HK1225407B - Microfluidic devices having isolation pens and methods of testing biological micro-objects with same - Google Patents

Description

Microfluidic device with isolation fence and method for testing biological micro-target using the same

Background Art

As the field of microfluidics continues to advance, microfluidic devices have become convenient platforms for handling and manipulating microscopic objects such as biological cells. Some embodiments of the present invention are directed to improving microfluidic devices and methods of operating the same.

Summary of the Invention

In some embodiments of the present invention, a microfluidic device may include a flow region and a microfluidic isolation fence. The flow region may be configured to contain a flow of a first fluid medium. The microfluidic isolation fence may 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 fluidically connect the isolation region to the flow region such that when the flow region and the microfluidic isolation fence are substantially filled with fluid medium: components of the second medium can diffuse into the first medium, or components of the first medium can diffuse into the second medium; and substantially no first medium flows from the flow region into the isolation region.

Some embodiments of the present invention include a process for analyzing biological microtargets in a microfluidic device, which may include at least one microfluidic isolation fence having a microfluidic channel fluidically connected thereto. At least one isolation fence may include a fluid isolation structure comprising an isolation region and a connection region fluidically connecting the isolation region to the channel. The process may include loading one or more biological microtargets into the at least one isolation fence, and culturing the loaded biological microtargets for a period of time sufficient for the biological microtargets to produce an analyte of interest. The process may also include positioning a capture microtarget in the channel adjacent to an opening from the connection region of the at least one isolation fence to the channel, and monitoring the binding of the capture microtarget to the analyte of interest. The capture microtarget may include at least one type of affinity agent capable of specifically binding to the analyte of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example of a process by which at least two tests may be performed on micro-targets in a microfluidic device according to some embodiments of the present invention.

2A is a perspective view of a microfluidic device by which the process of FIG. 1 may be performed, according to some embodiments of the present invention.

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

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

3A is a partial side cross-sectional view of the microfluidic device of FIGs. 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 .

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

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

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

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

6 is another example of an isolation fence including a connection region from a channel to an isolation area that is longer than a penetration depth of a medium flowing in the channel, according to some embodiments of the present invention.

7A to 7C illustrate yet another example of a configuration of an isolation fence according to some embodiments of the present invention.

FIG. 8 illustrates an example of loading a biological micro-target into the flow path of the microfluidic device of FIG. 2A to FIG. 2C , according to some embodiments of the present invention.

FIG. 9 illustrates an example of flowing a biological micro-target into a channel of the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

10 illustrates an example of testing a biological micro-target in a flow path of the microfluidic device of FIG. 2A through FIG. 2C for a first feature, according to some embodiments of the present invention.

11 is an example of a biological micro-target selected in the microfluidic device of FIG. 2A to FIG. 2C according to some embodiments of the present invention.

FIG. 12 illustrates an example of selecting a biological microtarget in the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

FIG. 13 illustrates an example of moving a selected biological micro-target into a holding fence in the microfluidic device of FIG. 2A to FIG. 2C , according to some embodiments of the present invention.

14 illustrates examples of biological micro-targets flushed from the flow path of the microfluidic device of FIG. 2A to FIG. 2C , according to some embodiments of the present invention.

15 illustrates an example of moving a selected biological micro-target from a channel into an isolation fence of the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

16 is an example of biological micro-targets flushed from a channel in the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

17 is an example of providing assay material to a biological microtarget in a holding enclosure of the microfluidic device of FIGs. 2A-2C, according to some embodiments of the present invention.

18 illustrates assay material diffusing into the retaining fence of the microfluidic device of FIG. 2A-2C , according to some embodiments of the present invention.

19 shows examples of assay materials in channels of the microfluidic devices of FIG. 4A-4C and biological microtargets producing analytes of interest in isolation enclosures, according to some embodiments of the present invention.

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

21 is an example of an assay material including labeled capture micro-targets in the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

22 is an example of an assay material including a mixture of capture micro-targets and tags in the microfluidic device of FIG. 4A to FIG. 4C , according to some embodiments of the present invention.

23 illustrates an example of capturing micro-targets, components of a tag, and an analyte of interest of FIG. 22 , according to some embodiments of the present invention.

FIG. 24 illustrates an example of a composite capture microtarget comprising multiple affinity agents according to some embodiments of the present invention.

25 is a flowchart illustrating an example process of detecting localized reactions and identifying isolation fences containing positive biological microtargets in a microfluidic device, such as the device shown in FIGs. 4A-4C, according to some embodiments of the present invention.

26 illustrates moving a negative biological microtarget from a holding enclosure into a flow path in the apparatus of FIGs. 2A-2C, according to some embodiments of the present invention.

27 illustrates flushing of negative biological microtargets from the flow paths in the microfluidic device of FIGs. 2A-2C, according to some embodiments of the present invention.

28 illustrates an example of clearing a channel of assay material in the microfluidic device of FIG. 4A-4C , according to some embodiments of the present invention.

29 is an example of separating negative biological micro-targets from positive biological micro-targets in the fluidic device of FIG. 4A-4C, according to some embodiments of the present invention.

30 illustrates an example of generating clonal biological micro-targets in isolation enclosures in the microfluidic device of FIG. 4A through FIG. 4C , according to some embodiments of the present invention.

Figures 31A through 31C depict a microfluidic device comprising a microchannel and multiple isolation enclosures that open the microchannel. Each isolation enclosure contains multiple mouse splenocytes. Figure 31A is a bright field image of a portion of the microchannel device. Figures 31B and 31C are fluorescence images obtained using a Texas Red filter. In Figure 31B, the image was obtained 5 minutes after the start of the antigen-specific assay described in Example 1. In Figure 31C, the image was obtained 20 minutes after the start of the antigen-specific assay described in Example 1. The white arrows in Figure 31C point to isolation enclosures that produced a positive signal in the assay.

DETAILED DESCRIPTION

This specification describes exemplary embodiments and applications of the present invention. However, the present invention is not limited to these exemplary embodiments and applications, or to the manner in which the exemplary embodiments and applications are described herein or operate. Moreover, the accompanying drawings may show simplified or partial views, and for the sake of clarity, the dimensions of the elements in the accompanying drawings may not be enlarged or reduced to scale. In addition, when the terms "on...", "attached to," or "coupled to" are used herein, an element (e.g., a material, layer, substrate, etc.) may be "on another element," "attached to another element," or "coupled to another element," regardless of whether the element is directly on, attached to, or coupled to the other element, or whether there are one or more intervening elements between the element and the other element. In addition, if provided, directions (e.g., above, below, top, bottom, side, up, down, below, above, below, horizontal, vertical, "x," "y," "z," etc.) are relative and are provided only as examples, for ease of illustration and discussion, and are not intended to be limiting. Furthermore, where reference numbers are given to a list of elements (eg, elements a, b, c), these reference numbers are intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or combinations of all of the listed elements.

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

As used herein, the term "microtarget" may include one or more of the following: inanimate microtargets, such as microparticles, microbeads (e.g., polystyrene beads, Luminex ^™ beads, etc.), magnetic beads, microrods, microwires, quantum dots, etc.; biological microtargets, such as cells (e.g., cells obtained from tissue or body fluid samples, blood cells, hybrid cells, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, etc.), liposomes (e.g., synthetic membrane preparations or derived from membrane preparations), nanolipid rafts, etc.; or a combination of inanimate microtargets and biological microtargets (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). Nanolipid rafts have been described, for example, in "Ritchie et al. (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, Mehot d Enzymol., 464:211-231".

As used herein, the term "cell" refers to a biological cell, which can be a plant cell, an animal cell (e.g., a mammalian cell), a bacterial cell, a fungal cell, etc. Animal cells can be, for example, from humans, mice, rats, horses, goats, sheep, 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, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, etc.

As used herein with respect to a fluid medium, "diffuse" and "diffusion" refer to the thermodynamic movement of a component of a fluid medium toward a direction of lower concentration gradient.

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

The phrase "substantially no flow" refers to a flow rate of the fluid medium that is less than the rate at which components of a material (e.g., an analyte of interest) diffuse into or within the fluid medium. The diffusion rate of components of such a material 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" means that when the different regions are substantially filled with a liquid (such as a fluid medium), the fluid in each region is connected to form a single body of fluid. This does not mean that the fluids (or fluid media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules), which are constantly changing due to the solutes moving in the direction of their respective lower concentration gradients and/or the fluid flowing through the device.

In some embodiments, a microfluidic device may include a "touched" region and an "untouched" region. The untouched region may be fluidically connected to the touched region, provided that the fluid connection is configured to allow diffusion between the touched and untouched regions, but substantially no flow of the medium between the touched and untouched regions. The microfluidic device may thus be configured to substantially isolate the untouched region from the flow of the medium in the touched region, while allowing only diffusional fluid communication between the touched and untouched regions.

The ability of biological micro-targets (e.g., biological cells) to produce specific biological materials can be measured in such a microfluidic device. For example, for the production of an analyte of interest, sample materials including the biological micro-target to be measured can be loaded into the affected area of the microfluidic device. A plurality of biological micro-targets can be selected for specific features and arranged in an unaffected area. The remaining sample material can then flow out of the affected area, and the assay material can flow into the affected area. Since the selected biological micro-target is in the unaffected area, the selected biological micro-target is substantially unaffected by the outflow from the remaining sample material or the inflow of the assay material. The selected biological micro-target can allow the production of an analyte of interest, which can diffuse into the affected area from the unaffected area, where the analyte of interest can react with the assay material to produce a locally detectable reaction, each locally detectable reaction being associated with a specific unaffected area. Any unaffected area associated with the detected reaction can be analyzed to determine (if any) which biological micro-targets in the unaffected area are sufficient producers of the analyte of interest.

FIG1 illustrates an example of a process 100 for testing a microtarget in a microfluidic device according to some embodiments of the present invention. FIG2A through FIG2C illustrate an example of a microfluidic device 200 by which process 100 may be performed, and FIG3A and FIG3B illustrate an example of a dielectrophoresis (DEP) device that may be part of microfluidic device 200. FIG4A through FIG4C illustrate another example of a microfluidic device 400 by which process 100 may also be performed. However, neither the device 200 of FIG2A through FIG2C nor the device 400 of FIG4A through FIG4C is limited to performing the process 100 of FIG1 . Nor is process 100 limited to being performed on devices 200 or 400.

As shown in Figure 1, process 100 can load a mixture of microtargets into a flow path in a microfluidic device at step 102. The mixture loaded at step 102 can include different types of microtargets as well as debris and other targets. At step 104, process 100 can test the microtargets in the flow path for a first feature, and at step 106, process 100 can separate microtargets that are positive for the first feature test from microtargets that are not positive for the first feature test (e.g., microtargets that are negative for the test). As shown, process 100 can repeat steps 102 to 106 any number of times. For example, steps 102 to 106 can be performed k times, and then the mixture of k microtargets loaded at step 102 is divided into an initial group of microtargets (all microtargets of the initial group are tested positive for the first feature) at steps 104 and 106. Quantity k can be 1 or any integer greater than 1. (Hereinafter, biological microtargets that test positive for a substance are sometimes referred to as "positive," while biological microtargets that do not test positive for a substance (e.g., test negative for a substance) are sometimes referred to as "negative" biological microtargets.)

Then, process 100 can continue to step 108, where process 100 can perform subsequent tests on the initial group of micro-targets. The subsequent tests performed at step 108 can be different from the first test performed at step 104. For example, the subsequent tests can test for a subsequent feature that is different from the first feature tested at step 104. As another example, the subsequent tests performed at step 108 can test for the same feature as step 104 (the first feature mentioned above), but the subsequent tests have different sensitivity, accuracy, precision, etc. For example, for the first feature, the subsequent tests performed at step 108 can be more sensitive than the first test performed at step 104. In any case, at step 110, process 100 can separate the micro-targets that are positive for the subsequent tests at step 108 from the micro-targets that are negative for the subsequent tests.

If the first test in step 104 and the subsequent test in step 108 test for the same characteristic, after steps 108 and 110, the microtargets that tested positive for that characteristic (referred to as the first characteristic in the discussion of step 104 above) in response to the two different tests have been separated from the mixture of k microtargets loaded into the microfluidic device at the kth execution of step 102. As shown, steps 108 and 110 can be repeated, and at each repetition, process 100 can apply a different subsequent test at step 108 that tests for the same characteristic. In fact, steps 108 and 110 can be repeated n times, after which process 100 has sorted out from the mixture of k microtargets loaded into the microfluidic device at step 102 those microtargets that tested positive for the first characteristic tested in steps 104 and 108 n+1 times. The number n can be any integer 1 or greater.

As mentioned, alternatively, process 100 can test for a subsequent characteristic at step 108 that is different from the first characteristic tested at step 104. In such an embodiment, microtargets having both the first characteristic and the subsequent characteristic have already been sorted from the mixture of k microtargets loaded into the microfluidic device at step 102. If steps 108 and 110 are repeated, then at each repetition, process 100 can test for a different subsequent characteristic at step 108. For example, at each execution of step 108, process 100 can test for a subsequent characteristic that is different not only from the first characteristic but also from any previous subsequent characteristic tested by steps 108 and 110. At each execution of step 110, process 100 can isolate microtargets that tested positive for the subsequent characteristic 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 microtargets having all n+1 features tested at steps 104 and 108 from the mixture of k microtargets loaded into the microfluidic device at step 102. The number n may be any integer of 1 or greater.

Variations of process 100 are contemplated. For example, in some embodiments, repetitions of step 108 may sometimes test for new features that were not tested at step 104 or any previous execution of step 108, and at other times may test for the same features that were tested at step 104 or any previous execution of step 108. As another example, at step 106 or any repetition of step 110, process 100 may separate micro-targets that tested positive from micro-targets that tested negative. As yet another example, process 100 may repeat step 104 multiple times before continuing to step 106. In such an example, process 100 may test for a different feature at each repetition of step 104, and then separate micro-targets that tested positive at each repetition of step 104 from micro-targets that tested negative at at least one repetition of step 104. Similarly, step 108 may be repeated multiple times before continuing to step 110.

Examples of microfluidic devices 200 and 400 are now discussed with respect to Figures 2A through 7C. Operational examples of process 100 utilizing devices 200 and 400, where the micro-targets include biological micro-targets such as biological cells, are then described with respect to Figures 8 through 30.

2A-2C illustrate an example of a microfluidic device 200 by which process 100 may be performed. As shown, 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. FIG2B illustrates an inner surface 242 of the flow region 240 on which the medium 244 may be disposed as uniform (e.g., flat) and featureless. Alternatively, however, the inner surface 242 may be non-uniform (e.g., non-flat) 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, an opening, a valve, a channel, a fluid connector, etc. 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 Ser. No. 13/856,781, filed April 4, 2013 (Attorney Docket No. BL1-US). All or part 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 also include multiple 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 can define a flow region 240. Although one flow region 240 is shown in Figures 2A to 2C, the microfluidic structure 204 and the base 206 can define multiple flow regions for a medium 244. The flow region 240 can include channels (252 and 253 in Figure 2C) and chambers that can be interconnected to form a microfluidic circuit. For enclosures that include more than one flow region 240, each flow region 240 can be associated with one or more inlets 108 and one or more outlets 110 for respectively inputting and removing the medium 244 from the flow region 240.

As shown in Figures 2B and 2C, the flow area 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. As shown again in the figure, a retaining fence 256 that defines a non-flow space (or isolation area) may be provided in the flow area 240. That is, a portion of the interior of each retaining fence 256 may be a non-flow space, and except when the empty flow area 240 is initially filled with the medium 244, the medium 244 from the channel 252 does not flow directly into the non-flow space. For example, each retaining fence 256 may include one or more barriers 254 that form a partial enclosure, the interior of which may include a non-flow space. When the flow area 240 is filled with the medium 244, the barriers 254 that define the retaining fence 256 may therefore prevent the medium 244 from flowing directly from the channel 252 into the protected interior of any retaining fence 256. For example, when the flow area 240 is filled with the media 244, the barrier 254 of the fence 256 can substantially prevent the bulk flow of the media 244 from the channel 252 from flowing into the dead flow space of the fence 256, and instead, substantially only allow the media in the dead flow space in the fence 256 to mix with the media from the channel 252 by diffusion. Thus, the exchange of nutrients and waste between the dead flow space in the fence 256 and the channel 252 can occur substantially only by diffusion.

The foregoing can be accomplished by orienting the fences 256 so that the openings into the fences 256 do not directly face the flow of the medium 244 in the channel 252. For example, if the flow of the medium is from the inlet 208 to the outlet 210 (and therefore from left to right) in the channel 252 in FIG2C , each of the fences 256 substantially blocks the flow of the medium 244 directly from the channel 252 into the fences 256 because the opening of each fence 256 does not face the left side in FIG2C (which would otherwise directly enter such flow).

There may be many such retaining fences 256 in the flow region 240 arranged in any pattern, and the retaining fences 256 may be any of many different sizes and shapes. Although shown as being disposed relative to the sidewalls of the microfluidic structure 204 in FIG. 2C , one or more (including all) of the fences 256 may be separate structures disposed outside the sidewalls of the microfluidic structure 204 in the channel 252. As shown in FIG. 2C , the openings of the retaining fences 256 may be disposed adjacent to the channel 252, which may be adjacent to the openings of more than one fence 256. Although one channel 252 adjacent to fourteen fences 256 is shown, there may be many more channels 252, and there may be more or fewer fences 256 adjacent to any particular channel 252.

The barriers 254 of the enclosure 256 may comprise any of the types of materials discussed above with respect to the microfluidic structure 204. The barriers 254 may comprise the same material as the microfluidic structure 204 or a different material. As shown in FIG2B , the barriers 254 may extend from the surface 242 of the base 206, through the entire flow region 240, to an upper wall (opposite the surface 242) of the microfluidic structure 204. Alternatively, one or more barriers 254 may extend only partially across the flow region 240, and thus not fully extend to the surface 242 or the upper wall of the microfluidic structure 204.

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

For example, the selector 222 may include one or more optical (e.g., laser) tweezers devices and/or one or more optoelectronic tweezers (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. BL9-US) (the entire contents of which are also incorporated herein by reference). As yet another example, the selector 222 may include one or more devices (not shown) for moving droplets of the medium 244 in which one or more micro-targets are suspended. Such devices (not shown) may include electrowetting devices, such as electroelectrowetting (OEW) devices (e.g., as disclosed in U.S. Patent No. 6,958,132). The selector 222 may therefore be characterized as a DEP device in some embodiments.

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

In the example shown in FIG3B , a light pattern 322′ directed onto the inner surface 242 illuminates the cross-hatched electrode areas 314a shown in the square pattern. The other electrode areas 314 are not illuminated and are hereinafter referred to as “dark” electrode areas 314. The relative electrical impedance from each dark electrode area 314 through the electrode activation base 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 areas 314. However, illuminating the electrode areas 314a reduces the relative impedance from the illuminated electrode areas 314a through the electrode activation base 308 to the second electrode 310, which is less than the relative impedance from the first electrode 304 through the medium 244 in the flow region 240 to the illuminated electrode areas 314a.

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

3B is merely an example. Any pattern of electrode areas 314 can be illuminated by a pattern of light 322 projected into device 200, and the pattern of illuminated electrode areas 322' can be repeatedly changed by varying the light pattern 322.

In some embodiments, the electrode activation base 308 can be a photoconductive material, and the inner surface 242 can be featureless. In such embodiments, the DEP electrodes 314 can be formed anywhere and in any pattern on the inner surface 242 of the flow region 240 according to the light pattern 322 (see FIG. 3A ). The number and pattern of electrode regions 314 are therefore not fixed, but rather correspond to the light pattern 322. Examples are described in the aforementioned U.S. Patent No. 7,612,355, in which the undoped amorphous silicon material 24 shown in the drawings of the aforementioned patent can be an example of a photoconductive material that can constitute the electrode activation base 308.

In other embodiments, the electrode activation substrate 308 may comprise a circuit substrate such as a semiconductor material, including a plurality of doped layers, electrically insulating layers, and conductive layers that form a semiconductor integrated circuit, such as is known in the semiconductor art. In such an embodiment, the circuit element may form an electrical connection between the electrode region 314 at the inner surface 242 of the flow region 240 and the second electrode 310, which can be selectively activated and deactivated by the light pattern 322. When unactivated, 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, thereby activating the DEP electrode at the corresponding electrode region 314 as discussed above. The DEP electrodes that attract or repel micro-targets (not shown) in the medium 244 can thus be selectively activated and deactivated at a number of different electrode regions 314 on the inner surface 242 of the flow region 240 by the light pattern 322. Non-limiting examples of such configurations of the electrode activation substrate 308 include the phototransistor-based OET device 300 shown in Figures 21 and 22 of U.S. Patent No. 7,956,339 and the OET devices shown in all figures of the aforementioned U.S. Patent Application Serial No. 14/051,004.

In some embodiments, the first electrode 304 may be part of the first wall 302 (or 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 FIG3A. 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, while one or both of the electrode activation base 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 base 308 and the second electrode 310. For example, the electrode activation base 308 may include the first electrode 304 and/or the second electrode 310. Furthermore, the light source 320 may alternatively be located below the housing 202.

3A and 3B , selector 222 can thus select micro-targets (not shown) in medium 244 in flow region 240 by projecting a light pattern 322 into device 200 to activate one or more DEP electrodes at electrode regions 314 on inner surface 242 of flow region 240 in a pattern that surrounds and captures the micro-targets. Selector 222 can then move the captured micro-targets by moving light pattern 322 relative to device 200. Alternatively, device 200 can be moved relative to light pattern 322.

While barrier 254 defining retention fence 256 is shown in FIGS. 2B and 2C and discussed above as a physical barrier, barrier 254 may alternatively comprise a virtual barrier that includes a DEP force activated by light pattern 322 .

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

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

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

The control module 230 can 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, the controller 232 may be a digital electronic controller (e.g., a microprocessor, a microcontroller, a computer, etc.) configured to operate according to machine-readable instructions (e.g., software, firmware, microcode, etc.) stored as non-transitory signals in the memory 234, which may be a digital electronic, optical, or magnetic storage device. Alternatively, the controller 232 may include hard-wired digital circuits and/or analog circuits, or a combination of a digital electronic controller and hard-wired digital circuits and/or analog circuits that operate according to machine-readable instructions. The controller 232 may be configured to perform all or any portion of the processes 100, 2500 disclosed herein.

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

Figures 4A through 4C illustrate another example of a microfluidic device 400. As shown, the microfluidic device 400 may include a microfluidic circuit 432 comprising a plurality of interconnected fluid circuit elements. In the example shown in Figures 4A through 4C, the microfluidic circuit 432 includes a flow region/channel 434 with isolation fences 436, 438, 440 fluidically connected thereto. One channel 434 and three isolation fences 436, 438, 440 are shown, but there may be more than one channel 434 and more or less than three isolation fences 436, 438, 440 connected to any particular channel. The channel 434 and isolation fences 436, 438, 440 are examples of fluid circuit elements. The microfluidic circuit 432 may also include additional or different fluid circuit elements, such as fluid chambers, reservoirs, and the like.

Each isolation fence 436, 438, 440 may include an isolation structure 446 (see FIG4C ) defining an isolation region 444 and a connection region 442 fluidically 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 the maximum penetration depth of a flow of a fluid medium (not shown) flowing at a maximum velocity (V _max ) in the channel 434 does not extend into the isolation region 444. Micro-targets (not shown) or other materials (not shown) disposed in the isolation region 444 of the fences 436, 438, 440 may 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 region, and the isolation regions of the isolation fences 436, 438, 440 may be examples of unswept regions. Before discussing the foregoing in more detail, a brief description of the microfluidic device 400 and an example of an associated control system 470 is provided.

The microfluidic device 400 may include an enclosure 402 surrounding a microfluidic circuit 432, which may contain one or more fluid media. However, the device 400 may be physically configured differently. In the examples shown in Figures 4A to 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 each other. For example, the microfluidic circuit structure 412 may be disposed on the support structure 404, and the cover 422 may be disposed above the microfluidic circuit structure 412. Through the support structure 404 and the cover 422, the microfluidic circuit structure 412 may define a microfluidic circuit 432. The inner surface of the microfluidic circuit 432 is identified as 406 in the figures.

As shown in Figures 4A and 4B, the support structure 404 can be located at the bottom, while the cover 422 can be located at the top of the device 400. Alternatively, the support structure 404 and the cover 422 can be located in other directions. For example, the support structure 404 can be located at the top, while the cover 422 is located at the bottom of the device 400. In any case, there can be one or more ports 424, each including a passage 426 that enters or exits the enclosure 402. Examples of passages 426 include valves, doors, through holes, etc. Two ports 424 are shown, but the device 400 can 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 within the enclosure 402. In an example, as shown in FIG4A to FIG4C , the microfluidic circuit structure 412 includes a frame 414 and a microfluidic circuit material 416.

The support structure 404 may include a substrate or multiple interconnected substrates. For example, the support structure 404 may include one or more interconnected semiconductor substrates, printed circuit boards, etc. The frame 414 may partially or completely surround 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 metal material.

The microfluidic circuit material 416 is patterned with cavities, etc., to define the microfluidic circuit elements and interconnections of the microfluidic circuit 432. The microfluidic circuit material 416 may include a flexible material such as rubber, plastic, elastomer, silicone (e.g., patternable silicone), PDMS, etc., which may be gas permeable. Other examples of materials that may comprise the microfluidic circuit material 416 include molded glass, etchable materials such as silicon, photoresist (e.g., SU8), etc. In some embodiments, such a material (and therefore the microfluidic circuit material 416) may be rigid and/or substantially gas impermeable. Regardless, the microfluidic circuit material 416 may be disposed on the support structure 404 and within the frame 414.

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

4A also shows a simplified block diagram depiction of an example of a control/monitoring system 470 that can be used in conjunction with the microfluidic device 400. As shown, the system 470 can include a control module 472 and a control/monitoring device 480. The control module 472 can be configured to control and monitor the device 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, a 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 according to such machine-executable instructions stored in the memory 476. Alternatively or additionally, the controller 474 may include hard-wired digital circuits and/or analog circuits. The control module 472 may thus be configured to perform all or part of any process (e.g., process 100 of FIG. 1 and/or process 2500 of FIG. 25 ), the steps, functions, actions, etc. of such processes being discussed herein.

The control/monitoring device 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 device 480 may include a power supply (not shown) for providing power to the microfluidic device 400, a fluid medium source (not shown, but may include a flow controller similar to 226 of FIG. 2A ) for providing fluid medium to 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-targets (not shown) within the microfluidic circuit 432, an image capture mechanism (not shown, but may include a detector similar to 224 of FIG. 2A ) for capturing images (e.g., of the micro-targets) within the microfluidic circuit 432, an actuation mechanism (not shown) for directing energy into the microfluidic circuit 432 to stimulate a reaction, and the like.

As mentioned, the control/monitoring device 480 may include a power module for selecting and moving micro-targets (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-targets (not shown) in the microfluidic circuit. The base 404 and/or the cover 422 of the microfluidic device 400 may include a DEP configuration for selectively directing a DEP force to micro-targets (not shown) in a fluid medium (not shown) in the microfluidic circuit 432 to select, capture, and/or move individual micro-targets. The control/monitoring device 480 may include one or more control modules for such a DEP configuration.

An example of such a DEP configuration for the support structure 404 or the cover 422 is an optoelectronic tweezers (OET) configuration. Examples of suitable OET configurations for the support structure 404 or the cover 422 and associated monitoring and control equipment are described in the following U.S. Patents: U.S. Patent No. 7,612,355, U.S. Patent No. 7,956,339, U.S. Patent Application Publication No. 2012/0325665, U.S. Patent Application Publication No. 2014/0124370, U.S. Patent Application Serial No. 14/262,140 (pending), and U.S. Patent Application Serial No. 14/262,200 (pending), the entire contents of which are incorporated herein by reference. Micro-targets (not shown) can thus be individually selected, captured, and moved within the microfluidic circuit 432 of the microfluidic device 400 using a DEP device and techniques such as OET.

As mentioned, channel 434 and fence 436,438,440 can be configured to contain one or more fluid media (not shown). In the example shown in Figures 4A to 4C, port 424 is connected to channel 434 and allows fluid media (not shown) to be introduced into or removed from microfluidic circuit 432. Once microfluidic circuit 432 contains fluid media (not shown), the flow of fluid media (not shown) can be selectively generated and stopped in channel 434. For example, as shown, port 424 can be set at different positions (e.g., opposite ends) of channel 434, and the flow of medium (not shown) can be formed from one port 424 acting as an inlet to another port 424 acting 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 does not extend into the isolation region 444.

FIG5 shows a detailed view of an example of an isolation fence 436. Fences 438 and 440 can be similarly configured. Also shown is an example of a microtarget 522 within fence 436. As is well known, flow 512 of fluid medium 502 in microfluidic channel 434 through proximal opening 452 of fence 436 can cause secondary flow 514 of medium 502 to flow into and/or out of the fence. To isolate microtarget 522 within isolation region 444 of fence 436 from secondary flow 514, the length _Lcon of connection region 442 of isolation fence 436, from proximal opening 452 to distal opening 454, can be greater than the maximum penetration depth _Dp of secondary flow 514 flowing into connection region 442 when the velocity of flow 512 in channel 434 is at a maximum ( _Vmax ). As long as flow 512 in channel 434 does not exceed the maximum velocity _Vmax , flow 512 and the resulting secondary flow 514 can be confined to channel 434 and connection region 442 and kept outside isolation region 444. Flow 512 in channel 434 will therefore not cause micro-target 522 to leave isolation region 444. Micro-target 522 in isolation region 444 will therefore remain in isolation region 444 regardless of flow 512 in channel 432.

Furthermore, the flow 512 does not move miscellaneous particles (e.g., microparticles and/or nanoparticles) from the channel 434 into the isolated region 444 of the enclosure 436, nor does the flow 512 carry miscellaneous particles from the isolated region 444 into the channel 434. Thus, making the length _Lcon of the connecting region 442 greater than the maximum penetration depth _Dp prevents one enclosure 436 from being contaminated by miscellaneous particles from the channel 434 or the other enclosure 438, 440.

Because the connection region 442 of the channel 434 and the enclosures 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 the swept (or flow) region of the microfluidic circuit 432. On the other hand, the isolated region 444 of the enclosures 436, 438, 440 can be considered as the unswept (or non-flow) region. For example, the first medium 502 in the channel 434 (e.g., components (not shown) in the first medium 502) can be mixed with the second medium 504 in the isolated region 444 (e.g., components (not shown) in the second medium 504) substantially only by diffusion of the first medium 504 from the channel 434 through the connection region 442 into the second medium 504 in the isolated region 444. Similarly, the second medium 504 in the isolation region 444 (e.g., components (not shown) in the second medium 504) can be mixed with the first medium 504 in the channel 434 (e.g., components (not shown) in the first medium 502) essentially simply by causing the second medium 502 to diffuse from the isolation region 444 through the connecting region 442 into the first medium 502 in the channel 434. The first medium 502 can be the same medium as the second medium 504 or a different medium. In addition, the first medium 502 and the second medium 504 can start out as the same and then become different (e.g., by modulation of the second medium by one or more biological microtargets in the isolation region 444, or by changing the medium flowing through the channel 434).

The maximum penetration depth _Dp of the secondary flow 514 caused by the flow 512 in the channel 434 can depend on a number of parameters. Examples of such parameters include: the shape of the channel 434 (e.g., the channel can direct the medium into the connecting region 442, deflect the medium away from the connecting region 442, or simply flow through the connecting region 442); the width _Wch (or cross-sectional area) of the channel 434 at the proximal opening 452; the width _Wcon (or cross-sectional area) of the connecting region 442 at the proximal opening 452; the maximum velocity _Vmax of the flow 512 in the channel 434; 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 fences 436, 438, 440 may be oriented relative to the flow 512 in the channel 434 as follows: the channel width _Wch (or the cross-sectional area of the channel 434) may be substantially perpendicular to the flow 512, the width _Wcon (or the cross-sectional area) of the connection region 442 at the proximal opening 552 may be substantially parallel to the flow 512, and the length _Lcon of the connection region may be substantially perpendicular to the flow 512. The foregoing are examples only, and the dimensions of the channel 434 and the isolation fences 436, 438, 440 may be oriented in other directions relative to each other.

In some embodiments, the width _Wch of the channel 434 at the proximal opening 452 can be in 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 are examples only, and the width W _ch of the channel 434 can be within other ranges (eg, a range defined by any of the endpoints listed above).

In some embodiments, the height _Hch of the channel 134 at the proximal opening 152 can be in 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 are examples only, and the height _Hch of the channel 434 can be in other ranges (e.g., ranges defined by any of the endpoints listed above).

In some embodiments, the cross-sectional area of the channel 434 at the proximal opening 452 can be in any of the following ranges: 500 to 50,000 square microns, 500 to 40,000 square microns, 500 to 30,000 square microns, 500 to 25,000 square microns, 500 to 20,000 square microns, 500 to 15,000 square microns, 500 to 10,000 square microns, 500 to 7,500 square microns, 500 to 5,000 square microns, 1,000 to 25,000 square microns, 1,000 to 20 ... 15,000 square microns, 1,000 to 10,000 square microns, 1,000 to 7,500 square microns, 1,000 to 5,000 square microns, 2,000 to 20,000 square microns, 2,000 to 15,000 square microns, 2,000 to 10,000 square microns, 2,000 to 7,500 square microns, 2,000 to 6,000 square microns, 3,000 to 20,000 square microns, 3,000 to 15,000 square microns, 3,000 to 10,000 square microns, 3,000 to 7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the passageway 434 at the proximal opening 452 can be within other ranges (e.g., ranges defined by any of the endpoints listed above).

In some embodiments, the length of the connection region _Lcon can be within any of the following ranges: 1 to 200 micrometers, 5 to 150 micrometers, 10 to 100 micrometers, 15 to 80 micrometers, 20 to 60 micrometers, 20 to 500 micrometers, 40 to 400 micrometers, 60 to 300 micrometers, 80 to 200 micrometers, and 100 to 150 micrometers. The foregoing are merely examples, and the length _Lcon of the connection region 442 can 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 _Wcon of the connection region 443 at the proximal opening 452 can be in 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, or 80 microns, 70 to 150 microns, 70 to 100 microns, and 80 to 100 microns. The foregoing are merely examples, and the width Wcon of the connection region 442 at the proximal opening 452 may be within a range different from the foregoing examples (e.g., a _range defined by any of the endpoints listed above).

In other embodiments, the width _Wcon of the connection region 442 at the proximal opening 452 can be in any range of 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 are merely examples, and the width _Wcon of the connection region 442 at the proximal opening 452 can be in a range different from the foregoing examples (e.g., a range defined by any of the endpoints listed above).

In some embodiments, the ratio of the length _Lcon of the connection region 442 at the proximal opening 452 to the width _Wcon of the connection region 442 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 are merely examples, and the ratio of the length _Lcon of the connection region 442 at the proximal opening 452 to the width _Wcon of the connection region 442 may be different from the foregoing examples.

5 , the width W _con of the connection region 442 can be uniform from the proximal opening 452 to the distal opening 454. The width W _con of the connection region 442 at the distal opening 454 can therefore be within any range determined above for the width W _con of the connection region 442 at the proximal opening 452. Alternatively, the width W _con of the connection region 442 at the distal opening 454 can be greater (e.g., as shown in FIG. 6 ) or less (e.g., as shown in FIG. 7A to FIG. 7C ) than the width W _con of the connection region 442 at the proximal opening 452.

5 , the width of the isolation region 444 at the distal opening 454 can be substantially the same as the width W _con of the connection region 442 at the proximal opening 452. The width of the isolation region 444 at the distal opening 454 can therefore be within any range determined above for the width W _con of the connection region 442 at the proximal opening 452. Alternatively, the width of the isolation region 444 at the distal opening 454 can be greater (e.g., as shown in FIG6 ) or less (not shown) than the width W _con of the connection region 442 at the proximal opening 452.

In some embodiments, the maximum velocity V _max of the flow 512 in the channel 434 is the maximum velocity that the channel can maintain without causing structural damage to the microfluidic device in which the channel is located. The maximum velocity that the channel can maintain depends on various factors, including the structural integrity of the microfluidic device and the cross-sectional area of the channel. For an exemplary microfluidic device of the present invention, the maximum flow velocity V _max in a channel having a cross-sectional area of approximately 3000 to 4000 square microns is approximately 10 μL/second. Alternatively, the maximum velocity V _max of the flow 512 in the channel 434 can be set to ensure that the isolation region 444 is separated from the flow 512 in the channel 434. In particular, based on the width W _con of the proximal opening 452 of the connection region 442 of the isolation fences 436, 438, 440, V _max can be set to ensure that the penetration depth D _p of the secondary flow 514 entering the connection region is less than L _con . For example, for an isolation fence having a connection region with a proximal opening 452 having a width _Wcon of approximately 30 to 40 microns, _Vmax can be set to approximately 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/second.

In some embodiments, the sum of the length _Lcon of the connection region 442 of the isolation fences 436, 438, 440 and the corresponding length of the isolation region 444 can be sufficiently short to allow components of the second medium 504 in the isolation region 444 to diffuse relatively quickly into the first medium 502 in the channel 434. For example, in some embodiments, the sum of (1) the length _Lcon of the connection region 442 and (2) the distance between the biological micro-target located in the isolation region 444 of the isolation fences 436, 438, 440 and the distal opening 454 of the connection region can be in the range of 40 microns to 300 microns, 50 microns to 550 microns, 60 microns to 500 microns, 70 microns to 180 microns, 80 microns to 160 microns, 90 microns to 140 microns, 100 microns to 120 microns, or any range including one of the aforementioned endpoints. The diffusion rate of a molecule (e.g., an analyte of interest, such as an antibody) depends on many factors, including temperature, viscosity of the medium, and the diffusion coefficient _D0 of the molecule. The _D0 of an IgG antibody in an aqueous solution at 20°C is approximately 4.4 x ^10-7 square centimeters per second ( ^cm2 /sec), while the viscosity of the biological micro-target culture medium is approximately 9 x ^10-4 square meters per second ( ^m2 /sec). Thus, for example, an antibody in a biological micro-target culture medium at 20°C can have a diffusion rate of approximately 0.5 micrometers per second. Thus, in some embodiments, the time period for diffusion of a biological micro-target located in the isolation region 444 into the channel 434 can be approximately 10 minutes or less (e.g., 9, 8, 7, 6, 5 minutes, or less). The time period for diffusion can be manipulated by varying parameters that affect the diffusion rate. For example, the temperature of the medium can be increased (e.g., to a physiological temperature such as 37°C) or decreased (e.g., to 15°C, 10°C, or 4°C) to increase or decrease the diffusion rate, respectively.

The configuration of isolation fence 436 shown in FIG5 is merely an example, and many variations are possible. For example, isolation region 444 can be configured to contain multiple micro-targets 522, but isolation region 444 can also be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-targets 522. Thus, the capacity of isolation region 444 can be, for example, at least 3×10 ³ , 6×10 ³ , 9×10 ³ , 1×10 ⁴ , 2×10 ⁴ , 4×10 4 , 8×10 4 , 1×10 5 , ² ×10 ⁵ , ⁴ ×10 ⁵ , 8×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ cubic ^microns , or more.

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

As another example, the connection region 442 and the isolation region 444 are shown as substantially rectangular in FIG5 , but one or both of the connection region 442 and the isolation region 444 may be other shapes. Examples of such shapes include oval, triangle, circle, hourglass, etc.

As yet another example, connecting region 442 and isolation region 444 are shown in FIG5 as having substantially uniform widths. That is, in FIG5 , width W _con of connecting region 442 is shown as being uniform from proximal opening 452 to distal opening 454; the corresponding width of isolation region 444 is similarly uniform; and width W _con of connecting region 442 and the corresponding width of isolation region 444 are considered equal. Any of the foregoing aspects may vary from that shown in FIG5 . For example, width W _con of connecting region 442 may vary from proximal opening 452 to distal opening 454 (e.g., in a trapezoidal or hourglass manner); the width of isolation region 444 may vary (e.g., in a triangular or flask-like manner); and width W _con of connecting region 442 may differ from the corresponding width of isolation region 444.

Figure 6 shows an example of an isolation fence showing examples of some of the aforementioned variations. The fence shown in Figure 6 can replace any fence 436, 438, 440 in any of the figures or discussed herein.

The isolation fence of FIG6 may include a connection region 642 and an isolation structure 646 including 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 FIG6 , the connection region 642 is enlarged such that its width _Wcon increases from the proximal opening 652 to the distal opening 654. However, other than their shapes, the connection region 642, isolation structure 646, and isolation region 644 may be substantially the same as the connection region 442, isolation structure 446, and isolation region 444 of FIG5 , as discussed above.

For example, the channel 434 and isolation fence of FIG6 can be configured so that the maximum penetration depth _Dp of the secondary flow 514 extends into the connection region 642 but not into the isolation region 644. The length _Lcon of the connection region 642 can therefore be greater than the maximum penetration depth _Dp , as generally discussed above with respect to FIG5. As also discussed above, as long as the velocity of the flow 512 in the channel 434 does not exceed the maximum flow velocity _Vmax , the micro-targets 522 in the isolation region 644 will therefore remain in the isolation region 644. The channel 434 and the connection region 642 are therefore examples of swept (or flowing) regions, while the isolation region 644 is an example of an unswept (or non-flowing) region.

7A through 7C illustrate examples of variations of the microfluidic circuit 432 and channel 434 of FIG. 4A through 4C , as well as additional examples of variations of the isolation fences 436, 438, and 440. The isolation fence 736 shown in FIG. 7A through 7C may replace any of the fences 436, 438, and 440 in any of the figures and discussed herein. Similarly, the microfluidic device 700 may replace the microfluidic device 400 in any of the figures and discussed herein.

The microfluidic device 700 of Figures 7A to 7C may include a support structure (not visible, but may be similar to 404 of Figures 4A to 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 or substantially similar to the frame 414 and microfluidic circuit material 416 of Figures 4A to 4C. As shown in Figure 7A, a microfluidic circuit 732 defined by the microfluidic circuit material 716 may include a plurality of isolation fences 736 and a plurality of channels 734 (two channels 734 are shown, but there may be more) fluidically connected thereto.

Each isolation fence 736 can include an isolation structure 746, an isolation region 744 within the isolation structure 746, and a connection region 742. The connection region 742 can fluidly connect the channel 734 to the isolation region 744 from a proximal opening 772 at the channel 734 to a distal opening 774 at the isolation structure 736. Generally, as discussed above with respect to FIG5 , a flow 782 of the first fluid medium 702 in the channel 734 can form a secondary flow 784 of the first fluid medium 702 from the channel 734 into and/or out of the connection region 742 of the fence 736 connected to the channel 734.

As shown in FIG7B , the connection region 742 may include the region between the proximal opening 772 to the channel 734 and the distal opening 774 to the isolation structure 746. The length _Lcon of the connection region 742 may be greater than the maximum penetration depth _Dp of the secondary flow 784, in which case the secondary flow 784 will extend into the connection region 742 and will not be redirected toward the isolation region 744 (as shown in FIG7A ). Alternatively, as shown in FIG7C , the connection region 742 may have a length _Lcon that is less than the maximum penetration depth _Dp , in which case the secondary flow 784 will extend through the connection region 742 and may be redirected toward the isolation region 744. In the latter case, the sum of the lengths _Lc1 and _Lc2 of the connection region 742 may be greater than the maximum penetration depth _Dp . In this manner, the secondary flow 784 will not extend into the isolation region 744. Regardless of whether the length _Lcon of the connecting region 742 is greater than the penetration depth _Dp or the sum of the lengths _Lc1 and _Lc2 of the connecting region 742 is greater than the penetration depth _Dp , the flow 782 of the first medium 702 in the channel 734 that does not exceed the maximum velocity _Vmax will generate a secondary flow having a penetration depth _Dp , and the micro-targets (not shown, but similar to 522 in FIG. 5 ) in the isolated region 744 of the fence 736 will not leave the isolated region 744 through the flow 782 of the first medium 702 in the channel 734. The flow 782 in the channel 734 also does not cause miscellaneous materials (not shown) to leave the channel 734 and enter the isolated region 744 of the fence 736, or to leave the isolated region 744 and enter the channel 734. Diffusion is the only means by which components in the first medium 702 in the channel 734 can move from the channel 734 to the second medium 704 in the isolated region 744 of the fence 736. Likewise, diffusion is the only means by which components in the second medium 704 in the isolation region 744 of the fence 736 can move from the isolation region 744 to the first medium 702 in the channel 734. The first medium 702 can be the same medium as the second medium 704, or the first medium 702 can be a different medium than the second medium 704. Alternatively, the first medium 702 and the second medium 704 can start out as the same and then become different (e.g., by modulation of the second medium by one or more biological microtargets in the isolation region 744, or by changing the medium flowing through the channel 734).

As shown in FIG7B , the width W _ch of the channel 734 perpendicular to the direction of flow 782 (see FIG7A ) in the channel 734 can be substantially perpendicular to the width W _con1 of the proximal opening 772 and, therefore, substantially parallel to the width W _con2 of the distal opening 774. However, the width W _con1 of the proximal opening 772 and the width W _con2 of the distal opening 774 do not need to be substantially perpendicular to each other. For example, the angle between an axis (not shown) on which the width W _con1 of the proximal opening 772 is oriented and another axis on which the width W _con2 of the distal opening 774 is oriented can be non-perpendicular and, therefore, 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.

With respect to the aforementioned discussion of a microfluidic device having a channel and one or more isolation fences, the fluid medium (e.g., the first medium and/or the second medium) can be any fluid capable of maintaining a biological microtarget in a substantially assayable state. The assayable state will depend on the biological microtarget and the assay being performed. For example, if the biological microtarget is a biological microtarget for which secretion of a protein of interest is being assayed, then assuming the biological microtarget is viable and capable of expressing and secreting the protein, the biological microtarget will be substantially assayable.

Figures 8 through 30 illustrate an example of process 100 of Figure 1 for testing biological microtargets (e.g., biological cells) in the microfluidic device 200 of Figures 2A through 2C or the microfluidic device 400 of Figures 4A through 4C. However, process 100 is not limited to classifying biological microtargets or operating microfluidic devices 200 and 400. Microfluidic devices 200 and 400 are also not limited to process 100. Furthermore, while aspects of the steps of process 100 may be discussed in connection with device 200 rather than device 400 (or vice versa), such aspects may be applied to other devices or any other similar microfluidic devices.

At step 102, process 100 may load a biological microtarget into a microfluidic device. FIG8 illustrates an example in which a biological microtarget 802 (e.g., a biological cell) is loaded into a flow region 240 (e.g., a channel 252) of a microfluidic device 200. FIG9 illustrates an example in which a sample material 902 including a biological microtarget 904 is flowed into a channel 434 of a microfluidic device 400.

As shown in FIG8 (which is similar to FIG10 , FIG11 , FIG13 , FIG14 , FIG17 , FIG18 , FIG26 , and FIG27 , showing a partial top cross-sectional view of the flow region 240 of the device 200), a mixture of biological micro-targets 802 can be loaded into the channel 252 of the microfluidic device 200. For example, the biological micro-targets 802 can be input into the device 200 through the inlet 208 (see FIG2A through FIG2C ), and the biological micro-targets 802 can be moved in the channel 252 by the flow 804 of the medium 244. The flow 804 can be a countercurrent. Once the biological micro-target 802 is in the channel 252 and adjacent to the fence 256, the flow 804 can be stopped or slowed to maintain the biological micro-target 802 in the flow channel 252 adjacent to the fence 256 for a period of time sufficient to perform steps 104 and 106. The mixture of biological micro-targets 802 loaded into the channel 252 may include different types of biological micro-targets and other components, such as debris, proteins, contaminants, particles, and the like.

FIG9 illustrates an example of a sample material 902 including a biological microtarget 904 flowing into a channel 434 of a microfluidic device 400. In addition to the biological microtarget 904, the sample material 902 may include other microtargets (not shown) or materials (not shown). In some embodiments, the channel 434 may have a cross-sectional area as disclosed herein, for example, approximately 3,000 to 6,000 square micrometers or approximately 2,500 to 4,000 square micrometers. The sample material 902 may flow into the channel 434 at a rate as disclosed herein, for example, approximately 0.05 to 0.25 μL/sec (e.g., approximately 0.1 to 0.2 μL/sec or approximately 0.14 to 0.15 μL/sec). In some embodiments, the control module 472 of FIG4A may cause the control/monitoring device 480 to cause a first fluid medium (not shown) containing the sample material 902 to flow into the channel 434 through the port 424. Once the sample material 902 is in the channel 434, the flow of the medium (not shown) in the channel 434 can be slowed or substantially stopped. Starting and stopping the flow of the medium (not shown) in the channel 434 can include opening and closing a valve (not shown) of the passage 426 including the port 424.

The biological microtargets 802, 904 can be any biological microtarget 802, 904 that is used to produce a specific analyte or analyte of interest to be assayed. Examples of biological microtargets 802, 904 include biological microtargets, such as mammalian biological microtargets, human biological microtargets, immune biological microtargets (e.g., T biological microtargets, B biological microtargets, macrophages, etc.), B biological microtarget hybridoma cells, stem biological microtargets (e.g., bone marrow stem biological microtargets, adipose stem biological microtargets, etc.), transformed biological microtarget lines (e.g., transformed CHO biological microtargets, HeLa biological microtargets, HEK biological microtargets, etc.), insect biological microtargets (e.g., Sf9, Sf21, HighFive, etc.), protozoan biological microtargets (e.g., Leishmania salpingii), yeast biological microtargets (e.g., S. cerevisiae, P. cerevisiae, etc.), bacterial biological microtargets (e.g., E. coli, B. subtilis, B. thuringiensis, etc.), any combination of the foregoing, etc. Examples of biological microtargets 904 also include embryos, such as mammalian embryos (e.g., human, primate, dog, cat, bear, cow, sheep, goat, horse, pig, etc.). Examples of analytes of interest include proteins, carbohydrates, lipids, nucleic acids, metabolites, etc. Other examples of analytes of interest include materials containing antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclasses), IgM, IgA, IgD, or IgE class antibodies.

At step 104, process 100 can perform a first test on the biological microtarget loaded into the microfluidic device at step 102. Step 104 can include selecting a plurality of biological microtargets based on the first test. Alternatively, step 104 can include selecting one of the biological microtargets without performing the first test. FIG. 10 illustrates an example of performing a first test on a biological microtarget 802 in a channel 252 of a microfluidic device 200, and FIG. 11 illustrates an example of selecting a biological microtarget 802 based on the first test. (The selected biological microtarget is labeled 1002 in FIG. 11 and subsequent figures.) FIG. 12 illustrates an example in which biological microtargets 1202, 1204, 1206 are selected from the microtarget 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 test for a first characteristic of the biological microtarget 802 or the biological microtarget 904. The first test performed at step 104 may be any test that tests for any desired characteristic. For example, the desired characteristic may relate to the size, shape, and/or morphology of the biological microtarget 802 or the biological microtarget 904. The first test may include capturing an image of the biological microtarget 802 or the biological microtarget 904 and analyzing the image to determine which biological microtarget 802 or the biological microtarget 904 has the desired characteristic. As another example, the first test performed at step 104 may determine which biological microtarget 802 or the biological microtarget 904 exhibits a specific detectable condition indicative of the first characteristic. For example, the first characteristic 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 microtargets 802, 904. By testing for appropriate cell surface markers or combinations of cell surface markers, specific cell types can 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, etc.

In the example shown in FIG10 , the detectable condition of the biological microtarget in the microfluidic device 200 is the radiation of energy 1006, which can be, for example, electromagnetic radiation. Before loading the biological microtarget into the microfluidic device 200 or the channel 252, the biological microtarget 802 can be pre-treated with an assay material (not shown) that causes the biological microtarget 802 having a first characteristic to radiate energy 1006.

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

As shown in FIG11 , a biological microtarget 1002 can be selected by capturing the microtarget 1002 using an optical trap 1102. As generally discussed with reference to FIG3A and FIG3B , the optical trap 1102 can be generated, moved, and closed in the channel 252 of the microfluidic device 200 by directing a varying pattern of light into the channel 252. The unselected biological microtargets in FIG11 are labeled 1004. In the example shown in FIG11 , no optical trap 1102 is generated for the unselected biological microtarget 1004.

FIG12 illustrates the selection of biological microtargets 1202, 1204, 1206 from the biological microtargets 904 in the channel 434 of the microfluidic device 400 at step 104. This selection can be responsive to the results of a first test performed at step 104. Alternatively, the selection of the microtargets 1202, 1204, 1206 can be random, and thus performed without performing a first test. If based on a first test, step 104 can, for example, include selecting the biological microtargets 1202, 1204, 1206 based on one or more observable physical characteristics or measurable characteristics as discussed above. For example, the biological microtargets 1202, 1204, 1206 can be selected from the microtargets 904 in the sample material 90 based on any of a plurality of possible detectable characteristics, such as biological microtarget type-specific characteristics and/or characteristics associated with biological microtarget viability and health. Examples of such characteristics include size, shape, color, texture, permeability, conductivity, capacitance, expression of biological microtarget type-specific markers, response to changes in the environment, and the like. In one particular embodiment, a biological microtarget 904 having a circular cross-section with a diameter within any of the following ranges can be selected from the sample material 602: 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, a biological microtarget 604 can be selected from the sample material 902 to have 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).

12 shows micro-targets 1202, 1204, 1206 selected in channel 434, sample material 902 may alternatively be at least partially in connection region 442 of enclosures 436, 438, 440. Micro-targets 1202, 1204, 1206 may thus be selected in connection region 442 simultaneously.

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

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

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

As mentioned above with respect to FIG. 11 , each biological microtarget 1002 can be selected by an optical trap 1102. For example, the selector 222 (see FIG. 2A through FIG. 2C ) configured as the DEP device of FIG. 3A and FIG. 3B can generate an optical trap 1102 that captures a single selected biological microtarget 1002. As shown in FIG. 13 , the DEP device 300 can then move the optical trap 1102 into the enclosure 256, which moves the captured selected biological microtarget 1002 into the enclosure 256. As shown, each selected biological microtarget 1002 can be individually captured and moved into the holding enclosure 256. Alternatively, more than one selected biological microtarget 1002 can be captured by a single trap, and/or more than one selected biological microtarget 1002 can be moved into any one enclosure 256. Regardless, two or more selected biological microtargets 1002 can be selected in the channel 252 and moved into the enclosure 256 in parallel.

3A and 3B , the light trap 1102 can be part of a changing pattern 322 of light projected onto the inner surface 242 of the flow region 240 of the microfluidic device 200. As shown in FIG14 , once a selected biological microtarget 1002 is within the enclosure 256, the light trap corresponding to the biological microtarget 1002 can be turned off. The detector 224 can capture images of all or part of the flow region 240, including images of selected and unselected biological microtargets 1002, 1004, the channel 252, and the enclosure 256, and these images can help identify, capture, and move individual selected biological microtargets 1002 to a particular enclosure 256. Detector 224 and/or selector 222 (e.g., configured as the DEP device of Figures 3A and 3B) can therefore be one or more examples of a separation device for separating microtargets that test positive for a first characteristic (e.g., the selected biological microtarget 1002) from microtargets that test negative for the first characteristic (e.g., the unselected biological microtarget 1004).

14 , with the selected biological microtarget 1002 within the enclosure 256, the flow 804 (e.g., bulk flow) of the medium 244 can flush the unselected biological microtargets 1004 out of the channel 252. As mentioned, after loading the biological microtarget 904 into the channel 252 at step 102, the flow 804 of the medium 252 can be stopped or slowed. As part of step 106, the flow 804 can be resumed or increased to flush the unselected biological microtargets 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 microtargets 1202, 1204, 1206 can be moved into the isolation region 444 of the isolation enclosures 436, 438, 440 of the microfluidic device 400 in any number of possible ways. For example, as discussed above, the enclosure 402 of the microfluidic device can include a DEP configuration that can be used to capture and move a specific plurality of biological microtargets 904 within the sample material 902.

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

15 shows one selected biological microtarget 1202, 1204, 1206 in each enclosure 436, 438, 444, more than one biological microtarget 1202, 1204, 1206 can be moved into a single enclosure. Examples of the number of biological microtargets that can be moved from the sample material 902 into a single enclosure 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 are merely examples, and other numbers of biological micro-targets 904 may be moved from the sample material 902 into a single enclosure 436 , 438 , 440 .

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

As shown in FIG16 , as part of step 106, the channel 434 can be cleared of sample material 902, including unselected microtargets 904, by flushing the channel 434 with a flushing medium (not shown). In FIG16 , the flow of flushing medium through the channel 134 is labeled 1602. The flow 1602 of flushing medium can be controlled so that the velocity of the flow 1602 is maintained below a maximum flow velocity _Vmax corresponding to the maximum penetration depth _Dp discussed above. As also discussed above, this will maintain the selected biological microtargets 1202, 1204, 1206 within the isolated region 444 of their respective enclosures 436, 438, 440 and prevent material from the channel 434 or one of the enclosures 436, 438, 440 from contaminating another of the enclosures. In some embodiments, the flushing medium is flowed into the channel 434 having a cross-sectional area as disclosed herein, for example, approximately 3000 to 6000 square micrometers or approximately 2500 to 4000 square micrometers. The flushing medium may be flowed into the channel at a rate as disclosed herein, for example, about 0.05 to 5.0 μL/second (e.g., about 0.1 to 2.0, 0.2 to 1.5, 0.5 to 1.0 μL/second, or about 1.0 to 2.0 μL/second). Purging the channel 434 as part of step 106 may include flushing the channel 434 multiple times.

In some embodiments, the control module 472 can cause the control/monitoring device 480 to purge the channel 434. For example, the control module 472 can cause the control/monitoring device 480 to cause the flushing medium to flow into the channel 434 through the port 424 and out of the other port 424. The control module 472 can maintain the velocity of the stream 1602 below the maximum flow velocity _Vmax . For example, for a channel 434 having a cross-sectional area of approximately 3000 to 6000 square microns (or approximately 2500 to 4000 square microns), the control module 472 can maintain the velocity of the stream 1602 below _Vmax of 5.0 μL/sec (e.g., 4.0, 3.0, or 2.0 μL/sec).

After steps 102 through 106, process 100 has separated the mixture of biological microtargets (e.g., 802, 904) in the microfluidic device (e.g., 200, 400) into selected biological microtargets (e.g., 1004, 1202, 1204, 1206) and unselected biological microtargets (e.g., 1004, 904). Process 100 also places the selected biological microtargets in holding enclosures (e.g., 256, 436, 438, 440) in the microfluidic device and rinses away the unselected biological microtargets. As discussed above, steps 102 through 106 may be repeated and thus performed k times, where k is 1 (in which case steps 102 through 106 are performed once but not repeated) or greater than 1. The result may be that there are many selected biological microtargets in the holding enclosures in the microfluidic device.

It should also be noted that before executing step 106, step 104 may perform 1 test for up to 1 different characteristics, where 1 is a positive integer 1 or greater than 1. For example, step 104 may test for a first characteristic of the biological microtarget (such as size, shape, morphology, texture, visible markers, etc.), and then step 104 may be repeated to test for subsequent characteristics (such as measurable characteristics). Therefore, the selected biological microtargets may include biological microtargets from the group(s) of biological microtargets loaded at step 102 that test positive for up to 1 different characteristic.

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

At step 108, process 100 can perform a test on the selected biological micro-target or biological micro-targets. If a first test was performed as part of step 104, the test can 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 it from the "first test" discussed above in step 104). As mentioned, the subsequent test performed at step 108 can test for the same feature (i.e., the first feature) as the first test of step 104 or a different feature. As also mentioned above, if the subsequent test performed at step 108 is for the first feature (and therefore for the same feature tested at step 104), the subsequent test can also be different from the first test. For example, the subsequent test may be more sensitive to detection of the first feature than the first test.

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

As shown in FIG17 , the assay material 1702 may be flowed into the channel 252 in a sufficient amount to expose the selected biological microtarget 1002 in the enclosure 256 to the flow 804 of the assay material 1702. For example, while the barrier 254 may prevent the assay material 1702 from flowing directly from the channel 252 into the interior space of the enclosure 256, the assay material 1702 may diffuse into the interior of the enclosure 256 and thereby reach the selected biological microtarget 1002 in the enclosure. The assay material 1702 may include a material that reacts with the selected biological microtarget 1002 having a subsequent characteristic to produce a different detectable condition. The assay material 1702 and the different detectable condition produced may be different from any assay material and condition discussed above for the first test at step 104. A wash buffer (not shown) may also be flowed into the channel 252 and allowed to diffuse into the enclosure 256 to wash the selected biological microtarget 1002.

The detectable condition can be radiation having an energy that meets one or more criteria (such as a threshold intensity, a frequency within a specific frequency band, etc.). The color of the biological microtarget 1002 is an example of electromagnetic radiation radiating within a specific frequency band. In the example shown in FIG18 , selected biological microtargets 1002 that test positive for the subsequent characteristic at step 18 continue to be labeled with label 1002, but biological microtargets that test negative for the subsequent characteristic at step 108 (e.g., do not test positive) are labeled with label 1802.

An example of a subsequent characteristic tested at step 410 may be the viability of biological microtarget 1002. For example, the subsequent characteristic may be whether biological microtarget 1002 is alive or dead, and the assay material may be a viability dye, such as 7-aminoactinomycin D. Such a dye can cause living biological microtargets 1002 to turn a particular color and/or cause dead biological microtargets to turn a different color. Detector 224 (see FIG. 2A through FIG. 2C ) may capture images of biological microtargets 1002 within holding enclosure 256, and control module 230 may be configured to analyze the images to determine which biological microtargets exhibit a color corresponding to living biological microtargets 1002 and/or which biological microtargets exhibit a color corresponding to dead biological microtargets 1002. Alternatively, a human operator may analyze the images from detector 224. Detector 224 and/or control module 230 configured in this manner may thus be one or more examples of a testing device for testing microtargets in a liquid medium within a flow path in a microfluidic device for a particular characteristic (e.g., a first characteristic or a subsequent characteristic).

FIG19 shows an example in which the test performed at step 108 is for an analyte of interest produced by a selected biological microtarget 1202, 1204, 1206 in the isolation enclosure 236, 238, 240 of the microfluidic device 400. In FIG19, components of the analyte of interest 1902 are labeled with a label 1904. The analyte of interest can be, for example, a protein, nucleic acid, carbohydrate, lipid, metabolite, or other molecule secreted or otherwise released by a specific cell type (e.g., healthy cells, cancer cells, virus- or parasite-infected cells, inflammatory response cells, etc.). Specific analytes of interest can be, for example, growth factors, cytokines (e.g., inflammatory or other), viral antigens, parasite antigens, cancer cell-specific antigens, or therapeutic drugs (e.g., therapeutic drugs such as hormones or therapeutic antibodies).

19, step 108 can include loading assay material 1910 into microfluidic device 400 and detecting a localized response, if any, of analyte component 1904. Step 108 can also include providing an incubation period after loading assay material 1910 into channel 434.

As shown in FIG19 , the assay material 1910 can substantially fill the channel 434 or at least the area adjacent to the proximal openings 442 of the fences 436 , 438 , and 440 . Additionally, the assay material 110 can extend into the connection areas 442 of at least some of the isolation fences 436 , 438 , and 440 . In some embodiments, the assay material flows into the channel 434 having a cross-sectional area as disclosed herein (e.g., approximately 3000 to 6000 square microns or approximately 2500 to 4000 square microns). The assay material can flow into the channel at a rate as disclosed herein (e.g., approximately 0.02 to 0.25 μL/sec (e.g., approximately 0.03 to 0.2 μL/sec, or approximately 0.05 to 0.15 μL/sec, with lower rates for biological cell assay materials and higher rates for non-cellular assay materials)). Once the assay material 1910 is loaded into position in the channel 434 , the flow in the channel 434 can be slowed or substantially stopped.

The assay material 1910 can flow into the channel 434 quickly enough to position the assay material 1910 adjacent the proximal opening 452 of the fences 436, 438, 440 before the analyte components 1904 generated in any of the fences 436, 438, 440 can diffuse into the channel 434. This can avoid the problem of analyte components from one fence 436, 438, 440 contaminating the channel 434 and/or other fences between the time the selected biological microtarget 1202, 1204, 1206 is positioned in the fence 436, 438, 440 and the time the assay material 1910 is loaded into the channel 434.

The rate at which the assay material 1910 is loaded into the channel 434 can therefore be at least a minimum flow rate, _Vmin , to completely _load the assay material 1910 into a position adjacent the proximal opening 452 within a time period, _Tload , that is less than a minimum time period, _Tdiff , for a sufficient amount of the analyte component 1904 to diffuse from the isolation region 444 of the enclosures 436, 438, 440 into the channel 434. As used herein, "sufficient amount" refers to a detectable amount of the analyte component that is sufficient to affect accurate detection of the analyte component from the isolation enclosure. The minimum flow rate, Vmin _, can be a function of various parameters. Examples of such parameters include the length of the channel 434, the length, _Lcon , of the connecting region 442 of the enclosures 436, 438, 440, the diffusivity of the analyte component 1904, the viscosity of the medium, the ambient temperature, and the like. Examples of minimum flow rates _Vmin include at least about 0.04 μL/sec (eg, at least about 0.10, 0.11, 0.12, 0.13, 0.14 μL/sec, or more).

The minimum flow rate _Vmin for loading the assay material 1910 into the channel 434 can be less than the maximum flow rate _Vmax corresponding to a penetration depth _Dp less than the length _Lcon of the connection region 442 of the rails 436, 438, 440 as discussed above. For example, the ratio of _Vmax / _Vmin can be in any range of approximately 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 can be sufficient to allow the biological microtargets 1202, 1204, 1206 to produce the analyte 1902 of interest and to allow the analyte components 1904 to diffuse from the isolation regions 444 of the enclosures 436, 438, 440 to the corresponding connection regions 442 or proximal openings 452. For example, the incubation period can provide sufficient time for the analyte components 1904 to diffuse into the channel 434.

The incubation period may include merely passively allowing the biological microtargets 1202, 1204, 1206 to naturally produce the analyte of interest 1902 within the isolation enclosures 436, 438, 440. Alternatively, the incubation period may include actively stimulating the biological microtargets 1202, 1204, 1206 to produce the analyte of interest 1902 by, for example, providing nutrients, growth factors, and/or induction factors to the biological microtargets 1202, 1204, 1206; controlling the temperature, chemical composition, pH, etc., of the medium within the isolation region 444 of the isolation enclosures 436, 438, 440; directing excitation energy, such as light, to the isolation region 444; etc.

As used herein, the term "culturing" covers the range described above from simply passively allowing the biological microtargets 1202, 1204, 1206 to naturally produce the analyte 1902 in the isolation enclosures 436, 438, 440 to actively stimulating the production of the analyte. Stimulating the production of the analyte 1902 can also include stimulating the growth of the biological microtargets 1202, 1204, 1206. Thus, for example, the biological microtargets 1202, 1204, 1206 can be stimulated to grow before and/or while they are stimulated to produce the analyte of interest 1902. If the biological microtargets 1202, 1204, 1206 have been loaded into the isolation enclosures 436, 438, 440 as a single biological microtarget, the growth stimulation can result in the generation of a population of clonal biological microtargets that express and/or secrete (or can be stimulated to express and/or secrete) the analyte of interest.

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

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

The assay material 1910 can 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 FIG20 , the analyte component 1904 from the biological microtargets 1202, 1204 in the isolation fences 436, 438 interacts with the assay material 1910 adjacent to the proximal openings 452 of the isolation fences 436, 438 to produce a localized detectable reaction. However, the biological microtarget 1206 in the isolation fence 440 does not produce the analyte 1902 of interest. Therefore, no such localized reaction (e.g., similar to 2002) occurs adjacent to the proximal opening 452 of the isolation fence 440.

The localized reaction 2002 can be a detectable reaction. For example, the reaction 2002 can be a localized luminescence (e.g., fluorescence). Furthermore, the localized reaction 2002 can be sufficiently localized or isolated so as to be individually detectable by a human observer, a camera in the control/monitoring device 480 of FIG. 4A , or the like. For example, the channel 434 can be sufficiently filled with the assay material 1910 that the reaction (e.g., similar to 2002 ) is localized, that is, confined to the space adjacent to the proximal openings 452 corresponding to the isolation fences 436 , 438 . As can be seen, the reaction 2002 can result from the aggregation of components of the plurality of assay materials 1910 adjacent to one or more proximal openings 452 of the isolation fences 436 , 438 , 440 .

The proximal openings 452 of consecutive isolation fences 436, 438, 440 can be separated by at least a distance _Ds (see FIG. 4C ) sufficient to distinguish, for example, by a human observer, an image captured by a camera, or the like, the localized reactions provided at adjacent proximal openings 452 (e.g., similar to 2002). Examples of suitable distances _Ds between the proximal openings 452 of consecutive isolation fences 436, 438, 440 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-targets, such as biological micro-targets, micro-beads, etc.) can be organized in front of the isolation fences. For example, using DEP forces, etc., the capture micro-targets can be grouped together and concentrated in the region of the channel 434 adjacent to the proximal openings 452 of the isolation fences 436, 438, 440.

As mentioned, the assay material 1910, including components such as capture microtargets (e.g., biological microtargets, microbeads, etc.), can enter and thus be partially disposed in the connection region 442 of the isolation fences 436, 438, 440. In this case, the reactions 2002, 2004 can occur completely, substantially completely, or partially in the connection region 442, as opposed to being substantially completely in the channel 434. In addition, the capture microtargets (e.g., biological microtargets, microbeads, etc.) in the assay material 1910 can be disposed in the isolation region 444. For example, DEP forces, etc., can be used to select and move the capture microtargets into the isolation region 444. For capture microtargets disposed in the isolation region of the isolation fence, the capture microtarget can be disposed proximate to the biological microtarget(s) and/or in a portion of the isolation region (e.g., a subchamber) that is different from the portion occupied by the biological microtarget(s).

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

FIG21 (which shows a portion of the channel 434 and the proximal opening 452 of the isolation fence 436) shows an example of an assay material 1910 of labeled capture micro-targets 2112. Each labeled capture micro-target 2112 may include a binding substance and a labeling substance capable of specifically binding to the analyte component 1904. As the analyte component 1904 diffuses toward the proximal opening 452 of the isolation fence 436, the labeled capture micro-targets 2112 proximate to the opening 452 (or within the isolation fence) may bind to the analyte component 1904, which may result in a localized reaction 2002 (e.g., aggregation of the labeled capture micro-targets 2112) proximate to the proximal opening 452 (or within the proximal opening 452).

When the tagged capture micro-target 2112 is immediately adjacent to or within the proximal opening 452, the binding of the analyte component 1904 to the tagged capture micro-target 2112 is greatest. This is because the concentration of the analyte component 1904 is highest in the isolation region 444 and the connection region 442, thereby facilitating the binding of the analyte component 1904 to the tagged capture micro-target 2112 and promoting their aggregation in these regions. As the analyte component 1904 diffuses out of the channel 234 and outside the proximal opening 252, their concentration decreases. Consequently, fewer analyte components 1904 bind to the tagged capture micro-target 2112 outside the proximal opening 252. This reduced binding of the analyte component 1904 to the tagged capture micro-target 2112, in turn, results in reduced aggregation of the tagged capture micro-target 2112 outside the proximal opening 452. A labeled capture microtarget 2112 that is not proximate to the proximal opening 452 of the fence 436, 438, 440 (or is not within the proximal opening 452 of the fence 436, 438, 440) therefore does not produce a detectable local reaction 2002 (or produces a local reaction 2002 that is detectably smaller in size than a local reaction 2002 that occurs proximate to the proximal opening 452 or within the proximal opening 452).

For analyte components that do not have two binding sites for the binding material on the tagged capture microtarget 2112, the tagged capture microtarget can include two different binding materials (as discussed and shown below in FIG23), each of which can be specifically bound by the analyte component. Alternatively, if the analyte components aggregate (e.g., form homodimers, trimers, etc.), then the assay is feasible.

Examples of labeled capture micro-targets 2112 include both inanimate micro-targets and biological micro-targets. Examples of inanimate micro-targets include microstructures such as microbeads (e.g., polystyrene beads), microrods, magnetic beads, quantum dots, etc. The microstructures can be large (e.g., 10 to 15 microns in diameter, or larger) or small (e.g., less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron in diameter, or smaller). Examples of biological microtargets include biological microtargets (e.g., reporter biological microtargets), liposomes (e.g., synthetic membrane preparations or derived from membrane preparations), microbeads coated with liposomes, nanolipid rafts (see, for example, "Ritchie et al. (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, Mehotd Enzymol., 464: 211-231 (Ritchie et al. (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, Methods Enzymol., 464: 211-231)"), etc.

FIG22 shows an example of an assay material 1910 comprising a mixture of a capture microtarget 2212 and a label, the component of which is identified as 2222 and hereinafter referred to as "label 2222." FIG23 shows an example of a configuration of a capture microtarget 2212, an analyte component 1904, and a label 2222. The capture microtarget 2212 may include a first affinity agent 2312 that specifically binds to a first region 2302 of the analyte component 1904. The label 2222 may include a second affinity agent 2322 that specifically binds to a second region 2304 of the analyte component 1904. As shown in FIG22 , a reaction 2002 occurs when the first region 2302 of the analyte component 1904 binds to the first affinity agent 2312 of the capture microtarget 2212 and the second region 2304 of the analyte component 1904 binds to the second affinity agent 2322 of the label 2222.

As the analyte component 1904 generated by the biological microtarget 1202 in the isolation region 444 of the isolation fence 436 diffuses toward the proximal opening 452, the analyte component 1904 can bind to the capture microtarget 2212 and the tag 2222 that are proximate to (or within) the opening 452, thereby causing the tag 2112 to accumulate on the surface of the capture microtarget 2212. When the capture microtarget 2212 is proximate to (or within) the proximal opening 452, the binding of the analyte component 1904 to the tagged capture microtarget 2212 is greatest. Similar to the above discussion, this is because the relatively high concentration of the analyte component 1904 in the isolation region 444 and the connection region 442 facilitates the binding of the analyte component 1904 to the capture microtarget 2212 and the corresponding association of the tag 2222 at the surface of the capture microtarget 2212. As the analyte components 1904 diffuse out of the channel 434 and outside the proximal opening 452, the concentration decreases and fewer analyte components 1904 bind to the capture micro-targets 2212 outside the proximal opening 452. The reduced binding of the analyte components 1904 to the capture micro-targets 2212 results in a reduced accumulation of labels 2222 at the surface of the capture micro-targets 2112 outside the proximal opening 452. Thus, capture micro-targets 2212 that are not immediately adjacent to the proximal opening 452 of the fences 436, 438, 440 (or within the proximal opening 452 of the fences 436, 438, 440) are not detectably labeled, or are not detectably labeled to the extent that they are detectably labeled, with the labeling being detectably lower in magnitude than labeling that occurs immediately adjacent to or within the proximal opening 452.

Examples of capture microtargets 2212 include all of the examples presented above for labeled capture microtargets 2112. Examples of first affinity agents 2312 include tags that specifically recognize analyte component 1904 or ligands that are specifically recognized by analyte component 1904. For example, in the case of an antibody analyte, first affinity agent 2312 can be an antigen of interest.

Examples of the label 2222 include a label having a luminescent tag (eg, a fluorescent tag) and a label having an enzyme capable of cleaving a signal molecule that emits fluorescence when cleaved.

Examples of assay materials 1910 include assay materials that include composite capture microtargets that include multiple affinity agents. FIG24 illustrates an example of a composite capture microtarget 2412 that includes 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 (see FIG23 ) of an analyte component 1904, 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. Furthermore, the first affinity agent 2402 and the second affinity agent 2404 can optionally bind to both the first region 2302 and the second region 2304 of the analyte component 1904 simultaneously.

Examples of first affinity agents 2402 include those discussed above. Examples of second affinity agents 2404 include receptors that specifically recognize the second region 2304 of the analyte component 1904 or ligands that are specifically recognized by the second region 2304 of the analyte component 1904. For example, in the case of an antibody analyte, the second affinity agent 2404 can bind to the constant region of the antibody. Examples of the aforementioned include Fc molecules, antibodies (e.g., anti-IgG antibodies), protein A, protein G, and the like.

Another example of an assay material 1910 is an assay material that includes multiple capture microtargets. For example, the assay material 1910 may include a first capture microtarget (not shown) having a first affinity agent 2402, and a second capture microtarget (not shown) having a second affinity agent 2404. The first capture microtarget may be different from the second capture microtarget. For example, the first capture microtarget may have a size, color, shape, or other feature that distinguishes the first capture microtarget from the second capture microtarget. Alternatively, the first capture microtarget and the second capture microtarget may be substantially the same type of capture microtarget, except for the type of affinity agent each includes.

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

Regardless of the specific contents 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 can maintain the flow of the assay material 1910 in the channel 434 between a minimum flow rate V _min and a maximum flow rate V _max , as discussed above. Once the assay material 1910 is in position adjacent the proximal opening 452 of the fence 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 localized reactions 2002 proximate to one or more proximal openings 452 of the isolation fences 436, 438, 440 that indicate a reaction with the analyte component 1904 of the assay material 1910 loaded into the channel 434. If localized reactions 2002 are detected proximate to any proximal opening 452 of the isolation fences 436, 438, 440, it may be determined whether any of those detected localized reactions 2002 indicate a positive reaction to one or more biological microtargets 1202, 1204, 1206 within the isolation fences 436, 438, 440. In some embodiments, a human user may observe the channel 434 or the connection region 442 of the fences 436, 438, 440 to monitor the localized reactions 2002 and determine whether the localized reactions 2002 indicate a positive reaction to the biological microtarget 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 operations performed by the control module 472 to monitor a local reaction 2002 and determine whether the local reaction 2002 indicates a positive property of a biological microtarget 1202 , 1204 , 1206 .

At step 2502, the control module 472 performing the process 2500 may capture at least one image of the corridor 434 or the connected area 442 of the isolation fences 436, 438, 440 using a camera or other image capture device (not shown, but may be a component 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 one image, that image may be referred to below as the last image. If the control module 472 captures multiple images, the control module 472 may combine two or more captured images into a final image. For example, the control module 472 may average the 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 generate the final image.

At step 2504, the control module 472 may identify any signs of a localized reaction 2002 in the final image. As discussed above, examples of a localized reaction 2002 include luminescence (e.g., fluorescence), and the control module 472 may therefore analyze the final image for luminescence proximate to any proximal openings 452 of the isolation fences 436, 438, 440. The control module 472 may be programmed to utilize any image processing technique to identify a localized reaction 2002 in the final image. In an example, as shown in FIG20 , the control module 472 may detect a localized reaction 2002 proximate to the proximal openings 452 of the isolation fences 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 do this by associating each local reaction 2002 detected at step 2504 with the isolation fence 436, 438, 440 having the proximal opening 452 closest to the reaction 1002. In the example of FIG. 20 , the control module 472 may associate the reaction 2002 with the isolation fences 436, 438.

The control module 472 may perform steps 2508 and 2510 of FIG. 25 for each isolation fence 436, 438, 440 for which a reaction associated therewith was detected at step 2506. With respect to the example of FIG. 20 , the control module 472 may therefore 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 detected reaction 1002 associated with the current isolation fence 436 indicates a positive result for the biological microtarget(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 brightness exceeding a threshold, luminescence color falling within a predetermined color range, etc. If, at step 2508, the control module 472 determines that the detected reaction is positive, the control module 472 may proceed to step 2510, where the control module 472 may identify the current isolation fence 436 as containing a positive biological microtarget 1202. If the determination at step 2508 is negative, the control module 472 may repeat step 2508 for the next isolation fence 438 with which the detected reaction was associated at step 2506.

In the example shown in FIG20 , assume that at step 2508, the local reaction 2002 associated with isolation fence 436 is determined to be positive, while the local reaction 2002 associated with isolation fence 438 is determined to be negative (e.g., luminescence is detected, but it is below the threshold used to determine that isolation fence 438 is positive). As previously described, no reaction is detected adjacent to the proximal opening 452 of isolation fence 440. Therefore, the control module 472 identifies only isolation fence 436 as having a positive biological microtarget. Although not shown in FIG25 , as part of process 2500, the control module 472 can identify isolation fences 438 and 440 as negative.

1 , at step 110, process 100 can separate biological microtargets that tested positive at step 108 from biological microtargets that tested negative. Figures 26 and 27 illustrate examples in which biological microtargets 1002 that tested negative for subsequent characteristics at step 108 are moved into channel 252 of microfluidic device 200 and then flushed out of channel 252 of microfluidic device 200. Figure 29 illustrates an example in which negative biological microtargets 1204, 1206 are separated from positive biological microtarget 1202 in microfluidic device 400.

As shown in FIG26 , each biological microtarget 1002 that tests negative at step 110 can be selected and captured by an optical trap 2602 in the holding enclosure 256. The negative microtargets are labeled 1802 in FIG26 . The optical trap 2602 can then be moved from the holding enclosure 256 into the channel 252. As shown in FIG27 , the trap 2602 can be closed in the channel 252, and the flow 804 (e.g., convection) of the medium 244 can flush the negative biological microtargets 1802 out of the channel 252 (and, optionally, out of the flow region 240). The assay material 1702 can diffuse out of the enclosure 256, and the flow 804 can also flush the assay material 1702 out of the channel 252.

The optical trap 2602 can be generated and operated as described above. For example, as shown, each negative biological microtarget 2602 can be individually captured and moved from the holding enclosure 256 into the channel 252. Alternatively, more than one negative biological microtarget 2602 can be captured by a single trap 2602. For example, there can be more than one biological microtarget 2602 in a single enclosure 256. In any case, two or more negative biological microtargets 2602 can be selected in the enclosure 256 and moved into the channel 252 in parallel.

Detector 224 can capture images of all or a portion of flow region 240 (including images of biological microtargets 1002 in enclosures 256), and these images can assist in identifying, capturing, and moving individual negative biological microtargets 2602 out of a particular enclosure 256 and into channel 252. Detector 224 and/or selector 222 (e.g., configured as the DEP device of Figures 3A and 3B) can thus be one or more examples of a separation device for separating microtargets that test positive for a characteristic from microtargets that test negative for the characteristic.

27 , for a negative biological microtarget 1802 in channel 252, flow 804 of medium 244 can flush the biological microtarget 1802 out of channel 252 and, in some examples, out of microfluidic device 200 (e.g., through outlet 210). For example, if flow 804 was previously stopped or slowed, flow 804 can be resumed or increased.

Alternatively, biological microtargets 1002 that test positive at step 108 can be moved from the enclosure 256 into the channel 252 and flushed from the channel 252 by the flow 804 at step 110. In such an example, biological microtargets 1002 that test positive at steps 104 and 108 can be collected elsewhere in the microfluidic device 200 for storage, further processing, transfer to another device (not shown), etc. Biological microtargets 1802 that test negative at step 108 can then be removed from the holding enclosure 256 and discarded.

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

Following steps 108 and 110, process 100 also classifies the microtargets selected at step 104 (e.g., 1002, 1202, 1204, 1206) according to the test performed at step 108. Additionally, microtargets selected at step 104 that also test positive for the subsequent test at step 108 can remain in the holding pen (e.g., 256, 436, 438, 440), while negative microtargets can be removed.

As discussed above, steps 108 and 110 can be repeated and thus performed n times, where n is an integer 1 (in which case steps 108 and 110 are performed once without repetition) or greater than 1. The subsequent test performed at each repetition of step 108 can be a different test. Alternatively, the subsequent test performed at the repetition of step 108 can be the same test as the test performed at the aforementioned step 104 or the previous execution of step 108. The biological microtarget (e.g., biological microtarget) loaded at step 102 can thus be subjected to a series of n+1 tests. In some embodiments, each of the n+1 tests can be a different test, and in some embodiments, each of the n+1 tests can test for a different characteristic. Process 100 can thus be capable of sorting out a group that tests positive for n+1 tests (each of which is different) from an initial mixture of biological microtargets, and in some embodiments, process 100 can sort out a group that tests positive for n+1 different characteristics from an initial mixture of biological microtargets.

Alternatively, process 100 can select biological micro-targets at step 104 and then classify the selected biological micro-targets according to the number of tests in step 108 in which the biological micro-targets tested positive (performing step 108 simultaneously or repeatedly). Evaluating multiple features in this manner is desirable for many applications, including antibody identification. For example, multiple assessments can aid in any of the following: identifying conformation-specific antibodies (e.g., different characteristics can be the ability of an antibody analyte to bind to different conformations of a particular antigen); epitope mapping of an antibody analyte (e.g., different characteristics can be the ability to bind to various genetically or chemically altered forms of the antigen); assessing species cross-reactivity of an antibody analyte (e.g., different characteristics can be the ability of an antibody analyte to bind to homologous antigens from different species, such as human, mouse, rat, and/or other animals (e.g., laboratory animals); and the IgG isotype of an antibody analyte. For example, the generation of chemically altered antigens for epitope mapping of antibodies has been described in Dhungana et al. (2009), Methods Mol. Biol. 524: 119-34.

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

Similarly, process 100 can be repeated multiple times on the microfluidic device 400. For example, process 100 can be repeated to retest or reanalyze the positive biological microtargets held in their isolation enclosures 436, 438, 440 at step 110; retest and reanalyze the positive biological microtargets at a low density (e.g., one biological microtarget per isolation enclosure) assuming that the initial testing was performed on multiple biological targets per isolation enclosure; test or analyze new biological microtargets loaded into the microfluidic device 400 at the next repetition of step 108; test or analyze the positive biological microtargets held in their isolation enclosures 436, 438, 440 at step 110 for a different analyte material (e.g., by repeating step 108 using assay material 1910 designed to detect a second or additional analyte of interest), etc.

FIG30 shows another example. As shown, after step 110 has been performed, one or more biological micro-targets (e.g., 1202) held in an isolation enclosure (e.g., 436) can be allowed to generate a clonal population 3002 of biological micro-targets in their isolation enclosure (e.g., 436). Population 3002 can then be tested or analyzed using all or part of process 100 (e.g., steps 108 and 110). Alternatively, as discussed above, the biological micro-targets can be isolated and retested. In yet another alternative, the biological micro-targets can be allowed to grow into a population before process 100 has been completed (e.g., after either step 106 or 108, but before step 110).

Although specific embodiments and applications of the present invention have been described in this specification, these embodiments and applications are exemplary only 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 steps of process 100 and/or process 2500 may be performed in an order different from that shown, and some steps may be performed simultaneously or may overlap with other steps. As other examples, processes 100, 2500 may include additional steps not shown or lack some of the steps shown.

Example

Example 1 - Screening for mouse splenocytes secreting IgG antibodies capable of binding human CD45.

A screen was performed to identify mouse splenocytes that secrete IgG antibodies that bind to human CD45. The experimental design included the following steps:

1. Produce microbeads coated with CD45 antigen;

2. Obtain mouse spleen cells;

3. Loading cells into the microfluidic device; and

4. Determine antigen specificity.

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

Table 1 - Reagents

Generation of microbeads coated with CD45 antigen

Microbeads coated with CD45 antigen were generated by:

50 μg of carrier-free CD45 was resuspended in 500 μL PBS (pH 7.2).

Rinse the slide-A-Lyzer ^™ mini cup with 500 μL of PBS and then add it to the microcentrifuge tube.

Add 50 μL of 0.1 μg/μL CD45 solution to the washed Slide-A-Lyzer ^™ mini cup.

170 μL of PBS was added to 2 mg of NHS-PEG4-biotin, followed by the addition of 4.1 μL of NHS-PEG4-biotin to the slide-A-Lyzer ^™ mini cup containing the CD45 antigen.

EZ-Link ^™ NHS-PEG4-Biotin was incubated with CD45 antigen for 1 hour at room temperature.

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

100 μL of biotinylated CD45 solution (approximately 50 ng/μL) was pipetted into the labeled tube.

500 μL of Spherotech streptavidin-coated microbeads were transferred to a microcentrifuge tube, washed three times in PBS (pH 7.4) (1000 μL/wash), and then centrifuged at 3000 RCF for 5 minutes.

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

50 μL of biotinylated protein was mixed with resuspended Spherotech streptavidin-coated microbeads. The mixture was incubated at 4°C with shaking for 2 hours and then centrifuged at 3000 RCF for 5 minutes at 4°C. The supernatant was discarded and the microbeads coated with CD45 were rinsed three times in 1 mL of PBS (pH 7.4). The microbeads were then centrifuged at 3000 RCF for another 5 minutes at 4°C. Finally, the CD45 microbeads were resuspended in 500 μL of PBS at pH 7.4 and stored at 4°C.

Obtain mouse spleen cells

Spleens from mice immunized with CD45 were obtained and placed in DMEM medium + 10% FBS. Scissors were used to mince the spleen.

The minced spleen was placed on a 40 μm cell strainer. A 10 ml pipette was used to flush single cells through the cell strainer. A glass rod was used to further disrupt the spleen and force single cells through the cell strainer, after which a 10 ml pipette was used to flush single cells through the cell strainer.

Red blood cells were lysed using a commercially available kit.

The cells were quickly centrifuged at 200 x G and the primary splenocytes were resuspended in DMEM medium + 10% FBS at a concentration of 2e ⁸ cells/ml using a 10 ml pipette.

Loading cells into the microfluidic device

Splenocytes were introduced into a microfluidic chip and loaded into enclosures, each containing 20 to 30 cells. 100 μL of medium was flowed through the device at 1 μL/second to remove unwanted cells. The temperature was set to 36°C, and culture medium was perfused at 0.1 μL/second for 30 minutes.

Antigen-specific assay

Prepare cell culture medium containing 1:2500 goat anti-mouse F(ab')2-Alexa568.

100 μL of CD45 microbeads were resuspended in 22 μL of cell culture medium containing 1:2500 dilution of goat anti-mouse F(ab')-Alexa568.

The resuspended CD45 microbeads were then flowed into the main channel of the microfluidic chip at a rate of 1 μL/sec until they were adjacent to, but just outside, the enclosure containing the splenocytes. The fluid flow was then stopped.

The microfluidic chip was then imaged in bright field to determine the position of the beads.

Next, images of the cells and beads were captured using a Texas Red filter. Images were taken every 5 minutes for 1 hour, with each exposure lasting 1000 milliseconds and a gain of 5.

result

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

Using the method of the present invention, each group of splenocytes associated with a positive signal can be isolated and moved as single cells to a new enclosure and re-assayed. In this way, single cells expressing anti-CD45 IgG antibodies can be detected.

In addition to any previously indicated modifications, many other variations and alternative arrangements may be envisioned by those skilled in the art without departing from the spirit and scope of the invention. Thus, although the information has been described above with particular examples and details in conjunction with what are currently considered to be the most practical and preferred solutions, it is apparent that many modifications may be made by those skilled in the art, including but not limited to form, function, mode of operation, and use, without departing from the principles and concepts of the invention as set forth herein. As used herein, the examples and embodiments are illustrative in all respects 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 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 limit in any other way.

Claims (69)

1. A microfluidic device including a boundary, comprising: A base, a microfluidic circuit structure disposed on the base, and a cap that together define the microfluidic circuit, wherein the microfluidic circuit includes: The flow region is configured to contain a flow of a first fluid medium; One or more inlets through which the first fluid medium can be introduced into the flow region; One or more outlets through which the first fluid medium can be removed; Microfluidic channels, including at least a portion of the flow region; and Microfluidic isolation fence, including: An isolation structure includes an isolation region configured to contain a second fluid medium, the isolation region having a single opening; and A connecting region fluidly connects the isolation region to the flow region, wherein the connecting region includes a proximal opening into the microfluidic channel and a distal opening into the isolation region, wherein the width W _{_con} of the proximal opening is in the range of 20 micrometers to 100 micrometers, and the length L _{_con} of the connecting region from the proximal opening to the distal opening is at least twice the width W _{_con} of the proximal opening of the connecting region. The isolated area of the microfluidic isolation fence is the unaffected area of the microfluidic device. 2. The apparatus of claim 1, wherein the width of the channel at the proximal opening in the connection region is between 50 micrometers and 500 micrometers. 3. The apparatus of claim 1, wherein the length L _{_con} of the connecting region from the proximal opening to the distal opening is at least 2.0 times the width W _{_con} of the proximal opening of the connecting region. 4. The apparatus of claim 1, wherein the length L _{_con} of the connecting region from the proximal opening to the distal opening and the width W _{_con} of the proximal opening of the connecting region are adjusted such that the penetration depth of the first fluid medium flowing through the microfluidic channel at a flow rate not greater than 5.0 μL/s into the isolation fence is less than the length L_{_con} . 5. The apparatus of claim 1, wherein the proximal opening of the connection region has a width W_{_con} between 20 micrometers and 60 micrometers. 6. The apparatus of claim 1, wherein the length L _{_con} of the connection region from the proximal opening to the distal opening is between 60 micrometers and 300 micrometers. 7. The device of claim 1, wherein the microfluidic device further comprises a dielectrophoretic structure including a first electrode, an electrode activation substrate, and a second electrode, wherein the first electrode is part of a first wall of the microfluidic device, and the electrode activation substrate and the second electrode are part of a second wall of the microfluidic device. The electrode activation substrate provides a dielectric region on its inner surface. 8. The apparatus of claim 7, wherein the electrode activation substrate is photoactivated. 9. The apparatus of claim 7, wherein the electrode activation substrate comprises: a. Optical guiding material; or b. Semiconductor materials, including multiple doped layers, electrically insulating layers, and conductive layers forming semiconductor integrated circuits. 10. The device of claim 7, wherein the first wall of the microfluidic device is the cap, and the second wall of the microfluidic device is the base. 11. The device of claim 10, wherein the cover and/or the base is light-transmitting. 12. The apparatus according to any one of claims 1-11, wherein the barrier defining the microfluidic isolation fence extends from the surface of the base of the microfluidic device through the entire flow region to the inner surface of the cap of the microfluidic device opposite to the surface. 13. A process for analyzing biological cells in a microfluidic device, said device comprising a boundary, said boundary comprising: A base, a microfluidic circuit structure disposed on the base, and a cap that together define the microfluidic circuit, wherein the microfluidic circuit includes: The flow region is configured to contain a flow of a first fluid medium; One or more inlets through which the first fluid medium can be introduced into the flow region; One or more outlets through which the first fluid medium can be removed; Microfluidic channels, including at least a portion of the flow region; and At least one microfluidic isolation fence, comprising: An isolation structure includes an isolation region configured to contain a second fluid medium, the isolation region having a single opening; and A connecting region fluidly connects the isolation region to the flow region, wherein the connecting region includes a proximal opening into the microfluidic channel and a distal opening into the isolation region, wherein the width W _{_con} of the proximal opening is in the range of 20 micrometers to 100 micrometers, and the length L _{_con} of the connecting region from the proximal opening to the distal opening is at least twice the width W _{_con} of the proximal opening of the connecting region. The isolated area of the at least one microfluidic isolation fence is the unaffected area of the microfluidic device. The process includes: Load one or more biological cells into at least one isolation fence; The loaded biological cells are cultured for a period of time sufficient to enable them to produce analytes of interest. A micro-target for trapping is disposed in the channel adjacent to the connecting region of the at least one isolation fence to the proximal opening of the channel. The micro-target for trapping includes at least one type of affinity agent capable of specifically binding the analyte of interest. The adhesion of the captured microtargets to the analyte of interest is monitored. 14. The process of claim 13, wherein loading includes loading the one or more biological cells into the isolation area of the at least one isolation fence. 15. The process of claim 13, wherein loading the one or more biological cells comprises: A group of biological cells flow into the channel of the microfluidic device; and Move one or more biological cells from the group into each of the at least one isolation fence. 16. The process of claim 15 further includes flushing to remove any biological cells remaining in the channel after the at least one isolation fence has been installed. 17. The process according to claim 15, wherein: Loading the one or more biological cells also includes selecting individual biological cells from the group that meet predetermined criteria; and The selection is performed when the biological cell is in the channel or the connection area or the isolation area of at least one isolation fence. 18. The process of claim 13, wherein setting the micro-target capture includes: The micro-targets are allowed to flow in the channel, and The flow is essentially stopped, such that the captured micro-target is adjacent to the proximal opening of the connecting area from the at least one isolation fence. 19. The process of claim 13, wherein the capture of micro-targets includes a tag. 20. The process of claim 13, wherein setting the micro-target in the channel comprises setting a mixture of the micro-target and the tag into the channel. 21. The process of claim 20, wherein the tag comprises a fluorescent tag. 22. The process of claim 13, wherein the analyte of interest is an antibody. 23. The process of claim 22, wherein the at least one type of affinity agent is an antigen specifically recognized by the antibody. 24. The process according to claim 23, wherein the antigen is a protein, carbohydrate, lipid, nucleic acid, metabolite, antibody, or a combination thereof. 25. The process of claim 22, wherein the at least one type of affinity agent is an Fc molecule, an antibody, protein A, or protein G. 26. The process according to claim 13, wherein: The microfluidic device includes multiple isolation fences, each of which includes a fluid isolation structure. The fluid isolation structure includes an isolation region and a connection region on the fluid surface connecting the isolation region to the channel. Setting up the micro-targets involves substantially filling the channel adjacent to the opening from the connection area of the plurality of isolation fences to the channel with the micro-targets or a mixture of micro-targets and tags. 27. The process according to claim 13, wherein: The length L _{_con} of the connecting area of the at least one isolation fence is in the range of 20 micrometers to 200 micrometers, and The process also includes keeping any flow in the channel less than the maximum permissible flow rate _Vmax . 28. The process according to claim 13, wherein the microfluidic device is the microfluidic device according to any one of claims 1-12. 29. The process of claim 13 further includes rinsing to remove the captured microtargets from the channel. 30. The process according to claim 16 or 29, wherein: The length L _{_con} of the connecting area of the at least one isolation fence is in the range of 20 micrometers to 200 micrometers, wherein the length L _{_con} is greater than the penetration depth D_{_p} of the medium flowing in the channel at the maximum permissible flow rate V _{_max}, and The flushing includes causing the flushing medium in the channel to flow at a rate less than the maximum permissible flow rate _Vmax . 31. A machine-readable storage device for storing non-transitory machine-readable instructions that cause a control device to perform a process in a microfluidic device, the microfluidic device including a boundary, the boundary comprising: A base, a microfluidic circuit structure disposed on the base, and a cap that together define the microfluidic circuit, wherein the microfluidic circuit includes: The flow region is configured to contain a flow of a first fluid medium; One or more inlets through which the first fluid medium can be introduced into the flow region; One or more outlets through which the first fluid medium can be removed; Microfluidic channels, including at least a portion of the flow region; and At least one isolation fence, including: An isolation structure includes an isolation region configured to contain a second fluid medium, the isolation region having a single opening; and A connecting region fluidly connects the isolation region to the flow region, wherein the connecting region includes a proximal opening into the microfluidic channel and a distal opening into the isolation region, wherein the width W _{_con} of the proximal opening is in the range of 20 micrometers to 100 micrometers, and the length L _{_con} of the connecting region from the proximal opening to the distal opening is at least twice the width W _{_con} of the proximal opening of the connecting region. The isolated area of the at least one isolation fence is the unaffected area of the microfluidic device. The process includes: Load one or more biological cells into at least one isolation fence; The loaded biological cells are cultured for a period of time sufficient to enable them to produce analytes of interest. The microtarget is disposed in the channel such that it is located at a proximal opening in the channel adjacent to the connecting region, the microtarget comprising at least one type of affinity agent capable of specifically binding the analyte of interest; and The adhesion of the captured micro-targets to the analyte of interest is monitored. 32. The storage device of claim 31, wherein the step of culturing the loaded biological cells is performed for a period of time sufficient to (1) cause the loaded biological cells to produce an analyte of interest, and (2) cause the analyte of interest to diffuse out of the isolation fence and into the channel. 33. The storage device of claim 31, wherein the step of loading the one or more biological cells comprises: Allowing a group of one or more biological cells to flow into the channel of the microfluidic device; and Move one or more biological cells from the group into the at least one isolation fence. 34. The storage device of claim 33, wherein the step of moving the one or more biological cells comprises moving the one or more biological cells of the group to the isolation area of the at least one isolation fence. 35. The storage device according to claim 33, wherein: The microfluidic device includes multiple isolation fences connected to the channel via a fluid equalization surface. Each isolation fence includes a fluid isolation structure, which includes an isolation region and a connection region on the fluid surface connecting the isolation region to the channel. The moving step includes moving a single one of the one or more biological cells into each of the plurality of isolation fences. 36. The storage device according to claim 33, wherein: The microfluidic device includes a dielectrophoretic structure; and The step of moving one or more biological cells is accomplished by controlling the dielectrophoretic structure. 37. The storage device of claim 33, wherein the step of moving the one or more biological cells comprises: Capture multiple images of the group of biological cells in the microfluidic device; Select one or more biological cells from the group that meet predetermined criteria; and Move one or more of the selected biological cells into the at least one isolation fence. 38. The storage device of claim 37, wherein the predetermined criterion for selecting biological cells is a size exceeding a threshold. 39. The storage device of claim 37, wherein the predetermined criterion for selecting biological cells is a circular shape having a cross-section having a diameter of 5 micrometers to 20 micrometers, 20 micrometers to 40 micrometers, or 100 micrometers to 500 micrometers. 40. The storage device of claim 33, wherein the step of moving the one or more selected biological cells comprises generating a dynamic dielectrophoretic force in the microfluidic device to capture and move the biological cells. 41. The storage device of claim 31, wherein the step of setting the micro-target capture includes: Controlling the flow of the medium in the channel to cause the captured micro-target to flow to a region in the channel adjacent to the opening in the channel from the connection region; and Essentially, the flow is stopped. 42. The storage device of claim 41, wherein the step of controlling the flow of the medium in the channel comprises driving valves on the inlet and outlet pipes fluidly connected to the channel of the microfluidic device. 43. The storage device of claim 33, wherein the process further comprises the following steps: Any biological cells remaining in the channel are flushed out of the microfluidic device, wherein the flushing step is performed after the step of loading the one or more biological cells into the at least one isolation fence is completed. 44. The storage device of claim 31, wherein the step of setting the capture micro-targets includes setting a plurality of capture micro-targets located in the connection area of the at least one isolation fence. 45. The storage device of claim 31, wherein the step of setting the micro-target capture comprises substantially filling the channel with the micro-target capture of the opening adjacent to the connection area of the at least one isolation fence. 46. The storage device of claim 31, wherein the step of setting the capture microtarget includes using dielectrophoresis. 47. The storage device of claim 46, wherein the dielectric force is provided by an opto-tweezers configuration. 48. The storage device of claim 31, wherein the capture microtarget comprises a tag. 49. The storage device of claim 48, wherein the tag is a fluorescent tag. 50. The storage device of claim 48, wherein the step of monitoring the adhesion of the captured microtarget to the analyte of interest includes capturing an image of cold light from the tag. 51. The storage device according to claim 31, wherein: The setting step includes setting a label, including a tag, and the mixture of the micro-target capture material adjacent to the opening from the connection area into the channel. 52. The storage device of claim 48, wherein monitoring the attachment of the captured microtarget to the analyte of interest comprises capturing images of multiple cold lights originating from the tag or a tag including the tag and averaging the detected cold lights. 53. The storage device of claim 50, wherein the step of monitoring the adhesion of the captured microtarget to the analyte of interest further comprises determining whether the cold light from a region adjacent to the opening of the connection region exceeds a threshold intensity. 54. The storage device of claim 53, wherein the process further comprises the step of: when the cold light from the adjacent region exceeds the threshold intensity, identifying the at least one isolation fence including the connection region as containing one or more biological cells expressing the analyte of interest. 55. The storage device according to claim 31, further comprising: After performing the step of monitoring the adhesion of the captured microtargets to the analyte of interest, the process further includes the step of rinsing to remove the captured microtargets from the channel. 56. The storage device according to claim 31, wherein: The length L _{_con} of the connecting region of the at least one isolation fence of the microfluidic device is in the range of 20 micrometers to 200 micrometers, and The process also includes the step of keeping any flow in the channel less than the maximum allowed flow rate _Vmax . 57. A process for classifying biological microtargets in a microfluidic device, the microfluidic device including a boundary, the boundary comprising: A base, a microfluidic circuit structure disposed on the base, and a cap that together define the microfluidic circuit, wherein the microfluidic circuit includes: The flow region is configured to contain a flow of a first fluid medium; One or more inlets through which the first fluid medium can be introduced into the flow region; One or more outlets through which the first fluid medium can be removed; Microfluidic channels, including at least a portion of the flow region; and Microfluidic isolation fence, including: An isolation structure includes an isolation region configured to contain a second fluid medium, the isolation region having a single opening; and A connecting region fluidly connects the isolation region to the flow region, wherein the connecting region includes a proximal opening into the microfluidic channel and a distal opening into the isolation region, wherein the width W _{_con} of the proximal opening is in the range of 20 micrometers to 100 micrometers, and the length L _{_con} of the connecting region from the proximal opening to the distal opening is at least twice the width W _{_con} of the proximal opening of the connecting region. The isolated area of the microfluidic isolation fence is the unaffected area of the microfluidic device. The process includes: Loading biological micro-targets into the flow path of a microfluidic device; Perform a first test on the first feature on the biological micro-target in the flow path; Multiple positive biological microtargets are moved into an unaffected space within the microfluidic device, wherein, in response to the first test, the multiple positive biological microtargets test positive for the first feature; and A second test targeting the second characteristic is performed on the plurality of positive biological micro-targets in the unaffected space. 58. The process according to claim 57, wherein the first feature is a specific biological state of the biological microtarget. 59. The process of claim 58, wherein the specific biological state corresponds to a specific appearance of the biological microtarget. 60. The process of claim 58, wherein the specific biological state corresponds to the expression of cell surface markers by the biological microtarget. 61. The process according to claim 57, wherein: Performing the first test includes detecting radiation from the plurality of positive biological microtargets in the flow path, wherein the detected radiation indicates the first characteristic. 62. The process according to claim 61, wherein: The plurality of positive biomicrotargets include assay materials bonded to the analyte of interest; and The material being measured emits the radiation. 63. The process of claim 62, wherein the step of performing the first test further includes identifying only a plurality of the biological microtargets emitting radiation having an intensity greater than an intensity threshold as positive for the test against the first feature. 64. The process of claim 62, wherein the step of performing the first test further includes identifying only a plurality of the biological microtargets emitting radiation having an intensity less than an intensity threshold as positive for the test against the first feature. 65. The process of claim 57, wherein the first feature or the second feature includes the expression of the analyte of interest. 66. The process of claim 65, wherein the analyte of interest is a protein. 67. The process of claim 57, wherein the moving step comprises moving only the plurality of positive biological microtargets from the flow path into the unaffected space. 68. The process of claim 57, wherein the unaffected space comprises an isolated area within one or more isolation fences inside the microfluidic device. 69. The process according to claim 57, wherein: The step of moving multiple positive biological microtargets to the unaffected space within the microfluidic device includes: A first light pattern is projected into the microfluidic device to activate one or more dielectrophoretic electrodes at an electrode region on the inner surface of the electrode activation substrate, thereby surrounding and trapping the positive biological microtarget; and The first light pattern projected onto the microfluidic device is moved to guide the captured positive biological microtarget from the flow path into the unaffected space.