TW201726540A - Microfluidic apparatus having an optimized electrowetting surface and related systems and methods - Google Patents

Abstract

Microfluidic devices having an electrowetting configuration are provided. The devices include a substrate having a dielectric layer, a droplet actuation surface, and a first electrode configured to be connected to an AC voltage source, wherein the dielectric layer is electrically coupled to the first electrode, and the droplet actuation surface comprises a hydrophobic layer covalently bonded to the dielectric layer. The devices also include a second electrode configured to be connected to the AC voltage source.

Description

Microfluidic device with optimized electrowetting surface and related systems and methods

Tiny objects such as biological cells can be processed in a microfluidic device. For example, droplets containing tiny objects or reagents can move freely and fuse within the microfluidic device. Embodiments of the present invention relate to improvements in microfluidic devices that facilitate robust manipulation of droplets, thereby allowing complex chemical and biological reactions to be performed in a precise and reproducible manner on a small scale. The droplets can be moved and fused within the microfluidic device by altering the effective wetting properties of the electrowetting surface in the microfluidic device. Such movements may facilitate the process of culturing cells in a microfluidic device to post-treat cells to evaluate various cellular properties, as appropriate. Existing solutions for electrowetting are extremely limited in nature and cannot be scaled or implemented for other functions. Therefore, there is a need in the industry for improved electrowetting surfaces, stabilization of substrates for microfluidic applications, and integration of other functions (eg, cell growth and characterization, followed by electrowetting to enable downstream processing), all of which contribute to additional medicine. Research applications.

In one aspect, the invention provides a microfluidic device comprising an electrowetting configuration comprising a substrate having a droplet-actuating surface comprising covalently bonded to an underlying dielectric layer (ie, within The hydrophobic layer (ie, the outer hydrophobic layer) of the surface of the dielectric layer (either consists of or consists essentially of). When the microfluidic device is operatively coupled to a voltage source, the aqueous droplets that reside on or otherwise contact the hydrophobic layer can wet in a reliable and robust manner and thereby move by electrowetting force. The microfluidic device can comprise a substrate comprising a substrate, and the substrate can further have at least one electrode (eg, a first electrode) configured to be coupled to a voltage source (eg, an AC voltage source), the at least one electrode being electrically coupled to the internal medium Electrical layer. In some embodiments, the microfluidic device further comprises a cover and at least one spacer element. The substrate and cover may be substantially parallel to one another and joined together by spacer elements to define an outer casing configured to receive a liquid medium. In such embodiments, the cover can include at least one electrode configured to connect to a voltage source (eg, an AC voltage source). In some embodiments, the microfluidic device can comprise a single-sided electrowetting configuration. In such embodiments, the microfluidic device need not include a cover. For example, the substrate can include a substrate and a first electrode configured to connect to a voltage source (eg, an AC voltage source), and the substrate can include a second electrode (eg, a mesh electrode configured to connect to a voltage source) ). In some embodiments, the outer hydrophobic layer comprises self-associating molecules that are covalently bonded to the inner dielectric layer to form a densely packed hydrophobic monolayer. In some embodiments, the self-associating molecules of the hydrophobic monolayer each comprise a oxoalkyl group. In other embodiments, the self-associating molecules of the hydrophobic monolayer each comprise a phosphonic acid group. The oxoalkyl group or phosphonic acid group can be covalently bonded to the surface of the inner dielectric layer. In some embodiments, the self-associating molecules of the hydrophobic monolayer each comprise a surface modifying ligand and a linking group that links the surface modifying ligand directly or indirectly to the surface of the inner dielectric layer. The surface modifying ligand can be any of the surface modifying ligands disclosed herein. For example, the surface modifying ligand can comprise an aliphatic group, such as an alkane group. Thus, for example, the self-associating molecule of the hydrophobic monolayer can be an alkyl-terminated oxime or an alkyl-terminated phosphonic acid molecule. The alkyl group can include a chain of at least 10 carbons (eg, at least 14, 16, 18, 20, 22 or more carbons) (eg, unbranched). In other embodiments, the surface modifying ligand may comprise a fluorine substituted aliphatic group, such as a fluoroalkyl group. Thus, for example, the self-associating molecule can be a fluoroalkyl-terminated oxime or a fluoroalkyl-terminated phosphonic acid molecule. The fluoroalkyl group can include a chain of at least 10 carbons (eg, at least 14, 16, 18, 20, 22 or more carbons) (eg, unbranched). In certain embodiments, the fluoroalkyl group comprises one or more (eg, at least 4, 6, 8, 10, 12 or more) perfluorocarbons. For example, the fluoroalkyl group can have the formula _{CF 3 - (CF 2) m-} (CH 2) n-, wherein m is at least 2, n is at least 2, and m + n is at least 9. In some embodiments, the surface modifying ligand comprises an ether linkage between the first aliphatic group and the second aliphatic group. For example, the first aliphatic group can be an alkyl group and the second aliphatic group can be a fluoroalkyl group (eg, a perfluoroalkyl group). In certain embodiments, the alkyl or fluoroalkyl group of the surface modifying ligand is not branched. In some embodiments, the alkyl or fluoroalkyl group of the surface modifying ligand does not contain any cyclic structure. In some embodiments, the hydrophobic layer outside the substrate has a thickness of less than 5 nanometers (eg, from about 1.5 nanometers to 3.0 nanometers). In some embodiments, the hydrophobic layer outside the substrate can be patterned such that the selected region is relatively hydrophilic compared to the remainder of the outer hydrophobic layer. In some embodiments, the inner dielectric layer of the substrate can comprise a first layer of dielectric material. For example, the inner dielectric layer can be composed of a single layer of dielectric material. The first layer of dielectric material may comprise an oxide, such as a metal oxide layer (eg, aluminum oxide, hafnium oxide, or the like). In some embodiments, the first oxide layer is formed by atomic layer deposition (ALD). Alternatively, the inner dielectric layer can be a dielectric stack comprising two or more layers of dielectric material. Thus, in some embodiments, the inner dielectric layer can comprise a first layer of dielectric material and a second layer of dielectric material. The first layer of dielectric material may comprise an oxide, such as a metal oxide (eg, aluminum oxide, hafnium oxide, or the like); and the second layer of dielectric material may comprise an oxide (eg, hafnium oxide) or a nitride (eg, nitride)矽). In such embodiments, the first layer of dielectric material can have a first surface that contacts the second layer of dielectric material and an opposite surface that is covalently bonded to the hydrophobic layer. In some embodiments, the second layer of dielectric material can have a thickness of from about 30 nm to about 100 nm, depending on the type of dielectric material used. For example, the second layer of dielectric material can comprise hafnium oxide and can have a thickness from about 30 nm to about 50 nm, or from about 30 nm to about 40 nm. Alternatively, the second layer of dielectric material may comprise tantalum nitride and may have a thickness of from about 50 nm to about 100 nm, or from about 80 nm to about 100 nm. In some embodiments, the second layer of dielectric material is formed by ALD. In other embodiments, the second layer of dielectric material is formed by plasma assisted chemical vapor deposition (PECVD) techniques. In certain embodiments, the first layer of dielectric material can have from about 10 nm to about 50 nm (eg, from about 10 nm to about 20 nm, from about 15 nm to about 25 nm, from about 20 nm to about 30 nm, about Thickness from 25 nm to about 35 nm, from about 30 nm to about 40 nm, from about 35 nm to about 45 nm, from about 40 nm to about 50 nm, or any range defined by either of the foregoing endpoints, and Formed by ALD. In other embodiments, the inner dielectric layer can comprise a third layer of dielectric material, wherein the third layer of dielectric material has a first surface that contacts the first layer of dielectric material and is covalently bonded to the opposite surface of the hydrophobic layer . In such embodiments, the first layer of dielectric material may comprise an oxide as described above (or elsewhere herein), and the second layer of dielectric material may comprise an oxidation as described above (or elsewhere herein) Matter or nitride. In certain embodiments, the third layer of dielectric material can comprise an oxide (eg, cerium oxide) or other dielectric material that is sufficiently bonded to the oxoalkyl group. In some embodiments, the third layer of dielectric material is deposited by ALD. In certain embodiments, the third layer of dielectric material has a thickness of from about 2 nm to about 10 nm, or from about 4 nm to about 6 nm. Regardless of the number of layers that make up the inner dielectric layer, the inner dielectric layer can have from about 40 nm to about 120 nm (eg, from about 40 nm to about 60 nm, from about 50 nm to about 70 nm, from about 60 nm to about 80 nm). a total thickness of from about 70 nm to about 90 nm, from about 80 nm to about 100 nm, from about 90 nm to about 110 nm, from about 100 nm to about 120 nm, or a range defined by either of the foregoing endpoints. Likewise, the dielectric layer can have from about 50 kilo ohms to about 150 kilo ohms (eg, from about 50 kilo ohms to about 75 kilo ohms, from about 75 kilo ohms to about 100 kilo ohms, from about 100 kilo ohms to about 125 kilo ohms, about The impedance of 125 kilo ohms to about 150 kilo ohms, or the range defined by either of the aforementioned endpoints. In some embodiments, the substrate can further comprise a photoreactive layer. The photoreactive layer can have a first side that contacts the inner dielectric layer and a second side that contacts the at least one electrode. In certain embodiments, the photoreactive layer can comprise a hydrogenated amorphous germanium. In such embodiments, irradiating any one of a plurality of regions of the photoreactive layer with a light beam reduces the electrical impedance of the illuminated region of the photoreactive layer. In other embodiments, the photoreactive layer comprises a plurality of conductors, each conductor being controllably connectable to at least one electrode of the substrate via a photonic crystal switch. For embodiments in which the microfluidic device includes a cover, the surface of the cover that faces inwardly toward the outer casing can include an inner layer and a hydrophobic layer (ie, an outer hydrophobic layer) covalently bonded to the inner layer. Similar to the hydrophobic layer outside the substrate, the hydrophobic layer outside the cover may comprise self-associating molecules covalently bonded to the inner layer to form a densely packed hydrophobic monolayer. Thus, the outer hydrophobic layer can comprise any of the self-associating molecules described above (or elsewhere herein) for the hydrophobic layer other than the substrate. In some embodiments, the hydrophobic layer outside the cap comprises the same self-associating molecules as the hydrophobic layer outside the substrate. In other embodiments, the hydrophobic layer outside the substrate has a different type (or types) of self-associating molecules than the hydrophobic layer outside the substrate. In some embodiments, the hydrophobic layer outside the inwardly facing surface of the cover has a thickness of less than 5 nanometers (eg, from about 1.5 nanometers to 3.0 nanometers). In some embodiments, the hydrophobic layer outside the inwardly facing surface of the cover can be patterned such that the selected area is relatively hydrophilic compared to the remainder of the outer hydrophobic layer. In some embodiments, a portion of the substrate can further comprise a dielectrophoresis (DEP) configuration. In other embodiments, the substrate may comprise an electrowetting configuration, but lacks a dielectrophoresis (DEP) configuration. Therefore, microfluidic devices may lack photoelectric clamps (OET configurations). In some embodiments, a microfluidic device can include a housing having at least one microfluidic channel. Additionally, the outer casing can include at least one microfluidic chamber (or barrier fence) fluidly coupled to the microfluidic channel. The defined microchannels and/or at least a portion of the substrate may have an electrowetting configuration. The electrowetting configuration can be coupled to the bias magnetic potential and, at the same time, thereby altering the effective wetting characteristics of any of a plurality of respective regions of the substrate surface (ie, the droplet actuation surface). The wetting characteristics of the substrate surface can be varied sufficiently to allow liquid droplets to pass through the substrate surface and move between the microfluidic channel and the chamber. In some embodiments, the chamber (or barrier fence) can include a containment region (eg, a separation region) configured to receive liquid droplets and one (or more) connection regions that fluidly connect the containment region to the microfluidic channel. The first attachment zone can be configured to allow droplets to move between the microfluidic channel and the chamber. When a second attachment zone is present, it can be configured to allow fluid flow and pressure relief as the droplet moves between the microfluidic channel and the containment zone. In some embodiments, the outer casing can further include a second microfluidic channel. In such embodiments, the chamber can be coupled to both the first microfluidic channel and the second microfluidic channel. In some embodiments, the microfluidic channel can have a height of from about 30 microns to about 200 microns, or from about 50 microns to about 150 microns, and the height is measured in a direction perpendicular to the direction of fluid flow through the channel. In some embodiments, the microfluidic channel has a width of from about 50 microns to about 1000 microns, or from about 100 microns to about 500 microns, and the width is measured in a direction perpendicular to the direction of fluid flow through the channel. In some embodiments, the height of the chamber (or barrier fence) is substantially the same as the height of the microfluidic channel. For example, the chamber height can be from about 30 microns to about 200 microns, or from about 50 microns to about 150 microns. In some embodiments, the chamber (or containment fence) has a cross-sectional area of from about 100,000 square microns to about 2,500,000 square microns, or from about 200,000 square microns to about 2,000,000 square microns. In some embodiments, the height of the attachment zone (first, second, etc.) is substantially the same as the height of the respective chamber and/or connection zone from which the microfluidic channel is open. In some embodiments, the attachment zone has a width of from about 50 microns to about 500 microns, or from about 100 microns to about 300 microns. In some embodiments, the microfluidic device can further comprise a droplet generator. The droplet generator can be configured to selectively provide droplets of one or more liquid media (e.g., aqueous liquid media) to the microfluidic channels within the outer casing or outer casing. The droplets may contain, for example, tiny objects such as biological microscopic objects (e.g., cells) or beads. Alternatively or additionally, the droplets may contain reagents such as lysis buffers, affinity reagents, detectable labels, enzyme mixtures, and the like. In some embodiments, the microfluidic device comprises a culture chamber (eg, a fence) suitable for culturing biological microscopic objects. The culture chamber can be located within the housing and can be connected to the microfluidic channel. When the culture chamber is located within the outer casing, the outer casing can include a perfusion microfluidic channel configured to allow fresh medium to flow through the culture chamber such that nutrients in the fresh medium and waste products in the culture chamber can be exchanged (eg, by Nutrients diffuse into the culture chamber and allow waste products to diffuse out of the culture medium). The perfusion channel can be separated from the microfluidic channel that is connected to the droplet generator. In another aspect, the invention provides a method of making a microfluidic device of the invention. The method can include: bonding a spacer element (eg, made from a microfluidic circuit material) to an inner surface of a cover having at least one electrode configured to connect to a voltage source; bonding the spacer element and the cover to have at least one Forming an internal dielectric surface of the substrate coupled to the electrode of the voltage source; and forming a hydrophobic layer by vapor deposition on at least a portion of the inner surface of the cover and at least a portion of the dielectric surface of the substrate. In some embodiments, the spacer element is sandwiched between the inner surface of the cover and the inner dielectric surface of the substrate such that the cover and the substrate are oriented substantially parallel to each other. The substrate, spacer element and cover may collectively define an outer casing configured to receive a liquid medium. In some embodiments, the hydrophobic layer is deposited on substantially all of the exposed areas of the inner surface of the cover and substantially all exposed areas of the inner dielectric surface of the substrate (ie, deposited in substantially all of the inward facing outer surface) On the surface). In some embodiments, the hydrophobic layer is further deposited on the surface of the spacer element that faces inwardly toward the outer casing. In certain embodiments, the hydrophobic layer comprises self-associating molecules covalently bonded to the inner surface of the cap and the inner dielectric surface of the substrate, wherein the self-associating molecules form a densely packed monolayer. In some embodiments, the self-associating molecules deposited by vapor deposition each comprise a surface modifying ligand and a linking group that directly or indirectly links the surface modifying ligand to the surface of the inner dielectric layer. Thus, the self-associating molecule can be any of the self-associating molecules described above or elsewhere herein. In another aspect, the invention provides a method of treating a material, such as a chemical and/or biological material, in a microfluidic device. In certain embodiments, the methods include: filling a housing or portion thereof of the microfluidic device with a first liquid medium, the microfluidic device comprising a substrate having an electrowetting configuration, a cover, and a spacer element that together define the outer casing; Applying an AC voltage potential between at least one electrode of the substrate and at least one electrode of the cover; introducing a first droplet of the liquid medium into the outer casing, the liquid medium of the droplet being immiscible in the first liquid medium; and An electrowetting force is applied to the first droplet to move the first droplet to a desired location within the outer casing. The first liquid medium can comprise any of the first liquid media described herein, such as polyoxyxides, fluorinated oils, or combinations thereof, and the first droplets can comprise an aqueous medium. In some embodiments, the methods can include dragging the first droplet from a first portion of the outer casing (eg, a microfluidic channel) to a second portion of the outer casing (eg, a chamber), or vice versa. The aforementioned dragging can include changing an effective electrowetting feature of the substrate surface area in contact with the first droplet and/or adjacent to the first droplet. Thus, filling the outer casing with the first liquid medium can include filling the microfluidic channel and chamber with the first liquid medium. In some embodiments, the microfluidic device comprises a droplet generator. The methods can include generating a first droplet using a droplet generator. Additionally, the droplet generator can introduce the first droplet into the outer casing. The resulting droplets can have a volume of from about 100 picoliters to 100 nanoliters, or from about 1 nanoliter to 50 nanoliters. In some embodiments, the first droplet can include a microscopic object, such as a bead or a biological microscopic object (eg, a cell, vesicle, etc.), a cell secretion, or an agent. The beads may have molecules with affinity for the material of interest, such as cell secretions (eg, antibodies) or other biomolecules (eg, nucleic acids such as DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA). Or any combination thereof). The droplets may comprise a single tiny object (eg a single biological cell) or multiple tiny objects. For example, the droplets can include from 2 to 20 or more tiny objects, such as beads. In some embodiments, the droplets can include reagents, such as cell lysis buffers, labels (eg, fluorescent labeling reagents), luminescent reagents, enzyme mixtures, or the like. In some embodiments, the methods further comprise introducing the second, third, fourth, etc. droplets into the outer shell, and applying the electrowetting force to the droplets to cause the second, third, fourth, etc. droplets Move to the desired location within the enclosure. The second droplet can be moved closer to the first droplet and then fused with the first droplet to form a first merged droplet; the third droplet can be moved closer to the first merged droplet and then The first merged droplets are fused to form a second merged droplet; the fourth droplet can be moved to a position near the second merged droplet and then fused with the second merged droplet to form a third merged droplet or the like. Each additional droplet may contain a fluid medium that is immiscible in the first liquid medium but miscible with the liquid medium of the first droplet. In some embodiments, the first droplet contains biological cells and the second droplet contains reagents. The reagent may be a cell lysis buffer that dissolves biological cells when the first and second droplets are fused. Alternatively, the reagent can be a fluorescent label (eg, a fluorescently labeled antibody or other affinity reagent) or a reagent for use in luminescence analysis. The third droplet may contain reagents, such as one or more (eg, 2 to 20) capture beads having an affinity for the material of interest. For example, the material of interest can be an antibody or nucleic acid (eg, DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof). The capture beads may optionally be discharged from the device for subsequent analysis. Like the second and third droplets, the fourth droplet may contain reagents such as a mixture of enzymes suitable for carrying out a reaction, such as a reverse transcriptase reaction or a whole genome amplification reaction. In some embodiments, the use of an electrowetting force to move and fuse the droplets comprises an effective electrowetting feature that changes the surface area of the substrate adjacent the droplets, thereby moving or fusing the droplets. In some embodiments, altering the effective electrowetting characteristics of the surface of the substrate can include activating the electrowetting electrode adjacent the surface area of the substrate of the droplet. In some embodiments, electrowetting the electrode that activates the surface area of the substrate adjacent to the droplet involves directing a light pattern onto the surface area of the substrate. In some embodiments, the electrowetting device is integrated with the electrical positioning device. Therefore, a single unitary device can combine the functions of both devices. Other aspects and embodiments of the invention will be apparent from the description and drawings.

priority U.S. Provisional Application No. 62/246,605, filed on October 27, 2015, and U.S. Provisional Application No. 62/247,725, filed on October 28, 2015, filed on October 27, 2015 The priority of U.S. Provisional Application No. 62/342,131, filed on Jan. 19, and U.S. Provisional Application No. 62/410,238, filed on October 19, 2016, the entire contents of each of which is incorporated by reference. Incorporated herein. The present application also claims priority to U.S. Patent Application Serial No. 15/135, 707, filed on Apr. This specification sets forth example embodiments and applications of the invention. However, the invention is not limited to the exemplary embodiments and applications or the manner in which the exemplary embodiments and applications are described or illustrated herein. In addition, the figures may show simplified or partial views, and the dimensions of the elements in the figures may be exaggerated or otherwise disproportionate. In addition, when the terms "upper", "attached to", "connected to", "coupled to" or the like are used herein, one element (eg, material, layer, substrate, etc.) can be "on another element. , "attached to another component", "connected to another component" or "coupled to another component", and one component is directly on the other component, attached to another component, connected to another Whether an element is coupled to another element or whether one or more intermediate elements are present between one element and another element. Moreover, unless the context indicates otherwise, directions are provided (eg, top, bottom, top, bottom, side, up, down, below, above, upper, lower, horizontal, vertical, "x", "y") , "z", etc.) are relative and are merely by way of example and are not to be construed as limiting. In addition, when referring to a list of elements (eg, elements a, b, c), the reference is intended to include any of the listed elements themselves, less than any combination of all listed elements, and/or all listed. A combination of components. Partial divisions in this specification are for ease of review only and do not limit any combination of the elements discussed. As used herein, "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for a sub-absolute or perfect state, size, measurement, result, or the like, such as a minor, insignificant change, such as would be expected by those skilled in the art, but would not significantly affect the overall performance. "Substantially" means within 10% when used for a numerical value or a parameter or feature that can be expressed as a numerical value. The term "multiple" means more than one. The term "plurality" as used herein may be two, three, four, five, six, seven, eight, nine, ten or more. The term "arranged" as used herein is used in its meaning "positioning". As used herein, a "microfluidic device" or "microfluidic device" is a device comprising one or more discrete microfluidic circuits configured to contain a fluid, each microfluidic circuit comprising a fluid interconnected loop element, including But not limited to) regions, flow zones, channels, chambers and/or fences and (for microfluidic devices including covers) at least two configured to allow fluid (and, as the case may be, suspended in a fluid) to flow in and/or Or flow out of the microfluidic device. Typically, the microfluidic circuit of the microfluidic device will include at least one microfluidic channel and at least one chamber, and will hold a volume of less than about 1 mL (eg, less than about 750 μL, 500 μL, 250 μL, 200 μL, 150 μL, 100 μL) , 75 μL, 50 μL, 25 μL, 20 μL, 15 μL, 10 μL, 9 μL, 8 μL, 7 μL, 6 μL, 5 μL, 4 μL, 3 μL or 2 μL). In certain embodiments, the microfluidic circuit holds about 1-2 μL, 1-3 μL, 1-4 μL, 1-5 μL, 2-5 μL, 2-8 μL, 2-10 μL, 2-12 μL, 2-15 μL, 2-20 μL, 5-20 μL, 5-30 μL, 5-40 μL, 5-50 μL, 10-50 μL, 10-75 μL, 10-100 μL, 20-100 μL, 20-150 μL, 20-200 μL, 50-200 μL, 50-250 μL or 50-300 μL. As used herein, a "nanofluidic device" or "nanofluidic device" is a type of microfluidic device having at least one configured to accommodate a volume of less than about 1 μL (eg, less than about 750 nL, 500 nL, 250 nL, 200 nL, 150 nL, 100 nL, 75 nL, 50 nL, 25 nL, 20 nL, 15 nL, 10 nL, 9 nL, 8 nL, 7 nL, 6 nL, 5 nL, 4 nL, 3 nL , a microfluidic circuit of a loop element of a fluid of 2 nL, 1 nL or less. The nanofluidic device can comprise a plurality of loop elements (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 , 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000 or more). In certain embodiments, one or more (eg, all) of the at least one loop element are configured to accommodate a volume of between about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL 1 nL to 10 nL, 1 nL to 15 nL, 1 nL to 20 nL, 1 nL to 25 nL, or 1 nL to 50 nL of fluid. In other embodiments, one or more (eg, all) of the at least one loop element are configured to accommodate a volume of between about 20 nL and 200 nL, 100 nL to 200 nL, 100 nL to 300 nL, and 100 nL to 400 nL. 100 nL to 500 nL, 200 nL to 300 nL, 200 nL to 400 nL, 200 nL to 500 nL, 200 nL to 600 nL, 200 nL to 700 nL, 250 nL to 400 nL, 250 nL to 500 nL, 250 Fluids from nL to 600 nL, or from 250 nL to 750 nL. As used herein, "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device that is significantly longer than both horizontal and vertical dimensions. For example, the length of the flow channel can be at least 5 times the horizontal or vertical dimension, for example, the length is at least 10 times, the length is at least 25 times, the length is at least 100 times, the length is at least 200 times, the length It is at least 500 times, the length is at least 1,000 times, and the length is at least 5,000 times or longer. In some embodiments, the length of the flow channel is in the range of from about 50,000 microns to about 500,000 microns, including any range therebetween. In some embodiments, the horizontal dimension ranges from about 100 microns to about 1000 microns (eg, from about 150 microns to about 500 microns) and the vertical dimension ranges from about 25 microns to about 200 microns, such as from about 40 microns to about 150. Within the micrometer range. It should be noted that the flow channel can have a plurality of different spatial configurations in the microfluidic device, and thus is not limited to a perfect linear element. For example, the flow channel can include one or more portions having any of the following configurations: curve, bend, helix, slope, downtilt, cross (eg, a plurality of different flow paths), and any combination thereof. Additionally, the flow channels may have different cross-sectional areas along their path, widening and contracting to provide a desired fluid flow therein. The term "blocking" as used herein generally refers to a bump or similar type of structure that is large enough to partially (but not completely) obstruct the movement of a target micro-object between two different regions or loop elements in a microfluidic device. The two different zone/loop elements can be, for example, a microfluidic barrier and a microfluidic channel, or a connection zone and a separation zone of the microfluidic barrier. The term "shrinkage" as used herein generally refers to narrowing the width of a loop element (or an interface between two loop elements) in a microfluidic device. The contraction can be located, for example, at the interface between the microfluidic barrier and the microfluidic channel or at the interface between the separation zone and the connection zone of the microfluidic barrier. The term "transparent" as used herein refers to a material that allows visible light to pass through but does not substantially alter light as it passes through. The term "microscopic object" as used herein generally refers to any microscopic object that can be separated and collected in accordance with the present invention. Non-limiting examples of minute objects include: inanimate tiny objects such as microparticles; microbeads (eg, polystyrene beads, LuminexTM beads, or the like); magnetic beads; microrods; microfilaments; quantum dots and Such as biological microscopic objects, such as cells (eg embryos, oocytes, eggs, sperm cells, self-organized dissociated cells, eukaryotic cells, protist biological cells, animal cells, mammalian cells, human cells, immune cells, hybridomas) , cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, prokaryotic cells, and the like; biological organelles; vesicles or complexes; a vesicle; a liposome (eg, synthetic or derived from a membrane preparation); a lipid nanoparticle (as described in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs", Methods Enzymol., 464:211-231. ) and the like; or a combination of inanimate tiny objects and biological microscopic objects (eg, microbeads attached to cells, liposomes coated microbeads) The liposome-coated beads or the like). The beads may further have other moieties/molecules that are covalently or non-covalently attached, such as fluorescent labels, proteins, small molecule signaling moieties, antigens or chemical/biological materials that can be used in the assay. The term "maintaining cells" as used herein refers to an environment that provides a surface comprising both fluid and gaseous components and, optionally, conditions necessary to maintain cell viability and/or expansion. The "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 or the like. As used herein, "diffuse" and "diffusion" as used in a fluid medium means that the components of the fluid medium move down the thermodynamically along the concentration gradient. The phrase "media flow" means that the fluid medium is primarily displaced by any mechanism other than diffusion. For example, media flow can involve the fluid medium moving from one point to another due to the pressure differential between the two points. The flow can include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When a fluid medium flows into another fluid medium, turbulence and mixing of the medium can occur. The phrase "substantially non-flowing" means that the flow rate of the fluid medium averaged over time is less than the rate of diffusion of the component of the material (eg, the analyte of interest) into or into the fluid medium. The rate of diffusion of the components of the material can depend, for example, on temperature, the size of the components, and the strength of the interaction between the components and the fluid medium. The phrase "fluid connection" as used herein with respect to different regions within a microfluidic device means that when different regions are substantially filled with a fluid (eg, a fluid medium), the fluids in each region are joined to form a single fluid entity. This does not mean that the composition of the fluid (or fluid medium) in different areas must be the same. Conversely, fluids in different fluid connection regions of a microfluidic device can have different compositions (eg, different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) as the solute moves down along its respective concentration gradients and/or These compositions can be constantly changing as the fluid flows through the device. Microfluidic (or nanofluidic) devices may include "overreach" regions and "unseen" regions. As used herein, a "fluctuation" region includes one or more fluid interconnected loop elements of a microfluidic circuit, each of which undergoes a flow of media as it flows through the microfluidic circuit. The loop elements of the sweeping zone may include, for example, zones, passages, and all or part of the chamber. As used herein, an "unflucated" region includes one or more fluid interconnected loop elements of a microfluidic circuit, each of which substantially does not undergo fluid flow as it flows through the microfluidic circuit. The uncontaminated region can be fluidly coupled to the sweeping region, with the condition that the fluidic connection is structured such that the medium between the intervening region and the uncontaminated region can diffuse but does not substantially flow. Thus, the microfluidic device can be structured to substantially separate the flow of the medium in the un-interpolated region from the undulation region while achieving substantially only diffusional fluid communication between the occluded region and the un-interpolated region. For example, a flow channel of a microfluidic device is an example of a region of the wave, and a separation region of the microfluidic device (described further below) is an example of an unaffected zone. As used herein, "flow zone" refers to one or more fluid-connected loop elements (eg, channels, regions, chambers, and the like) that define and are subject to a flow path of a medium. Thus, the flow zone is an example of a sweeping zone of a microfluidic device. Other loop elements (e.g., un-intermittent regions) may be in fluid connection with loop elements that include a flow zone but are not subjected to a flow of media in the flow zone. As used herein, "alkyl" refers to a straight or branched hydrocarbon chain radical (eg, C1-C6 alkyl) consisting solely of carbon and hydrogen atoms, free of unsaturation, having from 1 to 6 carbon atoms. Whenever it appears herein, a numerical range such as "1 to 6" means each integer in the given range; for example, "1 to 6 carbon atoms" means that the alkyl group may be 1 carbon atom, 2 A carbon atom, three carbon atoms, etc., up to and including six carbon atoms, but this definition also encompasses the term "alkyl" in the range of unspecified values. In some embodiments, it is a C1-C3 alkyl group. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl isobutyl, tert-butyl, pentyl, isopentyl , neopentyl, hexyl and the like. The alkyl group is attached to the remainder of the molecule by a single bond, such as methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl Base, 1,1-dimethylethyl (t-butyl), hexyl and the like. Unless expressly stated otherwise in the specification, an alkyl group may be optionally substituted with one or more substituents, which are independently: aryl, arylalkyl, heteroaryl, heteroarylalkyl, Hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethyldecyl, -OR', -SR', -OC(O)-R', -N(R' 2, -C(O)R', -C(O)OR', -OC(O)N(R')2, -C(O)N(R')2, -N(R')C (O)OR', -N(R')C(O)R', -N(R')C(O)N(R')2, N(R')C(NR')N(R' 2, -N(R')S(O)tR' (where t is 1 or 2), -S(O)tOR' (where t is 1 or 2), -S(O)tN(R') 2 (wherein t is 1 or 2) or PO3(R')2, wherein each R' is independently hydrogen, alkyl, fluoroalkyl, aryl, aralkyl, heterocycloalkyl or heteroaryl. As referred to herein, a fluorinated alkyl moiety is an alkyl moiety in which one or more of the alkyl moieties are replaced by a fluorine substituent. The perfluorinated alkyl moiety replaces all of the hydrogen attached to the alkyl moiety with a fluorine substituent. As referred to herein, a "halo" moiety is a bromine, chlorine or fluorine moiety. As referred to herein, an "olefin" compound is an organic molecule containing an "olefinic hydrocarbon" moiety. An olefinic moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond. The non-olefinic hydrocarbon moiety of the molecule can be any class of organic molecules, and in some embodiments can include an alkyl group or a fluorinated (including but not limited to, perfluorinated) alkyl moiety, either of which can Further replaced. As used herein, "densely stacked hydrophobic monolayer" means that a single layer of hydrophobic molecules is packed sufficiently tightly to resist insertion and/or intrusion of polar molecules such as water, ions, and other charged species. As used herein: "μm" (or "um") means micron; "μm³ “pL” means picoliter, “nL” means nanoliter; and “μL” (or “uL”) means microliter.Loading method . Loading tiny objects (eg, biological micro-objects and/or beads) into different regions of the microfluidic device can involve the use of fluid flow, gravity, dielectrophoresis (DEP) forces, electrowetting forces, magnetic forces, or any combination thereof, such as As described in this article. The DEP force can be generated optically, for example by photo-electrical clamp (OET) configuration and/or electrically, for example by activating the electrode/electrode region in a time/space mode. Similarly, the electrowetting force can be provided optically, for example by photo-wetting (OEW) configuration and/or electrically, for example by activating the electrode/electrode region in a time-space mode.Microfluidic devices and systems for operating and observing such devices . FIG. 1A illustrates a generalized example of a microfluidic device 100 and a system 150 that can be used to control the movement of the microfluidic device 100 and the tiny objects and/or droplets therein. A perspective view of the microfluidic device 100 is shown, partially trimming its cover 110 to provide a partial view into the microfluidic device 100. Microfluidic device 100 generally includes a microfluidic circuit 120 that includes a flow region 106 through which fluid medium 180 can flow, optionally carrying one or more tiny objects (not shown) into microfluidic circuit 120 and/or through Microfluidic circuit 120. Although a single microfluidic circuit 120 is illustrated in FIG. 1A, suitable microfluidic devices can include a plurality (eg, 2 or 3) of such microfluidic circuits. Nonetheless, the microfluidic device 100 can be configured as a nanofluidic device. In the embodiment illustrated in FIG. 1A, the microfluidic circuit 120 includes a plurality of microfluidic isolation fences 124, 126, 128, and 130 each having a single opening in fluid communication with the flow region 106. As discussed further below, the microfluidic isolation fence includes a number of features and structures that have been optimized to retain tiny objects in the microfluidic device (e.g., microfluidic device 100) even as the medium 180 flows through the flow region 106. However, prior to the above, a brief description of the microfluidic device 100 and system 150 is provided. As generally illustrated in FIG. 1A, the microfluidic circuit 120 is defined by a housing 102. Although the outer casing 102 can be physically structured in different configurations, in the example shown in FIG. 1A, the outer casing 102 is illustrated as including a support structure 104 (eg, a substrate), a microfluidic circuit structure 108, and a cover 110. However, in certain embodiments, the outer casing 102 may lack the cover 110 and the microfluidic circuit 120 may be defined by the support structure 104 and the microfluidic circuit structure 108. The support structure 104, the microfluidic circuit structure 108, and (as appropriate) the cover 110 can be attached to each other. For example, the microfluidic loop structure 108 can be disposed on the inner surface 109 of the support structure 104 and the cover 110 can be disposed over the microfluidic loop structure 108. The microfluidic loop structure 108 can define elements of the microfluidic circuit 120 with the support structure 104 and, as the case may be, the cover 110. The support structure 104 can be at the bottom of the microfluidic circuit 120 and the cover 110 is on top of the microfluidic circuit 120, as illustrated in Figure 1A. Alternatively, support structure 104 and cover 110 can be configured in other orientations. For example, the support structure 104 can be at the top of the microfluidic circuit 120 and the cover 110 is at the bottom of the microfluidic circuit 120. Nonetheless, there may be one or more turns 107 each containing a passage into or out of the outer casing 102. Examples of channels include valves, gates, through holes, or the like. As illustrated, the crucible 107 is a through-hole created by a void in the microfluidic loop structure 108. However, the crucible 107 can be located in other components of the housing 102 (eg, the cover 110). Only one crucible 107 is illustrated in FIG. 1A, but the microfluidic circuit 120 can have two or more crucibles 107. For example, there may be a first crucible 107 that serves as an inlet for fluid entering the microfluidic circuit 120, and there may be a second crucible 107 that serves as an outlet for fluid exiting the microfluidic circuit 120. The use of the crucible 107 as an inlet or outlet may depend on the direction of fluid flow through the flow zone 106. Support structure 104 can include one or more electrodes (not shown) and a substrate or a plurality of interconnect substrates. The substrate can be any suitable substrate known in the art. For example, the support structure 104 can include one or more semiconductor substrates, each of which is electrically coupled to at least one of the one or more electrodes (eg, all or a subset of the semiconductor substrates can be electrically connected to a single electrode ). Alternatively, support structure 104 can include a printed circuit board assembly ("PCBA") that includes one or more electrodes. In other embodiments, the support structure 104 can include a substrate (eg, a semiconductor substrate) mounted on the PCBA. The microfluidic loop structure 108 can define loop elements of the microfluidic circuit 120. The loop elements can include spaces or regions that can be fluidly interconnected when the microfluidic circuit 120 is filled with a fluid, such as a flow region (which can include or be one or more flow channels), a chamber, a fence, a well, and the like. In the microfluidic circuit 120 illustrated in FIG. 1A, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a structure that substantially surrounds the relatively rigid structure of the microfluidic circuit material 116. For example, the frame 114 can comprise a metallic material. Alternatively, the microfluidic loop structure 108 may lack a frame. For example, the microfluidic loop structure 108 can be composed of or consist essentially of the microfluidic loop material 116. The microfluidic loop material 116 can be patterned via a cavity or the like to define loop elements and interconnects of the microfluidic circuit 120. The microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (eg, rubber, plastic, elastomer, polyoxymethylene, polydimethyl siloxane ("PDMS"), or the like), which can be breathable . Other examples of materials that may form the microfluidic circuit material 116 include molded glass, etchable materials (e.g., polyfluorene oxide, such as photopatternable polyoxyl or "PPS"), photoresists (e.g., SU8), or the like. In some embodiments, the materials, and thus the microfluidic circuit material 116, can be rigid and/or substantially gas impermeable. Nonetheless, the microfluidic circuit material 116 can be disposed on the support structure 104 and (as appropriate) inside the frame 114. Cover 110 can be an integral part of microfluidic circuit material 116 and/or frame 114. Alternatively, cover 110 can be a structurally different component, as illustrated in Figure 1A. Cover 110 may comprise the same or a different material than frame 114 and/or microfluidic circuit material 116. Similarly, support structure 104 can be a separate structure from microfluidic circuit material 116 or frame 114, as illustrated, or be part of microfluidic circuit material 116 or frame 114. Likewise, the microfluidic circuit material 116 and the frame 114 (if present) can be a separate structure or a component of the same structure as shown in Figure 1A. In some embodiments, the cover 110 can comprise a rigid material. The rigid material can be glass or a material having similar properties. In some embodiments, the cover 110 can comprise a deformable material. The deformable material can be a polymer such as PDMS. In some embodiments, the cover 110 can comprise both rigid and deformable materials. For example, one or more portions of the cover 110 (eg, one or more portions above the isolation fences 124, 126, 128, 130) can include a deformable material that interfaces with the rigid material of the cover 110. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes may comprise a conductive oxide, such as indium-tin oxide (ITO), which may be applied to a glass or similar insulating material. Alternatively, the one or more electrodes may be flexible electrodes embedded in a deformable material, such as a polymer (eg, PDMS), such as single-walled nanotubes, multi-walled nanotubes, nanowires, conductive nanoparticles Cluster or a combination thereof. Flexible electrodes that can be used in microfluidic devices are described, for example, in U.S. Patent Application Serial No. 2012/032566, the entire disclosure of which is incorporated herein by reference. In some embodiments, the cover 110 can be modified (eg, by coating or conditionally facing all or a portion of the surface of the microfluidic circuit 120 inwardly) to support droplet movement and/or cell adhesion, cell viability, and/or Or cell growth. Modifications can include coating a synthetic or natural polymer or a conditional surface having a covalently bound molecule (eg, a self-associating molecule). In some embodiments, the cover 110 and/or the support structure 104 can be light transmissive. Cover 110 can also include at least one gas permeable material (e.g., PDMS or PPS). FIG. 1A also shows a system 150 for operating and controlling a microfluidic device, such as microfluidic device 100. System 150 includes a power source 192, an imaging device 194 (not shown, but may be part of imaging module 164), and tilting device 190 (not shown, but may be part of tilt module 166). Power source 192 can provide power to microfluidic device 100 and/or tilt device 190 to provide a bias voltage or current as needed. Power source 192 can, for example, include one or more alternating current (AC) and/or direct current (DC) voltage or current sources. Imaging device 194 can include a device for capturing an image of the interior of microfluidic circuit 120, such as a digital camera. In some cases, imaging device 194 further includes a detector having a high frame rate and/or high sensitivity (eg, for low light applications). Imaging device 194 may also include mechanisms for directing stimulating radiation and/or light beams into microfluidic circuit 120 and collecting radiation and/or light beams that are reflected or emitted from microfluidic circuit 120 (or tiny objects contained therein). The emitted light beam can be in the visible spectrum and can include, for example, fluorescent emissions. The reflected beam may comprise a reflected emission originating from an LED or a broad spectrum lamp, such as a mercury lamp (eg, a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to FIG. 3B, imaging device 194 can further include a microscope (or string of optical elements) that may or may not include an eyepiece. System 150 further includes a tilting device 190 configured to rotate microfluidic device 100 along one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or house the housing 102 including the microfluidic circuit 120 along at least one axis such that the microfluidic device 100 (and thus the microfluidic circuit 120) can be maintained in a horizontal orientation ( That is, 0°), vertical orientation (ie, 90° with respect to the x-axis and/or y-axis) relative to the x-axis and y-axis, or any orientation therebetween. The orientation of microfluidic device 100 (and microfluidic circuit 120) relative to the axis is referred to herein as the "inclination" of microfluidic device 100 (and microfluidic circuit 120). For example, tilting device 190 can tilt microfluidic device 100 by 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1° with respect to the x-axis or y-axis. 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 °, 75°, 80°, 90° or any degree therebetween. The horizontal orientation (and thus the x-axis and y-axis) is defined to be perpendicular to the vertical axis defined by gravity. The tilting device can also tilt the microfluidic device 100 (and the microfluidic circuit 120) to any extent greater than 90° relative to the x-axis and/or y-axis, or to cause the microfluidic device 100 (and the microfluidic circuit 120) to be relative to the x-axis. The shaft or y-axis is tilted by 180° to completely invert the microfluidic device 100 (and the microfluidic circuit 120). Similarly, in some embodiments, tilting device 190 tilts microfluidic device 100 (and microfluidic circuit 120) along a rotational axis defined by flow region 106/channel 122 or some other portion of microfluidic circuit 120. In some cases, the microfluidic device 100 is tilted into a vertical orientation such that the flow zone 106/channel 122 is located above or below one or more isolation fences. The term "above" as used herein means that the flow zone 106/channel 122 is positioned above one or more isolation fences on a vertical axis defined by gravity (ie, objects in the isolation fence above the flow zone 106/channel 122 will have Higher than the gravitational potential of the object in the flow zone/channel). The term "below" as used herein means that the flow zone 106/channel 122 is positioned below one or more isolation fences on a vertical axis defined by gravity (ie, objects in the isolation fence below the flow zone 106/channel 122 will have Below the gravitational potential of the object in the flow zone/channel). In some cases, tilting device 190 tilts microfluidic device 100 along an axis parallel to flow region 106/channel 122. Additionally, the microfluidic device 100 can be tilted to an angle of less than 90° such that the flow zone 106/channel 122 is above or below one or more isolation fences but not directly above or below the isolation fence. In other cases, tilting device 190 tilts microfluidic device 100 along an axis perpendicular to flow zone 106/channel 122. In other cases, tilting device 190 tilts microfluidic device 100 along an axis that is neither parallel nor perpendicular to flow region 106/channel 122. System 150 can further include a media source 178. Media source 178 (eg, a container, reservoir, or the like) can include multiple portions or containers that each serve to accommodate a different fluid medium 180. Thus, the dielectric source 178 can be a device external to and separate from the microfluidic device 100, as illustrated in Figure 1A. Alternatively, the media source 178 may be located wholly or partially within the outer casing 102 of the microfluidic device 100. For example, the media source 178 can include a reservoir that is part of the microfluidic device 100. FIG. 1A also illustrates a simplified block diagram illustration of an example of a control and monitoring device 152 that forms part of system 150 and that can be used in conjunction with microfluidic device 100. As shown, examples of the control and monitoring device 152 include a main controller 154, a media module 160 for controlling the media source 178, and for controlling tiny objects and/or media in the microfluidic circuit 120 (eg, microscopic media) a moving module 162 for moving and/or selecting, an imaging module 164 for controlling an imaging device 194 (eg, a camera, a microscope, a light source, or any combination thereof) to capture images (eg, digital images) and for controlling Tilting module 166 of tilting device 190. Control device 152 may also include other modules 168 for controlling, monitoring, or otherwise performing other functions of microfluidic device 100. As shown, device 152 can be operatively coupled to display device 170 and input/output device 172 (or further include display device 170 and input/output device 172). The main controller 154 can include a control module 156 and a digital memory 158. Control module 156 can include, for example, a digital processor that is configured to operate in accordance with machine-executable instructions (eg, software, firmware, source code, or the like) stored as non-transitory data or signals in memory 158. . Alternatively or additionally, the control module 156 can include a hardwired digital loop and/or an analog loop. The media module 160, the power module 162, the imaging module 164, the tilt module 166, and/or other modules 168 can be configured in a similar manner. Thus, the functions, process acts, acts or steps of the processes discussed herein as embodied for the microfluidic device 100 or any other microfluidic device may be configured by the main controller 154, the media module 160, the power module, as discussed above. Either or more of the group 162, the imaging module 164, the tilt module 166, and/or other modules 168 are implemented. Similarly, the main controller 154, the media module 160, the power module 162, the imaging module 164, the tilt module 166, and/or other modules 168 can be coupled to each other to communicate and receive any of the functions, processes, and processes discussed herein. Data used in actions, actions, or steps. The media module 160 controls the media source 178. For example, media module 160 can control media source 178 to input selected fluid media 180 into housing 102 (eg, via inlet port 107). The media module 160 can also control the removal of the media from the housing 102 (eg, via an exit port (not shown)). Thus, one or more media can be selectively input into and removed from the microfluidic circuit 120. The media module 160 can also control the flow of the fluid medium 180 in the flow zone 106/channel 122 inside the microfluidic circuit 120. For example, in some embodiments, the media module 160 is prior to loading the tiny objects or beads into the isolation fence (eg, using gravity, electrowetting (EW) forces, dielectrophoresis (DEP) forces, or a combination thereof). The flow of the medium 180 in the flow zone 106/channel 122 flows through the outer casing 102. The power module 162 can be configured to control the selection, capture, and movement of minute objects and/or media droplets in the microfluidic circuit 120. As discussed in detail herein, the outer casing 102 can comprise an electrowetting (EW) configuration, such as a photo-wetting (OEW) configuration, a dielectric wetting (EWOD) configuration, a one-sided electrowetting configuration, or the like. In certain embodiments, the housing 102 can further comprise a dielectrophoresis (DEP) configuration, such as an optoelectronic clamp (OET) configuration, an electrically actuated DEP configuration, and the like. The power module 162 can control the activation of electrodes and/or transistors (eg, optoelectronic crystals) included in the EW and/or DEP configuration to select and move the flow region 106/channel 122 and/or the isolation fences 124, 126, 128. , tiny objects in 130, and / or medium droplets. Imaging module 164 can control imaging device 194 (not shown). For example, imaging module 164 can receive and process image data from imaging device 194. Image data from imaging device 194 may include any type of information captured by imaging device 194 (eg, tiny objects, presence or absence of media droplets, accumulation of labels (eg, fluorescent markers), etc.). Using information captured by imaging device 194, imaging module 164 can further calculate the location of objects (eg, tiny objects, media droplets, or the like) and/or the rate of movement of such objects within microfluidic device 100. The tilt module 166 can control the tilting motion of the tilting device 190 (not shown). Additionally, the tilt module 166 can control the tilt rate and time, for example, to optimize the transfer of tiny objects to one or more fences via gravity. The tilt module 166 is coupled in communication with the imaging module 164 to receive data indicative of movement of the micro-objects and/or medium droplets in the microfluidic circuit 120. Using this data, tilt module 166 can adjust the tilt angle of microfluidic circuit 120 to adjust the rate at which fine objects and/or medium droplets move in microfluidic circuit 120. The tilt module 166 can also use this data to repeatedly adjust the position of the tiny objects and/or medium droplets in the microfluidic circuit 120. In the example shown in FIG. 1A, microfluidic circuit 120 is illustrated as including a single flow region 106 that is substantially comprised of microfluidic channels 122. Each of the barrier fences 124, 126, 128, and 130 includes a single opening to the flow zone 106/channel 122, but is otherwise encapsulated such that the fences substantially separate the tiny objects and flow areas 106/ within the fence. Tiny objects and/or fluid medium 180 in channel 122 or other enclosure. The wall of the barrier fence can extend from the inner surface 109 of the base to the inner surface of the cover 110, thereby facilitating the separation. The opening of the fence of the flow zone 106/channel 122 is oriented at an angle to the flow of the fluid medium 180 in the flow zone 106/channel 122 such that the flow of fluid medium 180 is not directed to the fence. The stream may be tangent or orthogonal to the plane of the opening, such as a fence. In some cases, the fences 124, 126, 128, and/or 130 are configured to physically enclose one or more tiny objects within the microfluidic circuit 120. The barrier fence of the present invention may include a variety of shapes, surfaces, and features that are optimized for use with EW forces, OEW forces, DEP forces, and/or OET forces, fluid flow, and/or gravity, as will be discussed in detail below. The microfluidic circuit 120 can include any number of microfluidic isolation fences. Although five isolation fences are shown, the microfluidic circuit 120 can have fewer or more isolation fences. As shown, the microfluidic barriers 124, 126, 128, and 130 of the microfluidic circuit 120 each include different features and shapes that provide one or more micro-objects and/or fluid media that can be manipulated by the microfluidic device 100. The benefits of dripping. Thus, in some embodiments, the microfluidic circuit 120 can include a plurality of microfluidic isolation enclosures, wherein two or more of the isolation enclosures contain different structures and/or features that provide different benefits. However, in some embodiments, the microfluidic circuit 120 includes a plurality of identical microfluidic isolation fences. Microfluidic devices that can be used to manipulate tiny objects and/or media droplets can include any of the isolation fences 124, 126, 128, and 130, or variations thereof, including as shown in Figures 2B, 2C, 2D, 2E, and 2F. They are shown in their configured fences as discussed below. In the embodiment illustrated in Figure 1A, a single flow zone 106 is shown. However, other embodiments of the microfluidic device 100 can include a plurality of flow regions 106 that are each configured to provide a separate path for fluid flow through the microfluidic device 100. The microfluidic circuit 120 includes an inlet valve or helium 107 in fluid communication with the flow zone 106 whereby the fluid medium 180 can reach the flow zone 106 / channel 122 via the inlet port 107. In some cases, flow zone 106 includes a single flow path. In other cases, flow region 106 includes a plurality of flow paths (eg, 2, 3, 4, 5, 6 or more), each of which may include a microchannel (eg, such as channel 122). Two or more of the plurality of flow paths (eg, all) may be substantially parallel to each other. For example, flow zone 106 can be divided into a plurality of parallel channels (eg, such as channel 122). In certain embodiments, the flow zone 106 (and one or more channels included in the flow zone) are arranged in a zigzag pattern whereby the flow zone 106 passes through the microfluidic device 100 in alternating directions two or more times. Times. In some cases, the fluid medium within each flow zone 106 flows in at least one of a forward or reverse direction. In some cases, the plurality of isolation fences are configured (eg, relative to the flow zone 106/channel 122) such that the isolation fence can be loaded with target microscopic objects in parallel. In some embodiments, the microfluidic circuit 120 further includes one or more micro object traps 132. The well 132 is typically formed in the wall forming the boundary of the flow region 106/channel 122 and is positionable relative to the opening of one or more of the microfluidic isolation fences 124, 126, 128, 130. In some embodiments, the well 132 is configured to receive or capture a single tiny object from the flow zone 106 / channel 122. In some embodiments, the well 132 is configured to receive or capture a plurality of tiny objects from the flow region 106/channel 122. In some cases, well 132 contains a volume that is approximately equal to the volume of a single target tiny object. The well 132 can further include an opening configured to assist in targeting a tiny object into the well 132. In some cases, the well 132 includes openings having a height and a width equal to the size of a single target micro-object, thereby preventing other tiny objects (or larger, larger objects) from entering the micro-object trap. The well 132 can further include other features configured to assist in targeting the retention of tiny objects within the well 132. In some cases, the well 132 is aligned with the channel 122 with respect to the opening of the microfluidic barrier and is located on the opposite side of the channel 122 such that the microfluidic device 100 captures tiny objects as they are tilted parallel to the axis of the channel 122. The well 132 is exited by a trajectory that causes the tiny object to fall into the opening of the isolation fence. In some cases, well 132 includes side channels 134 that are smaller than the target minute object to facilitate flow through well 132 and thereby increase the likelihood of capturing tiny objects in well 132. As discussed in more detail below, in some embodiments, an electrowetting (EW) force is applied to one of the surfaces of the support structure 104 (and/or cover 110) of the microfluidic device 100 via one or more electrodes (not shown). Or multiple locations (eg, locations within the flow zone and/or isolation fence) to manipulate, transport, separate, and sort the droplets located in the microfluidic circuit 120. For example, in some embodiments, an EW force is applied to one or more locations on the surface of support structure 104 (and/or cover 110) to transfer droplets from flow zone 106 to a desired microfluidic isolation fence. . In some embodiments, the EW force is used to prevent the droplets within the isolation fence (eg, the isolation fence 124, 126, 128, or 130) from being displaced therefrom. Moreover, in some embodiments, the EW force is used to selectively remove droplets previously collected in accordance with the teachings of the present invention from a fence. In some embodiments, the EW force comprises an opto-wetting (OEW) force. In some embodiments, a dielectrophoretic (DEP) force is applied to fluid medium 180 (eg, in a flow zone and/or isolation fence) via one or more electrodes (not shown) for manipulation, transfer, separation, and separation. Select the tiny objects in it. For example, in some embodiments, a DEP force is applied within one or more portions of the microfluidic circuit 120 to transfer a single tiny object from the flow zone 106 to the desired microfluidic isolation fence. In some embodiments, the DEP force is used to prevent the displacement of tiny objects within the isolation fence (eg, the isolation fence 124, 126, 128, or 130) therefrom. Moreover, in some embodiments, the DEP force is used to selectively remove tiny objects previously collected in accordance with the teachings of the present invention from a fence. In some embodiments, the DEP force comprises an optoelectronic clamp (OET) force. In some embodiments, DEP and/or EW forces are combined with other forces, such as flow and/or gravity, to manipulate, transfer, separate, and sort fine objects and/or droplets within microfluidic circuit 120. For example, the outer casing 102 can be tilted (eg, by tilting the device 190) to position the flow region 106/channel 122 and the tiny objects located therein over the microfluidic isolation fence, and gravity can cause tiny objects and/or micro Drops are sent to the fence. In some embodiments, the DEP force and/or EW force can be applied before other forces. In other embodiments, the DEP force and/or EW force may be applied after other forces. In other cases, the DEP and/or EW forces may be applied simultaneously with other forces or in an alternating manner with other forces.Microfluidic device dynamic configuration. As described above, the system's control and monitoring equipment can include a power module for selecting and moving objects (eg, tiny objects or droplets) in the microfluidic circuit of the microfluidic device. The microfluidic device of the present invention can have a variety of dynamic configurations depending on the type of moving object and other considerations. In particular, the support structure 104 and/or cover 110 of the microfluidic device 100 can include an electrowetting (EW) configuration that selectively introduces EW forces into the droplets in the fluid medium 180 in the microfluidic circuit 120, and Thereby, individual droplets or groups of droplets are selected, captured and/or moved. In certain embodiments, the microfluidic device of the present invention can comprise a first portion having an EW configuration and a second portion having a dielectrophoresis (DEP) configuration. Accordingly, at least a portion of the support structure 104 and/or the cover 110 of the microfluidic device 100 can include a DEP configuration for selectively introducing DEP forces into the tiny objects in the fluid medium 180 in the microfluidic circuit 120, and Selecting, capturing, and/or moving a group of individual tiny objects or small objects. In certain embodiments, the microfluidic device of the present invention can comprise an electrowetting configuration comprising a substrate having a dielectric layer and a droplet-actuating surface The droplet actuation surface comprises a hydrophobic layer covalently bonded to the dielectric layer. The dielectric layer can be located directly below the hydrophobic layer such that the droplets resting on the substrate directly contact the hydrophobic layer. Figure 2A illustrates an example of a portion of the microfluidic device. As shown, device 400 can include a substrate 104 that includes a substrate and at least one electrode (eg, first electrode) 418. The substrate can include a plurality of layers including an outer hydrophobic layer 412, an inner dielectric layer 414, a conductive layer 416, an electrode 418, and optionally a support 420. The hydrophobic layer 412 and the inner dielectric layer 414 can provide an inwardly facing portion of the substrate 102 that defines the outer casing. Device 400 also includes a cover 110 that includes an outer hydrophobic layer 422, an inner layer 428 that can include at least one electrode, and an optional support 430. Cover 110 and substrate 104 are substantially parallel to each other and are joined together by spacer elements 108 (e.g., microfluidic circuit materials) to define a housing 435 that is configured to receive a liquid medium. The liquid medium can be, for example, a hydrophobic liquid such as an oil. Additionally, the outer casing 435 can hold liquid droplets 440, such as an aqueous medium. Typically, the liquid medium and the liquid of the droplets are selected to be immiscible liquids. Spacer element 108 can comprise a polymer. The polymer can be, for example, a ruthenium based organic polymer such as polydimethyl siloxane (PDMS) or photopatternable poly oxirane (PPS), both available from Dow Corning. Alternatively, the spacer element 108 can comprise an epoxide-based adhesive. The epoxide-based adhesive can be, for example, SU-8 or a material of the same type. Spacer element 108 can have a thickness of at least 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns or more (ie, a gap between substrate 104 and the inner surface of cover 110) . Thus, for example, the spacer element 108 can have a thickness of 30-60 microns, 40-80 microns, 50-100 microns, 60-120 microns, 70-140 microns, 75-150 microns, 80-160 microns, 90- 180 microns or 100-200 microns. The spacer element 108 can define one or more microfluidic channels within the housing. Additionally, the spacer element 108 can further define a plurality of chambers (or barrier fences) within the housing, wherein each chamber is fluidly coupled to and open from the at least one microfluidic channel. Thus, for example, the spacer element 108 can define a single microfluidic channel and a plurality of chambers fluidly coupled thereto, or a plurality of microfluidic channels, wherein each channel is fluidly coupled to a plurality of chambers. Additionally, each chamber can be fluidly coupled to more than one microfluidic channel, as illustrated in Figures 6 and 7. When at least one electrode 418 of the substrate 104 and at least one electrode 428 of the cover 110 are connected to opposite terminals of an AC voltage source (not shown), the substrate 104 can apply an electrowetting force to the hydrophobic surface 412 outside the substrate 104 (ie, The droplets actuate the surface) contact with the aqueous droplets. In certain embodiments, the AC voltage used to achieve electrowetting based droplet movement in the microfluidic device is at least 20 volts peak to peak (ppV) (eg, from about 20 ppV to 80 ppV, from about 20 ppV to 60 ppV, Approximately 25 ppV to 50 ppV, approximately 25 ppV to 40 ppV, or approximately 25 ppV to 35 ppV). In certain embodiments, the frequency used to achieve the electrowetting-based droplet movement in the microfluidic device is between about 1 kHz and 100 kHz (eg, about 5 kHz to 90 kHz, about 10 kHz to 80 kHz, Approximately 15 kHz to 70 kHz, approximately 20 kHz to 60 kHz, approximately 25 kHz to 50 kHz, or approximately 30 kHz to 40 kHz). The hydrophobic layer 412 outside the substrate 104 and the hydrophobic layer 422 outside the cover 110 may each comprise a densely packed monolayer of self-associating molecules that are covalently bonded to the inner dielectric layer 414 of the substrate 104 or the inner layer 428 of the cover 110, respectively. The monolayer of self-associating molecules comprises a sufficient two-dimensional bulk density to create a hydrophobic barrier between the surface of the bonded monolayer and the hydrophilic liquid (ie, to prevent insertion and/or penetration of polar molecules or other chemicals into the monolayer). The bulk density of a densely packed monolayer will depend on the self-association molecule used. A monolayer comprising a densely packed alkyl terminated alkane will typically comprise at least 1 x 10¹⁴ Molecules/cm² (for example, at least 1.5×10¹⁴ , 2.0×10¹⁴ , 2.5×10¹⁴ One or more molecules/cm² ). As set forth in more detail below, the self-associating molecules can each comprise a linking group, such as a decyloxy group or a phosphonic acid group. The oxoalkyl group can be covalently bonded to the molecules of inner dielectric layer 414 or inner layer 428. Similarly, a phosphonic acid group can be covalently bonded to a molecule of inner dielectric layer 414 or inner layer 428. Self-associating molecules can comprise long chain hydrocarbons that can be unbranched. Thus, the self-associating molecule can comprise an alkyl-terminated oxime or an alkyl-terminated phosphonic acid. Long chain hydrocarbons may comprise chains of at least 10 carbons (eg, at least 16, 18, 20, 22 or more carbons). The self-associating molecule can comprise a fluorinated carbon chain. Thus, for example, the self-associating molecule can comprise a fluoroalkyl-terminated oxime or a fluoroalkyl-terminated phosphonic acid. Fluorinated carbon chain can have the chemical formula CF₃ -(CF₂ )m-(CH)₂ N-, where m is at least 2, n is 0, 1, 2 or greater, and m+n is at least 9. The monolayer of self-associating molecules can have a thickness of less than about 5 nanometers (e.g., from about 1.0 nanometers to about 4.0 nanometers, from about 1.5 nanometers to about 3.0 nanometers, or from about 2.0 nanometers to about 2.5 nanometers). . The hydrophobic layer 412 outside of the substrate 104 can be patterned such that the selected regions are relatively hydrophilic compared to the remainder of the outer hydrophobic layer. This can be achieved, for example, by increasing the voltage drop across the underlying dielectric layer 122 to 50 ppV or greater over a period of time (eg, 60 ppV, 65 ppV, 70 ppV, 75 ppV, 80 ppV or greater). . Without wishing to be bound by theory, it is believed that the relatively hydrophilic regions comprise water molecules that have been inserted into a single layer. In some embodiments, the inner dielectric layer of the substrate can include one or more oxide layers. For example, the inner dielectric layer can comprise or consist of a single oxide layer, such as a metal oxide layer. Alternatively, the inner dielectric layer can comprise or consist of two layers. In some embodiments, the layer can be hafnium oxide or tantalum nitride, and the other layer can be a metal oxide, such as aluminum oxide. In certain embodiments, the thickness of the metal oxide layer can range from about 15 nm to about 45 nm, or from about 30 nm to about 40 nm, or from about 33 nm to about 36 nm. The metal oxide layer can be deposited by atomic layer deposition (ALD) techniques and the layer comprising hafnium oxide or tantalum nitride can be deposited by plasma assisted chemical vapor deposition (PECVD) techniques. In another embodiment, the inner dielectric layer can comprise three layers of dielectric material. In some embodiments, the first layer can comprise a metal oxide, such as aluminum oxide, hafnium oxide, or the like, which can be sandwiched between the hafnium oxide layer and the tantalum nitride layer. In some embodiments, the thickness of the metal oxide layer can range from about 5 nm to about 20 nm, and the layer can be deposited by atomic layer deposition (ALD) techniques. The hafnium oxide layer can also be deposited by ALD and can have a thickness of from about 2 nm to about 10 nm. The tantalum nitride layer may be deposited by plasma assisted chemical vapor deposition (PECVD) techniques and may have a thickness of from about 80 nm to about 100 nm or a thickness of about 90 nm. The inner dielectric layer can have a thickness of between about 50 nanometers and 105 nanometers and/or an impedance of between about 50 kiloohms and 150 kiloohms, and the preferred embodiment is about 100 kiloohms, and the layer that forms the inner dielectric layer Nothing is relevant. The substrate 104 can include a photoreactive layer 146 that contacts the inner dielectric layer 414 with a first side. The second side of photoreactive layer 416 can contact at least one electrode 418. The photoreactive layer 416 may comprise hydrogenated amorphous germanium (a-Si:H). For example, a-Si:H may comprise from about 8% to 40% hydrogen (ie, calculated from the total number of 100* hydrogen atoms/hydrogen and deuterium atoms). The a-Si:H layer can have at least about 500 nanometers (eg, at least about 600 nanometers to 1400 nanometers, about 700 nanometers to 1300 nanometers, about 800 nanometers to 1200 nanometers, about 900 nanometers to 1100). The thickness of nano, or about 1000 nm). However, the thickness of the a-Si:H layer may vary with the thickness of the inner dielectric layer 414 to allow the substrate 104 to be in an open state (ie, illuminated and conductive) and a closed state (ie, dark and non-conductive). A suitable difference is achieved between the impedance of the inner dielectric layer 414 and the impedance of the a-Si:H layer. For example, the impedance of the inner dielectric layer 414 can be tuned to between about 50 kilo ohms and about 150 kilo ohms, and the impedance of the a-Si:H layer can be tuned to at least about 0.5 megaohms in the off state and on. The state is less than or equal to about 1 kohm. These are merely examples, but illustrate how the impedance can be tuned to achieve a photoreactive (in this case, photoconductive) layer 416 that exhibits robust on/off performance. In embodiments where the substrate 104 has a photoreactive layer 416 formed from an a-Si:H layer, the substrate 104 may optionally include a floating electrode pad between the photoreactive layer 416 and the inner dielectric layer 414. Such floating electrode pads are described, for example, in U.S. Patent No. 6,958,132, the disclosure of which is incorporated herein by reference. Alternatively, photoreactive layer 416 can comprise a plurality of conductors, each conductor being controllably connectable to at least one electrode of substrate 102 via a photonic crystal switch. The conductors controlled by the optoelectronic crystal switches are well known in the art and are described, for example, in U.S. Patent Application Serial No. 2014/0124370, the disclosure of which is incorporated herein by reference. Substrate 104 can include a single electrode 418 that is configured to connect to an AC voltage source. The single electrode 418 can comprise an indium-tin oxide (ITO) layer that can be formed, for example, on the glass support 420. Alternatively, single electrode 418 can comprise a conductive germanium layer. In other embodiments, substrate 104 may comprise a plurality of electrodes that are individually addressable in the manner of EWOD devices, as is well known in the art. The individually addressable electrodes can be connected to one or more AC voltage sources via respective transistor switches. The cover 110 may further include a dielectric layer (not shown) juxtaposed with the hydrophobic layer 422 and a conductive layer (not shown) juxtaposed between the dielectric layer and the electrode 428. Accordingly, the microfluidic device 400 can have both the substrate 104 and the cover 110 configured to provide an electrowetting force to the aqueous droplets 440 located within the outer casing 435. In these embodiments, the dielectric layer of the cap 110 can be configured in any manner disclosed herein for the inner dielectric layer 414 of the substrate 104, and the conductive layer of the cap 104 can be disclosed herein for the conductive layer 126 of the substrate 102. Any way of configuration. As discussed herein, the microfluidic devices of the present invention can include portions having a DEP configuration. An example of this portion is the microfluidic device 200 illustrated in Figures 1C and 1D. Although for the sake of clarity, Figures 1C and 1D respectively show a vertical cross-sectional view and a horizontal cross-sectional view of a portion of the outer casing 102 of the microfluidic device 200 having the open region/chamber 202, it will be understood that the region/chamber 202 may have a more detailed structure. One of the fluid circuit elements of a growth chamber, a fence, a flow zone, or a flow channel. Additionally, the microfluidic device 200 can include other fluid circuit components. For example, the microfluidic device 200 can include a plurality of growth chambers or isolation fences and/or one or more flow regions or flow channels, such as those described herein for the microfluidic device 100. The DEP configuration can be incorporated into any of the fluid circuit elements of the microfluidic device 200 or selected portions thereof. It should be further appreciated that any of the above or below microfluidic device components and system components can be incorporated into and/or used in combination with the microfluidic device 200. For example, the system 150 including the above-described control and monitoring device 152 can be combined with one or more of the media module 160, the power module 162, the imaging module 164, the tilt module 166, and other modules 168. Device 200 is used together. As seen in FIG. 1C, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activating substrate 206 covering the bottom electrode 204, and a cover 110 having a top electrode 210, wherein the top electrode 210 is spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activating substrate 206 define opposing surfaces of the regions/chambers 202. Thus, the medium 180 contained in the zone/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activating substrate 206. A power source 212 is also shown that is configured to connect to the bottom electrode 204 and the top electrode 210 and create a bias voltage between the electrodes, as required to generate a DEP force in the zone/chamber 202. Power source 212 can be, for example, an alternating current (AC) power source. In certain embodiments, the microfluidic device 200 illustrated in Figures 1C and 1D can have an optically actuated DEP configuration. Thus, the varying pattern of light 218 from source 216 that can be controlled by power module 162 can selectively activate and deactivate the pattern of changes in the DEP electrode at region 214 of inner surface 208 of electrode activation substrate 206. (The region 214 of the microfluidic device having the DEP configuration is hereinafter referred to as the "DEP electrode region.") As illustrated in Figure ID, the light pattern 218 directed into the inner surface 208 of the electrode activating substrate 206 can be illuminated in a pattern. The DEP electrode region 214a (shown in white) is selected (e.g., square). The unexposed DEP electrode region 214 (cross hatching) is hereinafter referred to as the "dark" DEP electrode region 214. The relative electrical impedance across the DEP electrode activating substrate 206 (i.e., from the bottom electrode 204 up to the inner surface 208 of the electrode activating substrate 206, the inner surface being bounded by the medium 180 in the flow region 106) is greater than each dark DEP electrode region. The relative electrical impedance of the medium 180 in the traversing zone/chamber 202 (i.e., from the inner surface 208 of the electrode activating substrate 206 to the top electrode 210 of the cap 110). However, the illuminated DEP electrode region 214a exhibits a reduced relative impedance across the electrode activating substrate 206 that is less than the relative impedance of the medium 180 in the passing region/chamber 202 of each irradiated DEP electrode region 214a. Under activation power source 212, the DEP configuration creates an electric field gradient in fluid medium 180 between irradiated DEP electrode region 214a and adjacent dark DEP electrode region 214, which in turn creates attraction or repelling of nearby tiny objects in fluid medium 180. Partial DEP force (not shown). Thus, a small object in the fluid medium 180 can be attracted or repelled at a plurality of such different DEP electrode regions 214 of the inner surface 208 of the region/chamber 202 by changing the light pattern 218 projected from the light source 216 into the microfluidic device 200. The DEP electrode is selectively activated and deactivated. The ability of the DEP force to attract or repel nearby tiny objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or tiny objects (not shown). The square pattern 220 of the illuminated DEP electrode region 214a illustrated in Figure 1C is merely an example. Any pattern of DEP electrode regions 214 can be illuminated (and thereby activated) by a pattern of light 218 projected into device 200, and the irradiated/activated DEP electrode region can be repeatedly altered by changing or moving light pattern 218 214 pattern. In some embodiments, the electrode activating substrate 206 can comprise or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activating substrate 206 may be featureless. For example, the electrode active substrate 206 can comprise or consist of a hydrogenated amorphous germanium (a-Si:H) layer. a-Si:H may contain, for example, about 8% to 40% hydrogen (calculated based on the number of 100* hydrogen atoms/hydrogen and the total number of germanium atoms). The a-Si:H layer may have a thickness of from about 500 nm to about 2.0 μm. In such embodiments, depending on the light pattern 218, the DEP electrode region 214 can be created anywhere on the inner surface 208 of the electrode activating substrate 206 and in any pattern. Therefore, the number and pattern of the DEP electrode regions 214 need not be fixed, but may correspond to the light pattern 218. An example of a microfluidic device having a DEP configuration, such as the photoconductive layer discussed above, is described in, for example, U.S. Patent No. 4, 711, issued to U.S. Patent No. 7,612,355. The entire contents of this article are incorporated herein by reference. In other embodiments, the electrode active substrate 206 can comprise a plurality of substrates including, for example, doped layers, electrically insulating layers (or regions), and conductive layers known in the semiconductor art to form semiconductor integrated circuits. For example, electrode activation substrate 206 can comprise a plurality of optoelectronic crystals including, for example, lateral bipolar optoelectronic crystals, each photocrystal corresponding to DEP electrode region 214. Alternatively, the electrode activating substrate 206 can include electrodes (eg, conductive metal electrodes) controlled by a photodiode switch, wherein each of the electrodes corresponds to the DEP electrode region 214. Electrode activated substrate 206 can include a pattern of such optoelectronic crystals or electrodes controlled by the optoelectronic crystal. The pattern can be, for example, a substantially square photonic crystal or a photonic crystal controlled electrode array arranged in columns and rows, such as shown in Figure 2B. Alternatively, the pattern can be a substantially hexagonal photonic crystal or a photonic crystal controlled electrode array forming a hexagonal lattice. Regardless of the pattern, the circuit components can form an electrical connection between the DEP electrode region 214 and the bottom electrode 210 of the inner surface 208 of the electrode activating substrate 206, and can be electrically connected by a light pattern 218 (ie, a photonic crystal or electrode). ) selective activation and deactivation. When not activated, each electrical connection can have a high impedance such that the substrate 206 is activated through the electrode (ie, from the bottom electrode 204 to the inner surface 208 of the electrode activating substrate 206, the inner surface and the medium in the region/chamber 202 The relative impedance of the 180 interface is greater than the relative impedance of the corresponding DEP electrode region 214 through the dielectric 180 (ie, from the inner surface 208 of the electrode activating substrate 206 to the top electrode 210 of the cover 110). However, when activated by light in the light pattern 218, the relative impedance across the electrode activating substrate 206 is less than the relative impedance of each of the irradiated DEP electrode regions 214 through the medium 180, thereby activating the corresponding DEP electrode regions as discussed above. DEP electrode at 214. Thus, the DEP of a small object (not shown) in the medium 180 can be attracted or repelled at a plurality of different DEP electrode regions 214 of the inner surface 208 of the electrode activating substrate 206 in the region/chamber 202 by means of the light pattern 218. The electrode is selectively activated and deactivated. An example of a microfluidic device having an electrode-activated substrate comprising an optoelectronic crystal is described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) (see, for example, the device 300 illustrated in Figures 21 and 22 and its description). The entire contents of the patent are incorporated herein by reference. An example of a microfluidic device having an electrode-activated substrate comprising an electrode controlled by a photo-electric crystal switch is described, for example, in U.S. Patent Publication No. 2014/0124370 (Short et al.) (for example, see the entire disclosure of the drawings) The devices 200, 400, 500, 600, and 900, and the description thereof, are incorporated herein by reference in their entirety. In some embodiments of the DEP-configured microfluidic device, the top electrode 210 is part of the first wall (or cover 110) of the outer casing 102, and the electrode activating substrate 206 and the bottom electrode 204 are the second wall of the outer casing 102 (or A portion of the support structure 104). Zone/chamber 202 can be between the first wall and the second wall. In other embodiments, electrode 210 is part of a second wall (or support structure 104) and one or both of electrode activation substrate 206 and/or electrode 210 is part of a first wall (or cover 110). Additionally, light source 216 can alternatively be used to illuminate housing 102 from below. Using the microfluidic device 200 having the DEP configuration of FIGS. 1C-1D, the power module 162 can activate the electrodes in the pattern (eg, square pattern 220) surrounding and capturing the tiny objects by projecting the light pattern 218 into the device 200. A first group of one or more DEP electrodes of the DEP electrode region 214a of the inner surface 208 of the substrate 206 is activated to select a fine object (not shown) in the medium 180 in the region/chamber 202. Subsequently, the power module 162 can move the captured minute object to activate one of the DEP electrode regions 214 or a second group of the plurality of DEP electrodes by moving the light pattern 218 relative to the device 200. Alternatively, device 200 can be moved relative to light pattern 218. In other embodiments, the microfluidic device 200 can have a light activated DEP configuration that is independent of the DEP electrode of the inner surface 208 of the electrode activating substrate 206. For example, electrode activation substrate 206 can include electrodes that are selectively addressable and energizable with a location that includes a surface (eg, cover 110) relative to at least one electrode. The switch can be selectively turned on and off (eg, a transistor switch in a semiconductor substrate) to activate or deactivate the DEP electrode of the DEP electrode region 214, thereby activating tiny objects in the region/chamber 202 adjacent the DEP electrode (not Show) produces a net DEP force. Depending on such characteristics as the frequency of the power source 212 and the dielectric properties of the medium (not shown) and/or tiny objects in the zone/chamber 202, the DEP force can attract or repel nearby tiny objects. By selectively activating and deactivating the DEP electrode sets (e.g., the set of DEP electrode regions 214 forming the square pattern 220), one or more of the micro-objects in the zone/chamber 202 can be captured and placed in the zone/ The chamber 202 moves within. The power module 162 of FIG. 1A can control the switches and thereby activate and deactivate individual instances of the DEP electrodes to select, capture, and move specific small objects (not shown) around the zone/chamber 202. Microfluidic devices having a DEP configuration comprising selectively addressable and energizable electrodes are known in the art and are described, for example, in U.S. Patent Nos. 6,294,063 (Becker et al.) and 6,942,776 (Medoro), which are incorporated herein by reference. All content is incorporated herein by reference.Isolation fence . Non-limiting examples of general isolation fences 224, 226, and 228 are shown in the microfluidic device 230 depicted in Figures 2A-2C. Each of the isolation fences 224, 226, and 228 can include a separation structure 232 that defines the separation zone 240 and a connection zone 236 that fluidly connects the separation zone 240 to the channel 122. The attachment zone 236 can include a proximal opening 234 of the channel 122 and a distal opening 238 of the separation zone 240. The attachment zone 236 can be configured such that the maximum penetration depth of the fluid medium flow (not shown) from the passage 122 into the isolation fences 224, 226, 228 does not extend into the separation zone 240. Thus, due to the connection zone 236, tiny objects (not shown) or other materials (not shown) disposed in the separation zone 240 of the isolation fences 224, 226, 228 can thus be separated from the flow of the medium 180 in the channel 122 and It is virtually unaffected by it. The isolation fences 224, 226, and 228 of Figures 2A-2C each have a single opening that is open directly toward the channel 122. The opening of the isolation fence is open laterally from the passage 122. The electrode activation substrate 206 is located below the channel 122 and the isolation fences 224, 226, and 228. An upper surface of the electrode activating substrate 206 forming a bottom plate of the isolation fence in the outer casing of the isolation fence is disposed in a flow channel (or a separate flow region) of the microfluidic device formed in the channel 122 (or a flow region if no channel exists) The electrodes of the bottom plate activate the same level or substantially the same level as the upper surface of the substrate 206. The electrode activating substrate 206 may be featureless or may have a variation from its highest height to its lowest depression of less than about 3 microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns. Or smaller or random or patterned surfaces. The height change of the surface above the substrate across the channel 122 (or flow zone) and the isolation fence may be less than about 3%, 2%, 1%, 0.9%, 0.8% of the height of the wall of the barrier fence or the wall of the microfluidic device, 0.5%, 0.3% or 0.1%. Although described in detail for microfluidic device 200, this also applies to any of the microfluidic devices 100, 230, 250, 280, 290, 600, 700 described herein. Thus, channel 122 can be an example of a flooding zone, and separation zone 240 of isolation fences 224, 226, 228 can be an example of an unaffected zone. As noted, the channel 122 and the isolation fences 224, 226, 228 can be configured to contain one or more fluid media 180. In the example shown in FIGS. 2A-2B, the crucible 222 is coupled to the channel 122 and allows the fluid medium 180 to be introduced into or removed from the microfluidic device 230. Prior to introduction of the fluid medium 180, the microfluidic device can be pretreated with a gas such as carbon dioxide gas. Once the microfluidic device 230 contains the fluid medium 180, the flow 242 of the fluid medium 180 in the channel 122 can be selectively generated and terminated immediately. For example, as shown, the turns 222 can be disposed at different locations (eg, opposite ends) of the channel 122, and the flow 242 of the media can be generated from one turn 222 that serves as an inlet to another turn 222 that serves as an outlet. 2C illustrates a detailed view of an example of the isolation fence 224 of the present invention. Examples of tiny objects 246 are also shown. As is known, the flow 242 of the fluid medium 180 in the microfluidic channel 122 in the proximal opening 234 of the isolation fence 224 can cause the medium 180 to flow 244 into and/or out of the isolation fence 224. In order to protect the minute object 246 in the separation zone 240 of the isolation fence 224 from the secondary flow 244, the length L of the connection zone 236 of the isolation fence 224_Con (ie, from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth D of the secondary flow 244 entering the connection zone 236._p . Penetration depth D of secondary flow 244_p Depending on the speed of the fluid medium 180 flowing in the passage 122 and various parameters associated with the configuration of the passage 122 and the proximal opening 234 of the connection region 236 of the passage 122. For a given microfluidic device, the configuration of channel 122 and opening 234 will be fixed, while the rate of flow 242 of fluid medium 180 in channel 122 will be variable. Thus, for each isolation fence 224, the maximum velocity V of the flow 242 of the fluid medium 180 in the channel 122 can be identified._Max To ensure the penetration depth D of the secondary flow 244_p Does not exceed the length L of the connection area 236_Con . As long as the rate of flow 242 of fluid medium 180 in passage 122 does not exceed the maximum speed V_Max The resulting secondary flow 244 can be limited to the channel 122 and the connection region 236 and away from the separation region 240. Thus, the flow 242 of the medium 180 in the channel 122 will not draw the tiny object 246 from the separation zone 240. Conversely, the tiny objects 246 located in the separation zone 240 will remain in the separation zone 240 regardless of the flow 242 of the fluid medium 180 in the channel 122. Moreover, as long as the velocity of the flow 242 of the medium 180 in the channel 122 does not exceed V_Max The flow 242 of the fluid medium 180 in the channel 122 does not move other particles (eg, particulates and/or nanoparticles) from the channel 122 into the separation zone 240 of the isolation fence 224. Therefore, the length L of the connection region 236 is made_Con Greater than the maximum penetration depth D of the secondary flow 244_p One isolation fence 224 can be prevented from being contaminated by other particles from the passage 122 or another isolation fence (e.g., the isolation fences 226, 228 in Figure 2D). Since the connection region 236 of the channel 122 and the isolation fences 224, 226, 228 can be affected by the flow 242 of the medium 180 in the channel 122, the channel 122 and the connection region 236 can be considered as the wave (or flow) region of the microfluidic device 230. Alternatively, the separation zone 240 of the isolation fences 224, 226, 228 can be considered as an unaffected (or non-flowing) zone. For example, a component (not shown) of the first fluid medium 180 in the channel 122 can diffuse from the channel 122 through the connection region 236 and into the second of the separation region 240 substantially only by the components of the first medium 180. The fluid medium 248 is mixed with the second fluid medium 248 in the separation zone 240. Similarly, a component (not shown) of the second medium 248 in the separation zone 240 can diffuse through the connection zone 236 and into the first medium in the channel 122 by substantially only the components of the second medium 248 from the separation zone 240. 180 is mixed with the first medium 180 in the channel 122. In some embodiments, the degree of fluid exchange between the separation zone and the flow zone of the isolation fence by diffusion is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater than about 99% fluid exchange. The first medium 180 can be the same medium as the second medium 248 or a different medium. Moreover, the first medium 180 and the second medium 248 may be intended to be the same at the beginning, and then become different (eg, via the one or more unit conditions in the separation zone 240 to process the second medium 248 or by changing the flow path) 122 medium 180). Maximum penetration depth D of secondary flow 244 caused by flow 242 of fluid medium 180 in passage 122_p It may depend on a number of parameters, as mentioned above. Examples of such parameters include the shape of the channel 122 (eg, the channel can direct the media into the connection region 236, deflecting the media away from the connection region 236, or in a direction substantially perpendicular to the proximal opening 234 of the connection region 236 Directing the media to the channel 122); the width W of the channel 122 at the proximal opening 234_Ch (or sectional area); and the width W of the connection area 236 at the proximal opening 234_Con (or sectional area); velocity V of flow 242 of fluid medium 180 in channel 122; viscosity of first medium 180 and/or second medium 248, or the like. In some embodiments, the dimensions of the channel 122 and the isolation fences 224, 226, 228 can be oriented relative to the vector of the flow 242 of the fluid medium 180 in the channel 122 as follows: channel width W_Ch (or the cross-sectional area of the channel 122) may be substantially perpendicular to the flow 242 of the medium 180; the width W of the connection region 236 at the opening 234_Con (or the cross-sectional area) may be substantially parallel to the flow 242 of the medium 180 in the channel 122; and/or the length L of the connection region_Con The flow 242 of the medium 180 in the channel 122 can be substantially perpendicular. The foregoing is merely an example, and the relative positions of the channels 122 and the isolation fences 224, 226, 228 may be otherwise oriented relative to one another. Width W of the connection region 236 as illustrated in Figure 2C_Con The outer opening 234 can be uniform from the proximal opening 234. Thus, the width W of the connection region 236 at the distal opening 238_Con This may be the width W of the connection region 236 at the proximal opening 234 herein._Con Any of the scope of identification. Alternatively, the width W of the connection region 236 at the distal opening 238_Con Can be greater than the width W of the connection region 236 at the proximal opening 234_Con . As illustrated in FIG. 2C, the width of the separation zone 240 at the distal opening 238 can be substantially the width of the attachment zone 236 at the proximal opening 234._Con the same. Thus, the width of the separation zone 240 at the distal opening 238 can be the width W of the attachment zone 236 herein for the proximal opening 234._Con Any of the scope of identification. Alternatively, the width of the separation zone 240 at the distal opening 238 may be greater or less than the width of the attachment zone 236 at the proximal opening 234._Con . Additionally, the distal opening 238 can be smaller than the proximal opening 234 and the width of the connecting region 236._Con It can be narrowed between the proximal opening 234 and the distal opening 238. For example, the attachment region 236 can be narrowed between the proximal opening and the distal opening using a variety of different geometries (eg, decoupling the connecting region, beveling the connecting region). Moreover, any portion or sub-portion of the connection region 236 can be narrow (eg, a portion of the connection region adjacent the proximal opening 234). 2D-2F illustrate another exemplary embodiment of a microfluidic device 250 including a microfluidic circuit 262 and a flow channel 264, which are variations of the respective microfluidic device 100, circuit 132, and channel 134 of FIG. The microfluidic device 250 also has a plurality of isolation fences 266 that are other variations of the isolation fences 124, 126, 128, 130, 224, 226 or 228 described above. In particular, it should be appreciated that the isolation fence 266 of the device 250 illustrated in Figures 2D-2F can be substituted for the isolation fences 124, 126, 128, 130, 224 of the device 100, 200, 230, 280, 290 or 320, Any of 226 or 228. Likewise, the microfluidic device 250 is another variation of the microfluidic device 100 and can also have any of the microfluidic devices 100, 200, 230, 280, 290, 320 described above, as well as other microfluidic system components described herein. The same or different DEP configurations. The microfluidic device 250 of Figures 2D-2F includes a support structure (not visible in Figures 2D-2F, but may be identical or substantially similar to the support structure 104 of the device 100 depicted in Figure 1A), a microfluidic loop structure 256 And the cover (not visible in Figures 2D-2F, but may be the same or substantially similar to the cover 122 of the device 100 depicted in Figure 1A). The microfluidic loop structure 256 includes a frame 252 and a microfluidic loop material 260 that may be the same or substantially similar to the frame 114 of the device 100 and the microfluidic loop material 116 shown in FIG. 1A. As shown in FIG. 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 can include a plurality of channels 264 (two shown, but more can be present) with a plurality of isolation fences 266 fluidly coupled thereto. Each isolation fence 266 can include a separation structure 272, a separation zone 270 within the separation structure 272, and a connection zone 268. From the proximal opening 274 of the passage 264 to the distal opening 276 at the separation structure 272, the connection region 268 fluidly connects the passage 264 to the separation zone 270. In general, according to the discussion of FIGS. 2B and 2C above, the flow 278 of the first fluid medium 254 in the channel 264 can create a first medium 254 from the secondary flow 282 of the channel 264 to the respective connection region 268 of the isolation fence 266 and/or From it. As illustrated in FIG. 2E, the attachment zone 268 of each barrier fence 266 generally includes an area extending between the proximal opening 274 of the channel 264 and the distal opening 276 of the separation structure 272. Length L of connection zone 268_Con Can be greater than the maximum penetration depth D of the secondary flow 282_p In this case, the secondary flow 282 will extend into the connection zone 268 without redirecting towards the separation zone 270 (as shown in Figure 2D). Alternatively, as illustrated in Figure 2F, the connection zone 268 can have a depth less than the maximum penetration depth D_p Length L_Con In this case, the secondary flow 282 will extend through the connection zone 268 and be redirected toward the separation zone 270. In this latter case, the length L of the connection zone 268_C1 And L_C2 The sum is greater than the maximum penetration depth D_p So that the secondary flow 282 will not extend into the separation zone 270. Regardless of the length L of the connection zone 268_Con Greater than penetration depth D_p Or the length of the connection zone 268 L_C1 and_Lc2 The sum is greater than the penetration depth D_p , does not exceed the maximum speed V_Max The flow 278 of the first medium 254 in the channel 264 will produce a depth of penetration D_p The second flow, and the tiny objects in the separation zone 270 of the isolation fence 266 (not shown, but may be the same or substantially similar to the tiny objects 246 shown in FIG. 2C) will not pass through the first medium 254 in the channel 264. The flow 278 is withdrawn from the separation zone 270. Flow 278 in passage 264 also does not draw other material (not shown) from passage 264 into separation zone 270 of isolation fence 266. Thus, the component of the first medium 254 in the diffusion system channel 264 can be moved from the channel 264 to the only mechanism in the second medium 258 in the separation zone 270 of the isolation fence 266. Likewise, the only mechanism in which the components of the second medium 258 in the separation zone 270 of the diffusion isolation fence 266 can move from the separation zone 270 to the first medium 254 in the channel 264. The first medium 254 can be the same medium as the second medium 258, or the first medium 254 can be a different medium than the second medium 258. Alternatively, the first medium 254 and the second medium 258 may be intended to be the same at the beginning, and then become different, for example, by processing the second medium by one or more unit conditions in the separation area 270 or by changing the flow path 264. Medium. Width W of channel 264 in channel 264 as illustrated in Figure 2E_Ch (i.e., taken in the direction of the fluid medium flowing through the channel as indicated by arrow 278 in Figure 2D) may be substantially perpendicular to the width of the proximal opening 274._Con1 And thus substantially parallel to the width W of the distal opening 276_Con2 . However, the width of the proximal opening 274 is W_Con1 Width W with the distal opening 276_Con2 It is not necessary to be substantially perpendicular to each other. For example, the width of the proximal opening 274 is W_Con1 Alignment axis (not shown) and width of distal opening 276 W_Con2 The angle between the other axis of the orientation may not be vertical and therefore not 90°. Examples of alternating orientation angles include the angles of any of the following ranges: from about 30° to about 90°, from about 45° to about 90°, from about 60° to about 90°, or the like. In various embodiments of the isolation fence (eg, 124, 126, 128, 130, 224, 226, 228, or 266), the separation zone (eg, 240 or 270) is configured to contain a plurality of tiny objects. In other embodiments, the separation zone can be configured to contain only 1, 2, 3, 4, 5, or a relatively small number of relatively small objects. Therefore, the volume of the separation zone can be, for example, at least 1×10⁶ Cubic micron, 2 x 10⁶ Cubic micron, 4×10⁶ Cubic micron, 6×10⁶ Cubic micrometers or larger. In various embodiments of the isolation fence, the width (eg, 122) of the channel (eg, 122) at the proximal opening (eg, 234)_Ch Can be in any of the following ranges: about 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 Micron, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 Micron, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120 microns. In some other embodiments, the width (eg, 122) of the channel (eg, 122) at the proximal opening (eg, 234)_Ch It can be in the range of about 200-800 microns, 200-700 microns, or 200-600 microns. The above is only an example, and the width of the channel 122 is W_Ch It may be within other ranges (eg, the range defined by any of the endpoints listed above). In addition, the channel 122 is W_Ch It can be selected to be in any of the ranges of the channel regions other than the proximal opening of the isolation fence. In some embodiments, the barrier fence has a height of from about 30 microns to about 200 microns, or from about 50 microns to about 150 microns. In some embodiments, the isolation fence has approximately 1 x 10⁴ - 3 × 10⁶ Square micron, 2 × 10⁴ - 2 × 10⁶ Square micron, 4 × 10⁴ - 1 × 10⁶ Square micron, 2 × 10⁴ - 5 × 10⁵ Square micron, 2 × 10⁴ - 1 × 10⁵ Square micron or about 2 × 10⁵ - 2×10⁶ The area of the square micrometer. In some embodiments, the tie region has a cross-sectional width of from about 100 microns to about 500 microns, from 200 microns to about 400 microns, or from about 200 microns to about 300 microns. In various embodiments of the isolation fence, the height H of the channel (eg, 122) at the proximal opening (eg, 234)_Ch Can be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 Micron. The above is only an example, and the height of the channel (for example, 122) H_Ch It may be within other ranges (eg, the range defined by any of the endpoints listed above). Height H of channel 122_Ch It can be selected to be in any of the ranges of the channel regions other than the proximal opening of the isolation fence. In various embodiments of the barrier fence, the cross-sectional area of the channel (e.g., 122) at the proximal opening (e.g., 234) can be within any of the following ranges: 500-50,000 square microns, 500-40,000 square microns, 500- 30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 squares Micron, 1,000-15,000 square micrometers, 1,000-10,000 square micrometers, 1,000-7,500 square micrometers, 1,000-5,000 square micrometers, 2,000-20,000 square micrometers, 2,000-15,000 square micrometers, 2,000-10,000 square micrometers, 2,000-7,500 square micrometers, 2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000 square microns to 6,000 square microns. The foregoing is merely an example, and the cross-sectional area of the channel (e.g., 122) at the proximal opening (e.g., 234) may be within other ranges (e.g., the range defined by any of the endpoints listed above). In various embodiments of the isolation fence, the length L of the connection zone (e.g., 236)_Con It can be in any of the following ranges: about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60- 300 microns, 80-200 microns, or about 100-150 microns. The above is only an example, and the length L of the connection area (for example, 236)_Con It may be within a range different from the above examples (eg, a range defined by any of the endpoints listed above). In various embodiments of the barrier fence, the width of the attachment zone (e.g., 236) at the proximal opening (e.g., 234)_Con Can be in any of the following ranges: 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 Micron, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 Micron, 40-100 microns, 40-80 microns, 40-60 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-200 microns, 60-150 Micron, 60-100 microns, 60-80 microns, 70-150 microns, 70-100 microns and 80-100 microns. The foregoing is merely an example, and the width of the connection region (eg, 236) at the proximal opening (eg, 234) is W_Con It may differ from the above examples (eg, the range defined by any of the endpoints listed above). In various embodiments of the barrier fence, the width of the attachment zone (e.g., 236) at the proximal opening (e.g., 234)_Con It may be at least as large as the largest size of the tiny object (e.g., biological cells, which may be T cells, B cells, or eggs or embryos) that the isolation fence is intended to use. For example, the width W of the connection region 236 at the proximal opening 234 of the isolation fence in which the droplet is placed_Con May be in any of the following ranges: about 100 microns, about 110 microns, about 120 microns, about 130 microns, about 140 microns, about 150 microns, about 160 microns, about 170 microns, about 180 microns, about 190 Micron, about 200 microns, about 225 microns, about 250 microns, about 300 microns or about 100-400 microns, about 120-350 microns, about 140-200-200 300 microns, or about 140-200 microns. The foregoing is merely an example, and the width of the connection region (eg, 236) at the proximal opening (eg, 234) is W_Con It may differ from the above examples (eg, the range defined by any of the endpoints listed above). In various embodiments of the isolation fence, the width of the proximal opening of the attachment zone is W_Pr It may be at least as large as the largest size of a small object (eg, a biological tiny object, such as a cell) that the isolation fence is intended to use. For example, width W_Pr Can be about 50 microns, about 60 microns, about 100 microns, about 200 microns, about 300 microns, or can be between about 50-300 microns, about 50-200 microns, about 50-100 microns, about 75-150 microns, about In various embodiments of the barrier fence in the range of 75-100 microns, or about 200-300 microns, the length L of the attachment zone (eg, 236) at the proximal opening 234_Con Width to the connection area (eg 236) W_Con The ratio can be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or greater. The foregoing is merely an example, and the length L of the connection region 236 at the proximal opening 234_Con The width W of the connection area 236_Con The ratio can be different from the above examples. In various embodiments of the microfluidic device 100, 200, 230, 250, 280, 290, 320, 600, 700, V_Max Can be set to approximately 0.2 μL/sec, 0.3 μL/sec, 0.4 μL/sec, 0.5 μL/sec, 0.6 μL/sec, 0.7 μL/sec, 0.8 μL/sec, 0.9 μL/sec, 1.0 μL/sec, 1.1 μL/sec, 1.2 μL/sec, 1.3 μL/sec, 1.4 μL/sec or 1.5 μL/sec. In various embodiments of the microfluidic device having a barrier fence, the volume of the separation zone (e.g., 240) of the barrier fence can be, for example, at least 5 x 10⁵ Cubic micron, 8×10⁵ Cubic micron, 1×10⁶ Cubic micron, 2 x 10⁶ Cubic micron, 4×10⁶ Cubic micron, 6×10⁶ Cubic micron, 8×10⁶ Cubic micron, 1×10⁷ Cubic micron, 5×10⁷ Cubic micron, 1×10⁸ Cubic micron, 5×10⁸ Cubic micron or 8 x 10⁸ Cubic micrometers or larger. In various embodiments of a microfluidic device having a barrier fence, the volume of the barrier fence can be about 5 x 10⁵ Cubic micron, 6×10⁵ Cubic micron, 8×10⁵ Cubic micron, 1×10⁶ Cubic micron, 2 x 10⁶ Cubic micron, 4×10⁶ Cubic micron, 8×10⁶ Cubic micron, 1×10⁷ Cubic micron, 3×10⁷ Cubic micron, 5×10⁷ Cubic micron or about 8×10⁷ Cubic micrometers or larger. In some other embodiments, the volume of the isolation fence may range from about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, and about 2 nanoliters to about 15 nanoliters. Or about 2 nanoliters to about 10 nanoliters. In various embodiments, the microfluidic device has an isolation fence configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 5 to about 10 isolation fences, from about 10 to about 50 Separating fences, from about 100 to about 500 isolation fences; from about 200 to about 1000 isolation fences, from about 500 to about 1500 isolation fences, from about 1000 to about 2,000 isolation fences or from about 1000 to about 3500 An isolation fence. The isolation fences need not all be the same size and can include a variety of configurations (eg, different widths, different features within the isolation fence). In some other embodiments, the microfluidic device has an isolation fence configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 1500 to about 3000 isolation barriers, from about 2000 to about 3,500 isolation fences, from about 2,500 to about 4,000 isolation fences, from about 3,000 to about 4,500 isolation fences, from about 3,500 to about 5,000 isolation fences, from about 4,000 to about 5,500 isolation fences, from about 4,500 to about 6,000 Separation fences, from about 5,000 to about 6,500 isolation fences, from about 5,500 to about 7,000 isolation fences, from about 6,000 to about 7,500 isolation fences, from about 6,500 to about 8,000 isolation fences, from about 7,000 to about 8,500 Isolated fences, from about 7,500 to about 9,000 barrier fences, from about 8,000 to about 9,500 barrier fences, from about 8,500 to about 10,000 barrier fences, from about 9,000 to about 10,500 barrier fences, from about 9,500 to about 11,000 Separation fences, from about 10,000 to about 11,500 isolation fences, from about 10,500 to about 12,000 isolation fences, from about 11,000 to about 12,500 isolation fences, from about 11,500 to about 13,000 isolation fences, from about 12,000 to about 13,500 Isolated Fences, from about 12,500 to about 14,000 isolation fences, from about 13,000 to about 14,500 isolation fences, from about 13,500 to about 15,000 isolation fences, from about 14,000 to about 15,500 isolation fences, from about 14,500 to about 16,000 isolations Fences, from about 15,000 to about 16,500 isolation fences, from about 15,500 to about 17,000 isolation fences, from about 16,000 to about 17,500 isolation fences, from about 16,500 to about 18,000 isolation fences, from about 17,000 to about 18,500 isolations Fences, from about 17,500 to about 19,000 barrier fences, from about 18,000 to about 19,500 barrier fences, from about 18,500 to about 20,000 barrier fences, from about 19,000 to about 20,500 fences, from about 19,500 to about 21,000 isolations Fence, or about 20,000 to about 21,500 isolation fences. FIG. 2G illustrates a microfluidic device 280 of an embodiment. The microfluidic device 280 illustrated in Figure 2G is a stylized diagram of the microfluidic device 100. In fact, the microfluidic device 280 and its constituent loop elements (e.g., channel 122 and isolation fence 128) will have the dimensions discussed herein. The microfluidic circuit 120 illustrated in Figure 2G has two turns 107 and a flow zone 106 having four different channels 122. The microfluidic device 280 further includes a plurality of isolation fences that are open from each of the channels 122. In the microfluidic device illustrated in Figure 2G, the barrier fence has a geometry similar to the fence illustrated in Figure 2C, and thus has both a junction zone and a separation zone. Thus, the microfluidic circuit 120 includes a sweeping zone (eg, the maximum penetration depth D of the secondary flow 244 of the channel 122 and the junction zone 236_p The inner part) and the non-fluctuating area (for example, the separation zone 240 and the connection zone 236 are not at the maximum penetration depth D of the secondary flow 244_p Both of the connection areas 236)). 3A-3B show various embodiments of system 150 that can be used to operate and view the microfluidic devices (e.g., 100, 200, 230, 280, 250, 290, 320) of the present invention. As illustrated in FIG. 3A, system 150 can include a structure ("nested") 300 configured to house microfluidic device 100 (not shown) or any other microfluidic device described herein. The nest 300 can include a receptacle 302 that can interface with the microfluidic device 320 (e.g., optically actuated electrodynamic device 100) and provide electrical connection of the power source 192 to the microfluidic device 320. The nest 300 can further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 can be configured to supply a bias voltage to the receptacle 302 such that when the microfluidic device 320 is held by the receptacle 302, a bias voltage is applied across the pair of electrodes in the microfluidic device 320. Thus, electrical signal generation subsystem 304 can be part of power supply 192. The ability to apply a bias voltage to the microfluidic device 320 does not imply that a bias voltage is always applied when the microfluidic device 320 is held by the receptacle 302. Conversely, in most cases, the bias voltage is applied intermittently, for example, only in accordance with facilitating the generation of electrical power (eg, dielectrophoresis or electrowetting) in the microfluidic device 320. As illustrated in FIG. 3A, the nest 300 can include a printed circuit board assembly (PCBA) 322. Electrical signal generation subsystem 304 can be mounted on PCBA 322 and electroformed into it. The example support also includes a receptacle 302 that is mounted to the PCBA 322. Typically, electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 can further include an oscilloscope (not shown) and/or a waveform amplification loop (not shown) configured to amplify the waveform received from the waveform generator. The oscilloscope, if present, can be configured to measure the waveform supplied to the microfluidic device 320 held by the receptacle 302. In some embodiments, the oscilloscope measures the waveform at the location of the proximal end of the microfluidic device 320 (and at the distal end of the waveform generator), thereby ensuring a greater accuracy of the waveform actually applied to the device. Data obtained from oscilloscope measurements can be provided, for example, as feedback from a waveform generator, and the waveform generator can adjust its output based on the feedback configuration. An example of a suitable combination of waveform generators and oscilloscopes is Red PitayaTM. In some embodiments, the nest 300 further includes a controller 308, such as a microprocessor for sensing and/or controlling the electrical signal generation subsystem 304. Examples of suitable microprocessors include ArduinoTM microprocessors such as the Arduino NanoTM. Controller 308 can be used to implement functions and analysis or can be in communication with external host controller 154 (shown in Figure 1A) to perform functions and analysis. In the embodiment illustrated in FIG. 3A, controller 308 is in communication with host controller 154 via interface 310 (eg, a plug or connector). In some embodiments, the nest 300 can include an electrical signal generation subsystem 304 that includes a Red PitayaTM waveform generator/oscilloscope unit ("Red Pitaya Unit") and amplifies the waveform generated by the Red Pitaya unit and passes the amplified voltage through the micro A waveform amplification loop of the fluidic device 100. In some embodiments, the Red Pitaya unit is configured to measure the amplified voltage of the microfluidic device 320, and then adjust its own output voltage as needed such that the measured voltage of the microfluidic device 320 is the desired value. In some embodiments, the waveform amplification loop can have a +6.5V to -6.5V power supply generated by a DC-DC converter pair mounted on PCBA 322 to produce a signal of up to 13 Vpp in microfluidic device 100. As illustrated in FIG. 3A, the support structure 300 can further include a thermal control subsystem 306. Thermal control subsystem 306 can be configured to adjust the temperature of microfluidic device 320 held by support structure 300. For example, thermal control subsystem 306 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device can have a first surface that is configured to interface with at least one surface of the microfluidic device 320. The cooling unit can be, for example, a cooling block (not shown), such as a liquid cooled aluminum block. The second surface of the Peltier thermoelectric device (e.g., the surface opposite the first surface) can be configured to interface with the surface of the cooling block. The cooling block can be coupled to a fluid path 314 that is configured to circulate the cooled fluid through the cooling block. In the embodiment illustrated in FIG. 3A, the support structure 300 includes an inlet 316 and an outlet 318 to receive a cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluid path 314, and pass through the cooling block. The cooled fluid is then returned to the external reservoir. In some embodiments, the Peltier thermoelectric device, cooling unit, and/or fluid path 314 can be mounted to the casing 312 of the support structure 300. In some embodiments, thermal control subsystem 306 is configured to adjust the temperature of the Peltier thermoelectric device to achieve a target temperature of microfluidic device 320. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power source such as PololuTM Thermoelectric Power Supply (Pololu Robotics and Electronics Corp.). Thermal control subsystem 306 can include a feedback loop, such as a temperature value provided by an analog loop. Alternatively, the feedback loop can be provided by a digital loop. In some embodiments, the nest 300 can include a thermal control subsystem 306 having a feedback loop that is analogous to a voltage divider loop (not shown) that includes a resistor (eg, a resistance of 1 kohm +/- 0.1) %, temperature coefficient +/- 0.02 ppm/C0) and NTC thermistor (for example, nominal resistance is 1 kohm +/- 0.01%). In some cases, thermal control subsystem 306 measures the voltage from the feedback loop and then uses the calculated temperature value as an input to the on-board PID control loop algorithm. The output of the PID control loop algorithm can drive a directional and pulse-width-adjusted signal pin on, for example, a PololuTM electric drive (not shown) to actuate the thermoelectric source, thereby controlling the Peltier thermoelectric device. The nest 300 can include a serial port 324 that allows the microprocessor of the controller 308 to communicate with the external host controller 154 via an interface 310 (not shown). Additionally, the microprocessor of controller 308 can be in communication with electrical signal generation subsystem 304 and thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, via the combination of controller 308, interface 310, and serial port 324, electrical signal generation subsystem 304 and thermal control subsystem 306 can be in communication with external host controller 154. In this manner, the main controller 154 can assist the electrical signal generation subsystem 304, inter alia, by implementing scaling calculations for output voltage regulation. A graphical user interface (GUI) (not shown) provided via display device 170 coupled to external host controller 154 can be configured to plot the temperature and waveform obtained from thermal control subsystem 306 and electrical signal generation subsystem 304, respectively. data. Alternatively or additionally, the GUI may allow for updates to controller 308, thermal control subsystem 306, and electrical signal generation subsystem 304. As discussed above, system 150 can include imaging device 194. In some embodiments, imaging device 194 includes light conditioning subsystem 330 (see Figure 3B). Light conditioning subsystem 330 can include a digital micromirror device (DMD) or a micro-goggle array system (MSA), any of which can be configured to receive light from source 332 and pass a subset of the received light to the microscope 350 in the optical component string. Alternatively, light conditioning subsystem 330 may include devices that generate light by themselves (and thus do not require light source 332), such as organic light emitting diode displays (OLEDs), liquid crystal on silicon (LCOS) devices, and ferroelectric liquid crystal devices (FLCOS). Or a transmissive liquid crystal display (LCD). Light conditioning subsystem 330 can be, for example, a projector. Thus, light conditioning subsystem 330 can be capable of emitting both structured and unstructured light. One example of a suitable light conditioning subsystem 330 is the MosaicTM system from Andor TechnologiesTM. In some embodiments, imaging module 164 and/or power module 162 of system 150 can control light conditioning subsystem 330. In some embodiments, imaging device 194 further includes a microscope 350. In such embodiments, the nest 300 and light conditioning subsystem 330 can be individually configured to be mounted to the microscope 350. Microscope 350 can be, for example, a standard research grade light microscope or a fluorescence microscope. Thus, the nest 300 can be configured to be mounted on the stage 344 of the microscope 350 and/or the light conditioning subsystem 330 can be configured to be mounted on the top of the microscope 350. In other embodiments, the nest 300 and light conditioning subsystem 330 described herein can be an integrated assembly of the microscope 350. In some embodiments, microscope 350 can further include one or more detectors 348. In some embodiments, detector 348 is controlled by imaging module 164. Detector 348 can include an eyepiece, a charge coupled device (CCD), a camera (eg, a digital camera), or any combination thereof. If there are at least two detectors 348, one detector can be, for example, a high frame rate camera and the other detector can be a high sensitivity camera. Additionally, microscope 350 can include a string of optical elements configured to receive light reflected and/or emitted from microfluidic device 320 and focus at least a portion of the reflected and/or emitted light onto one or more detectors 348 . The optical element string of the microscope can also include different tube lenses (not shown) for different detectors such that the final magnification on each detector can be different. In some embodiments, imaging device 194 is configured to use at least two light sources. For example, the first source 332 can be used to generate structured light (eg, via the light conditioning subsystem 330), and the second source 334 can be used to provide unstructured light. The first source 332 can generate structured light for optically controlled control and/or fluorescence excitation, and the second source 334 can be used to provide bright field illumination. In such embodiments, power module 164 can be used to control first light source 332 and imaging module 164 can be used to control second light source 334. The string of optical elements of microscope 350 can be configured to (1) receive structured light from light conditioning subsystem 330 and focus the structured light when the microfluidic device (eg, an optically actuated electrodynamic device) is held by nest 300 On at least a first region of the device, and (2) receiving light reflected and/or emitted from the microfluidic device and focusing at least a portion of the reflected and/or emitted light onto the detector 348. The string of optical elements can be further configured to receive unstructured light from the second source and focus the unstructured light onto at least a second region of the microfluidic device while the device is held by the nest 300. In some embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first zone can be a subset of the second zone. In FIG. 3B, a first light source 332 that supplies light to a light conditioning subsystem 330 is shown that provides structured light to a string of optical elements of a microscope 350 of system 355 (not shown). A second source 334 that provides unstructured light to the string of optical elements via beam splitter 336 is shown. The structured light from the light conditioning subsystem 330 and the unstructured light from the second source 334 are serially passed from the beam splitter 336 through the optical element to the second beam splitter (or dichroic filter 338, This end depends on the light provided by light conditioning subsystem 330, where light is reflected down through objective lens 336 to sample plane 342. Subsequently, light reflected and/or emitted from the sample plane 342 travels back up through the objective lens 340, through the beam splitter and/or dichroic color filter 338, and to the dichroic color filter 346. Only a portion of the light that reaches the dichroic color filter 346 passes through and reaches the detector 348. In some embodiments, the second source 334 emits blue light. With a suitable dichroic color filter 346, blue light reflected from the sample plane 342 can pass through the dichroic color filter 346 and reach the detector 348. In contrast, structured light from light conditioning subsystem 330 reflects from sample plane 342 but does not pass through dichroic filter 346. In this example, dichroic filter 346 filters visible light having a wavelength longer than 495 nm. If the light emitted from the light conditioning subsystem does not include any wavelength shorter than 495 nm, then the filtering of light from the light conditioning subsystem 330 will be only complete (as shown). In fact, if the light from the light conditioning subsystem 330 includes a wavelength that is shorter than 495 nm (eg, a blue wavelength), some of the light from the light conditioning subsystem will pass through the color filter 346 to reach the detector 348. In this embodiment, color filter 346 is used to vary the balance between the amount of light from first source 332 and second source 334 to detector 348. This may be beneficial if the first source 332 is significantly stronger than the second source 334. In other embodiments, the second source 334 can emit red light, and the dichroic filter 346 can filter out visible light other than red light (eg, visible light having a wavelength shorter than 650 nm).Surface Modification . The surface of the materials, devices, and/or devices used to manipulate and store the biological material may have natural properties that are not optimized for short-term and/or long-term contact with the material, and may include, but are not limited to, tiny objects ( These include, but are not limited to, biological microscopic objects, such as biological cells, biomolecules, biomolecules, or fragments of biological microscopic objects, and any combination thereof. It can be used to modify one or more surfaces of a material, device or device to reduce undesirable phenomena associated with one or more natural surfaces in contact with one or more biological materials. In other embodiments, it can be used to enhance the surface properties of materials, devices, and/or devices to introduce desired features into the surface, thereby broadening the handling, handling, or handling capabilities of the materials, devices, and/or devices. To this end, there is a need in the industry for molecules that can modify surfaces to reduce undesirable properties or introduce desirable properties.a compound that can be used to modify the surface . In various embodiments, the surface modifying compound can include a surface modifying ligand that can be a covalently modified non-polymeric moiety of the surface to which it is attached, such as an alkyl moiety or a substituted alkyl moiety, such as a fluoroalkyl moiety ( Including, but not limited to, perfluoroalkyl moieties). The surface modifying compound also includes a linking moiety which is a group which covalently attaches the surface modifying ligand to the surface, as schematically shown in Equation 1. The covalently modified surface has a surface modifying ligand attached via a linking group LG that is a product of a linking moiety that reacts with a functional group of the surface (including hydroxides, oxides, amines or sulfur).Equation 1. In some embodiments, the surface modifying compound can include a linear carbon atom (eg, at least 10 carbons, or a linear chain of at least 14, 16, 18, 20, 22, or more carbons) And it may be a non-branched alkyl moiety. In some embodiments, an alkyl group can include a substituted alkyl group (eg, some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group can include a first segment joined to the second segment, the first segment can include a perfluoroalkyl group, and the second segment can include an unsubstituted alkyl group, wherein The first and second sections can be joined directly or indirectly (e.g., via ether linkages). The first section of the alkyl group can be at the distal end of the linking group and the second section of the alkyl group can be located at the proximal end of the linking moiety. In various embodiments, the surface modifying compound can have the structure of Formula I:Wherein the linking moiety V is -P(O)(OH)Q- or -Si(T)₂ W; W-T, -SH or -NH₂ And is configured to be attached to a portion of the surface; Q-OH- and is configured to be attached to a portion of the surface; and T-series OH, OC_1-3 Alkyl or Cl. R is hydrogen or fluorine and M is hydrogen or fluorine. Each instance of h is independently an integer of 2 or 3; j is 0 or 1; k is 0 or 1; m is 0 or an integer from 1 to 25; and n is 0 or an integer from 1 to 25. In some other embodiments, the sum of (n + [(h + j)·k] + m) may be an integer from 11 to 25. In some embodiments, M is hydrogen. In various embodiments, m is two. In some embodiments, k is 0. In other embodiments, k is 1. In various embodiments, j is 1. For compounds of formula I, when k is an integer of 1, then m can be at least 2 and M is hydrogen. For compounds of formula I, when k is 0 and R is fluorine, then m can be at least 2 and M is hydrogen. In various embodiments, when the surface modifying compound has the structure of Formula I, the linking moiety V can be -Si(T)₂ W, wherein T and W are as defined above. W can be OC_1-3 Alkyl or Cl. W may be methoxy, ethoxy or propoxy. In some embodiments, W can be methoxy. T can be OC_1-3 Alkyl or Cl. In various embodiments, the connecting portion V-Si (OMe)₃ . In various other embodiments, V can be -P(O)(OH)Q, wherein Q is OH. The surface modifying compound of Formula 1 may have a preferred range of the number of atoms of the linear backbone of the constituent compound. As defined above, each segment constituting the compound of Formula 1 can have a range of sizes. Thus, a compound of Formula 1 may have a repeating unit attached to the linking moiety as defined above such that (n + [(h + j)·k] + m) is equal to 25, which will result in a total length of 26 atoms, including End CR₃ - group. In the case where (n + [(h + j)·k] + m) is equal to 25, a plurality of different compositions can be covered. For example, section - [CR₂ ]_n - can have n = 23; -[(CH₂ )_h -(O)_j ]_k - can have k = 0; and - [CM₂ ]_m - can have m = 2. Another case with the same total (n + [(h + j)·k] + m) equal to 25 may have a segment - [CR₂ ]_n - where n = 6; -[(CH2)h-(O)j]k-, where k = 3, and including j = 1 and h = 2; and - [CM₂ ]_m - can have m = 4. In some embodiments, the sum of (n + [(h + j)·k] + m) may be 11, 13, 15, 17, or 21. In other embodiments, the sum of (n + [(h + j)·k] + m) may be 15 or 17. In other embodiments, the sum of (n + [(h + j)·k] + m) may be 13 or 15. In some embodiments, one or more ether linkages may be present in the compound of Formula I. In some embodiments, j can be one. In some embodiments, when both k and j are 1, m can be at least 2. In other embodiments, the backbone carbon can be fluorinated. In some embodiments, the backbone carbon can be perfluorinated, wherein CR₃ -and/or-[CR₂ ]_n -and/or-[CM₂ ]_m - each R can be fluorinated. In some embodiments, one portion of the compound can have a fluorinated carbon backbone atom, and other portions of the compound can have a carbon backbone atom substituted with hydrogen. For example, in some embodiments, CR₃ -and-[CR₂ ]_n - the segment may have a fluorine non-backbone substituent (eg, R-based fluorine), while -[CM]_m The segment may have a hydrogen non-backbone substituent (eg, M-based hydrogen). In some embodiments, when R is fluorine, then k is zero. In other embodiments, R can be fluorine and k is 1, j is 1 and h is 2. In various embodiments, M can be hydrogen. In other embodiments, the compound of Formula 1 can be synthesized from a hydrogenated olefin, as described below, wherein m is at least 2 and M is hydrogen. In some embodiments, m is 2 and M is hydrogen. Some of the various compounds of formula I may be more readily found in the subgroup of compounds described in the formula below, but the equation in no way limits the width of formula I. In some embodiments, a compound of Formula I can include a compound of Formula 110: CH₃ (CH₂ )_m Si (OC_1-3 alkyl)₃ Wherein m is an integer from 9 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15. In other embodiments, the compound of Formula I may comprise a compound of Formula 111: CF₃ (CF₂ )_n (CH₂ )₂ Si (OC_1-3 alkyl)₃ Wherein n can be an integer from 9 to 22. Alternatively, n may be an integer from 11 to 17. In some other embodiments, n can be 9, 11, 13, or 15. In some embodiments, n can be 13 or 15. In other embodiments, the compound of Formula I may comprise a compound of Formula 112: CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si (OC_1-3 alkyl)₃ Wherein n is an integer from 3 to 19; h is an integer of 2 or 3; and m is an integer from 2 to 18. In some embodiments, R can be fluorine. In some embodiments, n can be an integer from 3 to 11, h can be 2, and m can be an integer from 2 to 15. Alternatively, the compound of formula I may comprise a compound of formula 113: CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ Wherein n is an integer from 3 to 21; and m is an integer from 2 to 21. In some embodiments of the compound of Formula 113, R can be fluoro. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13.Surface for decoration . The surface that can be modified by the surface modifying compounds described herein, including the compounds of Formula I, can be a metal, a metal oxide, a glass, or a polymer. Some materials into which a covalently modified surface can be introduced can include, but are not limited to, cerium and its oxides, polyoxyn oxide, aluminum or its oxides (Al)₂ O₃ ), indium bismuth oxide (ITO), titanium dioxide (TiO₂ ), zirconia (ZrO2), cerium (IV) oxide (HfO)₂ ), yttrium (V) oxide (Ta₂ O₅ ) or any combination thereof. The surface can be a wafer or plate of such materials or can be incorporated into a device or device. In some embodiments, a surface comprising any of the materials can be incorporated into a microfluidic device as described herein. The polymer can include any suitable polymer. Suitable polymers may include, but are not limited to, (e.g., rubber, plastic, elastomer, polyoxymethylene, organopolyoxygen (e.g., polydimethylsiloxane) ("PDMS") or the like), which may be breathable of. Other examples may include molded glass, patternable materials (eg, polyoxyl polymers such as photopatternable polyfluorene or "PPS"), photoresists (eg, epoxide-based photoresists such as SU8) ) or the like. In other embodiments, surfaces of materials such as natural fibers or wood may be functionalized with surface modifying compounds (including compounds of Formula I) as described herein to introduce covalently modified surfaces. The surface to be modified may include nucleophilic moieties including, but not limited to, hydroxides, amine groups, and mercaptans. A nucleophilic moiety on the surface (eg, a hydroxide (referred to as an oxide in some embodiments)) can be reacted with a surface modifying compound (including a compound of Formula I) described herein to pass a hydroxy linking group or a phosphine. The acid ester linking group covalently links the surface modifying ligand to the surface to provide a functionalized surface. The surface to be modified may comprise a natural nucleophilic moiety, or may be treated with a reagent (such as a piranha solution) or by plasma treatment to introduce a nucleophilic moiety (eg, a hydroxide (or oxide) ). In some embodiments, the surface can be formed from any of the above materials, either alone or in any combination. The surface can include a semiconductor substrate. In various embodiments, the surface comprising the semiconductor substrate can further comprise a DEP or EW substrate as described herein. In some embodiments, a surface comprising a semiconductor substrate having a DEP or EW substrate can be part of a microfluidic device as described herein. In some embodiments, the modified surface can be at least one inwardly facing surface of a microfluidic device as described herein. At least one surface may be part of a flow region of a microfluidic device (which may include a channel) or may include a surface of an encapsulation structure such as a fence, which may include an isolation fence as described herein.Covalently modified surface . The covalently modified surface can include a surface modifying ligand, which can be a non-polymeric moiety, such as an alkyl moiety, a substituted alkyl moiety (eg, a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety)), and The ligand may be a surface modification of any of the surfaces described above, which is covalently bonded to the surface via a linking group that is part of the reaction between the linking moiety and the surface. The linking group can be a decyloxy linking group or a phosphonate linking group. In some embodiments, the surface modifying ligand can include a linear carbon atom (eg, at least 10 carbons, or a linear chain of at least 14, 16, 18, 20, 22, or more carbons) And may be a non-branched alkyl moiety. In some embodiments, an alkyl group can include a substituted alkyl group (eg, some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group can include a first segment joined to the second segment, the first segment can include a perfluoroalkyl group, and the second segment can include an unsubstituted alkyl group, wherein The first and second sections can be joined directly or indirectly (e.g., via ether linkages). The first segment of the alkyl group can be at the distal end of the linking group and the second segment of the alkyl group can be at the proximal end of the linking group.formula II Covalently modified surface . In some embodiments, the covalently modified surface has the structure of Formula II:Where is the surface; V-P(O)(OY)W- or -Si(OZ)₂ W. W is -O-, -S- or -NH- and is attached to the surface. Z-bond to a bond attached to the surface adjacent to a ruthenium atom or a bond to the surface. Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface. For the covalently modified surface of Formula II, R, M, h, j, k, m, and n are as defined above. When k is an integer of 1, then m is at least 2 and M is hydrogen. When k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. The covalently modified surface of Formula II can be illustrated as a surface modifying ligand attached via a linking group LG, as in Formula IIA, wherein LG is linked to the surface:The covalently modified surface of Formula IIA can comprise any of Formula II surfaces in any combination as described above for the surface modifying compound of Formula I. In some embodiments, the covalently modified surface of Formula II can be the surface of Formula 210:; where 210The surface, the oxygen attached to the helium atom is also bonded to the surface, and m is an integer from 11 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15. In some other embodiments, the covalently modified surface of Formula II can be the surface of Formula 211:; 211 whereThe surface, the oxygen attached to the helium atom is also bonded to the surface, and n may be an integer from 9 to 22. Alternatively, n may be an integer from 11 to 17. In some other embodiments, n can be 7, 9, 11, 13, or 15. In some embodiments, n can be 13 or 15. In other embodiments, the covalently modified surface of Formula II can be the surface of Formula 212:; where 212The surface, the oxygen attached to the helium atom is also bonded to the surface, and n is an integer from 3 to 21, h is an integer of 2 or 3, and m is an integer from 2 to 21. In some embodiments, R can be fluorine. In some embodiments, n can be an integer from 3 to 11, h can be 2, and m can be an integer from 2 to 15. Alternatively, the covalently modified surface of Formula II can be the surface of Formula 213:; 213 whereThe surface, the oxygen attached to the phosphorus atom is also bonded to the surface, n is an integer from 3 to 21 and m is an integer from 2 to 21. In some embodiments of the compound of Formula 113, R can be fluoro. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13. In some embodiments, the microfluidic device includes a flow zone fluidly coupled to the first inlet and the first outlet, the flow zone configured to contain a flow of the first fluid medium. The microfluidic device can include one or more chamber openings to the flow zone. The covalently modified surface can be a covalently modified substrate of the microfluidic device and can be located below the flow zone and/or at least one chamber. In some embodiments, the microfluidic device is configured to have a covalently modified surface of Formula II for all or substantially all of the inner surface facing the fluid. 2H depicts a cross-sectional view of a microfluidic device 290 comprising an exemplary covalently modified surface 298. As illustrated, the covalently modified surface 298 (shown schematically) can comprise a single layer of molecules that are covalently bonded to both the inner surface 294 of the substrate 286 and the inner surface 292 of the lid 288 of the microfluidic device 290. The covalently modified surface 298 can be disposed on substantially all of the inner surfaces 294, 292 of the outer shell 284 proximate and inwardly facing the microfluidic device 290, and in some embodiments and as discussed above, includes for defining a microfluidic device The surface of the circuit element and/or structure of the microfluidic circuit material (not shown) within 290. In an alternate embodiment, the covalently modified surface 298 can be disposed on only one or some of the inner surfaces of the microfluidic device 290. In the embodiment shown in FIG. 2H, covalently modified surface 298 comprises a single layer of alkyl-terminated siloxane molecules, each molecule covalently bonded to microfluidic device 290 via oxirane linker 296 Surface 292, 294. For the sake of simplicity, other yttrium oxide bonds linked to the ruthenium atom are shown, but the invention is not limited thereto. In some embodiments, the covalently modified surface 298 can comprise a fluoroalkyl group (eg, a fluorinated alkyl or a perfluorinated alkyl group) at its end facing the outer shell (ie, a single layer of surface modifying ligand 298 is not bonded to Inner surfaces 292, 294 are adjacent to portions of outer casing 284). Although Figure 2H is discussed as having an alkyl terminated modified surface, any suitable surface modifying compound can be used, as described herein.Natural surface . At least one surface of the microfluidic device to be modified may be glass, metal, metal oxide or polymer. Some materials that can be incorporated into a microfluidic device and that can be modified to introduce a covalently modified surface of Formula II thereto can include, but are not limited to, ruthenium and its oxides, polyxime, aluminum, or oxides thereof (Al)₂ O₃ ), indium bismuth oxide (ITO), titanium dioxide (TiO₂ ), zirconia (ZrO2), cerium (IV) oxide (HfO)₂ ), yttrium (V) oxide (Ta₂ O₅ ) or any combination thereof. The polymer can include any suitable polymer. Suitable polymers may include, but are not limited to, (e.g., rubber, plastic, elastomer, polyoxymethylene, organopolyoxygen (e.g., polydimethylsiloxane) ("PDMS") or the like), which may be breathable of. Other examples may include molded glass, patternable materials (eg, polyoxyl polymers such as photopatternable polyfluorene or "PPS"), photoresists (eg, epoxide-based photoresists such as SU8) ) or the like.Physical and performance properties of covalently modified surfaces . In some embodiments, the covalently modified surface can have increased hydrophobic character. The increased hydrophobic character of the modified surface prevents fouling due to biological materials. Surface fouling as used herein refers to the amount of material that is deposited indiscriminately on the surface of a microfluidic device, which may include permanent or semi-permanent use of biological materials such as proteins and their degradation products, nucleic acids, and individual degradation products. Sex deposition. This fouling increases the amount of biological tiny objects that stick to the surface. In other embodiments, the increased hydrophobic character of the covalently modified surface reduces the adhesion of biological microscopic objects on the surface, independent of the adhesion initiated by surface fouling. Surface modification can increase the durability, functionality, and/or biocompatibility of the surface. Each of these features may further benefit the activity (including growth rate and/or cell multiplication rate) of tiny objects or biomolecules on the modified surface and devices and/or devices having covalently modified surfaces, The nature or portability (including vigor of the discharge) of a community formed on a covalently modified surface (including the surface having the structure of Formula II) as described herein. In some embodiments, the covalently modified surface can be any surface as described herein (including the surface of Formula II), which can have less than 10 nm (eg, less than about 7 nm, less than about 5 nm, or about 1.5 nm) Thickness up to 3.0 nm). This can advantageously be provided on the modified surface in particular with other hydrophobic materials (eg perfluorotetrahydrofuran-based polymer CYTOP)^® A different thin layer that is spin coated to produce a typical thickness of about 30 nm to 50 nm. The data shown in Table 1 pertains to the ruthenium/iridium oxide surface treated to have a covalently modified surface as shown in the table. The contact angle measurement system is obtained using a static drop method. (Drelich, J. Colloid Interface Sci.179 , 37-50, 1996. The thickness is measured by ellipsometry. Contact angle hysteresis measurements were performed using a Biolin Scientific contact angle goniometer. The chemically modified OEW surface was placed in 5 cSt polyoxyxide oil wrapped in a clear container. Phosphate buffered saline (PBS) droplets were then dispensed onto the surface in the oil. A platinum (Pt) wire electrode was inserted into the droplet and the fixation water contact angle was measured. Then, an applied AC voltage of 50 Vppk was applied between the OEW substrates at a frequency of 30 kHz and the Pt line was inserted into the PBS droplets for 10 seconds. Then, the applied voltage is removed and the contact angle is measured again. The contact angle hysteresis was calculated by subtracting the contact angle at zero bias after applying the voltage from the original contact angle at zero bias before the application of the 50 Vppk AC voltage.table 1 . Physical data of the selected surface. The T and Q systems are as described above. The contact angle observed for the modified surface is different from the contact angle of less than 10 degrees of water on the surface of the plasma cleaned. Each of these surfaces is less susceptible to wetting than the natural tantalum/yttria surface. Other analytical methods suitable for characterizing the surface may include infrared spectroscopy and/or X-ray photoelectron spectroscopy. Another desirable feature of the modified surface of the present invention is the lack of spontaneous fluorescence, which may depend on the chemical nature of the surface modifying compound. In some embodiments, the covalently modified surfaces described herein (including the surface of Formula II) can form a single layer. Especially when the single layer modified surface has other functional properties, the uniformity and uniformity of the single layer modified surface can provide advantageous properties. For example, the covalently modified surface (including the surface of Formula II) described herein can also include an electrode-activated substrate, and can optionally include a dielectric layer, such as can be found to have a dielectrophoretic configuration or an electrowetting configuration. In materials, devices, and/or devices. The lack of saturation of the perfluoroalkyl moiety of the modified surface minimizes "charge trapping" as compared to a monolayer containing, for example, an olefin or aromatic moiety. Additionally, the dense packing properties of the monolayers formed on the surfaces described herein (including the surface of Formula II) may minimize the potential for driving the cations through the monolayer to the underlying metal, metal oxide, glass or polymer substrate. Without being bound by theory, the destruction of the substrate surface by the addition of cations to the substrate composition can destroy the electrical properties of the substrate, thereby reducing its ability to function as an electrokinetic function. Furthermore, the ability to introduce a modified surface via a covalent bond can increase the dielectric strength of the modified surface and protect the underlying material from decomposition under an applied electric field. Uniformity and thinness of dielectrophoretic or electrowetting surfaces of materials, devices and/or devices having covalently modified surfaces (including surfaces of Formula II) as described herein when optically actuating materials, devices, and/or devices Further advantageous benefits can be provided for the modified dielectrophoresis and/or electrowetting surface.Method for preparing covalently modified surface . The surface of the material that can be used as a component of the device or device can be modified prior to assembly of the device or device. Alternatively, a partially or fully constructed device or device may be modified such that all surfaces that contact biological material, including biomolecules and/or microscopic objects (which may include biological microscopic objects), are simultaneously modified. In some embodiments, the entire interior of the device and/or device can be modified even if different materials are present on different surfaces within the device and/or device. In some embodiments, a partially or fully constructed device and/or device can be a microfluidic device or a component thereof as described herein. The surface to be modified can be cleaned prior to modification to ensure that the nucleophilic moiety on the surface is free to react, for example, without oil or adhesive. Cleaning can be accomplished by any suitable method, including treatment with solvents (including alcohol or acetone), ultrasonic treatment, steam cleaning, and the like. In some embodiments, the surface to be modified is treated with an oxygen plasma treatment, which removes contaminants while introducing other oxide (e.g., hydroxide) portions on the surface. This may advantageously provide more modification sites on the surface, thereby providing a more closely packed modified surface layer. The surface to be modified can be cleaned prior to modification to ensure that the nucleophilic moiety on the surface is free to react, for example, without oil or adhesive. Cleaning can be accomplished by any suitable method, including treatment with solvents (including alcohol or acetone), ultrasonic treatment, steam cleaning, and the like. In some embodiments, the surface to be modified is treated with an oxygen plasma treatment, which removes contaminants while introducing other oxide (e.g., hydroxide) portions on the surface. This may advantageously provide more modification sites on the surface, thereby providing a more closely packed modified surface layer. In some embodiments, a method of covalently modifying a surface comprises the step of contacting a surface with a compound of Formula I:; Formula I where V is -P(O)(OH)Q or -Si(T)₂ W. W series -T, -SH or -NH₂ And configured to connect to portions of the surface. Alternatively, when V is -P(O)(OH)Q, Q is -OH and is configured to attach to a portion of the surface. T system OH, OC_1-3 Alkyl or Cl. Each of R, M, h, j, k, m and n is as defined above for the compound of formula I. The sum of (n + [(h + j)·k] + m) is an integer of 11 to 25. In various embodiments, when k is an integer of 1, m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. The compound of formula I is reacted with a nucleophilic moiety of the surface; and a covalently modified surface is formed. Any combination or subcombination of the compounds of formula I can be used as described above. In various embodiments of the method, the covalently modified surface thus formed can be a single layer. In some embodiments of the method, the compound of formula I can be a compound of formula 110: CH₃ (CH₂ )_m Si (OC_1-3 alkyl)₃ Wherein m is an integer from 9 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15. In other embodiments of the method, the compound of formula I can be a compound of formula 111: CF₃ (CF₂ )_n (CH₂ )₂ Si (OC_1-3 alkyl)₃ Wherein n is an integer from 9 to 22. Alternatively, n may be an integer from 11 to 17. In other embodiments, n can be an integer from 11 to 17. In some other embodiments, n can be 9, 11, 13, or 15. In some embodiments, n can be 13 or 15. In other embodiments of the method, the compound of formula I can be a compound of formula 112: CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si (OC_1-3 alkyl)₃ Wherein n is an integer from 3 to 21; h is an integer of 2 or 3; and m is an integer from 2 to 21. In some embodiments, R can be fluorine. In some embodiments, n can be an integer from 3 to 11, h can be 2, and m can be an integer from 2 to 15. Alternatively, the surface can be contacted with a compound of formula I, which can be a compound of formula 113: CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ Wherein n is an integer from 3 to 21; and m is an integer from 2 to 21. In some embodiments of the compound of Formula 113, R can be fluoro. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13. The contacting step can be carried out by contacting the surface with a liquid solution containing a compound of formula I. For example, the surface can be exposed to a solution containing 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM, or 100 mM of a compound of formula I. The reaction can be carried out at ambient temperature and can be carried out over a period of time between about 2 h, 4 h, 8 h, 12 h, 18 h, 24 h or any value therebetween. Examples of solvents include, but are not limited to: toluene, 1,3 bistrifluorobenzene or Fluorinert^TM (3M) fluorinated solvent. An acid such as acetic acid can be added to the solution to increase the reaction rate by promoting hydrolysis of the trialkoxy group, if present. Alternatively, the surface can be contacted with a gas phase containing a compound of formula I. In some embodiments, a controlled amount of water vapor is also present when the reaction step is carried out by contacting the surface with a compound of formula I in the gas phase. A controlled amount of water vapor can be provided by placing a preselected amount of magnesium sulfate heptahydrate in the same chamber or enclosure with the object having the surface to be modified. In other embodiments, a controlled amount of water can be introduced into the reaction chamber or housing via an external water vapor feed. The reaction can be carried out under reduced pressure relative to atmospheric pressure. In some embodiments, the reduced pressure can be 100 Torr or less. In other embodiments, the reduced pressure can be less than 10 Torr or less than 1 Torr. The reaction can be carried out at a temperature ranging from about 150 ° C to about 200 ° C. In various embodiments, the reaction can be carried out at a temperature of about 150 ° C, 155 ° C, 160 ° C, 165 ° C, 170 ° C, 175 ° C, 180 ° C, 185 ° C, or about 190 ° C. The reaction can be tolerated for about 2 h, 6 h, 8 h, 18 h, 24 h, 48 h, 72 h, 84 h or longer. In some embodiments, the covalently modified surface can have the structure of Formula II:Wherein R, M, n, h, j, k, m and V in any combination are as described above. In some embodiments of the method, the covalently modified surface can have the formula of Formula 210, 211, 212, or 213 as described above, with any combination of elements that can be tolerated for each. In various embodiments of the method, the surface can include a nucleophilic moiety selected from the group consisting of hydroxides, amines, and thiols. The surface can be a metal, a metal oxide, a glass, a polymer, or any combination thereof. The metal surface can include tantalum, yttria, cerium oxide, indium lanthanum oxide, aluminum oxide, or any combination thereof. In various embodiments of the method, the step of forming a covalently modified surface can be performed on a DEP substrate or an EW substrate. The step of forming a covalently modified surface can include forming a covalently modified surface on at least one surface of the microfluidic circuit element of the microfluidic device. The microfluidic circuit element can include walls, flow zones, fences, and electrode activated substrates (including DEP or EW substrates). The surface that can be covalently modified within the microfluidic circuit can be all or substantially all of the surface facing the portion of the fluid-bearing microfluidic device. For example, in the microfluidic device 200, 230, the inner surface of the top electrode 210, the upper surface of the electrode activating substrate 206, and the surface of the microfluidic circuit material 116 (see FIGS. 1B, 2A, 2B) may be modified. Microfluidic channel 122 and fences 244, 246, 248. Similarly, in Figures 2D-2F, the inner surface of the microfluidic circuit material 260, the surface defining the separation structure 272 of the isolation fence 266, or all surfaces facing the microfluidic circuit 262 can be covalently modified by the methods described herein.Immiscible medium . The movement of the aqueous droplets on the surface of the substrate can be carried out in a water immiscible fluid medium that is regionally distributed in one or more flow zones (which can include flow channels) and, if present, fluidly connected to the interior of the flow zone. The water immiscible fluid medium can have a kinematic viscosity greater than that of pure water droplets. The water immiscible fluid medium can have a kinematic viscosity in the range of from about 1 centistoke (cSt) to about 15 cSt, wherein 1 cSt is equal to 1 millipascal or 1 centipoise (CPS). In some embodiments, the water immiscible fluid medium can have a viscosity ranging from about 3 cSt to about 10 cSt or from about 3 cSt to about 8 cSt. The water immiscible fluid medium can be non-flammable at temperatures of at least 100 °C. The water immiscible fluid medium is non-toxic to living biological cells for a duration of time during which the biological cells are treated, cultured or stored in aqueous microdroplets in a water immiscible fluid medium. Water immiscible fluid media can have low or very low water solubility. The water immiscible fluid medium can dissolve less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of its total volume of water when contacted with the aqueous layer (e.g., by water). The water immiscible fluid medium may not dissolve greater than about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% by volume at temperatures ranging from about 25 °C to about 38 °C. Aqueous droplets present in a water immiscible fluid medium. In some embodiments, the water immiscible fluid medium dissolves less than about 20% by volume of the aqueous droplets present in the water immiscible fluid medium. The water immiscible fluid medium can include at least one organic or organic phosphonium compound having a backbone structure comprising atoms selected from the group consisting of carbon, ruthenium and oxygen. In some embodiments, the water immiscible fluid medium can include more than one organic/organic cerium compound, wherein more than one compound is a polymeric organic/organic cerium compound having a molecular weight range of polymeric compound subunits. For example, a polymeric organic/organic cerium compound can have two different subunits that make up a polymer (eg, a copolymer), and each of the two different subunits can be a series of repeats having the general formula AaBb, wherein A and B are two different polymer subunits, and a and b are the number of repeats per subunit. The repetition numbers a and b may not be a single integer, but may be a series of repeating units. In other embodiments, the water immiscible fluid medium comprising more than one organic/organic hydrazine compound can comprise a mixture of organic compounds, a mixture of organic hydrazine compounds, or any combination thereof. The water immiscible fluid medium can include any suitable mixture of compounds having different chemical structures and/or molecular weights that will provide suitable properties. The compound of the water immiscible fluid medium can have a molecular weight of less than about 1000 Da, about 700 Da, about 500 Da, or about 350 Da. In other embodiments, the compound of the water immiscible medium can have a molecular weight greater than about 1000 Da and still provide suitable properties. In various embodiments, the organic/organophosphonium compound of the water immiscible fluid medium can have a backbone structure in which the atoms that make up the backbone are carbon, helium or oxygen. The substituent of the main chain carbon may be hydrogen or fluorine. In some embodiments, the water immiscible fluid medium can include one or more organic cerium compounds, wherein the backbone of the organic cerium compound can include cerium and oxygen atoms. The ruthenium atom of the organic ruthenium compound may have a carbon substituent, which in turn may have a hydrogen or fluorine substituent. In some embodiments, the carbon substituent of the organofluorene compound can be all fluorine (eg, perfluorinated). In other embodiments, the carbon substituent of the organofluorene compound can be partially fluorinated. In various embodiments, the substituent of the carbon atom of the organofluorene compound may be no more than about 90% fluorine, 80% fluorine, 70% fluorine, 60% fluorine, 50% fluorine, 40% fluorine, 30% fluorine, 20% fluorine or less. In other embodiments, the organic compound of the water immiscible fluid medium can have a backbone structure in which the atoms that make up the backbone are carbon or oxygen. In some embodiments, the substituent of the backbone carbon can be hydrogen or fluorine. In other embodiments, the substituent of the backbone carbon can include an oxygen containing moiety such as an ether, carbonyl or carbonate component. In some embodiments, the organic compound of the water immiscible fluid medium can have an all carbon backbone structure. In some embodiments of the all carbon backbone structure of the organic compound of the water immiscible fluid medium, it may have a perfluoro substituent on the carbon atom (e.g., perfluorinated). In other embodiments, the substituents of the organic compound can be partially fluorinated (eg, without perfluorination). In various embodiments, the substituent of the carbon atom of the organic compound (including the compound having an all carbon backbone) may not exceed about 90% fluorine, 80% fluorine, 70% fluorine, 60% fluorine, 50% fluorine, 40% fluorine. Or less. In some embodiments, suitable organic compounds of the water immiscible fluid medium may include or may be monofluoro substituted hydrocarbons such as 1-fluorooctane, 1-fluorodecane, 1-fluorododecane or 1-fluoro. Tetradecane. In other embodiments, the organic compound of the water immiscible fluid medium may have no fluorine substituents on the carbon, but may have a hydrogen substituent. In some embodiments, the organic compound of the water immiscible fluid medium can have an unsaturated carbon-carbon bond, such as an olefinic group within the backbone carbon or at a terminal position. In some embodiments, the selection of a suitable compound to be included in a water immiscible fluid medium will include consideration of other properties of the compound. In various embodiments, compounds that are suitable for use in a water immiscible fluid medium will not spontaneously fluoresce when irradiated, structured light, or daylight/laboratory illumination in a microfluidic device. In other embodiments, the nature of the covalently modified hydrophobic surface will affect the choice of compounds suitable for use in a water immiscible fluid medium. For example, the covalently modified surface can be sufficiently hydrophobic such that droplets of water in the perfluorinated water immiscible fluid medium can exhibit a sufficiently high surface tension such that the water droplets can be wetted without the use of light as described herein. Configuration to move. In some other embodiments, the nature of the microfluidic circuit material can affect the choice of compounds suitable for use in a water immiscible fluid medium. The swelling of the circuit material by the water immiscible fluid medium can be maintained within acceptable limits. For example, in some embodiments, if the microfluidic circuit material comprises SU8 or a photo-patternable aryl-substituted organopolyfluorene, a linear hydrocarbon comprising a ring, an aryl or a heteroaryl group, a linear chain may be selected. Fluorocarbon or carbon backbone compounds are used. In other embodiments, the microfluidic circuit material can include other materials, such as light-patternable organopolyfluorene without aryl substitution, and can be limited to swelling by using different compounds in a water immiscible fluid medium. Accepted limits. For example, swelling of less than about 40%, 30%, 20%, or 10% compared to prior to exposure to a water immiscible fluid medium can be acceptable. However, in some embodiments, a compound that causes swelling in a water immiscible fluid medium can still be selected for use. In some embodiments, the compound of the water immiscible fluid medium can be an organic compound having a backbone having a carbon or oxygen atom. In some embodiments, the organic compound can have a backbone that contains carbon atoms and is free of oxygen atoms, and that has a backbone of the other carbon atoms. In various embodiments, the branched carbon atom backbone of the organic compound of the water immiscible fluid medium is acyclic. The organic compound having a branched carbon backbone of the water immiscible fluid medium may additionally be free of any cyclized portion. Although the above selection criteria can be used to select one or more compounds to be included in the water immiscible fluid medium and to eliminate compounds that do not provide acceptable performance, acceptable water immiscible fluid media can be multicomponent mixtures, and Some portions of the individual organic or organic bismuth compounds that would not provide acceptable performance when used as the sole component of the water immiscible fluid medium can be included. For example, the components can be highly fluorinated or can cause unacceptable swelling of the microfluidic circuit material when used alone, but can be used in combination with other organic or organic bismuth compounds to form a water immiscible fluid medium. Some of the organic compounds suitable for use in the water immiscible fluid medium, alone or in any combination, may include isohexadecane, 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500). , 3MTM, NovecTM), heptamethylnonane (HMN), bis(2-ethylhexyl) carbonate (TEGOSOFT® DEC, (Evonik)) and (trifluoro-1,1,2,2,-four Hydrogen octyl) tetramethyldioxane (Gelest, Cat. No. SIB1816.0) or polyoxygenated oil (5 psi Stoker viscosity, Gelest catalog number DMS-T05). Aqueous microdroplets. Aqueous microdroplets may contain one or more microscopic objects, which may include biological cells or beads. Aqueous droplets can contain biological products, which can include nucleic acids or proteins. In some other embodiments, the aqueous droplets can contain an assay reagent, which can be any type of reagent, such as an enzyme, an antibody, a fluorescently labeled probe, or a chemical reagent. In some embodiments, the aqueous droplets can also include a surfactant. Surfactants increase the portability of aqueous droplets in a water immiscible fluid medium. In some embodiments, suitable surfactants can include nonionic surfactants. In various embodiments, the surfactant can be, but is not limited to, a Pluronic® alkylene oxide block copolymer, including F68 (ThermoFisher Cat. No. 2400032); a fatty ester ethoxylated sorbitan such as TWEEN® 20 (Signa Aldrich catalog number P1379) or TWEEN® 60 (Sigma Aldrich P1629); 2,4,7,9, tetramethyl-5-decyne-4,7,-diol ethoxylate (TET, Sigma Aldrich Cat. No. 9014-85-1); ethoxylated nonionic fluorosurfactant, such as Capstone® FS-30 (DuPontTM, Synquest Laboratories Cat. No. 2108-3-38). In some embodiments, sodium dodecyl sulfate (SDS) can be used as a surfactant. In various embodiments, phosphate buffered saline (PBS) can be used as a surfactant. The surfactant can be added to the aqueous droplets in the range of about 1%, 3%, 5%, 10%, 15%, 20%, about 25% v/v, or any value therebetween.Method of making a microfluidic device . The microfluidic device of the present invention (e.g., device 400) can be fabricated by (i) bonding spacer element 108 to inner surface 428 of cover 110 having at least one configuration configured to connect to an AC voltage source. (not shown) the electrode, (ii) bonding the spacer element 108 (and associated cover 110) to the dielectric surface 414 of the substrate 104, the dielectric surface having at least one configuration configured to connect to an AC voltage source (not shown) The electrode 418, whereby the spacer element 108 is sandwiched between the inner surface 428 of the cover 110 and the dielectric surface 414 of the substrate 104, wherein the cover 110 and the substrate 104 are substantially oriented parallel to each other, and the substrate 104, the spacer element 108, and The cover 110 collectively defines a housing 435 that is configured to contain a liquid, and (iii) an outer hydrophobic layer 412 that is vapor deposited on at least a portion of the inner surface 428 of the cover 110 and an inner dielectric layer 414 of the substrate 104. An outer hydrophobic layer 412 on at least a portion. By vapor deposition of the amphiphilic molecules, the hydrophobic layers 422 and 412 can achieve a densely packed monolayer in which the amphiphilic molecules are covalently bonded to the inner surface 428 of the cap 110 and the inner dielectric surface 414 of the substrate 104, respectively. . Any of the self-associating molecules described herein and their equivalents can be vapor deposited on the inner surface of the microfluidic device. To achieve the desired bulk density, a self-associating molecule comprising, for example, an alkyl-terminated siloxane can be vapor deposited at a temperature of at least 110 ° C (eg, at least 120 ° C, 130 ° C, 140 ° C, 150 ° C, 160 ° C, etc.) A period of at least 15 hours (eg, at least 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, or longer). The vapor deposition is usually carried out under vacuum and at a water source (eg magnesium sulfate heptahydrate (ie MgSO₄ ·7H₂ O)) Implementation in the presence. Generally, increasing the temperature and duration of vapor deposition results in improved features of the hydrophobic layers 422 and 412. The vapor deposition process can be modified, for example, by pre-cleaning the cover 110 (with spacer elements 108) and the substrate 104. For example, the pre-cleaning can include a solvent bath, such as an acetone bath, an ethanol bath, or a combination thereof. The solvent bath can include ultrasonic treatment. Alternatively or additionally, the pre-cleaning can include processing the lid 110 (with the spacer element 108) and the substrate 104 in an oxygen plasma cleaner. The oxygen plasma cleaner can be operated, for example, at 100 W under vacuum for 60 seconds. 6 illustrates an example of a microfluidic device 600 that includes a housing having microfluidic channels 612, 614 and a plurality of chambers 616, and a droplet generator 606 for providing fluid droplets 620 to the housing. The microfluidic channel 614 is configured to receive the first fluid medium 624. Typically, the first fluid medium is a hydrophobic fluid, such as an oil (eg, a polyoxygenated oil or a fluorinated oil). Microfluidic channel 614 is coupled to droplet generator 606 via interface 608, which allows channel 614 to receive droplet 620 produced by droplet generator 606. The received droplet 620 contains a liquid that is immiscible in the first fluid medium 624. Typically, the received droplets will comprise an aqueous medium which may contain fine objects (e.g., cells or beads) or reagents that are soluble in the aqueous medium. The microfluidic channel 614 is also coupled to each of the plurality of chambers 616, which facilitates movement of the received droplets 620 (and droplets 632 taken from a reservoir that is immiscible in the fluid in the first fluid medium 624) to The chamber 616 moves in and between the chambers 616. Microfluidic channel 612 of device 600 is coupled to a subset of chambers 616 and is thus indirectly coupled to microfluidic channel 614 via such chambers 616. As illustrated, the microfluidic channel 612 and chamber 616 coupled thereto contain a fluid medium 622 that is immiscible in the first fluid medium 624. Thus, for example, fluid medium 622 can be an aqueous medium, such as a cell culture medium. When the fluid medium 622 is a cell culture medium, the chamber 616 containing the medium can be used as a culture chamber for growing cells, and the microfluidic channel 612 can be a perfusion channel that provides a flow of fresh medium. As discussed herein, the fresh medium stream in the perfusion channel can provide nutrients to the chamber and diffuse the waste from the chamber by diffusing the molecules between the perfusion channel and the culture chamber, thereby promoting sustained cell growth. 7 illustrates another example of a microfluidic device 700 that includes a housing having microfluidic channels 612, 614, a first plurality of chambers 716, and a second plurality of chambers 616, and droplets for providing fluid droplets 620 to the housing Generator 606. 7 presents a variation of the microfluidic device 600 shown in FIG. 6, wherein the chamber 616 contains immiscible primary fluid medium 624 (located in the microfluidic channel 614) and is positioned directly from the respective chamber 716 through the microfluidic channel. Medium 622 of 614. This configuration facilitates movement of fluid droplets 632 (which optionally contain tiny objects 630 or biological material) from selection chamber 616 to respective chambers 716 where fluid droplets (and any minute objects 630 or biological material) can be processed. Another example of a microfluidic device includes a housing having microfluidic channels 612, 614, a first plurality of chambers 716 and a second plurality of chambers 616, and a droplet generator 606 for providing fluid droplets 620 to the housing. This embodiment presents a variation of the microfluidic device 700 shown in Figure 7, wherein one end of the chamber 616 tapers to cause the microfluidic device to tilt such that the tapered end of the chamber 616 has a lower relative to the non-tapered end The potential energy (when applied in the gravitational field) facilitates movement of the particles to the interface of the first fluid medium 624 and the second fluid medium 622. The microfluidic circuits formed by microfluidic channels 612, 614 and chambers 616, 716 are merely examples, and the present invention contemplates many other configurations of channels and chambers. For example, in each of the devices 600 and 700, the microfluidic channel 612 and the chamber 616 that is directly connected to the channel 612 are optional features. Thus, devices 600 and 700 can lack a perfusion channel and a culture chamber. In embodiments in which the microfluidic channel 612 is present, the substrate that helps define the channel 612 and/or the directly connected chamber 616 (e.g., by forming the substrate of the channel and/or chamber) can have an electrowetting configuration. Alternatively, however, the substrate that assists in defining the channel 612 and/or the directly connected chamber 616 may lack an electrowetting configuration (eg, may have a DEP configuration or neither an electrowetting configuration nor a DEP configuration). In embodiments where the microfluidic channel 612 is present and the substrate defining the channel 612 and/or the directly connected chamber 616 has an electrowetting configuration, the hydrophobic surface outside the substrate can be patterned to help define the channel 614 beyond the substrate. The hydrophobic surface is more hydrophilic. Increased hydrophilicity can be achieved, for example, as discussed above. The droplet generator 606 and any microfluidic circuit to which the droplets are provided may be part of (or connected to) a microfluidic device, which may be in any of the microfluidic devices illustrated in the figures or described herein. same. Although one droplet generator 606 is shown in FIGS. 6 and 7, more than one droplet generator 606 can provide droplets to the microfluidic circuits of devices 600 and 700. The droplet generator 606 itself may comprise an electrowetting configuration, and thus may comprise a substrate having a photoreactive layer that may comprise a-Si:H (e.g., as illustrated in U.S. Patent No. 6,958,132), a photo-actuated circuit A substrate (e.g., as illustrated in U.S. Patent Application Publication No. 2014/0124370), a substrate based on an optoelectronic crystal (e.g., as illustrated in U.S. Patent No. 7,956,339), or an electrically actuated circuit substrate (e.g., Illustrated in U.S. Patent No. 8,685,344). Alternatively, the droplet generator can have a T-shaped or a Y-shaped fluid dynamic structure (for example, as disclosed in U.S. Patent Nos. 7,708,949, 7,041,481 (Re-issued as RE 41,780), No. 2008/0014589, Illustrated in 2008/0003142, 2010/0137163 and 2010/0172803). The entire disclosure of the aforementioned U.S. Patent is incorporated herein by reference. As shown, the droplet generator 606 can include one or more fluid inputs 602 and 604 (two but fewer or more can be present) and a fluid output 208 that can be coupled to the microfluidic channel 614. Liquid media 622, 624, biological micro-objects 630, reagents, and/or other biological media may be loaded into droplet generator 606 via inputs 602 and 604. Drop generator 606 can generate droplets 620 of liquid medium 622 (which may, but need not contain one or more biological micro-objects 630), reagents or other biological media and output them into channel 614. If channel 614 has an electrowetting configuration, droplet 620 can be moved in channel 614 using electrowetting (or photo-wetting). Alternatively, the droplet 620 can be moved in the channel 614 by other means. For example, the droplets 620 can move in the channel 614 using fluid flow, gravity, or the like. As discussed above, the microfluidic channel 614 and the selection chamber 616/716 can be filled with a first fluid medium 624, and the microfluidic channel 612 and chamber 616 directly coupled thereto can be filled with a second fluid medium 622. The second fluid medium 622 (hereinafter referred to as "aqueous medium") may be an aqueous medium such as a sample medium for maintaining, cultivating or such a biological micro-object 630. The first fluid medium 624 (hereinafter referred to as "immiscible medium") may be a medium in which the aqueous medium 622 is immiscible. Examples of aqueous medium 622 and immiscible medium 624 include any of the examples discussed above for various media. The droplet generator 606 can be used to load biological microscopic objects onto the microfluidic device and/or to facilitate the operation of biochemical and/or molecular biological workflows on the microfluidic device. Figures 6 and 7 illustrate non-limiting examples. By using a droplet generator, the device can have an electrowetting configuration throughout the fluid circuit. 6 and 7 illustrate an example in which droplet generator 606 produces droplets 620 comprising reagents (or other biological materials). The reagent-containing droplet 620 can move through the microfluidic channel 614 and into one of the chambers 616/716 containing the immiscible medium 624. One or more of the one or more micro-objects 630 may be moved into the same chamber 616/716 before or after the reagent-containing droplet 620 is moved into one of the chambers 616/716. The reagent-containing droplet 620 can then be fused with the droplet 632 containing the fine object 630, allowing the reagent of the droplet 620 to mix with the contents of the droplet 632 and undergo a chemical reaction. Droplets 632 containing one or more minute objects may be supplied by droplet generator 606 (not shown) or may be obtained from containment fence 616, as shown in Figures 6 and 7. The tiny object 630 can be a biological microscopic object, such as a cell, that has been cultured (eg, in chamber 616) as appropriate prior to moving to the processing chamber 616/716. Alternatively, the micro-object 630 can be a bead, such as an affinity that can be bound to a molecule of interest in the sample (eg, cell secretion present in the sample material 622 after the sample material 622 has been used to culture one or more biological cells). Beads. In other alternatives, one or more of the droplets 632 may be free of minute objects but only aqueous media, such as sample material 622, for example, containing cell secretions after the sample material 622 has been used to culture one or more biological cells. FIG. 8 illustrates an example of a process 800 that can be implemented in a microfluidic device (eg, any of devices 600 and 700) that includes a microfluidic circuit. At step 802 of process 800, biological micro-objects can be cultured in a containment enclosure filled with a sample medium (e.g., cell culture medium). For example, the tiny object 630 of Figure 6 or 7 can be a biological product and can be cultured in its chamber 616. Culture can generally be as discussed above. For example, culturing can include perfusing medium 622 into channel 612. Step 802 can be implemented for a specified period of time. At step 804, the cultured biological micro-object can be moved from chamber 616 in which the sample medium is cultured to chamber 616/716 filled with the medium immiscible with the sample medium. For example, the cultured minute object 630 can be moved from one of the containment fences 616 to one of the containment fences 616/716 in the droplets 620 or 632 of the sample medium 622, as shown in FIGS. 6 and 7. Graphical illustration, as discussed above. At step 806, the cultured biological micro-object can be subjected to one or more treatments or processes in a containment enclosure filled with immiscible medium. For example, one or more droplets 620 containing one or more reagents can be generated by droplet generator 606 and moved into chambers 612/716 filled with immiscible medium and with droplets containing cultured biological micro-objects 630 632 fusion, as shown in Figures 6 and 7 and discussed above. For example, the first reagent-containing droplet 620 can contain a lysing reagent. The fusion of the droplet 632 containing the cultured biological micro-object 630 with the first reagent-containing droplet 620 containing the lysing reagent will dissolve the cultured biological micro-object 630. In other words, a combined droplet (not shown) containing cell lysate from the cultured biological micro-object 630 will be formed. Other (e.g., second, third, fourth, etc.) reagent-containing droplets 620 can then be fused to new droplets containing cell lysates to further process cell lysates as needed. Additionally or alternatively, it contains one or more secretions produced by cultured biological micro-objects 630 or one or more other materials of interest (eg, nucleic acids (eg, DNA or RNA), proteins, metabolites, or other organisms) One or more droplets of a labeled, captured microscopic object (not shown) having affinity may be generated by droplet generator 606 and moved into a fence 616 or 716 filled with immiscible medium and cultured in a similar manner. The droplets of the sample medium 622 of the biological micro-object 630 are fused. In the case where the cultured biological micro-object 630 has dissolved, the droplet 620 containing the captured micro-object may contain one or more affinity beads (eg, affinity for nucleic acids such as DNA, RNA, microRNA, or the like), It can be bound to the target molecule present in the lysate after fusion with the droplet containing the cell lysate in the containment enclosure 616 or 716. At step 808, the processed biological micro-objects are processed as appropriate. For example, if, at step 806, a capture object (not shown) is moved into chamber 616/716 containing the immiscible immiscible medium of cultured biological micro-object 630, then chamber 616/716 can be monitored at step 808. The indication indicates that the material of interest binds to a reaction (eg, a fluorescent signal) that is labeled to capture the amount of the fine object. Alternatively, the capture micro-object (not shown) can be removed from chamber 616/716 (eg, in droplet 622) and discharged from the microfluidic device (not shown in Figures 6 and 7) for subsequent analysis. As another example, the treated biological micro-object 630 can be removed from chamber 616/716 (eg, in droplet 632) and discharged from the microfluidic device (not shown) for subsequent analysis. Although specific embodiments and applications of the invention have been described in the specification, such embodiments and applications are only illustrative, and many variations are possible. For example, the method of Figure 8 can be performed on a sample material containing cell secretions (e.g., after sample material 682 has been used to culture one or more biological cells). In this embodiment, step 802 will remain the same, but step 804 will involve moving droplets 632 (eg, sample material 622 containing cell secretions) that may be free of small objects but only aqueous media to contain immiscible media. In chambers 616/716, steps 806 and 808 are performed on the droplets 632 containing the aqueous medium. Moreover, the electrowetting configurations discussed herein can be any type of optoelectronic wetting (OEW) device known in the art, an example of which is disclosed in U.S. Patent No. 6,958,132. Other examples of electrowetting configurations include electronically controllable dielectric wetting (EWOD) devices, examples of which are disclosed in U.S. Patent No. 8,685,344. The entire disclosure of the aforementioned U.S. Patent is incorporated herein by reference. Figure 9 is a method for forming a substrate of a microfluidic device. In the following methods, a process flow diagram is depicted to facilitate the formation of a substrate stack that can be used in a microfluidic device as illustrated in the above and below figures. In one embodiment, the substrate 104 illustrated in FIG. 1B is formed using the method illustrated for forming a substrate. 10-16 below will be a cross-sectional view of the action illustrated in the method illustrated in FIG. The first action illustrated by block 902 illustrates the fabrication of a substrate (in one embodiment, a conductive germanium substrate) using a thermal annealing process. Subsequently, the second action illustrated by block 904 depicts depositing a nitride layer on top of the germanium substrate. Subsequently, block 906 depicts a third action of applying the first pattern. Applying the pattern to the substrate is typically accomplished by a lithography process well known in the semiconductor processing industry, such as electron beam, X-ray, UV, and deep UV. Typically, polymers are used to define patterns of complex structures of transistors and metal lines. The polymer is then treated with a light and photoreactive layer and will be set forth in the following paragraphs. Subsequent actions to deposit the photoreactive layer are as depicted in block 908. The following actions, illustrated by block 910, illustrate etching the photoreactive layer to a first predetermined location. In one embodiment, the first predetermined location is a nitride layer. The next action is illustrated by block 912 to deposit at least one dielectric layer. As discussed previously, various embodiments having different combinations of dielectric layers have been discussed in connection with the description of FIG. 1B. The final step, as depicted in block 912, illustrates applying a second pattern on top of the at least one dielectric layer and etching the dielectric layer to a second predetermined location. In the same embodiment, the second predetermined location is a nitride layer. In one embodiment, the optional steps can be further processed. For example, a third pattern is deposited and stripping of the nitride layer is performed until etching to 10 um in the germanium substrate or tantalum oxide. In addition, another optional step of backside oxide stripping and silver backside metallization is performed. 10 is a cross-sectional view of the substrate processed in the middle of the method illustrated in FIG. In one embodiment, this figure depicts a cross-sectional view of process step 902 in conjunction with FIG. As previously mentioned, block 902 illustrates the preparation of a substrate (in one embodiment, a conductive germanium substrate) using a thermal annealing process. Note that mark 1001 depicts two arrows that target the location of the thermal annealing process. Figure 11 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 904 illustrated in connection with FIG. Block 904 depicts depositing a nitride layer on top of the germanium substrate, and this figure depicts layer 1102, which depicts a nitride layer formed on top of the germanium substrate. Figure 12 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 906 in conjunction with FIG. Block 906 illustrates the application of the pattern and subsequent removal of the nitride. Please note that the mark 1202 depicts the opening in the pattern from which the nitride layer is removed. Figure 13 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 908 in conjunction with FIG. Block 906 depicts the application of a photoreactive layer. Please note that reference numeral 1302 depicts the applied photoreactive layer. Figure 14 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 910, illustrated in conjunction with FIG. Block 910 illustrates etching the photoreactive layer added in process step 908 to a first predetermined location. In one embodiment, the etching of the first predetermined location to the nitride layer is stopped. Please note that the mark 1402 depicts the opening in the pattern that etches or removes the photoreactive layer. Figure 15 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 912 in conjunction with FIG. Block 912 illustrates depositing at least one dielectric layer. Please note that the marks 1501 and 1502 respectively depict the dielectric layer and the opening from the previous mark 1402 in FIG. 14 indicating the deposited dielectric layer. Figure 16 is a cross-sectional view showing the substrate processed in the middle of the method illustrated in Figure 9. In one embodiment, this figure depicts a cross-sectional view of process step 914 illustrated in conjunction with FIG. Block 914 illustrates applying a pattern to the dielectric layer and etching the dielectric layer to a second predetermined location. In one embodiment, the second predetermined location is a nitride layer. Please note that the mark 1602 depicts the opening in the dielectric layer that is removed by etching. 17 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. The system integrates two microfluidic operations, such as by the modules OEP and OEW that overlap the regions. The module performs microfluidic operations as previously discussed, specifically photo-alignment (hereinafter referred to as OEP) and photo-wetting (hereinafter referred to as OEW). In one embodiment, the modules are integrated by facilitating the integration of the two modules by utilizing at least one spacer (in one embodiment, a polyphenylene sulfide spacer) due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide spacers are thicker than the module and are capable of pressing and sealing the sides to allow for integration. The electrical operation of the two modules will be further discussed in conjunction with Figures 20A and 20B. 18 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. The system integrates two microfluidic operations, as illustrated by modules OEP and OEW having multiple zones. For example, in one embodiment, three OEW modules and a single OEP module are illustrated. However, the subject matter claimed is not limited to this particular combination. Those skilled in the art will appreciate the different configurations of the modules used based on design needs or the need for different light mask layers. The module performs microfluidic operations as previously discussed, in particular photovoltaic positioning and photo-wetting. In one embodiment, the modules are integrated by facilitating the integration of the two modules by utilizing at least one spacer (in one embodiment, a polyphenylene sulfide spacer) due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide spacers are thicker than the module and are capable of pressing and sealing the sides to allow for integration. 19 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. The system integrates two microfluidic operations, as illustrated by modules OEP and OEW with discrete zones per module. The module performs microfluidic operations as previously discussed, in particular photovoltaic positioning and photo-wetting. In one embodiment, the modules are integrated by facilitating the integration of the two modules by utilizing at least one spacer (in one embodiment, a polyphenylene sulfide spacer) due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide spacers are thicker than the module and are capable of pressing and sealing the sides to allow for integration. 20A and 20B are views of a functional representation of an electrical addressing operation in accordance with one of the functional aspects of the embodiment illustrated in FIG. As previously described in connection with Figure 17, the system incorporates two microfluidic operations, such as by the modules OEP and OEW that overlap the regions. In this embodiment, the OEP module has a lower impedance relative to the OEW module. During operation, the impedance of the OEW module overcomes the impedance of the OEP module and substantially shorts the OEP module. In one embodiment, as depicted in Figure 20A, the OEP module operates by applying a voltage in the range of 1-10 volts at a frequency ranging from 100 kHz to 10 mHz. In the same embodiment, as depicted in Figure 20B, the OEW module operates by applying a voltage in the range of 10-100 volts at a frequency ranging from 1 kHz to 300 kHz. In a preferred embodiment, the OEP module is operated by applying a voltage of 5 volts at a frequency of 1 Mhz, and the OEW module is operated by applying a voltage of 30 volts at a frequency of 30 kHz. Figure 20B is a view of a functional representation of an electrical addressing operation in conjunction with one of the embodiments of Figure 17;Instance System and microfluidic devices : Manufactured by Berkeley Lights, Inc. The system includes at least a flow controller, a temperature controller, a fluid medium condition processing and pump assembly, a light source for a photoactivated DEP or EW configuration, a microfluidic device, a mounting station, and a camera.Instance 1. Preparation of electrowetting microfluidic device with modified inner surface . A microfluidic device (Berkeley Lights, Inc.) has a first photosensitive semiconductor electrode-activated substrate (having a dielectric upper surface of alumina) and a second ITO substrate on the opposite wall and a light patterned poly layer separating the two substrates Oxygen microfluidic circuit material, the microfluidic device was treated with an oxygen plasma cleaner (Nordson Asymtek) using 100 W power, 240 mTorr pressure and 440 sccm oxygen flow rate for 1 min. In a vacuum reactor in the foil boat at the bottom of the vacuum reactor or in the presence of magnesium sulfate heptahydrate (0.5 g, Acros) as a source of water reactant in the bottom of the vacuum reactor in a separate foil boat, Use trimethoxy (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14 </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Said) treating a plasma treated microfluidic device. The chamber was then pumped to 750 mTorr using a vacuum pump and then sealed. The vacuum reactor was placed in an oven and heated at 180 ° C for 24-48 h. After cooling to room temperature and introducing argon into the vacuum chamber, the dimethoxy group (3,3,4,4,5,5,6,6,7,7,8,8,9, 9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-29-fluoro-hexadecyl) Fluid device. After cooling to room temperature and introducing argon into the vacuum chamber, the removal from the reactor has dimethoxy groups on all internal surfaces (3,3,4,4,5,5,6,6,7,7,8 ,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-trinylfluorohexadecyl) decyloxy moiety Layer( surface 202 ) The microfluidic device was pretreated with polyoxyxylene oil (5 psi Stoker viscosity, Gelest catalog number DMS-T05) prior to use. Figure 4A-C is a modified surface in an immiscible polyoxyxene oil phase202 A continuous photographic image of droplets of free moving water. The droplet display utilizes the excellent mobility of the optically actuated electrowetting configuration in a microfluidic device.

100‧‧‧Microfluidic devices
102‧‧‧Shell
104‧‧‧Support structure
106‧‧‧Flow zone
107‧‧‧Inlet 埠/埠/inlet valve or 埠
108‧‧‧ Spacer/microfluidic loop structure
109‧‧‧ inner surface
110 ‧‧‧ Cover
114 ‧‧‧Framework
116 ‧‧‧Microfluidic circuit materials
120‧‧‧Microfluidic circuit
122‧‧‧Microfluidic channel
124‧‧‧Microfluid isolation fence
126‧‧‧Microfluid isolation fence
128‧‧‧Microfluid isolation fence
130‧‧‧Microfluidic isolation fence
132‧‧‧Small object trap/loop
134‧‧‧ side channel
150‧‧‧ system
154‧‧‧Master controller
156‧‧‧Control Module
158‧‧‧ digital memory
160‧‧‧Media Module
162‧‧‧Power Module
164‧‧‧ imaging module
166‧‧‧ tilt module
168‧‧‧Other modules
170‧‧‧Display devices
172‧‧‧Input/Output Devices
178‧‧‧Media source
180‧‧‧Fluid medium
190‧‧‧ tilting device
192‧‧‧Power supply
194‧‧‧ imaging device
200‧‧‧Microfluidic devices
202‧‧‧Open area/room
204‧‧‧ bottom electrode
206‧‧‧Electrode activated substrate
208‧‧‧ inner surface
210‧‧‧Top electrode
212‧‧‧Power supply
214‧‧‧DEP electrode area
214a‧‧‧DEP electrode area
216‧‧‧Light source
218‧‧‧Light pattern
220‧‧‧ square pattern
222‧‧‧埠
224‧‧‧Isolation fence
226‧‧‧Isolation fence
228‧‧‧Isolation fence
230‧‧‧Microfluidic devices
232‧‧‧Separate structure
234‧‧‧ proximal opening
236‧‧‧Connected area
238‧‧‧ distal opening
240‧‧‧Separation zone
242‧‧‧ Flow
244‧‧‧Secondary flow
246‧‧‧ tiny objects
248‧‧‧Second fluid medium / second medium / fence
250‧‧‧Microfluidic devices
252‧‧‧Frame
254‧‧‧First medium/first fluid medium
256‧‧‧Microfluidic loop structure
258‧‧‧Second medium
260‧‧‧Microfluidic circuit materials
262‧‧‧Microfluidic circuit
264‧‧‧Microfluidic channel/flow channel
266‧‧‧Isolation fence
268‧‧‧Connected area
270‧‧‧Separation zone
272‧‧‧Separate structure
274‧‧‧ proximal opening
276‧‧‧ distal opening
278‧‧‧flow/arrow
280‧‧‧Microfluidic devices
282‧‧‧Secondary flow
284‧‧‧ Shell
286‧‧‧Substrate
288‧‧‧ Cover
290‧‧‧Microfluidic devices
292‧‧‧ inner surface
294‧‧‧ inner surface
296‧‧‧矽oxy linker
298‧‧‧Complex modified monolayer of surface/surface modifying ligands
300‧‧‧ Nest/support structure
302‧‧‧ socket
304‧‧‧Electrical Signal Generation Subsystem
306‧‧‧ Thermal Control Subsystem
308‧‧‧ Controller
310‧‧‧ interface
312‧‧ ‧ shell
314‧‧‧ Fluid path
316‧‧‧ entrance
318‧‧‧Export
320‧‧‧Microfluidic devices
322‧‧‧Printed circuit board assembly
324‧‧‧ tandem
330‧‧‧Light Regulation Subsystem
332‧‧‧Light source / first light source
334‧‧‧second light source
336‧‧‧beam splitter/objective lens
338‧‧‧beam splitter/two-color filter
340‧‧‧ objective lens
342‧‧‧sample plane
344‧‧
346‧‧‧ two-color filter
348‧‧‧Detector
350‧‧‧Microscope
355‧‧‧System
400‧‧‧Microfluidic device
412‧‧‧ outer hydrophobic layer
414‧‧‧Internal dielectric layer
416‧‧‧Conductive layer/photoreactive layer
418‧‧‧electrode
420‧‧‧Support/glass support
422‧‧‧ outer hydrophobic layer
428‧‧‧Inner/inner surface/electrode
430‧‧‧Support
435‧‧‧shell
440‧‧‧Liquid droplets/aqueous droplets
500‧‧‧ devices
600‧‧‧Microfluidic devices
602‧‧‧ fluid input
604‧‧‧ Fluid input
606‧‧‧Drop generator
608‧‧‧ interface
612‧‧‧Microfluidic channel
614‧‧‧Microfluidic channel
616‧‧‧room/accommodating fence
620‧‧‧ fluid droplets
622‧‧‧Fluid Medium / Second Fluid Medium / Liquid Medium / Aqueous Medium / Sample Material / Medium / Sample Medium
624‧‧‧First fluid medium/liquid medium/immiscible medium
630‧‧‧Small objects/biological objects
632‧‧‧ fluid droplets
700‧‧‧Microfluidic devices
716‧‧ ‧ room / accommodating fence
800‧‧‧ Process
1001‧‧‧ mark
1102‧‧ layer
1202‧‧‧ mark
1302‧‧‧ mark
1402‧‧‧ mark
1501‧‧‧ mark
1502‧‧‧ mark
1602‧‧‧ mark
L _con ‧‧‧ length
W _con ‧‧‧Width
W _ch ‧‧‧Width/channel width
D _p ‧‧‧ penetration depth / maximum penetration depth
W _{con1 ‧‧‧Width}
W _con2 ‧‧‧Width
L _c1 ‧‧‧ length
L _c2 ‧‧‧ length

1A illustrates a generalized microfluidic device and system having associated control devices for controlling and monitoring microfluidic devices, in accordance with some embodiments of the present invention. 1B is a vertical cross-sectional view of a microfluidic device having a substrate, a cover, and spacer elements that together form an outer casing configured to contain a liquid medium and liquid droplets that are immiscible in the liquid medium. The substrate has an electrowetting configuration that allows manipulation of the droplets within the housing. 1C and 1D illustrate a microfluidic device in accordance with some embodiments of the present invention. 2A and 2B illustrate a separation fence of some embodiments of the present invention. Figure 2C illustrates a detailed isolation fence of some embodiments of the present invention. 2D-F illustrate an isolation fence of some other embodiments of the present invention. Figure 2G illustrates a microfluidic device in accordance with an embodiment of the present invention. 2H illustrates a coated surface of a microfluidic device in accordance with an embodiment of the present invention. 3A illustrates a specific example of a system for use with a microfluidic device and associated control device in accordance with some embodiments of the present invention. Figure 3B illustrates an imaging device in accordance with some embodiments of the present invention. 4 illustrates an example of a microfluidic device having a two-sided (duolithic) substrate having an EW configuration and a DEP configuration. Figure 5 illustrates an example of a microfluidic device having a two-piece substrate having an EW configuration and a DEP configuration. 6 is a horizontal cross-sectional view of a microfluidic device that can include an electrowetting configuration as shown in FIG. 1B and includes a plurality of microfluidic channels, a chamber that is open from at least one microfluidic channel, and a droplet generator. In this embodiment, one microfluidic channel contains an aqueous medium (light color) and the microfluidic channel attached to the droplet generator contains a non-aqueous medium (dark color). Each chamber also contains an aqueous medium or a non-aqueous medium. 7 is a horizontal cross-sectional view of a microfluidic device that can include an electrowetting configuration as shown in FIG. 1B and includes a plurality of microfluidic channels, a chamber that is open from at least one microfluidic channel, and a droplet generator. In this embodiment, one microfluidic channel and the first set of chambers contain an aqueous medium (light color), while the microfluidic channels connected to the droplet generator and the second set of chambers contain a hydrophobic medium (dark color). Figure 6 presents a variation of the embodiment shown in Figure 5, wherein each chamber containing an aqueous medium is positioned directly through a passage containing a hydrophobic medium from a corresponding chamber containing a hydrophobic medium. Figure 8 is a diagram of a method of processing a biological micro-object in a microfluidic device. Figure 9 is a method for forming a substrate of a microfluidic device. 10 is a cross-sectional view of the substrate processed in the middle of the method illustrated in FIG. Figure 11 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. Figure 12 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. Figure 13 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. Figure 14 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. Figure 15 is a cross-sectional view of the substrate processed in the middle of the method illustrated in Figure 9. Figure 16 is a cross-sectional view showing the substrate processed in the middle of the method illustrated in Figure 9. 17 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. 18 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. 19 is a cross-sectional view of a system incorporating multiple microfluidic applications in accordance with an embodiment. Figure 20A is a view of a representation of an electrical addressing operation of one of the functional aspects of the embodiment illustrated in Figure 17; Figure 20B is a view of a functional representation of an electrical addressing operation in conjunction with one of the embodiments of Figure 17; 21A-21C are photographic representations of the movement of aqueous droplets on a modified microfluidic surface of an embodiment of the invention.

100‧‧‧Microfluidic devices

102‧‧‧Shell

104‧‧‧Support structure

106‧‧‧Flow zone

107‧‧‧Inlet 埠/埠/inlet valve or 埠

108‧‧‧ Spacer/microfluidic loop structure

109‧‧‧ inner surface

110‧‧‧ Cover

114‧‧‧Frame

116‧‧‧Microfluid circuit materials

120‧‧‧Microfluidic circuit

122‧‧‧Microfluidic channel

124‧‧‧Microfluid isolation fence

126‧‧‧Microfluid isolation fence

128‧‧‧Microfluid isolation fence

130‧‧‧Microfluidic isolation fence

132‧‧‧Small object trap/loop

134‧‧‧ side channel

150‧‧‧ system

154‧‧‧Master controller

156‧‧‧Control Module

158‧‧‧ digital memory

160‧‧‧Media Module

162‧‧‧Power Module

164‧‧‧ imaging module

166‧‧‧ tilt module

168‧‧‧Other modules

170‧‧‧Display devices

172‧‧‧Input/Output Devices

178‧‧‧Media source

180‧‧‧Fluid medium

190‧‧‧ tilting device

192‧‧‧Power supply

Claims (198)

A microfluidic device having an electrowetting configuration, the microfluidic device comprising: a substrate having a dielectric layer, a droplet actuation surface, and a first electrode configured to be coupled to an AC voltage source; and a second electrode Connected to the AC voltage source; wherein the dielectric layer is electrically coupled to the first electrode, and wherein the droplet actuation surface comprises a hydrophobic layer covalently bonded to the dielectric layer. The microfluidic device of claim 1, wherein the device has a single-sided electrowetting configuration. The microfluidic device of claim 2, wherein the second electrode is a mesh electrode included in the substrate. The microfluidic device of claim 1, wherein the device has a photo-wetting (OEW) configuration. The microfluidic device of claim 1, wherein the device has an electrowetting on dielectric configuration (EWOD). The microfluidic device of any one of claims 1 to 5, wherein the hydrophobic layer comprises a surface modifying ligand and a single layer linking the surface modifying ligand to a linking group of the surface, wherein the droplet is actuated The surface has the structure of formula II: Formula II Is the surface of the dielectric layer; V is -P(O)(OY)W- or -Si(OZ) ₂ W-; W is -O-, -S- or -NH- and is attached to the surface; a bond to an adjacent argon atom attached to the surface or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; H is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer from 1 to 20 is an integer of 0 or 1 to 20; (n + [(h + j)·k] + m) is an integer from 11 to 25; when k is 1, then m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 And M is hydrogen. The microfluidic device of any of claims 1 to 6, wherein the electrowetting configuration of the device is included in a first portion of the device, and wherein the device further comprises a second having a dielectrophoresis (DEP) configuration section. A microfluidic device comprising: a substrate having at least one electrode configured to be coupled to a voltage source; a cover having at least one electrode configured to connect to the voltage source; and at least one spacer element, wherein The substrate and the cover are substantially parallel to one another and are joined together by the spacer element to define an outer casing configured to contain a liquid, wherein the substrate has a droplet actuation surface that partially defines the outer casing, the droplet actuation The surface has an inner dielectric layer and an outer hydrophobic layer, wherein the outer hydrophobic layer comprises self-associating molecules covalently bonded to the surface of the inner dielectric layer, thereby forming a dense stack thereon a hydrophobic monolayer, and wherein when the at least one electrode of the substrate and the at least one electrode of the cap are connected to opposite terminals of the voltage source, the substrate is capable of applying an electrowetting force to the droplets of the substrate Aqueous droplets that contact the surface. The microfluidic device of claim 8, wherein the self-associating molecules of the hydrophobic monolayer each comprise a surface modifying ligand and a linking group linking the surface modifying ligand to the surface of the inner dielectric layer, wherein The droplet actuation surface has the structure of Formula II: Formula II: where Is the surface of the dielectric layer; V is -P(O)(OY)W- or -Si(OZ) ₂ W-; W is -O-, -S- or -NH- and is attached to the surface; a bond to an adjacent argon atom attached to the surface or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; H is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer from 1 to 20 is an integer of 0 or 1 to 20; (n + [(h + j)·k] + m) is an integer from 11 to 25; when k is 1, then m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 And M is hydrogen. A microfluidic device according to claim 9, wherein the V system is -Si(OZ) ₂ W-. A microfluidic device according to claim 9, wherein the V system is -P(O)(OY)W-. The microfluidic device of any one of claims 9 to 11, wherein n is an integer from 1 to 20, and wherein R is hydrogen. The microfluidic device of claim 12, wherein m is an integer from 1 to 20, and wherein M is hydrogen. The microfluidic device of claim 13, wherein m is 2. The microfluidic device of any one of clauses 9 to 11, wherein n is an integer from 1 to 20, and wherein R is fluorine. The microfluidic device of claim 15, wherein m is an integer from 1 to 20, and wherein M is hydrogen. The microfluidic device of claim 16, wherein m is 2. The microfluidic device of any one of claims 9 to 17, wherein k is 1 . The microfluidic device of any one of claims 9 to 17, wherein k is 0. The microfluidic device of any one of claims 9 to 19, wherein the sum of (n + [(h + j)·k] + m) is an integer from 13 to 19. The microfluidic device of any one of claims 8 to 20, wherein the outer hydrophobic layer of the droplet actuation surface of the substrate has a thickness of less than 5 nanometers. The microfluidic device of any one of claims 8 to 21, wherein the outer hydrophobic layer of the droplet actuation surface of the substrate is patterned such that the selected region is relatively hydrophilic compared to the remainder of the outer hydrophobic layer. The microfluidic device of any one of claims 8 to 22, wherein the inner dielectric layer of the droplet actuation surface of the substrate comprises a first layer of dielectric material comprising an oxide. The microfluidic device of any one of claims 8 to 23, wherein the oxide is a metal oxide. The microfluidic device of claim 24, wherein the metal oxide is alumina. The microfluidic device of any one of claims 23 to 25, wherein the first layer of dielectric material is formed by atomic layer deposition. The microfluidic device of any one of claims 23 to 26, wherein the inner dielectric layer of the droplet actuation surface of the substrate further comprises a second layer of dielectric material, and wherein the outer hydrophobic layer is covalently bonded To the first layer of dielectric material. The microfluidic device of claim 27, wherein the second layer of dielectric material comprises an oxide or a nitride. The microfluidic device of claim 28, wherein the second layer of dielectric material is selected from the group consisting of cerium oxide and cerium nitride. The microfluidic device of any one of claims 27 to 29, wherein the second layer of dielectric material is formed by plasma assisted chemical vapor deposition. The microfluidic device of any one of claims 23 to 30, wherein the first layer of dielectric material comprises first and second sub-layers of dielectric material, wherein the first sub-layer is covalently bonded to the hydrophobic layer. The microfluidic device of claim 31, wherein the first dielectric material sublayer comprises yttrium oxide. The microfluidic device of claim 31 or 32, wherein the first layer of dielectric material is deposited by ALD. The microfluidic device of any one of claims 31 to 33, wherein the first layer of dielectric material has a thickness of from about 10 nm to about 20 nm. The microfluidic device of claim 34, wherein the first dielectric material sublayer has a thickness of from about 2 nm to about 10 nm. The microfluidic device of any one of claims 8 to 35, wherein the inner dielectric layer of the droplet actuation surface of the substrate has a thickness of at least about 40 nanometers. The microfluidic device of claim 36, wherein the inner dielectric layer of the droplet actuation surface of the substrate has a thickness of from about 40 nanometers to about 120 nanometers. The microfluidic device of any one of claims 8 to 37, wherein the substrate further comprises a photoreactive layer having a first side contacting the inner dielectric layer and a second side contacting the at least one electrode. The microfluidic device of claim 38, wherein the photoreactive layer comprises hydrogenated amorphous germanium (a-Si:H). The microfluidic device of claim 38 or 39, wherein the photoreactive layer has a thickness of at least 900 nanometers. The microfluidic device of claim 40, wherein the photoreactive layer has a thickness of between about 900 nanometers and 1100 nanometers. The microfluidic device of claim 38, wherein the photoreactive layer comprises a plurality of conductors, each conductor being controllably connectable to the at least one electrode of the substrate via a phototransistor switch. The microfluidic device of any of claims 8 to 42, wherein the substrate comprises a single electrode configured to be connected to an AC voltage source, the single electrode comprising an indium-tin oxide (ITO) layer. The microfluidic device of any of claims 8 to 42, wherein the substrate comprises a single electrode configured to be coupled to an AC voltage source, the single electrode comprising a conductive germanium layer. The microfluidic device of any of claims 8 to 37, wherein the substrate comprises a plurality of electrodes, each electrode being configured to be coupled to one or more sources of AC voltage. The microfluidic device of claim 45, wherein each of the plurality of electrodes is connectable to one of the one or more AC voltage sources via a transistor switch. The microfluidic device of any one of claims 8 to 46, wherein the cover has an inwardly facing surface that partially defines the outer surface, the inwardly facing surface of the cover having an inner layer and an outer hydrophobic layer, wherein the cover is external The hydrophobic layer comprises self-associating molecules covalently bonded to the surface of the inner layer of the cap, thereby forming a densely packed hydrophobic monolayer thereon. The microfluidic device of claim 47, wherein the self-associating molecules of the hydrophobic monolayer of the cap each comprise a surface modifying ligand and a link linking the surface modifying ligand to the surface of the inner layer of the cap a group wherein the inwardly facing surface of the lid has the structure of Formula II: Formula II: where Is the surface of the dielectric layer; V is -P(O)(OY)W- or -Si(OZ) ₂ W-; W is -O-, -S- or -NH- and is attached to the surface; a bond to an adjacent argon atom attached to the surface or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; H is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer from 1 to 20; n is 0 or an integer from 1 to 20; (n + [( h + j)·k] + m) is an integer of 11 to 25; when k is 1, m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. The microfluidic device of claim 48, wherein the self-associating molecules of the hydrophobic monolayer of the cap are identical to the self-associating molecules of the hydrophobic monolayer of the droplet-actuating surface of the substrate. The microfluidic device of any one of claims 47 to 49, wherein the outer hydrophobic layer of the inwardly facing surface of the cap has a thickness of less than 5 nanometers. The microfluidic device of any one of claims 47 to 50, wherein the inner layer of the cover is an inner dielectric layer. The microfluidic device of claim 51, wherein the cover further comprises a photoreactive layer. The microfluidic device of claim 51, wherein the cover comprises a plurality of electrodes, each electrode being configured to be coupled to one or more AC voltage sources. The microfluidic device of any one of claims 8 to 53, wherein the at least one spacer element comprises a ruthenium-based organic polymer. The microfluidic device of claim 54, wherein the ruthenium-based organic polymer is selected from the group consisting of polydimethyl siloxane (PDMS) and photopatternable polyfluorene oxide (PPS). The microfluidic device of any of claims 8 to 53, wherein the at least one spacer element comprises SU-8. The microfluidic device of any one of claims 8 to 56, wherein the at least one spacer element has a thickness of at least 30 microns. The microfluidic device of any one of claims 8 to 57, wherein the at least one spacer element defines one or more microchannels within the housing. The microfluidic device of claim 58, wherein the at least one spacer element further defines a plurality of chambers within the housing, wherein each chamber is open from the at least one microchannel. A method of fabricating a microfluidic device, the method comprising: bonding a spacer element to an inner surface of a cover having at least one electrode configured to be coupled to a voltage source; bonding the spacer element and the cover to at least one configuration Forming a dielectric surface of the substrate connected to the electrode of the voltage source, whereby the spacer element is sandwiched between the inner surface of the cover and the dielectric surface of the substrate, wherein the cover and the substrate are substantially in contact with each other Parallelly oriented, and the substrate, the spacer element and the cover collectively define an outer casing configured to contain a liquid; forming a densely packed hydrophobic monolayer on at least a portion of the inner surface of the cover by vapor deposition, wherein The hydrophobic monolayer comprises a self-associating molecule covalently bonded to the inner surface of the cap; and a densely packed hydrophobic monolayer formed on at least a portion of the dielectric surface of the substrate by vapor deposition, wherein the hydrophobic layer The single layer comprises self-associating molecules covalently bonded to the dielectric surface of the substrate. The method of claim 60, wherein the self-associating molecules of the hydrophobic monolayer and the self-associating molecules of the hydrophobic monolayer of the substrate each comprise a surface modifying ligand and the surface modifying ligand Linking to the inner surface of the cover and the linking group of the dielectric surface of the substrate, wherein the resulting surface of the cover and the substrate has the structure of Formula II: Formula II: where Is the surface of the dielectric layer; V is -P(O)(OY)W- or -Si(OZ) ₂ W-; W is -O-, -S- or -NH- and is attached to the surface; a bond to an adjacent argon atom attached to the surface or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; H is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer from 1 to 20; n is 0 or an integer from 1 to 20; (n + [( h + j)·k] + m) is an integer of 11 to 25; when k is 1, m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. The method of claim 61, wherein the V system is -Si(OZ) ₂ W-. The method of claim 61, wherein the V is -P(O)(OY)W-. The method of any one of clauses 61 to 63, wherein n is an integer from 1 to 20, and wherein R is hydrogen. The method of claim 64, wherein m is an integer from 1 to 20, and wherein M is hydrogen. The method of claim 65, wherein m is 2. The method of any one of claims 61 to 63, wherein n is an integer from 1 to 20, and wherein R is fluorine. The method of claim 67, wherein m is an integer from 1 to 20, and wherein M is hydrogen. The method of claim 68, wherein m is 2. The method of any one of clauses 61 to 69, wherein k is 1. The method of any one of clauses 61 to 69, wherein k is 0. The microfluidic device of any one of claims 61 to 71, wherein the sum of (n + [(h + j)·k] + m) is an integer from 13 to 19. A microfluidic device comprising: a conductive germanium substrate having a dielectric stack and at least one electrode configured to be coupled to a voltage source; a cover having at least one electrode configured to connect to a voltage source; and at least a spacer element, wherein the conductive germanium substrate and the cover are substantially parallel to each other and joined together by the spacer element to define a housing configured to contain a liquid, wherein the conductive germanium substrate has an inwardly defined portion that partially defines the outer casing a surface, the inwardly facing surface comprising an outermost surface of the dielectric stack, and wherein the substrate is capable of being electrically charged when the at least one electrode of the substrate and the at least one electrode of the cover are connected to opposite terminals of an AC voltage source A wetting force is applied to the aqueous droplets that are in contact with the inwardly facing surface of the substrate. The microfluidic device of claim 73, wherein the conductive germanium substrate comprises an amorphous germanium. The microfluidic device of claim 73, wherein the conductive germanium substrate comprises a photovoltaic crystal array. The microfluidic device of claim 73, wherein the conductive germanium substrate comprises an array of electrodes. The microfluidic device of any one of claims 73 to 76, wherein the inwardly facing surface of the electrically conductive tantalum substrate further comprises an outer hydrophobic layer comprising a self-association of covalent bonding to the internal dielectric stack molecule. The microfluidic device of any one of claims 73 to 77, wherein the internal dielectric stack comprises a first layer of dielectric material and a second layer of dielectric material. The microfluidic device of claim 78, wherein the first layer of dielectric material has a first surface and an opposite surface, wherein the first surface of the first layer is adjacent to the second layer, and wherein the first layer is opposite A surface forms the outermost surface of the dielectric stack. The microfluidic device of claim 78 or 79, wherein the first layer of dielectric material comprises a metal oxide. The microfluidic device of claim 80, wherein the first layer of dielectric material comprises aluminum oxide or hafnium oxide. The microfluidic device of any one of clauses 78 to 81, wherein the second layer of dielectric material comprises an oxide or a nitride. The microfluidic device of claim 82, wherein the second layer of dielectric material comprises hafnium oxide or tantalum nitride. The microfluidic device of any one of claims 78 to 83, wherein the second layer is deposited by a plasma assisted chemical vapor deposition (PECVD) technique. The microfluidic device of any one of clauses 78 to 84, wherein the first layer is deposited by an atomic layer deposition (ALD) technique. The microfluidic device of any one of claims 78 to 85, wherein the internal dielectric stack comprises a third layer having a first surface and an opposite surface, wherein the first surface of the third layer is adjacent to the first layer The opposing surface, and wherein the opposing surface of the third layer forms the outermost surface of the dielectric stack. The microfluidic device of claim 86, wherein the third layer comprises cerium oxide. The microfluidic device of claim 86 or 87, wherein the third layer is deposited by atomic layer deposition (ALD) techniques. The microfluidic device of any one of claims 78 to 85, wherein the first layer of dielectric material has a thickness of from about 10 nm to about 50 nm. The microfluidic device of any one of claims 86 to 88, wherein the first layer of dielectric material has a thickness of from about 5 nm to about 20 nm, and the third layer of dielectric material has a thickness of from about 2 nm to about 10 nm The thickness. The microfluidic device of any one of claims 78 to 90, wherein the second layer of dielectric material has a thickness of from about 30 nm to about 100 nm. The microfluidic device of any one of claims 73 to 91, wherein the dielectric stack of the droplet actuation surface of the substrate has a thickness of at least about 40 nanometers. The microfluidic device of claim 92, wherein the dielectric stack of the droplet actuation surface of the substrate has a thickness of between about 40 nanometers and about 120 nanometers. The microfluidic device of any one of claims 73 to 93, wherein the dielectric layer has an impedance of between about 50 kilohms (kOhms) and about 150 kilohms. The microfluidic device of any one of claims 73 to 94, wherein the device comprises: a dielectrophoresis module that performs a first microfluidic operation in response to a first applied voltage of a first frequency; and an electrowetting module that receives An output of the dielectrophoresis module and performing a second microfluidic operation in response to a second applied voltage of the second frequency, wherein the electrowetting module comprises the dielectric stack of the conductive germanium substrate. The system of claim 95, further comprising a bridge between the first module and the second module. The system of claim 96, wherein the bridge does not perform the first or the second microfluidic operation. The system of claim 96 or 97, wherein the bridge is an electrically neutral zone. The system of any one of clauses 96 to 98, wherein the bridge comprises a tube. The system of any one of clauses 96 to 98, wherein the bridge comprises a polymer. The system of any one of clauses 95 to 100, wherein the output is a biological material. The system of any one of clauses 95 to 101, wherein the first frequency is in the range of 100 kHz to 10 mHz. The system of any one of clauses 95 to 102, wherein the second frequency is in the range of 1 kHz to 300 kHz. The system of any one of clauses 95 to 103, wherein the first voltage is in the range of 1 volt to 10 volts. The system of any one of clauses 95 to 104, wherein the second voltage is in the range of 10 volts to 100 volts. [Claim 105] The system of any one of claims 95 to 105, wherein the conductive germanium substrate is monolithic. The system of any one of clauses 95 to 105, wherein the conductive germanium substrate is in two pieces. The system of claim 105 or 106, wherein the conductive germanium substrate comprises an amorphous germanium. The system of claim 105 or 106, wherein the conductive germanium substrate comprises a photovoltaic crystal array. The system of claim 105 or 106, wherein the conductive germanium substrate comprises an array of electrodes. A system for transporting microscopic objects, biological products, and/or reagents that are compatible with and/or soluble in an aqueous medium, the system comprising: a microfluidic device having a substrate and a microfluidic loop structure An outer casing, wherein the substrate comprises a hydrophobic monolayer covalently bonded to at least a portion of an upper surface of the substrate; a first fluid medium that is immiscible with the aqueous medium; and at least one aqueous droplet. The system of claim 110, wherein the hydrophobic monolayer has a surface modifying ligand and a linking group linking the surface modifying ligand to the surface, wherein the hydrophobic surface has the structure of Formula II: Formula II Is the surface; V-system - P(O)(OY)W- or -Si(OZ)2W-; W-system -O-, -S- or -NH- and is attached to the surface; Z-line to attach to a bond of the surface adjacent to a ruthenium atom or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; M is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer from 1 to 20; n is 0 or an integer from 1 to 20; (n + [(h + j)· k] + m) is an integer of 11 to 25; when k is 1, m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, m is at least 2 and M is hydrogen . A system of claim 110 or 111, wherein the substrate comprises a conductive substrate. The system of any one of claims 1 to 72, wherein the microfluidic device is the microfluidic device of any one of claims 1 to 72. The system of claim 113, wherein the microfluidic device comprises an optically actuated EW configuration. The system of claim 113 or 114, wherein the microfluidic device further comprises a DEP configuration. The system of any one of claims 110 to 115, wherein the first fluid medium comprises at least one organic or organic ruthenium compound having a backbone structure comprising atoms selected from the group consisting of carbon, ruthenium and oxygen. The system of claim 116, wherein the backbone structure of the at least one organogermanium compound comprises a deuterium atom and optionally an oxygen atom. The system of claim 116, wherein the backbone structure of the at least one organic compound comprises a carbon atom and optionally an oxygen atom. The system of claim 118, wherein the backbone structure is branched. The system of any one of claims 116 to 119, wherein the first fluid medium comprises one or more acyclic organic or organic phosphonium compounds. The system of claim 120, wherein the first fluid medium consists of an acyclic organic or organic hydrazine compound. The system of any one of claims 110 to 121, wherein the first fluid medium does not comprise a perfluorocarbon atom. The system of any one of claims 110 to 121, wherein the substituent of the carbon atom of the compound of the first fluid medium comprises no more than 90% of the fluorine substituent. The system of any one of clauses 111 to 121, wherein the surface modifying ligand comprises at least a first portion comprising a perfluorocarbon atom located at an inward end of the hydrophobic monolayer. The system of claim 124, wherein all of the carbon atoms of the hydrophobic monolayer are perfluorinated. The system of any one of claims 110 to 125, wherein the first fluid medium comprises more than one organic or organic phosphonium compound. The system of any one of claims 110 to 126, wherein the outer casing further comprises a cover. The system of claim 127, wherein the cover is transparent. The system of claim 127 or 128, wherein the cover comprises glass and/or indium antimony oxide (ITO). The system of any one of claims 127 to 129, wherein the cover comprises an electrode. The system of any one of claims 110 to 130, wherein the aqueous droplets comprise a surfactant. The system of claim 131, wherein the surfactant comprises a nonionic surfactant. The system of claim 131 or 132, wherein the surfactant comprises a block alkylene oxide copolymer, a fatty ester ethoxylated sorbitan, an ethoxylated fluorosurfactant, sodium lauryl sulfate or 2,4,7,9, tetramethyl-5-decyne-4,7,-diol ethoxylate. The 131 to request entry system of any one of 133, wherein the surfactant comprises ^{Capstone® FS-30 (DuPont TM,} Synquest Laboratories). The system of any one of claims 110 to 134, wherein the droplets comprise a phosphate buffered saline solution. The system of any one of claims 110 to 135, wherein the aqueous droplet comprises at least one microscopic object. The system of claim 136, wherein the micro-organism system is a biological micro-object. The system of any one of claims 110 to 137, wherein the aqueous droplet comprises a biological product comprising a nucleic acid and/or a protein. The system of any one of claims 110 to 138, wherein the aqueous droplet comprises a reagent. A kit for transporting microscopic objects, biological products, and/or reagents that are compatible with and/or soluble in an aqueous medium, the kit comprising: a microfluidic device having a substrate and a microfluidic An outer casing of a loop structure, wherein the substrate comprises a hydrophobic monolayer covalently bonded to at least a portion of an upper surface of the substrate; and a first fluid medium that is immiscible with the aqueous medium. The system of claim 140, wherein the hydrophobic monolayer has a surface modifying ligand and a linking group linking the surface modifying ligand to the surface, wherein the hydrophobic surface has the structure of Formula II: Formula II Is the surface; V-system - P(O)(OY)W- or -Si(OZ)2W-; W-system -O-, -S- or -NH- and is attached to the surface; Z-line to attach to a bond of the surface adjacent to a ruthenium atom or a bond to the surface; Y is a bond to the adjacent phosphorus atom attached to the surface or a bond to the surface; R is hydrogen or fluorine; M is hydrogen or fluorine; h is independently an integer of 2 or 3; j is 1; k is 0 or 1; m is 0 or an integer of 1 to 20 is an integer of 0 or 1 to 20; (n + [(h + j)·k And m) is an integer of 11 to 25; when k is 1, m is at least 2 and M is hydrogen; and when k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. A system of claim 140 or 141, wherein the substrate comprises a conductive substrate. The system of any one of claims 1 to 72, wherein the microfluidic device is the microfluidic device of any one of claims 1 to 72. A method of operating a microfluidic device according to any one of claims 8 to 72, the method comprising: filling the outer casing or a portion thereof with a first liquid medium; at least one of the at least one electrode of the substrate and the cover Applying an AC voltage potential between the electrodes; introducing a first liquid droplet into the outer casing, wherein the first droplet is immiscible in the first liquid medium; and applying an electrowetting force to the first droplet The first droplet is moved to a desired location within the housing. The method of claim 144, wherein the first liquid medium is an oil. The method of claim 144, wherein the first liquid medium is a polyoxygenated oil, a fluorinated oil, or a combination thereof. The method of any one of claims 144 to 146, wherein the applied AC voltage potential is at least 20 ppV. The method of claim 147, wherein the applied AC voltage potential is between about 25 ppV and 35 ppV. The method of any one of claims 144 to 148, wherein the applied AC voltage potential has a frequency of between about 1 kHz and 100 kHz. The method of any one of claims 144 to 149, wherein the microfluidic device comprises a droplet generator, and wherein the droplet generator introduces the first droplet into the outer casing. The method of any one of claims 144 to 150, wherein the first droplet comprises an aqueous solution. The method of claim 151, wherein the first droplet comprises at least one micro object. The method of claim 152, wherein the at least one micro-organism system is a biological micro-object. The method of claim 153, wherein the biological widget system cells. The method of any one of clauses 151 to 154, wherein the aqueous solution is a cell culture medium. The method of claim 152, wherein the at least one micro-file system has affinity capture beads for the material of interest. The method of claim 156, wherein the first droplet comprises from 2 to 20 capture beads. The method of claim 156 or 157, wherein the material of interest is a biological cell secretion. The method of claim 156 or 157, wherein the material of interest is selected from the group consisting of: DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. The method of claim 151 or 152, wherein the first droplet comprises a reagent. The method of claim 160, wherein the reagent is a cell lysing reagent. The method of claim 161, wherein the reagent comprises a non-ionic detergent. [Item 162] The method of claim 162, wherein the concentration of the nonionic detergent is less than 0.2%. The method of claim 160, wherein the reagent is a proteolytic enzyme. The method of claim 163, wherein the proteolytic enzyme is inactivated. The method of any one of claims 144 to 164, further comprising: introducing a second liquid droplet into the outer casing, wherein the liquid of the second droplet is immiscible in the first liquid medium but is The liquid of the first droplet is miscible; moving the second droplet to the position within the outer casing adjacent to the first droplet by applying an electrowetting force to the second droplet; and fusing the second micro The first droplet is dropped with the first droplet to form a first combined droplet. The method of claim 165, wherein the second droplet and the first droplet are fused by applying an electrowetting force to the second and/or the first droplet. The method of claim 165 or 166, wherein the first droplet comprises a biological micro-object, and wherein the second droplet comprises a reagent. The method of claim 167, wherein the reagent contained in the second droplet is selected from the group consisting of a lysis buffer, a fluorescent label, and a luminescent assay reagent. The method of claim 167, wherein the reagent contained in the second droplet is a lysis buffer, and wherein the biological cell dissolves after the first droplet is fused with the second droplet. The method of any one of claims 165 to 169, further comprising: introducing a third liquid droplet into the outer casing, wherein the liquid of the third droplet is immiscible in the first liquid medium, but Miscible with the liquid of the first combined droplet; and moving the third droplet to a position adjacent to the first merged droplet within the outer shell by applying an electrowetting force to the third droplet; and fusion The third droplet merges with the first merged droplet to form a second merged droplet. The method of claim 170, wherein the third droplet and the first combined droplet are fused by applying an electrowetting force to the third droplet and/or the first combined droplet. The method of claim 170 or 171, wherein the third droplet comprises a reagent. The method of claim 172, wherein the third droplet comprises a protease inhibitor. The method of claim 172, wherein the third droplet comprises from 1 to 20 capture beads having an affinity for the material of interest. The method of claim 174, wherein the capture beads comprise an oligonucleotide capture agent. The method of claim 175, wherein the oligonucleotide capture agents are poly dT oligonucleotides. The method of any one of claims 174 to 176, wherein the material of interest is selected from the group consisting of: DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. The method of any one of claims 174 to 177, further comprising: discharging the 1 to 20 capture beads from the microfluidic device. The method of any one of claims 170 to 178, further comprising: introducing a fourth liquid droplet into the outer casing, wherein the liquid of the fourth droplet is immiscible in the first liquid medium, but Miscible with the liquid of the second combined droplet; moving the fourth droplet to the position within the outer casing adjacent to the second merged droplet by applying an electrowetting force to the fourth droplet; and fusing the The fourth droplet and the second merged droplet form a third merged droplet. The method of claim 179, wherein the fourth droplet and the second combined droplet are fused by applying an electrowetting force to the fourth droplet and/or the second combined droplet. The method of claim 179 or 180, wherein the fourth droplet comprises a reagent. The method of claim 181, wherein the reagent contained in the fourth droplet comprises a mixture comprising a buffer, dNTP, and a polymerase suitable for performing a reverse transcription reaction. The method of claim 181, wherein the reagent contained in the fourth droplet comprises a mixture comprising a buffer, dNTP, and a polymerase suitable for performing a whole genome amplification reaction. The method of any one of claims 144 to 183, wherein the first droplet, the second droplet, the third droplet, and the fourth droplet each have a volume of from about 5 nanoliters to 50 nanoliters. The method of claim 184, wherein the first droplet, the second droplet, and the third droplet each have a volume of between about 5 nanoliters and 20 nanoliters. The method of claim 185, wherein the volume of the second droplet and/or the third droplet is substantially equal to the volume of the first droplet. The method of claim 185 or 186, wherein the volume of the fourth droplet is about 1 to 3 times the volume of the first droplet. The method of claim 187, wherein the fourth droplet has a volume of from about 10 nanoliters to 30 nanoliters. The method of any one of claims 144 to 188, wherein the housing comprises at least one microchannel. The method of claim 189, wherein moving the first droplet to a desired location within the housing comprises moving the first droplet through the at least one microchannel. The method of claim 189 or 190, wherein the housing further comprises a plurality of chambers that are open from the at least one microchannel. The method of claim 191, wherein moving the first droplet to a desired location within the housing comprises moving the first droplet into one of the plurality of chambers. The method of any one of clauses 189 to 192, wherein moving the second droplet to a position adjacent to the first droplet comprises moving the second droplet through the at least one microchannel and optionally entering The first droplet is in the chamber. The method of claim 193, wherein moving the third droplet to a position adjacent to the first merged droplet comprises moving the third droplet through the at least one microchannel and optionally entering the first merged micro Drop it in the room. The method of claim 194, wherein moving the fourth droplet to a position adjacent to the second merged droplet comprises moving the fourth droplet through the at least one microchannel and optionally entering the second merged micro Drop it in the room. The method of any one of claims 144 to 195, wherein applying an electrowetting force to move and/or fuse the droplets comprises modifying an effective electrowetting characteristic of the surface of the substrate proximate to the region of the droplet. The method of claim 196, wherein changing the effective electrowetting feature comprises activating the electrowetting electrode of the substrate surface adjacent the region of the (etc.) droplet. The method of claim 197, wherein the substrate comprises a photoreactive layer, and wherein the electrowetting electrodes that activate the surface of the substrate proximate the region of the (etc.) droplets comprise directing a light pattern to the region of the electrowetting surface in.