at least a portion of the light emitted from the substrate and at least a portion of the light emitted from the light source, and wherein a portion of the light emitted from the light source is excluded from the portion of the light emitted from the substrate.109. The imaging system of any one of embodiments 90-108, further comprising a macroscope with a tandem macro lens system of at most lx magnification.110. The imaging system of any one of embodiments 90-109, further comprising a macroscope with a tandem macro lens system of less than or greater than lx magnification.111. The imaging system of embodiment 109 or embodiment 110, wherein the macroscope is configured for trans-illumination.112. The imaging system of any one of embodiments 109-111, wherein the macroscope is configured for side-on illumination.113. The imaging system of any one of embodiments 90-112, wherein the system incorporates a fly’s eye homogenizer configured to achieve near-uniform illumination.114. The imaging system of any one of embodiments 90-113, wherein the system includes a stacked arrangement of emission filters configured to enhance a signal-to-noise ratio of detected optical signals.115. The imaging system of any one of embodiments 90-114, wherein the system includes one or more emission filters positioned within an infinity space between lenses, configured to improve optical performance.116. The imaging system of any one of embodiments 90-115, wherein the system comprises an integrated hydraulic microfluidic control system.117. The imaging system of any one of embodiments 90-116, further comprising a subsystem for analyzing the kinetics of biochemical events involving surface immobilized fluorophores.Attorney Docket No. 69414-702601118. The imaging system of any one of embodiments 90-117, further configured to analyze the kinetics of biochemical events involving surface-immobilized fluorophores in real-time or near real-time.119. The imaging system of any one of embodiments 90-118, further comprising a subsystem configured to analyze the kinetics of biochemical events involving solution-phase fluorophores.120. The imaging system of any one of embodiments 90-119, further configured to analyze the kinetics of biochemical events involving solution-phase fluorophores in real-time or near real-time.121. The imaging system of any one of embodiments 90-120, further comprising a hydraulic microfluidic control system including one or more common reservoirs configured to supply one or more solenoid valves.122. The imaging system of embodiment 121, wherein at least one solenoid valve is filled with water or other liquids.123. The imaging system of embodiment 121 or embodiment 122, wherein the hydraulic microfluidic control system is scalable to accommodate one or more solenoid valves.124. A method for operating an imaging system, comprising: a. providing a substrate comprising a microfluidic device, wherein the microfluidic device includes a plurality of channels, wherein at least one channel of the plurality of channels comprises: i. one or more reaction chambers; ii. one or more reservoir chambers fluidically coupled to the one or more reaction chambers; and iii. one or more members positioned adjacent to the one or more reaction chambers and configured to move along an axis orthogonal to the one or more reaction chambers; and b. providing a transillumination subsystem comprising a light source and a detector, wherein the light source is configured to provide light to the substrate, wherein the substrate is configured to provide the light to the detector, and wherein the detector is configured to detect at least a portion of the light emitted from the substrate.125. The method of embodiment 124, wherein at least one reservoir chamber of the one or more reservoir chambers is an elongated reservoir chamber.126. A method for operating an imaging system, comprising:Attorney Docket No. 69414-702601 a. providing a substrate comprising a microfluidic device, wherein the microfluidic device includes a plurality of channels, wherein at least one channel of the plurality of channels comprises one or more reaction chambers, wherein the one or more reaction chambers comprises an elongated reaction chamber; wherein the surface is configured to display one or more target biomolecules over at least 3% of the surface and; b. providing a transillumination subsystem comprising a light source and a detector, wherein the light source is configured to provide light to the substrate, wherein the substrate is configured to provide the light to the detector, and wherein the detector is configured to detect at least a portion of the light emitted from the substrate.127. The method of embodiment 126, wherein the surface is configured to display the one or more target biomolecules over at least 6% of the surface.128. The method of embodiment 126 or embodiment 127, wherein the plurality of channels comprises one or more members positioned adjacent to the one or more reaction chambers and configured to move along an axis orthogonal to the one or more reaction chambers, and wherein the elongated reaction chamber comprises variable stretch values, resulting in one or more proportional fold changes to a chamber volume and a member area.129. The method of embodiment 128, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.130. The method of embodiment 128, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.131. The method of embodiment 128, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.132. The method of embodiment 128, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.133. The method of embodiment 128, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 1200 chambers per chip.Attorney Docket No. 69414-702601134. The method of embodiment 128, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 900 chambers per chip.135. The method of embodiment 128, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 700 chambers per chip.136. The method of embodiment 128, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 600 chambers per chip.137. The method of embodiment 128, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 6000 chambers per chip.138. The method of embodiment 128, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 4500 chambers per chip.139. The method of embodiment 128, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 3500 chambers per chip.140. The method of embodiment 128, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 3000 chambers per chip.141. The method of any one of embodiments 124-140, wherein the detector is configured to detect at least a portion of the light emitted from the substrate and at least a portion of the light emitted from the light source.142. The method of any one of embodiments 124-141, wherein the detector is configured to detect at least a portion of the light emitted from the substrate and at least a portion of the light emitted from the light source, and wherein a portion of the light emitted from the light source is excluded from the portion of the light emitted from the substrate.143. The method of any one of embodiments 124-142, wherein the detector is configured to detect photons from the light source.144. The method of any one of embodiments 124-143, wherein the detector is configured to detect photons from the substrate.Attorney Docket No. 69414-702601145. The method of any one of embodiments 124-144, wherein the detector is configured to detect the light from any other source rather than the light source.146. The method of any one of embodiments 124-145, wherein uniform transillumination is achieved using a Fly’s eye homogenizer.147. The method of any one of embodiments 124-146, further comprising detecting the transmitted light using a macroscope equipped with a macro lens system.148. The method of embodiment 147, further comprising creating an overview image of a sample illuminated by the Fly’s eye homogenizer, captured using the macroscope.149. The method of embodiment 148, further comprising capturing light from a shallow region centered on a sample plane using the macroscope.150. The method of any one of embodiments 124-149, further comprising analyzing the kinetics of biochemical events involving surface immobilized fluorophores.151. The method of any one of embodiments 124-150, further comprising analyzing the kinetics of biochemical events involving surface-immobilized fluorophores in realtime or near real-time.152. The method of any one of embodiments 124-151, further comprising analyzing the kinetics of biochemical events involving solution-phase fluorophores.153. The method of any one of embodiments 124-152, further comprising analyzing the kinetics of biochemical events involving solution-phase fluorophores in real-time or near real-time.154. The method of any one of embodiments 124-153, further comprising analyzing kinetics of enzyme reactions wherein an enzyme is surface immobilized.155. The method of any one of embodiments 124-154, further comprising analyzing kinetics of enzyme reactions wherein an enzyme is in solution.156. The method of any one of embodiments 124-155, further comprising controlling fluidic flow using a hydraulic microfluidic control system that includes one or more common reservoirs supplying one or more solenoid valves filled with water or other liquids.157. A microfluidic system comprising: a. one or more elongated reaction chambers; b. a plurality of reservoir chambers, wherein each reservoir chamber of the plurality of reservoir chambers is located on a same side of the elongated reaction chamber wherein each reservoir chamber of the plurality is connected in an angular orientation;Attorney Docket No. 69414-702601 c. one or more members positioned adjacent to the elongated reaction chamber and configured to move orthogonally to a surface of the reaction chamber; and d. a set of channels fluidically connecting at least two of the plurality of reservoir chambers to the reaction chamber.158. The microfluidic system of embodiment 157, wherein each reservoir chamber of the plurality is connected at a substantially 90° angle to the elongated reaction chamber.159. The microfluidic system of embodiment 157, wherein one or more reservoir chambers of the plurality of reservoir chambers is elongated.160. The microfluidic system of embodiment 157, wherein the plurality of reservoir chambers comprises a denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.161. The microfluidic system of embodiment 157, wherein the denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber is about 1.75-fold.162. The microfluidic system of embodiment 157, wherein the reaction chamber comprises a 2 to 10-fold greater member area than a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.163. The microfluidic system of embodiment 157, wherein the reaction chamber comprises a 10-fold greater member area compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.164. The microfluidic system of embodiment 157, wherein the one or more member is configured to move along an axis orthogonal to a surface of the elongated reaction chamber.165. The microfluidic system of embodiment 157, wherein the reservoir chambers of the plurality of reservoir chambers are reversibly separated or made contiguous with the reaction chamber by two neck valves.Attorney Docket No. 69414-702601166. The microfluidic system of embodiment 157, further comprising micro-chambers with volumes between 0.5 nL and 50 nL configured to perform incubations and reactions.167. The microfluidic system of embodiment 157, wherein the system is equipped with an array that allows for programming via microarray prints.168. The microfluidic system of embodiment 157, configured with a two-layer polydimethylsiloxane structure on top of a glass layer.169. The microfluidic system of embodiment 157, further comprising integrated pushdown, "Quake style" valves.170. The microfluidic system of embodiment 157, wherein the system may simultaneously express and purify a plurality of proteins, including enzymes or variants thereof, the plurality ranging from at least one to at least 1,000 proteins or variants thereof.171. The microfluidic system of embodiment 157, wherein the system may quantitatively assay all, or a subset of, proteins or protein variants at once.172. The microfluidic system of embodiment 157, wherein, when the one or more member is open, fluid flows through the reaction chamber.173. The microfluidic system of embodiment 157, wherein, when the one or more member is closed, fluid flows through the reaction chamber around the volume partitioned off by the member without disturbing purified variants or their interactors.174. The microfluidic system of embodiment 157, wherein the introduction of a second reservoir chamber allows for multiplexing to multiple protein variants by introducing a unique molecular barcode while chambers are still isolated from each other.175. The microfluidic system of embodiment 157, wherein the reaction chamber accommodates variable stretch values, leading to corresponding chamber volume and member area fold changes.176. The microfluidic system of embodiment 157, wherein a stretch value of 200 microns leads to a chamber volume fold change of about 2.1 and a member area fold change of about 2.8.177. The microfluidic system of embodiment 157, wherein a stretch value of 400 microns leads to a chamber volume fold change of about 3.25 and a member area fold change of about 4.6.Attorney Docket No. 69414-702601178. The microfluidic system of embodiment 157, wherein a stretch value of 600 microns leads to a chamber volume fold change of about 4.39 and a member area fold change of about 6.45.179. The microfluidic system of embodiment 157, wherein a stretch value of 800 microns leads to a chamber volume fold change of about 5.5 and a member area fold change of about 8.28.180. The microfluidic system of embodiment 157, wherein a stretch value of 200 microns leads to a configuration of approximately 1,200 chambers per chip.181. The microfluidic system of embodiment 157, wherein a stretch value of 400 microns leads to a configuration of approximately 900 chambers per chip.182. The microfluidic system of embodiment 157, wherein a stretch value of 600 microns leads to a configuration of approximately 700 chambers per chip.183. The microfluidic system of embodiment 157, wherein a stretch value of 800 microns leads to a configuration of approximately 600 chambers per chip.184. A method of identifying a plurality of binding sites of a plurality of proteins of interest, the method comprising a. providing a microchannel, wherein the microchannel comprises: i. the plurality of proteins of interest, and ii. a plurality of candidate binding partners configured to bind to one or more binding sites of the plurality of proteins of interest; b. binding the plurality of proteins of interest and the plurality of candidate binding partners to form a plurality of complexes; c. tagging the plurality of complexes formed in (b) to generate a plurality of tagged complexes; and d. identifying the plurality of tagged complexes, thereby identifying the plurality of binding sites.185. The method of embodiment 184, wherein the plurality of candidate binding partners each comprise a unique barcode, and said unique barcodes are used to identify the candidate binding partners following binding to the proteins of interest.186. The method of embodiment 184, further comprising predicting candidate binding partners that may bind to allosteric binding sites of the proteins of interest using bioinformatics or computational modeling.187. The method of embodiment 1, wherein the target biomolecule is identified.188. The method of embodiment 1, wherein the candidate binder partner is identified.Attorney Docket No. 69414-702601189. The method of embodiment 1, wherein the interaction between the target biomolecule and candidate binding partner is identified.190. A method of identifying one or more binding partners of one or more target biomolecules, the method comprising: a. providing a device, wherein the device comprises a plurality of reaction chambers on a surface, wherein the plurality of reaction chambers comprises one or more sets of reaction chambers; b. providing a target biomolecule of the one or more target biomolecules and a plurality of candidate binding partners of the target biomolecule to a set of reaction chambers, wherein the surface is configured to display the one or more target biomolecules over at least 3% of the surface; and c. identifying one or more binding partners of the target biomolecule from a subset of the plurality of candidate binding partners bound to the target biomolecule.191. The method of embodiment 190, further comprising (i) mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device, and (ii) generating one or more complexes comprising the target molecule and the subset of the plurality of candidate binding partners.192. The method of embodiment 190 or embodiment 191, wherein the plurality of candidate binding partners comprises at least 10,000 unique candidate binding partners.193. The method of embodiment 192, wherein the plurality of candidate binding partners comprises at least 1 billion unique candidate binding partners.194. The method of any one of embodiments 190-193, wherein the one or more target biomolecules comprises at least 8 unique target biomolecules.195. The method of embodiment 194, wherein the one or more target biomolecules comprises at least 32 unique target biomolecules.196. The method of embodiment 194 or embodiment 195, wherein a number of unique candidate binding partners in the plurality of candidate binding partners is at least 93 million-fold greater than a number of unique target biomolecules in the one or more target biomolecules.197. The method of any one of embodiments 191-196, wherein the method does not comprise tagging the one or more complexes with a barcode in the device.Attorney Docket No. 69414-702601198. The method of any one of embodiments 190-197, wherein the set of reaction chambers from the one or more sets of reaction chambers comprises 2 or more reaction chambers.199. The method of embodiment 198, wherein reaction chambers of the set of reaction chambers are fluidly connected during (b).200. The method of embodiment 199, wherein the reaction chambers of the set of reaction chambers are fluidly connected in series.201. The method of embodiment 199 or embodiment 200, wherein the reaction chambers of the set of reaction chambers are fluidly connected in parallel.202. The method of any one of embodiments 190-201, wherein the plurality of candidate binding partners is configured to interact with two or more binding sites on the target biomolecule.203. The method of any one or embodiments 190-202, wherein the one or more target biomolecules comprises one or more variants of the target biomolecule.204. The method of embodiment 203, wherein the one or more variants comprises a point mutation, an insertion, a deletion, or a combination thereof relative to the target biomolecule.205. The method of any one of embodiments 190-204, wherein the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during (b).206. The method of embodiment 205, further comprising providing a second target biomolecule of the one or more target biomolecules and the plurality of candidate binding partners of the second target biomolecule to the additional set of reaction chambers.207. The method of embodiment 206, further comprising identifying one or more binding partners of the second target biomolecule from the plurality of candidate binding partners.208. The method of embodiment 207, further comprising identifying a difference between the one or more binding partners of the target biomolecule and the one or more binding partners of the second target biomolecule.209. The method of any one of embodiments 190-208, further comprising identifying one or more binding sites of the one or more binding partners.Attorney Docket No. 69414-702601210. The method of embodiment 209, wherein the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing the target biomolecule and the second target biomolecule.211. The method of embodiment 210, wherein the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing a sequence of the target biomolecule and a sequence the second target biomolecule.212. The method of embodiment 210 or embodiment 211, wherein the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing a structure of the target biomolecule and a structure the second target biomolecule.213. The method of any one of embodiments 190-212, wherein the plurality of reaction chambers comprises at least 8 sets of reaction chambers, wherein reaction chambers of each of the at least 8 sets of reaction chambers are not fluidly connected to reaction chambers of a different set amongst the at least 8 sets of reaction chambers during (b).214. The method of any one of embodiments 190-213, wherein the plurality of candidate binding partners is provided to the set of reaction chambers at a concentration of at least 1 nM.215. The method of any one of embodiments 190-214, wherein the plurality of reaction chambers comprises at least 4200 reaction chambers.216. The method of any one of embodiments 190-215, wherein the plurality of reaction chambers covers at least 30% of the surface.217. The method of any one of embodiments 190-216, wherein the plurality of reaction chambers displays at least 0.3 pmol of protein.218. The method of any one of embodiments 190-217, wherein (b) does not comprise providing the target biomolecule from a reservoir chamber on the surface adjacent to a reaction chamber of the set of reaction chambers.219. The method of any one of embodiments 190-218, further comprising eluting the subset of the plurality of candidate binding partners from the one or more complexes.220. The method of embodiment 219, further comprising providing the subset of the plurality of candidate binding partners to a set of reaction chambers on a different surface.Attorney Docket No. 69414-702601221. The method of any one of embodiments 190-220, further comprising obtaining an unbound subset of the plurality of candidate binding partners, wherein the unbound subset of the plurality of candidate binding partners is not bound to the target biomolecule.222. The method of embodiment 221, further comprising providing the unbound subset of the plurality of candidate binding partners to the set of reaction chambers.223. The method of any one of embodiments 190-222, further comprising expressing the one or more target biomolecules external to the device.224. The method of embodiment 223, wherein expressing the one or more target biomolecules comprises cell-free expression.225. The method of any one of embodiments 190-224, wherein the plurality of candidate binding partners comprises tags.226. The method of embodiment 225, wherein the tags are unique for each of the unique candidate binding partners.227. The method of embodiment 225 or embodiment 226, wherein (c) comprises identifying the tags from the subset of the plurality of candidate binding partners.228. The method of any one of embodiments 225-227, wherein the tags comprise nucleic acids.229. The method of embodiment 228, wherein the tags comprise deoxyribonucleic acids (DNA).230. The method of embodiment 228, wherein the tags comprise single-stranded DNA.231. The method of embodiment 228, wherein the tags comprise double-stranded DNA.232. The method of embodiment 228, wherein the tags comprise ribonucleic acids (RNA).233. The method of any one of embodiments 227-232, wherein identifying the tags comprises sequencing the tags from the subset of the plurality of candidate binding partners.234. The method of any one of embodiments 190-233, wherein the plurality of candidate binding partners comprises small molecules.235. The method of any one of embodiments 190-234, wherein the plurality of candidate binding partners comprises proteins.236. The method of any one of embodiments 190-235, wherein the plurality of candidate binding partners comprises nucleic acids.Attorney Docket No. 69414-702601237. The method of any one of embodiments 190-236, wherein (b) comprises providing the target biomolecule and the plurality of candidate binding partners through a common inlet.238. The method of any one of embodiments 190-236, wherein (b) comprises providing the target biomolecule and the plurality of candidate binding partners from fluidically separate inlets.239. The method of any one of embodiments 190-238, wherein the target biomolecule comprises DNA, RNA, a protein, or a combination thereof.240. The method of any one of embodiments 191-239, wherein the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule.241. The method of embodiment 240, wherein the member is a button valve.242. The method of any one of embodiments 190-241, further comprising inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site.243. The method of any one of embodiments 190-242, wherein the method is configured to perform multiple forward and reverse screens simultaneously.244. The method of any one of embodiments 190-243, wherein the plurality of reaction chambers comprises reaction chambers having a volume between 0.5 nL and 50 nL.245. The method of any one of embodiments 190-244, further comprising quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor.246. The method of any one of embodiments 190-245, wherein the method is used for analyzing interactions between DNA sequences and transcription factors.247. The method of any one of embodiments 190-246, wherein the method is employed for analyzing small molecule interactions with the one or more target biomolecules.248. The method of any one of embodiments 190-247, wherein the method is employed for analyzing antibody interactions with the one or more target biomolecules.249. The method of any one of embodiments 190-248, wherein the method is employed for analyzing interactions of peptides or proteins with the one or more target biomolecules using mRNA display libraries.Attorney Docket No. 69414-702601250. The method of any one of embodiments 190-249, wherein the method is employed for analyzing interactions of peptides or proteins with the one or more target biomolecules using cDNA display libraries.251. The method of any one of embodiments 191-250, further comprising administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, or any combination thereof.252. The method of any one of embodiments 190-251, wherein the target biomolecule is engineered to contain one or more unnatural amino acids.253. The method of any one of embodiments 190-252, wherein the target biomolecule is post-translationally modified with one or more chemical entities.254. The method of any one of embodiments 191-253, wherein the one or more chemical entities is selected from the group consisting of phosphate, sugars, and ubiquitin protein.255. A microfluidic system comprising: a. a plurality of reaction chambers on a surface, wherein the surface is configured to display one or more target biomolecules over at least 3% of the surface; b. one or more members positioned adjacent to one or more reaction chambers of the plurality of reaction chambers configured to move along an axis orthogonal to the surface; and c. a hydraulic control module configured to actuate the one or more members.256. The microfluidic system of embodiment 255, wherein the plurality of reaction chambers covers at least 30% of the surface.257. The microfluidic system of embodiment 255 or embodiment 256, wherein the plurality of reaction chambers comprises at least 4200 reaction chambers.258. The microfluidic system of any one of embodiments 255-257, wherein the plurality of reaction chambers displays at least 0.3 pmol of protein.259. The microfluidic system of any one of embodiments 255-258, wherein the plurality of reaction chambers comprises a set of reaction chambers, wherein reaction chambers of the set of reaction chambers are fluidly connected.260. The microfluidic system of embodiment 259, wherein the reaction chambers of the set of reaction chambers are fluidly connected in series.Attorney Docket No. 69414-702601261. The microfluidic system of embodiment 259 or embodiment 260, wherein the reaction chambers of the set of reaction chambers are fluidly connected in parallel.262. The microfluidic system of any one of embodiments 255-261, wherein the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are configured to not be fluidly connected to the set of reaction chambers at any point during use of the microfluidic system.263. The microfluidic system of any one of embodiments 255-262, wherein the plurality of reaction chambers is not connected to a reservoir chamber adjacent to a reaction chamber of the plurality of reaction chambers on the surface.264. The microfluidic system of any one of embodiments 255-263, wherein the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface.265. The microfluidic system of any one of embodiments 255-264, wherein the plurality of reaction chambers comprises one or more reaction chambers having a volume between 0.5 nL and 50 nL.266. The microfluidic system of any one of embodiments 255-265, further comprising a two-layer elastomer structure positioned on top of a glass layer.267. The microfluidic system of any one of embodiments 255-266, further comprising integrated push-down valves configured in a Quake-style arrangement, or a pneumatically or hydraulically actuated elastomeric valve arrangement for controlling fluid flow.268. The microfluidic system of any one of embodiments 255-267, wherein the microfluidic system is configured to quantitatively assay the one or more target biomolecules, or a subset thereof, simultaneously.269. The microfluidic system of any one of embodiments 255-268, wherein, when the one or more members are open, fluid flows through the full volume of all reaction chambers in the plurality of reaction chambers.270. The microfluidic system of any one of embodiments 255-269, wherein at least one reaction chamber of the plurality of reaction chambers is an elongated reaction chamber.271. The microfluidic system of embodiment 270, wherein the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area.Attorney Docket No. 69414-702601272. The microfluidic system of embodiment 271, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.273. The microfluidic system of embodiment 271, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.274. The microfluidic system of embodiment 271, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.275. The microfluidic system of embodiment 271, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.276. The microfluidic system of embodiment 271 or embodiment 272, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 6000 chambers per chip.277. The microfluidic system of embodiment 271 or embodiment 273, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 4500 chambers per chip.278. The microfluidic system of embodiment 271 or embodiment 274, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 3500 chambers per chip.279. The microfluidic system of embodiment 271 or embodiment 275, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 3000 chambers per chip.280. The microfluidic system of any one of embodiments 255-279, wherein the surface has an area greater than 1100 mm2.281. The microfluidic system of any one of embodiments 255-280, wherein the surface has a dimension of 25 mm by 43 mm.

Claims

Attorney Docket No. 69414-702601CLAIMSWHAT IS CLAIMED IS:

1. A method of identifying one or more binding partners of one or more target biomolecules, the method comprising: a. providing a device, wherein the device comprises a plurality of reaction chambers on a surface, wherein the plurality of reaction chambers comprises one or more sets of reaction chambers; b. providing a target biomolecule of the one or more target biomolecules and a plurality of candidate binding partners of the target biomolecule to a set of reaction chambers, wherein the surface is configured to display the one or more target biomolecules over at least 3% of the surface; and c. identifying one or more binding partners of the target biomolecule from a subset of the plurality of candidate binding partners bound to the target biomolecule.

2. The method of claim 1, further comprising (i) mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device, and (ii) generating one or more complexes comprising the target molecule and the subset of the plurality of candidate binding partners.

3. The method of claim 1 or claim 2, wherein the plurality of candidate binding partners comprises at least 10,000 unique candidate binding partners.

4. The method of claim 2 or claim 3, wherein the method does not comprise tagging the one or more complexes with a barcode in the device.

5. The method of any one of claims 1-4, wherein the plurality of candidate binding partners is configured to interact with two or more binding sites on the target biomolecule.

6. The method of any one or claims 1-5, wherein the one or more target biomolecules comprises one or more variants of the target biomolecule.

7. The method of any one of claims 1-6, wherein the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are not fluidly connected to the set of reaction chambers during (b).Attorney Docket No. 69414-7026018. The method of claim 7, further comprising providing a second target biomolecule of the one or more target biomolecules and the plurality of candidate binding partners of the second target biomolecule to the additional set of reaction chambers.

9. The method of claim 8, further comprising identifying one or more binding partners of the second target biomolecule from the plurality of candidate binding partners.

10. The method of claim 9, further comprising identifying a difference between the one or more binding partners of the target biomolecule and the one or more binding partners of the second target biomolecule.

11. The method of any one of claims 1-10, further comprising identifying one or more binding sites of the one or more binding partners.

12. The method of claim 11, wherein the identifying the one or more binding sites of the one or more binding partners of the target biomolecule is based at least in part on comparing the target biomolecule and the second target biomolecule.

13. The method of any one of claims 1-12, wherein the plurality of reaction chambers comprises at least 4200 reaction chambers.

14. The method of any one of claims 1-13, wherein the plurality of reaction chambers covers at least 30% of the surface.

15. The method of any one of claims 1-14, wherein the plurality of reaction chambers displays at least 0.3 pmol of protein.

16. The method of any one of claims 1-15, wherein (b) does not comprise providing the target biomolecule from a reservoir chamber on the surface adjacent to a reaction chamber of the set of reaction chambers.

17. The method of any one of claims 1-16, further comprising eluting the subset of the plurality of candidate binding partners from the one or more complexes.

18. The method of claim 17, further comprising providing the subset of the plurality of candidate binding partners to a set of reaction chambers on a different surface.

19. The method of any one of claims 1-18, further comprising obtaining an unbound subset of the plurality of candidate binding partners, wherein the unbound subset of the plurality of candidate binding partners is not bound to the target biomolecule.

20. The method of claim 19, further comprising providing the unbound subset of the plurality of candidate binding partners to the set of reaction chambers.Attorney Docket No. 69414-70260121. The method of any one of claims 1-20, further comprising expressing the one or more target biomolecules external to the device.

22. The method of any one of claims 1-21, wherein the plurality of candidate binding partners comprises tags.

23. The method of claim 22, wherein the tags comprise nucleic acids.

24. The method of any one of claims 1-23, wherein the plurality of candidate binding partners comprises small molecules.

25. The method of any one of claims 1-24, wherein the plurality of candidate binding partners comprises proteins.

26. The method of any one of claims 1-25, wherein the plurality of candidate binding partners comprises nucleic acids.

27. The method of any one of claims 1-26, wherein the target biomolecule comprises DNA, RNA, a protein, or a combination thereof.

28. The method of any one of claims 2-27, wherein the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule.

29. The method of any one of claims 1-28, further comprising inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site.

30. The method of any one of claims 1-29, wherein the plurality of reaction chambers comprises reaction chambers having a volume between 0.5 nL and 50 nL.

31. The method of any one of claims 1-30, further comprising quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor.

32. The method of any one of claims 2-31, further comprising administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, or any combination thereof.

33. A method for identifying a candidate binding partner of a target biomolecule, the method comprising:Attorney Docket No. 69414-702601 a. providing a device, wherein the device comprises a reaction chamber; b. providing the target biomolecule and one or more candidate binding partners of the target biomolecule; c. mechanically trapping the target biomolecule and the one or more candidate binding partners of the target biomolecule between a first surface and a second surface of the device; d. subsequent to (c), tagging the target biomolecule, the candidate binding partners, or both with a barcode in the device; and e. identifying the barcode, thereby identifying the target biomolecule, the candidate binding partner, additional information about an interaction between the target biomolecule and the one or more candidate binding partners, or any combination thereof.

34. The method of claim 33, wherein the reaction chamber is amongst a plurality of reaction chambers, wherein the device comprises the plurality of reaction chambers.

35. The method of claim 33, wherein the one or more candidate binding partners comprise an additional barcode that encodes a unique identifier corresponding to the candidate binding partner.

36. The method of claim 33, wherein the one or more candidate binding partners comprises a small molecule.

37. The method of claim 33, wherein the second surface comprises a member configured to move along an axis orthogonal to a surface of a reaction zone, wherein the reaction zone comprises a surface of the reaction chamber coated by a coating, and wherein the coating is configured to immobilize the target biomolecule.

38. The method of claim 33, wherein the one or more candidate binding partners interact with one or more binding sites of the target biomolecule.

39. The method of claim 33, wherein the method further comprises identifying one or more allosteric binding sites of the target biomolecule.

40. The method of claim 33, wherein the method further comprises inputting data associated with an identified allosteric binding site of the target biomolecule into a bioinformatics model to identify one or more compounds that bind to the identified allosteric binding site.Attorney Docket No. 69414-70260141. The method of claim 33, wherein the method further comprises trapping a bound interaction between the target biomolecule and the one or more candidate binding partners, washing away unbound compounds, then eluting a solution from the reaction zone, thereby collecting the bound interaction.

42. The method of claim 33, wherein the reaction chamber comprises microchambers having a volume configured for screening of a plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule, wherein the volume comprises between 0.5 nL and 50 nL.

43. The method of claim 33, further comprising introducing single- or multi-mutant target biomolecules, including unnatural or extant allelic variants, to the target biomolecule, and assessing the effect of the introduced variants on identification of binding partners.

44. The method of claim 33, further comprising performing scanning mutagenesis on the target biomolecule to assess the effects on binding partner identification.

45. The method of claim 33, further comprising performing mutagenesis on the target biomolecule to localize binding sites of binding partners.

46. The method of claim 33, further comprising performing mutagenesis to introduce multiple mutations in the target biomolecule and assessing effects of the multiple mutations on binding partner identification.

47. The method of claim 33, further comprising performing a plurality of screens with a plurality of target biomolecules to identify a plurality of sets of binding partners of the plurality of target biomolecules, wherein the plurality of target biomolecules comprises the target biomolecule.

48. The method of claim 47, wherein the plurality of target biomolecules comprises one or more variants of the target biomolecule.

49. The method of claim 47 or claim 48, wherein performing the plurality of screens comprises identifying variation amongst the plurality of sets of binding partners.

50. The method of claim 33, further comprising quantifying bound interactors to determine binding affinities or relative binding affinities for each interactor.

51. The method of claim 33, further comprising administrating a coating to the first surface or second surface of the device, wherein the coating comprises a biotinylated albumin, a neutravidin, a biotinylated antibody, a biotinylated nanobody, a target in vitro expression plasmid, a protein-identifying barcode, or any combination thereof.Attorney Docket No. 69414-70260152. The method of claim 33, wherein the method includes the expression and immobilization of the target biomolecule in the device.

53. The method of claim 33, wherein the device further comprises an array configured to allow programming via microarray prints.

54. The method of claim 33, further comprising, prior to (b), disposing one or more nucleic acid molecules onto a surface adjacent to the device, wherein the one or more nucleic acid molecules encode the target biomolecule, the candidate binding partner, the barcode or any combination thereof.

55. The method of claim 54, wherein the one or more nucleic acid molecules comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes the target biomolecule, wherein the second nucleic acid molecule encode the barcode, and wherein the barcode corresponds to the target biomolecule.

56. The method of claim 55, wherein the first nucleic acid molecule is provided in a first chamber of the device, and wherein the second nucleic acid molecule is provided in a second chamber of the device, and wherein the first chamber of the device and the second chamber of the device are fluidically coupled to the reaction chamber.

57. The method of claim 33, wherein the device further comprises an array configured to introduce a DNA barcode encoding the target biomolecule via microarray prints, wherein the printed DNA barcode is in a reservoir chamber adjacent to the DNA encoding of the target biomolecule and connected to a same reaction chamber.

58. The method of claim 57, wherein the reservoir chamber is an elongated reservoir chamber.

59. The method of claim 33, wherein the reaction chamber is an elongated reaction chamber.

60. The method of claim 33, wherein the method includes expressing the target biomolecule or multiple proteins off the device, followed by immobilization of the target biomolecule or proteins on the device.

61. The method of claim 33, wherein the method involves flowing on DNA-tagged small molecules onto the device.

62. The method of claim 33, wherein the method involves flowing on DNA-tagged protein molecules onto the device.Attorney Docket No. 69414-70260163. The method of claim 33, further comprising attaching in situ barcodes to molecules on the device.

64. The method of claim 33, further comprising the elution of in situ barcoded small molecules.

65. The method of claim 33, further comprising the elution of in situ barcoded macro molecules, including DNA, RNA, and protein.

66. The method of claim 33, wherein protein interactors are introduced by a microarray print.

67. The method of claim 33, further comprising using spatial barcoding to map interactors to their respective target biomolecule.

68. The method of claim 33, further comprising using temporal barcoding to tag reaction products based on the order of generation.

69. The method of claim 33, further comprising sequentially flowing a first library of interactors onto the reaction zone, thereby causing at least one interactor of the first library of interactors to bind, flowing a first barcode to label bound interactors, eluting the first library of interactors, subsequently flowing a second library of interactors onto the reaction zone, flowing a second barcode to label bound interactors, and eluting the second library of interactors, wherein each barcode corresponds to a specific library of interactors.

70. The method of claim 33, further comprising employing temporal barcoding to tag interactors based on the order of introduction to the chip.

71. The method of claim 33, wherein barcoding is performed on-chip.

72. A microfluidic system comprising: a. one or more reaction chambers comprising a reaction surface; b. a plurality of reservoir chambers fluidically connected to the reaction chamber via a set of channels; c. one or more members positioned adjacent to the reaction chamber and configured to move along an axis orthogonal to the reaction surface; and d. a hydraulic control module configured to actuate the one or more members.

73. The microfluidic system of claim 72, wherein each reservoir chamber of the plurality of reservoir chambers is located on a same side of the reaction chamber.

74. The microfluidic system of claim 72, wherein at least one reaction chamber is an elongated reaction chamber.Attorney Docket No. 69414-70260175. The microfluidic system of claim 72, wherein at least one reservoir chamber of the plurality is an elongated chamber.

76. The microfluidic system of claim 72, wherein the set of channels fluidically connects at least two reservoir chambers of the plurality to the reaction chamber.

77. The microfluidic system of claim 72, wherein the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface.

78. The microfluidic system of claim 77, wherein the orthogonal movement of the member is configured to control fluidic mixing between at least one reservoir chamber of the plurality of reservoir chambers and one or more reaction chambers.

79. The microfluidic system of claim 72, wherein each of the plurality of reservoir chambers is oriented at an angle relative to at least one surface of a reaction chamber of the one or more reaction chambers.

80. The microfluidic system of claim 72, wherein the plurality of reservoir chambers has a denser packing efficiency compared to a microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.

81. The microfluidic system of claim 72, wherein the denser packing efficiency is at least 1.75-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.

82. The microfluidic system of claim 72, wherein a volume of at least one reaction chamber is at least 4.71-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.

83. The microfluidic system of claim 82, wherein a member area of the reaction chamber is at least 2 to 10-fold greater than that of the microfluidic system having a circular reaction chamber and a plurality of reservoir chambers located on opposite sides of the reaction chamber wherein the at least two reservoir chambers are located on opposite sides of the reaction chamber.Attorney Docket No. 69414-70260184. The microfluidic system of claim 72, wherein at least one reservoir chamber of the plurality of reservoir chambers are reversibly separated from the one or more reaction chambers, or made contiguous with the one or more reaction chambers by a plurality of neck valves.

85. The microfluidic system of claim 72, further comprising micro-chambers with volumes between 0.5 nL and 50 nL, wherein the micro-chambers are configured to house incubations and reactions.

86. The microfluidic system of claim 72, wherein the system is configured to simultaneously express and purify a plurality of proteins, including enzymes or variants thereof, wherein the plurality of proteins comprises from at least one to at least 1,000 proteins or variants thereof.

87. The microfluidic system of claim 72, wherein the system is configured to quantitatively assay all or a subset of proteins or protein variants simultaneously.

88. The microfluidic system of claim 72, wherein a second reservoir chamber is configured to introduce a unique molecular barcode for multiplexing across multiple protein variants, while maintaining isolation between chambers.

89. The microfluidic system of claim 74, wherein the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area.

90. The microfluidic system of claim 89, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.

91. The microfluidic system of claim 89, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.

92. The microfluidic system of claim 89, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.

93. The microfluidic system of claim 89, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.

94. The microfluidic system of claim 89, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 1200 chambers per chip.Attorney Docket No. 69414-70260195. The microfluidic system of claim 89, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 900 chambers per chip.

96. The microfluidic system of claim 89, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 700 chambers per chip.

97. The microfluidic system of claim 89, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 600 chambers per chip.

98. A microfluidic system comprising: a. a plurality of reaction chambers on a surface, wherein the surface is configured to display one or more target biomolecules over at least 3% of the surface; b. one or more members positioned adjacent to one or more reaction chambers of the plurality of reaction chambers configured to move along an axis orthogonal to the surface; and c. a hydraulic control module configured to actuate the one or more members.

99. The microfluidic system of claim 98, wherein the plurality of reaction chambers covers at least 30% of the surface.

100. The microfluidic system of claim 98 or claim 99, wherein the plurality of reaction chambers comprises at least 4200 reaction chambers.

101. The microfluidic system of any one of claims 98-100, wherein the plurality of reaction chambers displays at least 0.3 pmol of protein.

102. The microfluidic system of any one of claims 98-101, wherein the plurality of reaction chambers comprises a set of reaction chambers, wherein reaction chambers of the set of reaction chambers are fluidly connected.

103. The microfluidic system of any one of claims 98-102, wherein the plurality of reaction chambers comprises an additional set of reaction chambers, wherein the additional set of reaction chambers are configured to not be fluidly connected to the set of reaction chambers at any point during use of the microfluidic system.

104. The microfluidic system of any one of claims 98-103, wherein the plurality of reaction chambers is not connected to a reservoir chamber adjacent to a reaction chamber of the plurality of reaction chambers on the surface.Attorney Docket No. 69414-702601105. The microfluidic system of any one of claims 98-104, wherein the hydraulic control module is configured to actuate at least one member along the axis orthogonal to the reaction surface.

106. The microfluidic system of any one of claims 98-105, wherein the plurality of reaction chambers comprises one or more reaction chambers having a volume between 0.5 nL and 50 nL.

107. The microfluidic system of any one of claims 98-106, wherein the microfluidic system is configured to quantitatively assay the one or more target biomolecules, or a subset thereof, simultaneously.

108. The microfluidic system of any one of claims 98-107, wherein at least one reaction chamber of the plurality of reaction chambers is an elongated reaction chamber.

109. The microfluidic system of claim 108, wherein the elongated reaction chamber comprises variable stretch values, resulting in proportional fold changes to chamber volume and member area.

110. The microfluidic system of claim 109, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a chamber volume fold change of at least 2.1 and a member area fold change of at least 2.8.

111. The microfluidic system of claim 109, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a chamber volume fold change of at least 3.2 and a member area fold change of at least 4.6.

112. The microfluidic system of claim 109, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a chamber volume fold change of at least 4.3 and a member area fold change of at least 6.4.

113. The microfluidic system of claim 109, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a chamber volume fold change of at least 5.5 and a member area fold change of at least 8.3.

114. The microfluidic system of claim 109 or claim 110, wherein the application of a stretch value of 200 microns to the elongated reaction chamber results in a configuration of at least 6000 chambers per chip.

115. The microfluidic system of claim 109 or claim 111, wherein the application of a stretch value of 400 microns to the elongated reaction chamber results in a configuration of at least 4500 chambers per chip.Attorney Docket No. 69414-702601116. The microfluidic system of claim 109 or claim 112, wherein the application of a stretch value of 600 microns to the elongated reaction chamber results in a configuration of at least 3500 chambers per chip.

117. The microfluidic system of claim 109 or claim 113, wherein the application of a stretch value of 800 microns to the elongated reaction chamber results in a configuration of at least 3000 chambers per chip.

118. The microfluidic system of any one of claims 98-117, wherein the surface has an area greater than 1100 mm2.

119. The microfluidic system of any one of claims 98-118, wherein the surface has a dimension of 25 mm by 43 mm.

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