JP2021126656A - Microfluidic electrowetting device system having covalently bonded hydrophobic surface - Google Patents

Abstract

To solve the problem in which present solutions for electrowetting are extremely limited in nature and fail to scale or implement additional functionality.SOLUTION: Microfluidic devices have an electrowetting configuration and an optimized droplet actuation surface. The devices include a conductive substrate having a dielectric layer, a hydrophobic layer covalently bonded to the dielectric layer, and a first electrode electrically coupled to the dielectric layer and configured to be connected to a voltage source. The microfluidic devices also include a second electrode optionally included in a cover and configured to be connected to the voltage source. The hydrophobic layer features self-associating molecules covalently bonded to a surface of the dielectric layer to produce a densely-packed monolayer that resists interaction and/or penetration by polar molecules or species.SELECTED DRAWING: Figure 2H

Description

Mutual reference to related applications This application is a US provisional patent application No. 62 / 246,605 filed on October 27, 2015, and a US provisional patent application Nos. 62 / 247,725, 2016 filed on October 28, 2015. It claims the priority of US Provisional Patent Application No. 62 / 342,131 filed on May 26, 2016 and US Provisional Patent Application No. 62 / 410,238 filed on October 19, 2016. The contents of each of these are incorporated herein by reference in their entirety. This application is a partial continuation of US Patent Application Publication No. 15 / 135,707 filed April 22, 2016, the contents of which are incorporated herein by reference in their entirety.

Background In a microfluidic device, microscopic objects such as living cells can be processed. For example, droplets containing micro-objects or reagents can be moved and mixed within a microfluidic device. Embodiments of the present invention relate to improvements in microfluidic devices that help robust manipulation of droplets, thereby allowing complex chemical and biological reactions to be performed on a small scale, precisely and reproducibly. Droplets can move and mix within the microfluidic device by altering the effective wetting properties of the electrowetting surface within the microfluidic device. Such migration can be useful in workflows where cells are optionally cultured in a microfluidic apparatus and then processed to evaluate various cytological properties. Current solutions for electrowetting are extremely limited in nature and cannot scale or implement additional functionality. Therefore, there is a need for improved electrowetting surfaces, stable substrates for microfluidic applications, and integration of additional functions (eg, cell growth and characterization prior to downstream processing enabled by electrowetting). There are, all of which are useful for further medical research applications.

Overview of the Invention In one aspect, the invention provides a microfluidic device comprising an electrowetting configuration comprising a substrate having a droplet working surface, the droplet working surface being the surface of the underlying dielectric layer (ie, the dielectric inner layer). Includes (or consists of, or essentially consists of) a hydrophobic layer (ie, a hydrophobic outer layer) covalently attached to. When the microfluidic device is operably connected to a voltage source, it reliably and robustly wets aqueous droplets placed on the hydrophobic layer or otherwise in contact with the hydrophobic layer, thereby electrowetting. It can be moved by force.

The microfluidic device can include a base that includes a substrate, the substrate further comprising at least one electrode (eg, a first electrode) configured to be connected to a voltage source (eg, an AC voltage source). It can have at least one electrode that is electrically coupled to the dielectric inner layer. In some embodiments, the microfluidic device further comprises a cover and at least one separating element. The substrate and cover can define an enclosure that is substantially parallel to each other and is joined together by a separating element to hold the liquid medium. In such an embodiment, the cover can include at least one electrode configured to be connected to a voltage source (eg, an AC voltage source). In some embodiments, the microfluidic device can include a single-sided electrowetting configuration. In such an embodiment, the microfluidic device does not need to include a cover. For example, the base can include a substrate and a first electrode configured to be connected to a voltage source (eg, an AC voltage source), and the substrate is configured to be connected to the voltage source. A second electrode (eg, a mesh electrode) can be included.

In some embodiments, the hydrophobic outer layer comprises a self-associating molecule that covalently bonds to the dielectric inner layer to form a high density hydrophobic monolayer. In some embodiments, the self-associating molecules of the hydrophobic monolayer each contain a siloxane group. In other embodiments, the self-associating molecules of the hydrophobic monolayer each contain a phosphonic acid group. The siloxane group or phosphonic acid group can be covalently bonded to the surface of the dielectric inner layer. In some embodiments, the self-associating molecule of the hydrophobic monolayer comprises a surface-modifying ligand and a binding group that binds the surface-modifying ligand directly or indirectly to the surface of the dielectric inner layer, respectively. The surface-modifying ligand can be any surface-modifying ligand disclosed herein. For example, the surface modification ligand can include an aliphatic group such as an alkane group. Thus, for example, the self-associating molecule of the hydrophobic monolayer can be an alkyl-terminated siloxane molecule or an alkyl-terminated phosphonic acid molecule. Alkyl groups can include chains of at least 10 carbons (eg, at least 14, 16, 18, 20, 22, or more carbons) (eg, non-branched chains). .. In other embodiments, the surface modifying ligand can include a fluorine-substituted aliphatic group such as a fluoroalkyl group. Thus, for example, the self-associating molecule can be a fluoroalkyl-terminated siloxane molecule or a fluoroalkyl-terminated phosphonic acid molecule. Fluoroalkyl groups can include chains of at least 10 carbons (eg, at least 14, 16, 18, 20, 22, or more carbons) (eg, non-branched chains). can. In certain embodiments, the fluoroalkyl group comprises one or more (eg, at least 4, 6, 8, 10, 12, or more) total fluorine-substituted carbons. For example, a fluoroalkyl group can have the 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 modifying 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 (eg, a perfluoroalkyl group). In certain embodiments, the alkyl or fluoroalkyl groups of the surface modifying ligand are non-branched. In some embodiments, the alkyl or fluoroalkyl groups of the surface modifying ligand do not contain any cyclic structure.

In some embodiments, the hydrophobic outer layer of the substrate has a thickness of less than 5 nanometers (eg, about 1.5-3.0 nanometers). In some embodiments, the hydrophobic outer layer of the substrate can be patterned such that the selected region is relatively hydrophilic relative to the rest of the hydrophobic outer layer.

In some embodiments, the dielectric inner layer of the substrate can include a first dielectric material layer. For example, the dielectric inner layer can consist of a single layer of dielectric material. The first dielectric material layer can contain oxides such as a metal oxide layer (eg, aluminum oxide, hafnium oxide, etc.). In certain embodiments, the first oxide layer is formed by atomic layer deposition (ALD). Alternatively, the dielectric inner layer can be a dielectric laminate containing two or more dielectric material layers. Therefore, in certain embodiments, the dielectric inner layer can include a first dielectric material layer and a second dielectric material layer. The first dielectric material layer can contain an oxide such as a metal oxide layer (for example, aluminum oxide or hafnium oxide), and the second dielectric material layer is an oxide such as silicon oxide or nitride. It can contain nitrides such as silicon. In such an embodiment, the first dielectric material layer can have a first surface in contact with the second dielectric material layer and an inverted surface in which the hydrophobic layers are covalently bonded. In certain embodiments, the second dielectric material layer can 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 can contain silicon oxide and can 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 can contain silicon nitride and can have a thickness of about 50 nm to about 100 nm or about 80 nm to about 100 nm. In certain embodiments, the second dielectric material layer is formed by ALD. In another embodiment, the second dielectric material layer is formed by plasma chemical vapor deposition (PECVD) techniques. In certain embodiments, the first dielectric material layer is about 10 nm to about 50 nm (eg, 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. It can have a thickness of about 40 nm, about 35 nm to about 45 nm, about 40 nm to about 50 nm, or any range defined by the two endpoints above, and can be formed by an ALD.

In yet another embodiment, the dielectric inner layer may include a third dielectric material layer, the third dielectric material layer being hydrophobic with the first surface in contact with the first dielectric material layer. It has a sex layer and a covalently bonded inverted surface. In such an embodiment, the first dielectric material layer can contain oxides as described above (or described elsewhere herein), and the second dielectric material layer is Oxides or nitrides can be included as described above (or described elsewhere herein). In certain embodiments, the third dielectric material layer can include other dielectric materials that bind well to oxides such as silicon dioxide or siloxane groups. In certain embodiments, the third dielectric material layer is deposited by ALD. In certain 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 dielectric inner layer, the dielectric inner layer is 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 80 nm. It can have a total thickness of 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 endpoints). Similarly, the dielectric layer is about 50 k ohms to about 150 k ohms (eg, about 50 k ohms to about 75 k ohms, about 75 k ohms to about 100 k ohms, about 100 k ohms to about 125 k ohms, about 125 k ohms to about 150 k ohms, or It can have an impedance (range defined by any two of the endpoints).

In some embodiments, the substrate can further include a photoresponsive layer. The photoresponsive layer can have a first surface that includes an inner dielectric layer and a second surface that includes at least one electrode. In certain embodiments, the photoresponsive layer can include hydrogenated amorphous silicon. In such an embodiment, the electrical impedance of the light response layer in the illuminated region can be reduced by illuminating any of the plurality of regions of the light response layer with the light beam. In another embodiment, the photoresponsive layer comprises a plurality of conductors, each of which is controllably connectable to at least one electrode of the substrate via a phototransistor switch.

In embodiments where the microfluidic device comprises a cover, the surface of the cover facing inward towards the enclosure can include an inner layer and a hydrophobic layer covalently bonded to the inner layer (ie, a hydrophobic outer layer). Like the hydrophobic outer layer of the substrate, the hydrophobic outer layer of the cover can contain self-associating molecules that covalently bond to the inner layer to form a high density hydrophobic monolayer. Thus, the hydrophobic outer layer can include any of the self-associating molecules described above (or described elsewhere herein) for the hydrophobic outer layer of the substrate. In some embodiments, the hydrophobic outer layer of the cover contains the same self-associating molecules as the hydrophobic outer layer of the substrate. In other embodiments, the hydrophobic outer layer of the substrate has one type (or multiple types) of self-associating molecules that differ from the hydrophobic outer layer of the substrate.

In some embodiments, the hydrophobic outer layer of the inward facing surface of the cover has a thickness of less than about 5 nanometers (eg, about 1.5-3.0 nanometers). In some embodiments, the hydrophobic outer layer of the inward facing surface of the cover can be patterned such that the selected region is relatively hydrophilic relative to the rest of the hydrophobic outer layer.

In some embodiments, the microfluidic device can include an enclosure having at least one microfluidic channel. In addition, the enclosure can include at least one microfluidic chamber (or isolation pen) fluidly connected to the microfluidic device. At least a portion of the substrate defining the microchannel and / or chamber can have an electrowetting configuration. The electrowetting configuration can be connected to a bias potential, and while so connected, it modifies the electrowetting properties of any of the corresponding regions of the substrate surface (ie, the droplet working surface). Can be done. The wetting properties of the substrate surface can be modified sufficiently to allow the droplets to travel across the substrate surface and between the microfluidic channel and the chamber.

In some embodiments, the chamber (or isolation pen) is a holding region (eg, a separation zone) configured to hold the droplet and one that fluidly connects the holding region to the microfluidic channel (e.g.). Or it can include a plurality of) connection areas. The first contiguous zone may be configured to move the droplet between the microfluidic channel and the chamber. If a second contiguous zone is present, the second contiguous zone may be configured to allow the fluid to flow and relieve pressure as the droplet moves between the microfluidic channel and the retention zone. In some embodiments, the enclosure may further include a second microfluidic channel. In such an embodiment, the chamber can be connected to both the first microfluidic channel and the second microfluidic channel.

In some embodiments, the microfluidic channel can have a height of about 30 to about 200 μm or about 50 to about 150 μm, and the height is measured in a direction orthogonal to the direction of fluid flow through the channel. NS. In some embodiments, the microfluidic channel has a width of about 50 to about 1000 μm or about 100 to about 500 μm, the width being measured in a direction orthogonal to the direction of fluid flow through the channel.

In some embodiments, the chamber (or isolation pen) has approximately the same height as the microfluidic channel. For example, the height of the chamber can be from about 30 to about 200 μm or from about 50 to about 150 μm. In some embodiments, the chamber (or holding pen) has a cross-sectional area of ^{about 100,000 to about 2,500,000 μm 2} or about 200,000 to about 2,000,000 μm ^2. In some embodiments, the contiguous zone (first, second, etc.) has approximately the same height as the corresponding chamber and / or microfluidic channel where the contiguous zone opens at the end. In some embodiments, the contiguous zone has a width of about 50 to about 500 μm or about 100 to about 300 μm.

In some embodiments, the microfluidic device can further include a drop generator. The droplet generator may be configured to selectively deliver droplets of one or more liquid media (eg, an aqueous liquid medium) into or to a microfluidic channel within an enclosure. The droplets can include, for example, biological micro-objects (eg, cells) or micro-objects such as beads. Alternatively or additionally, the droplet can include reagents such as lysis buffers, affinity reagents, detectable labels, enzyme mixtures and the like.

In some embodiments, the microfluidic device comprises a culture chamber (eg, isolation pen) suitable for culturing biological microobjects. The culture chamber can be placed within an enclosure and connected to a microfluidic channel. When the culture chamber is placed within the enclosure, the enclosure allows the exchange of nutrients in the fresh culture medium and waste products in the culture chamber (eg, diffusion of nutrients into the culture chamber and into the culture medium). (By diffusion of waste products) can include perfused microfluidic channels configured to allow fresh culture medium to flow into the culture chamber. The perfusion channel can be separate from the microfluidic channel connected to the droplet generator.

In some embodiments, the electrowetting device is integrated with the electronic positioning device. For example, in some embodiments, the microfluidic device can include a substrate having an electrowetting configuration, and a portion of the substrate can further include a dielectrophoresis (DEP) configuration. Therefore, the substrate can be monolithic. Alternatively, the microfluidic device or apparatus has a first module or section with a first substrate having a dielectrophoresis (DEP) configuration and a second module or section with a second substrate containing an electrowetting configuration. Can include sections and. Such a device can be considered as having a dualistic board, and a bridge between each board and a bridge that provides integration of functions associated with its particular configuration is between the first module or section and the second module or section. It can be in. The bridge can include a tube or the like connecting two discrete devices as it is. Alternatively, the bridge can include a binder that is attached close to the substrate (eg, within 2 mm, within 1.5 mm, within 1.0 mm, within 0.5 mm, or less). In yet another alternative form, the bridge can be a non-functional region on the monolithic substrate, and the non-functional zone can be from one configuration (eg, electrowetting configuration) to another configuration (eg, DEP configuration) of the substrate configuration. It is a place to switch. Whether the microfluidic device has a monolithic substrate or a dualistic substrate (or even a multilithic substrate), the electrowetting configuration and the DEP configuration are known in the art, respectively, or the present. It can be any such configuration disclosed in the specification. For example, the electrowetting configuration may be an optical electrowetting (OEW) configuration, a dielectric electrowetting (EWOD) configuration, a single-sided electrowetting configuration, or the like. Similarly, the DEP configuration is provided by a photoconductive substrate containing a layer of amorphous silicon and / or an array of phototransistors, an array of electrodes controlled by phototransistors, or an array of electrically actuated electrodes, etc. It can be a photoelectron tweezers (OET) configuration. In certain alternative embodiments, the substrate may include an electrowetting configuration but no additional configuration (eg, no dielectrophoresis (DEP) configuration).

Thus, in some embodiments, a single monolithic device can combine the functions of both devices.

In another aspect, the invention provides a method of manufacturing the microfluidic device of the invention. The method involves joining a separating element (eg, made from a microfluidic circuit material) to the inner surface of a cover having at least one electrode configured to be connected to a voltage source and being connected to the voltage source. By joining the separating element and the cover to the dielectric inner surface of the substrate having at least one electrode configured as described above and by vapor deposition, the hydrophobic layer is attached to at least a part of the inner surface of the cover and at least a part of the dielectric inner surface of the substrate. It can include forming. In certain embodiments, the separating element is sandwiched between the inner surface of the cover and the dielectric inner surface of the substrate such that the cover and the substrate are oriented substantially parallel to each other. The substrate, separating elements, and cover can collectively define an enclosure configured to hold the liquid medium. In certain embodiments, the hydrophobic layer comprises substantially all exposed areas of the inner surface of the cover and substantially all exposed areas of the dielectric inner surface of the substrate (ie, substantially inward facing the enclosure). Accumulated on all surfaces). In certain embodiments, the hydrophobic layer is further deposited on the surface of the separating element facing inward towards the enclosure.

In certain embodiments, the hydrophobic layer comprises self-associating molecules covalently bonded to the inner surface of the cover and the dielectric inner surface of the substrate, which self-associating molecules form a dense monolayer. In some embodiments, the self-associating molecule deposited by vapor deposition comprises a surface-modifying ligand and a binding group that directly or indirectly binds the surface-modifying ligand to the surface of the dielectric inner layer, respectively. Thus, the self-associating molecule can be any self-associating molecule described above or elsewhere herein.

In another aspect, the invention provides a method of processing materials such as chemical and / or biomaterials in a microfluidic device. In certain embodiments, the method places a first fluid medium in or part of the enclosure of a microfluidic device that includes a substrate having an electrowetting configuration, a cover, and a separating element that together define the enclosure. Filling, applying an AC voltage potential between at least one electrode on the substrate and at least one electrode on the cover, and introducing a first droplet of liquid medium into the enclosure are liquids. The medium droplets are not mixed with the first liquid medium, they are introduced and the electrowetting force is applied to the first droplets to move the first droplets to the desired position within the enclosure. Including to let. The first liquid medium can include any first liquid medium described herein, such as silicone oils, fluorinated oils, or combinations thereof.

In some embodiments, the method may include drawing a first droplet from a first section of an enclosure, such as a microfluidic channel, into a second section of an enclosure, such as a chamber, or vice versa. The pull-in may include altering the effective electrowetting properties of the area of the substrate surface in contact with and / or adjacent to the first droplet. Therefore, filling the enclosure with a first liquid medium can include filling the microfluidic channels and chambers with a first liquid medium.

In some embodiments, the microfluidic device comprises a droplet generator. The method may include generating a first droplet using a droplet generator. In addition, the droplet generator can introduce the first droplet into the enclosure. The droplets produced can have a volume of about 100 picolitres to about 100 nanoliters or about 1 to 50 nanoliters. In some embodiments, the first droplet can include beads or micro-objects such as biological micro-objects (eg, cells, cysts, etc.), cell secretions, or reagents. Beads can be used as materials of interest such as cell secretions (eg, antibodies) or other biomolecules (eg, nucleic acids such as DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof). It can have a molecule that has an affinity for it. The droplet may contain a single micro-object such as a single living cell, or may contain a plurality of micro-objects. For example, a droplet can contain 2 to 20 or more microscopic objects, such as beads. In some embodiments, the droplet can include a reagent such as a cell lysis buffer, a label (eg, a fluorescent label reagent), a luminescent reagent, or an enzyme mixture.

In some embodiments, the method involves introducing second, third, fourth, etc. droplets into the enclosure and applying electrowetting force to the droplets to apply the second, third, third, etc. It further includes moving a fourth class droplet to a desired position within the enclosure. The second droplet can be moved to a position in the vicinity of the first droplet and then mixed with the first droplet to form the first combined droplet, making the third droplet It can be moved to a position near the first bonded droplet and then mixed with the first bonded droplet to form a second bonded droplet, which allows the fourth droplet to form a second bonded droplet. It can be moved to a position near the droplet and then mixed with the second bonded droplet to form a third bonded droplet, and so on. Each additional droplet may include a fluid medium that does not mix with the first liquid medium, but mixes with the liquid medium of the first droplet.

In some embodiments, the first droplet comprises a living cell and the second droplet comprises a reagent. The reagent can be a cell lysis buffer that lyses living cells when the first and second droplets are mixed. Alternatively, the reagent can be a fluorescent label (eg, a fluorescently labeled antibody or other affinity reagent) or a reagent used in a luminescence assay. The third droplet may contain a reagent, such as one or more (eg, 2 to 20) capture beads, that have an affinity for the material of interest. For example, the material of interest can be an antibody or nucleic acid such as DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. Such capture beads can optionally be removed from the device for subsequent analysis. The fourth droplet, like the second and third droplets, can contain a reagent such as an enzyme mixture suitable for performing a reaction such as a reverse transcription reaction or a whole genome amplification reaction.

In some embodiments, the movement and mixing of the droplets involves the use of electrowetting forces, altering the effective electrowetting properties of the area of the substrate surface in the vicinity of the droplets, thereby moving or moving the droplets. Including mixing. In certain embodiments, altering the effective electrowetting properties of the substrate surface can include activating electrowetting electrodes in the area of the substrate surface in the vicinity of the droplet. In certain embodiments, activating an electrowetting electrode in a region of the substrate surface in the vicinity of a droplet comprises directing a light pattern to that region of the substrate surface.

Additional embodiments and embodiments of the present invention will become apparent from the drawings and the detailed description below.

Demonstrates a generalized microfluidic device according to some embodiments of the present invention and a system having associated control devices for controlling and monitoring the microfluidic device. FIG. 5 is a vertical cross-sectional view of a microfluidic device having a substrate, a cover, and a separating element that together form an enclosure configured to hold a liquid medium and droplets of liquid that are immiscible with the liquid medium. The substrate has a droplet electrowetting configuration that allows the droplets to be manipulated within the enclosure. A microfluidic device according to some embodiments of the present invention is shown. A microfluidic device according to some embodiments of the present invention is shown. Separation pens according to some embodiments of the present invention are shown. Separation pens according to some embodiments of the present invention are shown. A detailed isolation pen according to some embodiments of the present invention is shown. The isolation pens according to some other embodiments of the present invention are shown. The isolation pens according to some other embodiments of the present invention are shown. The isolation pens according to some other embodiments of the present invention are shown. A microfluidic device according to an embodiment of the present invention is shown. The coated surface of the microfluidic device according to the embodiment of the present invention is shown. Specific examples of systems used in combination with microfluidic devices and related control devices according to some embodiments of the present invention are shown. An imaging device according to some embodiments of the present invention is shown. An example of a microfluidic device having an EW configuration and a DEP configuration using a dual chic substrate is shown. An example of a microfluidic device having an EW configuration and a DEP configuration using a monolithic substrate is shown. It is a horizontal sectional view of a microfluidic device, the microfluidic device can include an electrowetting configuration as shown in FIG. 1B, and a plurality of microfluidic channels, a chamber that opens at the end of at least one of the microfluidic channels. , And a droplet generator. In this embodiment, one microfluidic channel comprises an aqueous medium (light color), while the microfluidic channel connected to the droplet generator comprises a non-aqueous medium (dark color), as well as the chamber. Includes either aqueous or non-aqueous medium. It is a horizontal sectional view of a microfluidic device, the microfluidic device can include an electrowetting configuration as shown in FIG. 1B, and a plurality of microfluidic channels, a chamber that opens at the end of at least one of the microfluidic channels. , And a droplet generator. In this embodiment, one microfluidic channel and the first set of chambers contain an aqueous medium (bright color), while the microfluidic channel and the second set of chambers connected to the droplet generator are Contains hydrophobic media (dark color). FIG. 6 presents a variant of the embodiment shown in FIG. 5, in which each chamber containing the aqueous medium is directly opposite the corresponding chamber containing the hydrophobic medium across a channel having the hydrophobic medium. Is placed in. It is a figure of the method of processing a biological microobject in a microfluidic apparatus. It is a method that can be applied to manufacture substrates for microfluidic devices that have a first section with an electrowetting configuration and a second section with a dielectrophoretic configuration. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. A vertical cross section of a substrate processed according to the method shown in FIG. 9 is provided. FIG. 5 is a diagram of an operation expression for electrically coping with one functional aspect according to the embodiment shown together with FIG. FIG. 5 is a diagram of an operation expression for electrically coping with one functional aspect according to the embodiment shown together with FIG. It is a photographic representation of the movement of aqueous droplets on a modified microfluidic surface according to an embodiment of the present invention. It is a photographic representation of the movement of aqueous droplets on a modified microfluidic surface according to an embodiment of the present invention. It is a photographic representation of the movement of aqueous droplets on a modified microfluidic surface according to an embodiment of the present invention.

This specification describes exemplary embodiments and applications of the present invention. However, the invention is not limited to these exemplary embodiments and applications or the modes in which the exemplary embodiments and applications work or as described herein. In addition, the figure may show a simplified or partial view, and the dimensions of the elements in the figure may not be exaggerated or otherwise to a constant scale. Further, when the terms "above", "attached", "connected", "bonded" or similar terms are used herein, certain elements (eg, materials, layers, substrates, etc.) ) Is directly on another element, attached, connected, or combined, or there is one or more intervening elements between one element and another. Regardless, it is on another element "above" and can be "attached" to it, "connected" to it, or "bonded" to it. Also, unless the context indicates otherwise, the direction (eg, up, down, top, bottom, side, up, down, down, top, top, bottom, horizontal, vertical, "x", "y", “Z” etc.) are relative, if provided, and are provided only as an example, not as a limitation, and for ease of explanation and consideration. Further, when a list of elements (eg, elements a, b, c) is referenced, such a reference is any one of the elements listed by itself, any less than all of the listed elements. It is intended to include combinations and / or all combinations of listed elements. Sectional divisions herein are for ease of consideration only and do not limit any combination of elements considered.

As used herein, "substantially" means functioning well for the intended purpose. Therefore, the term "substantially" allows small and slight variations from absolute or perfect conditions, dimensions, measurements, results, etc., as expected by one of ordinary skill in the art, but does not significantly affect overall performance. .. When used with respect to a number or a parameter or feature that can be expressed as a number, "substantially" means within 10%.

The term "one" means more than one.

As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, the term "placed" includes "placed" within its meaning.

As used herein, a "microfluidic device" or "microfluidic device" is a device that includes one or more discrete microfluidic circuits configured to hold fluid, each microfluidic circuit. With fluidly interconnected circuit elements including, but not limited to, regions, flow regions, channels, chambers, and / or pens, and fluids (and optionally for microfluidic devices including covers). It consists of at least two ports configured to allow micro-objects suspended in the fluid to flow into and / or out of the micro-fluid device. Typically, the microfluidic circuit of a microfluidic device comprises at least one microfluidic channel and at least one chamber and has a volume of less than about 1 mL of fluid, such as less than about 750 μL, less than about 500 μL, less than about 250 μL, less than about 200 μL. Less than about 150 μL, less than about 100 μL, less than about 75 μL, less than about 50 μL, less than about 25 μL, less than about 20 μL, less than about 15 μL, less than about 10 μL, less than about 9 μL, less than about 8 μL, less than about 7 μL, less than about 6 μL, about Holds less than 5 μL, less than about 4 μL, less than about 3 μL, or less than about 2 μL of fluid. In certain embodiments, the microfluidic circuit is about 1-2 μL, about 1-3 μL, about 1-4 μL, about 1-5 μL, about 2-5 μL, about 2-8 μL, about 2-10 μL, about 2-12 μL. , About 2-15 μL, about 2-20 μL, about 5-20 μL, about 5-30 μL, about 5-40 μL, about 5-50 μL, about 10-50 μL, about 10-75 μL, about 10-100 μL, about 20-100 μL , About 20-150 μL, about 20-200 μL, about 50-200 μL, about 50-250 μL, or about 50-300 μL.

As used herein, a "nanofluid device" or "nanofluid device" refers to a fluid having a capacity of less than about 1 μL, eg, about 750 nL, about 500 nL, about 250 nL, about 200 nL, about 150 nL, about 100 nL. Fluids of about 75 nL, about 50 nL, about 25 nL, about 20 nL, about 15 nL, about 10 nL, about 9 nL, about 8 nL, about 7 nL, about 6 nL, about 5 nL, about 4 nL, about 3 nL, about 2 nL, about 1 nL, or less. A type of microfluidic device having a microfluidic circuit comprising at least one circuit element configured to hold the. Nanofluid devices include multiple circuit elements (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 15, 20, 25, 50). 75 pieces, 100 pieces, 150 pieces, 200 pieces, 250 pieces, 300 pieces, 400 pieces, 500 pieces, 600 pieces, 700 pieces, 800 pieces, 900 pieces, 1000 pieces, 1500 pieces, 2000 pieces, 2500 pieces, 3000 pieces, 3500 pieces, 4000 pieces, 4500 pieces, 5000 pieces, 6000 pieces, 7000 pieces, 8000 pieces, 9000 pieces, 10,000 pieces, or more) can be included. In certain embodiments, one or more (eg, all) of at least one circuit element is about 100 pL to 1 nL, about 100 pL to 2 nL, about 100 pL to 5 nL, about 250 pL to 2 nL, about 250 pL to 5 nL, about 250 pL. 10 nL, about 500 pL to 5 nL, about 500 pL to 10 nL, about 500 pL to 15 nL, about 750 pL to 10 nL, about 750 pL to 15 nL, about 750 pL to 20 nL, about 1 to 10 nL, about 1 to 15 nL, about 1 to 20 nL, about 1 It is configured to hold a volume of ~ 25 nL, or about 1-50 nL of fluid. In other embodiments, one or more (eg, all) of at least one circuit element is about 20 nL-200 nL, about 100-200 nL, about 100-300 nL, about 100-400 nL, about 100-500 nL, about 200. Holds a fluid with a volume of ~ 300 nL, about 200-400 nL, about 200-500 nL, about 200-600 nL, about 200-700 nL, about 250-400 nL, about 250-500 nL, about 250-600 nL, or about 250-750 nL. It is configured as follows.

"Microfluidic channel" or "flow channel" as used herein refers to the flow region of a microfluidic device having a length much longer than both horizontal and vertical dimensions. For example, the flow channel is at least 5 times the length of either the horizontal or vertical dimension, eg, at least 10 times the length, at least 25 times the length, at least 100 times the length, at least the length. It can be 200 times, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or more. In some embodiments, the length of the flow channel is in the range of about 50,000 μm to about 500,000 μm, including any range in between. In some embodiments, the horizontal dimensions range from about 100 μm to about 1000 μm (eg, about 150 to about 500 μm) and the vertical dimensions range from about 25 to about 200 μm, eg, from about 40 to about 150 μm. be. Note that flow channels can have a variety of different spatial configurations in microfluidic devices and are therefore not limited to perfectly linear elements. For example, a flow channel may include one or more sections having any of the following configurations: curves, curves, spirals, slopes, descents, forks (eg, different channels), and theirs. Any combination. In addition, the flow channels have different cross-sectional areas along the path and can expand and contract to provide the desired fluid flow internally.

As used herein, the term "obstacle" generally partially (but not completely) impedes the movement of a target microobject between two different regions or circuit elements within a microfluidic device. Refers to bumps large enough or similar types of structures. The two different regions / circuit elements can be, for example, a microfluidic isolation pen and a microfluidic channel or a connection and isolation region of the microfluidic isolation pen.

As used herein, the term "stenosis" generally refers to the narrowing of a circuit element (or interface between two circuit elements) in a microfluidic device. The constriction can be placed, for example, at the interface between the microfluidic isolation pen and the microfluidic channel or at the interface between the separation area and the connection area of the microfluidic isolation pen.

As used herein, the term "transparent" refers to a material that, when visible light is transmitted, allows visible light to pass through substantially unchanged.

As used herein, the term "microscopic object" generally refers to any microscopic object that can be separated and collected by the present invention. Non-limiting examples of microobjects include fine particles; microbeads (eg, polystyrene beads, Luminex ™ beads, etc.); magnetic beads; microrods; microwires; inanimate microobjects such as quantum dots, cells (eg, eg, quanta dots). Embryos, egg mother cells, eggs, sperm cells, cells dissociated from tissues, eukaryotic cells, progenitor cells, animal cells, mammalian cells, immune cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infections Cells, transfected cells and / or transformed cells, reporter cells, prokaryotic cells, etc.); biological cell organs; vesicles or complexes; synthetic vesicles; liposomes (eg, derived from synthetic or membrane specimens); lipid nanocrafts (eg, derived from synthetic or membrane specimens) Ritchie et al. (2009) “Reconstitution of Membrane Proteins”
Biological microobjects such as "in Phospholipid Bilayer Nanodiscs", Methods Enzymol., 464: 211-231), or a combination of inanimate microobjects and biological microobjects (eg, microadherent to cells) Beads, liposome-coated microbeads, liposome-coated magnetic beads, etc.). Beads share or do not share fluorescent labels, proteins, small molecule signaling moieties, antigens, or chemical / biological species that can be used in the assay. It may further have other attached moieties / molecules.

As used herein, the term "maintaining cells" includes both fluid and gaseous components and optionally refers to the conditions necessary to maintain cell survival and / or proliferation. Refers to providing an environment that includes the surface to be provided.

The "ingredients" of a fluid medium are present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, etc. Any chemical or biological molecule that

As used herein with reference to a fluid medium, "diffusing" and "diffusing" refer to the thermodynamic movement of the components of the fluid medium towards the lower concentration gradient.

The phrase "medium flow" means bulk transfer of fluid medium primarily due to any mechanism other than diffusion. For example, the flow of the medium can include the movement of the fluid medium from one location to another due to the pressure difference between the locations. Such flows can include continuous, pulsed, periodic, random, intermittent, or reciprocating flows of liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence or mixing of the medium can occur.

The phrase "substantially no flow" is, on average over time, a fluid medium that is less than the diffusivity of the components of the material (eg, the analyte of interest) into or in the fluid medium. Refers to the flow rate of. The diffusivity of the components of such a material can depend, for example, on the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein with reference to different regions within a microfluidic device, the phrase "fluidally connected" is used within each region if the different regions are substantially filled with a fluid such as a fluid medium. It means that the fluids of the above are communicated to form a single fluid. This does not mean that the fluids (or fluid media) in different regions are necessarily the same in the composition. Rather, fluids within different fluid connection regions of a microfluidic device can have different compositions within the flux (eg, solutes of different concentrations such as proteins, carbohydrates, iron, or other molecules), which The reason is that the solute moves down each concentration gradient and / or the fluid flows through the device.

Microfluidic (or nanofluidic) devices can include "sweep" and "non-sweep" regions. As used herein, a "sweep" region consists of one or more interconnected circuit elements of a microfluidic circuit, each of which is a fluid flowing through a microfluidic circuit. Experience the flow of medium when you are. The circuit elements of the sweep region can include, for example, all or parts of the region, channel, and chamber. As used herein, a "non-sweep" region consists of one or more fluidly interconnected circuit elements of a microfluidic circuit, each circuit element in which the fluid passes through a microfluidic circuit. Virtually no experience of flux when flowing. The non-swept area can be fluid-connected to the swept area if the fluid connection allows diffusion but is structured so that the medium is substantially non-flowing between the swept area and the non-sweep area. .. Thus, the microfluidic device is structured to substantially separate the non-sweep region from the flow of medium in the sweep region, while substantially allowing only diffusion communication between the sweep region and the non-sweep region. can do. For example, the flow channel of a microfluidic device is an example of a sweep region, while the isolation region of a microfluidic device (discussed in more detail below) is an example of a non-sweep region.

As used herein, a "flow region" is one or more fluid-connected circuit elements (eg, channels, regions, and, etc., that define the trajectory of a medium flow and receive the trajectory of a medium flow. Refers to a chamber, etc.). Therefore, the flow region is an example of a sweep region for a microfluidic device. Other circuit elements (eg, non-sweep regions) may be fluid connected to circuit elements that include the flow region without receiving the medium flow in the flow region.

As used herein, "alkyl" is a straight or branched chain consisting only of unsaturated carbon and hydrogen atoms having 1 to 6 carbon atoms (eg, C1 to C6 alkyl). Refers to hydrocarbon radicals. Whenever an alkyl appears herein, a numerical range such as "1-6" refers to each integer within a given range, for example, "1-6 carbon atoms" have an alkyl group. It means that it can consist of up to 6 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., but this definition is the generation of the term "alkyl" for which a numerical range is not specified. Also includes. In some embodiments, it is a C1-C3 alkyl group. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butylisobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl and the like. Alkyl can be single bonds such as methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (isopropyl) n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and It is bound to the rest of the molecule by hexyl or the like.

Unless otherwise specified herein, alkyl groups are optionally, independently, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoro. Methyl, 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'(here , 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 can be substituted with one or more substituents, PO3 (R') 2, where each R'is independently hydrogen, alkyl, fluoroalkyl, aryl, alalkyl, heterocyclo. It is alkyl or heteroaryl.

As referred to herein, an alkyl fluorinated moiety is an alkyl moiety in which one or more hydrogens of the alkyl moiety are substituted with a fluoro substituent. In the all-fluorine-substituted alkyl moiety, all hydrogen bonded to the alkyl moiety is substituted with a fluoro-substituted group.

As referred to herein, the "halo" moiety is the bromo, chloro, or fluoro moiety.

As referred to herein, an "olefinic" compound is an organic moiety that includes an "alkene" moiety. The alkene moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond. The non-alkene moiety of the molecule can be any type of organic molecule, and in some embodiments, an alkyl or a fluorinated (including, but not limited to, total fluorine substitution) alkyl capable of further substituting any moiety. May include parts.

As used herein, a "high density hydrophobic monolayer" is dense enough to be resistant to the interaction and / or invasion of polar molecules such as water, ions, and other charged species. Refers to a single layer of hydrophobic molecules packed together.

As used herein, "μm" (or "um") means micrometer, "μm ³ " means cubic micrometer, "pL" means picolitre, and "nL". Means nanoliters and "μL" (or "uL") means microliters.

Loading method. Loading of micro-objects such as biological micro-objects and / or micro-objects such as beads into different regions of microfluidic devices is as described herein in fluid flow, gravity, dielectrophoresis (DEP) forces, electrowetting forces. , Magnetic force, or the use of any combination thereof. The DEP force can be generated electrically, such as by photoelectron tweezers (OET) configuration, optically and / or by activation of the electrode / electrode region in a temporal / spatial pattern. Similarly, electrowetting forces can be provided electrically, such as by optical electrowetting (OEW) configuration, optically and / or by activation of the electrode / electrode region in a spatiotemporal pattern.

Microfluidic devices and systems that operate and observe such devices. FIG. 1A shows a generalized example of a microfluidic device 100 and a system 150 that can be used to control the movement of microobjects and / or droplets within the microfluidic device 100 and the microfluidic device 100. A perspective view of the microfluidic device 100 is shown, in which the cover 110 is partially cut out and a partial view of the inside of the microfluidic device 100 is provided. The microfluidic device 100 generally includes a microfluidic circuit 120 that includes a flow region 106, through which the fluid medium 180 can flow, optionally one or more microobjects (not shown). ) Is conveyed within the microfluidic circuit 120 and / or through the microfluidic circuit 120. Although one microfluidic circuit 120 is shown in FIG. 1A, suitable microfluidic devices can include multiple (eg, 2 or 3) such microfluidic circuits. Regardless, the microfluidic device 100 can be configured to be a nanofluidic device. In the embodiment shown in FIG. 1A, the microfluidic circuit 120 includes a plurality of microfluidic isolation pens 124, 126, 128, and 130, each isolation pen having a single opening that fluidly connects to the flow region 106. Have. Further discussed below, the microfluidic isolation pen is optimal for holding microobjects in a microfluidic device, such as the microfluidic device 100, even when the medium 180 is flowing through the flow region 106. Includes various features and structures that have been modified. However, before referring to the above, an overview of the microfluidic device 100 and the system 150 is provided.

As generally shown in FIG. 1A, the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in FIG. 1A, the enclosure 102 includes a support structure 104 (eg, a base), a microfluidic circuit structure 108, and a cover 110. It is shown as a thing. However, in certain embodiments, the enclosure 102 does not have a cover 110 and the microfluidic circuit 120 may be defined by a support structure 104 and a microfluidic circuit structure 108. The support structure 104, the microfluidic circuit structure 108, and (optionally) the cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be placed on the inner surface 109 of the support structure 104, and the cover 110 can be placed on the microfluidic circuit structure 108. Together with the support structure 104 and (optionally) the cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120.

As shown in FIG. 1A, the support structure 104 may be at the bottom of the microfluidic circuit 120 and the cover 110 may be at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and cover 110 may be configured in other orientations. For example, the support structure 104 may be at the top of the microfluidic circuit 120 and the cover 110 may be at the bottom of the microfluidic circuit 120. Regardless, there may be one or more ports 107, each containing a passage into or out of the enclosure 102. Examples of passages include valves, gates, through holes and the like. As shown, port 107 is a through hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can be located in other components of the enclosure 102, such as the cover 110. Although only one port 107 is shown in FIG. 1A, the microfluidic circuit 120 can have two or more ports 107. For example, there may be a first port 107 that acts as an inlet for the fluid to enter the microfluidic circuit 120 and a second port 107 that acts as an outlet for the fluid to exit the microfluidic circuit 120. Whether the port 107 functions as an inlet or an outlet may depend on the direction in which the fluid flows through the flow region 106.

The support structure 104 can include one or more electrodes (not shown) and one substrate or a plurality of interconnected substrates. The substrate can be any suitable substrate known in the art. For example, the support structure 104 can include one or more semiconductor substrates, each substrate being electrically connected to at least one of one or more electrodes (eg, all or all of the semiconductor substrates). The subset can be electrically connected to a single electrode). Alternatively, the support structure 104 can include a printed circuit board assembly (“PCBA”) that includes one or more electrodes. In yet another embodiment, the support structure 104 may include a substrate (eg, a semiconductor substrate) mounted on the PCBA.

The microfluidic circuit structure 108 can define the circuit elements of the microfluidic circuit 120. Such circuit elements may include a flow region (which may include one or more flow channels, or one or more flows) that can be fluidly interconnected when the microfluidic circuit 120 is filled with fluid. Can include spaces or areas such as (which can be channels), chambers, pens, and traps. In the microfluidic circuit 120 shown in FIG. 1A, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure that substantially surrounds the microfluidic circuit material 116. For example, the frame 114 can include a metallic material. Alternatively, the microfluidic circuit structure 108 does not have to have a frame. For example, the microfluidic circuit structure 108 can consist of the microfluidic circuit material 116, or can essentially consist of the microfluidic circuit material 116.

In the microfluidic circuit material 116, cavities and the like can be patterned to define circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 can include flexible materials such as flexible polymers that may be gas permeable (eg, rubber, plastics, elastomers, silicones, polydimethylsiloxane (“PDMS”), etc.). Other examples of materials that can constitute microfluidic circuit material 116 include molded glass, etchable materials such as silicone (photopatternable silicone or "PPS"), photoresists (eg, SU8), and the like. .. In some embodiments, such a material-thus the microfluidic circuit material 116-can be substantially impermeable to stiffness and / or gas. Regardless, the microfluidic circuit material 116 can be placed on the support structure 104 and (optionally) 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 can be a structurally distinct element, as shown in FIG. 1A. The cover 110 may contain the same or different material as the frame 114 and / or the microfluidic circuit material 116. Similarly, the support structure 104 may have a structure separate from the microfluidic circuit material 116 or frame 114 as shown, or may be an integral part of the microfluidic circuit material 116 or frame 114. Similarly, the microfluidic circuit material 116 and the frame 114, if present, may have separate structures, as shown in FIG. 1A, or may be integral parts 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 can be a polymer such as PDMS. In some embodiments, the cover 110 can include both rigid and deformable materials. For example, one or more parts of cover 110 (eg, one or more parts located on isolation pens 124, 126, 128, 130) include a deformable material that interfaces with the rigid material of cover 110. be able to. In some embodiments, the cover 110 may further include one or more electrodes. One or more electrodes can include conductive oxides such as indium-tin-oxide (ITO) that can be coated with glass or similar insulating material. Alternatively, one or more electrodes may be monolayer nanotubes, multilayer nanotubes, nanowires, clusters of conductive nanoparticles, or a combination thereof, embedded in a deformable polymer such as a polymer (eg, PDMS). It can be a flexible electrode. Flexible electrodes that can be used in microfluidic devices are described, for example, in US Patent Application Publication No. 2012/0325665 (Chiou et al.), The contents of which are incorporated herein by reference. In some embodiments, the cover 110 can be modified to support droplet migration and / or cell adhesion, cell survival, and / or cell growth (eg, to the microfluidic circuit 120). By coating or adjusting all or part of the surface facing inward toward you). Modifications can include coatings or conditioning surfaces of synthetic or natural polymers with covalently bonded molecules (eg, self-associating molecules). In some embodiments, the cover 110 and / or the support structure 104 is capable of transmitting light. The cover 110 may also contain at least one material that is gas permeable (eg, PDMS or PPS).

FIG. 1A also shows a system 150 that operates and controls a microfluidic device, such as the microfluidic device 100. The system 150 includes an electrical power supply 192, an imaging device 194 (not shown, but can be part of the imaging module 164), and a tilting device 190 (not shown, but can be part of the tilting module 166). including.

The power supply 192 can provide power to the microfluidic device 100 and / or tilt device 190 and provide a bias voltage or current as needed. The power supply 192 can include, for example, one or more alternating current (AC) and / or direct current (DC) voltage sources or current sources. The imaging device 194 can include a device such as a digital camera that captures an image inside the microfluidic circuit 120. In some cases, the imaging device 194 further includes a detector with a high frame rate and / or high sensitivity (eg, for low light applications). The imaging device 194 directs the stimulating radiation and / or the light beam into the microfluidic circuit 120, and the radiation and / or emitted from the microfluidic circuit 120 (or a small object contained in the microfluidic circuit 120). It can also include a mechanism for collecting light rays. The emitted light can be in the visible spectrum and may include, for example, fluorescent radiation. The reflected light may include reflected radiation emitted from a wide spectrum lamp such as an LED or a mercury lamp (eg, 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 optical column), which may or may not include an eyepiece.

The system 150 further includes a tilting device 190 configured to rotate the microfluidic device 100 around one or more axes of rotation. In some embodiments, the tilt device 190 orients the microfluidic device 100 (and thus the microfluidic circuit 120) horizontally (ie, 0 ° relative to the x-axis and y-axis) and vertically (ie, x-axis). And / or 90 ° relative to the y-axis), or support and / or support the enclosure 102 containing the microfluidic circuit 120 around at least one axis so that it can be held in any orientation between them. Configured to hold. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and the microfluidic circuit 120). For example, the tilt device 190 is 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 ° relative to the x-axis or y-axis. , 0.8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, The microfluidic device 100 can be tilted at 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 tilts the microfluidic device 100 (and the microfluidic circuit 120) at any angle greater than 90 ° relative to the x-axis and / or the y-axis, or the microfluidic device 100 (and the microfluidic circuit 120). The microfluidic device 100 (and the microfluidic circuit 120) can also be reversed by tilting 120) 180 ° relative to the x-axis or y-axis. Similarly, in some embodiments, the tilt device 190 is a microfluidic device 100 (and a microfluidic circuit 120) around a rotation axis defined by a flow region 106 / channel 122 or any other part of the microfluidic circuit 120. ) Is tilted.

In some cases, the microfluidic device 100 is tilted vertically so that the flow region 106 / channel 122 is located above or below one or more isolation pens. As used herein, the term "upper" means that the flow region 106 / channel 122 is located higher than one or more isolation pens on the vertical axis defined by gravity (ie, the flow region). 106 / The object in the isolation pen above the channel 122 has a higher gravitational potential energy than the object in the flow region / channel). As used herein, the term "downward" means that the flow region 106 / channel 122 is located below one or more isolation pens on a vertical axis defined by gravity (ie, flow). The object in the isolation pen below the region 106 / channel 122 has a lower gravitational potential energy than the object in the flow region / channel).

In some cases, the tilt device 190 tilts the microfluidic device 100 around an axis parallel to the flow region 106 / channel 122. Further, the microfluidic device 100 is less than 90 ° such that the flow region 106 / channel 122 is located above or below one or more isolation pens rather than directly above or below the isolation pens. Can be tilted to the angle of. In other cases, the tilt device 190 tilts the microfluidic device 100 around an axis orthogonal to the flow region 106 / channel 122. In yet other cases, the tilt device 190 tilts the microfluidic device 100 around an axis that is neither parallel nor orthogonal to the flow region 106 / channel 122.

System 150 can further include medium source 178. The medium source 178 (eg, container, reservoir, etc.) can include multiple sections or containers, each holding a different fluid medium 180. Therefore, the medium source 178 can be a device that is external to the microfluidic device 100 and is separate from the microfluidic device 100, as shown in FIG. 1A. Alternatively, the medium source 178 can be placed, in whole or in part, inside the enclosure 102 of the microfluidic device 100. For example, medium source 178 can include a reservoir that is part of the microfluidic device 100.

FIG. 1A also shows a simplified block diagram representation of an example of a control and monitoring device 152 that constitutes a portion of the system 150 and can be used in conjunction with the microfluidic device 100. As shown, examples of such control and monitoring equipment 152 include a master controller 154, a medium module 160 that controls a medium source 178, and micro-objects and / or media (eg, medium) in the microfluidic circuit 120. Imaging that controls a prime mover module 162 that controls the movement and / or selection of (droplets) and an imaging device 194 (eg, a camera, microscope, light source, or any combination thereof) that captures an image (eg, a digital image). It includes a module 164 and a tilt module 166 that controls the tilt device 190. The control device 152 may also include other modules 168 that control, monitor, or perform other functions relating to the microfluidic device 100. As shown, the device 152 can be functionally coupled (or further included) with the display device 170 and the input / output device 172.

The master controller 154 can include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate according to machine executable instructions (eg, software, firmware, source code, etc.) stored as non-temporary data or signals in memory 158. can. Alternatively or additionally, the control module 156 can include hard-wired digital and / or analog circuits. The medium module 160, the prime mover module 162, the imaging module 164, the tilt module 166, and / or the other module 168 can be configured similarly. Therefore, the function, process behavior, operation, or process step considered herein as being performed with respect to the microfluidic device 100 or any other microfluidic device is a master controller 154 configured as described above. , Media module 160, prime mover module 162, imaging module 164, tilt module 166, and / or any one or more of other modules 168. Similarly, the master controller 154, medium module 160, prime mover module 162, imaging module 164, tilt module 166, and / or other modules 168 are communicably coupled to any function discussed herein. It can send and receive data used in processes, actions, actions, or steps.

The medium module 160 controls the medium source 178. For example, the medium module 160 can control the medium source 178 to put the selected fluid medium 180 into the enclosure 102 (eg, via the inlet 107). The medium module 160 can also control the removal of medium from the enclosure 102 (eg, through an outlet (not shown)). Therefore, one or more media can be selectively put into the microfluidic circuit 120 and carried out of the microfluidic circuit 120. The medium module 160 can also control the flow of the fluid medium 180 in the flow region 106 / channel 122 inside the microfluidic circuit 120. For example, in some embodiments, the medium module 160 stops the flow of the medium 180 through the flow region 106 / channel 122 and through the enclosure 102 before loading the micro-objects or beads into the isolation pen (eg, gravity, Elect Wetting (EW) forces, Dielectrophoresis (DEP) forces, or a combination thereof).

The prime mover module 162 can be configured to control the selection, capture, and / or movement of droplets of microobjects and / or media within the microfluidic circuit 120. As detailed herein, the enclosure 102 includes an electrowetting configuration, such as an optical electrowetting (OEW), dielectric electrowetting (EWOD) configuration, or single-sided electrowetting configuration. Can be done. In certain embodiments, the enclosure 102 may further include a dielectrophoresis (DEP) configuration, such as a photoelectron tweezers (OET) configuration, an electrically actuated DEP configuration, and the like. The prime mover module 162 controls the activation of electrodes and / or transistors (eg, phototransistors) included in such EW and / or DEP configurations to control the flow region 106 / channel 122 and / or isolation pen 124, Droplets of microobjects and / or media within 126, 128, 130 can be selected and moved.

The image pickup module 164 can control the image pickup device 194 (not shown). For example, the image pickup module 164 can receive and process image data from the image pickup device 194. The image data from the imaging device 194 can include any type of information captured by the imaging device 194 (eg, the presence or absence of accumulation of labels such as microscopic objects, medium droplets, fluorescent labels, etc.). Using the information captured by the imaging device 194, the imaging module 164 determines the location of objects (eg, microobjects, droplets of medium, etc.) and / or the speed of movement of such objects within the microfluidic device 100. Further calculations can be made.

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 ratio and timing to optimize the transfer of small objects to one or more isolation pens, for example via gravity. The tilt module 166 is communicably coupled with the imaging module 164 to receive data describing the movement of microobjects and / or medium droplets in the microfluidic circuit 120. Using this data, the tilt module 166 can adjust the tilt of the microfluidic circuit 120 to adjust the rate at which microobjects and / or medium droplets move within the microfluidic circuit 120. The tilt module 166 can also use this data to iteratively adjust the position of microobjects and / or medium droplets within the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 is shown as including a single flow region 106 essentially consisting of the microfluidic channel 122. Each of the isolation pens 124, 126, 128, and 130 contains a single opening to the flow area 106 / channel 122, but the pen is a micro-object and / or other tiny object within the flow area 106 / channel 122 or other pen. Alternatively, the other parts are enclosed so that the small objects inside the pen can be substantially separated from the fluid medium 180. The wall of the isolation pen extends from the inner surface 109 of the base to the inner surface of the cover 110, thereby facilitating such separation. The opening of the pen into the flow region 106 / channel 122 can be tilted with respect to the flow of the fluid medium 180 in the flow region 106 / channel 122 so that the flow of the fluid medium 180 is not directed into the pen. .. The flow can be, for example, tangential or orthogonal to the plane of the opening of the pen. In some cases, the pens 124, 126, 128, and / or 130 are configured to physically enclose one or more microobjects within the microfluidic circuit 120. Isolation pens according to the invention are various shapes, surfaces, and optimized for use with EW, OEW, DEP, and / or OET forces, fluid flow, and / or gravity, as described in detail below. Features can be included.

The microfluidic circuit 120 may include any number of microfluidic isolation pens. Although five isolation pens are shown, the microfluidic circuit 120 may have a smaller number or more isolation pens. As shown, the microfluidic isolation pens 124, 126, 128, and 130 of the microfluidic circuit 120 may be one or more useful for manipulating droplets of microobjects and / or fluid media using the microfluidic device 100. Each contains different features and shapes that may provide the benefits of. Thus, in some embodiments, the microfluidic circuit 120 may include a plurality of microfluidic isolation pens, the two or more of the isolation pens comprising different structures and / or features that provide different advantages. However, in some embodiments, the microfluidic circuit 120 includes a plurality of identical microfluidic isolation pens. Manipulating droplets of micro-objects and / or media Useful microfluidic devices are shown in isolation pens 124, 126, 128, and 130, or FIGS. 2B, 2C, 2D, 2E, and 2F described below. It may include any of the variants of the pen, including the pen configured as such.

In the embodiment shown in FIG. 1A, a single flow region 106 is shown. However, other embodiments of the microfluidic device 100 may include multiple flow regions 106, each flow region 106 being configured to provide a separate path for fluid to flow through the microfluidic device 100. The microfluidic circuit 120 includes an inflow valve or port 107 that flows through the flow region 106 so that the fluid medium 180 can access the flow region 106 / channel 122 through the inflow port 107. In some cases, the flow region 106 comprises a single flow path. In other cases, the flow regions 106 include a plurality of channels (eg, 2, 3, 4, 5, 6, or more), and each flow region 106 is a microchannel (eg, number). , Such as channel 122). Two or more (eg, all) of the plurality of channels can be substantially parallel to each other. For example, the flow region 106 can be divided into a plurality of parallel channels (eg, channels 122, etc.). In certain embodiments, the flow regions 106 (and one or more channels contained within the flow regions) are arranged in a zigzag pattern, whereby the flow regions 106 alternate the microfluidic device 100 twice in alternating directions. Pass above. In some cases, the fluid medium in each flow region 106 flows in at least one of the forward and reverse directions. In some cases, the plurality of isolation pens are configured so that they can be placed in parallel with the target micro-object (eg, relative to flow region 106 / channel 122).

In some embodiments, the microfluidic circuit 120 further comprises one or more microobject traps 132. The trap 132 is generally formed on the wall forming the boundary of the flow region 106 / channel 122 and may be located opposite to one or more openings of the microfluidic isolation pens 124, 126, 128, and 130. In some embodiments, the trap 132 is configured to receive or capture a microobject from the flow region 106 / channel 122. In some embodiments, the trap 132 is configured to receive or capture a plurality of microobjects from the flow region 106 / channel 122. In some cases, the trap 132 contains a volume approximately equal to the volume of one target microobject.

The trap 132 may further include an opening configured to assist the flow of the target microobject to the trap 132. In some cases, the trap 132 includes an opening having a height and width corresponding to the dimensions of one target microobject, thereby making other microobjects (or larger microobjects) microscopic. Be prevented from entering object traps. The trap 132 may further include other features configured to assist in the retention of the target microobject within the trap 132. In some cases, the trap 132 is aligned with the opening of the microfluidic isolation pen and is located opposite the channel 122 with respect to the opening of the microfluidic isolation pen, thereby around an axis parallel to the channel 122. When the microfluidic device 100 is tilted, the captured micro-object exits the trap 132 in a trajectory that drops the micro-object into the opening of the isolation pen. In some cases, the trap 132 includes a side passage 134 that is smaller than the target micro-object and facilitates flow through the trap 132, thereby increasing the probability of capturing the micro-object into the trap 132.

As will be described in more detail below, in some embodiments, the electrowetting (EW) force is applied to the support structure 104 (and / /) of the microfluidic device 100 via one or more electrodes (not shown). Alternatively, the droplets placed in the microfluidic circuit 120 are manipulated, transported, separated, applied to one or more positions on the surface of the cover 110) (eg, in the flow area and / or isolation pen). And sort. For example, in some embodiments, an EW force is applied to one or more locations on the surface of the support structure 104 (and / or cover 110) to eject droplets from the flow region 106 into the desired microfluidics. Transfer to isolation pen. In some embodiments, EW forces are used to prevent droplets in the isolation pen (eg, isolation pen 124, 126, 128, or 130) from displacing from it. Further, in some embodiments, EW forces are used to selectively remove the previously collected droplets from the isolation pen according to the teachings of the present invention. In some embodiments, the EW force includes an optical electrowetting (OEW) force.

In some embodiments, dielectrophoretic (DEP) forces are applied over the fluid medium 180 via one or more electrodes (not shown) (eg, in the flow region and / or isolation pen) and internally. Manipulates, transports, separates, and sorts micro-objects placed in. For example, in some embodiments, the DEP force is applied within one or more parts of the microfluidic circuit 120 to transport one microobject from the flow region 106 to the desired microfluidic isolation pen. In some embodiments, a DEP force is used to prevent microscopic objects in the isolation pen (eg, isolation pen 124, 126, 128, or 130) from being displaced from the isolation pen. Further, in some embodiments, DEP forces are used to selectively remove microobjects previously collected according to the teachings of the present invention from the isolation pen. In some embodiments, the DEP force includes a photoelectron tweezers (OET) force.

In some embodiments, the DEP and / or EW forces are combined with other forces such as flow and / or gravity to manipulate, transport, and separate micro-objects and / or droplets within the microfluidic circuit 120. , And sort. For example, the enclosure 102 can be tilted (eg, by tilting device 190) to position micro-objects located within the flow region 106 / channel 122 and the flow region 106 onto the microfluidic isolation pen, and gravity , Micro-objects and / or droplets can be transported into the pen. In some embodiments, the DEP force and / or the EW force can be applied before other forces. In other embodiments, the DEP force and / or the EW force can be applied after the other force. In yet other cases, the DEP force and / or the EW force can be applied simultaneously with or alternately with the other forces.

The prime mover configuration of a microfluidic device. As mentioned above, the control and monitoring equipment of the system can include a prime mover module that selects and moves objects such as micro-objects or droplets in the microfluidic circuit of the microfluidic device. The microfluidic devices of the present invention can have different prime mover configurations depending on the type of object being moved and other considerations. In particular, the support structure 104 and / or cover 110 of the microfluidic device 100 selectively induces an electron wetting (EW) force on the droplets in the fluid medium 180 in the microfluidic circuit 120, thereby causing an electron wetting (EW) force. , Can include electronic wetting (EW) configurations that select, capture, and / or move individual droplets or groups of droplets. In certain embodiments, the microfluidic device of the present invention can include a first section having an EW configuration and a second section having a dielectrophoresis (DEP) configuration. Thus, at least one section of the support structure 104 and / or cover 110 of the microfluidic device 100 selectively induces a DEP force on a microobject in the fluid medium 180 in the microfluidic circuit 120, thereby individually. Can include a DEP configuration that selects, captures, and / or moves micro-objects or groups of micro-objects.

Electrowetting configuration. In certain embodiments, the microfluidic device of the invention can include an electrowetting configuration, the configuration comprising a substrate having a dielectric layer and a droplet working surface, the droplet working surface being a dielectric layer. Contains a covalently bonded hydrophobic layer. The dielectric layer can be placed under the hydrophobic layer so that the droplets placed on the substrate are in direct contact with the hydrophobic layer. FIG. 2A shows an example of some of such microfluidic devices.

As shown, the device 400 can include a base 104, which includes a substrate and at least one electrode (eg, a first electrode) 418. The substrate can include various layers, including a hydrophobic outer layer 412, a dielectric inner layer 414, a conductive layer 416, an electrode 418, and optionally a support 420. The hydrophobic layer 412 and the dielectric inner layer 414 can provide an inward surface of the substrate 102 that partially defines the enclosure.

The device 400 also includes a cover 110, which includes a hydrophobic outer layer 422, an inner layer 428 that may include at least one electrode, and an optional support 430. The cover 110 and the base 104 are substantially parallel to each other and are joined together by a separating element 108 (eg, a microfluidic circuit material) to define an enclosure 435 configured to hold a liquid medium. The liquid medium can be, for example, a hydrophobic liquid such as oil. In addition, the enclosure 435 can hold droplets of liquid 440, such as an aqueous medium. Generally, the liquid medium and the liquid in the droplets are selected to be immiscible liquids.

The separating element 108 can include a polymer. The polymers can be, for example, silicon-based organic polymers such as polydimethylsiloxane (PDMS) or photopatternable silicone (PPS), both available from Dow Corning. Alternatively, the separating element 108 can include an epoxy-based adhesive. The epoxy adhesive can be, for example, SU-8 or an equivalent type of material. The separating element 108 has a thickness of at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, or greater (ie, between the inner surface of the substrate 104 and the cover 110). Can have a gap). Thus, for example, the thickness of the separating element 108 is 30-60 μm, 40-80 μm, 50-100 μm, 60-120 μm, 70-140 μm, 75-150 μm, 80-160 μm, 90-180 μm, or 100-200 μm. obtain.

The separating element 108 can define one or more microfluidic channels within the enclosure. In addition, the separation element 108 can further define multiple chambers (or isolation pens) within the enclosure, each chamber being fluidly connected to at least one microfluidic channel and at least one microfluidic channel. Open at the end of. Thus, for example, the separating element 108 can define a single microfluidic channel and a plurality of chambers fluidly connected to it or a plurality of microfluidic channels fluidly connected to each of the plurality of chambers. In addition, each chamber can be fluidly connected to two or more microfluidic channels, as shown in FIGS. 6 and 7.

When at least one electrode 418 of the substrate 104 and at least one electrode 428 of the cover 110 are connected to opposite terminals of an AC voltage source (not shown), the substrate 104 is a hydrophobic outer surface 412 (ie, liquid) of the substrate 104. It is possible to apply electrowetting force to aqueous droplets in contact with the droplet working surface). In certain embodiments, the AC voltage used to achieve electrowetting-based migration of droplets within a microfluidic device is at least 20 volts peak-to-peak (ppV) (eg, about 20-80 ppV, about 20-80 ppV, about). 20-60 ppV, about 25-50 ppV, about 25-40 ppV, or about 25-35 ppV). In certain embodiments, the frequency of the AC voltage used to achieve electrowetting-based migration of droplets within a microfluidic device is about 1-100 kHz (eg, about 5 to 90 kHz, about 10 to 80 kHz). , About 15-70 kHz, about 20-60 kHz, about 25-50 kHz, or about 30-40 kHz).

The hydrophobic outer layer 412 of the substrate 104 and the hydrophobic outer layer 422 of the cover 110 can each include a high density monolayer of self-associating molecules covalently bonded to the dielectric inner layer 414 of the substrate 104 or the inner layer 428 of the cover 110, respectively. The monolayer self-associating molecule contains sufficient two-dimensional packing density and creates a hydrophobic barrier between the surface to which the monolayer is attached and the hydrophobic liquid (eg, polar molecules or other species). To avoid interaction and / or invasion into the monolayer). The packing density of the high density monolayer depends on the self-associating molecules used. High-density monolayers containing alkyl-terminated siloxanes typically contain at least 1 × 10 ¹⁴ molecules / cm ² (eg, at least 1.5 × 10 ¹⁴ molecules / cm ² , 2.0 × 10 ¹⁴ molecules / cm ² , 2. Contains 5 × 10 ¹⁴ molecules / cm ² or higher densities).

As will be described in more detail later, the self-associating molecule can contain a binding group such as a siloxane group or a phosphonic acid group, respectively. The siloxane group can be covalently bonded to the molecule of the dielectric inner layer 414 or inner layer 428. Similarly, the phosphonic acid group can be covalently bonded to the molecule of the dielectric inner layer 414 or inner layer 428. Self-associating molecules can include long chain hydrocarbons, which can be non-branched. Thus, the self-associating molecule can include an alkyl-terminated siloxane or an alkyl-terminated phosphonic acid. Long-chain hydrocarbons can include chains of at least 10 carbons (eg, at least 16, at least 18, at least 20, at least 22, or more carbons). The self-associating molecule can include a fluorinated carbon chain. Thus, for example, the self-associating molecule can include a fluoroalkyl-terminated siloxane or a fluoroalkyl-terminated phosphonic acid. The fluorinated carbon chain can have the formula CF _3- (CF ₂ ) m- (CH ₂ ) n-, where m is at least 2, n is 0, 1, 2, or it. And m + n is at least 9.

Single layers of self-associating molecules are less than about 5 nanometers (eg, about 1.0 to about 4.0 nm, about 1.5 to about 3.0 nm, or about 2.0 to about 2.5 nm). Can have

The hydrophobic outer layer 412 of the substrate 104 can be patterned such that the selected region is relatively hydrophilic relative to the rest of the hydrophobic outer layer. This can be achieved, for example, by increasing the voltage drop over the lower dielectric inner layer 122 to 50 ppV or higher (eg, 60 ppV, 65 ppV, 70 ppV, 75 ppV, 80 ppV, or higher) over a period of time. .. Unintentionally constrained by theory, the relatively hydrophilic regions are thought to contain water molecules inserted into the monolayer.

In some embodiments, the dielectric inner layer of the substrate can include one or more oxide layers. For example, the dielectric inner layer may include a single oxide layer such as a metal oxide layer, or may consist of a single oxide layer. Alternatively, the dielectric inner layer may include two layers or may consist of two layers. In some embodiments, the layer can be silicon dioxide or silicon nitride and the outer layer can be a metal oxide such as aluminum oxide. In certain embodiments, the thickness of the metal oxide layer can range from about 15 nm to about 45 nm, from about 30 nm to about 40 nm, or from about 33 nm to about 36 nm. Metal oxide layers can be deposited by atomic layer deposition (ALD) techniques, and layers containing silicon dioxide or silicon nitride can be deposited by plasma chemical vapor deposition (PECVD) techniques.

In yet another embodiment, the dielectric inner layer can include three dielectric material layers. In some embodiments, the first layer can contain a metal oxide such as aluminum oxide or hafnium oxide, and the first layer can be sandwiched between the silicon dioxide layer and the silicon nitride layer. .. In certain embodiments, the thickness of the metal oxide layer can range from about 5 nm to about 20 nm, and the layer can be deposited by atomic layer deposition (ALD) techniques. The 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 chemical vapor deposition (PECVD) techniques and can have a thickness of about 80 nm to about 100 nm or about 90 nm.

Regardless of the number of layers constituting the dielectric inner layer, the dielectric inner layer can have a thickness of about 50 to 105 nm and / or an impedance of about 50 to 150 kΩ, and in a preferred embodiment about 100 kΩ.

The substrate 104 can include a photoreactive layer 146 having a first surface in contact with the dielectric inner layer 414. The second surface of the photoreactive layer 416 can be in contact with at least one electrode 418. The photoreaction layer 416 can include hydrogenated amorphous silicon (a-Si: H). For example, a-Si: H can contain from about 8% to about 40% hydrogen (ie, ^{calculated as 100 *} number of hydrogen atoms / total number of hydrogen and silicon atoms). The a-Si: H layer can have a thickness of at least about 500 nm (eg, at least about 600 to 1400 nm, at least about 700 to 1300 nm, at least about 800 to 1200 nm, at least about 900 to 1100 nm, or about 1000 nm). .. However, the thickness of the a-Si: H layer is changed according to the thickness of the dielectric inner layer 414, and the substrate 104 is in the on state (that is, illuminated and conductive) and in the off state (that is, dark and not conductive). At some point, a suitable difference can be achieved between the impedance of the dielectric inner layer 414 and the impedance of the a-Si: H layer. For example, the impedance of the dielectric inner layer 414 can be adjusted to be about 50 kΩ to about 150 kΩ, and the impedance of the a-Si: H layer is at least about 0.5 MΩ in the off state and about 1 kΩ in the on state. It can be adjusted as follows. These are just examples, but exemplify how the impedance can be adjusted to achieve a photoresponsive (in this case, photoconducting) layer 416 that exhibits robust on / off performance. In an embodiment in which the substrate 104 has a photoresponsive layer 416 formed of an a-Si: H layer, the substrate 104 is optionally a floating electrode arranged between the photoresponsive layer 416 and the dielectric inner layer 414. Pads can be included. Such floating electrode pads are described, for example, in US Pat. No. 6,958,132, the contents of which are incorporated herein by reference.

The optical response layer 416 can optionally include a plurality of conductors, each of which can be controllably connected to at least one electrode of the substrate 102 via a phototransistor switch. Conductors controlled by phototransistor switches are well known in the art and are described in US Patent Application Publication No. 2014/01/24370, the content of which is incorporated herein by reference. ..

The substrate 104 can include a single electrode 418 configured to be connected to an AC voltage source. The single electrode 418 can include a layer of indium tin oxide (ITO), which layer can be formed, for example, on the glass support 420. Alternatively, the single electrode 418 can include a layer of conductive silicon. In another embodiment, the substrate 104 may include a plurality of individually addressable electrodes, such as EWOD devices well known in the art. The individually addressable electrodes may be connectable to one or more AC voltage sources via the corresponding transistor switches.

The cover 110 further includes a dielectric layer (not shown) attached to the hydrophobic layer 422 and a conductive layer (not shown) attached between the dielectric layer and the electrode 428, such as a substrate. Can be done. Thus, the microfluidic device 400 can have both a substrate 104 and a cover 110 configured to provide electrowetting forces to the aqueous droplets 440 disposed within the enclosure 435. In such an embodiment, the dielectric layer of the cover 110 can be configured by any method disclosed herein for the dielectric inner layer 414 of the substrate 104, and the conductive layer of the cover 104 is the conductive layer 126 of the substrate 102. Can be configured by any method disclosed herein.

Dielectrophoresis (DEP) configuration. As discussed herein, the microfluidic devices of the invention can include sections with a DEP configuration. An example of such a section is the microfluidic device 200 shown in FIGS. 1C and 1D. For brevity, FIGS. 1C and 1D show a vertical and horizontal cross section of a portion of the enclosure 102 of the microfluidic device 200 having an open region / chamber 202, respectively, where the region / chamber 202 is the growth chamber. , Isolation pens, flow regions, or parts of fluid circuit elements with more detailed structures such as flow channels. In addition, the microfluidic device 200 may include other fluid circuit elements. For example, the microfluidic device 200 can include multiple growth chambers, such as those described herein for the microfluidic device 100, or isolation pens and / or one or more flow regions or flow channels. The DEP configuration can be incorporated into any such fluid circuit element of the microfluidic device 200, or parts thereof can be selected. It should be further understood that any of the above or below microfluidic device and system components can be incorporated within the microfluidic device 200 and / or used in combination with the microfluidic device 200. For example, a system 150 including the above-mentioned control and monitoring device 152 including one or more of a medium module 160, a prime mover module 162, an imaging module 164, a tilt module 166, and another module 168 is used in combination with the microfluidic device 200. obtain.

As seen in FIG. 1C, the microfluidic device 200 includes a support structure 104 having an electrode activating substrate 206 overlapping the lower electrode 204 and the lower electrode 204, and a cover 110 having the upper electrode 210, the upper electrode 210. Is separated from the lower electrode 204. The upper electrode 210 and the electrode activating substrate 206 define both sides of the region / chamber 202. Therefore, the medium 180 contained in the region / chamber 202 provides a resistance connection between the upper electrode 210 and the electrode activating substrate 206. Also shown is a power supply 212 connected between the lower electrode 204 and the upper electrode 210 and configured to generate a bias voltage between the electrodes as needed for the generation of DEP force in the region / chamber 202. There is. The power supply 212 can be, for example, an alternating current (AC) power supply.

In certain embodiments, the microfluidic device 200 shown in FIGS. 1C and 1D can have an optically actuated DEP configuration. Therefore, the change pattern of the light 218 from the light source 216, which can be controlled by the prime mover module 162, can selectively activate or deactivate the change pattern of the DEP electrode in the region 214 of the inner surface 208 of the electrode activation substrate 206. can. (Hereinafter, the region 214 of the microfluidic device having the DEP configuration is referred to as a "DEP electrode region"). As shown in FIG. 1D, the light pattern 218 directed at the inner surface 208 of the electrode activating substrate 206 can illuminate the selected DEP electrode region 214a (shown in white) with a pattern such as a square. The unilluminated DEP electrode region 214 (shaded) is hereinafter referred to as the "dark" DEP electrode region 214. The relative electrical impedance through the DEP electrode activation substrate 206 (ie, from the lower electrode 204 to the inner surface 208 of the electrode activation substrate 206 interfaceing with the medium 180 in the flow region 106) is the region in each dark DEP electrode region 214. It is greater than the relative electrical impedance through the medium 180 in the / chamber 202 (ie, from the inner surface 208 of the electrode activating substrate 206 to the upper electrode 210 of the cover 110). However, the illuminated DEP electrode region 214a exhibits a reduction in relative impedance through the electrode activated substrate 206 that is less than the relative impedance through the medium 180 in the region / chamber 202 in each illuminated DEP electrode region 214a.

When the power supply 212 is activated, the DEP configuration creates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, and then the electric field gradient is: Generates a local DEP force that attracts or rejects nearby microscopic objects (not shown) in the fluid medium 180. Therefore, the DEP electrode that attracts or rejects microscopic objects in the fluid medium 180 often by altering the light pattern 218 projected from the light source 216 to the microfluidic device 200 on the inner surface 208 of the region / chamber 202. It can be selectively activated and deactivated in different such DEP electrode regions 214. Whether the DEP force attracts or rejects nearby micro-objects may depend on parameters such as the frequency of the power supply 212 and the dielectric properties of the medium 180 and / or micro-objects (not shown).

The square pattern 220 of the illuminated DEP electrode region 214a shown in FIG. 1C is merely an example. The light pattern 218 projected onto the device 200 can illuminate (and thereby activate) the DEP electrode region 214 of any pattern, and the illuminated / activated pattern of the DEP electrode region 214 is a light pattern. It can be changed repeatedly by changing or moving 218.

In some embodiments, the electrode activating substrate 206 comprises or can be made of a photoconductive material. In such an embodiment, the inner surface 208 of the electrode activated substrate 206 may have no features. For example, the electrode activating substrate 206 may include or consist of a layer of hydrogenated amorphous silicon (a—Si: H) or a layer of a—Si: H. a-Si: H can contain, for example, about 8% to 40% hydrogen (calculated as the number of hydrogen atoms / the total number of hydrogen and silicon atoms multiplied by 100). The layer a—Si: H can have a thickness of about 500 nm to about 2.0 μm. In such an embodiment, the DEP electrode region 214 can be created in any pattern at any location on the inner surface 208 of the electrode activation substrate 206 by the optical pattern 218. Therefore, the number and pattern of the DEP electrode regions 214 need not be fixed and can correspond to the optical pattern 218. An example of a microfluidic device having a DEP configuration including a photoconductive layer as described above is, for example, US Pat. No. RE44,711 (Wu et al.) (Originally issued as US Pat. No. 7,612,355). Incorporated herein by reference in its entirety.

In another embodiment, the electrode activating substrate 206 can include a substrate including a plurality of dope layers, insulating layers (or regions), and conductive layers that form semiconductor integrated circuits known in the semiconductor field. For example, the electrode activation substrate 206 can include, for example, a plurality of phototransistors including a horizontal bipolar phototransistor, and each phototransistor corresponds to a DEP electrode region 214. Alternatively, the electrode activation substrate 206 can include electrodes controlled by a phototransistor switch (eg, conductive metal electrodes), each such electrode corresponding to the DEP electrode region 214. The electrode activation substrate 206 can include such a patterned phototransistor or phototransistor-controlled electrode. The pattern can be a substantially square phototransistor or an array of phototransistor-controlled electrodes arranged in a matrix, for example as shown in FIG. 2B. Alternatively, the pattern can be a substantially hexagonal phototransistor forming a hexagonal lattice or an array of phototransistor-controlled electrodes. Regardless of the pattern, the electrical circuit element can form an electrical connection between the DEP electrode region 214 and the lower electrode 210 on the inner surface 208 of the electrode activating substrate 206 and their electrical connection (ie, phototransistor or The electrode) can be selectively activated or deactivated by the light pattern 218. If not activated, each electrical connection has a relative impedance through the electrode activating substrate 206 (ie, from the lower electrode 204 to the inner surface 208 of the electrode activating electrode 206 interfaceing with the medium 180 in the region / chamber 202). It can have a high impedance that is greater than the relative impedance through the medium 180 in the corresponding DEP electrode region 214 (ie, from the inner surface 208 of the electrode activating substrate 206 to the upper electrode 210 of the cover 110). However, when activated by light within the light pattern 218, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 in each illuminated DEP electrode region 214, thereby as described above. In addition, it activates the DEP electrode in the corresponding DEP electrode region 214. Therefore, many DEP electrodes that attract or reject microscopic objects (not shown) in medium 180 are on the inner surface 208 of the electrode activation substrate 206 in the region / chamber 202, as determined by the light pattern 218. It can be selectively activated and deactivated in different DEP electrode regions 214.

An example of a microfluidic device having an electrode activated substrate containing a phototransistor is described, for example, in US Pat. No. 7,965,339 (Ohta et al.) (Eg, device 300 shown in FIGS. 21 and 22). Also see its description), the entire contents of which are incorporated herein by reference. An example of a microfluidic device having an electrode activating substrate comprising an electrode controlled by a phototransistor switch is described, for example, in US Patent Application Publication No. 2014/0124370 (Short et al.) (Eg, shown throughout the drawings. Devices 200, 400, 500, 600, and 900 and their descriptions), the entire contents of which are incorporated herein by reference.

In some embodiments of the DEP configuration microfluidic device, the upper electrode 210 is part of the first wall (or cover 110) of the enclosure 102, and the electrode activation substrate 206 and the lower electrode 204 are of the enclosure 102. It is part of the second wall (or support structure 104). The area / chamber 202 can be between the first wall and the second wall. In other embodiments, the electrode 210 is part of a second wall (or support structure 104) and one or both of the electrode activating substrate 206 and / or the electrode 210 is the first wall (or cover 110). ) Is part of. Further, the light source 216 can be used as an alternative to illuminate the enclosure 102 from below.

Using the microfluidic device 200 of FIGS. 1C and 1D having a DEP configuration, the prime mover module 162 projects an optical pattern 218 onto the device 200 and electrodes in a pattern (eg, a square pattern 220) that surrounds and captures a small object. Microobjects in medium 180 in region / chamber 202 by activating one or more pairs of DEP electrodes in the DEP electrode region 214a of the inner surface 208 of the activation substrate 206 (not shown). Can be selected. The prime mover module 162 was then captured by moving the light pattern 218 relative to the device 200 to activate one or more of the second set of DEP electrodes in the DEP electrode region 214. Small objects can be moved. Alternatively, the device 200 can be moved relative to the light pattern 218.

In another embodiment, the microfluidic device 200 can have a DEP configuration that is independent of photoactivation of the DEP electrode on the inner surface 208 of the electrode activation substrate 206. For example, the electrode activating substrate 206 can include selectively addressable and energizable electrodes located opposite the surface containing at least one electrode (eg, cover 110). A switch (eg, a transistor switch on a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode in the DEP electrode region 214, thereby activating or deactivating the region / region in the vicinity of the activated DEP electrode. Generates a net DEP force on a small object (not shown) in the chamber 202. Depending on features such as the frequency of the power supply 212 and the medium (not shown) and / or the dielectric properties of the micro-objects in the region / chamber 202, the DEP force can attract or reject nearby micro-objects. .. By selectively activating or deactivating a set of DEP electrodes (eg, in a set of DEP electrode regions 214 forming a square pattern 220), one or more microobjects in the region / chamber 202 are captured. , Can be moved within the area / room 202. The prime mover module 162 of FIG. 1A controls such a switch, thus activating and deactivating the individual electrodes of the DEP electrode to deactivate certain micro-objects (not shown) around the region / chamber 202. It can be selected, captured, and moved. Microfluidic devices having a DEP configuration that includes selectively addressable and energizable electrodes are known in the art and are described, for example, in US Pat. Nos. 6,294,063 (Becker et al.) And 6. , 942, 776 (Medoro), the entire contents of which are incorporated herein by reference.

A microfluidic device having an electrowetting configuration and a dielectrophoresis (DEP) configuration. FIG. 4 is a vertical cross-sectional view of a microfluidic device or device 450 that integrates a plurality of microfluidic applications according to various embodiments. The device 450 comprises two different sections (although there may be three or more different sections), each section having a single microfluidic configuration. Section 460 includes an electrowetting configuration, which includes a base 104 that includes a substrate. The substrate includes various layers, including a hydrophobic outer layer 412, a dielectric inner layer 414, a conductive layer 416, and an electrode 418. The hydrophobic layer 412 and the dielectric inner layer 414 can provide an inward surface of the substrate that partially defines the enclosure 435. Section 460 also includes a cover 110 containing an electrode 428 and a hydrophobic outer layer 422 as well as a microfluidic circuit material 108 so that the microfluidic circuit material 108 connects the base 104 to the cover 110 to hold an immiscible fluid. It further helps define the microfluidic circuit of the electrowetting section, including the configured enclosure 435.

Section 470 of the microfluidic device 450 includes a dielectrophoretic DEP configuration, which includes an inward surface that partially defines a base 104, a first electrode 479, an electrode activation substrate 474, and an enclosure 475. Section 470 further includes a cover 110 that includes electrodes 468 and a microfluidic circuit material 108 that connects the base 104 to the cover 110 and further aids in defining the macrofluidic circuit of the DEP section.

As shown in FIG. 4, the electrowetting section 460 and the DEP section 470 can share the same base 104 and cover 110, while the substrate and electrodes are not. The electrowetting section 460 and DEP section 470 of the device 450 can be joined by a bridge 465, which can be a tube, an adhesive material, etc., or any combination thereof.

FIG. 5 is a vertical cross-sectional view of a microfluidic device or device 500 that integrates a plurality of microfluidic applications according to various embodiments. Like device 400, device 500 includes two different sections (but there may be three or more different sections), each section having a single microfluidic configuration. In particular, section 460 includes an electrowetting configuration and section 470 includes a DEP configuration. The various components of the device 500 have components that correspond to the components in the device 400, as indicated by the corresponding reference numerals. However, the device 500 has a monolithic substrate having a conductive layer 416, a first electrode 418, and a second electrode 428, all shared by both sections 460 and 470.

19A and 19B provide diagrams of electrically coping motion representations in one functional aspect according to the embodiments shown in conjunction with FIG. As previously described with FIG. 5, the system integrates two microfluidic operations, as shown by the DEP module and the EW module sharing the monolithic substrate 416. In this embodiment, the DEP (which can be OET) module has a lower impedance than the EW module. During operation, the impedance of the EW module overcomes the impedance of the DEP module and basically shorts the DEP module.

In one embodiment shown in FIG. 19A, the OEP module operates by applying a voltage in the range of 1 volt to 10 volt at frequencies in the range of 100 kHz to 10 MHz. In the same embodiment, as shown in FIG. 19B, the OEW module operates by applying an operation in the range of 10 to 100 volts at a frequency in the range of 1 kHz to 300 kHz. In a preferred embodiment, the OEP module operates by applying a voltage of 5 volts at a frequency of 1 Mhz and the OEW module operates by applying a voltage of 30 volts at a frequency of 30 kHz.

Isolation pen. Non-limiting examples of common isolation pens 224, 226, and 228 are shown within the microfluidic device 230 shown in FIGS. 2A-2C. Each isolation pen 224, 226, and 228 can include a separation structure 232 that defines a separation region 240 and a connection region 236 that fluidly connects the separation region 240 to the channel 122. Contiguous zone 236 can include a proximal opening 234 to the channel 122 and a distal opening 238 to the separation zone 240. Contiguous zone 236 may be configured such that the maximum penetration depth of the flow of fluid medium (not shown) flowing from channel 122 into isolation pens 224, 226, 228 does not reach into isolation zone 240. Thus, due to the contiguous zone 236, micro-objects (not shown) or other materials (not shown) placed within the isolation zone 240 of the isolation pens 224, 226, 228 are medium 180 in channel 122. Can be separated from the flow of and substantially unaffected by the flow of medium 180 in channel 122.

Isolation pens 224, 226, and 228 of FIGS. 2A-2C each have a single opening that opens directly to channel 122. The opening of the isolation pen opens laterally from channel 122. The electrode activation substrate 206 is under both the channel 122 and the isolation pens 224, 226, and 228. The top surface of the electrode activation substrate 206 in the enclosure of the isolation pen, which forms the floor of the isolation pen, forms the floor of the flow channel (or flow region, respectively) of the microfluidic device, channel 122 (or if the channel is absent). , Flow region) and at the same height as or substantially the same height as the upper surface of the electrode activation substrate 206. The electrode activated substrate 206 may be featureless or less than about 3 μm, less than about 2.5 μm, less than about 2 μm, less than about 1.5 μm, less than about 1 μm, less than about 0.9 μm, about 0. It may have an irregular or patterned surface that varies from less than 5 μm, less than about 0.4 μm, less than about 0.2 μm, less than about 0.1 μm, or less, from the highest ridge to the lowest depression. Fluctuations in the elevation of the top surface of the substrate across both the channel 122 (not shown) and the isolation pen are less than about 3%, less than about 2%, about the height of the wall of the isolation pen or the wall of the microfluidic device. It can be less than 1%, less than about 0.9%, less than about 0.8%, less than about 0.5%, less than about 0.3%, or less than about 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, 700 described herein.

Thus, channel 122 can be an example of a sweep area, and isolation area 240 of isolation pens 224, 226, 228 can be an example of a non-sweep area. As mentioned, channels 122 and isolation pens 224, 226, 228 may be configured to include one or more fluid media 180. In the example shown in FIGS. 2A-2B, port 222 can be connected to channel 122 to allow fluid medium 180 to be introduced into or out of microfluidic device 230. Prior to introducing the fluid medium 180, the microfluidic device can be primed with a gas such as carbon dioxide gas. When the microfluidic device 230 includes the fluid medium 180, the flow 242 of the fluid medium 180 in the channel 122 can be selectively generated and stopped. For example, as shown, port 222 can be located at different locations on channel 122 (eg, at both ends) and of medium from one port 222 acting as an inlet to another port 222 acting as an outlet. Flow 242 can be generated.

FIG. 2C shows a detailed view of an example of the isolation pen 224 according to the present invention. An example of a micro object 246 is also shown.

As is known, the flow 242 of the fluid medium 180 in the microfluidic channel 122 beyond the proximal opening 234 of the isolation pen 224 results in a secondary flow 244 of the medium 180 in and / or out of the isolation pen 224. Can be made to. _{Length L con} of connection area 236 of isolation pen 224 (ie, from proximal opening 234 to tip opening 238) to separate microobjects 246 in isolation area 240 of isolation pen 224 from secondary flow 244. Should be greater than _{the penetration depth D p} of the secondary flow 244 into the contiguous zone 236. The depth _{D p} penetration of the secondary flow 244 will depend on various parameters associated with the configuration of the proximal end opening portion 234 of the connection region 236 to the speed and the channel 122 and channel 122 of the fluid medium 180 flowing through the channel 122 .. In a given microfluidic device, the configuration of the channel 122 and the opening 234 is fixed, while the velocity of the flow 242 of the fluid medium 180 in the channel 122 is variable. Therefore, for each isolation pen 224, the maximum velocity Vmax of the flow 242 of the fluid medium 180 in the channel 122 is identified, which ensures that _{the penetration depth D p} of the secondary flow 244 does not exceed the length L _{con of the contiguous zone 236.} can do. As long as the flow rate of the flow 242 of the fluid medium 180 in the channel 122 does not exceed the maximum velocity Vmax, the resulting secondary flow 244 to the channel 122 and the contiguous zone 236 can be restricted to the separation zone 240. You can prevent it from entering. Therefore, the flow 242 of the medium 180 in the channel 122 does not draw the micro-object 246 out of the separation region 240. Rather, the microobjects 246 disposed within the separation region 240 remain within the separation region 240 regardless of the flow 242 of the fluid medium 180 within the channel 122.

Further, as long as the flow rate of the flow 242 of the medium 180 in the channel 122 does not exceed Vmax, the flow 242 of the fluid medium 180 in the channel 122 isolates various particles (eg, microparticles and / or nanoparticles) from the channel 122. Do not move the pen 224 into the separation area 240. Therefore, by making the length L _con of the connection area 236 greater than the maximum penetration depth D _{p of the} secondary flow 244, the isolation pen of one isolation pen 224, channel 122 or another isolation pen (eg, the isolation pen of FIG. 2D). Contamination by various particles from 226, 228) can be avoided.

The channel 122 and the contiguous zone 236 are swept (or swept) of the microfluidic device 230 because the contiguous zone 236 of the channels 122 and the isolation pens 224, 226, 228 can be affected by the flow 242 of the medium 180 in the channel 122. It can be regarded as a flow) region. On the other hand, the isolation region 240 of the isolation pens 224, 226, 228 can be considered as a non-sweep (or non-flow) region. For example, the components (not shown) in the first fluid medium 180 in the channel 122 are substantially the first from the channel 122 to the second fluid medium 248 in the separation zone 240 through the contiguous zone 236. Only by diffusion of the components of medium 180 can it be mixed with the second fluid medium 248 in the separation zone 240. Similarly, a component (not shown) of the second medium 248 within the separation zone 240 substantially passes from the separation zone 240 through the contiguous zone 236 to a second medium 180 within the channel 122. Only by diffusion of the components of medium 248 can it be mixed with the first medium 180 in channel 122. In some embodiments, the degree of fluid medium exchange between the separation and flow regions of the isolation pen by diffusion is about 90%, about 91%, about 92%, about 93%, about 94 of the fluid exchange. %, About 95%, about 96%, about 97%, about 98%, or higher than about 99%. The first medium 180 may be the same medium as the second medium 248, or may be a different medium. In addition, the first medium 180 and the second medium 248 can start as the same medium and be different (eg, flow by one or more cells within the isolation region 240 or through channel 122). (Through adjusting the second medium 248 by modifying the medium 180).

Maximum penetration depth _{D p} of the secondary flow 244 resulting from the flow 242 of the fluid medium 180 in the channel 122, as described above, may depend on several parameters. Examples of such parameters are the shape of the channel 122 (eg, the channel can direct the medium to the connecting region 236 and divert the medium from the connecting region 236, or the connecting region 236 to the channel 122. The medium can be directed in a direction substantially orthogonal to the proximal opening 234), the width _Wch (or cross-sectional area) of the channel 122 at the proximal opening 234 and the connecting area 236 at the proximal opening 234. Width W _con (or cross-sectional area), velocity V of flow 242 of fluid medium 180 in channel 122, viscosity of first medium 180 and / or second medium 248, and the like.

In some embodiments, the dimensions of channel 122 and isolation pens 224, 226, 228 can be oriented as follows with respect to the vector of flow 242 of fluid medium 180 within channel 122: channel width W _ch (channel width W ch ( Or the cross-sectional area of the channel 122) can be approximately orthogonal to the flow 242 of the medium 180, and the width W _con (or cross-sectional area) of the connecting region 236 at the opening 234 is the flow 242 of the medium 180 in the channel 122. And / or the length of the connecting area L _con can be approximately orthogonal to the flow 242 of medium 180 in channel 122. The above is merely an example, and the relative positions of the channels 122 and the isolation pens 224, 226, 228 may be in other orientations with respect to each other.

As shown in FIG. 2C, the width W _con of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. _{Thus, the width W con} of the connection region 236 at the tip opening 238 can be within any range identified herein for _{the width W con} of the connection region 236 at the proximal opening 234. _{Alternatively, the width W con} of the connection region 236 at the tip opening 238 can be greater than _{the width W con} of the connection region 236 at the proximal opening 234.

As shown in FIG. 2C, the width of the separation region 240 at the tip opening 238 can be substantially the same as _{the width W con of the proximal region 236 at the proximal opening 234.} Thus, the width of the separation zone 240 at the tip opening 238 can be within any range identified herein for _{the width W con of the connection region 236 at the proximal opening 234.} Alternatively, the width of the separation zone 240 at the tip opening 238 may be greater than or smaller than _{the width W con of the connection region 236 at the proximal opening 234.} Further, the tip opening 238 may be smaller than the proximal opening 234, and the width W _con of the connection region 236 may be narrowed between the proximal opening 234 and the distal opening 238. For example, the contiguous zone 236 can be narrowed between the proximal and distal openings using a variety of different geometries, such as chamfering the contiguous zone and grading the contiguous zone. Further, any portion or sub portion of the connection region 236 may be narrowed (eg, portion of the connection region adjacent to the proximal opening 234).

2D-2F show another exemplary embodiment of the microfluidic device 250 including the microfluidic circuit 262 and the flow channel 264, which are variants of each microfluidic device 100, circuit 132, and channel 134 of FIG. show. The microfluidic device 250 also has a plurality of isolation pens 266, which are additional variants of the isolation pens 124, 126, 128, 130, 224, 226, or 228 described above. In particular, the isolation pens 266 of device 250 shown in FIGS. 2D-2F are the isolation pens 124, 126, 128, 130, 224, 226, or 228 described above in devices 100, 200, 230, 280, 290, or 320. Please understand that it can be replaced by any of. Similarly, the microfluidic device 250 is another variant of the microfluidic device 100 and described herein with the same or different DEP configurations as the microfluidic devices 100, 200, 230, 280, 290, 320 described above. It can also have any other microfluidic system component.

The microfluidic device 250 of FIGS. 2D-2F is a support structure (not visible in FIGS. 2D-2F, but may be the same as or generally similar to the support structure 104 of device 100 shown in FIG. 1A), microfluidics. Includes a circuit structure 256 and a cover (not visible in FIGS. 2F-2F, but may be the same as or generally similar to the cover 122 of 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 generally similar to the frame 114 and microfluidic circuit material 116 of device 100 shown in FIG. 1A. As shown in FIG. 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 can include multiple channels 264 (two are shown, but there can be more channels), with channel 264 being A plurality of isolation pens 266 are fluidly connected.

Each isolation pen 266 can include a separation structure 272, a separation area 270 within the separation structure 272, and a connection area 268. From the proximal opening 274 of the channel 264 to the distal opening 276 in the separation structure 272, the contiguous zone 268 fluidly connects the channel 264 to the separation zone 270. In general, according to the above considerations of FIGS. 2B and 2C, the flow 278 of the first fluid medium 254 in channel 264 is the first in and / or out of each connection region 268 of the isolation pen 266 from channel 264. A secondary flow 282 of medium 254 can be provided.

As shown in FIG. 2E, the connection area 268 of each isolation pen 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. The length L _con of the contiguous zone 268 can be greater than the maximum penetration depth D _{p of the} secondary flow 282, in which case the secondary flow 282 is not redirected towards the demarcation zone 270 and is in the contiguous zone. Extends within 268 (as shown in FIG. 2D). Alternatively, as shown in FIG. 2F, the connection area 268 _{can have a length L con} that is less than _{the maximum penetration depth D p} , in which case the secondary flow 282 passes through the connection area 268. Extends and redirects towards the demarcation zone 270. In this latter situation, _{the sum of the lengths L c1} and L _c2 of the contiguous zone 268 is greater than the maximum penetration depth D _p , so the secondary flow 282 does not extend into the demarcation zone 270. _{Regardless of whether the length L con} of the connection area 268 is greater than the penetration depth D _{p or whether the sum of the lengths L c1} and L _c2 of the connection area 268 is greater than the penetration depth D _p. Flow 278 of the first medium 254 in channel 264 not exceeding the maximum velocity V _{max results in a secondary flow with penetration depth D p} and micro-objects within the separation region 270 of the isolation pen 266 (not shown). However, it may be the same as or generally similar to the micro-object 246 shown in FIG. 2C) is not pulled out of the separation region 270 by the flow 278 of the first medium 254 in channel 264. The flow 278 in the channel 264 also does not draw various materials (not shown) from the channel 264 into the isolation region 270 of the isolation pen 266. Therefore, the only mechanism by which the components in the first medium 254 in the channel 264 can be moved from the channel 264 into the second medium 258 in the separation region 270 of the isolation pen 266 is diffusion. Similarly, diffusion is also the only mechanism by which the components in the second medium 258 in the separation region 270 of the isolation pen 266 can be moved from the separation region 270 into the first medium 254 in the channel 264. The first medium 254 can be the same medium as the second medium 258, or the first medium 254 can be a different medium than the second medium 258. Alternatively, the first medium 254 and the second medium 258 start from the same medium and, for example, by changing the medium flowing by one or more cells within the isolation region 270 or through channel 264. It can be different through adjusting the second medium.

As shown in FIG. 2E, the width W _{ch (i.e.,} taken across the direction of fluid medium flow through the channel indicated by the arrow 278 in FIG. 2D) of the channel 264 in the channel 264, proximal end opening portion It can be approximately orthogonal to the width W _con1 of 274 and therefore can be substantially parallel to _{the width W con 2 of the tip opening 276. However, the width con1 of} the base end opening 274 and the width W _con2 of the tip end opening 276 do not have to be substantially orthogonal to each other. For example, a shaft directed width W _con1 the proximal end opening portion 274 (not shown), the angle between the another axis directed width W _con2 the distal opening 276 can be a non-orthogonal, thus , Can be other than 90 °. Examples of alternatively directed angles include angles within any of the following ranges: about 30 ° to about 90 °, about 45 ° to about 90 °, about 60 ° to about 90 °, and the like.

In various embodiments of the isolation pen (eg, 124, 126, 128, 130, 224, 226, 228, or 266), the separation region (eg, 240 or 270) is configured to include a plurality of microobjects. NS. In other embodiments, the separation region may be configured to include only one, two, three, four, five, or a similar relatively small number of microobjects. Thus, the volume of the separation region is, for example, at least 1 × 10 ⁶ cubic μm, at least 2 × 10 ⁶ cubic μm, at least 4 × 10 ⁶ cubic μm, at least 6 × 10 ⁶ cubic μm, or greater. obtain.

In various embodiments of the isolation pen width _{W ch} of the channel at the proximal end opening portion (e.g., 234) (e.g., 122) may be in any range of: about 50 to 1000 [mu] m, from about 50 500 μm, about 50-400 μm, about 50-300 μm, about 50-250 μm, about 50-200 μm, about 50-150 μm, about 50-100 μm, about 70-500 μm, about 70-400 μm, about 70-300 μm, about 70- 250 μm, about 70-200 μm, about 70-150 μm, about 90-400 μm, 90-300 μm, about 90-250 μm, about 90-200 μm, about 90-150 μm, about 100-300 μm, about 100-250 μm, about 100-200 μm , About 100-150 μm, and about 100-120 μm. In some other embodiments, the width _{W ch} of the channel at the proximal end opening portion (e.g., 234) (e.g., 122) is about 200～800Myuemu, range from about 200～700Myuemu, or about 200~600μm obtain. The above is merely an example, and the width _{Wch of the} channel 122 may be in another range (eg, the range defined by any of the endpoints listed above). Further, the width _{Wch of the} channel 122 can be selected to be in any of these ranges in the region of the channel other than the proximal opening of the isolation pen.

In some embodiments, the isolation pen has a height of about 30 to about 200 μm or about 50 to about 150 μm. In some embodiments, the isolation pen is about 1x10 ⁴ to about ^{3x10 6} square μm, about 2x10 ⁴ to about ^{2x10 6} square μm, about 4x10 ⁴ to about 1x10 ⁶ It has a cross-sectional area of about 2 × 10 ⁴ to about 5 × 10 ⁵ square μm, about 2 × 10 ⁴ to about 1 × 10 ⁵ square μm, or about 2 × 10 ⁵ to about 2 × 10 ^{6 square μm.} .. In some embodiments, the contiguous zone has a cross-sectional width of about 100-about 500 μm, 200-about 400 μm, or about 200-about 300 μm.

In various embodiments of the isolation pen, proximal end opening portion (e.g., 234) channels in (e.g., 122) the height _{H ch} of, may be within any range below: 20~100μm, 20~90μm, 20-80 μm, 20-70 μm, 20-60 μm, 20-50 μm, 30-100 μm, 30-90 μm, 30-80 μm, 30-70 μm, 30-60 μm, 30-50 μm, 40-100 μm, 40-90 μm, 40- 80 μm, 40-70 μm, 40-60 μm, or 40-50 μm. The above is only an example, the channel (e.g., 122) the height H _ch of the other ranges (e.g., a range defined by any endpoint that is listed above) may be used. The height H _ch of the channel 122, in the region of the channel other than the proximal end opening portion of the isolation pen, can be selected to be any of these ranges.

In various embodiments of the isolation pen, the cross-sectional area of the channel (eg, 122) at the proximal opening (eg, 234) can be within any of the following ranges: 500-50,000 square μm, 500- 40,000 square μm, 500 to 30,000 square μm, 500 to 25,000 square μm, 500 to 20,000 square μm, 500 to 15,000 square μm, 500 to 10,000 square μm, 500 to 7, 500 square μm, 500 to 5,000 square μm, 1,000 to 25,000 square μm, 1,000 to 20,000 square μm, 1,000 to 15,000 square μm, 1,000 to 10,000 square μm, 1,000 to 7,500 square μm, 1,000 to 5,000 square μm, 2,000 to 20,000 square μm, 2,000 to 15,000 square μm, 2,000 to 10,000 square μm, 2,000 to 7,500 square μm, 2,000 to 6,000 square μm, 3,000 to 20,000 square μm, 3,000 to 15,000 square μm, 3,000 to 10,000 square μm, 3,000 to 7,500 square μm, or 3,000 to 6,000 square μm. The above is merely an example, and the cross-sectional area of the channel (eg, 122) at the proximal opening (eg, 234) is in another range (eg, the range defined by any of the endpoints listed above). May be good.

In various embodiments of the isolation pen, the length L _con of the contiguous zone (eg, 236) can be in any of the following ranges: about 1-600 μm, 5-550 μm, 10-500 μm, 15-400 μm, 20-300 μm, 20-500 μm, 40-400 μm, 60-300 μm, 80-200 μm, or about 100-150 μm. The above is merely an example, and the length L _con of the continental zone (eg, 236) can be within a range different from that of the above example (eg, the range defined by any of the listed endpoints above).

_{In various embodiments of the isolation pen, the width W con} of the contiguous zone (eg, 236) at the proximal opening (eg, 234) can be within any of the following ranges: 20-500 μm, 20-400 μm. 20, 300 μm, 20 to 200 μm, 20 to 150 μm, 20 to 100 μm, 20 to 80 μm, 20 to 60 μm, 30 to 400 μm, 30 to 300 μm, 30 to 200 μm, 30 to 150 μm, 30 to 100 μm, 30 to 80 μm, 30 -60 μm, 40-300 μm, 40-200 μm, 40-150 μm, 40-100 μm, 40-80 μm, 40-60 μm, 50-250 μm, 50-200 μm, 50-150 μm, 50-100 μm, 50-80 μm, 60-200 μm , 60-100 μm, 60-100 μm, 60-80 μm, 70-150 μm, 70-100 μm, and 80-100 μm. _{The above is merely an example, and the width W con} of the contiguous zone (eg, 236) of the proximal opening (eg, 234) can be different from the example above (eg, defined by any of the endpoints listed above). Range).

_{In various embodiments of the isolation pen, the width W con} of the connection area (eg, 236) at the proximal opening (eg, 234) is such that the micro-object (eg, T cells, B cells, eggs) for which the isolation pen is intended. , Or a living cell that can be an embryo), which can be at least as large as the maximum size. _{For example, the width W con} of the connection area 236 at the proximal opening 234 of the isolation pen on which the droplets are placed can be in any of the following ranges: about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, About 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm, about 225 μm, about 250 μm, about 300 μm, or about 100-400 μm, about 120-350 μm, about 140-200-200 300 μm, or about 140- 200 μm. _{The above is merely an example, and the width W con} of the contiguous zone (eg, 236) at the proximal opening (eg, 234) may be different from the example above (eg, defined by any of the endpoints listed above). Range).

In various embodiments of the isolation pen, the width _Wpr of the proximal opening of the connection area is at least as large as the maximum size of the micro-object intended for the isolation pen (eg, a biological micro-object such as a cell). Can be. For example, the width W _pr is about 50 μm, about 60 μm, about 100 μm, about 200 μm, about 300 μm, or about 50-300 μm, about 50-200 μm, about 50-100 μm, about 75-150 μm, about 75-100 μm, or about. It can be in the range of 200-300 μm.

In various embodiments of the isolation pen, _{the ratio of the length L con of the connection area (eg, 236) to the width W con} of the connection area (eg, 236) at the proximal opening 234 is greater than or equal to any of the following ratios: Can be: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7. A ratio of 0, 8.0, 9.0, 10.0, or more. The above is merely an example, _{and the ratio of the length L con of the connection region 236 to the width W con} of the connection region 236 at the proximal opening 234 may differ from the above example.

In various embodiments of the microfluidic device 100, 200, _{230, 250, 280, 290, 320, 600, 700, the V max} is about 0.2 μL / sec, about 0.3 μL / sec, about 0.4 μL / sec. Seconds, about 0.5 μL / sec, about 0.6 μL / sec, about 0.7 μL / sec, about 0.8 μL / sec, about 0.9 μL / sec, about 1.0 μL / sec, about 1.1 μL / sec , About 1.2 μL / sec, about 1.3 μL / sec, about 1.4 μL / sec, or about 1.5 μL / sec.

In various embodiments of a microfluidic device having an isolation pen, the volume of the isolation region (eg, 240) of the isolation pen is, for example, at least 5 × 10 ⁵ cubic μm, at least 8 × 10 ⁵ cubic μm, at least 1 × 10 ⁶ cubic μm, at least 2 × 10 ⁶ cubic μm, at least 4 × 10 ⁶ cubic μm, at least 6 × 10 ⁶ cubic μm, at least 8 × 10 ⁶ cubic μm, at least 1 × 10 ⁷ cubic μm, at least 5 × 10 ⁷ cubic The volume can be μm, at least 1 × 10 ⁸ cubic μm, at least 5 × 10 ⁸ cubic μm, at least 8 × 10 ⁸ cubic μm, or more. In various embodiments of a microfluidic device having an isolation pen, the volume of the isolation pen is about 5 × 10 ⁵ cubic μm, about 6 × 10 ⁵ cubic μm, about 8 × 10 ⁵ cubic μm, about 1 × 10 ⁶ cubic. μm, about 2 × 10 ⁶ cubic μm, about 4 × 10 ⁶ cubic μm, about 8 × 10 ⁶ cubic μm, about 1 × 10 ⁷ cubic μm, about 3 × 10 ⁷ cubic μm, about 5 × 10 ⁷ cubic μm, Alternatively, it can have a volume of about 8 × 10 ^{7 cubic μm or more.} In some other embodiments, the volume of the isolation pen is from about 1 nanoliter to about 50 nanolitre, 2 nanoliters to about 25 nanolitre, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 It can be nanoliters, or about 2 nanoliters to about 10 nanoliters.

In various embodiments, the microfluidic device has an isolation pen configured as in any of the embodiments described herein, in which case the microfluidic device has about 5 to about 10 pieces. Isolation pens, about 10 to about 50 isolation pens, about 100 to about 500 isolation pens, about 200 to about 1000 isolation pens, about 500 to about 1500 isolation pens, about 1000 to about 2000 It has a quarantine pen, or about 1000 to about 3500 quarantine pens. The isolation pens do not have to be all the same size and may include different configurations (eg, different widths, different features within the isolation pen).

In some other embodiments, the microfluidic device is such that the microfluidic device has about 1500 to about 3000 isolation pens, about 2000 to about 3500 isolation pens, about 2500 to about 4000 isolation pens, about 3000. ~ Approximately 4500 quarantine pens, Approximately 3500 to Approximately 5000 Quarantine pens, Approximately 4000 to Approximately 5500 Quarantine pens, Approximately 4000 to Approximately 6000 Quarantine pens, Approximately 5000 to Approximately 6500 Quarantine pens, Approximately 5500 ~ About 7,000 quarantine pens, about 6000 ~ about 7500 quarantine pens, about 6500 ~ about 8000 quarantine pens, about 7000 ~ about 8500 quarantine pens, about 7500 ~ about 9000 quarantine pens, about 8000 ~ About 9,500 isolation pens, about 8500 ~ about 10,000 isolation pens, about 9000 ~ about 10,500 isolation pens, about 9,500 ~ about 11,000 isolation pens, about 10,000 ~ about 11,500 isolation pens, about 10,500 to about 12,000 isolation pens, about 11,000 to about 12,500 isolation pens, about 11,500 to about 13,000 isolation pens, About 12,000 to about 13,000 isolation pens, about 12,500 to about 14,000 isolation pens, about 13,000 to about 14,500 isolation pens, about 13,500 to about 15, 000 isolation pens, about 14,000 to about 15,500 isolation pens, about 14,500 to about 16,000 isolation pens, about 15,000 to about 16,500 isolation pens, about 15 , 500 to about 17,000 isolation pens, about 16,000 to about 17,500 isolation pens, about 16,000 to about 18,000 isolation pens, about 17,000 to about 18,500 Isolation pens, about 17,500 to about 19,000 isolation pens, about 18,000 to about 19,500 isolation pens, about 18,500 to about 20,000 isolation pens, about 19,000 Any of the isolation pens discussed herein having from about 20,500 isolation pens, from about 19,500 to about 21,000 isolation pens, or from about 20,000 to about 21,500 isolation pens. It has an isolation pen configured as in the embodiment.

FIG. 2C shows a microfluidic device 280 according to one embodiment. The microfluidic device 280 is shown in FIG. 2G and is a stylized view of the microfluidic device 100. In practice, the microfluidic device 280 and its components (eg, channel 122 and isolation pen 128) have the dimensions discussed herein. The microfluidic circuit 120 shown in FIG. 2G has a flow region 106 with two ports and four separate channels 122. The microfluidic device 280 further includes a plurality of isolation pens leading to each channel 122. In the microfluidic device shown in FIG. 2G, the isolation pen has a geometry similar to that of the pen shown in FIG. 2C and therefore has both a contiguous zone and a separation zone. Thus, the microfluidic circuit 120, sweep areas (e.g., the maximum penetration depth _D portion of the connection region 236 in the _p channel 122 and secondary flow 244) and non-sweeping region (e.g., isolation region 240 and the secondary flow 244 including both the maximum penetration depth D _p no part of the connection region 236 in) of.

3A-3B show various implementations of system 150 that can be used to operate and observe microfluidic devices according to the invention (eg, 100, 200, 230, 280, 250, 290, 320). Shows the morphology. As shown in FIG. 3A, the system 150 is a structure (“nested”) configured to hold the microfluidic device 100 (not shown) or any other microfluidic device described herein. ) 300 can be included. The nest 300 can interface with the microfluidic device 320 (eg, an optically actuated electrokinetic device 100) and can provide an electrical connection from the power supply 192 to the microfluidic device 320. 302 can be included. The nest 300 may further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 supplies a bias voltage to the socket 302 so that when the microfluidic device 320 is held by the socket 302, a bias voltage is applied across a pair of electrodes in the microfluidic device 320. Can be configured in. Therefore, the electrical signal generation subsystem 304 can be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 320 does not mean that a bias voltage is applied whenever the microfluidic device 320 is held by the socket 302. Rather, in most cases, the bias voltage is applied intermittently only when necessary to facilitate the generation of dynamic power, such as electrophoresis or electron wetting within the microfluidic device 320.

As shown in FIG. 3A, the nest 300 can include a printed circuit board assembly (PCBA) 322. The electrical signal generation subsystem 304 is mounted on the PCBA 322 and can be electrically integrated in the PCBA 322. An exemplary support also includes a socket 302 mounted on PCBA 322 as well.

Typically, the electrical signal generation subsystem 304 includes a waveform generator (not shown). The electrical signal generation subsystem 304 may further include an oscilloscope (not shown) and / or a waveform amplification circuit (not shown) configured to amplify the waveform received from the waveform generator. The oscilloscope, if present, may be configured to measure the waveform supplied to the microfluidic device 320 held by the socket 302. In certain embodiments, the oscilloscope measures the waveform at the proximal position of the microfluidic device 320 (and at the tip of the waveform generator), thereby being larger in measuring the waveform actually applied to the device. Guarantee accuracy. The data obtained from the oscilloscope measurements may be provided to the waveform generator as feedback, for example, and the waveform generator may be configured to adjust the output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is Red Pitaya ™.

In certain embodiments, the nest 300 further includes a controller 308, such as a microprocessor, used to detect and / or control the electrical signal generation subsystem 304. Examples of suitable microprocessors include Arduino ™ microprocessors such as the Arduino Nano ™. The controller 308 may be used to perform function and analysis, or it may communicate with an external master controller 154 (shown in FIG. 1A) to perform function and analysis. In the embodiment shown in FIG. 3A, the controller 308 communicates with the master controller 154 through an interface 310 (eg, a plug or connector).

In some embodiments, the nest 300 amplifies the waveform generated by the electrical signal generation subsystem 304, including a Red Pitaya ™ waveform generator / oscilloscope unit (“Red Pitaya unit”), and the Red Pitaya unit. , A waveform amplifier circuit that passes the amplification voltage to the microfluidic device 100 can be included. In some embodiments, the Red Pitaya unit measures the amplified voltage at the microfluidic device 320 and then itself as needed so that the measured voltage at the microfluidic device 320 is at the desired value. It is configured to adjust the output voltage of. In some embodiments, the waveform amplifier circuit can have a + 6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in the microfluidic device 100. Signals up to 13 Vpp are generated.

As shown in FIG. 3A, the support structure 300 can further include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to regulate the temperature of the microfluidic device 320 held by the support structure 300. For example, the thermal control subsystem 306 can include a Perche thermoelectric device (not shown) and a cooling unit (not shown). The Perche thermoelectric device can have a first surface configured to interface with at least one surface of the microfluidic device 320. The cooling unit can be, for example, a cooling block (not shown) such as a liquid-cooled aluminum block. The second surface of the Pelche thermoelectric device (eg, the surface opposite to the first surface) may be configured to interface with the surface of such a cooling block. The cooling block can be connected to a fluid path 314 configured to circulate the cooling fluid through the cooling block. In the embodiment shown in FIG. 3A, the support structure 300 receives a cooled fluid from an external reservoir (not shown), including an inlet 316 and an outlet 318, and introduces the cooled fluid into the fluid path 314. Then pass through the cooling block and then return the cooled fluid to the external reservoir. In some embodiments, the Perche thermoelectric device, cooling unit, and / or fluid path 314 can be mounted in case 312 of support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Pelche thermoelectric device to achieve the target temperature of the microfluidic device 320. Temperature control of the Pelche thermoelectric device can be achieved, for example, with a thermoelectric power source such as the Pololu ™ thermoelectric power source (Pololu Robotics and Electronics Corp.). The thermal control subsystem 306 can include a feedback circuit such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit may be provided by a digital circuit.

In some embodiments, the nest 300 is a resistor (eg, resistance 1 k ohm +/- 0.1%, temperature coefficient +/- 0.02 ppm / C0) and an NTC thermistor (eg, nominal resistance 1 k ohm +/-). A thermal control subsystem 306 with a feedback circuit that is an analog voltage divider circuit (not shown) including (having 0.01%) can be included. In some cases, the thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to the onboard PID control loop algorithm. The output from the PID control loop algorithm can, for example, drive both directional and pulse width modulated signal pins on a Pololu ™ motor drive device (not shown) to activate a thermoelectric power supply. , Control the Perche thermoelectric device.

The nest 300 can include a serial port 350, which allows the microprocessor of controller 308 to communicate with the external master controller 154 via interface 310 (not shown). In addition, the microprocessor of controller 308 can communicate with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (eg, via the Plink tool (not shown)). Therefore, the electrical signal generation subsystem 304 and the thermal control subsystem 306 can communicate with the external master controller 154 via the combination of the controller 308, the interface 310, and the serial port 324. In this way, the master controller 154 can assist the electrical signal generation subsystem 304, in particular by performing scaling calculations for output voltage regulation. A graphical user interface (GUI) (not shown) provided via a display device 170 coupled to an external master controller 154 provides temperature data and waveforms obtained from a thermal control subsystem 306 and an electrical signal generation subsystem 304, respectively. It can be configured to plot the data. Alternatively or additionally, the GUI can allow updates to the controller 308, thermal control subsystem 306, and electrical signal generation subsystem 304.

As mentioned above, the system 150 can include an imaging device 194. In some embodiments, the imaging device 194 includes an optical modulation subsystem 330 (see FIG. 3B). The light modulation subsystem 330 can include a Digital Mirror Device (DMD) or a Microshutter Array System (MSA), either of which receives light from a light source 332 and a subset of the light received by the optics of the microscope 350. It can be configured to send in columns. Alternatively, the optical modulation subsystem 330 is itself such as an organic emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). A device that produces light (and thus eliminates the need for a light source 332) can be included. The light modulation subsystem 330 can be, for example, a projector. Therefore, the photomodulation subsystem 330 may be capable of emitting both structured and unstructured light. An example of a suitable optical modulation subsystem 422 is the Mosaic ™ system of Andor Technologies ™. In certain embodiments, the imaging module 164 and / or the prime mover module 162 of the system 150 can control the light modulation subsystem 330.

In certain embodiments, the imaging device 194 further includes a microscope 350. In such an embodiment, the nest 300 and the optical modulation subsystem 330 may be individually configured to be mounted on the microscope 350. The microscope 350 can be configured to be, for example, a standard study grade optical or fluorescence microscope. Thus, the nest 300 may be configured to mount on the stage 344 of the microscope 350, and / or the light modulation subsystem 330 may be configured to mount on the port of the microscope 350. In other embodiments, the nesting 300 and optical modulation subsystem 330 described herein can be integral 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 can include an eyepiece, a charge-coupled device (CCD), a camera (eg, a digital camera), or any combination thereof. If at least two detectors 348 are present, one detector can be, for example, a high frame rate camera, while the other detector can be a sensitive camera. Further, the microscope 350 receives the light reflected and / or emitted from the microfluidic device 320 so that at least a portion of the reflected and / or synchrotron radiation is imaged on one or more detectors 348. It can include configured optical columns. The optical column of the microscope can also include different tube lenses (not shown) for different detectors so that the final magnification at each detector can be different.

In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, the first light source 332 can be used to generate structured light (eg, via a light modulation subsystem 330) and the second light source 334 can be used to provide unstructured light. .. The first light source 332 can generate structured light for optically actuated electrodynamic and / or fluorescence excitation, and the second light source 334 is used to provide brightfield illumination. be able to. In these embodiments, the prime mover 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 columns of the microscope 350 (1) receive structured light from the optical modulation subsystem 330 when the device is held by the nest 300 and receive the structured light from a microfluidic device such as an optically actuated electrokinetic device. It forms an image in at least the first region of the (2) receives the light reflected and / or emitted from the microfluidic device, and detects at least a portion of such reflected and / or emitted light in the detector 348. It can be configured to form an image on top. The optical column is further configured to receive unstructured light from a second light source and image unstructured light in at least a second region of the microfluidic device when the device is held by the nest 300. obtain. In certain embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first region can be a subset of the second region.

FIG. 3B shows a first light source 332 that supplies light to the light modulation subsystem 330, where the light modulation subsystem 330 is an optical column of the microscope 350 of system 355 (not shown) with structured light. To provide. A second light source 334 that provides unstructured light to the optical column via the beam splitter 336 is shown. As shown in and the structured light from the light modulation subsystem 330, the unstructured light from the second light source 334 travels together from the beam splitter 336 through the optical column and into the second beam splitter. (Or depending on the light provided by the light modulation subsystem 330, the dicroic filter 338) is reached, where the light is reflected and descends through the objective lens 336 to the sample surface 342. Next, the reflected light and / or the emitted light from the sample surface 342 moves again through the objective lens 340, passes through the beam splitter and / or the dichroic filter 338, and reaches the dichroic filter 346. Only part of the light that reaches the dichroic filter 346 is transmitted 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 surface 342 can pass through the dichroic filter 346 and reach the detector 348. In contrast, structured light coming from the photomodulation subsystem 330 is reflected at the sample surface 342 but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 filters out visible light having a wavelength longer than 495 nm. Such a filter of light from the light modulation subsystem 330 is completed (as shown) only if the light emitted from the light modulation subsystem does not contain any wavelengths shorter than 495 nm. In practice, if the light coming from the light modulation subsystem 330 contains wavelengths shorter than 495 nm (eg, blue wavelength), some of the light from the light modulation subsystem will pass through the filter 346 and reach the detector 348. do. In such an embodiment, the filter 346 serves to change the balance between the amount of light reaching the detector 348 from the first light source 332 and the amount of light reaching the detector 348 from the second light source 334. Fulfill. This can be beneficial if the first light source 332 is much more powerful than the second light source 334. In another embodiment, the second light source 334 can emit red light, and the dichroic filter 346 filters out visible light other than red light (eg, visible light having a wavelength shorter than 650 nm). can do.

Surface modification. The surfaces of materials, devices, and / or devices for manipulating and storing biomaterials are not limited, but include micro-objects (including, but not limited to, biological micro-objects such as living cells), biomolecules, biomolecules. Or they may have innate properties that are not optimized for short-term and / or long-term contact with such materials, which may include fragments of biological microobjects, and any combination thereof. It is useful to modify one or more surfaces of a material, device, or device so as to reduce one or more unwanted reductions associated with the innate surface that comes into contact with one or more biomaterials. could be. In other embodiments, the surface of the material, device, and / or device introduces the desired properties to the surface, thereby extending the handling, manipulating, or processing capacity of the material, device, and / or device. It can be useful to improve the properties. To that end, molecules are needed whose surface can be modified to reduce unwanted properties or introduce desirable properties.

A compound useful for surface modification. In various embodiments, the surface-modifying compound can be a non-polymeric moiety such as an alkyl moiety that co-modifies the bound surface or a substituted alkyl moiety such as a fluoroalkyl moiety (including, but not limited to, perfluoroalkyl). May include. The surface-modifying compound also includes a connecting moiety, which is a group in which a surface-modifying ligand is commonly attached to the surface, as schematically shown in Formula 1. The co-modified surface has a surface-modifying ligand bound via a linking group LG, which is the product of the reaction of the connecting moiety with the functional group on the surface (hydroxides, oxides, amines). , Or contains sulfur).

In some embodiments, the surface-modifying compound is linear (eg, at least 10 carbons, or at least 14, at least 16, at least 18, at least 20, at least 22, or more. It can contain carbon atoms forming a straight chain of carbon) and can be a non-branched alkyl moiety. In some embodiments, the alkyl group may comprise a substituted alkyl group (eg, some of the carbons in the alkyl group can be fluorinated or totally fluorinated). In some embodiments, the alkyl group may comprise a first segment and the first segment may comprise a perfluoroalkyl group attached to a second segment that may comprise an unsubstituted alkyl group, wherein the first segment may comprise a perfluoroalkyl group. The first segment and the second segment can be bonded directly or indirectly (eg, by ether bonding). The first segment of the alkyl group may be located at the tip of the linking group and the second segment of the alkyl group may be located near the connection.

の構造を有し得、式中、接続部分Ｖは、−Ｐ（Ｏ）（ＯＨ）Ｑ−又は−Ｓｉ（Ｔ）_２Ｗであり、Ｗは、−Ｔ、−ＳＨ、又は−ＮＨ_２であり、且つ表面に接続するように構成された部分であり、Ｑは、−ＯＨであり、且つ表面に接続するように構成された部分であり、Ｔは、ＯＨ、ＯＣ_１−３アルキル、又はＣｌである。Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素である。ｈの各インスタンスは、独立して２又は３の整数であり、ｊは、０又は１であり、ｋは、０又は１であり、ｍは、０又は１〜２５の整数であり、ｎは、０又は１〜２５の整数である。幾つかの実施形態では、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり得る。幾つかの実施形態では、Ｍは、水素である。様々な実施形態では、ｍは、２である。幾つかの実施形態では、ｋは、０である。他の実施形態では、ｋは、１である。様々な実施形態では、ｊは、１である。式Ｉの化合物では、ｋが１の整数である場合、ｍは、少なくとも２であり得、且つＭは、水素である。式Ｉの化合物では、ｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり得、且つＭは、水素である。 In various embodiments, the surface-modifying compound is of formula I:

In the equation, the connecting portion V is -P (O) (OH) Q- or -Si (T) ₂ W, where W is -T, -SH, or -NH ₂ . Yes and a part configured to connect to the surface, Q is -OH and a part configured to connect to the surface, T is OH, OC _1-3 alkyl, or Cl. R is hydrogen or fluorine, and M is hydrogen or fluorine. Each instance of h is independently an integer of 2 or 3, j is 0 or 1, k is 0 or 1, m is an integer of 0 or 1-25, and n is an integer of 0 or 1-25. , 0 or an integer of 1 to 25. In some embodiments, the sum of (n + [(h + j) · k] + m) can be an integer of 11-25. In some embodiments, M is hydrogen. In various embodiments, m is 2. In some embodiments, k is 0. In other embodiments, k is 1. In various embodiments, j is 1. In the compound of formula I, if k is an integer of 1, m can be at least 2 and M is hydrogen. In the compound of formula I, if k is 0 and R is fluorine, m can be at least 2 and M is hydrogen.

In various embodiments, if the surface-modifying compound has the structure of formula I, the connecting portion V can be −Si (T) ₂ W, where T and W are defined as described above. W can be OC _1-3 alkyl or Cl. W can be methoxy, ethoxy, or propoxy. In some embodiments, W can be methoxy. T can be OC _1-3 alkyl or Cl. In various embodiments, the connecting portion V is −Si (OMe) ₃ . In various other embodiments, V can be −P (O) (OH) Q, where Q is OH in the formula.

The surface-modified compound of formula I may have a preferred range of atomic numbers constituting the linear skeleton of the compound. As defined above, each segment constituting a compound of formula I can have various sizes. Thus, a compound of formula I can have a repeating unit as defined above such that (n + [(h + j) · k] + m) is equal to 25, which is the terminal attached to the connecting moiety. It brings the total length of 26 atoms including the _{CR 3-group.} If equal to 25 (n + [(h + j) · k] + m), it can contain a variety of different compositions. For example, the segment − [CR ₂ ] _n − can have n = 23, − [(CH ₂ ) _h − (O) _j ] _k − can have k = 0, − [CM ₂ ] _m − Can have m = 2. In another case where the same sum (n + [(h + j) · k] + m) is equal to 25, − [CR ₂ ] _n − (n = 6 in the equation); − [(CH2) h− (O) j] k-wherein (where, k = a 3), and may have an a j = 1 and _{h = 2; - [CM 2} ] m - may have m = 4.

In some embodiments, the sum of (n + [(h + j) · k] + m) can be 11, 13, 15, 17, or 21. In other embodiments, the sum of (n + [(h + j) · k] + m) can be 15 or 17. In yet another embodiment, the sum of (n + [(h + j) · k] + m) can be 13 or 15.

In some embodiments, one or more ether bonds may be present in the compound of formula I. In some embodiments, j can be 1. In some embodiments, m can be at least 2 if both k and j are 1.

In yet other embodiments, the skeletal carbon can be fluorinated. In some embodiments, the skeletal carbon can be totally fluorinated, in which case _{each R of CR 3-} and / or-[CR ₂ ] _n- and / or-[CM ₂ ] _m- can be fluorinated. .. In some embodiments, some sections of the compound may have carbon skeleton atoms that are fluorinated, and other sections of the compound may have carbon skeleton atoms that are replaced by hydrogen. For example, in some embodiments, the CR ₃ -segment and- [CR ₂ ] _n -segment may have a fluorine non-skeleton substituent (eg, R is fluorine), while- [CM] _m. -The segment can have a hydrogen non-skeleton substituent (eg, M is hydrogen). In some embodiments, if R is fluorine, then k is 0. In other embodiments, R can be fluorine, k is 1, j is 1, and h is 2. In various embodiments, M can be hydrogen.

In yet another embodiment, the compound of formula I can be synthesized from hydrosilylation of the olefin, 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 subset of compounds described by the formulas below, but these formulas never limit the scope of formula I.

In some embodiments, the compound of formula I is of formula 110 :.
CH ₃ (CH ₂ ) _m Si (OC _1-3 alkyl) ₃
Equation 110
In the formula, m is an integer of 9 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15.

In another embodiment, the compound of formula I is of formula 111:
CF ₃ (CF ₂ ) _n (CH ₂ ) ₂ Si (OC _1-3 alkyl) ₃
Equation 111
In the formula, n is an integer of 9 to 22. Alternatively, n can be an integer of 11-17. In some other embodiments, n can be 9, 11, 13, or 15. In some embodiments, n can be 13 or 15.

In yet another embodiment, the compound of formula I is of formula 112:
CR ₃ (CR ₂ ) _n (CH2) _h O (CH ₂ ) _m Si (OC _1-3 alkyl) ₃
Equation 112
In the formula, n is an integer of 3 to 19, h is 2 or 3, and m is an integer of 2 to 18. In some embodiments, R can be fluorine. In some embodiments, n can be an integer of 3 to 11, h can be 2, and m can be an integer of 2 to 15.

Alternatively, the compounds of formula I have formula 113:
CR ₃ (CR ₂ ) _n (CM ₂ ) _m P (O) (OH) ₂
Equation 113
In the equation, n is an integer of 3 to 21, and m is an integer of 2 to 21. In some embodiments of the compound of formula 113, R can be fluorine. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13.

Modified surface. Surfaces that can be modified by the surface-modifying compounds described herein, including compounds of formula I, can be metals, metal oxides, glass, or polymers. Some materials that may have a covalently modified surface therein are, but are not limited to, silicon and its oxides, silicones, aluminum or its oxides (Al ₂ O ₃ ), indium tantalum oxide (ITO), and more. It may include titanium dioxide (TiO ₂ ), zirconium oxide (ZrO2), hafnium oxide (IV) (HfO ₂ ), tantalum oxide (V) (Ta ₂ O ₅ ), or any combination thereof. The surface may be a wafer or sheet of these materials, or may be incorporated within an apparatus or device. In some embodiments, the surface containing any of these materials may be incorporated within the microfluidic device described herein.

The polymer may include any suitable polymer. Suitable polymers may include, but are not limited to (eg, rubber, plastics, elastomers, silicones, or organic silicones such as polydimethylsiloxane (“PDMS”)) and may be gas permeable. Other examples include molded glass, patternable materials such as silicone polymers (eg, photopatternable silicone or "PPS"), or photoresists (eg, epoxy-based photoresists such as SU8). Can be done. In other embodiments, the surface of a material such as natural fiber or wood can be functionalized to introduce a co-modified surface with the surface-modified compounds described herein, including compounds of formula I.

The surface to be modified may include nucleophilic moieties including, but not limited to, hydroxides, aminos, and thiols. The nucleophilic portion of the surface (eg, a hydroxide (referred to as an oxide in some embodiments)) reacts with the surface modification compounds described herein, including compounds of formula I, to syroxy. A surface converging ligand may be covalently attached to the surface via a binding or phosphonate binding group to provide a functionalized surface. The surface to be modified may contain a natural nucleophile, or may be treated with a reagent (eg, piranha solution) or by plasma treatment to include a nucleophile (eg, hydroxide (eg, hydroxide (alternatively)). ))) May be introduced.

In some embodiments, the surface can be formed from any single or any combination of materials described above. The surface may include a semiconductor substrate. In various embodiments, the surface comprising the semiconductor substrate may further include the DEP substrate or EW substrate described herein. In some embodiments, the surface comprising a semiconductor substrate having a DEP substrate or an EW substrate can be part of the microfluidic device described herein.

In some embodiments, the modified surface can be at least one inward surface of the microfluidic device described herein. At least one surface may be a portion of the flow region (which may include a channel) of the microfluidic device, or may include a surface of an enclosed structure such as a pen, which may include an isolation pen as described herein. It may be included.

Shared modified surface. The covalently modified surface may comprise a surface modifying ligand, which may be a non-polymeric moiety such as an alkyl moiety, a substituted alkyl moiety such as a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety), and the like. It can be any of the surface-modifying ligands described above that covalently attach to the surface via a linking group that is the moiety produced from the reaction between the connecting moiety and the surface. The binding group can be a syroxy binding group or a phosphonate binding group.

In some embodiments, the surface-modifying ligand is linear (eg, at least 10 carbons, or at least 14, at least 16, at least 18, at least 20, at least 22, or more. It can contain carbon atoms forming a straight chain of carbon) and can be a non-branched alkyl moiety. In some embodiments, the alkyl group may comprise a substituted alkyl group (eg, some of the carbons in the alkyl group can be fluorinated or totally fluorinated). In some embodiments, the alkyl group may comprise a first segment and the first segment may comprise a perfluoroalkyl group attached to a second segment that may comprise an unsubstituted alkyl group, wherein the first segment may comprise a perfluoroalkyl group. The first segment and the second segment can be bonded directly or indirectly (eg, by ether bonding). The first segment of the alkyl group may be located at the tip of the linking group and the second segment of the alkyl group may be located near the connecting portion.

の構造を有し、式中、表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）_２Ｗである。Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続する。Ｚは、表面に結合した隣接ケイ素原子への結合であるか、又は表面への結合である。Ｙは、表面に結合した隣接リン原子への結合であるか、又は表面への結合である。式ＩＩの共有修飾表面では、Ｒ、Ｍ、ｈ、ｊ、ｋ、ｍ、及びｎは、上記で定義されるようなものである。ｋが１の整数である場合、ｍは、少なくとも２であり、且つＭは、水素である。ｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である。式ＩＩの共有修飾表面は、式ＩＩＡでのように、結合基ＬＧを介して結合した表面修飾リガンドとして説明することができ、ここで、ＬＧは、表面に結合する。

共有修飾表面は、式Ｉの表面修飾化合物について上述したように、式ＩＩの任意の表面を任意の組み合わせで含み得る。 Co-modified surface of formula II. In some embodiments, the covalently modified surface is of formula II :.

In the formula, it is a surface, and 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 bonded to the surface or a bond to the surface. Y is a bond to an adjacent phosphorus atom bonded to the surface or a bond to the surface. On the co-modified surface of formula II, R, M, h, j, k, m, and n are as defined above. If k is an integer of 1, m is at least 2 and M is hydrogen. When k is 0 and R is fluorine, m is at least 2 and M is hydrogen. The co-modified surface of formula II can be described as a surface-modified ligand bound via a binding group LG, as in formula IIA, where LG binds to the surface.

The co-modified surface may comprise any surface of formula II in any combination, as described above for the surface-modified compound of formula I.

の表面であり得、式中、

は、表面であり、ケイ素原子に結合した酸素も表面に結合し、ｍは、１１〜２３の整数である。幾つかの実施形態では、ｍは、１１、１３、１５、１７、又は１９であり得る。幾つかの他の実施形態では、ｍは、１３又は１５であり得る。 In some embodiments, the co-modified surface of formula II is of formula 210:

Can be the surface of, in the formula,

Is a surface, oxygen bonded to a silicon atom is also bonded to the surface, and m is an integer of 11 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15.

の表面であり得、式中、

は、表面であり、ケイ素原子に結合した酸素も表面に結合し、ｎは、９〜２２の整数であり得る。代替的に、ｎは、１１〜１７の整数であり得る。幾つかの他の実施形態では、ｎは、７、９、１１、１３、又は１５であり得る。幾つかの実施形態では、ｎは、１３又は１５であり得る。 In some other embodiments, the co-modified surface of formula II is of formula 211:

Can be the surface of, in the formula,

Is a surface, oxygen bonded to a silicon atom is also bonded to the surface, and n can be an integer of 9-22. Alternatively, n can be an integer of 11-17. In some other embodiments, n can be 7, 9, 11, 13, or 15. In some embodiments, n can be 13 or 15.

の表面であり得、式中、

は、表面であり、ケイ素原子に結合した酸素も表面に結合し、ｎは、３〜２１の整数であり、ｈは、２又は３の整数であり、ｍは、２〜２１の整数である。幾つかの実施形態では、Ｒは、フッ素であり得る。幾つかの実施形態では、ｎは、３〜１１であり得、ｈは、２であり得、ｍは、２〜１５の整数であり得る。 In yet another embodiment, the co-modified surface of formula II is of formula 212:

Can be the surface of, in the formula,

Is a surface, oxygen bonded to a silicon atom is also bonded to the surface, n is an integer of 3 to 21, h is an integer of 2 or 3, and m is an integer of 2 to 21. .. In some embodiments, R can be fluorine. In some embodiments, n can be 3-11, h can be 2, and m can be an integer of 2-15.

の表面を有し得、式中、

は、表面であり、リン原子に結合した酸素も表面に結合し、ｎは、３〜２１の整数であり、ｍは、２〜２１の整数である。式１１３の化合物の幾つかの実施形態では、Ｒは、フッ素であり得る。幾つかの実施形態では、Ｍは、水素であり得る。様々な実施形態では、ｎは、５、７、９、又は１１であり得る。他の実施形態では、ｍは、２、４、５、７、９、１１、又は１３であり得る。 Alternatively, the co-modified surface of formula II is: formula 213:

Can have a surface of, in the formula,

Is a surface, oxygen bonded to a phosphorus atom is also bonded to the surface, n is an integer of 3 to 21, and m is an integer of 2 to 21. In some embodiments of the compound of formula 113, R can be fluorine. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13.

In some embodiments, the microfluidic device comprises a flow region that is fluidly connected to a first inlet and a first outlet so that the flow region includes the flow of the first fluid medium. It is composed. The microfluidic device may include one or more chamber openings to the flow region. The co-modifying surface can be a co-modifying substrate for a microfluidic device and can be under a flow region and / or at least one chamber. In some embodiments, all or nearly all inner surfaces of microfluidic devices configured to face the fluid have a covalently modified surface of formula II.

FIG. 2H shows a cross-sectional view of a microfluidic device 290 including an exemplary shared modified surface 298. As shown, the covalently modified surface 298 (schematically shown) may contain a single layer of high density molecules covalently bonded to both the inner surface 294 of the substrate 286 of the microfluidic device 290 and the inner surface 292 of the cover 288. can. The covalently modified surface 298, in some embodiments and as described above, is a surface of a microfluidic circuit material (not shown) used to define circuit elements and / or structures within the microfluidic device 290. Including, it can be placed in the vicinity of the enclosure 284 of the microfluidic device 290 and on substantially all inner surfaces 294, 292 facing inward towards the enclosure 284. In an alternative embodiment, the covalently modified surface 298 can be located on only one or several of the plurality of inner surfaces of the microfluidic device 290.

In the embodiment shown in FIG. 2H, the covalently modified surface 298 comprises a single layer of alkyl-terminated siloxane molecules, each molecule covalently attached to the inner surface 292, 294 of the microfluidic device 290 via a siloxylinker 296. For brevity, additional silicon oxide bonds attached to adjacent silicon atoms are shown, but the invention is not so limited. In some embodiments, the covalently modified surface 298 has a fluoroalkyl group at the end facing the enclosure (ie, a monolayer portion of the surface modified ligand 298 that is not attached to the inner surface 292, 294 and is in the vicinity of the enclosure 284). (For example, an alkyl fluorinated group or an alkyl fluorinated group) can be included. Although FIG. 2H is considered to have an alkyl-terminated surface, any suitable surface-modified compound can be used as described herein.

Natural surface. At least one surface of the modified microfluidic device can be glass, metal, metal oxide, or polymer. Some materials that can be incorporated into microfluidic devices and that can be modified to introduce the covalently modified surface of Formula II into the interior are not limited to silicon and its oxides, silicones, aluminum or its oxides ( Al ₂ O ₃ ), indium tantalum oxide (ITO), titanium dioxide (TIO ₂ ), zirconium oxide (ZrO2), hafnium oxide (IV) (HfO ₂ ), tantalum oxide (V) (Ta ₂ O ₅ ), or It may include any combination thereof. The polymer may include any suitable polymer. Suitable polymers may include, but are not limited to (eg, rubber, plastics, elastomers, silicones, or organic silicones such as polydimethylsiloxane (“PDMS”)) and may be gas permeable. Other examples include molded glass, patternable materials such as silicone polymers (eg, photopatternable silicone or "PPS"), or photoresists (eg, epoxy-based photoresists such as SU8). Can be done.

Physical and performance characteristics of the covalently modified surface. In some embodiments, the covalently modified surface can increase hydrophobic properties. Increased hydrophobic properties of the modified surface can prevent fouling by biomaterials. As used herein, surface fouling is indiscriminate on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biomaterials such as proteins and degradation products, nucleic acids, and each degradation product. Refers to the amount of material deposited in. Such fouling can increase the amount of biological microobjects attached to the surface. In other embodiments, the increased hydrophobicity of the co-modified surface can reduce the attachment of biological micro-objects to the surface, independent of the adhesion caused by surface fouling.

Surface modifications can increase surface durability, functionality, and / or biocompatibility. Each of these properties includes viability (including growth rate and / or cell doubling), the nature of colonies formed on the co-modified surfaces described herein, including surfaces with the structure of formula II. Alternatively, it may be more beneficial for the portability (including survival on export) of micro-objects or biomolecules on and / or in devices having modified surfaces and co-modified surfaces.

In some embodiments, the covalently modified surface, which can be any surface described herein, including the surface of Formula II, is less than 10 nm (eg, less than about 7 nm, less than about 5 nm, or about 1.5 nm. It can have a thickness of ~ 3.0 nm). This is advantageously thin, especially in contrast to other hydrophobic materials such as CYTOP®, which is a spin-coated perfluorotetrahydrofuranyl polymer that produces a typical thickness of about 30-50 nm. A layer can be provided on the modified surface. The data shown in Table 1 are for silicon / silicon oxide surfaces that are treated to have a co-modified surface as shown in the table. Static droplets (static sessile)
Contact angle measurements were obtained using the drop) method (Drelich, J. Colloid Interface Sci. 179, 37-50, 1996). The thickness was measured by ellipsometry.

Contact angle hysteresis measurements were performed using a Biolin Scientific contact angle goniometer. The chemically modified OEW surface was placed in a bath of 5cSt silicone oil placed in a transparent holder. The phosphate buffered saline (PBS) droplets were then dispensed onto the surface in the oil. A platinum (Pt) wire was inserted into the droplet and the water droplet contact angle was measured. Next, an applied AC voltage of 50 Vppk at a frequency of 30 kHz was applied between the OEW substrate and the Pt wire inserted in the PBS droplet for 10 seconds. Next, the applied voltage was eliminated and the contact angle was measured again. The contact angle hysteresis was calculated by subtracting the zero bias contact angle after the application of the AC voltage of 50 Vppk from the original contact angle at the zero bias before the voltage application.

The contact angle observed on the modified surface is less than 10 degrees, in contrast to the contact angle for water on plasma-cleaned silicon surfaces. Each of these surfaces is less wet than the native silicon / silicon oxide surface.

Other analytical methods suitable for surface characterization include infrared spectroscopy and / or X-ray photoelectron spectroscopy.

Another desirable feature of the modified surface of the present invention is the absence of autofluorescence, which can depend on the chemistry of the surface-modified compound.

In some embodiments, the covalently modified surfaces described herein, including the surface of formula II, may form a monolayer. The uniformity and uniformity of the monolayer modified surface can provide advantageous performance, especially if the monolayer modified surface has other functional attributes. For example, the covalently modified surfaces described herein, including the surface of formula II, may also include an electrode activated substrate, with materials, devices, and / or devices having a dielectrophoretic or electrowetting configuration. As can be seen, it may optionally further include a dielectric layer. The absence of unsaturated perfluoroalkyl moieties on the modified surface can minimize "charge capture" as compared to, for example, a monolayer containing an olefin moiety or an aromatic moiety. In addition, the high density of the monolayers formed on the surfaces described herein, including the surface of Formula II, allows cations to pass through the monolayer and underneath the metal, metal oxide, glass, or glass. The potential to reach polymer materials can be minimized. Without being limited by theory, the destruction of the substrate surface by the addition of cations to the substrate composition can destroy the electrical properties of the substrate, thereby reducing the ability of the substrate to function electrodynamically.

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 fracture under conditions where an electric field is applied. The uniformity and thinness of the dielectrophoretic or electrowetting surfaces of materials, devices, and / or devices with co-modified surfaces described herein, including the surfaces of formula II, are the materials, devices, and / or If the device operates optically, it may further provide advantageous advantages for such modified dielectrophoresis and / or electrowetting surfaces.

How to prepare a shared modified surface. The surface of the material that can be used as a device or component of the device can be modified prior to assembly of the device or device. Alternatively, a partially or fully constructed device or device simultaneously modifies all surfaces that will come into contact with biomaterials and / or micro-objects (which may include biological micro-objects), including biomolecules. Can be modified to be. In some embodiments, the entire interior of the device and / or device may be modified, even if there are different materials on different surfaces within the device and / or device. In some embodiments, the partially or fully constructed device and / or device can be a microfluidic device or a component thereof as described herein.

The surface to be modified can be cleaned prior to modification to ensure that the nucleophile on the surface is freely available for the reaction, eg, not covered with oil or adhesive. Cleaning can be accomplished by any suitable method, including treatment with a solvent containing alcohol or acetone, sonication, steam cleaning and the like. In some embodiments, the surface to be modified is treated with an oxygen plasma treatment that can remove contaminants while at the same time introducing additional oxide (eg, hydroxide) moieties into the surface. NS. This can advantageously provide more space on the surface for modification, thereby providing a denser modified surface layer.

Cleaning prior to modification can ensure that the nucleophilic moiety on the surface is freely available for the reaction, eg, not covered with oil or adhesive. Cleaning can be accomplished by any suitable method, including treatment with a solvent containing alcohol or acetone, sonication, steam cleaning and the like. In some embodiments, the surface to be modified is treated with an oxygen plasma treatment that can remove contaminants while at the same time introducing additional oxide (eg, hydroxide) moieties into the surface. NS. This can advantageously provide more space on the surface for modification, thereby providing a denser modified surface layer.

の化合物に表面を接触させるステップを含み、式中、Ｖは、−Ｐ（Ｏ）（ＯＨ）Ｑ又は−Ｓｉ（Ｔ）_２Ｗである。Ｗは、−Ｔ、−ＳＨ、又は−ＮＨ_２であり、且つ表面に接続するように構成される部分である。代替的に、Ｖは、−Ｐ（Ｏ）（ＯＨ）Ｑであり、Ｑは、−ＯＨであり、且つ表面に接続するように構成される部分である。Ｔは、ＯＨ、ＯＣ_１−３アルキル又はＣｌである。Ｒ、Ｍ、ｈ、ｊ、ｋ、ｍ、及びｎのそれぞれは、式Ｉの化合物について上記で定義したようなものである。（ｎ＋［（ｈ＋ｊ・ｋ］＋ｍ）の和は、１１〜２５の整数である。様々な実施形態では、ｋが１の整数である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である。式Ｉの化合物は、表面の求核部分と反応し、共有修飾表面が形成される。上述したように、式Ｉの化合物の任意の組み合わせ又は部分的組み合わせが使用可能である。 In some embodiments, the method of co-modifying the surface is Formula I:

In the formula, V is -P (O) (OH) Q or -Si (T) ₂ W, including the step of bringing the surface into contact with the compound of. W is -T, -SH, or -NH ₂ , and is a portion configured to connect to the surface. Alternatively, V is −P (O) (OH) Q, where Q is −OH and is a portion configured to connect to the surface. T is OH, OC _1-3 alkyl or Cl. Each of R, M, h, j, k, m, and n is as defined above for the compounds of formula I. The sum of (n + [(h + j · k] + m) is an integer of 11-25. In various embodiments, if k is an integer of 1, m is at least 2 and M is hydrogen. And k is 0 and R is fluorine, then m is at least 2 and M is hydrogen. The compounds of formula I react and share with the nucleophilic portion of the surface. A modified surface is formed. As mentioned above, any combination or partial combination of compounds of formula I can be used.

In various embodiments of the method, the covalently modified surface thus formed is monolayer.

In some embodiments of the method, the compound of formula I is of formula 110 :.
CH ₃ (CH ₂ ) _m Si (OC _1-3 alkyl) ₃
Equation 110
In the formula, m is an integer of 9 to 23. In some embodiments, m can be 11, 13, 15, 17, or 19. In some other embodiments, m can be 13 or 15.

In another embodiment of the method, the compound of formula I is of formula 111:
CF ₃ (CF ₂ ) _n (CH ₂ ) ₂ Si (OC _1-3 alkyl) ₃
Equation 111
In the formula, n is an integer of 9 to 22. Alternatively, n can be an integer of 11-17. In other embodiments, n can be an integer of 11-17. In some other embodiments, n can be 9, 11, 13, or 15. In some embodiments, n can be 13 or 15.

In yet another embodiment of the method, the compound of formula I is of formula 112:
CR ₃ (CR ₂ ) _n (CH2) _h O (CH ₂ ) _m Si (OC _1-3 alkyl) ₃
Equation 112
In the formula, n is an integer of 3 to 21, h is an integer of 2 or 3, and m is an integer of 2 to 21. In some embodiments, R can be fluorine. In some embodiments, n can be an integer of 3-11, h can be 2, and m can be an integer of 2-15.

Alternatively, it can be a compound of formula 113:
CR ₃ (CR ₂ ) _n (CM ₂ ) _m P (O) (OH) ₂
The compound of formula 113 may contact the surface, where n is an integer of 3 to 21 and m is an integer of 2 to 21. In some embodiments of the compound of formula 113, R can be fluorine. In some embodiments, M can be hydrogen. In various embodiments, n can be 5, 7, 9, or 11. In other embodiments, m can be 2, 4, 5, 7, 9, 11, or 13.

The contact step can be performed by contacting the surface with a liquid solution containing a compound of formula I. For example, the surface can be exposed to a solution containing a compound of formula I of 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM, or 100 mM. The reaction can be carried out at ambient temperature and over a time period of about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours, or any range of values in between. obtain. Examples of the solvent include, but are not limited to, toluene, 1,3 bistrifluorobenzene, or Fluorinert ™ (3M) fluorinated solvent. The reaction rate can be increased by adding an acid such as acetic acid to the solution to promote the hydrolysis of the trialkoxy group in the presence of the trialkoxy group.

Alternatively, the surface can be contacted with a gas phase containing a compound of formula I. In some embodiments, a controlled amount of water vapor is also present if the reaction step is carried out by contacting the surface with a compound of formula I of the gas phase. A controlled amount of water vapor can be provided by placing a preselected amount of magnesium sulphate heptahydrate in the same chamber or enclosure as the object with the surface to be modified. In other embodiments, a controlled amount of water may be introduced into the reaction chamber or enclosure via an external steam supply. The reaction can be carried out under reduced pressure compared to atmospheric pressure. In some embodiments, the decompression can be less than 100 tolls. In other embodiments, the decompression can be less than 10 torr or less than 1 torr.

The reaction can be carried out at a temperature in the range of about 150 ° C to about 200 ° C. In various embodiments, the reaction can be carried out at temperatures of about 150 ° C, about 155 ° C, about 160 ° C, about 165 ° C, about 170 ° C, about 175 ° C, about 180 ° C, about 185 ° C, or about 190 ° C. .. The reaction may continue to run for about 2 hours, about 6 hours, about 8 hours, about 18 hours, about 24 hours, about 48 hours, about 72 hours, about 84 hours, or more.

の構造を有し得、式中、Ｒ、Ｍ、ｎ、ｈ、ｊ、ｋ、ｍ、及びＶは、任意の組み合わせでの上述したようなものである。方法の幾つかの実施形態では、共有修飾表面は、各式で許容可能な要素の任意の組み合わせを有する上述した式２１０、２１１、２１２、又は２１３の式を有し得る。 In some embodiments, the covalently modified surface is of formula II :.

In the formula, R, M, n, h, j, k, m, and V are as described above in any combination. In some embodiments of the method, the covalently modified surface may have the formulas 210, 211, 212, or 213 described above having any combination of elements acceptable in each formula.

In various embodiments of the method, the surface may contain a nucleophilic moiety selected from the group consisting of hydroxides, aminos, and thiols. The surface can be metal, metal oxide, glass, polymer, or any combination thereof. The metal surface may contain silicon, silicon oxide, hafnium oxide, indium tantalum oxide, alumina, or any combination thereof.

In various embodiments of the method, the steps of forming a covalently modified surface can be performed on a DEP substrate or an EW substrate. The step of forming a co-modified surface may include forming a co-modified surface on at least one surface of a microfluidic circuit element of a microfluidic device. Microfluidic circuit elements can include walls, flow regions, pens, and electrode activation substrates, including DEP substrates or EW substrates. The surfaces in the microfluidic circuit that can be co-modified can be all or nearly all surfaces facing the fluid carrier of the microfluidic device. For example, in the microfluidic devices 200 and 230, the inner surface of the upper electrode 210 facing the microfluidic channel 122 and the pens 244, 246, 248, the upper surface of the electrode activation substrate 206, and the surface of the microfluidic circuit material 116 (FIG. 1B, FIG. 1C, 2A, 2B) can be modified. Similarly, in FIGS. 2D-2F, the inner surface of the microfluidic circuit material 260, the surface of the separation structure 272 defining the isolation pen 266, or all surfaces facing the microfluidic circuit 262 are described herein. It can be shared and modified by the method.

Miscible medium. The movement of aqueous droplets on the surface of the substrate is locally distributed within one or more flow regions (which may include flow channels) and, where chambers are present, within the chambers fluidly connected to the flow regions. It can be performed in a water-immiscible fluid medium. Water-immiscible fluid media can have a higher kinematic viscosity than droplets of pure water. Water-immiscible fluid media can have kinematic viscosity in the range of about 1 centimeter Stokes (cSt) to about 15 cSt, where 1 cSt equals 1 millipascal or 1 centipores (CPS). In some embodiments, the water immiscible fluid medium may have viscosities in the range of about 3 cSt to about 10 cSt or about 3 cSt to about 8 cSt. The water immiscible fluid medium can be nonflammable at a temperature of at least 100 ° C. A water-immiscible fluid medium can be non-toxic to living living cells for the duration of treatment, culturing, or storage of living cells in aqueous droplets within the water-immiscible fluid medium.

Water-immiscible fluid media can have low or negligible solubility in water. A water-immiscible fluid medium, when in contact with a layer of water (partitioned by water), is less than about 5%, about 4%, about 3%, about 2%, about 1%, or about 1% of the total amount of water. Can dissolve in less than 1%. The water immiscible fluid medium is about 5%, about 10%, about 15%, about 20% of the volume of aqueous droplets present in the water immiscible fluid medium at temperatures in the range of about 25 ° C to about 38 ° C. Not soluble in excess of%, about 25%, or about 30%. In some embodiments, the water immiscible fluid medium is soluble in less than about 20% of the volume of aqueous droplets present in the water immiscible fluid medium.

The water immiscible fluid medium may comprise at least one organosilicon or organosilicon compound having a backbone structure containing atoms selected from carbon, silicon, and oxygen. In some embodiments, the water immiscible fluid medium may comprise more than one organic compound / organic silicon compound, in which case the two or more compounds contain subsystems of various molecular weights of the polymeric compound. It is a high molecular weight organic compound / organic silicon compound. For example, a polymeric organic compound / organic silicon compound may have two different subunits that make up a polymer (eg, a copolymer), and the two different subunits are presented in various iterations, each with the general formula AaBb. Obtained, in the formula, A and B are two different polymer subunits, and a and b are the number of iterations for each subunit. The iteration numbers a and b do not have to be a single integer and can be various iteration units.

In other embodiments, the water immiscible fluid medium comprising two or more organosilicon / organosilicon compounds may comprise a mixture of organic compounds, a mixture of organosilicon compounds, or any combination thereof. The water immiscible fluid medium may contain any suitable mixture of compounds having different chemical structures and / or molecular weights that provide suitable performance.

Compounds in water-immiscible fluid media can have a molecular weight of less than about 1000 Da, less than about 700 Da, less than about 500 Da, or less than about 350 Da. In other embodiments, the compound of the water immiscible medium may have a molecular weight higher than about 1000 Da and may still provide suitable performance.

In various embodiments, the organocompound / organosilicon compound in the water-immiscible fluid medium may have a backbone structure in which the atoms constituting the skeleton are carbon, silicon, or oxygen. Substitution of skeletal carbon can be hydrogen or fluorine. In some embodiments, the water immiscible fluid medium may comprise one or more organosilicon compounds, in which case the backbone of the organosilicon compound may comprise silicon and oxygen atoms. The silicon atom of the organosilicon compound can have a carbon substituent, and the carbon substituent can have a hydrogen substituent or a fluorine substituent. In some embodiments, the carbon substituent of the organosilicon compound can be all fluorines (eg, can be totally fluorinated). In other embodiments, the carbon substituents of the organosilicon compound can be partially fluorinated. In various embodiments, the substituents on the carbon atom of the organosilicon compound are about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30%. Hereinafter, about 20% or less, or less than that, may be fluorine.

In other embodiments, the organic compound in a water-immiscible fluid medium may have a backbone structure in which the atoms constituting the skeleton are carbon or oxygen. In some embodiments, the substituent of the backbone carbon can be hydrogen or fluorine. In other embodiments, the substituents on the skeletal carbon may include oxygen-containing moieties such as an ether component, a carbonyl component, or a carbonate component. In some embodiments, the organic compound in the water immiscible fluid medium may have a total carbon backbone structure. In some embodiments of the total carbon backbone structure of an organic compound in a water immiscible fluid medium, it may have a total fluorine substituent on a carbon atom (eg, total fluorination). In other embodiments, the substituents of the organic compound can be partially fluorinated (eg, not fully fluorinated). In various embodiments, the carbon atom substituents of organic compounds, including compounds having a total carbon skeleton, are about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, Approximately 40% or less, or less, can be fluorine. In some embodiments, suitable organic compounds in water-immiscible fluid media may include monofluorosubstituted hydrocarbons such as 1-fluorooctane, 1-fluorodecane, 1-fluorododecane, or 1-fluorotetradecane. Alternatively, it can be a monofluorosubstituted hydrocarbon.

In other embodiments, the organic compounds in the water-immiscible fluid medium do not have fluorine substituents on carbon and may have hydrogen substituents. In some embodiments, the organic compound in the water immiscible fluid medium may have an unsaturated carbon-carbon bond, eg, an olefin group within or at the terminal position in the skeleton carbon.

In some embodiments, the selection of a suitable compound for inclusion in a 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 do not self-fluoresce when illuminated by lasers, structured light projected onto microfluidic devices, or sunlight / laboratory lighting. ..

In other embodiments, the properties of the covalently modified hydrophobic surface influence the selection of suitable compounds for use in water-immiscible fluid media. For example, a covalently modified surface may exhibit a surface tension sufficiently high that water droplets in a totally fluorinated water immiscible fluid medium are not mobile using the photoelectrowetting configurations described herein. It can have sufficient hydrophobicity.

In some other embodiments, the nature of the microfluidic circuit material can influence the selection of suitable compounds for use in water-immiscible fluid media. Expansion of the circuit material by the water-immiscible fluid medium can be maintained within acceptable limits. For example, in some embodiments, if the microfluidic circuit material comprises SU8 or an optical patternable aryl-substituted organic silicone, a linear hydrocarbon, a linear fluorocarbon, or a cyclic group, an aryl group, or a heteroaryl. Group-containing carbon skeleton compounds may be selected for use.

In other embodiments, the microfluidic circuit material may include other materials such as photopatternable organic silicones that do not contain aryl substituents and the expansion uses different compounds in a water immiscible fluid medium. Can be limited to the permissible limit. For example, expansion of less than about 40%, less than about 30%, less than about 20%, or less than about 10% may be acceptable compared to pre-exposure to a water-immiscible fluid medium. However, in some embodiments, compounds in water-immiscible fluid media that cause swelling may still be selected for use.

In some embodiments, the compound in the water immiscible fluid medium can be an organic compound having a skeleton containing carbon or oxygen atoms. In some embodiments, the organic compound may contain carbon atoms, no oxygen atoms, and may have a skeleton in which the carbon atom skeleton does not branch. In various embodiments, the branched-chain carbon atomic skeleton of the organic compound in the water-immiscible fluid medium is acyclic. Organic compounds in water-immiscible fluid media with a branched chain carbon backbone can further be free of any cyclic moieties.

The selection criteria described above may be used to select one or more compounds to be incorporated into a water immiscible fluid medium, eliminating compounds that may not provide acceptable performance, but tolerable water immobility. The admixture fluid medium can be a multi-component mixture and contains some portion of an individual organic compound or organic silicon compound that does not provide acceptable performance when used as a single component of a water immiscible fluid medium. obtain. For example, the components, when used alone, may be over-fluorinated or may unacceptably expand the microfluidic circuit material, but may be used in combination with other organic or organic silicon compounds. Can form a water-immiscible fluid medium.

Some organic compounds suitable for use in water-impermeable fluid media, alone or in any combination of types, include isocetane, 2- (trifluoromethyl) -3-ethoxydodecafluorohexane (HFE-7500). , 3MTM, NovecTM), heptamethylnonane (HMN), bis (2-ethylhexyl) carbonate (TEGOSOFT® DEC, (Evonik)), and (tridecafluoro-1,1,2,2,-tetrahydrooctyl). ) Tetramethyldisiloxane (Gelest Catalog No. SIB1816.0) or silicone oil (5 cm stroke viscosity, Gelest Catalog No. DMS-T05).

Aqueous droplets. Aqueous droplets may contain one or more microobjects that may contain living cells or beads. Aqueous droplets may contain biological material which may contain nucleic acids or proteins. In some other embodiments, the aqueous droplet may comprise a reagent for the assay, which may be any type of reagent, such as an enzyme, antibody, fluorescently labeled probe, or chemical reagent.

In some embodiments, the aqueous droplet can also contain a surfactant. 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, surfactants include, but are not limited to, F68 (Thermo Fisher Catalog No. 2400032).
Block alkylene oxide copolymer; fatty acid ester ethoxylated sorbitan such as TWEEN® 20 (Signa Aldrich Catalog No. P1379) or TWEEN® 60 (Sigma Aldrich P1629); 2,4,7,9, tetra Methyl-5-decyne-4,7, -diol ethoxylate (TET, Sigma Aldrich Catalog No. 9014-
85-1); Capstone® FS-30 (a nonionic ethoxylated fluorine-based surfactant such as DuPont ™, Synquest Laboratories Catalog No. 2108-3-38). In some embodiments, sodium dodecyl sulfate (SDS) can be used as a surfactant. In various embodiments, phosphate buffered saline (PBS) can be used as the surfactant. Surfactants are in the range of about 1% by volume, about 3% by volume, about 5% by volume, about 10% by volume, about 15% by volume, about 20% by volume, about 25% by volume, or any value between them. Can be added to aqueous droplets.

system. The present invention provides a system for transporting microobjects, biological substances, and / or reagents that are compatible with and / or soluble in aqueous media. The system is, for example, a microfluidic device having an enclosure comprising any microfluidic device disclosed herein (eg, a base and a microfluidic circuit structure, wherein the base is at least one of the top surfaces of the base. Microfluidic devices) that include a hydrophobic monolayer covalently attached to the moiety can be included. In addition, the system includes a fluid medium and aqueous droplets, which are immiscible fluids. The fluid medium can be any immiscible medium described herein, and the aqueous droplets can contain any biomaterial and / or chemicals described herein (eg,). Proteins, nucleic acids, detergents, surfactants, etc.).

kit. The present invention also provides kits that are compatible with aqueous media and / or suitable for transporting microobjects, biological substances, and / or reagents that are soluble in aqueous media. A kit is a microfluidic device having an enclosure comprising any microfluidic device disclosed herein (eg, a base and a microfluidic circuit structure, with the base on at least a portion of the top surface of the base. Microfluidic devices, including covalently bonded hydrophobic monolayers) can be included. In addition, the system includes a fluid medium and aqueous droplets, which are immiscible fluids. The kit can further include a fluid medium immiscible with the aqueous medium and other useful reagents (eg, surfactants, etc.).

How to make a microfluidic device. Microfluidic devices of the invention, such as device 400, (i) join a separating element 108 to an inner surface 428 of a cover 110 having at least one electrode configured to be connected to an AC voltage source (not shown). And (ii) join the separating element 108 (and associated cover 110) to the dielectric surface 414 of the substrate 104 having at least one electrode 418 configured to be connected to an AC voltage source (not shown). That is, the separating element 108 is sandwiched between the inner surface 428 of the cover 110 and the dielectric surface 414 of the substrate 104, with the cover 110 and the substrate 104 oriented substantially parallel to each other. The substrate 104, the separating element 108, and the cover 110 collectively define and join the enclosure 435 configured to hold the liquid, and (iii) by vapor deposition to cover the hydrophobic outer layer 412 of the cover 110. It can be manufactured by forming it on at least a part of the inner surface 428 and forming the hydrophobic outer layer 412 on at least a part of the dielectric inner layer 414 of the substrate 104.

Through the deposition of amphipathic molecules, the hydrophobic layers 422 and 412 can achieve a high density monolayer in which the amphipathic molecules are covalently bonded to the molecules of the inner surface 428 of the cover 110 and the dielectric inner layer 414 of the substrate 104, respectively. .. Any self-associating molecule described herein and its equivalents can be deposited on the inner surface of the microfluidic device. To achieve the desired packing density, for example, the self-associating molecule containing the alkyl-terminated siloxane is at least 110 ° C. (eg, at least 120, 130, 140, 150, 160, etc.) for at least 15 hours (eg, at least 120, 130, 140, 150, 160, etc.). Deposition can be carried out over at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, or more). Such deposition is usually under vacuum, is carried out in the presence of a water source, such as magnesium sulfate heptahydrate (i.e. MgSO 4 · _7H 2 _0). Generally, increasing the temperature and duration of the deposition improves the properties of the hydrophobic layers 422 and 412. The vapor deposition process can be optionally improved, for example, by pre-cleaning the cover 110 (having the separating element 108) and the substrate 104. For example, such pre-cleaning can include solvent baths such as acetone baths, ethanol baths, or combinations thereof. The solvent bath can include sonication. Alternatively or additionally, such pre-cleaning may include treating the cover 110 (having the separating element 108) and the substrate 104 in an oxygen plasma cleaner. The oxygen plasma cleaner can be operated, for example, at 100 W for 60 seconds under vacuum conditions.

FIG. 6 shows an example of a microfluidic device 600 including an enclosure with microfluidic channels 612, 614 and a plurality of chambers 616 and a droplet generator 606 that provides fluid droplets 620 to the enclosure. The microfluidic channel 614 is configured to hold the first fluid medium 624. Usually, the first fluid medium is a hydrophobic fluid such as an oil (eg, silicone oil or fluorinated oil). The microfluidic channel 614 is connected to the droplet generator 606 via an interface 608, which causes the channel 614 to receive the droplet 620 produced by the droplet generator 606. The droplet 620 received contains a liquid immiscible with the first fluid medium 624. Generally, the droplets received include microscopic objects such as cells or beads or aqueous media which may contain reagents soluble in aqueous media. Microfluidic channels 614 are also connected to each of the plurality of chambers 616 and are received from droplets 620 (and immiscible fluid reservoirs in the first fluid medium 624) within and between chambers 616. Promotes the movement of the pulled droplet 632).

The microfluidic channel 612 of the device 600 is connected to a subset of chambers 616 and is therefore indirectly connected to the microfluidic channel 614 through such chamber 616. As shown, the microfluidic channel 612 and the chamber 616 connected thereto include a fluid medium 622 that is immiscible with the first fluid medium 624. Thus, for example, the fluid medium 622 can be an aqueous medium such as a cell culture medium. When the fluid medium 622 is a cell culture medium, the chamber 616 containing the culture medium can be used as a culture chamber for growing cells, and the microfluid channel 612 is a perfusion channel that provides a flow of fresh culture medium. could be. As discussed herein, the flow of fresh culture medium within the perfusion channel provides nutrients to the chamber through the diffusion of molecules between the perfusion channel and the culture chamber, as well as waste products from the chamber. Can be removed and thus promote continued cell growth.

FIG. 7 is a micro containing microfluidic channels 612, 614, an enclosure having a first plurality of chambers 716, and a second plurality of chambers 616, and a droplet generator 606 that provides fluid droplets 620 to the enclosure. Another example of the fluid device 700 is shown. FIG. 7 presents a variant of the microfluidic device 600 shown in FIG. 6, in which the chamber 616 is immiscible with the first fluid medium 624 (located within the microfluidic channel 614). 622 is included and is located directly from the corresponding chamber 716 across the microfluidic channel 614. This configuration facilitates the movement of the fluid droplets 632 (optionally including microobjects 630 or biomaterial) from the selection chamber 616 to the corresponding chambers 716, and in the corresponding chambers 716 the fluid droplets (and optionally). 630 or biomaterial) can be processed.

Another example of a microfluidic device is an enclosure with microfluidic channels 612, 614, a first plurality of chambers 716, and a second plurality of chambers 616, and a droplet generator that provides fluid droplets 620 to the enclosure. 606 and is included. This embodiment presents a variant of the microfluidic device 700 shown in FIG. 7, in which the chamber 616 has a tapered shape at one end and the microfluidic device has a tapered end of the chamber 616. Of microparticles to the interface between the first fluid medium 624 and the second fluid medium 622 when is tilted to have a lower potential (in an applicable gravitational field) compared to the non-tapered end. Promote movement.

The microfluidic circuit formed by the microfluidic channels 612, 614 and chambers 616, 716 is merely an example, and many other configurations of channels and chambers are included by the present invention. For example, in devices 600 and 700, the chamber 616 directly connected to the microfluidic channel 612 and channel 612 is an optional feature. Therefore, devices 600 and 700 do not have to have a perfusion channel and a culture chamber.

In embodiments where the microfluidic channel 612 is present, the substrate facilitates the demarcation of the channel 612 and / or the directly connected chamber 616 (eg, by forming the base of the channel and / or chamber) in an electrowetting configuration. Can have. However, alternatively, the substrate that facilitates the demarcation of the channels 612 and / or the directly connected chamber 616 may not have an electrowetting configuration (eg, instead can have a DEP configuration, or It does not have to have an electrowetting configuration or a DEP configuration). In embodiments where the microfluidic channel 612 is present and the substrate facilitates the demarcation of the channel 612 and / or the directly connected chamber 616 has an electrowetting configuration, the hydrophobic outer layer of the substrate is more than the hydrophobic outer surface of the substrate. It can be patterned to have high hydrophilicity, which helps define the channel 614. Increased hydrophilicity can be achieved, for example, as described above.

The droplet generator 606 and any microfluidic circuit in which the droplet generator 606 provides droplets can be like any microfluidic device shown in the drawings or described herein. It can be part of a device (integral or connected). One droplet generator 606 is shown in FIGS. 6 and 7, but two or more such droplet generators 606 can provide droplets to the microfluidic circuits of devices 600 and 700. The droplet generator 606 itself can include an electrowetting configuration and thus can include a substrate having a photoresponsive layer, the photoresponsive layer being a-Si: H (eg, US Pat. Optical actuated circuit boards (as shown in US Patent Application Publication No. 2014/0124370), phototransistor-based boards (eg, US Pat. No. 7,965,339), as shown in 958,132. (As shown in No. 8) or electrically actuated circuit boards (eg, as shown in US Pat. No. 8,685,344) can be included. Alternatively, the droplet generator has a T-shaped or Y-shaped hydrodynamic structure (eg, US Pat. No. 7,708,949, US Pat. No. 7,041,481 (reissued as RE41,780), Having U.S. Patent Application Publication No. 2008/0014589, U.S. Patent Application Publication No. 2008/0003142, U.S. Patent Application Publication No. 2010/0137163, and U.S. Patent Application Publication No. 2010/0172383) can. All of the above US patent documents are incorporated herein by reference in their entirety.

As shown, the droplet generator 606 is connected to one or more fluid inlets 602 and 604 (two are shown, but may be less or more) and the microfluidic channel 614. Can include a fluid outlet 208 and the like. Through inlets 602 and 604, liquid media 622, 624, biological microobjects 630, reagents, and / or other biological media can be loaded into the droplet generator 606. The droplet generator 606 can contain droplets of a liquid medium 622 (which can include, but does not have to be, one or more biological microobjects 630), a reagent, or another biological medium. It can be generated and output to channel 614. If the channel 614 has an electrowetting configuration, the droplet 620 can move within the channel 614 using electrowetting (or optical electrowetting). Alternatively, the droplet 620 can be moved within the channel 614 by other means. For example, the droplet 620 can move within the channel 614 using fluid flow, gravity, or the like.

As described 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 are filled with the second fluid medium 622. can do. The second fluid medium 622 (hereinafter, “aqueous medium”) can be an aqueous medium such as a sample medium for maintaining or culturing the biological microobject 630. The first fluid medium 624 (hereinafter, "immiscible medium") can be a medium in which the aqueous medium 622 is immiscible. Examples of the aqueous medium 622 and the immiscible medium 624 include any of the examples described above for various media.

A droplet generator 606 can be utilized to load biological microobjects and / or facilitate the execution of biological and / or molecular biological workflows in microfluidic devices. 6 and 7 show non-limiting examples. By using a drop generator, the device can have an electrowetting configuration throughout the fluid circuit.

6 and 7 show an example in which the droplet generator 606 produces a droplet 620 containing a reagent (or other biomaterial). The reagent-containing droplet 620 can travel through the microfluidic channel 614 to one of the chambers 616/716 containing the immiscible medium 624. Before or after moving the reagent-containing droplet 620 to one of the chambers 616/716, one or more microobjects 630 in one or more droplets 632 may move into the same chamber 616/716. can. The reagent-containing droplet 620 can then be mixed with the droplet 632 containing the microscopic object 630 to mix the reagent of the droplet 620 with the contents of the droplet 632 and chemically react. One or more microobject-containing droplets 632 can be supplied by the droplet generator 606 (not shown) or obtained from the retention pen 616 as shown in FIGS. 6 and 7. can. The microobject 630 can be a biological microobject such as a cell that has been optionally cultured (eg, in chamber 616) prior to moving to processing chamber 616/716. Alternatively, the microobject 630 is directed to the molecule of interest in the sample (eg, cell secretions present in the sample material 622 after culturing one or more living cells using the sample material 622). It can be a bead, such as an affinity bead, that can be bound. In yet another alternative, one or more droplets 632 can be micro-object free, eg, cell secretions after culturing one or more living cells using sample material 622. Including, only aqueous media such as sample material 622 can be included.

FIG. 8 shows an example of process 800 that can be performed in a microfluidic device that includes a microfluidic circuit such as any of the devices 600 and 700.

In step 802 of process 800, the biological microobject can be cultured in a retention pen filled with a sample medium (eg, a cell culture medium). For example, the micro-object 630 of FIG. 6 or 7 can be a biological micro-object and can be cultured in chamber 616. The culture can generally be as described above. For example, culturing may include perfusing channel 612 with culture medium 622. Step 802 can be performed over a specified time period.

In step 804, the cultured biological microobject can be moved from the sample medium filling chamber 616 in which it was cultured to the chamber 616/716 filled with a medium in which the sample medium is immiscible. For example, as described above, as shown in FIGS. 6 and 7, the cultured microobject 630 is from one of the holding pens 616 to one of the holding pens 616/716 in the droplet 620 or 632 of the sample medium 622. You can move to one.

In step 806, the cultured biological microobjects can undergo one or more treatments or processes in an immiscible medium-filled retention pen. For example, as shown in FIGS. 6 and 7, as described above, one or more droplets 620 containing one or more reagents are generated by the droplet generator 606 and the immiscible medium filling chamber 612 /. It can be moved into 716 and mixed with a droplet 632 containing the cultured biological microobject 630. For example, the first reagent-containing droplet 620 can contain a dissolution reagent. Mixing the droplet 632 containing the cultured biological microobject 630 with the first reagent-containing droplet 620 containing the lysis reagent dissolves the cultured biological microobject 630. In other words, bound droplets (not shown) containing cytolysates from the cultured biological microobjects 630 are formed. The additional (eg, second, third, fourth, etc.) reagent-containing droplets 620 are then mixed with new droplets containing the cytolysate to further treat the cytolyte as needed. be able to.

In addition or as another example, one or more other materials (eg, nucleic acids such as DNA or RNA, proteins, metabolites, or) that produced the secretion or cultured biological microobject 630 of interest. One or more droplets containing one or more labeled trapped micro-objects (not shown) that have an affinity for (other biomolecules) are generated by the droplet generator 606 and immiscible. It can be moved to a sex medium filling pen 616 or 716 and mixed with droplets of sample medium 622 containing the similarly cultured biological microobjects 630. If the cultured biological microobjects 630 have already been lysed, the captured microobject-containing droplets 620 have an affinity for one or more affinity beads (eg, DNA, RNA, or nucleic acids such as microRNA). Affinity beads can bind to target molecules present in the lysate when mixed with cell lysate-containing droplets in a retention pen 616 or 716.

In step 808, the target biological microobject can be optionally processed. For example, in step 806, if the capture object (not shown) moves into the immiscible medium filling chamber 616/716 with the cultured biological microobject 630, the labeled capture micron is in step 808. Chambers 616/716 can be monitored for reactions (eg, fluorescent signals) that indicate the quantity of material of interest bound to the object. Alternatively, such trapped microobjects (not shown) are removed from chamber 616/716 (eg, in droplet 622) and microfluidic devices (not shown in FIGS. 6 and 7) for subsequent analysis. Can be carried out from. As yet another example, the treated biological microobject 630 is removed from chamber 616/716 (eg, in droplet 632) and removed from a microfluidic device (not shown) for subsequent analysis. Can be done.

FIG. 9 outlines a method of forming a substrate for a microfluidic device that includes both an electrowetting configuration and a dielectrophoresis (DEP) configuration. For example, the method shown in FIG. 9 can be used to form the type of monolithic substrate shown in the microfluidic device of FIG. 10 to 18 show cross-sectional views of the intermediate structure formed after the various steps in the method of FIG. 9 have been performed. A substrate having a DEP configuration including an array of phototransistors is a starting point in FIGS. 10-18. Of course, as those skilled in the art will understand, the starting substrate is not limited to a DEP-configured substrate having an array of phototransistors, but rather a substrate containing a layer of amorphous silicon or an array of electrically actuated electrodes, or the like. It can also be applied to other types of substrates. In addition, the steps in the method of FIG. 9 can be used independently and / or in other combinations to include other types of microfluidics having a conductive substrate, including the other microfluidic devices described herein. You can also generate a device.

Step 902 in the method of FIG. 9 involves preparing an initial substrate for further processing. As shown in the vertical cross section of FIG. 10, the initial substrate 1000 includes a high concentration doped layer 1010 of conductive silicon on which an array of phototransistors 1020 is formed. The step of preparing substrate 1000 can include a thermal annealing process. The process of step 902 can prepare the surface of substrate 1000 to ensure proper bonding of materials that are subsequently deposited on substrate 1000.

Step 904 in the method of FIG. 9 involves depositing a selective etching resistant material on the top surface of the initial substrate. As shown in the vertical cross section in FIG. 11, the layer 1130 of the conditional etching resistant material is deposited on the upper surface of the substrate 1000 so as to cover the surface of the arrayed phototransistors 1020. In some embodiments, the conditional etching resistant material 1130 can be a nitride.

Step 906 in the method of FIG. 9 comprises applying a first pattern onto the conditional etching resistant material deposited on the substrate during step 904. As shown in FIG. 12, the pattern allows the conditional etching resistant material 1130 to be removed from the selected region of the substrate 1000 (eg, the surface of the phototransistor array on the left side of the substrate 1000). Coating of the pattern on the conditional etching resistant material 1130 deposited on the substrate 1000 during step 904 can be achieved by a lithography process, as is well known in the semiconductor processing industry. Such lithographic processes include, for example, E-beam, X-ray, UV, and deep UV. Polymers are typically used to define the pattern.

As described in step 908 of the method of FIG. 9, the pattern (eg, polymer) deposited in step 906 subsequently deposits a photoresponsive layer on the pattern and then selects a portion of the photoresponsive layer. It is treated by exposing it to light (eg, light having a wavelength and intensity suitable for the material of the photoresponsive layer).

Step 910 in the method of FIG. 9 comprises etching the photoresponsive layer (and any conditional etching resistant material beneath the etchable portion of the photoresponsive layer) downward to a first predetermined position. As shown in FIG. 12, the first predetermined position can be, for example, the surface of the substrate (eg, the surface of the phototransistor 1020).

A set of optional subsequent steps (not shown) in the method of FIG. 9 is to deposit, pattern, and etch a layer of conductive material after the conditional etching resistant layer is patterned on the substrate. That is. As shown in FIG. 13, both the surface of the substrate (eg, the surface of the phototransistor 1020 on the left side of the substrate 1000) and the portion of the conditional etching resistant layer 1130 that was not removed during steps 908 and 910. Can be deposited on top. The conductive material 1330 can be, for example, conductive silicon such as amorphous silicon or high concentration doped silicon. As shown in FIG. 14, the conductive material 1330 is then directly deposited on the top (eg, on the surface of the phototransistor 1020 on the left side of the substrate 1000) by patterning and etching the conductive material 1330. The portion 1 and the layer 1130 of the conditional etching resistant material can produce a second portion of the substrate 1000 deposited directly on top (eg, on the surface of the phototransistor 1020 on the right side of the substrate 1000).

Step 912 in the method of FIG. 9 comprises depositing at least one dielectric layer on the substrate (and any material already deposited on the substrate and not removed by etching). As described elsewhere in the specification (eg, in connection with the device of FIG. 1B), the individual layers of the dielectric layer stack (eg, the first layer of the dielectric material, the second layer of the dielectric material). Layer, a third layer of dielectric material, etc.) can be sequentially deposited on the substrate. For example, as shown in FIG. 15, a dielectric laminate 1530 made of two layers of dielectric material can be deposited on the substrate 1000. For consistency with the other sections herein, the first layer of the dielectric laminate 1530 need not be the first layer deposited on the substrate 1000. Rather, the terms first and second can be used with respect to the order of layers of dielectric material, starting arbitrarily or from the surface and moving toward the inside of the substrate. Therefore, with respect to FIG. 15, the first layer of the dielectric material deposited on the substrate 1000 can be the "second layer" of the dielectric material and the second layer of the dielectric material deposited on the substrate 1000. Can be the "first layer" of the dielectric material.

Step 914 in the method of FIG. 9 comprises applying a second pattern onto at least one dielectric layer and etching at least one dielectric layer to a second predetermined position. In some embodiments, the second predetermined location may be the surface of layer 1130 of conditional etching resistant material. Thus, as shown in FIG. 16, the layer of the dielectric laminate 1530 can be etched down to the surface of the conditional etching resistant material 1130 away from the selected portion of the substrate 1000. As mentioned above, the conditional etching resistant material 1130 can be a nitride. Therefore, the etching material used in step 914 may be a material suitable for etching and removing the dielectric material but not the nitride.

In various embodiments, further optional steps may be performed. For example, a third pattern can be deposited and the conditional etching resistant layer can be stripped (optionally, up to 10 um can be etched into the silicon substrate). As shown in FIG. 17, the conditional etching resistant layer 1130 is etched and removed from the right side of the substrate 1000, resulting in re-exposing the surface of the phototransistor 1020 on the right side. In addition, as shown in FIG. 18, the steps of stripping the oxide at the bottom of the substrate 1000, metallizing the back surface, and adding a layer 1830 of a conductive metal (eg, silver or gold) to the substrate can be performed. can. The resulting substrate, shown in FIG. 18, has a first section configured to generate a DEP force (eg, right side) and a second section configured to generate an electwetting force. (For example, on the left side) and can have. At the junction of the first section and the second section, the substrate can be electrically inactive with respect to the generation of at least DEP and electrowetting forces. The thickness of the inactive region depends on the accuracy of the masking and etching steps, for example, with a thickness of less than 2 mm (eg, less than 1.5 mm, less than 1.0 mm, less than 0.5 mm, or less). could be.

Although specific embodiments and applications of the present invention have been described herein, these embodiments and applications are merely examples and many variations are possible. For example, the method of FIG. 8 can be performed on a sample material containing cell secretions (eg, after the sample material 682 has been used to culture one or more living cells). In such an embodiment, step 802 remains the same, but step 804 is immiscible with droplet 632, which is free of microobjects and can contain only aqueous media such as sample material 622 containing cell secretions. Steps 806 and 808 are performed on such aqueous medium-containing droplets 632, including moving to a sex medium-containing chamber 616/716. Further, the electrowetting configurations discussed herein can be any type of electrowetting configuration known in the art, examples of which are US Pat. No. 6,958,132 (for OEW configurations). ) And US Patent Application Publication No. 2016/0158748 (for single-sided OEW configuration). Another example of an electrowetting configuration is a dielectric electrowetting (EWOD) device that can be electronically controlled, an example of which is disclosed in US Pat. No. 8,685,344. Similarly, the dielectrophoretic configurations discussed herein can be of any type of dielectrophoretic configuration known in the art, examples of which are US Pat. Nos. RE44,711 (Wu et al.), 7. , 956,339 (Ohta et al.), 6,294,063 (Becker et al.), 6,942,776 (Medoro), and 9,403,172 (Wu et al.). Has been done. All of the above US patent documents are incorporated herein by reference in their entirety.

Systems and Microfluidic Devices: Microfluidic devices and equipment to operate microfluidic devices were manufactured by Berkeley Lights, Inc. The system included at least a flow controller, a temperature controller, fluid medium conditioning and pump components, a light source for a photoactivated DEP or EW configuration, a mount stage, and a camera. The microfluidic device included an EW configuration with a surface described below.

Example 1. Preparation of electrowetting microfluidic device with modified inner surface A base containing an electrode activated substrate having a semiconductor layer of photosensitive silicon and a dielectric layer having an upper surface of alumina, and a cover having a glass support having an ITO electrode. , Microfluidic device (Berkeley Lights, Inc.) with an optical patternable silicon microfluidic circuit material that separates the base and cover, 100 W power, 240 m toll (about 31.9 Pa).
Treatment was performed in an oxygen plasma cleaner (Nordson Asymtek) for 1 minute using pressure and 440 sccm oxygen flow. Trimethoxy (3,3,4) in a foil boat at the bottom of the vacuum reaction chamber
, 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) -Nonaicosafluorohexadecyl) Silane (0.3 g, details of synthesis are described in US Provisional Patent Application No. 62/410238, filed October 19, 2016), vacuum. Plasma treated microfluidic devices are treated in a vacuum reaction chamber in the presence of magnesium sulphate heptahydrate (0.5 g, Acros) as a water reactant source in a separate foil boat at the bottom of the reaction chamber. bottom. Next, a vacuum pump was used to bring the chamber to 750 m toll (about 99.9 Pa) and seal it. The vacuum reaction chamber was placed in an oven heated to 180 ° C. for 24 to 48 hours. After cooling to room temperature and introducing argon into the vacuum chamber, dimethoxy (3,3,4,5,5,6,6,7,7,8,8,9,9,10,10) on all inner surfaces , 11,11,12,12,13,13,14,14,15,15,16,16,16-nonacosafluoro-hexadecyl) A microfluidic device with a hydrophobic outer layer of the siloxy moiety was removed from the reaction chamber. .. After removal, the microfluidic device was primed with silicone oil (5 cm stalk (5 mm ² / s) viscosity, Gelest catalog number DMS-T05) prior to use. 20A-20C are continuous photographic images of water droplets moving on a hydrophobic layer (ie, droplet working surface) within an immiscible silicone oil phase. The droplets demonstrated excellent mobility using the optically actuated electrowetting configuration and droplet actuating surface of the microfluidic device.

Listing of embodiments 1. A microfluidic device with an electrowetting configuration
A substrate having a dielectric layer, a droplet working surface, and a first electrode configured to be connected to an AC voltage source.
Includes a second electrode configured to be connected to an AC voltage source
The dielectric layer is electrically coupled to the first electrode and
A microfluidic device in which the droplet working surface contains a hydrophobic layer covalently bonded to a dielectric layer.

2. The microfluidic device according to embodiment 1, which has a single-sided electrowetting configuration.

3. 3. The microfluidic device according to the second embodiment, wherein the second electrode is a mesh electrode included in the substrate.

4. The microfluidic device according to embodiment 1, which has an optical electrowetting (OEW) configuration.

5. The microfluidic device according to embodiment 1, which has a dielectric electrowetting (EWOD) configuration.

の構造を有し、式中、

は、誘電層の表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）_２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態１〜５のいずれか１つに記載のマイクロ流体デバイス。 6. The hydrophobic layer is a monolayer containing a surface modifying ligand and a binding group that binds the surface modifying ligand to the surface, and the droplet working surface is of formula II :.

Has the structure of

Is the surface of the dielectric layer, V is -P (O) (OY) W- or -Si (OZ) ₂ W-, and W is -O-, -S-, or -NH-. Yes, and connected to the surface, Z is a bond to an adjacent silicon atom attached to the surface, or is a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface. Or a bond to the surface, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is. It is 0 or 1, m is an integer of 0 or 1 to 20, n is an integer of 0 or 1 to 20, and the sum of (n + [(h + j) · k] + m) is 11 to 25. If k is 1, then m is at least 2, and if M is hydrogen, and k is 0, and R is fluorine, then m is at least 2. The microfluidic device according to any one of embodiments 1 to 5, wherein M is hydrogen.

7. 13. Microfluidic device.

8. A microfluidic device having a substrate having at least one electrode configured to be connected to a voltage source and a cover having at least one electrode configured to be connected to a voltage source and at least one. Including separating elements
The substrate and cover are substantially parallel to each other and are joined together by a separating element to define an enclosure configured to hold the liquid, and the substrate is a droplet actuation that partially defines the enclosure. It has a surface, the droplet working surface has a dielectric inner layer and a hydrophobic outer layer,
The hydrophobic outer layer contains self-associating molecules covalently bonded to the surface of the dielectric inner layer, thereby forming a dense hydrophobic monolayer on it.
When at least one electrode on the substrate and at least one electrode on the cover are connected to the opposite terminals of the voltage source, the substrate shall apply electrowetting force to the aqueous droplets in contact with the droplet working surface of the substrate. Is possible, microfluidic device.

の構造を有し、式中、

は、誘電層の表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）_２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態８に記載のマイクロ流体装置。 9. The self-associating molecule of the hydrophobic monolayer contains a surface modifying ligand and a binding group that binds the surface modifying ligand to the surface of the dielectric inner layer, respectively, and the droplet working surface is of formula II :.

Has the structure of

Is the surface of the dielectric layer, V is -P (O) (OY) W- or -Si (OZ) ₂ W-, and W is -O-, -S-, or -NH-. Yes, and connected to the surface, Z is a bond to an adjacent silicon atom attached to the surface, or is a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface. Or a bond to the surface, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is. It is 0 or 1, m is an integer of 0 or 1 to 20, n is an integer of 0 or 1 to 20, and the sum of (n + [(h + j) · k] + m) is 11 to 25. If k is 1, then m is at least 2, and if M is hydrogen, and k is 0, and R is fluorine, then m is at least 2. The microfluidic apparatus according to the eighth embodiment, wherein M is hydrogen.

10. The microfluidic device according to embodiment 9, wherein V is −Si (OZ) _{2 W−.}

11. The microfluidic device according to embodiment 9, wherein V is −P (O) (OY) W−.

12. The microfluidic device according to any one of embodiments 9 to 11, wherein n is an integer of 1 to 20 and R is hydrogen.

13. The microfluidic device according to embodiment 12, wherein m is an integer of 1 to 20 and M is hydrogen.

14. The microfluidic device according to the thirteenth embodiment, wherein m is 2.

15. The microfluidic device according to any one of embodiments 9 to 11, wherein n is an integer of 1 to 20 and R is fluorine.

16. The microfluidic device according to embodiment 15, wherein m is an integer of 1 to 20 and M is hydrogen.

17. The microfluidic device according to embodiment 16, wherein m is 2.

18. The microfluidic device according to any one of embodiments 9 to 17, wherein k is 1.

19. The microfluidic device according to any one of embodiments 9 to 17, wherein k is 0.

20. The microfluidic device according to any one of embodiments 9 to 19, wherein the sum of (n + [(h + j) · k] + m) is an integer of 13 to 19.

21. The microfluidic device according to any one of embodiments 8 to 20, wherein the hydrophobic outer layer of the droplet working surface of the substrate has a thickness of less than 5 nanometers.

22. The hydrophobic outer layer of the droplet working surface of the substrate is patterned such that the selected region is relatively hydrophilic relative to the rest of the hydrophobic outer layer, any one of embodiments 8-21. The microfluidic device described in.

23. The microfluidic device according to any one of embodiments 8 to 22, wherein the dielectric inner layer of the droplet working surface of the substrate comprises a first layer of a dielectric material containing an oxide.

24. The microfluidic device according to any one of embodiments 8 to 23, wherein the oxide is a metal oxide.

25. The microfluidic device according to embodiment 24, wherein the metal oxide is aluminum oxide.

26. The microfluidic device according to any one of embodiments 23-25, wherein the first layer of the dielectric material is formed by atomic layer deposition.

27. The dielectric inner layer of the droplet working surface of the substrate further comprises a second layer of the dielectric material, and the hydrophobic outer layer is covalently bonded to the first layer of the dielectric material, any one of embodiments 23-26. One of the microfluidic devices described.

28. The microfluidic device of embodiment 27, wherein the second layer of dielectric material comprises an oxide or nitride.

29. 28. The microfluidic device of embodiment 28, wherein the second layer of dielectric material is selected from the group consisting of silicon dioxide and silicon nitride.

30. The microfluidic device according to any one of embodiments 27-29, wherein the second layer of the dielectric material is formed by plasma chemical vapor deposition.

31. The first layer of the dielectric material comprises the first and second sublayers of the dielectric material, the first sublayer being covalently bonded to the hydrophobic layer, any one of embodiments 23-30. The microfluidic device described in.

32. The microfluidic device according to embodiment 31, wherein the first sublayer of the dielectric material comprises silicon oxide.

33. The microfluidic device of embodiment 31, wherein the first adrenoleukodys of the dielectric material is deposited by ALD.

34. The microfluidic device according to any one of embodiments 31 to 33, wherein the first layer of the dielectric material has a thickness of about 10 nm to about 20 nm.

35. The microfluidic device according to embodiment 34, wherein the first sublayer of the dielectric material has a thickness of about 2 nm to about 10 nm.

36. The microfluidic device according to any one of embodiments 8 to 35, wherein the dielectric inner layer of the droplet working surface of the substrate has a thickness of at least about 40 nanometers.

37. The microfluidic device of embodiment 36, wherein the dielectric inner layer of the droplet working surface of the substrate has a thickness of about 40 nanometers to about 120 nanometers.

38. The microfluidic device according to any one of embodiments 8 to 37, wherein the substrate further comprises a photoresponsive layer having a first surface in contact with the dielectric inner layer and a second surface in contact with at least one electrode.

39. The microfluidic device according to embodiment 38, wherein the photoresponsive layer comprises hydrogenated amorphous silicon (a-Si: H).

40. The microfluidic device according to embodiment 38 or 39, wherein the photoresponsive layer has a thickness of at least 900 nanometers.

41. The microfluidic device of embodiment 40, wherein the photoresponsive layer has a thickness of about 900 nanometers to 1100 nanometers.

42. 38. The microfluidic device of embodiment 38, wherein the photoresponsive layer comprises a plurality of conductors, each of which is controllably connectable to at least one electrode of the substrate via a phototransistor switch.

43. The substrate comprises a single electrode configured to be connected to an AC voltage source, the single electrode comprising a layer of indium tin oxide (ITO), any one of embodiments 8-42. The microfluidic device described in.

44. The micro according to any one of embodiments 8 to 42, wherein the substrate comprises a single electrode configured to be connected to an AC voltage source, wherein the single electrode comprises a layer of conductive silicon. Fluid device.

45. The microfluidic device according to any one of embodiments 8 to 37, wherein the substrate comprises a plurality of electrodes, each electrode being configured to be connected to one or more AC voltage sources.

46. The microfluidic device of embodiment 45, wherein each electrode of the plurality is connectable to one or more AC voltage sources via a transistor switch.

47. The cover has an inward surface that partially defines the enclosure, the inward surface of the cover has an inner layer and a hydrophobic outer layer, and the hydrophobic outer layer of the cover is covalently bonded to the surface of the inner layer of the cover. The microfluidic device according to any one of embodiments 8 to 46, wherein the microfluidic device comprises an associative molecule, thereby forming a dense hydrophobic monolayer on it.

の構造体を有し、式中、

は、誘電層の表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）_２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態４７に記載のマイクロ流体デバイス。 48. The self-associating molecule of the hydrophobic monolayer of the cover contains a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface of the inner layer of the cover, respectively, and the inward surface of the cover is of formula II :.

Has the structure of

Is the surface of the dielectric layer, V is -P (O) (OY) W- or -Si (OZ) ₂ W-, and W is -O-, -S-, or -NH-. Yes, and connected to the surface, Z is a bond to an adjacent silicon atom attached to the surface, or is a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface. Or a bond to the surface, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is. It is 0 or 1, m is an integer of 0 or 1 to 20, n is an integer of 0 or 1 to 20, and the sum of (n + [(h + j) · k] + m) is 11 to 25. If k is 1, then m is at least 2, and if M is hydrogen, and k is 0, and R is fluorine, then m is at least 2. The microfluidic device according to embodiment 47, wherein M is hydrogen.

49. The microfluidic device of embodiment 48, wherein the hydrophobic monolayer self-associating molecule of the cover is the same as the hydrophobic monolayer self-associating molecule of the droplet working surface of the substrate.

50. The microfluidic device according to any one of embodiments 47-49, wherein the hydrophobic outer layer of the inward facing surface of the cover has a thickness of less than 5 nanometers.

51. The microfluidic device according to any one of embodiments 47 to 50, wherein the inner layer of the cover is a dielectric inner layer.

52. The microfluidic device according to embodiment 51, wherein the cover further comprises a photoresponsive layer.

53. 51. The microfluidic device of embodiment 51, wherein the cover comprises a plurality of electrodes, each electrode being configured to be connected to one or more AC voltage sources.

54. The microfluidic device of embodiment 8, wherein the at least one separating element comprises a silicon-based organic polymer.

55. The microfluidic device of embodiment 54, wherein the silicon-based organic polymer is selected from the group consisting of polydimethylsiloxane (PDMS) and photopatternable silicone (PPS).

56. The microfluidic device according to any one of embodiments 8 to 53, wherein at least one separating element comprises SU-8.

57. The microfluidic device according to any one of embodiments 8 to 56, wherein the at least one separating element has a thickness of at least 30 μm.

58. The microfluidic device of any one of embodiments 8-57, wherein the at least one separating element defines one or more microchannels within the enclosure.

59. 58. The microfluidic device of embodiment 58, wherein the at least one separating element further defines a plurality of chambers within the enclosure, each chamber opening at the end of at least one microchannel.

60. A method of manufacturing a microfluidic device, in which a separating element is joined to the inner surface of a cover having at least one electrode configured to be connected to a voltage source.
By joining the separating element and cover to the dielectric surface of a substrate having at least one electrode configured to be connected to a voltage source, the separating element is such that the cover and substrate are substantially parallel to each other. In an oriented state, sandwiched between the inner layer of the cover and the dielectric surface of the substrate, the substrate, separating elements, and cover collectively define an enclosure configured to hold liquid. By joining and vapor deposition, a dense hydrophobic monolayer is formed on at least a portion of the inner surface of the cover, the hydrophobic monolayer containing self-associating molecules covalently attached to the inner surface of the cover. , Forming and
By vapor deposition, a high density hydrophobic monolayer is formed on at least a portion of the dielectric surface of the substrate, the hydrophobic monolayer containing self-associating molecules covalently bonded to the dielectric surface of the substrate. Methods, including with and to do.

の構造体を有し、式中、

は、誘電層の表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）_２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態６０に記載の方法。 61. The self-associating molecule of the hydrophobic molecule of the cover and the self-associating molecule of the hydrophobic monolayer of the substrate have a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the inner surface of the cover and the dielectric surface of the substrate, respectively. Containing, the resulting surface of the cover and substrate is formulated in Formula II :.

Has the structure of

Is the surface of the dielectric layer, V is -P (O) (OY) W- or -Si (OZ) ₂ W-, and W is -O-, -S-, or -NH-. Yes, and connected to the surface, Z is a bond to an adjacent silicon atom attached to the surface, or is a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface. Or a bond to the surface, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is. It is 0 or 1, m is an integer of 0 or 1 to 20, n is an integer of 0 or 1 to 20, and the sum of (n + [(h + j) · k] + m) is 11 to 25. If k is 1, then m is at least 2, and if M is hydrogen, and k is 0, and R is fluorine, then m is at least 2. The method according to embodiment 60, wherein M is hydrogen.

62. The method according to embodiment 61, wherein V is −Si (OZ) _{2 W−.}

63. The method according to embodiment 61, wherein V is −P (O) (OY) W−.

64. The method according to any one of embodiments 61 to 63, wherein n is an integer of 1 to 20 and R is hydrogen.

65. The method according to embodiment 64, wherein m is an integer of 1 to 20 and M is hydrogen.

66. The method according to embodiment 65, wherein m is 2.

67. The method according to any one of embodiments 61 to 63, wherein n is an integer of 1 to 20 and R is fluorine.

68. The method of embodiment 67, wherein m is an integer of 1 to 20 and M is hydrogen.

69. The method according to embodiment 68, wherein m is 2.

70. The method according to any one of embodiments 61-69, wherein k is 1.

71. The method according to any one of embodiments 61-69, wherein k is 0.

72. The method according to any one of embodiments 61 to 71, wherein the sum of (n + [(h + j) · k] + m) is an integer of 13 to 19.

73. A microfluidic device, a conductive silicon substrate having a dielectric laminate and at least one electrode configured to be connected to a voltage source, and at least one configured to be connected to a voltage source. Includes a cover with electrodes and at least one separating element
The conductive silicon substrate and cover define an enclosure that is substantially parallel to each other and joined together by separating elements to hold the liquid.
The conductive silicon substrate has an inward facing surface that partially defines the enclosure, the inward facing surface including the outermost surface of the dielectric laminate.
When at least one electrode on the substrate and at least one electrode on the cover are connected to the opposite terminals of the AC voltage source, the substrate shall apply electrowetting forces to the aqueous droplets in contact with the inward surface of the substrate. Is possible, microfluidic device.

74. The microfluidic apparatus according to embodiment 73, wherein the conductive silicon substrate contains amorphous silicon.

75. The microfluidic apparatus according to embodiment 73, wherein the conductive silicon substrate comprises a phototransistor array.

76. The microfluidic apparatus according to embodiment 73, wherein the conductive silicon substrate comprises an array of electrodes.

77. 13. Microfluidic device.

78. The microfluidic apparatus according to any one of embodiments 73-77, wherein the internal dielectric laminate comprises a first layer of dielectric material and a second layer of dielectric material.

79. The first layer of the dielectric material has a first surface and an inverted surface, the first surface of the first layer is adjacent to the second layer, and the inverted surface of the first layer is dielectric. The microfluidic apparatus according to embodiment 78, which forms the outermost surface of the body laminate.

80. The microfluidic apparatus according to embodiment 78 or 79, wherein the first layer of the dielectric material comprises a metal oxide.

81. The microfluidic apparatus according to embodiment 80, wherein the first layer of the dielectric material comprises aluminum oxide or hafnium oxide.

82. The microfluidic apparatus according to any one of embodiments 78-81, wherein the second layer of the dielectric material comprises an oxide or a nitride.

83. The microfluidic apparatus according to embodiment 82, wherein the second layer of the dielectric material comprises silicon oxide or silicon nitride.

84. The microfluidic apparatus according to any one of embodiments 78-83, wherein the second layer is deposited by a plasma chemical vapor deposition (PECVD) technique.

85. The microfluidic apparatus according to any one of embodiments 78-84, wherein the first layer is deposited by an atomic layer deposition (ALD) technique.

86. The internal dielectric laminate comprises a third layer having a first surface and a reverse surface, the first surface of the third layer is adjacent to the first layer, and the reverse surface of the third layer is The microfluidic apparatus according to any one of embodiments 78 to 85, which forms the outermost surface of the dielectric laminate.

87. The microfluidic apparatus according to embodiment 86, wherein the third layer contains silicon oxide.

88. The microfluidic apparatus according to embodiment 86 or 87, wherein the third layer is deposited by an atomic layer deposition (ALD) technique.

89. The microfluidic apparatus according to any one of embodiments 78-85, wherein the first layer of the dielectric material has a thickness of about 10 nm to about 50 nm.

90. Any of embodiments 86-88, the first layer of the dielectric material has a thickness of about 5 nm to about 20 nm, and the third layer of the dielectric material has a thickness of about 2 nm to 10 nm. The microfluidic device according to one.

91. The microfluidic apparatus according to any one of embodiments 78-90, wherein the second layer of the dielectric material has a thickness of about 30 nm to about 100 nm.

92. The microfluidic apparatus according to any one of embodiments 73-91, wherein the dielectric stacking of the droplet working surface of the substrate has a thickness of at least about 40 nanometers.

93. The microfluidic apparatus according to embodiment 92, wherein the dielectric stacking of the droplet working surface of the substrate has a thickness of about 40 nanometers to about 120 nanometers.

94. The microfluidic apparatus according to any one of embodiments 73 to 93, wherein the dielectric layer has an impedance of about 50 k ohms to about 150 k ohms.

95.
A dielectrophoresis module that performs a first microfluidic operation in response to a first applied voltage at a first frequency,
Includes an electrowetting module that receives output from the dielectrophoresis module and performs a second microfluidic operation in response to a second applied voltage at a second frequency.
The microfluidic device according to any one of embodiments 73 to 94, wherein the electrowetting module comprises a dielectric lamination of a conductive silicon substrate.

96. The microfluidic apparatus according to embodiment 95, further comprising a bridge between the first module and the second module.

97. The microfluidic device according to embodiment 96, wherein the bridge does not perform a first or second microfluidic operation.

98. The microfluidic device according to embodiment 96 or 97, wherein the bridge is electrically neutral.

99. The microfluidic device according to any one of embodiments 96-98, wherein the bridge comprises a tube.

100. The microfluidic device according to any one of embodiments 96-98, wherein the bridge comprises a polymer.

101. The microfluidic apparatus according to any one of embodiments 95-100, wherein the output comprises a biomaterial.

102. The microfluidic apparatus according to any one of embodiments 95 to 101, wherein the first frequency is in the range of 100 kHz to 10 MHz.

103. The microfluidic apparatus according to any one of embodiments 95 to 102, wherein the second frequency is in the range of 1 kHz to 300 kHz.

104. The microfluidic device according to any one of embodiments 95 to 103, wherein the first voltage is in the range of 1 to 10 volts.

105. The microfluidic device according to any one of embodiments 95 to 104, wherein the second voltage is in the range of 10 to 100 volts.

106. The microfluidic apparatus according to any one of embodiments 95 to 105, wherein the conductive silicon substrate is monolithic.

107. The microfluidic apparatus according to any one of embodiments 95 to 106, wherein the conductive silicon substrate is dual chic.

108. The microfluidic apparatus according to embodiment 106, wherein the conductive silicon substrate contains amorphous silicon.

109. The microfluidic apparatus according to embodiment 107, wherein the conductive silicon substrate contains amorphous silicon.

110. The microfluidic apparatus according to embodiment 106, wherein the conductive silicon substrate comprises a phototransistor array.

111. The microfluidic apparatus according to embodiment 107, wherein the conductive silicon substrate comprises a phototransistor array.

112. The microfluidic apparatus according to embodiment 106, wherein the conductive silicon substrate comprises an array of electrodes.

113. The microfluidic apparatus according to embodiment 107, wherein the conductive silicon substrate comprises an array of electrodes.

114. A system for transporting microobjects, biological substances, and / or reagents that are compatible with and / or meltable in aqueous media.
A microfluidic device having an enclosure containing a base and a microfluidic circuit structure, wherein the base comprises a hydrophobic monolayer covalently bonded to at least a portion of the upper surface of the base.
A first fluid medium that does not mix with the aqueous medium,
A system comprising at least one aqueous droplet.

の構造体を有し、式中、

は、表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態１１４に記載のシステム。 115. The hydrophobic monolayer has a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface, and the hydrophobic surface is of formula II :.

Has the structure of

Is the surface, V is -P (O) (OY) W- or -Si (OZ) 2W-, W is -O-, -S-, or -NH-, and the surface. In connection with, Z is a bond to an adjacent silicon atom attached to the surface or a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface or to the surface. Bond, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is 0 or 1. Yes, m is an integer of 0 or 1-20, n is an integer of 0 or 1-20, and the sum of (n + [(h + j) · k] + m) is an integer of 11-25. , K is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. , Hydrogen, the system according to embodiment 114.

116. The system according to embodiment 114 or 115, wherein the base comprises a conductive substrate.

117. The system according to any one of embodiments 114 to 116, wherein the microfluidic device is the microfluidic device according to any one of embodiments 1-59.

118. The system according to embodiment 117, wherein the microfluidic device comprises an optically actuated EW configuration.

119. The system according to embodiment 117 or 118, wherein the microfluidic device further comprises a DEP configuration.

120. 13. system.

121. The system according to embodiment 120, wherein the main chain structure of the at least one organosilicon compound comprises a silicon atom and optionally an oxygen atom.

122. The system according to embodiment 120, wherein the main chain structure of at least one organic compound comprises a carbon atom and optionally an oxygen atom.

123. The system according to embodiment 122, wherein the main chain structure is branched.

124. The system according to any one of embodiments 120-123, wherein the first fluid medium comprises one or more acyclic organic compounds or organosilicon compounds.

125. The system according to embodiment 124, wherein the first fluid medium comprises an acyclic organic compound or an organosilicon compound.

126. The system according to any one of embodiments 114-125, wherein the first fluid medium is free of perfluorocarbon atoms.

127. The system according to any one of embodiments 114 to 125, wherein the substituent of the carbon atom of the compound of the first fluid medium contains 90% or less of a fluorine substituent.

128. The system according to any one of embodiments 115-125, wherein the surface-modifying ligand comprises at least a first moiety comprising a perfluorocarbonated carbon atom at the inward end of the hydrophobic monolayer.

129. The system according to embodiment 128, wherein all carbon atoms in the hydrophobic monolayer are perfluorolated.

130. The system according to any one of embodiments 114 to 129, wherein the first fluid medium comprises two or more organic compounds or organosilicon compounds.

131. The system according to any one of embodiments 114-130, wherein the enclosure further comprises a cover.

132. The system according to embodiment 131, wherein the cover transmits light.

133. The system according to embodiment 131 or 132, wherein the cover comprises glass and / or indium tantalum oxide (ITO).

134. The system according to any one of embodiments 131-133, wherein the cover comprises electrodes.

135. The system according to any one of embodiments 114-134, wherein the aqueous droplet comprises a surfactant.

136. The system according to embodiment 135, wherein the surfactant comprises a nonionic surfactant.

137. Surfactants include block alkylene oxide copolymers, fatty acid ester ethoxylated sorbitan, ethoxylated fluorine-based surfactants, sodium dodecyl sulfate, or 2,4,7,9, tetramethyl-5-decyne-4,7, -diol. The system according to embodiment 135 or 136, comprising ethoxylate.

138. The system according to any one of embodiments 135-137, wherein the surfactant comprises Capstone® FS-30 (DuPont Trademark, Synquest Laboratories).

139. The system according to any one of embodiments 114-139, wherein the droplet comprises a phosphate buffered physiological saline solution.

140. The system according to any one of embodiments 114-139, wherein the aqueous droplet comprises at least one microobject.

141. The system according to embodiment 140, wherein the micro-object is a biological micro-object.

142. The system according to any one of embodiments 114-141, wherein the aqueous droplet comprises a biological material comprising nucleic acid and / or protein.

143. The system according to any one of embodiments 114-142, wherein the aqueous droplet comprises a reagent.

144. A kit for transporting micro-objects, biological substances, and / or reagents that are compatible with and / or meltable in an aqueous medium and that have an enclosure containing a base and a microfluidic circuit structure. A kit comprising a microfluidic device comprising a hydrophobic monolayer covalently attached to at least a portion of the upper surface of the base and a first fluid medium that is immiscible with an aqueous medium.

の構造体を有し、式中、

は、表面であり、Ｖは、−Ｐ（Ｏ）（ＯＹ）Ｗ−又は−Ｓｉ（ＯＺ）２Ｗ−であり、Ｗは、−Ｏ−、−Ｓ−、又は−ＮＨ−であり、且つ表面に接続し、Ｚは、表面に付着した隣接ケイ素原子への結合であるか、又は表面への結合であり、Ｙは、表面に付着した隣接リン原子への結合であるか、又は表面への結合であり、Ｒは、水素又はフッ素であり、Ｍは、水素又はフッ素であり、ｈは、独立して２又は３の整数であり、ｊは、１であり、ｋは、０又は１であり、ｍは、０又は１〜２０の整数であり、ｎは、０又は１〜２０の整数であり、（ｎ＋［（ｈ＋ｊ）・ｋ］＋ｍ）の和は、１１〜２５の整数であり、ｋが１である場合、ｍは、少なくとも２であり、且つＭは、水素であり、及びｋが０であり、且つＲがフッ素である場合、ｍは、少なくとも２であり、且つＭは、水素である、実施形態１４４に記載のキット。 145. The hydrophobic monolayer has a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface, and the hydrophobic surface is of formula II :.

Has the structure of

Is the surface, V is -P (O) (OY) W- or -Si (OZ) 2W-, W is -O-, -S-, or -NH-, and the surface. In connection with, Z is a bond to an adjacent silicon atom attached to the surface or a bond to the surface, and Y is a bond to an adjacent phosphorus atom attached to the surface or to the surface. Bond, R is hydrogen or fluorine, M is hydrogen or fluorine, h is an independently integer of 2 or 3, j is 1, and k is 0 or 1. Yes, m is an integer of 0 or 1-20, n is an integer of 0 or 1-20, and the sum of (n + [(h + j) · k] + m) is an integer of 11-25. , K is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. , Hydrogen, the kit according to embodiment 144.

146. The kit according to embodiment 144 or 145, wherein the base comprises a conductive substrate.

147. The kit according to embodiment 144 or 145, wherein the microfluidic device is the microfluidic device according to any one of embodiments 1-59.

148. A process of operating the microfluidic apparatus according to any one of embodiments 8 to 59.
Filling the enclosure or part of it with a first fluid medium,
Applying an AC voltage potential between at least one electrode on the substrate and at least one electrode on the cover,
Introducing a first droplet of liquid into the enclosure, where the first droplet is immiscible with the first fluid medium.
A process comprising moving the first droplet to a desired position within the enclosure by applying an electrowetting force to the first droplet.

149. The process of embodiment 148, wherein the first liquid medium is oil.

150. The process according to embodiment 148, wherein the first liquid medium is a silicone oil, a fluorinated oil, or a combination thereof.

151. The process according to any one of embodiments 148-150, wherein the applied AC voltage potential is at least 20 ppV.

152. The process of embodiment 151, wherein the applied AC voltage potential is about 25-35 ppV.

153. The process according to any one of embodiments 148-152, wherein the applied AC voltage potential has a frequency of about 1-100 kHz.

154. The process according to any one of embodiments 148-153, wherein the microfluidic apparatus comprises a droplet generator, the droplet generator introducing a first droplet into an enclosure.

155. The process according to any one of embodiments 148-154, wherein the first droplet comprises an aqueous solution.

156. The process of embodiment 155, wherein the first droplet comprises at least one microobject.

157. 156. The process of embodiment 156, wherein the at least one micro-object is a biological micro-object.

158. 157. The process of embodiment 157, wherein the biological microobject is a cell.

159. The process according to any one of embodiments 155-158, wherein the aqueous solution is a cell medium.

160. 156. The process of embodiment 156, wherein the at least one microobject is a capture bead that has an affinity for the material of interest.

161. The process of embodiment 160, wherein the first droplet comprises 2 to 20 capture beads.

162. The process according to embodiment 160, wherein the material of interest is a living cell secretion.

163. The process according to embodiment 160 or 161, wherein the material of interest is selected from the group consisting of DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof.

164.164. The process according to embodiment 155 or 156, wherein the first droplet comprises a reagent.

165. The process according to embodiment 164, wherein the reagent is a cytolytic reagent.

166. The process of embodiment 165, wherein the reagent comprises a nonionic detergent.

167. The process according to embodiment 166, wherein the nonionic detergent has a concentration of less than 0.2%.

168. The process according to embodiment 164, wherein the reagent is a proteolytic enzyme.

169. The process of embodiment 168, wherein the proteolytic enzyme can be deactivated.

170. Introducing a second droplet of liquid into the enclosure, where the liquid in the second droplet does not mix in the first liquid medium, but mixes with the liquid in the first droplet. That and
By applying an electrowetting force to the second droplet, moving the second droplet to a position adjacent to the first droplet in the enclosure and
The process according to any one of embodiments 148-169, further comprising mixing the second droplet with the first droplet to form a first combined droplet.

171. The process of embodiment 170, wherein the second droplet is mixed with the first droplet by applying an electrowetting force to the second droplet and / or the first droplet.

172. The process according to embodiment 170 or 171 wherein the first droplet comprises a biological microobject and the second droplet comprises a reagent.

173. The process according to embodiment 172, wherein the reagent contained in the second droplet is selected from the group consisting of a lysis buffer, a fluorescent label, and a luminescent assay reagent.

174. The process according to embodiment 172, wherein the reagent contained in the second droplet is a lysis buffer and the living cells are lysed upon mixing the first and second droplets.

175. Introducing a third droplet of liquid into the enclosure, where the liquid in the third droplet does not mix with the first liquid medium, but mixes with the liquid in the first bound droplet. That and
By applying an electrowetting force to the third droplet, moving the third droplet to a position adjacent to the first combined droplet in the enclosure and
The process according to any one of embodiments 170-174, further comprising mixing the third droplet with the first combined droplet to form a second bonded droplet.

176. The process of embodiment 175, wherein the third droplet is mixed with the first combined droplet by applying an electrowetting force to the third droplet and / or the first bonded droplet. ..

177. The process according to embodiment 175 or 176, wherein the third droplet comprises a reagent.

178. The process of embodiment 177, wherein the third droplet comprises a protease inhibitor.

179. The process of embodiment 177, wherein the third droplet comprises 1 to 20 capture beads that have an affinity for the material of interest.

180. The process of embodiment 179, wherein the capture beads include an oligonucleotide capture agent.

181. The process of embodiment 180, wherein the oligonucleotide scavenger is a poly-dT oligonucleotide.

182. The process according to any one of embodiments 179-181, wherein the material of interest is selected from the group consisting of DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof.

The process according to any one of embodiments 179-182, further comprising removing 183.1-20 capture beads from the microfluidic apparatus.

184. Introducing a fourth droplet of liquid into the enclosure, where the liquid in the fourth droplet does not mix with the first liquid medium, but mixes with the liquid in the second bound droplet. That and
By applying an electrowetting force to the fourth droplet, moving the fourth droplet to a position adjacent to the second coupling droplet in the enclosure and
The process according to any one of embodiments 175-183, further comprising mixing a fourth droplet with a second bonded droplet to form a third bonded droplet.

185. The process of embodiment 184, wherein the fourth droplet is mixed with the second combined droplet by applying an electrowetting force to the fourth droplet and / or the second bonded droplet. ..

186. The process according to embodiment 184 or 185, wherein the fourth droplet comprises a reagent.

187. 186. The process of embodiment 186, wherein the reagent contained in the fourth droplet comprises a mixture containing a buffer, dNTP, and a polymerase suitable for performing a reverse transcription reaction.

188. The process of embodiment 186, wherein the reagent contained in the fourth droplet comprises a mixture containing a buffer, dNTP, and a polymerase suitable for performing a whole genome amplification reaction.

189. The first droplet, the second droplet, the third droplet, and the fourth droplet each have a capacity of about 5 to 50 nanoliters, according to any one of embodiments 148 to 188. Process.

190. 189. The process of embodiment 189, wherein the first droplet, the second droplet, and the third droplet each have a volume of about 5 to 20 nanoliters.

191. The process of embodiment 190, wherein the second and / or third droplet has a volume substantially equal to that of the first droplet.

192. The process according to embodiment 190 or 191 wherein the fourth droplet has a volume about 1-3 times that of the first droplet.

193. The process of embodiment 192, wherein the fourth droplet has a volume of about 10-30 nanoliters.

194. The process according to any one of embodiments 148-193, wherein the enclosure comprises at least one microchannel.

195. The process of embodiment 194, wherein moving the first droplet to a desired location within the enclosure comprises moving the first droplet through at least one microchannel.

196. The process of embodiment 194 or 195, wherein the enclosure further comprises a plurality of chambers that open at the end of at least one microchannel.

197. The process of embodiment 196, wherein moving the first droplet to a desired position within the enclosure comprises moving the first droplet into one of a plurality of chambers.

198. Moving the second droplet to a position adjacent to the first droplet causes the second droplet to pass through at least one microchannel and optionally into a chamber containing the first droplet. The process according to any one of embodiments 194 to 197, comprising moving.

199. Moving the third droplet to a position adjacent to the first bound droplet is a third liquid through at least one microchannel and optionally into a chamber containing the first bound droplet. 198. The process of embodiment 198, which comprises moving the droplets.

200. Moving the fourth droplet to a position adjacent to the second bound droplet is a fourth liquid through at least one microchannel and optionally into a chamber containing the second bound droplet. 199. The process of embodiment 199, which comprises moving the droplets.

201. The application of electrowetting forces to move and / or mix the droplets comprises altering the effective electrowetting properties of the area of the substrate surface in the vicinity of the droplets, embodiments 148-200. The process described in any one of.

202. The process of embodiment 201, wherein altering the effective electrowetting properties comprises activating the electrowetting electrodes in a region of the substrate surface in the vicinity of the droplet.

203. The substrate comprises a photoresponsive layer, and activating the electrowetting electrodes in the region of the substrate surface in the vicinity of the droplet comprises directing a pattern of light to the region of the electrowetting surface, according to embodiment 202. Described process.

Equivalents The above specification is considered sufficient to allow one of ordinary skill in the art to implement the embodiment. The above description and examples detail specific embodiments and describe the best possible embodiments. However, it is understood that the embodiments can be implemented in many ways and should be construed according to the appended claims and any equivalent thereof, regardless of how detailed the above may appear in the text. Will be done.

Claims (203)

A microfluidic device with an electrowetting configuration
A substrate having a dielectric layer, a droplet working surface, and a first electrode configured to be connected to an AC voltage source.
Includes a second electrode configured to be connected to the AC voltage source.
The dielectric layer is electrically coupled to the first electrode.
The droplet working surface comprises a hydrophobic layer covalently bonded to the dielectric layer, a microfluidic device. The microfluidic device according to claim 1, which has a single-sided electrowetting configuration. The microfluidic device according to claim 2, wherein the second electrode is a mesh electrode included in the substrate. The microfluidic device of claim 1, which has an optical electrowetting (OEW) configuration. The microfluidic device of claim 1, which has a dielectric electrowetting (EWOD) configuration. The hydrophobic layer is a monolayer comprising a surface modifying ligand and a binding group that binds the surface modifying ligand to the surface, and the droplet working surface is of formula II :.

Has the structure of

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 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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
When k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The microfluidic device of claim 1, which is hydrogen. The electrowetting configuration of the device is configured by a first section of the device, wherein the device further comprises a second section having a dielectrophoresis (DEP) configuration, any one of claims 1-6. The microfluidic device described in the section. It ’s a microfluidic device,
A substrate having at least one electrode configured to be connected to a voltage source,
A cover having at least one electrode configured to be connected to the voltage source.
Including at least one separating element
The substrate and the cover define an enclosure that is substantially parallel to each other and is joined together by the separating elements to hold the liquid.
The substrate has a droplet working surface that partially defines the enclosure, and the droplet working surface has a dielectric inner layer and a hydrophobic outer layer.
The hydrophobic outer layer contains self-associating molecules covalently bonded to the surface of the dielectric inner layer, thereby forming a dense hydrophobic monolayer on it.
When the at least one electrode on the substrate and the at least one electrode on the cover are connected to opposite terminals of the voltage source, the substrate becomes an aqueous droplet in contact with the droplet working surface of the substrate. A microfluidic device to which electrowetting forces can be applied. The self-associating molecule of the hydrophobic monolayer comprises a surface modifying ligand and a binding group that binds the surface modifying ligand to the surface of the dielectric inner layer, respectively, and the droplet working surface is of formula II:

Has the structure of

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 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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
When k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The microfluidic apparatus according to claim 8, which is hydrogen. The microfluidic device of claim 9, wherein V is −Si (OZ) _{2 W−.} The microfluidic device according to claim 9, wherein V is −P (O) (OY) W−. The microfluidic device according to any one of claims 9 to 11, wherein n is an integer of 1 to 20 and R is hydrogen. The microfluidic device according to claim 12, wherein m is an integer of 1 to 20 and M is hydrogen. The microfluidic device according to claim 13, wherein m is 2. The microfluidic device according to any one of claims 9 to 11, wherein n is an integer of 1 to 20 and R is fluorine. The microfluidic device of claim 15, wherein m is an integer of 1-20 and M is hydrogen. The microfluidic device according to claim 16, wherein m is 2. The microfluidic device according to claim 9, wherein k is 1. The microfluidic device of claim 9, wherein k is 0. The microfluidic device according to claim 9, wherein the sum of (n + [(h + j) · k] + m) is an integer of 13 to 19. The microfluidic device of claim 8, wherein the hydrophobic outer layer of the droplet working surface of the substrate has a thickness of less than 5 nanometers. 8. The hydrophobic outer layer of the droplet working surface of the substrate is patterned such that the selected region is relatively hydrophilic relative to the rest of the hydrophobic outer layer. Microfluidic device. The microfluidic device of claim 8, wherein the dielectric inner layer of the droplet working surface of the substrate comprises a first layer of a dielectric material containing an oxide. 23. The microfluidic device of claim 23, wherein the oxide is a metal oxide. The microfluidic device of claim 24, wherein the metal oxide is aluminum oxide. 23. The microfluidic device of claim 23, wherein the first layer of the dielectric material is formed by atomic layer deposition. 23 to 23. The dielectric inner layer of the droplet working surface of the substrate further comprises a second layer of the dielectric material, and the hydrophobic outer layer is covalently bonded to the first layer of the dielectric material. The microfluidic device according to any one of 26. 27. The microfluidic device of claim 27, wherein the second layer of the dielectric material comprises an oxide or nitride. 28. The microfluidic device of claim 28, wherein the second layer of the dielectric material is selected from the group consisting of silicon dioxide and silicon nitride. The microfluidic device of claim 27, wherein the second layer of the dielectric material is formed by plasma chemical vapor deposition. 23. The micro according to claim 23, wherein the first layer of the dielectric material comprises first and second sublayers of the dielectric material, the first sublayer covalently bonding to the hydrophobic layer. Fluid device. The microfluidic device of claim 31, wherein the first sublayer of the dielectric material comprises silicon oxide. 31. The microfluidic device of claim 31, wherein the first adrenoleukodys of the dielectric material is deposited by ALD. The microfluidic device according to any one of claims 31 to 33, wherein the first layer of the dielectric material has a thickness of about 10 nm to about 20 nm. The microfluidic device of claim 34, wherein the first sublayer of the dielectric material has a thickness of about 2 nm to about 10 nm. The microfluidic device of claim 8, wherein the dielectric inner layer of the droplet working surface of the substrate has a thickness of at least about 40 nanometers. 36. The microfluidic device of claim 36, wherein the dielectric inner layer of the droplet working surface of the substrate has a thickness of about 40 nanometers to about 120 nanometers. The microfluidic device of claim 8, wherein the substrate further comprises a photoresponsive layer having a first surface in contact with the dielectric inner layer and a second surface in contact with the at least one electrode. 38. The microfluidic device of claim 38, wherein the photoresponsive layer comprises hydrogenated amorphous silicon (a-Si: H). The microfluidic device of claim 38 or 39, wherein the photoresponsive layer has a thickness of at least 900 nanometers. The microfluidic device of claim 40, wherein the photoresponsive layer has a thickness of about 900 to 1100 nanometers. 38. The microfluidic device of claim 38, wherein the photoresponsive layer comprises a plurality of conductors, each of which is controllably connectable to said at least one electrode of the substrate via a phototransistor switch. The microfluide of claim 8, wherein the substrate comprises a single electrode configured to be connected to an AC voltage source, the single electrode comprising a layer of indium tin oxide (ITO). device. The microfluidic device of claim 8, wherein the substrate comprises a single electrode configured to be connected to an AC voltage source, wherein the single electrode comprises a layer of conductive silicon. The microfluidic device of claim 8, wherein the substrate comprises a plurality of electrodes, each electrode being configured to be connected to one or more AC voltage sources. The microfluidic device of claim 45, wherein each electrode of the plurality is connectable to one of the one or more AC voltage sources via a transistor switch. The cover has an inward surface that partially defines the enclosure, the inward surface of the cover has an inner layer and a hydrophobic outer layer, and the hydrophobic outer layer of the cover is said to the cover. The microfluidic device of claim 8, wherein the microfluidic device comprises a covalently bonded self-associating molecule on the surface of the inner layer, thereby forming a dense hydrophobic monolayer on it. The self-associating molecule of the hydrophobic monolayer of the cover comprises a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface of the inner layer of the cover, respectively, and the inward facing of the cover. The surface is equation II:

Has the structure of

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 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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
If k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The microfluidic device of claim 47, which is hydrogen. 48. The microfluidic device of claim 48, wherein the self-associating molecule of the hydrophobic monolayer of the cover is the same as the self-associating molecule of the hydrophobic monolayer of the droplet working surface of the substrate. .. The microfluidic device of any one of claims 47-49, wherein the hydrophobic outer layer of the inward facing surface of the cover has a thickness of less than 5 nanometers. The microfluidic device of claim 47, wherein the inner layer of the cover is a dielectric inner layer. The microfluidic device of claim 51, wherein the cover further comprises a photoresponsive layer. 51. The microfluidic device of claim 51, wherein the cover comprises a plurality of electrodes, each electrode being configured to be connected to one or more AC voltage sources. The microfluidic device of claim 8, wherein the at least one separating element comprises a silicon-based organic polymer. The microfluidic device of claim 54, wherein the silicon-based organic polymer is selected from the group consisting of polydimethylsiloxane (PDMS) and photopatternable silicone (PPS). The microfluidic device of claim 8, wherein the at least one separating element comprises SU-8. The microfluidic device of claim 8, wherein the at least one separating element has a thickness of at least 30 μm. The microfluidic device of claim 8, wherein the at least one separating element defines one or more microchannels within the enclosure. 58. The microfluidic device of claim 58, wherein the at least one separating element further defines a plurality of chambers within the enclosure, each chamber opening at the end of at least one microchannel. A method of manufacturing microfluidics
Joining a separating element to the inner surface of a cover having at least one electrode configured to be connected to a voltage source
The separating element and the cover are joined to the dielectric surface of a substrate having at least one electrode configured to be connected to a voltage source, whereby the separating element is such that the cover and the substrate are attached to each other. It is sandwiched between the inner layer of the cover and the dielectric surface of the substrate in a substantially parallel orientation so that the substrate, the separating elements, and the cover collectively hold the liquid. Demarcate, join, and
By vapor deposition, a high density hydrophobic monolayer is formed on at least a portion of the inner surface of the cover, the hydrophobic monolayer comprising self-associating molecules covalently bonded to the inner surface of the cover. , Forming and
By vapor deposition, a high-density hydrophobic single layer is formed on at least a part of the dielectric surface of the substrate, and the hydrophobic single layer is covalently bonded to the dielectric surface of the substrate. Methods that include, form, and include molecules. The self-associating molecule of the hydrophobic molecule of the cover and the self-associating molecule of the hydrophobic monolayer of the substrate are composed of a surface-modifying ligand and the surface-modifying ligand on the inner surface of the cover and the substrate. The resulting cover and the resulting surface of the substrate, each containing a binding group to be attached to the dielectric surface, are of formula II :.

Has the structure of

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 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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
When k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The method of claim 60, which is hydrogen. The method of claim 61, wherein V is −Si (OZ) _{2 W−.} The method of claim 61, wherein V is −P (O) (OY) W−. The method of claim 61, wherein n is an integer of 1-20 and R is hydrogen. The method of claim 64, wherein m is an integer of 1-20 and M is hydrogen. The method according to claim 65, wherein m is 2. The method of claim 61, wherein n is an integer of 1-20 and R is fluorine. 67. The method of claim 67, wherein m is an integer of 1-20 and M is hydrogen. The method according to claim 68, wherein m is 2. The method according to any one of claims 61 to 69, wherein k is 1. The method according to any one of claims 61 to 69, wherein k is 0. The method of claim 61, wherein the sum of (n + [(h + j) · k] + m) is an integer of 13-19. It ’s a microfluidic device,
A conductive silicon substrate having a dielectric laminate and at least one electrode configured to be connected to a voltage source.
A cover with at least one electrode configured to be connected to a voltage source,
Including at least one separating element
The conductive silicon substrate and the cover define an enclosure that is substantially parallel to each other and is joined together by the separating elements to hold a liquid.
The conductive silicon substrate has an inward facing surface that partially defines the enclosure, the inward facing surface including the outermost surface of the dielectric laminate.
When the at least one electrode of the substrate and the at least one electrode of the cover are connected to the opposite terminals of the AC voltage source, the substrate is electrowetting the aqueous droplets in contact with the inward surface of the substrate. A microfluidic device to which wetting forces can be applied. The microfluidic device according to claim 73, wherein the conductive silicon substrate contains amorphous silicon. The microfluidic device according to claim 73, wherein the conductive silicon substrate includes a phototransistor array. The microfluidic device of claim 73, wherein the conductive silicon substrate comprises an array of electrodes. 33. The microfluidic of claim 73, wherein the inward facing surface of the conductive silicon substrate further comprises a hydrophobic outer layer, the hydrophobic outer layer containing self-associating molecules covalently bonded to the internal dielectric laminate. Device. The microfluidic device of claim 73, wherein the internal dielectric laminate comprises a first layer of dielectric material and a second layer of dielectric material. The first layer of the dielectric material has a first surface and an inverted surface, and the first surface of the first layer is adjacent to the second layer and is of the first layer. The microfluidic apparatus according to claim 78, wherein the reverse surface forms the outermost surface of the dielectric laminate. The microfluidic device of claim 78, wherein the first layer of the dielectric material comprises a metal oxide. The microfluidic device of claim 80, wherein the first layer of the dielectric material comprises aluminum oxide or hafnium oxide. The microfluidic device of claim 78, wherein the second layer of the dielectric material comprises an oxide or nitride. The microfluidic device of claim 82, wherein the second layer of the dielectric material comprises silicon oxide or silicon nitride. 28. The microfluidic apparatus of claim 78, wherein the second layer is deposited by a plasma chemical vapor deposition (PECVD) technique. The microfluidic apparatus of claim 78, wherein the first layer is deposited by an atomic layer deposition (ALD) technique. The internal dielectric laminate comprises a third layer having a first surface and an inverted surface, the first surface of the third layer being adjacent to the first layer and the third layer. The microfluidic apparatus according to claim 78, wherein the reverse surface of the above forms the outermost surface of the dielectric laminate. The microfluidic apparatus according to claim 86, wherein the third layer contains silicon oxide. The microfluidic apparatus of claim 86, wherein the third layer is deposited by an atomic layer deposition (ALD) technique. The microfluidic apparatus of claim 78, wherein the first layer of the dielectric material has a thickness of about 10 nm to about 50 nm. 86. The first layer of the dielectric material has a thickness of about 5 nm to about 20 nm, and the third layer of the dielectric material has a thickness of about 2 nm to 10 nm. Microfluidic device. The microfluidic apparatus of claim 78, wherein the second layer of the dielectric material has a thickness of about 30 nm to about 100 nm. The microfluidic apparatus of claim 73, wherein the dielectric laminate on the droplet working surface of the substrate has a thickness of at least about 40 nanometers. The microfluidic apparatus according to claim 92, wherein the dielectric laminate on the droplet working surface of the substrate has a thickness of about 40 nanometers to about 120 nanometers. The microfluidic device of claim 73, wherein the dielectric layer has an impedance of about 50 k ohms to about 150 k ohms. A dielectrophoresis module that performs a first microfluidic operation in response to a first applied voltage at a first frequency,
Includes an electrowetting module that receives output from the dielectrophoresis module and performs a second microfluidic operation in response to a second applied voltage at a second frequency.
The microfluidic device according to any one of claims 73 to 94, wherein the electrowetting module includes the dielectric lamination of the conductive silicon substrate. The microfluidic device of claim 95, further comprising a bridge between the first module and the second module. The microfluidic device of claim 96, wherein the bridge does not perform the first or second microfluidic operation. The microfluidic device of claim 96, wherein the bridge is electrically neutral. The microfluidic device of claim 96, wherein the bridge comprises a tube. The microfluidic device of claim 96, wherein the bridge comprises a polymer. The microfluidic device of claim 95, wherein the output comprises a biomaterial. The microfluidic apparatus according to claim 95, wherein the first frequency is in the range of 100 kHz to 10 MHz. The microfluidic apparatus according to claim 95, wherein the second frequency is in the range of 1 kHz to 300 kHz. The microfluidic device of claim 95, wherein the first voltage is in the range of 1-10 volts. The microfluidic device of claim 95, wherein the second voltage is in the range of 10 to 100 volts. The microfluidic apparatus according to claim 95, wherein the conductive silicon substrate is monolithic. The microfluidic device according to claim 95, wherein the conductive silicon substrate is dual chic. The microfluidic device according to claim 106, wherein the conductive silicon substrate contains amorphous silicon. The microfluidic device according to claim 107, wherein the conductive silicon substrate contains amorphous silicon. The microfluidic device according to claim 106, wherein the conductive silicon substrate includes a phototransistor array. The microfluidic device according to claim 107, wherein the conductive silicon substrate includes a phototransistor array. The microfluidic device of claim 106, wherein the conductive silicon substrate comprises an array of electrodes. The microfluidic device of claim 107, wherein the conductive silicon substrate comprises an array of electrodes. A system for transporting microobjects, biological substances, and / or reagents that are compatible with and / or meltable in aqueous media.
A microfluidic device having an enclosure comprising a base and a microfluidic circuit structure, wherein the base comprises a hydrophobic monolayer covalently bonded to at least a portion of the upper surface of the base.
A first fluid medium that does not mix with the aqueous medium,
A system comprising at least one aqueous droplet. The hydrophobic monolayer has a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface, and the hydrophobic surface is of formula II :.

Has the structure of

Is the surface
V is −P (O) (OY) W− or −Si (OZ) 2W−.
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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
When k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The system of claim 114, which is hydrogen. The system of claim 114, wherein the base comprises a conductive substrate. The system of claim 114, wherein the microfluidic device is the microfluidic device of any one of claims 1-59. The system of claim 117, wherein the microfluidic device comprises an optically actuated EW configuration. The system of claim 117, wherein the microfluidic device further comprises a DEP configuration. The system of claim 114, wherein the first fluid medium comprises at least one organosilicon or organosilicon compound having a backbone structure comprising atoms selected from carbon, silicon, and oxygen. The system according to claim 120, wherein the main chain structure of the at least one organosilicon compound comprises a silicon atom and optionally an oxygen atom. The system of claim 120, wherein the backbone structure of the at least one organic compound comprises a carbon atom and optionally an oxygen atom. The system according to claim 122, wherein the main chain structure is branched. The system according to claim 120, wherein the first fluid medium comprises one or more acyclic organic compounds or organosilicon compounds. The system according to claim 124, wherein the first fluid medium comprises an acyclic organic compound or an organosilicon compound. The system of claim 114, wherein the first fluid medium is free of perfluorocarbon atoms. The system according to claim 114, wherein the substituent of the carbon atom of the compound of the first fluid medium contains 90% or less of a fluorine substituent. 15. The system of claim 115, wherein the surface-modifying ligand comprises at least a first moiety comprising a perfluorocarbonated carbon atom at the inward end of the hydrophobic monolayer. The system of claim 128, wherein all carbon atoms in the hydrophobic monolayer are perfluorolated. The system according to claim 114, wherein the first fluid medium comprises two or more organic compounds or organosilicon compounds. The system of claim 114, wherein the enclosure further comprises a cover. The system according to claim 131, wherein the cover transmits light. 13. The system of claim 131, wherein the cover comprises glass and / or indium tantalum oxide (ITO). The system according to any one of claims 131 to 133, wherein the cover comprises electrodes. The system of claim 114, wherein the aqueous droplet comprises a surfactant. The system of claim 135, wherein the surfactant comprises a nonionic surfactant. The surfactant may be a block alkylene oxide copolymer, a fatty acid ester ethoxylated sorbitan, an ethoxylated fluorine-based surfactant, sodium dodecyl sulfate, or 2,4,7,9, tetramethyl-5-decyne-4,7,-. The system according to claim 135 or 136, which comprises a diol ethoxylate. The system of claim 135, wherein the surfactant comprises Capstone® FS-30 (DuPont Trademark, Synquest Laboratories). The system of claim 114, wherein the droplet comprises a phosphate buffered physiological saline solution. The system of claim 114, wherein the aqueous droplet comprises at least one microobject. The system of claim 140, wherein the micro-object is a biological micro-object. The system of claim 114, wherein the aqueous droplet comprises a biological material, including nucleic acids and / or proteins. The system of claim 114, wherein the aqueous droplet comprises a reagent. A kit for transporting microobjects, biological substances, and / or reagents that are compatible with and / or meltable in aqueous media.
A microfluidic device having an enclosure comprising a base and a microfluidic circuit structure, wherein the base comprises a hydrophobic monolayer covalently bonded to at least a portion of the upper surface of the base.
A kit comprising a first fluid medium that is immiscible with an aqueous medium. The hydrophobic monolayer has a surface-modifying ligand and a binding group that binds the surface-modifying ligand to the surface, and the hydrophobic surface is of formula II :.

Has the structure of

Is the surface
V is −P (O) (OY) W− or −Si (OZ) 2W−.
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.
R is hydrogen or fluorine,
M is hydrogen or fluorine,
h is an independently integer of 2 or 3
j is 1,
k is 0 or 1 and
m is an integer of 0 or 1-20,
n is an integer of 0 or 1-20,
The sum of (n + [(h + j) · k] + m) is an integer of 11 to 25.
When k is 1, m is at least 2, and M is hydrogen, and k is 0, and R is fluorine, then m is at least 2, and M is. The kit of claim 144, which is hydrogen. The kit of claim 144, wherein the base comprises a conductive substrate. The kit according to any one of claims 144 to 146, wherein the microfluidic device is the microfluidic device according to any one of claims 1 to 72. A process for operating the microfluidic apparatus according to any one of claims 8 to 59.
Filling the enclosure or a part thereof with a first fluid medium,
Applying an AC voltage potential between the at least one electrode on the substrate and the at least one electrode on the cover,
Introducing a first droplet of liquid into the enclosure, the first droplet being immiscible with the first fluid medium.
A process comprising applying an electrowetting force to the first droplet to move the first droplet to a desired position within the enclosure. The process of claim 148, wherein the first liquid medium is oil. The process of claim 148, wherein the first liquid medium is a silicone oil, a fluorinated oil, or a combination thereof. 148. The process of claim 148, wherein the applied AC voltage potential is at least 20 ppV. 15. The process of claim 151, wherein the applied AC voltage potential is about 25-35 ppV. 148. The process of claim 148, wherein the applied AC voltage potential has a frequency of about 1-100 kHz. 148. The process of claim 148, wherein the microfluidic apparatus comprises a droplet generator, which introduces the first droplet into the enclosure. The process of claim 148, wherein the first droplet comprises an aqueous solution. The process of claim 155, wherein the first droplet comprises at least one microobject. 156. The process of claim 156, wherein the at least one micro-object is a biological micro-object. 157. The process of claim 157, wherein the biological microobject is a cell. The process of claim 155, wherein the aqueous solution is a cell medium. 156. The process of claim 156, wherein the at least one microobject is a capture bead that has an affinity for the material of interest. The process of claim 160, wherein the first droplet comprises 2 to 20 capture beads. The process of claim 160, wherein the material of interest is a living cell secretion. The process of claim 160, wherein the material of interest is selected from the group consisting of DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. The process of claim 155, wherein the first droplet comprises a reagent. The process of claim 164, wherein the reagent is a cytolytic reagent. The process of claim 165, wherein the reagent comprises a nonionic detergent. 166. The process of claim 166, wherein the nonionic detergent has a concentration of less than 0.2%. The process of claim 164, wherein the reagent is a proteolytic enzyme. 168. The process of claim 168, wherein the proteolytic enzyme can be deactivated. Introducing a second droplet of liquid into the enclosure, wherein the liquid in the second droplet is not mixed into the first liquid medium, but the liquid in the first droplet. To mix with, to introduce,
By applying an electrowetting force to the second droplet, the second droplet can be moved to a position adjacent to the first droplet in the enclosure.
148. The process of claim 148, further comprising mixing the second droplet with the first droplet to form a first combined droplet. 170. The second droplet is mixed with the first droplet by applying an electrowetting force to the second droplet and / or the first droplet. Process. The process of claim 170, wherein the first droplet comprises a biological microobject and the second droplet comprises a reagent. 172. The process of claim 172, wherein the reagent contained in the second droplet is selected from the group consisting of a lysis buffer, a fluorescent label, and a luminescent assay reagent. 172. The reagent according to claim 172, wherein the reagent contained in the second droplet is a lysis buffer, and the living cell is lysed when the first droplet and the second droplet are mixed. Process. Introducing a third droplet of liquid into the enclosure, wherein the liquid of the third droplet does not mix with the first liquid medium, but with the liquid of the first bound droplet. Mixing, introducing, and
By applying an electrowetting force to the third droplet, the third droplet is moved to a position adjacent to the first bonded droplet in the enclosure, and the third droplet is moved.
The process of claim 170, further comprising mixing the third droplet with the first binding droplet to form a second binding droplet. The third droplet is mixed with the first binding droplet by applying an electrowetting force to the third droplet and / or the first binding droplet, claim 175. The process described in. The process of claim 175, wherein the third droplet comprises a reagent. The process of claim 177, wherein the third droplet comprises a protease inhibitor. The process of claim 177, wherein the third droplet comprises 1 to 20 capture beads that have an affinity for the material of interest. 179. The process of claim 179, wherein the capture beads include an oligonucleotide capture agent. The process of claim 180, wherein the oligonucleotide scavenger is a poly-dT oligonucleotide. 179. The process of claim 179, wherein the material of interest is selected from the group consisting of DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. 179. The process of claim 179, further comprising removing the 1-20 capture beads from the microfluidic apparatus. Introducing a fourth droplet of liquid into the enclosure, wherein the liquid in the fourth droplet does not mix with the first liquid medium, but with the liquid in the second bound droplet. Mixing, introducing, and
By applying an electrowetting force to the fourth droplet, the fourth droplet is moved to a position adjacent to the second binding droplet in the enclosure.
The process of claim 175, further comprising mixing the fourth droplet with the second binding droplet to form a third binding droplet. 184. The fourth droplet is mixed with the second binding droplet by applying an electrowetting force to the fourth droplet and / or the second binding droplet. The process described in. The process of claim 184, wherein the fourth droplet comprises a reagent. 186. The process of claim 186, wherein the reagent contained in the fourth droplet comprises a mixture containing a buffer, dNTP, and a polymerase suitable for performing a reverse transcription reaction. 186. The process of claim 186, wherein the reagent contained in the fourth droplet comprises a mixture containing a buffer, dNTP, and a polymerase suitable for performing a whole genome amplification reaction. 148. The process of claim 148, wherein the first droplet, the second droplet, the third droplet, and the fourth droplet each have a capacity of about 5 to 50 nanoliters. 189. The process of claim 189, wherein the first, second, and third droplets each have a volume of about 5 to 20 nanoliters. The process of claim 190, wherein the second and / or third droplet has a volume substantially equal to that of the first droplet. The process of claim 190, wherein the fourth droplet has a volume about 1-3 times that of the first droplet. 192. The process of claim 192, wherein the fourth droplet has a volume of about 10-30 nanoliters. 148. The process of claim 148, wherein the enclosure comprises at least one microchannel. The process of claim 194, wherein moving the first droplet to a desired location within the enclosure comprises moving the first droplet through the at least one microchannel. 194. The process of claim 194, wherein the enclosure further comprises a plurality of chambers that open at the end of the at least one microchannel. 196. The movement of the first droplet to a desired position within the enclosure comprises moving the first droplet into one of the plurality of chambers. Process. Moving the second droplet to a position adjacent to the first droplet is said through the at least one microchannel and optionally into the chamber containing the first droplet. The process of claim 194, which comprises moving a second droplet. Moving the third droplet to a position adjacent to the first binding droplet is through the at least one microchannel and optionally within the chamber containing the first binding droplet. 198. The process of claim 198, comprising moving the third droplet. Moving the fourth droplet to a position adjacent to the second bound droplet is through the at least one microchannel and optionally within the chamber containing the second bound droplet. 199. The process of claim 199, comprising moving the fourth droplet. Claim 148, wherein applying an electrowetting force to move and / or mix the droplets changes the effective electrowetting properties of the area of the substrate surface in the vicinity of the droplets. The process described in. The process of claim 201, wherein altering the effective electrowetting properties comprises activating an electrowetting electrode in the region of the substrate surface in the vicinity of the droplet. The substrate comprises a photoresponsive layer, and activating the electrowetting electrode in the region of the substrate surface in the vicinity of the droplet comprises directing a pattern of light to the region of the electrowetting surface. 202. The process of claim 202.

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