HK1227098B - Micro-fluidic devices for assaying biological activity - Google Patents

Description

Microfluidic device for determining biological activity

Background

In the field of bioscience and related technology, it may be useful to measure the biological activity of micro-objects, such as cells. Some embodiments of the invention include devices and processes for determining biological activity in a holding pen of a microfluidic device.

Disclosure of Invention

In some embodiments, the present invention provides a process for determining biological activity in a microfluidic device. The biological activity may be indicative of production of a biological material of interest, such as by a biological cell. Thus, the process can include culturing one or more biological cells that produce the biological material of interest in a holding pen of the microfluidic device. The process can also include introducing one or more capture micro-objects into the holding pens and allowing the biological material of interest produced by the one or more biological cells to bind to the one or more capture micro-objects. The capture micro-objects may comprise, for example, a binding substance that specifically binds the biological material of interest. The process can also include evaluating the capture micro-objects for the bound biological material of interest.

In certain embodiments, after allowing the biological material of interest to bind to the one or more capture micro-objects, but before evaluating the capture micro-objects for bound biological material of interest, the one or more capture micro-objects are removed from the holding pens. Removing the one or more capture micro-objects may include moving the one or more capture micro-objects to an assay region located within the microfluidic device. In certain embodiments, the assay region is a stop (stop) located within a channel of the microfluidic device, or a chamber located within the microfluidic device, or the like. Regardless, the assay region can be adjacent to a holding pen from which one or more capture micro-objects are removed. Alternatively, or in addition, removing one or more capture micro-objects may comprise moving one or more capture micro-objects to a channel in the microfluidic device and then outputting one or more capture micro-objects from the microfluidic device.

In certain embodiments, removing one or more capture micro-objects includes forming an optical trap that traps at least one of the capture micro-objects when the optical trap is in the holding pen. The optical trap may include a light pattern projected onto an inner surface of the microfluidic device that encompasses at least one capture micro-object and an activation electrode, such as a Dielectrophoresis (DEP) electrode, within the microfluidic device. Moving the optical traps from the holding pen to the channel and/or assay region of the microfluidic device can cause the trapped capture micro-objects to move accordingly.

In certain embodiments, the one or more capture micro-objects are magnetic. In a related embodiment, removing the one or more capture micro-objects may include applying a magnetic field to the microfluidic device.

In certain embodiments, a capture micro-object that has been removed from a holding pen can remain associated with the holding pen. For example, a correlation may be maintained between a capture micro-object and a holding pen from which the capture micro-object has been removed. In this manner, when a microfluidic device contains multiple holding pens, data obtained from capture micro-objects that have been removed from their holding pens can be traced back to the appropriate holding pen.

In certain embodiments, evaluating the capture micro-objects is performed for the bound biological material of interest while the capture micro-objects are in the holding pens.

In certain embodiments, evaluating the capture micro-objects for bound biological material of interest may include determining the type of biological material of interest bound to the capture micro-objects. In certain embodiments, evaluating the capture micro-objects for bound biological material of interest may include determining the activity of the biological material of interest bound to the capture micro-objects. In certain embodiments, evaluating the capture micro-objects for bound biological material of interest may include determining an amount of biological material of interest bound to the capture micro-objects. Any such determination may include mixing (and/or binding) the assay material with the biological material of interest bound to the capture micro-objects and detecting a correlation between the capture micro-objects and the assay material. For example, if the assay material is capable of producing detectable radiation, the determining can include detecting a correlation between the capture micro-objects and the radiation from the assay material. The determining may also include washing unbound and/or unreacted assay material from the capture micro-objects prior to detecting a correlation between the micro-objects and radiation from the assay material. Alternatively, or in addition, the determining may further include determining whether the radiation associated with capturing the micro-objects corresponds to a predetermined characteristic. For example, the radiation may have a characteristic wavelength.

In certain embodiments, the biological material of interest is a protein, such as a therapeutic protein, an antibody, a growth factor, a cytokine, a cancer antigen, an infectious antigen associated with a virus or other pathogen, a secreted protein, or any other protein produced and/or released by a biological cell. In certain embodiments, the biological material of interest is a protein, nucleic acid, carbohydrate, lipid, hormone, metabolite, small molecule, polymer, or any combination thereof. In certain embodiments, the binding substance that captures the micro-objects has an affinity for the biological material of interest of at least 1 μ Μ, 100nM, 50nM, 25nM, 10nM, 5nM, 1nM or more.

In certain embodiments, a single biological cell is present in the holding pen. In other embodiments, two or more biological cells are present in the holding pen. In certain embodiments, the biological cells in the holding pen are clonal populations. In certain embodiments, a single capture micro-object is introduced into the holding pen. In other embodiments, two or more (e.g., a plurality of) capture micro-objects are introduced into the holding pens. In these latter embodiments, each capture micro-object of the plurality of capture micro-objects may have a binding substance that is different from the binding substance of other capture micro-objects of the plurality of capture micro-objects.

In certain embodiments, the biological material of interest is an antibody, such as a candidate therapeutic antibody. In a related embodiment, the process can include a plurality of capture micro-objects, each having a binding substance that binds to a different isotype of antibody. In other related embodiments, the process may include a plurality of capture micro-objects, each having a binding substance corresponding to a different epitope of the antigen recognized by the antibody. In yet other related embodiments, the process may include a plurality of capture micro-objects, one of which has a binding substance corresponding to an antigen recognized by the antibody or epitope thereof. The remaining capture micro-objects of the plurality of capture micro-objects may have a binding substance corresponding to a homolog of the antigen or epitope thereof. The homologous antigens or epitopes thereof may be from different species.

In some embodiments, the present invention provides a process for assaying the production of n different biological materials of interest in a microfluidic device. The process can include culturing one or more biological cells in a holding pen of a microfluidic device, wherein the one or more cells produce n different biological materials of interest. The process may further comprise introducing n different types of capture micro-objects into the holding pens, each type having a binding substance that specifically binds to one of the n different biological materials of interest, and allowing the n different biological materials of interest produced by the biological cells to bind to the n different types of capture micro-objects. The process may also include evaluating the n different types of capture micro-objects for the bound biological material of interest. In certain embodiments, such an assessment is positive if at least one of the n different biological materials of interest specifically binds to one of the n different types of capture micro-objects. In other embodiments, such an assessment is positive if at least two n different biological materials of interest each specifically bind to one of the n different types of capture micro-objects. In still other embodiments, such an assessment is positive if all n different biological materials of interest each specifically bind to one of the n different types of capture micro-objects.

In certain embodiments, n different types of capture micro-objects are introduced into the holding pens simultaneously. In other embodiments, n different types of capture micro-objects are introduced sequentially into the holding pens.

In some embodiments, a process for assaying the production of n different biological materials of interest in a microfluidic device comprises introducing one or more y-material capture micro-objects into a holding pen, each y-material capture micro-object having y different binding substances, each of which specifically binds to one of the n different biological materials of interest produced by one or more biological cells. The process may further comprise allowing n different biological materials of interest produced by one or more biological cells to bind to the y-material capture micro-objects. Further, the process can include evaluating the y-material capture micro-objects for bound biological material of interest.

For any of the foregoing processes, the microfluidic device can comprise a plurality of holding pens, each of which contains one or more biological cells that can be assayed sequentially or in parallel.

In some embodiments, the present disclosure provides a microfluidic device. The microfluidic device can include an enclosure having a channel, a holding pen, and an assay region. The holding pen may include an isolation region and a connection region having a proximal opening to the channel and a distal opening to the isolation region. The assay region can be adjacent to a holding pen. For example, the assay region can include a stop located within the channel. The stop may extend directly through the channel from the proximal opening of the connection region or may extend directly through the channel just beyond the proximal opening of the connection region. Alternatively, the assay region may comprise an assay chamber. The assay chamber can be located alongside the holding pen or directly through a channel from the proximal opening of the connection region of the holding pen. In some embodiments, the assay chamber is substantially devoid of an isolation region (e.g., less than 50% of the volume of the assay chamber may be isolated from the bulk flow of the medium being flowed through the channel). In certain embodiments, the microfluidic device may further comprise a means for generating a magnetic force within the enclosure. Such means may be, for example, magnets.

Drawings

Fig. 1 is an example of a process for determining biological activity in a holding pen of a microfluidic device according to some embodiments of the invention.

Fig. 2A is a perspective view of a microfluidic device with which the process of fig. 1 may be performed, according to some embodiments of the invention.

Fig. 2B is a side cross-sectional view of the microfluidic device of fig. 2A.

Fig. 2C is a top cross-sectional view of the microfluidic device of fig. 2A.

Fig. 3A is a partial cross-sectional side view (for ease of illustration) of the microfluidic device of fig. 2A-2C lacking barriers and stops, wherein the selector is configured as a Dielectrophoresis (DEP) device, according to some embodiments of the invention.

Fig. 3B is a partial top cross-sectional view of fig. 3A.

Fig. 4 is an example of a process according to some embodiments of the invention in which the biological activity of cells in a holding pen can be determined.

Fig. 5A illustrates an example of the culturing step of fig. 4 according to some embodiments of the invention.

Fig. 5B shows an example of the culturing step of fig. 4, wherein biological cells are cultured in a holding pen having an isolation region and a connecting region.

Fig. 6 illustrates an example of the moving step of fig. 4 according to some embodiments of the invention.

Fig. 7A illustrates another example of the moving step of fig. 4 according to some embodiments of the invention.

Fig. 7B illustrates a variation of the microfluidic device of fig. 7A, wherein deflectors are used to direct capture objects into holding pens as they flow through channels adjacent to the holding pens.

Fig. 8 and 9 illustrate examples of the steps of continuing the culture in fig. 4 according to some embodiments of the invention.

Fig. 10 illustrates an example of the removal step of fig. 4, according to some embodiments of the invention.

Fig. 11A illustrates another example of the removal step of fig. 4 according to some embodiments of the invention.

Fig. 11B shows a variation of the removal step of fig. 4, wherein the capture objects are removed from the holding pens containing the biological cells and placed in the assay pens.

Fig. 11C shows another variation of the removal step of fig. 4, wherein the capture objects are removed from the holding pens containing the biological cells and placed in the assay pens.

Fig. 12-14 illustrate examples of the evaluation steps of fig. 4 according to some embodiments of the invention.

Fig. 15 illustrates an example of a process for testing biological activity in a holding pen in a microfluidic device for a first number (n) of features and a second number (m) of features according to some embodiments of the invention.

Fig. 16 is an example of a process for testing n and/or m features for the process of fig. 15, according to some embodiments of the invention.

Fig. 17 is another example of a process for testing n and/or m features for the process of fig. 15, according to some embodiments of the invention.

Fig. 18 illustrates an example of x capture objects each configured to bind a different material of interest to move in series or in parallel into a holding pen, according to some embodiments of the invention.

Fig. 19 illustrates an example of moving capture objects configured to bind a plurality of y different materials of interest into a holding pen, according to some embodiments of the invention.

Fig. 20A to 20C show examples of holding pens having an area for culturing biological cells and a separate area for placing capture micro-objects.

Detailed Description

This specification describes exemplary embodiments and applications of the invention. However, the invention is not limited to these exemplary embodiments and applications or 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 not be exaggerated or minimized to scale for clarity. Further, when the terms "on …," "attached to," or "coupled to" are used herein, one element (e.g., material, layer, substrate, etc.) may be "on," "attached to," or "coupled to" another element, whether the one element is directly on, attached or coupled to the other element, or whether there are one or more intervening elements between the one element and the other element. Further, 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, for ease of illustration and discussion, and not by way of limitation. Further, where a series of elements (e.g., elements a, b, c) are referenced, such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or combinations of all of the listed elements.

As used herein, "substantially" means sufficient to achieve the intended purpose. The term "substantially" thus allows for minor, unimportant variations such as would be expected by one of ordinary skill in the art, but which have no significant impact on overall performance, in terms of absolute or perfect states, dimensions, measurements, results, and the like. The term "substantially" when used with respect to a numerical value or a parameter or feature that may be represented as a numerical value means within ten percent. The term "plurality" means more than one.

As used herein, the terms "capture target" and "capture micro-target" are used interchangeably and may include one or more of the following: inanimate micro-objects such as microparticles, microbeads (e.g., polystyrene beads, Luminex)^TMBeads, etc.), magnetic beads, nanorods, microwires, quantum dots, etc.; biological micro-objects such as cells (e.g., cells obtained from tissue or bodily fluid samples, blood cells, hybrid cells, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, etc.), liposomes (e.g., synthetic or derived from membrane preparations), nano-lipid rafts, and the like; or inanimate micro-meshA combination of target and biological micro-objects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). For example, in "Ritchie et al (2009) Regulation of Membrane Proteins in phospholipid membranes Nanodiscs, Mehotd enzymol.,464:211-231 (Ricke et al (2009), recombination of Membrane Proteins in phospholipid Bilayer nano-discs, methodological, 464: 211-231)", a description has been given of nanolipid rafts.

As used herein, the terms "specific binding" and "specific binding" refer to an interaction between a ligand and a receptor, wherein a particular surface of the ligand binds to a particular surface of the receptor, such that ionic bonds, hydrogen bonds, and/or van der waals forces cause the ligand and receptor to bind together in a particular configuration. The ligand may be a biological material of interest, such as a protein (e.g., a therapeutic protein, an antibody, a growth factor, a cytokine, a cancer antigen, an infectious antigen associated with a virus or other pathogen, a secreted protein, or any other protein produced and/or released by a biological cell), a nucleic acid, a carbohydrate, a lipid, a hormone, a metabolite, or any combination thereof. The receptor may be a binding substance, for example, a biological or chemical molecule, such as a protein (e.g., a therapeutic protein, an antibody, a growth factor, a cytokine, a cancer antigen, an infectious antigen associated with a virus or other pathogen, a secreted protein, or any other protein produced and/or released by a biological cell), a nucleic acid, a carbohydrate, a lipid, a hormone, a metabolite, a small molecule, a polymer, or any combination thereof. Specific binding of ligand to receptor is associated with quantifiable affinity. Affinity can be expressed, for example, as the dissociation constant Kd.

As used herein with respect to liquids, the term "flow" refers to the bulk movement of the liquid caused by any mechanism other than diffusion. For example, the flow of the medium may include movement of the fluid medium from one point to another due to a pressure difference between the points. Such a flow may comprise a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the media may result.

The phrase "substantially no flow" refers to a flow rate of a liquid that is less than the rate at which components of a material (e.g., an analyte of interest) diffuse into or within the liquid. 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 a fluid medium, "diffusing …" and "diffusion" refer to the thermodynamic movement of a component of the fluid medium in a direction of low concentration gradient.

As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" refers to the fluids in each of the regions being connected to form a single body of fluid when the different regions are substantially filled with a liquid (such as a fluidic medium). This does not mean that the fluids (or fluid media) in the different regions must be identical in composition. In contrast, fluids in different fluidly connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) that are constantly changing due to the movement of solutes in the direction of their respective concentration gradients that are low and/or fluid flow through the device.

The microfluidic devices or apparatus of the present invention may include "swept" regions and "unswept" regions. The unswept region may be fluidly connected to the swept region, provided that the fluid connection is configured to enable diffusion between the swept and unswept regions, but substantially no flow of media between the swept and unswept regions. The microfluidic device may thus be configured to substantially isolate the unswept region from the flow of the medium in the swept region, while only enabling diffusive fluid communication between the swept and unswept regions.

A population of biological cells is "clonal" if all living cells in the population that are capable of regeneration are daughter cells from a single mother cell. The term "clonal cells" refers to cells of the same clonal population.

In some embodiments of the invention, biological activity in a holding pen in a microfluidic device can be determined by placing capture objects bound to a particular material of interest resulting from the biological activity in the holding pen. The material of interest bound to each capture object can then be evaluated in the microfluidic device. Embodiments of the invention can thus be effective to determine biological activity occurring in a holding pen in a microfluidic device. Further, where biological activity includes cloning populations of cells, each producing a particular biological material of interest in one of the holding pens, some embodiments of the invention can assess the ability of each population to produce the material of interest in a microfluidic device while keeping each population cloned (e.g., without mixing cells that can be regenerated from either population with any other population).

Fig. 1 shows an example of an assay process 100. Fig. 2A-2C illustrate an example of a microfluidic device 200 for performing the process 100, and fig. 3A and 3B illustrate an example of a Dielectrophoresis (DEP) device that may be part of the microfluidic device 200.

As shown in fig. 1, at step 102, the process 100 can move capture objects into holding pens in a microfluidic device, and at step 104, the process 100 can incubate biological activities that produce a particular biological material of interest in each of the holding pens. The holding pens can include unswept regions, and the biological activity can be located or placed in such unswept regions. The biological activity can be part of or consist of one or more cells, such as one, oocytes, sperm, cells isolated from tissue, blood cells (e.g., B cells, T cells, macrophages, etc.), hybrid cells, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. The capture objects may include one or more binding substances, each of which specifically binds to a particular biological material of interest. For example, a binding substance that captures a target can have an affinity (e.g., Kd) for a particular biological material of interest of at least about 1mM or more (e.g., about 100 μ M, 10 μ M, 1 μ M, 500nM, 400nM, 300nM, 200nM, 100nM, 75nM, 50nM, 25nM, 15nM, 10nM, 5nM, 2.5nM, 1nM or more). Such affinity can be two, three, four, five, ten, or more times greater than, for example, the affinity for any material other than the particular biological material of interest (or at least any other biological material of interest present in the holding pens and/or microfluidic devices). Thus, it can be said that each capture object binds one or more particular biomaterials of interest, but does not substantially bind other biomaterials in the holding pens. After a period of time, capture objects can be removed from the holding pens at step 106, and correlations between the removed capture objects and the pens from which each removed capture object was taken can be maintained at step 108. At step 110, the biological activity in each holding pen can be assessed by analyzing the biological material bound to the capture objects removed from the holding pen. For example, at step 110, the process 100 can evaluate the biological activity in each holding pen by determining the amount of biological material bound by the capture objects removed from the holding pen. The evaluation can include, for example, determining whether the population in each holding pen produces the material of interest at or above a threshold rate. As another example, the evaluation may quantify the amount of material of interest produced by the population in each holding pen.

Fig. 1 is an example, and many variations of process 100 are contemplated. For example, process 100 can evaluate biological activity at step 110 while the capture object is in the holding pen, and in some variations, process 100 need not therefore include steps 106, 108 or steps 106 and 108 can be skipped. As another example, steps 102 through 110 need not be performed in the order shown in FIG. 1. For example, steps 102 and 104 may be reversed.

Fig. 2A-2C illustrate an example of a microfluidic device 200 on which process 100 may be performed. As shown, the microfluidic device 200 may include a housing 202, a selector 222, a detector 224, a flow controller 226, and a control module 230.

As shown, the housing 202 may include one or more flow regions 240 for holding a liquid medium 244. Fig. 2B shows an inner surface 242 of the flow region 240 on which the medium 244 may be disposed as uniform (e.g., flat) and featureless. However, alternatively, the inner surface 242 may be non-uniform (e.g., non-planar) and include features such as electrode terminals (not shown).

The housing 202 may include one or more inlets 208, and the medium 244 may be input into the flow region 240 through the one or more inlets 208. The inlet 208 may be, for example, an input port, an opening, a valve, another channel, a fluid connector, etc. The housing 202 may also include one or more outlets 210 through which the medium 244 may be removed through the one or more outlets 210. The outlet 210 may be, for example, an output port, an opening, a valve, a channel, a fluid connector, or the like. As another example, outlet 210 may include a droplet output mechanism such as any of the output mechanisms disclosed in U.S. patent application serial No. 13/856,781 (attorney docket No. BL1-US), filed on 4/2013. All or a portion of the housing 202 may be gas permeable to allow gas (e.g., ambient air) to enter and exit the flow region 240.

The housing 202 may also include a microfluidic structure 204 disposed on a base (e.g., substrate) 206. The microfluidic structure 204 may include a flexible material, such as rubber, plastic, elastomer, silicone (e.g., patternable silicone), polydimethylsiloxane ("PDMS"), etc., which may be gas permeable. Alternatively, the microfluidic structure 204 may comprise other materials including rigid materials. The base 206 may include one or more substrates. Although shown as a single structure, the base 206 may include a plurality of interconnected structures, such as a plurality of substrates. The microfluidic structure 204 may likewise comprise a plurality of structures that may be interconnected. For example, the microfluidic structure 204 may also include a cover (not shown) made of the same or different material as the other materials in the structure.

The microfluidic structure 204 and the base 206 may define a flow region 240. Although one flow region 240 is shown in fig. 2A-2C, the microfluidic structure 204 and the base 206 may define multiple flow regions for the medium 244. The flow region 240 may include channels (252 and 253 in fig. 2C) and chambers that may be interconnected to form a microfluidic circuit. For an enclosure including more than one flow region 240, each flow region 240 may be associated with one or more inlets 208 and one or more outlets 210 for respectively inputting and removing media 244 from the flow region 240.

As shown in fig. 2B and 2C, a holding pen 256 can be disposed in the flow region 240. For example, each retention pen 256 can include a barrier 254 that forms a partial enclosure. The partial enclosure may define a non-flow space (or isolation region). Thus, a portion of the interior of each holding pen 256 can be a non-flow space into which medium 244 from the channel 252 does not flow directly, except when the empty flow region 240 is initially filled with medium 244. For example, each holding pen 256 can include one or more barriers 254 that form part of an enclosure, the interior of which can include a non-flow space. When the flow region 240 is filled with the medium 244, the barriers 254 defining the retention pens 256 can thus prevent the medium 244 from flowing directly from the channel 252 into the protected interior of any retention pens 256. For example, when the flow region 240 is filled with the medium 244, the barrier 254 of the pens 256 can substantially prevent the entire flow of the medium 244 from the channel 252 from flowing into the non-flow spaces of the pens 256, and instead substantially only allow diffusive mixing of the medium in the non-flow spaces in the pens 256 with the medium from the channel 252. Thus, the exchange of nutrients and waste between the non-flow space in the holding pens 256 and the channel 252 can occur substantially only by diffusion.

The foregoing can be accomplished by orienting the pens 256 such that the openings into the pens 256 do not directly face the flow of the medium 244 in the channel 252. For example, if the flow of media is from the inlet 208 to the outlet 210 (and thus from left to right) in the channel 252 in fig. 2C, each of the pens 256 substantially prevents the media 244 from flowing directly from the channel 252 into the pen 256 because the opening of each pen 256 does not face the left in fig. 2C (which would otherwise directly enter such a flow).

There may be many such holding pens 256 in the flow region 240 arranged in any pattern, and the holding pens 256 may be any of many different sizes and shapes. As shown in fig. 2C, the opening of the holding pen 256 can be disposed adjacent to the channels 252, 253, which can be a space adjacent to the openings of more than one pen 256. The opening of each pen 256 can allow for natural exchange of liquid medium 244 flowing in the channels 252, 253, but the opening of each holding pen 256 can additionally be sufficiently enclosed to prevent micro-objects (not shown), such as biological cells, in any one pen 256 from mixing with micro-objects in any other pen 256. Although eight pens 256 and two channels 252, 253 are shown, there can be more or fewer. The medium 244 can flow in the channels 252, 253 through openings in the holding pens 256. The flow of medium 244 in the channels 252, 253 can, for example, provide nutrients to biological targets (not shown) in the holding pens 256. As another example, the flow of media 252, 253 in the common flow spaces 252, 253 can also provide for removal of waste from the holding pens 256.

As shown in fig. 2C, stops 258 may also be provided in the flow region 240, e.g., in the channels 252, 253. Each stop 258 may be configured to hold a micro-object (not shown) in place against the flow of media 244 in the channels 252, 253. The stops 258 and barriers 254 of pens 256 can comprise any of the types of materials discussed above with respect to microfluidic structure 204. The stop 258 and barrier 254 may comprise the same material as the microfluidic structure 204 or a different material. As shown in fig. 2B, the barrier 254 may extend from the surface 242 of the base 206 across the entire flow region 240 to an upper wall (opposite the surface 242) of the microfluidic structure 204. Alternatively, one or more barriers 254 may extend only partially through the flow region 240, and thus not extend completely to the surface 242 or the upper wall of the microfluidic structure 204. Although not shown, the stop 258 and/or the barrier 254 may include additional features, such as one or more relatively small openings through which the medium 244 may pass. Such openings (not shown) may be smaller than the micro-objects (not shown) to prevent the micro-objects from passing through.

The selector 222 may be configured to selectively create an electromotive force on micro-objects (not shown) in the medium 244. For example, the selector 222 may be configured to selectively activate (e.g., turn on) and deactivate (e.g., turn off) electrodes at the inner surface 242 of the flow region 240. The electrodes may create a force in the medium 244 that attracts or repels micro-objects (not shown) in the medium 244, and the selector 222 may thus select and move one or more micro-objects in the medium 244. The electrodes may be, for example, Dielectrophoresis (DEP) electrodes.

For example, the selector 222 may include one or more optical (e.g., laser) tweezer devices and/or one or more Optical Electrical Tweezer (OET) devices (e.g., as disclosed in U.S. patent No. 7,612,355, which is hereby incorporated by reference in its entirety, or as disclosed in U.S. patent application serial No. 14/051,004 (attorney docket No. BL9-US), the entire contents of which are also incorporated herein by reference, as yet another example, the selector 222 may include one or more devices (not shown) for moving droplets of a medium 244 in which one or more micro-objects are suspended, such devices (not shown) may include electrowetting devices, such as an opto-electrowetting (OEW) device (e.g., as disclosed in U.S. patent No. 6,958,132), or other electrowetting devices the selector 222 may thus be characterized in some embodiments as a DEP device.

Fig. 3A and 3B show an example in which the selector 222 comprises an OET DEP apparatus 300. As shown, DEP device 300 can include a first electrode 304, a second electrode 310, an electrode activation substrate 308, a power source 312 (e.g., an Alternating Current (AC) power source), and a light source 320. The medium 244 and the electrode activation substrate 308 in the flow region 240 may separate the electrodes 304, 310. Varying the pattern of light 322 from the light source 320 selectively activates and deactivates the pattern of DEP electrodes that is varied at the region 314 of the inner surface 242 of the flow region 240. (hereinafter, the region 314 is referred to as an "electrode region")

In the example shown in fig. 3B, light pattern 322' directed onto inner surface 242 illuminates cross-hatched electrode areas 314a in the square pattern shown. The other electrode region 314 is not illuminated and is therefore referred to hereinafter as the "dark" electrode region 314. The relative electrical impedance from each dark electrode region 314 through the electrode activation substrate 308 to the second electrode 310 is greater than the relative impedance from the first electrode 304 through the medium 244 in the flow region 240 to the dark electrode region 314. However, illuminating electrode region 314a reduces the relative impedance from illuminated electrode region 314a through electrode activation substrate 308 to second electrode 310, which is less than the relative impedance from first electrode 304 through medium 244 in flow region 240 to illuminated electrode region 314 a.

With the power source 312 activated, the foregoing creates an electric field gradient in the medium 244 between the illuminated electrode region 314a and the adjacent dark electrode region 314, which in turn creates a local DEP force that attracts or repels nearby micro-objects (not shown) in the medium 244. DEP electrodes that attract or repel micro-objects in the medium 244 can thus be selectively activated or deactivated at many different such electrode regions 314 of the inner surface 242 of the flow region 240 by varying the light pattern 322 projected from the light source 320 (e.g., a laser source, a high-intensity gas discharge lamp, or other type of light source) to the DEP apparatus 300. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of the power source 312 and the dielectric properties of the medium 244 and/or micro-objects (not shown).

The square pattern 322' of the illuminated electrode regions 314a shown in fig. 3B is merely an example. Any pattern of electrode regions 314 can be illuminated by a pattern of light 322 projected into the device 300, and the pattern of illuminated electrode regions 322' can be repeatedly changed by changing the light pattern 322.

In some embodiments, the electrode activation substrate 308 may be a photoconductive material, and the inner surface 242 may be featureless. In such embodiments, the DEP electrodes 314 may be formed anywhere and in any pattern on the inner surface 242 of the flow region 240 according to the light pattern 322 (see fig. 3A). The number and pattern of electrode areas 314 is therefore not fixed, but corresponds to the light pattern 322. Examples are illustrated in the aforementioned U.S. patent No. 7,612,355, wherein the undoped amorphous silicon material 24 shown in the figures of the aforementioned patent may be an example of a photoconductive material that may constitute the electrode activation substrate 308.

In other embodiments, the electrode activation substrate 308 may comprise a circuit substrate, such as a semiconductor material, including a plurality of doped layers, electrically insulating layers, and conductive layers that form a semiconductor integrated circuit, such as is known in the semiconductor art. In such embodiments, the circuit element may form an electrical connection between the electrode region 314 at the inner surface 242 of the flow region 240 and the second electrode 310 that may be selectively activated and deactivated by the light pattern 322. When inactive, each electrical connection may have a high impedance such that the relative impedance from the corresponding electrode region 314 to the second electrode 310 is greater than the relative impedance from the first electrode 204 through the medium 244 to the corresponding electrode region 314. However, when activated by light in the light pattern 322, each electrical connection may have a low impedance such that the relative impedance from the corresponding electrode region 314 to the second electrode 310 is less than the relative impedance from the first electrode 304 through the medium 244 to the corresponding electrode region 314, which activates the DEP electrode at the corresponding electrode region 314 as discussed above. DEP electrodes that attract or repel micro-objects (not shown) in the medium 244 can thus be selectively activated and deactivated at many different electrode regions 314 of the inner surface 242 of the flow region 240 by the light pattern 322. Non-limiting examples of such configurations of the electrode activation substrate 308 include the phototransistor-based OET device 300 shown in figures 21 and 22 of U.S. patent No. 7,956,339 and the OET device shown in all of the figures of the aforementioned U.S. patent application serial No. 14/051,004.

In some embodiments, the first electrode 304 may be part of the first wall 302 (or cover) of the housing 202, and the electrode activation substrate 308 and the second electrode 310 may be part of the second wall 306 (or base) of the housing 202, as generally shown in fig. 3A. As shown, the flow region 240 may be between the first wall 302 and the second wall 306. However, the foregoing are merely examples. In other embodiments, the first electrode 304 may be part of the second wall 306, while one or both of the electrode activation substrate 308 and/or the second electrode 310 may be part of the first wall 302. As another example, the first electrode 304 may be a portion of the same wall 302 or 306 as the electrode activation substrate 308 and the second electrode 310. For example, the electrode activation substrate 308 may include the first electrode 304 and/or the second electrode 310. Further, the light source 320 may alternatively be located below the housing 202.

Configured as the DEP device 300 shown in fig. 3A and 3B, the selector 222 can thus select micro-objects (not shown) in the medium 244 in the flow region 240 by projecting a light pattern 322 into the device 300 to activate one or more DEP electrodes at the electrode regions 314 of the inner surface 242 of the flow region 240 in a pattern that encompasses and captures the micro-objects. The selector 222 may then move the capture micro-object by moving the light pattern 322 relative to the device 300. Alternatively, the device 300 may be moved relative to the light pattern 322.

Although the barrier 254 defining the holding pens 256 is shown in fig. 2B and 2C and discussed above as a physical barrier, the barrier 254 may alternatively comprise a virtual barrier that includes DEP forces activated by the light patterns 322. The stop 258 may likewise comprise a physical barrier and/or a virtual barrier comprising DEP forces activated by the light pattern 322.

Referring again to fig. 2A-2C, detector 224 may be a mechanism for detecting events in flow region 240. For example, detector 224 may include a photodetector capable of detecting one or more radiation characteristics (e.g., due to fluorescence or luminescence) of a micro-object (not shown) in the medium. Such a detector 224 may be configured to detect, for example, that one or more micro-objects (not shown) in the medium 244 are radiating electromagnetic radiation and/or the approximate wavelength, brightness, intensity, etc. of the radiation. Examples of suitable light detectors include, but are not limited to, photomultiplier tube detectors and avalanche photodetectors.

The detector 224 may alternatively or additionally include an imaging device for capturing a digital image of the flow region 240 of micro-objects (not shown) included in the medium 244. Examples of suitable imaging devices that detector 224 may comprise include digital cameras or light sensors, such as charge coupled devices, complementary metal oxide semiconductor imagers. Images may be captured and analyzed by such devices (e.g., by control module 230 and/or an operator).

The flow controller 226 may be configured to control the flow of the medium 244 in the flow region 240. For example, the flow controller 226 may control the direction and/or speed of the flow. Non-limiting examples of flow controllers 226 include one or more pumps or fluid actuators. In some embodiments, the flow controller 226 may include additional elements, such as one or more sensors (not shown) for sensing the velocity of the flow of the medium 244, for example, in the flow region 240.

Control module 230 may be configured to receive signals from selector 222, detector 224, and/or flow controller 226 and control selector 222, detector 224, and/or flow controller 226. As shown, the control module 230 may include a controller 232 and a memory 234. In some embodiments, the controller 232 may be a digital electronic controller (e.g., a microprocessor, microcontroller, computer, etc.) configured to operate in accordance with machine-readable instructions (e.g., software, firmware, microcode, etc.) stored as non-transitory signals in a memory 234, which memory 234 may be a digital electronic, optical, or magnetic storage device. Alternatively, the controller 232 may comprise hardwired digital and/or analog circuitry, or a combination of a digital electronic controller operating in accordance with machine-readable instructions and hardwired digital and/or analog circuitry.

As mentioned, microfluidic device 200 is an example of a device that may be used to perform process 100. For example, at step 102, the selector 222 (e.g., configured as shown in fig. 3A and 2B) can select a capture object (not shown) in the medium 244 in the flow region 240 and move the selected capture object into the holding pen 256. At step 104, nutrients can be provided to biological micro-objects (not shown) in pens 256 in the flow of medium 244 in channels 252, 253. At step 106, the selector 222 can select and remove a capture object (not shown) from the pens 256, and at step 108, the detector 224 and controller 232 can associate each removed capture object (not shown) with a pen 256 from which it was retrieved. For example, the detector 224 can capture an image of a capture target (not shown) and the pens 256, and the controller 232 can store the correlation as digital data in the memory 234. At step 110, the biological material bound to each removed capture object (not shown) may be evaluated in the microfluidic device 200. For example, the detector 224 may capture an image or detect a feature of a removed capture object (not shown) to evaluate biological material bound to the removed capture object.

Fig. 4 shows another example of a process 400 for determining biological activity in a holding pen of a microfluidic device. Process 400 can be a more narrow example of a more general process 100, where, in process 400 of fig. 4, maintaining biological activity in a pen is the production of a biological material of interest by a clonal population of cells. For ease of illustration and discussion, the process 400 is discussed below with reference to the microfluidic device 200 of fig. 2A-2C, where the selector 222 may be configured as shown in fig. 3A and 3B. However, process 400 is not so limited and may therefore be performed on other microfluidic devices.

As shown in fig. 4, at step 402, the process 400 can culture the production of a population of clonal cells in the holding pens 256 of the microfluidic device 200. Fig. 5A (which, like fig. 6, 7A, 8-11B, and 12-14, show top cross-sectional views of a portion of the flow region 240 of the microfluidic device 200 of fig. 2A-2C) and 5B illustrate examples.

As shown in fig. 5A and 5B, biological cells 502 can be cultured in one or more holding pens 256 by flowing a liquid medium 244 in a channel 252 adjacent to the openings of at least some of the pens 256. The nutrients in the stream 506 can culture biological activity in the holding pens 256. The flow 506 can also provide for removal of waste from the pens 256. Similar flow can be provided in other channels (e.g., 253 shown in fig. 2C) adjacent to the openings of other pens 256 in the device 200.

Fig. 5B shows a fence with isolation regions 508 and connection regions 510. As is well known, a flow 506 of fluidic medium 244 in the microfluidic channel 252 through the proximal opening of the pens 256 can cause a secondary flow of medium 244 into and/or out of the pens. To isolate the micro-objects 502 in the isolation region 508 of the pen 256 from the secondary flow, the length of the connection region 510 of the isolation pen 256 from the proximal opening to the distal opening can be greater than when the velocity of the flow 506 in the channel 252 is at a maximum (V)_max) Maximum penetration depth D of the secondary flow into the connecting region 510_P. As long as the flow 506 in the channel 252 does not exceed the maximum velocity V_maxThe flow 506 and the resulting secondary flow may thus be confined in the channel 252 and the junction region 510 and maintained outside of the isolation region 508. The flow 506 in the channel 252 will therefore not cause the biological micro-objects 502 (or any other micro-objects) to exit the isolation region 508. The biological micro-objects 502 in the isolation region 508 will thus remain in the isolation region 508 regardless of the flow 506 in the channel 252.

Culturing at step 402 can facilitate increasing one or more cells 502 in each pen 256 to produce a population 500 of cells 502 in each pen 256. Each pen 256 can isolate its cells 502 from the cells 502 in all other pens 256 to substantially prevent the cells 502 in any one pen 256 from mixing with the cells 502 in any other pen 256. In addition, the population 500 generated in each holding pen 256 can begin with a single cell 502 in the pen 256. The population 500 of cells 502 in each pen 256 can thus be clonal (clonogenic).

The incubation at step 402 may also aid in the production of the particular material of interest 504 to be assayed. Non-limiting examples of material of interest 504 include proteins, nucleic acids, carbohydrates, lipids, hormones, metabolites, or any combination thereof. The protein of interest may include, for example, a therapeutic protein, an antibody, a growth factor, a cytokine, a cancer cell specific antigen, an infectious antigen associated with a virus or other pathogen, a secreted protein, or any other protein produced and/or released by a biological cell. Thus, for example, the cell 502 can be a cell that produces a protein (e.g., an antibody), and the material of interest 504 can be a particular protein (e.g., a particular antibody). For example, the material of interest may be an antibody of the immunoglobulin g (igg) type. Materials including biological materials other than the material of interest 504 can be in the pen. For example, the cells 502 may produce other materials in addition to the material of interest 504.

In some embodiments, the culturing at step 402 may include multiple types of culturing. For example, a first flow 506 of a first type of medium 244 can culture the growth and division of cells 502 in each pen 256. Thereafter, the second flow of the second type of medium 244 can culture the material of interest 504 produced by the cells 502 in each pen 256.

At step 404 of fig. 4, the process 400 can move the capture object 602 into the holding pen 256 (see fig. 6). The capture objects 602 may be, for example, inanimate micro-objects such as microparticles, microbeads (e.g., polystyrene beads, Luminex)^TMBeads, etc.), magnetic beads, nanorods, microwires, quantum dots, etc. In some cases, the capture objects 602 may be a combination of inanimate micro-objects and biological micro-objects (e.g., liposome-coated microbeads, liposome-coated magnetic beads, microbeads attached to cells, etc.). In other cases, capture objects 602 may be biological micro-objects, such as cells, liposomes, nano-lipid rafts, and the like. In addition, each capture object 602 may include a specific binding substance that specifically binds a specific biological material of interest. Capture targets 602 may include, for example, having a sense of use for a particularA specific binding substance that has an affinity (e.g., Kd) of at least about 1mM or greater (e.g., about 100 μ M, 10 μ M, 1 μ M, 500nM, 400nM, 300nM, 200nM, 100nM, 75nM, 50nM, 25nM, 15nM, 10nM, 5nM, 2.5nM, 1nM or greater) for the biological material of interest. Such affinity can be two, three, four, five, ten, or more times greater than, for example, the affinity for any material other than the particular biological material of interest (or at least any other biological material of interest present in the holding pens and/or microfluidic devices). For example, if the material of interest 504 is a particular antibody, the capture objects 602 can include a binding substance (e.g., an antigen or epitope thereof) that has an affinity for the particular antibody that is greater than the affinity for the holding pens 256 and/or any other material in the microfluidic device. As noted, the material of interest 504 may be an IgG antibody, in which case the binding substance of the capture objects 602 may include a material having an IgG Fc receptor for binding the IgG antibody. Fig. 6 to 8 show an example of step 404.

As shown in fig. 6, a capture object 602 can be disposed in the channel 252 adjacent to the opening of the pen 256. As shown in fig. 7A-7B and 8, an individual capture object 602 can be moved into a particular pen 256.

The capture objects 602 can be introduced into the microfluidic device 200 through the inlet 208 (see fig. 2A-2C) and moved to the channel 252 by the flow 506, as shown in fig. 6. Fig. 7A illustrates an example in which a selector 222 (see fig. 2A-2C) configured like the DEP device 300 of fig. 3A-3B produces an optical trap 702 that can trap an individual capture object 602. The DEP apparatus 300 can then move the light trap 702 into one of the pens 256, which can move the trapped capture objects 602 into the pen 256. The light trap 702 can be part of the varying pattern 322 of light projected onto the inner surface 242 of the flow region 240 of the DEP device 300, as discussed above with reference to fig. 3A and 3B. Once the capture object 602 is in the pen 256, the optical trap 602 corresponding to the capture object 602 can be turned off, as shown in fig. 8. The detector 224 may capture images of all or part of the flow region 240, and these images may facilitate the capture and movement of individual capture objects 602 into particular pens 256. Thus, a particular number (e.g., one or more) of capture objects 602 can be identified, selected, and moved into each pen 256.

As shown in FIG. 7A, the flow 506 of the medium 244 may be stopped after the flow 506 brings the capture object 602 into the channel 252. Stopping the flow 506 may facilitate identification and selection of individual capture objects 602. As shown in fig. 8, once the capture object 602 is in the pen 256, the flow 506 can be restored. Alternatively, rather than stopping the flow 506, the flow 506 may be slowed only to a speed that is slow enough for the detector 224 polarity detection and the selector 222 to capture and move the individual capture objects 602 in the channel 252. As yet another alternative, the stream 506 may be initiated and maintained at a substantially steady rate that is slow enough for the detector 224 to detect and the selector 222 to capture and move the individual capture objects 602. In such a case, the flow 506 may be maintained at a substantially constant velocity in each of fig. 6, 7A, and 8.

Although fig. 7A shows one capture object 602 per well 702, a well 702 may capture more than one capture object 602. Similarly, although fig. 8 shows one capture object 602 in each pen 256, more than one capture object 602 may be moved to the pens 256. Regardless, a particular, known number of capture objects 602 (e.g., one or more) can be moved into each pen 256. In general, the order of the steps in processes 100 and 400 is not critical, and thus, for example, the order of steps 404 and 402 may be reversed. For example, the capture objects 602 can be placed in the holding pens 256 before the first cells 502 are placed in the pens 256. In such a case, the process can include a step for moving the biological activity (e.g., biological cells 502) into the holding pens 256.

As an alternative to actively selecting capture objects 602 and moving capture objects 602 into holding pens 256, fig. 7B illustrates a relatively passive method for loading capture objects 602 into holding pens 256. The microfluidic device of fig. 7B is similar to that shown in fig. 7A, except that there is a deflector 754 in the channel 252 just outside the holding pens 256. When the capture objects 602 flow into the microfluidic device through the channel 252, a small portion of the capture objects 602 will be carried to the periphery of the channel 252. The capture objects 602 carried by the stream 506 at the periphery of the channel 252 can be captured by the deflector 754 and deflected into the holding pen 256. Unlike methods that use optical traps to select a particular capture object 602 and move a particular capture object 602 into a particular holding pen 256, the use of deflectors as shown in fig. 7B cannot provide specific which capture objects 602 are carefully controlled or how many capture objects 602 are moved into each holding pen 256. However, the use of deflector 754 may facilitate loading of a large number of holding pens at the same time.

The deflector 754 shown in fig. 7B may be made of the same material as the barrier 254, or any other suitable material discussed herein. Further, the deflector 754 may be spaced apart from or connected to the barrier 254 (as shown). The deflector 754 can extend the entire height of the channel 252, or it can extend only partially up through the channel, potentially reducing the number of capture objects 602 (or biological micro-objects, such as cells) deflected into the holding pens 256. Further, deflector 754 may be a virtual barrier formed by light focused on surface 242 of channel 252. Such light may activate electrodes (e.g., DEP electrodes) to form a barrier for capturing the target 602 (or cell 502) in the manner of the optical traps discussed above. Such a virtual deflector can be beneficial because it can be turned off once a threshold number of capture objects 602 have been deflected into the holding pen 256. For example, a user or controller 232 can monitor the number of capture objects 602 deflected into any particular holding pen 256 and then turn off the light that is activating the electrodes (and thus creating the deflector) once a threshold number of capture objects 602 is reached.

As yet another alternative for actively selecting capture objects 602 and moving capture objects 602 into holding pens 256, channel 2The high rate of flow 506 of the medium 244 in 52 can be used to increase the penetration depth D of the secondary flow into the holding pen 256_p. Thus, by increasing the rate of flow 506 of the medium 244 in the channel 252, the capture objects 602 can be pushed into the holding pens 256. In some embodiments, the microfluidic device has a channel 252, the channel 252 having a cross-sectional area of about 3,000 to 6,000 square microns, or about 2,500 to 4,000 square microns. The rate of flow 506 of medium 244 suitable for loading capture objects 602 into holding pens 256 in such microfluidic devices can be, for example, about 0.05 to 5.0 μ Ι/sec (e.g., about 0.1 to 2.0, 0.2 to 1.5, 0.5 to 1.0 μ Ι/sec, or about 1.0 to 2.0 μ Ι/sec).

At step 406 of fig. 4, the cells 502 in the pens 256 can be cultured for a period of time during which the cells 502 can continue to multiply and/or produce the material of interest 504. As shown in fig. 9, capture objects 602 in a particular pen 256 can bind to the material of interest 504 produced by the cells 502 in the pen 256. Fig. 9 thus shows the material of interest 504 bonded to capture objects 602 in the pens 256.

In some embodiments, the purpose of the assay process 400 of fig. 4 can be to identify a population of cells 500 in a pen 256 that produces a material of interest 504 at a minimum threshold rate. In such an embodiment, the amount of material of interest 504 that can be bound by one or more capture objects 602 in any of the pens 256 and the time period of step 406 can be such that producing a population 500 of material of interest 504 at or above a minimum threshold rate will produce sufficient material of interest 504 to saturate the capture object(s) 602 in the pens 256.

In other embodiments, the purpose of the assay process 400 can be to determine the amount of material of interest 504 produced in each pen 256. In such embodiments, one or more capture objects 602 in a pen 256 can bind the material of interest 504 and the time period of step 406 can be such that even producing the population 500 of the material of interest 504 at the highest possible rate does not saturate the capture object(s) 602 in the pen 256.

As shown in the holding pens 256 on the right side of the page in fig. 5-14, the process 400 can assay for a single biological target 502 (e.g., a single biological cell) in the holding pen 256. The ability to assay a single biological target 502 in a holding pen 256 is significant because the known techniques for assaying biological cells are not believed to be sensitive enough to enable the assay of materials produced by, for example, a single cell.

As shown in fig. 4, after the time period of step 406 discussed above, at step 408, the process 400 may select an individual capture object 602 from a particular pen 256 and remove the selected capture object 602 from the pen 256. In some embodiments, the removed capture objects 602 may be moved into the channel 252. Fig. 10 and 11A show an example of step 408.

As shown in fig. 10, individual capture objects 602 can be selected in a particular pen 256 using optical traps 1002, which can be similar to the optical traps 702 discussed above. As shown in fig. 11A, the captured capture objects 602 can be removed from the pens 256 and placed in the channels 252 adjacent to the openings of the pens 256. For example, the light trap 1002 can be moved from the pen 256 to the channel 252. As also shown in fig. 11A, the capture objects 602 may be moved to a stop 258 in the channel 252, which, as discussed, may hold the capture objects 602 in place against the flow 506 of the medium 244 in the flow region 240. Once the removed capture objects 602 are moved to the stop 258, the optical trap 1002 may be closed. Alternatively, the optical trap 1002 may be held open to hold the removed capture objects 602 in place, for example, against the flow 506 of the medium 244. In such a case, the stop 258 need not be included in the flow region 240 of the device 200. Regardless, the flow 506 may be slowed or even stopped during step 408. As yet another alternative, once moved into the channel 252, the capture objects 602 may be output from the device for subsequent analysis. Suitable methods for outputting capture targets have been disclosed, for example, in U.S. patent application serial No. 14/520,150 filed on 22/10/2014, the entirety of whichThe contents of which are incorporated herein by reference. The capture objects 602 may be output individually, in groups of outputs from the same holding pen 256, or in groups of outputs including capture objects 602 from multiple holding pens 256. In the latter case, the capture object 602 may have an identifier that facilitates its identification and association with the holding pen 256 from which the capture object 602 is removed. For example Luminex^TMBeads may be used as capture objects 602, allowing capture objects 602 from a particular holding pen 256 to be distinguished from capture objects from other holding pens 256.

As shown in FIG. 11B, an alternative to the stop 258 for moving the capture objects 602 into the channel 252 includes moving the capture objects 602 to the assay region 1156. The assay region 1156 can be adjacent to the holding pens 256, thereby reducing the time required to move the capture objects 602 and facilitating correlation between the holding capture objects 602 and the holding pens 256 from which the capture objects 602 are removed. The assay region may be defined by a barrier 1154, which barrier 1154 may be made of the same material as barrier 254, or any other suitable material discussed herein. Although shown as having the same size and shape as the holding pens 256, the assay region 1156 can be smaller and/or have a different shape. For example, the assay region 1156 can be smaller and may or may not include an isolation region. Thus, for example, the assay region 1156 may be substantially devoid of an isolation region (e.g., less than 50% of the volume of the assay region may be isolated from the secondary flow of the flow 506 of the medium 244 in the channel 25). In certain embodiments, the substantial absence of an isolation region can facilitate removal of assay material from the capture object 602 (discussed further below).

As an alternative to using the optical trap 1002 to move the capture objects 602 out of the holding pens 256, magnetic forces (such as magnets) can be used to force the magnetic capture objects 602 out of the pens 256. As shown in fig. 11C, the microfluidic device 1100 can include an assay region 1156 located opposite the channel 252 relative to the opening of the holding pen 256. To move the capture objects 602 out of the holding pens 256 or into the assay region 1156, a magnetic force can be applied to the microfluidic device such that the magnetic capture objects 602 are pulled or pushed into the assay region 1156. During this time, the flow 506 of the medium 244 in the channel 252 may be slowed or stopped.

As described above, although removal of one capture object 602 from each pen 256 is shown in fig. 10 and 11A-11C, more than one capture object 602 may be placed into a pen 256 at step 404, and in such cases, more than one capture object 602 may be removed from a pen 256 at step 408 accordingly.

Returning again to fig. 4, at step 410, the process 400 can maintain a correlation between each capture object 602 removed from a pen 256 at step 408 and the pen 256 from which that capture object 602 was removed. For example, the controller 232 may identify and track the locations of the capture objects 602 and pens 256 from the images provided by the detector 224, and the controller 232 may store in the memory 234 the correlations between the respective capture objects 602 removed into the channel 252 and the pens 256 from which each capture object 602 was taken. Table 1 is an example of a digital table that may be stored in the memory 234 that associates the location of a capture object 602 in the channel 252 with the pen 256 from which the capture object 602 was removed. In the example of table 1, the capture object 602 at the stop 258 identified as stop a is taken out of the fence 256 numbered 3. Similarly, capture objects 602 at stop 258B are removed from pen 256 number 1, and capture objects 602 at stop 258C are removed from pen 256 number 2. A corresponding table may be used to store data regarding the position of the capture objects 602 in the assay region 1156 and the holding pens 256 from which the capture objects 602 were removed. Similarly, for capture objects 602 output from the microfluidic device for analysis, a table can be used to store data regarding the identifier associated with a particular capture object 602 and the holding pen 256 from which that capture object 602 was removed.

At step 412 of fig. 4, the process 400 may evaluate the material of interest 504 bound to the removed capture objects 602 in the channel 252. For example, the process 400 can evaluate the material of interest 504 at step 412 by determining the quantity and/or quality of the material of interest 504 produced by the population of cells 500 or the individual cells 502 in the pen 256. As another example, the process 400 can assess the type of material 504 produced by a population 500 of cells 502 or a single cell 502 in a pen 256 at step 412. As yet another example, the process 400 can assess the activity of the material 504 produced by the population 500 of cells 502 or individual cells 502 in the pen 256 at step 412. Because the material of interest 504 bound to the capture object 602 results from the biological activity in the pens 256 from which the capture object 602 was removed at step 408, evaluating the material of interest 504 bound to the removed capture object 602 at step 412 can provide information that enables evaluation of the biological activity from the pens 256. Fig. 12 to 14 show an example of step 412.

As shown in fig. 12, at step 412, the assay material 1202 may be a stream 506 flowing through the channel 252. The assay material 1202 may bond to the material of interest 504 located on the removed capture objects 602 and exhibit a distinct, detectable behavior. For example, the assay material 1202 can include a label that includes a binding substance that specifically binds the biological material of interest 504 (e.g., at a location on the material of interest 504 that is different from the location bound by the capture objects 602). In the case where the biological material of interest 504 is an antibody, the tag may comprise an Fc receptor and the capture object 602 may comprise an antigen bound by the antibody, or vice versa. In the example shown in fig. 12-14, the assay material 1202 can include a label that adheres to the material of interest 504 located on the capture object and radiates energy 1402, as shown in fig. 14. Thus, for example, the assay material 1202 can include a binding substance that specifically binds the biological material of interest 504 and is attached to a chromophore. In other embodiments, the assay material 1202 can include, for example, a binding substance linked to luciferase that specifically binds to the biological material of interest 504. In the latter case, the assay material 1202 may also include a suitable luciferase substrate (e.g., a luciferin substrate). Thus, the assay material 1202 can fluoresce or emit light. Regardless, the assay material 1202 can be provided to the removed capture object 602 in a sufficient amount and for a sufficient time to cause the assay material 1202 to bond to substantially all of the material of interest 504 that is bonded to the removed capture object 602.

As shown in fig. 13, thereafter, assay material 1202 that is not bound to one of the removed capture objects 602 can be washed out of the channel 252. For example, the flow 506 of assay material 1202 may then be a flow of medium 244 (or any washing material) in the channel 252, which may wash substantially all of the assay material 1202 that is not bound to the material of interest 504 located on the removed capture objects 602 away from the channel 252. As shown in fig. 13, the capture objects 602 in the channel 252 may now include the material of interest 504 bonded to the capture objects 602 and the assay material 1202 bonded to the material of interest 504.

As shown in fig. 14, the assay material 1202 may radiate energy 1402 that may be examined by detector 224. In some embodiments, the assay material 1202 may require stimulation (e.g., by light or other radiation, or a chemical catalyst or substrate (which may flow through the channel 252)) to trigger the irradiation of the energy 1402. The detectable characteristic of the energy 1402 radiated from the removed capture objects 602, such as level, brightness, color (e.g., a particular wavelength), etc., may correspond to the amount of assay material 1202 bound to the removed capture objects 602, may correspond to the amount of biological material bound to the removed capture objects 602, and may in turn correspond to the ability of the cell population 500 in the pen 256 from which the capture objects 602 were removed to produce the material of interest 504. In some embodiments, the assay material may be repeatedly stimulated. For example, the optical stimulation may be performed periodically and any radiation generated detected after each stimulation. Alternatively, a chemical catalyst or substrate (e.g., luciferin) may flow into the channel 252, upon which detectable radiation may be detected. After an appropriate period of time, the chemical catalyst may be purged from passage 252, after which the process may be repeated.

Step 412 can include detecting a level of energy 1402 radiated from each individual capture object 602 removed from the pen 256 at step 408. For example, the detector 224 may detect the level of energy 1402 from each removed capture object 602 in the channel 252. As noted with respect to step 410, a correlation may be maintained between each removed capture object 602 and the pen 256 from which the capture object 602 was taken, e.g., in a numerical table like table 1 above. The level of energy 1402 radiated from each removed capture object 602 detected as part of step 412 may be stored in a table, as shown in table 2 below, which may include a column for the detected energy level.

In step 414 of fig. 4, the process 400 can identify holding pens 256 with desired cell populations 500 and, at least by default, can also identify holding pens 256 with undesired cell populations 500. For example, in step 414, the process 400 may determine which removed capture objects 602 radiate energy above (or below) a threshold level, and the holding pens associated with those removed capture objects 602 may be identified as having the desired cell population 500. The holding pens 256 associated with the removed capture objects 602 radiating 1402 below the threshold level can be identified as containing an undesirable cell population 500.

In other embodiments, the process 400 can quantitatively evaluate the cell population 500 in each holding pen 256 corresponding to the removed capture objects 602, as opposed to only identifying holding pens 256 with desired and undesired cell populations 500 at step 414. For example, the process 400 can detect and quantify the energy 1402 radiated by each removed capture object 602 and thus evaluate the ability of the cell population 500 in each holding pen 256 from which the removed capture object 602 was removed to produce the material of interest 504.

In some embodiments, the detector 224 can capture an image from which an operator or controller 232 can calculate or roughly estimate the number of cells 502 in each holding pen 256 from which one of the removed capture objects 602 is to be removed. In such embodiments, the process 400 may utilize the level of radiant energy 1402 (or other characteristic, such as color, brightness, etc.) detected as part of step 412 and the number of cells in the holding pens 256 to determine the ability of the population 500 of cells 502 in a particular holding pen 256 to produce the material of interest 504 as a per cell 502 ratio. The process 400 can then utilize the foregoing to identify the holding pens 256 with the desired cell populations 500 at step 414.

In any event, after step 414, the desired cell population 500 can be removed from its respective holding pen 256 to other locations in the device 200, or other devices (not shown) for further processing, analysis, testing, or use. For example, a desired cell population 500 may be selected and moved as shown in U.S. patent application serial No. 14/520,150, filed 10/22/2014, assigned to the same assignee as the instant application.

Fig. 4 is an example, and many variations of process 400 are contemplated. For example, the process 400 can evaluate biological activity at step 412 while the capture object 602 is in the holding pen 256. In some variations, process 400 therefore need not include steps 408, 410 or steps 408 and 410 may be skipped. As another example, all of steps 402 through 414 need not be performed in the order shown in FIG. 4.

Fig. 15 shows yet another example of a process 1500 for determining biological activity in a holding pen of a microfluidic device. Process 1500 may be a more narrow example of a more general process 100, where in process 1500 of fig. 15, biological activity is tested for a first number (n) of features and then for a second number (m) of features, where n and m (which may be the same number or different numbers) may each be equal to or greater than any integer value. For ease of illustration and discussion, the process 1500 is discussed below with reference to the microfluidic device 200 of fig. 2A-2C, and the selector 222 can be configured as shown in fig. 3A and 3B. However, the process 1500 is not so limited and can therefore be performed on other microfluidic devices.

As shown in fig. 15, at step 1502, process 1502 can culture biological activity in a holding pen in a microfluidic device. Step 1502 may be performed similarly to step 104 of fig. 1 or step 402 in fig. 4. For example, biological activity can produce one or more different materials of interest by one or more biological cells in each pen 256 of the microfluidic device 200 of fig. 2A-2C, generally in accordance with the discussion above of fig. 4. The culturing of step 1502 may be performed continuously throughout the execution of process 1500, and the culturing of step 1502 may thus be continued during steps 1504 and/or 1506.

At step 1504, the process 1500 can test the biological activity in each retention pen 256 for n features (each feature can be a different feature). The n features may be any feature or other feature of biological activity tested for process 100 or process 400 of fig. 1 and 4, as discussed above. Evaluating multiple characteristics in this manner is desirable for a number of applications including antibody identification. Thus, for example, multiple evaluations may contribute to any of the following: recognition of conformation-specific antibodies (e.g., the different characteristics may be the ability to bind antibody analytes of different conformations of a particular antigen); epitope mapping of antibody analytes (e.g., the different characteristic may be the ability of various genetic or chemically altered forms to bind to an antigen); other applications that may benefit from assessing multiple characteristics include, for example, detection of cellular health, cancer, infection (e.g., viruses, bacteria, etc.), for example, detection of antibodies to cell health, cancer, and infection (e.g., viruses, bacteria, and bacteria, etc.), for example, the ability of antibody analytes to bind to homologous antigens from different species (e.g., humans, mice, rats, and/or other animals (e.g., laboratory animals)), and the IgG isotype of antibody analytes (e.g., the ability of different characteristics to bind to IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE, and/or IgD), for example, the generation of chemically altered antigens for epitope localization of antibodies has been described in "Dhungana et al (2009), methods in molecular biology 524: 119-34)" Parasites, etc.), inflammation, response to therapeutic agents, etc.

At step 1504, process 1500 can perform a test that indicates whether the biological activity in each pen 256 has any one or more of the n characteristics. Thus, in some embodiments, if the biological activity is only one of the n features, the biological activity in the pens 256 is considered to test positive at step 1504. In other embodiments, the biological activity in pen 256 is considered positive for testing at step 1504 only if the biological activity has all n characteristics, and in other embodiments, the biological activity in pen 256 is considered positive for testing at step 1504 if the biological activity has q of the n characteristics, where q is greater than 1 but less than n.

At step 1506, the process 1500 can test the biological activity in each retention pen 256 that tested positive at step 1504 for m different features (each of which can be a different feature). The m features tested at step 1506 may be different from the n features tested at step 1504. The m features may include any feature or other feature of biological activity tested for process 100 or process 400 of fig. 1 and 4, as described above. Alternatively, there may be overlap between the m features tested at step 1506 and the n features tested at step 1504.

Step 1506 may be performed in any manner discussed above for performing step 1504. For example, at step 1506, the process 1500 can perform a test that indicates whether the biological activity in the pens 256 that tested positive at step 1504 has any one or more of the m characteristics. Thus, in some embodiments, if the biological activity has only one of the m features, the biological activity in pen 256 is considered to test positive at step 1506. In other embodiments, the biological activity in pen 256 is considered positive for the test at step 1506 only if the biological activity has all m characteristics, and in other embodiments, the biological activity in pen 256 is considered positive for the test at step 1506 if the biological activity has p of the m characteristics, where p is greater than 1 but less than m.

Fig. 16 and 17 illustrate examples of processes 1600, 1700 that may perform step 1504 and/or step 1506 of fig. 15.

Turning first to fig. 16, at step 1602, the process 1602 can move a number x of capture objects into each pen 256 of the microfluidic device 200. For example, the number x may be all numbers between 1 and n (including 1 and n). Fig. 18 (which shows a top cross-sectional view of a portion of the flow region 240 of the microfluidic device 200 of fig. 2A-2C) illustrates an example. As shown, x capture objects 1812 can be moved into the pens 256. The capture objects 1812 can be moved into the pens 256 serially, in parallel, or in a combination of serial and parallel. As also shown, a biological micro-object 1802 can be in pen 256. While three biological micro-objects 1802 are shown in pen 256, there may be one, two, or more than three. Biological micro-object 1802 can be, for example, a biological cell that produces one or more materials of interest.

Each capture object 1812 can include a binding substance that specifically binds to a particular biological material of interest. For example, the binding substance can have an affinity (e.g., Kd) for a particular biological material of interest of at least about 1mM or more (e.g., about 100 μ M, 10 μ M, 1 μ M, 500nM, 400nM, 300nM, 200nM, 100nM, 75nM, 50nM, 25nM, 15nM, 10nM, 5nM, 2.5nM, 1nM or more). Such affinity can be two, three, four, five, ten, or more times greater than, for example, the affinity for any material other than the particular biological material of interest (or at least any other biological material of interest present in the holding pens and/or microfluidic devices). Thus, for example, each capture object 1812 can comprise a different binding substance with such a predominant affinity for a different material of interest that can be present or result from a biological activity being cultured in pen 256 by step 1502 of fig. 15. Otherwise, the capture target 1812 may be generally similar to the capture target 602, and the capture target 1812 may be selected and moved in any of the manners discussed above for selecting and moving the capture target 602.

At step 1604, the process can evaluate the biological material captured by each of the x capture objects moved into the pen 256 at step 1602. Step 1604 may be similar to step 110 of fig. 1 or step 414 of fig. 4, and may be performed in any of the ways discussed above with reference to step 110 of fig. 1 or step 414 of fig. 4.

As shown in fig. 16, process 1600 may optionally be repeated any number of times. The number x may be the same or different for each iteration of step 1602. Thus, for example, the process 1600 can be performed one or more times until n capture objects (each of which can have a different binding substance) have been moved into the pen at step 1602 and evaluated at step 1604. Thus, performing process 1600 one or more times can cause a total of n capture objects to be moved into each pen by performing step 1602 one or more times, and the biological material captured by the n capture objects to be evaluated by performing step 1604 one or more times. For example, at each iteration of step 1602, the value of x can be any number between 1 and n-1 (including n-1), and process 1600 can be repeated until the sum of the values of x at each iteration of step 1602 is at least n.

As noted, step 1504 and/or step 1506 of fig. 15 may be performed by process 1600 of fig. 16. If step 1506 is executed, the number n is replaced by m in the above discussion of FIG. 16.

Referring now to fig. 17, at step 1702, the process 1700 can move y material capture objects, each of which can include y different bonding substances, into the pens 256 of the microfluidic device 200. The number y may be a number between 2 and n (including 2 and n). Fig. 19 (which shows a top cross-sectional view of a portion of the flow region 240 of the microfluidic device 200 of fig. 2A-2C) illustrates an example. As shown, the y-material capture objects 1912 can be moved into the pens 256 with one or more biological micro-objects 1802, which can be as discussed above.

The y-material capture objects 1912 may include y different binding substances, each of which specifically binds to a particular biological material of interest. For example, each binding substance can have an affinity (e.g., Kd) for a particular biological material of interest of at least about 1mM or more (e.g., about 100 μ M, 10 μ M, 1 μ M, 500nM, 400nM, 300nM, 200nM, 100nM, 75nM, 50nM, 25nM, 15nM, 10nM, 5nM, 2.5nM, 1nM or more). Such affinity can be two, three, four, five, ten, or more times greater than, for example, the affinity for any material other than the particular biological material of interest (or at least any other biological material of interest present in the holding pens and/or microfluidic devices). In addition, the y-material capture objects 1912 may be generally similar to the capture objects 602, and the capture objects 1912 may be selected and moved in any of the manners discussed above for selecting and moving the capture objects 602.

At step 1704, the process 1700 can evaluate the biological material captured by the y-material capture objects 1912 in the pens 256. Step 1704 may be similar to step 110 of FIG. 1 or step 414 of FIG. 4, and may be performed in any of the ways discussed above with reference to step 110 of FIG. 1 or step 414 of FIG. 4.

As shown in fig. 17, process 1700 may optionally be repeated any number of times. The number y may be the same or different for each iteration of step 1702. Thus, for example, the process 1700 may be performed one or more times until the value of y at each execution of step 1702 is added to at least n. For example, at each iteration of step 1702, the value of y may be any number between 2 and n-2, and the process 1700 may be repeated until the sum of the values of y at each iteration of step 1702 is at least n.

As noted, step 1504 and/or step 1506 of fig. 15 may be performed by process 1700 of fig. 17. If step 1506 is executed, the number n is replaced by m in the above discussion of FIG. 17.

Fig. 20A-20C illustrate variations in the shape of holding pens that can be used with the microfluidic devices and methods of the present invention. In each case, the holding pen includes a region that can be used to contain biological activity (e.g., one or more biological cells) and another region that can be used to contain capture objects 602. For example, in fig. 20A, the holding pen 256 has an isolation region 508 that includes a left portion that can contain biological cells 502 and a right portion that can contain capture objects 602. The holding pen 256 also includes a connection region 510 having a proximal opening to the channel 252 and a distal opening to the isolation region 508. In fig. 20B, a similar structure exists, but the holding pens 256 are longer and shallower (in terms of the depth of the connecting region 510). In fig. 20C, the holding pen 256 includes a thin wall separating the left portion that can contain the biological cells 502 from the right portion that can contain the capture objects 602. The thin wall is leaky, thus allowing diffusion of biological material of interest between the left and right portions of the holding pen 256, thereby preventing biological activity (e.g., biological cells 502) from contacting the capture object 602.

While specific embodiments and applications of the invention have been described in this specification, these embodiments and applications are exemplary only, and many variations are possible.

Claims (39)

1. A process for determining biological activity in a microfluidic device, the process comprising:

culturing one or more biological cells in a holding pen of a microfluidic device, wherein the one or more cells produce a biological material of interest, wherein the microfluidic device comprises a housing, wherein the housing comprises:

a base;

a microfluidic structure disposed on the base; and

the holding pens are arranged so that, when the holding pens are in use,

wherein the base and the microfluidic structure define a flow region, wherein the holding pen comprises an isolation region having a single opening and a connection region having a proximal opening to the flow region and a distal opening to the isolation region, wherein the isolation region of the holding pen is an unswept region of the microfluidic device;

introducing one or more capture micro-objects into the holding pen, wherein each of the capture objects comprises a binding substance that specifically binds the biological material of interest;

allowing the biological material of interest produced by the one or more biological cells to bind to the one or more capture micro-objects in the holding pens; and

evaluating the biological material of interest bound to the capture micro-objects.

2. The process of claim 1, further comprising removing said one or more capture micro-objects from said holding pen after allowing said biological material of interest to bind to said one or more capture micro-objects but before evaluating said biological material of interest bound to said one or more capture micro-objects.

3. The process of claim 2, wherein removing the one or more capture micro-objects comprises moving the one or more capture micro-objects to an assay region located within the microfluidic device.

4. The process of claim 3, wherein the assay region is a stop located in a channel in the microfluidic device.

5. The process of claim 3, wherein the assay region is a chamber located within the microfluidic device.

6. The process of claim 2, wherein removing the one or more capture micro-objects comprises:

moving the one or more capture micro-objects to a channel in the microfluidic device; and

outputting the one or more capture micro-objects from the microfluidic device.

7. The process of any one of claims 2 to 6, wherein removing the one or more capture micro-objects comprises:

forming a light trap in the holding pen that traps at least one of the capture micro-objects, the light trap formed by projecting a light pattern that encompasses the at least one of the capture micro-objects onto an interior surface of the microfluidic device; and

moving the light trap from the holding pen to a channel in the microfluidic device.

8. The process of any one of claims 2 to 6, wherein the one or more capture micro-objects are magnetic, and wherein removing the one or more capture micro-objects comprises applying a magnetic field to the microfluidic device.

9. The process of any one of claims 2 to 6, further comprising maintaining an association between each said removed capture micro-object and said holding pen from which said removed capture micro-object was removed, thereby enabling data obtained from each said capture micro-object removed from said holding pen to be traced back to said holding pen.

10. The process of claim 1, wherein said evaluating comprises determining a type of said biological material of interest bound to said one or more capture micro-objects.

11. The process of claim 1, wherein said evaluating comprises determining an activity of said biological material of interest bound to said one or more capture micro-objects.

12. The process of claim 1, wherein said evaluating comprises determining an amount of said biological material of interest bound to said one or more capture micro-objects.

13. The process of any of claims 10-12, wherein the determining comprises:

binding an assay material to the biological material of interest bound to the one or more capture micro-objects, wherein the assay material is capable of generating detectable radiation; and

detecting a correlation between the one or more capture micro-objects and radiation from the assay material.

14. The process of claim 13, wherein said determining further comprises, after binding said assay material to said biological material of interest, but before detecting a correlation between said one or more capture micro-objects and radiation from said assay material, washing unbound assay material from said one or more capture micro-objects.

15. The process of claim 13, further comprising determining whether the radiation associated with each of said capture micro-objects corresponds to a predetermined characteristic.

16. The process of claim 1, wherein the biological material of interest is a protein.

17. The process of claim 16, wherein the protein is an antibody.

18. The process of claim 1, wherein said evaluating is performed while said one or more capture micro-objects are in said holding pens.

19. The process of any one of claims 1 to 6 or 16 to 18, wherein the binding substance of the one or more capture micro-objects has an affinity for the biological material of interest of at least 1 μ Μ.

20. The process of any one of claims 1-6 or 16-18, wherein the one or more biological cells in the holding pen comprise a clonal population of biological cells.

21. The process of any one of claims 1-6 or 16-18, wherein the one or more biological cells in the holding pen are single cells.

22. The process of any one of claims 1 to 6 or 16 to 18, wherein said one or more capture micro-objects is a single capture micro-object.

23. The process of any one of claims 1 to 6 or 16 to 18, wherein said one or more capture micro-objects comprise a plurality of capture micro-objects, each of which comprises a binding substance that is different from the binding substance of other capture micro-objects of said plurality of capture micro-objects.

24. The process of claim 23, wherein said biological material of interest is an antibody, and wherein each of said plurality of capture micro-objects comprises a binding substance that binds to an isotype antibody that is different from the isotype antibody bound by the binding substance of the other capture micro-objects of said plurality of capture micro-objects.

25. The process of claim 23, wherein the biological material of interest is an antibody, and wherein each of the plurality of capture micro-objects comprises a binding substance corresponding to an epitope of an antigen recognized by the antibody.

26. The process of claim 23, wherein said biological material of interest is an antibody, and wherein one capture micro-object of said plurality of capture micro-objects comprises a binding substance corresponding to an antigen recognized by said antibody or epitope thereof, and wherein said other capture micro-objects of said plurality of capture micro-objects each comprise a binding substance corresponding to a homolog of said antigen from a different species or epitope thereof.

27. The process of any one of claims 2 to 6, wherein removing the one or more capture micro-objects comprises:

activating a light-induced DEP electrode adjacent to at least one of the capture micro-objects in the holding pen by projecting a light pattern onto an interior surface of the microfluidic device adjacent to the at least one capture micro-object, and

moving the light pattern from the holding pen into a channel in the microfluidic device, whereby the activated DEP electrode repels the at least one of the capture micro-objects into the channel.

28. A process for determining biological activity in a microfluidic device, the process comprising:

culturing one or more biological cells in a holding pen of a microfluidic device, wherein the microfluidic device comprises a housing, wherein the housing comprises:

a base;

a microfluidic structure disposed on the base; and

the holding pens are arranged so that, when the holding pens are in use,

wherein the base and the microfluidic structure define a flow region, wherein the holding pen comprises an isolation region having a single opening and a connection region having a proximal opening to the flow region and a distal opening to the isolation region, wherein the isolation region of the holding pen is an unswept region of the microfluidic device, wherein the one or more cells produce n different biological materials of interest;

introducing n different types of capture micro-objects into the holding pens, each of the types of capture micro-objects comprising a binding substance that specifically binds to one of the n different biological materials of interest;

allowing the n different biological materials of interest produced by the one or more biological cells to bind to the n different types of capture micro-objects; and

evaluating the binding between the n different biological materials of interest and the n different types of capture micro-objects.

29. The process of claim 28, wherein the result of said assessment is positive if at least one of said n different biological materials of interest specifically binds to one of said n different types of capture micro-objects.

30. The process of claim 28, wherein the result of said assessment is positive if at least two of said n different biological materials of interest each specifically binds to one of said n different types of capture micro-objects.

31. The process of claim 28, wherein the result of said assessment is positive if all n different biological materials of interest each specifically bind to one of said n different types of capture micro-objects.

32. The process of any one of claims 28 to 31, wherein said n different types of capture micro-objects are introduced into said pen simultaneously.

33. The process of any one of claims 28 to 31, wherein said n different types of capture micro-objects are introduced into said pen sequentially.

34. A microfluidic device comprising:

an enclosure comprising a channel, a holding pen and a measurement area,

wherein the holding pen comprises an isolation region having a single opening and a connection region having a proximal opening to the channel and a distal opening to the isolation region, wherein the isolation region of the holding pen is an unswept region of the microfluidic device; and

wherein the assay region is adjacent to the holding pen.

35. The microfluidic device of claim 34, wherein the assay region comprises a stop located within the channel.

36. The microfluidic device of claim 34, wherein the assay region comprises an assay chamber having an opening to the channel, wherein the assay chamber is located next to the holding pen.

37. The microfluidic device of claim 34, wherein the assay region comprises an assay chamber having an opening to the channel, wherein the opening to the assay chamber is located directly opposite the channel relative to the proximal opening of the connection region of the holding pen.

38. The microfluidic device of claim 36 or 37, wherein the assay chamber is substantially devoid of an isolation region.

39. The microfluidic device of any one of claims 34 to 37, wherein the device further comprises means for generating a magnetic force within the enclosure.