JP7068384B2 - Microfluidic device for assaying biological activity - Google Patents

Description

Cross-reference to related applications (s) This application is not provisional of US Modified Provisional Patent Application No. 14 / 060,423 (thus claiming its benefits and / or priority over it).

Background In biological sciences and related disciplines, assaying the biological activity of microscopic objects such as cells can be useful. Some aspects of the invention include devices and treatments for assaying biological activity in holding pens of microfluidic devices.

Overview In some embodiments, the invention provides a process for assaying biological activity in microfluidic devices. Biological activity can be the production of a biomaterial of interest, such as by living cells. Thus, the treatment may include culturing one or more living cells that produce the desired biomaterial in the holding enclosure of the microfluidic device. The treatment further introduces one or more capture micro-objects into the holding enclosure and the target biomaterial produced by one or more living cells is one or more captured micros. It may include making it possible to bond to an object. Capturing microobjects may include, for example, a binding substance that specifically binds the biomaterial of interest. The treatment may also include evaluating captured microobjects for the bound biomaterial of interest.

In some embodiments, one or more captured micro-objects are after the biomaterial of interest is made to bind to one or more captured micro-objects, but for the biomaterial of interest that is bound. Before evaluating the object, it is removed from the holding enclosure. Removing one or more captured micro-objects may include moving one or more captured micro-objects into an assay region located within the microfluidic device. In some embodiments, the assay region is a stop located within a channel in the microfluidic device, a chamber located within the microfluidic device, or the like. Regardless, the assay region may be positioned adjacent to a retention enclosure from which one or more trapped microobjects are removed. Alternatively or additionally, removing one or more captured micro-objects moves one or more captured micro-objects to a channel in the micro-fluid device and then one or more captured micro-objects from the micro-fluid device. May include carrying out.

In some embodiments, removing one or more captured micro-objects comprises creating an optical trap that captures at least one of the captured micro-objects while it is in the holding enclosure. The optical trap may include a light pattern that is projected onto the inner surface of the microfluidic device, surrounds at least one captured microobject, and activates an electrode such as a dielectrophoresis (DEP) electrode within the microfluidic device. Moving the optical trap from the holding enclosure to the channel and / or assay region of the microfluidic device can cause the captured micro-object to move accordingly.

In some embodiments, the one or more captured microobjects are magnetic. In a related embodiment, removing one or more captured microobjects may involve applying a magnetic field to the microfluidic device.

In some embodiments, the captured micro-object removed from the holding enclosure may remain associated with the retaining enclosure. For example, the interrelationship can be maintained between the captured micro-object and the retaining enclosure removed from it. In this way, when the microfluidic device houses a plurality of holding enclosures, the data obtained from the captured micro-objects removed from the holding enclosure can be traced back to the appropriate holding enclosure.

In some embodiments, the evaluation of the captured micro-object for the biomaterial of interest to be bound is performed while the captured micro-object is in the holding enclosure.

In some embodiments, evaluating a captured microobject for the biomaterial of interest to be bound may involve determining the type of biomaterial of interest to be bound to the captured microobject. In some embodiments, assessing a captured microobject for the biomaterial of interest to be bound may involve determining the activity of the biomaterial of interest to be bound to the captured microobject. In some embodiments, evaluating a captured microobject for the biomaterial of interest to be bound may involve determining the amount of the biomaterial of interest to be bound to the captured microobject. Any such determination is to mix (and / or bind) the biomaterial of interest to the assay material to be bound to the capture micro-object and to detect the association between the capture micro-object and the assay material. May include. For example, if the assay material is capable of producing detectable radiation, the determination may involve detecting the association between the captured micro-object and the radiation originating from the assay material. The determination may further involve flushing the unbound and / or unreacted assay material from the captured micro-object before detecting the association between the micro-object and the radiation generated from the assay material. Alternatively, or in addition, the determination may further accompany determining whether the radiation associated with the captured micro-object corresponds to a given feature. For example, radiation may have a characteristic wavelength.

In some embodiments, the biological material of interest is produced by an infectious antigen, secretory protein, or living cell associated with a therapeutic protein, antibody, growth factor, cytokine, cancer antigen, virus or other pathogen, and /. Or a protein, such as any other protein that is released. In some embodiments, the biomaterial of interest is a protein, nucleic acid, carbohydrate, lipid, hormone, metabolite, small molecule, polymer or any combination thereof. In some embodiments, the binding material of the captured microobject has at least 1 μM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 1 nM or stronger binding affinity for the biomaterial of interest.

In some embodiments, there is a single living cell in the retention enclosure. In another embodiment, there are two or more living cells in the retaining enclosure. In some embodiments, the living cell in the retention enclosure is a clonal colony. In some embodiments, a single trapped micro-object is introduced into the holding enclosure. In another embodiment, two or more (eg, a plurality) captured micro-objects are introduced into the holding enclosure. In these latter aspects, each of the plurality of captured micro-objects may have a binding material that is different from the binding material of the other captured micro-objects in the plurality.

In some embodiments, the biomaterial of interest is an antibody, such as a candidate therapeutic antibody. In a related embodiment, the treatment may include multiple capture micro-objects, each of which has a binding agent that binds to a different antibody isotype. In other related embodiments, the treatment may include multiple capture micro-objects, each of which has a binding agent corresponding to a different epitope of the antigen recognized by the antibody. In yet another related embodiment, the treatment may comprise a plurality of captive micro-objects, one of which has a binding agent corresponding to the antigen or epitope thereof recognized by the antibody. The remaining captive microobjects in the plurality may have a binding agent corresponding to the homologue of the antigen or its epitope. Homological antigens or epitopes thereof can be of different species.

In some embodiments, the invention provides a process for an assay for the production of n different biomaterials of interest in a microfluidic device. The treatment may comprise culturing one or more living cells in a holding enclosure of the microfluidic device, where the one or more cells produce n different biomaterials of interest. The treatment is to introduce n different types of trapped micro-objects into the holding enclosure, each type of binding specifically binding to one of the n different types of biomaterial of different purpose. It further comprises having the substance and allowing the n different biomaterials of interest produced by the living cell to bind to n different types of captured microobjects. The treatment may also include evaluating n different types of captured microobjects for the biomaterial of interest to be bound. In some embodiments, the result of such an assessment is positive if at least one of the n different biomaterials of interest specifically binds to one of the n different types of captured microobjects. In another embodiment, the result of such an assessment is positive if at least two of each of the n different target biomaterials specifically bind to one of the n different types of captured microobjects. In yet another embodiment, the result of such an assessment is positive if each of the n different types of biomaterials of different purpose specifically binds to one of the n different types of captured microobjects.

In some embodiments, n different types of captured microobjects are simultaneously introduced into the holding enclosure. In another embodiment, n different types of captured microobjects are continuously introduced into the holding enclosure.

In some embodiments, processing for an assay for the production of n different biomaterials in a microfluidic device comprises introducing one or more y-material capture microobjects into a holding enclosure, respectively. The y-material capture microobject has y different binding substances, each of which specifically binds to one of n different biomaterials of interest produced by one or more living cells. The treatment may further comprise allowing n different biomaterials of interest produced by one or more living cells to bind to the y-material trapping microobject. In addition, the treatment may include evaluating y-material capture microobjects for the biomaterial of interest to be bound.

For all of the aforementioned processes, the microfluidic device may include multiple retention enclosures, each containing one or more living cells and assayed sequentially or in parallel.

In some embodiments, the invention provides a microfluidic device. Microfluidic devices can include enclosures with channels, retention enclosures and assay regions. The retaining enclosure may include a separation area and a connection area with a connection area having a proximal opening to the channel and a distal opening to the separation area. The assay region may be located adjacent to the holding enclosure. For example, the assay region may include a stop located within the channel. The anchorage can be located directly from the proximal opening of the connection area or across the channel just outside it. Alternatively, the assay region may include an assay chamber. The assay chamber can be positioned side by side with the holding enclosure or directly across the channel from the proximal opening of the connecting area of the holding enclosure. In some embodiments, the assay chamber substantially lacks a separation region (eg, less than 50% of the volume of the assay chamber is separated from the bulk flow of medium flowing through the channel). In some embodiments, the microfluidic device may also include means for generating a magnetic force within the enclosure. Such means can be, for example, a magnet.

A brief description of the drawing
FIG. 1 is an example of a process for assaying biological activity in a holding enclosure of a microfluidic device according to some embodiments of the invention.

FIG. 2A is a perspective view of a microfluidic device comprising which the process of FIG. 1 can be performed according to some aspects of the invention.

FIG. 2B is a vertical cross-sectional view of the microfluidic device of FIG. 2A.

FIG. 2C is a horizontal cross-sectional view of the microfluidic device of FIG. 2A.

FIG. 3A is the microfluidic device of FIGS. 2A-2C in which the selector is configured as a dielectrophoresis (DEP) device according to some aspects of the invention, without barriers and stalls (for ease of explanation). It is a partial vertical sectional view.

FIG. 3B is a partial horizontal cross-sectional view of FIG. 3A.

FIG. 4 is an example of a process in which the biological activity of the cells in the retention enclosure can be assayed according to some embodiments of the invention.

FIG. 5A illustrates an example of the culturing step of FIG. 4 according to some aspects of the invention.

FIG. 5B illustrates an example of the culturing step of FIG. 4 in which the living cells are cultured in a retention enclosure having a separation area and a connection area.

FIG. 6 illustrates an example of the moving step of FIG. 4 according to some aspects of the invention.

FIG. 7A illustrates another example of the moving step of FIG. 4 according to some aspects of the invention.

FIG. 7B illustrates the variation of the microfluidic device of FIG. 7A in which the deflector is used to guide the capture object into the retention enclosure as the capture object flows through a channel adjacent to it. do.

8 and 9 show examples of steps to continue culturing in FIG. 4 according to some aspects of the invention. 8 and 9 show examples of steps to continue culturing in FIG. 4 according to some aspects of the invention.

FIG. 10 illustrates an example of the removal step of FIG. 4 according to some aspects of the invention.

FIG. 11A illustrates another example of the removal step of FIG. 4 according to some aspects of the invention.

FIG. 11B illustrates a variation in the removal step of FIG. 4 in which the capture object is removed from the holding enclosure containing the living cells and placed in the assay enclosure.

FIG. 11C illustrates another variation in removing the step of FIG. 4 in which the capture object is removed from the holding enclosure containing the living cells and placed in the assay enclosure.

12-14 show examples of the steps to be evaluated in FIG. 4 according to some aspects of the invention. 12-14 show examples of the steps to be evaluated in FIG. 4 according to some aspects of the invention. 12-14 show examples of the steps to be evaluated in FIG. 4 according to some aspects of the invention.

FIG. 15 is a process for testing biological activity in a retention enclosure in a microfluidic device for a first number n of features and then a second number m of features according to some embodiments of the invention. An example of is described.

FIG. 16 is an example of a process for testing n features and / or m features in the process of FIG. 15 according to some aspects of the invention.

FIG. 17 is another example of a process for testing n features and / or m features in the process of FIG. 15 according to some aspects of the invention.

FIG. 18 is an example of moving a number x of capture objects, each of which is configured to bond different materials of interest according to some aspects of the invention, into a holding enclosure continuously or in parallel. To explain.

FIG. 19 shows an example of moving a captive object configured to bond a plurality of y different materials of interest according to some aspects of the invention into a holding enclosure.

20A-20C illustrate examples of holding enclosures with regions for culturing living cells and separation regions for placing capture micro-objects. 20A-20C illustrate examples of holding enclosures with regions for culturing living cells and separation regions for placing capture micro-objects. 20A-20C illustrate examples of holding enclosures with regions for culturing living cells and separation regions for placing capture micro-objects.

Detailed Description of Illustrated Embodiments This specification describes exemplary embodiments and applications of the present invention. However, the invention is not limited to these exemplary embodiments and applications, or to the manner in which the exemplary embodiments and applications work or are described herein. The figure may also show a simplified or partial view, the dimensions of the elements in the figure may appear larger for clarity, or otherwise balanced. It does not have to be. In addition, when the terms "on", "attached to" or "coupled to" are used herein, one. An element (eg, material, layer, substrate, etc.) is one or more elements, whether or not one element is directly attached to or coupled to it on top of another. Whether the intervening element is between one element and the other, it is "on", "attached to", or "coupled" with another element. It may be ". If provided, directions (eg above, below, top, bottom, side, up, down, down) Also (under), up (over), up (upper), down (lower), horizontal (horizontal), vertical (vertical), "x", "y", "z", etc.) It is relative and is provided merely with examples for ease of explanation and review and has no purpose of limitation. In addition, when referring to a list of elements (eg, elements a, b, c), such reference consists of any one of the loaded elements itself, less than all of the loaded elements. It is intended to include combinations and / or combinations consisting of all of the mounted elements.

As used herein, "substantially" means sufficient to work as intended. Thus, the term "substantially" may be expected to be in perfect or perfect condition, size, size, insignificant small variation from the results, or, for example, those skilled in the art, but overall. It enables the same kind of things that do not affect the performance in a perceptible manner. When used with respect to numbers or features that can be expressed as parameters or numbers, the term "substantially" means within 10 percent. The term "one" means more than one.

The terms "captured object" and "captured microobject" used herein are used interchangeably and may include one or more of the following: microparticles, microbeads (eg, polystyrene beads, Luminex ™ beads). Or similar), inanimate microobjects such as magnetic beads, microrods, microwires, quantum dots; from cells (eg tissue or fluid samples, blood cells, hybridomas, cultured cells, cell lines) Biological microscopic objects such as cells, cancer cells, infected cells, transgenic and / or transformed cells, reporter cells, etc., liposomes (eg from synthetic or membrane preparations), lipids. For example, lipid nanoraft; or a combination of inanimate microobjects and biological microobjects (eg, microbeads attached to cells, microbeads coated with liposomes, magnetic beads coated with liposomes). Or the same kind). Lipid nanorafts are described, for example, in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods Enzymol., 464: 211-231.

As used herein, the terms "specifically binding" and "specifically binding" mean that ionic bonds, hydrogen bonds and / or van der Waals forces are in a specific conformation. It refers to the interaction between the ligand and the receptacle, in which the specific surface of the ligand binds to the specific surface on the receptacle, as it holds the ligand and the receptacle together. Ligands are produced and / or released by proteins (eg, therapeutic proteins, antibodies, growth factors, cytokines, cancer antigens, infectious antigens associated with viruses or other pathogens, secretory proteins, or living cells. It can be the biological material of interest, such as any other protein), nucleic acids, carbohydrates, lipids, hormones, metabolites or any combination thereof. Receptors are produced and produced by binding agents, such as proteins (eg, therapeutic proteins, antibodies, growth factors, cytokines, cancer antigens, infectious antigens associated with viruses or other pathogens, secreted proteins, or living cells. / Or any other protein released), a biological or chemical molecule such as a nucleic acid, carbohydrate, lipid, hormone, metabolite, small molecule, polymer, or any combination thereof. The specific binding of the ligand to the receptor is associated with a quantifiable binding affinity. The binding affinity can be expressed, for example, as the dissociation constant Kd.

The term "flow" as used herein in accordance with a liquid refers primarily to the bulk movement of a liquid due to any mechanism other than diffusion. For example, the flow of the medium can be accompanied by the movement of the fluid medium from one point to another due to the pressure difference between the points. Such flows may include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or a combination thereof. Turbulence and mixing of media can occur when one fluid medium flows into another.

The phrase "substantially no flow" refers to the rate of flow of a liquid that is less than the rate of diffusion of a component of the material (eg, the analyst of interest) into or within the liquid. The rate of diffusion of the components of such a material may depend, for example, on the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein in accordance with a fluid medium, "diffuse" and "diffuse" refer to the thermodynamic movement of components of a fluid medium below a concentration gradient.

The phrase "fluidically connected" as used herein in accordance with different regions within a microfluidic device is a region of the region when the different regions are substantially filled with a fluid such as a fluid medium. It means that the fluids in each are connected so as to form a single body of the fluid. This does not mean that the composition of the fluid (or fluid medium) in the different regions is necessarily the same. Rather, the fluids in the different regions of the microfluidic device that are fluidly connected are fluid so that the solute moves down and / or the fluid flows through the device. It may have different compositions (eg, solutes such as different concentrations of proteins, carbohydrates, ions or other molecules).

The microfluidic device or device of the present invention may include a "sweep" region and a "non-sweep" region. The non-swept area is fluidly connected to the swept area, provided that the fluid connection allows diffusion, but is constructed with virtually no media flow between the swept area and the non-swept area. obtain. Thus, the microfluidic device substantially separates the non-sweep region from the flow of media in the sweep region, but is substantially the only fluid communication between the sweep region and the non-sweep region. ) Can be constructed to enable.

A colony of living cells is a "clonal" if all of the living cells in the colony that are capable of replication are daughter cells derived from a single parent cell. The term "cloned cell" refers to a cell in the same cloned colony.

In some embodiments of the invention, biological activity in a retention enclosure in a microfluidic device holds a trapping object in the retention enclosure that binds a particular material of interest produced by the biological activity. By placing, it can be assayed. The material of interest then coupled to each trapped object can then be evaluated in a microfluidic device. Accordingly, aspects of the invention can efficiently assay biological activity that occurs in a retention enclosure in a microfluidic device. Also, if biological activity involves each cloned cell colony that produces a particular biomaterial in one of the retention enclosures, some embodiments of the invention retain the clone of each colony (eg,). (Without mixing cells capable of replicating from any one colony with any other colony), the ability of each colony to produce the material of interest in a microfluidic device can be assessed.

FIG. 1 illustrates an example of assay processing 100. 2A-2C illustrate examples of microfluidic devices 200 for performing process 100, FIGS. 3A and 3B illustrate examples of dielectrophoresis (DEP) devices that may be part of microfluidic device 200. ..

As shown in FIG. 1, process 100 may move the captured object into the holding enclosure in the microfluidic device in step 102, and process 100 produces biomaterial of particular interest in step 104. Biological activity can be cultured in each of the retention enclosures. The retaining enclosure may include a non-swept area and biological activity may be located or placed in such a non-swept area. Biological activities include egg mother cells, sperm, cells dissociated from tissues, blood cells (eg B cells, T cells, macrophages and the like), hybridomas, cultured cells, cells from cell lines, It can be, or can consist of, one or more cells such as cancer cells, infected cells, transgenic and / or transformed cells, reporter cells and allogeneic ones. The captured object may contain one or more binding substances, each of which specifically binds to a particular biomaterial of interest. For example, the binding material of the capture object is at least about 1 mM or stronger (eg, about 100 μM, 10 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 2. It may have an affinity (eg, Kd) for a particular biomaterial of interest (5 nM, 1 nM or stronger). Such affinity is greater than affinity for any other material other than the biomaterial of a particular purpose (or at least any other biomaterial present in the holding enclosure and / or microfluidic device). Can also be stronger, for example in multiples of 2x, 3x, 4x, 5x, 10x or more. Thus, it can be said that each trapping object binds one or more specific biomaterials of interest, but does not substantially bind other biomaterials in the holding enclosure. After a while, the captured objects may be removed from the holding enclosure in step 106, and the interrelationship between the captured objects removed and the enclosure from which each captured object was taken is a step. Can be maintained at 108. Biological activity in each retention enclosure can be assessed in step 110 by analyzing the biomaterial bound to the trapped object removed from the retention enclosure. For example, process 100 obtains a rate of biological activity in each retention enclosure by determining in step 110 the amount of biomaterial bound by the capture object removed from the retention enclosure. Estimates may include, for example, determining whether the colonies in each retention enclosure produce the material of interest at or above the threshold. As another example, estimates can quantify the amount of material of interest produced by colonies in each retention enclosure.

FIG. 1 is an example, and many variations of the process 100 are considered. For example, process 100 may assess biological activity in step 110 while the capture object is in the holding enclosure, and therefore process 100 does not need to include steps 106, 108 in some variation. , Or steps 106, 108 may be skipped. As another example, steps 102-110 need not be performed in the order shown in FIG. For example, steps 102 and 104 can be reversed.

2A-2C illustrate an example of a microfluidic device 200 on which the process 100 can 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 the liquid medium 244. FIG. 2B describes the inner surface 242 of the flow region 240 in which the medium 244 can be placed as being flat (eg, flat) and featureless. However, the inner surface 242 may instead be non-flat (eg, non-planar) and may include features (not shown) such as electrical terminals.

The housing 202 may include one or more inlets 208 through which the medium 244 may be loaded into the flow region 240. The inlet 208 may be, for example, an inlet, an opening, a valve, another channel, a fluid connector or the like. The housing 202 may also include one or more outlets 210 through which the medium 244 may be removed. The outlet 210 can be, for example, an outlet, an opening, a valve, a channel, a fluid connector or the like. As another example, Exit 210 is a droplet ejection mechanism such as any of the ejection mechanisms disclosed in U.S. Patent Application No. 13 / 856,781 (attorney docket no. BL1-US) filed April 4, 2013. It can also include a mechanism. All or part of the housing 202 may be gas permeable to allow gas (eg, ambient air) to enter and exit the flow region 240.

The housing 202 may also include a microfluidic structure 204 located on a base (eg, a substrate) 206. The microfluidic structure 204 is a gas permeable, flexible material such as rubber, plastic, elastomer, silicone (eg, patterned silicone), polydimethylsiloxane (“PDMS”) or the like. May include. Alternatively, the microfluidic structure 204 may include other materials, including rigid materials. The base 206 may include one or more substrates. Although described as a single structure, the base 206 may include a large number of interconnected structures such as a large number of substrates. The microfluidic structure 204 may similarly include a number of structures that can be interconnected. For example, the microfluidic structure 204 may additionally 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 FIGS. 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, 253 in FIG. 2C) and chambers that may be interconnected to form a microfluidic circuit. In an enclosure containing more than one flow region 240, each flow region 240 has one or more inlets 208 and one or more for charging the medium 244 and removing it from the flow region 240, respectively. Can be associated with exit 210 of.

As shown in FIGS. 2B and 2C, the holding enclosure 256 may be placed in the flow region 240. For example, each holding enclosure 256 may include a barrier 254 forming a partial enclosure. The partial enclosure may define a flow-free space (or separation area). Therefore, a portion of the interior of each retaining enclosure 256 does not allow the medium 244 from the channel 252 to flow directly into it, except when the empty flow area 240 is initially filled with the medium 244. It can be a space. For example, each holding enclosure 256 may include one or more barriers 254 forming a partial enclosure, the inside of which may include a flow-free space. Thus, the barrier 254 defining the retention enclosure 256 ensures that the medium 244 flows directly from the channel 252 into the protected interior of any retention enclosure 256 while the flow region 240 is filled with the medium 244. Can hinder. For example, the barrier 254 of enclosure 256 can substantially prevent bulk flow of medium 244 from channel 252 to the flow-free space of enclosure 256 while the flow region 240 is filled with medium 244, instead in enclosure 256. Only diffusible mixing of the medium in the flow-free space with the medium from the channel 252 is substantially possible. Accordingly, the exchange of nutrients and waste between the flow-free space in the retention enclosure 256 and the channel 252 can be substantially the only result of diffusion.

The above can be achieved by orienting the enclosure 256 so that any opening into the enclosure 256 does not directly face the flow of the medium 244 in the channel 252. For example, if the flow of medium in channel 252 in FIG. 2C is from inlet 208 to exit 210 (hence left to right), each of the enclosures 256 will not face to the left in FIG. 2C because each opening of the enclosure 256 does not. It substantially impedes the direct flow of the medium 244 from the channel 252 into the enclosure 256, which would have been direct into such a flow.

Within the flow region 240 arranged in any pattern, there may be many such holding enclosures 256, which may be of any of many different sizes and shapes. As shown in FIG. 2C, the opening of the holding enclosure 256 may be located adjacent to channels 252 and 253, and the channels 252 and 253 may be spaces adjacent to more than one opening of the enclosure 256. The opening of each retention enclosure 256 may allow the natural replacement of the liquid medium 244 flowing through channels 252 and 253, otherwise each retention enclosure 256 is a living cell in any one enclosure 256. Small objects such as (not shown) can be sufficiently closed to prevent them from mixing with the small objects in another enclosure 256. Eight enclosures 256 and two channels 252, 253 are shown, but may be more or less. The medium 244 can pass through the openings in the holding enclosure 256 and flow into channels 252 and 253. The flow of media in channels 252 and 253 may provide nutrients, for example, to biological objects (not shown) in the retention enclosure 256. As another example, the flow of media 252, 253 in the common flow space 252, 253 may also provide the removal of waste from the holding enclosure 256.

As shown in FIG. 2C, the anchorage 258 may also be located in the flow region 240, eg, in channels 252 and 253. Each of the anchorages 258 may be configured to keep micro-objects (not shown) in place with respect to the flow of medium 244 in channels 252 and 253. The anchorage 258 and barrier 254 of enclosure 256 may include any of the materials discussed above for the microfluidic structure 204. The anchorage 258 and the barrier 254 may contain the same or different materials as the microfluidic structure 204. The barrier 254 may extend from the surface 242 of the base 206 beyond the entire flow region 240 to the upper wall of the microfluidic structure 204 (opposite the surface 242), as shown in FIG. 2B. Instead, one or more barriers 254 can only partially extend beyond the flow region 240 and thus do not extend entirely to the surface 242 or the upper wall of the microfluidic structure 204. Although not shown, the anchorage 258 and / or the barrier 254 may include additional features such as one or more relatively small openings through which the medium 244 can pass. Such an opening (not shown) may be smaller than the micro object (not shown) to prevent the micro object from passing through.

The selector 222 may be configured to selectively generate an electrokinetic force on a tiny object (not shown) in the medium 244. For example, the selector 222 may be configured to selectively activate (eg, turn on) and start and stop (eg, turn off) the electrodes on the inner surface 242 of the flow region 240. The electrodes may generate a force in the medium 244 that attracts or moves away micro-objects (not shown) in the medium 244, so the selector 222 may select and move one or more micro-objects in the medium 244. .. The electrode can be, for example, a dielectrophoresis (DEP) electrode.

For example, the selector 222 is one or more optical (eg, laser) tweezers devices and / or one or more photoelectric tweezers (OET) devices (eg, U.S. Patent No. 7,612,355, which is hereby in its entirety by reference. (Incorporated in) or U.S. Patent Application No. 14 / 051,004 (attorney docket no. BL9-US) (also as disclosed herein in its entirety 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 of microobjects are suspended. Such devices (not shown) may include electrowetting devices such as photoelectric wetting (OEW) devices (eg, as disclosed in U.S. Patent No. 6,958,132) or other electrowetting devices. Thus, the selector 222 can be characterized as a DEP device in some embodiments.

3A and 3B illustrate an example in which the selector 222 includes an OET DEP device 300. As shown, the DEP device 300 may include a first electrode 304, a second electrode 310, an electrode activation substrate 308, a power source 312 (eg, 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. Changing the pattern 322 of the light from the light source 320 may selectively start and stop the changing pattern of the DEP electrode in the region 314 of the inner surface 242 of the flow region 240. (Hereinafter, the region 314 is also referred to as an "electrode region".)

In the example illustrated in FIG. 3B, the light pattern 322'directed onto the inner surface 242 illuminates the oblique light parallel electrode region 314a in the square pattern shown. The other electrode region 314 is not exposed to light, which is sometimes referred to as the "dark" electrode region 314. The relative electrical impedance from each dark electrode region 314 to the second electrode 310 beyond the electrode activation substrate 308 is from the relative impedance from the first electrode 304 to the dark electrode region 314 beyond the medium 244 in the flow region 240. big. However, shining light on the electrode region 314a causes the relative impedance from the lighted electrode region 314a to the second electrode 310 beyond the electrode activation substrate 308 from the first electrode 304 to the flow region 240. It is reduced beyond the medium 244 to less than the relative impedance to the electrode region 314a to which the light is applied.

Upon activation of the power supply 312, the aforementioned causes an electric field gradient in the medium 244 between the lighted electrode region 314a and the adjacent dark electrode region 314, which in turn near the medium 244. Generates a local DEP force that attracts or moves away small objects (not shown). Thus, the DEP electrode that attracts or moves away micro-objects in the medium 244 is a light pattern 322 projected into the microfluidous device 300 from a light source 320 (eg, a laser source, a high-intensity discharge lamp or other type of light source). Can be selectively started and stopped at many different such electrode areas 314 on the inner surface 242 of the flow area 240. Whether the DEP force attracts or moves away nearby micro-objects may depend on parameters such as the frequency of the power supply 312 and the dielectric properties of the medium 244 and / or micro-objects (not shown).

The square pattern 322'of the illuminated electrode region 314a, described in FIG. 3B, is only an example. Any pattern of the electrode region 314 can be illuminated by the pattern 322 of light projected into the device 300, and the pattern of the illuminated electrode region 322'is repeated by varying the optical pattern 322. Can be changed.

In some embodiments, the electrode activation substrate 308 may be a light conductive material and the inner surface 242 may be featureless. In such an embodiment, the DEP electrode 314 can be generated anywhere and in any pattern on the inner surface 242 of the flow region 240 according to the optical pattern 322 (see FIG. 3A). Therefore, the number and pattern of the electrode regions 314 are not fixed, but correspond to the optical pattern 322. An example is described in U.S. Patent No. 7,612,355 above. Here, the non-doped amorphous silicon material 24 shown in the drawings of the patent described above may be an example of a light conductive material that can constitute the electrode activation substrate 308.

In another embodiment, the electrode activation substrate 308 may include a circuit board such as a semiconductor material including a plurality of dope layers, an electrically insulating layer and a conductive layer forming a semiconductor integrated circuit known in the semiconductor field and the like. In such an embodiment, the electrical circuit element forms an electrical connection between the electrode region 314 on the inner surface 242 of the flow region 240 and the second electrode 310 which can be selectively started and stopped by the optical pattern 322. obtain. When not activated, each electrical connection is high so 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 304 to the corresponding electrode region 314 via the medium 244. Can have impedance. However, when activated by the light in the light pattern 322, the relative impedance from the corresponding electrode region 314 to the second electrode 310 is smaller than the relative impedance from the first electrode 304 to the corresponding electrode region 314 via the medium 244. As such, each electrical connection can have a low impedance, which activates the DEP electrode in the corresponding electrode region 314, as described above. Thus, the DEP electrodes that attract or alienate microscopic objects (not shown) in the medium 244 are selectively activated and stopped by the optical pattern 322 at many different electrode regions 314 on the inner surface 242 of the flow region 240. Can be done. Non-limiting examples of such configurations of the electrode activation substrate 308 are described across the optical transistor-based OET device 300 described in FIGS. 21 and 22 of U.S. Patent No. 7,956,339 and the drawings in U.S. Patent Application No. 14 / 051,004 above. Includes OET devices to be used.

In some embodiments, the first electrode 304 may be part of the first wall 302 (or cover) of the housing 202, the electrode activation substrate 308 and the second electrode 310, as generally described in FIG. 3A. It can be part of the second wall 306 (or base) of the housing 202. As shown, the flow area 240 can be between the first wall 302 and the second wall 306. However, the above is nothing but an example. In another embodiment, the first electrode 304 may be part of the second wall 306 and 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 part 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 a first electrode 304 and / or a second electrode 310. Also, the light source 320 is instead positioned below the housing 202.

Therefore, when configured as the DEP device 300 of FIGS. 3A and 3B, the optical pattern 322 is a pattern that surrounds and captures a minute object in order to activate one or more DEP electrodes in the electrode region 314 of the inner surface 242 of the flow region 240. By projecting into the device 300 at, the selector 222 may select a small object (not shown) in the medium 244 in the flow region 240. The selector 222 can then move the captured micro-object by moving the light pattern 322 relative to the device 300. Instead, the device 300 can be moved relative to the light pattern 322.

The barrier 254 defining the holding enclosure 256 is described in FIGS. 2B and 2C and described above as a physical barrier, but the barrier 254 is instead a virtual barrier containing the DEP force activated by the light pattern 322. obtain. Similarly, the anchorage 258 may include physical and / or virtual barriers including DEP forces activated by the optical pattern 322.

With reference to FIGS. 2A-2C again, the detector 224 can be a mechanism for detecting an event in the flow region 240. For example, the detector 224 may include an optical detector capable of detecting one or more radiating features (eg, by fluorescence or cold light) of a small object (not shown) in the medium. For example, such detector 224 is configured to detect that one or more microscopic objects (not shown) in the medium 244 emit electromagnetic radiation and / or radiation of similar wavelength, brightness, brightness or the like. Can be done. Examples of suitable optical detectors include, but are not limited to, photomultiplier tube detectors and avalanche optical detectors.

The detector 224 may optionally or additionally include an imaging device for capturing a digital image of the flow region 240 containing micro-objects (not shown) in the medium 244. Examples of suitable imaging devices that the detector 224 may include include digital cameras or optical sensors such as charge-coupled devices and complementary metal oxide semiconductor imaging devices. Images can be captured and analyzed by such devices (eg by control module 230 and / or human operators).

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 controller 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 flow speed of the medium 244 in the flow region 240.

The control module 230 may be configured to receive and control signals from the 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 is a machine-readable instruction (eg, software, eg, software, eg, stored as a non-transient signal in memory 234, which can be a digital electronic, optical or magnetic memory device. It can be a digital electronic controller (eg, a microprocessor, a microcontroller, a computer or the like) configured to operate according to firmware, a microcontroller, or the like. Instead, the controller 232 comprises a combination of a real-wired digital network and / or an analog network or a digital electronic controller that operates according to machine-readable instructions and a real-wired digital and / or analog network. obtain.

As discussed, the microfluidic device 200 is an example of a device that can be used to perform the process 100. For example, in step 102, the selector 222 (configured as shown, eg, FIGS. 3A and 2B) may select a trapped object (not shown) in the medium 244 in the flow region 240 and of the holding enclosure 256. The selected capture object can be moved into it. At step 104, nutrients may be provided to biological micro-objects (not shown) in enclosure 256 in the flow of medium 244 in channels 252 and 253. In step 106, the selector 222 may select and remove the captured object (not shown) from the enclosure 256, and in step 108 the detector 224 and the controller 232 are removed with the enclosure 256 from which the captured object was taken. Each captured object (not shown) can be associated with each other. For example, the detector 224 may capture images of the captured object (not shown) and the enclosure 256, and the controller 232 may store the interrelationships as digital data in memory 234. At step 110, the biomaterial is bound to each captured object (not shown) that can be evaluated in the microfluidic device 200. For example, the detector 224 may capture an image or detect the characteristics of the captured object (not shown) to be removed in order to evaluate the biomaterial bound to the captured object to be removed.

FIG. 4 illustrates another example of processing 400 for assaying biological activity in a holding enclosure of a microfluidic device. Treatment 400 may be a narrower example of the more general treatment 100, where the biological activity in the retention enclosure is the production of the desired biomaterial by the clonal colonies of the cells in treatment 400 of FIG. For ease of description and discussion, process 400 is discussed below with respect to the microfluidic device 200 of FIGS. 2A-2C, in which the selector 222 may be configured as described in FIGS. 3A and 3B. However, the process 400 is not so limited and can therefore be performed on other microfluidic devices.

As shown in FIG. 4, treatment 400 may culture the product of clonal cell colonies in the holding enclosure 256 of the microfluidic device 200 in step 402. 5A (showing a horizontal cross-sectional view of a portion of the flow region 240 of the microfluidic device 200 of FIGS. 2A-2C, as in FIGS. 6, 7A, 8-11B and 12-14) and FIG. 5B illustrate examples. do.

As shown in FIGS. 5A and 5B, living cells 502 can be cultured in one or more retention enclosures 256 by a liquid medium 244 flowing into channels 252 adjacent to at least some openings in enclosure 256. The nutrients in the stream 506 can cultivate the biological activity in the retention enclosure 256. Flow 506 may also provide waste removal from enclosure 256. A similar flow may be provided in another channel adjacent to the opening of the other enclosure 256 of the device 200 (eg, 253 shown in FIG. 2C).

FIG. 5B shows an enclosure having a separation area 508 and a connection area 510. As is known, the flow 506 of the fluid medium 244 in the microfluidic channel 252 through the proximal opening of the enclosure 256 can cause a second flow of the medium 244 into and / or out of the enclosure. .. In order to separate the micro-object 502 in the isolation region 508 of the enclosure 256 from the second flow, the length of the connection region 510 of the isolation enclosure 256 from the proximal opening to the distal opening is the flow 506 in the channel 252. When the speed of is maximum (V _max ), it may be larger than the maximum penetration depth _Dp of the second flow into the connection region 510. Thus, unless the flow 506 in the channel 252 exceeds the maximum speed _Vmax , the flow 506 and the resulting second flow can be limited to the channel 252 and the connection region 510 and are outside the separation region 508. Can continue. Thus, the flow 506 in channel 252 will not pull the biological micro-object 502 (or any other micro-object) out of the separation region 508. Thus, the biological micro-object 502 in the separation region 508 will remain in the separation region 508, independent of the flow 506 in the channel 252.

Culturing in step 402 may facilitate cell propagation or cell 502 in each enclosure 256 to produce colonies 500 of cells 502 in each enclosure 256. Each enclosure 256 from cells 502 in all of the other enclosures 256 to sufficiently prevent cells 502 in any one enclosure 256 from mixing with cells 502 in any other enclosure 256. 502 can be separated. Also, the colony 500 produced in each retention enclosure 256 can begin with a single cell 502 in the enclosure 256. Thus, the colony 500 of cells 502 in each enclosure 256 can be a clone.

Culturing in step 402 can also facilitate the production of material 504 of particular interest to be assayed. Non-limiting examples of the material 504 of interest include proteins, nucleic acids, carbohydrates, lipids, hormones, metabolites or any combination thereof. The protein of interest is produced and / or released by, for example, therapeutic proteins, antibodies, growth factors, cytokines, cancer antigens, infectious antigens associated with viruses or other pathogens, secretory proteins or living cells. It may contain proteins such as any other protein. Thus, for example, the cell 502 can be a protein that produces the cell (eg, an antibody) and the material 504 of interest can be a particular protein (eg, a particular antibody). For example, the material of interest can be an immunoglobulin G (IgG) isotype antibody. Materials containing biomaterials other than the material of interest 504 may be in the enclosure. For example, cell 502 may produce other materials in addition to the material of interest 504.

In some embodiments, culturing in step 402 may involve culturing multiple types . For example, a first stream 506 of the first type of medium 244 may culture the growth and division of cells 502 in each enclosure 256. A second stream of the second type of medium 244 can then culture the production of material 504 of interest by cells 502 in each enclosure 256.

Process 400 may move the capture object 602 into the holding enclosure 256 in step 404 of FIG. 4 (see FIG. 6). The capture object 602 is an inanimate microobject such as microparticles, microbeads (eg, polystyrene beads, Luminex ™ beads or the like), magnetic beads, microrods, microwires, quantum dots or the like. Can be. In some cases, the capture object 602 is an inanimate microbead and a biological microbead (eg, liposome-coated microbeads, liposome-coated magnetic beads, cell-attached microbeads, or It can be a combination of the same kind). In yet other cases, the capture object 602 can be a biological microobject such as a cell, liposome, lipid nanoraft or the like. Also, each capture object 602 may include a particular binding substance that specifically binds to a particular biomaterial of interest. The captured object 602, for example, is at least about 1 mM or stronger (eg, about 100 μM, 10 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, It may contain a particular binding agent (1 nM or stronger), having an affinity (eg, Kd) for a particular biomaterial of interest. Such affinity is greater than affinity for any other material other than the biomaterial of a particular purpose (or at least any other biomaterial present in the holding enclosure and / or microfluidic device). Can also be stronger, for example in multiples of 2x, 3x, 4x, 5x, 10x or more. For example, if the material 504 of interest is a particular antibody, the capture object 602 has a greater affinity for that particular antibody than for any other material in the retention enclosure 256 and / or microfluidic device. It may contain a binding agent having sex (eg, an antigen or an epitope thereof). As mentioned, the material 504 of interest may be an IgG antibody, where the binding agent of the capture object 602 may include a material with an IgG Fc receptor for binding the IgG antibody. 6-8 show an example of step 404.

As shown in FIG. 6, the capture object 602 may be placed in the channel 252 adjacent to the opening to the enclosure 256. As shown in FIGS. 7A-7B and 8, individual capture objects 602 can be moved into a particular enclosure 256.

The capture object 602 can be introduced into the microfluidic device 200 through the inlet 208 (see FIGS. 2A-2C) and can be moved to channel 252 with the flow 506 as shown in FIG. FIG. 7A illustrates an example of generating an optical trap 702, wherein a selector 222 (see FIGS. 2A-2C) configured like the DEP device 300 of FIGS. 3A and 3B provides a separate capture object 602. Can be caught. The DEP device 300 may then move the light trap 702 into one of the enclosures 256, which moves the captured capture object 602 into the enclosure 256. The light trap 702 may be part of altering the pattern 322 of light projected onto the inner surface 242 of the flow region 240 of the microfluidic device 300, as described above with respect to FIGS. 3A and 3B. When the capture object 602 is in the enclosure 256, the light trap 602 corresponding to the capture object 602 can be turned off as described in FIG. The detector 224 may capture images of all or part of the flow region 240, which may facilitate the capture and movement of the individual capture object 602 into a particular enclosure 256. Thus, a particular number (eg, 1 or more) of captured objects 602 can be identified, selected, and moved into each enclosure 256.

As shown in FIG. 7A, the flow 506 of the medium 244 may be stationary after the flow 506 brings the trapped object 602 into the channel 252. Restoring the flow 506 can facilitate the identification and selection of individual capture objects 602. As shown in FIG. 8, if the capture object 602 is in enclosure 256, flow 506 can be resumed. Instead, rather than stagnating the flow 506, the flow 506 simply detects the individual trapped object 602 in channel 252 by the detector 224 and is slow enough for the selector 222 to catch and move it. Can be delayed. Yet another alternative, the flow 506 can be started and maintained at a generally stable ratio slow enough for the detector 224 to detect the individual capture object 602 and the selector 222 to capture and move it. In such cases, the flow 506 can generally be maintained at a constant rate in each of FIGS. 6, 7A and 8.

Although FIG. 7A illustrates capturing one capture object 602 for each trap 702, the trap 702 may capture more than one capture object 602. Similarly, FIG. 8 shows one capture object 602 in each enclosure 256, but more than one capture object 602 can be moved into the enclosure 256. Regardless of that, a particular, known number of capture objects 602 (eg, 1 or more) can be moved into each enclosure 256. Generally speaking, the order of steps in processes 100 and 400 is not significant, so for example the order of steps 404 and 402 can be reversed. For example, the capture object 602 can be placed in the holding enclosure 256 even before the first cell 502 is placed in the enclosure 256. In such cases, the treatment may include steps to transfer biological activity (eg, living cell 502) into the retention enclosure 256.

FIG. 7B illustrates a more passive approach to loading capture object 602 into retention enclosure 256 instead of actively selecting and moving capture object 602 into retention enclosure 256. do. The microfluidic device of FIG. 7B is similar to the microfluidic device shown in FIG. 7A, except that there is a deflector 754 located in the channel 252 just outside the holding enclosure 256. A small portion of the capture object 602 will be carried into the microfluidic device and around the channel 252 as it is flushed through the channel 252. The captured object 602 carried by the flow 506 around the channel 252 can be captured by the deflector 754 and deflected into the holding enclosure 256. The use of a deflector is different from the approach of using an optical trap to select a particular capture object 602 and move it into a particular retention enclosure 256, as shown in FIG. 7B, exactly which capture object 602. Or, it does not allow close control of how many capture objects 602 are moved into each holding enclosure 256. However, the use of the deflector 754 can facilitate the loading of a large number of holding enclosures at the same time.

The deflector 754 shown in FIG. 7B can be made of the same material as the barrier 254 or any other suitable material considered herein. In addition, the deflector 754 can be separated or adhered to the barrier 254 (as shown). The deflector 754 can extend to the full height of the channel 252, or it can only partially extend upward through the channel, thereby distracting the trapping object 602 (or cell or other organism) into the holding enclosure 256. Potentially reduce the number of (physical micro-objects). Also, the deflector 754 can be a virtual barrier created by light focused on the surface 242 of channel 252. Such light can activate an electrode (eg, a DEP electrode), thereby creating a barrier to the capture object 602 (or cell 502) in the manner of a light trap as described above. Such a virtual deflector can be advantageous as it can be turned off if the threshold number of the capture object 602 is deflected into the holding enclosure 256. For example, a human user or controller 232 may monitor the number of captured objects 602 deflected into any particular holding enclosure 256 and then activate the electrodes when the threshold number of captured objects 602 is reached. It can turn off the light that is turning (and thereby causing the deflector).

A high proportion of the flow 506 of the medium 244 in the channel 252 enters the retention enclosure 256 as yet another alternative to actively selecting the capture object 602 and moving it into the retention enclosure 256. It can be used to increase the penetration depth _Dp of the flow. Thus, the captured object 602 can be pushed into the holding enclosure 256 by increasing the ratio of the flow 506 of the medium 244 in the channel 252. In some embodiments, the microfluidic device has a channel 252 with a cross-sectional area of about 3,000 to 6,000 square microns or about 2,500 to 4,000 square microns. The ratio of the flow 506 of the medium 244 suitable for loading the capture object 602 into the holding enclosure 256 in such a microfluidic device is, for example, about 0.05 to 5.0 μE / sec (eg, about 0. 1 to 2.0, 0.2 to 1.5, 0.5 to 1.0 μE / sec or about 1.0 to 2.0 μE / sec).

Culturing the cells 502 in the enclosure 256 in step 406 of FIG. 4 may continue for as long as the cells 502 can continue to propagate and / or produce the material 504 of interest in the meantime. As described in FIG. 9, the capture object 602 in a particular enclosure 256 may bind the material 504 of interest produced by the cells 502 in that enclosure 256. Thus, FIG. 9 shows the material 504 of interest coupled to the trapped object 602 in enclosure 256.

In some embodiments, the purpose of assay treatment 400 of FIG. 4 may be to identify cell colonies 500 in enclosure 256 that produce the material 504 of interest at the minimum threshold. In such an embodiment, the amount of the material 504 of interest capable of binding one or more capture objects 602 in any one enclosure 256, and the period of step 406, to produce the material 504 of interest at a minimum threshold or greater. The colony 500 may be in an amount and duration of production, such as material 504 of interest, sufficient to immerse the trapped object (s) 602 in the enclosure 256.

In another aspect, the purpose of assay processing 400 may be to determine the quantity of material 504 of interest produced in each enclosure 256. In such an embodiment, the amount of the material 504 of interest to which one or more capture objects 602 in the enclosure 256 can bind, and the period of step 406, in the colony 500 producing the material 504 of interest in the highest possible proportion. Even the amount and duration can be such that the trapped object (s) 602 or the like in the enclosure 256 is not soaked.

Treatment 400 assayed a single biological object 502 (eg, a single living cell) in the retention enclosure 256, as described in the retention enclosure 256 on the right of pages in FIGS. 5-14. Can be. The ability to assay a single biological object 502 in a retention enclosure 256 is sufficient, for example, for known techniques for assaying living cells to assay materials produced by, for example, a single cell. It is significant because it is believed that there is no sensitivity.

Process 400 may select individual capture objects 602 from a particular enclosure 256 and capture objects 602 selected from enclosure 256 in step 408 after the period of step 406 described above, as described in FIG. Can be removed. In some embodiments, the captured object 602 to be removed can be moved into channel 252. 10 and 11A show an example of step 408.

As shown in FIG. 10, the individual capture object 602 may be selected in a particular enclosure 256 with a possible optical trap 1002, such as the optical trap 702, as described above. As shown in FIG. 11A, the captured object 602 can be removed from enclosure 256 and placed in channel 252 adjacent to the opening of enclosure 256. For example, the optical trap 1002 can be moved from the enclosure 256 into the channel 252. Also, as shown in FIG. 11A, the captured object 602, as examined, is moved to the anchorage 258 in the channel 252, which can hold the captured object 602 in place with respect to the flow 506 of the medium 244 in the flow region 240. Can be. The optical trap 1002 can be turned off if the removed captured object 602 is moved to the anchorage 258. Alternatively, the optical trap 1002 may continue to hold the removed captured object 602 in place, eg, against flow 506 of medium 244. In such a case, the stop 258 does not need to be included in the flow area 240 of the device 200. Regardless of that, the flow 506 can be slowed down or even stagnated during step 408. Yet another alternative, the capture object 602 may be removed from the device for subsequent analysis if moved into channel 252. A suitable method for carrying out a captured object is disclosed, for example, in U.S. Patent Application No. 14 / 520,150, filed October 22, 2014, the entire contents of which are herein by reference. Be incorporated. The capture objects 602 may be individually carried out with a group of capture objects from the same retention enclosure 256, or with a group containing capture objects 602 from a plurality of retention enclosures 256. In the latter case, the captured objects 602 may have an identifier that facilitates their identification and association with the retaining enclosure 256 removed from them. For example, Luminex ™ beads can be used as a capture object 602, which allows the capture object 602 from a particular retention enclosure 256 to be distinguished from the capture objects from other retention enclosures 256. Become.

As shown in FIG. 11B, instead of moving the capture object 602 to the anchorage 258 in the channel 252 involves moving the capture object 602 to the assay region 1156. The assay region 1156 may be adjacent to the retention enclosure 256, thereby reducing the time required to move the capture object 602, with the capture object 602 and the retention enclosure 256 removed from it. Facilitates the maintenance of interrelationships between. The assay region can be defined by a barrier 254 or a barrier 1154 which can be made of the same material as any other suitable material considered herein. The assay region 1156 is shown to have the same size and shape as the holding enclosure 256, but may be smaller and / or may have a different shape. For example, assay region 1156 may be smaller and may or may not include a separation region. Thus, for example, assay region 1156 may substantially lack a separation region (eg, less than 50% of the volume of assay region may be separated from a second stream of stream 506 of medium 244 in channel 252). In some embodiments, the substantial lack of separation region may facilitate flushing of assay material from capture object 602 (further discussed below).

Instead of using the optical trap 1002 to move the capture object 602 out of the holding enclosure 256, the magnetic capture object 602 can be forced out of the enclosure 256 using a magnetic force such as a magnet. As shown in FIG. 11C, the microfluidic device 1100 may include an assay region 1156 positioned beyond the channel 252 from the aperture to the retention enclosure 256. To move the magnetic capture object 602 out of the holding enclosure 256 and into the assay region 1156, a magnetic force is applied so that the magnetic capture object 602 is pulled or pushed into the assay region 1156. Can be given to microfluidic devices. During such a step, the flow 506 of the medium 244 in the channel 252 can be slowed down or stagnant.

In FIGS. 10 and 11A-11C, one capture object 602 is shown as being removed from each enclosure 256, but as discussed above, more than one capture object 602 is placed in enclosure 256 in step 404. Obtained, in such cases, more than one capture object 602 may be removed from the enclosure 256 accordingly in step 408.

Returning to FIG. 4 again, in step 410, process 400 maintains the interrelationship between each captured object 602 removed from the enclosure 256 in step 408 and the enclosure 256 from which the captured object 602 is removed. obtain. For example, the controller 232 may identify and trace the location of the capture object 602 and enclosure 256 from the image provided by the detector 224, and the controller 232 may identify the individual removed capture objects in the channel 252. The interrelationship between 602 and the enclosure 256 from which each captured object 602 was taken may be stored in memory 234. Table 1 is an example of a digital table that can be stored in memory 234, relating the position in channel 252 of the capture object 602 and the enclosure 256 from which the capture object 602 has been removed. In the example of Table 1, the captured object 602 at the stop 258 identified as the stop A was taken from the enclosure 256 numbered 3. Similarly, the captured object 602 at the anchorage 258B is taken from the enclosure 256 numbered 1 and the captured object 602 at the anchorage 258C is taken from the enclosure 256 numbered 2 rice field. The corresponding table can be used to store data about the location of the capture object 602 in the assay region 1156 and the retention enclosure 256 from which the capture object 602 was removed. Similarly, the table for the capture object 602 carried out of the microfluidic device for analysis relates to the particular capture object 602 and the identifier associated with the holding enclosure 256 from which the capture object 602 was removed. Can be used to store data.

In step 412 of FIG. 4, process 400 may evaluate the material 504 of interest bound to the removed trapped object 602 in channel 252. For example, treatment 400 evaluates the material 504 of interest in step 412 by determining the quantity and / or quality of the material 504 of interest produced by a single cell 502 in the cell colony 500 or enclosure 256. Can be. As another example, treatment 400 may evaluate in step 412 the type of material 504 produced by a colony 500 of cells 502 or a single cell 502 in enclosure 256. As yet another example, treatment 400 may assess the activity of material 504 produced by a single cell 502 in a colony 500 of cells 502 or enclosure 256 in step 412. In step 408, the material 504 of interest bound to the capture object 602 was produced by biological activity in the enclosure 256 from which the capture object 602 was removed, so that the capture object 602 removed in step 412. The evaluation of the material 504 of interest to be bound may then provide information on which the biological activity in the enclosure 256 can be evaluated. FIGS. 12-14 explain an example of step 412.

As shown in FIG. 12, in step 412, assay material 1202 can be flushed through channel 252. Assay material 1202 can both bind to material 504 of interest on the removed capture object 602 and present separate, detectable behavior. For example, assay material 1202 may contain a label comprising a binding material that specifically binds to the biomaterial 504 of interest (eg, at a position on the material 504 that is different from the position bound by the capture object 602). .. Where the biomaterial 504 of interest is an antibody, the label may include an Fc receptor, the capture object 602 may contain an antigen bound by the antibody, and vice versa. In the examples shown in FIGS. 12-14, assay material 1202 may include a label that binds to material 504 of interest on the capture object and radiates energy 1402, as shown in FIG. Thus, for example, assay material 1202 may contain a binding substance that specifically binds to and binds to the chromophore of the biomaterial 504 of interest. In another embodiment, assay material 1202 may comprise, by way of example, a luciferase-linked binding material that specifically binds to the biomaterial 504 of interest. In the latter case, assay material 1202 may additionally contain a suitable luciferase substrate (eg, a luciferin substrate). Thus, assay material 1202 may fluoresce or emit cold light. Regardless, assay material 1202 is in sufficient quantity and for sufficient time to bind substantially all of the material 504 of interest to which assay material 1202 is bound to the removed capture object 602. During that time, it may be provided to the captured object 602 that has been removed.

As shown in FIG. 13, assay material 1202 that was not subsequently bound to one of the removed capture objects 602 can be flushed out of channel 252. For example, assay material 1202 flow 506 may be followed by media flow 244 (or any cleaning material) in channel 252, which did not bind to the material of interest 504 on the removed capture object 602. All of 1202 can be flushed out of channel 252 substantially. As shown in FIG. 13, the capture object 602 in the channel 252 may now include the target material 504 bound to the capture object 602 and the assay material 1202 bound to the target material 504.

As shown in FIG. 14, assay material 1202 can radiate energy 1402 that can be detected by detector 224. In some embodiments, assay material 1202 is stimulated (with, for example, light or other radiation, or a chemical catalyst or substrate (which can be shed through channel 252)) to induce radiation of energy 1402. It may need to be done. Detectable features such as the level, brightness, color (eg, specific wavelength) or similar of energy 1402 emitted from the removed captured object 602 are the assay material bound to the removed captured object 602. It could correspond to an amount of 1202, which could correspond to the amount of biomaterial bound to the removed capture object 602, which in turn removed the capture object 602 from which to produce the material 504 of interest. It may correspond to the performance of the cell colony 500 in the enclosure 256. In some embodiments, the assay material may be repeatedly stimulated. For example, light stimuli can be given periodically following each stimulus, detecting any of the resulting radiation. Alternatively, the chemical catalyst or substrate (eg, luciferin) can be flushed into channel 252, where detectable radiation can be detected. The channel 252 may remove the chemical catalyst after a suitable period and the treatment may be repeated thereafter.

Step 412 may include detecting the level of energy 1402 radiating from each individual capture object 602 removed from enclosure 256 in step 408. For example, the detector 224 may detect the level of energy 1402 from each captured object 602 removed in channel 252. As mentioned with respect to step 410, the interrelationship between each captured object 602 removed and the enclosure 256 from which the captured object 602 was taken can be maintained, for example, in a digital table such as Table 1 above. The level of energy 1402 emitted from each removed captured object 602, detected as part of step 412, can be stored in such a table, which is the detected energy level as shown in Table 2 below. May include columns for.

In step 414 of FIG. 4, treatment 400 may identify a retention enclosure 256 with the desired cell colony 500, and at least by default setting it also identifies a retention enclosure 256 with the unwanted cell colony 500. Can be. For example, in step 414, process 400 may determine which removed capture object 602 radiated energy above (or below) the threshold level, and of those removed capture objects 602. , The retaining enclosure 256 associated with each other can be identified as having the desired cell colony 500. Retention enclosures 256 associated with the removed capture object 602, which emit 1402 below the threshold level, can be identified as accommodating unwanted cell colonies 500.

Rather than simply identifying the retention enclosure 256 with the desired and unwanted cell colonies 500 in step 414, the treatment 400, in other embodiments, corresponds to each retention enclosure 256 corresponding to the removed capture object 602. The cell colonies 500 inside can be estimated quantitatively. For example, processing 400 may detect and quantify the energy 1402 emitted by each captured object 602 removed, whereby the captured object 602 removed from it is taken in order to produce the material 504 of interest. The performance of the cell colony 500 in each of the holding enclosures 256 can be estimated.

In some embodiments, the detector 224 may capture an image from which the human operator or controller 232 is in each of the holding enclosures 256 from which one of the capture objects 602 removed is taken. The number of cells 502 can be counted or approximated. In such an embodiment, the treatment 400 is part of step 412 to determine the performance of the colony 500 of the cells 502 in a particular retention enclosure 256 for producing the material 504 of interest, as a ratio per cell 502. The level of emitted energy 1402 detected (or other features such as color, brightness or the like) and the number of cells in the holding enclosure 256 may be utilized. Treatment 400 may then utilize those described above to identify the retention enclosure 256 with the desired cell colonies 500 in step 414.

Regardless, after step 414, the desired cell colonies 500 move from their respective retention enclosures 256 to other locations in the device 200, or to other locations for further processing, analysis, testing or use. Can be removed to device (not shown). For example, the desired cell colony 500 may be selected and moved as shown in U.S. Patent Application No. 14 / 520,150 filed October 22, 2014, which is the same assignment as in this application. Transferred to a person.

FIG. 4 is an example, and many variations of the process 400 are considered. For example, process 400 may assess biological activity in step 412 while the capture object 602 is in the holding enclosure 256. Thus, process 400 does not need to include steps 408, 410 in some variation, or steps 408, 410 may be skipped. As another example, all of steps 402-414 need not be performed in the order shown in FIG.

FIG. 15 illustrates yet another example of processing 1500 for assaying biological activity in a holding enclosure of a microfluidic device. The treatment 1500 may be a narrower example of the more general treatment 100, in which the biological activity in the treatment 1500 of FIG. 15 is tested for a first number n of features and then a second number m of features. Where n and m (they can be the same or different numbers) can be any integer value greater than or equal to 1 each. For ease of description and discussion, process 1500 is discussed below with respect to the microfluidic device 200 of FIGS. 2A-2C, in which the selector 222 may be configured as described in FIGS. 3A and 3B. However, processing 1500 is not so limited and may thus be performed on other microfluidic devices.

As shown in FIG. 15, at step 1502, treatment 1502 may culture biological activity in the retention enclosure in the microfluidic device. Step 1502 may be performed as in step 104 of FIG. 1 or step 402 of FIG. For example, generally according to the above study of FIG. 4, the biological activity is the production of one or more different purpose materials by one or more living cells in each enclosure 256 of the microfluidic device 200 of FIGS. 2A-2C. obtain. The culturing of step 1502 can be carried out sequentially throughout the run of treatment 1500, so the culturing of step 1502 can be continued during steps 1504 and / or 1506.

At step 1504, processing 1500 may test the biological activity in each retention enclosure 256 for n features, each of which may be a different feature. The n features can be any of the features tested during treatment 100 or 400 of FIGS. 1 and 4 as described above or other features of biological activity. Evaluating a large number of features in this manner is desired for many applications, including antibody characterization. Thus, for example, a number of assessments can help any of the following: Identifying a coordination-specific antibody (for example, different features for binding to a different configuration of a particular antigen). It can be the ability of Wright); epitope mapping of antibody-analytes (eg, different features can be the ability to bind to various genetically or chemically altered forms of the antigen); of antibody-analytes. Assessing interspecific cross-reactivity (antibody ana for binding to homologous antigens originating from different species, such as humans, mice, rats and / or other animals (eg, experimental animals), for example with different characteristics). It can be the ability of Wright); and the IgG isotyping of antibody analysts (eg different features can be the ability to bind to IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE and / or IgD. ). Generation of chemically recombined antigens for epitope mapping of antibodies is described, for example, in Dhungana et al. (2009), Methods Mol. Biol. 524: 119-34. Other applications that can benefit from assessing a number of features are, for example, cell health, cancer, infections (eg viruses, bacteria, parasites, etc.), inflammation, response to therapeutic agents and the like. Includes detecting markers that relate to one another.

At step 1504, treatment 1500 may perform a test suggesting whether the biological activity in each enclosure 256 has any one or more of the n features. Thus, in some embodiments, the biological activity in enclosure 256 is likely to test positive in step 1504 if the biological activity has only one of n features. In another embodiment, the biological activity in the enclosure 256 appears to give a positive test in step 1504 only if the biological activity has all of the n features, yet in another embodiment. In, the biological activity in enclosure 256 is likely to give a positive test in step 1504 if the biological activity has a number of q out of n features, where q is 1. Greater and smaller than n.

At step 1506, treatment 1500 may test for biological activity in each retention enclosure 256 that tested positive in step 1504 for different m features, each of which may be a different feature. The m features tested in step 1506 may differ from the n features tested in step 1504. The m features may include any of the features tested during treatment 100 or treatment 400 of FIGS. 1 and 4 or other features of biological activity as described above. Alternatively, there may be overlap between the m features tested in step 1506 and the n features tested in step 1504.

Step 1506 can be performed in any of the ways described above for carrying out step 1504. For example, in step 1506, treatment 1500 performed a test suggesting whether the biological activity in the enclosure 256 that tested positive in step 1504 had any one or more m features. obtain. Thus, in some embodiments, the biological activity in enclosure 256 appears to have tested positive in step 1506 if the biological activity had only one of the m features. In another embodiment, the biological activity in the enclosure 256 appears to give a positive test in step 1506 only if the biological activity has all of the m features, yet in another embodiment. In, biological activity in enclosure 256 is likely to give a positive test in step 1506 if the biological activity has a number of p out of m features, where p is 1 Greater and smaller than m.

16 and 17 illustrate examples of processes 1600 and 1700 that may perform step 1504 and / or step 1506 of FIG.

First, looking at FIG. 16, process 1602 may move a number x of captured objects in each enclosure 256 of the microfluidic device 200 in step 1602. For example, the number x can be between them, including 1 and n. FIG. 18 (showing a horizontal cross-sectional view of a portion of the flow region 240 of the microfluidic device 200 of FIGS. 2A-2C) illustrates an example. As shown, x capture objects 1812 can be moved into enclosure 256. The capture object 1812 can be moved into the enclosure 256 continuously, in parallel or in a combination of continuous and parallel. As further shown, the biological microobject 1802 can be in enclosure 256. Three biological micro-objects 1802 are shown in the enclosure 256, but there can be one, two or more. The biological microobject 1802 can be, for example, a living cell that produces one or more materials of interest.

Each trapped object 1812 may contain a binding agent that specifically binds to a particular biomaterial of interest. For example, the binding agent is at least about 1 mM or stronger (eg, about 100 μM, 10 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM or It may have an affinity (eg, Kd) for a biomaterial of a particular purpose (stronger). Such affinity is greater than affinity for any other material other than the biomaterial of a particular purpose (or at least any other biomaterial present in the holding enclosure and / or microfluidic device). Can also be stronger, for example, in multiples of 2x, 3x, 4x, 5x, 10x or more. Thus, for example, each capture object 1812 may be present or produced by biological activity cultured in enclosure 256 by step 1502 of FIG. 15 such predominant affinity for different target materials. May include different binding agents with. Otherwise, the capture object 1812 may generally resemble the capture object 602, and the capture object 1812 may be selected and moved in any of the above ways for selecting and moving the capture object 602.

At step 1604, the process may evaluate the biomaterial captured by each of the x capture objects moved into the enclosure 256 at step 1602. Step 1604 may be similar and may be carried out in any of the above methods with respect to step 110 of FIG. 1 or step 414 of FIG.

As described in FIG. 16, process 1600 can be arbitrarily repeated any number of times. The number x may be the same or different for each repeated performance of step 1602. Thus, for example, process 1600 is one or more times until n trapped objects, each of which may have different binding materials, are moved into the enclosure in step 1602 and evaluated in step 1604. Can be carried out at. Therefore, when the treatment 1600 is performed 1 or more times, the biological material captured by n captured objects is obtained by performing step 1602 1 or more times and step 1604 1 or more times. Evaluation can result in the total movement of n trapped objects into each enclosure. For example, in each of the repeated performances of step 1602, the value of x can be any number of 1 or more and n-1 or less, and in process 1600, the value of x in each repeated performance of step 1602 is at least up to n. It can be repeated all the way to the sum.

As mentioned, step 1504 and / or step 1506 in FIG. 15 can be performed by process 1600 in FIG. When step 1506 is performed, a few meters are substituted for n in the above study of FIG.

Now referring to FIG. 17, in step 1702, process 1700 may move the y-material capture object into the enclosure 256 of the microfluidic device 200, where each y-material capture object has y different binding materials. May include. The number y can be 2 or more and n or less. FIG. 19 (showing a horizontal cross-sectional view of a part of the flow region 240 of the microfluidic device 200 of FIGS. 2A-2C) illustrates an example. As shown, the y-material capture object 1912 can be moved into the enclosure 256 with one or more biological microobjects 1802, which can be as described above.

The y-material capture object 1912 may contain y different binding substances, each of which specifically binds to a particular biomaterial of interest. For example, each binding agent is at least about 1 mM or stronger (eg, about 100 μM, 10 μM, 1 μM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM). Or stronger) may have an affinity (eg, Kd) for a particular biomaterial of interest. Such affinity is greater than affinity for any other material other than the biomaterial of a particular purpose (or at least any other biomaterial present in the holding enclosure and / or microfluidic device). Can also be stronger, for example, in multiples of 2x, 3x, 4x, 5x, 10x or more. Otherwise, the y-material capture object 1912 may generally resemble the capture object 602, and the capture object 1912 may be selected and moved in any of the above ways for selecting and moving the capture object 602. Can be.

At step 1704, process 1700 may evaluate the biomaterial captured by the y-material capture object 1912 in each enclosure 256. Step 1704 may be similar and may be carried out in any of the methods described above with respect to step 110 of FIG. 1 or step 414 of FIG.

As described in FIG. 17, process 1700 can be arbitrarily repeated any number of times. The number y may be the same or different for each repeated performance of step 1702. Thus, for example, process 1700 may be performed one or more times until the value of y in each performance of step 1702 is added to at least n. For example, in each of the repeated performances of step 1702, the value of y can be any number of 2 or more and n-2 or less, and in the process 1700, the value of y in each repeated performance of step 1702 is at least up to n. It can be repeated all the way to the sum.

As mentioned, step 1504 and / or step 1506 in FIG. 15 can be performed by process 1700 in FIG. When step 1506 is performed, a few meters are substituted for n in the above study of FIG.

20A-20C show the shape variations of the retaining enclosure and the methods of the invention that can be used in microfluidic devices. In each case, the retention enclosure comprises an area that can be used to contain biological activity (eg, one or more living cells) and another area that can be used to contain the capture object 602. .. For example, in FIG. 20A, the retention enclosure 256 has a separation region 508, including a left portion capable of accommodating a living cell 502 and a right portion capable of accommodating a capture object 602. The retaining enclosure 256 further comprises a connecting region 510 having a proximal opening to the channel 252 and a distal opening to the separation region 508. There is a similar sequence in FIG. 20B, but the retaining enclosure 256 is longer and shallower (in terms of the depth of the connection region 510). In FIG. 20C, the retention enclosure 256 includes a thin wall that separates the left portion, which can accommodate the living cell 502, from the right portion, which can accommodate the capture object 602. The thin wall is vulnerable and therefore allows the diffusion of the desired biomaterial between the left and right parts of the retention enclosure 256, thereby allowing biological activity (eg, living cell 502) to the capture object 602. Prevent contact.

Specific embodiments and applications of the invention are described herein, but these embodiments and applications are for illustration purposes only and may vary in many ways.

Claims (30)

A microfluidic device comprising a flow region and a housing with an enclosure having a holding enclosure open in the flow region.
The holding enclosure comprises a wall that separates the first portion, the second portion, and the first portion of the holding enclosure from the second portion of the holding enclosure.
The first portion is open to the flow area and
The second part is open to the flow area and
The wall prevents the living cells placed in the first portion of the holding enclosure from coming into contact with the capture object placed in the second portion of the holding enclosure, and the retaining enclosure. It is configured to allow diffusion to occur between the first portion and the second portion of the holding enclosure.
Micro fluid device. The housing is
At the base,
The microfluidic structure placed on the base and
Equipped with
The microfluidic structure and the base define the flow region and the holding enclosure.
The holding enclosure is located on the base,
The microfluidic device according to claim 1. The microfluidic device of claim 1 or 2, wherein the flow region comprises a channel. The holding enclosure comprises an isolated area and a connecting area.
The connecting region has a proximal opening to the channel and a distal opening to the isolated region.
The isolated region of the retaining enclosure is the non-swept region of the microfluidic device.
The microfluidic device according to claim 3. The microfluidic device of claim 4, wherein the proximal opening of the connection area of the holding enclosure is open beside the channel. The microfluidic device according to any one of claims 3 to 5, further comprising an assay region. The microfluidic device of claim 6, wherein the holding enclosure is open to the channel and the assay region comprises a stop located within the channel. The microfluidic device of claim 6, wherein the assay region comprises an assay chamber having an opening to the channel, wherein the assay chambers are arranged side by side in the holding enclosure. 6. The microfluidic device described. The microfluidic device of claim 8 or 9, wherein the assay chamber has substantially no isolated region. The microfluidic device of claim 1 or 2, further comprising an assay region. The microfluidic device according to any one of claims 1 to 11, further comprising a dielectrophoresis (DEP) electrode on the inner surface of the flow region. Further equipped with a first electrode, a second electrode, an electrode starting board, a power supply, and a light source,
The flow region and the electrode activation substrate are arranged between the first electrode and the second electrode.
12. The microfluidic device of claim 12, wherein changing the pattern of light from the light source selectively activates and stops the changing pattern of the DEP electrode on the inner surface of the flow region. The microfluidic device according to any one of claims 1 to 13, further comprising a plurality of holding enclosures. The microfluidic device according to any one of claims 1 to 14, further comprising means for generating a magnetic force in the enclosure. A process of assaying biological activity in the microfluidic device according to any one of claims 1 to 15.
The biological activity involves the production of the desired biomaterial.
Culturing one or more living cells that produce the biomaterial of interest in the first portion of the holding enclosure of the microfluidic device.
In the second portion of the holding enclosure of the microfluidic device, one or more capture microobjects, each comprising a binding substance that specifically binds to the biomaterial of interest. Introducing an object and
Binding the biomaterial of interest produced by the one or more living cells in the first part of the holding enclosure to the one or more captive microobjects in the second part of the holding enclosure. When,
To evaluate the biomaterial of interest bound to the captured micro-object,
Processing to assay biological activity, including. 16. The process of claim 16, wherein the evaluation is performed while the one or more captured micro-objects are in the second portion of the holding enclosure. From the holding enclosure after binding the biomaterial of interest to the one or more capture micro-objects and before evaluating the biomaterial of interest bound to the one or more capture micro-objects. 16. The process of claim 16, further comprising removing the one or more captured microobjects. Removing the one or more captured micro-objects removes the one or more captured micro-objects.
Including moving to an assay region located within the microfluidic device.
The assay region is at least one selected from the anchorage and chamber.
The stop is located within the channel of the microfluidic device.
The anchor is configured to hold a small object in place against the flow of media in the channel.
The chamber is located within the microfluidic device.
The process according to claim 18. Removing the one or more captured micro-objects
Moving the one or more captured microobjects to a channel within the microfluidic device,
Carrying out the one or more captured micro-objects from the micro-fluid device
18. The process of claim 18. Removing the one or more captured micro-objects
(I) Light that captures at least one of the captured micro-objects in the second portion of the holding enclosure by projecting a light pattern surrounding the at least one captured micro-object onto the inner surface of the micro-fluid device. Creating a trap and moving the optical trap from the second portion of the holding enclosure to the channel of the microfluidic device;
(Ii) Adjacent to at least one of the captured micro-objects in the second portion of the holding enclosure by projecting a light pattern onto the inner surface of the micro-fluid device adjacent to the at least one captured micro-object. By activating the photoinduced DEP electrode to move the optical pattern from the second portion of the holding enclosure to the channel of the microfluidic device, the activated DEP electrode is subjected to the at least one capture micro. Moving an object away from the channel; and (iii) the one or more captured microobjects being magnetic and applying a magnetic field to the microfluidic device;
The process according to any one of claims 18 to 20, which is one selected from the group consisting of. Evaluating the biomaterial of interest bound to the trapped micro-object can be
Determining the type of biomaterial of interest bound to the one or more capture micro-objects;
Determining the activity of the biomaterial of interest bound to the one or more capture micro-objects; and determining the amount of the biomaterial of interest bound to the one or more capture micro-objects;
The process according to any one of claims 16 to 21, which is one or more selected from the group consisting of. The above decision can be made
Binding an assay material capable of detectable radiation to the biomaterial of interest bound to the one or more capture micro-objects.
To detect the association between the one or more captured micro-objects and the radiation generated from the assay material.
22. The process of claim 22. The process according to any one of claims 16 to 23, wherein the biomaterial of interest is a protein containing an antibody. The treatment according to any one of claims 16 to 24, wherein the binding substance of the one or more captured microobjects has a binding affinity of at least 1 μM for the biomaterial of interest. The one according to any one of claims 16 to 25, wherein the one or more living cells in the holding enclosure are one selected from the group consisting of cloned colonies of living cells and a single cell. process. The one or more captured micro-objects may be a single captured micro-object or include a plurality of captured micro-objects, each of which is different from the binding material of the other captured micro-objects in the plurality. The process according to any one of claims 16 to 26, comprising a binding substance. The target biomaterial is an antibody.
(I) Each of the plurality of captured micro-objects comprises a binding substance that binds to an antibody isotype different from the antibody isotype bound by the binding substance of the other captured micro-objects in the plurality of objects;
(Iii) Each of the plurality of captured micro-objects comprises a binding substance corresponding to an epitope of an antigen recognized by the antibody; and (iii) one of the plurality of captured micro-objects is the antibody. The other captive micro-objects of the plurality of capture micro-objects are the homologues of the antigen or homologues of the antigen, respectively, which are provided with a binding substance corresponding to the antigen recognized by the above-mentioned antigen or an epitope of the antigen. Equipped with a binding substance corresponding to the epitope;
It is one selected from the group consisting of
The process of claim 27. The one or more cells produce n different biomaterials of interest,
The one or more captured micro-objects comprises different n types of captured micro-objects, and each species of the captured micro-objects specifically binds to one of the different n species of biomaterial of interest. Equipped with
It comprises binding the different n species of biomaterial of interest produced by the one or more biological cells to the different n species of captured microobjects.
Evaluating the biomaterial of interest bound to the captured micro-object comprises detecting the bond between the different n species of biomaterial of interest and the different n species of captured micro-object.
The process according to any one of claims 16 to 28. The microfluidic device comprises a plurality of holding enclosures.
Each of the plurality of retention enclosures has one or more living cells.
Evaluate the one or more living cells in each of the plurality of retention enclosures.
The process according to any one of claims 16 to 29.

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