HK40016859B - Microfluidic devices having isolation pens and methods of testing biological micro-objects with same - Google Patents

Description

Microfluidic device with isolated pens and method of testing biological micro-objects using same

The present application is a divisional application of application number CN 2014800584449, filed on 22 days of 2014, 10 months of application number CN 2014800584449, entitled "microfluidic device with isolation pens" and method of testing biological micro-objects with it.

Background

As the field of microfluidics continues to advance, microfluidic devices have become a convenient platform for processing and manipulating micro-objects such as biological cells. Some embodiments of the invention are directed to improvements in microfluidic devices and methods of operating the same.

Disclosure of Invention

In some embodiments of the invention, a microfluidic device can include a flow region and a microfluidic isolation rail. The flow region may be configured to contain a flow of the first fluid medium. The microfluidic isolation rail can include an isolation structure and a connection region. The isolation structure may include an isolation region configured to contain a second fluid medium. The connection region may fluidly connect the isolation region to the flow region such that when the flow region and the microfluidic isolation rail are substantially filled with a fluidic medium: the component of the second medium can diffuse into the first medium or the component of the first medium can diffuse into the second medium; and substantially no first medium from the flow region flows into the isolation region.

Some embodiments of the invention include a process for analyzing biological micro-objects in a microfluidic device that may include at least one microfluidic channel fluidly connected thereto by a microfluidic isolation pen. The at least one isolation fence may include a fluid isolation structure including an isolation region and a connection region fluidly connecting the isolation region to the channel. The process may include loading one or more biological micro-objects into at least one sequestration pen, and incubating the loaded biological micro-objects for a period of time sufficient for the biological micro-objects to produce an analyte of interest. The process may further include disposing capture micro-objects in the channel adjacent to the opening (the opening from the connection region of the at least one isolation pen to the channel), and monitoring binding the capture micro-objects to the analyte of interest. The capture micro-objects may include at least one type of affinity agent capable of specifically binding to the analyte of interest.

Drawings

Fig. 1 is an example of a process that may perform at least two tests on a micro-object in a microfluidic device according to some embodiments of the invention.

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

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

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

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

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

Fig. 4A is a perspective view of another example of a microfluidic device according to some embodiments of the present disclosure.

Fig. 4B is a side cross-sectional view of the microfluidic device of fig. 4A.

Fig. 4C is a top cross-sectional view of the microfluidic device of fig. 4A.

Fig. 5 illustrates an example of an isolation fence according to some embodiments of the present invention in which the length of the connection region from the channel to the isolation region is greater than the penetration depth of the medium flowing in the channel.

Fig. 6 is another example of an insulated rail according to some embodiments of the invention that includes a connection region from a channel to an insulated region that is longer than the penetration depth of a medium flowing in the channel.

Fig. 7A-7C illustrate yet another example of a configuration of an insulated pen according to some embodiments of the invention.

Fig. 8 illustrates an example of loading biological micro-objects into the flow path of the microfluidic device of fig. 2A-2C according to some embodiments of the invention.

Fig. 9 illustrates an example of flowing biological micro-objects into channels of the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 10 illustrates an example of testing biological micro-objects in the flow path of the microfluidic device of fig. 2A-2C for a first feature according to some embodiments of the invention.

Fig. 11 is an example of selecting biological micro-objects in the microfluidic device of fig. 2A-2C, according to some embodiments of the invention.

Fig. 12 illustrates an example of selecting biological micro-objects in the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 13 illustrates an example of moving selected biological micro-objects into a holding pen in the microfluidic device of fig. 2A-2C, according to some embodiments of the invention.

Fig. 14 illustrates an example of biological micro-objects flushed from the flow path of the microfluidic device of fig. 2A-2C, according to some embodiments of the invention.

Fig. 15 illustrates an example of moving selected biological micro-objects from a channel into an isolation pen of the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 16 is an example of biological micro-objects flushed from channels in the microfluidic devices of fig. 4A-4C, according to some embodiments of the invention.

Fig. 17 is an example of providing assay material to biological micro-objects in a holding pen of the microfluidic device of fig. 2A-2C, according to some embodiments of the invention.

Fig. 18 illustrates assay material diffusing into a holding pen of the microfluidic device of fig. 2A-2C, according to some embodiments of the present invention.

Fig. 19 illustrates an example of assay materials in the channels of the microfluidic device of fig. 4A-4C and biological micro-objects in the isolation pen that produce an analyte of interest, according to some embodiments of the invention.

Fig. 20 illustrates an example of components of an analyte of interest diffusing out of an isolation region of an isolation pen and reacting with an assay material adjacent to a proximal opening of a channel in the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 21 is an example of assay materials including labeled capture micro-objects in the microfluidic device of fig. 4A-4C according to some embodiments of the invention.

Fig. 22 is an example of an assay material including a mixture of capture micro-objects and labels in the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

FIG. 23 illustrates an example of the capture micro-objects, the components of the tags, and the analytes of interest of FIG. 22, according to some embodiments of the invention.

FIG. 24 illustrates an example of a composite capture micro-object including multiple affinity agents, according to some embodiments of the invention.

Fig. 25 is a process illustrating an example of detecting local reactions and identifying an isolation fence containing positive biological micro-objects in a microfluidic device (such as the device shown in fig. 4A-4C) according to some embodiments of the invention.

Fig. 26 illustrates moving negative biological micro-objects from a holding pen into a flow path in the device of fig. 2A-2C according to some embodiments of the invention.

Fig. 27 illustrates flushing away negative biological micro-objects from the flow path in the microfluidic device of fig. 2A-2C, according to some embodiments of the invention.

Fig. 28 illustrates an example of cleaning channels of assay material in the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 29 is an example of separating negative biological micro-objects from positive biological micro-objects in the fluidic devices of fig. 4A-4C, according to some embodiments of the invention.

Fig. 30 illustrates an example of creating cloned biological micro-objects in an isolation pen in the microfluidic device of fig. 4A-4C, according to some embodiments of the invention.

Fig. 31A-31C depict a microfluidic device including a microchannel and a plurality of isolation pens opening the microchannel. Each sequestration pen contains a plurality of mouse spleen cells. Fig. 31A is a bright field image of a portion of a microchannel apparatus. Fig. 31B and 31C are fluorescence images obtained using a texas red filter. In 31B, images were obtained 5 minutes after the start of the antigen-specific assay described in example 1. In fig. 31C, images were obtained 20 minutes after the start of the antigen-specific assay described in example 1. The white arrows in fig. 31C point to the spacer pens that produced a positive signal in the assay.

Detailed Description

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 operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may not be exaggerated or reduced on scale for clarity. Furthermore, when the terms "on," "attached to," or "coupled to" are used herein, an element (e.g., a material, layer, substrate, etc.) can be "on," "attached to," or "coupled to" another element, whether the one element is directly on, attached to, or coupled to the other element, or there are one or more intervening elements between the one element and the other element. Further, directions (e.g., above, below, top, bottom, sides, upper, lower, below …, above …, above, below, horizontal, vertical, "x", "y", "z", etc.) are relative and are provided by way of example only for ease of illustration and discussion and not limitation, if provided. Furthermore, where a series of elements (e.g., elements a, b, c) is referred to, such reference numerals are intended to include any one of the listed elements per se, any combination of less than all of the listed elements, and/or combinations of all of the listed elements.

As used herein, "substantially" means sufficient to achieve the intended purpose. The term "plurality" means more than one.

As used herein, the term "micro-object" may include one or more of the following: inanimate micro-objects such as microparticles, microbeads (e.g., polystyrene beads, luminex) ^TM Beads, etc.), magnetic beads, micro rods, microwires, quantum dots, etc.; biological micro-objects such as cells (e.g., cells obtained from a tissue or body fluid sample, blood cells, hybrid cells, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, etc.), liposomes (e.g., synthetic membrane preparations or derived from membrane preparations), nanolipid rafts, etc.; or a combination of inanimate and biological micro-objects (e.g., cell-attached microbeads, liposome-coated magnetic beads, etc.). Nanolipid rafts have been described, for example, in "ritche et al (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, mehotd enzymol 464:211-231 (richi et al (2009), methozymology 464:211-231)" for the recombination of membrane proteins in phospholipid bilayer nanodiscs.

As used herein, the term "cell" refers to a biological cell, which may be a plant cell, an animal cell (e.g., a mammalian cell), a bacterial cell, a fungal cell, and the like. The animal cells may be, for example, from humans, mice, rats, horses, goats, mianyang, cattle, primates, etc.

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

As used herein with respect to the fluid medium, "diffusing …" and "diffusing" refer to thermodynamic movement of a component of the fluid medium toward a direction of low concentration gradient.

The phrase "flow of medium" refers to the overall movement of the fluid medium caused by any mechanism other than diffusion. For example, the flow of medium may include movement of fluid medium from one point to another due to pressure differences between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the media may result.

The phrase "substantially free of flow" refers to a flow rate of a fluid medium that is less than a rate at which a component of a material (e.g., an analyte of interest) diffuses into or within the fluid medium. The diffusion rate of the components of such materials may depend on, for example, temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

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

In some embodiments, the microfluidic device may include "swept" regions and "unswept" regions. The unswept region may be fluidly connected to the swept region, provided that the fluid connection is configured to enable diffusion between the swept region and the unswept region, but there is substantially no flow of medium between the swept region and the unswept region. The microfluidic device may thus be configured to substantially isolate the unswept region from the flow of medium in the swept region, while enabling only diffuse fluid communication between the swept region and the unswept region.

The ability of biological micro-objects (e.g., biological cells) to produce a particular biological material can be determined in such a microfluidic device. For example, sample material for the production of an analyte of interest, including biological micro-objects to be assayed, may be loaded into the swept area of the microfluidic device. Multiple biological micro-objects may be selected for a particular feature and placed in the unswept area. The remaining sample material may then flow out of the swept area, and the assay material may flow into the swept area. Since the selected biological micro-objects are in the unswept region, the selected biological micro-objects are substantially unaffected by the outflow of the remaining sample material or inflow of the assay material. The selected biological micro-objects may allow for the production of analytes of interest that may diffuse from the unswept regions into the swept regions, wherein the analytes of interest may react with the assay material to produce locally detectable reactions, each of which may be associated with a particular unswept region. Any unswept regions associated with the detected reaction may be analyzed to determine which biological micro-objects, if any, in the unswept regions are sufficient producers of the analyte of interest.

Fig. 1 illustrates an example of a process 100 for testing micro-objects in a microfluidic device according to some embodiments of the invention. Fig. 2A to 2C illustrate examples of microfluidic devices 200 by which the process 100 may be performed, and fig. 3A and 3B illustrate examples of Dielectrophoresis (DEP) devices that may be part of the microfluidic devices 200. Fig. 4A-4C illustrate another example of a microfluidic device 400 through which the process 100 may also be performed. However, neither the apparatus 200 of fig. 2A-2C nor the apparatus 400 of fig. 4A-4C is limited to performing the process 100 of fig. 1. The process 100 is also not limited to being performed on the apparatus 200 or 400.

As shown in fig. 1, a process 100 may load a mixture of micro-objects into a flow path in a microfluidic device at step 102. The mixture loaded at step 102 may include different types of micro-objects as well as fragments and other objects. At step 104, the process 100 may test for micro-objects in the flow path for the first feature, and at step 106, the process 100 may isolate micro-objects that are positive for the first feature from micro-objects that are not positive for the first feature (e.g., micro-objects that are negative for the test). As shown, process 100 may repeat steps 102 through 106 any number of times. For example, steps 102-106 may be performed k times, and then the mixture of k micro-objects that have been loaded at step 102 is divided into an initial group of micro-objects at steps 104, 106 (all micro-objects of the initial group are tested positive for the first feature). The number k may be 1 or any integer greater than 1. (hereinafter, biological micro-objects that are positive for the test are sometimes referred to as "positive", while biological micro-objects that are not positive for the test (e.g., negative for the test) are sometimes referred to as "negative" biological micro-objects.)

Process 100 may then continue to step 108, where process 100 may perform subsequent tests on the initial group of micro-objects. The subsequent test performed at step 108 may be different from the first test performed at step 104. For example, the subsequent test may be performed on a different subsequent feature than the first feature tested at step 104. As another example, the subsequent test performed at step 108 may be performed for the same feature as step 104 (the first feature mentioned above), but with different sensitivity, accuracy, precision, etc. For example, for a first feature, the subsequent test performed at step 108 may be more sensitive than the first test performed at step 104. Regardless, at step 110, the process 100 may isolate the micro-objects that are positive for the subsequent test at step 108 from the micro-objects that are negative for the subsequent test.

If the first test of step 104 and the subsequent test of step 108 are for the same feature test, after steps 108 and 110, in response to two different tests, the micro-objects that test positive for that feature (referred to as the first feature in the discussion of step 104 above) have been separated from the mixture of k micro-objects that were loaded into the microfluidic device at the time of the k executions of step 102. As shown, steps 108 and 110 may be repeated, and with each repetition, process 100 may apply different subsequent tests at step 108 that test for the same feature. In practice, steps 108 and 110 may be repeated n times, and then process 100 has sorted out the k micro-objects of the mixture loaded into the microfluidic device at step 102 micro-objects that have been tested positive n+1 times for the first features tested at steps 104 and 108. The number n may be 1 or any integer greater than 1.

As mentioned, alternatively, the process 100 may test at step 108 for a subsequent feature that is different from the first feature tested at step 104. In such an embodiment, a micro-object having both a first feature and a subsequent feature has been sorted from a mixture of k micro-objects loaded into the microfluidic device at step 102. If steps 108 and 110 are repeated, then at each repetition, process 100 may test for different subsequent features at step 108. For example, at each execution of step 108, process 100 may test for a subsequent feature that is not only different from the first feature, but also different from any previous subsequent feature that was previously tested by steps 108 and 110. At each execution of step 110, the process 100 may isolate micro-objects that are positive for subsequent feature tests at step 108.

As mentioned, steps 108 and 110 may be repeated n times. After performing steps 108 and 110 n times, process 100 has sorted out the micro-objects having all n+1 features tested at steps 104 and 108 from the mixture of k micro-objects loaded into the microfluidic device at step 102. The number n may be 1 or any integer greater than 1.

Variations of process 100 are contemplated. For example, in some embodiments, the repetition of step 108 may be tested at times for new features that were not tested at step 104 or any prior execution of step 108, while other times may be tested for the same features that were tested at step 104 or the prior execution of step 108. As another example, at step 106 or any repeated step 110, the process 100 may isolate test positive micro-objects from test negative micro-objects. As yet another example, the process 100 may repeat step 104 multiple times before continuing with step 106. In such an example, the process 100 may test for different features at each repetition of step 104 and then isolate micro-objects that test positive at each repetition of step 104 from micro-objects that test negative at least one repetition of step 104. Likewise, step 108 may be repeated multiple times before continuing with step 110.

Examples of microfluidic devices 200 and 400 are now discussed with respect to fig. 2A-7C. Next, an example of operation of the process 100 (where the micro-objects include biological micro-objects such as biological cells) using the devices 200 and 400 is described with respect to fig. 8-30.

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

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

The housing 202 may include one or more inlets 208 through which a medium 244 may be input into the flow region 240. The inlet 208 may be, for example, an input port, an opening, a valve, another channel, a fluid connector, etc. The housing 202 may also include one or more outlets 210 through which the medium 244 may be removed. The outlet 210 may be, for example, an output port, opening, valve, channel, fluid connector, or the like. As another example, the outlet 210 may include a droplet output mechanism such as any of the output mechanisms disclosed in U.S. patent application serial No. 13/856,781 (attorney docket No. BL 1-US) filed on 4/2013. All or a portion of the housing 202 may be gas permeable to allow gas (e.g., ambient air) to enter and exit the flow region 240.

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

The microfluidic structure 204 and the base 206 may define a flow region 240. Although one flow region 240 is shown in fig. 2A-2C, the microfluidic structure 204 and the base 206 may define multiple flow regions for the medium 244. The flow region 240 may include channels (252 and 253 in fig. 2C) and chambers that may be interconnected to form a microfluidic circuit. For a perimeter that includes more than one flow region 240, each flow region 240 may be associated with one or more inlets 108 and one or more outlets 110 for respectively inputting and removing medium 244 from the flow region 240.

As shown in fig. 2B and 2C, the flow region 240 may include one or more channels 252 for the medium 244. For example, the channel 252 may generally extend from the inlet 208 to the outlet 210. Also as shown, a holding pen 256 defining a non-flow space (or isolation zone) can be provided in the flow zone 240. That is, a portion of the interior of each holding pen 256 can be a non-flow space into which the medium 244 from the channel 252 does not flow directly except when the empty flow region 240 is initially filled with the medium 244. For example, each holding pen 256 can include one or more barriers 254 that form part of an enclosure, the interior of which can include a non-flow space. When the flow region 240 is filled with media 244, the barrier 254 defining the holding pens 256 can thus prevent the media 244 from flowing directly from the channel 252 into the protected interior of any holding pens 256. For example, when the flow area 240 is filled with media 244, the barrier 254 of the pen 256 can substantially prevent the entire flow of media 244 from the channel 252 from flowing into the non-flow space of the pen 256, rather, substantially only allowing diffusive mixing of media in the non-flow space in the pen 256 with media from the channel 252. Thus, exchange of nutrients and waste between the non-flowing space in the holding pen 256 and the channel 252 can occur substantially only by diffusion.

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

There may be many such holding pens 256 in the flow region 240 arranged in any pattern, and the holding pens 256 may be any of a number of different sizes and shapes. Although shown disposed opposite the sidewalls of the microfluidic structure 204 in fig. 2C, one or more (including all) of the pens 256 can be stand alone structures disposed outside the sidewalls of the microfluidic structure 204 in the channel 252. As shown in fig. 2C, the opening of the holding pen 256 can be disposed adjacent to the channel 252, which can be adjacent to the opening of more than one pen 256. Although one channel 252 is shown adjacent fourteen pens 256, there may be more channels 252 and there may be more or fewer pens 256 adjacent any particular channel 252.

The barrier 254 of the pen 256 can comprise any of the types of materials discussed above with respect to the microfluidic structure 204. The barrier 254 may comprise the same material as the microfluidic structure 204 or a different material. As shown in fig. 2B, the barrier 254 may extend from the surface 242 of the base 206 across the entire flow region 240 to the upper wall (opposite the surface 242) of the microfluidic structure 204. Alternatively, one or more of the barriers 254 may extend only partially through the flow region 240 and thus not extend completely to the surface 242 or the upper wall of the microfluidic structure 204.

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

For example, the selector 222 may include one or more optical (e.g., laser) tweezer devices and/or one or more optoelectronic tweezer (OET) devices (e.g., as disclosed in U.S. patent No. 7,612,355, the entire contents of which are incorporated herein by reference), or as disclosed in U.S. patent application serial No. 14/051,004 (attorney docket No. BL 9-US), as yet another example, the selector 222 may include one or more devices (not shown) for moving droplets of the medium 244 in which one or more micro-objects are suspended, such devices (not shown) may include an electrowetting device, such as an optoelectronic wetting (OET) device (e.g., as disclosed in U.S. patent No. 6,958,132).

Fig. 3A and 3B illustrate examples in which the selector 222 includes a DEP apparatus 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 (e.g., an Alternating Current (AC) power source), and a light source 320. The medium 244 in the flow region 240 and the electrode activation substrate 308 may separate the electrodes 304, 310. Changing the pattern of light 322 from the light source 320 selectively activates and deactivates the pattern of DEP electrodes that change at the region 314 of the inner surface 242 of the flow region 240. (hereinafter, the region 314 is referred to as "electrode region")

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

With the power supply 312 activated, the foregoing creates an electric field gradient in the medium 244 between the illuminated electrode region 314a and the adjacent dark electrode region 314, which in turn creates a localized DEP force that attracts or repels nearby micro-objects (not shown) in the medium 244. The DEP electrodes that attract or repel micro-objects in the medium 244 can thus be selectively activated or deactivated at many different such electrode regions 314 of the inner surface 242 of the flow region 240 by varying the light pattern 322 projected from the light source 320 (e.g., a laser source or other type of light source) to the microfluidic device 200. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of the power source 312 and the dielectric properties of the medium 244 and/or micro-objects (not shown).

The square pattern 322' of illuminated electrode region 314a shown in fig. 3B is merely an example. Any pattern of electrode regions 314 may be illuminated by the pattern of light 322 projected into device 200, and the pattern of illuminated electrode regions 322' may be repeatedly changed by changing light pattern 322.

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

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

In some embodiments, the first electrode 304 may be part of the first wall 302 (or lid) of the housing 202, and the electrode activation base 308 and the second electrode 310 may be part of the second wall 306 (or base) of the housing 202, as generally shown in fig. 3A. As shown, the flow region 240 may be between the first wall 302 and the second wall 306. However, the foregoing is merely an example. In other embodiments, 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 the first electrode 304 and/or the second electrode 310. Further, the light source 320 may alternatively be located below the housing 202.

Configured as the DEP device 300 shown in fig. 3A and 3B, the selector 222 can thus select a micro-object (not shown) in the medium 244 in the flow region 240 by projecting a light pattern 322 into the device 200 to activate one or more DEP electrodes at the electrode region 314 surrounding and capturing the inner surface 242 of the flow region 240 in the pattern of micro-objects. The selector 222 may then move the capture micro-objects by moving the light pattern 322 relative to the device 200. Alternatively, the device 200 may be moved relative to the light pattern 322.

Although the barrier 254 defining the holding pens 256 is shown in figures 2B and 2C and discussed above as a physical barrier, the barrier 254 may alternatively comprise a virtual barrier comprising DEP forces activated by the light pattern 322.

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

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

The flow controller 226 may be configured to control the flow of the medium 244 in the flow region 240. For example, the flow controller 226 may control the direction and/or speed of 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, for example, the velocity of the flow of the medium 244 in the flow region 240.

The control module 230 may be configured to receive signals from the selector 222, the detector 224, and/or the flow controller 226 and control the selector 222, the detector 224, and/or the flow controller 226. As shown, the control module 230 may include a controller 232 and a memory 234. In some embodiments, controller 232 may be a digital electronic controller (e.g., microprocessor, microcontroller, computer, etc.) configured to operate according to machine readable instructions (e.g., software, firmware, microcode, etc.) stored as non-transitory signals in memory 234, which memory 234 may be a digital electronic, optical, or magnetic storage device. Alternatively, controller 232 may comprise hardwired digital and/or analog circuitry, or a combination of digital electronic controllers operating according to machine-readable instructions and hardwired digital and/or analog circuitry. The controller 232 may be configured to perform all or any portion of the processes 100, 2500 disclosed herein.

In some embodiments, the pen 256 can be shielded from illumination (e.g., by the detector 224 and/or the selector 222) or can be selectively illuminated for only a brief period of time. After the biological micro-objects are moved into the pens 256, the biological micro-objects can thus be protected from further illumination or further illumination of the biological micro-objects can be minimized.

Fig. 4A to 4C illustrate another example of a microfluidic device 400. As shown, the microfluidic device 400 may include a microfluidic circuit 432, the microfluidic circuit 432 including a plurality of interconnected fluidic circuit elements. In the example shown in fig. 4A-4C, the microfluidic circuit 432 includes flow regions/channels 434 fluidly connected thereto by isolation pens 436, 438, 440. One channel 434 and three spacer pens 436, 438, 440 are shown, but there may be more than one channel 434 and more or less than three spacer pens 436, 438, 440 connected to any particular channel. The channel 434 and isolation pens 436, 438, 440 are examples of fluid circuit elements. Microfluidic circuit 432 may also include additional or different fluidic circuit elements, such as fluid chambers, reservoirs, and the like.

Each isolation fence 436, 438, 440 may include an isolation structure 446 (see fig. 4C) defining an isolation region 444 and a connection region 442 fluidly connecting the isolation region 444 to the channel 434. The connection region 442 may include a proximal opening 452 to the channel 434 and a distal opening 454 to the isolation region 444. The connection region 442 may be configured such that at a maximum velocity (V _max ) The maximum penetration depth of the flow of the flowing fluid medium (not shown) does not extend into the isolation region 444. Micro-objects (not shown) or other materials (not shown) disposed in the isolation regions 444 of the pens 436, 438, 440 can thus be isolated from and substantially unaffected by the flow of the medium (not shown) in the channel 434. The channel 434 may thus be an example of a swept area, and the isolated area of the isolated pens 436, 438, 440 may be an example of an unswept area. Before proceeding to the foregoing discussion in greater detail, a brief description of the microfluidic device 400 and an example of a related control system 470 is provided.

The microfluidic device 400 may include an enclosure 402 surrounding a microfluidic circuit 432, which enclosure 402 may contain one or more fluidic media. However, the apparatus 400 may be physically configured in a different manner, in the example shown in fig. 4A-4C, the enclosure 402 is depicted as including a support structure 404 (e.g., a base), a microfluidic circuit structure 412, and a cover 422. The support structure 404, the microfluidic circuit structure 412, and the cover 422 may be connected to one another. For example, the microfluidic circuit structure 412 may be disposed on the support structure 404, and the cover 422 may be disposed over the microfluidic circuit structure 412. The microfluidic circuit structure 412 may define a microfluidic circuit 432 by the support structure 404 and the cover 422. The inner surface of microfluidic circuit 432 is identified as 406 in the figures.

As shown in fig. 4A and 4B, the support structure 404 may be located at the bottom and the cover 422 may be located at the top of the device 400. Alternatively, the support structure 404 and the cover 422 may be located in other orientations. For example, the support structure 404 may be located at the top and the cover 422 at the bottom of the device 400. In any event, there may be one or more ports 424, each including a passageway 426 into or out of the enclosure 402. Examples of passages 426 include valves, gates, through-holes, and the like. Two ports 424 are shown, but the apparatus 400 may have only one or more than two.

The microfluidic circuit structure 412 may define a circuit element of the microfluidic circuit 432 or a circuit in the enclosure 402. In an example, as shown in fig. 4A-4C, the microfluidic circuit structure 412 includes a frame 414 and a microfluidic circuit material 416.

The support structure 404 may include a substrate or a plurality of interconnected substrates. For example, the support structure 404 may include one or more interconnected semiconductor substrates, printed circuit boards, and the like. The frame 414 may partially or fully enclose the microfluidic circuit material 416. The frame 414 may be, for example, a relatively rigid structure that substantially surrounds the microfluidic circuit material 416. For example, the frame 414 may include a metallic material.

The microfluidic circuit material 416 is patterned with cavities or the like to define microfluidic circuit elements and interconnections of microfluidic circuits 432. The microfluidic circuit material 416 may comprise a flexible material, such as rubber, plastic, elastomer, silicone (e.g., patternable silicone), PDMS, or the like, which may be breathable. Other examples of materials from which the microfluidic circuit material 416 may be composed include molded glass, etchable materials such as silicon, photoresist (e.g., SU 8), and the like. In some embodiments, such material (and thus the microfluidic circuit material 416) may be rigid and/or substantially impermeable to air. Regardless, the microfluidic circuit material 416 may be disposed on the support structure 404 and within the frame 414.

The cover 422 may be an integrated component of the frame 414 and/or the microfluidic circuit material 416. Alternatively, the cover 422 may be a structurally different element (as shown in fig. 4A and 4B). The cover 422 may comprise the same or different material as the frame 414 and/or the microfluidic circuit material 416. Similarly, the support structure 404 may be a separate structure from the frame 414 or microfluidic circuit material 416 or an integrated component of the frame 414 or microfluidic circuit material 416 as shown. Likewise, the frame 414 and the microfluidic circuit material 416 may be separate structures as shown in fig. 4A-4C or integrated portions of the same structure. In some embodiments, the cover 422 and/or the support structure 404 may be transparent to light.

Fig. 4A also shows a simplified block diagram depiction of an example of a control/monitoring system 470 that may be used in conjunction with the microfluidic device 400. As shown, the system 470 may include a control module 472 and a control/monitoring device 480. The control module 472 may be configured to control and monitor the apparatus 400 directly or through the control/monitoring device 480.

The control module 472 may include a digital controller 474 and a digital memory 476. The controller 474 may be, for example, a digital processor, computer, etc., and the digital memory 476 may be a non-transitory digital memory for storing data and machine-executable instructions (e.g., software, firmware, microcode, etc.) as non-transitory data or signals. The controller 474 may be configured to operate in accordance with such machine-executable instructions stored in the memory 476. Alternatively or additionally, the controller 474 may include hardwired digital circuitry and/or analog circuitry. The control module 472 may thus be configured to perform all or a portion of any process (e.g., process 100 of fig. 1 and/or process 2500 of fig. 25), the steps, functions, actions, etc. of such a process are discussed herein.

The control/monitoring apparatus 480 may include any number of different types of devices for controlling or monitoring the microfluidic device 400 and the processes performed by the microfluidic device 400. For example, the apparatus 480 may include a power source (not shown) for providing power to the microfluidic device 400; a source of fluidic medium (not shown, but may include a flow controller similar to 226 of fig. 2A) for providing fluidic medium to the microfluidic device 400 or removing medium from the microfluidic device 400; a power module (not shown, but may include a selector similar to 222 of fig. 2A) for controlling the selection and movement of micro-objects (not shown) in the microfluidic circuit 432; an image capture mechanism (not shown, but may be a detector similar to 224 of fig. 2A) for capturing an image (e.g., of a micro-object) inside the microfluidic circuit 432; an excitation mechanism (not shown) for directing energy into the microfluidic circuit 432 to excite a reaction or the like.

As mentioned, the control/monitoring device 480 may include a power module for selecting and moving micro-objects (not shown) in the microfluidic circuit 432. Various power mechanisms may be utilized. For example, a Dielectrophoresis (DEP) mechanism (e.g., similar to the selector 222 of fig. 2A) may be utilized to select and move micro-objects (not shown) in a microfluidic circuit. The base 404 and/or cover 422 of the microfluidic device 400 may include a DEP configuration for selectively directing DEP forces to micro-objects (not shown) in a fluidic medium (not shown) in the microfluidic circuit 432 to select, capture, and/or move individual micro-objects. The control/monitoring device 480 may include one or more control modules for such a DEP configuration.

An example of such a DEP configuration of the support structure 404 or cover 422 is an optoelectronic tweezers (OET) configuration. Examples of suitable OET configurations of the support structure 404 or cover 422 and associated monitoring and control devices are shown in the following U.S. patent documents: U.S. Pat. No. 7,612,355, U.S. Pat. No. 7,956,339, U.S. patent application publication No. 2012/0325675, U.S. patent application publication No. 2014/0124370, U.S. patent application Ser. No. 14/262,140 (not yet decided) and U.S. patent application Ser. No. 14/262,200 (not yet decided), the entire contents of which are incorporated herein by reference. Micro-objects (not shown) may thus be individually selected, captured and moved within the microfluidic circuit 432 of the microfluidic device 400 using DEP devices and techniques such as OET.

As mentioned, the channels 434 and pens 436, 438, 440 may be configured to contain one or more fluid media (not shown). In the example shown in fig. 4A-4C, the port 424 is connected to a channel 434 and allows for the introduction of a fluidic medium (not shown) into the microfluidic circuit 432 or removal from the microfluidic circuit 432. Once microfluidic circuit 432 contains a fluidic medium (not shown), the flow of the fluidic medium (not shown) can be selectively generated and stopped in channels 434. For example, as shown, ports 424 may be disposed at different locations (e.g., opposite ends) of channel 434, and a flow of medium (not shown) may be established from one port 424 functioning as an inlet to another port 424 functioning as an outlet.

As discussed above, each isolation fence 436, 438, 440 may include a connection region 442 and an isolation region 444. The connection region 442 may include a proximal opening 452 to the channel 434 and a distal opening 454 to the isolation region 444. The channel 434 and each isolation fence 436, 438, 440 may be configured such that a maximum penetration depth of a flow of a medium (not shown) flowing in the channel 434 extends into the connection region 442 but not into the isolation region 444.

Fig. 5 shows a detailed view of an example of an isolation fence 436. The pens 438, 440 can be similarly configured. An example of micro-objects 522 in pen 436 is also shown. As is well known, the flow 512 of the fluid medium 502 in the microfluidic channel 434 through the proximal opening 452 of the pen 436 may cause a secondary flow 514 of the medium 502 to flow into and/or out of the pen. To isolate the micro-objects 522 in the isolation region 444 of the pen 436 from the secondary flow 514, the length L of the connection region 442 of the isolation pen 436 from the proximal opening 452 to the distal opening 454 _con May be greater than when the velocity of stream 512 in channel 434 is at a maximum (V _max ) Maximum penetration depth D of secondary flow 514 into connection region 442 _p . As long as the flow 512 in the channel 434 does not exceed the maximum velocity V _max The flow 512 and the resulting secondary flow 514 may thus be confined in the channel 434 and the connection region 442 and maintained outside the isolation region 444. The flow 512 in the channel 434 will thus not cause the micro-objects 522 to leave the isolation region 444. The micro-objects 522 in the isolation region 444 will thus remain in the isolation region 444 regardless of the flow 512 in the channel 432.

In addition, the flow 512 does not move miscellaneous particles (e.g., microparticles and/or nanoparticles) from the channel 434 into the isolation region 444 of the pen 436, nor does the flow 512 carry miscellaneous particles from the isolation region 444 into the channel 434. Thus, the length of the connection region 442 L _con Greater than the maximum penetration depth D _p One rail 436 is prevented from being contaminated with miscellaneous particles from the channel 434 or the other rail 438, 440.

Since the connection region 442 of the channel 434 and the pens 436, 438, 440 can be affected by the flow 512 of the medium 502 in the channel 434, the channel 434 and the connection region 442 can be considered as sweep (or flow) regions of the microfluidic circuit 432. On the other hand, the isolation regions 444 of the pens 436, 438, 440 can be considered unswept (or non-flowing) regions. For example, a first medium 502 (e.g., a component (not shown) in the first medium 502) in the channels 434 may be substantially mixed with a second medium 504 (e.g., a component (not shown) in the second medium 504) in the isolation region 444 merely by diffusing the first medium 504 from the channels 434 through the connection region 442 into the second medium 504 in the isolation region 444. Similarly, the second medium 504 in the isolation region 444 (e.g., a component (not shown) in the second medium 504 may be mixed with the first medium 504 in the channel 434 (e.g., a component (not shown) in the first medium 502)) substantially only by diffusing the second medium 502 from the isolation region 444 into the first medium 502 in the channel 434 through the connection region 442. The first medium 502 may be the same medium as the second medium 504 or a different medium. Furthermore, the first medium 502 and the second medium 504 may begin to be the same and then become different (e.g., by modulating the second medium by one or more biological micro-objects in the isolation region 444, or by altering the medium flowing through the channel 434).

Maximum penetration depth D of secondary stream 514 caused by stream 512 in channel 434 _p May depend on a number of parameters. Examples of such parameters include: the shape of the channels 434 (e.g., the channels may direct media into the connection region 442, deflect media away from the connection region 442, or simply flow through the connection region 442); width W of channel 434 at proximal opening 452 _ch (or cross-sectional area); width W of the connection region 442 at the proximal opening 452 _con (or cross-sectional area); maximum velocity V of flow 512 in channel 434 _max The method comprises the steps of carrying out a first treatment on the surface of the Viscosity of the first medium 502 and/or the second medium 504, etc.

In some embodiments, the dimensions of the channel 434 and the isolation pens 436, 438, 440 may be oriented with respect to the flow 512 in the channel 434 as follows: channel width W _ch (or cross-sectional area of channel 434) may be substantially perpendicular to flow 512, width W of connection region 442 at proximal opening 552 _con (or cross-sectional area) may be substantially parallel to flow 512, and the length L of the connection region _con May be substantially perpendicular to flow 512. The foregoing is merely an example, and the dimensions of the channel 434 and the spacer pens 436, 438, 440 may be in other directions relative to each other.

In some embodiments, the width W of the channel 434 at the proximal opening 452 _ch Can be within any of the following ranges: 50 to 1000 microns, 50 to 500 microns, 50 to 400 microns, 50 to 300 microns, 50 to 250 microns, 50 to 200 microns, 50 to 150 microns, 50 to 100 microns, 70 to 500 microns, 70 to 400 microns, 70 to 300 microns, 70 to 250 microns, 70 to 200 microns, 70 to 150 microns, 90 to 400 microns, 90 to 300 microns, 90 to 250 microns, 90 to 200 microns, 90 to 150 microns, 100 to 300 microns, 100 to 250 microns, 100 to 200 microns, 100 to 150 microns, and 100 to 120 microns. The foregoing is merely an example, and the width W of the channel 434 _ch May be within other ranges (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the height H of the channel 134 at the proximal opening 152 _ch Can be within any of the following ranges: 20 to 100 microns, 20 to 90 microns, 20 to 80 microns, 20 to 70 microns, 20 to 60 microns, 20 to 50 microns, 30 to 100 microns, 30 to 90 microns, 30 to 80 microns, 30 to 70 microns, 30 to 60 microns, 30 to 50 microns, 40 to 100 microns, 40 to 90 microns, 40 to 80 microns, 40 to 70 microns, 40 to 60 microns, or 40 to 50 microns. The foregoing is merely an example, and the height H of the channel 434 _ch May be within other ranges (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the cross-sectional area of the channel 434 at the proximal opening 452 may be within any of the following ranges: 500 to 50000 square micrometers, 500 to 40000 square micrometers, 500 to 30000 square micrometers, 500 to 25000 square micrometers, 500 to 20000 square micrometers, 500 to 15000 square micrometers, 500 to 10000 square micrometers, 500 to 7500 square micrometers, 500 to 5000 square micrometers, 1000 to 25000 square micrometers, 1000 to 20000 square micrometers, 1000 to 15000 square micrometers, 1000 to 10000 square micrometers, 1000 to 7500 square micrometers, 1000 to 5000 square micrometers, 2000 to 20000 square micrometers, 2000 to 15000 square micrometers, 2000 to 10000 square micrometers, 2000 to 7500 square micrometers, 2000 to 6000 square micrometers, 3000 to 20000 square micrometers, 3000 to 15000 square micrometers, 3000 to 10000 square micrometers, 3000 to 7500 square micrometers, or 3000 to 6000 square micrometers. The foregoing is merely an example, and the cross-sectional area of the channel 434 at the proximal opening 452 may be within other ranges (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the connection region L _con The length of (c) may be in any of the following ranges: 1 to 200 microns, 5 to 150 microns, 10 to 100 microns, 15 to 80 microns, 20 to 60 microns, 20 to 500 microns, 40 to 400 microns, 60 to 300 microns, 80 to 200 microns, and 100 to 150 microns. The foregoing is merely an example, and the length L of the connection region 442 _con May be within a range different from the foregoing examples (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the width W of the connection region 443 at the proximal opening 452 _con Can be within any of the following ranges: 20 to 500 microns, 20 to 400 microns, 20 to 300 microns, 20 to 200 microns, 20 to 150 microns, 20 to 100 microns, 20 to 80 microns, 20 to 60 microns, 30 to 400 microns, 30 to 300 microns, 30 to 200 microns, 30 to 150 microns, 30 to 100 microns, 30 to 80 microns, 30 to 60 microns, 40 to 300 microns, 40 to 200 microns, 40 to 150 microns, 40 to 100 microns, 40 to 80 microns, 40 to 60 microns, 50 to 250 microns, 50 to 200 microns, 50 to 150 microns, 50 to 100 microns, 50 to 80 microns, 60 to 200 microns, 60 to 150 microns, 60 to 100 microns, 60 to 80 microns, 70 to 150 microns, 70 to 100 microns, and 80 to 100 microns. The foregoing is merely an example, and the width W of the connection region 442 at the proximal opening 452 _con May be in a range different from the foregoing examples(e.g., a range defined by any of the endpoints listed above).

In other embodiments, the width W of the connection region 442 at the proximal opening 452 _con Can be within any of the following ranges: 2 to 35 microns, 2 to 25 microns, 2 to 20 microns, 2 to 15 microns, 2 to 10 microns, 2 to 7 microns, 2 to 5 microns, 2 to 3 microns, 3 to 25 microns, 3 to 20 microns, 3 to 15 microns, 3 to 10 microns, 3 to 7 microns, 3 to 5 microns, 3 to 4 microns, 4 to 20 microns, 4 to 15 microns, 4 to 10 microns, 4 to 7 microns, 4 to 5 microns, 5 to 15 microns, 5 to 10 microns, 5 to 7 microns, 6 to 15 microns, 6 to 10 microns, 6 to 7 microns, 7 to 15 microns, 7 to 10 microns, 8 to 15 microns, and 8 to 10 microns. The foregoing is merely an example, and the width W of the connection region 442 at the proximal opening 452 _con May be within a range different from the foregoing examples (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the length L of the connection region 442 at the proximal opening 452 _con Width W of connection region 442 _con The ratio of (c) may be greater than or equal to any of the following ranges: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or more. The foregoing is merely an example, and the length L of the connection region 442 at the proximal opening 452 _con Width W of connection region 442 _con May be different from the previous examples.

As shown in fig. 5, the width W of the connection region 442 _con The uniformity may be from the proximal opening 452 to the distal opening 454. Width W of the connection region 442 at the distal opening 454 _con May thus be as above for the width W of the connection region 442 at the proximal opening 452 _con Within any range determined. Alternatively, the width W of the connection region 442 at the distal opening 454 _con May be greater than (e.g., as shown in fig. 6) or less than (e.g., as shown in fig. 7A-7C) the width W of the connection region 442 at the proximal opening 452 _con 。

As also shown in fig. 5, the width of the isolation region 444 at the distal opening 454 may be substantially the same as the width of the connection region 442 at the proximal opening 452 W _con The same applies. The width of the isolation region 444 at the distal opening 454 may thus be as described above for the width W of the connection region 442 at the proximal opening 452 _con Within any range determined. Alternatively, the width of the isolation region 444 at the distal opening 454 may be greater than (e.g., as shown in fig. 6) or less than (not shown) the width W of the connection region 442 at the proximal opening 452 _con 。

In some embodiments, the maximum velocity V of flow 512 in channel 434 _max Is the maximum speed at which a channel can be maintained without causing structural damage in the microfluidic device in which the channel is located. The maximum speed at which a channel can be maintained depends on various factors including the structural integrity of the microfluidic device and the cross-sectional area of the channel. For the exemplary microfluidic device of the present invention, the maximum flow velocity V in the channel has a cross-sectional area of about 3000 to 4000 square microns _max About 10 μl/sec. Alternatively, the maximum velocity V of flow 512 in channel 434 _max May be provided to ensure that the isolation region 444 is isolated from the flow 512 in the channel 434. In particular, the width W of the proximal opening 452 of the connecting region 442 of the spacer pens 436, 438, 440 _con ，V _max May be set to ensure penetration depth D of secondary stream 514 into the connection region _p Less than L _con . For example, for a film having a width W with a width of about 30 to 40 microns _con Isolation fence, V, of the connection area of the proximal opening 452 of (c) _max May be set to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 μl/sec.

In some embodiments, the length L of the connection region 442 of the isolation pens 436, 438, 440 _con And the sum of the corresponding lengths of the isolation regions 444 may be short enough to allow the composition of the second medium 504 in the isolation regions 444 to diffuse relatively quickly into the first medium 502 in the channels 434. For example, in some embodiments, (1) the length L of the connection region 442 _con And (2) the sum of the distances between the biological micro-objects located in the isolation region 444 of the isolation pens 436, 438, 440 and the distal opening 454 of the connection region can be within the following range:40 to 300 microns, 50 to 550 microns, 60 to 500 microns, 70 to 180 microns, 80 to 160 microns, 90 to 140 microns, 100 to 120 microns, or any range including one of the foregoing endpoints. The diffusion rate of a molecule (e.g., an analyte of interest, such as an antibody) depends on a number of factors, including temperature, viscosity of the medium, and diffusion coefficient D of the molecule ₀ . D of IgG antibodies in aqueous solution at 20 DEG C ₀ Is about 4.4x10 ^-7 Square centimeter per second (cm) ² /sec), while the viscosity of the biological micro-object culture medium is about 9x10 ^-4 Square meter/second (m) ² /sec). Thus, for example, an antibody in a biological micro-object culture medium at 20 ℃ can have a diffusion rate of about 0.5 microns/sec. Thus, in some embodiments, the period of time for diffusing biological micro-objects located in the isolation region 444 into the channel 434 may be about 10 minutes or less (e.g., 9, 8, 7, 6, 5 minutes or less). The period of time for diffusion may be manipulated by varying parameters that affect the diffusion rate. For example, the temperature of the medium may be increased (e.g., to a physiological temperature such as 37 ℃) or decreased (e.g., to 15 ℃, 10 ℃, or 4 ℃) to increase or decrease the diffusion rate, respectively.

The configuration of the insulated fence 436 shown in fig. 5 is merely an example, and many variations are possible. For example, the isolation region 444 may be adapted to contain a plurality of micro-objects 522, but the isolation region 444 may be adapted to contain only one, two, three, four, five, or similar relatively small number of micro-objects 522. Thus, the capacity of the isolation region 444 may be, for example, at least 3x10 ³ 、6x10 ³ 、9x10 ³ 、1x10 ⁴ 、2x10 ⁴ 、4x10 ⁴ 、8x10 ⁴ 、1x10 ⁵ 、2x10 ⁵ 、4x10 ⁵ 、8x10 ⁵ 、1x10 ⁶ 、2x10 ⁶ Cubic microns or more.

As another example, the spacer rail 436 is shown extending generally perpendicularly from the channel 434 and thus forming a generally 90 ° angle with the channel 434. The isolation fence 436 may alternatively extend from the channel 434 at other angles (such as any angle between 30 ° and 150 °).

As yet another example, the connection region 442 and the isolation region 444 are shown as being substantially rectangular in fig. 5, but one or both of the connection region 442 and the isolation region 444 may be other shapes. Examples of such shapes include oval, triangular, circular, hourglass, and the like.

As yet another example, the connection region 442 and the isolation region 444 are shown in fig. 5 as having substantially uniform widths. That is, in fig. 5, the width W of the connection region 442 _con Is shown as being uniform from the proximal opening 452 to the distal opening 454; the corresponding width of the isolation regions 444 is similarly uniform; and the width W of the connection region 442 _con And the corresponding widths of the isolation regions 444 are considered to be equal. Any of the foregoing aspects may differ from that shown in fig. 5. For example, the width W of the connection region 442 _con May vary (e.g., in a trapezoidal or hourglass fashion) from proximal opening 452 to distal opening 454; the width of the isolation region 444 may vary (e.g., in a triangular or flask fashion); width W of connecting region 442 _con May be different than the corresponding width of the isolation region 444.

Fig. 6 shows an example of an insulated fence showing an example of some of the foregoing variations. The pens shown in fig. 6 may be substituted for any of the pens 436, 438, 440 in any of the figures or discussed herein.

The isolation fence of fig. 6 can include a connection region 642 and an isolation structure 646 that includes an isolation region 644. The connection region 642 may include a proximal opening 652 to the channel 434 and a distal opening 654 to the isolation region 644. In the example shown in FIG. 6, the connecting region 642 is enlarged such that its width W _con Increasing from proximal opening 652 to distal opening 654. However, except for the shape, the connection region 642, isolation structure 646 and isolation region 644 may be substantially identical to the connection region 442, isolation structure 446 and isolation region 444 of fig. 5 as discussed above.

For example, the channel 434 and isolation fence of fig. 6 may be configured such that the maximum penetration depth D of the secondary stream 514 _p Extends to the connection region 642 but not to the isolation region 644. Length L of the connection region 642 _con Can thus be greater than the maximum penetration depth D _p As generally discussed above with respect to fig. 5. As also discussed above, so long as the velocity of stream 512 in channel 434 does not exceed the maximum flow velocity V _max The micro-objects 522 in the isolation region 644 will thus remain in the isolation region 644. The channels 434 and connecting regions 642 are thus examples of swept (or flowing) regions, while the isolation regions 644 are examples of unswept (or non-flowing) regions.

Fig. 7A-7C illustrate examples of variations of the microfluidic circuit 432 and channel 434 of fig. 4A-4C, as well as additional examples of variations of the isolation pens 436, 438, 440. The spacer rail 736 shown in fig. 7A-7C can replace any of the rails 436, 438, 440 in any of the figures and discussed herein. Likewise, the microfluidic device 700 may replace the microfluidic device 400 in any of the figures and discussion herein.

The microfluidic device 700 of fig. 7A-7C may include a support structure (not visible, but may be similar to 404 of fig. 4A-4C), a microfluidic circuit structure 712, and a cover (not visible, but may be similar to 422). The microfluidic circuit structure 712 may include a frame 714 and a microfluidic circuit material 716, which may be the same as the frame 414 and microfluidic circuit material 416 of fig. 4A-4C or substantially similar to the frame 414 and microfluidic circuit material 416 of fig. 4A-4C. As shown in fig. 7A, the microfluidic circuit 732 defined by the microfluidic circuit material 716 can include a plurality of channels 734 (two channels 734 are shown, but there can be more) to which a plurality of isolation pens 736 are fluidly connected.

Each isolation fence 736 can include an isolation structure 746, an isolation region 744 within the isolation structure 746, and a connection region 742. From the proximal opening 772 at the channel 734 to the distal opening 774 at the isolation structure 736, the connection region 742 may fluidly connect the channel 734 to the isolation region 744. Generally, in accordance with the discussion above of FIG. 5, the flow 782 of the first fluid medium 702 in the channel 734 can form a secondary flow 784 of the first medium 702 from the channel 734 into and/or out of the connection region 742 of the rail 736 connected to the channel 734.

As shown in FIG. 7B, the connection region 742 may include a region between the proximal opening 772 to the channel 734 and the distal opening 774 to the isolation structure 746. Length L of connection region 742 _con Can be greater than the maximum penetration depth D of the secondary flow 784 _p In this case, secondary flow 784 would extend into connection region 742 without being redirected toward isolation region 744 (as shown in fig. 7A). Alternatively, as shown in FIG. 7C, the connection region 742 may have a depth D less than the maximum penetration depth D _p Length L of (2) _con In this case, secondary flow 784 would extend through connection region 742 and may be redirected toward isolation region 744. In the latter case, the length L of the connection region 742 _c1 And L _c2 May be greater than the maximum penetration depth D _p . In this way, secondary flow 784 will not extend into isolation region 744. Regardless of the length L of the attachment region 742 _con Greater than penetration depth D _p Or length L of connection region 742 _c1 And L _c2 Is greater than the penetration depth D _p Not exceeding maximum speed V _max The stream 782 of the first medium 702 in the channel 734 of (1) will result in a channel with a penetration depth D _p And micro-objects (not shown, but which may be similar to 522 in fig. 5) in the isolation region 744 of the rail 736 will not leave the isolation region 744 by the flow 782 of the first medium 702 in the channel 734. The flow 782 in the channel 734 does not cause stray material (not shown) to leave the channel 734 into the isolation region 744 of the rail 736 or leave the isolation region 744 into the channel 734. Diffusion is the only way in which components in the first medium 702 in the channel 734 can move from the channel 734 into the second medium 704 in the isolation region 744 of the rail 736. Likewise, diffusion is the only way in which components in the second medium 704 in the isolation region 744 of the rail 736 can move from the isolation region 744 to the first medium 702 in the channel 734. The first medium 702 may be the same medium as the second medium 704, or the first medium 702 may be a different medium than the second medium 704. Alternatively, the first medium 702 and the second medium 704 may initially be the same and then become different (e.g., by one or more organisms in the isolation region 744 The micro-object modulates the second medium, or by changing the medium flowing through the channel 734).

As shown in fig. 7B, the width W of the channel 734 perpendicular to the direction of the flow 782 (see fig. 7A) in the channel 734 _ch May be substantially perpendicular to the width W of the proximal opening 772 _con1 And thus is substantially parallel to the width W of the distal opening 774 _con2 . However, the width W of the proximal opening 772 _con1 And the width W of distal opening 774 _con2 It need not be substantially perpendicular to each other. For example, width W of opening 772 at proximal end _con1 The width W of the shaft (not shown) and distal opening 774 oriented thereon _con2 The angle between the other axis on which it is oriented may be non-perpendicular and thus not 90 °. Examples of alternative angles include angles within any of the following ranges: between 30 ° and 90 °, between 45 ° and 90 °, between 60 ° and 90 °, etc.

For the foregoing discussion regarding microfluidic devices having a channel and one or more isolation pens, the fluidic medium (e.g., first medium and/or second medium) can be any fluid capable of enabling biological micro-objects to remain substantially in an determinable state. The determinable state will depend on the biological micro-object and the assay being performed. For example, if the biological micro-object is a biological micro-object that performs an assay on the secretion of a protein of interest, the biological micro-object will be essentially determinable provided that the biological micro-object is viable and capable of expressing (express) and secreting the protein.

Fig. 8 to 30 illustrate examples of the process 100 of fig. 1 for testing biological micro-objects (e.g., biological cells) in the microfluidic device 200 of fig. 2A to 2C or the microfluidic device 400 of fig. 4A to 4C. However, the process 100 is not limited to classifying biological micro-objects or operating the microfluidic device 200, 400. The microfluidic device 200, 400 is also not limited to the process 100. Furthermore, while aspects of the steps of process 100 may be discussed in connection with apparatus 200 rather than apparatus 400 (or vice versa), such aspects may be used with other apparatuses or any other similar microfluidic devices.

At step 102, the process 100 may load biological micro-objects into a microfluidic device. Fig. 8 illustrates an example in which biological micro-objects 802 (e.g., biological cells) are loaded into the flow region 240 (e.g., channel 252) of the microfluidic device 200. Fig. 9 illustrates an example in which sample material 902 including biological micro-objects 904 flows into channels 434 of a microfluidic device 400.

As shown in fig. 8 (which is similar to fig. 10, 11, 13, 14, 17, 18, 26, and 27, showing a partial top cross-sectional view into the flow region 240 of the device 200), a mixture of biological micro-objects 802 may be loaded into the channels 252 of the microfluidic device 200. For example, biological micro-objects 802 may be input into the device 200 through the inlet 208 (see fig. 2A-2C), and the biological micro-objects 802 may be moved in the channels 252 through the flow 804 of the medium 244. Stream 804 may be a convection stream. Once the biological micro-objects 802 are in the channel 252 and adjacent to the pen 256, the flow 804 can be stopped or slowed such that the biological micro-objects 802 remain in the flow channel 252 adjacent to the pen 256 for a period of time sufficient to perform steps 104 and 106. The mixture of biological micro-objects 802 loaded in the channel 252 may include different types of biological micro-objects and other components, such as fragments, proteins, contaminates, particles, and the like.

Fig. 9 illustrates an example in which sample material 902 including biological micro-objects 904 flows into channels 434 of a microfluidic device 400. In addition to biological micro-objects 904, sample material 902 may include other micro-objects (not shown) or materials (not shown). In some embodiments, the channels 434 may have a cross-sectional area as disclosed herein, for example, about 3000 to 6000 square micrometers or about 2500 to 4000 square micrometers. The sample material 902 may flow into the channel 434 at a rate disclosed herein, for example, about 0.05 to 0.25 μl/sec (e.g., about 0.1 to 0.2 μl/sec or about 0.14 to 0.15 μl/sec). In some embodiments, the control module 472 of fig. 4A may cause the control/monitoring device 480 to flow a first fluid medium (not shown) containing the sample material 902 through the port 424 into the channel 434. Once sample material 902 is in channel 434, the flow of media (not shown) in channel 434 may be slowed or substantially stopped. Starting and stopping the flow of media (not shown) in the channel 434 may include opening and closing a valve (not shown) that includes the passageway 426 of the port 424.

The biological micro-objects 802, 904 may be any biological micro-object 802, 904 to be determined for producing a particular analyte or analyte of interest. Examples of biological targets 802, 904 include biological targets such as mammalian biological targets, human biological targets, immune biological targets (e.g., T biological targets, B biological targets, macrophages, etc.), B biological target hybridoma cells, stem biological targets (e.g., bone marrow stem biological targets, adipose stem biological targets, etc.), transformed biological targets (e.g., transformed CHO biological targets, heLa biological targets, HEK biological targets, etc.), insect biological targets (e.g., sf9, sf21, highFive, etc.), protozoan biological targets (e.g., lizard leishmania), yeast biological targets (e.g., s, p, etc.), bacterial biological targets (e.g., e, B, bacillus subtilis, B, bacillus thuringiensis, etc.), any combination of the foregoing, and the like. Examples of biological micro-objects 904 also include embryos, such as mammalian embryos (e.g., human, primate, canine, feline, bear, bovine, ovine, caprine, equine, porcine, etc.), and the like. Examples of analytes of interest include, proteins, carbohydrates, lipids, nucleic acids, metabolites, and the like. Other examples of analytes of interest include materials comprising antibodies, such as IgG (e.g., subclass IgG1, igG2, igG3, or IgG 4), igM, igA, igD, or IgE class antibodies.

At step 104, the process 100 may perform a first test on the biological micro-objects loaded into the microfluidic device at step 102. Step 104 may include selecting a plurality of biological micro-objects based on the first test. Alternatively, step 104 may include selecting one of the biological micro-objects without performing the first test. Fig. 10 illustrates an example of performing a first test on biological micro-objects 802 in channels 252 of a microfluidic device 200, and fig. 11 illustrates an example of selecting biological micro-objects 802 according to the first test. (selected biological micro-objects are labeled 1002 in FIG. 11 and subsequent figures). Fig. 12 shows an example in which biological micro-objects 1202, 1204, 1206 are selected from micro-objects 904 in a channel 434 of a microfluidic device 400.

The first test may include any number of possible tests. For example, whether the first test is performed in the microfluidic device 200 or 400, the first test may be performed against a first feature of the biological micro-object 802 or the biological micro-object 904. The first test performed at step 104 may be any test that tests for any desired feature. For example, the desired features may relate to size, shape, and/or morphology of the biological micro-objects 802 or 904. The first test may include capturing an image of the biological micro-object 802 or the biological micro-object 904 and analyzing the image to determine which biological micro-object 802 or biological micro-object 904 has the desired characteristics. As another example, the first test performed at step 104 may determine which biological micro-objects 802 or 904 exhibit a particular detectable condition indicative of the first characteristic. For example, the first feature may represent one or more cell surface markers and the first test performed at step 104 may detect the presence or absence of such cell surface markers on the biological micro-objects 802, 904. By testing for an appropriate cell surface marker or combination of cell surface markers, a particular cell type may be identified and selected at step 104. Examples of such specific cell types may include healthy cells, cancer cells, infected cells (e.g., infected with a virus or parasite), immune cells (e.g., B cells, T cells, macrophages), stem cells, and the like.

In the example shown in fig. 10, the detectable condition of the biological micro-objects in the microfluidic device 200 is radiation of energy 1006, which may be, for example, electromagnetic radiation. The biological micro-objects 802 may be pre-treated with an assay material (not shown) that causes the biological micro-objects 802 having the first characteristic to radiate energy 1006 prior to loading the biological micro-objects into the microfluidic device 200 or channel 252.

Examples of the first feature tested at step 104 may include, but are not limited to, a biological state (e.g., cell type) or a particular biological activity of the biological micro-object 802. For example, the first feature may be an observable physical feature such as size, shape, color, texture, surface topography, identifiable sub-assembly, or other feature markers. Alternatively, the first characteristic may be a measurable characteristic, such as permeability, conductivity, capacitance, response to a change in environment, or production (e.g., expression, secretion, etc.) of a particular biological material of interest. The particular biological material of interest may be a cell surface marker (e.g., annexin, glycoprotein, etc.). Another example of a particular biological material of interest is a therapeutic protein, such as an antibody (e.g., an IgG-type antibody) that specifically binds to an antigen of interest. Thus, the selected biological micro-objects 1002 may be one or more biological micro-objects 802 that are positive for the production (e.g., expression) of a particular biological material test, such as a cell surface marker, and the unselected biological micro-objects 1004 may be biological micro-objects 802 that are not positive for the foregoing test. Suitable assay materials through which biological micro-objects 802 can be pre-treated include reactants that bind to a particular biological material of interest and include tags that radiate energy 1006.

As shown in fig. 11, biological micro-objects 1002 may be selected by trapping micro-objects 1002 using optical traps 1102. As generally discussed with reference to fig. 3A and 3B, by directing a varying pattern of light to the channel 252, an optical trap 1102 may be created, moved, and closed in the channel 252 of the microfluidic device 200. Unselected biological micro-objects in fig. 11 are labeled 1004. In the example shown in fig. 11, no optical trap 1102 is created for the unselected biological micro-objects 1004.

Fig. 12 shows the selection of biological micro-objects 1202, 1204, 1206 from the biological micro-objects 904 in the channel 434 of the microfluidic device 400 at step 104. The selection may be in response to the result of the first test performed at step 104. Alternatively, the selection of the micro-objects 1202, 1204, 1206 may be a random selection, and thus performed without performing the first test. If based on the first test, step 104 may include, for example, selecting biological micro-objects 1202, 1204, 1206 for one or more observable physical or determinable features as discussed above. For example, biological micro-objects 1202, 1204, 1206 may be selected from micro-objects 904 in the sample material 90 based on any of a plurality of possible detectable features, such as biological micro-object type-specific features and/or features related to biological micro-object viability and health. Examples of such features include size, shape, color, texture, permeability, conductivity, capacitance, expression of biological micro-object-specific markers, response to changes in the environment, and the like. In one particular embodiment, biological micro-objects 904 having a cross-section with a circular shape may be selected from the sample material 602, the cross-section having a diameter within any of the following ranges: 0.5 to 2.5 microns, 1 to 5 microns, 2.5 to 7.5 microns, 5 to 10 microns, 5 to 15 microns, 5 to 20 microns, 5 to 25 microns, 10 to 15 microns, 10 to 20 microns, 10 to 25 microns, 10 to 30 microns, 15 to 20 microns, 15 to 25 microns, 15 to 30 microns, 15 to 35 microns, 20 to 25 microns, 20 to 30 microns, 20 to 35 microns, or 20 to 40 microns. As another example, biological micro-objects 604 may be selected from the sample material 902, the biological micro-objects 604 having a size between 100 and 500 microns (e.g., between 100 and 200 microns, between 150 and 300 microns, 200 to 400 microns, or 250 to 500 microns).

Although the example shown in fig. 12 illustrates the selection of micro-objects 1202, 1204, 1206 in the channel 434, the sample material 902 may alternatively be at least partially in the connection region 442 of the pens 436, 438, 440. The micro-objects 1202, 1204, 1206 may thus be selected in the connection region 442 at the same time.

In some embodiments, the control module 472 may perform a first test at step 104 by having the control/monitoring device 480 capture an image of biological micro-objects 904 in the sample material 902. The control module 472, which may be configured with known image analysis algorithms, may analyze the image and identify a plurality of biological micro-objects 904 having desired characteristics. Alternatively, a human user may analyze the captured image.

To determine characteristics of biological micro-objects, a human user and/or control module 472 may control the determination. For example, biological micro-objects, such as biological micro-objects, can be determined for magnetic permeability, conductivity, or biological micro-object-specific markers (e.g., using antibodies to biological micro-object surface proteins).

At step 106, the process 100 may isolate the selected biological micro-objects or select biological micro-objects as part of step 104. However, if the biological micro-objects are selected without performing the first step at step 104, step 106 may be skipped or may include simply flushing the unselected biological micro-objects out of the channel 252 (and, optionally, the flow region 240 as well). Fig. 13 and 14 illustrate examples in which selected biological micro-objects 1002 are moved into holding pens 256 in a microfluidic device 200 and unselected biological micro-objects 1004 are flushed out of a channel 252. Fig. 15 and 16 illustrate examples in which selected biological micro-objects 1202, 1204, 1206 are moved into the isolated region 444 of the pens 436, 438, 440 of the microfluidic device 400, and then unselected micro-objects 904 are rinsed out of the flow channel 434.

As mentioned above with respect to fig. 11, each biological micro-object 1002 may be selected by an optical trap 1102. For example, the selector 222 (see fig. 2A-2C) configured as the DEP device of fig. 3A and 3B may produce an optical trap 1102 that traps individual selected biological micro-objects 1002. As shown in fig. 13, the DEP apparatus 300 can then move the light trap 1102 into the pen 256, which moves the captured selected biological micro-objects 1002 into the pen 256. As shown, each selected biological micro-object 1002 can be individually captured and moved into the holding pen 256. Alternatively, more than one selected biological micro-object 1002 can be captured by a single well and/or more than one selected biological micro-object 1002 can be moved into any pen 256. Regardless, two or more selected biological micro-objects 1002 can be selected in the channel 252 and moved in parallel into the pen 256.

As discussed above with reference to fig. 3A and 3B, the light trap 1102 may be part of the pattern of variation 322 of light projected onto the inner surface 242 of the flow region 240 of the microfluidic device 200. As shown in fig. 14, once a selected biological micro-object 1002 is in a pen 256, the optical trap corresponding to the biological micro-object 1002 can be turned off. The detector 224 can capture all or part of the images of the flow area 240, including images of selected and unselected biological micro-objects 1002, 1004, channels 252, and pens 256, and these images can help identify, capture, and move individual selected biological micro-objects 1002 to a particular pen 256. The detector 224 and/or the selector 222 (e.g., configured as the DEP device of fig. 3A and 3B) may thus be one or more examples of a separation device for separating micro-objects that are positive for the first characteristic test (e.g., selected biological micro-objects 1002) from micro-objects that are negative for the first characteristic test (e.g., unselected biological micro-objects 1004).

As shown in fig. 14, with the selected biological micro-objects 1002 in the pens 256, the flow 804 (e.g., bulk flow) of medium 244 can flush the unselected biological micro-objects 1004 out of the channel 252. As mentioned, after loading biological micro-objects 904 into the channel 252 at step 102, the flow 804 of the medium 252 may be stopped or slowed. As part of step 106, the flow 804 may be resumed or increased to flush unselected biological micro-objects 1004 out of the channel 252, and in some examples, out of the microfluidic device 200 (e.g., through the outlet 210).

The selected biological micro-objects 1202, 1204, 1206 may be moved into the isolation pens 436, 438, 440 of the isolation region 444 microfluidic device 400 in any number of possible ways. For example, as discussed above, the enclosure 402 of the microfluidic device may include a DEP configuration that may be used to capture and move a particular plurality of biological micro-objects 904 in the sample material 902.

For example, as shown in fig. 15, the control module 472 may draw a path 1512, 1514, 1516 from the channel 434 to the isolation region 444 of one of the isolation pens 436, 438, 440 for each of the selected biological micro-objects 1202, 1204, 1206. The control module 472 can then cause a DEP module (not shown) of the control/monitoring device 480 to generate and direct a pattern of light variations into the microfluidic circuit 432 to capture the selected biological micro-objects 1202, 1204, 1206 and move the selected biological micro-objects 1202, 1204, 1206 along paths 1512, 1514, 1516 into the isolation region 444 of the isolation pens 436, 438, 440. The control module 472 may also store data in the memory 476 identifying each of the selected biological micro-objects and the particular isolation pens 436, 438, 440 to which each of the selected biological micro-objects is moved.

Although one selected biological micro-object 1202, 1204, 1206 in each pen 436, 438, 444 is shown in the example of fig. 15, more than one biological micro-object 1202, 1204, 1206 may be moved into a single pen. Examples of the number of biological micro-objects that can be moved from the sample material 902 into the individual pens 136, 138, 140 include the following: 1. 2, 3, 4, 5, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 10, 5 to 50, 5 to 40, 5 to 30, 5 to 20, and 5 to 10. The foregoing is merely an example, and other numbers of biological micro-objects 904 may be moved from the sample material 902 into the individual pens 436, 438, 440.

In some embodiments, at least a portion of the sample material 902 may be loaded into the isolation region 444 of the pens 436, 438, 440 at step 104. Additionally, as part of step 104, micro-objects 1202, 1204, 1206 may be selected in isolation region 144. In such embodiments, the sample material 902 including the unselected micro-objects 904 may be removed from the isolation region 444 at step 106, leaving only the selected micro-objects 1202, 1204, 1206 in the isolation region 444.

As shown in fig. 16, as part of step 106, the channel 434 may be purged of sample material 902 including unselected micro-objects 904 by flushing the channel 434 with a flushing medium (not shown). In fig. 16, the flow of flushing medium through the channel 134 is marked 1602. The flow 1602 of flushing medium may be controlled such that the velocity of the flow 1602 is maintained to correspond to the maximum penetration depth D as discussed above _p Maximum flow velocity V of (2) _max Below. As also discussed above, this will be in the selected living beings held in the isolation region 444 of their respective pens 436, 438, 440Micro-objects 1202, 1204, 1206 and prevent material from one of channel 434 or pens 436, 438, 440 from contaminating the other of the pens. In some embodiments, the flushing medium flows to the channels 434 having the cross-sectional areas disclosed herein, for example, about 3000 to 6000 square microns or about 2500 to 4000 square microns. The flushing medium may flow into the channel at the rates disclosed herein, for example, about 0.05 to 5.0 μl/sec (e.g., about 0.1 to 2.0, 0.2 to 1.5, 0.5 to 1.0 μl/sec, or about 1.0 to 2.0 μl/sec). Purging the passage 434 may include flushing the passage 434 multiple times as part of step 106

In some embodiments, the control module 472 may cause the control/monitoring device 480 to clear the channel 434. For example, the control module 472 may cause the control/monitoring device 480 to flow flushing medium into the channel 434 through the port 424 and out of another port 424. The control module 472 may maintain the velocity of the stream 1602 below the maximum flow velocity V _max . For example, for a channel 434 having a cross-sectional area of about 3000 to 6000 square microns (or about 2500 to 4000 square microns), the control module 472 may maintain the velocity of the stream 1602 at a V of less than 5.0 μl/sec _max (e.g., 4.0, 3.0, or 2.0 μl/sec).

After steps 102 through 106, the process 100 has separated the mixture of biological micro-objects (e.g., 802, 904) in the microfluidic device (e.g., 200, 400) into selected biological micro-objects (e.g., 1004, 1202, 1204, 1206) and unselected biological micro-objects (e.g., 1004, 904). The process 100 also places selected biological micro-objects in holding pens (e.g., 256, 436, 438, 440) in the microfluidic device and flushes to remove unselected biological micro-objects. As discussed above, steps 102 to 106 may be repeated and thus performed k times, where k is 1 (in which case steps 102 to 106 are performed once but not repeated) or greater than 1. The result may be a number of selected biological micro-objects in the holding pen in the microfluidic device.

It should also be noted that step 104 may perform l tests for up to l different features, where l is a positive integer 1 or greater than 1, before performing step 106. For example, step 104 may be tested for a first feature (such as size, shape, morphology, texture, visible markers, etc.) of the biological micro-object, after which step 104 may be repeated to test for a subsequent feature (such as a determinable feature). Thus, the selected biological micro-objects may comprise biological micro-objects from the (poly) group of biological micro-objects loaded at step 102 that are positive for up to l different feature tests.

As mentioned, moving selected biological micro-objects from the channel (e.g., 252, 434) into the pen and flushing out unselected biological micro-objects from the channel is just one example of how step 106 may be performed. Other examples include moving unselected biological micro-objects from the channel into the pen and flushing out selected biological micro-objects from the channel. For example, selected biological micro-objects are flushed out of the channel and collected elsewhere in the microfluidic device or transferred to other devices (not shown), where the selected biological micro-objects may be further processed. Unselected biological micro-objects can then be removed from the holding pen and discarded.

At step 108, the process 100 may perform a test on the selected biological micro-object or biological micro-objects. If the first test is performed as part of step 104, the test may be a subsequent test (e.g., a second test). (hereinafter, the test performed at step 108 is referred to as a "subsequent test" to distinguish the "first test" discussed above in step 104). As mentioned, the subsequent test performed at step 108 may be tested for the same feature (i.e., first feature) or a different feature than the first test of step 104. As also mentioned above, if the subsequent test performed at step 108 is for a first feature (and thus for the same feature tested at step 104), the subsequent test may also be different from the first test. For example, for the detection of a first feature, the subsequent test may be more sensitive than the first test.

Fig. 17 and 18 illustrate examples in which the subsequent test performed at step 108 is performed in the microfluidic device 200 for a measurable feature different from the first feature tested at step 104. Fig. 19 to 25 show examples in which the test of step 108 is performed in a microfluidic device 400.

As shown in FIG. 17, the assay material 1702 can be flowed into the channel 252 in an amount sufficient to expose selected biological micro-objects 1002 in the pens 256 to the flow 804 of the assay material 1702. For example, while the barrier 254 can prevent the assay material 1702 from flowing directly from the channel 252 into the interior space of the pen 256, the assay material 1702 can enter the interior of the pen 256 in a diffuse manner and thus reach selected biological micro-objects 1002 in the pen. Assay material 1702 may include a material that reacts with selected biological micro-objects 1002 having subsequent characteristics to produce different detectable conditions. The assay material 1702 and the resulting different detectable conditions may be different from any of the assay materials and conditions discussed above for the first test at step 104. A wash buffer (not shown) can also flow into the channel 252 and allow diffusion into the pens 256 to wash selected biological micro-objects 1002.

The detectable condition may be radiation of energy having one or more criteria, such as a threshold intensity, frequencies in a particular frequency band, etc. The color of biological micro-objects 1002 is an example of radiating electromagnetic radiation in a particular frequency band. In the example shown in fig. 18, selected biological micro-objects 1002 that are positive for the subsequent feature test at step 18 continue to be labeled with labels 1002, but biological micro-objects that are negative for the subsequent feature test at step 108 (e.g., test non-positive) are labeled with labels 1802.

An example of a subsequent feature tested at step 410 may be the viability of biological micro-objects 1002. For example, the subsequent feature may be whether the biological micro-object 1002 is alive or dead, and the assay material may be a reactive dye, such as 7-amino actinomycin D. Such dyes may turn living biological micro-objects 1002 into a particular color and/or dead biological micro-objects into a different color. The detector 224 (see fig. 2A-2C) can capture an image of the biological micro-objects 1002 in the holding pen 256, and the control module 230 can be configured to analyze the image to determine which biological micro-objects exhibit a color corresponding to living biological micro-objects 1002 and/or which biological micro-objects exhibit a color corresponding to dead biological micro-objects 1002. Alternatively, a human operator may analyze the image from the detector 224. The detector 224 and/or the control module 230 so configured may thus be one or more examples of a testing device for testing micro-objects in a liquid medium in a flow path in a microfluidic device for a particular feature (e.g., a first feature or a subsequent feature).

Fig. 19 illustrates an example in which the test performed at step 108 is for an analyte of interest produced by a selected biological micro-object 1202, 1204, 1206 in an isolation pen 236, 238, 240 of a microfluidic device 400. In fig. 19, components of analyte 1902 of interest are labeled with tag 1904. The analyte of interest may be, for example, a protein, nucleic acid, carbohydrate, lipid, metabolite, or other molecule secreted or otherwise released by a particular cell type (e.g., a healthy cell, a cancer cell, a virus or parasite infected cell, an inflammatory response cell, etc.). The particular analyte of interest may be, for example, a growth factor, a cytokine (e.g., inflammatory or otherwise), a viral antigen, a parasitic antigen, a cancer cell-specific antigen, or a therapeutic drug (e.g., a therapeutic drug such as a hormone or therapeutic antibody).

In the example shown in fig. 19, step 108 may include loading assay material 1910 into microfluidic device 400 and detecting a local reaction of analyte component 1904, if any. Step 108 may also include providing a incubation period after loading assay material 1910 into channel 434.

As shown in fig. 19, the assay material 1910 may substantially fill the channel 434 or at least fill the area adjacent the proximal opening 442 of the pens 436, 438, 440. In addition, the assay material 110 can extend into the connection region 442 of at least some of the isolation pens 436, 438, 440. In some embodiments, the assay material flows into channels 434 having the cross-sectional areas disclosed herein (e.g., about 3000 to 6000 square microns or about 2500 to 4000 square microns). The assay material can flow into the channel at a rate disclosed herein (e.g., about 0.02 to 0.25 μl/sec (e.g., about 0.03 to 0.2 μl/sec, or about 0.05 to 0.15 μl/sec, with a lower velocity for biological cell assay material and a higher velocity for non-cell assay material)). Once assay material 1910 is loaded into place in channel 434, the flow in channel 434 may be slowed or substantially stopped.

The assay material 1910 can flow into the channel 434 sufficiently quickly to place the assay material 1910 in a position adjacent the proximal opening 452 of the pens 436, 438, 440 before the analyte component 1904 generated in any of the pens 436, 438, 440 can diffuse into the channel 434. This can avoid problems with contamination of the channel 434 and/or other pens with analyte components from one pen 436, 438, 440 between the time that the selected biological micro-object 1202, 1204, 1206 is disposed in the pen 436, 438, 440 and the time that loading of the assay material 1910 into the channel 434 is completed.

The speed at which the assay material 1910 is loaded into the channel 434 may thus be at least a minimum flow rate V _min To be in a period of time T _load Completely loading the assay material 1910 into a position adjacent to the proximal opening 452 for a period of time T _load Less than a minimum period of time T for a sufficient amount of analyte component 1904 to diffuse from the isolation region 444 of the pens 436, 438, 440 into the channel 434 _diff . As used herein, "sufficient amount" refers to an amount of a detectable analyte component that is sufficient to affect accurate detection of the analyte component from the sequestration pen). Minimum flow velocity V _min May be a function of a variety of different parameters. Examples of such parameters include the length of the channel 434, the length L of the connection region 442 of the pens 436, 438, 440 _con Diffusivity of analyte component 1904, media viscosity, ambient temperature, etc. Minimum flow velocity V _min Examples of (a) include at least about 0.04 μl/sec (e.g., at least about 0.10, 0.11, 0.12, 0.13, 0.14 μl/sec, or higher).

Minimum flow velocity V for loading assay material 1910 into channel 434 _min May be smaller than the connection area 44 corresponding to less than the pens 436, 438, 440 as discussed aboveLength L of 2 _con Penetration depth D of (2) _p Maximum flow velocity V of (2) _max . For example, V _max /V _min The ratio of (c) may be in any of the following ranges: about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, or more.

The incubation period provided after loading the assay material 1910 may be sufficient to generate the analyte 1902 of interest to the biological micro-objects 1202, 1204, 1206 and to diffuse the analyte components 1904 from the isolation regions 444 of the pens 436, 438, 440 to the corresponding connection regions 442 or proximal openings 452. For example, the incubation period may provide sufficient time for analyte component 1904 to diffuse into channel 434.

The incubation period may include simply passively causing the biological micro-objects 1202, 1204, 1206 to naturally produce the analyte of interest 1902 in the sequestration pens 436, 438, 440. Alternatively, the incubation period may include actively stimulating the biological micro-objects 1202, 1204, 1206 to produce the analyte 1902 of interest by, for example, providing nutrients, growth factors, and/or induction factors to the biological micro-objects 1202, 1204, 1206; controlling the temperature, chemical composition, pH, etc. of the medium in the isolation region 444 of the isolation pens 436, 438, 440; directing excitation energy, such as light, into the isolation region 444, etc.

The term "culturing" as used herein covers the range previously described from just passively causing biological micro-objects 1202, 1204, 1206 to naturally produce analyte 1902 in isolation pens 436, 438, 440 to actively stimulating the production of analyte. Stimulating the production of the analyte 1902 may also include stimulating the growth of biological micro-objects 1202, 1204, 1206. Thus, for example, the biological micro-objects 1202, 1204, 1206 may be stimulated to grow before and/or while they are stimulated to produce the analyte of interest 1902. If the biological micro-objects 1202, 1204, 1206 have been loaded into the sequestration pens 436, 438, 440 as single biological micro-objects, the growth stimulus may result in the production of a clonal population of biological micro-objects that express and/or secrete (or may be stimulated to express and/or secrete) the analyte of interest.

In some embodiments, control module 472 may cause control/monitoring device 480 to perform one or more actions during incubation period 150. For example, the control module 472 may cause the control/monitoring device 480 to provide the growth medium and/or the induction medium periodically or in a continuous flow. Alternatively, the control module 472 may cause the control/monitoring device 480 to incubate the biological micro-objects for a period of time sufficient to allow the analyte of interest to diffuse into the channels 434. For example, in the case of a protein analyte such as an antibody, the control module 472 may provide a diffusion time equal to about 2 seconds for every 1 micron of distance that the biological micro-object is spaced from the channel 434. For proteins and other analytes that are significantly smaller than antibodies, the time required for diffusion may be less, such as 1.5 seconds or less per 1 micron (e.g., 1.25s/μm, 1.0s/μm, 0.75s/μm, 0.5s/μm or less). Conversely, for proteins or other analytes that are significantly larger than antibodies, the time allocated to diffusion may be greater, such as 2.0 seconds per micron or more (e.g., 2.25s/μm, 2.5s/μm, 2.75s/μm, 3.0s/μm or more).

It should be noted that the incubation period may continue during subsequent steps of the execution process 100. In addition, the incubation period may begin prior to completion of step 106 (e.g., during any of steps 102 through 106).

The assay material 1910 may be configured to interact with the analyte component 1904 of the analyte 902 of interest and produce a detectable reaction from the interaction. As shown in fig. 20, analyte components 1904 from biological micro-objects 1202, 1204 in the sequestration pens 436, 438 interact with assay material 1910 adjacent the proximal openings 452 of the sequestration pens 436, 438 to produce locally detectable reactions. However, the biological micro-objects 1206 in the isolation pen 440 do not produce the analyte 1902 of interest. Thus, no such localized reaction occurs (e.g., similar to 2002) adjacent the proximal opening 452 of the isolation pen 440.

The local reaction 2002 may be a detectable reaction. For example, the reaction 2002 can be localized luminescence (e.g., fluorescence). Furthermore, the local reactions 2002 may be sufficiently localized or separated to be able to be detected individually by a human observer, a camera in the control/monitoring device 480 of fig. 4A, or the like. For example, the channel 434 may be substantially filled with assay material 1910, the reaction of which (e.g., like 2002) is localized, that is, the space adjacent the proximal opening 452 corresponding to the isolation pens 436, 438 is restricted. As can be seen, the reaction 2002 can result from the aggregation of components of a plurality of assay materials 1910 adjacent to one or more proximal openings 452 of the sequestration pens 436, 438, 440.

The proximal openings 452 of the successive spacer pens 436, 438, 440 may be separated by at least a distance D _s (see fig. 4C), which is sufficient to distinguish the local reaction provided at the adjacent proximal opening 452, e.g., by a human observer, an image captured by a camera, etc. (e.g., similar to 2002). A suitable distance D between proximal openings 452 of successive spacer pens 436, 438, 440 _s Examples of (a) include at least 20, 25, 30, 35, 40, 45, 50, 55, 60 microns or more. Alternatively or additionally, components of the assay material 910 (e.g., capture micro-objects, such as biological micro-objects, microbeads, etc.) may be organized in front of the sequestration pen. For example, using DEP forces, etc., capture micro-objects can be grouped together and concentrated in a region of the channel 434 adjacent the proximal opening 452 of the isolation pens 436, 438, 440.

As mentioned, assay material 1910 including components such as capture micro-objects (e.g., biological micro-objects, microbeads, etc.) can enter and thus be partially disposed in the connection region 442 of the isolation pens 436, 438, 440. In this case, the reactions 2002, 2004 may occur entirely, substantially entirely, or partially in the connection region 442 as opposed to substantially entirely in the channel 434. Further, capture micro-objects (e.g., biological micro-objects, microbeads, etc.) in the assay material 1910 can be disposed in the isolation region 444. For example, DEP forces, etc. may be used to select capture micro-objects and move the capture micro-objects into the isolation region 444. For capture micro-objects disposed in the isolation region of the isolation pen, the capture micro-objects may be disposed proximate to the biological micro-object(s) and/or in a portion (e.g., sub-chamber) of the isolation region that is different from the portion occupied by the biological micro-object(s).

The assay material 1910 can be any material that specifically interacts directly or indirectly with the analyte 1902 of interest to produce a detectable reaction (e.g., 2002). Fig. 19-23 illustrate examples in which the analyte includes an antigen having two binding sites. Those skilled in the art will appreciate that the same example can readily accommodate situations where the analyte of interest is sometimes different from the two binding site antigens.

Fig. 21 (which shows a channel 434 and a portion of the proximal opening 452 of the isolation pen 436) shows an example of assay material 1910 with labeled capture micro-objects 2112. Each labeled capture micro-object 2112 may include a binding substance capable of specifically binding to analyte component 1904 and a label substance. As the analyte component 1904 diffuses toward the proximal opening 452 of the sequestration pen 436, labeled capture micro-objects 2112 in close proximity to (or within) the opening 452 may bind to the analyte component 1904, which may result in localized reactions 2002 (e.g., aggregation of labeled capture micro-objects 2112) immediately adjacent to (or within) the proximal opening 452.

Analyte component 1904 and labeled capture micro-objects 2112 are maximally bound when labeled capture micro-objects 2112 are immediately adjacent to end opening 452 or within proximal end opening 452. This is because the concentration of analyte component 1904 is highest in isolation region 444 and junction region 442, thereby facilitating the binding of analyte component 1904 and labeled capture micro-objects 2112 and facilitating their aggregation in these regions. As analyte components 1904 diffuse out of channel 234 and out of proximal opening 252, their concentration decreases. Thus, fewer analyte components 1904 bind to labeled capture micro-objects 2112 located outside of proximal opening 252. The reduced binding of analyte component 1904 to labeled capture micro-objects 2112 in turn results in reduced aggregation of labeled capture micro-objects 2112 outside of proximal opening 452. Labeled capture micro-objects 2112 that are not immediately adjacent to the proximal opening 452 of the pens 436, 438, 440 (or that are not within the proximal opening 452 of the pens 436, 438, 440) therefore do not produce a detectable localized reaction 2002 (or produce a localized reaction 2002 that is detectably smaller in size than occurs immediately adjacent to the proximal opening 452 or within the proximal opening 452).

For analyte components that do not have two binding sites for binding substances on the tagged capture micro-objects 2112, the tagged capture micro-objects can include two different binding substances (as discussed and illustrated below in fig. 23), each of which is capable of specifically binding to the analyte component. Alternatively, an assay is feasible if the analyte components aggregate (e.g., form homodimers, trimers, etc.).

Examples of tagged capture micro-objects 2112 include both inanimate micro-objects and biological micro-objects. Examples of inanimate micro-objects include microstructures such as microbeads (e.g., polystyrene beads), micro-rods, magnetic beads, quantum dots, and the like. The microstructures can be large (e.g., 10 to 15 microns in diameter, or greater) or small (e.g., less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 microns in diameter, or less). Examples of biological micro-objects include biological micro-objects (e.g., reporter biological micro-objects), liposomes (e.g., synthetic membrane preparations or derived from membrane preparations), liposome-coated microbeads, nanolipid rafts (see, e.g., "ritche et al (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, mehotd enzymol, 464:211-231 (li et al (2009), recombination of membrane proteins in phospholipid bilayer nanodiscs, method enzymology, 464:211-231)"), and the like.

Fig. 22 shows an example of an assay material 1910 that includes a mixture of capture micro-objects 2212 and a label whose composition is identified as 2222 and is hereinafter referred to as "label 2222". Fig. 23 shows an example of a configuration of capture micro-objects 2212, analyte components 1904, and labels 2222. Capture micro-objects 2212 can include a first affinity 2312 that specifically binds to first region 2302 of analyte component 1904. The label 2222 may include a second affinity reagent 2322 that specifically binds to the second region 2304 of the analyte component 1904. As shown in fig. 22, reaction 2002 occurs when a first region 2302 of analyte component 1904 binds to a first affinity agent 2312 that captures a micro-object 2212 and a second region 2304 of analyte component 1904 binds to a second affinity agent 2322 of label 2222.

As the analyte component 1904 generated by the biological micro-objects 1202 in the isolation region 444 of the isolation pen 436 diffuses toward the proximal opening 452, the analyte component 1904 can bind to the capture micro-objects 2212 and the labels 2222 in close proximity to the opening 452 (or within the opening 452), thereby causing the labels 2112 to accumulate on the surface of the capture micro-objects 2212. The binding of analyte component 1904 to labeled capture micro-objects 2212 is maximized when capture micro-objects 2212 are immediately adjacent to end opening 452 (or within proximal end opening 452). Similar to that discussed above, this is because the relatively high concentration of analyte component 1904 in isolation region 444 and connection region 442 facilitates the binding of analyte component 1904 to capture micro-objects 2212 and the corresponding association of labels 2222 at the surface of capture micro-objects 2212. As analyte component 1904 diffuses out of channel 434 and out of proximal opening 452, the concentration decreases and less analyte component 1904 binds to capture micro-objects 2212 located out of proximal opening 452. The reduction in binding of analyte component 1904 and capture micro-objects 2212 results in a reduction in accumulation of labels 2222 at the surface of capture micro-objects 2112 outside of proximal opening 452. Thus, capture micro-objects 2212 that are not immediately adjacent to the proximal opening 452 of the pens 436, 438, 440 (or within the proximal opening 452 of the pens 436, 438, 440) are not detectably labeled, or are labeled to an extent that they are not detectably labeled, which is detectably lower in size than the labels that occur immediately adjacent to the proximal opening 452 or within the proximal opening 452.

Examples of capture micro-objects 2212 include all of the examples set forth above for tagged capture micro-objects 2112. Examples of first affinity agent 2312 include a tag that specifically recognizes analyte component 1904 or a ligand that is specifically recognized by analyte component 1904. For example, in the case of an antibody analyte, the first affinity agent 2312 may be an antigen of interest.

Examples of labels 2222 include labels having luminescent labels (e.g., fluorescent labels) and labels having enzymes capable of cleaving signal molecules that fluoresce upon cleavage.

Examples of assay materials 1910 include assay materials that include a composite capture micro-object that includes a plurality of affinity agents. Fig. 24 shows an example of a composite capture micro-object 2412 comprising a first affinity agent 2402 and a second affinity agent 2404. The first affinity agent 2402 is capable of specifically binding to a first region 2302 of an analyte component 1904 (see fig. 23), and the second affinity agent 2404 is capable of specifically binding to a second region 2304 of the same analyte component 1904 or a different analyte component. In addition, first affinity agent 2402 and second affinity agent 2404 optionally bind simultaneously to first region 2302 and second region 2304 of analyte component 1904.

Examples of first affinity 2402 include those discussed above. Examples of second affinity agent 2404 include a receptor that specifically recognizes second region 2304 of analyte component 1904 or a ligand that specifically recognizes second region 2304 of analyte component 1904. For example, in the case of an antibody analyte, second affinity agent 2404 may bind to a constant region of the antibody. Examples of the foregoing include Fc molecules, antibodies (e.g., anti-IgG antibodies), protein a, protein G, and the like.

Another example of an assay material 1910 is one such assay material that includes a plurality of capture micro-objects. For example, assay material 1910 can include a first capture micro-object (not shown) with a first affinity agent 2402, and a second capture micro-object (not shown) with a second affinity agent 2404. The first capture micro-object may be different from the second capture micro-object. For example, the first capture micro-objects may have a size, color, shape, or other characteristic that distinguishes the first capture micro-objects from the second capture micro-objects. Alternatively, the first capture micro-objects and the second capture micro-objects may be substantially the same type of capture micro-objects, except for the type of affinity agent that each comprises.

Another example of an assay material 1910 is one that includes multiple types of capture micro-objects, each of which is designed to bind to a different analyte of interest. For example, the assay material 1910 may include a first capture micro-object (not shown) with a first affinity agent and a second capture micro-object (not shown) with a second affinity agent, wherein the first and second affinity agents do not bind to the same analyte of interest. The first capture micro-object may have a size, color, shape, label, or other characteristic that distinguishes the first capture micro-object from the second capture micro-object. In this way, multiple analytes of interest can be screened simultaneously.

Regardless of the specific content of the assay material 1910, in some embodiments, the control module 472 can cause the control/monitoring device 480 to load the assay material 1910 into the channel 434. The control module 472 may maintain the flow of assay material 1910 in the channel 434 at the minimum flow velocity V discussed above _min And maximum flow velocity V _max Between them. Once the assay material 1910 is in a position adjacent the proximal opening 452 of the pens 436, 438, 440, the control module 472 can substantially stop the flow of the assay material 1910 in the channel 434.

Step 108 performed in the microfluidic device 400 may include detecting a local reaction 2002 proximate to one or more proximal openings 452 of the isolation pens 436, 438, 440, which indicates a reaction of an analyte component 1904 with an assay material 1910 loaded into the channel 434. If a local reaction 2002 is detected proximate any proximal opening 452 of an isolation pen 436, 438, 440, it may be determined whether any of those detected local reactions 2002 is indicative of a positive performance of one or more biological micro-objects 1202, 1204, 1206 in the isolation pen 436, 438, 440. In some embodiments, a human user may view the channel 434 or the junction region 442 of the pens 436, 438, 440 to monitor the local response 2002 and determine whether the local response 2002 is indicative of a positive performance of the biological micro-objects 1202, 1204, 1206. In other embodiments, the control module 472 may be configured to perform this function. The process 2500 of fig. 25 is an example of the operation of the control module 472 for performing monitoring of a local reaction 2002 and determining whether the local reaction 2002 indicates positive performance of a biological micro-object 1202, 1204, 1206.

At step 2502, a control module 472 executing process 2500 may capture at least one image of the connection region 442 of the aisle 434 or the isolation pens 436, 438, 440 by a camera or other image capture device (not shown, but may be an element of the control/monitoring device 480 of fig. 4A). Examples of exposure times for capturing each image include 10 milliseconds to 2 seconds, 10 milliseconds to 1.5 seconds, 10 milliseconds to 1 second, 50 to 500 milliseconds, 50 to 400 milliseconds, 50 to 300 milliseconds, 100 to 500 milliseconds, 100 to 400 milliseconds, 100 to 300 milliseconds, 150 to 500 milliseconds, 150 to 400 milliseconds, 150 to 300 milliseconds, 200 to 500 milliseconds, 200 to 400 milliseconds, or 200 to 300 milliseconds. The control module 472 may capture one such image or multiple images. If the control module 472 captures an image, the image may be the last image referred to below. If the control module 472 captures multiple images, the control module 472 may incorporate two or more captured images into the final image. For example, the control module 472 may average two or more captured images. In some embodiments, the control module 472 may capture and average at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more captured images to produce a final image.

At step 2504, the control module 472 may identify any evidence of a local reaction 2002 in the final image. As discussed above, examples of the local reaction 2002 include luminescence (e.g., fluorescence), and the control module 472 can thus analyze the final image for luminescence proximate any proximal opening 452 of the isolation pens 436, 438, 440. The control module 472 can be programmed to identify the local reaction 2002 in the final image using any image processing technique. In an example, as shown in fig. 20, the control module 472 can detect a local reaction 2002 proximate to the proximal opening 452 of the isolation pens 436, 438.

At step 2506, the control module 472 may associate each local reaction 2002 detected at step 2504 with a corresponding isolation fence 436, 438, 440. For example, the control module 472 may perform this step as follows: each local reaction 2002 detected at step 2504 is associated with an isolation fence 436, 438, 440 having a proximal opening 452 nearest to the reaction 1002. In the example of fig. 20, the control module 472 may associate the reaction 2002 with the sequestration pens 436, 438.

The control module 472 may perform steps 2508 and 2510 of fig. 25 for each isolation fence 436, 438, 440 with which a reaction associated is detected at step 2506. With respect to the example of fig. 20, control module 472 can thus perform steps 2508 and 2510 for isolation fence 436, and then repeat steps 2508 and 2510 for isolation fence 438.

At step 2508, the control module 472 may determine whether the needle detected reaction 1002 associated with the current quarantine fence 436 indicates a positive result of the biological micro-object(s) 1202 in the current fence 436. For example, the control module 472 may extract data regarding the detected reaction 1002 from the last image obtained at step 2502 and determine whether the extracted data indicates a positive result. Any number of different criteria may be used. For example, the detected reaction 2002 may be luminescence, and the criteria for determining a positive result may include luminescence intensity exceeding a threshold, luminescence color falling within a predetermined color range, and so forth. If at step 2508, the control module 472 determines that the detected reaction is positive, the control module 472 may proceed to step 2510, wherein the control module 472 may identify the current isolation fence 436 as containing positive biological micro-objects 1202. If the determination at step 2508 is negative, the control module 472 may repeat step 2508 for the next sequestration pen 438 with which the detected reaction is associated at step 2506.

In the example shown in fig. 20, assume that the local reaction 2002 associated with the sequestration pen 436 is determined to be positive at step 2508, while the local reaction 2002 associated with the sequestration pen 438 is negative (e.g., luminescence is detected but it is below a threshold for determining that the sequestration pen 438 is positive). As previously described, no reaction is detected adjacent the proximal opening 452 of the isolation pen 440. Thus, the control module 472 recognizes only the isolation pen 436 as having a positive biological micro-object. Although not shown in fig. 25, as part of process 2500, control module 472 can identify the sequestration pens 438, 440 as negative.

Returning to fig. 1, at step 110, the process 100 may isolate biological micro-objects that test positive from biological micro-objects that test negative at step 108. Fig. 26 and 27 illustrate examples in which biological micro-objects 1002 that are negative for subsequent feature tests at step 108 are moved into channels 252 of microfluidic device 200 and then flushed out of channels 252 of microfluidic device 200. Fig. 29 shows an example in which negative biological micro-objects 1204, 1206 are separated from positive biological micro-objects 1202 in the microfluidic device 400.

As shown in fig. 26, each biological micro-object 1002 that tests negative at step 110 can be selected and trapped in the holding pen 256 by the optical trap 2602. Negative micro-objects are labeled 1802 in fig. 26. The light trap 2602 can then be moved from the holding pen 256 into the channel 252. As shown in fig. 27, the trap 2602 may be closed in the channel 252 and the flow 804 (e.g., convection) of the medium 244 may flush the negative biological micro-objects 1802 out of the channel 252 (and, optionally, out of the flow region 240). The assay material 1702 can diffuse out of the pen 256 and the flow 804 can also flush the assay material 1702 out of the channel 252.

The optical trap 2602 may be created and operated as described above. For example, as shown, each negative biological micro-object 2602 can be individually captured and moved from the holding pen 256 into the channel 252. Alternatively, more than one negative biological micro-object 2602 may be captured by a single well 2602. For example, there can be more than one biological micro-object 2602 in a single pen 256. Regardless, two or more negative biological micro-objects 2602 can be selected in the pen 256 and moved into the channel 252 in parallel.

The detector 224 can capture images of all or part of the flow region 240 (including images of biological micro-objects 1002 in the pens 256) and these images can help identify, capture, and move individual negative biological micro-objects 2602 out of a particular pen 256 and into the channel 252. The detector 224 and/or the selector 222 (e.g., configured as the DEP device of fig. 3A and 3B) may thus be one or more examples of a separation device for separating micro-objects that are positive for the feature test from micro-objects that are negative for the feature test.

As shown in fig. 27, for negative biological micro-objects 1802 in the channel 252, the flow 804 of the medium 244 may flush the biological micro-objects 1802 out of the channel 252 and, in some examples, out of the microfluidic device 200 (e.g., through the outlet 210). For example, if the flow 804 was previously stopped or slowed down, the flow 804 may be resumed or increased.

Alternatively, biological micro-objects 1002 that test positive at step 108 can be moved from the pen 256 into the channel 252 and flushed from the channel 252 at step 110 by the flow 804. In such examples, biological micro-objects 1002 that test positive at steps 104 and 108 may be collected elsewhere in the microfluidic device 200 for storage, further processing, transfer to another device (not shown), and so forth. Biological micro-objects 1802 that are negative for testing at step 108 can then be removed from the holding pens 256 and discarded.

As shown in fig. 28 and 29, assay material 1910 can be punched (2802) out of channel 434 (fig. 28). Then, as shown in fig. 29, biological micro-objects 1204, 1206 in the microfluidic device 400 tested negative at step 108 may be moved from the isolation pens 438, 440 into the channel 434, and the negative biological micro-objects 1204, 1206 may be cleared from the channel 434 (e.g., by a flow of medium in the channel 434 (not shown, but may be similar to 2802 of fig. 28)). The biological micro-objects 1204, 1206 may be moved from the isolation pens 438, 440 into the channel 434 in any manner (e.g., DEP, gravity, etc.) as discussed above for moving the biological micro-objects 1202, 1204, 1206 from the channel 434 into the isolation pens 436, 438, 440.

After steps 108 and 110, the process 100 also classifies the micro-objects (e.g., 1002, 1202, 1204, 1206) selected at step 104 according to the test performed at step 108. Further, the micro-objects selected at step 104 and also tested positive for the subsequent test at step 108 can remain in the holding pens (e.g., 256, 436, 438, 440) while the negative micro-objects can be removed.

As discussed above, steps 108 and 110 may be repeated and thus performed n times, where n is an integer of 1 (in which case steps 108 and 110 are performed once without repetition) or greater than 1. The subsequent tests performed at each repeated step 108 may be different tests. Alternatively, the subsequent test performed at the repeated step 108 may be the same test as previously performed at step 104 or the previous execution of step 108. The biological micro-objects (e.g., biological micro-objects) loaded at step 102 may thus be subjected to a series of n+1 tests. In some embodiments, each of the n+1 tests may be a different test, and in some embodiments, each of the n+1 tests may be for a different feature test. The process 100 may thus be able to sort out the set of tests positive for n+1 tests (which are different each time) from the initial mixture of biological micro-objects, and in some embodiments, the process 100 may sort out the set of tests positive for n+1 different features from the initial mixture of biological micro-objects.

Alternatively, the process 100 may select biological micro-objects at step 104, and then sort the selected biological micro-objects according to the number of tests of step 108 in which the biological micro-objects test positive (either simultaneously executing or repeating step 108). Evaluating multiple features in this manner is desirable for numerous applications including antibody identification. For example, multiple evaluations may contribute to any of the following: a conformation-specific antibody (e.g., the different feature may be the ability of an antibody analyte of different conformations to bind a particular antigen); epitope localization of antibody analytes (e.g., the different feature may be the ability to bind to various genes or chemically altered forms of an antigen); the ability to assess species cross-reactivity of antibody analytes (e.g., different characteristics may be the ability of antibody analytes to bind to homologous antigens from different species such as humans, mice, rats, and/or other animals (e.g., experimental animals)), and IgG isotypes of antibody analytes, for example, the production of chemically altered antigens for epitope localization of antibodies has been described in "Dhungana et al (2009), methods mol. Biol.524:119-34 (dungana et al (2009), methods of molecular biology 524:119-34)".

The entire process 100 may be repeated one or more times. Thus, after n steps 108 and 110 are performed, steps 102 through 106 may also be performed k more times, followed by n more steps 108 and 110. The number k need not be the same number for each repetition of process 100. Similarly, the number n need not be the same number for each repetition of process 100. For example, for the last iteration of steps 108 and 110 of a particular iteration of process 100, flow 804 shown in fig. 27 may load a new mixture of biological micro-objects into channel 252 of microfluidic device 200 as shown in fig. 8, and thus may be part of step 102 of the next process 100 performed on microfluidic device 200.

Similarly, the process 100 may be repeated multiple times on the microfluidic device 400. For example, the process 100 may be repeated to retest or reanalyze the positive biological micro-objects held in their sequestration pens 436, 438, 440 at step 110; retests and reanalyzes positive biological micro-objects at low density (e.g., one biological micro-object per sequestration pen) assuming that the initial test is performed on multiple biological objects per sequestration pen; testing or analyzing new biological micro-objects loaded into the microfluidic device 400 at the next iteration of step 108; testing or analyzing positive biological micro-objects held in their isolation pens 436, 438, 440 for different analyte materials at step 110 (e.g., by repeating step 108 using assay material 1910 designed to detect a second or additional analyte of interest), etc.

Fig. 30 shows another example. As shown, after step 110 has been performed, one or more biological micro-objects (e.g., 1202) held in the sequestration pen (e.g., 436) may be allowed to generate a clonal population 3002 of biological micro-objects in its sequestration pen (e.g., 436). All or a portion of process 100 (e.g., steps 108 and 110) may then be used to test or analyze population 3002. Alternatively, biological micro-objects may be isolated and retested as discussed above. In yet another alternative, biological micro-objects may be allowed to grow into a population before process 100 has been completed (e.g., after either of steps 106 or 108, but before step 110).

While specific embodiments and applications of the invention have been described in this specification, these embodiments and applications are merely exemplary and many variations are possible. For example, process 100 of fig. 1 and process 2500 of fig. 25 are merely examples, and variations are contemplated. Thus, for example, at least some of the steps of process 100 and/or process 2500 may be performed in an order different than shown, and some of the steps may be performed concurrently or may overlap with other steps. As other examples, the processes 100, 2500 may include additional steps not shown or lack some of the steps shown.

Example

Example 1-screening of mouse spleen cells secreting IgG antibodies capable of binding to human CD 45.

Screening was performed to identify mouse spleen cells that secrete IgG-type antibodies that bind to human CD 45. The experimental design comprises the following steps:

1. producing microbeads coated with CD45 antigen;

2. obtaining mouse spleen cells;

3. loading cells into a microfluidic device; and

4. the antigen specificity was determined.

The reagents used in the experiments include those shown in Table 1

TABLE 1 reagents

Production of microbeads coated with CD45 antigen

Microbeads coated with CD45 antigen were produced by:

50 μg of unsupported CD45 was resuspended in 500 μl PBS (pH 7.2).

slide-A-Lyzer was rinsed with 500. Mu.L PBS ^TM Mini-cup and then add microcentrifuge tube.

50. Mu.L of CD45 solution with the concentration of 0.1. Mu.g/. Mu.L is added to the slide-A-Lyzer after washing ^TM In the mini cup.

Will be 170 muL PBS was added to 2mg NHS-PEG 4-biotin, followed by 4.1. Mu.L NHS-PEG 4-biotin to the slide-A-Lyzer containing CD45 antigen ^TM In the mini cup.

EZ-Link incubation with CD45 antigen at RT ^TM NHS-PEG 4-Biotin for 1 hour.

After culturing, slide-A-Lyzer ^TM The mini-cup was removed from the microcentrifuge tube and placed into 1.3ml PBS (pH 7.2) in a second microcentrifuge tube and incubated at 4℃for the first 1 hour period with shaking. slide-A-Lyzer ^TM The mini-cup was then transferred to a third microcentrifuge tube containing 1.3ml of fresh PBS (pH 7.2) and incubated at 4℃for a second period of 1 hour with shaking. This last step was repeated three more times for a total of 5 1 hour incubations.

100. Mu.L of biotinylated CD45 solution (approximately 50 ng/. Mu.L) was transferred into labeled tubes.

500. Mu.L of streptavidin-coated microbeads from Spherech were transferred into microcentrifuge tubes, washed 3 times (1000. Mu.L/wash) in PBS (pH 7.4) and then centrifuged at 3000RCF for 5 minutes.

The beads were resuspended in 500. Mu.l PBS (pH 7.4) to give a bead concentration of 5 mg/ml.

50. Mu.L of biotinylated protein was mixed with resuspended Spheretech streptavidin-coated microbeads. The mixture was incubated at 4℃for 2 hours with shaking and then centrifuged at 3000RCF for 5 minutes at 4 ℃. The supernatant was discarded and the CD45 coated microbeads were washed 3 times in 1mL PBS (pH 7.4). The beads were then centrifuged at 3000RCF for an additional 5 minutes at 4 ℃. Finally, the microbead coated CD45 was resuspended in 500 μl of PBS pH7.4 and stored at 4 ℃.

Obtaining spleen cells of mice

Spleens of mice immunized with CD45 were obtained and placed in DMEM medium+10% fbs. Scissors were used to chop the spleens.

The crushed spleen was placed in a 40 μm cell filter. Individual cells were rinsed through the cell filter with a 10ml pipette. The glass rod was used to further rupture the spleen and force the single cells through the cell filter, after which the single cells were rinsed through the cell filter with a 10ml pipette.

Red blood cells were lysed by commercial kits.

Cells were centrifuged rapidly at 200xG and the primordial spleen cells were pelleted at 2e by 10ml pipette ⁸ The cells/ml concentration was resuspended in DMEM medium+10% fbs.

Loading cells into microfluidic devices

Splenocytes were introduced into microfluidic chips and loaded into pens, each pen containing 20 to 30 cells. 100. Mu.L of medium was flowed through the device at 1. Mu.L/sec to remove unwanted cells. The temperature was set at 36℃and the culture medium was perfused at a rate of 0.1. Mu.L/sec for 30 minutes.

Antigen-specific assay

Preparation of F (ab') 2-Alexa containing 1:2500 sheep anti-mice568, a cell medium.

100. Mu.L of CD45 microbeads contained 1:2500 diluted goat anti-mouse F (ab') -Alexa at 22. Mu.L568 was resuspended in the cell medium.

The resuspended CD45 microbeads then flowed into the main channel of the microfluidic chip at a rate of 1 μl/sec until they were adjacent to, but only outside of, the rail containing the splenocytes. Then, the fluid flow is stopped.

The microfluidic chip is then imaged in the bright field to determine the location of the microbeads.

Next, a texas red filter was used to capture images of cells and microbeads. An image was taken every 5 minutes for 1 hour, each exposure lasting 1000 milliseconds and with a gain of 5.

Results

The development of positive signals on the microbeads was observed, reflecting the diffusion of IgG isotype antibodies out of the specific pens and into the main channel where they were able to bind to the CD45 coated microbeads. The binding of the anti-CD 45 antibody to the microbeads allows the goat anti-mouse IgG-568 to associate with the microbeads and generate a detectable signal. See fig. 31A to 31C and white arrows.

Using the methods of the invention, each group of spleen cells associated with a positive signal can be isolated and moved as individual cells into a new pen and re-assayed. In this way, individual cells expressing anti-CD 45IgG antibodies can be detected.

In addition to any previously indicated modifications, many other variations and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention. Thus, while the information has been described above in connection with what is presently considered to be the most practical and preferred embodiment, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, forms, functions, manner of operation, and use, may be made without departing from the principles and concepts of the invention as set forth herein. As used herein, examples and embodiments are illustrative in various aspects and should not be construed as limiting in any way. It should also be noted that although the term "step" is used herein, the term may be used to simply draw attention to the different parts of the described method and is not meant to delineate the starting or ending points of any part of the method or to be limited in any other way.

Claims (32)

1. A microfluidic device, comprising:

a flow region configured to contain a flow of a first fluid medium; and

a microfluidic isolation fence comprising:

an isolation structure comprising an isolation region configured to contain a second fluid medium; and

a connection region fluidly connecting the isolation region to the flow region,

wherein, when the flow region and the microfluidic isolation rail are substantially filled with a fluidic medium:

the component of the second fluid medium is capable of diffusing into the first fluid medium or the component of the first fluid medium is capable of diffusing into the second fluid medium; and is also provided with

Substantially no flow of the first fluid medium from the flow region into the isolation region; and

a microfluidic channel comprising at least a portion of the flow region, wherein the connection region comprises a proximal opening into the microfluidic channel and a distal opening into the isolation region and the microfluidic channel is configured to comprise a plurality of capture micro-objects, and wherein the microfluidic channel has a cross-sectional height of 20 micrometers to 100 micrometers.

2. The device of claim 1, wherein a width of the channel at the proximal opening of the connection region is between 50 microns and 500 microns.

3. The device of claim 1, wherein a length L of the connection region from the proximal opening to the distal opening _con Greater than or equal to the penetration depth D of the first fluid medium into the connection region _p 。

4. A device according to any one of claims 1 to 3, wherein the length L of the connection region from the proximal opening to the distal opening _con And the width W of the proximal opening of the attachment region _con Sufficient to prevent micro-objects in the isolation region from moving from the isolation region through the connection region into the channel.

5. A device according to any one of claims 1 to 3, wherein the length L of the connection region of the proximal opening to the distal opening _con At least equal to the width W of the proximal opening of the attachment region _con 。

6. According to claim 1 to3, wherein the length L of the connection region from the proximal opening to the distal opening _con Width W of the proximal opening of the connection region _con At least 1.5 times.

7. A device according to any one of claims 1 to 3, the length L of the connection region of the proximal opening to the distal opening _con Width W of the proximal opening of the connection region _con At least 2.0 times.

8. A device according to any one of claims 1 to 3, wherein the proximal opening of the connection region has a width W of between 20 and 100 microns _con 。

9. A device according to any one of claims 2 to 3, wherein the length L of the connection region from the proximal opening to the distal opening _con Is sufficiently short that the composition of the second fluid medium is able to diffuse from the isolation region through the connection region into the microfluidic channel within ten minutes at 25 ℃.

10. A device according to any one of claims 1 to 3, wherein the length L of the connection region from the proximal opening to the distal opening _con Between 20 microns and 500 microns.

11. A process of analyzing biological micro-objects in a microfluidic device, the device comprising at least one microfluidic channel fluidically connected thereto by a microfluidic isolation pen, the at least one isolation pen comprising a fluidic isolation structure comprising an isolation region and a connection region fluidically connecting the isolation region to the channel, and the microfluidic channel having a cross-sectional height of 20 microns to 100 microns, the process comprising:

Loading one or more biological micro-objects into the at least one sequestration pen;

culturing the loaded biological micro-objects for a period of time sufficient for the biological micro-objects to produce analytes of interest;

disposing a capture micro-object in the channel adjacent to an opening from the connection region of the at least one sequestration pen to the channel, the capture micro-object comprising at least one type of affinity agent capable of specifically binding the analyte of interest; and

monitoring binding of the capture micro-objects to the analyte of interest.

12. The process of claim 11, wherein loading comprises loading the one or more biological micro-objects into the isolation region or regions of the at least one isolation pen.

13. The process of claim 11, wherein the biological micro-object is a biological cell.

14. The process of any one of claims 11 to 13, wherein loading the biological micro-objects comprises:

flowing a set of biological cells into the channel of the microfluidic device; and

moving the set of one or more biological cells into each of the at least one sequestration pen.

15. The process of any one of claims 11 to 13, further comprising rinsing to remove any biological cells remaining in the channel after loading the at least one sequestration pen.

16. The process of claim 15, wherein the flushing removes biological micro-objects from the channel without removing any of the loaded biological micro-objects from the at least one sequestration pen.

17. The process according to claim 14, wherein:

loading the biological micro-objects further comprises selecting individual biological micro-objects from the group that meet a predetermined criterion; and

selection is performed when the biological micro-object is within the connection region or the isolation region of the channel or the at least one isolation pen.

18. The process of any one of claims 11 to 13, wherein setting the capture micro-objects comprises:

flowing the capture micro-objects in the channel, and

stopping the flow such that the capture micro-objects are adjacent to the opening or openings from the connection region or regions of the at least one isolation fence.

19. The process of any one of claims 11 to 13, wherein the capture micro-objects comprise tags.

20. The process of any one of claims 11 to 13, wherein binding the capture micro-objects to the analyte of interest is monitored by monitoring the aggregation of the capture micro-objects.

21. The process of any one of claims 11 to 13, wherein disposing capture micro-objects in the channel comprises disposing a mixture of capture micro-objects and tags into the channel.

22. The process of claim 21, wherein the taggant comprises a fluorescent tag.

23. The process of any one of claims 11 to 13, wherein the analyte of interest is an antibody.

24. The process of claim 23, wherein the at least one type of affinity agent is an antigen specifically recognized by the antibody.

25. The process of claim 24, wherein the antigen is one of a protein, a carbohydrate, a lipid, a nucleic acid, a metabolite, an antibody, or a combination thereof.

26. The process of claim 23, wherein the at least one type of affinity agent is an Fc molecule, an antibody, protein a, or protein G.

27. The process of any one of claims 11 to 13, wherein the analyte of interest produced by the one or more biological micro-objects diffuses from the at least one sequestration pen into the channel.

28. The process of any one of claims 11 to 13, wherein the analyte of interest produced by the one or more biological micro-objects diffuses from the isolation region of the at least one isolation pen into the connection region.

29. The process of any one of claims 11 to 13, wherein:

the microfluidic device comprises a plurality of the isolation pens, each comprising a fluidic isolation structure comprising an isolation region and a connection region fluidically connecting the isolation region to the channel; and

providing the capture micro-objects includes providing the capture micro-objects or a mixture of capture micro-objects and tags in the channel adjacent to an opening from the connection area of the plurality of isolation pens to the channel.

30. The process of any one of claims 11 to 13, wherein:

length L of the connection region of the at least one isolation fence _con Greater than the maximum allowable flow rate V in the channel _max Penetration depth D of flowing medium _p And (b)

The process further includes maintaining any flow of the channel less than the maximum allowable flow rate V _max 。

31. The process of any one of claims 11 to 13, further comprising rinsing to remove the capture micro-objects from the channel after monitoring binding of the capture micro-objects to the analyte of interest.

32. The process according to claim 31, wherein:

length L of the connection region of the at least one isolation fence _con Greater than the maximum allowable flow rate V in the channel _max Penetration depth D of flowing medium _p And (b)

The flushing comprises causing flushing medium in the channel to flow at a rate less than the maximum allowable flow rate V _max And (3) flowing.