TW202126571A - 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 system and method

Small 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. The embodiment of the present invention relates to an improvement of a microfluidic device that facilitates the robust manipulation of droplets, thereby allowing complex chemical and biological reactions to be performed on a small scale in an accurate and reproducible manner. By changing the effective wetting properties of the electrowetting surface in the microfluidic device, the droplets can move and fuse in the microfluidic device. These movements can facilitate the workflow of culturing cells in the microfluidic device and processing the cells to evaluate various cell properties as appropriate. Current solutions for electrowetting are extremely limited in nature and cannot scale or implement other functions. Therefore, the industry needs an improved electrowetting surface, a stable substrate for microfluidic applications, and the integration of other functions (such as cell growth and characterization, and then downstream processing by electrowetting), which all contribute to additional medicine Research applications.

In one aspect, the present invention provides a microfluidic device comprising an electrowetting configuration, which includes a substrate having a droplet actuation surface, the droplet actuation surface comprising covalent bonding to an underlying dielectric layer (ie, internal The hydrophobic layer (ie, the outer hydrophobic layer) on the surface of the dielectric layer (or consists of or consists essentially of it). When the microfluidic device is operatively connected to a voltage source, the aqueous droplets that stay on the hydrophobic layer or otherwise contact the hydrophobic layer can be wetted in a reliable and robust manner, and thus move by electrowetting force. The microfluidic device may include a base including a substrate, and the substrate may further have at least one electrode (such as a first electrode) configured to be connected to a voltage source (such as an AC voltage source), the at least one electrode being electrically coupled to the inner dielectric Electric layer. In some embodiments, the microfluidic device further includes a cover and at least one spacer element. The substrate and the cover can be substantially parallel to each other and joined together by spacer elements to define a housing that is configured to contain the liquid medium. In such embodiments, the cover may include at least one electrode that is configured to be connected to a voltage source (for example, an AC voltage source). In some embodiments, the microfluidic device may include a single-sided electrowetting configuration. In these embodiments, the microfluidic device need not include a cover. For example, the base may include a substrate and a first electrode that is configured to be connected to a voltage source (such as an AC voltage source), and the substrate may include a second electrode that is configured to be connected to a voltage source (such as a mesh electrode). ). In some embodiments, the outer hydrophobic layer includes self-associated 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 include a siloxane group. In other embodiments, the self-associating molecules of the hydrophobic monolayer each contain a phosphonic acid group. The siloxyalkyl group or the 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 include a surface modification ligand and a linking group that directly or indirectly links the surface modification ligand 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 modification ligand may include an aliphatic group, such as an alkane group. Therefore, for example, the self-associating molecules of the hydrophobic monolayer can be alkyl-terminated siloxanes or alkyl-terminated phosphonic acid molecules. Alkyl groups may include chains (e.g., unbranched) of at least 10 carbons (e.g., at least 14, 16, 18, 20, 22, or more carbons). In other embodiments, the surface modification ligand may include a fluorine-substituted aliphatic group, such as a fluoroalkyl group. Thus, for example, the self-associating molecule can be a fluoroalkyl-terminated siloxane or a fluoroalkyl-terminated phosphonic acid molecule. The fluoroalkyl group may include a chain (e.g., unbranched) of at least 10 carbons (e.g., at least 14, 16, 18, 20, 22, or more carbons). In certain embodiments, the fluoroalkyl group includes one or more (e.g., at least 4, 6, 8, 10, 12, or more) perfluorocarbons. For example, the fluoroalkyl group may have the chemical formula CF ₃ -(CF ₂ )m-(CH ₂ )n-, where m is at least 2, n is at least 2, and m+n is at least 9. In some embodiments, the surface modification ligand comprises an ether bond 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 (e.g., a perfluoroalkyl group). In some embodiments, the alkyl or fluoroalkyl group of the surface modification ligand does not have a branch. In some embodiments, the alkyl or fluoroalkyl group of the surface modification ligand does not contain any cyclic structure. In some embodiments, the hydrophobic layer outside the substrate has a thickness of less than 5 nanometers (for example, about 1.5 nanometers to 3.0 nanometers). In some embodiments, the outer hydrophobic layer of the substrate may be patterned so that the selected area is relatively hydrophilic compared to the rest of the outer hydrophobic layer. In some embodiments, the inner dielectric layer of the substrate may include a first dielectric material layer. For example, the inner dielectric layer may be composed of a single dielectric material layer. The first dielectric material layer may include an oxide, such as a metal oxide layer (e.g., aluminum oxide, hafnium dioxide, or the like). In some embodiments, the first oxide layer is formed by atomic layer deposition (ALD). Alternatively, the inner dielectric layer may be a dielectric stack including two or more dielectric material layers. Therefore, in some embodiments, the inner dielectric layer may include a first dielectric material layer and a second dielectric material layer. The first dielectric material layer may include an oxide, such as a metal oxide (e.g., aluminum oxide, hafnium dioxide, or the like); and the second dielectric material layer may include an oxide (e.g., silicon oxide) or a nitride (e.g., nitride). Silicon). In these embodiments, the first dielectric material layer may have a first surface contacting the second dielectric material layer and an opposite surface covalently bonded to the hydrophobic layer. In some embodiments, the second dielectric material layer may have a thickness of about 30 nm to about 100 nm, depending on the type of dielectric material used. For example, the second dielectric material layer may include silicon oxide and may have a thickness of about 30 nm to about 50 nm, or about 30 nm to about 40 nm. Alternatively, the second dielectric material layer may include silicon nitride and may have a thickness of about 50 nm to about 100 nm, or about 80 nm to about 100 nm. In some embodiments, the second dielectric material layer is formed by ALD. In other embodiments, the second dielectric material layer is formed by plasma assisted chemical vapor deposition (PECVD) technology. In some embodiments, the first dielectric material layer may have about 10 nm to about 50 nm (for example, about 10 nm to about 20 nm, about 15 nm to about 25 nm, about 20 nm to about 30 nm, about 25 nm to about 35 nm, about 30 nm to about 40 nm, about 35 nm to about 45 nm, about 40 nm to about 50 nm, or any range defined by both of the foregoing endpoints) thickness, and can It is formed by ALD. In other embodiments, the inner dielectric layer may include a third dielectric material layer, wherein the third dielectric material layer has a first surface contacting the first dielectric material layer and an opposite surface covalently bonded to the hydrophobic layer . In these embodiments, the first dielectric material layer may include the oxide described above (or elsewhere in this document), and the second dielectric material layer may include the oxide described above (or elsewhere in this document).物 or nitride. In some embodiments, the third dielectric material layer may include an oxide (such as silicon dioxide) or other dielectric material sufficiently bonded to the siloxane group. In some embodiments, the third dielectric material layer is deposited by ALD. In some embodiments, the third dielectric material layer has a thickness of about 2 nm to about 10 nm, or about 4 nm to about 6 nm. Regardless of the number of layers constituting the inner dielectric layer, the inner dielectric layer may have about 40 nm to about 120 nm (for example, about 40 nm to about 60 nm, about 50 nm to about 70 nm, about 60 nm to about 80 nm). , About 70 nm to about 90 nm, about 80 nm to about 100 nm, about 90 nm to about 110 nm, about 100 nm to about 120 nm, or a range defined by any two of the foregoing endpoints) total thickness. Similarly, the dielectric layer may have about 50 kiloohms to about 150 kiloohms (e.g., about 50 kiloohms to about 75 kiloohms, about 75 kiloohms to about 100 kiloohms, about 100 kiloohms to about 125 kiloohms, about 125 kilohms to about 150 kilohms, or the range defined by any two of the foregoing endpoints). In some embodiments, the substrate may further include a photoreactive layer. The photoreactive layer may have a first side contacting the inner dielectric layer and a second side contacting at least one electrode. In some embodiments, the photoreactive layer may include hydrogenated amorphous silicon. In these embodiments, irradiating any of the plurality of regions of the photoreactive layer with a light beam can reduce the electrical impedance of the irradiated region of the photoreactive layer. In other embodiments, the photoreactive layer includes a plurality of conductors, and each conductor can be controllably connected to at least one electrode of the substrate via a photoelectric crystal switch. For embodiments in which the microfluidic device includes a cover, the inwardly facing surface of the cover may include an inner layer and a hydrophobic layer covalently bonded to the inner layer (ie, an outer hydrophobic layer). Similar to the hydrophobic layer outside the substrate, the hydrophobic layer outside the cover may include self-associating molecules covalently bonded to the inner layer to form a densely packed hydrophobic monolayer. Therefore, the outer hydrophobic layer may comprise any of the self-associating molecules described above (or elsewhere herein) for the outer hydrophobic layer of the substrate. In some embodiments, the hydrophobic layer outside the cover contains 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 from the hydrophobic layer outside the substrate. In some embodiments, the hydrophobic layer outside the inner surface of the cover has a thickness of less than 5 nanometers (for example, about 1.5 nanometers to 3.0 nanometers). In some embodiments, the outer hydrophobic layer of the inner-facing surface of the cover may be patterned so that selected areas are relatively hydrophilic compared to the rest of the outer hydrophobic layer. In some embodiments, a portion of the substrate may further include a dielectrophoresis (DEP) configuration. In other embodiments, the substrate may include an electrowetting configuration, but lack a dielectrophoresis (DEP) configuration. Therefore, the microfluidic device may lack the photoelectric tweezers (OET configuration). In some embodiments, the microfluidic device may include a housing having at least one microfluidic channel. In addition, the housing may include at least one microfluidic chamber (or isolation fence) fluidly connected to the microfluidic channel. At least a portion of the substrate defining microchannels and/or chambers may have an electrowetting configuration. The electrowetting configuration can be connected to the magnetic bias potential, and at the same time, to change the effective wetting characteristics of any one of a plurality of corresponding regions of the substrate surface (ie, the droplet actuation surface). The wetting characteristics of the substrate surface can be changed 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 isolation fence) may include a containment area (such as a separation zone) configured to contain liquid droplets and one (or more) connection areas that fluidly connect the containment area and the microfluidic channel. The first connection zone can be configured to allow droplets to move between the microfluidic channel and the chamber. When a second connection zone is present, it can be configured to allow fluid flow and pressure relief when the droplet moves between the microfluidic channel and the containment area. In some embodiments, the housing may further include a second microfluidic channel. In these embodiments, the chamber can be connected to both the first microfluidic channel and the second microfluidic channel. In some embodiments, the microfluidic channel may have a height of about 30 microns to about 200 microns, or about 50 microns to about 150 microns, and the height is measured in a direction perpendicular to the direction in which the fluid flows through the channel. In some embodiments, the microfluidic channel has a width of about 50 microns to about 1000 microns, or about 100 microns to about 500 microns, and the width is measured in a direction perpendicular to the direction in which the fluid flows through the channel. In some embodiments, the height of the chamber (or isolation fence) is substantially the same as the height of the microfluidic channel. For example, the chamber height can be about 30 microns to about 200 microns, or about 50 microns to about 150 microns. In some embodiments, the chamber (or containment fence) has a cross-sectional area of about 100,000 square microns to about 2,500,000 square microns, or about 200,000 square microns to about 2,000,000 square microns. In some embodiments, the height of the connection zone (first, second, etc.) is substantially the same as the height of the corresponding chamber and/or the microfluidic channel from which the connection zone opens. In some embodiments, the connection region has a width of about 50 microns to about 500 microns, or about 100 microns to about 300 microns. In some embodiments, the microfluidic device may further include a droplet generator. The droplet generator can be configured to selectively provide droplets of one or more liquid media (for example, aqueous liquid media) into the housing or microfluidic channels within the housing. The droplets may contain, for example, tiny objects, such as biological tiny objects (such as cells) or beads. Alternatively or in addition, the droplets may contain reagents, such as lysis buffer, affinity reagents, detectable labels, enzyme mixtures, and the like. In some embodiments, the microfluidic device includes a culture chamber (for example, an isolation fence) suitable for culturing small biological objects. The culture chamber can be located in the housing and can be connected to the microfluidic channel. When the culture chamber is located in the housing, the housing may include perfusion microfluidic channels that are configured to allow fresh medium to flow through the culture chamber so that nutrients in the fresh medium and waste products in the culture chamber can be exchanged (for example, by making Nutrients diffuse into the culture chamber and allow waste products to diffuse out into the culture medium). The perfusion channel can be separated from the microfluidic channel connected to the droplet generator. In another aspect, the present invention provides a method of manufacturing the microfluidic device of the present invention. The method may include: adhering a spacer element (for example, made from a microfluidic circuit material) to the inner surface of a cover having at least one electrode configured to connect to a voltage source; adhering the spacer element and the cover to have at least one via The internal dielectric surface of the substrate is configured to be connected to the electrode of the voltage source; and a hydrophobic layer is formed on at least a part of the inner surface of the cover and at least a part of the internal dielectric surface of the substrate by vapor deposition. In some embodiments, the spacer element is sandwiched between the inner surface of the cover and the inner dielectric surface of the substrate so that the cover and the substrate are oriented substantially parallel to each other. The substrate, spacer element, and cover can collectively define a housing that is configured to contain the liquid medium. In some embodiments, the hydrophobic layer is deposited on substantially all exposed areas of the inner surface of the cover and substantially all exposed areas of the internal dielectric surface of the substrate (ie, deposited on substantially all of the inner surface of the outer shell). On the surface). In some embodiments, the hydrophobic layer is further deposited on the surface of the spacer element facing the housing inwardly. In some embodiments, the hydrophobic layer includes self-associating molecules covalently bonded to the inner surface of the cover and the inner dielectric surface of the substrate, wherein the self-associating molecules form a densely packed monolayer. In some embodiments, the self-associated molecules deposited by vapor deposition each include a surface modification ligand and a linking group that directly or indirectly links the surface modification ligand to the surface of the inner dielectric layer. Therefore, the self-associating molecule can be any self-associating molecule described above or elsewhere herein. In another aspect, the present invention provides methods for processing materials (such as chemicals and/or biological materials) in a microfluidic device. In some embodiments, the methods include: filling a housing or a portion of a microfluidic device with a first liquid medium, the microfluidic device including a substrate having an electrowetting configuration, a cover, and a spacer, which together define the housing; Apply an AC voltage potential between at least one electrode of the substrate and at least one electrode of the cover; introduce the first droplet of the liquid medium into the housing, the liquid medium of the droplet is not miscible in the first liquid medium; and The electrowetting force is applied to the first droplet to move the first droplet to a desired position within the housing. The first liquid medium may include any of the first liquid medium described herein, such as silicone oil, fluorinated oil, or a combination thereof, and the first droplet may include an aqueous medium. In some embodiments, the methods may include dragging the first droplet from the first portion of the housing (e.g., microfluidic channel) to the second portion (e.g., chamber) of the housing, or vice versa. The aforementioned dragging may include changing the effective electrowetting characteristics of the substrate surface area in contact with and/or adjacent to the first droplet. Therefore, filling the housing with the first liquid medium may include filling the microfluidic channels and chambers with the first liquid medium. In some embodiments, the microfluidic device includes a droplet generator. The methods may include using a droplet generator to generate the first droplet. In addition, the droplet generator may introduce the first droplet into the housing. The droplets produced may have a volume of about 100 picoliters to 100 nanoliters, or about 1 nanoliter to 50 nanoliters. In some embodiments, the first droplet may include tiny objects, such as beads or biological tiny objects (such as cells, vesicles, etc.), cell secretions, or reagents. Beads may have molecules with affinity for the material of interest, such as cell secretions (such as antibodies) or other biological molecules (such as nucleic acids, such as DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA) Or any combination thereof). The droplet may include a single tiny object (for example, a single biological cell) or multiple tiny objects. For example, the droplet may include 2 to 20 or more tiny objects, such as beads. In some embodiments, the droplets may include reagents, such as lysis buffer, labels (e.g., fluorescent labeling reagents), luminescent reagents, enzyme mixtures, or the like. In some embodiments, the methods further include introducing second, third, fourth, etc. droplets into the housing, and applying electrowetting force to the droplets to make the second, third, fourth, etc. droplets Move to the desired position in the housing. The second droplet can be moved to a position close to the first droplet, and then merged with the first droplet to form a first combined droplet; the third droplet can be moved to a position close to the first combined droplet, and then with The first merged droplet merges to form a second merged droplet; the fourth droplet can be moved to a position close to the second merged droplet, and then merges with the second merged droplet to form a third merged droplet, etc. Each additional droplet may contain a fluid medium that is not miscible in the first liquid medium but is 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 lysis buffer that lyses biological cells when the first and second droplets are fused. Alternatively, the reagent may be a fluorescent label (such as a fluorescently labeled antibody or other affinity reagent) or a reagent used in luminescence analysis. The third droplet may contain a reagent, such as one or more (e.g., 2 to 20) capture beads that have affinity for the material of interest. For example, the material of interest can be antibodies or nucleic acids (eg, DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof). The capture beads can be discharged from the device for subsequent analysis depending on the situation. Like the second and third droplets, the fourth droplet may contain reagents, such as an enzyme mixture suitable for carrying out a reaction, such as a reverse transcriptase reaction or a whole-genome amplification reaction. In some embodiments, involving the use of electrowetting force to move and fuse droplets includes changing the effective electrowetting characteristics of the substrate surface area close to the droplets, thereby causing the droplets to move or fuse. In some embodiments, changing the effective electrowetting characteristics of the substrate surface may include activating electrowetting electrodes in areas of the substrate surface near the droplets. In certain embodiments, activating the electrowetting electrode near the surface area of the substrate of 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 integrated device can combine the functions of the two devices. The accompanying drawings and detailed description will clarify other aspects and embodiments of the present invention.

在一些實施例中，表面修飾化合物可包括形成直鏈之碳原子(例如，至少10個碳、或至少14個、16個、18個、20個、22個或更多個碳之直鏈)且可為不具支鏈烷基部分。在一些實施例中，烷基可包括經取代烷基(例如，烷基中之一些碳可經氟化或全氟化)。在一些實施例中，烷基可包括接合至第二區段之第一區段，該第一區段可包括全氟烷基，該第二區段可包括未經取代之烷基，其中第一及第二區段可直接或間接接合(例如藉助醚鍵)。烷基之第一區段可位於鏈接基團之遠端，且烷基之第二區段可位於連接部分之近端。 在各個實施例中，表面修飾化合物可具有式I之結構：

； 式I 其中連接部分V係-P(O)(OH)Q-或-Si(T)₂ W；W係-T、-SH或-NH₂ 且係經構形以連接至表面之部分；Q係-OH且係經構形以連接至表面之部分；且T係OH、OC_1-3 烷基或Cl。R係氫或氟及M係氫或氟。h之每一實例獨立地係2或3之整數；j係0或1；k係0或係1；m為0或1至25之整數；且n為0或1至25之整數。在一些其他實施例中，(n + [(h + j)·k] + m)之和可為11至25之整數。在一些實施例中，M係氫。在各個實施例中，m為2。在一些實施例中，k係0。在其他實施例中，k係1。在各個實施例中，j係1。對於式I化合物，當k係整數1時，則m可為至少2且M係氫。對於式I化合物，當k係0且R係氟時，則m可為至少2且M係氫。 在各個實施例中，當表面修飾化合物具有式I之結構時，連接部分V可為-Si(T)₂ W，其中T及W係如上文所定義。W可為OC_1-3 烷基或Cl。W可為甲氧基、乙氧基或丙氧基。在一些實施例中，W可為甲氧基。T可為OC_1-3 烷基或Cl。在各個實施例中，連接部分V係-Si(OMe)₃ 。在各個其他實施例中，V可為-P(O)(OH)Q，其中Q係OH。 式1之表面修飾化合物可具有較佳範圍之構成化合物之直鏈主鏈之原子數。如上文所定義，構成式1化合物之每一區段可具有一定範圍之大小。因此，式1化合物可具有如上文所定義附接至連接部分之重複單元，使得(n + [(h + j)·k] + m)等於25，其將產生26個原子之總長度，包括末端CR₃ -基團。在(n + [(h + j)·k] + m)等於25之情況下，可涵蓋多種不同組成。例如，區段-[CR₂ ]_n -可具有n = 23；-[(CH₂ )_h -(O)_j ]_k -可具有k= 0；且-[CM₂ ]_m -可具有m= 2。具有相同總(n + [(h + j)·k] + m)等於25之另一情況可具有區段-[CR₂ ]_n -，其中n = 6；-[(CH2)h-(O)j]k-，其中k= 3，且包括j= 1及h= 2；且-[CM₂ ]_m -可具有m= 4。 在一些實施例中，(n + [(h + j)·k] + m)之和可為11、13、15、17或21。在其他實施例中，(n + [(h + j)·k] + m)之和可為15或17。在其他實施例中，(n + [(h + j)·k] + m)之和可為13或15。 在一些實施例中，在式I化合物中可存在一或多個醚鍵。在一些實施例中，j可為1。在一些實施例中，當k及j皆為1時，m可為至少2。 在其他實施例中，主鏈碳可經氟化。在一些實施例中，主鏈碳可經全氟化，其中CR₃ -及/或-[CR₂ ]_n -及/或-[CM₂ ]_m -之每一R可經氟化。在一些實施例中，化合物之一部分可具有經氟化之碳主鏈原子，且化合物之其他部分可具有經氫取代之碳主鏈原子。舉例而言，在一些實施例中，CR₃ -及-[CR₂ ]_n -區段可具有氟非主鏈取代基(例如，R係氟)，而-[CM]_m -區段可具有氫非主鏈取代基(例如，M係氫)。在一些實施例中，當R係氟時，則k係0。在其他實施例中，R可為氟且k係1，j係1且h係2。在各個實施例中，M可為氫。 在其他實施例中，式1化合物可自矽氫化烯烴來合成，如下文所述，其中m為至少2且M係氫。在一些實施例中，m係2且M係氫。 多種式I化合物中之一些可更易見於下式中所述之化合物亞組中，但該等式決不限制式I之寬度。 在一些實施例中，式I化合物可包括式110化合物： CH₃ (CH₂ )_m Si(OC_1-3 烷基)₃ ； 式110 其中m係9至23之整數。在一些實施例中，m可為11、13、15、17或19。在一些其他實施例中，m可為13或15。 在其他實施例中，式I化合物可包括式111化合物： CF₃ (CF₂ )_n (CH₂ )₂ Si(OC_1-3 烷基)₃ ； 式111 其中n可為9至22之整數。或者，n可為11至17之整數。在一些其他實施例中，n可為9、11、13或15。在一些實施例中，n可為13或15。 在其他實施例中，式I化合物可包括式112化合物： CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si(OC_1-3 烷基)₃ ； 式112 其中n係3至19之整數；h係2或3之整數；且m係2至18之整數。在一些實施例中，R可為氟。在一些實施例中，n可為3至11之整數，h可為2，且m可為2至15之整數。 或者，式I化合物可包括式113化合物： CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ ； 式113 其中n係3至21之整數；且m係2至21之整數。在式113化合物之一些實施例中，R可為氟。在一些實施例中，M可為氫。在各個實施例中，n可為5、7、9或11。在其他實施例中，m可為2、4、5、7、9、11或13。用於修飾之表面 . 能夠經本文所述之表面修飾化合物(包括式I化合物)修飾之表面可為金屬、金屬氧化物、玻璃或聚合物。其中可引入共價修飾表面之一些材料可包括(但不限於)矽及其氧化物、聚矽氧、鋁或其氧化物(Al₂ O₃ )、銦鉭氧化物(ITO)、二氧化鈦(TiO₂ )、氧化鋯(ZrO2)、鉿(IV)氧化物(HfO₂ )、鉭(V)氧化物(Ta₂ O₅ )或其任一組合。表面可為該等材料之晶圓或板，或可納入裝置或器件內。在一些實施例中，包括該等材料中之任一者之表面可納入如本文所述之微流體器件內。 聚合物可包括任何適宜聚合物。適宜聚合物可包括(但不限於) (例如橡膠、塑膠、彈性體、聚矽氧、有機聚矽氧(例如聚二甲基矽氧烷(「PDMS」))或諸如此類)，其可為透氣的。其他實例可包括模製玻璃、可圖案化材料(例如聚矽氧聚合物，例如光可圖案化聚矽氧或「PPS」)、光阻劑(例如基於環氧化物之光阻劑，例如SU8)或諸如此類。在其他實施例中，諸如天然纖維或木材等材料之表面可經本文所述之表面修飾化合物(包括式I化合物)官能化，以引入共價修飾表面。 欲修飾之表面可包括親核部分，包括(但不限於)氫氧化物,胺基及硫醇。表面上之親核部分(例如氫氧化物(在一些實施例中稱為氧化物))可與本文所述之表面修飾化合物(包括式I化合物)反應，以經由矽氧基鏈接基團或膦酸酯鏈接基團將表面修飾配體共價鏈接至表面來提供官能化表面。欲修飾之表面可包括天然親核部分，或可用試劑(例如食人魚溶液(piranha solution))或藉由電漿處理來處理以引入親核部分(例如，氫氧化物(或者稱為氧化物))。 在一些實施例中，表面可自上述材料中之任一者單獨或以任一組合形成。表面可包括半導體基板。在各個實施例中，包括半導體基板之表面可進一步包括如本文所述之DEP或EW基板。在一些實施例中，具有DEP或EW基板之包括半導體基板之表面可為如本文所述微流體器件之一部分。 在一些實施例中，經修飾表面可為如本文所述微流體器件之至少一個朝內表面。至少一個表面可為微流體器件(其可包括通道)之流動區之一部分或可包括諸如圍欄等包封結構之表面，該包封結構可包括如本文所述之隔離圍欄。共價修飾表面 . 共價修飾表面可包括表面修飾配體，其可為非聚合部分，例如烷基部分、經取代烷基部分(例如氟烷基部分(包括(但不限於)全氟烷基部分))，且可為上文所述之任何表面修飾配體，其經由鏈接基團共價結合至表面，該鏈接基團係自連接部分與表面之反應產生之部分。鏈接基團可為矽氧基鏈接基團或膦酸酯鏈接基團。 在一些實施例中，表面修飾配體可包括形成直鏈之碳原子(例如至少10個碳、或至少14個、16個、18個、20個、22個或更多個碳之直鏈)，且可為不具支鏈烷基部分。在一些實施例中，烷基可包括經取代烷基(例如，烷基中之一些碳可經氟化或全氟化)。在一些實施例中，烷基可包括接合至第二區段之第一區段，該第一區段可包括全氟烷基，該第二區段可包括未經取代之烷基，其中第一及第二區段可直接或間接接合(例如藉助醚鍵)。烷基之第一區段可位於鏈接基團之遠端，且烷基之第二區段可位於鏈接基團之近端。式 II 之共價修飾表面 . 在一些實施例中，共價修飾表面具有式II之結構：

； 式II 其中係表面；V係-P(O)(OY)W-或-Si(OZ)₂ W。W係-O-、-S-或-NH-且連接至表面。Z係至附接至表面之毗鄰矽原子之鍵或係至表面之鍵。Y係至附接至表面之毗鄰磷原子之鍵或係至表面之鍵。對於式II之共價修飾表面，R、M、h、j、k、m及n係如上文所定義。當k係整數1時，則m為至少2且M係氫。當k係0且R係氟時，則m為至少2且M係氫。式II之共價修飾表面可闡述為經由鏈接基團LG附接之表面修飾配體，如在式IIA中，其中LG鏈接至表面：

式IIA 共價修飾表面可包括如上文針對式I之表面修飾化合物所述呈任一組合之任何式II表面。 在一些實施例中，式II之共價修飾表面可為式210之表面：

； 式210 其中

係表面，附接至矽原子之氧亦結合至表面，且m係11至23之整數。在一些實施例中，m可為11、13、15、17或19。在一些其他實施例中，m可為13或15。 在一些其他實施例中，式II之共價修飾表面可為式211之表面：

； 式211 其中

係表面，附接至矽原子之氧亦結合至表面，且n可為9至22之整數。或者，n可為11至17之整數。在一些其他實施例中，n可為7、9、11、13或15。在一些實施例中，n可為13或15。 在其他實施例中，式II之共價修飾表面可為式212之表面：

； 式212 其中

係表面，附接至矽原子之氧亦結合至表面，且n係3至21之整數，h係2或3之整數，且m係2至21之整數。在一些實施例中，R可為氟。在一些實施例中，n可為3至11之整數，h可為2，且m可為2至15之整數。 或者，式II之共價修飾表面可為式213之表面：

； 式213 其中

係表面，附接至磷原子之氧亦結合至表面，n係3至21之整數且m係2至21之整數。在式113化合物之一些實施例中，R可為氟。在一些實施例中，M可為氫。在各個實施例中，n可為5、7、9或11。在其他實施例中，m可為2、4、5、7、9、11或13。 在一些實施例中，微流體器件包含流體連接至第一入口及第一出口之流動區，該流動區經構形以含有第一流體介質之流。微流體器件可包括至流動區之一或多個室開口。共價修飾表面可為微流體器件之共價修飾基板且可位於流動區及/或至少一個室之下。在一些實施例中，微流體器件之經構形以面向流體之所有或實質上所有的內表面具有式II之共價修飾表面。 圖2H繪示包含實例性共價修飾表面298之微流體器件290之剖視圖。如所圖解說明，共價修飾表面298 (示意性顯示)可包含共價結合至基板286之內表面294及微流體器件290之蓋288之內表面292二者之緻密堆疊之分子之單層。共價修飾表面298可佈置於靠近且向內面向微流體器件290之外殼284之實質上所有的內表面294、292上，在一些實施例中且如上文所論述，包括用於界定微流體器件290內之迴路元件及/或結構之微流體迴路材料(未顯示)的表面。在替代實施例中，共價修飾表面298可佈置於微流體器件290之內表面中之僅一者或一些上。 在圖2H中所示之實施例中，共價修飾表面298包含烷基封端矽氧烷分子之單層，每一分子經由矽氧基鏈接體296共價鍵結至微流體器件290之內表面292、294。為簡明起見，顯示鏈接至毗鄰矽原子之其他氧化矽鍵，但本發明並不限於此。在一些實施例中，共價修飾表面298可包含在其面向外殼之末端之氟烷基(例如氟化烷基或全氟化烷基) (即表面修飾配體298之單層之未結合至內表面292、294且靠近外殼284之部分)。儘管圖2H論述為具有烷基封端修飾表面，但可使用任何適宜的表面修飾化合物，如本文所述。天然表面 . 微流體器件之欲修飾之至少一個表面可為玻璃、金屬、金屬氧化物或聚合物。可納入微流體器件內且可經修飾以向其中引入式II之共價修飾表面之一些材料可包括(但不限於)矽及其氧化物、聚矽氧、鋁或其氧化物(Al₂ O₃ )、銦鉭氧化物(ITO)、二氧化鈦(TiO₂ )、氧化鋯(ZrO2)、鉿(IV)氧化物(HfO₂ )、鉭(V)氧化物(Ta₂ O₅ )或其任一組合。聚合物可包括任何適宜聚合物。適宜聚合物可包括(但不限於) (例如橡膠、塑膠、彈性體、聚矽氧、有機聚矽氧(例如聚二甲基矽氧烷(「PDMS」))或諸如此類)，其可為透氣的。其他實例可包括模製玻璃、可圖案化材料(例如聚矽氧聚合物，例如光可圖案化聚矽氧或「PPS」)、光阻劑(例如基於環氧化物之光阻劑，例如SU8)或諸如此類。共價修飾表面之物理及性能性質 . 在一些實施例中，共價修飾表面可具有增加的疏水特徵。經修飾表面之增加的疏水特徵可防止因生物材料而結垢。如本文所用之表面結垢係指無差別地沈積於微流體器件之表面上之材料的量，其可包括生物材料(例如蛋白質及其降解產物、核酸及各別降解產物)之永久性或半永久性沈積。該結垢可增加生物微小物體黏著至表面之量。在其他實施例中，共價修飾表面之增加的疏水特徵可減少表面上生物微小物體之黏著，此獨立於由表面結垢起始之黏著。 表面之修飾可增加表面之耐久性、功能性及/或生物相容性。該等特徵中之每一者可進一步有益於在經修飾表面上及具有共價修飾表面之器件及/或裝置內之微小物體或生物分子之活力(包括生長速率及/或細胞倍增速率)、在如本文所述之共價修飾表面(包括具有式II之結構之表面)上形成之群落之性質或可攜性(包括排出時之活力)。 在一些實施例中，共價修飾表面可為如本文所述之任何表面(包括式II之表面)，其可具有小於10 nm (例如，小於約7 nm、小於約5 nm、或約1.5 nm至3.0 nm)之厚度。此可有利地在經修飾表面上提供具體而言與其他疏水材料(例如全氟四氫呋喃基聚合物CYTOP^® )不同之薄層，其經旋塗以產生約30 nm至50 nm之典型厚度。表1中所示之數據係關於經處理以具有共價修飾表面之矽/氧化矽表面，如表中所示。接觸角量測係使用靜態坐滴方法來獲得。(Drelich, J. Colloid Interface Sci.179 , 37-50, 1996。)厚度係藉由橢圓偏光術來量測。 接觸角滯後量測係使用Biolin Scientific接觸角測角器來實施。將經化學修飾之OEW表面置於包裹於透明容器中之5 cSt聚矽氧油中。然後將磷酸鹽緩衝鹽水(PBS)微滴分配至油中之表面上。將鉑(Pt)線電極插入微滴中，且量測固著水接觸角。然後，在OEW基板之間以30 kHz頻率施加50 Vppk之施加AC電壓且將Pt線插入PBS微滴中達10秒。然後，移除施加電壓，且再量測接觸角。藉由自在施加50Vppk AC電壓之前零偏壓下之原始接觸角減去在施加該電壓後零偏壓下之接觸角來計算接觸角滯後。表 1 . 所選表面之物理數據. 編號 經修飾表面 接觸角 ( 水或水性溶液 ) 接觸角滯後 厚度 201 CH₃ (CH₂ )₁₇ -Si(OT)₂ O-(表面) 110-112° 5°，小於10° 約2 nm 202 CF₃ (CF₂ )₁₃ (CH₂ )₂ Si(OT)₂ O-(表面) 110-115° 數據 約2 nm 203 CF₃ (CF₂ )₇ (CH₂ )₂ Si(OT)₂ O-(表面) 110-115° ＞10° 約1 nm 204 CH₃ (CH₂ )₂₁ Si(OT)₂ O-(表面) 110-112° n/a 約2-2.5nm 205 CH₃ (CH₂ )₁₅ Si(OT)₂ O-(表面) 110-112° ＞10° n/a 206 CF₃ (CF₂ )₅ (CH₂ )₂ O(CH₂ )₁₁ Si(OT)₂ O-(表面) 110-114° n/a 約2 nm 207 CH₃ (CH₂ )₁₇ P(O)(OQ)O-(表面) 110° n/a n/a 208 CF₃ (CF₂ )₇ (CH₂ )₁₁ Si(OT)₂ O-(表面) 113° n/a 約2 nm 209 CF3(CF2)₁₁ (CH₂ )₂ Si(OT)₂ O-(表面) 112° n/a 約1.5-2 nm T及Q係如上文所述。 對經修飾表面所觀察到之接觸角與電漿清潔之矽表面上水之小於10度之接觸角不同。該等表面中之每一者與天然矽/氧化矽表面相比不太易濕潤。 適於表徵表面之其他分析方法可包括紅外光譜法及/或X射線光電子光譜法。 本發明經修飾表面之另一期望特徵係缺少自發螢光，其可取決於表面修飾化合物之化學性質。 在一些實施例中，本文所述之共價修飾表面(包括式II之表面)可形成單層。尤其在單層經修飾表面具有其他功能屬性時，單層經修飾表面之均一性及均勻度可提供有利的性能。舉例而言，本文所述之共價修飾表面(包括式II之表面)亦可包括電極活化基板，且可視情況進一步包括介電層，如可發現於具有介電泳構形或電濕潤構形之材料、器件及/或裝置中。與含有例如烯烴或芳香族部分之單層相比，缺少經修飾表面之全氟烷基部分之不飽和可最小化「電荷捕集」。另外，在本文所述之表面(包括式II之表面)上形成之單層之緻密堆積性質可最小化驅動陽離子穿過單層至下伏金屬、金屬氧化物、玻璃或聚合物基板之可能。在不受限於理論下，藉由將陽離子添加至基板組合物中破壞基板表面可破壞基板之電性質，由此降低其發揮電動力學功能之能力。 此外，經由共價鍵引入經修飾表面之能力可增加經修飾表面之介電強度且保護下伏材料免於在施加電場下分解。當光學致動材料、器件及/或裝置時，具有本文所述之共價修飾表面(包括式II之表面)之材料、器件及/或裝置之介電泳或電濕潤表面的均一性及薄度可進一步為該經修飾之介電泳及/或電濕潤表面提供有利的益處。製備共價修飾表面之方法 . 可用作器件或裝置之組件之材料表面可在組裝器件或裝置之前經修飾。或者，部分或完全構造之器件或裝置可經修飾，使得將接觸生物材料(包括生物分子及/或微小物體(其可包括生物微小物體))之所有表面同時經修飾。在一些實施例中，即使在器件及/或裝置內之不同表面存在不同材料，仍可修飾器件及/或裝置之整個內部。在一些實施例中，部分或完全構造之器件及/或裝置可為如本文所述之微流體器件或其組件。 欲修飾表面可在修飾之前經清潔以確保表面上之親核部分可自由用於反應，例如不經油或黏著劑覆蓋。清潔可藉由任何適宜方法來完成，包括用溶劑(包括醇或丙酮)處理、超音波處理、蒸汽清潔及諸如此類。在一些實施例中，欲修飾表面係用氧電漿處理來處理，此移除污染物，同時可在表面上引入其他氧化物(例如氫氧化物)部分。此可有利地提供表面上之更多修飾位點，由此提供更緊密堆積之經修飾表面層。 欲修飾表面可在修飾之前經清潔以確保表面上之親核部分可自由用於反應，例如不經油或黏著劑覆蓋。清潔可藉由任何適宜方法來完成，包括用溶劑(包括醇或丙酮)處理、超音波處理、蒸汽清潔及諸如此類。在一些實施例中，欲修飾表面係用氧電漿處理來處理，此移除污染物，同時可在表面上引入其他氧化物(例如氫氧化物)部分。此可有利地提供表面上之更多修飾位點，由此提供更緊密堆積之經修飾表面層。 在一些實施例中，共價修飾表面之方法包括以下步驟：使表面與式I化合物接觸：

； 式I 其中V係-P(O)(OH)Q或-Si(T)₂ W。W係-T、-SH或-NH₂ 且係經構形以連接至表面之部分。或者，當V係-P(O)(OH)Q時，Q係-OH且係經構形以連接至表面之部分。T係OH、OC_1-3 烷基或Cl。R、M、h、j、k、m及n中之每一者係如上文對於式I化合物所定義。(n + [(h + j)·k] + m)之和係11至25之整數。在各個實施例中，當k係整數1時，則m為至少2且M係氫；且當k係0且R係氟時，則m為至少2且M係氫。使式I化合物與表面之親核部分反應；及形成共價修飾表面。可使用式I化合物之任何組合或子組合，如上文所述。 在該方法之各個實施例中，如此形成之共價修飾表面可為單層。 在該方法之一些實施例中，式I化合物可為式110化合物： CH₃ (CH₂ )_m Si(OC_1-3 烷基)₃ ； 式110 其中m係9至23之整數。在一些實施例中，m可為11、13、15、17或19。在一些其他實施例中，m可為13或15。 在該方法之其他實施例中，式I化合物可為式111化合物： CF₃ (CF₂ )_n (CH₂ )₂ Si(OC_1-3 烷基)₃ ； 式111 其中n係9至22之整數。或者，n可為11至17之整數。在其他實施例中，n可為11至17之整數。在一些其他實施例中，n可為9、11、13或15。在一些實施例中，n可為13或15。 在該方法之其他實施例中，式I化合物可為式112化合物： CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si(OC_1-3 烷基)₃ ； 式112 其中n係3至21之整數；h係2或3之整數；且m係2至21之整數。在一些實施例中，R可為氟。在一些實施例中，n可為3至11之整數，h可為2，且m可為2至15之整數。 或者，可使表面與式I化合物接觸，該式I化合物可為式113化合物： CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ ； 式113 其中n係3至21之整數；且m係2至21之整數。在式113化合物之一些實施例中，R可為氟。在一些實施例中，M可為氫。在各個實施例中，n可為5、7、9或11。在其他實施例中，m可為2、4、5、7、9、11或13。 接觸步驟可藉由使表面與含有式I化合物之液體溶液接觸來實施。舉例而言，可將表面暴露於含有0.01 mM、0.1 mM、0.5 mM、1 mM、10 mM或100 mM式I化合物之溶液下。該反應可在環境溫度下實施且可經介於約2 h、4 h、8 h、12 h、18 h、24 h或其間之任一值之範圍內之時段實施。溶劑之實例包括(但不限於)：甲苯、1,3雙三氟苯或Fluorinert^TM (3M)氟化溶劑。可將諸如乙酸等酸添加至溶液中以藉由促進三烷氧基(若存在)之水解來增加反應速率。 或者，可使表面與含有式I化合物之氣相接觸。在一些實施例中，當藉由使表面與氣相中之式I化合物接觸來實施反應步驟時，亦存在受控量之水蒸氣。受控量之水蒸氣可藉由將預選量之硫酸鎂七水合物與具有欲修飾表面之物體一起置於相同室或外殼中來提供。在其他實施例中，可經由外部水蒸氣進料將受控量之水引入反應室或外殼中。反應可在相對於大氣壓之減壓下進行。在一些實施例中，減壓可為100托或更小。在其他實施例中，減壓可小於10托或小於1托。 反應可在介於約150℃至約200℃範圍內之溫度下實施。在各個實施例中，反應可在約150℃、155℃、160℃、165℃、170℃、175℃、180℃、185℃或約190℃之溫度下實施。可容許反應持續約2 h、6 h、8 h、18 h、24 h、48 h、72 h、84 h或更長時間。 在一些實施例中，共價修飾表面可具有式II之結構：

； 式II 其中呈任一組合之R、M、n、h、j、k、m及V係如上文所述。在該方法之一些實施例中，共價修飾表面可具有如上文所述式210、211、212或213之式，其具有可容許用於每一式之要素之任一組合。 在該方法之各個實施例中，表面可包括選自由氫氧化物、胺基及硫醇組成之群之親核部分。表面可為金屬、金屬氧化物、玻璃、聚合物或其任一組合。金屬表面可包括矽、氧化矽、二氧化鉿、銦鉭氧化物、氧化鋁或其任一組合。 在該方法之各個實施例中，其中形成共價修飾表面之步驟可在DEP基板或EW基板上實施。形成共價修飾表面之步驟可包括在微流體器件之微流體迴路元件之至少一個表面上形成共價修飾表面。微流體迴路元件可包括壁、流動區、圍欄及電極活化基板(包括DEP或EW基板)。微流體迴路內可經共價修飾之表面可為所有或實質上所有的面向帶有流體之微流體器件部分之表面。舉例而言，在微流體器件200、230中，可修飾頂部電極210之內表面、電極活化基板206之上表面、微流體迴路材料116之表面(參見圖1B、2A、2B)，其皆面向微流體通道122及圍欄244、246、248。類似地，在圖2D-2F中，可藉由本文所述之方法共價修飾微流體迴路材料260之內表面、界定隔離圍欄266之分離結構272之表面或面向微流體迴路262之所有表面。不可混溶介質 . 可在區域性分佈於一或多個流動區(其可包括流動通道)及(若存在)流體連接至流動區之室內的水不可混溶流體介質內實施水性微滴在基板表面上之移動。水不可混溶流體介質可具有大於純水微滴之運動黏度。水不可混溶流體介質可具有介於約1厘斯托克(cSt)至約15 cSt範圍內之運動黏度，其中1 cSt等於1毫帕斯卡或1厘泊(CPS)。在一些實施例中，水不可混溶流體介質可具有介於約3 cSt至約10 cSt或約3cSt至約8 cSt範圍內之黏度。水不可混溶流體介質在至少100℃之溫度下可為不可燃的。水不可混溶流體介質可在生物細胞經處理、培養或儲存於水不可混溶流體介質內之水性微滴內之持續時間內對活生物細胞無毒。 水不可混溶流體介質可具有低或極低之水溶解度。當與水層接觸(例如用水分配)時，水不可混溶流體介質可溶解小於約5%、4%、3%、2%、1%或小於1%之其總體積之水。在介於約25℃至約38℃範圍內之溫度下，水不可混溶流體介質可不溶解大於約5%、約10%、約15%、約20%、約25%或約30%體積之存在於水不可混溶流體介質內之水性微滴。在一些實施例中，水不可混溶流體介質溶解小於約20%體積之存在於水不可混溶流體介質內之水性微滴。 水不可混溶流體介質可包括至少一種具有包含選自碳、矽及氧之原子之主鏈結構的有機或有機矽化合物。在一些實施例中，水不可混溶流體介質可包括一種以上之有機/有機矽化合物，其中一種以上之化合物係具有聚合化合物亞單位之分子量範圍之聚合有機/有機矽化合物。舉例而言，聚合有機/有機矽化合物可具有兩個構成聚合物(例如共聚物)之不同亞單位，且兩個不同亞單位中之每一者可以一系列具有通式AaBb之重複存在，其中A及B係兩個不同的聚合物亞單位，且a及b係每一亞單位之重複數。重複數a及b可不為單一整數，但可為一系列重複單元。 在其他實施例中，包括一種以上之有機/有機矽化合物之水不可混溶流體介質可包括有機化合物之混合物、有機矽化合物之混合物或其任一組合。水不可混溶流體介質可包括將提供適宜性能之具有不同化學結構及/或分子量之化合物之任何適宜混合物。 水不可混溶流體介質之化合物可具有小於約1000 Da、約700 Da、約500 Da或約350 Da之分子量。在其他實施例中，水不可混溶介質之化合物可具有高於約1000Da之分子量且仍提供適宜性能。 在各個實施例中，水不可混溶流體介質之有機/有機矽化合物可具有主鏈結構，其中構成主鏈之原子係碳、矽或氧。主鏈碳之取代基可為氫或氟。在一些實施例中，水不可混溶流體介質可包括一或多種有機矽化合物，其中有機矽化合物之主鏈可包括矽及氧原子。有機矽化合物之矽原子可具有碳取代基，其進而可具有氫或氟取代基。在一些實施例中，有機矽化合物之碳取代基可皆為氟(例如全氟化)。在其他實施例中，有機矽化合物之碳取代基可經部分氟化。在各個實施例中，有機矽化合物之碳原子之取代基可不超過約90%氟、80%氟、70%氟、60%氟、50%氟、40%氟、30%氟、20%氟或更少。 在其他實施例中，水不可混溶流體介質之有機化合物可具有主鏈結構，其中構成主鏈之原子係碳或氧。在一些實施例中，主鏈碳之取代基可為氫或氟。在其他實施例中，主鏈碳之取代基可包括含氧部分，例如醚、羰基或碳酸根組份。在一些實施例中，水不可混溶流體介質之有機化合物可具有全碳主鏈結構。水不可混溶流體介質之有機化合物的全碳主鏈結構之一些實施例中，可具有碳原子上之全氟取代基(例如經全氟化)。在其他實施例中，有機化合物之取代基可經部分氟化(例如不經全氟化)。在各個實施例中，有機化合物(包括具有全碳主鏈之化合物)之碳原子之取代基可不超過約90%氟、80%氟、70%氟、60%氟、50%氟、40%氟或更少。在一些實施例中，水不可混溶流體介質之適宜有機化合物可包括或可為單氟取代之烴，例如1-氟辛烷、1-氟癸烷、1-氟十二烷或1-氟十四烷。 在其他實施例中，水不可混溶流體介質之有機化合物可不具碳上之氟取代基，但可具有氫取代基。在一些實施例中，水不可混溶流體介質之有機化合物可具有不飽和碳-碳鍵，例如在主鏈碳內或在末端位置之烯烴基團。 在一些實施例中，欲納入水不可混溶流體介質中之適宜化合物之選擇將包括考慮該化合物之其他性質。在各個實施例中，當經雷射、投射至微流體器件中之結構化光或日光/實驗室照明照射時，適用於水不可混溶流體介質內之化合物將不自發螢光。 在其他實施例中，共價修飾疏水表面之性質將影響適用於水不可混溶流體介質內之化合物之選擇。舉例而言，共價修飾表面可足夠疏水，使得全氟化水不可混溶流體介質內水之微滴可展示足夠高之表面張力，此使得水之微滴可不利用如本文所述之光電濕潤構形來移動。 在一些其他實施例中，微流體迴路材料之性質可影響適用於水不可混溶流體介質內之化合物之選擇。水不可混溶流體介質對迴路材料之溶脹可保持在可接受限值內。舉例而言，在一些實施例中，若微流體迴路材料包括SU8或光可圖案化芳基取代之有機聚矽氧，則可選擇包括環、芳基或雜芳基之直鏈烴、直鏈氟碳或碳主鏈化合物來使用。 在其他實施例中，微流體迴路材料可包括其他材料，例如不含芳基取代之光可圖案化有機聚矽氧，且可藉由在水不可混溶流體介質中使用不同化合物使溶脹限於可接受之限值。舉例而言，與暴露於水不可混溶流體介質之前相比小於約40%、30%、20%或10%之溶脹可為可接受的。然而，在一些實施例中，仍可選擇在水不可混溶流體介質內引起溶脹之化合物來使用。 在一些實施例中，水不可混溶流體介質之化合物可為具有含碳或氧原子之主鏈之有機化合物。在一些實施例中，有機化合物可具有含碳原子且不含氧原子、且另外碳原子主鏈具支鏈之主鏈。在各個實施例中，水不可混溶流體介質之有機化合物之具支鏈碳原子主鏈為非環狀。水不可混溶流體介質之具有具支鏈碳主鏈之有機化合物另外可不含任何環化部分。 儘管可使用上述選擇標準來選擇一或多種欲納入水不可混溶流體介質內之化合物及消除無法提供可接受性能之化合物，但可接受之水不可混溶流體介質可為多組份混合物，且可包括在用作水不可混溶流體介質之唯一組份時將不提供可接受性能之個別有機或有機矽化合物之一些部分。舉例而言，組份可經過高氟化或在單獨使用時可使微流體迴路材料不可接受地溶脹，但可與其他有機或有機矽化合物組合使用以形成水不可混溶流體介質。 單獨或以任何種類之組合適用於水不可混溶流體介質中之一些有機化合物可包括異十六烷、2-(三氟甲基)-3-乙氧基十二氟己烷(HFE-7500, 3MTM, NovecTM)、七甲基壬烷(HMN)、碳酸雙(2-乙基己基)酯(TEGOSOFT® DEC, (Evonik))及(十三氟-1,1,2,2,-四氫辛基)四甲基二矽氧烷(Gelest，目錄號SIB1816.0)或聚矽氧油(5厘斯托克黏度，Gelest目錄號DMS-T05)。 水性微滴. 水性微滴可含有一或多個微小物體，其可包括生物細胞或珠粒。水性微滴可含有生物產物，其可包括核酸或蛋白質。在一些其他實施例中，水性微滴可含有分析用試劑，其可為任一種類之試劑，例如酶、抗體、經螢光標記之探針或化學試劑。 在一些實施例中，水性微滴亦可包括表面活性劑。表面活性劑可增加水不可混溶流體介質內之水性微滴之可攜性。在一些實施例中，適宜表面活性劑可包括非離子型表面活性劑。在各個實施例中，表面活性劑可為(但不限於) Pluronic®環氧烷嵌段共聚物，包括F68 (ThermoFisher目錄號2400032)；脂肪酯乙氧基化去水山梨醇，例如TWEEN® 20 (Signa Aldrich目錄號P1379)或TWEEN® 60 (Sigma Aldrich P1629)；2,4,7,9,四甲基-5-癸炔-4,7,-二醇乙氧基化物(TET, Sigma Aldrich目錄號9014-85-1)；乙氧基化非離子型氟表面活性劑，例如Capstone® FS-30 (DuPontTM, Synquest Laboratories目錄號2108-3-38)。在一些實施例中，可使用十二烷基硫酸鈉(SDS)作為表面活性劑。在各個實施例中，可使用磷酸鹽緩衝鹽水(PBS)作為表面活性劑。可將表面活性劑以約1%、3%、5%、10%、15%、20%、約25% v/v或其間之任一值之範圍添加至水性微滴中。製造微流體器件之方法 . 本發明之微流體器件(例如裝置400)可藉由以下方法來製造：(i) 將間隔元件108黏合至蓋110之內表面428，該內表面具有至少一個經構形以連接至AC電壓源(未顯示)之電極，(ii) 將間隔元件108 (及相關蓋110)黏合至基板104之介電表面414，該介電表面具有至少一個經構形以連接至AC電壓源(未顯示)之電極418，藉此間隔元件108變得夾在蓋110之內表面428與基板104之介電表面414之間，其中蓋110及基板104實質上彼此平行定向，且基板104、間隔元件108及蓋110共同界定經構形以容納液體之外殼435，及(iii) 藉由氣相沈積在形成蓋110之內表面428之至少一部分上的外疏水層412及基板104之內介電層414之至少一部分上的外疏水層412。 經由氣相沈積兩親性分子，疏水層422及412可達成緻密堆積之單層，其中兩親性分子分別共價鍵結至蓋110之內表面428及基板104之內部介電表面414的分子。可將本文所述之任一自締合分子及其等效物氣相沈積在微流體裝置之內表面上。為達成期望堆積密度，包含例如烷基封端矽氧烷之自締合分子可在至少110℃(例如至少120℃、130℃、140℃、150℃、160℃等)之溫度下氣相沈積達至少15小時(例如，至少20小時、25小時、30小時、35小時、40小時、45小時或更長時間)之時段。該氣相沈積通常係在真空下及在水源(例如硫酸鎂七水合物(即MgSO₄ ·7H₂ O))存在下實施。通常，增加氣相沈積之溫度及持續時間會產生疏水層422及412之經改良之特徵。氣相沈積過程可視情況例如藉由預清潔蓋110 (具有間隔元件108)及基板104來改良。舉例而言，該預清潔可包括溶劑浴，例如丙酮浴、乙醇浴或其組合。溶劑浴可包括超音波處理。或者或另外，該預清潔可包括於氧電漿清潔器中處理蓋110 (具有間隔元件108)及基板104。氧電漿清潔器可例如在真空條件下在100W下操作達60秒。 圖6圖解說明微流體裝置600之實例，其包含具有微流體通道612、614及複數個室616之外殼，及用於向外殼提供流體微滴620之微滴產生器606。微流體通道614經構形以容納第一流體介質624。通常，第一流體介質係疏水流體，例如油(例如，聚矽氧油或氟化油)。微流體通道614經由界面608連接至微滴產生器606，此允許通道614接收由微滴產生器606產生之微滴620。所接收之微滴620包含不可混溶於第一流體介質624中之液體。通常，所接收之微滴將包含水性介質，其可含有可溶於水性介質中之微小物體(例如細胞或珠粒)或試劑。微流體通道614亦連接至複數個室616中之每一者，此促進所接收之微滴620 (以及自不可混溶於第一流體介質624中之流體之儲存器取出的微滴632)移動至室616中及在其之間移動。 裝置600之微流體通道612連接至室616之亞組，且因此經由該等室616間接連接至微流體通道614。如所圖解說明，微流體通道612及與其連接之室616含有不可混溶於第一流體介質624中之流體介質622。因此，舉例而言，流體介質622可為水性介質，例如細胞培養基。當流體介質622係細胞培養基時，含有培養基之室616可用作用於生長細胞之培養室，且微流體通道612可為提供新鮮培養基流之灌注通道。如本文所論述，灌注通道中之新鮮培養基流可經由使分子擴散於灌注通道與培養室之間將營養素提供至室並自室移除廢棄物，由此促進持續性細胞生長。 圖7圖解說明微流體裝置700之另一實例，其包含具有微流體通道612、614、第一複數個室716及第二複數個室616之外殼，及用於向外殼提供流體微滴620之微滴產生器606。圖7呈現圖6中所示微流體裝置600之變化形式，其中室616含有不可混溶於第一流體介質624 (位於微流體通道614中)中且自相應室716直接定位穿過微流體通道614之介質622。此構形有助於流體微滴632 (視情況含有微小物體630或生物材料)自選擇室616移動至相應室716，其中可處理流體微滴(及任何微小物體630或生物材料)。 微流體裝置之另一實例包含具有微流體通道612、614、第一複數個室716及第二複數個室616之外殼，及用於向外殼提供流體微滴620之微滴產生器606。此實施例呈現圖7中所示微流體裝置700之變化形式，其中室616之一端逐漸變細，以當微流體裝置傾斜使得室616之逐漸變細端具有相對於未逐漸變細端較低之勢能(在適用重力場中)時，促進微粒移動至第一流體介質624及第二流體介質622之界面。 由微流體通道612、614及室616、716形成之微流體迴路僅係實例，且本發明涵蓋通道及室之許多其他構形。舉例而言，在裝置600及700中之每一者中，微流體通道612及直接連接至通道612之室616係可選特徵。因此，裝置600及700可缺少灌注通道及培養室。 在存在微流體通道612之實施例中，幫助界定通道612及/或直接連接之室616 (例如，藉由形成通道及/或室之基底)之基板可具有電濕潤構形。或者，然而，幫助界定通道612及/或直接連接之室616之基板可缺少電濕潤構形(例如，而是可具有DEP構形或既非電濕潤構形亦非DEP構形)。在存在微流體通道612且幫助界定通道612及/或直接連接之室616之基板具有電濕潤構形之實施例中，基板之外疏水表面可經圖案化以比幫助界定通道614之基板之外疏水表面更親水。可例如如上文所論述達成增加的親水性。 微滴產生器606及向其提供微滴之任何微流體迴路可為微流體器件之一部分(組成部分或與其連接)，此可與圖式中所圖解說明或本文所述之任一微流體器件一樣。儘管圖6及7中顯示一個微滴產生器606，但一個以上之該微滴產生器606可將微滴提供至裝置600及700之微流體迴路。微滴產生器606自身可包括電濕潤構形，且因此可包含具有可包含a-Si:H之光反應層之基板(例如，如美國專利第6,958,132號中所圖解說明)、光致動迴路基板(例如，如美國專利申請公開案第2014/0124370號中所圖解說明)、基於光電晶體之基板(例如，如美國專利第7,956,339號中所圖解說明)或電致動迴路基板(例如，如美國專利第8,685,344號中所圖解說明)。或者，微滴產生器可具有T形或Y形流體動力學結構(例如，如美國專利及專利申請公開案第7,708,949號、第7,041,481號(重新頒佈為RE41,780)、第2008/0014589號、第2008/0003142號、第2010/0137163號及第2010/0172803號中所圖解說明)。上述美國專利文件之全文皆以引用方式併入本文中。 如所示，微滴產生器606可包含一或多個流體輸入602及604 (顯示兩個但可存在更少或更多)及流體輸出208，其可連接至微流體通道614。可經由輸入602及604將液體介質622、624、生物微小物體630、試劑及/或其他生物介質裝載至微滴產生器606中。微滴產生器606可產生液體介質622之微滴620 (其可但無需含有一或多個生物微小物體630)、試劑或其他生物介質並將其輸出至通道614中。若通道614具有電濕潤構形，則微滴620可利用電濕潤(或光電濕潤)在通道614中移動。或者，微滴620可藉由其他方式在通道614中移動。舉例而言，微滴620可利用流體流動、重力或諸如此類在通道614中移動。 如上文所論述，微流體通道614及選擇室616/716可填充有第一流體介質624，且與其直接連接之微流體通道612及室616可填充有第二流體介質622。第二流體介質622 (下文稱為「水性介質」)可為水性介質，例如用於維持、培養或諸如此類生物微小物體630之樣品介質。第一流體介質624 (下文稱為「不可混溶介質」)可為水性介質622不可混溶之介質。水性介質622及不可混溶介質624之實例包括上文針對各種介質論述之任一實例。 微滴產生器606可用於將生物微小物體裝載於微流體裝置上及/或促進微流體裝置上生物化學及/或分子生物學工作流程之運行。圖6及7圖解說明非限制性實例。藉由使用微滴產生器，該裝置在整個流體迴路中可具有電濕潤構形。 圖6及7圖解說明微滴產生器606產生包含試劑(或其他生物材料)之微滴620之實例。含試劑之微滴620可移動穿過微流體通道614且進入含有不可混溶介質624之室616/716中之一者中。在含試劑之微滴620移動至室616/716中之一者中之前或之後，一或多個微滴632中之一或多個微小物體630可移動至相同室616/716中。含試劑之微滴620隨後可與含微小物體630之微滴632融合，從而允許微滴620之試劑與微滴632之內容物混合且進行化學反應。含一或多個微小物體之微滴632可由微滴產生器606 (未顯示)供應或可自容納圍欄616獲得，如圖6及7中所示。微小物體630可為生物微小物體，例如細胞，其在移動至處理室616/716之前已視情況經培養(例如，在室616中)。或者，微小物體630可為珠粒，例如能夠結合至樣品中之所關注分子(例如，在樣品材料622已用於培養一或多種生物細胞後存在於樣品材料622中之細胞分泌物)之親和珠粒。在其他替代中，一或多個微滴632可不含微小物體但僅含水性介質，例如樣品材料622，例如其在樣品材料622已用於培養一或多種生物細胞後含有細胞分泌物。 圖8圖解說明製程800之實例，其可在包含微流體迴路之微流體器件(如裝置600及700中之任一者)中實施。 在製程800之步驟802下，可在填充有樣品介質(例如細胞培養基)之容納圍欄中培養生物微小物體。舉例而言，圖6或7之微小物體630可為生物製品且可培養於其室616中。培養通常可如上文所論述。舉例而言，培養可包括向通道612灌注培養基622。步驟802可在指定時間段內實施。 在步驟804下，經培養之生物微小物體可自培養其之填充樣品介質之室616移動至填充有樣品介質不可混溶之介質之室616/716。舉例而言，經培養之微小物體630可在樣品介質622之微滴620或632中自容納圍欄616中之一者移動至容納圍欄616/716中之一者中，如圖6及7中所圖解說明，如上文所論述。 在步驟806下，可使經培養之生物微小物體在填充不可混溶介質之容納圍欄中經受一或多個處理或過程。舉例而言，一或多個含有一或多種試劑之微滴620可由微滴產生器606產生且移動至填充不可混溶介質之室612/716中並與含有經培養生物微小物體630之微滴632融合，如圖6及7中所示及上文所論述。舉例而言，第一含試劑微滴620可含有溶解試劑。含有經培養生物微小物體630之微滴632與含有溶解試劑之第一含試劑微滴620之融合將使經培養之生物微小物體630溶解。換言之，將形成含有來自經培養生物微小物體630之細胞溶解物之合併微滴(未顯示)。其他(例如，第二、第三、第四等)含試劑微滴620隨後可與含有細胞溶解物之新微滴融合，以視需要進一步處理細胞溶解物。 另外或作為另一實例，含有一或多個對經培養之生物微小物體630產生之分泌物或一或多種其他所關注材料(例如，核酸(例如DNA或RNA)、蛋白質、代謝物或其他生物分子)具有親和力的經標記捕獲微小物體(未顯示)之一或多個微滴可由微滴產生器606產生且移動至填充不可混溶介質之圍欄616或716中並以類似方式與含有經培養生物微小物體630之樣品介質622之微滴融合。在經培養之生物微小物體630已溶解之情形下，含有捕獲微小物體之微滴620可含有一或多個親和珠粒(例如，對諸如DNA、RNA、微小RNA或諸如此類等核酸具有親和力)，其在與容納圍欄616或716中含有細胞溶解物之微滴融合後可結合至存在於溶解物中之目標分子。 在步驟808下，可視情況處理經處理之生物微小物體。舉例而言，若在步驟806下，捕獲物體(未顯示)移動至含有經培養之生物微小物體630之填充不可混溶介質之室616/716中，則可在步驟808下監測室616/716之指示所關注材料結合至經標記捕獲微小物體之量的反應(例如螢光信號)。或者，該捕獲微小物體(未顯示)可自室616/716移除(例如於微滴622中)且自微流體器件排出(在圖6及7中未顯示)用於後續分析。作為另一實例，經處理之生物微小物體630可自室616/716移除(例如於微滴632中)且自微流體器件排出(未顯示)用於後續分析。 儘管在本說明書中已闡述本發明之特定實施例及應用，但該等實施例及應用僅具有實例性，且許多變化形式係可能的。舉例而言，可對含有細胞分泌物之樣品材料實施圖8之方法(例如，在樣品材料682已用於培養一或多種生物細胞後)。在該實施例中，步驟802將保持一致，但步驟804將涉及使可不含微小物體但僅含水性介質之微滴632 (例如含有細胞分泌物之樣品材料622)移動至含有不可混溶介質之室616/716中，且將對該等含有水性介質之微滴632實施步驟806及808。此外，本文所論述之電濕潤構形可為業內已知之任一類型之光電子濕潤(OEW)器件，其實例揭示於美國專利第6,958,132號中。電濕潤構形之其他實例包括可經電子控制之介電濕潤(EWOD)器件，其實例揭示於美國專利第8,685,344號中。上述美國專利文件之全文皆以引用方式併入本文中。 圖9係用於形成微流體裝置之基板之方法。在以下方法中，繪示製程流程圖以有助於形成可用於微流體裝置之已闡述於上圖及下圖中之基板堆疊。在一個實施例中，使用經繪示用於形成基板之方法來形成結合圖1B繪示之基板104。下圖10-16將繪示在圖9中所示之方法中繪示之動作之剖視圖。 由方塊902繪示之第一動作繪示利用熱退火製程製備基板(在一個實施例中為導電矽基板)。隨後，由方塊904繪示之第二動作繪示將氮化物層沈積在矽基板之頂部上。 隨後，方塊906繪示施加第一圖案之第三動作。將圖案施加至基板通常係藉由半導體處理業內所熟知之微影製程來達成，例如電子束、X射線、UV及深UV。通常，使用聚合物來界定電晶體及金屬線之複雜結構之圖案。隨後，用光及光反應層處理聚合物且將闡述於以下段落中。 沈積光反應層之後續動作如方塊908中所繪示。由方塊910繪示之以下動作圖解說明將光反應層蝕刻至第一預定位置。在一個實施例中，第一預定位置係氮化物層。 由方塊912繪示之下一動作繪示沈積至少一個介電層。如先前所論述，已結合圖1B之描述論述具有不同介電層組合之多個實施例。 如方塊912中所繪示之最後步驟繪示將第二圖案施加在至少一個介電層之頂部所及將介電層蝕刻至第二預定位置。在相同實施例中，第二預定位置係氮化物層。 在一個實施例中，可進一步處理可選步驟。舉例而言，沈積第三圖案且實施氮化物層之剝離直至蝕刻至矽基板或氧化矽中10 um。此外，進行背側氧化物剝離及銀背側金屬化之另一可選步驟。 圖10係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟902之剖視圖。如先前所提及，方塊902繪示利用熱退火製程製備基板(在一個實施例中為導電矽基板)。請注意，標記1001繪示靶向熱退火製程之位置之兩個箭頭。 圖11係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟904之剖視圖。方塊904繪示將氮化物層沈積在矽基板之頂部上，且此圖繪示層1102，其繪示在矽基板之頂部上形成之氮化物層。 圖12係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟906之剖視圖。方塊906繪示施加圖案及隨後移除氮化物。請注意，標記1202繪示圖案中移除氮化物層之開口。 圖13係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟908之剖視圖。方塊908繪示施加光反應層。請注意，標記1302繪示施加之光反應層。 圖14係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟910之剖視圖。方塊910繪示將在處理步驟908中添加之光反應層蝕刻至第一預定位置。在一個實施例中，第一預定位置係至氮化物層之蝕刻停止。請注意，標記1402繪示圖案中蝕刻或移除光反應層之開口。 圖15係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟912之剖視圖。方塊912繪示沈積至少一個介電層。請注意，標記1501及1502分別繪示介電層及指示沈積介電層之來自圖14中之先前標記1402之開口。 圖16係繪示結合圖9繪示之方法之中間處理的基板之剖視圖。在一個實施例中，此圖繪示結合圖9繪示之處理步驟914之剖視圖。方塊914繪示將圖案施加至介電層及將介電層蝕刻至第二預定位置。在一個實施例中，第二預定位置為氮化物層。請注意，標記1602繪示介電層中藉由蝕刻移除其之開口。 圖17係根據實施例整合多個微流體應用之系統之剖視圖。該系統整合兩種微流體操作，如藉由使各區域重疊之模組OEP及OEW所繪示。模組執行如先前所論述之微流體操作，特定而言光電定位(下文稱為OEP)及光電濕潤(下文稱為OEW)。在一個實施例中，模組係藉由利用至少一個間隔件(在一個實施例中為聚苯硫組成間隔件)因模組之間之厚度差異而幫助整合兩個模組來整合。在此實施例中，聚苯硫組成間隔件厚於模組且能夠按壓並密封兩側以允許整合。將結合圖20A及20B進一步論述兩個模組之電操作。 圖18係根據實施例整合多個微流體應用之系統之剖視圖。該系統整合兩種微流體操作，如藉由具有多個區域之模組OEP及OEW所繪示。舉例而言，在一個實施例中，繪示三個OEW模組及單一OEP模組。然而，所主張之標的物並不限於此特定組合。熟習此項技術者瞭解基於設計需要或對不同光遮罩層之需要來使用模組之不同構形。模組執行如先前所論述之微流體操作，特定而言光電定位及光電濕潤。在一個實施例中，模組係藉由利用至少一個間隔件(在一個實施例中為聚苯硫組成間隔件)因模組之間之厚度差異而幫助整合兩個模組來整合。在此實施例中，聚苯硫組成間隔件厚於模組且能夠按壓並密封兩側以允許整合。 圖19係根據實施例整合多個微流體應用之系統之剖視圖。該系統整合兩種微流體操作，如藉由每一模組具有離散區之模組OEP及OEW所繪示。模組執行如先前所論述之微流體操作，特定而言光電定位及光電濕潤。在一個實施例中，模組係藉由利用至少一個間隔件(在一個實施例中為聚苯硫組成間隔件)因模組之間之厚度差異而幫助整合兩個模組來整合。在此實施例中，聚苯硫組成間隔件厚於模組且能夠按壓並密封兩側以允許整合。 圖20A及20B係結合圖17繪示之實施例之一功能態樣的電定址操作代表圖之視圖。如先前結合圖17所述，該系統整合兩種微流體操作，如藉由使各區域重疊之模組OEP及OEW所繪示。在此實施例中，OEP模組具有相對於OEW模組較低之阻抗。在操作期間，OEW模組之阻抗克服OEP模組之阻抗且基本上使OEP模組短路。 在一個實施例中，如圖20A中所繪示，OEP模組係藉由以介於100 kHz至10 mHz範圍內之頻率施加介於1-10伏特範圍內之電壓來操作。在相同實施例中，如圖20B中所繪示，OEW模組係藉由以介於1 kHz至300 kHz範圍內之頻率施加介於10-100伏特範圍內之電壓來操作。在一個較佳實施例中，OEP模組係藉由以1 Mhz之頻率施加5伏特之電壓來操作，且OEW模組係藉由以30 kHz之頻率施加30伏特之電壓來操作。 圖20B係結合圖17繪示之實施例之一功能態樣的電定址操作代表圖之視圖。實例 系統及微流體器件 ： 由Berkeley Lights, Inc製造。該系統包括至少流動控制器、溫度控制器、流體介質條件處理及幫浦組件、用於光活化DEP或EW構形之光源、微流體器件、安裝台及照相機。實例 1. 具有經修飾內表面之電濕潤微流體器件之製備 . 微流體器件(Berkeley Lights, Inc.)具有基於矽之第一光敏半導體電極活化基板(具有氧化鋁介電上表面)及相對壁上之第二ITO基板以及分開兩個基板之光圖案化聚矽氧微流體迴路材料，用氧電漿清潔器(Nordson Asymtek)使用100W功率、240毫托壓力及440 sccm氧流速將該微流體器件處理1 min。在真空反應器中在箔舟皿中在真空反應器底部或在硫酸鎂七水合物(0.5 g, Acros) (作為在單獨箔舟皿中在真空反應器底部之水反應物來源)存在下，用三甲氧基(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-二十九氟十六烷基)矽烷(0.3 g，合成之細節如於2016年10月19日提出申請之美國臨時申請案62/410238中所述)處理經電漿處理之微流體器件。然後使用真空幫浦將室泵送至750毫托，且然後密封。將真空反應器置於烘箱內，在180℃下加熱24-48 h。冷卻至室溫且將氬引入抽真空室後，自反應器移除具有二甲氧基(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-二十九氟-十六烷基)矽氧基部分之層之微流體器件。冷卻至室溫且將氬引入抽真空室後，自反應器移除在所有內表面上具有二甲氧基(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-二十九氟十六烷基)矽氧基部分之層( 表面 202 ) 之微流體器件，且在使用前用聚矽氧油(5厘斯托克黏度，Gelest目錄號DMS-T05)預處理。圖21A-C係在不可混溶聚矽氧油相內在經修飾表面202 上自由移動之水之微滴之連續照相影像。微滴展示利用微流體器件中之光學致動之電濕潤構形之極佳移動能力。 priority This application claims U.S. Provisional Application No. 62/246,605 filed on October 27, 2015, U.S. Provisional Application No. 62/247,725 filed on October 28, 2015, and on May 26, 2016 Priority of U.S. Provisional Application No. 62/342,131 filed on October 19, 2016 and U.S. Provisional Application No. 62/410,238 filed on October 19, 2016. The full content of each of these applications is by reference Incorporated into this article. This application also claims the priority of U.S. Patent Application No. 15/135,707 filed on April 22, 2016, and the full content of the application is incorporated herein by reference. This specification describes exemplary embodiments and applications of the present invention. However, the present invention is not limited to the exemplary embodiments and applications or the manner in which the exemplary embodiments and applications are operated or described herein. In addition, each figure may show simplified or partial views, and the size of the elements in the figures may be enlarged or otherwise out of proportion. In addition, when the terms "on", "attached to", "connected to", "coupled to" or similar words are used herein, one element (such as a material, layer, substrate, etc.) can be "on another element". "", "attached to another component", "connected to another component" or "coupled to another component", and a component is directly on another component, attached to another component, connected to another It does not matter whether an element is coupled to another element or whether there are one or more intermediate elements between one element and another element. Also, unless the context dictates otherwise, if directions are provided (e.g., above, below, top, bottom, side, up, down, below, above, top, bottom, horizontal, vertical, "x", "y" , "Z", etc.) are relative and are only provided by way of example for ease of explanation and discussion and not provided in a restrictive manner. In addition, when referring to a list of elements (such as elements a, b, c), the reference is intended to include any one of the listed elements by itself, any combination of less than all listed elements, and/or all listed elements Show the combination of components. Part of the division in this specification is only for the purpose of easy review and does not limit any combination of the discussed elements. As used herein, "substantially" means sufficient for the intended purpose. Therefore, the term "substantially" allows minor insignificant changes from absolute or perfect conditions, dimensions, measurements, results, or the like, such as those familiar with the technology would expect but not significantly affect overall performance. When used for a numerical value or a parameter or feature that can be expressed as a numerical value, "substantially" means within 10%. The term "multiple" means more than one. The term "plurality" as used herein can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. As used herein, the term "deployed" is included in its meaning "positioning". As used herein, a "microfluidic device" or "microfluidic device" is a device that includes one or more discrete microfluidic circuits configured to contain fluids, each microfluidic circuit includes fluidly interconnected circuit elements, including ( But not limited to) areas, flow areas, channels, chambers, and/or fences and (for microfluidic devices including a cover) at least two configured to allow fluid (and, as appropriate, tiny objects suspended in the fluid) to flow in and/ Or out of the port of the microfluidic device. Generally, the microfluidic circuit of the microfluidic device will include at least one microfluidic channel and at least one chamber, and will contain a volume less than about 1 mL (for example, 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 some embodiments, the microfluidic circuit contains 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, "nanofluidic device" or "nanofluidic device" is a type of microfluidic device that has at least one structured to contain a volume less than about 1 µL (e.g., 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 , 2 nL, 1 nL or less) of the microfluidic circuit of the fluid circuit element. The nanofluidic device may include a plurality of loop elements (for example, 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 (e.g., all) of the at least one loop element is configured to contain a volume of 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. In other embodiments, one or more (eg, all) of the at least one loop element is configured to contain a volume of about 20 nL to 200 nL, 100 nL to 200 nL, 100 nL to 300 nL, 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 nL to 600 nL, or 250 nL to 750 nL of fluid. As used herein, "microfluidic channel" or "flow channel" refers to the flow area of a microfluidic device, the length of which is significantly longer than both the horizontal and vertical dimensions. For example, the length of the flow channel may 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 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 ranges from about 50,000 microns to about 500,000 microns, including any range therebetween. In some embodiments, the horizontal size ranges from about 100 microns to about 1000 microns (eg, from about 150 microns to about 500 microns), and the vertical size ranges from about 25 microns to about 200 microns, such as from about 40 microns to about 150 microns. Within the micron range. It should be noted that the flow channel can have a number of different spatial configurations in the microfluidic device, and therefore is not limited to a perfectly linear element. For example, the flow channel may include one or more portions having any of the following configurations: curved, curved, spiral, inclined, downwardly inclined, crossing (e.g., multiple different flow paths), and any combination thereof. In addition, the flow channel may have different cross-sectional areas along its path, widened and contracted to provide a desired fluid flow therein. The term "blocking" as used herein generally refers to bumps or similar types of structures that are large enough to partially (but not completely) hinder the movement of target tiny objects between two different areas or circuit elements in the microfluidic device. The two different regions/circuit elements can be, for example, a microfluidic isolation fence and a microfluidic channel, or a connection area and a separation area of a microfluidic isolation fence. The term "shrinking" as used herein generally refers to narrowing the width of a circuit element (or the interface between two circuit elements) in a microfluidic device. The shrinkage can be located, for example, at the interface between the microfluidic isolation fence and the microfluidic channel or the interface between the separation zone and the connection zone of the microfluidic isolation fence. The term "transparent" as used herein refers to a material that allows visible light to pass through but does not substantially change the light when it passes through. The term "tiny object" as used herein generally refers to any microscopic object that can be separated and collected according to the present invention. Non-limiting examples of tiny objects include: inanimate tiny objects, such as particles; microbeads (for example, polystyrene beads, Luminex™ beads or the like); magnetic beads; microrods; microwires; quantum dots and And so on; biological tiny objects, such as cells (such as embryos, oocytes, eggs, sperm cells, self-organized dissociated cells, eukaryotic cells, protist 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; synthetic vesicles Bubbles; liposomes (for example, synthetic or derived from membrane preparations); lipid nano-rafts (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 tiny objects (for example, microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, or the like). The beads may further have other moieties/molecules attached covalently or non-covalently, such as fluorescent labels, proteins, small molecule signaling moieties, antigens, or chemical/biological substances that can be used in analysis. As used herein, the term "maintaining cells" refers to an environment that provides a surface that includes both fluid and gaseous components and optionally provides conditions for maintaining cell viability and/or expansion. The "component" of the 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" for fluid media refer to the thermodynamic movement of components of the fluid media downward along the concentration gradient. The phrase "medium flow" refers to the overall movement of the fluid medium mainly due to any mechanism other than diffusion. For example, the flow of the medium may involve the movement of the fluid medium from one point to another due to the pressure difference between two points. The flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of these mediums can be generated. The phrase "substantially non-flowing" means that the flow rate of the fluid medium averaged over time is less than the diffusion rate of the components of the material (such as the analyte of interest) into or into the fluid medium. The diffusion rate of the components of the material can depend on, for example, the 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 for different areas in a microfluidic device means that when the different areas are substantially filled with fluid (such as a fluid medium), the fluids in each area are connected to form a single fluid entity. This does not mean that the composition of the fluid (or fluid medium) in different regions must be the same. On the contrary, the fluids in the different fluid connection areas of the microfluidic device may have different compositions (for example, different concentrations of solutes, such as proteins, carbohydrates, ions or other molecules), when the solutes move down along their respective concentration gradients and/or The composition can change continuously as the fluid flows through the device. Microfluidic (or nanofluidic) devices can include "swept" areas and "unswept" areas. As used herein, the "swept" area includes one or more fluidly interconnected circuit elements of the microfluidic circuit, and each of these elements undergoes a medium flow when fluid flows through the microfluidic circuit. The loop elements in the affected zone can include, for example, areas, channels, and all or part of the chamber. As used herein, the "unswept" area includes one or more fluidly interconnected circuit elements of the microfluidic circuit. When fluid flows through the microfluidic circuit, each of these elements does not substantially experience fluid flow. The unswept zone can be fluidly connected to the affected zone, and the condition is that the fluid connection is structured so that the medium between the affected zone and the unswept zone can diffuse but does not flow substantially. Therefore, the microfluidic device can be structured to substantially separate the medium flow in the unswept zone and the affected zone, while realizing the diffusion fluid communication between essentially only the affected zone and the unswept zone. For example, the flow channel of the microfluidic device is an example of the affected area, and the separation area of the microfluidic device (described in further detail below) is an example of the unaffected area. As used herein, "flow zone" refers to one or more fluid-connected circuit elements (eg, channels, regions, chambers, and the like) that define and undergo medium flow trajectories. Therefore, the flow zone is an example of the affected zone of a microfluidic device. Other circuit elements (such as the unswept zone) can be fluidly connected to circuit elements that contain the flow zone but are not subjected to the flow of the medium in the flow zone. As used herein, "alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing no unsaturation, and having 1 to 6 carbon atoms (for example, C1-C6 alkyl). Whenever it appears in this text, a numerical range such as "1 to 6" refers to each integer in the given range; for example, "1 to 6 carbon atoms" means that the alkyl group can be composed of 1 carbon atom, 2 It consists of six carbon atoms, three carbon atoms, etc., up to and including six carbon atoms, but this definition also covers the term "alkyl" in which an unspecified numerical range appears. 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, second butyl isobutyl, tertiary butyl, pentyl, isopentyl , Neopentyl, hexyl and the like. The alkyl group is attached to the rest of the molecule by a single bond, such as methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl Group, 1,1-dimethylethyl (tertiary butyl), hexyl and the like. Unless expressly stated otherwise in this specification, the alkyl group may be substituted by one or more substituents as appropriate, and these substituents are independently: aryl, arylalkyl, heteroaryl, heteroarylalkyl, Hydroxyl, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -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 (where t is 1 or 2) or PO3(R')2, where each R'is independently hydrogen, alkyl, fluoroalkyl, aryl, aralkyl, heterocycloalkyl, or heteroaryl. As mentioned herein, a fluorinated alkyl moiety is an alkyl moiety in which one or more hydrogens of the alkyl moiety are replaced by a fluorine substituent. The perfluorinated alkyl moiety replaces all hydrogens attached to the alkyl moiety with fluorine substituents. As mentioned herein, the "halo" moiety is a bromine, chlorine or fluorine moiety. As mentioned herein, "olefin" compounds are organic molecules containing "olefinic hydrocarbons". The olefinic hydrocarbon moiety refers to a group composed of at least two carbon atoms and at least one carbon-carbon double bond. The non-olefinic hydrocarbon portion of the molecule can be any type of organic molecule, and in some embodiments can include an alkyl group or a fluorinated (including but not limited to) perfluorinated alkyl portion, any of which can be Further replaced. As used herein, "densely packed hydrophobic monolayer" refers to a single layer of hydrophobic molecules that is closely packed together to resist the insertion and/or intrusion of polar molecules (such as water, ions, and other charged substances). As used herein: "µm" (or "um") means micrometers; "µm³ "Means cubic micron; "pL" means picoliter, "nL" means nanoliter; and "μL" (or "uL") means microliter.Loading method . Loading tiny objects (such as biological tiny objects and/or beads) into different areas of the microfluidic device may involve the use of fluid flow, gravity, dielectrophoresis (DEP) force, electrowetting force, magnetic force, or any combination thereof, such as Described in this article. The DEP force can be generated optically, for example by optical tweezers (OET) configuration and/or electrically, for example by activating the electrode/electrode area in a time/space pattern. Similarly, the electrowetting force can be provided optically, for example, by electro-wetting (OEW) configuration and/or electrically, for example, by activating the electrode/electrode area in a temporal and spatial pattern.Microfluidic device and system for operating and observing the device . 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 its tiny objects and/or droplets. A perspective view of the microfluidic device 100 is shown, with the cover 110 partially cut away to provide a partial view of the access to the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic circuit 120, which includes a flow zone 106 through which a fluid medium 180 can flow, and optionally carries one or more tiny objects (not shown) into the microfluidic circuit 120 and/or passes through it. Microfluidic circuit 120. Although a single microfluidic circuit 120 is illustrated in FIG. 1A, a suitable microfluidic device may include a plurality (e.g., 2 or 3) of such microfluidic circuits. Nevertheless, 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 of which has a single opening in fluid communication with the flow zone 106. As discussed further below, the microfluidic isolation fence contains a number of features and structures that have been optimized to retain tiny objects in the microfluidic device (such as the microfluidic device 100) even when the medium 180 flows through the flow zone 106. However, before referring 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 bounded by the housing 102. Although the housing 102 can be physically structured in different configurations, in the example shown in FIG. However, in some embodiments, the housing 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 may be attached to each other. For example, the microfluidic circuit structure 108 can be arranged on the inner surface 109 of the support structure 104, and the cover 110 can be arranged above the microfluidic circuit structure 108. The microfluidic circuit structure 108 may define the elements of the microfluidic circuit 120 together with the support structure 104 and (as appropriate) the cover 110. The support structure 104 may be at the bottom of the microfluidic circuit 120 and the cover 110 at the top of the microfluidic circuit 120, as illustrated in FIG. 1A. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 can be on the top of the microfluidic circuit 120 and the cover 110 can be on the bottom of the microfluidic circuit 120. Nonetheless, there may be one or more ports 107 each containing a passage into or out of the housing 102. Examples of channels include valves, gates, through holes, or the like. As illustrated, the port 107 is a through hole created by the void in the microfluidic circuit structure 108. However, the port 107 may be located in other components of the housing 102 (such as the cover 110). Only one port 107 is illustrated in FIG. 1A, but the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 used as an inlet for the fluid to enter the microfluidic circuit 120, and there may be a second port 107 used as an outlet for the fluid to leave the microfluidic circuit 120. Whether the port 107 is used as an inlet or an outlet may depend on the direction in which the fluid flows through the flow zone 106. The support structure 104 may include one or more electrodes (not shown) and a substrate or a plurality of interconnection substrates. The substrate can be any suitable substrate known in the industry. For example, the support structure 104 may include one or more semiconductor substrates, each of which is electrically connected to at least one of the one or more electrodes (for example, all or a subset of the semiconductor substrates may be electrically connected to a single electrode ). Alternatively, the support structure 104 may include a printed circuit board assembly ("PCBA") including one or more electrodes. In other embodiments, the supporting structure 104 may include a substrate (such as a semiconductor substrate) mounted on the PCBA. The microfluidic circuit structure 108 can define the circuit elements of the microfluidic circuit 120. The circuit elements may include spaces or regions that can be fluidly interconnected when the microfluidic circuit 120 is filled with fluid, such as flow areas (which may include or be one or more flow channels), chambers, fences, traps, 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 may partially or completely encapsulate the microfluidic circuit material 116. The frame 114 may be a relatively rigid structure that substantially surrounds the microfluidic circuit material 116, for example. For example, the frame 114 may include a metal material. Alternatively, the microfluidic circuit structure 108 may lack a frame. For example, the microfluidic circuit structure 108 may consist of or consist essentially of the microfluidic circuit material 116. The microfluidic circuit material 116 may be patterned by cavities or the like to define the circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 may include a flexible material, such as a flexible polymer (such as rubber, plastic, elastomer, polysiloxane, polydimethylsiloxane ("PDMS") or the like), which may be breathable . Other examples of materials that can form the microfluidic circuit material 116 include molded glass, etchable materials (such as polysiloxane, such as photo-patternable polysiloxane or "PPS"), photoresist (such as SU8), or the like. In some embodiments, the materials and therefore the microfluidic circuit material 116 may be rigid and/or substantially impermeable to air. Nevertheless, the microfluidic circuit material 116 can be arranged on the support structure 104 and (as appropriate) inside the frame 114. The cover 110 can be an integral part of the microfluidic circuit material 116 and/or the frame 114. Alternatively, the cover 110 may be a structurally different element, as illustrated in FIG. 1A. The cover 110 may include the same or different material as the frame 114 and/or the microfluidic circuit material 116. Similarly, the support structure 104 may be a structure separate from the microfluidic circuit material 116 or the frame 114, as illustrated, or a component of the microfluidic circuit material 116 or the frame 114. Similarly, the microfluidic circuit material 116 and the frame 114 (if present) can be a separate structure as shown in FIG. 1A or a part of the same structure. In some embodiments, the cover 110 may include a rigid material. The rigid material can be glass or a material with similar properties. In some embodiments, the cover 110 may include a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the cover 110 may include both rigid and deformable materials. For example, one or more parts of the cover 110 (for example, one or more parts located above the isolation fences 124, 126, 128, 130) may include a deformable material that interfaces with the rigid material of the cover 110. In some embodiments, the cover 110 may further include one or more electrodes. One or more electrodes may include conductive oxides, such as indium-tin oxide (ITO), which may be coated on glass or similar insulating materials. Alternatively, one or more electrodes can be flexible electrodes embedded in deformable materials, such as polymers (eg PDMS), such as single-walled nanotubes, multi-walled nanotubes, nanowires, conductive nanoparticles Clusters or combinations thereof. Flexible electrodes that can be used in microfluidic devices have been described in, for example, U.S. 2012/0325665 (Chiou et al.), and the content of the patent is incorporated herein by reference. In some embodiments, the cover 110 may be modified (for example, by coating or conditioning all or part of the surface facing the microfluidic circuit 120 inwardly) to support droplet movement and/or cell adhesion, cell viability and/ Or cell growth. Modifications can include coating synthetic or natural polymers or conditioned surfaces with covalently bound molecules (e.g., self-associating molecules). In some embodiments, the cover 110 and/or the support structure 104 can transmit light. The cover 110 may also include at least one gas permeable material (for example, PDMS or PPS). Figure 1A also shows a system 150 for operating and controlling a microfluidic device (such as the microfluidic device 100). The system 150 includes a power supply 192, an imaging device 194 (not shown, but may be part of the imaging module 164), and a tilt device 190 (not shown, but may be a part of the tilt module 166). The power supply 192 can provide power to the microfluidic device 100 and/or the tilt device 190, thereby providing a bias voltage or current as needed. The power source 192 may, for example, include one or more alternating current (AC) and/or direct current (DC) voltage or current sources. The imaging device 194 may include a device for capturing images inside the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device 194 further includes a detector with a high frame rate and/or high sensitivity (e.g., for low-light applications). The imaging device 194 may also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting the radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or small objects contained therein). The emitted light beam may be in the visible spectrum, and may, for example, include fluorescent emission. The reflected light beam may include reflected emission from an LED or a broad-spectrum lamp (such as a mercury lamp (such as a high-pressure mercury lamp) or a xenon arc lamp). As discussed with respect to FIG. 3B, the imaging device 194 may further include a microscope (or string of optical elements), which may or may not include eyepieces. The system 150 further includes a tilting device 190 configured to rotate the microfluidic device 100 along one or more rotation axes. In some embodiments, the tilting device 190 is configured to support and/or contain the housing 102 containing the microfluidic circuit 120 along at least one axis so that the microfluidic device 100 (and therefore the microfluidic circuit 120) can be maintained in a horizontal orientation ( That is, 0° with respect to the x-axis and y-axis), vertical orientation (ie 90° with respect to the x-axis and/or y-axis), or any orientation in between. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) with respect to the axis is referred to herein as the "tilt angle" of the microfluidic device 100 (and the microfluidic circuit 120). For example, the tilting device 190 can tilt the 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 the 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 in between. The horizontal orientation (and therefore the x-axis and y-axis) is defined as perpendicular to the vertical axis defined by gravity. The tilting device can also tilt the microfluidic device 100 (and the microfluidic circuit 120) relative to the x-axis and/or y-axis to any degree greater than 90°, or make the microfluidic device 100 (and the microfluidic circuit 120) relative to x The axis or y axis is tilted by 180° so that the microfluidic device 100 (and the microfluidic circuit 120) is completely inverted. Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) along the axis of rotation defined by the flow zone 106/channel 122 of the microfluidic circuit 120 or some other part. 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. As used herein, the term "above" means that the flow zone 106/channel 122 is positioned higher than one or more isolation fences on the 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 energy of objects in the flow zone/channel). As used herein, the term "below" means that the flow zone 106/channel 122 is positioned below one or more isolation fences on the vertical axis defined by gravity (ie, objects in the isolation fence below the flow zone 106/channel 122 will have Lower than the gravitational potential energy of objects in the flow zone/channel). In some cases, the tilting device 190 tilts the microfluidic device 100 along an axis parallel to the flow zone 106/channel 122. In addition, the microfluidic device 100 can be inclined to an angle less than 90°, so that the flow area 106/channel 122 is located above or below one or more isolation fences but not directly above or directly below the isolation fences. In other cases, the tilting device 190 tilts the microfluidic device 100 along an axis perpendicular to the flow zone 106/channel 122. In other cases, the tilting device 190 tilts the microfluidic device 100 along an axis that is neither parallel nor perpendicular to the flow zone 106/channel 122. The system 150 may further include a media source 178. The medium source 178 (eg, a container, a reservoir, or the like) may include multiple parts or containers, each of which is used to contain a different fluid medium 180. Therefore, the medium source 178 may be a device external to and separate from the microfluidic device 100, as illustrated in FIG. 1A. Alternatively, the medium source 178 may be entirely or partially located inside the housing 102 of the microfluidic device 100. For example, the medium source 178 may include a reservoir that is part of the microfluidic device 100. FIG. 1A also illustrates a simplified block diagram depiction of an example of a control and monitoring device 152 that forms part of the system 150 and can be used in conjunction with the microfluidic device 100. As shown, examples of the control and monitoring device 152 include a main controller 154, a medium module 160 for controlling a medium source 178, and a small object and/or medium (such as a medium of a medium) in the microfluidic circuit 120. The power module 162 for the movement and/or selection of the drop), the imaging module 164 for controlling the imaging device 194 (such as a camera, a microscope, a light source, or any combination thereof) to capture images (such as digital images), and an imaging module 164 for controlling The tilt module 166 of the tilt device 190. The control device 152 may also include other modules 168 for controlling, monitoring, or implementing other functions of the microfluidic device 100. As shown, the device 152 may be operatively coupled with the display device 170 and the input/output device 172 (or further include the display device 170 and the input/output device 172). The main controller 154 may include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor 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 the memory 158 . Alternatively or additionally, the control module 156 may include hard-wired digital circuits and/or analog circuits. 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. Therefore, the functions, process actions, actions or steps of the process discussed herein as implemented for the microfluidic device 100 or any other microfluidic device can be configured by the main controller 154, the medium module 160, and the power module configured as discussed above. Any one 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 the other modules 168 can be connected and coupled to transmit and receive any functions and processes discussed herein. Data used in, action, function or step. The media module 160 controls the media source 178. For example, the media module 160 can control the media source 178 to input the selected fluid media 180 into the housing 102 (eg, via the inlet port 107). The media module 160 can also control the removal of the media from the housing 102 (for example, via an exit port (not shown)). Therefore, one or more media can be selectively input into the microfluidic circuit 120 and removed therefrom. The medium module 160 can also control the fluid medium 180 to flow in the flow area 106/channel 122 inside the microfluidic circuit 120. For example, in some embodiments, before loading tiny objects or beads into the isolation enclosure (for example, using gravity, electrowetting (EW) force, dielectrophoresis (DEP) force, or a combination thereof), the dielectric module 160 The flow of the medium 180 in the flow zone 106/channel 122 and the flow through the housing 102 are terminated. The power module 162 can be configured to control the selection, capture, and movement of small objects and/or medium droplets in the microfluidic circuit 120. As discussed in detail herein, the housing 102 may include an electrowetting (EW) configuration, such as an electrowetting (OEW) configuration, a dielectric wetting (EWOD) configuration, a single-sided electrowetting configuration, or the like. In some embodiments, the housing 102 may further include a dielectrophoresis (DEP) configuration, such as an optical tweezers (OET) configuration, an electrically actuated DEP configuration, and the like. The power module 162 can control the activation of the electrodes and/or transistors (such as photoelectric crystals) included in the EW and/or DEP configuration to select and move the flow area 106/channel 122 and/or the isolation fence 124, 126, 128 , Small objects and/or medium droplets in 130. The imaging module 164 can control the imaging device 194 (not shown). For example, the imaging module 164 can receive and process image data from the imaging device 194. The image data from the imaging device 194 may include any type of information captured by the imaging device 194 (for example, the presence or absence of tiny objects, medium droplets, accumulation of markers (such as fluorescent markers), etc.). Using the information captured by the imaging device 194, the imaging module 164 can further calculate the position of objects (such as tiny objects, medium droplets, or the like) and/or the speed of movement of these objects within the microfluidic device 100. The tilt module 166 can control the tilt movement of the tilt device 190 (not shown). In addition, the tilt module 166 can control the tilt rate and time, for example, to optimize the transfer of small objects to one or more isolation fences via gravity. The tilt module 166 is connected to the imaging module 164 to receive data describing the movement of the small objects and/or medium droplets in the microfluidic circuit 120. Using this data, the inclination module 166 can adjust the inclination angle of the microfluidic circuit 120 to adjust the speed at which the small objects and/or medium droplets move in the microfluidic circuit 120. The tilt module 166 can also use this data to repeatedly adjust the position of the small objects and/or medium droplets in the microfluidic circuit 120. In the example shown in FIG. 1A, the microfluidic circuit 120 is illustrated as including a single flow zone 106 consisting essentially of microfluidic channels 122. Each of the isolation fences 124, 126, 128, and 130 includes a single opening to the flow zone 106/channel 122, but is originally enclosed so that the fences can substantially separate the tiny objects inside the fence from the flow zone 106/ Small objects and/or fluid medium 180 in the channel 122 or other enclosures. The wall of the isolation fence may 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 and the flow of the fluid medium 180 in the flow zone 106/channel 122 are oriented at an angle so that the flow of the fluid medium 180 is not directed to the fence. The flow may be tangent to or orthogonal to the plane of the opening of the fence, for example. 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 isolation fence of the present invention may include a variety of shapes, surfaces, and features optimized for use with EW force, OEW force, DEP force and/or OET force, fluid flow, and/or gravity, as will be discussed in detail below. The microfluidic circuit 120 may include any number of microfluidic isolation fences. Although five isolation fences are shown, the microfluidic circuit 120 may have fewer or more isolation fences. As shown, the microfluidic isolation fences 124, 126, 128, and 130 of the microfluidic circuit 120 each include different features and shapes, which can provide one or more microfluidic devices 100 that can be used to manipulate microscopic objects and/or fluid media. The benefits of drops. Therefore, in some embodiments, the microfluidic circuit 120 may include a plurality of microfluidic isolation fences, wherein two or more of the isolation fences include 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. The microfluidic device that can be used to manipulate tiny objects and/or medium droplets can include any one of isolation fences 124, 126, 128, and 130, or variations thereof, including those shown in Figures 2B, 2C, 2D, 2E, and 2F. Show the fences of their configuration, as discussed below. In the embodiment illustrated in FIG. 1A, a single flow zone 106 is shown. However, other embodiments of the microfluidic device 100 may contain multiple flow regions 106, each of which is configured to provide an independent path for fluid to flow through the microfluidic device 100. The microfluidic circuit 120 includes an inlet valve or port 107 in fluid communication with the flow zone 106, whereby the fluid medium 180 can reach the flow zone 106/channel 122 through the inlet port 107. In some cases, the flow zone 106 contains a single flow path. In other cases, the flow zone 106 includes a plurality of flow paths (e.g., 2, 3, 4, 5, 6, or more), each of which may include a microchannel (e.g., such as channel 122). Two or more (eg, all) of the plurality of flow paths may be substantially parallel to each other. For example, the flow area 106 may be divided into a plurality of parallel channels (for example, such as channels 122). In some 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 twice or more in alternating directions Second-rate. In some cases, the fluid medium in each flow zone 106 flows in at least one of a forward or reverse direction. In some cases, a plurality of isolation fences are configured (for example, relative to the flow area 106/channel 122) so that the isolation fences can be loaded with target minute objects in parallel. In some embodiments, the microfluidic circuit 120 further includes one or more small object traps 132. The well 132 is generally formed in the wall forming the boundary of the flow zone 106/channel 122, and can be positioned relative to the opening of one or more of the microfluidic isolation fences 124, 126, 128, and 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 small objects from the flow zone 106/channel 122. In some cases, the well 132 contains a volume approximately equal to the volume of a single target tiny object. The well 132 may further include openings that are configured to assist in the flow of targeted small objects into the well 132. In some cases, the well 132 includes an opening whose height and width are equal to the size of a single target small object, thereby preventing other small objects (or small objects of larger size) from entering the small object well. The well 132 may further include other features that are configured to assist in targeting the small objects retained in the well 132. In some cases, the well 132 is aligned with the channel 122 with respect to the opening of the microfluidic isolation fence and is located on the opposite side of the channel 122, so that when the microfluidic device 100 is tilted along an axis parallel to the channel 122, small objects are trapped Leave the well 132 in a trajectory that causes the tiny object to fall into the opening of the isolation fence. In some cases, the well 132 includes a side channel 134 smaller than the target small object to facilitate flow through the well 132 and thereby increase the possibility of trapping the small object in the 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 the cover 110) of the microfluidic device 100 via one or more electrodes (not shown) Or multiple locations (for example, locations within the flow area and/or isolation fence) to manipulate, transfer, separate, and sort the droplets located in the microfluidic circuit 120. For example, in some embodiments, the EW force is applied to one or more positions on the surface of the support structure 104 (and/or the cover 110) to transfer the droplets from the flow zone 106 to the desired microfluidic isolation enclosure . In some embodiments, the EW force is used to prevent droplets within an isolation fence (eg, isolation fence 124, 126, 128, or 130) from being displaced therefrom. Furthermore, in some embodiments, the EW force is used to selectively remove droplets previously collected in accordance with the teachings of the present invention from the isolation fence. In some embodiments, the EW force includes electro-optic wetting (OEW) force. In some embodiments, dielectrophoresis (DEP) forces are applied to the fluid medium 180 (for example, in the flow zone and/or isolation fence) via one or more electrodes (not shown) to manipulate, transfer, separate, and divide Select the tiny objects located in it. For example, in some embodiments, a DEP force is applied to one or more parts of the microfluidic circuit 120 to transfer a single tiny object from the flow zone 106 to the desired microfluidic isolation enclosure. In some embodiments, the DEP force is used to prevent small objects within an isolation fence (eg, isolation fence 124, 126, 128, or 130) from being displaced from it. In addition, 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 the isolation fence. In some embodiments, the DEP force includes an optical tweezers (OET) force. In some embodiments, the DEP and/or EW force is combined with other forces, such as flow and/or gravity, to manipulate, transfer, separate, and sort small objects and/or droplets in the microfluidic circuit 120. For example, the housing 102 can be tilted (for example, by tilting the device 190) so that the flow area 106/channel 122 and the small objects located therein are positioned above the microfluidic isolation fence, and gravity can cause the small objects and/or micro Drops are sent to the fence. In some embodiments, DEP force and/or EW force may be applied before other forces. In other embodiments, DEP force and/or EW force may be applied after other forces. In other cases, DEP and/or EW forces can be applied simultaneously with other forces or alternately with other forces.Dynamic configuration of microfluidic devices. As mentioned above, the control and monitoring equipment of the system may include a power module for selecting and moving objects (such as 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 the moving object and other considerations. Specifically, the support structure 104 and/or the cover 110 of the microfluidic device 100 may include an electrowetting (EW) configuration, which selectively introduces EW forces on 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 some embodiments, the microfluidic device of the present invention may include a first part having an EW configuration and a second part having a dielectrophoresis (DEP) configuration. Therefore, at least a part of the supporting structure 104 and/or the cover 110 of the microfluidic device 100 may include a DEP configuration, which is used to selectively introduce DEP force to the micro-objects in the fluid medium 180 in the microfluidic circuit 120, and This selection, capture and/or movement of individual tiny objects or groups of tiny objects In some embodiments, the microfluidic device of the present invention may include an electrowetting configuration, which includes a substrate having a dielectric layer and a droplet actuation surface, the droplet actuation surface including covalent bonding to the dielectric layer The hydrophobic layer. The dielectric layer may be located directly below the hydrophobic layer, so that the droplets remaining on the substrate directly contact the hydrophobic layer. Figure 2A illustrates an example of a part of the microfluidic device. As shown, the device 400 may include a substrate 104 including a substrate and at least one electrode (eg, a first electrode) 418. The substrate may include multiple layers, including an outer hydrophobic layer 412, an inner dielectric layer 414, a conductive layer 416, an electrode 418, and a support 420 as appropriate. The hydrophobic layer 412 and the inner dielectric layer 414 may provide a portion of the substrate 102 that defines the inner facing surface of the outer shell. The device 400 also includes a cover 110, which includes an outer hydrophobic layer 422, an inner layer 428 that may include at least one electrode, and a support 430 as appropriate. The cover 110 and the base 104 are substantially parallel to each other and are joined together by a spacer 108 (for example, a microfluidic circuit material) to define a housing 435 that is configured to contain a liquid medium. The liquid medium may be, for example, a hydrophobic liquid, such as oil. In addition, the housing 435 may contain liquid droplets 440, such as an aqueous medium. Generally, the liquid medium and the liquid of the droplets are selected as immiscible liquids. The spacer element 108 may comprise a polymer. The polymer can be, for example, a silicon-based organic polymer, such as polydimethylsiloxane (PDMS) or photopatternable polysiloxane (PPS), both of which are commercially available from Dow Corning. Alternatively, the spacer element 108 may include an epoxy-based adhesive. The epoxy-based adhesive can be, for example, SU-8 or the same type of material. The spacer element 108 may have a thickness of at least 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers or more (ie, the gap between the inner surface of the substrate 104 and the cover 110) . Therefore, for example, the thickness of the spacer element 108 can be 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 may define one or more microfluidic channels in the housing. In addition, the spacer element 108 may further define a plurality of chambers (or isolation fences) within the housing, wherein each chamber is fluidly connected to and opened from at least one microfluidic channel. Thus, for example, the spacer element 108 may define a single microfluidic channel and a plurality of chambers fluidly connected to it, or a plurality of microfluidic channels, wherein each channel is fluidly connected to a plurality of chambers. In addition, each chamber can be fluidly connected 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 the opposite terminal of an AC voltage source (not shown), the substrate 104 can apply electrowetting force to the hydrophobic surface 412 (ie, The droplet actuates the aqueous droplet in contact with the surface). In some embodiments, the AC voltage used to achieve electrowetting-based droplet movement in the microfluidic device is at least 20 volts peak-to-peak (ppV) (e.g., about 20 ppV to 80 ppV, about 20 ppV to 60 ppV, About 25 ppV to 50 ppV, about 25 ppV to 40 ppV, or about 25 ppV to 35 ppV). In some embodiments, the frequency of the AC voltage used to achieve electrowetting-based droplet movement in the microfluidic device is about 1 kHz to 100 kHz (e.g., about 5 kHz to 90 kHz, about 10 kHz to 80 kHz, About 15 kHz to 70 kHz, about 20 kHz to 60 kHz, about 25 kHz to 50 kHz, or about 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 single layer of self-associating molecules covalently bonded to the inner dielectric layer 414 of the substrate 104 or the inner layer 428 of the cover 110, respectively. The self-associated molecules of the monolayer contain sufficient two-dimensional packing density to create a hydrophobic barrier between the surface of the bound monolayer and the hydrophilic liquid (ie, to prevent the insertion and/or penetration of polar molecules or other chemicals into the monolayer). The packing density of densely packed monolayers will depend on the self-associated molecules used. A densely packed monolayer containing alkyl-terminated siloxanes will usually contain at least 1×10¹⁴ Molecules/cm² (E.g. at least 1.5×10¹⁴ Pcs, 2.0×10¹⁴ Pcs, 2.5×10¹⁴ Or more molecules/cm² ). As explained in more detail below, the self-associating molecules may each contain a linking group, such as a siloxyalkyl group or a phosphonic acid group. The siloxane groups can be covalently bonded to molecules of the inner dielectric layer 414 or the inner layer 428. Similarly, the phosphonic acid group can be covalently bonded to the molecules of the inner dielectric layer 414 or the inner layer 428. Self-associated molecules may include long chain hydrocarbons that may or may not have branches. Therefore, self-associating molecules may contain alkyl-terminated siloxanes or alkyl-terminated phosphonic acids. Long chain hydrocarbons may contain chains of at least 10 carbons (e.g., at least 16, 18, 20, 22, or more carbons). The self-associating molecule may comprise a fluorinated carbon chain. Thus, for example, the self-associating molecule may comprise fluoroalkyl-terminated siloxane or fluoroalkyl-terminated phosphonic acid. The fluorinated carbon chain can have the chemical formula CF₃ -(CF₂ )m-(CH₂ ) 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-associated molecules may have a thickness of less than about 5 nanometers (for example, about 1.0 nanometers to about 4.0 nanometers, about 1.5 nanometers to about 3.0 nanometers, or about 2.0 nanometers to about 2.5 nanometers). . The outer hydrophobic layer 412 of the substrate 104 may be patterned so that selected areas are relatively hydrophilic compared to the rest of the outer hydrophobic layer. This can be achieved, for example, by increasing the voltage drop on the underlying inner dielectric layer 122 to 50 ppV or more (for example, 60 ppV, 65 ppV, 70 ppV, 75 ppV, 80 ppV or more) over a period of time . Without wishing to be bound by theory, it is believed that the relatively hydrophilic region contains water molecules that have been inserted into the monolayer. In some embodiments, the inner dielectric layer of the substrate may include one or more oxide layers. For example, the inner dielectric layer may include or consist of a single oxide layer (such as a metal oxide layer). Alternatively, the inner dielectric layer may include or consist of two layers. In some embodiments, the layer may be silicon dioxide or silicon nitride, and the other layer may be a metal oxide, such as aluminum oxide. In some embodiments, the thickness of the metal oxide layer may be in the range of about 15 nm to about 45 nm, or about 30 nm to about 40 nm, or about 33 nm to about 36 nm. The metal oxide layer can be deposited by atomic layer deposition (ALD) technology and the layer containing silicon dioxide or silicon nitride can be deposited by plasma assisted chemical vapor deposition (PECVD) technology. In another embodiment, the inner dielectric layer may include three dielectric material layers. In some embodiments, the first layer may include a metal oxide, such as aluminum oxide, hafnium dioxide, or the like, which may be sandwiched between the silicon dioxide layer and the silicon nitride layer. In some embodiments, the thickness of the metal oxide layer may range from about 5 nm to about 20 nm, and the layer may be deposited by atomic layer deposition (ALD) technology. The silicon oxide layer can also be deposited by ALD, and can have a thickness of about 2 nm to about 10 nm. The silicon nitride layer can be deposited by plasma-assisted chemical vapor deposition (PECVD) technology, and can have a thickness of about 80 nm to about 100 nm or a thickness of about 90 nm. The inner dielectric layer may have a thickness of about 50 nanometers to 105 nanometers and/or an impedance of about 50 kiloohms to 150 kiloohms, and a preferred embodiment is about 100 kiloohms, and the layer constituting the inner dielectric layer The number is irrelevant. The substrate 104 may include a photoreactive layer 146 having a first side contact with the inner dielectric layer 414. The second side of the photoreactive layer 416 can contact at least one electrode 418. The photoreactive layer 416 may include hydrogenated amorphous silicon (a-Si:H). For example, a-Si:H may contain about 8% to 40% hydrogen (ie, calculated based on 100*number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si:H layer may have at least about 500 nanometers (e.g., at least about 600 nanometers to 1400 nanometers, about 700 nanometers to 1300 nanometers, about 800 nanometers to 1200 nanometers, and about 900 nanometers to 1100 nanometers. Nanometers, or about 1000 nanometers). However, the thickness of the a-Si:H layer can vary with the thickness of the inner dielectric layer 414, so that when the substrate 104 is in the open state (i.e., irradiated and conductive) and closed state (i.e., 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 about 50 kohms to about 150 kohms, and the impedance of the a-Si:H layer can be tuned to at least about 0.5 Mohms in the closed state and in the open state. Less than or equal to about 1 kiloohm in the state. These are only examples, but they illustrate how the impedance can be tuned to achieve the photoreactive (photoconductive in this case) layer 416 that exhibits robust on/off performance. In the embodiment where the substrate 104 has a photoreactive layer 416 formed from the 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. These floating electrode pads have been described in, for example, US Patent No. 6,958,132, the content of which is incorporated herein by reference. Alternatively, the photoreactive layer 416 may include a plurality of conductors, and each conductor may be controllably connected to at least one electrode of the substrate 102 via a photoelectric crystal switch. The conductor controlled by the photoelectric crystal switch is well known in the industry and has been described in, for example, US Patent Application No. 2014/0124370, the content of which is incorporated herein by reference. The substrate 104 may include a single electrode 418 that is configured to connect to an AC voltage source. The single electrode 418 may include an indium-tin oxide (ITO) layer, which may be formed on the glass support 420, for example. Alternatively, the single electrode 418 may include a conductive silicon layer. In other embodiments, the substrate 104 may include a plurality of electrodes that can be individually addressed in the manner of an EWOD device, which is well known in the industry. Individually addressable electrodes can be connected to one or more AC voltage sources via corresponding 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 in the form of a substrate. Therefore, the microfluidic device 400 may have a configuration to provide electrowetting force to both the substrate 104 and the cover 110 of the aqueous droplet 440 located in the housing 435. In these embodiments, the dielectric layer of the cover 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 cover 104 can be disclosed herein for the conductive layer 126 of the substrate 102 Any way of configuration. As discussed herein, the microfluidic device of the present invention may include a portion having a DEP configuration. An example of this part is the microfluidic device 200 illustrated in FIGS. 1C and 1D. Although for the sake of brevity, FIGS. 1C and 1D respectively show a vertical cross-sectional view and a horizontal cross-sectional view of a part of the housing 102 of the microfluidic device 200 with an open area/chamber 202, it should be understood that the area/chamber 202 may have a more detailed structure (Such as growth chamber, isolation fence, flow area or flow channel) part of the fluid circuit element. In addition, the microfluidic device 200 may include other fluidic circuit elements. For example, the microfluidic device 200 may include a plurality of growth chambers or isolation fences and/or one or more flow areas 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 parts thereof. It should be further understood that any of the microfluidic device components and system components described above or below can be incorporated into the microfluidic device 200 and/or used in combination therewith. For example, the system 150 including the above-mentioned control and monitoring device 152 can be combined with microfluidics including one or more of the media module 160, the power module 162, the imaging module 164, the tilt module 166, and other modules 168 The device 200 is used together. As can be seen in FIG. 1C, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activation substrate 206 covering the bottom electrode 204, and a cover 110 having a top electrode 210, wherein the top electrode 210 and the bottom electrode 204 are spaced apart. The top electrode 210 and the electrode activation substrate 206 define opposite surfaces of the region/chamber 202. Therefore, the medium 180 contained in the zone/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. The power supply 212 is also shown, which is configured to connect to the bottom electrode 204 and the top electrode 210 and generate a bias voltage between the electrodes, as required to generate the DEP force in the zone/chamber 202. The power source 212 may be, for example, an alternating current (AC) power source. In some embodiments, the microfluidic device 200 illustrated in FIGS. 1C and 1D may have an optically actuated DEP configuration. Therefore, the changing pattern of the light 218 from the light source 216 that can be controlled by the power module 162 can selectively activate and deactivate the changing pattern of the DEP electrode at the region 214 of the inner surface 208 of the electrode activation substrate 206. (The region 214 of the microfluidic device with the DEP configuration is hereinafter referred to as the "DEP electrode region".) As illustrated in FIG. 1D, the light pattern 218 guided into the inner surface 208 of the electrode activation substrate 206 can be illuminated in a pattern (For example, a square) select the DEP electrode area 214a (shown in white). The unirradiated DEP electrode area 214 (cross-hatched) is hereinafter referred to as the "dark" DEP electrode area 214. The relative electrical impedance across the DEP electrode activation substrate 206 (that is, from the bottom electrode 204 up to the inner surface 208 of the electrode activation substrate 206, which borders the medium 180 in the flow zone 106) is greater than the relative electrical impedance of each dark DEP electrode zone The relative electrical impedance of 214 passing through the medium 180 in the zone/chamber 202 (ie, from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, the irradiated DEP electrode region 214a exhibits a reduced relative impedance through the electrode activation substrate 206, which is less than the relative impedance of each irradiated DEP electrode region 214a through the medium 180 in the zone/chamber 202. Under the activation power source 212, the DEP configuration described above generates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode area 214a and the adjacent dark DEP electrode area 214, which in turn attracts or repels nearby small objects in the fluid medium 180 (Not shown) local DEP force. Therefore, by changing the light pattern 218 projected from the light source 216 into the microfluidic device 200, the small objects in the fluid medium 180 can be attracted or repelled at the many different DEP electrode regions 214 on the inner surface 208 of the zone/chamber 202. The DEP electrode is selectively activated and deactivated. Whether the DEP force attracts or repels nearby small objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or the small objects (not shown). The square pattern 220 of the illuminated DEP electrode region 214a illustrated in FIG. 1C is only an example. Any pattern of the DEP electrode area 214 can be illuminated (and thereby activated) by the pattern of the light 218 projected into the device 200, and the illuminated/activated DEP electrode area can be repeatedly changed by changing or moving the light pattern 218 The pattern of 214. In some embodiments, the electrode activation substrate 206 may include or consist of a photoconductive material. In these embodiments, the inner surface 208 of the electrode activation substrate 206 may be featureless. For example, the electrode activation substrate 206 may include or consist of a hydrogenated amorphous silicon (a-Si:H) layer. The a-Si:H may contain, for example, about 8% to 40% hydrogen (calculated based on 100*number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si:H layer may have a thickness of about 500 nm to about 2.0 μm. In these embodiments, according to the light pattern 218, the DEP electrode region 214 can be generated anywhere on the inner surface 208 of the electrode activation substrate 206 and in any pattern. Therefore, the number and pattern of the DEP electrode regions 214 need not be fixed, but can correspond to the light pattern 218. An example of a microfluidic device with a DEP configuration including the photoconductive layer discussed above has been described in, for example, U.S. Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355). All of the contents are incorporated into this article by reference. In other embodiments, the electrode activation substrate 206 may include a plurality of substrates including a plurality of doped layers, electrically insulating layers (or regions), and conductive layers that are known in the semiconductor field to form semiconductor integrated circuits, for example. For example, the electrode activation substrate 206 may include a plurality of photoelectric crystals, including, for example, a lateral bipolar photoelectric crystal, and each photoelectric crystal corresponds to the DEP electrode region 214. Alternatively, the electrode activation substrate 206 may include electrodes (for example, conductive metal electrodes) controlled by a photoelectric crystal switch, wherein each of the electrodes corresponds to the DEP electrode region 214. The electrode activation substrate 206 may include the photoelectric crystals or patterns of electrodes controlled by the photoelectric crystals. The pattern can be, for example, a substantially square photoelectric crystal or an electrode array controlled by a photoelectric crystal arranged in columns and rows, for example, as shown in FIG. 2B. Alternatively, the pattern may be a substantially hexagonal photoelectric crystal or an electrode array controlled by the photoelectric crystal forming a hexagonal crystal lattice. Regardless of the pattern, the circuit element can form an electrical connection between the DEP electrode area 214 of the inner surface 208 of the electrode activation substrate 206 and the bottom electrode 210, and they can be electrically connected by the light pattern 218 (ie, photoelectric crystal or electrode). ) Selective activation and deactivation. When not activated, each electrical connection may have a high impedance, so that it passes through the electrode activation substrate 206 (ie, from the bottom electrode 204 to the inner surface 208 of the electrode activation substrate 206, which is the same as the medium in the zone/chamber 202). The relative impedance of the corresponding DEP electrode region 214 is greater than the relative impedance of the corresponding DEP electrode region 214 through the medium 180 (ie, from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, when activated by light in the light pattern 218, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 of each irradiated DEP electrode region 214, thereby activating the corresponding DEP electrode region as discussed above DEP electrode at 214. Therefore, the DEP that attracts or repels the small objects (not shown) in the medium 180 at the many different DEP electrode regions 214 on the inner surface 208 of the electrode activation substrate 206 in the zone/chamber 202 can be determined by the light pattern 218. Electrode is selectively activated and deactivated. Examples of microfluidic devices with electrode-activated substrates containing photoelectric crystals have been described in, for example, U.S. Patent No. 7,956,339 (Ohta et al.) (see, for example, the device 300 illustrated in FIGS. 21 and 22 and its description). The entire content of the patent is incorporated herein by reference. Examples of microfluidic devices with electrode-activated substrates containing electrodes controlled by photoelectric crystal switches have been described in, for example, U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, for example, the illustrations illustrated throughout the drawings Devices 200, 400, 500, 600, and 900 and their descriptions), the entire contents of the patent are incorporated herein by reference. In some embodiments of the microfluidic device in the DEP configuration, the top electrode 210 is a part of the first wall (or cover 110) of the housing 102, and the electrode activation substrate 206 and the bottom electrode 204 are the second wall (or cover 110) of the housing 102 Support structure 104) part of. The zone/chamber 202 may be between the first wall and the second wall. In other embodiments, the electrode 210 is a part of the second wall (or the support structure 104), and one or both of the electrode activation substrate 206 and/or the electrode 210 is a part of the first wall (or the cover 110). In addition, the light source 216 may alternatively be used to illuminate the housing 102 from below. Using the microfluidic device 200 with the DEP configuration of FIGS. 1C-1D, the power module 162 can project the light pattern 218 into the device 200 to activate the electrodes in the pattern (for example, the square pattern 220) surrounding and capturing small objects The first group of one or more DEP electrodes of the DEP electrode area 214a of the inner surface 208 of the substrate 206 is activated to select the minute objects (not shown) in the medium 180 in the zone/chamber 202. Subsequently, the power module 162 can move the captured minute objects by moving the light pattern 218 relative to the device 200 to activate one of the DEP electrode regions 214 or a second set of more DEP electrodes. Alternatively, the device 200 can be moved relative to the light pattern 218. In other embodiments, the microfluidic device 200 may have a light-activated DEP configuration that does not rely on the DEP electrode of the inner surface 208 of the electrode-activated substrate 206. For example, the electrode activation substrate 206 may include and include selectively addressable and energizable electrodes positioned relative to a surface (eg, cover 110) of at least one electrode. The switch (for example, a transistor switch in a semiconductor substrate) can be selectively turned on and off to activate or inactivate the DEP electrode of the DEP electrode region 214, thereby activating the tiny objects (not shown in the region/chamber 202 near the DEP electrode) Shown) produces a net DEP force. Depending on characteristics such as the frequency of the power source 212 and the medium (not shown) in the zone/chamber 202 and/or the dielectric properties of the tiny objects, the DEP force can attract or repel the nearby tiny objects. By selectively activating and deactivating the DEP electrode group (for example, the group of DEP electrode regions 214 forming the square pattern 220), one or more minute objects in the zone/chamber 202 can be trapped and placed in the zone/chamber 202. Move inside the room 202. The power module 162 in FIG. 1A can control the switches and thus activate and deactivate individual multiples of DEP electrodes to select, trap, and move specific tiny objects around the area/chamber 202 (not shown). Microfluidic devices with DEP configurations that include selectively addressable and energized electrodes are known in the industry and have been described in, for example, U.S. Patent Nos. 6,294,063 (Becker et al.) and No. 6,942,776 (Medoro). All contents are incorporated into this article by reference.Isolation fence . Non-limiting examples of general isolation fences 224, 226, and 228 are shown in the microfluidic device 230 depicted in FIGS. 2A-2C. Each isolation fence 224, 226, and 228 may include a separation structure 232 that defines a separation area 240 and a connection area 236 that fluidly connects the separation area 240 to the channel 122. The connecting region 236 may include a proximal opening 234 of the channel 122 and a distal opening 238 of the separating region 240. The connection area 236 may be configured so that the maximum penetration depth of the fluid flow (not shown) flowing from the channel 122 into the isolation fences 224, 226, 228 does not extend into the separation area 240. Therefore, due to the connection area 236, the tiny objects (not shown) or other materials (not shown) arranged in the separation area 240 of the isolation fences 224, 226, 228 can be separated from the flow of the medium 180 in the channel 122 and Substantially unaffected by it. The isolation fences 224, 226, and 228 of FIGS. 2A-2C each have a single opening that opens directly toward the channel 122. The opening of the isolation fence is laterally opened from the channel 122. The electrode activation substrate 206 is located under the channel 122 and the isolation fences 224, 226, and 228. The upper surface of the electrode activation substrate 206 in the shell of the isolation fence forming the bottom plate of the isolation fence is arranged in the channel 122 (or if there is no channel, the flow zone) to form the flow channel (or flow zone respectively) of the microfluidic device The electrodes of the bottom plate activate the same level or substantially the same level on the upper surface of the substrate 206. The electrode activation 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 microns, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns Or smaller random or patterned surface. The height change of the upper surface of the substrate across the channel 122 (or flow zone) and the isolation fence can be less than about 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3% or 0.1%. Although the microfluidic device 200 has been described in detail, this also applies to any of the microfluidic devices 100, 230, 250, 280, 290, 600, and 700 described herein. Therefore, the channel 122 may be an example of an affected area, and the separation area 240 of the isolation fences 224, 226, 228 may be an example of an unaffected area. As mentioned, the channel 122 and the isolation fences 224, 226, 228 may be configured to contain one or more fluid media 180. In the example shown in FIGS. 2A-2B, the port 222 is connected to the channel 122 and allows the fluid medium 180 to be introduced into or removed from the microfluidic device 230. Before introducing the fluid medium 180, the microfluidic device may be pretreated with a gas such as carbon dioxide gas. After 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 ports 222 can be arranged at different positions (such as opposite ends) of the channel 122, and the flow of the medium 242 can be generated from one port 222 used as an inlet to another port 222 used as an outlet. Figure 2C illustrates a detailed view of an example of the isolation fence 224 of the present invention. An example of a tiny object 246 is also shown. As is known, the flow 242 of the fluid medium 180 in the microfluidic channel 122 through the proximal opening 234 of the isolation fence 224 can cause a secondary flow 244 of the medium 180 into and/or out of the isolation fence 224. In order to prevent the small objects 246 in the separation area 240 of the isolation fence 224 from secondary flow 244, the length L of the connection area 236 of the isolation fence 224_con (I.e., 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 connecting region 236_p . Penetration depth D of secondary flow 244_p It depends on the speed of the fluid medium 180 flowing in the channel 122 and various parameters related to the configuration of the channel 122 and the proximal opening 234 of the connecting region 236 to the channel 122. For a given microfluidic device, the configuration of the channel 122 and the opening 234 will be fixed, and the flow rate 242 of the fluid medium 180 in the channel 122 will be variable. Therefore, 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 connecting area 236_con . As long as the flow 242 of the fluid medium 180 in the channel 122 does not exceed the maximum speed V_max , The resulting secondary flow 244 can be limited to the channel 122 and the connection area 236 and far away from the separation area 240. Therefore, the flow 242 of the medium 180 in the channel 122 will not draw out the small objects 246 from the separation zone 240. On the contrary, 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. In addition, 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 will not move other particles (for example, particles and/or nanoparticles) from the channel 122 to the separation zone 240 of the isolation fence 224. Therefore, the length L of the connecting area 236_con Greater than the maximum penetration depth D of the secondary flow 244_p It is possible to prevent one isolation fence 224 from being contaminated by other particles from the channel 122 or another isolation fence (for example, the isolation fences 226, 228 in FIG. 2D). Since the connection area 236 between 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 area 236 can be regarded as the affected (or flow) area of the microfluidic device 230. On the other hand, the separation area 240 of the isolation fences 224, 226, 228 can be regarded as an unaffected (or non-flowing) area. For example, the components (not shown) of the first fluid medium 180 in the channel 122 can be substantially diffused from the channel 122 through the connection region 236 and into the second separation region 240 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, the components (not shown) of the second medium 248 in the separation zone 240 can be substantially diffused from the separation zone 240 through the connection zone 236 and into the first medium in the channel 122 only by the components of the second medium 248. 180 is mixed with the first medium 180 in the channel 122. In some embodiments, the degree of fluid medium exchange by diffusion between the separation zone and the flow zone of the isolation fence is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater than about 99% fluid exchange. The first medium 180 may be the same medium as the second medium 248 or a different medium. In addition, the first medium 180 and the second medium 248 may be intended to be the same at the beginning, and then become different (for example, by conditioning the second medium 248 by one or more units in the separation zone 240 or by changing the flow through the channel 122 of the medium 180). The maximum penetration depth D of the secondary flow 244 caused by the flow 242 of the fluid medium 180 in the channel 122_p It may depend on multiple parameters, as mentioned above. Examples of these parameters include: the shape of the channel 122 (for example, the channel can guide the medium into the connection area 236, divert the medium away from the connection area 236, or in a direction substantially perpendicular to the proximal opening 234 of the connection area 236 Guide the medium to the channel 122); the width W of the channel 122 at the proximal opening 234_ch (Or cross-sectional area); and the width W of the connecting area 236 at the proximal opening 234_con (Or cross-sectional area); the velocity V of the flow 242 of the fluid medium 180 in the channel 122; the viscosity of the first medium 180 and/or the 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 with respect 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) can be substantially perpendicular to the flow 242 of the medium 180; the width W of the connecting area 236 at the opening 234_con (Or cross-sectional area) can be substantially parallel to the flow 242 of the medium 180 in the channel 122; and/or the length L of the connecting area_con The flow 242 of the medium 180 in the channel 122 may be substantially perpendicular. The foregoing is only an example, and the relative positions of the passage 122 and the isolation fences 224, 226, 228 can be in other orientations with respect to each other. As illustrated in FIG. 2C, the width W of the connecting area 236_con It can be uniform from the proximal opening 234 to the distal opening 238. Therefore, the width W of the connecting area 236 at the distal opening 238_con It can be the width W of the connection area 236 at the proximal opening 234 in this article_con Any one of the scope of identification. Or, the width W of the connecting region 236 at the distal opening 238_con Can be greater than the width W of the connection area 236 at the proximal opening 234_con . As illustrated in FIG. 2C, the width of the separation region 240 at the distal opening 238 may be substantially the same as the width W of the connection region 236 at the proximal opening 234_con same. Therefore, the width of the separation area 240 at the distal opening 238 may be the width W of the connection area 236 at the proximal opening 234 herein._con Any one of the scope of identification. Alternatively, the width of the separation zone 240 at the distal opening 238 may be greater or smaller than the width W of the connection zone 236 at the proximal opening 234_con . In addition, the distal opening 238 may be smaller than the proximal opening 234 and the width W of the connecting region 236_con It may be narrowed between the proximal opening 234 and the distal opening 238. For example, the connecting area 236 may use a variety of different geometric structures (such as chamfering the connecting area, beveling the connecting area) to narrow between the proximal opening and the distal opening. In addition, any portion or sub-portion of the connecting region 236 can be narrowed (e.g., the portion of the connecting region adjacent to the proximal opening 234). 2D-2F show another exemplary embodiment of a microfluidic device 250 including a microfluidic circuit 262 and a flow channel 264, which is a variation of the respective microfluidic device 100, circuit 132, and channel 134 of FIG. 1. The microfluidic device 250 also has a plurality of isolation fences 266, which are other variations of the aforementioned isolation fences 124, 126, 128, 130, 224, 226, or 228. Specifically, it should be understood that the isolation fence 266 of the device 250 shown in FIGS. 2D-2F can replace the aforementioned isolation fence 124, 126, 128, 130, 224, Either 226 or 228. Similarly, the microfluidic device 250 is another variant of the microfluidic device 100, and can also be compatible with any of the aforementioned microfluidic devices 100, 200, 230, 280, 290, 320 and other microfluidic system components described herein. The same or different DEP configuration. The microfluidic device 250 of FIGS. 2D-2F includes a supporting structure (not visible in FIGS. 2D-2F, but may be the same as or substantially similar to the supporting structure 104 of the device 100 shown in FIG. 1A), a microfluidic circuit structure 256 And the cover (not visible in FIGS. 2D-2F, but can be the same as or substantially similar to the cover 122 of the device 100 shown in FIG. 1A). The microfluidic circuit structure 256 includes a frame 252 and a microfluidic circuit material 260, which may be the same as or substantially similar to the frame 114 and the microfluidic circuit material 116 of the device 100 shown in FIG. 1A. As shown in FIG. 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 may include a plurality of channels 264 (two are shown, but there may be more) to which a plurality of isolation fences 266 are fluidly connected. Each isolation fence 266 may include a separation structure 272, a separation area 270 and a connection area 268 within the separation structure 272. From the proximal opening 274 of the channel 264 to the distal opening 276 at the separation structure 272, the connecting region 268 fluidly connects the channel 264 to the separation region 270. Generally, according to the above discussion of FIGS. 2B and 2C, the flow 278 of the first fluid medium 254 in the channel 264 can generate the second flow 282 of the first medium 254 from the channel 264 to the respective connection area 268 of the isolation fence 266 and/or Flow from it. As illustrated in FIG. 2E, the connection area 268 of each isolation 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 of connection area 268 L_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 being redirected towards the separation zone 270 (as shown in Figure 2D). Alternatively, as illustrated in Figure 2F, the connecting zone 268 may have a depth less than the maximum penetration D_p The length L_con In this case, the secondary flow 282 will extend through the connection zone 268 and redirect towards the separation zone 270. In the latter case, the length L of the connecting area 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 connecting area 268_con Greater than penetration depth D_p Or the length L of the connecting area 268_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 penetration depth D_p The secondary flow, and the small objects in the separation zone 270 of the isolation fence 266 (not shown, but can be the same as or substantially similar to the small objects 246 shown in FIG. 2C) will not be passed through the first medium 254 in the channel 264 The flow 278 is drawn from the separation zone 270. The flow 278 in the channel 264 also does not draw other materials (not shown) from the channel 264 into the separation area 270 of the isolation fence 266. Therefore, the diffusion system is the only mechanism in which the components of the first medium 254 in the channel 264 can move from the channel 264 to the second medium 258 in the separation area 270 of the isolation fence 266. Similarly, diffusion is the only mechanism by which the components in the second medium 258 in the separation zone 270 of the isolation fence 266 can move from the separation zone 270 to the first medium 254 in the channel 264. The first medium 254 may be the same medium as the second medium 258, or the first medium 254 may be a different medium from the second medium 258. Alternatively, the first medium 254 and the second medium 258 may initially have the same meaning and then become different, for example, by conditioning the second medium by one or more units in the separation zone 270 or by changing the flow through the channel 264 The medium. As illustrated in Figure 2E, the width W of the channel 264 in the channel 264_ch (That is, it can be obtained by crossing the direction in which the fluid medium flows through the channel indicated by the arrow 278 in FIG. 2D) can be substantially perpendicular to the width W of the proximal opening 274_con1 , And therefore substantially parallel to the width W of the distal opening 276_con2 . However, the width W of the proximal opening 274_con1 And the width W of the distal opening 276_con2 It does not have to be substantially perpendicular to each other. For example, the width W of the proximal opening 274_con1 Orientation axis (not shown) and width W of distal opening 276_con2 The angle between the other axes of orientation may not be perpendicular and therefore not 90°. Examples of alternate orientation angles include angles in any of the following ranges: about 30° to about 90°, about 45° to about 90°, about 60° to about 90°, or the like. In various embodiments of the isolation fence (e.g., 124, 126, 128, 130, 224, 226, 228, or 266), the separation area (e.g., 240 or 270) is configured to contain a plurality of tiny objects. In other embodiments, the separation zone may be configured to contain only one, two, three, four, five, or similar relatively small number of tiny objects. Therefore, the volume of the separation zone can be, for example, at least 1×10⁶ Cubic microns, 2×10⁶ Cubic micrometer, 4×10⁶ Cubic microns, 6×10⁶ Cubic microns or larger. In the various embodiments of the isolation fence, the width W of the channel (for example 122) at the proximal opening (for example 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 W of the channel (e.g., 122) at the proximal opening (e.g., 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 W_ch It can be within other ranges (for example, a range defined by any of the endpoints listed above). In addition, the W of channel 122_ch It can be selected to be in any of these ranges of the passage area except the proximal opening of the isolation fence. In some embodiments, the isolation fence has a height of about 30 microns to about 200 microns, or about 50 microns to about 150 microns. In some embodiments, the isolation fence has about 1×10⁴ -3 ×10⁶ Square microns, 2 × 10⁴ -2 ×10⁶ Square microns, 4 × 10⁴ -1 ×10⁶ Square microns, 2 × 10⁴ -5 ×10⁵ Square microns, 2 × 10⁴ -1 ×10⁵ Square microns or about 2 × 10⁵ -2×10⁶ The cross-sectional area of square microns. In some embodiments, the connection region has a cross-sectional width of about 100 micrometers to about 500 micrometers, 200 micrometers to about 400 micrometers, or about 200 micrometers to about 300 micrometers. In the various embodiments of the isolation fence, the height H of the channel (for example 122) at the proximal opening (for example 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 Micrometers. The above is only an example, and the height of the channel (such as 122) is H_ch It can be within other ranges (for example, a range defined by any of the endpoints listed above). The height of channel 122 H_ch It can be selected to be in any of these ranges of the passage area except the proximal opening of the isolation fence. In various embodiments of the isolation fence, the cross-sectional area of the channel (such as 122) at the proximal opening (such as 234) can be in 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 square microns Micron, 1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500 square microns, 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 3,000-6,000 square microns. The foregoing is only an example, and the cross-sectional area of the passage (for example, 122) at the proximal opening (for example, 234) may be within other ranges (for example, the range defined by any of the end points listed above). In the various embodiments of the isolation fence, the length of the connecting area (for example 236) is L_con 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 of the connecting area (such as 236) is L_con It can be in a range different from the above example (for example, a range defined by any of the endpoints listed above). In various embodiments of the isolation fence, the width W of the connecting area (for example 236) at the proximal opening (for example 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 above is only an example, and the width W of the connecting area (such as 236) at the proximal opening (such as 234)_con It may be different from the above examples (e.g., a range defined by any of the endpoints listed above). In various embodiments of the isolation fence, the width W of the connecting area (for example 236) at the proximal opening (for example 234)_con It can be at least as large as the largest size of the tiny object (for example, biological cells, which can be T cells, B cells, or eggs or embryos) for which the isolation fence is intended to be used. For example, the width W of the connecting area 236 at the proximal opening 234 of the isolation fence in which the droplet is placed_con Can 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 Micrometers, about 200 micrometers, about 225 micrometers, about 250 micrometers, about 300 micrometers, or about 100-400 micrometers, about 120-350 micrometers, about 140-200-200 300 micrometers, or about 140-200 micrometers. The above is only an example, and the width W of the connecting area (such as 236) at the proximal opening (such as 234)_con It may be different from the above examples (e.g., a range defined by any of the endpoints listed above). In each embodiment of the isolation fence, the width W of the proximal opening of the connecting area_pr It can be at least as large as the maximum size of the tiny objects (such as biological tiny objects, such as cells) for which the isolation fence is intended to be used. For example, the 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 75-100 microns, or within the range of about 200-300 microns In various embodiments of the isolation fence, the length L of the connecting area (for example 236) at the proximal opening 234_con For the width W of the connecting area (e.g. 236)_con The ratio of may 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 above is only an example, and the length L of the connecting area 236 at the proximal opening 234_con The width W of the connecting area 236_con The ratio can be different from the above example. In the various embodiments of the microfluidic device 100, 200, 230, 250, 280, 290, 320, 600, 700, V_max Can be set to about 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 with an isolation fence, the volume of the separation area (for example 240) of the isolation fence may be, for example, at least 5×10⁵ Cubic microns, 8×10⁵ Cubic micrometer, 1×10⁶ Cubic microns, 2×10⁶ Cubic micrometer, 4×10⁶ Cubic microns, 6×10⁶ Cubic microns, 8×10⁶ Cubic micrometer, 1×10⁷ Cubic micrometer, 5×10⁷ Cubic micrometer, 1×10⁸ Cubic micrometer, 5×10⁸ Cubic microns or 8×10⁸ Cubic microns or larger. In various embodiments of the microfluidic device with an isolation fence, the volume of the isolation fence may be about 5×10⁵ Cubic microns, 6×10⁵ Cubic microns, 8×10⁵ Cubic micrometer, 1×10⁶ Cubic microns, 2×10⁶ Cubic micrometer, 4×10⁶ Cubic microns, 8×10⁶ Cubic micrometer, 1×10⁷ Cubic micrometer, 3×10⁷ Cubic micrometer, 5×10⁷ Cubic microns or about 8×10⁷ Cubic microns or larger. In some other embodiments, the volume of the isolation fence may be 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 about 5 to about 10 isolation fences, about 10 to about 50 Two isolation fences, about 100 to about 500 isolation fences; about 200 to about 1000 isolation fences, about 500 to about 1500 isolation fences, about 1000 to about 2000 isolation fences, or about 1000 to about 3500 isolation fences Isolation fence. The isolation fences need not all have the same size and can include multiple configurations (for example, 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 about 1500 to about 3000 isolation fences, about 2000 to about 3500 isolation fences, about 2500 to about 4000 isolation fences, about 3000 to about 4500 isolation fences, about 3500 to about 5000 isolation fences, about 4000 to about 5500 isolation fences, about 4500 to about 6000 isolation fences Two isolation fences, about 5000 to about 6500 isolation fences, about 5500 to about 7000 isolation fences, about 6000 to about 7500 isolation fences, about 6500 to about 8000 isolation fences, about 7000 to about 8500 isolation fences One isolation fence, about 7500 to about 9000 isolation fences, about 8000 to about 9500 isolation fences, about 8500 to about 10,000 isolation fences, about 9000 to about 10,500 isolation fences, about 9500 to about 11,000 isolation fences One isolation fence, about 10,000 to about 11,500 isolation fences, about 10,500 to about 12,000 isolation fences, about 11,000 to about 12,500 isolation fences, about 11,500 to about 13,000 isolation fences, about 12,000 to about 13,500 isolation fences 1 isolation fence, about 12,500 to about 14,000 isolation fences, about 13,000 to about 14,500 isolation fences, about 13,500 to about 15,000 isolation fences, about 14,000 to about 15,500 isolation fences, about 14,500 to about 16,000 isolation fences 1 isolation fence, about 15,000 to about 16,500 isolation fences, about 15,500 to about 17,000 isolation fences, about 16,000 to about 17,500 isolation fences, about 16,500 to about 18,000 isolation fences, about 17,000 to about 18,500 isolation fences Isolation fences, about 17,500 to about 19,000 isolation fences, about 18,000 to about 19,500 isolation fences, about 18,500 to about 20,000 isolation fences, about 19,000 to about 20,500 isolation fences, about 19,500 to about 21,000 isolation fences Two isolation fences, or about 20,000 to about 21,500 isolation fences. Figure 2G illustrates a microfluidic device 280 according to an embodiment. The microfluidic device 280 illustrated in FIG. 2G is a stylized view of the microfluidic device 100. In fact, the microfluidic device 280 and its constituent circuit elements (such as the channel 122 and the isolation fence 128) will have the dimensions discussed herein. The microfluidic circuit 120 illustrated in FIG. 2G has two ports 107 and a flow zone 106 with four different channels 122. The microfluidic device 280 further includes a plurality of isolation fences opened from each channel 122. In the microfluidic device illustrated in FIG. 2G, the isolation fence has a geometric structure similar to the fence illustrated in FIG. 2C, and therefore has both a connection zone and a separation zone. Therefore, the microfluidic circuit 120 includes the maximum penetration depth D in the secondary flow 244 of the affected area (such as the channel 122 and the connecting area 236)_p Inner part) and non-swept areas (e.g. separation area 240 and connection area 236 where the maximum penetration depth D of the secondary flow 244 is not_p Part of the inner connecting area 236) both. 3A to 3B show various embodiments of the system 150, which can be used to operate and observe the microfluidic device of the present invention (for example, 100, 200, 230, 280, 250, 290, 320). As illustrated in Figure 3A, the system 150 may include a structure ("nest") 300 that is configured to house the microfluidic device 100 (not shown) or any other microfluidic device described herein. The nest 300 may include a socket 302 that can interface with the microfluidic device 320 (for example, the optically actuated electrodynamic device 100) and provide an electrical connection from the power source 192 to the microfluidic device 320. The nest 300 may further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 may be configured to supply a bias voltage to the socket 302 such that when the microfluidic device 320 is held by the socket 302, a bias voltage is applied to the electrode pair in the microfluidic device 320. Therefore, the electrical signal generation subsystem 304 can be a part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 320 does not mean that the bias voltage is always applied when the microfluidic device 320 is held by the socket 302. On the contrary, in most cases, the bias voltage is applied intermittently, for example, only as needed to facilitate the generation of electromotive force (for example, dielectrophoresis or electrowetting) in the microfluidic device 320. As illustrated in FIG. 3A, the nest 300 may include a printed circuit board assembly (PCBA) 322. The electrical signal generating subsystem 304 can be installed on the PCBA 322 and electrically integrated into it. The exemplary support also includes a socket 302 mounted on the PCBA 322. Generally, the electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 may further include an oscilloscope (not shown) and/or a waveform amplification loop (not shown) configured to amplify the waveform received from the waveform generator. An oscilloscope (if present) can be configured to measure the waveform supplied to the microfluidic device 320 held by the socket 302. In some embodiments, the oscilloscope measures the waveform at the proximal end of the microfluidic device 320 (and at the far end of the waveform generator), thereby ensuring greater accuracy in measuring the waveform actually applied to the device. For example, the data obtained from the oscilloscope measurement can be provided as the feedback of the waveform generator, and the waveform generator can adjust its output based on the feedback configuration. An example of a suitable combination of waveform generator and oscilloscope is Red Pitaya™. 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 Arduino™ microprocessors, such as Arduino Nano™. The controller 308 can be used to implement functions and analysis or can communicate with an external main controller 154 (shown in FIG. 1A) to implement functions and analysis. In the embodiment illustrated in FIG. 3A, the controller 308 communicates with the main controller 154 via an interface 310 (such as a plug or connector). In some embodiments, the nest 300 may include an electrical signal generation subsystem 304, which includes a Red Pitaya™ 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 The waveform amplification circuit of the fluid 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, so that the measured voltage of the microfluidic device 320 is the desired value. In some embodiments, the waveform amplifying circuit may have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, thereby generating a signal up to 13 Vpp in the microfluidic device 100. As illustrated in FIG. 3A, the support structure 300 may further include a thermal control subsystem 306. The thermal control subsystem 306 can be configured to adjust the temperature of the microfluidic device 320 held by the support structure 300. For example, the thermal control subsystem 306 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface that is configured to interface with at least one surface of the microfluidic device 320. The cooling unit may be, for example, a cooling block (not shown), such as a liquid-cooled aluminum block. The second surface (for example, the surface opposite to the first surface) of the Peltier thermoelectric device may be configured to interface with the surface of the cooling block. The cooling block may be connected to a fluid path 314 that is configured to circulate a 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 the cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluid path 314 and pass through the cooling block, And then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluid path 314 may be installed on the casing 312 of the support structure 300. In some embodiments, the thermal control subsystem 306 is configured to adjust the temperature of the Peltier thermoelectric device to achieve the target temperature of the microfluidic device 320. The temperature adjustment of the Peltier thermoelectric device can be achieved by, for example, a thermoelectric power supply (such as Pololu™ thermoelectric power supply (Pololu Robotics and Electronics Corp.)). The thermal control subsystem 306 may 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 may include a thermal control subsystem 306 with a feedback loop that is analogous to a voltage divider loop (not shown), which includes a resistor (for example, a resistance of 1 kiloohm +/- 0.1 %, the temperature coefficient is +/-0.02 ppm/C0) and NTC thermistor (for example, the nominal resistance is 1 kiloohm +/-0.01%). In some cases, the thermal control subsystem 306 measures the voltage from the feedback loop, and then uses the calculated temperature value as input to the on-board PID control loop algorithm. The output of the PID control loop algorithm can drive, for example, the orientation and pulse-width-adjusted signal pins on the Pololu™ electric drive device (not shown) to activate the thermoelectric power source, thereby controlling the Peltier thermoelectric device. The nest 300 may include a serial port 324 that allows the microprocessor of the controller 308 to communicate with the external main controller 154 via the interface 310 (not shown). In addition, the microprocessor of the controller 308 can communicate with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (for example, via a Plink tool (not shown)). Therefore, through the combination of the controller 308, the interface 310, and the serial port 324, the electrical signal generation subsystem 304 and the thermal control subsystem 306 can communicate with the external main controller 154. In this way, the main controller 154 can assist the electrical signal generation subsystem 304 by implementing scaling calculations for output voltage adjustment. The graphical user interface (GUI) (not shown) provided via the display device 170 coupled to the external main controller 154 can be configured to draw the temperature and waveform obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 304, respectively data. Alternatively or additionally, the GUI may allow updates to the controller 308, the thermal control subsystem 306, and the electrical signal generation subsystem 304. As discussed above, the system 150 may include an imaging device 194. In some embodiments, the imaging device 194 includes a light conditioning subsystem 330 (see FIG. 3B). The light conditioning subsystem 330 can include a digital micromirror device (DMD) or a micro shutter array system (MSA), either of which can be configured to receive light from the light source 332 and transfer a subset of the received light to the microscope 350 in the optical element string. Alternatively, the light conditioning subsystem 330 may include a device that generates light by itself (and therefore does not require the light source 332), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS) ) Or transmissive liquid crystal display (LCD). The light adjustment subsystem 330 may be, for example, a projector. Therefore, the light conditioning subsystem 330 may be capable of emitting structured and unstructured light. An example of a suitable light conditioning subsystem 330 is the Mosaic™ system from Andor Technologies™. In some embodiments, the imaging module 164 and/or the power module 162 of the system 150 can control the light adjustment subsystem 330. In certain embodiments, the imaging device 194 further includes a microscope 350. In these embodiments, the nest 300 and the light adjustment subsystem 330 can be individually configured to be mounted on the microscope 350. The microscope 350 may be, for example, a standard research-grade light microscope or a fluorescence microscope. Therefore, the nest 300 can be configured to be mounted on the stage 344 of the microscope 350 and/or the light adjustment subsystem 330 can be configured to be mounted on the port of the microscope 350. In other embodiments, the nest 300 and the light conditioning subsystem 330 described herein may be integrated components of the microscope 350. In certain embodiments, the microscope 350 may further include one or more detectors 348. In some embodiments, the detector 348 is controlled by the imaging module 164. The detector 348 may include an eyepiece, a charge coupled device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two detectors 348, one detector may be, for example, a high frame rate camera, and the other detector may be a high sensitivity camera. In addition, the microscope 350 may include a string of optical elements configured to receive light reflected and/or emitted from the microfluidic device 320, and focus at least a portion of the reflected and/or emitted light on one or more detectors 348 . The optical element string of the microscope can also include different tube lenses (not shown) for different detectors, so that the final magnification on each detector can be different. In some embodiments, the imaging device 194 is configured to use at least two light sources. For example, the first light source 332 may be used to generate structured light (eg, via the light conditioning subsystem 330), and the second light source 334 may be used to provide unstructured light. The first light source 332 can generate structured light for electrical control and/or fluorescent excitation of optical actuation, and the second light source 334 can be used to provide bright field illumination. In these embodiments, the power module 164 can be used to control the first light source 332 and the imaging module 164 can be used to control the second light source 334. The optical element string of the microscope 350 can be configured to (1) receive the structured light from the light conditioning subsystem 330 and focus the structured light when the microfluidic device (such as an optically actuated electrodynamic device) is held by the nest 300 On at least the first area of the device, and (2) receive the light reflected and/or emitted from the microfluidic device and focus at least a part of the reflected and/or emitted light onto the detector 348. The optical element string may be further configured to receive unstructured light from the second light source and focus the unstructured light on at least the second region of the microfluidic device when the device is held by the nest 300. In some embodiments, the first and second regions of the microfluidic device may be overlapping regions. For example, the first zone can be a subgroup of the second zone. In FIG. 3B, a first light source 332 that supplies light to the light conditioning subsystem 330 is shown, which provides structured light to the string of optical elements of the microscope 350 of the system 355 (not shown). The second light source 334 that provides unstructured light to the optical element string via the beam splitter 336 is shown. The structured light from the light conditioning subsystem 330 and the unstructured light from the second light source 334 travel together from the beam splitter 336 through the optical element to reach the second beam splitter (or dichroic filter 338, This end depends on the light provided by the light adjustment subsystem 330), in which the light is reflected down to the sample plane 342 through the objective lens 336. Subsequently, the light reflected and/or emitted from the sample plane 342 travels back upward through the objective lens 340, through the beam splitter and/or the dichroic filter 338, and reaches the dichroic filter 346. Only a part of the light reaching the two-color filter 346 passes through and reaches the detector 348. In some embodiments, the second light source 334 emits blue light. With a suitable dichroic filter 346, the blue light reflected from the sample plane 342 can pass through the dichroic filter 346 and reach the detector 348. In contrast, the structured light from the light conditioning subsystem 330 reflects from the sample plane 342, but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 filters out visible light with a wavelength longer than 495 nm. If the light emitted from the light conditioning subsystem does not include any wavelengths shorter than 495 nm, the filtering of the light from the light conditioning subsystem 330 will only be complete (as shown). In fact, if the light from the light conditioning subsystem 330 includes a wavelength shorter than 495 nm (for example, a blue wavelength), some light from the light conditioning subsystem will pass through the color filter 346 to reach the detector 348. In this embodiment, the color filter 346 is used to change the balance between the amount of light reaching the detector 348 from the first light source 332 and the second light source 334. This may be beneficial if the first light source 332 is significantly stronger than the second light source 334. In other embodiments, the second light source 334 can emit red light, and the two-color filter 346 can filter out visible light (for example, visible light with a wavelength shorter than 650 nm) other than red light.Surface Modification . The surface of materials, devices, and/or devices used to manipulate and store biological materials may have natural properties that are not optimized for short-term and/or long-term contact with the material. Such materials may include (but are not limited to) tiny objects ( Including (but not limited to) biological micro-objects, such as biological cells, biological molecules, biological molecules or fragments of biological micro-objects, and any combination thereof. It can be used to modify one or more surfaces of materials, devices, or devices to reduce one or more undesirable phenomena associated with 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, manipulation, or processing capabilities of the materials, devices, and/or devices. For this reason, the industry needs to modify the surface to reduce undesirable properties or introduce molecules with desired properties.Compounds that can be used to modify the surface . In various embodiments, the surface modification compound may include a surface modification ligand, which may be a non-polymeric moiety that covalently modifies 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 modification compound also includes a linking moiety, which is a group that allows the surface modification ligand to be covalently attached to the surface, as shown schematically in Equation 1. The covalently modified surface has a surface-modifying ligand attached via a linking group LG, which is a product (including hydroxide, oxide, amine, or sulfur) of the reaction between the linking part and the functional group of the surface.Equation 1.

In some embodiments, the surface modification compound may include carbon atoms that form a straight chain (for example, at least 10 carbons, or at least 14, 16, 18, 20, 22 or more carbons in a straight chain) And may not have a branched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group may be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first section joined to a second section, the first section may include a perfluoroalkyl group, and the second section may include an unsubstituted alkyl group. The first and second segments can be joined directly or indirectly (for example, via an ether bond). The first section of the alkyl group can be located 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 group. In various embodiments, the surface modification compound may have the structure of Formula I:

； Formula I The connecting part of the V series -P(O)(OH)Q- or -Si(T)₂ W; W series -T, -SH or -NH₂ And is the part that is configured to be connected to the surface; Q is -OH and is the part that is configured to be connected to the surface; and T is OH, OC_1-3 Alkyl group 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 of 1-25; and n is an integer of 0 or 1-25. 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 each embodiment, m is 2. In some embodiments, k is zero. In other embodiments, k is 1. In each embodiment, j is 1. For compounds of formula I, when k is an integer 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 modification compound has the structure of Formula I, the connecting portion V may be -Si(T)₂ W, where T and W are as defined above. W can be OC_1-3 Alkyl group or Cl. W may be methoxy, ethoxy or propoxy. In some embodiments, W may be methoxy. T can be OC_1-3 Alkyl group or Cl. In each embodiment, the connection part V-Si (OMe)₃ . In various other embodiments, V may be -P(O)(OH)Q, where Q is OH. The surface-modified compound of Formula 1 may have a preferable range of the number of atoms constituting the linear main chain of the compound. As defined above, each segment constituting the compound of Formula 1 may have a certain range of sizes. Therefore, the 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 produce a total length of 26 atoms, including Terminal CR₃ -Groups. In the case where (n + [(h + j)·k] + m) is equal to 25, a variety of different compositions can be covered. For example, the section-[CR₂ ]_n -Can have n = 23; -[(CH₂ )_h -(O)_j ]_k -May have k=0; and -[CM₂ ]_m -May have m=2. Another case with the same total (n + [(h + j)·k] + m) equal to 25 may have the segment -[CR₂ ]_n -, where n = 6; -[(CH2)h-(O)j]k-, where k = 3, including j = 1 and h = 2; and -[CM₂ ]_m -May 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, there may be one or more ether linkages in the compound of formula I. In some embodiments, j may be 1. In some embodiments, when k and j are both 1, m may be at least 2. In other embodiments, the backbone carbon can be fluorinated. In some embodiments, the backbone carbon can be perfluorinated, where CR₃ -And/or-[CR₂ ]_n -And/or-[CM₂ ]_m -Each R can be fluorinated. In some embodiments, one part of the compound may have fluorinated carbon backbone atoms, and the other part of the compound may have hydrogen substituted carbon backbone atoms. For example, in some embodiments, CR₃ -And-[CR₂ ]_n -The segment may have fluorine non-main chain substituents (for example, R is fluorine), and -[CM]_m -The segment may have hydrogen non-backbone substituents (e.g., 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 may be hydrogen. In other embodiments, the compound of formula 1 can be synthesized from hydrosilation olefins, as described below, where 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 can be more easily found in the subgroup of compounds described in the following formulas, but these formulas in no way limit the breadth of formula I. In some embodiments, the compound of formula I may include a compound of formula 110: CH₃ (CH₂ )_m Si(OC_1-3 alkyl)₃ ； Formula 110 Wherein m is an integer from 9 to 23. In some embodiments, m may be 11, 13, 15, 17, or 19. In some other embodiments, m may be 13 or 15. In other embodiments, the compound of formula I may include a compound of formula 111: CF₃ (CF₂ )_n (CH₂ )₂ Si(OC_1-3 alkyl)₃ ； Formula 111 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 may be 9, 11, 13, or 15. In some embodiments, n may be 13 or 15. In other embodiments, the compound of formula I may include a compound of formula 112: CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si(OC_1-3 alkyl)₃ ； Formula 112 Wherein n is an integer from 3 to 19; h is an integer from 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 include the compound of formula 113: CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ ； Formula 113 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 fluorine. In some embodiments, M may be hydrogen. In various embodiments, n may be 5, 7, 9, or 11. In other embodiments, m may be 2, 4, 5, 7, 9, 11, or 13.Surface for modification . The surface that can be modified by the surface modification compounds described herein (including compounds of formula I) can be a metal, metal oxide, glass, or polymer. Some of the materials that can be introduced to covalently modify the surface can include (but are not limited to) silicon and its oxides, polysiloxy, aluminum or its oxides (Al₂ O₃ ), indium tantalum oxide (ITO), titanium dioxide (TiO₂ ), zirconium oxide (ZrO2), hafnium (IV) oxide (HfO₂ ), tantalum (V) oxide (Ta₂ O₅ ) Or any combination thereof. The surface can be a wafer or board of these materials, or can be incorporated into a device or device. In some embodiments, a surface that includes any of these 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, silicone, organopolysiloxane (e.g. polydimethylsiloxane ("PDMS")) or the like), which can be breathable of. Other examples may include molded glass, patternable materials (such as polysiloxane polymers, such as photo-patternable polysiloxane or "PPS"), photoresists (such as epoxy-based photoresists, such as SU8 ) Or something like that. In other embodiments, the surface of materials such as natural fibers or wood can be functionalized with surface modification compounds (including compounds of formula I) 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 thiols. Nucleophilic moieties on the surface (such as hydroxides (called oxides in some embodiments)) can be reacted with the surface modification compounds described herein (including compounds of formula I) to pass through siloxy linking groups or phosphines. The acid ester linking group covalently links the surface modification ligand to the surface to provide a functionalized surface. The surface to be modified can include natural nucleophilic moieties, or can be treated with reagents (such as piranha solution) or by plasma treatment to introduce nucleophilic moieties (such as hydroxides (or oxides)) ). In some embodiments, the surface can be formed from any of the aforementioned materials alone or in any combination. The surface may include a semiconductor substrate. In various embodiments, the surface including the semiconductor substrate may further include a DEP or EW substrate as described herein. In some embodiments, a surface including a semiconductor substrate with a DEP or EW substrate can be part of a microfluidic device as described herein. In some embodiments, the modified surface may be at least one inwardly facing surface of a microfluidic device as described herein. At least one surface may be a part of the flow area of the microfluidic device (which may include a channel) or may include the surface of an encapsulated structure such as a fence, which may include an isolated fence as described herein.Covalently modified surface . The covalently modified surface may include a surface-modifying ligand, which may be a non-polymeric moiety, such as an alkyl moiety, a substituted alkyl moiety (e.g., a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety)), and It can be any of the surface modification ligands described above, which is covalently bound to the surface via a linking group, which is a part generated from the reaction between the linking part and the surface. The linking group may be a siloxy linking group or a phosphonate linking group. In some embodiments, the surface modification ligand may include carbon atoms that form a straight chain (for example, at least 10 carbons, or at least 14, 16, 18, 20, 22 or more carbons in a straight chain) , And may not have a branched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group may be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first section joined to a second section, the first section may include a perfluoroalkyl group, and the second section may include an unsubstituted alkyl group. The first and second segments can be joined directly or indirectly (for example, via an ether bond). The first segment of the alkyl group can be located at the distal end of the linking group, and the second segment of the alkyl group can be located at the proximal end of the linking group.Mode II Covalently modified surface . In some embodiments, the covalently modified surface has the structure of Formula II:

； Formula II Which is the surface; V is -P(O)(OY)W- or -Si(OZ)₂ W. W is -O-, -S- or -NH- and is connected to the surface. Z is a bond to an adjacent silicon atom attached to the surface or a bond to the surface. Y is a bond to an 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 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 described as a surface-modified ligand attached via a linking group LG, as in formula IIA, where LG is linked to the surface:

Formula IIA The covalently modified surface can include any surface of formula II in any combination as described above for the surface modified compound of formula I. In some embodiments, the covalently modified surface of formula II can be the surface of formula 210:

； Formula 210 in

It is the surface, the oxygen attached to the silicon atom is also bound to the surface, and m is an integer from 11 to 23. In some embodiments, m may be 11, 13, 15, 17, or 19. In some other embodiments, m may be 13 or 15. In some other embodiments, the covalently modified surface of formula II can be the surface of formula 211:

； Formula 211 in

It is the surface, the oxygen attached to the silicon atom is also bound to the surface, and 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 7, 9, 11, 13, or 15. In some embodiments, n may be 13 or 15. In other embodiments, the covalently modified surface of formula II may be the surface of formula 212:

； Formula 212 in

It is the surface, the oxygen attached to the silicon atom is also bound to the surface, and n is an integer from 3 to 21, h is an integer from 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:

； Formula 213 in

It is the surface, the oxygen attached to the phosphorus atom is also bound 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 fluorine. In some embodiments, M may be hydrogen. In various embodiments, n may be 5, 7, 9, or 11. In other embodiments, m may be 2, 4, 5, 7, 9, 11, or 13. In some embodiments, the microfluidic device includes a flow zone fluidly connected to the first inlet and the first outlet, the flow zone being configured to contain the flow of the first fluid medium. The microfluidic device may include one or more chamber openings to the flow zone. The covalently modified surface can be the covalently modified substrate of the microfluidic device and can be located under 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 on all or substantially all of the inner surface facing the fluid. FIG. 2H shows a cross-sectional view of a microfluidic device 290 including an exemplary covalently modified surface 298. FIG. As illustrated, the covalently modified surface 298 (shown schematically) may comprise a monolayer of densely stacked molecules covalently bonded to both the inner surface 294 of the substrate 286 and the inner surface 292 of the cover 288 of the microfluidic device 290. The covalently modified surface 298 may be arranged on substantially all of the inner surfaces 294, 292 of the housing 284 of the microfluidic device 290 that are close to and inwardly facing, in some embodiments and as discussed above, including those used to define the microfluidic device The surface of the microfluidic circuit material (not shown) of the circuit element and/or structure in 290. In alternative embodiments, the covalently modified surface 298 may be disposed on only one or some of the inner surfaces of the microfluidic device 290. In the embodiment shown in FIG. 2H, the covalently modified surface 298 comprises a monolayer of alkyl-terminated siloxane molecules, each of which is covalently bonded to the microfluidic device 290 via a siloxy linker 296 Surfaces 292, 294. For the sake of brevity, other silicon oxide bonds linked to adjacent silicon atoms are shown, but the present invention is not limited to this. In some embodiments, the covalently modified surface 298 may include a fluoroalkyl group (such as a fluorinated alkyl group or a perfluorinated alkyl group) at its end facing the outer shell (ie, the monolayer of the surface-modified ligand 298 is not bound to The inner surface 292, 294 and the part close to the housing 284). Although Figure 2H is discussed as having an alkyl-terminated modified surface, any suitable surface modification compound can be used, as described herein.Natural surface . At least one surface of the microfluidic device to be modified can be glass, metal, metal oxide, or polymer. Some materials that can be incorporated into the microfluidic device and can be modified to introduce the covalently modified surface of formula II therein can include, but are not limited to, silicon and its oxides, polysiloxy, aluminum or its oxides (Al₂ O₃ ), indium tantalum oxide (ITO), titanium dioxide (TiO₂ ), zirconium oxide (ZrO2), hafnium (IV) oxide (HfO₂ ), tantalum (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, silicone, organopolysiloxane (e.g. polydimethylsiloxane ("PDMS")) or the like), which can be breathable of. Other examples may include molded glass, patternable materials (such as polysiloxane polymers, such as photo-patternable polysiloxane or "PPS"), photoresists (such as epoxy-based photoresists, such as SU8 ) Or something like that.Physical and performance properties of covalently modified surfaces . In some embodiments, the covalently modified surface may have increased hydrophobic characteristics. 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 deposited on the surface of the microfluidic device indiscriminately, which may include the permanent or semi-permanent properties of biological materials (such as proteins and their degradation products, nucleic acids and individual degradation products) Sexual deposition. The fouling can increase the amount of tiny biological objects sticking to the surface. In other embodiments, the increased hydrophobicity of the covalently modified surface can reduce the adhesion of tiny biological objects on the surface, independent of adhesion initiated by surface fouling. Surface modification can increase the durability, functionality, and/or biocompatibility of the surface. Each of these features can further benefit the vitality (including growth rate and/or cell doubling rate) of small objects or biomolecules on the modified surface and in devices and/or devices with covalently modified surfaces, The nature or portability (including the vitality of excretion) of the community formed on the covalently modified surface (including the surface having the structure of Formula II) as described herein. In some embodiments, the covalently modified surface may be any surface as described herein (including the surface of Formula II), which may have a diameter of less than 10 nm (for example, less than about 7 nm, less than about 5 nm, or about 1.5 nm). To 3.0 nm) thickness. This can advantageously provide on the modified surface in particular with other hydrophobic materials (such as perfluorotetrahydrofuran polymer CYTOP^® ) Different thin layers, which are spin-coated to produce a typical thickness of about 30 nm to 50 nm. The data shown in Table 1 relates to the silicon/silica surface treated to have a covalently modified surface, as shown in the table. The contact angle measurement is obtained using the static sitting drop method. (Drelich, J. Colloid Interface Sci.179 , 37-50, 1996. ) The thickness is measured by ellipsometry. The contact angle hysteresis measurement system uses Biolin Scientific contact angle goniometer to implement. Place the chemically modified OEW surface in 5 cSt silicone oil wrapped in a transparent container. Then phosphate buffered saline (PBS) droplets were dispensed onto the surface in the oil. A platinum (Pt) wire electrode was inserted into the droplet, and the fixed 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 wire was inserted into the PBS droplet for 10 seconds. Then, the applied voltage was removed, and the contact angle was measured again. The contact angle hysteresis was calculated by subtracting the contact angle under zero bias after applying the voltage from the original contact angle under zero bias before applying the 50Vppk AC voltage.surface 1 . The physical data of the selected surface. serial number Modified surface Contact angle ( water or aqueous solution ) Contact angle hysteresis thickness 201 CH ₃ (CH ₂ ) ₁₇ -Si(OT) ₂ O- (surface) 110-112° 5°, less than 10° About 2 nm 202 CF ₃ (CF ₂ ) ₁₃ (CH ₂ ) ₂ Si(OT) ₂ O-(surface) 110-115° data About 2 nm 203 CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ Si(OT) ₂ O-(surface) 110-115° ＞10° About 1 nm 204 CH ₃ (CH ₂ ) ₂₁ Si(OT) ₂ O- (surface) 110-112° n/a About 2-2.5nm 205 CH ₃ (CH ₂ ) ₁₅ Si(OT) ₂ O-(surface) 110-112° ＞10° n/a 206 CF ₃ (CF ₂ ) ₅ (CH ₂ ) ₂ O(CH ₂ ) ₁₁ Si(OT) ₂ O-(surface) 110-114° n/a About 2 nm 207 CH ₃ (CH ₂ ) ₁₇ P(O)(OQ)O-(surface) 110° n/a n/a 208 CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₁₁ Si(OT) ₂ O-(surface) 113° n/a About 2 nm 209 CF3(CF2) ₁₁ (CH ₂ ) ₂ Si(OT) ₂ O-(surface) 112° n/a About 1.5-2 nm T and Q are as described above. The contact angle observed for the modified surface is different from the contact angle of water on the plasma-cleaned silicon surface of less than 10 degrees. Each of these surfaces is less wettable than natural silica/silica surfaces. Other analytical methods suitable for surface characterization 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 properties of the surface-modifying compound. In some embodiments, the covalently modified surfaces described herein (including the surface of Formula II) can form a monolayer. Especially when the single-layer modified surface has other functional properties, the uniformity and uniformity of the single-layer modified surface can provide advantageous performance. For example, the covalently modified surface described herein (including the surface of formula II) may also include an electrode-activated substrate, and optionally may further include a dielectric layer, such as can be found in a dielectrophoretic configuration or an electrowetting configuration. Materials, devices and/or devices. The lack of unsaturation of the perfluoroalkyl moiety of the modified surface can minimize "charge trapping" compared to monolayers containing, for example, alkene or aromatic moieties. In addition, the dense packing properties of monolayers formed on the surfaces described herein (including those of Formula II) can minimize the possibility of driving cations through the monolayer to the underlying metal, metal oxide, glass, or polymer substrate. Without being bound by theory, adding cations to the substrate composition to destroy the surface of the substrate can destroy the electrical properties of the substrate, thereby reducing its ability to perform electrodynamic functions. In addition, 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. When optically actuating materials, devices and/or devices, the uniformity and thinness of the dielectrophoresis or electrowetting surface of the materials, devices and/or devices with covalently modified surfaces (including the surface of formula II) described herein The modified dielectrophoretic and/or electrowetting surface can further provide advantageous benefits.Method for preparing covalently modified surface . The surface of the material that can be used as a component of a device or device can be modified before the device or device is assembled. Alternatively, a partially or fully constructed device or device may be modified so that all surfaces that will contact biological materials (including biomolecules and/or micro-objects (which may include biological micro-objects)) are simultaneously modified. In some embodiments, even if different materials exist on different surfaces of the device and/or the device, the entire interior of the device and/or the device can still be modified. In some embodiments, the partially or fully constructed device and/or device may be a microfluidic device or a component thereof as described herein. The surface to be modified can be cleaned before modification to ensure that the nucleophilic portion on the surface is free for reaction, for example, not covered by 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 oxygen plasma treatment, which removes contaminants and at the same time introduces other oxides (such as hydroxides) on the surface. This can 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 before modification to ensure that the nucleophilic portion on the surface is free for reaction, for example, not covered by 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 oxygen plasma treatment, which removes contaminants and at the same time introduces other oxides (such as hydroxides) on the surface. This can advantageously provide more modification sites on the surface, thereby providing a more closely packed modified surface layer. In some embodiments, the method of covalently modifying a surface includes the following steps: contacting the surface with a compound of formula I:

； Formula I Wherein V series -P(O)(OH)Q or -Si(T)₂ W. W series -T, -SH or -NH₂ And it is configured to connect to the part of the surface. Alternatively, when V is -P(O)(OH)Q, Q is -OH and is configured to be connected to the part of the surface. T series OH, OC_1-3 Alkyl group 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 from 11 to 25. In each embodiment, when k is an integer 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. Reacting the compound of formula I with the nucleophilic moiety of the surface; and forming a covalently modified surface. Any combination or sub-combination of the compounds of formula I can be used, as described above. In various embodiments of the method, the covalently modified surface thus formed may be a single layer. In some embodiments of the method, the compound of formula I may be a compound of formula 110: CH₃ (CH₂ )_m Si(OC_1-3 alkyl)₃ ； Formula 110 Wherein m is an integer from 9 to 23. In some embodiments, m may be 11, 13, 15, 17, or 19. In some other embodiments, m may be 13 or 15. In other embodiments of the method, the compound of formula I may be a compound of formula 111: CF₃ (CF₂ )_n (CH₂ )₂ Si(OC_1-3 alkyl)₃ ； Formula 111 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 may be 9, 11, 13, or 15. In some embodiments, n may be 13 or 15. In other embodiments of the method, the compound of formula I may be a compound of formula 112: CR₃ (CR₂ )_n (CH2)_h O(CH₂ )_m Si(OC_1-3 alkyl)₃ ； Formula 112 Wherein n is an integer from 3 to 21; h is an integer from 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 may be contacted with a compound of formula I, which may be a compound of formula 113: CR₃ (CR₂ )_n (CM₂ )_m P(O)(OH)₂ ； Formula 113 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 fluorine. In some embodiments, M may be hydrogen. In various embodiments, n may be 5, 7, 9, or 11. In other embodiments, m may be 2, 4, 5, 7, 9, 11, or 13. The contacting step can be performed by contacting the surface with a liquid solution containing the 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 the compound of formula I. The reaction can be carried out at ambient temperature and can be carried out over a period of time within the range of about 2 h, 4 h, 8 h, 12 h, 18 h, 24 h, or any value in between. Examples of solvents include (but are not limited to): toluene, 1,3 bistrifluorobenzene or Fluorinert^TM (3M) Fluorinated solvents. An acid such as acetic acid can be added to the solution to increase the reaction rate by promoting the hydrolysis of the trialkoxy group (if present). Alternatively, the surface can be brought into contact with a gas phase containing a compound of formula I. In some embodiments, when the reaction step is carried out by contacting the surface with the compound of formula I in the gas phase, a controlled amount of water vapor is also present. A controlled amount of water vapor can be provided by placing a preselected amount of magnesium sulfate heptahydrate in the same chamber or enclosure together with the object with the surface to be modified. In other embodiments, a controlled amount of water can be introduced into the reaction chamber or enclosure via an external steam feed. The reaction can be carried out under reduced pressure relative to atmospheric pressure. In some embodiments, the reduced pressure may be 100 Torr or less. In other embodiments, the reduced pressure may 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 may have the structure of Formula II:

； Formula II 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 a formula of formula 210, 211, 212, or 213 as described above, which has any combination of elements that can be allowed for each formula. In various embodiments of the method, the surface may include a nucleophilic moiety selected from the group consisting of hydroxides, amine groups, and thiols. The surface can be metal, metal oxide, glass, polymer, or any combination thereof. The metal surface may include silicon, silicon oxide, hafnium dioxide, indium tantalum oxide, aluminum oxide, or any combination thereof. In various embodiments of the method, the step in which the covalently modified surface is formed can be implemented on a DEP substrate or an EW substrate. The step of forming a covalently modified surface may include forming a covalently modified surface on at least one surface of the microfluidic circuit element of the microfluidic device. Microfluidic circuit elements can include walls, flow areas, fences, and electrode activation substrates (including DEP or EW substrates). The surface that can be covalently modified in the microfluidic circuit can be all or substantially all of the surfaces facing the part of the microfluidic device with the fluid. For example, in the microfluidic devices 200 and 230, the inner surface of the top electrode 210, the upper surface of the electrode activation substrate 206, and the surface of the microfluidic circuit material 116 (see FIGS. 1B, 2A, and 2B) can be modified, all of which face Microfluidic channel 122 and fences 244, 246, 248. Similarly, in FIGS. 2D-2F, the inner surface of the microfluidic circuit material 260, the surface of the separation structure 272 defining the isolation fence 266, or all the surfaces facing the microfluidic circuit 262 can be covalently modified by the methods described herein.Immiscible medium . The movement of aqueous droplets on the surface of the substrate can be implemented in a water-immiscible fluid medium distributed regionally in one or more flow zones (which may include flow channels) and (if present) fluidly connected to the flow zone chamber. The water-immiscible fluid medium may have a kinematic viscosity greater than that of pure water droplets. The water immiscible fluid medium may have a kinematic viscosity ranging from about 1 centistoke (cSt) to about 15 cSt, where 1 cSt is equal to 1 mPa or 1 centipoise (CPS). In some embodiments, the water-immiscible fluid medium may 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 may be non-flammable at a temperature of at least 100°C. The water-immiscible fluid medium can be non-toxic to living biological cells for the duration of the biological cells being processed, cultured, or stored in aqueous droplets in the water-immiscible fluid medium. The water-immiscible fluid medium may have low or very low water solubility. When in contact with a water layer (for example, for dispensing with water), 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. At a temperature ranging from about 25°C to about 38°C, the water-immiscible fluid medium may not dissolve more than about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% by volume. Aqueous droplets that exist 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 may include at least one organic or organosilicon compound having a main chain structure containing atoms selected from carbon, silicon and oxygen. In some embodiments, the water-immiscible fluid medium may include more than one organic/organosilicon compound, wherein more than one compound is a polymeric organic/organosilicon compound having a molecular weight range of the subunits of the polymeric compound. For example, a polymeric organo/organosilicon compound can have two different subunits constituting a polymer (such as a copolymer), and each of the two different subunits can exist in a series of repetitions with the general formula AaBb, where A and B are two different polymer subunits, and a and b are the number of repetitions of each subunit. The repeating 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 including more than one organic/organosilicon compound may include a mixture of organic compounds, a mixture of organosilicon compounds, or any combination thereof. The water immiscible fluid medium may 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 may 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 may have a molecular weight higher than about 1000 Da and still provide suitable properties. In various embodiments, the organic/organosilicon compound of the water-immiscible fluid medium may have a main chain structure, in which the atoms constituting the main chain are carbon, silicon or oxygen. The substituent of the main chain carbon may be hydrogen or fluorine. In some embodiments, the water-immiscible fluid medium may include one or more organosilicon compounds, wherein the backbone of the organosilicon compounds may include silicon and oxygen atoms. The silicon atom of the organosilicon compound may have a carbon substituent, which in turn may have a hydrogen or fluorine substituent. In some embodiments, the carbon substituents of the organosilicon compound may all be fluorine (for example, perfluorinated). In other embodiments, the carbon substituent of the organosilicon compound may be partially fluorinated. In each embodiment, the substituent of the carbon atom of the organosilicon compound may not exceed 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 may have a main chain structure, in which the atoms constituting the main chain are carbon or oxygen. In some embodiments, the substituent of the backbone carbon may be hydrogen or fluorine. In other embodiments, the substituents of the main chain carbons may include oxygen-containing moieties, such as ether, carbonyl or carbonate components. In some embodiments, the organic compound of the water-immiscible fluid medium may have an all-carbon backbone structure. In some embodiments of the all-carbon backbone structure of the organic compound in the water-immiscible fluid medium, it may have a perfluorinated substituent on the carbon atom (for example, perfluorinated). In other embodiments, the substituents of the organic compound may be partially fluorinated (for example, not perfluorinated). In various embodiments, the substituents of carbon atoms of organic compounds (including compounds with a full-carbon backbone) may not exceed about 90% fluorine, 80% fluorine, 70% fluorine, 60% fluorine, 50% fluorine, and 40% fluorine. Or less. In some embodiments, the suitable organic compound for the water immiscible fluid medium may include or may be a monofluoro-substituted hydrocarbon, 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 not have fluorine substituents on carbon, but may have hydrogen substituents. In some embodiments, the organic compound of the water-immiscible fluid medium may have an unsaturated carbon-carbon bond, such as an olefin group within the main chain carbon or at a terminal position. In some embodiments, the selection of a suitable compound to be included in the water-immiscible fluid medium will include consideration of other properties of the compound. In various embodiments, compounds suitable for use in water-immiscible fluid media will not fluoresce spontaneously when illuminated by lasers, structured light projected into the microfluidic device, or sunlight/laboratory lighting. In other embodiments, the properties of the covalently modified hydrophobic surface will affect the choice of compounds suitable for use in water-immiscible fluid media. For example, the covalently modified surface can be sufficiently hydrophobic so that the water droplets in the perfluorinated water immiscible fluid medium can exhibit sufficiently high surface tension, so that the water droplets can not be wetted by the photoelectric as described herein. Configure to move. In some other embodiments, the properties of the microfluidic circuit material can influence 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 kept within acceptable limits. For example, in some embodiments, if the microfluidic circuit material includes SU8 or a photo-patternable aryl-substituted organopolysiloxane, a straight-chain hydrocarbon including a ring, an aryl group, or a heteroaryl group, and a straight-chain hydrocarbon group can be selected. Fluorocarbon or carbon backbone compound is used. In other embodiments, the microfluidic circuit material can include other materials, such as photo-patternable organopolysiloxanes without aryl substitution, and the swelling can be limited to the water-immiscible fluid medium by using different compounds. Acceptable limit. For example, a swelling of less than about 40%, 30%, 20%, or 10% compared to before exposure to the water-immiscible fluid medium may 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 may be an organic compound having a main chain containing carbon or oxygen atoms. In some embodiments, the organic compound may have a main chain that contains carbon atoms and does not contain oxygen atoms, and in addition, the carbon atom main chain is branched. In each embodiment, the branched carbon atom backbone of the organic compound in the water-immiscible fluid medium is acyclic. The organic compound with a branched carbon backbone in the water-immiscible fluid medium may additionally not contain any cyclization moieties. Although the above selection criteria can be used to select one or more compounds to be included in the water-immiscible fluid medium and eliminate compounds that cannot provide acceptable performance, the acceptable water-immiscible fluid medium can be a multi-component mixture, and It may include parts of individual organic or organosilicon compounds that will not provide acceptable performance when used as the sole component of a water-immiscible fluid medium. For example, the components can be highly fluorinated or can unacceptably swell the microfluidic circuit material when used alone, but can be used in combination with other organic or organosilicon compounds to form a water-immiscible fluid medium. Some organic compounds suitable for water-immiscible fluid media 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 (tridecafluoro-1,1,2,2,-tetrafluoroethylene) Hydroxyoctyl) tetramethyldisiloxane (Gelest, catalog number SIB1816.0) or polysiloxane oil (5 centistokes viscosity, Gelest catalog number DMS-T05). Aqueous droplets. Aqueous droplets can contain one or more tiny objects, which can include biological cells or beads. The aqueous droplets may contain biological products, which may include nucleic acids or proteins. In some other embodiments, the aqueous droplets may contain reagents for analysis, which may be any kind of reagents, such as enzymes, antibodies, fluorescently labeled probes, or chemical reagents. In some embodiments, the aqueous droplets may also include surfactants. Surfactants can increase the portability of aqueous droplets in water-immiscible fluid media. In some embodiments, suitable surfactants may include nonionic surfactants. In various embodiments, the surfactant can be, but is not limited to, Pluronic® alkylene oxide block copolymers, including F68 (ThermoFisher catalog number 2400032); fatty ester ethoxylated sorbitol, 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 Catalog number 9014-85-1); ethoxylated nonionic fluorosurfactant, such as Capstone® FS-30 (DuPontTM, Synquest Laboratories catalog number 2108-3-38). In some embodiments, sodium dodecyl sulfate (SDS) may be used as a surfactant. In various embodiments, phosphate buffered saline (PBS) may be used as the 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 in between.Method of manufacturing microfluidic device . The microfluidic device (such as the device 400) of the present invention can be manufactured by the following method: (i) bonding the spacer element 108 to the inner surface 428 of the cover 110, the inner surface having at least one configuration to be connected to an AC voltage source (Not shown) electrodes, (ii) bonding the spacer element 108 (and related cover 110) to the dielectric surface 414 of the substrate 104, the dielectric surface having at least one configuration to connect to an AC voltage source (not shown) The electrode 418, whereby the spacer element 108 becomes 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 oriented substantially parallel to each other, and the substrate 104, the spacer element 108 and the dielectric surface 414 are substantially parallel to each other. The cover 110 collectively defines an outer shell 435 that is configured to contain liquid, and (iii) the outer hydrophobic layer 412 formed by vapor deposition on at least a portion of the inner surface 428 of the cover 110 and the inner dielectric layer 414 of the substrate 104 At least a portion of the outer hydrophobic layer 412. By vapor deposition of amphiphilic molecules, the hydrophobic layers 422 and 412 can achieve a densely packed monolayer, in which the amphiphilic molecules are covalently bonded to the molecules on the inner surface 428 of the cover 110 and the inner dielectric surface 414 of the substrate 104, respectively. . Any of the self-associated molecules described herein and their equivalents can be vapor-deposited on the inner surface of the microfluidic device. To achieve the desired bulk density, self-associated molecules containing, for example, alkyl-terminated siloxanes can be vapor deposited at a temperature of at least 110°C (for example, at least 120°C, 130°C, 140°C, 150°C, 160°C, etc.) For a period of at least 15 hours (for example, at least 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours or more). The vapor deposition is usually under vacuum and in a water source (such as magnesium sulfate heptahydrate (ie MgSO)₄ ·7H₂ O)) Existence. Generally, increasing the temperature and duration of the vapor deposition will result in improved characteristics of the hydrophobic layers 422 and 412. The vapor deposition process can be improved by, for example, pre-cleaning the cover 110 (with the spacer element 108) and the substrate 104 according to the situation. For example, the pre-cleaning may include a solvent bath, such as an acetone bath, an ethanol bath, or a combination thereof. The solvent bath may include ultrasonic treatment. Alternatively or additionally, the pre-cleaning may include processing the cover 110 (with the spacer element 108) and the substrate 104 in an oxygen plasma cleaner. The oxygen plasma cleaner can be operated at 100 W for 60 seconds under vacuum conditions, for example. Figure 6 illustrates an example of a microfluidic device 600, which includes a housing with 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 contain the first fluid medium 624. Generally, the first fluid medium is a hydrophobic fluid, such as oil (for example, silicone oil or fluorinated oil). The microfluidic channel 614 is connected to the droplet generator 606 via an interface 608, which allows the channel 614 to receive the droplets 620 generated by the droplet generator 606. The received droplet 620 contains a liquid that is immiscible in the first fluid medium 624. Generally, the received droplets will contain an aqueous medium, which may contain tiny objects (such as cells or beads) or reagents that are soluble in the aqueous medium. The microfluidic channel 614 is also connected to each of the plurality of chambers 616, which facilitates the movement of the received droplet 620 (and the droplet 632 taken from the reservoir of fluid immiscible in the first fluid medium 624) to Move in and between chamber 616. The microfluidic channels 612 of the device 600 are connected to a subset of the chambers 616, and therefore indirectly connected to the microfluidic channels 614 via the chambers 616. As illustrated, the microfluidic channel 612 and the chamber 616 connected to it contain a fluid medium 622 that is immiscible in the first fluid medium 624. Thus, for example, the fluid medium 622 may be an aqueous medium, such as a cell culture medium. When the fluid medium 622 is a cell culture medium, the medium-containing chamber 616 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 flow of fresh media in the perfusion channel can provide nutrients to the chamber and remove waste from the chamber by diffusing molecules between the perfusion channel and the culture chamber, thereby promoting continuous cell growth. FIG. 7 illustrates another example of a microfluidic device 700, which includes a housing with 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 Producer 606. Fig. 7 presents a variation of the microfluidic device 600 shown in Fig. 6, in which the chamber 616 contains the immiscible first fluid medium 624 (located in the microfluidic channel 614) and is positioned directly through the microfluidic channel from the corresponding chamber 716 614 of the medium 622. This configuration facilitates the movement of fluid droplets 632 (containing small objects 630 or biological materials as appropriate) from the selection chamber 616 to the corresponding chamber 716, where the fluid droplets (and any small objects 630 or biological materials) 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 FIG. 7, in which one end of the chamber 616 is tapered, so that when the microfluidic device is tilted, the tapered end of the chamber 616 has a lower end than the non-tapered end. The potential energy (in the applicable gravitational field) promotes the movement of particles to the interface between the first fluid medium 624 and the second fluid medium 622. The microfluidic circuit formed by the microfluidic channels 612, 614 and the chambers 616, 716 is only an example, and the present invention covers 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 directly connected to the channel 612 are optional features. Therefore, the devices 600 and 700 may lack perfusion channels and culture chambers. In embodiments where there is a microfluidic channel 612, the substrate that helps define the channel 612 and/or the directly connected chamber 616 (e.g., by forming the base of the channel and/or chamber) may have an electrowetting configuration. Alternatively, however, the substrate that helps define the channel 612 and/or the directly connected chamber 616 may lack an electrowetting configuration (for example, but may have a DEP configuration or neither an electrowetting configuration nor a DEP configuration). In embodiments where there is a microfluidic channel 612 and the substrate that helps define the channel 612 and/or the directly connected chamber 616 has an electrowetting configuration, the hydrophobic surface outside of the substrate can be patterned to be better than the outside of the substrate that helps define the channel 614 Hydrophobic surfaces are more hydrophilic. Increased hydrophilicity can be achieved, for example, as discussed above. The droplet generator 606 and any microfluidic circuit that provides droplets to it can be a part of (a part of or connected to) the microfluidic device, which can be similar to any of the microfluidic devices illustrated in the drawings 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 the devices 600 and 700. The droplet generator 606 itself may include an electrowetting configuration, and therefore may include a substrate with a photoreactive layer that may include a-Si:H (for example, as illustrated in US Patent No. 6,958,132), a photo-actuated circuit Substrate (e.g., as illustrated in U.S. Patent Application Publication No. 2014/0124370), photoelectric crystal-based substrate (e.g., as illustrated in U.S. Patent No. 7,956,339) or electrically actuated circuit substrate (e.g., as Illustrated in U.S. Patent No. 8,685,344). Alternatively, the droplet generator may have a T-shaped or Y-shaped hydrodynamic structure (for example, as US Patent and Patent Application Publication No. 7,708,949, No. 7,041,481 (reissued as RE41,780), No. 2008/0014589, Illustrated in No. 2008/0003142, No. 2010/0137163 and No. 2010/0172803). The full texts of the aforementioned US patent documents are incorporated herein by reference. As shown, the droplet generator 606 may include one or more fluid inputs 602 and 604 (two are shown but there may be fewer or more) and a fluid output 208, which may be connected to the microfluidic channel 614. The liquid media 622, 624, biological micro-objects 630, reagents and/or other biological media can be loaded into the droplet generator 606 via the inputs 602 and 604. The droplet generator 606 can generate droplets 620 of the liquid medium 622 (which may but need not contain one or more biological micro-objects 630), reagents or other biological media and output them to the channel 614. If the channel 614 has an electrowetting configuration, the droplet 620 can move in the channel 614 using electrowetting (or photo-wetting). Alternatively, the droplet 620 can be moved in the channel 614 by other means. For example, the droplet 620 may 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 the first fluid medium 624, and the microfluidic channel 612 and the chamber 616 directly connected thereto can be filled with the 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 the like of biological micro-objects 630. The first fluid medium 624 (hereinafter referred to as "immiscible medium") may be an immiscible medium for the aqueous medium 622. 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 small biological objects on the microfluidic device and/or to facilitate the operation of the biochemical and/or molecular biology workflow on the microfluidic device. Figures 6 and 7 illustrate non-limiting examples. By using a droplet generator, the device can have an electrowetting configuration in the entire fluid circuit. 6 and 7 illustrate an example of the droplet generator 606 generating droplets 620 containing 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. Before or after the reagent-containing droplet 620 moves into one of the chambers 616/716, one or more of the one or more tiny objects 630 of the one or more droplets 632 may move into the same chamber 616/716. The reagent-containing droplet 620 can then be fused with the droplet 632 containing the tiny object 630, thereby allowing the reagent of the droplet 620 to mix with the contents of the droplet 632 and perform a chemical reaction. The droplet 632 containing one or more tiny objects can be supplied by the droplet generator 606 (not shown) or can be obtained from the containment fence 616, as shown in FIGS. 6 and 7. The tiny object 630 may be a biological tiny object, such as a cell, which has optionally been cultured before moving to the processing chamber 616/716 (for example, in the chamber 616). Alternatively, the tiny objects 630 may be beads, such as the affinity of molecules of interest that can bind to the sample (for example, cell secretions 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, the one or more droplets 632 may contain no tiny objects but only an aqueous medium, such as the sample material 622, which, for example, contains cellular secretions after the sample material 622 has been used to culture one or more biological cells. Figure 8 illustrates an example of a process 800, which can be implemented in a microfluidic device (such as any of the devices 600 and 700) that includes a microfluidic circuit. In step 802 of the process 800, the tiny biological objects can be cultured in a containment enclosure filled with a sample medium (e.g., cell culture medium). For example, the tiny object 630 in FIG. 6 or 7 can be a biological product and can be cultured in the chamber 616 thereof. Cultivation can generally be as discussed above. For example, culturing may include perfusing the channel 612 with medium 622. Step 802 can be implemented within a specified time period. In step 804, the cultured biological micro-object can move from the chamber 616 filled with the sample medium in which it is cultured to the chamber 616/716 filled with the immiscible medium of the sample medium. For example, the cultured micro-object 630 can move 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 Illustrated, as discussed above. In step 806, the cultured biological micro-objects can be subjected to one or more treatments or processes in the containment enclosure filled with immiscible medium. For example, one or more droplets 620 containing one or more reagents can be generated by the droplet generator 606 and moved to the chamber 612/716 filled with immiscible medium and combined with the droplets containing the 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 may contain a dissolving reagent. The fusion of the droplet 632 containing the cultured biological micro-object 630 and the first reagent-containing droplet 620 containing the dissolving reagent will dissolve the cultured biological micro-object 630. In other words, merged droplets (not shown) containing cell lysates from the cultured biological micro-objects 630 will be formed. Other (eg, second, third, fourth, etc.) reagent-containing droplets 620 can then be fused with the new droplet containing the cell lysate to further process the cell lysate as needed. In addition or as another example, contains one or more secretions or one or more other materials of interest (e.g., nucleic acid (e.g., DNA or RNA), proteins, metabolites, or other biological One or more droplets can be generated by the droplet generator 606 and moved to the enclosure 616 or 716 filled with immiscible medium and in a similar manner with the cultured objects (not shown). The droplets of the sample medium 622 of the biological micro-object 630 are fused. When the cultured biological micro-object 630 has dissolved, the droplet 620 containing the captured micro-object may contain one or more affinity beads (for example, having affinity for nucleic acids such as DNA, RNA, microRNA, or the like), It can bind to the target molecule present in the lysate after fusion with the droplet containing the cell lysate in the containment enclosure 616 or 716. In step 808, the processed tiny biological objects can be processed as appropriate. For example, if in step 806, the captured object (not shown) is moved to the chamber 616/716 filled with immiscible medium containing the cultured biological micro-object 630, then the chamber 616/716 can be monitored in step 808 The indicator indicates the response (e.g., fluorescent signal) of the material of interest binding to the amount of the labeled captured tiny object. Alternatively, the captured tiny object (not shown) can be removed from the chamber 616/716 (e.g., in the droplet 622) and discharged from the microfluidic device (not shown in Figures 6 and 7) for subsequent analysis. As another example, the processed biological micro-object 630 can be removed from the chamber 616/716 (e.g., in the droplet 632) and discharged from the microfluidic device (not shown) for subsequent analysis. Although specific embodiments and applications of the present invention have been described in this specification, these embodiments and applications are only examples, and many variations are possible. For example, the method of FIG. 8 can be performed on a sample material containing cell secretions (for example, after the 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 that may contain no micro-objects but only an aqueous medium (such as sample material 622 containing cell secretions) to a medium containing immiscible medium. In the chamber 616/716, steps 806 and 808 will be performed on the droplets 632 containing the aqueous medium. In addition, the electrowetting configuration discussed herein can be any type of optoelectronic wetting (OEW) device known in the industry, an example of which is disclosed in US Patent No. 6,958,132. Other examples of electrowetting configurations include electronically controllable dielectric wetting (EWOD) devices, examples of which are disclosed in US Patent No. 8,685,344. The full texts of the aforementioned US patent documents are incorporated herein by reference. Figure 9 is a method for forming a substrate of a microfluidic device. In the following method, a process flow chart is drawn to help form the substrate stack described in the upper and lower figures that can be used in a microfluidic device. In one embodiment, the method illustrated for forming a substrate is used to form the substrate 104 illustrated in conjunction with FIG. 1B. Figures 10-16 below show a cross-sectional view of the action shown in the method shown in Figure 9. The first action illustrated by block 902 illustrates the preparation of a substrate (a conductive silicon substrate in one embodiment) using a thermal annealing process. Subsequently, the second action illustrated by block 904 illustrates depositing a nitride layer on top of the silicon substrate. Subsequently, block 906 illustrates the third action of applying the first pattern. The application of the pattern to the substrate is usually achieved by a lithography process well known in the semiconductor processing industry, such as electron beam, X-ray, UV, and deep UV. Generally, polymers are used to define the pattern of complex structures of transistors and metal wires. Subsequently, the polymer is treated with light and a photoreactive layer and will be described in the following paragraphs. The subsequent actions of depositing the photoreactive layer are as depicted in block 908. The following action illustrated by block 910 illustrates the etching of the photoreactive layer to the first predetermined position. In one embodiment, the first predetermined position is a nitride layer. The next action represented by block 912 is to deposit at least one dielectric layer. As previously discussed, multiple embodiments with different combinations of dielectric layers have been discussed in conjunction with the description of FIG. 1B. The final step as depicted in block 912 illustrates applying the second pattern on top of the at least one dielectric layer and etching the dielectric layer to a second predetermined position. In the same embodiment, the second predetermined position is the nitride layer. In one embodiment, optional steps can be further processed. For example, the third pattern is deposited and the nitride layer is stripped until it is etched to 10 um into the silicon substrate or silicon oxide. In addition, another optional step of backside oxide stripping and silver backside metallization is performed. FIG. 10 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 902 shown in conjunction with FIG. 9. As mentioned previously, block 902 illustrates the preparation of a substrate (in one embodiment, a conductive silicon substrate) using a thermal annealing process. Please note that the mark 1001 depicts two arrows targeting the location of the thermal annealing process. FIG. 11 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 904 shown in conjunction with FIG. 9. Block 904 shows the deposition of a nitride layer on top of the silicon substrate, and this figure shows layer 1102, which shows the nitride layer formed on the top of the silicon substrate. FIG. 12 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 906 shown in conjunction with FIG. 9. Block 906 illustrates the pattern application and subsequent removal of the nitride. Please note that mark 1202 depicts the opening in the pattern where the nitride layer is removed. FIG. 13 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 908 shown in conjunction with FIG. 9. Block 908 shows the application of the photoreactive layer. Please note that mark 1302 shows the applied photoreactive layer. FIG. 14 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 910 shown in conjunction with FIG. 9. Block 910 shows that the photoreactive layer added in the processing step 908 is etched to the first predetermined position. In one embodiment, the first predetermined position is to stop the etching of the nitride layer. Please note that the mark 1402 depicts the opening in the pattern where the photoreactive layer is etched or removed. FIG. 15 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 912 shown in conjunction with FIG. 9. Block 912 illustrates depositing at least one dielectric layer. Please note that marks 1501 and 1502 respectively depict the dielectric layer and indicate the opening of the deposited dielectric layer from the previous mark 1402 in FIG. 14. FIG. 16 is a cross-sectional view of a substrate combined with the intermediate processing method shown in FIG. 9. In one embodiment, this figure shows a cross-sectional view of the processing step 914 shown in conjunction with FIG. 9. Block 914 illustrates applying a pattern to the dielectric layer and etching the dielectric layer to a second predetermined position. In one embodiment, the second predetermined position is a nitride layer. Please note that the mark 1602 depicts the opening in the dielectric layer that was removed by etching. Figure 17 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. The system integrates two microfluidic operations, as shown by the modules OEP and OEW that overlap each area. The module performs microfluidic operations as previously discussed, in particular photoelectric positioning (hereinafter referred to as OEP) and photoelectric wetting (hereinafter referred to as OEW). In one embodiment, the modules are integrated by using at least one spacer (in one embodiment, the spacer composed of polyphenylene sulfide) to help integrate the two modules due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide component spacer is thicker than the module and can be pressed and sealed on both sides to allow integration. The electrical operation of the two modules will be further discussed in conjunction with FIGS. 20A and 20B. Figure 18 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. The system integrates two types of microfluidic operations, as illustrated by modules OEP and OEW with multiple regions. For example, in one embodiment, three OEW modules and a single OEP module are shown. However, the claimed subject matter is not limited to this specific combination. Those who are familiar with this technology understand the use of different configurations of modules based on design needs or the needs of different light shielding layers. The module performs microfluidic operations as previously discussed, specifically photoelectric positioning and photowetting. In one embodiment, the modules are integrated by using at least one spacer (in one embodiment, the spacer composed of polyphenylene sulfide) to help integrate the two modules due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide component spacer is thicker than the module and can be pressed and sealed on both sides to allow integration. Figure 19 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. The system integrates two types of microfluidic operations, as illustrated by the modules OEP and OEW with discrete areas in each module. The module performs microfluidic operations as previously discussed, specifically photoelectric positioning and photowetting. In one embodiment, the modules are integrated by using at least one spacer (in one embodiment, the spacer composed of polyphenylene sulfide) to help integrate the two modules due to the difference in thickness between the modules. In this embodiment, the polyphenylene sulfide component spacer is thicker than the module and can be pressed and sealed on both sides to allow integration. 20A and 20B are views of representative diagrams of electrical addressing operations in one of the functional aspects of the embodiment shown in FIG. 17. As previously described in conjunction with FIG. 17, the system integrates two microfluidic operations, as illustrated by the modules OEP and OEW that overlap each area. In this embodiment, the OEP module has a lower impedance than the OEW module. During operation, the impedance of the OEW module overcomes the impedance of the OEP module and basically short-circuits the OEP module. In one embodiment, as shown in FIG. 20A, the OEP module is operated by applying a voltage in the range of 1-10 volts at a frequency in the range of 100 kHz to 10 mHz. In the same embodiment, as shown in FIG. 20B, the OEW module is operated by applying a voltage in the range of 10-100 volts at a frequency in the range of 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. FIG. 20B is a view of a representative diagram of an electrical addressing operation in a functional aspect of the embodiment shown in FIG. 17.Instance System and microfluidic device : Manufactured by Berkeley Lights, Inc. The system includes at least a flow controller, a temperature controller, fluid medium condition processing and pump components, a light source for light-activated DEP or EW configuration, a microfluidic device, a mounting platform and a camera.Instance 1. Preparation of electrowetting microfluidic device with modified inner surface . The microfluidic device (Berkeley Lights, Inc.) has a silicon-based first photosensitive semiconductor electrode activation substrate (with an aluminum oxide dielectric upper surface), a second ITO substrate on the opposite wall, and a photo-patterned polysilicon that separates the two substrates Oxygen microfluidic circuit material, the microfluidic device was treated with an oxygen plasma cleaner (Nordson Asymtek) using 100W power, 240 mtorr pressure and 440 sccm oxygen flow rate for 1 min. In a vacuum reactor in a 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 at 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 ,14,15,15,16,16,16-Nineafluorohexadecyl)silane (0.3 g, the details of the synthesis are as described in the U.S. Provisional Application 62/410238 filed on October 19, 2016 Description) Treating microfluidic devices treated with plasma. The chamber was then pumped to 750 mtorr using a vacuum pump, and then sealed. Place the vacuum reactor in an oven and heat it 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-Nineteen fluoro-hexadecyl) siloxy moiety layer Fluid devices. After cooling to room temperature and introducing argon into the evacuated chamber, remove the dimethoxy group (3,3,4,4,5,5,6,6,7,7,8) from the reactor on all inner surfaces ,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-nonafluorohexadecyl)siloxy moiety Layer of( surface 202 ) The microfluidic device is pretreated with silicone oil (5 centistokes viscosity, Gelest catalog number DMS-T05) before use. Figure 21A-C is the modified surface in the immiscible silicone oil phase202 Continuously photographed images of droplets of freely moving water on top. The droplets demonstrate the excellent mobility of the electrowetting configuration using the optical actuation in the microfluidic device.

100: Microfluidic device 102: Housing 104: Support structure 106: Flow area 107: Inlet port/port/inlet valve or port 108: Spacer element/Microfluidic circuit structure 109: Inner surface 110: Cover 114: Frame 116: Microfluidic Circuit material 120: Microfluidic circuit 122: Microfluidic channel 124: Microfluidic isolation fence 126: Microfluidic isolation fence 128: Microfluidic isolation fence 130: Microfluidic isolation fence 132: Tiny object trap/circuit 134: Side channel 150: System 154: main controller 156: control module 158: digital memory 160: media module 162: power module 164: imaging module 166: tilt module 168: other modules 170: display device 172: input/output device 178: Medium source 180: Fluid medium 190: Tilting device 192: Power supply 194: Imaging device 200: Microfluidic device 202: Open area/chamber 204: Bottom electrode 206: Electrode activation 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: port 224: isolation fence 226: isolation fence 228: isolation fence 230: microfluidic device 232: separation structure 234: proximal opening 236: Connection zone 238: Distal opening 240: Separation zone 242: Flow 244: Secondary flow 246: Small objects 248: Second fluid medium/second medium/fence 250: Microfluidic device 252: Frame 254: First medium /First fluid medium 256: microfluidic circuit structure 258: second medium 260: microfluidic circuit material 262: microfluidic circuit 264: microfluidic channel/flow channel 266: isolation fence 268: connection area 270: separation area 272: separation Structure 274: proximal opening 276: distal opening 278: flow/arrow 280: microfluidic device 282: secondary flow 284: housing 286: substrate 288: cover 290: microfluidic device 292: inner surface 294: inner surface 296: Siloxy linker 298: a single layer of covalently modified surface/surface modified ligand 300: nest/support structure 302: socket 304: electrical signal generation subsystem 306: thermal control subsystem 308: controller 310: interface 312: Housing 314: fluid path 316: inlet 318: outlet 320: microfluidic device 322: printed circuit board assembly 324: serial port 330: light conditioning subsystem 332: light source/first light source 334: second light source 336: beam separation 338: beam splitter/two-color filter 340: objective 342: sample plane 344: stage 346: two-color filter 348: detector 350: microscope 355: system 400: microfluidic device 412: external hydrophobic Layer 414: inner dielectric layer 416: conductive layer/photoreactive layer 418: electrode 420: support/glass Support 422: outer hydrophobic layer 428: inner layer/inner surface/electrode 430: support 435: shell 440: liquid droplet/aqueous drop 500: device 600: microfluidic device 602: fluid input 604: fluid input 606: Droplet generator 608: interface 612: microfluidic channel 614: microfluidic channel 616: chamber/accommodating fence 620: fluid droplet 622: fluid medium/second fluid medium/liquid medium/aqueous medium/sample material/medium/sample Medium 624: first fluid medium/liquid medium/immiscible medium 630: tiny object/biological tiny object 632: fluid droplet 700: microfluidic device 716: chamber/containment 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/passage width D _p : penetration depth/maximum penetration depth W _con1 : width W _con2 : width L _c1 : Length L _c2 : length

Figure 1A illustrates a generalized microfluidic device of some embodiments of the present invention and a system with related control equipment for controlling and monitoring the microfluidic device. Figure 1B is a vertical cross-sectional view of a microfluidic device with a substrate, a cover, and spacer elements, which together form a housing that is configured to contain a liquid medium and liquid droplets that are immiscible in the liquid medium. The substrate has an electrowetting configuration that allows the manipulation of droplets within the housing. Figures 1C and 1D illustrate microfluidic devices according to some embodiments of the present invention. Figures 2A and 2B illustrate the separation fence of some embodiments of the present invention. Figure 2C illustrates a detailed isolation fence of some embodiments of the present invention. Figures 2D-F illustrate the isolation fences of some other embodiments of the present invention. Figure 2G illustrates a microfluidic device according to an embodiment of the present invention. Figure 2H illustrates the coated surface of the microfluidic device of an embodiment of the present invention. Figure 3A illustrates a specific example of a system for use with a microfluidic device and related control equipment according to some embodiments of the present invention. Figure 3B illustrates imaging devices of some embodiments of the present invention. FIG. 4 illustrates an example of a microfluidic device having an EW configuration and a DEP configuration with a two-piece (duolithic) substrate. FIG. 5 illustrates an example of a microfluidic device having an EW configuration and a DEP configuration with a two-piece substrate. Fig. 6 is a horizontal cross-sectional view of a microfluidic device, which may include the electrowetting configuration as shown in Fig. 1B, and includes a plurality of microfluidic channels, a chamber opened 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 connected to the droplet generator contains a non-aqueous medium (dark color). Each compartment also contains an aqueous medium or a non-aqueous medium. Fig. 7 is a horizontal cross-sectional view of a microfluidic device, which may include an electrowetting configuration as shown in Fig. 1B, and includes a plurality of microfluidic channels, a chamber opened 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), and the microfluidic channel 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, in which each chamber containing the aqueous medium is positioned directly through the channel containing the hydrophobic medium from the corresponding chamber containing the hydrophobic medium. Fig. 8 is a diagram of a method of processing tiny biological objects in a microfluidic device. Figure 9 is a method for forming a substrate of a microfluidic device. FIG. 10 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. FIG. 11 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. FIG. 12 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. FIG. 13 is a cross-sectional view of a substrate combined with the intermediate processing of the method shown in FIG. 9. FIG. 14 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. FIG. 15 is a cross-sectional view of the substrate in the intermediate processing combined with the method shown in FIG. 9. FIG. 16 is a cross-sectional view of a substrate combined with the intermediate processing method shown in FIG. 9. Figure 17 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. Figure 18 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. Figure 19 is a cross-sectional view of a system integrating multiple microfluidic applications according to an embodiment. FIG. 20A is a view of a representative diagram of an electrical addressing operation in a functional aspect of the embodiment shown in FIG. 17. FIG. 20B is a view of a representative diagram of an electrical addressing operation in a functional aspect of the embodiment shown in FIG. 17. Figures 21A to 21C are representative photographs of water droplets moving on the modified microfluidic surface of an embodiment of the present invention.

100: Microfluidic device

102: Shell

104: support structure

106: Mobility Zone

107: inlet port/port/inlet valve or port

108: Spacer element/microfluidic circuit structure

109: inner surface

110: cover

114: Frame

116: Microfluidic circuit material

120: Microfluidic circuit

122: Microfluidic channel

124: Microfluidic isolation fence

126: Microfluidic isolation fence

128: Microfluidic isolation fence

130: Microfluidic isolation fence

132: Tiny object trap/loop

134: Side Channel

150: System

154: Main Controller

156: Control Module

158: Digital memory

160: Media module

162: Power Module

164: Imaging Module

166: Tilt module

168: other modules

170: display device

172: input/output devices

178: Media Source

180: fluid medium

190: Tilting device

192: Power

Claims (13)

A method of manufacturing a microfluidic device, the method comprising: Bonding the spacer element to the inner surface of the cover having at least one electrode configured to be connected to the voltage source; The spacer element and the cover are bonded to the dielectric surface of a substrate having at least one electrode configured to connect to a voltage source, whereby the spacer element becomes sandwiched between the inner surface of the cover and the substrate Between the electrical surfaces, where the cover and the substrate are oriented substantially parallel to each other, and the substrate, the spacer element and the cover jointly define a housing that is configured to contain 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 includes self-associating molecules covalently bonded to the inner surface of the cover; and A densely packed hydrophobic monolayer is formed on at least a part of the dielectric surface of the substrate by vapor deposition, wherein the hydrophobic monolayer includes self-associated molecules covalently bonded to the dielectric surface of the substrate. The method of claim 1, wherein the self-associating molecules of the hydrophobic monolayer of the cover and the self-associating molecules of the hydrophobic monolayer of the substrate each comprise a surface-modifying ligand and the surface-modifying ligand Linking groups respectively linked to the inner surface of the cover and the dielectric surface of the substrate, wherein the resulting surfaces of the cover and the substrate have 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 connected to the surface; Z The bond to the adjacent silicon atom attached to the surface or the bond to the surface; Y is the bond to the adjacent phosphorus atom attached to the surface or the 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; n is an integer of 0 or 1 to 20; (n + [( The sum of 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. Such as the method of claim 2, wherein V is -Si(OZ) ₂ W-. Such as the method of claim 2, where V is -P(O)(OY)W-. Such as the method of claim 2, wherein n is an integer from 1 to 20, and wherein R is hydrogen. Such as the method of claim 5, wherein m is an integer from 1 to 20, and wherein M is hydrogen. Such as the method of claim 6, where m is 2. Such as the method of claim 2, wherein n is an integer from 1 to 20, and wherein R is fluorine. Such as the method of claim 8, wherein m is an integer from 1 to 20, and wherein M is hydrogen. Such as the method of claim 8, where m is 2. Such as the method of any one of claims 2 to 10, where k is 1. Such as the method of any one of claims 2 to 10, where k is 0. Such as the method of claim 2, where the sum of (n + [(h + j)·k] + m) is an integer from 13 to 19.

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