HK40076603A - Methods for screening b cell lymphocytes - Google Patents

Description

Method for screening B cell lymphocytes

The application is a divisional application of Chinese patent application with the application number of 2017800800812, the application date of 2017, 10 and 23 and the name of the invention of a method for screening B cell lymphocytes.

This application is a non-provisional application claiming priority from U.S. provisional application No. 62/411,690 filed 2016, month 10, 23 and U.S. provisional application No. 62/412,092 filed 2016, month 10, 24, the disclosures of each of which are incorporated herein by reference in their entireties in accordance with 35 u.s.c.119 (e).

Background

It is of interest to screen and identify cells that produce antibodies capable of specifically binding to an antigen of interest, including in the field of hybridoma development. Furthermore, it is of interest to identify highly expressed antibody producing cells. Providing a suitable environment suitable for the growth environment of the antibody producing cells and providing an environment in which binding/expression assays can be easily monitored has been a formidable challenge. Furthermore, it is desirable to provide a correlation of the assay results with a particular cell that demonstrates the desired expression/binding characteristics of the antibody it secretes. Improvements to these aspects of the field of antibody development are provided herein.

Disclosure of Invention

The present invention is based in part on the following findings: b cell lymphocytes, including primary B cells, can be screened within a microfluidic device to determine whether the B cell lymphocytes express antibodies that specifically bind to an antigen of interest. Accordingly, in one aspect, a method of detecting that an antibody-producing cell expresses an antibody that specifically binds to an antigen of interest is provided. The method comprises the step of introducing antibody-producing cells into a microfluidic device. The antibody-producing cell may be, for example, a B cell lymphocyte, such as a memory B cell or a plasma cell.

For example, a microfluidic device may include a flow region, which may include a microfluidic channel, and at least one microfluidic isolation dock (e.g., a plurality of isolation docks). Each isolation dock may include a separation region and a connection region that fluidly connects the separation region to a flow region (e.g., a microfluidic channel).

Some of the methods of the present disclosure include the additional steps of: loading antibody-producing cells into a separate area of a sequestration dock; introducing a target antigen into the microfluidic device such that the target antigen is in proximity to the antibody-producing cells; and monitoring binding of the antigen of interest to the antibody expressed by the antibody-producing cell. The loaded cell may be one of a population of cells (e.g., B cells) loaded into a microfluidic device having a plurality of isolated docks. In such embodiments, one or more antibody-producing cells can be loaded into the isolated region of each of the plurality of isolated docks. In some embodiments, a single antibody-producing cell is loaded into each isolation dock. When provided in the vicinity of an antibody-producing cell, the antigen of interest may be solubilized or attached to a micro-object, such as a cell, a liposome, a lipid nanoraft (lipid nanoraft), or a synthetic bead (e.g., a microbead or nanobead). These micro objects can be visualized under a microscope. Monitoring binding between the antigen of interest and the antibody produced by the antibody-producing cell may comprise: providing a labeled target antigen and detecting direct binding of the target antigen (e.g., labeled target antigen); providing a labeled antibody binding agent, and detecting indirect binding of the labeled antibody binding agent to the antigen of interest (e.g., to a micro-object presenting the antigen of interest); and providing an antibody binding agent and detecting indirect binding of the labeled target antigen to the antibody binding agent (e.g., a micro-object linked to a plurality of antibody binding agents). The antibody binding agent can be isotype specific (e.g., an anti-IgG antibody or IgG-binding fragment thereof). The label on the antigen or target or antibody binding agent may be a fluorescent label.

For antibody-producing cells that are recognized as expressing antigen-binding antibodies, the disclosed methods can further comprise the steps of: lysing the identified cells (e.g., B cells); reverse transcription of V from lysed cells_HmRNA and/or V_LmRNA to form V respectively_HcDNA and/or V_LcDNA; and for the V_HcDNA and/or V_LAt least a portion of the cDNA is sequenced. The lysis and reverse transcription steps may be performed within the microfluidic device or external to the microfluidic device. For example, the identified cells may be exported (e.g., as individual cells) for cell lysis and further processing. Alternatively, the recognized cells are lysed in the sequestration dock loaded with it, the V released after lysis_HmRNA and/or V_LThe mRNA can be captured on beads (i.e., beads having oligonucleotides attached to their surfaces, wherein the oligonucleotides are capable of specifically binding to V_HmRNA and/or V_LmRNA) capture. The capture beads may be at the captured V_HmRNA and/or captured V_LThe mRNA is output from the microfluidic device either before or after reverse transcription.

These and other features and advantages of the method of the present invention will be set forth or will become more fully apparent in the description that follows and in the appended claims. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended embodiments and claims. Furthermore, the features and advantages of the described systems and methods may be learned by the practice or will be obvious from the description, as set forth hereinafter.

Brief description of the drawings

Fig. 1A illustrates an example of a microfluidic device and system for use with the microfluidic device, including associated control apparatus according to some embodiments disclosed herein.

Fig. 1B and 1C illustrate vertical and horizontal cross-sectional views, respectively, of a microfluidic device according to some embodiments disclosed herein.

Fig. 2A and 2B show vertical and horizontal cross-sectional views, respectively, of a microfluidic device with isolated docks, according to some embodiments of the present invention.

Fig. 2C illustrates a detailed horizontal cross-sectional view of an isolation dock, according to some embodiments disclosed herein.

Fig. 2D illustrates a partial horizontal cross-sectional view of a microfluidic device with isolated docks according to some embodiments disclosed herein.

Fig. 2E and 2F show detailed horizontal cross-sectional views of an isolation dock according to some embodiments disclosed herein.

Fig. 2G illustrates a microfluidic device having a flow region comprising a plurality of flow channels, each flow channel fluidically connected to a plurality of isolation docks, according to embodiments disclosed herein.

Fig. 2H illustrates a partial vertical cross-sectional view of a microfluidic device according to embodiments disclosed herein, wherein the inward-facing surface of the base and the inward-facing surface of the lid are conditioned surfaces.

Fig. 3A illustrates a specific example of a system nest configured to operably connect with a microfluidic device and associated control apparatus, according to some embodiments disclosed herein.

Fig. 3B illustrates an optical train of a system for controlling a microfluidic device according to some embodiments disclosed herein.

Fig. 4 illustrates steps in an exemplary workflow for detecting B cell lymphocytes expressing antibodies that specifically bind a target antigen, according to some embodiments disclosed herein.

Fig. 5A-5C are photographic illustrations of a microfluidic device comprising a plurality of microfluidic channels, each microfluidic channel being fluidically connected to a plurality of sequestration docks, and showing a method of screening for B-cell lymphocytes according to some embodiments disclosed herein.

Fig. 6A is a schematic illustration of a method for activating and screening memory B cells according to embodiments disclosed herein.

Fig. 6B is an image of individual memory B cells moved into an isolation dock according to embodiments disclosed herein.

Fig. 6C is a flow diagram of a multiplex assay according to some embodiments disclosed herein.

Fig. 6D is a fluorescence image of memory B cells determined according to embodiments disclosed herein.

Fig. 6E is a schematic diagram of steps in a method for screening memory B cells, according to embodiments disclosed herein, that begins with assaying a polyclonal group of memory B cells, and then segregating the memory B cell component into individual sequestration docks for subsequent assays.

Fig. 7A is a schematic illustration of a method for screening plasma cells according to embodiments disclosed herein.

Fig. 7B is a set of bright field and corresponding fluorescence images of plasma cells determined according to embodiments disclosed herein.

FIGS. 8A-8H are schematic diagrams of methods for generating BCR sequencing libraries.

Figure 9A is a graphical representation of electropherogram analysis of the size distribution of cDNA generated by single cell export and mRNA capture.

FIG. 9B is a photographic illustration of an electropherogram generated from single-cell amplicons generated by one embodiment of the methods described herein.

FIGS. 10A-10C are photographic illustrations of amplicon electrophoretograms generated from 19 single cell captured mRNA according to one embodiment of the methods described herein.

Detailed description of the invention

This specification describes exemplary embodiments and applications of the invention. However, the invention is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may be exaggerated or not in proportion. In addition, as the terms "on," "attached," "connected," "coupled" or similar terms are used herein, an element (e.g., a material, a layer, a substrate, etc.) may be "on," "attached to," "connected to" or "coupled to" another element, whether the element is directly on, attached to, connected to or coupled to the other element, or one or more intervening elements may be present between the element and the other element. Further, unless the context dictates otherwise, directions (e.g., above, below, top, bottom, side, up, down, below … …, above … …, above, below, horizontal, vertical, "x", "y", "z", etc.) if provided are relative and provided by way of example only and for ease of illustration and discussion and not by way of limitation. In addition, where a list of elements (e.g., elements a, b, c) is recited, such reference is intended to include any one of the recited elements themselves, any combination of fewer than all of the recited elements, and/or combinations of all of the recited elements. The division of the sections in the description is for ease of review only and does not limit any combination of the elements discussed.

As used herein, "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from absolute or perfect states, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art without significantly affecting overall performance. "substantially" when used in relation to a numerical value or a parameter or characteristic that may be expressed as a numerical value means within ten percent.

The terms "a" and "an" mean more than one.

As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, the term "disposed" includes within its meaning "located".

As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that: comprising one or more separate microfluidic conduits configured to contain a fluid, each microfluidic conduit comprising fluidly interconnected conduit elements including, but not limited to, regions, flow paths, channels, chambers, and/or docks; and at least one port configured to allow fluid (and optionally micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will include a flow region (which flow path may include a microfluidic channel) and at least one chamber, and will accommodate a fluid volume of less than about 1mL (e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μ Ι _). In certain embodiments, the microfluidic circuit contains about 1-2, 1-3, 1-4, 1-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-30, 5-40, 5-50, 10-75, 10-100, 20-150, 20-200, 50-250, or 50-300 μ L. The microfluidic circuit may be configured to have a first end in fluid connection with a first port (e.g., inlet) in the microfluidic device and a second end in fluid connection with a second port (e.g., outlet) in the microfluidic device.

As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a microfluidic device having microfluidic circuit containing at least one circuit element configured to accommodate a fluid volume of less than about 1 μ L, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1nL or less. The nanofluidic device may include a plurality of tubing elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one piping element is configured to hold a fluid volume of about 100pL to 1nL, 100pL to 2nL, 100pL to 5nL, 250pL to 2nL, 250pL to 5nL, 250pL to 10nL, 500pL to 5nL, 500pL to 10nL, 500pL to 15nL, 750pL to 10nL, 750pL to 15nL, 750pL to 20nL, 1 to 10nL, 1 to 15nL, 1 to 20nL, 1 to 25nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one piping element is configured to hold a volume of fluid of about 20nL to 200nL, 100 to 300nL, 100 to 400nL, 100 to 500nL, 200 to 300nL, 200 to 400nL, 200 to 500nL, 200 to 600nL, 200 to 700nL, 250 to 400nL, 250 to 500nL, 250 to 600nL, or 250 to 750 nL.

Microfluidic devices or nanofluidic devices may be referred to herein as "microfluidic chips" or "chips"; or "nanofluidic chip" or "chip".

As used herein, "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device having a length that is significantly longer than the horizontal and vertical dimensions. For example, the flow channel can be at least 5 times the length of the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of the flow channel is in the range of about 50,000 micrometers to about 500,000 micrometers, including any range therebetween. In some embodiments, the horizontal dimension is in a range from about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is in a range from about 25 microns to about 200 microns, e.g., about 40 to about 150 microns. It should be noted that the flow channels may have various different spatial configurations in the microfluidic device and are therefore not limited to perfectly linear elements. For example, the flow channel may include one or more portions having any of the following configurations: curved, bent, spiral, inclined, descending, forked (e.g., multiple distinct flow paths), and any combination thereof. In addition, the flow channel may have different cross-sectional areas along its path, widening and narrowing to provide the desired fluid flow therein.

As used herein, the term "obstruction" generally refers to a protrusion or similar type of structure that is large enough to partially (but not completely) impede movement of a target micro-object between two different regions or conduit elements in a microfluidic device. The two different regions/piping elements may for example be a microfluidic isolation dock and a microfluidic channel, or a connection region and a separation region of a microfluidic isolation dock.

As used herein, the term "constriction" generally refers to a narrowing of the width of a conduit element (or the interface between two conduit elements) in a microfluidic device. The constriction may be located, for example, at the interface between the microfluidic isolation dock and the microfluidic channel, or at the interface between the separation region and the connection region of the microfluidic isolation dock.

As used herein, the term "transparent" refers to a material that allows the passage of visible light without substantially altering the light upon passage.

As used herein, the term "micro-object" generally refers to any micro-object that can be separated and/or processed according to the present invention. Non-limiting examples of micro-objects include: inanimate micro-objects, such as microparticles; microbeads (e.g., polystyrene beads, Luminex)^TMBeads, etc.); magnetic beads; a micron rod; microfilaments; quantum dots, and the like; biological micro-objects, such as cells; a biological organelle; a vesicle or complex; synthesizing vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts (nanorafts), etc.; or a combination of inanimate and biological micro-objects (e.g., cell-attached microbeads, liposome-coated magnetic beads, etc.). The beads may include covalently or non-covalently attached moieties/molecules, such as fluorescent markers, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological substances that can be used in assays. Lipid nanorafts have been described, for example, in Ritchie et al (2009) "Regulation of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods enzymol.,464: 211-231.

As used herein, the term "cell" may be used interchangeably with the term "biological cell". "non-limiting examples of biological cells include eukaryotic cells; a plant cell; animal cells such as mammalian cells, crawling animal cells, avian cells, fish cells, and the like; a prokaryotic cell; a bacterial cell; a fungal cell; protozoan cells, etc.; cells dissociated from tissue (e.g., muscle, cartilage, fat, skin, liver, lung, neural tissue, etc.); immune cells, such as T cells, B cells, natural killer cells, macrophage cells, and the like; embryos (e.g., fertilized eggs); an oocyte; an ovum; a sperm cell; a hybridoma; a cultured cell; cells from a cell line; cancer cells; infected cells; transfected and/or transformed cells; reporter cells, etc. The mammalian cell can be, for example, a human, mouse, rat, horse, goat, sheep, cow, primate, and the like.

A colony of biological cells is "clonal" if all living cells in the colony that are capable of multiplying are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in the clonal colony are derived from a single parent cell no more than 10 divisions. In other embodiments, all daughter cells in the clonal colony are derived from a single parent cell no more than 14 divisions. In other embodiments, all daughter cells in the clonal colony are from a single parent cell for no more than 17 divisions. In other embodiments, all daughter cells in the clonal colony are derived from a single parent cell no more than 20 divisions. The term "clonal cells" refers to cells of the same clonal colony.

As used herein, a "colony" of biological cells refers to 2 or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells).

As used herein, the term "maintaining the cell(s)" refers to providing an environment comprising fluid and gas components and optionally surfaces that provide the conditions necessary to keep the cells alive and/or expanded.

As used herein, the term "expansion" when referring to cells refers to an increase in the number of cells.

A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, and the like.

As used herein with respect to a fluid medium, "diffusion" and "diffusion" refer to the thermodynamic movement of components of the fluid medium down a concentration gradient.

The phrase "flow of the medium" means that the fluid medium moves as a whole primarily due to any mechanism other than diffusion. For example, the flow of the medium may include the fluid medium moving from one point to another due to a pressure difference between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into the other fluid medium, turbulence and mixing of the media may result.

The phrase "substantially no flow" refers to a flow rate of the fluid medium that is, on average over time, less than the rate at which a component of the material (e.g., the analyte of interest) diffuses into or within the fluid medium. The diffusion rate of the components of such materials may depend on, for example, the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein with respect to different regions within a microfluidic device, the phrase "fluidically coupled" refers to the fluids in each region being coupled to form a single liquid when the different regions are substantially filled with a fluid (e.g., a fluidic medium). This does not mean that the fluids (or fluid media) in the different regions are necessarily identical in composition. In contrast, fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) that are in change as the solutes move down their respective concentration gradients and/or the fluid flows through the device.

Microfluidic (or nanofluidic) devices may include "swept" regions and "unswept" regions. As used herein, a "swept-out" region includes one or more fluidically interconnected conduit elements of microfluidic conduit, each conduit element being subjected to a flow of a medium as fluid flows through the microfluidic conduit. The conduit elements that sweep the region may include, for example, regions, channels, and all or part of the chamber. As used herein, an "unswept" region includes one or more fluidically interconnected conduit elements of a microfluidic conduit, each conduit element being substantially free of the flow of fluid as fluid flows through the microfluidic conduit. The unswept region can be fluidly connected to the swept region, provided that the fluid connection is configured to enable diffusion but substantially no media flow between the swept region and the unswept region. Thus, the microfluidic device may be configured to substantially separate the unswept region from the flow of the medium in the swept region, while substantially only diffusive fluid communication is enabled between the swept region and the unswept region. For example, the flow channel of a microfluidic device is an example of a swept area, while the separation area of a microfluidic device (described in further detail below) is an example of an unswept area.

As used herein, "flow path" refers to one or more fluidly connected conduit elements (e.g., channels, regions, chambers, etc.) that define and are constrained by the trajectory of the media flow. Thus, the flow path is an example of a swept (swept) region of a microfluidic device. Other conduit elements (e.g., unswept areas) may be in fluid connection with the conduit elements comprising the flow path, independent of the flow of the medium in the flow path.

As used herein, "B" is used to denote a single nucleotide, being a nucleotide selected from G (guanosine), C (cytidine), and T (thymidine) nucleotides, but excluding a (adenine).

As used herein, "H" is used to denote a single nucleotide, being a nucleotide selected from A, C and T, but excluding G.

As used herein, "D" is used to denote a single nucleotide, being a nucleotide selected from A, G and T, but excluding C.

As used herein, "K" is used to denote a single nucleotide, being a nucleotide selected from G and T.

As used herein, "N" is used to denote a single nucleotide, being a nucleotide selected from A, C, G and T.

As used herein, "R" is used to denote a single nucleotide, being a nucleotide selected from a and G.

As used herein, "S" is used to denote a single nucleotide, being a nucleotide selected from G and C.

As used herein, "V" is used to denote a single nucleotide, is a nucleotide selected from A, G and C, and does not include T.

As used herein, "Y" is used to denote a single nucleotide, a nucleotide selected from C and T.

As used herein, "I" is used to indicate that the mononucleotide is inosine.

As used herein, the 'A, C, T, G heel' indicates a phosphorothioate substitution in the phosphate linkage of the nucleotide.

As used herein IsoG is isoguanosine; IsoC is isocytidine; IsodG is isoguanosine deoxyribonucleotide and IsodC deoxyribonucleotide. Each isoguanosine and isocytidine ribonucleotide or deoxyribonucleotide contains a nucleobase that is isomeric with a guanine nucleobase or a cytosine nucleobase, respectively, which is normally incorporated into RNA or DNA.

As used herein, rG denotes a ribonucleotide contained within a nucleic acid, and otherwise contains a deoxyribonucleotide. A nucleic acid containing all ribonucleotides may not include a label to indicate that each nucleotide is a ribonucleotide, but it is clear from the context.

As used herein, a "priming sequence" is an oligonucleotide sequence that is part of a larger oligonucleotide and, when separated from the larger oligonucleotide such that the priming sequence includes a free 3' terminus, can serve as a primer in a DNA (or RNA) polymerization reaction.

As used herein: μ m means micron, μm³Refers to cubic micrometers, pL refers to picoliters, nL refers to nanoliters, and μ L (or uL) refers to microliters.

And (4) a loading method. Loading biological micro-objects or micro-objects (such as, but not limited to, beads) may involve the use of fluid flow, gravity, Dielectrophoresis (DEP) forces, electrowetting, magnetic forces, or any combination thereof, as described herein. DEP forces can be generated optically (e.g., by an optoelectronic tweezers (OET) configuration) and/or electrically (e.g., by activating electrodes/electrode regions in a temporal/spatial manner). Similarly, electrowetting forces may be provided optically (e.g. by an opto-electro-wetting (OEW) configuration) and/or electrically (e.g. by activating electrodes/electrode areas in a time-space manner).

Microfluidic devices and systems for operating and viewing such devices. Fig. 1A shows an example of a microfluidic device 100 and system 150 that can be used to screen and detect antibody-producing cells that secrete antibodies that bind (e.g., specifically bind) to an antigen of interest. A perspective view of the microfluidic device 100 is shown with the cover 110 partially cut away to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally includes microfluidic circuit 120 having a flow path 106, and a fluidic medium 180 may optionally carry one or more micro-objects (not shown) into and/or through the microfluidic circuit 120 via the flow path 106. Although a single microfluidic circuit 120 is shown in fig. 1A, a suitable microfluidic device may include a plurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, the microfluidic device 100 may be configured as a nanofluidic device. In the embodiment shown in fig. 1A, the microfluidic circuit 120 includes a plurality of microfluidic isolation docks 124, 126, 128, and 130, each of which has an opening (e.g., a single opening) in fluid communication with the flow path 106. As discussed further below, the microfluidic sequestration dock includes various features and structures that have been optimized for retaining micro-objects in a microfluidic device (e.g., microfluidic device 100) even as the medium 180 flows through the flow path 106. However, before the above is described, a brief description of the microfluidic device 100 and system 150 is provided.

As shown generally in fig. 1A, microfluidic circuit 120 is defined by housing 102. Although the housing 102 can be physically configured in different configurations, in the example shown in fig. 1A, the housing 102 is depicted as including a support structure 104 (e.g., a base), a microfluidic conduit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108 and the cover 110 may be attached to each other. For example, the microfluidic circuit structure 108 may be arranged on an inner surface 109 of the support structure 104, and the cover 110 may be arranged over the microfluidic circuit structure 108. The microfluidic circuit structure 108, together with the support structure 104 and the lid 110, may define elements of a microfluidic circuit 120.

As shown in fig. 1A, the support structure 104 may be located at the bottom of the microfluidic circuit 120 and the lid 110 may be located at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 may be located at the top of the microfluidic circuit 120 and the lid 110 may be located at the bottom of the microfluidic circuit 120. In any event, there may be one or more ports 107, each port 107 including a passageway into or out of the housing 102. Examples of passageways include valves, gates, through-holes, and the like. As shown, the port 107 is a through hole created by a gap in the microfluidic conduit structure 108. However, the port 107 may be located in other components of the housing 102 (e.g., the cover 110). Only one port 107 is shown in fig. 1A, but the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 that serves as an inlet for fluid into the microfluidic circuit 120, and there may be a second port 107 that serves as an outlet for fluid out of the microfluidic circuit 120. Whether the port 107 serves as an inlet or an outlet may depend on the direction of fluid flow through the flow path 106.

The support structure 104 may include one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 may include one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a portion of the semiconductor substrate may be electrically connected to a single electrode). The support structure 104 may further include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrate can be mounted on a PCBA.

The microfluidic circuit structure 108 may define circuit elements of a microfluidic circuit 120. When microfluidic circuit 120 is filled with a fluid, such circuit elements may include spaces or regions that may be fluidically interconnected, such as flow regions (which may include or be one or more flow channels), chambers, docks, wells (traps), and the like. In the microfluidic circuit 120 shown in fig. 1A, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 may partially or completely surround the microfluidic circuit material 116. The frame 114 may be, for example, a relatively rigid structure that substantially surrounds the microfluidic tubing material 116. For example, the frame 114 may comprise a metallic material.

The microfluidic circuit material 116 may be patterned with cavities or the like to define circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 may include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane ("PDMS"), etc.), which may be gas permeable. Other examples of materials from which the microfluidic circuit material 116 may be constructed include molded glass; etchable materials such as silicone (e.g., photo-patternable silicone or "PPS"), photoresist (e.g., SU8), and the like. In some embodiments, such materials (and thus the microfluidic circuit material 116) may be rigid and/or substantially gas impermeable. Regardless, the microfluidic circuit material 116 may be disposed on the support structure 104 and within the frame 114.

The lid 110 may be an integral component of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 may be a structurally different element, as shown in FIG. 1A. The cover 110 may comprise the same or different material as the frame 114 and/or the microfluidic circuit material 116. Similarly, the support structure 104 may be a separate structure from the frame 114 or the microfluidic circuit material 116 (as shown), or an integral part of the frame 114 or the microfluidic circuit material 116. Likewise, the frame 114 and the microfluidic circuit material 116 may be separate structures as shown in fig. 1A or integrated components of the same structure.

In some embodiments, the cover 110 may comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 may include a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the cover 110 may include both a rigid material and a deformable material. For example, one or more portions of the cover 110 (e.g., one or more portions located above the isolation docks 124, 126, 128, 130) may include a deformable material that interfaces with the rigid material of the cover 110. In some embodiments, the cover 110 may further include one or more electrodes. The one or more electrodes may comprise a conductive oxide, such as Indium Tin Oxide (ITO), which may be coated on glass or similar insulating material. Alternatively, the one or more electrodes may be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles embedded in a deformable material such as a polymer (e.g., PDMS), or a combination thereof. Flexible electrodes that may be used in microfluidic devices have been described, for example, in US 2012/0325665(Chiou et al), the contents of which are incorporated herein by reference. In some embodiments, the lid 110 may be modified (e.g., by adjusting all or a portion of the surface facing inward toward the microfluidic circuit 120) to support cell adhesion, viability, and/or growth. The modification may include a coating of a synthetic or natural polymer. In some embodiments, the cover 110 and/or the support structure 104 may be light transmissive. The cap 110 may also include at least one gas permeable material (e.g., PDMS or PPS).

Fig. 1A also shows a system 150 for operating and controlling a microfluidic device (e.g., microfluidic device 100). System 150 includes a power source 192, an imaging device 194 (incorporated within imaging module 164, where device 194 is not shown in fig. 1A itself), and a tilt device 190 (incorporated into tilt module 166, where device 190 is not shown in fig. 1).

The power supply 192 can provide power to the microfluidic device 100 and/or the tilting device 190 to provide a bias voltage or current as desired. The power supply 192 may, for example, include one or more Alternating Current (AC) and/or Direct Current (DC) voltage or current sources. The imaging device 194 (part of the imaging module 164, as discussed below) may include a device for capturing images within the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device 194 further includes a detector with a fast frame rate and/or high sensitivity (e.g., for low light applications). The imaging device 194 may also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beam may be in the visible spectrum and may, for example, include fluorescent emissions. The reflected light beam may comprise reflected emissions from an LED or a broad spectrum lamp such as a mercury lamp (e.g. a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to fig. 3B, the imaging device 194 may further include a microscope (or optical train), which may or may not include an eyepiece.

The system 150 further includes a tilting device 190 (part of the tilting module 166, as discussed below) configured to rotate the microfluidic device 100 about one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or hold the housing 102 including the microfluidic circuit 120 about at least one axis such that the microfluidic device 100 (and thus the microfluidic circuit 120) can be held in a horizontal orientation (i.e., 0 ° with respect to the x-axis and y-axis), a vertical orientation (i.e., 90 ° with respect to the x-axis and/or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and microfluidic circuit 120). For example, the tilting device 190 can tilt the microfluidic device 100 relative to the x-axis by 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 °, 0.8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 90 °, or any angle therebetween. The horizontal orientation (and thus the x-axis and y-axis) is defined as being perpendicular to the vertical axis defined by gravity. The tilting device may also tilt the microfluidic device 100 (and microfluidic circuit 120) by any degree greater than 90 ° with respect to the x-axis and/or the y-axis, or tilt the microfluidic device 100 (and microfluidic circuit 120) by 180 ° with respect to the x-axis or the y-axis, to completely invert the microfluidic device 100 (and microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) about an axis of rotation defined by the flow path 106 or some other portion of the microfluidic circuit 120.

In some cases, the microfluidic device 100 is tilted into a vertical orientation such that the flow path 106 is located above or below one or more isolation docks. The term "above" as used herein means that the flow path 106 is positioned higher than the one or more isolation docks on a vertical axis defined by gravity (i.e., an object in an isolation dock above the flow path 106 will have a higher gravitational potential energy than an object in the flow path). The term "below" as used herein means that the flow path 106 is positioned below the one or more isolation docks on a vertical axis defined by gravity (i.e., an object in an isolation dock below the flow path 106 will have a lower gravitational potential energy than an object in the flow path).

In some cases, the tilting device 190 tilts the microfluidic device 100 about an axis parallel to the flow path 106. Furthermore, the microfluidic device 100 may be tilted to an angle of less than 90 ° such that the flow path 106 is located above or below one or more isolation docks, rather than directly above or below the isolation docks. In other cases, the tilting device 190 tilts the microfluidic device 100 about an axis perpendicular to the flow path 106. In still other cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is neither parallel nor perpendicular to the flow path 106.

The system 150 may further include a media source 178. The media source 178 (e.g., container, reservoir, etc.) may include multiple portions or containers, each portion or container for holding a different fluid medium 180. Thus, the media source 178 can be a device that is external to and separate from the microfluidic device 100, as shown in fig. 1A. Alternatively, the media source 178 may be located in whole or in part inside the housing 102 of the microfluidic device 100. For example, the media source 178 can include a reservoir that is part of the microfluidic device 100.

Fig. 1A also shows a simplified block diagram depicting an example of a control and monitoring apparatus 152 that forms part of the system 150 and that may be used in conjunction with the microfluidic device 100. As shown, examples of such control and monitoring devices 152 include a master controller 154 that may control other controller and monitoring devices, such as a media module 160 for controlling a media source 178; a motion module 162 for controlling movement and/or selection of micro-objects (not shown) and/or media (e.g., drops of media) in the microfluidic circuit 120; an imaging module 164 for controlling an imaging device 194 (e.g., a camera, a microscope, a light source, or any combination thereof) to capture an image (e.g., a digital image); and a tilt module 166 for controlling the tilting device 190. The control apparatus 152 may also include other modules 168 for controlling, monitoring or performing other functions with respect to the microfluidic device 100. As shown, the device 152 may further include a display 170 and an input/output device 172.

The main controller 154 may include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate in accordance with machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-transitory data or signals in a memory 158. Alternatively or additionally, the control module 156 may include hard-wired digital circuitry and/or analog circuitry. Media module 160, motion module 162, imaging module 164, tilt module 166, and/or other modules 168 may be similarly configured. Accordingly, the functions, processes, actions, acts, or steps of the processes discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus may be performed by any one or more of the master controller 154, the substrate module 160, the motion module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 configured as discussed above. Similarly, the master controller 154, the media module 160, the motion module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 may be communicatively coupled to send and receive data used in any of the functions, processes, actions, or steps discussed herein.

The media module 160 controls the media source 178. For example, the media module 160 may control the media source 178 to input a selected fluidic media 180 into the housing 102 (e.g., through the inlet port 107). The media module 160 may also control the removal of media from the housing 102 (e.g., through an outlet port (not shown)). Thus, one or more media may be selectively input into and removed from the microfluidic circuit 120. The media module 160 may also control the flow of fluidic media 180 in the flow path 106 within the microfluidic circuit 120. For example, in some embodiments, media module 160 stops the flow of media 180 in flow path 106 and through housing 102 before tilt module 166 causes tilt device 190 to tilt microfluidic device 100 to a desired tilt angle.

The motion module 162 may be configured to control the selection, trapping, and movement of micro-objects (not shown) in the microfluidic circuit 120. As discussed below with reference to fig. 1B and 1C, the enclosure 102 can include a Dielectrophoresis (DEP), optoelectronic tweezers (OET), and/or optoelectronic wetting (OEW) configuration (not shown in fig. 1A), and the motion module 162 can control activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or media drops (not shown) in the flow path 106 and/or the isolation docks 124, 126, 128, 130.

The imaging module 164 may control an imaging device 194. For example, the imaging module 164 may receive and process image data from the imaging device 194. The image data from imaging device 194 may include any type of information captured by imaging device 194 (e.g., the presence or absence of micro-objects, drops of media, accumulation of markers (e.g., fluorescent markers), etc.). Using the information captured by imaging device 194, imaging module 164 may further calculate the position of objects (e.g., micro-objects, drops of media) within microfluidic device 100 and/or the rate of motion of those objects.

The tilt module 166 may control the tilting motion of the tilting device 190. Alternatively or additionally, the tilting module 166 may control the tilting rate and timing to optimize the transfer of micro-objects to one or more isolation docks via gravity. Tilt module 166 is communicatively coupled with imaging module 164 to receive data describing the movement of micro-objects and/or media drops in microfluidic circuit 120. Using this data, tilt module 166 can adjust the tilt of microfluidic circuit 120 in order to adjust the rate at which micro-objects and/or media droplets move in microfluidic circuit 120. Tilt module 166 may also use this data to iteratively adjust the position of micro-objects and/or media drops in microfluidic circuit 120.

In the example shown in fig. 1A, microfluidic circuit 120 is shown to include microfluidic channel 122 and isolation docks 124, 126, 128, 130. Each dock includes a single opening to the channel 122, but otherwise is enclosed so that the dock can substantially separate micro-objects within the dock from the fluid medium 180 and/or micro-objects in the flow path 106 of the channel 122 or other dock. The walls of the isolation dock extend from the inner surface 109 of the base to the inner surface of the cover 110 to provide an enclosure. The opening of the dock to channel 122 is oriented at an angle to the flow 106 of fluid medium 180 so that the flow 106 is not directed into the dock. The flow may be tangential or perpendicular to the plane of the opening of the dock. In some cases, the docks 124, 126, 128, 130 are configured to physically enclose one or more micro-objects within the microfluidic circuit 120. An isolating dock according to the present invention may include various shapes, surfaces and features, as will be discussed and illustrated in detail below, that are optimized for interaction with DEP, OET, OEW, fluid flow and/or gravity.

The microfluidic circuit 120 may include any number of microfluidic isolation docks. Although five isolation docks are shown, microfluidic circuit 120 may have fewer or more isolation docks. As shown, the microfluidic isolation docks 124, 126, 128, and 130 of the microfluidic circuit 120 each include different features and shapes that may provide one or more benefits for screening antibody-producing cells (e.g., separating one antibody-producing cell from another). Microfluidic sequestration docks 124, 126, 128, and 130 may provide other benefits, such as facilitating single cell loading and/or growth of colonies of antibody producing cells (e.g., clonal colonies). In some embodiments, the microfluidic circuit 120 includes a plurality of identical microfluidic isolation docks.

In some embodiments, microfluidic circuit 120 comprises a plurality of microfluidic isolation docks, wherein two or more of the isolation docks comprise different structures and/or features that provide different benefits of screening for antibody-producing cells. The microfluidic device for screening antibody-producing cells may include any of the isolation docks 124, 126, 128 and 130, or variations thereof, and/or may include docks similar to the dock configurations shown in fig. 2B, 2C, 2D, 2E and 2F, as discussed below.

In the embodiment shown in fig. 1A, a single channel 122 and flow path 106 are shown. However, other embodiments may contain multiple channels 122, each configured to include a flow path 106. The microfluidic circuit 120 further includes an inlet valve or port 107 in fluid communication with the flow path 106 and the fluidic medium 180, whereby the fluidic medium 180 may enter the channel 122 via the inlet port 107. In some cases, the flow path 106 includes a single path. In some cases, the single paths are arranged in a zigzag pattern whereby the flow paths 106 pass through the microfluidic device 100 two or more times in alternating directions.

In some cases, microfluidic circuit 120 includes a plurality of parallel channels 122 and flow paths 106, wherein fluidic medium 180 within each flow path 106 flows in the same direction. In some cases, the fluid medium within each flow path 106 flows in at least one of a forward or reverse direction. In some cases, multiple isolation docks are configured (e.g., relative to channel 122) such that the isolation docks can load the target micro-object in parallel.

In some embodiments, microfluidic circuit 120 further comprises one or more micro-object wells 132. The wells 132 are generally formed in the walls that border the channel 122 and may be disposed opposite the openings of one or more of the microfluidic sequestration docks 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture a single micro-object from the flow path 106. In some embodiments, the trap 132 is configured to receive or capture a plurality of micro-objects from the flow path 106. In some cases, well 132 includes a volume approximately equal to the volume of a single target micro-object.

The well 132 may also include an opening configured to assist the flow of the target micro-object into the well 132. In some cases, well 132 includes an opening having a height and width approximately equal to the dimensions of a single target micro-object, thereby preventing larger micro-objects from entering the micro-object well. The well 132 may further include other features configured to help retain the target micro-object within the well 132. In some cases, wells 132 are aligned with respect to the opening of the microfluidic isolation dock and are located on opposite sides of channel 122 such that when microfluidic device 100 is tilted about an axis parallel to channel 122, trapped micro-objects exit wells 132 in a trajectory that causes the micro-objects to fall into the opening of the isolation dock. In some cases, the well 132 includes side channels 134 that are smaller than the target micro-object in order to facilitate flow through the well 132, thereby increasing the likelihood of micro-objects being trapped in the well 132.

In some embodiments, Dielectrophoretic (DEP) forces are exerted on the fluidic medium 180 (e.g., in the flow path and/or in the isolation dock) via one or more electrodes (not shown) to manipulate, transport, separate, and sort micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of the microfluidic circuit 120 in order to transfer individual micro-objects from the flow path 106 into a desired microfluidic isolation dock. In some embodiments, DEP forces are used to prevent micro-objects within an isolation dock (e.g., isolation dock 124, 126, 128, or 130) from being displaced therefrom. Further, in some embodiments, DEP forces are used to selectively remove micro-objects previously collected according to the teachings of the present invention from the isolation dock. In some embodiments, the DEP force comprises an optoelectronic tweezers (OET) force.

In other embodiments, an electro-optical wetting (OEW) force is applied to one or more locations (e.g., locations that help define a flow path and/or a plurality of isolation docks) in the support structure 104 (and/or lid 110) of the microfluidic device 100 by one or more electrodes (not shown) to manipulate, transport, separate, and sort droplets located in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more locations in the support structure 104 (and/or the lid 110) to transfer individual droplets from the flow path 106 into a desired microfluidic isolation dock. In some embodiments, OEW forces are used to prevent a droplet within a isolation dock (e.g., isolation dock 124, 126, 128, or 130) from being displaced therefrom. Furthermore, in some embodiments, OEW forces are used to selectively remove droplets previously collected according to the teachings of the present invention from the isolating dock.

In some embodiments, DEP and/or OEW forces are combined with other forces (e.g., flow and/or gravity) in order to manipulate, transport, separate, and sort micro-objects and/or droplets within the microfluidic circuit 120. For example, the housing 102 can be tilted (e.g., by the tilting device 190) to position the flow path 106 and micro-objects located therein above the microfluidic isolation dock, and gravity can transport the micro-objects and/or droplets into the dock. In some embodiments, DEP and/or OEW forces may be applied before other forces are applied. In other embodiments, DEP and/or OEW forces may be applied after other forces are applied. In other cases, DEP and/or OEW forces may be applied simultaneously with or in an alternating manner with other forces.

Figures 1B, 1C, and 2A-2H illustrate various embodiments of microfluidic devices that can be used to implement the present invention. Fig. 1B depicts an embodiment of an electrokinetic device in which the microfluidic device 200 is configured to be optically actuated. A variety of optically actuated electrokinetic devices are known in the art, including devices having an opto-electronic tweezers (OET) configuration and devices having an opto-electronic wetting (OEW) configuration. Examples of suitable OET configurations are shown in the following U.S. patent documents, all of which are incorporated herein by reference in their entirety: U.S. Pat. No. RE 44,711(Wu et al) (originally issued in U.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339(Ohta et al). Examples of OEW configurations are shown in U.S. patent No. 6,958,132(Chiou et al) and U.S. patent application publication No. 2012/0024708(Chiou et al), both of which are incorporated herein by reference in their entirety. Another example of a light actuated electrodynamic device includes a combined OET/OEW configuration, examples of which are shown in U.S. patent publication nos. 20150306598(Khandros et al) and 20150306599(Khandros et al) and their corresponding PCT publications WO2015/164846 and WO2015/164847, both of which are incorporated herein by reference in their entirety.

Examples of microfluidic devices with docks in which antibody-producing cells may be placed, cultured, monitored and/or screened have been described, for example, in US application nos. 14/060,117, 14/520,568 and 14/521,447, each of which is incorporated herein by reference in its entirety. Each of the aforementioned applications further describes a microfluidic device configured to generate Dielectrophoretic (DEP) forces, such as optoelectronic tweezers (OET), or configured to provide optoelectronic wetting (OEW). For example, the optoelectronic tweezers device shown in fig. 2 of US application No. 14/060,117 is an example of a device that may be used to select and move a single biological micro-object or a group of biological micro-objects in an embodiment of the present invention.

A microfluidic device motion configuration. As mentioned above, the control and monitoring device of the system may comprise a motion module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. Microfluidic devices may have various motion configurations depending on the type of object being moved and other considerations. For example, a Dielectrophoresis (DEP) configuration can be used to select and move micro-objects in a microfluidic circuit. Accordingly, the support structure 104 and/or the lid 110 of the microfluidic device 100 may comprise a DEP configuration for selectively inducing DEP forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120, thereby selecting, capturing and/or moving individual micro-objects or groups of micro-objects. Alternatively, the support structure 104 and/or the cover 110 of the microfluidic device 100 can comprise an Electrowetting (EW) configuration for selectively inducing EW forces on droplets in the fluidic medium 180 in the microfluidic circuit 120 to select, capture, and/or move individual droplets or groups of droplets.

One example of a microfluidic device 200 containing a DEP configuration is shown in fig. 1B and IC. While fig. 1B and IC show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of the housing 102 of the microfluidic device 200 with an open region/chamber 202 for purposes of simplicity, it should be understood that the region/chamber 202 may be part of a fluidic tubing element having a more detailed structure, such as a growth chamber, isolation dock, flow region, or flow channel. In addition, the microfluidic device 200 may include other fluid conduit elements. For example, the microfluidic device 200 may include multiple growth chambers or isolation docks and/or one or more flow regions or flow channels, such as those described herein with respect to the microfluidic device 100. DEP configurations can be incorporated into any such fluid circuit elements of the microfluidic device 200, or selected portions thereof. It should also be understood that any of the microfluidic device components and system components described above or below may be incorporated into the microfluidic device 200 and/or used in combination with the microfluidic device 200. For example, the system 150 described above including the control and monitoring apparatus 152 may be used with a microfluidic device 200, the microfluidic device 200 including one or more of a media module 160, a motion module 162, an imaging module 164, a tilt module 166, and other modules 168.

As shown in fig. 1B, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activation substrate 206 covering the bottom electrode 204, and a lid 110 having a top electrode 210, the top electrode 210 being spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activation substrate 206 define opposing surfaces of the region/chamber 202. Thus, the dielectric 180 contained in the region/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. Also shown is a power supply 212 configured to connect to the bottom electrode 204 and the top electrode 210 and generate a bias voltage between these electrodes as required to generate DEP forces in the region/chamber 202. The power supply 212 may be, for example, an Alternating Current (AC) power supply.

In certain embodiments, the microfluidic device 200 shown in fig. 1B and 1C can have an optically-actuated DEP configuration. Thus, changing the pattern of light 218 from the light source 216 (which may be controlled by the motion module 162) may selectively activate and deactivate the changing pattern of DEP electrodes at the regions 214 of the interior surface 208 of the electrode activation substrate 206. (hereinafter, the region 214 of the microfluidic device having the DEP configuration is referred to as the "DEP electrode region") as shown in fig. 1C, a light pattern 218 directed at the inner surface 208 of the electrode activation substrate 206 may illuminate a selected DEP electrode region 214a (shown in white) in a pattern such as a square. The non-illuminated DEP electrode regions 214 (cross-hatched) are referred to hereinafter as "dark" DEP electrode regions 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., from the bottom electrode 204 up to the inner surface 208 of the electrode activation substrate 206 interfacing with the medium 180 in the flow region 106) is greater than the relative electrical impedance through the medium 180 in the region/chamber 202 at each dark DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the lid 110). However, the illuminated DEP electrode regions 214a exhibit a reduced relative impedance through the electrode activation substrate 206 that is less than the relative impedance through the medium 180 in the region/chamber 202 at each illuminated DEP electrode region 214 a.

With the power supply 212 activated, the aforementioned DEP configuration creates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, which in turn creates a local DEP force that attracts or repels nearby micro-objects (not shown) in the fluid medium 180. Thus, by varying the light pattern 218 projected from the light source 216 into the microfluidic device 200, DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 214 at the inner surface 208 of the region/chamber 202. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or micro-objects (not shown).

The square pattern 220 of the illuminated DEP electrode regions 214a shown in fig. 1C is merely an example. Any pattern of DEP electrode regions 214 can be illuminated (and thus activated) by a light pattern 218 projected into the apparatus 200, and the pattern of the illuminated/activated DEP electrode regions 214 can be repeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 may include or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 may be featureless. For example, the electrode activation substrate 206 may include or consist of a hydrogenated amorphous silicon (a-Si: H) layer. H may comprise, for example, about 8% to 40% hydrogen (calculated as 100 x number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 μm. In such embodiments, DEP electrode regions 214 may be formed in any pattern anywhere on the inner surface 208 of the electrode activation substrate 206, according to the light pattern 218. Thus, the number and pattern of DEP electrode regions 214 need not be fixed, but may correspond to the light pattern 218. Examples of microfluidic devices having DEP configurations comprising a photoconductive layer (such as those described above) have been described, for example, in U.S. patent No. RE 44,711(Wu et al) (originally issued as U.S. patent No. 7,612,355), the entire contents of which are incorporated herein by reference.

In other embodiments, electrode activation substrate 206 may comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and conductive layers forming a semiconductor integrated circuit, such as are known in the semiconductor arts. For example, the electrode activation substrate 206 may include a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each corresponding to a DEP electrode region 214. Alternatively, electrode activation substrate 206 can include electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, wherein each such electrode corresponds to a DEP electrode region 214. The electrode activation substrate 206 may include a pattern of such phototransistors or phototransistor-controlled electrodes. For example, the pattern may be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns, as shown in FIG. 2B. Alternatively, the pattern may be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes forming a hexagonal lattice (hexagonal lattice). Regardless of the pattern, the circuit elements can form electrical connections between the DEP electrode regions 214 and the bottom electrode 210 at the inner surface 208 of the electrode activation substrate 206, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 218. When not activated, each electrical connection can have a high impedance such that the relative impedance through the electrode activation substrate 206 (i.e., from the bottom electrode 204 to the inner surface 208 of the electrode activation substrate 206 interfacing with the medium 180 in the region/chamber 202) is greater than the relative impedance through the medium 180 at the corresponding DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, when activated by light in the light pattern 218, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 at each illuminated DEP electrode region 214, thereby activating the DEP electrode at the respective DEP electrode region 214, as described above. Thus, DEP electrodes that attract or repel micro-objects (not shown) in the medium 180 can be selectively activated and deactivated at a number of different DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 in the region/chamber 202 in a manner determined by the light pattern 218.

Examples of microfluidic devices having electrode-activated substrates including phototransistors have been described, for example, in U.S. Pat. No. 7,956,339(Ohta et al) (see, e.g., device 300 shown in fig. 21 and 22 and the description thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode-activated substrates including electrodes controlled by phototransistor switches have been described, for example, in U.S. patent publication No. 2014/0124370(Short et al) (see, e.g., devices 200, 400, 500, 600, and 900 and the description thereof shown throughout the figures), the entire contents of which are incorporated herein by reference.

In some embodiments of DEP configured microfluidic devices, the top electrode 210 is part of a first wall (or lid 110) of the housing 102, and the electrode activation substrate 206 and the bottom electrode 204 are part of a second wall (or support structure 104) of the housing 102. The region/chamber 202 may be located between the first wall and the second wall. In other embodiments, the electrode 210 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 206 and/or the electrode 210 is part of the first wall (or cover 110). Further, the light source 216 may alternatively be used to illuminate the housing 102 from below.

With the microfluidic device 200 of fig. 1B-1C having a DEP configuration, the motion module 162 can select a micro-object (not shown) in the medium 180 in the region/chamber 202 by projecting a light pattern 218 into the device 200 to activate a first set of one or more DEP electrodes at the DEP electrode region 214a of the inner surface 208 of the electrode activation substrate 206 in a pattern (e.g., a square pattern 220) that surrounds and captures the micro-object. The motion module 162 can then move the captured micro-object by moving the light pattern 218 relative to the device 200 to activate the second set of one or more DEP electrodes at the DEP electrode region 214. Alternatively, the device 200 may be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEP configuration that does not rely on photo-activation of DEP electrodes at the inner surface 208 of the electrode activation substrate 206. For example, the electrode activation substrate 206 can include selectively addressable and energizable electrodes located opposite a surface (e.g., the lid 110) that includes at least one electrode. A switch (e.g., a transistor switch in a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode at the DEP electrode region 214, thereby creating a net DEP force on a micro-object (not shown) in the region/chamber 202 near the activated DEP electrode. Depending on characteristics such as the frequency of the power source 212 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 202, DEP forces may attract or repel nearby micro-objects. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrode regions 214 forming a square pattern 220), one or more micro-objects in the region/chamber 202 can be captured and moved in the region/chamber 202. The motion module 162 in fig. 1A can control such switches to activate and deactivate individual DEP electrodes to select, trap, and move specific micro-objects (not shown) around the area/chamber 202. Microfluidic devices having DEP configurations comprising selectively addressable and excitable electrodes are known in the art and have been described, for example, in U.S. patent nos. 6,294,063(Becker et al) and 6,942,776 (Medoro), the entire contents of which are incorporated herein by reference.

As yet another example, the microfluidic device 200 can have an Electrowetting (EW) configuration, which can replace the DEP configuration, or can be located in a section of the microfluidic device 200 separate from the section having the DEP configuration. The EW configuration can be either an electro-wetting configuration or an electro-wetting on dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and the bottom electrode 204. The dielectric layer may comprise and/or may be coated with a hydrophobic material. For microfluidic devices 200 having an EW configuration, the inner surface 208 of the support structure 104 is an inner surface of a dielectric layer or hydrophobic coating thereof.

The dielectric layer (not shown) may include one or more oxide layers and may have a thickness of about 50nm to about 250nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer may include an oxide layer, such as a metal oxide (e.g., aluminum oxide or hafnium oxide) layer. In certain embodiments, the dielectric layer may include a dielectric material other than a metal oxide, such as a silicon oxide or a nitride. Regardless of the exact composition and thickness, the dielectric layer may have an impedance of about 10k ohms to about 50k ohms.

In some embodiments, the surface of the dielectric layer facing inward toward region/chamber 202 is coated with a hydrophobic material. The hydrophobic material may comprise, for example, carbon fluoride molecules. Examples of fluorinated carbon molecules include perfluoropolymers, such as polytetrafluoroethylene (e.g.,) Or poly (2, 3-difluoromethylene-perfluorotetrahydrofuran) (e.g., CYTOP)^TM). Molecules constituting the hydrophobic material may be covalently bonded to the surface of the dielectric layer. For example, molecules of the hydrophobic material may be covalently bonded to the surface of the dielectric layer by means of a linking group such as a siloxane group, a phosphonic acid group or a thiol group. Thus, in some embodiments, the hydrophobic material may comprise an alkyl-terminated siloxane, an alkyl-terminated phosphonic acid, or an alkyl-terminated thiol. The alkyl group can be a long chain hydrocarbon (e.g., a chain having at least 10 carbons or at least 16, 18, 20, 22 or more carbons). Alternatively, fluorinated (or perfluorinated) carbon chains may be used in place of alkyl groups. Thus, for example, the hydrophobic material may comprise a fluoroalkyl terminated siloxane, a fluoroalkyl terminated phosphonic acid, or a fluoroalkyl terminated thiol. In some embodiments, the hydrophobic coating has a thickness of about 10nm to about 50 nm. In other embodiments, the hydrophobic coating The thickness of the layer is less than 10nm (e.g., less than 5nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of the microfluidic device 200 having an electrowetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material may be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating may have a thickness that is substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. In addition, the lid 110 may include an electrode activation substrate 206 sandwiched between a dielectric layer and a top electrode 210 in the manner of the support structure 104. The dielectric layers of the electrode activation substrate 206 and the cap 110 may have the same composition and/or dimensions as the dielectric layers of the electrode activation substrate 206 and the support structure 104. Thus, the microfluidic device 200 may have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 may include a photoconductive material, such as those described above. Thus, in certain embodiments, the electrode activation substrate 206 may comprise or consist of a hydrogenated amorphous silicon layer (a-Si: H). H may contain, for example, about 8% to 40% hydrogen (calculated as 100 x the number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 μm. Alternatively, as described above, the electrode activation substrate 206 may include an electrode (e.g., a conductive metal electrode) controlled by a phototransistor switch. Microfluidic devices having electro-optical wetting configurations are known in the art and/or may be constructed with electrode-activated substrates known in the art. For example, U.S. Pat. No. 6,958,132(Chiou et al), the entire contents of which are incorporated herein by reference, discloses a electrowetting configuration having a photoconductive material such as a-Si: H, while U.S. Pat. No. 2014/0124370(Short et al), cited above, discloses an electrode activated substrate having electrodes controlled by phototransistor switches.

Thus, microfluidic device 200 can have a photo-electro-wetting configuration, and light pattern 218 can be used to activate a photoconductive EW region or a photo-responsive EW electrode in electrode activation substrate 206. Such activated EW regions or EW electrodes of the electrode activation substrate 206 can generate electrowetting forces at the inner surface 208 of the support structure 104 (i.e., the inner surface that covers the dielectric layer or hydrophobic coating thereof). By varying the light pattern 218 incident on the electrode-activated substrate 206 (or moving the microfluidic device 200 relative to the light source 216), droplets (e.g., containing an aqueous medium, solution, or solvent) in contact with the inner surface 208 of the support structure 104 can be moved through an immiscible fluid (e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic device 200 may have an EWOD configuration, and electrode activation substrate 206 may include selectively addressable and excitable electrodes that do not rely on light for activation. Thus, the electrode activation substrate 206 can include a pattern of such Electrowetting (EW) electrodes. For example, the pattern can be an array of substantially square EW electrodes arranged in rows and columns, as shown in figure 2B. Alternatively, the pattern can be an array of substantially hexagonal EW electrodes forming a hexagonal lattice of dots. Regardless of the pattern, the EW electrode can be selectively activated (or deactivated) by an electrical switch (e.g., a transistor switch in a semiconductor substrate). By selectively activating and deactivating the EW electrodes in the electrode activation substrate 206, droplets (not shown) in contact with the inner surface 208 of the covered dielectric layer or hydrophobic coating thereof can be moved within the region/chamber 202. The motion module 162 in figure 1A can control such switches to activate and deactivate individual EW electrodes to select and move specific droplets around the region/chamber 202. Microfluidic devices having EWOD configurations with selectively addressable and excitable electrodes are known in the art and have been described, for example, in U.S. patent No. 8,685,344(Sundarsan et al), the entire contents of which are incorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, the power supply 212 may be used to provide a potential (e.g., an AC voltage potential) that powers the circuitry of the microfluidic device 200. The power supply 212 may be the same as or a component of the power supply 192 referenced in FIG. 1. The power supply 212 may be configured to provide an AC voltage and/or current to the top electrode 210 and the bottom electrode 204. For AC voltages, the power supply 212 may provide a range of frequencies and a range of average or peak powers (e.g., voltages or currents): which, as described above, is sufficient to generate a net DEP force (or electrowetting force) strong enough to trap and move individual micro-objects (not shown) in the region/chamber 202, and/or which, as also described above, is sufficient to alter the wetting properties of the inner surface 208 of the support structure 104 (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) in the region/chamber 202. Such frequency ranges and average or peak power ranges are known in the art. See, for example, U.S. Pat. No. 6,958,132(Chiou et al), U.S. Pat. No. RE44,711(Wu et al) (originally issued as U.S. Pat. No. 7,612,355), and U.S. patent application publication Nos. US2014/0124370(Short et al), US2015/0306598 (Khandros et al), and US2015/0306599(Khandros et al).

Isolating the dock. Non-limiting examples of general isolation docks 224, 226 and 228 are shown within microfluidic device 230 depicted in fig. 2A-2C. Each isolation dock 224, 226, and 228 may include a separation structure 232 defining a separation region 240 and a connecting region 236 fluidly connecting the separation region 240 to the channel 122. The connecting region 236 may include a proximal opening 234 leading to the channel 122 and a distal opening 238 leading to the separating region 240. The connection region 236 may be configured such that a maximum penetration depth of a flow of fluid medium (not shown) from the channel 122 into the isolation dock 224, 226, 228 does not extend into the separation region 240. Thus, due to the connection region 236, micro-objects (not shown) or other materials (not shown) disposed in the separation region 240 of the isolation dock 224, 226, 228 may be separated from and substantially unaffected by the flow of the medium 180 in the channel 122.

The isolation docks 224, 226 and 228 of fig. 2A-2C each have a single opening that leads directly to the channel 122. The opening of the isolation dock opens laterally from the channel 122. Electrode activation substrate 206 is below both channel 122 and isolation docks 224, 226 and 228. The upper surface of the electrode activation substrate 206 within the housing of the isolation dock (forming the floor of the isolation dock) is disposed at the same or substantially the same level as the upper surface of the electrode activation substrate 206 within the channel 122 (or flow area if no channel is present) (forming the floor of the flow channel (or flow area) of the microfluidic device). The electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.8 microns, 0.7 microns, 0.6 microns, 0.5 microns, 0.4 microns, 0.3 microns, 0.2 microns, 0.1 microns, or less. The height variation in the upper surface of the substrate across the channel 122 (or flow region) and isolation dock may be less than about 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3%, or 0.1% of the height of the wall of the isolation dock or the wall of the microfluidic device. Although the microfluidic device 200 is described in detail, this also applies to any of the microfluidic devices 100, 230, 250, 280, 290 described herein.

Thus, the channel 122 may be an example of a swept area, and the separate area 240 separating the docks 224, 226, 228 may be an example of an unswept area. It should be noted that the channel 122 and the partitions 224, 226, 228 may be configured to contain one or more fluid mediums 180. In the example shown in fig. 2A-2B, port 222 is connected to channel 122 and allows for the introduction or removal of fluidic media 180 into or from microfluidic device 230. Prior to introduction of the fluid medium 180, the microfluidic device may be loaded with a gas, such as carbon dioxide gas. Once microfluidic device 230 contains fluidic medium 180, stream 242 of fluidic medium 180 in channel 122 may be selectively generated and stopped. For example, as shown, the ports 222 may be arranged at different locations (e.g., opposite ends) of the channel 122, and a flow 242 of media may be formed from one port 222 serving as an inlet to another port 222 serving as an outlet.

Fig. 2C shows a detailed view of an example of an isolated dock 224 according to the present invention. An example of a micro-object 246 is also shown.

As is known, the passage of a flow 242 of fluidic media 180 in the microfluidic channel 122 through the proximal opening 234 of the isolation dock 224 may cause a secondary flow 244 of media 180 to enter and/or exit the isolation dock 224. To separate micro-objects 246 in separation region 240 of isolation dock 224 from secondary flow 244, length L of connection region 236 of isolation dock 224 _con(i.e., from proximal opening 234 to distal opening 238) should be greater than the secondary flow 244 entering the union region 236Depth of penetration D_p. Depth of penetration D of secondary flow 244_pDepending on the velocity of the fluidic media 180 flowing in the channel 122 and various parameters related to the configuration of the channel 122 and the proximal opening 234 to the connection region 236 of the channel 122. For a given microfluidic device, the configuration of channel 122 and opening 234 will be fixed, while the rate of flow 242 of fluidic media 180 in channel 122 will be variable. Thus, for each isolation dock 224, the maximum velocity V of the flow 242 of fluid medium 180 in the channel 122 may be identified_maxEnsuring the penetration depth D of the secondary flow 244_pNot exceeding the length L of the connecting region 236_con. As long as the velocity of the flow 242 of fluid medium 180 in the passage 122 does not exceed the maximum velocity V_maxThe resulting secondary flow 244 may be confined to the passage 122 and the union region 236 and remain outside of the separation region 240. Thus, the flow 242 of medium 180 in the channel 122 will not drag the micro-objects 246 out of the separation region 240. In contrast, micro-objects 246 located in separation region 240 will reside in separation region 240 regardless of the flow 242 of fluid medium 180 in channel 122.

Furthermore, as long as the velocity of the stream 242 of the medium 180 in the channel 122 does not exceed V_maxThe flow 242 of fluid medium 180 in the channel 122 does not move the intermixed particles (e.g., particulates and/or nanoparticles) from the channel 122 into the separation region 240 of the isolation dock 224. Thus, the length L of the connecting region 236 is made_conGreater than the maximum penetration depth D of the secondary flow 244_pOne isolation dock 224 may be prevented from contamination by stray particles from channel 122 or another isolation dock (e.g., isolation docks 226, 228 in fig. 2D).

Because the channel 122 and the connection region 236 of the isolation docks 224, 226, 228 may be affected by the flow 242 of the medium 180 in the channel 122, the channel 122 and the connection region 236 may be considered a swept (or flow) region of the microfluidic device 230. On the other hand, the separation area 240 of the isolation docks 224, 226, 228 may be considered an unswept (or no-flow) area. For example, a component (not shown) in first fluid medium 180 in channels 122 may mix with second fluid medium 248 in separation zone 240 substantially only by diffusion of the component of first medium 180 from channels 122 through connection zone 236 and into second fluid medium 248 in separation zone 240. Similarly, the components (not shown) of the second media 248 in the separation zone 240 may mix with the first media 180 in the channels 122 substantially only by diffusion of the components of the second media 248 from the separation zone 240, through the connection zone 236, and into the first media 180 in the channels 122. In some embodiments, the degree of fluid medium exchange by diffusion between the separation region and the flow region of the isolation dock is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total fluid exchange. The first media 180 may be the same media as the second media 248 or a different media. In addition, the first media 180 and the second media 248 may begin to be the same and then become different (e.g., by conditioning the second media 248 by separating one or more cells in the region 240, or by altering the media 180 flowing through the channel 122).

As described above, the maximum penetration depth D of the secondary flow 244 caused by the flow 242 of the fluid medium 180 in the passage 122_pMay depend on a number of parameters. Examples of such parameters include: the shape of the channel 122 (e.g., the channel may direct media into the connection region 236, divert media from the connection region 236, or direct media in a direction substantially perpendicular to the proximal opening 234 to the connection region 236 of the channel 122); width W of channel 122 at proximal opening 234_ch(or cross-sectional area); and the width W of the attachment region 236 at the proximal opening 234_con(or cross-sectional area); velocity V of flow 242 of fluid medium 180 in passage 122; the viscosity of the first medium 180 and/or the second medium 248, and so on.

In some embodiments, the dimensions of the channel 122 and isolation docks 224, 226, 228 may be oriented relative to the vector of the flow 242 of the fluid medium 180 in the channel 122 as follows: width W of channel_ch(or cross-sectional area of the channel 122) may be substantially perpendicular to the flow 242 of the medium 180; width W of connecting region 236 at opening 234_con(or cross-sectional area) may be substantially parallel to the media 180 in the channel 122Stream 242; and/or length L of the connecting region_conMay be substantially perpendicular to the flow 242 of the medium 180 in the channel 122. The foregoing are examples only, and the relative positions of the channel 122 and isolation docks 224, 226, 228 may be otherwise oriented with respect to one another.

As shown in FIG. 2C, the width W of the connecting region 236_conMay be uniform from the proximal opening 234 to the distal opening 238. Thus, the width W of the attachment region 236 at the distal opening 238_conMay be referred to herein as the width W of the attachment region 236 at the proximal opening 234_conAny range identified. Alternatively, the width W of the attachment region 236 at the distal opening 238_conMay be greater than the width W of the union region 236 at the proximal opening 234_con。

As shown in FIG. 2C, the width W of the separation region 240 at the distal opening 238_isoMay be connected to the width W of the connection region 236 at the proximal opening 234_conAre substantially the same. Thus, the width W of the separation region 240 at the distal opening 238_isoMay be referred to herein as the width W of the connecting region 236 at the proximal opening 234_conAny range identified. Alternatively, the width W of the separation region 240 at the distal opening 238_isoMay be greater than or less than the width W of the union region 236 at the proximal opening 234_con. Further, the distal opening 238 may be smaller than the proximal opening 234, and the width W of the connecting region 236_conMay narrow between the proximal opening 234 and the distal opening 238. For example, using a variety of different geometries (e.g., beveling, etc.) the connection region 236 may narrow between the proximal and distal openings. Further, any portion or sub-portion of the connection region 236 may be narrowed (e.g., a portion of the connection region adjacent the proximal opening 234).

Fig. 2D-2F depict another exemplary embodiment of a microfluidic device 250 comprising microfluidic channels 262 and flow channels 264 that are variations of the respective microfluidic device 100, channels 132, and channels 134 of fig. 1. The microfluidic device 250 also has a plurality of isolation docks 266, which are additional variations of the isolation docks 124, 126, 128, 130, 224, 226 or 228 described above. In particular, it should be understood that the isolating dock 266 of the device 250 shown in fig. 2D-2F may replace any of the isolating docks 124, 126, 128, 130, 224, 226 or 228 described above in the devices 100, 200, 230, 280, 290 or 320. Similarly, the microfluidic device 250 is another variation of the microfluidic device 100, and may also have the same or different DEP configuration as the microfluidic devices 100, 200, 230, 280, 290, 320 described above, as well as any of the other microfluidic system components described herein.

The microfluidic device 250 of fig. 2D-2F includes a support structure (not visible in fig. 2D-2F, but can be the same as or substantially similar to the support structure 104 of the device 100 depicted in fig. 1A), a microfluidic conduit structure 256, and a lid (not visible in fig. 2D-2F, but can be the same as or substantially similar to the lid 122 of the device 100 depicted in fig. 1A). The microfluidic circuit structure 256 includes a frame 252 and a microfluidic circuit material 260, which may be the same as or substantially similar to the frame 114 and the microfluidic circuit material 116 of the device 100 depicted in fig. 1A. As shown in fig. 2D, the microfluidic tubing 262 defined by the microfluidic tubing material 260 may include a plurality of channels 264 (two are shown, but there may be more) to which a plurality of isolation docks 266 are fluidly connected.

Each isolation dock 266 may include a separation structure 272, a separation region 270 within separation structure 272, and a connection region 268. The connection region 268 fluidly connects the channel 264 to the separation region 270 from a proximal opening 274 at the channel 264 to a distal opening 276 at the separation structure 272. Generally, the flow 278 of the first fluidic medium 254 in the channel 264 may generate a secondary flow 282 of the first medium 254 from the channel 264 into and/or out of the respective connection regions 268 of the isolation dock 266, in accordance with the discussion of fig. 2B and 2C above.

As shown in fig. 2E, the connection region 268 of each isolation dock 266 generally includes a region extending between a proximal opening 274 to the channel 264 and a distal opening 276 to the separation structure 272. Length L of connecting region 268_conMay be greater than the maximum penetration depth D of the secondary flow 282_pIn this case, the secondary flow 282 would extend into the connecting region 268 without being redirected toward the separating region 270 (as in FIG. 2)Shown as D). Alternatively, as shown in FIG. 2F, the connection region 268 may have a depth D less than the maximum penetration depth_pLength L of_conIn this case, the secondary flow 282 would extend through the connecting region 268 and be redirected toward the separation region 270. In the latter case, the length L of the connecting region 268 _c1And L_c2Is greater than the maximum penetration depth D_pSuch that secondary flow 282 does not extend into separation region 270. Regardless of the length L of the connecting region 268_conWhether greater than the penetration depth D_pOr length L of connecting region 268_c1And L_c2Whether the sum of (A) and (B) is greater than the penetration depth D_pNo more than a maximum velocity V of first medium 254 in passage 264_maxWill produce a flow 278 having a penetration depth D_pAnd micro-objects (not shown, but may be the same as or substantially similar to micro-objects 246 shown in fig. 2C) in separation region 270 of isolation dock 266 are not dragged out of separation region 270 by flow 278 of first medium 254 in channel 264. The flow 278 in the channel 264 also does not drag mixed material (not shown) from the channel 264 into the separation region 270 of the isolation dock 266. As such, diffusion is the only mechanism by which a component in first medium 254 in channel 264 can move from channel 264 into second medium 258 in separation region 270 of isolation dock 266. Similarly, diffusion is the only mechanism by which the components in the second medium 258 in the separation region 270 of the isolation dock 266 can move from the separation region 270 into the first medium 254 in the channel 264. First media 254 may be the same media as second media 258 or first media 254 may be a different media than second media 258. Alternatively, first medium 254 and second medium 258 may be initially the same and then become different, for example, by conditioning the second medium with one or more cells in separation region 270, or by altering the medium flowing through channel 264.

As shown in FIG. 2E, the width W of the channels 264_ch(i.e., transverse to the direction of fluid medium flow through the channel, as indicated by arrow 278 in FIG. 2D) may be substantially perpendicular to the width W of proximal opening 274_con1And thus substantially parallel to the width W of the distal opening 276_con2. However, width W of proximal opening 274_con1And width W of distal opening 276_con2Need not be substantially perpendicular to each other. For example, width W of proximal opening 274_con1A shaft (not shown) oriented thereon and a width W of the distal opening 276_con2The angle between the further axes oriented thereon may be different from vertical and thus different from 90 deg.. Examples of selectable orientation angles include angles in any of the following ranges: about 30 ° to about 90 °, about 45 ° to about 90 °, about 60 ° to about 90 °, and the like.

In various embodiments of an isolated dock chamber (e.g., 124, 126, 128, 130, 224, 226, 228, or 266), an isolation region (e.g., 240 or 270) is configured to contain a plurality of micro-objects. In other embodiments, the separation region may be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. Thus, the volume of the separation region may be, for example, at least 5 × 10 ⁵、8×10⁵、1×10⁶、2×10⁶、4×10⁶、6×10⁶Cubic microns or greater.

In various embodiments of the isolation dock, the width W of the channel (e.g., 122) at the proximal opening (e.g., 234)_chMay be in any of the following ranges: about 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns, 100-200 microns, 100-150 microns and 100-120 microns. In some other embodiments, the width W of the channel (e.g., 122) at the proximal opening (e.g., 234)_chMay be in the range of about 200-800 microns, 200-700 microns, or 200-600 microns. The above are examples only, and the width W of the channel 122_chMay be within other ranges (e.g., within a range defined by any of the endpoints listed above). Further, W of channel 122 is in an area of the channel other than the proximal opening that isolates the dock_chCan be selected asWithin any of these ranges.

In some embodiments, the height of the isolation dock is about 30 to about 200 microns or about 50 to about 150 microns. In some embodiments, the cross-sectional area of the isolation dock is about 1 x 10 ⁴To about 3X 10⁶Square micron, about 2X 10⁴To about 2X 10⁶Square micron, about 4 x 10⁴To about 1X 10⁶Square micron, about 2 x 10⁴To about 5X 10⁵Square micron, about 2 x 10⁴To about 1X 10⁵Square micron and about 2 x 10⁵To about 2X 10⁶Square micron. In some embodiments, the connecting region has a cross-sectional width of about 20 to about 100 microns, about 30 to about 80 microns, or about 40 to about 60 microns.

In various implementations of the isolation dock, the height H of the channel (e.g., 122) at the proximal opening (e.g., 234)_chMay be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H of the channel (e.g., 122)_chMay be within other ranges (e.g., ranges defined by any of the endpoints listed above). Height H of channel 122 in the area of the channel other than the proximal opening that isolates the dock_chMay be selected to be within any of these ranges.

In various embodiments of the isolation dock, the cross-sectional area of the channel (e.g., 122) at the proximal opening (e.g., 234) can be within any of the following ranges: 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-containing 10,000 square microns, 1,000-containing 7,500 square microns, 1,000-containing 5,000 square microns, 2,000-containing 20,000 square microns, 2,000-containing 15,000 square microns, 2,000-containing 10,000 square microns, 2,000-containing 7,500 square microns, 2,000-containing 6,000 square microns, 3,000-containing 20,000 square microns, 3,000-containing 15,000 square microns, 3,000-containing 10,000 square microns, 3,000-containing 7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the channel (e.g., 122) at the proximal opening (e.g., 234) can be within other ranges (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the length L of the connection area (e.g., 236)_conMay be in any of the following ranges: about 20 to about 300 microns, about 40 to about 250 microns, about 60 to about 200 microns, about 80 to about 150 microns, about 20 to about 500 microns, about 40 to about 400 microns, about 60 to about 300 microns, about 80 to about 200 microns, or about 100 to about 150 microns. The foregoing are examples only, and the length L of the connection region (e.g., 236)_conMay be in a different range than the preceding examples (e.g., a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width W of the connection region (e.g., 236) at the proximal opening (e.g., 234)_conMay be in any of the following ranges: about 20 to about 150 microns, about 20 to about 100 microns, about 20 to about 80 microns, about 20 to about 60 microns, about 30 to about 150 microns, about 30 to about 100 microns, about 30 to about 80 microns, about 30 to about 60 microns, about 40 to about 150 microns, about 40 to about 100 microns, about 40 to about 80 microns, about 40 to about 60 microns, about 50 to about 150 microns, about 50 to about 100 microns, about 50 to about 80 microns, about 60 to about 150 microns, about 60 to about 100 microns, about 60 to about 80 microns, about 70 to about 150 microns, about 70 to about 100 microns, about 80 to about 150 microns, and about 80 to about 100 microns. The foregoing are examples only, and the width W of the connection region (e.g., 236) at the proximal opening (e.g., 234) _conMay be different from the foregoing examples (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width W of the connection region (e.g., 236) at the proximal opening (e.g., 234)_conCan be at least docked with the isolation dockThe largest size of the micro-object (e.g., a biological cell, which may be an immune cell, such as a B cell or T cell, or a hybridoma cell, etc.) to be used is as large. For example, the width W of the connection region 236 at the proximal opening 234 of the isolation dock where immune cells (e.g., B cells) will be placed_conAny of the following widths may be used: about 20 microns, about 25 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 60 microns, about 65 microns, about 70 microns, about 75 microns, or about 80 microns. The foregoing are examples only, and the width W of the connection region (e.g., 236) at the proximal opening (e.g., 234)_conMay be different from the foregoing examples (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the length L of the connection area (e.g., 236)_conWidth W of the connection region (e.g., 236) at the proximal opening 234 _conThe ratio of (d) may be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or greater. The above are examples only, and the length L of the connecting region 236_conWidth W of connection region 236 at proximal opening 234_conThe ratio of (c) may be different from the previous examples.

In various embodiments of the microfluidic devices 100, 200, 230, 250, 280, 290, 320, V_maxCan be set to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 μ L/sec.

In various embodiments of microfluidic devices with a isolation dock, the volume of the isolation region (e.g., 240) of the isolation dock can be, for example, at least 5 x 10⁵、8×10⁵、1×10⁶、2×10⁶、4×10⁶、 6×10⁶、8×10⁶、1×10⁷Cubic microns or greater. In various embodiments of microfluidic devices with isolation docks, the volume of the isolation dock may be about 5 x 10⁵、6×10⁵、8×10⁵、1×10⁶、2×10⁶、 4×10⁶、8×10⁶、1×10⁷Cubic microns or greater. In some other embodiments, the volume of the isolation dock can be about 0.5 nanoliter to about 10 nanoliters, about 1.0 nanoliter to about 5.0 nanoliters, about 1.5 nanoliters to about 4.0 nanoliters, about 2.0 nanoliters to about 3.0 nanoliters, or about 2.5 nanoliters, or any range defined by the two aforementioned endpoints.

In various embodiments, the microfluidic device has isolated docks configured as in any of the embodiments discussed herein, wherein the microfluidic device has about 5 to about 10 isolated docks, about 10 to about 50 isolated docks, about 100 to about 500 isolated docks; about 200 to about 1000 isolated docks, about 500 to about 1500 isolated docks, about 1000 to about 2000 isolated docks, or about 1000 to about 3500 isolated docks. The isolation docks need not all be the same size and may include multiple configurations (e.g., different widths, different features within the isolation dock).

In some other embodiments, the isolation dock of the microfluidic device is configured as any of the embodiments discussed herein, wherein the microfluidic device has from about 1500 to about 3000 isolation docks, from about 2000 to about 3500 isolation docks, from about 2500 to about 4000 isolation docks, from about 3000 to about 4500 isolation docks, from about 3500 to about 5000 isolation docks, from about 4000 to about 5500 isolation docks, from about 4500 to about 6000 isolation docks, from about 5000 to about 6500 isolation docks, from about 5500 to about 7000 isolation docks, from about 6000 to about 7500 isolation docks, from about 6500 to about 8000 isolation docks, from about 7000 to about 8500 isolation docks, from about 7500 to about 9000 isolation docks, from about 8000 to about 9500 isolation docks, from about 8500 to about 10,000 isolation docks, from about 9000 to about 10,500 isolation docks, from about 10,500 to about 11,000 isolation docks, from about 10,000 to about 9500 isolation docks, from about 11,500,000 isolation docks, from about 11,000 to about 11,000 isolation docks, from about 11,500,000 isolation docks, from about 11,000 isolation docks, about 11,000 to about 11,000 isolation docks, about 11,500,000 isolation docks, about 11,000 isolation docks, about 11,000 isolation docks, about 11,000, about 200, about 11,000, about 200, about 11,000, about 200, about 11,000, about 200, about 12,000 to about 13,500 isolating docks, about 12,500 to about 14,000 isolating docks, about 13,000 to about 14,500 isolating docks, about 13,500 to about 15,000 isolating docks, about 14,000 to about 15,500 isolating docks, about 14,500 to about 16,000 isolating docks, about 15,000 to about 16,500 isolating docks, about 15,500 to about 17,000 isolating docks, about 16,000 to about 17,500 isolating docks, about 16,500 to about 18,000 isolating docks, about 17,000 to about 18,500 isolating docks, about 17,500 to about 19,000 isolating docks, about 18,000 to about 19,500 isolating docks, about 18,500 to about 20,000 isolating docks, about 19,000 to about 20,500 isolating docks, about 19,500 to about 21,000 isolating docks, or about 20,500,000 isolating docks.

Fig. 2G illustrates a microfluidic device 280 according to an embodiment. The microfluidic device 280 shown in fig. 2G is a stylized schematic of the microfluidic device 100. In implementation, microfluidic device 280 and its constituent plumbing components (e.g., channel 122 and isolation dock 128) will have dimensions as discussed herein. The microfluidic circuit 120 shown in fig. 2G has two ports 107, four different channels 122, and four different flow paths 106. Microfluidic device 280 further includes a plurality of isolation docks that open into each channel 122. In the microfluidic device shown in fig. 2G, the isolation dock has a similar geometry to the dock shown in fig. 2C, and thus has both a connection region and a separation region. Thus, microfluidic circuit 120 includes a swept area (e.g., channel 122 and connecting area 236 at maximum penetration depth D of secondary flow 244_pInner portion) and non-swept areas (e.g., separation area 240 and union area 236 are not at the maximum penetration depth D of the secondary stream 244_pInner portion) of the two.

Fig. 3A-3B illustrate various embodiments of a system 150 that can be used to operate and view microfluidic devices (e.g., 100, 200, 230, 280, 250, 290, 320) according to the present invention. As shown in fig. 3A, the system 150 may include a structure ("nest") 300 configured to hold a microfluidic device 100 (not shown) or any other microfluidic device described herein. Nest 300 can include a socket 302 that can interface with a microfluidic device 320 (e.g., a light-actuated electrokinetic device 100) and provide an electrical connection from power source 192 to microfluidic device 320. Nest 300 may also include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 may be configured to provide a bias voltage to the receptacle 302 such that when the receptacle 302 holds the microfluidic device 320, a bias voltage is applied across a pair of electrodes in the microfluidic device 320. Thus, the electrical signal generation subsystem 304 may be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 320 does not mean that the bias voltage is always applied when the socket 302 holds the microfluidic device 320. In contrast, in most cases, the bias voltage will be applied intermittently, e.g., only when needed to facilitate generation of an electrokinetic force (e.g., dielectrophoresis or electrowetting) in the microfluidic device 320.

As shown in fig. 3A, nest 300 may include a Printed Circuit Board Assembly (PCBA) 322. The electrical signal generation subsystem 304 may be mounted on the PCBA 322 and electrically integrated therein. The example support also includes the socket 302 mounted on the PCBA 322.

Typically, electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 can also include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify waveforms received from the waveform generator. The oscilloscope (if any) may be configured to measure the waveform supplied to the microfluidic device 320 held by the receptacle 302. In certain embodiments, the oscilloscope measures the waveform at a location near the microfluidic device 320 (and away from the waveform generator), thereby ensuring a more accurate measurement of the waveform actually applied to the device. Data obtained from the oscillometric measurements may be provided, for example, as feedback to a waveform generator, and the waveform generator may be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is Red Pitaya^TM。

In certain embodiments, nest 300 further includes a controller 308, such as a microprocessor for detecting and/or controlling electrical signal generating subsystem 304. Examples of suitable microprocessors include Arduino ^TMMicroprocessors, e.g. Arduino Nano^TM. The controller 308 may be used to perform functions and analysis, or may communicate with the external master controller 154 (shown in FIG. 1A) to perform functions and analysis. In the embodiment shown in fig. 3A, the controller 308 communicates with the master controller 154 via an interface 310 (e.g., a plug or connector).

In some embodiments, the nest300 may include an electrical signal generation subsystem 304, which includes Red Pitaya^TMA waveform generator/oscilloscope cell ("Red Pitaya cell") and a waveform amplification circuit that amplifies the waveform generated by the Red Pitaya cell and transmits the amplified voltage to the microfluidic device 100. In some embodiments, the Red Pitaya cell is configured to measure the amplified voltage at the microfluidic device 320 and then adjust its own output voltage as needed so that the voltage measured at the microfluidic device 320 is a desired value. In some embodiments, the waveform amplification circuit may have a +6.5V to-6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, producing signals up to 13Vpp at the microfluidic device 100.

As shown in fig. 3A, the support structure 300 may further include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to regulate the temperature of the microfluidic device 320 held by the support structure 300. For example, the thermal control subsystem 306 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface configured to interface with at least one surface of the microfluidic device 320. The cooling unit may be, for example, a cooling block (not shown), such as a liquid cooled aluminum block. A second surface (e.g., a surface opposite the first surface) of the Peltier thermoelectric device may be configured to interface with a surface of such a cooling block. The cooling block may be connected to a fluid path 314, the fluid path 314 configured to circulate a cooled fluid through the cooling block. In the embodiment shown in fig. 3A, the support structure 300 includes an inlet 316 and an outlet 318 to receive cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluid path 314 and through the cooling block, and return the cooled fluid to the external reservoir. In some embodiments, a Peltier thermoelectric device, a cooling unit, and/or a fluid path 314 may be mounted on the housing 312 of the support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Peltier thermoelectric device to achieve a target temperature for the microfluidic device 320. Temperature regulation of a Peltier thermoelectric device may be achieved, for example, by a thermoelectric power supply E.g. by Pololu^TMThermoelectric power supply (Pololu semiconductors and Electronics Corp.) was implemented. Thermal control subsystem 306 may include feedback circuitry, such as temperature values provided by analog circuitry. Alternatively, the feedback circuit may be provided by a digital circuit.

In some embodiments, nest 300 may include a thermal control subsystem 306 having a feedback circuit that is an analog voltage divider circuit (not shown) including a resistor (e.g., having an impedance of 1k ohms +/-0.1% +/-0.02 ppm/C0 temperature coefficient) and an NTC thermistor (e.g., having a nominal impedance of 1k ohms +/-0.01%). In some cases, thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to the on-board PID control loop algorithm. The output from the PID control loop algorithm may drive, for example, Pololu^TMDirectional and pulse width modulated signal pins on a motor driver (not shown) to actuate the thermoelectric power supply to control the Peltier thermoelectric device.

Nest 300 may include a serial port 324 that allows the microprocessor of controller 308 to communicate with external master controller 154 via interface 310 (not shown). Additionally, the microprocessor of the controller 308 may be in communication with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, the electrical signal generation subsystem 304 and the thermal control subsystem 306 may communicate with the external master controller 154 via a combination of the controller 308, the interface 310, and the serial port 324. In this manner, the main controller 154 may assist the electrical signal generation subsystem 304 by performing, among other things, scaling calculations for output voltage regulation. A Graphical User Interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154 may be configured to plot temperature and waveform data obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 304, respectively. Alternatively or additionally, the GUI may allow for updating the controller 308, the thermal control subsystem 306, and the electrical signal generation subsystem 304.

As discussed above, the system 150 may include an imaging device 194.In some embodiments, the imaging device 194 includes a light modulation subsystem 330 (see fig. 3B). The light modulation subsystem 330 may include a Digital Mirror Device (DMD) or a micro-shutter array system (MSA), either of which may be configured to receive light from the light source 332 and send a portion of the received light into the optical train of the microscope 350. Alternatively, light modulation subsystem 330 may include a device that generates its own light (and thus does not require light source 332), such as an organic light emitting diode display (OLED), a Liquid Crystal On Silicon (LCOS) device, a liquid crystal on ferroelectric substrate device (FLCOS), or a transmissive Liquid Crystal Display (LCD). Light modulation subsystem 330 can be, for example, a projector. Thus, the light modulation subsystem 330 is capable of emitting structured light and unstructured light. One example of a suitable light modulation subsystem 330 is from Andor Technologies^TMMosaic of (1)^TMAnd (4) a system. In certain embodiments, the imaging module 164 and/or the motion module 162 of the system 150 may control the light modulation subsystem 330.

In certain embodiments, the imaging device 194 further comprises a microscope 350. In such embodiments, the nest 300 and the light modulation subsystem 330 may be individually configured to be mounted on the microscope 350. The microscope 350 may be, for example, a standard research grade optical microscope or a fluorescent microscope. Thus, the nest 300 may be configured to mount on the stage 344 of the microscope 350 and/or the light modulation subsystem 330 may be configured to mount on a port of the microscope 350. In other embodiments, the nest 300 and the light modulation subsystem 330 described herein may be integrated components of the microscope 350.

In certain embodiments, the microscope 350 may further include one or more detectors 348. In some embodiments, detector 348 is controlled by imaging module 164. Detector 348 may include an eyepiece, a Charge Coupled Device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two detectors 348, one detector may be, for example, a fast frame rate camera and the other detector may be a high sensitivity camera. Further, the microscope 350 can include an optical train configured to receive light reflected and/or emitted from the microfluidic device 320 and focus at least a portion of the reflected and/or emitted light onto the one or more detectors 348. The optical train of the microscope may also include different tube lenses (not shown) for different detectors so that the final magnification on each detector may be different.

In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, a first light source 332 may be used to generate structured light (e.g., via the light modulation subsystem 330), and a second light source 334 may be used to provide unstructured light. The first light source 332 may generate structured light for optically driven electrical motion and/or fluorescence excitation, and the second light source 334 may be used to provide bright field illumination. In these embodiments, the motion module 164 may be used to control the first light source 332, and the imaging module 164 may be used to control the second light source 334. The optics group of the microscope 350 can be configured to (1) receive structured light from the light modulation subsystem 330 and focus the structured light onto at least a first area in a microfluidic device (e.g., a light-actuated electro-kinetic device) when the device is held by the nest 300, and (2) receive light reflected and/or emitted from the microfluidic device and focus at least a portion of such reflected and/or emitted light onto the detector 348. The optical train can be further configured to receive unstructured light from the second light source and focus the unstructured light on at least a second area of the microfluidic device when the microfluidic device is held by the nest 300. In certain embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first region may be a subset of the second region.

In fig. 3B, the first light source 332 is shown providing light to the light modulation subsystem 330, which provides structured light to an optical train (not shown) of the microscope 350 of the system 355. The second light source 334 is shown providing unstructured light to the optical train via a beam splitter 336. The structured light from light modulation subsystem 330 and the unstructured light from second light source 334 travel together from beam splitter 336 through the optics group to the second beam splitter (or dichroic filter 338, depending on the light provided by light modulation subsystem 330), where the light is reflected down through objective lens 336 to sample plane 342. The light reflected and/or emitted from the sample plane 342 is then returned back up through the objective lens 340, through the beam splitter and/or dichroic filter 338, and back to the dichroic filter 346. Only a portion of the light that reaches the dichroic filter 346 passes through and reaches the detector 348.

In some embodiments, the second light source 334 emits blue light. With an appropriate dichroic filter 346, blue light reflected from the sample plane 342 can pass through the dichroic filter 346 and reach a detector 348. In contrast, structured light from the light modulation subsystem 330 reflects from the sample plane 342, but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 filters out visible light having a wavelength longer than 495 nm. This filtering of light from the light modulation subsystem 330 is accomplished (as shown) only if the light emitted from the light modulation subsystem does not include any wavelengths shorter than 495 nm. In implementation, if the light from the light modulation subsystem 330 includes wavelengths shorter than 495nm (e.g., a blue wavelength), some of the light from the light modulation subsystem passes through the filter 346 to the detector 348. In such embodiments, the filter 346 acts to change the balance between the amount of light reaching the detector 348 from the first light source 332 and the second light source 334. This may be beneficial if the first light source 332 is significantly stronger than the second light source 334. In other embodiments, the second light source 334 may emit red light, and the dichroic filter 346 may filter out visible light other than red light (e.g., visible light having a wavelength shorter than 650 nm).

Coating solution and coating agent. Without wishing to be bound by theory, when one or more interior surfaces of the microfluidic device have been conditioned or coated so as to present a layer of organic and/or hydrophilic molecules that provides a primary interface between the microfluidic device and a micro-object (e.g., a biological cell) maintained therein, culturing of the biological cell (e.g., an immune cell, such as a B cell or a T cell) within the microfluidic device may be facilitated (i.e., the micro-object exhibits increased viability, greater expansion, and/or greater portability within the microfluidic device). In some embodiments, one or more internal surfaces of the microfluidic device (e.g., the internal surfaces of the electrode-activated substrate of the microfluidic device in a DEP configuration, the surfaces of the cover and/or tubing material of the microfluidic device) are treated with a coating solution and/or a coating agent to produce the desired layer of organic and/or hydrophilic molecules. In some embodiments, the amplified micro-objects (e.g., biological cells) are cultured in a microfluidic device and optionally allowed to input into a coating solution comprising one or more coating agents.

In other embodiments, the interior surface of a microfluidic device (e.g., a DEP-configured microfluidic device) is treated or "primed" with a coating solution comprising a coating agent prior to introducing the micro-objects (e.g., biological cells) into the microfluidic device. Any convenient coating agent/solution may be used, including but not limited to: serum or serum factors, Bovine Serum Albumin (BSA), polymers, detergents, enzymes, and any combination thereof. In some specific embodiments, the coating agent will be used to treat the interior surface of the microfluidic device. In one example, a polymer containing alkylene ether moieties is included in the coating solution as a coating agent. Many alkylene ether-containing polymers may be suitable. One non-limiting exemplary class of alkylene ether-containing polymers is amphiphilic nonionic block copolymers comprising blocks of Polyoxyethylene (PEO) and polyoxypropylene (PPO) subunits having different proportions and positions within the polymer chain. Polymers (BASF) are such block copolymers and are known in the art to be suitable for use when in contact with living cells. Average molecular weight M of the Polymer_wAnd may range from about 2000Da to about 20 KDa. In some embodiments, the PEO-PPO block copolymer may have a hydrophilic-lipophilic balance (HLB) of greater than about 10 (e.g., 12-18). Specific for producing coated surfacesThe polymer comprisesL44, L64, P85 and F127 (including F127 NF). Another class of alkylene-containing ethersThe polymer of (A) is polyethylene glycol (PEG M)_w<100,000Da) or alternatively, polyoxyethylene (PEO, M)_w>100,000). In some embodiments, the PEG can have an M of about 1000Da, 5000Da, 10,000Da, or 20,000Da_w。

In some embodiments, the coating solution may comprise a plurality of proteins and/or peptides as coating agents. In a particular embodiment, the coating solution used in the present disclosure comprises as coating agent a protein, such as albumin (e.g. BSA) and/or a serum (or a combination of different sera) comprising albumin and/or one or more other similar proteins. The serum may be from any convenient source, including but not limited to fetal bovine serum, sheep serum, goat serum, horse serum, and the like. In certain embodiments, BSA is present in a blocking solution (blocking solution) at a concentration ranging from about 1mg/mL to about 100mg/mL, including 5mg/mL, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, or any value therebetween. In certain embodiments, serum may be present in the coating solution at a concentration ranging from about 20% (v/v) to about 50% v/v, including 25%, 30%, 35%, 40%, 45%, or higher or any value in between. In some embodiments, BSA may be present as a coating agent in the coating solution at 5mg/mL, while in other embodiments, BSA may be present as a coating agent in the coating solution at 70 mg/mL. In certain embodiments, serum is present at 30% in the coating solution as a coating agent.

And (3) coating materials. Depending on the embodiment, any of the above-described coating agents/coating solutions may be replaced by, or used in combination with, a variety of coating materials for coating one or more interior surfaces of microfluidic devices (e.g., DEP-configured and/or EW-configured microfluidic devices). In some embodiments, at least one surface of the microfluidic device comprises a coating material that provides an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects, such as cells, e.g., immune cells (e.g., B cells) or hybridoma cells. In some embodiments, substantially all of the interior surfaces of the microfluidic device comprise a coating material. The coated inner surface may include a flow area (e.g., a channel), a surface of a chamber or isolation dock, or a combination thereof. In some embodiments, each of the plurality of isolated docks has at least one interior surface coated with a coating material. In other embodiments, each of the plurality of flow regions or channels has at least one interior surface coated with a coating material. In some embodiments, at least one interior surface of each of the plurality of isolated docks and each of the plurality of channels is coated with a coating material.

A polymer-based coating material. At least one of the inner surfaces may include a coating material comprising a polymer. The polymer may be covalently or non-covalently bound (or linked) to at least one surface. The polymers may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for use in the methods disclosed herein.

The polymer may comprise a polymer comprising alkylene ether moieties. A wide variety of alkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein. One non-limiting exemplary class of alkylene ether-containing polymers is the amphoteric nonionic block copolymers, which comprise blocks of Polyoxyethylene (PEO) and polyoxypropylene (PPO) subunits having different proportions and positions within the polymer chain.Polymers (BASF) are such block copolymers and are known in the art to be suitable for use when in contact with living cells. Average molecular weight M of the Polymer_wFrom about 2000Da to about 20 kDa. In some embodiments, the PEO-PPO block copolymer may have a hydrophilic-lipophilic balance (HLB) of greater than about 10 (e.g., 12-18). Specific for producing coated surfaces The polymer comprisesL44, L64, P85 and F127 (including F127 NF). Another class of alkylene ether-containing polymers is polyethylene glycol (PEG M)_w<100,000Da) or alternatively, polyoxyethylene (PEO, M)_w>100,000). In some embodiments, the PEG may have a M of about 1000Da, 5000Da, 10,000Da, or 20,000 Da_w。

In other embodiments, the coating material may include a polymer containing carboxylic acid moieties. The carboxylic acid subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polylactic acid (PLA).

In other embodiments, the coating material may include a polymer containing sulfonic acid moieties. The sulfonate subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polystyrene sulfonic acid (PSSA) or polyanetholesulfonic acid. These latter exemplary polymers are polyelectrolytes and may alter the characteristics of the surface to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells).

In some embodiments, the coating material may include a urethane moiety-containing polymer, such as, but not limited to, a polyurethane.

In other embodiments, the coating material can include a polymer that includes phosphate moieties at the ends of or pendant from the backbone of the polymer.

In other embodiments, the coating material may include a polymer containing sugar moieties. In one non-limiting example, a polysaccharide (e.g., derived from an alga or a fungal polysaccharide, such as xanthan or dextran) may be suitable for forming a material that can reduce or prevent cell adhesion in a microfluidic device. For example, dextran polymers of about 3kDa in size can be used to provide a coating material for surfaces within a microfluidic device.

In other embodiments, the coating material may include a polymer containing nucleotide moieties, i.e., nucleic acids, which may have ribonucleotide moieties or deoxyribonucleotide moieties. Nucleic acids may contain only natural nucleotide moieties or may contain non-natural nucleotide moieties comprising nucleobase, ribose, or phosphate moiety analogs, such as 7-deazaadenine, pentose, methylphosphonate, or phosphorothioate moieties, but are not limited thereto. The nucleic acid-containing polymer may include a polyelectrolyte that may provide a layer of organic and/or hydrophilic molecules suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells).

In other embodiments, the coating material may include a polymer containing amino acid moieties. Polymers containing amino acid moieties can include polymers containing natural amino acids or polymers containing unnatural amino acids, which can each include peptides, polypeptides, or proteins. In one non-limiting example, the protein can be Bovine Serum Albumin (BSA). In some embodiments, extracellular matrix (ECM) proteins may be provided within the coating material for optimal cell adhesion to promote cell growth. Cell matrix proteins that may be included in the coating material may include, but are not limited to, collagen, elastin, RGD-containing peptides (e.g., fibronectin), or laminin. In other embodiments, growth factors, cytokines, hormones, or other cell signaling substances may be provided within the coating material of the microfluidic device.

In further embodiments, the coating material can include a polymer containing amine moieties. The polyamino polymer may comprise a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine.

In some embodiments, the coating material can include a polymer that includes more than one of an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a sugar moiety, a nucleotide moiety, or an amino acid moiety. In other embodiments, the polymer conditioned surface may comprise a mixture of more than one polymer, each polymer having alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, sugar moieties, nucleotide moieties, and/or amino acid moieties, which may be incorporated into the coating material independently or simultaneously.

A covalently linked coating material. In some embodiments, at least one internal surface comprises covalently linked molecules that provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) within a microfluidic device to provide a conditioned surface for such cells. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device. The linking group is also covalently linked to a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell). The surface to which the linking group is attached may comprise a substrate surface of a microfluidic device, which may comprise silicon and/or silicon dioxide for embodiments in which the microfluidic device comprises a DEP configuration. In some embodiments, the covalently attached coating material coats substantially all of the interior surfaces of the microfluidic device.

In some embodiments, a moiety configured to provide covalent attachment of an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) may include an alkyl or fluoroalkyl (including perfluoroalkyl) moiety; monosaccharides or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino groups (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing nonaromatic nitrogen ring atoms such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide carboxylate anionic surfaces); phosphonic acids, including but not limited to ethynylphosphonic acid (which may provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid.

The moieties configured to provide covalent attachment of an organic and/or hydrophilic molecular layer suitable for maintaining and/or amplifying a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device can be any of the polymers described herein, and can include polymers comprising alkylene oxide moieties, carboxylic acid moieties, sugar moieties, sulfonic acid moieties, phosphate moieties, amino acid moieties, nucleotide moieties, or amino moieties.

In other embodiments, a moiety configured to provide covalent attachment of an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device can include a non-polymeric moiety, such as an alkyl moiety, a substituted alkyl moiety (e.g., a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety)), an amino acid moiety, an alcohol moiety, an amino moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonic acid moiety, an aminosulfonic acid moiety, or a sugar moiety.

In some embodiments, the covalently linked moiety can be an alkyl group that comprises carbon atoms that form a straight chain (e.g., a straight chain of at least 10 carbons or at least 14, 16, 18, 20, 22 or more carbons). Thus, the alkyl group may be an unbranched alkyl group. In some embodiments, alkyl groups may include substituted alkyl groups (e.g., some carbons in an alkyl group may be fluorinated or perfluorinated). The alkyl group may include a linear chain of substituted (e.g., fluorinated or perfluorinated) carbons bonded to an unsubstituted carbon. For example, an alkyl group can include a first segment (which can include a perfluoroalkyl group) that is linked to a second segment (which can include an unsubstituted alkyl group). The first and second segments may be linked together directly or indirectly (e.g., via an ether linkage). The first segment of the alkyl group may be located distal to the linking group and the second segment of the alkyl group may be located proximal to the linking group. In other embodiments, the alkyl group may include branched alkyl groups, and may also have one or more arylene groups interrupting the alkyl backbone of the alkyl group. In some embodiments, the branched or arylene interrupted portion of the alkyl or fluoroalkyl group is located distal to the linking group to the surface and the covalent bond.

In other embodiments, the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid. Thus, the covalently linked moiety may comprise a peptide or a protein. In some embodiments, the covalently attached moiety may include amino acids that may provide a zwitterionic surface to support cell growth, viability, portability (transportability), or any combination thereof.

The covalently linked moiety may include one or more sugars. The covalently linked saccharide may be a monosaccharide, disaccharide or polysaccharide. The covalently linked sugar may be modified to introduce reactive pairing moieties that allow coupling or processing for attachment of the surface. Exemplary reactive partner moieties may include aldehyde, alkyne, or halogen moieties. The polysaccharide may be modified in a random manner, wherein each saccharide monomer or only a portion of the saccharide monomers within the polysaccharide may be modified to provide reactive partner moieties that may be coupled directly or indirectly to a surface. One example may include dextran polysaccharides, which may be indirectly coupled to a surface via an unbranched linker moiety.

The covalently linked moiety may include one or more amino groups. The amino group can be a substituted amine moiety, guanidine moiety, nitrogen-containing heterocyclic moiety, or heteroaryl moiety. The amino-containing moiety can have a structure that allows for pH modification of the environment within the microfluidic device and optionally within the sequestration dock and/or flow region (e.g., channel).

The coating material may comprise only one type of covalently linked moiety, or may comprise more than one different type of covalently linked moiety. For example, a fluoroalkyl-conditioned surface (including perfluoroalkyl) can have multiple covalently attached moieties that are all the same, e.g., having the same linking group and covalent attachment to the surface, the same total length, and the same number of fluoromethylene units, including fluoroalkyl moieties. Alternatively, the coating material may have more than one type of covalently linked moiety attached to the surface. For example, the coating material can include molecules having covalently attached alkyl or fluoroalkyl moieties having a specified number of methylene or fluoromethylene units, and can also include another group of molecules having charged moieties covalently attached to an alkyl or fluoroalkyl chain having a greater number of methylene or fluoromethylene units. In some embodiments, coating materials having more than one covalently linked moiety can be designed such that a first set of molecules with a greater number of backbone atoms and thus a longer distance from the covalent link to the surface can provide the ability to present a larger portion on the coated surface, while a second set of molecules with different, less spatially demanding ends and fewer backbone atoms can help functionalize the entire substrate surface, preventing undesirable adhesion or contact with the silicon or aluminum oxide comprising the substrate itself. In another example, the covalently linked moieties may provide a zwitterionic surface that exhibits alternating charges on the surface in a random manner.

Conditioned surface properties. In some embodiments, the covalently linked moieties can form a monolayer when covalently linked to a surface of a microfluidic device (e.g., a DEP configured substrate surface). In some embodiments, the conditioned surface formed by covalently attached moieties can have a thickness of less than 10nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). In other embodiments, the conditioned surface formed by covalently attached moieties may have a thickness of about 10nm to about 50 nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to function properly for operation within a DEP configured microfluidic device.

In various embodiments, the coating material of the microfluidic device can provide desired electrical properties. Without wishing to be bound by theory, one factor that affects the robustness of a surface coated with a particular coating material is inherent charge trapping. Different coating materials may trap electrons, which may lead to destruction of the coating material. Defects in the coating material may increase charge trapping and lead to further damage of the coating material. Similarly, different coating materials have different dielectric strengths (i.e., minimum applied electric field that results in dielectric breakdown), which may affect charge trapping. In certain embodiments, the coating material can have a bulk structure (e.g., a close-packed monolayer structure) that reduces or limits the amount of charge trapping.

In addition to the composition of the coating material, other factors, such as the physical (and electrical) thickness of the coating material, can influence the generation of DEP and electrowetting forces by the substrate of the microfluidic device. Various factors may alter the physical and electrical thickness of the coating material, including the manner in which the coating material is deposited on the substrate (e.g., vapor deposition, liquid deposition, spin coating, and electrostatic coating). The physical thickness and uniformity of the coating material can be measured using an ellipsometer.

In addition to its electrical properties, the coating material may have properties that are beneficial for use with biomolecules. For example, coating materials containing fluoro (or perfluoro) alkyl groups may provide benefits in terms of reducing the amount of surface fouling relative to unsubstituted alkyl groups. Surface fouling as used herein refers to the amount of any substance deposited on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biological substances (e.g., proteins and their degradation products, nucleic acids and their degradation products). Such fouling can increase the amount of adhesion of biological micro-objects to the surface.

In addition to the composition of the conditioned surface, other factors (e.g., the physical thickness of the hydrophobic material) may affect the DEP force. Various factors may alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid deposition, spin coating, flooding, and electrostatic coating). The physical thickness and uniformity of the conditioned surface can be measured using an ellipsometer.

In addition to its electrical properties, the conditioned surface may also have properties that are beneficial for use with biomolecules. For example, conditioned surfaces containing fluorinated (or perfluorinated) carbon chains may provide benefits in reducing the amount of surface fouling relative to alkyl terminated chains. Surface fouling, as used herein, refers to the amount of any substance deposited on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biological materials (e.g., proteins and their degradation products, nucleic acids and respective degradation products, etc.).

The following table includes a number of properties of the conditioned surface that can be used in DEP. As shown, for entries 1 to 7 (all of which are covalently attached to the conditioned surface as described herein), the thickness as measured by the ellipsometric technique is always thinner than that of entry 8 (a CYTOP surface formed by non-covalent spin coating) (N/a represents the unavailable data throughout the table). Fouling was found to be more dependent on the chemistry of the surface than the mode of formation, since fluorinated surfaces are generally less prone to fouling than alkyl (hydrocarbon) conditioned surfaces.

Table 1. properties of various conditioned surfaces prepared by covalently modifying a surface compared to a non-covalently formed surface CYTOP.

1. Spin coating, non-covalent.

A surface linking group. The covalent linking moieties forming the coating material are attached to the surface via a linking group. The linking group can be a siloxy linking group formed by reaction of a siloxane-containing reagent with an oxide of the substrate surface, which can include silicon oxide (e.g., a substrate for DEP configuration) or aluminum oxide or hafnium oxide (e.g., a substrate for EW configuration). In some other embodiments, the linking group can be a phosphate ester formed by reacting a phosphonic acid-containing reagent with an oxide of the substrate surface.

A multi-part conditioned surface. As described below, the covalently linked coating material may be formed by a reaction of molecules (e.g., alkyl siloxane reagents or fluoro-substituted alkyl siloxane reagents, which may include perfluoroalkyl siloxane reagents) that have been configured to provide a moiety suitable for maintaining and/or expanding an organic and/or hydrophilic molecular layer of a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device. Alternatively, the covalently linked coating material may be formed by coupling a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) to a surface modifying ligand, which itself is covalently linked to the surface.

A method of making a covalently linked coating material. In some embodiments, a coating material covalently attached to a surface of a microfluidic device (e.g., at least one surface comprising a spacer dock and/or a flow region) has a structure of formula 1.

The coating material can be covalently attached to the oxide on the surface of the DEP configured substrate. The DEP configured substrate may comprise silicon or aluminum oxide or hafnium oxide, and the oxide may be present as part of the initial chemistry of the substrate, or may be introduced as discussed below.

The coating material may be attached to the oxide via a linking group ("LG"), which may be a siloxy or phosphonate group formed from the reaction of a siloxane or phosphonic acid group with the oxide. The portion of the layer of organic and/or hydrophilic molecules configured to provide a layer suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) in a microfluidic device may be any portion described herein. The linking group LG may be directly or indirectly linked to a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device. When the linking group LG is directly connected to the moiety, there is no optional linking moiety ("L"), and n is 0. When the linking group LG is indirectly linked to the moiety, there is a linking moiety L, and n is 1. The linking moiety L may have a linear portion, wherein the backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, subject to the limitations of chemical bonding known in the art. In some non-limiting examples, it may be interrupted by any combination of one or more moieties that may be selected from ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L may have one or more arylene, heteroarylene, or heterocyclyl groups that interrupt the backbone of the linking group. In some embodiments, the backbone of the linking moiety L may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L may comprise from about 5 atoms to about 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, limited by chemical linkages known in the art.

When a portion configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) in a microfluidic device is added to the substrate surface in a one-step process, the molecules of formula 2 can be used to introduce a coating material:

part of- (L) n-LG.

Formula 2

In some embodiments, the portion configured to provide the layer of organic and/or hydrophilic molecules suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) in a microfluidic device may be added to the surface of the substrate in a multi-step process. When the moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects, e.g. cells, such as immune cells (e.g. B-cells) or hybridoma cells, is coupled to the surface in a stepwise manner, the linking moiety L may further comprise a coupling group CG, as shown in formula 3.

In some embodiments, the coupling group CG is represented by a reactive moiety R_xAnd a reactive partner R_px(i.e., configured to react with the reactive moiety R)_xPart of the reaction). For example, a typical coupling group CG may include a carboxamido group, which is the result of reacting an amino group with a carboxylic acid derivative (e.g., an activated ester, an acid chloride, etc.). Other CGs may include triazolylene, carboxamido, thioamido, oxime, mercapto, disulfide, ether or alkenyl groups, or any other suitable group that may be formed upon reaction of a reactive moiety with its corresponding reactive partner moiety. The coupling group CG may be located at the second end of the linking group L (i.e. the end adjacent to the portion configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or amplifying a biological micro-object (e.g. a cell, such as an immune cell (e.g. a B cell) or a hybridoma cell) in a microfluidic device). In some other embodiments, the coupling group CG may interrupt the backbone of the linking group L. In some embodiments, the coupling group CG is a triazolylene group, which may be obtained by reaction between an alkyne group and an azide group, any of which may be a reactive moiety R _xAnd a reactive partner moiety R_pxAs known in the art for Click coupling reactions. For example, the dibenzocyclooctenyl-fused triazolylene group may be conjugated to a dibenzocyclooctynyl reactive partner R_px(ii) an azido-reactive moiety R with a surface-modifying molecule_xThe reaction of (a) is obtained, which is described in more detail in the following paragraphs. A variety of dibenzocyclooctynyl-modified molecules are known in the art, or can be synthesized to incorporate moieties that are configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells).

When the coating material is formed in a multi-step process, moieties configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or amplifying biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) in a microfluidic device may be introduced by reacting a reagent containing the moiety (formula 5) with a substrate having a surface modifying ligand covalently attached thereto (formula 6).

The modified surface of formula 4 has a surface modifying ligand attached thereto having the formula-LG- (L') j-R_xWhich is connected to the oxide of the substrate and is formed similarly as described above for the conditioned surface of equation 1. The surface of the substrate may be a substrate surface of the DEP configuration as described above, and may comprise the substrate itself or an oxide incorporated therein. The linking group LG is as described above. The linking moiety L "may be present (j ═ 1) or absent (j ═ 0). The linking moiety L "may have a linear portion, wherein the backbone of the linear portion may comprise from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety. In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L "can comprise from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

Reactive moiety R_xIs present at the end of the surface modifying ligand remote from the covalent attachment of the surface modifying ligand to the surface. Inverse directionStress moiety R_xIs any suitable reactive moiety that can be used in a coupling reaction to introduce a moiety that provides an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device. In some embodiments, the reactive moiety R_xCan be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety.

A reagent containing a moiety. The reagent containing the moiety (formula 5) is configured to supply a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) in a microfluidic device.

Moiety- (L')_m-R_px

Formula 5

A moiety that is configured to provide a layer of organic and/or hydrophilic molecules suitable for maintaining and/or expanding biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) in a reagent containing the moiety is passed through a reactive pairing moiety R_pxWith reactive moieties R _xIs linked to a surface modifying ligand. Reactive pair moiety R_pxIs any suitable reactive group configured to react with a corresponding reactive moiety R_xAnd (4) reacting. In a non-limiting example, a suitable reactive partner R_pxCan be an alkyne, a reactive moiety R_xMay be an azide. Reactive partner R_pxMay alternatively be an azide moiety, the corresponding reactive moiety R_xMay be an alkyne. In other embodiments, the reactive partner R_pxCan be an active ester functional group, a reactive moiety R_xMay be an amino group. In other embodiments, the reactive partner R_pxMay be an aldehyde, a reactive moiety R_xMay be an amino group. Other reactive moiety-reactive partner combination are possible, and these examples are in no way limiting.

The portion of the reagent containing moiety of formula 5 configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., a B cell) or a hybridoma cell) can include any of the moieties described herein, including alkyl or fluoroalkyl (including perfluoroalkyl) moieties; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidinium salts, and heterocyclic groups containing a nitrogen ring atom that is not aromatic, such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which can provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonic anion surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; (ii) sulfamic acid; or an amino acid.

The moiety of the reagent comprising the moiety of formula 5 configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g. a cell, such as an immune cell (e.g. a B cell) or a hybridoma cell) may be linked directly to (i.e. L', wherein m ═ 0) or indirectly to the reactive partner R moiety_px. When the reactive partner R is_pxReactive partner moiety R when indirectly linked to a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for maintaining and/or expanding a biological micro-object (e.g., a cell, such as an immune cell (e.g., B cell) or a hybridoma cell)_pxMay be connected to the connecting portion L' (m ═ 1). Reactive partner R_pxA moiety that can be attached to a first end of the linking moiety L 'and that is configured to reduce surface fouling and/or prevent or reduce cell adhesion can be attached to a second end of the linking moiety L'. The linking moiety L' may have a linear portion, wherein the main chain of the linear portion comprises 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to the limitations of chemical bonding known in the art. In some non-limiting examples, it may be substituted with ether, amino, carbonyl, amido or Any combination of phosphonate groups is interrupted. In addition, linker L 'may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of linker L'. In some embodiments, the backbone of the linking moiety L' may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L' may comprise from about 5 atoms to about 100 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

When the reagent containing moieties (formula 5) reacts with the surface having surface modifying ligands (formula 3), a substrate having a conditioned surface of formula 2 is formed. The linking moiety L 'and linking moiety L' are then formally part of the linking moiety L, and the reactive partner R_pxWith a reactive moiety R_xThe reaction of (a) gives the coupling group CG of formula 2.

A surface modifier. The surface modifier is of the structure LG- (L') _j-R_xA compound of (formula 4). The linking group LG is covalently linked to the oxide on the substrate surface. The substrate may be a DEP configured substrate and may comprise silicon or aluminum oxide or hafnium oxide, and the oxide may be present as part of the native chemical structure of the substrate or may be incorporated as discussed herein. The linking group LG can be any linking group described herein, such as a siloxy or phosphonate group, formed from the reaction of a siloxane or phosphonate group with an oxide on the substrate surface. Reactive moiety R_xAs described above. Reactive moiety R_xThe linking group LG may be directly linked to (L ", j ═ 0) or indirectly linked to (j ═ 1) through a linking moiety L". The linking group LG may be linked to a first end of the linking moiety L' and the reactive moiety R_xCan be attached to the second end of the linking moiety L' once the surface modifying agent has been attached to the surface as shown in formula 6Reactive moieties R_xWill be located distally of the substrate surface.

The linking moiety L "may have a linear portion, wherein the backbone of the linear portion comprises 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety L". In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L "can comprise from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

In some embodiments, the coating material (or surface-modified ligand) is deposited on the inner surface of the microfluidic device using chemical vapor deposition. By chemical vapor deposition, the coating material can achieve a tightly packed monolayer in which molecules comprising the coating material are covalently bonded to molecules of the inner surface of the microfluidic device. To achieve the desired packing density, molecules comprising, for example, an alkyl-terminated siloxane can be vapor deposited at a temperature of at least 110 ℃ (e.g., at least 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, etc.) for at least 15 hours (e.g., at least 20, 25, 30, 35, 40, 45, or more hours). Such vapor deposition is typically carried out under vacuum and in a water source (e.g., hydrated sulfate salts (e.g., MgSO)₄·7H₂O)) in the presence of oxygen. Generally, the temperature and duration of the vapor deposition is increasedResulting in improved properties of the hydrophobic coating material.

The vapor deposition process can optionally be modified, for example, by pre-cleaning the lid 110, the microfluidic conduit material 116, and/or the substrate (e.g., the inner surface 208 of the electrode activation substrate 206 of a DEP configured substrate, or the dielectric layer of the support structure 104 of an EW configured substrate). For example, the pre-clean may include a solvent bath, such as an acetone bath, an ethanol bath, or a combination thereof. The solvent bath may include sonication. Alternatively or additionally, such pre-cleaning may include treating the cover 110, the microfluidic circuit material 116, and/or the substrate in an oxygen plasma cleaner, which may remove various impurities while introducing an oxidized surface (e.g., an oxide on the surface, which may be covalently modified as described herein). The oxygen plasma cleaner may be operated, for example, at 100W under vacuum for 60 seconds. Alternatively, a liquid phase treatment, which includes an oxidizing agent (e.g., hydrogen peroxide) to oxidize the surface, may be used in place of the oxygen plasma cleaner. For example, a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., a piranha solution, which may have a ratio range of sulfuric acid to hydrogen peroxide of about 3:1 to about 7: 1) is substituted for the oxygen plasma cleaner.

In some embodiments, vapor deposition is used to coat the inner surfaces of the microfluidic device 200 after the microfluidic device 200 has been assembled to form the housing 102 defining the microfluidic circuit 120. Depositing a coating material comprising a close-packed monolayer on a fully assembled microfluidic circuit 120 may be beneficial in providing a variety of functional properties. Without wishing to be bound by theory, depositing such a coating material on the fully assembled microfluidic circuit 120 may be beneficial to prevent delamination caused by weakened bonding between the microfluidic circuit material 116 and the electrode activation substrate 206 dielectric layer and/or the cap 110.

Fig. 2H depicts a cross-sectional view of a microfluidic device 290, the microfluidic device 290 including an example class of coating materials. As shown, the coating material 298 (shown schematically) may comprise a close-packed molecular monolayer covalently bonded to both the inner surface 294 of the substrate 286 and the inner surface 292 of the cover 288 of the microfluidic device 290. The coating material 298 may be disposed on all of the interior surfaces 294, 292 of the housing 284 adjacent and inwardly facing the microfluidic device 290, in some embodiments and as discussed above, including surfaces of microfluidic circuit material (not shown) for defining circuit elements and/or structures within the microfluidic device 290. In alternative embodiments, the coating material 298 may be disposed only on one or some of the interior surfaces of the microfluidic device 290.

In the embodiment shown in fig. 2H, the coating material 298 comprises a monolayer of alkyl-terminated siloxane molecules, each molecule covalently bonded to the interior surfaces 292, 294 of the microfluidic device 290 via a siloxy linker 296. However, any of the above-described coating materials 298 (e.g., alkyl-terminated phosphonate molecules) can be used. More specifically, the alkyl group can comprise a straight chain of at least 10 carbon atoms (e.g., 10, 12, 14, 16, 18, 20, 22, or more carbon atoms) and, optionally, can be a substituted alkyl group. As described above, the coating material 298 comprising a tightly packed molecular monolayer may have beneficial functional properties of the microfluidic device 290 for DEP configuration, such as minimal charge trapping, reduced physical/electrical thickness, and a substantially uniform surface.

In another particular embodiment, the coating material 298 may include a fluoroalkyl group (e.g., fluoroalkyl or perfluoroalkyl) at its end facing the housing (i.e., the portion of the monolayer of coating material 298 that is not bonded to the inner surfaces 292, 294 and is proximate to the housing 284). As described above, the coating material 298 may comprise a single layer of fluoroalkyl terminated siloxane or fluoroalkyl terminated phosphonate, where fluoroalkyl groups are present at the end of the coating material 298 facing the housing. Such a coating material 298 provides improved functional benefits of maintenance and/or expansion of biological micro-objects (e.g., cells, such as immune cells (e.g., B cells) or hybridoma cells) by separating or "shielding" the biological micro-objects from non-biological molecules (e.g., silicon and/or silicon oxide of the substrate).

In other particular embodiments, the coating material 298 used to coat the interior surfaces 292, 294 of the microfluidic device 290 may include anionic, cationic, or zwitterionic moieties, or any combination thereof. Without wishing to be limited by theory, by providing a cationic moiety, an anionic moiety, and/or a zwitterionic moiety on the inner surface of the housing 284 of the microfluidic circuit 120, the coating material 298 may form strong hydrogen bonds with water molecules, such that the resulting hydrated water acts as a layer (or "shield") separating the core (core) from interactions with non-biological molecules (e.g., silicon and/or silicon oxide of the substrate). Additionally, in embodiments where the coating material 298 is used in combination with a blocking agent, the anion, cation, and/or zwitterion of the coating material 298 may form an ionic bond with a charged portion of a non-covalent coating agent (e.g., a protein in solution) present in the medium 180 (e.g., a coating solution) in the housing 284.

In another specific embodiment, the coating material may comprise or be chemically modified to provide a hydrophilic coating agent at its end facing the housing. In some embodiments, the coating agent may be an alkylene ether-containing polymer, such as PEG. In some embodiments, the coating agent may be a polysaccharide, such as dextran. As with the charged moieties discussed above (e.g., anionic, cationic, and zwitterionic moieties), the hydrophilic coating agent can form strong hydrogen bonds with water molecules, such that the resulting water of hydration acts as a layer (or "shield") separating the core from interactions with non-biological molecules (e.g., silicon and/or silicon oxide of the substrate).

Methods for detecting expression of an antibody. The methods disclosed herein include methods of detecting or identifying biological cells that express antibodies that specifically bind to an antigen of interest. The antigen of interest may be a protein, a carbohydrate group or chain, a biological or chemical agent other than a protein or carbohydrate, or any combination thereof. The antigen of interest can be, for example, an antigen associated with a pathogen (e.g., a virus, a bacterial pathogen, a fungal pathogen, a protozoan pathogen, etc.). Alternatively, the antigen of interest may be associated with a cancer (e.g., lung cancer, breast cancer, melanoma, etc.). In another alternative, the antigen may be associated with an autoimmune disease (e.g., multiple sclerosis or type I diabetes). As used herein, the term "associated with a pathogen" when used with an antigen of interest means that the antigen of interest is produced directly by the pathogen or by an interaction between the pathogen and the host.

Methods of detecting biological cells expressing antibodies that specifically bind to an antigen of interest can be performed in the microfluidic devices described herein. In particular, the microfluidic device may comprise a housing having a flow region that may comprise one or more microfluidic channels and an isolation dock (or multiple isolation docks). The isolation dock may comprise a separation region and a connection region providing a fluidic connection between the separation region and the flow region/microfluidic channel. The volume of the isolation dock can be about 0.5nL to about 5.0nL or any range therein (e.g., about 0.5nL to about 1.0nL, about 0.5nL to about 1.5nL, about 0.5nL to about 2.0nL, about 1.0nL to about 1.5nL, about 1.0nL to about 2.0nL, about 1.0nL to about 2.5nL, about 1.5nL to about 2.0nL, about 1.5nL to about 2.5nL, about 1.5nL to about 3.0nL, about 2.0nL to about 2.5nL, about 2.0nL to about 3.0nL, about 2.0nL to about 3.5nL, about 2.5nL to about 3.0nL, about 2.5nL to about 3.5nL, about 5nL to about 3.5nL, about 3.5nL to about 3.0nL, about 3.5nL to about 3.5nL, about 3.5nL to about 3.0 to about 3.5nL, about 3.5nL to about, about 3.0 to about 3.5nL, about 3.0 to about 4.0 to about 3.5nL, or any range of about 4 to about 4 nL). The connection region may have a width W as generally described herein _con(e.g., from about 20 microns to about 100 microns, or from about 30 microns to about 60 microns). Width W of the separation region_isoMay be greater than the width W of the connection region_con. In certain embodiments, the width W of the separation region_isoFrom about 50 microns to about 250 microns.

The flow region, the isolation dock, and/or the isolation area of the isolation dock may comprise at least one surface coated with a coating material that promotes viability of the biological cells and/or reduces interaction with the biological cells. Thus, for example, the coating material can promote the viability of hybridoma cells, and/or promote the viability of B-cell lymphocytes (e.g., memory B cells or plasma cells), and/or the ability to move any such cells into the microfluidic device. As used herein, "promoting viability" means that the viability of the antibody-expressing biological cells is better on the coated surface compared to an uncoated equivalent surface. In certain embodiments, the flow region, isolation dock, and/or isolation region has a plurality of surfaces, each surface coated with a coating material that promotes viability of and/or reduces interaction with antibody-expressing cells. The coating material can be any suitable coating material known in the art and/or described herein. The coating material may for example comprise hydrophilic molecules. The hydrophilic molecule may be selected from the group consisting of a polymer comprising polyethylene glycol (PEG), a polymer comprising carbohydrate groups, a polymer comprising amino acids (e.g., a protein, such as BSA), and combinations thereof.

The flow region, the isolating dock and/or the separation region of the isolating dock may comprise at least one conditioned surface that promotes viability of the antibody expressing biological cells and/or reduces interaction with the antibody expressing biological cells. Thus, for example, the conditioned surface may promote the viability of hybridoma cells, and/or promote the viability of B cell lymphocytes (e.g., memory B cells or plasma cells), and/or promote the ability to move any cells into the microfluidic device. As used herein, "promoting viability" means that the viability of antibody-expressing biological cells is better on a conditioned surface compared to an equivalent surface that is not conditioned. In certain embodiments, the flow region, the spacer dock, and/or the separation region has a plurality of conditioned surfaces, each conditioned surface capable of promoting viability of the antibody-expressing cell and/or reducing interaction with the antibody-expressing cell. The conditioned surface may comprise covalently attached molecules. The covalently linked molecule can be any suitable molecule known in the art and/or disclosed herein, including, for example, covalently linked hydrophilic molecules. The hydrophilic molecule may be selected from the group consisting of a polymer comprising polyethylene glycol (PEG), a polymer comprising a carbohydrate group, a polymer comprising an amino acid, and combinations thereof. As described herein, the hydrophilic molecules may form a covalently linked layer of hydrophilic molecules. Alternatively, the covalently linked molecules may comprise perfluoroalkanes (e.g., covalently linked perfluoroalkane layers).

A method of detecting a biological cell expressing an antibody that specifically binds to an antigen of interest may comprise the steps of: introducing a sample containing antibody-expressing biological cells into a microfluidic device; loading antibody-expressing biological cells into a separation region of a separation dock in a microfluidic device; introducing a target antigen into the microfluidic device such that the target antigen is located in the vicinity of the antibody-expressing biological cells; and monitoring the binding of the antigen of interest to the antibody expressed by the biological cell.

The antibody-expressing biological cell can be, for example, a hybridoma cell. Alternatively, the antibody-expressing biological cell may be a B cell lymphocyte. The B cell lymphocyte may be, for example, CD27⁺B cells or CD138⁺B cells. In some embodiments, the B cell is a memory B cell. In other embodiments, the B cell is a plasma cell.

Introducing the antibody-expressing biological cells into the microfluidic device can involve obtaining a sample containing the antibody-expressing cells. For embodiments in which the antibody-expressing biological cells are B cell lymphocytes, the sample containing B cell lymphocytes can be obtained from a mammal, such as a human, a rodent (e.g., mouse, rat, guinea pig, gerbil, hamster), a rabbit, a ferret, a livestock (e.g., goat, sheep, pig, horse, cow), a llama, a camel, a monkey, or from an avian species, such as a chicken and turkey. In some embodiments, the mammal has been immunized against an antigen of interest. In some embodiments, the animal has been exposed to or infected with a pathogen associated with the antigen of interest. In some embodiments, the animal has a cancer associated with the antigen of interest. In other embodiments, the animal has an autoimmune disease associated with an antigen of interest. The sample containing B cell lymphocytes may be a peripheral blood sample (e.g., PBMCs), spleen biopsy, bone marrow biopsy, lymph node biopsy, tumor biopsy, or any combination thereof.

A sample containing B cell lymphocytes can be treated (e.g., sorted, negative and/or positive) to enrich for desired B cell lymphocytes. In some embodiments, the desired B cell lymphocyte is a memory B cell. In other embodiments, the desired B cell lymphocyte is a plasma cell. In some implementationsIn the protocol, the desired B cell lymphocytes express antibodies of the IgG type. Thus, for example, a sample may be depleted of cell types other than B cell lymphocytes. Methods for depleting non-B cell types from a sample are well known in the art and include, for example, using DYNABEADS^TMUnmodified B cell reagent (DYNABEADS)^TMUntouched Human B Cells reagent, Thermo Fisher), B Cell Isolation Kit (Miltenyi), easy Sep B Cell Enrichment Kit (easy Sep), Rosetesep Human B Cell Enrichment Cocktain (Rosetesep Human B Cell Enrichment Cocktain, Stem Cell Technologies), and the like. Alternatively, or in addition, samples containing B cell lymphocytes can be sorted by fluorescence-related cell sorting (FACS) to remove unwanted cell types and enrich for desired cell types. FACS sorting may be negative and/or positive. For example, FACS sorting can deplete B cell lymphocyte samples expressing IgM antibodies, IgA antibodies, IgD antibodies, IgG antibodies, or any combination thereof. Alternatively, or in addition, FACS sorting may enrich a sample for B cell lymphocytes expressing CD27 (or some other memory B cell marker) or for B cell lymphocytes expressing CD138 (or some other plasma cell marker). The sample containing B cell lymphocytes can be provided in an enriched state (i.e., pretreated) such that a treatment to enrich for desired B cell lymphocytes is not required as part of the method. Alternatively, treatment of a sample containing B cell lymphocytes to enrich for desired B cell lymphocytes can be performed as part of the method of the invention.

The sample containing B cell lymphocytes can be treated to reduce cell adhesion to the microfluidic device in the sample. For example, DNase may be used (e.g.Nucleic (Millipore)) treatment of the sample. Preferably, the dnase contains minimal protease activity.

Introduction of the antibody-expressing biological cells into the microfluidic device can be performed by flowing a sample containing the biological cells into an inlet of the microfluidic device and through a portion of a flow region of the microfluidic device. The flow of sample through the microfluidic device can then be stopped to allow loading of antibody-expressing biological cells (e.g., B-cell lymphocytes) into the separation region of the sequestration dock. Loading of antibody-expressing cells into the separation region can be performed by any technique known in the art or disclosed herein, for example, using gravitational forces and/or DEP forces. In certain embodiments, a single antibody-expressing cell (e.g., a B cell lymphocyte) is loaded into the isolation region. In certain embodiments, a single antibody-expressing cell (e.g., a B-cell lymphocyte) is loaded into a separate region of each of a plurality of isolated docks in a microfluidic device.

Methods of detecting biological cells that express an antibody that specifically binds to an antigen of interest can include the step of contacting a B cell lymphocyte with a stimulating agent that stimulates B cell activation. The stimulating agent may be a CD40 agonist, such as CD40L, a derivative thereof, or an anti-CD 40 antibody. The stimulant may comprise CD40L ⁺A feeder cell consisting essentially of, or consisting of. CD40L⁺The feeder cells may be T cells (e.g., Jurkat D1.1 cells) or derivatives thereof. Alternatively, the feeder cells may be cell lines transfected/transformed with the CD40L expression construct (e.g., NIH-3T3 cells). The stimulating agent may further comprise a B Cell Receptor (BCR) superantigen, such as protein a, protein G, or any other BCR superantigen. The BCR superantigen may be attached to a micro-object, such as a bead, a lipid vesicle, a lipid nanoraft, or the like. Thus, the micro-objects coated with superantigens may be contacted with CD40L⁺Feeder cells were mixed. The mixture may have a ratio of about 1:1 feeder cells to micro-objects, or a ratio of about 1:5 feeder cells to micro-objects, or any ratio therebetween. Alternatively, the mixture may have a ratio of about 1:2 feeder cells to micro-objects, or a ratio of about 2:10 feeder cells to micro-objects, or any ratio therebetween. The stimulating agent may further comprise a toll-like receptor (TLR) agonist (e.g., a TLR9 agonist), which may be combined with a CD40 agonist and optionally a BCR superantigen. The TLR agonist can be, for example, a CpG oligonucleotide (e.g., CpG 2006). The CpG oligonucleotide may be used at a concentration of about 1 microgram/mL to about 20 microgram/mL (e.g., about 1.5 to about 15 microgram/mL, about 2.0 to about 10 microgram/mL, or about 2.5 to about 5.0 microgram/mL). The B cell lymphocytes can be contacted (e.g., substantially continuously, or periodically/intermittently) with the stimulating agent for 1 to 10 days (e.g., 2 to 8 days, 3 to 7 days, or 4 to 6 days). The B cell lymphocytes may be contacted with a stimulating agent in a sequestration dock into which the B cell lymphocytes are loaded. This contact may occur after the B cell lymphocyte is loaded into the isolation dock.

The method of detecting a biological cell expressing an antibody that specifically binds to an antigen of interest can further include the step of providing a culture/activation medium to the biological cell expressing the antibody (e.g., a B cell lymphocyte), the culture/activation medium comprising one or more growth inducers that promote B cell activation and/or expansion. The one or more growth-inducing agents may comprise at least one agent selected from the group consisting of CpG oligonucleotides, IL-2, IL-4, IL-6, IL-10, IL-21, BAFF, and April. IL-2 may be provided at a concentration of about 2ng/mL to about 5ug/mL, or about 50ng/mL to about 2ug/mL, or about 100ng/mL to about 1.5ug/mL, or about 500ng/mL to about 1ug/mL, or about 1 ug/mL. IL-4 may be provided at a concentration of about 2ng/mL to about 20ng/mL, or about 5ng/mL to about 10ng/mL, or about 5 ng/mL. IL-6, IL-10 and/or IL-21 may be provided at a concentration of from about 2ng/mL to about 50ng/mL, or from about 5ng/mL to about 20ng/mL, or about 10 ng/mL. BAFF and/or April may be provided in a concentration of about 10ng/mL to about 100ng/mL, or about 10ng/mL to about 50ng/mL, or about 10ng/mL to about 20ng/mL, or about 10 ng/mL. The CpG oligonucleotide may be used at a concentration of about 1 microgram/mL to about 20 microgram/mL, about 1.5 to about 15 microgram/mL, about 2.0 to about 10 microgram/mL, or about 2.0 microgram/mL. In certain embodiments, the antibody-expressing biological cells are provided with culture medium for 1 to 10 days (e.g., 2 to 8 days, 3 to 7 days, or 4 to 6 days). The culture medium may comprise a stimulating agent (e.g., a CD40 agonist and/or a BCR superantigen). Thus, for example, where the antibody-producing cell is a B cell lymphocyte, the providing of the culture medium to the B cell lymphocyte can be performed while contacting the B cell lymphocyte with the activating agent. In certain embodiments, the steps of contacting the B cell lymphocyte with the stimulating agent and providing the culture medium to the B cell lymphocyte are performed over an overlapping period of time (e.g., over a substantially coextensive period of time).

In certain embodiments, introducing the antigen of interest into the microfluidic device such that the antigen of interest is located in the vicinity of the antibody-expressing biological cell comprises locating the antigen of interest within 1 millimeter (mm) of the biological cell (e.g., within 750 microns, within 600 microns, within 500 microns, within 400 microns, within 300 microns, within 200 microns, within 100 microns, or within 50 microns of the biological cell). In certain embodiments, the method can include introducing a micro-object or a plurality of micro-objects into a flow region/microfluidic channel connected to an isolation dock. The micro-objects can comprise an antibody-specific binding agent, such as an anti-IgG antibody or other IgG binding agent. See, for example, fig. 6C. In such embodiments, monitoring binding of the antigen of interest to the antibody expressed by the biological cell comprises detecting indirect binding of the labeled antigen of interest to the micro-object via the antibody expressed by the antibody expressing biological cell. The labeled target antigen may be soluble and may include a detectable label, such as a fluorescent label. The micro-objects may be any suitable micro-objects (e.g., cells, liposomes, lipid nanorafts, or beads) known in the art and/or described herein. The step of providing the target antigen may comprise placing such micro-objects near or within the connection region of the isolation dock, wherein the antibody-expressing biological cell is located. Alternatively, the step of providing the antigen of interest may comprise loading such micro-objects into a separate region of the sequestration dock, in which the antibody-expressing biological cells are located. The micro-objects and the target antigen may be provided simultaneously as a mixture, or sequentially (if the micro-objects are first placed in a separate dock).

Alternatively, in certain embodiments, the method can include introducing a micro-object or a plurality of micro-objects into a flow region/microfluidic channel connected to a separation dock, wherein the antigen of interest binds to the micro-object. In such embodiments, a soluble labeled antibody-specific binding agent, such as an anti-IgG antibody or other IgG binding agent, may also be provided, and monitoring binding of the antigen of interest to the antibody expressed by the biological cell comprises detecting indirect binding of the labeled antibody-specific binding agent to the micro-object by the antibody expressed by the antibody-expressing biological cell. The labeled antibody-specific binding agent may comprise a detectable label, such as a fluorescent label. The micro-objects may be any suitable micro-objects (e.g., cells, liposomes, lipid nanorafts, or beads) known in the art and/or described herein. The step of providing the target antigen may comprise placing such a micro-object near or inside the connection region of the sequestration dock, wherein the antibody-expressing biological cell is located. The step of providing the target antigen may further comprise loading such micro-objects into a separate region of the sequestration dock, wherein the antibody-expressing biological cells are located. The micro-objects and antibody-specific binding agent may be provided simultaneously as a mixture, or sequentially (if the micro-objects are first placed in a separate dock). Methods of screening for expression of a molecule of interest (e.g., an antibody) have been described, for example, in U.S. patent publication No. US2015/0151298, which is incorporated by reference herein in its entirety.

In some embodiments, the method further comprises providing a second antibody-specific binding agent prior to or concurrently with the first antibody-specific binding agent. See, e.g., fig. 6C. The second antibody binding agent may be an anti-IgG antibody or other type of antibody binding agent, and may be labeled (e.g., with a fluorescent label). In certain embodiments, the labeled second antibody-specific binding agent is provided in a mixture with the antigen of interest and the first antibody-specific binding agent. In other embodiments, the labeled second antibody-specific binding agent is provided after the antigen of interest and/or the first antibody-specific binding agent is provided.

In certain embodiments, providing the antigen of interest can include flowing a solution comprising a soluble antigen of interest through a flow region of the microfluidic device and diffusing the soluble antigen into a separation dock in which the antibody-expressing biological cell is located. Such soluble antigen may be covalently bound to a detectable label (e.g., a fluorescent label). A general method of screening for expression of molecules of interest (including antibodies) in this manner has been described in, for example, international application PCT/US2017/027795 filed on 2017, 4/14, which is incorporated herein by reference in its entirety.

In certain embodiments, the method can further comprise detecting binding of an antigen of interest to an antibody expressed by a biological cell (e.g., a B cell lymphocyte), and recognizing an antibody-expressing biological cell (e.g., a B cell lymphocyte) that expresses an antibody that specifically binds the antigen of interest.

Antibody sequences were obtained from the recognized B cell lymphocytes. Also disclosed herein are methods of providing sequencing libraries and/or obtaining heavy and light chain antibody sequences from antibody-expressing cells. Alternatively, obtaining a sequencing library from target B-cell lymphocytes can be performed by methods other than those described herein. Other suitable, but non-limiting, methods are described in PCT/US2017/054628, filed on 29/9/2017, and the entirety of which is incorporated herein by reference for all purposes.

Capture/priming oligonucleotides. The capture/priming oligonucleotide may comprise a first priming sequence and a capture sequence. The capture/priming oligonucleotide may comprise a 5 'endmost nucleotide and a 3' endmost nucleotide.

And (5) capturing the sequence. The capture sequence is an oligonucleotide sequence configured to capture nucleic acids from lysed cells. In various embodiments, the capture sequence may be near or include the 3' endmost nucleotide of the capture/priming oligonucleotide. The capture sequence can have from about 6 to about 50 nucleotides. In some embodiments, the capture sequence captures the nucleic acid by hybridization to nucleic acid released from the target cell. In some of the methods described herein, the nucleic acid released from the target B cell may be mRNA. The capture sequence that can capture and hybridize to the mRNA can include a polyT sequence, which mRNA has a PolyA sequence at the 3' end of the mRNA. The polyT sequence may have from about 20T nucleotides to more than 100T nucleotides. In some embodiments, the polyT sequence may have from about 30 to about 40 nucleotides. The polyT sequence may further contain two nucleotides VI at its 3' end.

A first priming sequence. The first priming sequence of the capture/priming oligonucleotide may be: 5 'of the capture sequence, proximal to the 5' most terminal nucleotide of the capture/priming oligonucleotide; or the 5' endmost nucleotide comprising a capture/priming oligonucleotide. The first priming sequence may be a universal sequence or a sequence-specific priming sequence. The first priming sequence may be bound to a primer that, upon binding, primes the reverse transcriptase. The first priming sequence may comprise from about 10 to about 50 nucleotides.

Additional priming and/or adaptor sequences. The capture oligonucleotide may optionally have one or more additional priming/adaptor sequences that provide a landing site for primer extension (which may include extension by a polymerase) or a site for immobilization to a complementary hybridization anchor site within a massively parallel sequencing array or flow cell. In the methods herein, the second (or additional) priming sequence may be a P1 sequence (e.g., AAGCAGTGGTATCAACGCAGAGT (SEQ ID No.1), as used in Illumina sequencing chemistry), but the methods are not so limited. Any suitable priming sequence for use in the preparation of other types of NGS libraries may be included. In some embodiments, when the P1 sequence is included as an additional priming sequence, it may be 5' to the first priming sequence. The P1 additional priming sequence may also be 5' to the capture sequence.

Template switching oligonucleotides. Template switch oligonucleotide as used herein refers to an oligonucleotide that allows terminal transferase activity of a suitable reverse transcriptase, such as but not limited to Moloney Murine Leukemia Virus (MMLV), to anchor the template switch oligonucleotide with added deoxycytidine nucleotides. After base pairing between the template switch oligonucleotide and the additional deoxycytidine, the reverse transcriptase "switches" the template strand from the captured RNA to the template switch oligonucleotide and proceeds to copy to the 5' end of the template switch oligonucleotide. Thus, the entire 5' end of the transcribed NA is included, and additional priming sequences for further amplification can be introduced. In addition, the cDNA is transcribed in a sequence-independent manner.

BCR gene sequence. The B cell receptor gene sequence includes several subregions to include variable (V), diversity (D), junction (J) and constant (C) segments in 5 'to 3' order in the released RNA. The constant region is exactly 5' to the polyA sequence. In many methods of sequencing BCRs, it may be desirable to construct a selection strategy that results in amplicons for sequencing that are free of poly a sequences (tails). Furthermore, it may be desirable to generate amplicons that retain few constant regions. Limiting amplification to exclude these segments of released nucleic acid sequence may allow for more robust sequencing of V, D (if present) and J segments of BCR.

To better understand this approach, turning to FIGS. 8A-8H, each of FIGS. 8A-8H represents a single species or a set of related double stranded species present at different points in a method of obtaining a BCR sequencing library from a single cell. The method can be multiplexed such that many individual cells can be processed to provide a sequencing library that can be traced back to a particular start site within the well plate. Knowledge of this location can be further traced back to a single isolation dock within the microfluidic device from which the cells have been exported. Thus, a biological cell can be assayed for a desired ability to produce a desired product or stimulate other cells, and then traceably output, traceably processed to provide a sequencing library, and the resulting genomic data resulting from the sequencing can be identifiably correlated back to the source cell in the microfluidic device.

In fig. 8A, mRNA 810 is released by lysing the biological cells. The released mRNA 810 may include the target gene sequence 805 and have a poly A stretch 815 at its 3' end. Capture/priming oligonucleotide 820 may include P1 priming sequence 825 and a PolyT capture sequence (shown in fig. 8A as T30NI, which represents a sequence of 30T nucleotides and has the double nucleotide sequence NI at the 3' end of capture/priming oligonucleotide 820). In some embodiments, the N nucleotides in the T30NI sequence of the PolyT capture sequence can be selected from G, C and a (e.g., T nucleotides can be excluded). The capture/priming oligonucleotides can bind to the polyA sequence 815 of the released mRNA 810.

In fig. 8B, the initial process of reverse transcription is shown, wherein reverse transcriptase extends the capture/priming oligonucleotide using mRNA 810 as template, thereby introducing the target gene 805 into the transcript. Upon reaching the 3' end of mRNA 810, the reverse transcriptase adds several C (shown here as three C) nucleotides.

In fig. 8C, a transcript conversion oligonucleotide (TSO)835 is present in the reverse transcription reaction mixture, where the TSO includes P1 sequence 825 and may also include a tetranucleotide (N4) first barcode 802. In the embodiments described below, TSO 835 can be an oligonucleotide having the sequence of SEQ ID No. 3. The first barcode 802 may be used for several experiments for multiplexing during sequencing, and the method is not limited to requiring the presence of the first barcode 802. The first barcode 802 is not limited to having four nucleotides, but can have any suitable number of nucleotides to make the sequencing library products identifiable. In some embodiments, the first (multiplex) barcode may have from about 3 nucleotides to about 10 nucleotides. The TSO may also include biotin attached to the 5' end of the oligonucleotide to improve efficiency.

In reverse transcription, the TSO aligns with the 5 'end of mRNA 810 and allows reverse transcriptase to "switch templates" and extends cDNA 830 beyond the three C nucleotides of its 3' end using the deoxynucleotides of the TSO as a template to incorporate the first four nucleotide barcode 802(N4) and P1 sequence 825 of the TSO as shown in fig. 8D. The fully extended cDNA product 840 now includes the P1 priming sequence, the target gene 805 and optionally the first barcode 802(N4) at both ends. cDNA product 840 also includes polyT-NI sequence incorporated from the capture sequence of capture/priming oligonucleotide 820.

In FIG. 8E, cDNA 840 can be amplified using forward and reverse P1 primers 845 to amplify the captured intact mRNA. In the examples described below, the P1 primer may have biotin at its 5' end and may have the sequence of SEQ ID No. 4. The sequence of the amplified product (amplified cDNA 840) retains all of the features of the transcript resulting from the reverse transcription step, including the 5 'and 3' end P1 sequences, the target gene sequence 805 and the first barcode 802. The first barcode was incorporated 5 ' to the target gene sequence 805 and 3 ' to the P1 priming sequence at the 5 ' end of the amplification product 840.

A first Polymerase Chain Reaction (PCR) is then performed. Fig. 8F shows a schematic of the primer arrangement used to selectively amplify the focus region of the BCR region. The forward primer 850 was designed to bind to the portion of the 5 'region of the cDNA amplification product 840 that was 3' to the P1 sequence 825 that had been used for the amplification of fig. 8E. The forward primer 850 may also include a second 6-nucleotide barcode 804, which may be used as part of a system to identify the source wells of a well plate. Although a 6 nucleotide second barcode 804 is shown in fig. 8E-8H, the second barcode can have any suitable number of nucleotides and can have from about 3 nucleotides to about 10 nucleotides. The reverse primer 855 is directed to bind a subregion of the large constant region of the BCR, near the 5 'end of the constant region, where it follows the 3' end of the junction (J) region of the BCR. This ensures that all variable (V), diversity (D) (if present) and junction (J) sub-regions fall within the sequencing portion of the amplicon. This removes the T30NI sequence and the P1 sequence 825 introduced from the capture/priming sequence 820. To ensure coverage of the heavy chain and the kappa and lambda light chains, a mixture of reverse primers 855 was used.

The selected and truncated amplification product 860 was subjected to a second PCR amplification, as shown in FIG. 8G. The amplification product 860 contains the target gene sequence 805, optionally a first (multiplex) barcode sequence 802(N4) and a second (well plate) barcode 804 (N6). The forward primer 865 for the second PCR binds to the sequence 5' of the second (well plate) barcode 804 introduced by the forward primer 850. Reverse primer 870 binds to a consensus sequence introduced by reverse primers 855 (the 5' portion of each reverse primer 855). The reverse primer 870 also includes a third (well plate) barcode 806 having 6 nucleotides (N6). Although fig. 8G-8H show a 6 nucleotide third (well plate) barcode 806, the third barcode can have any suitable number of nucleotides and can have from about 3 nucleotides to about 10 nucleotides.

The final amplicon 880 is shown in fig. 8H and contains a first optional multiplex barcode 802, a target gene sequence 805, a second (well plate) barcode 804, and a third (well plate) barcode 806. Additional adapters may be present for a particular sequencing chemistry.

Barcodes 2 and 3 are used across the wells of the output well plate to unambiguously identify each source well to determine the cells that generated the sequencing library. One economical approach may be to use unique barcodes distributed across 8X12 of wells of a well plate, thus only 20 unique barcodes are required to identify each well. The first (multiplex) barcode may be used if multiple well plate samples are combined in a sequencing run, but is not necessary if only one well plate is sequenced in a sequencing run.

Examples

Example 1 screening for secretionMouse splenocytes capable of binding IgG antibodies to human CD 45.

Screening was performed to identify mouse splenocytes that secreted IgG-type antibodies that bound to human CD 45. The experimental design comprises the following steps:

1. producing CD45 antigen coated beads;

2. harvesting mouse splenocytes;

3. loading cells into a microfluidic device; and

4. the antigen specificity was determined.

TABLE 1 reagents for example 1

Producing CD45 antigen coated beads. CD45 antigen coated beads were produced in the following manner:

50. mu.g of carrier-free CD45 were resuspended in 500. mu.l PBS (pH 7.2).

Slide-A-Lyzer dialysis mini-cups were rinsed with 500 microliters of PBS and then added to the microcentrifuge tubes.

50 microliters of 0.1 micrograms/microliter CD45 solution was added to the rinsed dialysis mini-cup.

170 microliters of PBS was added to 2mg HS-PEG 4-biotin, and then 4.1 microliters HS-PEG 4-biotin was added to the dialysis mini-cup containing CD45 antigen.

NGS-PEG 4-biotin was incubated with CD45 antigen for 1 hour at room temperature.

After incubation, the dialysis mini-cups were removed from the microcentrifuge tube, placed in 1.3ml PBS (pH7.2) in a second microcentrifuge tube, and incubated at 4 ℃ for 1 hour with shaking. The dialyzed mini-cups were then transferred to a third microcentrifuge tube containing 1.3ml of fresh PBS (pH7.2) and incubated at 4 ℃ for 1 hour with shaking. The last step was repeated three times for a total of 5 incubations of 1 hour.

100 microliters of biotinylated CD45 solution (. about.50 ng/microliter) was pipetted into a labeled tube.

500 microliters of Spherotech streptavidin-coated beads were pipetted into a microcentrifuge tube, washed 3 times (1000 microliters/wash) in PBS (pH7.4), and then centrifuged at 3000RCF for 5 minutes.

The beads were resuspended in 500. mu.l PBS (pH7.4) to a bead concentration of 5 mg/ml.

Biotinylated CD45 protein solution (50. mu.l) was mixed with resuspended Spherotech streptavidin-coated beads. The mixture was incubated at 4 ℃ with shaking for 2 hours and then centrifuged at 3000RCF for 5 minutes at 4 ℃. The supernatant was discarded, and the CD 45-coated beads were washed 3 times in 1mL PBS (pH 7.4). The beads were then centrifuged at 3000RCF for an additional 5 minutes at 4 ℃. Finally, the CD45 beads were resuspended in 500 microliters PBS pH7.4 and stored at 4 ℃.

Mouse splenocytes were harvested. Spleens from mice immunized with CD45 were harvested and placed in DMEM medium + 10% FBS. Scissors are used for cutting spleen.

The minced spleen was placed in a 40 micron cell filter. The single cells were washed through the cell filter with a 10ml pipette. The spleen was further disintegrated using a glass rod and the single cells were forced through a cell filter, and then washed again through the cell filter with a 10ml pipette.

Erythrocytes were lysed using a commercial kit.

The cells were centrifuged at 200 XG (spun down) and the raw spleen cells were resuspended in DMEM medium + 10% FBS using a 10ml pipette at a concentration of 2e⁸Individual cells/ml.

Cells are loaded into a microfluidic device. The microfluidic device is an OptoSelect^TMDevice (Berkeley Lights, Inc.) is provided with Opto Electro Positioning (OEP)^TM)Provided is a technique. The microfluidic device includes a flow region and a plurality of NanoPens fluidically connected thereto^TMChamber having a volume of about 7X 10⁵Cubic microns. The microfluidic device was operated on a prototype system (Berkeley Lights, Inc.) that included at least a flow controller, a temperature controller, a fluid medium conditioning and pump assembly, a light source for light activated DEP configuration, a mounting stage for the microfluidic device, and a camera.

Splenocytes were input to a microfluidic device and loaded into NanoPen chambers, each containing 20-30 cells. 100 microliters of medium was flowed through the device at 1 microliter/second to remove unwanted cells. The temperature was set at 36 ℃ and the medium was perfused at 0.1. mu.l/sec for 30 minutes. Bright field imaging as shown in fig. 5A shows the location of the cells within the NanoPen chamber.

And (3) antigen specificity determination. A medium containing 1:2500 goat anti-mouse F (ab') 2-Alexa 568 was prepared.

100 microliters of CD45 beads were resuspended in 22 microliters of a suspension containing 1: goat anti-mouse F (ab') 2-Alexa 568 secondary antibody at 2500 dilution in medium.

The resuspended CD45 beads were then flowed into the main channel of the microfluidic chip at a rate of 1 μ l/sec until they were located near, but just outside, the NanoPen chamber containing the splenocytes. The fluid flow is then stopped.

The microfluidic chip is then imaged in a bright field to determine the position of the beads (not shown). Next, images of the cells and beads were captured using a texas red filter. Images were taken every 5 minutes for 1 hour, each exposure lasting 1000ms and a gain of 5. As shown in fig. 5B, imaging at a time point of 5 minutes after introduction of the bead/labeled secondary antibody mixture showed that the fluorescent signal became apparent at some of the cell sites within the dock. Cell labeling indicated the presence of IgG on the cell surface. Faint labeling of the beads was observed at this time point.

And (6) obtaining the result. Positive signals observed at time points 20 minutes after the bead/antibody mixture was introduced were shown on the beads, reflecting the diffusion of IgG-isotype antibodies that diffuse away from certain docks and into the main channel of the microfluidic device where they are able to bind to CD 45-coated beads. The binding of the anti-CD 45 antibody to the beads allowed the second goat anti-mouse IgG-568 to bind to the beads and generate a detectable signal. See fig. 5C, white arrows.

Using the methods of the invention, each group of splenocytes associated with a positive signal can be isolated and engrafted as a single cell in a new dock and re-assayed. In this way, single cells expressing anti-CD 45 IgG antibodies can be detected and isolated.

Example 2: activation and screening of memory B cells in microfluidic devices

A general method for screening memory B cells in a microfluidic device is outlined in fig. 6A. The above method focuses on human memory B cells, but the method can be used to screen B cells from other animals.

Memory B cells were harvested. Frozen human Peripheral Blood Mononuclear Cells (PBMCs) were thawed and mixed with 6X volumes of RPMI 1640(Gibco) supplemented with 10% fbs (seradigm), counted, and centrifuged at 500g for 5 minutes. The supernatant was aspirated off and the cell pellet resuspended to 5X 10 in FACS buffer (PBS, 2% BSA, 1mM EDTA)⁷Concentration of individual cells/mL.

Next, B cell Enrichment was performed using the EasySep Human B cell Enrichment Kit (EasySep, # 19054). 50 microliters of the B cell enrichment mixture was added per mL of human PBMC, and the resulting mixture was incubated at room temperature for 10 minutes. Then 75 microliters of magnetic particles per mL of human PBMC were added, the mixture was mixed well and incubated at room temperature for 10 minutes. A volume of about 1.1 uL of the PBMC cell suspension was brought to 2.4mL by adding FACS buffer, and then mixed well by pipetting up and down. The tube containing the PBMC suspension was then placed in an EasySep magnet (without a cover) and incubated for 5 minutes. The enriched B cell suspension was poured into a new clean tube while holding the tube in the EasySep magnet. Cell counts of the enriched B cell suspensions were performed, after which the cells were centrifuged at 300g for 5 minutes. The supernatant was aspirated.

The enriched B cell pellet was resuspended to 5X 10 in FACS buffer containing anti-CD 27 antibody⁷Individual cells/mL, then incubated in the dark at 4 ℃ for 20 minutes. After incubation, the cells were washed 2 times with 3mL FACS buffer and the suspension was centrifuged at 300g for 5 minutes. Resuspend the final enriched B cell pellet in FACS buffer to a concentration of 5X 10⁷Individual cells/mL, then passed through the unicell filter with the pipette tip pressed against (and perpendicular to) the mesh of the filter. The filtered cell suspension was kept on ice until FACS sorting. CD27 was analyzed using a FACS Aria instrument⁺B cell sorting into B cell activation/culture Medium (RPM)I1640 (Gibco), 10% FBS (Seradigm), 2ug/mL CpG (Invivogen), 1ug/mL IL-2(Peprotek), 5ng/mL IL-4(Peprotek), 10ng/mL IL-6(Peprotek), 10ng/mL IL-21(Peprotek), and 10ng/mL BAFF (Peprotek)).

Modulation of isolated memory B cells to 2X 10⁶Individual cells/mL concentration, then incubated at 37 ℃ until input into the microfluidic device, which is done as quickly as possible.

Preparation of the microfluidic device and input of memory B cells. The microfluidic device is an OptoSelect^TMDevice (Berkeley Lights, Inc.) is configured with OptoElectro Positioning (OEP) ^TM) A technique having a conditioned inner surface comprising a layer of covalently attached polyethylene glycol (PEG) polymer. The microfluidic device comprises a flow region having a plurality of microfluidic channels and a plurality of isolated docks (or NanoPen) in fluid connection with each microfluidic channel^TMChamber), the volume of the isolation dock is about 5 x 10⁵Cubic microns. Microfluidic devices operate on Beacon platforms (Berkeley Lights, Inc.) or prototype Alpha platforms (Berkeley Lights, Inc.), which include flow controllers, temperature controllers, fluidic medium conditioning and pump assemblies, light sources for light activated DEP configurations, mounting stages for microfluidic devices, and cameras.

250 microliters of 100% carbon dioxide were flowed into the microfluidic device at a rate of 12 microliters/second. Followed by 250 microliters of priming medium containing 1000mL of Iscove modified Dulbecco's medium (ATCC), 200mL fetal bovine serum (ATCC), 10mL of pen-strep (Life Technologies), and 10mL of Pluronic F-127(Life Technologies). B cell culture medium containing RPMI 1640(Gibco) supplemented with 10% FBS (Seradigm), 1X Pen-strep (Gibco) and 1X kanamycin sulfate (Gibco) was then introduced.

The isolated memory B cell suspension prepared as above is then introduced into a microfluidic device by flowing the suspension into an inlet and stopping the flow while the memory B cells are located within the flow region/microfluidic channel. Memory B-cells are then loaded into the segregating dock, with the goal of one B-cell per dock. Memory B cells were moved from the flow region/microfluidic channel into the separation region of the isolation dock using light-activated DEP force (OEP technique). Parameters for operating the OEP included applying an AC potential (voltage 3.5V, frequency 2MHz) across the microfluidic device, using structured light to form an optical trap (as shown in fig. 6B) that trapped a single cell, and moving the optical trap at 8 microns/sec. Cells that remain in the flow region/microfluidic channel after entering the dock are washed out of the microfluidic device.

Memory B-cell activation. The protein a (spheriotech) coated beads were mixed with irradiated Jurkat D1.1 feeder cells in a ratio of about 1: 1. The bead/feeder cell mixture is then flowed into the microfluidic device and feeder cells and beads are bulk loaded into each isolated dock containing memory B cells. Batch loading is achieved by tilting the microfluidic device at the end and allowing gravity to pull the cells and beads down into the isolation dock. The bead/feeder cell mixture was mixed at approximately 1.5X 10⁷Individual feeder cells and beads per mL concentration were flowed into the microfluidic device, and the isolation docks, which were bulk loaded in this manner, received an expected average of about 10 feeder cells and about 10 beads per dock.

The microfluidic device is then moved to a culture station and the B-cell activation/culture medium (described above) is perfused through the flow path of the microfluidic device for four (4) days. The microfluidic device is held tilted in the end position while being held on the incubation station. The perfusion method is as follows: perfusing B cell activation/culture medium at 0.02 μ l/sec for 100 sec; the flow was stopped for 500 seconds; b cell activation/culture medium was perfused at 2 μ l/sec for 64 sec; and repeated.

Activated memory B cells were assayed. After 4 days of culture/activation, the microfluidic device was removed from the culture station and returned to the Beacon/Alpha system, followed by multiplex assays to detect IgG secretion and antigen specificity. The multiplex assay shown in fig. 6C includes anti-human IgG antibody-coated capture beads (sphenotech), anti-human IgG-Alexa Fluor 488 secondary antibody (Invitrogen), and target antigen labeled with Alexa Fluor 647 carboxylic acid succinimidyl ester. The capture beads, secondary antibody and labeled target antigen are mixed together in the B cell activation/culture medium and flow into the flow region/microfluidic channel of the microfluidic device. Flow was stopped and images of the isolated dock were taken periodically for 10 to 25 minutes using appropriate filters to visualize the fluorescent markers. Memory B cells secreting antibodies that bind to the target antigen will induce binding between the labeled target antigen and the IgG antibody coated capture beads. As a result, a "plume" (plume) of fluorescently labeled capture beads will appear at the opening between the flow region/microfluidic channel and the sequestration dock where the memory B cells are located. FIG. 6D shows typical assay results for memory B cells.

Outputting and further processing. Memory B cells recognized to secrete antibodies that bind to the target antigen were then used to open one dock at a time using DEP force (using the OEP parameters described above) and by passing output media (DPBS with Ca2+ and Mg2+ (Lonza), 5Mg/mL BSA (Sigma), and 1:100 Pluronic^TMF-127(Thermo Fisher)) flowed through the flow path of the microfluidic device, out of the microfluidic device into the wells of a 96 well plate. After export, memory B cells are lysed and transcripts encoding heavy and light chain antibody sequences are reverse transcribed into cDNA and sequenced.

As a result: using essentially the same protocol as the previous protocol, B cell activation rates (as measured by detecting IgG secretion) have reached about 12% for human memory B cells. The cell activation rate of non-human mammalian memory B cells has reached as high as 40%. The detection rate of activated memory B cells expressing antibodies that bind to the antigen of interest depends on the antigen of interest, but is typically about 1% or less. Testing putative Ag obtained from human memory B cells by such screening protocols⁺In one experiment of correlation of antibodies, a set of 20 memory B cells recognized as secreting Ag-binding antibodies was exported from the microfluidic device and their antibody heavy and light chain sequences were determined. After re-expression in HEK 393T cells and ELISA analysis with the target antigen used in the on-chip assay, 16 out of 20 antibodies (or 80%) detected the target antigen in the ELISA assay. This confirms the relevance of activating and screening memory B cells according to the methods disclosed herein.

And (4) changing. The foregoing methods can be varied in a number of ways and still achieve the goal of direct screening of memory B cells. These variations include:

1. screening for memory B cells isolated from non-human animals, including other mammalian species, such as rodents (e.g., mice, rats, guinea pigs, gerbils, hamsters), rabbits, ferrets, livestock (e.g., goats, sheep, pigs, horses, cows), llamas, camels, and birds, such as chickens and turkeys.

2. The OEP operating parameters used to dock and undock memory B cells can vary. For example, the AC potential on the microfluidic device can be set to about 2 to about 5 volts with a frequency of about 1 to about 3 MHz. Specific examples include (i) a voltage of about 2.5V and a frequency of about 3MHz, and (ii) a voltage of about 4.5V and a frequency of about 1 MHz. Additionally, the speed at which the structured light (or light cage) moves may vary from about 5 to about 10 microns/second.

3. As described above, the microfluidic device is held on the culture stage with the chip tilted at the end. Alternatively, the microfluidic device may be replaced on the Beacon/Alpha system in a standard position (i.e., the microfluidic device lies substantially flat). Memory B-cell culture/activation medium was then perfused through the flow region of the microfluidic device according to the following protocol: perfusing B cell culture/activation medium at 0.01 μ l/sec for 2 hours; perfusing B cell culture/activation medium at 2 microliters/second for 64 seconds; and repeated.

4. The assay can be further multiplexed to include a second antigen of interest or even second and third antigens of interest. See, e.g., fig. 6C. In this way, memory B cells can be simultaneously screened for antibodies that bind to different epitopes on the same target protein/molecule, for antibodies that exhibit different levels of cross-species reactivity to the target protein/molecule, or simply for antibodies that bind to disparate antigens of interest. Furthermore, the use of the second and/or third target antigen allows for high throughput screening.

5. The size of the isolation docks in a microfluidic device may be increased to, for example, about 1.1 x 10⁶Cubic microns. Typically, the dock will have a volume of about 5 x 10⁶Cubic micrometers or less (e.g., about 4 x 10)⁶Cubic micron, about 3 x 10⁶Cubic micron, about 2.5 x 10⁶Cubic micron, about 2 x 10⁶Cubic micron, about 1.5 x 10⁶Cubic microns or less). The larger size can be used to initially assay the polyclonals of memory B cells,but larger sizes also delay multiplexed assays and may potentially negatively impact activation and growth of memory B cells.

6. The assay can be started with a polyclonal assay and then converted to a monoclonal assay. In this approach, the sequestration dock is initially loaded with a plurality of memory B-cells (e.g., 2 to 10 or 4 to 10). When this method is used, it is necessary to further analyze that Ag appears in the initial assay ⁺To determine which memory B cells in the isolation dock produce the Ag⁺An antibody. For this purpose, Ag^-The cells in the dock are opened and output (e.g., by flowing the output medium through the flow region/microfluidic channel to a waste tube). Next, Ag is added⁺The cells in the dock open and reload the individual memory B cells into the empty dock, which is located near or near the source dock on the microfluidic device. Then repeating Ag⁺Multiplex assay of dock and identify any location as Ag in duplicate assay⁺Memory B cells in the dock are exported for further processing. This higher throughput, polyclonal to monoclonal approach adds the additional steps shown in fig. 6E to the method shown in fig. 6A.

Example 3: screening of plasma cells in a microfluidic device

A general method for screening plasma cells in a microfluidic device is outlined in fig. 7A. The foregoing method focuses on human plasma cells, but the method can be used to screen plasma cells from other animals.

And (5) harvesting the plasma cells. Frozen human Bone Marrow (BM) cells were rapidly thawed in a 37 ℃ water bath and then added dropwise to 5mL of a pre-warmed (37 ℃) Plasma Cell Culture Medium (RPMI 1640(Gibco), 10% FCS (Hyclone), 1X non-essential amino acid (NEAA) solution (Gibco), 1X sodium pyruvate (Gibco), 50uM β -mercaptoethanol (Gibco), and 1X pen-strep (Gibco)) supplemented with 1X DNase (DNase: (1X)) Nuclean 1000X stock containing 25,000U/mL, Millipore). The resulting mixture was centrifuged at 300g for 10 min and the cell pellet was washed with FACS buffer (PBS, 2% BSA, 1mM EDTA)) Washing is carried out for 2 times.

Cell pellets obtained after washing in FACS buffer were resuspended to 1X 10 in FACS buffer containing anti-CD 138 antibody⁷Individual cells/mL, then incubated for 20 minutes at 4 ℃ in the dark. After incubation, cells were washed 2 times in FACS buffer and resuspended in FACS buffer to a concentration of 1 × 10⁷Individual cells/mL. The cell suspension was kept on ice until FACS sorting. CD138 Using FACS Aria Instrument⁺Plasma cells were sorted to supplement with 40ug/mL IL-6 (R)&D Systems) in plasma cell culture medium (above).

Conditioning the isolated plasma cells to 2X 10⁶Individual cells/mL concentration, then incubated at 37 ℃ until input into the microfluidic device, which is done as quickly as possible.

Preparation of the microfluidic device and input of plasma cells. The microfluidic device is an OptoSelect^TMDevice (Berkeley Lights, Inc.) is configured with OptoElectro Positioning (OEP)^TM) Techniques, having a conditioned inner surface, include a layer of covalently attached polyethylene glycol (PEG) polymer. The microfluidic device includes a flow region having a plurality of microfluidic channels and a plurality of isolated docks (or NanoPen) in fluid connection with each microfluidic channel ^TMChamber), the volume of the isolation dock is about 5 x 10⁵Cubic microns. The microfluidic device was operated on a Beacon platform (Berkeley Lights, Inc.) or a prototype Alpha platform (Berkeley Lights, Inc.) that included flow controllers, temperature controllers, fluidic medium conditioning and pump assemblies, light sources for light-activated DEP configuration, a mounting stage for the microfluidic device, and a camera.

250 microliters of 100% carbon dioxide were flowed into the microfluidic device at a rate of 12 microliters/second. Followed by 250 microliters of priming medium containing 1000mL of Iscove modified Dulbecco's medium (ATCC), 200mL fetal bovine serum (ATCC), 10mL pen-strep (Life Technologies), and 10mL Pluronic F-127(Life Technologies). Plasma cell culture medium (described above) was then introduced, supplemented with 40ug/mL IL-6(R & D Systems).

The isolated plasma cell suspension prepared as above is then introduced into a microfluidic device by flowing the suspension into an inlet and stopping the flow while the plasma cells are located within the flow region/microfluidic channel. The plasma cells are then loaded into the sequestration dock with the goal of one plasma cell per dock. Light activated DEP forces (OEP technique) were used to move plasma cells from the flow region/microfluidic channel into the separation region of the isolation dock. Parameters for operating the OEP included applying an AC potential (voltage 3.5V, frequency 2MHz) to the microfluidic device, using structured light to form an optical trap (similar to that shown in figure 6B) to capture individual cells, and moving the optical trap at 8 microns/sec. The cells that remain in the flow region/microfluidic channel after entering the dock are washed out of the microfluidic device.

Plasma cells were assayed. Immediately after docking, plasma cells were assayed to detect IgG secretion and antigen specificity. The multiplex assay shown in fig. 6C includes anti-human IgG antibody-coated capture beads (sphenotech), anti-human IgG-Alexa Fluor 488 secondary antibody (Invitrogen), and target antigen labeled with Alexa Fluor 647 carboxylic acid succinimidyl ester. The capture beads, secondary antibody and labeled target antigen are mixed together in plasma cell culture medium (described above) supplemented with 40ug/mL IL-6(R & D Systems) and flowed into the flow region/microfluidic channel of the microfluidic device. The flow was stopped and images of the isolated dock were taken periodically using appropriate filters for 10 to 25 minutes to visualize the fluorescent markers. Plasma cells secreting antibodies that bind to the target antigen will induce binding between the labeled target antigen and the IgG antibody coated capture beads. As a result, a "plume" (plume) of fluorescently labeled capture beads will appear at the opening between the flow region/microfluidic channel and the sequestration dock where the plasma cells are located. Fig. 7B shows typical measurement results of plasma cells, with a bright field image on the left, a fluorescence image of an anti-human IgG secondary antibody in the middle, and a fluorescence image of a labeled target antigen on the right.

Outputting and further processing. Plasma cells recognized to secrete antibodies that bind to the target antigen were then used to open one dock at a time using DEP force (using the OEP parameters described above) and by passing export media (dpbs with Ca2+ and Mg2+ (lonza), 5Mg/mL BSA (Sigma) and 1:100 Pluronic^TMF-127(Thermo Fisher)) flows through the flow path of the microfluidic device fromThe microfluidic device was exported into the wells of a 96-well plate. After export, plasma cells were lysed and transcripts encoding heavy and light chain antibody sequences were reverse transcribed into cDNA and sequenced.

As a result: the above scheme is completed in less than one day. The detection rate of plasma cells expressing an antibody binding to a target antigen (which depends on the target antigen) was generally about 1% or less using substantially the same protocol as previously described, and in one study, recognized Ag obtained from plasma cells by such a protocol⁺The antibody, when re-expressed at a rate up to 82%, exhibited Ag specific binding.

And (4) changing. The foregoing methods may be varied in a number of ways and still achieve the goal of direct screening of plasma cells. These variations include:

1. screening for plasma cells isolated from non-human animals, includes other mammalian species, such as rodents (e.g., mice, rats, guinea pigs, gerbils, hamsters), rabbits, ferrets, livestock (e.g., goats, sheep, pigs, horses, cows), llamas, camels, and birds, such as chickens and turkeys.

2. B cell Enrichment can be performed prior to FACS separation of plasma cells, for example, using the EasySep Human B cell Enrichment Kit (EasySep, # 19054).

3. The OEP operating parameters used to dock and undock plasma cells can vary. For example, the AC potential on the microfluidic device can be set to about 2 to about 5 volts with a frequency of about 1 to about 3 MHz. Specific examples include (i) a voltage of about 3.5V and a frequency of about 2MHz, and (ii) a voltage of about 4.5V and a frequency of about 1 MHz. Additionally, the speed at which the structured light (or light cage) moves may vary from about 5 to about 10 microns/second.

4. The size of the plasma cell loaded sequestration dock may vary. For example, the dock may have a volume of about 1.1 x 10⁶Cubic microns. Typically, the dock will have a volume of about 5 x 10⁶Cubic microns or less (e.g., about 4 x 10)⁶Cubic micron, about 3 x 10⁶Cubic micron, about 2.5 x 10⁶Cubic micron, about 2 x 10⁶Cubic micron, about 1.5X 10⁶Cubic microns or less). By reducing the size of the isolation dock, the isolation dock can be made of a metal materialMultiplex assays are performed more rapidly, thereby avoiding extended screening periods during which plasma cells can undergo cell death.

5. In addition to exporting plasma cells expressing Ag + antibodies, the plasma cells can be lysed in the presence of barcoded beads designed to capture mRNA released by the lysed cells in isolated docks. The captured mRNA can then be reverse transcribed into a cDNA library attached to the barcoded beads, and the barcoded beads can be output for subsequent off-chip cDNA library sequencing. Methods of cell lysis, mRNA capture and cDNA library generation on a chip have been described, for example, in PCT International application No. PCT/US17/54628, filed 2017, 9/29, which is incorporated herein by reference in its entirety.

Example 4: single cell export of antibody-expressing B lymphocytes and sequencing libraries directed to B cell receptor regions Generation of

TABLE 2 primers used in this experiment. All primers were provided at 10 micromolar.

Cell: OKT3 cells (a murine myeloma hybridoma cell line) were obtained from ATCC ( Cat.#CRL-8001^TM). The cells are provided as a suspension cell line. By mixing about 1X 10⁵To about 2X 10⁵The cultures were maintained by seeding and incubating the cells/mL at 37 ℃ using air containing 5% carbon dioxide as the gaseous environment. Cells were bottled every 2-3 days. OKT3 cell number and viability were counted and cell density was adjusted to 1X 10⁶Ml, to load into a microfluidic device.

An output plate: a96-well full skirt (VWR Cat. # 95041-. Each plate was prepared by dispensing 10 microliters of mineral oil (Sigma Cat. # M5904) followed by 5 microliters of 2 xtcl buffer (Qiagen Cat. # 1070498). (other Lysis buffers such as Single Cell Lysis Kit (Single Cell Lysis Kit), Ambion Cat. #4458235 or Clontech Lysis buffer, Cat. #635013 may also be used as appropriate). The output plate was centrifuged at 200g for 1 min at room temperature and stored at room temperature until use.

And (3) outputting a buffer solution: dulbecco Phosphate Buffered Saline (DPBS) + calcium + magnesium (1000mL, Lonza Cat. # 17-513F); bovine Serum Albumin (BSA) (powder, 5g, Fisher Scientific Cat. # BP 9706-100); pluronic F-127(10ml, Life Technologies Cat. # 50-310-); and recombinant ribonuclease inhibitor (RNaseOUT) (Life Technologies Cat. #107777019) at a final concentration of 1. mu.l/ml. The export buffer was filtered prior to use using a 0.22 micron filtration device (VWR Cat. # 73520-.

Cell export and lysis: OKT3 cells (any kind of primary B cells can be used) were flowed into the microfluidic device and introduced into the NanoPen chambers using the OptoElectro localization (OEP) function of the system to provide a final distribution of one cell per NanoPen chamber. An IgG assay was performed as described in example 1 (antigen-specific assays may also be used). Cells identified as having expression of the target IgG (and optionally antigen-specific antibody) are exported into a 96-well output plate, one cell per well, in 5-microliter output volumes, respectively. The exportation was performed using a mixture of OEP forces to export the selected cells out of the NanoPen chamber in which they were already present, and then each selected cell was individually exported in a volume of 5 microliters by the flow of the culture medium in the flow region/microfluidic channel. The output well plate was centrifuged at 200g for 5 minutes at 4 ℃ immediately after output. Plates were frozen at-80 ℃ until RNA isolation and cDNA synthesis were performed. Under these conditions, the well plate suitably remains for at least one month. In some cases, overnight storage or storage may be performed for a period of up to one week.

And (4) RNA isolation. The single cell output plate was thawed on ice for 15 minutes and then warmed to room temperature. RNAclean XP SPRI beads (Beckman Coulter # A63987) were brought to room temperature and 10 microliters of bead mixture (1 volume) was added to each well (1 volume SPRI beads showed higher RNA recovery compared to the standard 1.8 to 2.2).

The lysate and bead mixture were incubated for 15 minutes at room temperature. This extended incubation period provides improved binding of released RNA. Plates were then transferred to 96-well plate magnets (MagWell)^TMMagnetic Separator (Magnetic Separator)96, Cat. #57624) and incubated for 5 minutes. The supernatant was carefully removed and washed with ethanol by adding 100 μ l 80% ethanol (Sigma Cat. # E7023, freshly prepared). After about 30 seconds, the ethanol was aspirated and the ethanol wash was repeated. After the last aspiration, the plate was removed from the 96-well plate magnet and the beads were dried for 5 minutes.

And (3) cDNA synthesis. The plate was transferred to 4 ℃ and the beads were resuspended in 4ul "RT mix 1": containing 0.8. mu.l RNase-free water (Ambion Cat no AM 9937); 1 microliter 1:5M ERCC control RNA (ThermoFisher Scientific Cat. # 4456740); 1 microliter dNTP (10 mM each, NEB, # N0447L); 1 microliter biotin-dTVI RNA capture/priming oligonucleotide (SEQ ID NO. 2); and 0.2. mu.l RNaseOUT (4U/l, Life Technologies Cat. # 107777-. The 3' inosine of the capture sequence of the biotin-dTVI RNA capture/priming oligonucleotide provided increased binding to released RNA, as inosine could bind any natural nucleotide. A capture sequence with 3' inosine may capture the released RNA better than a capture sequence including the final "N" nucleotides, which may bind mRNA only 25% of the time. A schematic of capture/priming sequence capture is shown in FIG. 8A, described above. The ERCC RNA control provides an internal RT control and also provides a carrier RNA that increases the efficiency of reverse transcription. The plates were incubated at 72 ℃ for 5 minutes and immediately transferred to 4 ℃. Add 4 microliters of "RT mix 2" to each well, which contains 1 microliter of betaine (5M, Sigma Cat. # B030075 VL); 1.5 microliters of 5 XT mix (Thermo, # EP0753), 0.5 microliters of biotinylated barcoded-template switch oligonucleotide (biot barcoded TSO; SEQ ID NO.3), 0.5 microliters of 120mM MgCl ₂(125mM, Life Technologies Cat. # AM9530G), 0.4. mu.l RNase OUT and 0.1. mu.l Maxima RNaseH-reverse transcriptase (200U/. mu.l, Thermo Fisher Cat. # EP 0753). After addition of "RT mix 2", reverse transcription was performed at 42 ℃ for 90 min, followed by 10 cycles: 50 ℃ 2 min/42 ℃ 2 min. The last thermal cycle was followed by heat inactivation at 75 ℃ for 15 minutes. The four nucleotide barcode "NNNN" of the biot barcoded TSO provides internal barcoding for potential multiplex sequencing experiments on multiplexed output plates and is not an essential feature of the method. A schematic of the initial strand extension is shown in fig. 8B, and TSO association is shown in fig. 8c, and the complete transcript of reverse transcription is shown in fig. 8D.

Amplification of intact mRNA. After cDNA synthesis, the output plate was centrifuged at 200g for 5 minutes and 17 microliters of PCR mix containing 12.5 microliters of 2X Kapa Hi Fi hot start ready mix (Roche Cat. # KK2602), 1 microliter of P1 primer (biot _ P1, SEQ ID No.4), and 3.5 microliters of nucleotide-free water (Ambion Cat. # AM9937) was added and PCR was performed at 98 ℃ for 3 minutes, followed by 20 cycles of: 15 seconds at 98 ℃, 30 seconds at 65 ℃ and 5 minutes at 72 ℃; and a final extension was performed at 72 ℃ for 5 minutes. The final extension period is long enough for the polymerase to amplify a long cDNA molecule (greater than 2 kb). A schematic of this amplification is shown in fig. 8E.

And (4) PCR purification. Add 25 μ l (1x volume) of DNAClean SPRI beads (Beckman Coulter, Cat. # a62881) to each well and mix well, removing primer dimers and short degraded RNA products that can contaminate downstream amplification. The mixture was incubated at room temperature for 10 minutes. After incubation, plates were placed on well plate magnets for 5 minutes. The supernatant was carefully removed and washed with ethanol by adding 100 μ l of 80% ethanol (freshly prepared). After about 30 seconds, the ethanol is aspirated. The ethanol washing procedure was repeated once. After final aspiration, the output well plate was removed from the well plate magnet and the beads were dried for 5 minutes. DNA was eluted from the dried beads using 15 uL of nuclease-free H2O. FIG. 9A shows a BioAnalyzer electropherogram trace of the product, showing the expected distribution of the expected average length of the full-length cDNA, which is about 1800 bp. After amplification and purification, typical amounts of cDNA were estimated to be about 2ng to about 20 ng.

BCR amplification and barcoding. B Cell Receptor (BCR) amplification and barcoding of single cells was performed in a 2-step Polymerase Chain Reaction (PCR).

And (3) PCR 1. In the first PCR, the forward primer (FP1, SEQ ID NO.5) was designed to hybridize to the 3' end of the P1 sequence introduced from the bio-barcoded TSO, thereby eliminating the P1 sequence introduced in the above-described whole RNA amplification step. The reverse primer (RP1, SEQ ID NO.6, 7, 8) binds to the constant region of the B cell receptor gene segment, which is adjacent to the joining (J) gene segments of the heavy and light chains. A schematic of PCR 1 is shown in FIG. 8F. The forward primers contained 12 different 6 nucleotide barcodes (nnnnnnnn) spanning 12 columns of the plate. Thus, PCR 1 selects RNA sequences containing BCR from the whole RNA amplification product and further selects regions of BCR that focus on the variable, junction, and diversity segments of BCR. The selectively amplified amplification product (shown as amplification product 860 of fig. 8F) further contains no polyA/polyT capture sequence, nor most of the constant region of the BCR, neither of which provides target data.

PCR 1 was performed in a 10. mu.l reaction containing 1. mu.l of amplified transcriptome, 5. mu.l of 2X Kapa Hi Fi HotStart ReadyMix, 0.1. mu.l of forward barcoded primer (FP1), 0.2. mu.l of reverse heavy chain constant primer (RP 1 of murine Hc, SEQ ID NO.6), 0.1. mu.l of light chain constant primer (which is a 1:1 mixture of RP1 of murine kc and RP1 of murine λ c (SEQ. ID NO.6 and 7)), and 3.6. mu.l of nuclease-free water. Circulation conditions are as follows: 3 minutes at 98 ℃; then 5 cycles of: 20 seconds at 98 ℃, 45 seconds at 70 ℃ and 45 seconds at 72 ℃; then 10 cycles of: 20 seconds at 98 ℃, 45 seconds at 68 ℃ and 45 seconds at 72 ℃; then 10 cycles of: 20 seconds at 98 ℃, 45 seconds at 65 ℃ and 45 seconds at 72 ℃; finally, extension was carried out at 72 ℃ for 5 minutes.

And (4) carrying out PCR 2. In a second PCR, a single forward primer (FP2, SEQ ID NO.9) was bound to barcoded FP1 and 8 barcoded across 8 rows of plates (FP2, SEQ ID NO.9) ((C))NNNNNN) The reverse primer (RP2, SEQ ID NO. 10). This strategy allows the use of only 20 barcodes to have a unique barcode for each well in the entire 96-well plate. Multiple plates can be combined with an internal plate barcode incorporated from the biot _ barcoded TSO used in cDNA synthesis. A schematic of PCR 2 is shown in FIG. 8G. A schematic of the final product amplicon 880 is shown in fig. 8H.

PCR 2 was performed using the PCR 1 product as a template (e.g., product 860 of fig. 8G). A10. mu.l reaction was performed containing 1. mu.l of PCR 1 product, 5. mu.l of 2X Kapa Hi Fi HotStart ReadyMix, 0.1. mu.l of forward primer FP2, 0.1. mu.l of reverse barcoded primer RP2, and 3.8. mu.l of nuclease-free water. FIG. 9B shows the results of gel electrophoresis of single cell amplicons provided by this method. The arrow points to a band on the reference size gradient (lane M), which represents 500 bp. In lane 1, a single cell amplified for the heavy chain (He), and in lane 2, a single cell amplified for the light chain Kc each showed a band of appropriate length for each amplification product. Circulation conditions are as follows: PCR was performed at 98 ℃ for 3 minutes; then 5 cycles of: 20 seconds at 98 ℃, 45 seconds at 68 ℃ and 45 seconds at 72 ℃; then, performing 15 cycles or less at 98 ℃ for 20 seconds, 65 ℃ for 45 seconds and 72 ℃ for 45 seconds; finally, extension was carried out at 72 ℃ for 5 minutes.

Amplicon pooling, purification and sequencing. Amplicons were pooled and 1x volume of DNAClean SPRI beads (Beckman Coulter, Cat. # a62881) was added to each well and mixed well. The mixture was incubated at room temperature for 10 minutes. After incubation, plates were placed on well plate magnets for 5 minutes. The supernatant was carefully removed and washed with ethanol by adding 100 μ l 80% ethanol (freshly prepared). After about 30 seconds, the ethanol was aspirated and the ethanol wash repeated again. After the last aspiration, the plate was removed from the magnet and the beads were dried for 5 minutes. DNA was eluted from the dried beads with 15. mu.l nuclease-free water.

Although the experiment is applicable to Illumina TruSeq, other adaptors may alternatively be used and libraries suitable for any other Next Generation Sequencing (NGS) instrument may be provided, or may alternatively be amplified to be suitable for Sanger sequencing.

Alternatively, when amplicons from individual wells of a well plate are not pooled, after two rounds of PCR and purification at the Qubit^TMQuantification above showed that about 25ng to about 90ng of amplification product was produced per well. This is sufficient for sequencing, as described above.

In FIGS. 10A-10C, the results of gel electrophoresis are shown, confirming the ability to detect a particular amplification product. In this experiment, 19 individual OKT3 cells were exported and the intact RNA amplification product prepared as above was divided into three parts. Each of the three fractions was amplified separately for heavy chain Hc, light chain κ Kc, and light chain λ c using the primers and methods described above. In fig. 10A, the heavy chain is shown to be amplified from all 19 individual cells (faint bands were confirmed by analysis with Qubit). In fig. 10B and 10C, light chain κ (fig. 10B) or light chain λ (fig. 10C) shows that each cell has either κ or λ, but not both. For example, lanes 8, 12, 13, 14 have a lambda light chain instead of kappa, while lanes 9, 10, 11 have a kappa light chain instead of lambda. The arrow at the left edge of the gel points to a band in a size gradient of 500bp, confirming the size of the expected product.

List of alternative embodiments

1. A method of detecting B cell lymphocytes expressing an antibody that specifically binds an antigen of interest, the method comprising: introducing a sample comprising B-cell lymphocytes into a microfluidic device comprising: a housing having a flow region and a docking station, wherein the docking station includes a separation region having a single opening and a connection region providing a fluidic connection between the separation region and the flow region, and wherein the docking station-holding separation region is an unswept region of the microfluidic device; loading B-cell lymphocytes from the sample into an isolated region of a sequestration dock; introducing an antigen of interest into the flow region of the housing such that the antigen of interest is in proximity to the B cell lymphocytes; and, monitoring binding of the antigen of interest to an antibody expressed by a B cell lymphocyte.

2. The method of embodiment 1, wherein the isolated region of the isolation dock comprises at least one conditioned surface. In some embodiments, the at least one conditioned surface may comprise a plurality of conditioned surfaces.

3. The method of embodiment 2, wherein the conditioned surface is substantially non-reactive with B cell lymphocytes.

4. The method of embodiment 2 or 3, wherein the at least one conditioned surface (or each of the plurality of conditioned surfaces) comprises a layer of covalently linked hydrophilic molecules.

5. The method of embodiment 4, wherein the hydrophilic molecule comprises a polyethylene glycol (PEG) -containing polymer.

6. The method of any of embodiments 1-5, wherein the housing of the microfluidic device further comprises a Dielectrophoresis (DEP) configuration.

7. The method of any of embodiments 1-6, wherein the housing of the microfluidic device further comprises a base, a microfluidic conduit structure, and a lid, which together define a microfluidic conduit, and wherein the microfluidic conduit comprises a flow region and an isolation dock.

8. The method of embodiment 7, wherein: the base includes a first electrode; the cover comprises a second electrode; the base or the lid comprises an electrode activation substrate, wherein the electrode activation substrate has a surface comprising a plurality of DEP electrode regions, and wherein the surface of the electrode activation substrate provides an inner surface of a flow region.

9. The method of any one of embodiments 1 to 8, wherein the width W of the attachment region_conFrom about 20 microns to about 60 microns.

10. The method of any one of embodiments 1 to 9, wherein the linker region has a length L_conAnd wherein the length L of the connection region_conWidth W of connection region_conHas a value of at least 1.5.

11. The method of embodiment 9 or 10, wherein the width W of the separation region_isoIs greater than the width W of the connecting region_con。

12. The method of any one of embodiments 9 to 11, wherein the width W of the separation region_isoFrom about 50 microns to about 250 microns.

13. The method of any one of embodiments 1-12, wherein the isolating dock comprises a volume of about 0.5nL to about 2.5 nL.

14. The method of any of embodiments 1-13, wherein the isolated area of the isolation dock comprises at least one surface (e.g., a plurality of surfaces) coated with a coating material.

15. The method of embodiment 14, wherein the coating material comprises a hydrophilic molecule that is substantially non-reactive with B cell lymphocytes.

16. The method of embodiments 14 or 15, wherein the coating material comprises a polyethylene glycol (PEG) -containing polymer (e.g., in some embodiments, the PEG-containing polymer comprises a PEG-PPG block copolymer).

17. The method of any one of embodiments 1 to 16, wherein the sample comprising B cell lymphocytes is a sample of peripheral blood, spleen biopsy, bone marrow biopsy, lymph node biopsy or tumor biopsy.

18. The method of any one of embodiments 1 to 16, wherein the sample comprising B cell lymphocytes is a peripheral blood sample.

19. The method of any one of embodiments 1 to 16, wherein the sample comprising B cell lymphocytes is a bone marrow biopsy.

20. The method of embodiment 17 or 18, wherein the B cell lymphocyte is a memory B cell.

21. The method of embodiment 17 or 19, wherein the B cell lymphocyte is a plasma B cell.

22. The method of any one of embodiments 1 to 21, wherein the sample comprising B cell lymphocytes is obtained from a mammal or an avian animal.

23. The method of embodiment 22, wherein the sample comprising B cell lymphocytes is obtained from a human, mouse, rat, guinea pig, gerbil, hamster, rabbit, goat, sheep, llama, chicken, ferret, pig, horse, cow, or turkey.

24. The method of embodiment 22 or 23, wherein the mammal has been immunized against an antigen of interest.

25. The method of embodiment 22 or 23, wherein the mammal has been exposed to or infected with a pathogen associated with an antigen of interest.

26. The method of embodiment 22 or 23, wherein the mammal has cancer and the cancer is associated with an antigen of interest.

27. The method of embodiment 22 or 23, wherein the mammal has an autoimmune disease and the autoimmune disease is associated with an antigen of interest.

28. The method of any one of embodiments 1 to 27, wherein the sample comprising B cell lymphocytes has been depleted of cell types other than B cell lymphocytes.

29. The method of any one of embodiments 1 to 28, wherein the sample comprising B cell lymphocytes has been depleted of B cell lymphocytes expressing IgM antibodies, IgA antibodies, IgD antibodies, or any combination thereof.

30. The method of any one of embodiments 1 to 19 and 21 to 29, wherein the sample comprising B cell lymphocytes is enriched for B cell lymphocytes expressing CD 27.

31. The method of any one of embodiments 1 to 20 and 22 to 29, wherein the sample comprising B cell lymphocytes is enriched for CD138 expressing B cell lymphocytes.

32. The method of any one of embodiments 1 to 31, wherein the sample comprising B cell lymphocytes has been contacted with a dnase prior to introduction into the microfluidic device.

33. The method of any one of embodiments 1 to 32, wherein single B cell lymphocytes are loaded into the separation region.

34. The method of any one of embodiments 1 to 32, wherein a plurality of B cell lymphocytes are loaded into the isolation region.

35. The method of any one of embodiments 1 to 34, further comprising: contacting the B cell lymphocyte with a stimulating agent that stimulates B cell activation.

36. The method of embodiment 35, wherein the stimulating agent comprises a CD40 agonist.

37. The method of embodiment 36, wherein the CD40 agonist comprises CD40L, a derivative thereof, or an anti-CD 40 antibody, and optionally wherein the CD40 agonist is attached to a micro-object (e.g., a bead).

38. The method of embodiment 35, wherein the stimulating agent comprises one or more CD40L⁺Feeder cells (e.g., irradiated T cells) or derivatives thereof.

39. The method of any one of embodiments 35 to 38, wherein the stimulating agent further comprises a B Cell Receptor (BCR) -linked molecule.

40. The method of embodiment 39, wherein the BCR-linked molecule comprises protein A or protein G.

41. The method of embodiment 39 or 40, wherein the BCR-linked molecule is linked to a micro-object (e.g., a bead).

42. The method of embodiment 35, wherein contacting the B cell lymphocyte with a stimulating agent comprises contacting the B cell lymphocyte with CD40L ⁺Feeder cells are contacted with a mixture of protein a conjugated to beads (e.g., in a ratio of about 1:1 to about 1: 10).

43. The method of embodiment 42, wherein contacting the B cell lymphocyte with the stimulating agent comprises loading the mixture into a separate region of the sequestration dock (e.g., using gravity or DEP force).

44. The method of any one of embodiments 35 to 43, wherein the stimulating agent further comprises a toll-like receptor (TLR) agonist.

45. The method of embodiment 44, wherein the TLR agonist is a CpG oligonucleotide.

46. The method of any one of embodiments 35 to 45, wherein the B cell lymphocytes are contacted with the stimulating agent for a period of 1 to 10 days (in some embodiments, the contact time may be 3 to 5 days).

47. The method of embodiment 46, wherein the B cell lymphocytes are contacted with the stimulating agent substantially continuously for a period of 1 to 10 days (in some embodiments, the contact time may be 3 to 5 days).

48. The method of any one of embodiments 35 to 47, further comprising:

providing a culture medium to the B cell lymphocytes, wherein the culture medium comprises one or more agents that promote B cell expansion and/or activation.

49. The method of embodiment 48, wherein said medium comprises at least one agent selected from the group consisting of IL-2, IL-4, IL-6, IL-10, IL-21, BAFF, and April.

50. The method of embodiment 48 or 49, wherein said culture medium comprises a TLR agonist.

51. The method of any one of embodiments 48 to 50, wherein the B cell lymphocytes are provided with culture medium for a period of 1 to 10 days (in some embodiments, the contact time may be 3 to 5 days).

52. The method of any one of embodiments 48 through 50, wherein contacting with the stimulating agent and providing the culture medium are performed over a substantially coextensive period.

53. The method of any one of embodiments 35 to 52, wherein the B cell lymphocytes are contacted with the stimulating agent prior to introducing the B cell lymphocytes into the microfluidic device.

54. The method of any one of embodiments 3f to 53, wherein contacting the B-cell lymphocytes with the stimulating agent occurs after introducing the B-cell lymphocytes into the microfluidic device (e.g., after loading the B-cell lymphocytes into the isolation region of the isolation dock).

55. The method of any one of embodiments 35 to 54, wherein the B-cell lymphocytes are contacted with the stimulating agent during the monitoring step.

56. The method of embodiment 6, wherein loading the B-cell lymphocytes into the separation region of the isolation dock comprises moving the B-cell lymphocytes into the separation region using DEP forces.

57. The method of embodiment 56, wherein said B cell lymphocytes move from said flow region to said isolation region.

58. The method of any one of embodiments 1-57, wherein providing an antigen of interest comprises flowing a solution comprising a soluble antigen of interest into or through the flow region.

59. The method of embodiment 58, wherein the antigen of interest is covalently bound to a first detectable label (e.g., a fluorescent label).

60. The method of embodiment 58 or 59, further comprising providing a micro-object comprising a first antibody binding agent, wherein the first antibody binding agent binds to an antibody expressed by the B-cell lymphocytes without inhibiting binding of a target antigen to the antibody expressed by the B-cell lymphocytes, and wherein monitoring binding of a target antigen to the antibody expressed by the B-cell lymphocytes comprises detecting indirect binding of a target antigen to the micro-object.

61. The method of embodiment 60, wherein the first antibody binding agent binds to the Fc domain of an antibody expressed by the B cell lymphocyte.

62. The method of embodiment 60 or 61, wherein the micro objects are beads.

63. The method of any of embodiments 60-62, wherein providing micro-objects comprises flowing a solution comprising micro-objects into a flow region and stopping flow when the micro-objects are in proximity to the isolation dock.

64. The method of embodiment 63, wherein providing the micro-objects further comprises loading the micro-objects into the isolation dock.

65. The method of embodiment 63 or 64, wherein the solution comprising the micro-objects and the solution comprising the soluble target antigen are the same solution.

66. The method of embodiment 63, wherein the solution comprising the micro-objects and the solution comprising the soluble antigen of interest are different solutions, and wherein the micro-objects are provided before the antigen of interest is provided.

67. The method of any one of embodiments 60 to 65, further comprising: providing a second antibody binding agent, wherein the second antibody binding agent comprises a second detectable label (e.g., a fluorescent label); and monitoring the indirect binding of the second antibody binding agent to the micro-object.

68. The method of embodiment 67 wherein the second antibody binding agent binds (which may optionally specifically bind) an IgG antibody (e.g., an anti-IgG secondary antibody).

69. The method of embodiment 67 or 68, wherein the first detectable label is different from (and can be differentially detected by) the second detectable label.

70. The method of any one of embodiments 67 to 69 wherein providing the second antibody binding agent comprises flowing a solution comprising a soluble second antibody binding agent into or through a flow region.

71. The method of embodiment 70, wherein the solution comprising a soluble secondary antibody binding agent and the solution comprising a soluble target antigen are the same solution.

72. The method of embodiment 70, wherein the solution comprising a soluble secondary antibody binding agent and the solution comprising a soluble target antigen are different solutions (e.g., provided sequentially).

73. The method of any one of embodiments 1 through 57, wherein providing the antigen of interest comprises providing a micro-object comprising the antigen of interest, wherein the micro-object is a cell, a liposome, a lipid nanoraft, or a bead.

74. The method of embodiment 73, further comprising: providing a labeled antibody binding agent prior to or concurrently with the antigen of interest, wherein monitoring binding of the antigen of interest to the antibody expressed by the B cell lymphocytes comprises detecting indirect binding of the labeled antibody binding agent to the antigen of interest.

75. The method of embodiment 74, wherein the labeled antibody binding agent binds (which may optionally specifically bind) an anti-IgG antibody (e.g., is an anti-IgG secondary antibody).

76. The method of embodiment 74 or 75 wherein the labeled antibody binding agent is covalently bound to a fluorescent label.

77. The method of any one of embodiments 74 to 76 wherein the labeled antibody binding agent is provided in a mixture with the antigen of interest.

78. The method of any one of embodiments 74 to 76 wherein a labeled antibody binding agent is provided after the antigen of interest is provided.

79. The method of any one of embodiments 1 to 78, wherein monitoring binding of an antigen of interest to an antibody expressed by the B cell lymphocyte comprises imaging all or a portion of a sequestration dock of the microfluidic device.

80. The method of embodiment 79, wherein the imaging comprises fluorescence imaging.

81. The method of embodiment 79 or 80, wherein said imaging comprises taking a plurality of images.

82. The method of embodiment 81, wherein the plurality of images are taken at fixed time intervals.

83. The method of any of embodiments 1-82, wherein the microfluidic device comprises a plurality of isolated docks, each isolated dock having a separation region and a connection region, each connection region providing a fluidic connection between the separation region and a flow region, the method further comprising: loading one or more of the plurality of B-cell lymphocytes into a separate region of each of two or more of the plurality of isolation docks; introducing an antigen of interest into the microfluidic device such that the antigen of interest is proximate to each of the two or more isolated docks loaded with one or more B cell lymphocytes; and monitoring binding of the antigen of interest to the antibody expressed by each loaded B cell lymphocyte.

84. The method of embodiment 83, wherein a single B-cell lymphocyte is loaded into the isolated region of each of two or more of the plurality of isolation docks.

85. The method of any one of embodiments 1 to 84, further comprising: detecting binding of the antigen of interest to antibodies expressed by the loaded B cell lymphocyte or several of the loaded B cell lymphocytes; identifying a loaded B cell lymphocyte or several of a plurality of loaded B cell lymphocytes expressing an antibody that specifically binds to an antigen of interest.

86. A method of characterizing an antibody that specifically binds to an antigen of interest, the method comprising: identifying a B cell lymphocyte, or clonal population thereof, that expresses an antibody that specifically binds to an antigen of interest, wherein the identifying is according to the method of embodiment 85; isolating the variable region encoding the immunoglobulin heavy chain (V) from said B cell lymphocytes or clonal populations thereof_H) And/or immunoglobulin light chain variable region (V)_L) The nucleic acid of (1); and encoding immunoglobulin heavy chain variable region (V)_H) At least a portion of the nucleic acid of (a) and/or encoding an immunoglobulin light chain variable region (V)_L) Sequencing at least a portion of the nucleic acid of (a).

87. The method of embodiment 86, wherein the immunoglobulin heavy chain variable region (V) is _H) Performing sequencing comprises: lysing the identified B cell lymphocytes or the B cell lymphocytes thereof(ii) a clonal population of B-cell lymphocytes; reverse transcribing mRNA isolated from B cell lymphocytes of said B cell lymphocytes or clonal populations thereof, wherein mRNA encodes an immunoglobulin heavy chain variable region (V)_H) Thereby forming V_HcDNA; and to V_HAt least a portion of the cDNA is sequenced.

88. The method of embodiment 86 or 87, wherein the immunoglobulin light chain variable region (V) is_L) Performing sequencing comprises: lysing the identified B cell lymphocytes or B cell lymphocytes of a clonal population thereof; reverse transcribing mRNA isolated from said B cell lymphocyte or clonal population thereof, wherein the mRNA encodes an immunoglobulin light chain variable region (V)_L) Thereby forming V_LcDNA; and to V_LAt least a portion of the cDNA is sequenced.

89. The method of embodiment 87 or 88, wherein reverse transcribing the mRNA comprises contacting the mRNA with a capture/priming oligonucleotide.

90. The method of embodiment 89, wherein said reverse transcription is performed in the presence of a transcript conversion oligonucleotide.

91. The method of any one of embodiments 87 to 90, wherein the identified B-cell lymphocytes or B-cell lymphocytes of the clonal population thereof are exported from the microfluidic device prior to lysis.

92. The method of embodiment 91, wherein outputting the identified B-cell lymphocytes or clonal populations thereof comprises: moving the identified B-cell lymphocytes or B-cell lymphocytes of the clonal population thereof from the isolation region of the sequestration dock into a flow region of the microfluidic device; and flowing the identified B cell lymphocytes, or B cell lymphocytes of a clonal population thereof, through the flow region and out of the microfluidic device.

93. The method of embodiment 92, wherein mobilizing the identified B-cell lymphocytes or the clonal population of B-cell lymphocytes from the isolated region of the sequestration dock comprises capturing and mobilizing the identified B-cell lymphocytes or the clonal population thereof using DEP forces.

94. The method of any one of embodiments 91 to 93, wherein the identified B-cell lymphocytes are exported as a single cell.

95. The method of any one of embodiments 91 to 93, wherein the identified B cell lymphocytes of the clonal population are exported as a panel.

96. The method of embodiment 87 or 88, wherein the identified B cell lymphocytes or B cell lymphocytes of the clonal population thereof are lysed within the microfluidic device.

97. The method of embodiment 96, further comprising: providing one or more capture beads in close proximity to the B cell lymphocytes of the identified B cell lymphocytes or clonal population thereof, wherein each of the one or more capture beads comprises a bead capable of binding V _HmRNA and/or V_LAn oligonucleotide of mRNA; lysing the identified B cell lymphocytes or clonal populations thereof; and allowing the V from lysed B cell lymphocytes or lysed B cell lymphocytes from a clonal population thereof_HmRNA and/or V_LThe mRNA is bound by one or more capture beads.

98. The method of embodiment 94, wherein each capture bead of the one or more capture beads comprises a plurality of capture/priming oligonucleotides.

99. The method of embodiment 97 or 98, wherein the one or more capture beads are provided prior to lysing the B cell lymphocytes of the identified B cell lymphocytes or clonal population thereof.

100. The method of any one of embodiments 97-99, wherein each of the one or more capture beads is loaded into a separate dock comprising the identified B-cell lymphocytes or the B-cell lymphocytes of the clonal population thereof.

101. The method of any one of embodiments 97 to 100, further comprising: moving the one or more capture beads to a substantially RNA-free region of the microfluidic device.

102. The method of embodiment 101, wherein the substantially RNA-free region does not comprise any B cell lymphocytes prior to moving the one or more capture beads to the substantially RNA-free region of the microfluidic device.

103. The method of embodiment 101 or 102, wherein the substantially RNA-free region is located within a separate dock from the separate dock in which the recognized B cell lymphocytes are loaded.

104. The method of any one of embodiments 97 to 103 wherein bound V_HmRNA and/or bound V_LReverse transcription of mRNA into V upon binding to one or more capture beads_HcDNA and/or V_L cDNA。

105. The method of embodiment 104 wherein bound V_HmRNA and/or bound V_LmRNA is reverse transcribed into V when one or more capture beads are contained within the microfluidic device (e.g., within a sequestration dock)_HcDNA and/or V_LcDNA。

106. The method of embodiment 104 or 105, wherein the bound V is bound by flowing a reverse transcriptase, nucleotides and appropriate buffers into or through a flow region of a microfluidic device_HAnd/or bound V_LReverse transcription of mRNA into V_HcDNA and/or V_L cDNA。

107. The method of any one of embodiments 104 to 106, wherein V_HcDNA and/or V_LThe cDNA is exported from the microfluidic device while bound to one or more capture beads.

108. The method of any one of embodiments 97 to 104, further comprising: in the process of mixing V_HmRNA and/or V_LReverse transcription of mRNA into V_HcDNA and/or V_LPrior to cDNA, one or more capture beads are output from the microfluidic device.

109. The method of embodiment 87 or 88, further comprising amplifying V prior to sequencing_HcDNA and/or V_L cDNA。

110. The method of embodiment 109, wherein amplifying comprises PCR amplification.

111. The method of embodiment 109 or 110, wherein amplifying comprises increasing V in reverse transcribed mRNA isolated from a B cell lymphocyte_HcDNA and/or V_LExpression of cDNA or fragments thereof.

112. The method of embodiment 111, wherein said amplifying comprises: first round of expansion, which increases isolation from B cell lymphocytesReverse transcription of mRNA V_HcDNA and/or V_LExpression of cDNA or fragments thereof; and a second round of amplification which introduces barcode sequences into the V amplified in the first round_HcDNA and/or V_LcDNA or a fragment thereof.

113. The method of embodiment 87 or 88, wherein V_HcDNA and/or V_LThe cDNA was sequenced without prior PCR amplification.

Enumeration of alternative paragraphs:

1. a method of detecting B cells expressing an antibody that specifically binds to an antigen of interest, the method comprising:

introducing a sample comprising memory B cells into a microfluidic device, wherein the microfluidic device comprises:

a housing having a flow region and a isolation dock, wherein the isolation dock comprises a separation region having a single opening and a connection region providing a fluidic connection between the separation region and the flow region, and wherein the separation region of the isolation dock is an unswept region of the microfluidic device;

Loading a single memory B cell from the sample to the isolation dock;

providing said memory B cells with a medium comprising IL-6 and BAFF;

introducing the antigen of interest into the flow region of the housing such that the antigen of interest is in proximity to the memory B cells; and also,

monitoring binding of the antigen of interest to the antibody expressed by the memory B cells, thereby detecting antibody-expressing memory B cells of interest.

2. The method of paragraph 1, further comprising lysing the memory B cells of interest, thereby releasing RNA of sufficient quality to prepare a cDNA library.

3. The method of paragraph 2, wherein lysing the target memory B cells comprises outputting the target memory B cells from the microfluidic device prior to lysing.

4. The method of paragraph 1, wherein the isolated region of the isolation dock comprises at least one conditioned surface, and wherein the at least one conditioned surface comprises a covalently attached layer of hydrophilic molecules; and further wherein the hydrophilic molecule comprises a polyethylene glycol (PEG) -containing polymer.

5. The method of paragraph 4, wherein the hydrophilic molecule further comprises a carbohydrate group-containing polymer, an amino acid-containing polymer, and combinations thereof.

6. The method of paragraph 1, wherein the sample comprising memory B cells is a sample of peripheral blood, spleen biopsy, bone marrow biopsy, lymph node biopsy, or tumor biopsy.

7. The method of paragraph 1, wherein the sample comprising memory B cells is obtained from a human, mouse, rat, guinea pig, gerbil, hamster, rabbit, goat, sheep, llama, chicken, ferret, pig, horse, cow, or turkey.

8. The method of paragraph 1, wherein the sample comprising memory B cells is obtained from a mammal and the mammal has been immunized against the antigen of interest, wherein the mammal has been exposed to or immunized against a pathogen associated with the antigen of interest, wherein the mammal has a cancer and the cancer is associated with the antigen of interest, or wherein the mammal has an autoimmune disease and the autoimmune disease is associated with the antigen of interest.

9. The method of paragraph 1, wherein the sample comprising memory B cells has been contacted with dnase prior to introduction into the microfluidic device.

10. The method of paragraph 1, wherein the memory B cells are provided with culture medium for a period of 3 to 5 days.

11. The method of paragraph 1, further comprising: contacting the memory B-cells with a stimulating agent that stimulates B-cell activation.

12. The method of paragraph 11, wherein the stimulating agent comprises a CD40 agonist, a B Cell Receptor (BCR) -linked molecule, a toll-like receptor (TLR) agonist, or a combination thereof.

13. The method of paragraph 1, wherein introducing the antigen of interest comprises flowing a solution comprising a soluble antigen of interest into or through the flow region, wherein the antigen of interest is covalently bound to a first detectable label.

14. The method of paragraph 13, further comprising providing a micro-object comprising a first antibody binding agent, wherein the first antibody binding agent binds the antibody expressed by the memory B cells without inhibiting binding of the target antigen to the antibody expressed by the memory B cells, and wherein monitoring binding of the target antigen to the antibody expressed by the memory B cells comprises detecting indirect binding of the target antigen to the micro-object.

15. The method of paragraph 14, wherein the first antibody binding agent binds to the Fc domain of the antibody expressed by the memory B cells.

16. The method of paragraph 14, wherein providing the micro-objects comprises flowing a solution containing the micro-objects into the flow region and stopping flow while the micro-objects are in proximity to the isolation dock.

17. The method of paragraph 14, wherein the solution comprising the micro-objects and the solution comprising the soluble target antigen are the same solution.

18. The method of paragraph 14, further comprising: providing a second antibody binding agent, wherein the second antibody binding agent comprises a second detectable label; and monitoring indirect binding of the second antibody binding agent to the micro-object, wherein the first detectable label is different from the second detectable label.

19. The method of paragraph 18 wherein the second antibody binding agent binds to an IgG antibody.

20. The method of paragraph 1, wherein providing the antigen of interest comprises providing a micro-object comprising the antigen of interest, wherein the micro-object is a cell, a liposome, a lipid nanoraft, or a bead; and are

Providing a labeled antibody binding agent prior to or concurrently with the antigen of interest, wherein the monitoring of binding of the antigen of interest to the antibody expressed by the memory B cells comprises detecting indirect binding of the labeled antibody binding agent to the antigen of interest.

21. The method of paragraph 20, wherein the labeled antibody binding agent binds to an anti-IgG antibody.

22. The method of paragraph 1, wherein monitoring binding of the target antigen to the antibody expressed by the memory B cells comprises imaging all or a portion of the sequestration dock of the microfluidic device.

23. The method of paragraph 22 wherein said imaging comprises fluorescence imaging.

24. A method as paragraph 22 recites, wherein the imaging comprises taking a plurality of images.

25. The method of paragraph 1, wherein the microfluidic device comprises a plurality of the isolation docks, each isolation dock having a separation region and a connection region, each connection region providing a fluidic connection between the separation region and the flow region, the method further comprising:

loading one or more of the plurality of memory B cells into the separation region of each of two or more of the plurality of isolated docks; introducing the antigen of interest into the microfluidic device such that the antigen of interest is proximate to each of the two or more isolated docks loaded with one or more memory B cells; and is

Monitoring binding of said antigen of interest to said antibody expressed by each of said loaded memory B cells.

26. The method of paragraph 25, further comprising: detecting binding of the antigen of interest to the antibody expressed by the loaded memory B cell or by several of the plurality of loaded memory B cells; identifying the several of the loaded memory B cells or the plurality of loaded memory B cells that express an antibody that specifically binds the antigen of interest.

27. The method of paragraph 1, wherein lysing the target memory B cells comprises outputting the target memory B cells from the microfluidic device prior to lysing.

28. The method of any of paragraphs 1 to 27, wherein said housing of said microfluidic device further comprises a Dielectrophoresis (DEP) configuration.

29. The method of paragraph 28, wherein loading the memory B cells into the separation region of the isolated dock comprises moving the memory B cells into the separation region using DEP forces.

30. A method of detecting B cells expressing an antibody that specifically binds to an antigen of interest, the method comprising:

introducing a sample comprising B cell lymphocytes into a microfluidic device, wherein the microfluidic device comprises:

A housing having a flow region and a isolating dock, wherein the isolating dock includes a splitting region having a single opening and a connecting region providing a fluid connection between the splitting region and the flow region;

wherein the separation region of the separation dock comprises at least one conditioned surface comprising a layer of covalently attached hydrophilic molecules, and further wherein the hydrophilic molecules comprise a polyethylene glycol (PEG) -containing polymer;

loading single B-cell lymphocytes from the sample into the isolation region of the isolation dock;

introducing the antigen of interest into the flow region of the housing such that the antigen of interest is in proximity to the B cell lymphocytes; and the number of the first and second electrodes,

monitoring binding of said antigen of interest to said antibody expressed by said B cell lymphocytes, wherein said isolated region of said spacer dock comprises at least one conditioned surface, thereby detecting antibody-expressing B cell lymphocytes of interest.

Numerous other modifications and alternative arrangements may be devised by those skilled in the art in addition to any previously indicated modifications without departing from the spirit and scope of the present specification, and the appended claims are intended to cover such modifications and arrangements. Thus, while information has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred aspects, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, form, function, manner of operation and use may be made without departing from the principles and concepts set forth herein. Furthermore, as used herein, the examples and embodiments are in all respects only illustrative and should not be construed as being limiting in any way. It should also be noted that although the term step is used herein, the term may be used to simply draw attention to different parts of the described method, and is not meant to delineate the beginning or stopping points of any part of the method, or be limited in any other way.

Informal SEQ ID NO. Listing

Sequence listing

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Claims (10)

1. A method of detecting B cells expressing an antibody that specifically binds to an antigen of interest, the method comprising:

Introducing a sample comprising memory B cells into a microfluidic device, wherein the microfluidic device comprises:

a housing having a flow region and a isolation dock, wherein the isolation dock comprises a separation region having a single opening and a connection region providing a fluidic connection between the separation region and the flow region, and wherein the separation region of the isolation dock is an unswept region of the microfluidic device;

loading a single memory B cell from the sample to the isolation dock;

providing said memory B cells with a medium comprising IL-6 and BAFF;

introducing the antigen of interest into the flow region of the housing such that the antigen of interest is in proximity to the memory B cells; and the number of the first and second electrodes,

monitoring binding of the target antigen to the antibody expressed by the memory B cells, thereby detecting antibody-expressing target memory B cells.

2. The method of claim 1, further comprising lysing the memory B cells of interest, thereby releasing RNA of sufficient quality to prepare a cDNA library.

3. The method of claim 2, wherein lysing the target memory B-cells comprises outputting the target memory B-cells from the microfluidic device prior to lysing.

4. The method of claim 1, wherein the isolated region of the isolation dock comprises at least one conditioned surface, and wherein the at least one conditioned surface comprises a layer of covalently linked hydrophilic molecules; and further wherein the hydrophilic molecule comprises a polyethylene glycol (PEG) -containing polymer.

5. The method of claim 4, wherein the hydrophilic molecule further comprises a polymer comprising a carbohydrate group, a polymer comprising an amino acid, and combinations thereof.

6. The method of claim 1, wherein the sample comprising memory B cells is a sample of peripheral blood, spleen biopsy, bone marrow biopsy, lymph node biopsy, or tumor biopsy.

7. The method of claim 1, wherein the sample comprising memory B cells is obtained from a human, mouse, rat, guinea pig, gerbil, hamster, rabbit, goat, sheep, llama, chicken, ferret, pig, horse, cow, or turkey.

8. The method of claim 1, wherein the sample comprising memory B cells is obtained from a mammal and the mammal has been immunized against the antigen of interest, wherein the mammal has been exposed to or immunized against a pathogen associated with the antigen of interest, wherein the mammal has a cancer and the cancer is associated with the antigen of interest, or wherein the mammal has an autoimmune disease and the autoimmune disease is associated with the antigen of interest.

9. The method of claim 1, wherein the sample comprising memory B cells has been contacted with dnase prior to introduction into the microfluidic device.

10. The method of claim 1, wherein the memory B cells are provided with culture medium for a period of 3 to 5 days.