Covalently modified surfaces, kits, and methods for preparation and use.

Abstract

The present invention provides a method for modifying the surface of devices, equipment, and materials that come into contact with biomaterials such as biomolecules and biological microorganisms, in order to provide improved or modified performance. [Solution] A method for forming a covalently modified surface on at least one inner surface of a microfluidic device, comprising: contacting at least one inner surface with a first modifying reagent and a second modifying reagent; reacting the first modifying reagent with a first nucleophilic portion on at least one inner surface; reacting the second modifying reagent with a second nucleophilic portion on at least one inner surface; and forming at least one covalently modified surface including a first covalent surface modification comprising a first bonding group and a first portion which is a first surface contact portion or a first reactive portion, and a second covalent surface modification comprising a second bonding group and a second portion which is a second surface contact portion or a second reactive portion.

Description

[Technical Field]

[0001] This application is a non-provisional application claiming under Section 119(e) of the United States Patent Act the interests of U.S. Provisional Patent Application No. 62 / 342,131 filed on 26 May 2016; U.S. Provisional Patent Application No. 62 / 345,603 filed on 3 June 2016; U.S. Provisional Patent Application No. 62 / 353,938 filed on 23 June 2016; U.S. Provisional Patent Application No. 62 / 411,191 filed on 21 October 2016; and U.S. Provisional Patent Application No. 62 / 410,238 filed on 19 October 2016 (each of the disclosures thereof is incorporated herein by reference in whole). [Background technology]

[0002] Background of the Invention In biological sciences and related fields, it may be useful to modify the surfaces of devices, equipment, and materials that come into contact with biomaterials such as biomolecules and biological microorganisms. One embodiment of the present invention includes a siloxane reagent, its preparation, and a method for modifying a surface to provide improved or modified performance in conjunction with a biomaterial. [Overview of the Initiative] [Means for solving the problem]

[0003] Summary of the Invention In a first embodiment, a microfluidic device is provided, comprising a base, a cover, and a microfluidic circuit material, wherein the enclosure includes the microfluidic circuit material defining a fluid circuit, and at least one inner surface of the base, cover, and microfluidic circuit material has a first covalent surface modification including a first bonding group and a first portion which is a first surface contact portion or a first reactive portion; and at least one inner surface of the base, cover, and microfluidic circuit material has a second covalent surface modification including a second bonding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first bonding group and the second bonding group are different from each other, and / or the first portion is different from the second portion. In a certain embodiment, a common inner surface of the base, cover, and microfluidic circuit material has the first covalent surface modification and the second covalent surface modification.

[0004] In another embodiment, a method for forming a covalently modified surface on at least one inner surface of a microfluidic device comprising a base, a cover, and a microfluidic circuit material, wherein the enclosure has a microfluidic circuit material defining a fluid circuit, the method comprising: contacting at least one inner surface with a first modifying reagent and a second modifying reagent; reacting the first modifying reagent with a first nucleophilic portion on at least one inner surface; reacting the second modifying reagent with a second nucleophilic portion on at least one inner surface; and forming at least one covalently modified surface comprising a first covalent surface modification including a first binding group and a first portion which is a first surface contact portion or a first reactive portion, and a second covalent surface modification including a second binding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first binding group is different from the second binding group, or the first portion is different from the second portion. In one embodiment, the first and second covalent surface modifications may be formed on a common inner surface of the base, cover, and microfluidic circuit material.

[0005] In another embodiment, a method is provided for positionally forming different covalently modified surfaces within a microfluidic device. The microfluidic device comprises a base, a cover, and a microfluidic circuit material, wherein a microfluidic circuit is defined therein. The microfluidic circuit may include an enclosure having a path material, and the microfluidic circuit includes a flow region and an isolation pen, the isolation pen includes a separation region and a connection region, the connection region includes a base opening to the flow region, and the separation region is fluidly connected to the flow region. The method may include the steps of: flowing a first modifying reagent through the flow region under conditions that the first modifying reagent does not enter the separation region of the isolation pen; reacting the first modifying reagent with a nucleophilic portion on at least one surface of the flow region to form a first modified surface within the flow region, wherein the first modified surface does not extend into the separation region of the isolation pen; flowing a second modifying reagent through the flow region under conditions that the second modifying reagent enters the separation region of the isolation pen; and reacting the second modifying reagent with a nucleophilic portion on at least one surface of the separation region of the isolation pen to form a second modified surface within the separation region of the isolation pen. Typically, the first modifying reagent does not have the same structure as the second modifying reagent.

[0006] In another embodiment, a kit comprising the microfluidic device described herein is provided. The kit comprises formula XII: RP-L-Surface contact part Formula XII The surface-modifying reagent may further comprise the structure of the formula, where RP is the reaction pair portion; and the surface contact portion supports cell growth, survival, portability, or any combination thereof. A portion configured such that; L is a linker, which can be 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and may further include 0 or 1 bonding group CG.

[0007] In another embodiment, formula XIII: [ka] A compound having the structure is provided, where h is an integer from 1 to 19, and R is independently selected from the group consisting of H and C1-C6 alkyl groups. In one embodiment, h is from 5 to 19.

[0008] In yet another embodiment, formula XIII: [ka] A method for synthesizing a compound having the structure shown below, wherein in the presence of a catalyst or initiator, the following formula is used: [ka] A compound having the structure of formula HSi(OR)3 is reacted with a compound having the structure of formula HSi(OR)3, and A method is provided for producing a compound of formula XIII (wherein h is an integer from 1 to 19, and each of R is independently H or a C1 to C6 alkyl).

[0009] In a further embodiment, Equation IV: [ka] (wherein n is an integer from 3 to 21, and R is independently H or a C1 to C6 alkyl group.) A compound having the structure is provided. In one embodiment, n is 9, 14, or 16.

[0010] In another embodiment, formula IV: [ka] A method for synthesizing the compound of formula XIII: [ka] (In the formula, h is between 1 and 19.) A method is provided which includes the step of reacting a compound having the structure with an azide ion to produce a compound of formula IV (wherein n is 3 to 21, and R is H or C1-C6 alkyl). [Brief explanation of the drawing]

[0011] Brief explanation of the drawing [Figure 1A] Examples of systems for use with microfluidic devices and associated control equipment according to certain embodiments of this disclosure are shown. [Figure 1B] This disclosure shows a microfluidic device according to one embodiment. [Figure 1C] This disclosure shows a microfluidic device according to one embodiment. [Figure 2A] A separation pen according to one embodiment of this disclosure is shown. [Figure 2B] A separation pen according to one embodiment of this disclosure is shown. [Figure 2C] Detailed isolation pens according to one embodiment of this disclosure are shown. [Figure 2D] An isolation pen according to another embodiment of this disclosure is shown. [Figure 2E] An isolation pen according to another embodiment of this disclosure is shown. [Figure 2F] An isolation pen according to another embodiment of this disclosure is shown. [Figure 2G] This disclosure shows a microfluidic device according to one embodiment. [Figure 2H] This shows the coating surface of a microfluidic device according to one embodiment of the present disclosure. [Figure 3A] Specific examples of systems for use with microfluidic devices and associated control equipment according to one embodiment of this disclosure are shown. [Figure 3B] An imaging device according to one embodiment of this disclosure is shown. [Figure 4]This is a graphical representation of the FTIR spectrum of a modified microfluidic circuit material according to one embodiment of the present disclosure. [Figure 5A] This is a graph representation of the overlay FTIR for a modified surface according to one embodiment of the present disclosure. [Figure 5B] This is a graph representation of the overlay FTIR for a modified surface according to one embodiment of the present disclosure. [Figure 6A] This is a photographic representation of a cell culture according to one embodiment of the present invention. [Figure 6B] This is a photographic representation of cell unpenning according to one embodiment of the present invention. [Figure 7A] This is a photographic representation of a cell culture according to another embodiment of the present invention. [Figure 7B] This is a photographic representation of cell extraction according to another embodiment of the present invention. [Modes for carrying out the invention]

[0012] Detailed description of the invention This specification describes exemplary embodiments and applications of the present disclosure. However, this disclosure is not limited to these exemplary embodiments and applications, or the modes in which the exemplary embodiments and applications operate or the modes described herein. Furthermore, figures may be simplified or partial, and the dimensions of elements in the figures may be exaggerated or not to scale. Furthermore, as used herein, the terms “on,” “attached,” “connected,” “linked,” or similar terms mean that one element (e.g., material, layer, substrate, etc.) may be “on,” “attached,” “connected,” or “linked” to another element, whether one element is directly on another element, attached to it, connected to it, or linked, or whether there are one or more intervening elements between one element and the other. Furthermore, unless otherwise indicated in the context, directions (e.g., up, down, top, bottom, side, above, below, above, upper side, lower side, horizontal, vertical, "x", "y", "z", etc.) are relative, where provided, and are provided merely as examples and to facilitate illustration and explanation, not as limitations. In addition, where a list of elements (e.g., elements a, b, c) is referenced, such reference is intended to include any one of the enumerated elements themselves, any combination of less than all of the enumerated elements, and / or all combinations of the enumerated elements. The division of sections in this specification is solely for the purpose of facilitating consideration and does not limit any combination of elements described.

[0013] When the dimensions of a microfluidic feature are described as having a certain width or area, the dimensions are typically given with respect to the x-axis and / or y-axis (both in a plane parallel to the substrate and / or cover of the microfluidic device). The height of a microfluidic feature may be given with respect to the z-axis direction perpendicular to the plane parallel to the substrate and / or cover of the microfluidic device. In some cases, the cross-sectional area of ​​a microfluidic feature, such as a channel or passage, may be related to the x-axis / z-axis, y-axis / z-axis, or x-axis / y-axis area.

[0014] As used herein, “substantially” means sufficient to function for its intended purpose. Therefore, the term “substantially” allows for small, slight variations from absolute or perfect conditions, dimensions, measurements, results, etc., that are predictable to a person skilled in the art but do not significantly affect the overall performance. When used in relation to numerical values, parameters, or characteristics that can be expressed numerically, “substantially” means within 10 percent. It means.

[0015] The term "one" means two or more. As used herein, the term "plural" may mean two, three, four, five, six, seven, eight, nine, ten or more.

[0016] As used herein, “alkyl” refers to a non-unsaturated, linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, having 1 to 6 carbon atoms (e.g., C1-C6 alkyl). Whenever alkyl appears herein, a numerical range such as “1 to 6” refers to each integer within a given range; for example, “1 to 6 carbon atoms” means that an alkyl group can consist of up to 6 (including 6) carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. However, this definition also includes appearances of the term “alkyl” where no numerical range is given. In some embodiments, the alkyl group is a C1-C3 alkyl group. Typical alkyl groups, but are not limited to, include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butylisobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, and hexyl. Alkyl atoms are attached to the rest of the molecule by single bonds, such as methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and hexyl.

[0017] Unless otherwise specified herein, alkyl groups are independently aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -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, and -N(R')S(O) t R'(where t is 1 or 2), -S(O) t OR' (where t is 1 or 2), -S(O) t It may be optionally substituted with one or more substituents that are N(R')2 (where t is 1 or 2) or PO3(R')2, where each R' is independently hydrogen, alkyl, fluoroalkyl, aryl, aralkyl, heterocycloalkyl, or heteroaryl.

[0018] Where referred to herein, a fluorinated alkyl moiety is an alkyl moiety in which one or more hydrogens of the alkyl moiety are substituted with fluoro substituents. A perfluorinated alkyl moiety is in which all hydrogens bonded to the alkyl moiety are substituted with fluoro substituents.

[0019] Where referred to herein, the "halo" portion is the bromo, chloro, or fluoro portion.

[0020] As used herein, “olefinic” compounds are organic molecules containing an “alkene” moiety. An 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 a molecule may be any type of organic molecule and, in some embodiments, may include an alkyl or fluorinated (but not limited to perfluorinated) alkyl moiety, either of which may be further substituted.

[0021] As used herein, “air” refers to the composition of the dominant gases in the Earth’s atmosphere. The four most abundant gases are nitrogen (typically present at about 78% by volume, e.g., in concentrations ranging from about 70 to 80%), oxygen (typically present at about 20.95% by volume at sea level, e.g., in concentrations ranging from about 10% to about 25%), and argon (typically present at about 1.0% by volume, e.g., about 0%). These are carbon dioxide (present in amounts ranging from 0.1% to approximately 3%) and carbon dioxide (typically present in amounts ranging from approximately 0.04%, e.g., from approximately 0.01% to approximately 0.07%). Air may also contain other trace gases such as methane, nitrous oxide, or ozone, trace pollutants such as pollen and diesel particulate matter, and organic materials. Air may also contain water vapor (typically present in amounts ranging from approximately 0.25%, or in amounts ranging from approximately 10 ppm to approximately 5 volume%). Air may be provided as a filtered and controlled composition for use in culture experiments and may be prepared as described herein.

[0022] As used herein, the term “plural” may mean two, three, four, five, six, seven, eight, nine, ten or more.

[0023] As used herein, the term “to be positioned” includes the meaning of “to be located.”

[0024] As used herein, “microfluidic device” or “microfluidic apparatus” is a device comprising one or more separate microfluidic circuits configured to hold fluid, each microfluidic circuit comprising, but not limited to, fluid-connected circuit elements including regions, flow paths, channels, chambers and / or pens, and at least one port configured to allow fluid (and optionally microscopic objects suspended in the fluid) to flow in and / or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device comprises a flow region which may include a microfluidic channel and at least one chamber, and holds a volume of fluid less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 μL. In certain embodiments, the microfluidic circuit holds approximately 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. The microfluidic circuit may be configured to have a first end that is fluidly connected to a first port (e.g., an inlet) in the microfluidic device, and a second end that is fluidly connected to a second port (e.g., an outlet) in the microfluidic device.

[0025] As used herein, “nanofluid device” or “nanofluid apparatus” is a microfluidic device of the type having a microfluidic circuit that includes at least one circuit element configured to hold a volume of fluid less than about 1 μL, for example, less than or equal to about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nL. Nanofluidic devices may include multiple circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000 or more). In a particular embodiment, one or more (e.g., all) of at least one circuit element is configured to hold a fluid in a volume of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of at least one circuit element has a pulse range of approximately 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, and 200 nL. It is configured to hold a fluid in a volume of 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.

[0026] Microfluidic devices or nanofluidic devices may be referred to herein as "microfluidic chips" or "chips" or "nanofluidic chips" or "chips."

[0027] As used herein, “microfluidic channel” or “flow channel” refers to a flow region of a microfluidic device having a length significantly longer than both the horizontal and vertical dimensions. For example, a flow channel may be at least five times, e.g., at least ten times, at least 25 times, at least 100 times, at least 200 times, at least 500 times, at least 1,000 times, at least 5,000 times, or more than the length of either the horizontal or vertical dimension. In one embodiment, the length of the flow channel is about 100,000 μm to about 500,000 μm (including any value in between). In one embodiment, the horizontal dimension is about 100 μm to about 1,000 μm (e.g., about 150 to about 500 μm), and the vertical dimension is about 25 μm to about 200 μm (e.g., about 40 to about 150 μm). It should be noted that the flow channel may have a variety of different spatial shapes in the microfluidic device and is therefore not limited to a perfectly linear element. For example, a flow channel may be or include one or more parts having the following shapes: curved, curved, helical, inclined, descending, fork-shaped (e.g., multiple different flow paths) and any combination thereof. Furthermore, a flow channel may have different cross-sectional areas along its path and may expand and contract to provide a desired fluid flow within it. A flow channel may include a valve, which may be any type known in the art of microfluidics. Examples of microfluidic channels including valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is incorporated herein by reference in whole.

[0028] As used herein, the term “obstacle” generally refers to a bump or similar structure large enough to partially (but not completely) obstruct the movement of a target microobject between two different regions or circuit elements in a microfluidic device. The two different regions / circuit elements may be, for example, a microfluidic isolation pen and a microfluidic channel, or a connection region and a separation region of a microfluidic isolation pen.

[0029] As used herein, the term “constriction” generally refers to a narrowing of the width of a circuit element (or the interface between two circuit elements) in a microfluidic device. Constriction may be located, for example, at the interface between a microfluidic isolation pen and a microfluidic channel, or at the interface between the isolation region and the connection region of a microfluidic isolation pen.

[0030] As used herein, the term "transparent" refers to a material that transmits visible light without substantially altering the light as it passes through.

[0031] As used herein, the term “micro-objects” generally refers to any microscopic objects that can be separated and / or manipulated in accordance with this disclosure. Non-limiting examples of micro-objects include fine particles; microbeads (e.g., polystyrene beads, Luminex® beads, etc.) Examples include: electromagnetic beads; microrods; microwires; inanimate microobjects such as quantum dots; cells; organelles of living cells; vesicles or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane specimens); biological microobjects such as lipid nanorafts; or combinations of inanimate and biological microobjects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated electromagnetic beads, etc.). Beads may contain covalent or non-covalently bonded moieties / molecules such as fluorescent labels, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical / biological species usable in assays. For example, this is described in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs”, Methods Enzymol., 464:211-231.

[0032] In this specification, the term “cell” is used synonymously with the term “living cell.” Non-exclusive examples of living cells include eukaryotic cells, plant cells, animal cells such as mammalian cells, reptile cells, avian cells, and fish cells; prokaryotic cells, bacterial cells, fungal cells, and protozoan cells; cells dissociated from tissues such as muscle, cartilage, fat, skin, liver, lung, and nerve tissue; immune cells such as T cells, B cells, natural killer cells, and macrophages; embryos (e.g., zygotes), oocytes, eggs, spermatids, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and / or transformed cells, and reporter cells. Mammalian cells may originate from, for example, humans, mice, rats, horses, goats, sheep, cattle, primates, and the like.

[0033] A colony of living cells is a "clone" if all living cells within the reproductively capable colony are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in a cloned colony are derived from a single parent cell by 10 divisions or less. In other embodiments, all daughter cells in a cloned colony are derived from a single parent cell by 14 divisions or less. In other embodiments, all daughter cells in a cloned colony are derived from a single parent cell by 17 divisions or less. In other embodiments, all daughter cells in a cloned colony are derived from a single parent cell by 20 divisions or less. The term "cloned cell" refers to cells in the same cloned colony.

[0034] As used herein, the term “colony” of living cells refers to two or more cells (for example, about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000 or more than 1000 cells).

[0035] As used herein, the term “maintain cells” means providing an environment that includes both fluid and gaseous components, and optionally includes a surface that provides the conditions necessary to maintain cell survival and / or proliferation.

[0036] As used herein, the term “proliferation” refers to an increase in the number of cells when referring to cells.

[0037] As used herein, “gas permeability” means that a material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, a gas permeable material or structure may be permeable to two or more of oxygen, carbon dioxide, and nitrogen, and may also be permeable to all three of these gases.

[0038] The "components" of a fluid culture medium are any chemical or biochemical molecules 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.

[0039] In this specification, when used with respect to fluid culture media, "diffuse" and "diffusion" refer to the thermodynamic transfer of components of the fluid culture medium toward the lower concentration gradient.

[0040] The term "culture medium flow" refers to the bulk movement of a fluid culture medium primarily due to any mechanism other than diffusion. For example, culture medium flow may include the movement of a fluid culture medium from one point to another due to a pressure difference between those points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flows of a liquid, or any combination thereof. When one fluid culture medium flows into another fluid culture medium, turbulence and mixing of the media may occur.

[0041] The phrase "virtually no flow" refers to a flow rate of the fluid medium that, when averaged over time, is less than the diffusion rate of components of the material (e.g., the analyte in question) into or within the fluid medium. The diffusion rate of such material components may depend, for example, on temperature, component size, and the strength of the interaction between the components and the fluid medium.

[0042] When used herein in reference to different regions within a microfluidic device, the phrase “fluid-connected” means that if different regions are substantially filled with a fluid, such as a fluid medium, the fluids in each region are connected in such a way that they form a single fluid. This does not necessarily mean that the fluids (or fluid mediums) in different regions are identical in composition. Rather, because solutes reduce their respective concentration gradients and / or the fluids flow through the device, the fluids in different fluid-connected regions of a microfluidic device may have different compositions within the flux (e.g., different concentrations of solutes such as proteins, carbohydrates, ions, or other molecules).

[0043] As used herein, “flow channel” refers to one or more fluid-connected circuit elements (e.g., channels, regions, chambers, etc.) that define and are exposed to the trajectory of a culture medium flow. Thus, a flow channel is an example of a swept region in a microfluidic device. Other circuit elements (e.g., non-swept regions) may be fluid-connected to the circuit elements containing the flow channel without being exposed to the culture medium flow in the flow channel.

[0044] As used herein, "separating micro-objects" means confining micro-objects to a defined region within a microfluidic device.

[0045] Microfluidic (or nanofluidic) devices may include “swept” and “unswept” regions. As used herein, a “swept” region consists of one or more fluid-connected circuit elements of a microfluidic circuit, each of which receives a flow of culture medium as the fluid flows through the microfluidic circuit. The circuit elements of a swept region may include, for example, all or part of regions, channels, and chambers. As used herein, an “unswept” region consists of one or more fluid-connected circuit elements of a microfluidic circuit, each of which receives substantially no fluid flux as the fluid flows through the microfluidic circuit. An unswept region may be fluid-connected to a swept region if the fluid connection is structured to allow diffusion but not substantially allow the flow of culture medium between the swept and unswept regions. Thus, a microfluidic device may be structured to substantially separate the unswept region from the flow of culture medium within the swept region while substantially allowing only diffusive fluid communication between the swept and unswept regions. For example, the flow channel of a microfluidic device is an example of a swept region, while the isolation region of a microfluidic device (described in more detail below) is an example of a non-swept region.

[0046] As used herein, the “non-sweeping” rate of the fluid medium flow means a flow rate sufficient to diffuse components of the second fluid medium in the isolation area of ​​the isolation pen into the first fluid medium in the flow area, and / or diffuse components of the first fluid medium into the second fluid medium in the isolation area, furthermore, the first medium does not substantially flow into the isolation area.

[0047] Surface modification. Surfaces of materials, devices and / or apparatus for handling and storing biomaterials may have natural properties that are not optimized for short-term and / or long-term contact with such materials, including, but not limited to, micro-objects (including, but not limited to, biological micro-objects such as living cells), biomolecules, fragments of biomolecules or biological micro-objects, and any combination thereof. Related to natural surfaces in contact with one or more biomaterials. To reduce one or more undesirable phenomena, it may be useful to modify one or more surfaces of a material, device, or apparatus. In other embodiments, it may be useful to enhance the surface properties of a material, device, and / or apparatus to introduce desired characteristics to the surface, thereby expanding the handling, operation, or processing capabilities of the material, device, and / or apparatus. For this purpose, molecules are required that can modify the surface to reduce undesirable properties or introduce desirable properties.

[0048] A microfluidic device is described herein, having an enclosure comprising a base, a cover, and a microfluidic circuit material, wherein the microfluidic circuit material defines a fluid circuit, and at least one inner surface of the base, cover, and microfluidic circuit material has a first covalent surface modification comprising a first binding group and a first portion which is a first surface contact portion or a first reactive portion; and at least one inner surface of the base, cover, and microfluidic circuit material has a second covalent surface modification comprising a second binding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first binding group and the second binding group are different from each other, or the first covalent portion is different from the second covalent portion. The first surface modification may be a covalently modified surface, and the second surface modification may be a functionalized surface. In other embodiments, the first surface modification may be a first covalently modified surface, and the second surface modification may be a second covalently modified surface having either different binding groups or different surface modification ligands.

[0049] Modifying reagent: Surface-modifying compound. In various embodiments, the surface-modifying compound may include a surface-modifying ligand which may be a nonpolymeric moiety such as an alkyl moiety, a substituted alkyl moiety such as a fluoroalkyl moiety (including, but not limited to, a perfluoroalkyl moiety), or an alkylene oxide moiety, amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety that covalently modifies the surface to which it is bound. The surface-modifying compound also includes a binding moiety which is a group that covalently bonds the surface-modifying ligand to the surface, as schematically shown in Formula 1. Depending on the surface composition, the binding moiety may be a silicon-containing moiety such as -Si(T)2W, where W is -T, -SH, or -NH2; and T is independently OH, OC 1~6 Alkyl or halo or a combination thereof; a phosphonic acid moiety or its activated form, a maleimide moiety, a terminal olefin, or any preferred binding moiety known in the art. The surface-modifying ligand is covalently bonded to the modified surface via a binding group LG, which is the product of the reaction between the binding moiety and the functional group of the surface (including hydroxides, oxides, amines, or sulfur). The binding group LG may include siloxy, phosphonates, alkyl sulfides, etc. In some embodiments, the binding group LG may be a siloxy or phosphonate group.

[0050] Formula 1. [ka]

[0051] In one embodiment, the surface modification compound is of formula XXXII: VL sm - Surface-modified ligand, formula XXXII The structure is such that the bonding portion V is -P(O)(OH)2 or -Si(T)2W; W is -T, -SH or -NH2, and is a portion configured to bond to the surface. Yes; each of T is independently OH, OC 1~6 It is alkyl or halo. smis a linker containing from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and further contains 0, 1, 2, 3 or 4 linking groups CG. The number of non-hydrogen atoms forming CG is not included in the size of L sm and is not limited by the size of L sm . The surface-modifying ligand may contain 0, 1, 2 or 3 CGs.

[0052] In certain embodiments, the surface-modified compound of formula XXXII is a compound of formula I: V-(CH2) n -Surface-modifying ligand Formula I where the linking moiety V is -P(O)(OH)Q- or -Si(T)2W; W is -T, -SH or -NH2 and is a moiety configured to bind to the surface; Q is -OH and is a moiety configured to bind to the surface; and n is an integer from about 3 to 21. In certain embodiments, n is an integer from about 7 to 21. Each of T is independently OH, OC 1~6 alkyl or halo, where alkyl includes, but is not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, etc. In certain embodiments, T is OH, OC 1~3 alkyl or Cl. The surface-modifying ligand may contain 0, 1, 2 or 3 CGs.

[0053] In certain embodiments, the compound of formula I is a compound of formula II:

Chemical formula

[0054] In other embodiments, the compound of formula I is a compound of formula III:

Chemical formula

[0055] The surface modification compounds used to covalently modify the inner surface of microfluidic devices described herein introduce surface modification ligands having a surface contact portion that support the growth, survival, or portability of living cells. Surface modification ligands containing a surface contact portion may include anionic, cationic, or amphoteric portions, or any combination thereof. While not intended to be theoretically constrained, by presenting cationic, anionic, and / or amphoteric portions on the inner surface of the microfluidic device enclosure, the surface modification ligands of the covalently modified surface may form strong hydrogen bonds with water molecules, such that the resulting hydrated water acts as a layer (or "shield") separating the biological microorganism from interactions with non-biomolecules (e.g., silicon and / or silicon oxide of the substrate). Furthermore, in embodiments where the covalently modified surface is used with a coating agent, the anionic and cationic portions of the surface contact portion of the surface modification ligand may also exist. And / or the zwitterions may form ionic bonds with the charged portion of a non-covalent coating agent (e.g., a protein in solution) present in the culture medium (e.g., a coating solution and / or fluid medium for supporting living cells) within the enclosure. In other embodiments, the surface-modifying ligand may comprise at least one amino acid, which may comprise two or more types of amino acids. Thus, the surface-modifying ligand may comprise a peptide or a protein. In one embodiment, the surface-modifying ligand may comprise an amino acid that can provide a zwitterionic surface to support cell growth, survival, portability, or any combination thereof.

[0056] In further embodiments, the surface-modifying ligand may represent a hydrophilic surface-contact portion at the opposite end of its enclosure, comprising, but not limited to, at least one alkylene oxide moiety. One useful type of alkylene ether-containing polymer is polyethylene glycol (PEG M w (<100,000 Da). In one embodiment, the PEG is M of about 100 Da, 300 Da, 500 Da, 1000 Da or 5000 Da. w It may have the following characteristics. In other embodiments, the hydrophilic surface-modified ligand may comprise one or more saccharides. The covalently bonded saccharides may be monosaccharides, disaccharides, or polysaccharides. Similar to the charged moieties described above, the hydrophilic surface-modified ligand may form strong hydrogen bonds with water molecules so that the resulting hydration water acts as a layer (or "shield") separating the biological microorganism from interactions with non-biomolecules (e.g., silicon and / or silicon oxide of the substrate).

[0057] Surface-modified ligands may instead contain one or more amino groups as surface contact moieties. The amino groups may be substituted amine moieties, guanidine moieties, nitrogen-containing heterocyclic moieties, or heteroaryl moieties. The amino-containing moieties may have a structure that allows for pH regulation of the environment within the microfluidic device. In some embodiments of the microfluidic devices described herein, the environment may be regulated within a pen (which may be the same as or different from the isolation pen described herein) and / or within the flow region (which may include channels) that opens into the flow region.

[0058] In various embodiments, the surface-modified compound comprises a linear skeleton of 8 to 26 atoms (where the atoms are carbon, oxygen, nitrogen, or sulfur); and a bonding moiety selected from -P(O)(OH)2 and -Si(Y)3 (where Y is Cl, OC). 1~3The linear skeleton may contain alkyl or OH groups, and the non-skeletal substituents of the carbon atoms in the linear skeleton are hydrogen or fluorine. The surface-modified compound may be bonded to functional groups (including hydroxides, oxides, amines, or sulfur) on the surface via bonding sites. The first end of the linear skeleton is bonded to the bonding site via bonding to phosphorus or silicon, and the second end of the linear skeleton is distal to the surface and not bonded to the surface. For each carbon in the linear skeleton independently, the non-skeletal substituents are either all hydrogen or all fluorine. In some embodiments, the linear skeleton may be all carbon atoms. A linear skeleton having all carbon skeleton atoms may have non-skeletal substituents that are all hydrogen atoms.

[0059] In one embodiment, the linear skeleton of the surface-modified compound is the linker L described above. sm This can be a portion and may include two carbon atoms located at the first end of the linear skeleton (e.g., directly bonded to the bonding portion), and the non-skeletal substituent for each of the two carbons may be hydrogen. In some embodiments, the linear skeleton may include sulfur atoms. In some embodiments, the linear skeleton may include two sulfur atoms, the two sulfur atoms arranged adjacent to each other. When two adjacent sulfur atoms are present in the linear skeleton, the two sulfur atoms are not located at the first end (e.g., neither of the two sulfur atoms is directly bonded to the bonding portion) or at the second end of the linear skeleton (e.g., distal to bonding to the surface, located at the end of the modified compound). In some embodiments, the disulfide of the linear skeleton The do portion may be a cleavable motif, which may allow for the removal of part or all of the surface-modifying ligand. Other cleavable motifs are linkers L of the surface-modifying compounds described herein. sm It can be included in this.

[0060] In one embodiment, the surface-modified compound may contain 0, 1, 2, 3, or 4 of the linking groups CG described herein. The surface-modified compound may be formed from two or more parts linked together to provide a linking group and a surface-modifying ligand, where CG is a linker L smThis could be the portion of the surface-modifying ligand (which may also include the surface-contact portion).

[0061] In some embodiments, the surface-modified compound may contain carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons or at least 14, 16, 18, 20, 22 or more carbons) and may be an unbranched alkyl moiety. In some embodiments, the alkyl group may contain a substituted alkyl group (e.g., some of the carbons in the alkyl group may be fluorinated or perfluorinated). In some embodiments, the alkyl group may contain a first segment that may contain a perfluoroalkyl group, bonded to a second segment that may contain an unsubstituted alkyl group, and the first and second segments may be bonded directly or indirectly (e.g., by an ether bond). The first segment of the alkyl group may be distal to the bonding group, and the second segment of the alkyl group may be proximal to the bonding moiety.

[0062] In other embodiments of the surface-modified compound, the linear skeleton may contain one or more oxygen atoms. Each of the one or more oxygen atoms may not be directly bonded to another oxygen, sulfur, or nitrogen, and may not be located at the first end of the linear skeleton. In some embodiments, if the linear skeleton contains one or more oxygen atoms, each of the one or more oxygen atoms may not be located at the second end of the linear skeleton. In some embodiments, each of the one or more oxygen atoms may be located within the linear skeleton such that at least two skeleton atoms adjacent to each oxygen atom proximal to the first end of the linear skeleton are carbon atoms containing a hydrogen non-skeleton substituent, and at least two skeleton atoms adjacent to each oxygen atom distal to the first end of the linear skeleton are carbon atoms containing a hydrogen substituent.

[0063] Covalent modification is introduced to the surface during reaction with the compound of formula XXXII, and formula XXXI: [ka] A surface having the structure of the formula can be provided, where LG is -W-Si(OZ)2O- or -OP(O)2O-; W is O, S or N, and Z is a bond to an adjacent silicon atom or a bond to the surface; L sm is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, 2, 3, or 4 bonding groups CG; and [ka] This is the surface. In one embodiment, n is an integer from 7 to 21.

[0064] In one embodiment, the covalent modification is given by formula VIII: [ka] It may have the structure, where W is O, S or N; Z is a bond to an adjacent silicon atom or a bond to the surface; n is an integer from 3 to 21; and [ka] is the surface. In one embodiment, W is O. In various embodiments, n is an integer from 7 to 21. The surface modification ligand may contain 0, 1, 2, or 3 CGs.

[0065] In other embodiments, the covalent modification is given by formula IX: [ka] It has the structure, in the formula, n and [ka] These are defined as described above. Z is either a bond to an adjacent phosphorus atom or a bond to the surface. Surface-modified ligands may contain 0, 1, 2, or 3 CGs.

[0066] In one embodiment, the surface-modified ligand is given by formula X: [ka] The formula may have the following structure, where L is a linker; and the surface contact portion is a portion that provides improved contact characteristics to biological microorganisms as described herein.

[0067] In another embodiment, the surface modification ligand of the modified surface is given by formula X: [ka] The structure may be such that L is a linker, and the surface contact portion is a portion that provides improved contact characteristics with biological microorganisms.

[0068] In accordance with the limitations of chemical bonding as known in the art, the linker L may be a bond or may contain 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In one embodiment, in accordance with the limitations of chemical bonding as known in the art, the linker L may contain 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. The linker L or surface contact portion may contain 0, 1, or 3 bonding groups CG.

[0069] Bonding group CG. CG is a binding group that may result from the binding of its surface contact portion to the remainder of a surface modification reagent of formula XXXII or a surface modification compound of formula I, II, or III (for example, formed as part of the synthesis of a surface modification ligand), including, but not limited to, triazolylenyl, carboxamide, imide, ether, ester, keto, and sulfone compounds. It may be any part such as an amide, sulfonate, cyclooctyl condensed diazine, alkene, or aromatic moiety.

[0070] In some other embodiments, CG is a portion obtained from the reaction between the reactive portion of a functionalizing reagent of formula XXXIII, formula IV, or formula VI and the respective reaction-pair portions of the surface modification reagents described herein. For example, a functionalizing reagent having an azide-reactive portion may form a triazolirenyl CG portion when forming a covalently modified surface of formula XXXI, formula VIII, or formula IX.

[0071] The binding group CG may be a triazolilenyl moiety, which may be further substituted and have one or more further ring systems condensed with the triazolilenyl moiety. The further condensed ring system may itself be further substituted with further condensed rings and may provide a binding site to the linker L-surface contact moiety. In some embodiments, the triazolilenyl moiety is condensed with a cyclooctinyl ring system, which may be further substituted with any of the following, but not limited to, a further condensed ring containing dibenzocyclooctinyl or other substitutions such as fluorine (difluorinated cyclooctin (DIFO)).

[0072] In some embodiments, the CG can be a non-covalent bond. For example, the non-covalent bond of biotin with streptavidin provides a very stable bond pair and can be a CG. Furthermore, since streptavidin has four binding sites, two parts of a surface-modifying ligand, surface-modifying reagent, or functionalized surface can be bound by a series of biotin / streptavidin / biotin. For example, the functionalized surface has a biotin-reactive moiety, in which streptavidin is introduced to bind to the biotin-reactive moiety, and here, finally, a second biotinylated moiety (such as biotin-fibronectin) is introduced and bound to another binding site in streptavidin. The product is a covalent surface modification with a surface-contacting moiety of fibronectin, and the series of biotin / streptavidin / biotin is considered a single binding group CG. Streptavidin plays the role of linking the two similarly functionalized moieties together.

[0073] Surface contact part. The surface contact portion of a surface-modified ligand may be any surface contact portion described herein and in other parts of this disclosure, and may include nonpolymer or polymer portions. The surface contact portion may include alkyl or fluoroalkyl (perfluoroalkyl) portions; monosaccharides or polysaccharides (but not limited to dextran); alcohols (but not limited to propargyl alcohol); and polysaccharides (but not limited to polyvinyl alcohol). The material may include: alcohols; alkylene ethers, but not limited to polyethylene glycol; polyelectrolytes (but not limited to polyacrylic acid or polyvinylphosphonic acid); amino groups (but not limited to alkylated amines, hydroxyalkylated amino groups, guanidium and their derivatives, and heterocyclic groups containing non-aromatic nitrogen ring atoms, such as morpholinyl or piperazinyl); carboxylic acids, but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, but not limited to ethynylphosphonic acid (which may provide a phosphonate anionic surface); sulfonic acid anions; carboxybetaine; sulfobetaine; sulfamic acid; or amino acids. The alkyl or perfluoroalkyl moiety may have a skeletal chain length of more than 10 carbon atoms. In other embodiments, the surface contact moiety may include a saccharide moiety and may be dextran. In other embodiments, the surface contact moiety may include an alkylene ether moiety. The alkylene ether moiety may be polyethylene glycol.

[0074] In various embodiments, the surface contact portion may be a surface-modified ligand comprising a nonpolymer portion such as an alkyl portion, a substituted alkyl portion such as a fluoroalkyl portion (including, but not limited to, a perfluoroalkyl portion), an amino acid portion, an alcohol portion, an amino portion, a carboxylic acid portion, a phosphonic acid portion, a sulfonic acid portion, a sulfamic acid portion, or a saccharide portion. Alternatively, the surface contact portion may include a polymer portion which may be any of the portions described above.

[0075] In some embodiments, the surface contact portion may include carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons or at least 14, 16, 18, 20, 22 or more carbons) and may be an unbranched alkyl portion. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group may be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first segment that may include a perfluoroalkyl group, bonded to a second segment that may include an unsubstituted alkyl group, and the first and second segments may be bonded directly or indirectly (e.g., by an ether bond). The first segment of the alkyl group may be distal to the bonding group, and the second segment of the alkyl group may be proximal to the bonding group.

[0076] Cuttable part. Surface-modified ligands are linkers L of surface-modified compounds. sm It may further include cleavable portions that may be located within, and the linker L of the surface-modifying ligand may be a portion of the surface-contact portion of the surface-modifying compound or surface-modifying reagent. In one embodiment, the cleavable portion is the linker L of the functionalized surface of formula XXX, formula V, or formula VII. mIt may be contained within. The cleavable portion may be configured to allow cleavage of the covalently modified surface. In some embodiments, cleavage may be useful in promoting the portability of one or more living cells after the culture period. The cleavable portion may be a UV-cleavable portion such as a nitro-substituted benzyl ester (e.g., a photocleavable portion such as BroadPharm catalog no. BP-22675); a substituted 1,2-diphenylethyl ketoester portion (e.g., a benzyl derivative such as BroadPharm catalog no. BP 22689); or a portion that can be cleaved under specific chemical conditions. For example, a disulfide bond may be cleaved under conditions that will not impede the growth or survival of living cells on the covalently modified surface (e.g., reducing conditions such as dithiothreitol). Other useful cleavable portions that can be incorporated into the surface-modifying ligand or functionalized surface may include adjacent diol portions that can be cleaved by sodium periodate. Sodium periodate cleavage is another non-cytotoxic cleavage reagent. Diazo portions that can be cleaved by dithionite may also be useful cleavable portions. Furthermore, the 5,5,dimethyl-exo-cyclohexenyl-1,3,dione moiety may be a cleavable moiety useful for use as a surface modification ligand or functionalized surface of formulas XXX, V, or VII, and can be cleaved by a hydrazine solution. .

[0077] Modification reagents: Surface functionalization reagents. The surface can be covalently modified with a functionalizing reagent to introduce a functionalized surface modification to one or more surfaces of a microfluidic device.

[0078] The functionalizing reagent is formula XXXIII: VL fm -R x Formula XXXIII The compound is such that V is -P(O)(OH)2 or -Si(T)2W; W is -T, -SH or -NH2 and is a portion configured to bond to the surface; and T is independently OH, OC 1~6 Alkyl or halo; L fmis a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, or 2 bonding groups CG; and R x This is the reactive part.

[0079] The reactive part. The reactive moiety may be any of the following: alkyne moiety, azide moiety, amine moiety, carboxylic acid moiety, biotin moiety, streptavidin moiety, olefin moiety, transcyclooctene moiety, s-tetrazine moiety, thiol moiety, maleimide moiety, halide moiety, cyano moiety, isocyanate moiety, epoxide moiety, hydroxyamine moiety, masked hydroxyl, such as acetate, or sulfonyl fluoride moiety. This list of reactive moieties is not limiting, and any suitable reactive moiety may be selected for use with a suitable reaction partner moiety. Most reactive moieties react with their respective reaction partner moieties to form covalently bonded CGs, while the high binding affinity between biotin and streptavidin enables their use as a reactive moiety / reaction partner moiety.

[0080] The functionalized surface formed by the reaction of functionalizing reagent XXXIII is given by formula XXX: [ka] The structure is such that, in the formula, LG is -W-Si(OZ)2O- or -OP(O)2O-; W is O, S or N, Z is a bond to an adjacent silicon atom or a bond to the surface, and L fm and R x This is as defined for formula XXXIII.

[0081] In one embodiment, the functionalizing reagent of formula XXXIII is formula IV: [ka] It may be a compound of OC, where R is OC 1~6It is alkyl, and n is an integer between 3 and 21. The azide is the reactive part R x In some embodiments of the compound of formula IV, n can be an integer from 7 to 21. For the compound of formula IV, each of R can be independently selected from H or C1-C6 alkyl groups, where alkyl groups are not limited to methyl, ethyl, n-propyl, 2-propyl, n-butyl, etc. In one embodiment, R may be a C1-C3 alkyl group. In another embodiment, R may be methyl or ethyl. In various embodiments, each of the three cases of R is either methyl or each of the three cases of R is ethyl. In yet another embodiment, n may be 9, 14, or 16. In yet another embodiment, n may be 9.

[0082] The functionalized surface formed by the reaction of the surface with the surface functionalization reagent of formula IV is expressed by formula V: [ka] The formula may have the following structure, where W is O, S, or N, and Z is another surface-functionalizing ligand (-WSi(OZ)2(CH2)) that is also bound to the surface. n -N3) is a bond to an adjacent silicon atom or a bond to the surface, where n is an integer from 3 to 21, and [ka] is the surface. In one embodiment, n can be an integer from about 7 to 21. In one embodiment, W can be O. In various embodiments, each of R can be independently selected from H or C1-C6 alkyl, where alkyls include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, etc. In one embodiment, R can be a C1-C3 alkyl. In one embodiment, R can be methyl or ethyl. In various embodiments, each of the three cases of R is either methyl or each of the three cases of R is ethyl. In other embodiments, n can be an integer from 7 to 21. In one embodiment, n can be an integer from 9 to 21, 10 to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, or 18 to 21. In yet another embodiment, n may be an integer between 10 and 18, 12 and 18, 13 and 18, or 14 and 18. In yet another embodiment, n may be 9, 14, or 16. In yet another embodiment, n may be 9.

[0083] In other embodiments, the surface functionalizing reagent of formula XXXIII is formula VI: [ka] The compound may be, where n is an integer from 3 to 21, and each of R is independently H or C1-C6 alkyl. Alkynes are the reactive part R of formula VI. x In some embodiments, n can be an integer from about 7 to 21. In various embodiments, each of R can be independently selected from H or C1-C6 alkyl groups, where alkyl groups are not limited to methyl, ethyl, n-propyl, 2-propyl, n-butyl, etc. In some embodiments, R can be a C1-C3 alkyl group. In some embodiments, R can be methyl or ethyl. In various embodiments, In each of the three cases of R, either methyl or ethyl. In one embodiment, n can be an integer from 9 to 21, 10 to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, or 18 to 21. In yet another embodiment, n can be an integer from 10 to 18, 12 to 18, 13 to 18, or 14 to 18. In yet another embodiment, n can be 9, 14, or 16. In yet another embodiment, n can be 9.

[0084] The compound of formula VI is formed by a reaction between the siloxane moiety and the nucleophilic group on the surface, resulting in a covalent bond on the surface, which is then formed by formula VII: [ka] A functionalized surface having the structure of can be provided, where W, Z, and n are defined as above for formula V, and [ka] This is the surface.

[0085] A covalently modified surface formed from a functionalized surface. Once the surface functionalization reagent is bonded to the surface, the reactive portion of the functionalized surface obtained from formula XXX, formula V, or formula VII can then be reacted with a surface modification reagent having a reaction partner portion selected to be a suitable reaction partner for the reactive portion of the functionalized surface. The surface modification reagent is formula XII: RP-L-Surface contact part Formula XII The structure is as follows: RP is the reaction pair portion; L is the linker; and the surface contact portion is the portion that provides improved contact properties to biological microorganisms. According to the limitations of chemical bonding as known in the art, the linker L may be a bond or may comprise 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments, according to the limitations of chemical bonding as known in the art, the linker L may comprise 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. The linker L or surface contact portion may comprise 0, 1, 2, or 3 bonding groups CG. The surface contact portion is any surface contact portion described herein.

[0086] Reaction pair portion. The reaction-pair portion RP is a portion of the functionalized surface that can react with the reactive portion. For example, the reactive portion R x It can be an alkyne, and the corresponding reaction pair partial RP can be an azide. Alternatively, R x The reactive part R can be an azide, and the reactive part R can be an alkyne. x Other pairs of reaction pairs for partial RP include, but are not limited to, cyanos and azides; carboxylic acids and amines; olefins and nucleophiles; amines and sulfonyl fluorides; transcyclooctene and s-tetrazine, thiols and maleimides; halides and nucleophiles; isocyanates and amines; epoxides and nucleophiles; hydroxyamines and aldehydes or esters; and masked hydroxyls, such as acetates and nucleophiles. Rx: Special cases of RP pairs are biotin and streptavidin, but they are not covalent pairs. x This is because it is an exemplary stable non-covalent pair that can be used as an RP pair.

[0087] Functionalized surface is R xIf the surface modification reagent has an azide or alkynyl moiety, the surface modification reagent has a reaction pair moiety RP which is an alkyne or azide, respectively, which can react to form a triazolirenyl moiety by a cyclization reaction ("click reaction"), as is known in the art. In one embodiment, the reactive moiety R x Alternatively, the reactive RP moiety is an acyclic alkyne. In other embodiments, the reactive moiety R x Alternatively, the reaction-to-RP moiety is a cyclized alkyne, which may be a portion of cyclooctin. In some embodiments, cyclooctin may be strained. Cyclooctin may have a further cyclic ring condensed to it, such as a benzo group, and may be dibenzocyclooctin. In other embodiments, cyclooctin may have a fluoro substituent. When the alkyne of the surface-modifying reagent is cyclooctin, the surface-contact portion of the reagent is bound to cyclooctin via a linker L, which can be bound at any suitable position on cyclooctin. When the alkyne of the functionalized surface is cyclooctin, the binding group that binds cyclooctin to the surface is bound to cyclooctin at any suitable position on cyclooctin.

[0088] Formula XXX: [ka] The covalently modified surface obtained from the reaction of the functionalized surface with the surface modification reagent of formula XII is expressed by formula XXXI: [ka] It may have a structure in which LG and L sm , surface-modified ligands and [ka] L is defined above, and sm Alternatively, the surface-modified ligand may contain at least one CG and may further have two, three, or four CGs.

[0089] In one embodiment, a covalently modified surface formed from a functionalized surface of formula XXXI, formula V, or formula VII is a surface of formula VIII: [ka] It may have a structure in which W, Z, n and [ka] These are defined as described above. A surface-modified ligand may contain 0, 1, 2, or 3 CGs.

[0090] In one embodiment, a covalently modified surface formed from a functionalized surface of formula XXXI is a surface of formula IX: [ka] It may have a structure in the formula, where Z, n and [ka] These are defined as described above. A surface-modified ligand may contain 0, 1, 2, or 3 CGs.

[0091] Further functionalization of functionalized surfaces. In yet another embodiment, the functionalized surface of formula XXXIV is: RP-L fm -R x2 Formula XXXIV It may have a further functionalization added by reaction with a secondary functionalization reagent, where RP is the reaction pair part for reacting with the reactive part of formula XXX; R x2 L is a reactive portion selected not to react with the reactive portion of the functionalized surface of formula XXX; and L fmR is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, or 2 bonding groups CG. x2 R of the functionalized surface x The orthogonal reaction pairs are selected so as not to hinder the bonding of RP to the portion. In some non-limiting examples, the R of the functionalized surface x If R is Azid, x2 This can be selected to be an amine, epoxide, or sulfonyl fluoride. This capability allows for control in further refining of the functionalized surface.

[0092] The product is a functionalized surface of formula XXXV, where the second functionalized surface is 1, 2, or 3 CGs: [ka] Includes, in the formula, R x2 This is defined for formula XXXIV, and L fm And LG are as defined above for formula XXX. When a functionalized surface of formula V or formula VII is reacted with a secondary functionalizing reagent of formula XXXIV, the product is a functionalized surface of formula XXXV, where LG is -W-Si(OZ)2O- and W is O, S, or N. In some embodiments, W is O.

[0093] The functionalized surface of formula XXXV is formula XXXI: [ka] It can be converted to a covalently modified surface, where LG, L sm The surface modification ligand is defined as described above by further reaction with the surface modification reagent of formula XII. In this embodiment, the surface modification (e.g., a covalently modified surface) is L sm It includes at least two CG images.

[0094] Figure 2H shows a cross-sectional view of a microfluidic device 290 including an exemplary covalently modified surface 298. As illustrated, the covalently modified surface 298 (circularly shown) may include a monolayer of densely packed molecules covalently bonded to both the inner surface 294 of the substrate 286 and the inner surface 292 of the cover 288 of the microfluidic device 290. The covalently modified surface 298 may be located on substantially all inner surfaces 294, 292 of the microfluidic device 290, proximal to the enclosure 284 and facing inward thereto, including the surface of a microfluidic circuit material (not shown) used to define circuit elements and / or structures within the microfluidic device 290, as in some embodiments and as described above. In alternative embodiments, the covalently modified surface 298 may be located on only one or more of the inner surfaces of the microfluidic device 290.

[0095] In an embodiment schematically shown in Figure 2H, the covalently modified surface 298 comprises a monolayer of substituted siloxane molecules, each molecule covalently bonded to the inner surfaces 292, 294 of the microfluidic device 290 via a siloxylinker 296. For brevity, further silicon oxide bonds linking to adjacent silicon atoms are shown, but the invention is not limited thereto. In some embodiments, the surface-modifying ligand 298 may contain any type of nonpolymer molecule described herein (e.g., fluorinated alkyl groups, polyethylene glycol-containing groups, alkyl groups containing carboxylic acid substituents) at its enclosure-opposite end (i.e., the monolayer portion of the surface-modifying ligand 298 that is not bonded to the inner surfaces 292, 294 and is proximal to the enclosure 284). Although Figure 2H is described as having a nonpolymer surface-modifying ligand, polymer portions are also preferred surface contact portions and / or surface-modifying ligands that can be incorporated into the covalently modified surface described herein.

[0096] In other embodiments, the surface modification ligand 298 used to covalently modify the inner surfaces 292, 294 of the microfluidic device 290 may include anionic, cationic, or amphoteric moieties or any combination thereof. While not intended to be theoretically constrained, the surface modification ligand for the covalently modified surface 298, by presenting cationic, anionic, and / or amphoteric moieties on the inner surface of the enclosure 284 of the microfluidic circuit 120, forms strong hydrogen bonds with water molecules so that the resulting hydrated water acts as a layer (or "shield") separating biological microorganisms from interactions with non-biomolecules (e.g., silicon and / or silicon oxide in the substrate). It can be formed.

[0097] The surface to be modified. Surfaces modifiable with any of the compounds of formulas XXXII, I, II, III, XXXIII, IV, VI, XII, or XXXIV may be metals, metal oxides, glass, or polymers. Some materials that may have covalently modified or functionalized surfaces therein include, but are not limited to, silicon and its oxides, silicone, aluminum or its oxide (Al2O3), indium tantalum oxide (ITO), titanium dioxide (TiO2), zirconium oxide (ZrO2), hafnium(IV) oxide (HfO2), tantalum(V) oxide (Ta2O5), or any combination thereof. Polymers may include any suitable polymer. Suitable polymers, but are not limited to, rubber, plastics, elastomers, silicones, and organosilicones such as polydimethylsiloxane ("PDMS"), which may be gas permeable. Other examples include patternable materials such as cast glass and silicone polymers (e.g., photopatternable silicone or "PPS"), and photoresists (e.g., SU8). Examples include epoxy-based photoresists. In other embodiments, the surface of a material such as natural fibers or wood may be modified with any of the compounds of formulas XXXII, I, II, III, XXXIII, IV, VI, XII, or XXXIV to introduce a covalently modified surface of formula XXXI, formula VIII, or formula IX, or a functionalized surface of formulas XXX, V, VII, or formula XXXV.

[0098] The modified surface may, but is not limited, contain nucleophilic moieties including hydroxides, aminos, and thiols. The nucleophilic moieties on the surface (e.g., hydroxides (referred to as oxides in some embodiments)) may react with any of the compounds of formula XXXII, I, II, III, XXXIII, IV, or VI to covalently bond the compound to the surface via siloxy or phosphonate groups, thereby providing a functionalized surface. The modified surface may contain naturally occurring nucleophilic moieties, or may be introduced with reagents (e.g., piranha solution) or by plasma treatment to introduce nucleophilic moieties (e.g., hydroxides (alternatively referred to as oxides)).

[0099] Physical and performance properties of covalently modified surfaces. In one embodiment, a covalently modified surface of formula XXXI, formula VIII, or formula IX may have a thickness of less than 10 nm (e.g., less than about 7 nm, less than about 5 nm, or about 1.5 to 3.0 nm). This is particularly true for spin-coated perfluorotetrahydrofuranyl polymers such as CYTOP®, which typically result in a thickness of about 30 to 50 nm. In contrast to other hydrophobic materials, it can favorably provide thin layers on modified surfaces. The data shown in Table 1 are for silicon / silicon oxide natural surfaces that have been converted to functionalized surfaces (e.g., specific members of the type of formula XV (a particular member of formula V)) or surface-modified with surface contact portions (e.g., specific embodiments of modified surfaces of formulas XVI, XVII, and VIII). Contact angle measurements were taken using the static droplet method (Drelich, J. Colloid Interface Sci. 179, 37-50, 1996). The thickness was measured using polarization analysis.

[0100] [Table 1]

[0101] As predicted, modification of the silicon / silicon oxide surface to have a functionalized surface of formula XV resulted in a modified surface with an increased contact angle to water of approximately 80°. This is in contrast to the contact angle to water of plasma-cleaned silicon surfaces, which is less than 10°. Further refinement of the functionalized surface to provide a modified surface of formula XVI (including the PEG portion) yields a much more hydrophilic surface with a reduced contact angle of 35°. Modified surfaces having a surface of formula XVII (including dextran) had a contact angle of 40°.

[0102] Other suitable analytical methods for characterizing surfaces include infrared spectroscopy and / or X-ray photoelectron spectroscopy.

[0103] In one embodiment, the modified surface of formula XXXI, formula VIII, or formula IX forms a single layer. It is possible. The uniformity and homogeneity of the monolayer modified surface can provide advantageous performance, especially if the monolayer modified surface has other functional attributes. For example, the modified surfaces of formulas XXXI, VIII, or IX may also include an electrode-activated substrate and optionally further include a dielectric layer, as seen in materials, devices, and / or apparatus having a dielectrophoretic or electrowetting configuration. The lack of unsaturation of the perfluoroalkyl moiety of the modified surface can minimize "charge trapping" compared to monolayers containing, for example, olefinic or aromatic moieties. Furthermore, the densely packed nature of the monolayer formed on the surface of formulas XXXI, VIII, or IX can minimize the possibility of cations passing through the monolayer to the underlying metal, metal oxide, glass, or polymer substrate. Although not limited by theory, the breakdown of the substrate surface due to the addition of cations to the substrate composition can disrupt the electrical properties of the substrate, thereby reducing its ability to function electrokinetically.

[0104] Furthermore, the ability to introduce a modified surface by covalent bonding can increase the dielectric strength of the modified surface and protect the underlying material from fracture under the application of an electric field. The uniformity and thinness of the dielectrophoretic or electrowetting surfaces of materials, devices and / or apparatus having covalently modified structures of formula XXXI, formula VIII, or formula IX may further provide favorable benefits to such modified dielectrophoretic and / or electrowetting surfaces when the material, device and / or apparatus is optically operated.

[0105] In one embodiment, the modified surface does not require a single layer that is fully formed to function suitably for operation. The physical thickness and uniformity of the layer on any of the surfaces of formulas XXXI, VIII, IX, XXX, V, VII, or XXXV can be measured using an ellipsometer.

[0106] Multiple covalent surface modifications and multilayer surfaces. A microfluidic device may have two or more regions within the microfluidic device having covalently modified surface modifications, each region having only one type of covalently bonded portion. Alternatively, a microfluidic device may have two or more different types of covalently bonded portions on a single selected surface (e.g., a common inner surface of the microfluidic device) or on all of the inner surfaces of the microfluidic device.

[0107] For example, a first covalent surface modification of a surface may have a predetermined number of non-hydrogen atoms as part of the linker and / or surface modification ligand. A second covalent surface modification of this surface may potentially include a surface contact portion having one or more charged moieties covalently bonded to a linker having a larger number of non-hydrogen atoms, which may provide the ability to present charged moieties further away from the thus modified surface in closer contact with biological microorganisms in a microfluidic environment.

[0108] In another case, the modified surface can have a first covalent surface modification that attaches to the surface via a linker having a first type of sterically unencumbered surface contact portion and fewer non-hydrogen atoms. The modified surface can have a second covalent surface modification having a linker with a sterically encumbered surface contact portion and a greater number of non-hydrogen atoms. This mixture of covalent surface modifications can serve to present a sterically encumbered surface contact portion while preventing unwanted interactions with the silicon / silicon oxide, hafnium oxide, or alumina that makes up the surface itself. In another example, the covalently attached moiety can provide an amphoteric surface that randomly presents oppositely charged surface contact portions at the surface.

[0109] In other embodiments, the covalently modified surface can have increased hydrophilic and / or amphiphilic properties by introducing a combination of a first covalent surface modification and a second covalent surface modification. The introduction of the combination of the first and second covalent surface modifications can provide a regulated or customizable hydrophilic, amphiphilic, or hydrophobic property to the surface (including the common inner surface of a microfluidic device). The increased hydrophilic and / or amphiphilic nature of the covalently modified surface can provide hydrophilic functional and / or hydrophobic moieties to which biological microparticles can bind without irreversibly adhering. These bindings can provide a beneficial environment during cell culture compared to the native unmodified surface of a microfluidic device.

[0110] Each of these properties may increase the durability, functionality, and / or biocompatibility of the modified surface. Each of these properties may further benefit viability (including growth rate and / or cell doubling acceleration) and the properties of colonies formed on covalently modified surfaces having the structure of formula XXXI, formula VIII, or formula IX. Improved viability may include providing surface contact portions that offer adherent cells suitable fixation sites that provide sufficient mechanical resistance to promote growth. Covalently modified surfaces of formula XXXI, formula VIII, or formula IX may improve the portability (including viability during export) of microobjects or biomolecules on and within devices and / or apparatus having covalently modified surfaces. In another embodiment, covalently modified surfaces having the structure of formula XXXI, formula VIII, or formula IX may provide surface contact portions that prevent motile cells from moving from a specific area of ​​a microfluidic device (e.g., an isolation pen), thereby minimizing cell migration from the selected area. Cell portability is inhibited in this case, preventing the self-propulsive movement of cells from one isolation pen to another, and minimizing contamination from one isolation pen to the other. However, this inhibitory effect can be regulated by selecting different ratios of covalent surface modifications such that the cells can still be removed from the isolation pen at the desired time under a force such as gravity or dielectrophoresis (which can be actuated by light).

[0111] The combination of covalent surface modifications may be any combination of covalently modified surfaces and / or functionalized surfaces or secondary functionalized surfaces as described herein. Any combination of bonding groups, linkers, reactive portions and / or surface contact portions may be selected for microfluidic devices having first and second covalent surface modifications, and the first and second covalent surface modifications may be distinct from each other. The first and second covalent surface modifications may be any of formulas XXX, V, VII, XXXI, VIII and / or IX.

[0112] In certain embodiments, the microfluidic device can have one or both of the first and second covalent surface modifications that are functionalized surfaces for further modification by the user. A microfluidic device having one or two functionalized surfaces with different reactive moieties, linkers, and / or binding groups can be reacted with a surface modification reagent (e.g., a reagent of formula XII) to provide a covalently modified surface or further functionalized by reaction with a secondary functionalization reagent (e.g., a reagent of formula XXXIV) to provide a secondary functionalized surface. Orthogonal chemistries (e.g., reactive moieties and reaction counter moieties and reaction conditions) can be selected to enable selective reaction of one functionalized surface in the presence of the second functionalized surface or in the presence of the covalently modified surface, as is known in the art. In one non-limiting example, when an alkyne is present as the first reactive moiety (Rx or Rx2) of the first covalent surface modification, it is designed to react with an azide as the reaction counter moiety. The second covalent surface modification can have a second reactive moiety selected such that it is an amine or carboxylic acid that does not participate in a "click" type reaction.

[0113] In certain embodiments, the covalently modified surface can include a combination of a functionalized surface of formula XXX, formula V, or formula VII; and a first covalent surface modification of formula XXXI, formula VIII, or formula IX. The combination of the functionalized surface and the first covalent surface modification of formula XXXI, formula VIII, or formula IX can be randomly distributed on the covalently modified surface. In other embodiments, the covalently modified surface is a functionalized one of formula XXX, formula V, or formula VII adjacent to a second region that includes the first covalent surface modification of formula XXXI, formula VIII, or formula IX It may have a first region having a surface. In other embodiments, the covalently modified surface may include a plurality of regions having a first covalent surface modification of formula XXX, formula VIII, or formula IX, separated from each other by functionalized surfaces of formula XXXI, formula V, or formula VII. In yet another embodiment, the covalently modified surface may include a plurality of regions having functionalized surfaces of formula XXX, formula V, or formula VII, separated from each other by a first covalent surface modification of formula XXXI, formula VIII, or formula IX.

[0114] In other embodiments, a covalently modified surface may have a combination of a first covalent surface modification of formula XXXI, formula VIII, or formula IX; and a second covalent surface modification of formula XXXI, formula VIII, or formula IX, wherein the first and second covalent surface modifications are different. In one embodiment, the different first and second covalent surface modifications may be randomly distributed on the covalently modified surface. In another embodiment, the covalently modified surface may have a first region having the first covalent surface modification of formula XXXI, formula VIII, or formula IX adjacent to a second region having the second covalent surface modification of formula XXXI, formula VIII, or formula IX. In yet another embodiment, the covalently modified surface may have multiple regions having the first covalent surface modification of formula XXXI, formula VIII, or formula IX, separated from each other by the second covalent surface modification of formula XXXI, formula VIII, or formula IX.

[0115] In further embodiments, a covalently modified surface may have a combination of a first covalent surface modification of formula XXX, formula V, or formula VII; and a second covalent surface modification of formula XXX, formula V, or formula VII, wherein the first and second covalent surface modifications are distinct, and the reactive portion of the first covalent surface modification does not react with the reactive portion of the second covalent surface modification. In one embodiment, the first and second covalent surface modifications may be randomly distributed on the surface. In another embodiment, a covalently modified surface may include a first region having the first covalent surface modification of formula XXX, formula V, or formula VII adjacent to a second region having the second covalent surface modification of formula XXX, formula V, or formula VII. In yet another embodiment, a covalently modified surface may have a plurality of regions containing the first covalent surface modification of formula XXX, formula V, or formula VII, separated from each other by the second covalent surface modification of formula XXX, formula V, or formula VII.

[0116] Multiple surface modifications to adjust adhesion. In some embodiments, it may be useful to modulate the ability of cells to adhere to a surface within a microfluidic device. Surfaces that are substantially hydrophilic may not provide anchoring points for cells that require the mechanical stress of adhesion to grow and proliferate properly. Surfaces exhibiting an excessive amount of such anchoring portions may prevent adherent cells that are growing without problems from being removed from the isolation pen and from the microfluidic device. The second covalent surface modification of the composite surface includes surface contact portions that help to anchor adherent cells. The surface structures described herein and the methods for fabricating them provide the ability to select the amount of anchoring portions that would be desirable for a particular use. It has been surprisingly found that a very small percentage of adhesive motifs may be required to provide a sufficient adhesion-enhancing environment. In some embodiments, the adhesion-enhancing portions are fabricated before the cells are introduced into the microfluidic device. Alternatively, the adhesion-enhancing modified surface may be provided before the cells are introduced, and further addition of another adhesion-enhancing portion may be made, which is designed to bond covalently or noncovalently (e.g., as the base of a biotin / streptavidin bond) to the first modified surface.

[0117] In one embodiment, adhesion-enhancing surface modification may modify the surface with a random pattern of individual molecules of surface-modifying ligands. In another embodiment, adhesion-enhancing surface modification with a more concentrated pattern may have hydrophilic surface modifications to modulate adhesion enhancement, generating small areas of surface modification surrounded by the rest of the surface, such as positively charged lysine side chains. This can be introduced by using polymers containing multiple adhesion-enhancing motifs. This can be further refined by using dendritic polymers having multiple adhesion-enhancing ligands. Dendritic polymer-type surface modification compounds or reagents may be present in much smaller proportions than a second surface modification having only hydrophilic surface contact portions, while still providing adhesion enhancement. Furthermore, the dendritic polymer-type surface modification compounds or reagents themselves may have a mixed combination of final functionalities that can further modulate the overall behavior of the surface.

[0118] In one embodiment, it would be desirable to provide regioselective introduction of a surface. It would be desirable to provide a surface in an isolation pen leading to a channel that provides the ability to culture adherent cells without issue and to easily extract them using dielectrophoretic force when needed, while providing a first type of surface within a microfluidic channel. In one embodiment, the adhesion-enhancing modification may include a cleavable portion. The cleavable portion may be cleavable under conditions compatible with the cells cultured therein, such that the cleavable portion can be cut at any desired time, and the surface properties may change so as not to enhance adhesion. Since the lower cut surface may be usefully uncontaminated, extraction is facilitated at that point. While the examples described herein focus on modulating adhesion and motility, the use of these regioselectively modified surfaces is not limited thereto. Various surface modifications can be incorporated into surfaces having the first and second surface modifications according to this disclosure for any kind of benefit of the cells cultured therein.

[0119] Adhesive motif. In general, surface modifications having positively charged surface contact moieties, such as poly-L-lysine and amines, can be used within the modified surfaces of this disclosure. Another motif that can be used comprises the tripeptide sequence RGD, which is available as a biotinylation reagent and readily adapts to the methods described herein. Other larger biomolecules that can be used include, among others, fibronectin, laminin, or collagen. Surprisingly, a surface modification having the structure of formula XXVI, comprising a polyglutamic acid surface contact moiety, has shown the ability to induce adherent cells to bind and grow viably. Another motif that may help provide adhesion sites is an elastin-like peptide (ELP), which comprises a repeating sequence VPGXG, where X is a variable amino acid that can modulate the effect of the motif.

[0120] Position-selective introduction to different surfaces. In one embodiment, the surface of a flow region (e.g., a microfluidic channel) may be modified with a first covalent surface modification, and the surface of at least one isolation pen may be modified with a second covalent surface modification, the first and second covalent surface modifications having different surface contact portions, different reactive portions, or a combination thereof. The first and second covalent surface modifications may be selected from any of formulas XXX, V, VII, XXXI, VIII, and / or IX. If both the first and second covalent surface modifications include functionalized surfaces of formula XXX, V, or VII, an orthogonal reaction chemistry is selected for the selection of the first and second reactive portions. In various embodiments, all surfaces of a flow region may be modified with a first covalent surface modification, and all surfaces of at least one isolation pen may be modified with a second covalent modification.

[0121] In one embodiment, the microfluidic device may have a surface with a combination of first and second covalent surface modifications selected from surfaces of formulas V, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, XXXVII, XXXVIII, XXXIX, or XL. In another embodiment, the microfluidic device may have a region of the microfluidic device having the first covalent surface modification, as well as surfaces of formulas V, XVI, XVII, XVIII, XIX, XX, XXI, XXII The microfluidic device may have a second region having a second covalent surface modification, which can be selected from the surfaces of formulas XXIII, XXIV, XXV, XXVI, XXVII, XXVIII, XXIX, XXXVI, XXXVII, XXXVIII, XXXIX, or XL (for example, a flow region having a first covalent surface modification and an isolation pen having a second covalent surface modification).

[0122] A method for preparing a covalently modified surface. The surfaces of materials that may be used as components of a device or apparatus may be modified before assembly of the device or apparatus. Alternatively, a partially or fully assembled device or apparatus may be modified so that all surfaces in contact with biomaterials, including biomolecules and / or microobjects (which may include biological microobjects), are modified simultaneously. In some embodiments, the entire interior of a device and / or apparatus may be modified, even if different materials are present on different surfaces within the device and / or apparatus. In some embodiments, a partially or fully assembled device and / or apparatus may be a microfluidic device or its components as described herein.

[0123] The surface to be modified may be cleaned before modification to ensure that the nucleophilic portions on the surface are not covered by, for example, oil or adhesive and are freely available for the reaction. Cleaning may be carried out by any suitable method, including treatment with a solvent containing alcohol or acetone, sonication, or vapor cleaning. Alternatively or in addition, such pre-cleaning may be performed in an oxygen plasma cleaner capable of removing various impurities while simultaneously introducing an oxidized surface (e.g., oxides on a surface that can be covalently modified as described herein). Alternatively, liquid-phase treatment such as a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., a piranha solution which may have a sulfuric acid to hydrogen peroxide ratio of about 3:1 to about 7:1) may be used instead of an oxygen plasma cleaner.

[0124] This advantageously provides more areas for surface modification, thereby enabling a more densely packed modified surface layer.

[0125] A method for covalently modifying a surface includes modifying the surface with a surface modification reagent of formula XXXII, formula I, or formula III. Introducing a covalently modified surface means modifying the surface using formula XXXII: VL sm - Surface-modified ligand, formula XXXII Contacting the surface-modified compound (wherein V, Lsm, and surface-modifying ligand are defined above) with the reagent of formula XXXII with the nucleophilic portion of the surface; formula XXXI: [ka] (In the formula, it is LG) This may include forming a covalently modified surface of the compound of formula XXXII. In one embodiment, the surface modification compound of formula XXXII is a compound and the surface [ka] is of formula I or formula III: V-(CH2) n -Surface-modifying ligand Formula I

Chem.

Chem.

Chem.

[0126] In other embodiments, the surface generated by the reaction of the surface-modifying compound of formula XXXI is of formula IX:

Chem.

Chem.

[0128] In one embodiment, the functionalizing reagent of formula XXXIII is given by the following formula: [ka] A functional reagent having one structure of formula IV or formula VI; formula V or formula VII is [ka] Each provides a functionalized surface; W, Z and n are as defined above, and [ka] is the surface. In one embodiment, W is O. Each of R can independently be H or a C1-C6 alkyl group. In one embodiment, n can be an integer from 7 to 21. In another embodiment, n can be 9, 14, or 16. In yet another embodiment, n is 9. In one embodiment, R is a C1-C3 alkyl group. In another embodiment, R is methyl or ethyl. In yet another embodiment, R is methyl.

[0129] In the case of surface modification reactions and surface functionalization reactions. In one embodiment, the nucleophilic portion of the surface is a hydroxide, amino acid, or thiol. In another embodiment, the nucleophilic portion of the surface may be a hydroxide. The surface may be a metal, metal oxide, glass, polymer, or any combination thereof. The surface material that can be modified by this method may be any material described herein.

[0130] The contact step may be carried out by bringing the surface into contact with a liquid solution containing a modifying reagent of formula XXXIII, formula IV, formula VI, formula XXXII, formula I and / or formula III, which may be any combination as described herein. For example, the surface may be exposed to a solution containing a modifying reagent of formula XXXIII, formula IV, formula VI, formula XXXII, formula I and / or formula III at a concentration of 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM, or 100 mM. The reaction may be carried out at ambient temperature for about 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, or The reaction can be carried out over a period of any value within that range. Examples of solvents include, but are not limited to, dimethylformamide (DMF), acetonitrile (ACN), toluene, 1,3-bistrifluorobenzene, or Fluorinert® (3M) fluorinating solvents. Acids such as acetic acid may be added to the solution, if present, to increase the reaction rate by promoting the hydrolysis of the trialkoxy group.

[0131] Alternatively, the surface may be contacted with a gas phase containing modifying reagents of formulas XXXIII, IV, VI, XXXII, I, and / or III, which may be any combination described herein. In one embodiment, when the reaction step is carried out by contacting the surface with modifying reagents of formulas XXXIII, IV, VI, XXXII, I, and / or III in the gas phase, a controlled amount of water vapor is also present. The controlled amount of water vapor may be provided by introducing a pre-selected amount of magnesium sulfate heptahydrate into the same chamber or enclosure containing the object having the surface to be modified. In other embodiments, a controlled amount of water may be introduced into the reaction chamber or enclosure by an external water vapor supply. The reaction may be carried out under a pressure reduced compared to atmospheric pressure.

[0132] The reaction may take place at temperatures above about 95°C or between about 100°C and about 200°C. In various embodiments, the reaction may take place at temperatures of about 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, or about 200°C. The reaction may continue for about 2 hours, 6 hours, 8 hours, 18 hours, 24 hours, 48 ​​hours, 72 hours, 84 hours, or longer.

[0133] In one embodiment, the modified and / or functionalized surface may be a single layer. In one embodiment, the modified and / or functionalized surface may include at least one surface of a microfluidic circuit element of a microfluidic chip. In another embodiment, the modified and / or functionalized surface may include all surfaces facing the fluid-holding portion of a microfluidic device. For example, in exemplary microfluidic devices 200, 230, the inner surface of the upper electrode 210, the inner surface 208 of the electrode activation substrate 206, and the surface of the microfluidic circuit material 116 (see Figures 1B, 2A, 2B) all face the microfluidic channel 122, and pens 224, 226, 228 can be functionalized. Similarly, in Figures 2D-2F, the inner surface of the microfluidic circuit material 260, the surface of the separation structure 272 defining the isolation pen 270, or all surfaces facing the microfluidic circuit 262 can be modified by reaction with a modifying reagent of formula XXXIII, formula IV, formula VI, formula XXXII, formula I, and / or formula III.

[0134] Further modification of the functionalized surface. A method for covalently modifying a surface is given by formula XXX: [ka] (In the formula, LG, L fm and R x These are defined as described above, and [ka] (This is the surface.) To provide a functionalized surface having the structure of a reactive portion R xEquation XII: RP-L-Surface contact part Formula XII This may involve reacting a surface-modifying reagent having the structure of (wherein RP is the reaction pair portion; L is the linker, and the surface contact portion is as defined above). According to the limitations of chemical bonding as known in the art, the linker L may be a bond or may comprise 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In one embodiment, according to the limitations of chemical bonding as known in the art, the linker L may comprise 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. The linker L or surface contact portion may comprise 0, 1, 2, or 3 bonding groups CG, thereby forming formula XXXI: [ka] A covalently modified surface having the structure of is generated, where L sm It is defined as above.

[0135] In one embodiment, the functionalized surface of formula XXX is formula V or formula VII: [ka] The functionalized surface may be, in formula VIII: W is O, S, or N, Z is a bond to an adjacent silicon atom bonded to the surface or a bond to the surface, and n is an integer from about 3 to 21, and in some embodiments, n is an integer from 7 to 21. The adjacent silicon atom to which Z is bonded may be incorporated into another surface modification molecule described above. The resulting covalently modified surface is given by formula VIII: [ka] It may be a surface-modified molecule having the structure, where S, Z, n and [ka] These are defined above for formula V or formula VII, respectively. If Z is a bond to an adjacent silicon atom, the silicon atom is given by the following formula: [ka] It could be part of another surface modification molecule.

[0136] In one embodiment, n is an integer from 9 to 21. In another embodiment, n is 9, 14, or 16. In yet another embodiment, n is 9. In one embodiment, W is O.

[0137] In other embodiments, a covalently modified surface which is a product having the structure of formula XXXI is a product of formula IX: [ka] The structure may be such that Z is either a bond to an adjacent phosphorus atom or a bond to the surface, and the surface [ka] It is defined as described above.

[0138] Alkynes functionalized surfaces (R xIf present in the surface modification reagent of formula XII (RP reaction pair portion), it may be an acyclic alkyne, and its reaction with the azide in the "click" cyclization reaction may be catalyzed by a copper(I) salt. When a copper(I) salt is used to catalyze the reaction, the reaction mixture may optionally contain other reagents that can accelerate the rate or degree of the reaction. If the alkyne of the surface modification reagent or the functionalized surface is cyclooctyne, the "click" cyclization reaction with the azide of the corresponding functionalized surface or surface modification reagent may be copper-free. The "click" cyclization reaction thereby binds the surface modification ligand to the functionalized surface to form a covalently modified surface. The cyclization reaction may be catalyzed by a copper(I) salt and may optionally contain other reagents that can accelerate the rate or degree of the reaction. As described above for the functionalized surface, a covalently modified surface may be at least one surface of a microfluidic device. In some embodiments, a covalently modified surface may include a surface facing substantially all of the fluid inside the microfluidic device.

[0139] copper catalyst. Any suitable copper(I) catalyst may be used. In some embodiments, this may be copper(I) iodide, copper(I) chloride, copper(I) bromide, or another copper(I) salt. In other embodiments, a copper(II) salt may be used in combination with a reducing agent such as an ascorbate to produce copper(I) species in situ. Copper sulfate or copper acetate are non-limiting examples of suitable copper(II) salts. In other embodiments, a reducing agent such as an ascorbate may be present in combination with a copper(I) salt to ensure sufficient copper(I) species during the reaction. Copper metal may be used to provide Cu(I) species in the redox reaction and to produce Cu(II) species. Copper coordination complexes such as [CuBr(PPh3)3], copper silicotungstate complexes, [Cu(CH3CN)4]PF6, or (Eto)3P CuI may be used. In yet another embodiment, silica-supported copper catalysts, copper nanoclusters, or copper / cuprous oxide nanoparticles may be used as catalysts.

[0140] Other reaction accelerators. As described above, even if oxygen is not strictly excluded from the reaction, the copper(I) species can be maintained throughout the reaction using reducing agents such as sodium ascorbate. Other auxiliary ligands may be included in the reaction mixture to stabilize the copper(I) species. Triazolyl-containing ligands, including but not limited to tris(benzyl-1H-1,2,3-triazol-4-yl)methylamine (TBTA) or 3[tris(3-hydroxypropyltriazolmethyl)amine (THPTA)], may be used. Another type of auxiliary ligand that may be used to accelerate the reaction is sulfonated bathophenanthroline, which is also water-soluble and may be used when oxygen can be excluded.

[0141] Surface modification reagents can be bonded to the functionalized surface described for the reaction pair portion using other chemical bonds known in the art.

[0142] Solvent and reaction conditions. If the inner surface of a microfluidic device is a functionalized surface that reacts with a surface modification reagent, the reaction may be carried out by flowing a solution of the surface modification reagent into and through the microfluidic device. In various embodiments, the surface modification reagent solution may be an aqueous solution. Other useful solvents include aqueous dimethyl sulfoxide (DMSO), DMF, acetonitrile, or alcohols. The reaction may be carried out at room temperature or at high temperatures. In some embodiments, the reaction is carried out at temperatures in the range of about 15°C to about 60°C; about 15°C to about 55°C; about 15°C to about 50°C; and about 20°C to about 45°C. In some embodiments, the reaction to convert the functionalized surface of a microfluidic device into a covalently modified surface is carried out at temperatures of about 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or about 60°C.

[0143] A method for generating combined surfaces. A method for preparing a covalently modified surface on at least one inner surface of a microfluidic device having a base, a cover, and a microfluidic circuit material, wherein the enclosure includes a microfluidic circuit material defining a fluid circuit, comprises: contacting at least one inner surface with a first modification reagent and a second modification reagent; reacting the first modification reagent with a first nucleophilic portion of at least one inner surface; reacting the second modification reagent with a second nucleophilic portion of at least one inner surface; and forming at least one covalently modified surface comprising a first covalent surface modification including a first binding group and a first portion which is a first surface contact portion or a first reactive portion, and a second covalent surface modification including a second binding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first binding group is different from the second binding group, or the first portion is different from the second portion.

[0144] In one embodiment, the reaction of the first modifying reagent with the surface may occur simultaneously with the reaction of the second modifying reagent. For example, if both the first and second modifying reagents are surface modification compounds (e.g., formulas XXXII, I, II, III), a mixture of two surface modification reagents, such as two different siloxane reagents, may be reacted simultaneously by chemical vapor deposition. The ratio of the two reagents may be varied as needed to obtain two surface modifications (e.g., surface modification ligands) in different percentages. In another example, the surface may be a functionalized surface, and the first and second modifying reagents are surface modification reagents (e.g., formula XII) and / or secondary functionalizing reagents (formula XXXIV), and a mixture of the two modifying reagents may react simultaneously with the reactive portion of the functionalized surface.

[0145] In other embodiments, the reaction of the first modifying reagent with the surface may occur before or after the reaction of the second modifying reagent with at least one inner surface of the microfluidic device. For example, the surface may be a functionalized surface (having a surface of formula XXX, formula V, or formula VII), and the first modifying reagent The drug is orthogonal R x2Alternatively, it may be a secondary functionalizing reagent that can introduce a surface-modifying reagent. The reaction may be carried out using a limited amount of the secondary functionalizing reagent, thereby introducing the reactive portion R of the functionalized surface. x This is only a portion of it. Alternatively, the first reaction may be carried out using a limited amount of surface modification reagent, so not all reactive parts may be bonded. This may be done, for example, to introduce a longer linker region through these first introduced surface modifications. The next reaction may introduce a second surface modification by using a surface modification reagent that can introduce the desired surface contact portion to all of the exposed reactive parts, or react only with the unreacted original reactive part sites. Orthogonal R x2 If R is introduced, further responses will be, x2 It reacts only with the reactive portion R of the original functionalized surface. x This can be carried out using a suitable surface modification reagent that does not react with the target substance.

[0146] In one embodiment, the reaction of the first and second modifying reagents with the surface may occur at random locations on the surface. In another embodiment, the reaction of the first modifying reagent may occur within a first region of the surface, and the reaction of the second modifying reagent may occur within a second region of the surface adjacent to the first region. For example, a surface within a channel of a microfluidic device may be selectively modified with a first surface modification, and a surface within an isolation pen adjacent to the channel surface may be selectively modified with a second, different surface modification.

[0147] In yet another embodiment, the reaction of the first modifying reagent may occur in a plurality of first regions separated from each other on at least one surface, and the reaction of the second modifying reaction may occur in a second region surrounding the plurality of first regions separated from each other.

[0148] In various embodiments, the modification of one or more surfaces of a microfluidic device to introduce a combination of a first and a second surface modification may be performed after the microfluidic device has been assembled. In one non-limiting example, the first and second surface modifications may be introduced by chemical vapor deposition after the assembly of the microfluidic device. In another non-limiting example, a functionalized surface may be introduced having a first surface modification having a first reactive portion and a second surface modification having a second orthogonal reactive portion. Different conversions to two different surface modification ligands having two different surface contact portions may then occur. In another embodiment, the microfluidic device may have a single functionalized surface of formula XXX, formula V, or formula VII, which may be differently modified by a mixture of two surface modification reagents or a mixture of a surface modification reagent and a secondary functionalization reagent (followed by the conversion of the secondary functionalized surface to a surface modification ligand having a second different surface contact portion).

[0149] In one embodiment, at least one of the combinations of the first and second surface modifications may be performed before the assembly of the microfluidic device. In one embodiment, at least one surface modification may be performed after the assembly of the microfluidic device.

[0150] In one embodiment, a method for fabricating a microfluidic device includes: forming a first modified surface on a base or cover before assembling the microfluidic device; assembling the microfluidic device, which includes assembling the first covalently modified surface on the base or cover together with the microfluidic circuit material and other unmodified parts of the cover or base; and forming a second modified surface on the unmodified surface of the assembled microfluidic device. For example, the first surface modification may be introduced on a portion of the cover of the microfluidic device before assembly, and there may still be unreacted portions of the cover remaining. The microfluidic device may be assembled and then reacted with a second surface modification (e.g., surface modification compounds of formulas XXXII, I, II, and III), the second surface modification reacting not only with all of the remaining unmodified areas on the inner surface of the cover, but also with all of the remaining inner surfaces of the base and microfluidic circuit material.

[0151] In one embodiment, the covalently modified surface is a surface of formula XXX, formula V, or formula VII The method comprises a functionalized surface of formula XXXI, formula VIII, or formula IX arranged therein, and a combination of first covalently modified surfaces of formula XXXIV: RP-L fm -R x2 Formula XXXIV This may further include reacting it with a secondary functionalizing reagent.

[0152] A secondary functionalized surface of formula XXX is generated in the presence of a first covalently modified surface of formula XXXI, formula VIII, or formula IX. The method may further include reacting the secondary functionalized surface of formula XXX with a surface modification reagent having the structure of formula XII, thereby generating a second covalently modified surface of formula XXXI, formula VIII, or formula IX in the presence of a first covalently modified surface of formula XXXI, formula VIII, or formula IX.

[0153] Alternatively, the method may further include reacting a first functionalized surface of formula XXX, formula V, or formula VII with a surface modification reagent having the structure of formula XII, thereby generating a second covalently modified surface of formula XXXI, formula VIII, or formula IX in the presence of a first covalently modified surface of formula XXXI, formula VIII, or formula IX.

[0154] use. Materials, devices and / or apparatus having one or more surfaces suitable for modification to introduce the structures of formulas XXXI, VIII, or IX described above include, but are not limited to, flow cytometry cells, apheresis centrifuges, tubing and containers; or microfluidic devices for handling cells, cell fragments, proteins or nucleic acids for any type of biological analysis process or biomaterial sorting process. Surfaces having the structures of formulas XXXI, VIII, or IX may be used in, in some non-limiting examples, large-scale biological production equipment, medical equipment or water purification equipment and analytical equipment, without being limited to micro-materials, devices and / or apparatus.

[0155] Filling method. The filling of biological micromaterials or micromaterials (including, but not limited to, beads) may include the use of flow rate, gravity, dielectrophoretic (DEP) force, electrowetting, magnetic force, or any combination thereof, as described herein. DEP force may be generated optically, such as by a photoelectron tweezers (OET) configuration, and / or electrically, such as by the activation of one or more electrode regions in a temporal / spatial pattern. Similarly, electrowetting force may be provided optically, such as by a photoelectrowetting (OEW) configuration, and / or electrically, such as by the activation of one or more electrode regions in a temporal / spatial pattern.

[0156] Microfluidic devices and systems for manipulating and observing such devices. Figure 1A shows an example of a microfluidic device 100 and system 150, which may be used to maintain, isolate, assay, or culture biological microobjects. A perspective view of the microfluidic device 100 is shown with a partial cutaway of its cover 110 to provide a partial view of the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic circuit 120, which includes a channel 106 through which a fluid medium 180 can flow, and the channel 106 optionally transports one or more microobjects (not shown) into and / or through the microfluidic circuit 120. Although a single microfluidic circuit 120 is shown in Figure 1A, a suitable microfluidic device may include multiple (e.g., two or three) such microfluidic circuits. Nevertheless, the microfluidic device 100 may be configured to be a nanofluidic device. As shown in Figure 1A, the microfluidic circuit 120 may include a plurality of microfluidic isolation pens 124, 126, 128, and 130, each isolation pen may have one or more openings that are in fluid communication with the flow path 106. In one embodiment of the device shown in Figure 1A, the isolation pen may have only one opening that is in fluid communication with the channel 106. As will be further described below, the microfluidic isolation pen includes various features and structures optimized to retain microscopic objects within a microfluidic device such as the microfluidic device 100, even when the culture medium 180 is flowing through the channel 106. However, before moving on to that, a brief description of the microfluidic device 100 and system 150 is provided.

[0157] As generally shown in Figure 1A, the microfluidic circuit 120 is defined by an enclosure 102. The enclosure 102 can be physically structured in different configurations, but in the example shown in Figure 1A, the enclosure 102 is shown as including a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108, and the cover 110 can be mounted to one another. 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 over the microfluidic circuit structure 108. Together with the support structure 104 and the cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120.

[0158] As shown in Figure 1A, the support structure 104 may be located below the microfluidic circuit 120, and the cover 110 may be located above the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 may be located above the microfluidic circuit 120, and the cover 110 may be located below the microfluidic circuit 120. Regardless, there may be one or more ports 107, each containing a passage to or from the enclosure 102. Examples of passages include valves, gates, through-holes, etc. As shown, the port 107 is a through-hole created by a gap in the microfluidic circuit structure 108. However, the port 107 may be located in other components of the enclosure 102, such as the cover 110. Although only one port 107 is shown in Figure 1A, the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 that functions as an inlet for the fluid to enter the microfluidic circuit 120, and a second port 107 that functions as an outlet for the fluid to exit the microfluidic circuit 120. Whether a port 107 functions as an inlet or an outlet may depend on the direction in which the fluid flows through the channel 106.

[0159] The support structure 104 may include one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 may include one or more semiconductor substrates, each of which is electrically connected to an electrode (for example, all or a subset of semiconductor substrates may be electrically connected to one electrode). The support structure 104 may further include a printed circuit board assembly ("PCBA"). For example, semiconductor substrates may be mounted on a PCBA.

[0160] The microfluidic circuit structure 108 can define the circuit elements of the microfluidic circuit 120. Such circuit elements may include spaces or regions such as flow regions (which may include one or more flow channels), chambers, pens, traps, etc., which can be fluidically interconnected when the microfluidic circuit 120 is filled with fluid. In the microfluidic circuit 120 shown in Figure 1A, the microfluidic circuit 108 includes a frame 114 and microfluidic circuit material 116. The frame 114 may partially or completely enclose the microfluidic circuit material 116. The frame 114 may be a relatively rigid structure that substantially encloses the microfluidic circuit material 116, for example. For example, the frame 114 may be made of a metallic material.

[0161] The microfluidic circuit material 116 can be patterned with cavities and other features to define the circuit elements and interconnections of the microfluidic circuit 120. This may include flexible materials such as flexible polymers that may be gas permeable (e.g., rubber, plastics, elastomers, silicones, polydimethylsiloxane ("PDMS"), etc.). Other examples of materials that can constitute the microfluidic circuit material 116 include molded glass, etchable materials such as silicone (photopatternable silicone or "PPS"), and photoresists (e.g., SU8). In some embodiments, such materials—and therefore the microfluidic circuit material 116—may be rigid and / or substantially gas-impermeable. Regardless, the microfluidic circuit material 116 can be placed on the support structure 104 and inside the frame 114.

[0162] The cover 110 may be an integral part of the frame 114 and / or the microfluidic circuit material 116. Alternatively, the cover 110 may be a structurally separate element, as shown in Figure 1A. The cover 110 may contain the same or different materials as the frame 114 and / or the microfluidic circuit material 116. Similarly, the support structure 104 may be a separate structure from the frame 114 or the microfluidic circuit material 116, as shown, or it may be an integral part of the frame 114 or the microfluidic circuit material 116. Likewise, the frame 114 and the microfluidic circuit material 116 may be separate structures, as shown in Figure 1A, or they may be an integral part of the same structure.

[0163] In some embodiments, the cover 110 may include a rigid material. The rigid material may be a material having properties similar to glass or the like. In some embodiments, the cover 110 may include a deformable material. The deformable material may be a polymer such as PDMS. In some embodiments, the cover 110 may include both a rigid material and a deformable material. For example, one or more parts of the cover 110 (e.g., one or more parts located on the isolation pens 124, 126, 128, 130) may include a deformable material that interfacially contacts the rigid material of the cover 110. In some embodiments, the cover 110 may further include one or more electrodes. One or more electrodes may include a conductive oxide such as indium-tin oxide (ITO), which may be coated with glass or the like insulating material. Alternatively, one or more electrodes may be flexible electrodes such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles, or combinations thereof, embedded in a deformable polymer such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices are described, for example, in U.S. Patent Application Publication No. 2012 / 0325665 (Chiou et al.), which is incorporated herein by reference. In some embodiments, the cover 110 can be modified to support cell adhesion, survival, and / or growth (for example, by modifying all or part of the surface facing inward toward the microfluidic circuit 120). Modifications may include coating with synthetic or natural polymers. In some embodiments, the cover 110 and / or support structure 104 can transmit light. The cover 110 may also include at least one gas-permeable material (e.g., PDMS or PPS).

[0164] Figure 1A also shows a system 150 that operates and controls microfluidic devices such as the microfluidic device 100. The system 150 includes a power supply 192, an imaging device (integrated into the imaging module 164 and not explicitly shown in Figure 1A), and a tilting device 190 (part of the tilting module 166 and not explicitly shown in Figure 1A).

[0165] The power supply 192 provides power to the microfluidic device 100 and / or the gradient device 190 and can provide a bias voltage or current as needed. The power supply 192 may include, for example, one or more alternating current (AC) and / or direct current (DC) voltage sources or current sources. The imaging device 194 (part of the imaging module 164 described below) may include a device such as a digital camera that captures images inside the microfluidic circuit 120. In some cases, the imaging device 194 may have a high frame rate and / or high sensitivity (e.g., The imaging device 194 may further include a detector having, for example, a low-light detector. The imaging device 194 may also include a mechanism for directing stimulating radiation and / or rays into the microfluidic circuit 120 and collecting radiation and / or rays reflected from or emitted from the microfluidic circuit 120 (or minute objects contained within the microfluidic circuit 120). Emitted rays may be in the visible spectrum and may include, for example, fluorescence radiation. Reflected rays may include reflected radiation emitted from broad-spectrum lamps such as LEDs or mercury lamps (e.g., high-pressure mercury lamps) or xenon arc lamps. As considered with respect to Figure 3B, the imaging device 194 may further include a microscope (or optical column), which may or may not include an eyepiece.

[0166] The system 150 further includes a tilt device 190 (part of the tilt module 166 described below) configured to rotate the microfluidic device 100 around one or more axes of rotation. In some embodiments, the tilt device 190 is configured to support and / or hold an enclosure 102 containing the microfluidic circuit 120 around at least one axis so that the microfluidic device 100 (and thus the microfluidic circuit 120) can be held in a horizontal orientation (i.e., 0° relative to the x and y axes), a vertical orientation (i.e., 90° relative to the x and / or y axes), or any orientation in between. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to an axis is referred to herein as the “tilt” of the microfluidic device 100 (and the microfluidic circuit 120). For example, the tilting device 190 can tilt the microfluidic device 100 relative to the x-axis by 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90°, or any degree in between. The horizontal orientation (and therefore the x and y axes) is defined as perpendicular to the vertical axis defined by gravity. The tilting device can tilt the microfluidic device 100 (and microfluidic circuit 120) to any angle greater than 90° relative to the x and / or y axes, or it can tilt the microfluidic device 100 (and microfluidic circuit 120) to 180° relative to the x or y axis, thereby reversing the microfluidic device 100 (and microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and microfluidic circuit 120) around an axis of rotation defined by the flow path 106 or some other part of the microfluidic circuit 120.

[0167] In some cases, the microfluidic device 100 is inclined vertically such that the channel 106 is located above or below one or more isolation pens. The term “above” as used herein indicates that the channel 106 is located above one or more isolation pens on a vertical axis defined by gravity (i.e., objects in isolation pens above the channel 106 have higher gravitational potential energy than objects in the channel). The term “below” as used herein indicates that the channel 106 is located below one or more isolation pens on a vertical axis defined by gravity (i.e., objects in isolation pens below the channel 106 have lower gravitational potential energy than objects in the channel).

[0168] In some cases, the tilting device 190 tilts the microfluidic device 100 around an axis parallel to the flow path 106. Furthermore, the microfluidic device 100 can be tilted at an angle of less than 90° such that the flow path 106 is positioned above or below one or more isolation pens, rather than directly above or below an isolation pen. In other cases, the tilting device 190 tilts the microfluidic device 100 around an axis perpendicular to the flow path 106. In yet another case, the tilting device 190 tilts the microfluidic device 100 around an axis that is neither parallel nor perpendicular to the flow path 106.

[0169] System 150 may further include a culture medium source 178. Culture medium source 178 (e.g., container The medium source (including reservoirs, etc.) may include multiple sections or containers, each holding a different fluid medium 180. Therefore, the medium source 178 may be a separate device located outside the microfluidic device 100, as shown in Figure 1A. Alternatively, the medium source 178 may be located entirely or partially inside the enclosure 102 of the microfluidic device 100. For example, the medium source 178 may include a reservoir, which is part of the microfluidic device 100.

[0170] Figure 1A also shows a simplified block diagram representation of an example of a control and monitoring device 152 that constitutes part of the system 150 and can be used in conjunction with the microfluidic device 100. As shown, an example of such a control and monitoring device 152 includes a master controller 154 which includes a culture module 160 that controls a culture medium source 178, a drive module 162 that controls the movement and / or selection of micro-objects (not shown) and / or culture medium (e.g., droplets of culture medium) in the microfluidic circuit 120, an imaging module 164 that controls an imaging device 194 (e.g., a camera, microscope, light source, or any combination thereof) that captures an image (e.g., a digital image), and a tilt module 166 that controls a 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 may further include a display device 170 and an input / output device 172.

[0171] The master controller 154 may include a control module 156 and a digital memory 158. The control module 156 may include a digital processor configured to operate according to machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-temporary data or signals in the memory 158. Alternatively or additionally, the control module 156 may include hardwired digital and / or analog circuits. The culture module 160, the driving module 162, the imaging module 164, the tilt module 166, and / or other modules 168 may be configured similarly. Thus, any function, process, action, operation, or step of process considered herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus may be performed by any one or more of the master controller 154, culture module 160, driving module 162, imaging module 164, tilt module 166, and / or other modules 168 configured as described above. Similarly, the master controller 154, culture module 160, drive module 162, imaging module 164, tilt module 166, and / or other modules 168 are communicatively coupled to send and receive data used in any function, process, action, movement, or step considered herein.

[0172] The culture medium module 160 controls the culture medium source 178. For example, the culture medium module 160 can control the culture medium source 178 to introduce a selected fluid culture medium 180 into the enclosure 102 (e.g., via the inlet 107). The culture medium module 160 can also control the removal of the culture medium from the enclosure 102 (e.g., via an outlet (not shown)). Thus, one or more culture media can be selectively introduced into and removed from the microfluidic circuit 120. The culture medium module 160 can also control the flow of the fluid culture medium 180 in the channel 106 inside the microfluidic circuit 120. For example, in some embodiments, the culture medium module 160 stops the flow of the culture medium 180 in the channel 106 and through the enclosure 102 before the tilt module 166 tilts the microfluidic device 100 to a desired tilt angle on the tilt device 190.

[0173] The driving module 162 may be configured to control the selection, capture, and movement of micro-objects (not shown) in the microfluidic circuit 120. As will be described later with respect to Figures 1B and 1C, the enclosure 102 may be configured with a dielectrophoretic (DEP) configuration, a photoelectron tweezers (OET) configuration The configuration may include a photoelectron wetting (OEW) configuration (not shown in Figure 1A), in which the driving module 162 controls the activation of electrodes and / or transistors (e.g., phototransistors) to selectively move micro-objects (not shown) and / or droplets of culture medium (not shown) in the channel 106 and / or isolation pens 124, 126, 128, 130.

[0174] The imaging module 164 can control the imaging device 194. For example, the imaging module 164 can receive and process image data from the imaging device 194. The image data from the imaging device 194 can include any type of information captured by the imaging device 194 (e.g., whether or not there is accumulation of labels such as micro-objects, culture medium droplets, or fluorescent labels). Using the information captured by the imaging device 194, the imaging module 164 can further calculate the position of an object (e.g., a micro-object, a culture medium droplet) and / or the movement speed of such an object within the microfluidic device 100.

[0175] The tilt module 166 can control the tilt movement of the tilt device 190. Alternatively or additionally, the tilt module 166 can control the tilt rate and timing to optimize the transfer of microobjects to one or more isolation pens via gravity. The tilt module 166 is communicatively coupled with the imaging module 164 to receive data describing the movement of microobjects and / or culture 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 culture 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 culture droplets within the microfluidic circuit 120.

[0176] In the example shown in Figure 1A, the microfluidic circuit 120 is shown as including a microfluidic channel 122 and isolation pens 124, 126, 128, and 130. Each pen includes an opening to the channel 122 but is otherwise closed so that the pen can substantially separate microorganisms inside the pen from the fluid medium 180 and / or the flow path 106 of the channel 122 or microorganisms in other pens. The walls of the isolation pens extend from the inner surface 109 of the base to the inner surface of the cover 110, providing an enclosure. The openings of the pens to the microfluidic channel 122 are directed at an inclination relative to the flow 106 so that the flow 106 is not directed into the pen. The flow may be tangential or perpendicular to the plane of the pen opening. In some cases, the pens 124, 126, 128, and 130 are configured to physically enclose one or more microorganisms within the microfluidic circuit 120. The isolation pens provided in this disclosure may include a variety of shapes, surfaces, and features optimized for use in conjunction with DEP, OET, OEW, fluid flow, and / or gravity, as will be discussed and described in detail below.

[0177] The microfluidic circuit 120 may include any number of microfluidic isolation pens. Although five isolation pens are shown, the microfluidic circuit 120 may have fewer or more isolation pens. As shown, the microfluidic isolation pens 124, 126, 128, and 130 of the microfluidic circuit 120 each include different features and shapes that may provide one or more advantages useful for the maintenance, isolation, assay, or culture of biological microorganisms. In some embodiments, the microfluidic circuit 120 includes multiple identical microfluidic isolation pens.

[0178] In the embodiment shown in Figure 1A, one channel 122 and a flow path 106 are shown. However, other embodiments may include multiple channels 122, each configured to include a flow path 106. The microfluidic circuit 120 further includes an inlet valve or port 107 that fluidly communicates with the flow path 106 and the fluid medium 180, thereby allowing the fluid medium 180 to access the channel 122 via the inlet 107. In some cases, there is one flow path 106. This includes the paths. In some cases, one path is arranged in a zigzag pattern, thereby causing the channel 106 to travel across the microfluidic device 100 two or more times in alternating directions.

[0179] In some cases, the microfluidic circuit 120 includes a plurality of parallel channels 122 and flow paths 106, where the fluid medium 180 in each flow path 106 flows in the same direction. In some cases, the fluid medium in each flow path 106 flows in at least one of the following directions: forward or reverse. In some cases, a plurality of isolation pens are configured so that the isolation pens can be positioned in parallel with the target microobject (for example, relative to the channels 122).

[0180] In some embodiments, the microfluidic circuit 120 further includes one or more microobject traps 132. The traps 132 are generally formed in the walls forming the boundary of the channel 122 and may be located opposite one or more openings of the microfluidic isolation pens 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture one microobject from the channel 106. In some embodiments, the trap 132 is configured to receive or capture multiple microobjects from the channel 106. In some cases, the trap 132 contains a volume approximately equal to the volume of one target microobject.

[0181] The trap 132 may further include an opening configured to assist the flow of target microobjects into the trap 132. In some cases, the trap 132 includes an opening having a height and width approximately equal to the dimensions of one target microobject, thereby preventing larger microobjects from entering the microobject trap. The trap 132 may further include other features configured to assist in retaining target microobjects within the trap 132. In some cases, the trap 132 is aligned with the opening of a microfluidic isolation pen and positioned on the opposite side of the channel 122 with respect to the opening of the microfluidic isolation pen, thereby, when the microfluidic device 100 is tilted around an axis parallel to the microfluidic channel 122, the captured microobject exits the trap 132 in a trajectory that drops the microobject into the opening of the isolation pen. In some cases, the trap 132 includes a side passage 134 smaller than the target microobject that facilitates flow through the trap 132, thereby increasing the probability of capturing microobjects within the trap 132.

[0182] In some embodiments, dielectrophoretic (DEP) forces are applied across a fluid medium 180 via one or more electrodes (not shown) to manipulate, transport, separate, and sort microobjects placed within it (e.g., in channels and / or isolation pens). For example, in some embodiments, DEP forces are applied to one or more parts of a microfluidic circuit 120 to transport a single microobject from a channel 106 to a desired microfluidic isolation pen. In some embodiments, DEP forces are used to prevent microobjects within an isolation pen (e.g., isolation pens 124, 126, 128, or 130) from being displaced from the isolation pen. Furthermore, in some embodiments, DEP forces are used to selectively remove microobjects previously collected by an embodiment of this disclosure from the isolation pen. In some embodiments, DEP forces include photoelectron tweezers (OET) forces.

[0183] In other embodiments, photoelectron wetting (OEW) forces are applied via one or more electrodes (not shown) to one or more locations on the support structure 104 (and / or cover 110) of the microfluidic device 100 (e.g., locations useful for defining channels and / or isolation pens) to manipulate, transport, separate, and sort droplets placed in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more locations on the support structure 104 (and / or cover 110) to transport a single droplet from channel 106 to a desired microfluidic isolation pen. In some embodiments, OEW forces are used to displace droplets within an isolation pen (e.g., isolation pens 124, 126, 128, or 130) away from the isolation pen. To prevent this from happening. Furthermore, in some embodiments, OEW force is used to selectively remove droplets previously collected by the embodiments of this disclosure from the isolation pen.

[0184] In some embodiments, DEP forces and / or OEW forces are combined with other forces such as flow and / or gravity to manipulate, transport, separate, and sort micro-objects and / or droplets within the microfluidic circuit 120. For example, the enclosure 102 can be tilted (e.g., by a tilting device 190) to position the flow path 106 and the micro-objects placed within the flow path 106 onto the microfluidic isolation pen, and gravity can transport the micro-objects and / or droplets into the pen. In some embodiments, DEP forces and / or OEW forces can be applied before other forces. In other embodiments, DEP forces and / or OEW forces can be applied after other forces. In yet another case, DEP forces and / or OEW forces can be applied simultaneously with or alternately with other forces.

[0185] Figures 1B, 1C, and 2A–2H illustrate various embodiments of a microfluidic device that can be used in embodiments of the present disclosure. Figure 1B shows an embodiment in which the microfluidic device 200 is configured as an optically actuated electrokinetic device. Various optically actuated electrokinetic devices are known in the art, including devices having an optoelectronic tweezers (OET) configuration and devices having an optoelectronic wetting (OEW) configuration. Examples of suitable OET configurations are shown in the following U.S. patent documents, each of which is incorporated herein by reference in whole: U.S. Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355); and U.S. Patent No. 7,956,339 (Ohta et al.). Examples of OEW configurations are U.S. Patent No. 6,958,132 (Chiou et al.) and U.S. Patent Application Publication No. 2012 / 0024 This is shown in No. 708 (Chiou et al.), and both are referred to herein by reference. Further examples of optically actuated electrodynamic devices include OET / OEW coupled configurations, examples of which are shown in U.S. Patent Application Publications 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT publications, International Publication 2015 / 164846 and International Publication 2015 / 164847, all of which are incorporated herein by reference in their entirety.

[0186] Examples of microfluidic devices having isolation pens capable of positioning, culturing, and / or monitoring biological microobjects are described, for example, in U.S. Patent Application Publication 2014 / 0116881 (Application No. 14 / 060,117, filed October 22, 2013), U.S. Patent Application Publication 2015 / 0151298 (Application No. 14 / 520,568, filed October 22, 2014), and U.S. Patent Application Publication 2015 / 0165436 (Application No. 14 / 521,447, filed October 22, 2014), which are respectively incorporated by reference in their entirety. U.S. Patent Application Publications 14 / 520,568 and 14 / 521,447 also describe exemplary methods for analyzing secretions from cells cultured in a microfluidic device. Each of the above applications further describes microfluidic devices configured to generate dielectrophoretic (DEP) forces, such as photoelectron tweezers (OETs), or to provide photoelectron wetting (OEW). For example, the photoelectron tweezer device shown in Figure 2 of U.S. Patent Application Publication No. 2014 / 0116881 is an example of a device that can be used in embodiments of the present disclosure to selectively move individual biological microorganisms or groups of biological microorganisms.

[0187] Driving microfluidic device configuration. As described above, the system's control and monitoring equipment may include a driving module that selectively moves objects such as micro-objects or droplets within the microfluidic circuit of a microfluidic device. Microfluidic devices can have various driving configurations depending on the type of object being moved and other considerations. For example, a dielectrophoretic (DEP) configuration can be used to selectively move micro-objects within a microfluidic circuit. Therefore, micro The support structure 104 and / or cover 110 of the microfluidic device 100 may include a DEP configuration that selectively induces DEP forces on microscopic objects in the fluid medium 180 within the microfluidic circuit 120, thereby enabling the selection, capture, and / or movement of individual microscopic objects or groups of microscopic objects. Alternatively, the support structure 104 and / or cover 110 of the microfluidic device 100 may include an electron wetting (EW) configuration that selectively induces EW forces on droplets in the fluid medium 180 within the microfluidic circuit 120, thereby enabling the selection, capture, and / or movement of individual droplets or groups of droplets.

[0188] An example of a microfluidic device 200 including a DEP configuration is shown in Figures 21B and 1C. For brevity, Figures 1B and 1C show side and top cross-sectional views, respectively, of a portion of the enclosure 102 of the microfluidic device 200 having a region / chamber 202, but it should be understood that the region / chamber 202 may be a portion of a fluid circuit element having a more detailed structure, such as a growth chamber, isolation pen, flow region, or flow channel. Furthermore, the microfluidic device 200 may include other fluid circuit elements. For example, the microfluidic device 200 may include multiple growth chambers or isolation pens and / or one or more flow regions or flow channels, as described herein with respect to the microfluidic device 100. The DEP configuration may be incorporated into any such fluid circuit element of the microfluidic device 200, or a portion thereof may be selected. It should be further understood that any of the above or below microfluidic device components and system components may be incorporated into the microfluidic device 200 and / or used in combination with the microfluidic device 200. For example, a system 150 including the control and monitoring equipment 152 described above, which includes one or more of the culture medium module 160, the drive module 162, the imaging module 164, the tilt module 166, and other modules 168, can be used in conjunction with a microfluidic device 200.

[0189] As seen in Figure 1B, the microfluidic device 200 includes a support structure 104 having a lower electrode 204 and an electrode activation substrate 206 overlapping the lower electrode 204, and a cover 110 having an upper electrode 210, the upper electrode 210 being spaced apart from the lower electrode 204. The upper electrode 210 and the electrode activation substrate 206 define both sides of the region / chamber 202. Thus, the culture medium 180 contained in the region / chamber 202 provides a resistive connection between the upper electrode 210 and the electrode activation 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 generating a DEP force in the region / chamber 202. The power supply 212 may be, for example, an alternating current (AC) power supply.

[0190] In certain embodiments, the microfluidic device 200 shown in Figures 1B and 1C may have an optically operated DEP configuration. Thus, the changing pattern of light 218 from a light source 216, which can be controlled by the driving module 162, can selectively activate or deactivate the changing pattern of the DEP electrodes in a region 214 on the inner surface 208 of the electrode activation substrate 206. (Hereinafter, the region 214 of a microfluidic device having a DEP configuration will be referred to as the "DEP electrode region"). As shown in Figure 1C, the light pattern 218 directed onto the inner surface 208 of the electrode activation substrate 206 can illuminate a selected DEP electrode region 214a (shown in white) in a pattern such as a square. The unilluminated DEP electrode region 214 (shown with diagonal lines) will be referred to below as the "dark" DEP electrode region 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., from the lower electrode 204 to the inner surface 208 of the electrode activation substrate 206 that is in interface with the culture medium 180 in the flow region 106) is greater than the relative electrical impedance through the culture medium 180 in the region / chamber 202 in each dark DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the upper electrode 210 of the cover 110). However, the illumination DEP electrode region 214a has an impedance less than the relative impedance through the culture medium 180 in the region / chamber 202 in each illumination DEP electrode region 214a. This demonstrates a reduction in relative impedance passing through the electrode activation substrate 206.

[0191] When power supply 212 is activated, the above DEP configuration creates an electric field gradient within the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214. This electric field gradient then generates a local DEP force that attracts or repels nearby micro-objects (not shown) within the fluid medium 180. Thus, the DEP electrodes that attract or repel micro-objects within the fluid medium 180 can be selectively activated and deactivated in many different such DEP electrode regions 214 on the inner surface 208 of the region / chamber 202 by changing the light pattern 218 projected from the light source 216 onto the microfluidic device 200. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of power supply 212 and the dielectric properties of the medium 180 and / or micro-objects (not shown).

[0192] The square pattern 220 of the illuminated DEP electrode region 214a shown in Figure 1C is merely an example. The light pattern 218 projected onto the microfluidic device 200 can illuminate (and thereby activate) any pattern of the DEP electrode region 214, and the pattern of the illuminated / activated DEP electrode region 214 can be repeatedly changed by changing or moving the light pattern 218.

[0193] In some embodiments, the electrode activation substrate 206 may include or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 may be featureless. For example, the electrode activation substrate 206 may include or consist of a layer of hydrogenated amorphous silicon (a-Si:H). a-Si:H may contain, for example, about 8% to 40% hydrogen (calculated by multiplying the number of hydrogen atoms by 100 in the total number of hydrogen and silicon atoms). The a-Si:H layer may have a thickness of about 500 nm to about 2.0 μm. In such embodiments, the DEP electrode regions 214 can be created in any pattern at any location on the inner surface 208 of the electrode activation substrate 206 by the light pattern 218. Therefore, the number and pattern of the DEP electrode regions 214 do not need to be fixed and can correspond to the light pattern 218. Examples of microfluidic devices having a DEP configuration including the photoconductive layer described above are described, for example, in U.S. Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355), the entire contents of which are incorporated herein by reference.

[0194] In other embodiments, the electrode activation substrate 206 may include a substrate comprising a plurality of doped layers, insulating layers (or regions), and conductive layers that form a semiconductor integrated circuit, such as those known in the semiconductor field. For example, the electrode activation substrate 206 may include a plurality of phototransistors, such as lateral bipolar phototransistors, each phototransistor corresponding to a DEP electrode region 214. Alternatively, the electrode activation substrate 206 may include electrodes controlled by a phototransistor switch (e.g., conductive metal electrodes), each such electrode corresponding to a DEP electrode region 214. The electrode activation substrate 206 may include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern may be a substantially square array of phototransistors or phototransistor-controlled electrodes arranged in a matrix, such as shown in Figure 2B. Alternatively, the pattern may be a substantially hexagonal array of phototransistors or phototransistor-controlled electrodes forming a hexagonal grid. Regardless of the pattern, electrical circuit elements can form electrical connections between the DEP electrode region 214 and the lower electrode 210 on the inner surface 208 of the electrode activation substrate 206, and these electrical connections (i.e., phototransistors or electrodes) can be selectively activated or deactivated by the light pattern 218. If not activated, each electrical connection passes through the electrode activation substrate 206 (i.e., from the lower electrode 204 to the inner surface 20 of the electrode activation electrode 206 that is in contact with the culture medium 180 in the region / chamber 202). Up to 8) The relative impedance can be high such that it is greater than the relative impedance through the medium 180 in the corresponding DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the upper electrode 210 of the cover 110). However, when activated by light in the light pattern 218, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 in each illuminated DEP electrode region 214, thereby activating the DEP electrode in the corresponding DEP electrode region 214 as described above. Thus, the DEP electrode that attracts or repels micro-objects (not shown) in the medium 180 can be selectively activated and deactivated in many different DEP electrode regions 214 on the inner surface 208 of the electrode activation substrate 206 in the region / chamber 202, as determined by the light pattern 218.

[0195] Examples of microfluidic devices having an electrode-activated substrate including a phototransistor are described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) (see, for example, device 300 shown in Figures 21 and 22 and its description), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having an electrode-activated substrate including an electrode controlled by a phototransistor switch are described, for example, in U.S. Patent Application Publication No. 2014 / 0124370 (Short et al.) (see, for example, throughout the drawings) See devices 200, 400, 500, 600, and 900 and their descriptions), the entire contents of which are incorporated herein by reference.

[0196] 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 part of the second wall (or support structure 104) of the enclosure 102. The region / chamber 202 may be between the first and second walls. In other embodiments, the electrode 210 is part of the second wall (or support structure 104), and one or both of the electrode activation substrate 206 and / or the electrode 210 are part of the first wall (or cover 110). Furthermore, the light source 216 can alternatively be used to illuminate the enclosure 102 from below.

[0197] Using the microfluidic device 200 shown in Figures 1B and 1C, which has a DEP configuration, the driving module 162 can select microobjects (not shown) in the culture medium 180 in a region / chamber 202 by projecting a light pattern 218 onto the microfluidic device 200 to activate one or more DEP electrodes of a first set in the DEP electrode region 214a on the inner surface 208 of the electrode activation substrate 206 with a pattern (e.g., a square pattern 220) that surrounds and captures microobjects. Next, the driving module 162 can move the in-situ generated and captured microobjects by moving the light pattern 218 relative to the microfluidic device 200 to activate one or more DEP electrodes of a second set in the DEP electrode region 214. Alternatively, the microfluidic device 200 can be moved relative to the light pattern 218.

[0198] In other embodiments, the microfluidic device 200 may have a DEP configuration that does not depend on the photoactivation of the DEP electrodes on the inner surface 208 of the electrode activation substrate 206. For example, the electrode activation substrate 206 may include selectively addressable and energy-donor electrodes located opposite a surface (e.g., cover 110) containing at least one electrode. A switch (e.g., a transistor switch on a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrodes in the DEP electrode region 214, thereby generating a net DEP force on micro-objects (not shown) in the region / chamber 202 near the activated DEP electrodes. Depending on the frequency of the power supply 212 and characteristics such as the dielectric properties of the culture medium (not shown) and / or the micro-objects in the region / chamber 202, the DEP force may attract or repel nearby micro-objects. A set of DEP electrodes (e.g., a square pattern 220) By selectively activating or deactivating the set of DEP electrode regions 214 formed, one or more micro-objects in the region / chamber 202 can be captured and moved within the region / chamber 202. The driving module 162 in Figure 1A controls such switches and thus activates and deactivates individual electrodes of the DEP electrodes to select, capture, and move specific micro-objects (not shown) around the region / chamber 202. Microfluidic devices having a DEP configuration including selectively addressable and energy-delivering electrodes are known in the art, for example, described in U.S. Patent No. 6,294,063 (Becker et al.) and U.S. Patent No. 6,942,776 (Medro), the entire contents of which are incorporated herein by reference.

[0199] As a further example, the microfluidic device 200 may have an electron wetting (EW) configuration, which may replace the DEP configuration or be located in a separate part of the microfluidic device 200 from the part having the DEP configuration. The EW configuration may be an optoelectronic wetting configuration or an electron wetting on a dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and a lower electrode 204, as described below. The dielectric layer may contain and / or be coated with a hydrophobic material. In the case of the microfluidic device 200 having an EW configuration, the inner surface 208 of the support structure 104 is the inner surface of the dielectric layer or its hydrophobic coating.

[0200] The dielectric layer (not shown) may include one or more oxide layers and may have a thickness of about 50 nm to about 250 nm (e.g., about 125 nm to about 175 nm). In certain embodiments, the dielectric layer may include layers of oxides such as metal oxides (e.g., aluminum oxide or hafnium oxide). In certain embodiments, the dielectric layer may include dielectric materials other than metal oxides, such as silicon oxide or nitrides. Regardless of the exact composition and thickness, the dielectric layer may have an impedance of about 10 k ohms to about 50 k ohms.

[0201] In some embodiments, the surface of the dielectric layer facing inward toward region / chamber 202 is coated with a hydrophobic material. The hydrophobic material may include, for example, fluorinated carbon molecules. Examples of fluorinated carbon molecules include perfluoropolymers such as polytetrafluoroethylene (e.g., TEFLON®) or poly(2,3-difluoromethyleneyl-perfluorotetrahydrofuran) (e.g., CYTOP®). The molecules constituting the material can be covalently bonded to the surface of the dielectric layer. For example, molecules of a hydrophobic material can be covalently bonded to the surface of the dielectric layer by linkers such as siloxane groups, phosphonic acid groups, or thiol groups. Therefore, in some embodiments, the hydrophobic material may include alkyl-terminated siloxanes, alkyl-terminated phosphonic acids, or alkyl-terminated thiols. The alkyl group may be a long-chain hydrocarbon (e.g., having a chain of at least 10 carbon atoms or at least 16, 18, 20, 22, or more carbon atoms). Alternatively, a fluorinated (or perfluorinated) carbon chain can be used instead of an alkyl group. Therefore, for example, the hydrophobic material may include fluoroalkyl-terminated siloxanes, fluoroalkyl-terminated phosphonic acids, or fluoroalkyl-terminated thiols. In some embodiments, the hydrophobic coating has a thickness of about 10 nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10 nm (e.g., less than 5 nm or about 1.5 to 3.0 nm).

[0202] In some embodiments, the cover 110 of the microfluidic device 200 having an electronic wetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material may be the same hydrophobic material used for coating the dielectric layer of the support structure 104, and the hydrophobic coating is approximately the same thickness as the hydrophobic coating of the dielectric layer of the support structure 104. It can have a certain thickness. Furthermore, the cover 110 may include an electrode activation substrate 206 sandwiched between the dielectric layer and the upper electrode 210 in the manner of the support structure 104. The dielectric layers of the electrode activation substrate 206 and the cover 110 may have the same composition and / or dimensions as the dielectric layers of the electrode activation substrate 206 and the support structure 104. Thus, the microfluidic device 200 may have two electron wetting surfaces.

[0203] In some embodiments, the electron activation substrate 206 may include photoconductive materials such as the photoconductive materials described above. Therefore, in certain embodiments, the electrode activation substrate 206 may include a layer of hydrogenated amorphous silicon (a-Si:H) or consist of a layer of a-Si:H. a-Si:H may contain, for example, about 8% to 40% hydrogen (calculated as the total number of hydrogen atoms and the total number of silicon atoms multiplied by 100). The a-Si:H layer may have a thickness of about 500 nm to about 2.0 μm. Alternatively, the electron activation substrate 206 may include electrodes (e.g., conductive metal electrodes) controlled by a phototransistor switch, as described above. Microfluidic devices having a photoelectron wetting configuration are known in the art and / or can be constructed using electrode activation substrates known in the art. For example, U.S. Patent No. 6,958,132 (Chiou et al.), whose entire content is incorporated herein by reference, describes photoconductive materials such as a-Si:H. A photoelectron wetting configuration having a material is disclosed, while U.S. Patent Application Publication No. 2014 / 0124370 (Short et al.), cited above, describes a phototransistor swimsuit An electrode activation substrate having electrodes controlled by a switch is disclosed.

[0204] Therefore, the microfluidic device 200 can have a photoelectron wetting configuration, and a photoresponsive EW region or photoresponsive EW electrode on the electrode activation substrate 206 can be activated using the light pattern 218. Such activated EW region or EW electrode on the electrode activation substrate 206 can generate an electron wetting force on the inner surface 208 of the support structure 104 (i.e., the inner surface of the overlapping dielectric layers or its hydrophobic coating). By changing the light pattern 218 incident on the electron activation substrate 206 (or moving the microfluidic device 200 relative to the light source 216), droplets in contact with the inner surface 208 of the support structure 104 (e.g., including aqueous culture media, aqueous solutions or aqueous solvents) can move through immiscible fluids (e.g., oil media) present in the region / chamber 202.

[0205] In other embodiments, the microfluidic device 200 may have an EWOD configuration, and the electrode activation substrate 206 may include selectively addressable and energy-donorable electrodes that are light-independent for activation. Thus, the electrode activation substrate 206 may include such electron wetting (EW) electrodes in a pattern. The pattern may be an array of substantially square EW electrodes arranged in a matrix, such as shown in Figure 2B. Alternatively, the pattern may be an array of substantially hexagonal EW electrodes forming a hexagonal grid. Regardless of the pattern, the EW electrodes can be selectively activated (or deactivated) by an electrical switch (e.g., a transistor switch on a semiconductor substrate). By selectively activating and deactivating the EW electrodes on the electrode activation substrate 206, droplets (not shown) in contact with the inner surface 208 of the overlapping dielectric layers or its hydrophobic coating can move within the region / chamber 202. The driving module 162 in Figure 1A can control such switches and thus activate and deactivate individual EW electrodes to selectively move specific droplets around the region / chamber 202. Microfluidic devices having an EWOD configuration with selectively addressable and energy-delivering electrodes are known in the art, for example, described in U.S. Patent No. 8,685,344 (Sundarsan et al.), the entirety of which is incorporated herein by reference.

[0206] Regardless of the configuration of the microfluidic device 200, the power supply 212 is used to control the microfluidic A potential (e.g., AC power potential) can be provided to supply power to the electrical circuit of device 200. Power supply 212 may be the same as or a component of power supply 192 referenced in Figure 1. Power supply 212 may be configured to provide AC voltage and / or current to the upper electrode 210 and the lower electrode 204. In the case of AC voltage, power supply 212 may provide a frequency range and average or peak power (e.g., voltage or current) sufficient to generate a net DEP force (or electronic wetting force) strong enough to capture and move individual micro-objects (not shown) within the region / chamber 202 and / or to change the wetting characteristics of the inner surface 208 of the support structure 104 within the region / chamber 202 (i.e., the dielectric layer and / or hydrophobic coating on the dielectric layer), as also described above. Such frequency ranges and average or peak power ranges are known in the art. For example, U.S. Patent No. 6,958,132 (Chiou et al.), U.S. Patent No. RE 44,711 (Wu et al.) (originally) (published as U.S. Patent No. 7,612,355), and U.S. Patent Application Publication No. 2014 / 0124370 (Short et al.), and U.S. Patent Application Publication No. 2015 / 0306598 (Khandros et al.) See also issue 2015 / 0306599 (Khandros et al.).

[0207] Isolation pen. Non-limiting examples of typical isolation pens 224, 226, and 228 are shown within the microfluidic device 230 in Figures 2A–2C. Each isolation pen 224, 226, and 228 may include an isolation structure 232 defining an isolation region 240 and a connection region 236 that fluidly connects the isolation region 240 to the channel 122. The connection region 236 may include a proximal opening 234 to the microfluidic channel 122 and an distal opening 238 to the isolation region 240. The connection region 236 may be configured such that the maximum penetration depth of the fluid culture medium (not shown) flowing from the microfluidic channel 122 into the isolation pens 224, 226, and 228 does not extend into the isolation region 240. Therefore, due to the connection region 236, micro-objects (not shown) or other materials (not shown) placed within the separation region 240 of the isolation pens 224, 226, and 228 can be separated from the flow of the culture medium 180 in the channel 122 and can be substantially unaffected by the flow of the culture medium 180 in the microfluidic channel 122.

[0208] The isolation pens 224, 226, and 228 in Figures 2A and 2C each have a single opening that opens directly into the microfluidic channel 122. The openings of the isolation pens open laterally from the microfluidic channel 122. The electrode activation substrate 206 is located beneath both the microfluidic channel 122 and the isolation pens 224, 226, and 228. The upper surface of the electrode activation substrate 206 within the isolation pen enclosure, which forms the floor of the isolation pens, is positioned at the same height as, or approximately the same height as, the upper surface of the electrode activation substrate 206 within the microfluidic channel 122 (or the flow region, if no channel exists), which forms the floor of the flow channel (or flow region, respectively) of the microfluidic device. The electrode activation substrate 206 may be featureless, or it may have an irregular or patterned surface that varies from the highest ridge to the lowest depression, or is less than approximately 3 μm, less than approximately 2.5 μm, less than approximately 2 μm, less than approximately 1.5 μm, less than approximately 1 μm, less than approximately 0.9 μm, less than approximately 0.5 μm, less than approximately 0.4 μm, less than approximately 0.2 μm, less than approximately 0.1 μm, or less than that. The variation in the ridge on the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and the isolation pen may be less than approximately 3%, less than approximately 2%, less than approximately 1%, less than approximately 0.9%, less than approximately 0.8%, less than approximately 0.5%, less than approximately 0.3%, or less than approximately 0.1% of the wall height of the isolation pen or the wall height of the microfluidic device. Although the microfluidic device 200 has been described in detail, this also applies to any of the microfluidic devices 100, 230, 250, 280, and 290 described herein.

[0209] Therefore, the microfluidic channel 122 may be an example of a swept region, and the isolation region 240 of the isolation pens 224, 226, and 228 may be an example of a non-swept region. As stated above, the microfluidic channel 122 and the isolation pens 224, 226, and 228 may be configured to contain one or more fluid media 180. In the example shown in Figures 2A and 2B, port 222 is a microfluidic channel 122. A chlorofluid channel 122 is connected, allowing the fluid medium 180 to be introduced into or removed from the microfluidic device 230. Before introducing the fluid medium 180, the microfluidic device may be primed with a gas such as carbon dioxide. Once the microfluidic device 230 contains the fluid medium 180, the flow 242 of the fluid medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, ports 222 can be located at different positions on the microfluidic channel 122 (e.g., at both ends), generating a flow 242 of the medium from one port 222 acting as an inlet to another port 222 acting as an outlet.

[0210] Figure 2C shows a detailed view of an example of the isolation pen 224 according to this disclosure. An example of a micro-object 246 is also shown.

[0211] As is known, the flow 242 of fluid medium 180 in the microfluidic channel 122 beyond the proximal opening 234 of the isolation pen 224 can cause a secondary flow 244 of medium 180 into and / or out of the isolation pen 224. To separate the micro-objects 246 in the isolation region 240 of the isolation pen 224 from the secondary flow 244, the length L of the connection region 236 of the isolation pen 224 is used. con (That is, from the proximal opening 234 to the tip opening 238) the penetration depth D of the secondary flow 244 into the connection region 236 p It should be a larger value than this. Intrusion depth D of secondary flow 244 p This depends on various parameters related to the velocity of the fluid medium 180 flowing through the microfluidic channel 122 and the configuration of the microfluidic channel 122 and the proximal opening 234 of the connection region 236 to the microfluidic channel 122. In a given microfluidic device, the configuration of the microfluidic channel 122 and the opening 234 is fixed, while the velocity of the flow 242 of the fluid medium 180 within the microfluidic channel 122 is variable. Therefore, for each isolation pen 224, the penetration depth D of the secondary flow 244 p The length L of the connection region 236 conThe maximum velocity Vmax of the flow 242 of the fluid medium 180 in channel 122 can be identified, ensuring that it does not exceed a certain limit. As long as the flow rate of the fluid medium 180 flow 242 in the microfluidic channel 122 does not exceed the maximum velocity Vmax, the resulting secondary flow 244 to the microfluidic channel 122 and connection region 236 can be limited and prevented from entering the separation region 240. Therefore, the flow 242 of the medium 180 in the microfluidic channel 122 does not draw the micro-objects 246 out of the separation region 240. Rather, micro-objects 246 placed within the separation region 240 remain within the separation region 240 regardless of the flow 242 of the fluid medium 180 in the microfluidic channel 122.

[0212] Furthermore, as long as the flow rate of the culture medium 180 flow 242 in the microfluidic channel 122 does not exceed Vmax, the flow of the fluid culture medium 180 flow 242 in the microfluidic channel 122 will not move various particles (e.g., microparticles and / or nanoparticles) from the microfluidic channel 122 into the isolation region 240 of the isolation pen 224. Therefore, the length L of the connection region 236 con The maximum penetration depth D of the secondary flow 244 p By making it larger, contamination of one isolation pen 224 by various particles from the microfluidic channel 122 or from another isolation pen (e.g., isolation pens 226 and 228 in Figure 2D) can be avoided.

[0213] The microfluidic channel 122 and the connection region 236 of the isolation pens 224, 226, and 228 can be influenced by the flow 242 of the culture medium 180 in the microfluidic channel 122, and therefore the microfluidic channel 122 and the connection region 236 can be considered as sweeping (or flowing) regions of the microfluidic device 230. On the other hand, the separation regions 240 of the isolation pens 224, 226, and 228 can be considered as non-sweeping (or non-flowing) regions. For example, components (not shown) in the first fluid culture medium 180 in the microfluidic channel 122 can be substantially absorbed only by the diffusion of components of the first culture medium 180 from the microfluidic channel 122 through the connection region 236 to the second fluid culture medium 248 in the separation region 240. The first medium 180 may be mixed with the second fluid medium 248. Similarly, components of the second medium 248 (not shown) in the isolation region 240 may be mixed with the first medium 180 in the microfluidic channel 122 only by diffusion of the components of the second medium 248 from the isolation region 240 through the connection region 236 to the first medium 180 in the microfluidic channel 122. In some embodiments, the degree of fluid medium exchange between the isolation region and the flow region of the isolation pen by diffusion is higher than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the fluid exchange. The first medium 180 may be the same medium as the second medium 248, or it may be a different medium. Furthermore, the first medium 180 and the second medium 248 can be started as the same medium and become different (for example, by adjusting the second medium 248 by altering the medium 180 flowing through the microfluidic channel 122, or by the presence of one or more cells in the isolation region 240).

[0214] The maximum penetration depth D of the secondary flow 244 generated by the flow 242 of the fluid medium 180 within the microfluidic channel 122. pAs mentioned above, this can depend on several parameters. Examples of such parameters include the shape of the microfluidic channel 122 (for example, the channel can direct the culture medium towards the connection region 236, divert the culture medium away from the connection region 236, or direct the culture medium in a direction substantially perpendicular to the proximal opening 234 of the connection region 236 of the microfluidic channel 122), and the width W of the microfluidic channel 122 at the proximal opening 234. ch (or cross-sectional area) and width W of the connection area 236 at the base opening 234. con Examples include (or cross-sectional area), the flow rate V of the fluid medium 180 in the microfluidic channel 122, and the viscosity of the first medium 180 and / or the second medium 248.

[0215] In some embodiments, the dimensions of the microfluidic channel 122 and the isolation pens 224, 226, 228 can be oriented with respect to the vector of the flow 242 of the fluid medium 180 in the microfluidic channel 122 as follows: microfluidic channel width W ch (or the cross-sectional area of ​​the microfluidic channel 122) can be approximately perpendicular to the flow of the culture medium 180 242, and the width W of the connection region 236 at the opening 234 con The (or cross-sectional area) may be substantially parallel to the flow 242 of the culture medium 180 in the microfluidic channel 122, and / or the length L of the connection region. con The flow 242 of the culture medium 180 in the microfluidic channel 122 can be approximately orthogonal to it. The above is merely an example, and the relative positions of the microfluidic channel 122 and the isolation pens 224, 226, and 228 may be oriented in other directions relative to each other.

[0216] As shown in Figure 2C, the width W of the connection region 236 con The width can be uniform from the base opening 234 to the tip opening 238. Therefore, the width W of the connection region 236 at the tip opening 238. con The width W of the connection region 236 at the base opening 234 is con This can be any value identified herein. Alternatively, the width W of the connection area 236 at the tip opening 238. concan be a value greater than the width W of the connection region 236 at the proximal opening 234. con

[0217] As shown in FIG. 2C, the width of the separation region 240 at the distal opening 238 can be substantially the same as the width W of the proximal region 236 at the proximal opening 234. Thus, the width of the separation region 240 at the distal opening 238 can be any value identified herein for the width W of the connection region 236 at the proximal opening 234. Alternatively, the width of the separation region 240 at the distal opening 238 can be greater than or less than the width W of the connection region 236 at the proximal opening 234. Further, the distal opening 238 can be smaller than the proximal opening 234, and the width W of the connection region 236 con can be tapered between the proximal opening 234 and the distal opening 238. For example, the connection region 236 can be tapered between the proximal and distal openings using various different geometries (e.g., chamfering the connection region, applying a gradient to the connection region). Further, any portion or sub-portion of the connection region 236 can be tapered (e.g., the portion of the connection region adjacent to the proximal opening 2 con 34). con con

[0218] ​​​​Figures 2D to 2F show another exemplary embodiment of a microfluidic device 250, which includes a microfluidic circuit 262 and a flow channel 264, which are variations of the microfluidic devices 100, circuit 132, and channel 134 of Figure 1A. The microfluidic device 250 also has a plurality of isolation pens 266, which are additional variations of the isolation pens 124, 126, 128, 130, 224, 226, or 228 described above. In particular, it should be understood that the isolation pens 266 of device 250 shown in Figures 2D to 2F can be replaced with any of the isolation pens 124, 126, 128, 130, 224, 226, or 228 described above in devices 100, 200, 230, 280, and 290. Similarly, the microfluidic device 250 is another variation of the microfluidic device 100 and may have the same or different DEP configuration as the microfluidic devices 100, 200, 230, 280, and 290 described above, as well as any other microfluidic system components described herein.

[0219] The microfluidic device 250 in Figures 2D to 2F includes a support structure (not visible in Figures 2D to 2F, but may be the same as or generally similar to the support structure 104 of device 100 shown in Figure 1A), a microfluidic circuit structure 256, and a cover (not visible in Figures 2F, but may be the same as or generally similar to the cover 122 of device 100 shown in Figure 1A). The microfluidic circuit structure 256 includes a frame 252 and 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 Figure 1A. As shown in Figure 2D, the microfluidic circuit 262 defined by the microfluidic circuit material 260 may include multiple channels 264 (two are shown, but there may be more channels), to which multiple isolation pens 266 are fluidly connected.

[0220] Each isolation pen 266 can include a separation structure 272, a separation region 270 within the separation structure 272, and a connection region 268. From the proximal opening 274 of the microfluidic channel 264 to the distal opening 276 in the separation structure 272, the connection region 268 fluidly connects the microfluidic channel 264 to the separation region 270. Generally, according to the above considerations of FIGS. 2B and 2C, the flow 278 of the first fluid medium 254 within the channel 264 can result in a secondary flow 282 of the first medium 254 from the microfluidic channel 264 into and / or out of each connection region 268 of the isolation pen 266.

[0221] As shown in FIG. 2E, the connection region 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 of the connection region 268 con can be a value greater than the maximum penetration depth D of the secondary flow 282, in which case the secondary flow 282 extends into the connection region 268 without being redirected towards the separation region 270 (as shown in FIG. 2D). Alternatively, as shown in FIG. 2F, the connection region 268 can have a length L p less than the maximum penetration depth D, in which case the secondary flow 282 extends through the connection region 268 and is redirected towards the separation region 270. In this latter situation, the sum of the lengths L p and L con of the connection region 268 is greater than the maximum penetration depth D, and thus the secondary flow 282 does not extend into the separation region 270. Whether the length L of the connection region 268 c1 is greater than the penetration depth D c2 or whether the sum of the lengths L p and L con of the connection region 268 is greater than the penetration depth D p , regardless of whether the flow 278 of the first medium 254 within the channel 264 that does not exceed the maximum velocity V c1 and L c2 is greater than the penetration depth D p , the penetration depth D max of the first medium 254 within the channel 264 that does not exceed the maximum velocity V pThis results in a secondary flow, and microorganisms within the isolation region 270 of the isolation pen 266 (not shown, but may be the same as or generally similar to the microorganisms 246 shown in Figure 2C) are not drawn out of the isolation region 270 by the flow 278 of the first medium 254 in channel 264. The flow 278 in channel 264 also does not draw various materials (not shown) from channel 264 into the isolation region 270 of the isolation pen 266. Therefore, the only mechanism by which components in the first medium 254 within the microfluidic channel 264 can be moved from the microfluidic channel 264 to the second medium 258 within the isolation region 270 of the isolation pen 266 is diffusion. Similarly, the only mechanism by which components in the second medium 258 within the isolation region 270 of the isolation pen 266 can be moved from the isolation region 270 to the first medium 254 within the microfluidic channel 264 is also diffusion. The first medium 254 may be the same medium as the second medium 258, or the first medium 254 may be a different medium from the second medium 258. Alternatively, the first medium 254 and the second medium 258 may start from the same medium and become different, for example, by the preparation of the second medium by one or more cells in the isolation region 270 or by changing the medium flowing through the microfluidic channel 264.

[0222] As shown in Figure 2E, the width W of the microfluidic channel 264 within the microfluidic channel 264. ch (That is, taken across the direction of fluid medium flow through the microfluidic channel indicated by arrow 278 in Figure 2D) is the width W of the proximal opening 274. con1 It can be approximately perpendicular to the direction, and therefore the width W of the tip opening 276 con2 It may be approximately parallel. However, the width of the base opening 274 con1 and width W of the tip opening 276 con2 They do not need to be approximately orthogonal to each other. For example, the width W of the base opening 274. con1 The axis to which it is directed (not shown), and the width W of the tip opening 276. con2The angle between the axis to which it is directed and the other axis to which it is directed can be other than orthogonal, and therefore can be other than 90°. Examples of alternative angles to which it can be directed include: approximately 30° to approximately 90°, approximately 45° to approximately 90°, approximately 60° to approximately 90°, etc.

[0223] In various embodiments of the isolation pen (e.g., 124, 126, 128, 130, 224, 226, 228, or 266), the isolation region (e.g., 240 or 270) is configured to contain multiple micro-objects. In other embodiments, the isolation region may be configured to contain only one, two, three, four, five, or a similarly small number of micro-objects. Thus, the volume of the isolation region is, for example, at least 1 × 10⁻⁶ 6 Cubic μm, at least 2 × 10⁻⁶ 6 Cubic μm, at least 4 × 10⁻⁶ 6 Cubic μm, at least 6 × 10⁻⁶ 6 It may be cubic micrometers or larger.

[0224] In various embodiments of the isolation pen, the width W of the microfluidic channel (e.g., 122) at the base opening (e.g., 234) ch These are approximately 50-1000 μm, approximately 50-500 μm, approximately 50-400 μm, approximately 50-300 μm, approximately 50-250 μm, approximately 50-200 μm, approximately 50-150 μm, approximately 50-100 μm, approximately 70-500 μm, approximately 70-400 μm, approximately 70-300 μm, approximately 70-250 μm, approximately 70- The width W of the microfluidic channel (e.g., 122) at the proximal opening (e.g., 234) may be 200 μm, approximately 70-150 μm, approximately 90-400 μm, 90-300 μm, approximately 90-250 μm, approximately 90-150 μm, approximately 100-300 μm, approximately 100-250 μm, approximately 100-200 μm, approximately 100-150 μm, or approximately 100-120 μm. In some other embodiments, the width W of the microfluidic channel (e.g., 122) at the proximal opening (e.g., 234) may be... ch The width W of the microfluidic channel 122 can be approximately 200-800 μm, approximately 200-700 μm, or approximately 200-600 μm. The above is merely an example. ch The width may be any width within any of the endpoints listed above. Furthermore, the width W of the microfluidic channel 122 may also be a width.ch These widths can be selected to be any width in the region of the microfluidic channel other than the proximal opening of the isolation pen.

[0225] 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 has a height of about 1 × 10 4 ~Approx. 3×10 6 square μm, approximately 2×10 4 ~Approx. 2×10 6 square μm, approximately 4×10 4 ~Approx. 1×10 6 square μm, approximately 2×10 4 ~Approx. 5×10 5 square μm, approximately 2×10 4 ~Approx. 1×10 5 Square micrometer, or approximately 2 × 10⁻⁶ 5 ~Approx. 2×10 6 It has a cross-sectional area of ​​square micrometers.

[0226] In various embodiments of the isolation pen, the height H of the microfluidic channel (e.g., 122) at the proximal opening (e.g., 234) ch The height can be any of the following: 20-100 μm, 20-90 μm, 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 are merely examples of height H of a microfluidic channel (e.g., 122). ch This may be the height within any of the endpoints listed above. Height H of the microfluidic channel 122 ch These can be selected to be any range within the region of the microfluidic channel other than the proximal opening of the isolation pen.

[0227] In various embodiments of the isolation pen, the cross-sectional area of ​​the microfluidic channel (e.g., 122) at the proximal opening (e.g., 234) is approximately 500-50,000 sq μm, 500-40,000 sq μm, 500-30,000 sq μm, 500-25,000 sq μm, 500-20,000 sq μm, 500-15,000 sq μm, 500-10,000 sq μm, 500-7,500 sq μm, 500-5,000 sq μm, 1,000-25,000 sq μm, 1,000-20,000 sq μm, and 1,000 ~15,000 square μm, 1,000~10,000 square μm, 1,000~7,500 square μm, 1,000~5,000 square μm, 2,000~20,000 square μm, 2,000~15,000 square μm, 2,000~10,000 square μm, 2,000 It can be ~7,500 sq. um, 2,000-6,000 sq. um, 3,000-20,000 sq. um, 3,000-15,000 sq. um, 3,000-10,000 sq. The above is merely an example, and the cross-sectional area of ​​the microfluidic channel (e.g., 122) at the proximal opening (e.g., 234) may be the area within any of the endpoints listed above.

[0228] In various embodiments of the isolation pen, the length L of the connection area (e.g., 236) con This can be approximately 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 approximately 100-150 μm. The above are merely examples, and the length L of the connecting region (e.g., 236) con This can be any length within any of the endpoints listed above.

[0229] In various embodiments of the isolation pen, the width W of the connection area (e.g., 236) at the base opening (e.g., 234) conThese are approximately 20-500 μm, 20-400 μm, 20-300 μm, 20-200 μm, 20-150 μm, 20-100 μm, 20-80 μm, 20-60 μm, 30-400 μm, 30-300 μm, 30-200 μm, 30-150 μm, 30-100 μm, 30-80 μm, 30-60 μm, 40-300 μm, 40-200 The width W of the connection region (e.g., 236) of the proximal opening (e.g., 234) is also possible. The above are merely examples, and the width W of the connection region (e.g., 236) of the proximal opening (e.g., 234) is also possible. con This can differ from the above example (for example, any value within any of the endpoints listed above).

[0230] In various embodiments of the isolation pen, the width W of the connection region (e.g., 236) at the base opening (e.g., 234) con The width W of the connection region (e.g., 236) at the basal opening (e.g., 234) can be made at least as large as the maximum dimensions of the micro-objects (e.g., living cells that may be T cells or B cells) targeted by the isolation pen. The above is merely an example, and the width W of the connection region (e.g., 236) at the basal opening (e.g., 234) can also be used. con This refers to the example above (for instance, the width within the range of any of the endpoints listed above). It may differ from that.

[0231] In various embodiments of the isolation pen, the width W of the base end opening of the connection region pr The size of the isolation pen may be at least the same as the maximum dimensions of the micro-object (e.g., a biological micro-object such as a cell) in which it is intended. For example, width W pr These can be approximately 50 μm, 60 μm, 100 μm, 200 μm, 300 μm, or approximately 50-300 μm, 50-200 μm, 50-100 μm, 75-150 μm, 75-100 μm, or 200-300 μm.

[0232] In various embodiments of the isolation pen, the length L of the connection area (e.g., 236) con and the width W of the connection region (e.g., 236) at the base opening 234. con The ratio may be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or a ratio greater than or equal to these. The above is merely an example, and the length L of the connection region 236. con and the width W of the connection region 236 at the base end opening 234 con The ratio may differ from that in the example above.

[0233] In various embodiments 100, 200, 23, 250, 280, and 290 of the microfluidic device, V max These are approximately 0.2 microliters / second, 0.5 microliters / second, 0.7 microliters / second, 1.0 microliters / second, 1.3 microliters / second, 1.5 microliters / second, 2.0 microliters / second, 2.5 microliters / second, 3.0 microliters / second, 3.5 microliters / second, 4.0 microliters / second, 4.5 microliters / second, 5.0 microliters / second, and 5.5 microliters / second. It can be set to 10 microliters / second, approximately 6.0 microliters / second, approximately 6.7 microliters / second, approximately 7.0 microliters / second, approximately 7.5 microliters / second, approximately 8.0 microliters / second, approximately 8.5 microliters / second, approximately 9.0 microliters / second, approximately 10 microliters / second, approximately 11 microliters / second, approximately 12 microliters / second, approximately 13 microliters / second, approximately 14 microliters / second, or approximately 15 microliters / second.

[0234] In various embodiments of microfluidic devices having an isolation pen, the volume of the isolation region of the isolation pen (e.g., 240) is, for example, at least 5 × 10 5 Cubic μm, at least 8 × 10⁻¹⁶ 5 Cubic μm, at least 1 × 10⁻⁶ 6 Cubic μm, at least 2 × 10⁻⁶ 6 Cubic μm, at least 4 × 10⁻⁶6 Cubic μm, at least 6 × 10⁻⁶ 6 Cubic μm, at least 8 × 10⁻¹⁶ 6 Cubic μm, at least 1 × 10⁻⁶ 7 Cubic μm, at least 5 × 10⁻⁶ 7 Cubic μm, at least 1 × 10⁻⁶ 8 Cubic μm, at least 5 × 10⁻⁶ 8 Cubic μm, at least 8 × 10⁻¹⁶ 8 The volume may be cubic μm or larger. In various embodiments of microfluidic devices having an isolation pen, the volume of the isolation pen is approximately 5 × 10⁻¹⁶ 5 cubic μm, approximately 6×10 5 cubic μm, approximately 8×10 5 cubic μm, approximately 1×10 6 cubic μm, approximately 2×10 6 cubic μm, approximately 4×10 6 cubic μm, approximately 8×10 6 cubic μm, approximately 1×10 7 cubic μm, approximately 3×10 7 cubic μm, approximately 5×10 7 cubic μm, or approximately 8 × 10⁻⁶ 7 The volume may be cubic micrometers or larger. In some other embodiments, the volume of the isolation pen may be about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters.

[0235] In various embodiments, the microfluidic device has isolation pens configured as in any embodiment described herein, in which case the microfluidic device has about 5 to about 10 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 isolation pens, about 1000 to about 3500 isolation pens, about 3000 to about 7000 isolation pens, about 5000 to about 10,000 isolation pens, and about 9000 to about 15,000 It may have 0 isolation pens, or approximately 12,000 to 20,000 isolation pens. The isolation pens do not all need to be the same size and may include various configurations (e.g., different widths, different features within the isolation pens).

[0236] Figure 2G shows a microfluidic device 280 according to one embodiment. The microfluidic device 280 shown in Figure 2G is a stylized diagram of the microfluidic device 100. In practice, the microfluidic device 280 and its constituent circuit elements (e.g., channels 122 and isolation pens 128) have the dimensions considered herein. The microfluidic circuit 120 shown in Figure 2G has two ports 107, four separate channels 122, and four separate flow paths 106. The microfluidic device 280 further includes a plurality of isolation pens leading to each channel 122. In the microfluidic device shown in Figure 2G, the isolation pens have a geometry similar to the pens shown in Figure 2C, and therefore have both a connecting region and a separation region. Thus, the microfluidic circuit 120 has a sweeping region (e.g., the maximum penetration depth D of channels 122 and secondary flow 244). p (Part of the connection area 236) and the non-swept area (e.g., the separation area 240 and the maximum penetration depth D of the secondary flow 244) p This includes both (the portion of the connection area 236 that is not contained within).

[0237] Figures 3A and 3B illustrate various embodiments of System 150 that can be used to operate and observe microfluidic devices (e.g., 100, 200, 230, 250, 280, 290) according to the present disclosure. As shown in Figure 3A, System 150 may include a structure ("nest") 300 configured to hold a microfluidic device 100 (not shown) or any other microfluidic device described herein. The nest 300 may include a socket 302 that can interface with a microfluidic device 320 (e.g., an optically actuated electrodynamic device 100) and provide an electrical connection from a power supply 192 to the microfluidic device 320. The nest 300 may further include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 may be configured to supply a bias voltage to the socket 302 such that a bias voltage is applied across pairs of electrodes in the microfluidic device 320 when the microfluidic device 320 is held by the socket 302. Therefore, the electrical signal generation subsystem 304 may 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 always applied when 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.

[0238] As shown in Figure 3A, the nest 300 may include a printed circuit board assembly (PCBA) 322. The electrical signal generation subsystem 304 can be mounted on the PCBA 322 and electrically integrated into the PCBA 322. The exemplary support also includes a socket 302, which is similarly mounted on the PCBA 322.

[0239] 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 may be configured to measure the waveform supplied to the microfluidic device 320, which is held by the socket 302, if present. In certain embodiments, the oscilloscope measures the waveform at the base end position of the microfluidic device 320 (and the tip position of the waveform generator) to ensure greater accuracy in measuring the waveform actually applied to the device. Data obtained from the oscilloscope measurement may be provided to the waveform generator, for example, as feedback, and the waveform generator will process such feedback. It can be configured to adjust the output based on [the specified parameters]. An example of a suitable coupled waveform generator and oscilloscope is Red Pitaya®.

[0240] In certain embodiments, the nest 300 further includes a controller 308, such as a microprocessor, used for sensing and / or controlling the electrical signal generation subsystem 304. Examples of suitable microprocessors include the Arduino® Micro, such as the Arduino Nano®. A processor is one example. It may perform functions and analyses using the controller 308, or by communicating with an external master controller 154 (shown in Figure 1A). In the embodiment shown in Figure 3A, the controller 308 communicates with the master controller 154 through an interface 310 (e.g., a plug or connector).

[0241] In some embodiments, Nest 300 may include an electrical signal generation subsystem 304, which includes a Red Pitaya® waveform generator / oscilloscope unit ("Red Pitaya Unit"), and a waveform amplification circuit that amplifies the waveform generated by the Red Pitaya Unit and passes the amplified voltage to the microfluidic device 100. In some embodiments, the Red Pitaya Unit is configured to measure the amplified voltage at the microfluidic device 320 and then adjust its own output voltage as needed so that the measured voltage at the microfluidic device 320 is a desired value. In some embodiments, the waveform amplification circuit may have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on PCBA 322, resulting in the generation of signals up to 13Vpp at the microfluidic device 100.

[0242] As shown in Figure 3A, the support structure 300 (e.g., a nest) may 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 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface configured to interface with at least one surface of the microfluidic device 320. The cooling unit may be a cooling block (not shown), such as a liquid-cooled aluminum block. A second surface of the Peltier thermoelectric device (e.g., the surface opposite to the first surface) may be configured to interface with the surface of such a cooling block. The cooling block may be connected to a fluid passage 314 configured to circulate a cooling fluid through the cooling block. In the embodiment shown in Figure 3A, the support structure 300 receives cooled fluid from an external reservoir (not shown), including an inlet 316 and an outlet 318, introduces the cooled fluid into a fluid channel 314, passes it through a cooling block, and then returns the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, cooling unit, and / or fluid channel 314 can be mounted in the case 312 of the support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Peltier thermoelectric device to achieve a target temperature for the microfluidic device 320. Temperature regulation of the Peltier thermoelectric device can be achieved by a thermoelectric power supply, for example, a Pololu® thermoelectric power supply (Pololu Robotics and Electronics Corp.). The thermal control subsystem 306 is configured to analyze The system may include a feedback circuit for temperature values ​​provided by the logging circuit. Alternatively, the feedback circuit may be provided by a digital circuit.

[0243] In some embodiments, the nest 300 may include a thermal control subsystem 306 having a feedback circuit which is an analog voltage divider circuit (not shown) including a resistor (e.g., resistance 1 k ohm ±0.1%, temperature coefficient ±0.02 ppm / C0) and an NTC thermistor (e.g., having a nominal resistance 1 k ohm ±0.01%). In some cases, the thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as input to an onboard PID control loop algorithm. PID control The output from the loop algorithm can, for example, drive both the direction signal and pulse width modulation signal pins on a Pololu® motor drive device (not shown) to operate a thermoelectric power supply, thereby controlling a Peltier thermoelectric device.

[0244] Nest 300 may include serial port 350, and serial port 324 allows the microprocessor of controller 308 to communicate with an 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 (for example, via a Plink tool (not shown)). Therefore The electrical signal generation subsystem 304 and the thermal control subsystem 306 can communicate with an external master controller 154 via a combination of controller 308, interface 310, and 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 adjustment. A graphical user interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154 may be configured to plot temperature data and waveform data obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 304, respectively. Alternatively or additionally, the GUI may enable updates to controller 308, thermal control subsystem 306, and electrical signal generation subsystem 304.

[0245] As described above, system 150 may include an imaging device 194. In some embodiments, the imaging device 194 includes an optical modulation subsystem 330 (see Figure 3B). The optical modulation subsystem 330 may include a digital mirror device (DMD) or a microshutter array system (MSA), either of which may be configured to receive light from a light source 332 and send a subset of the received light to the optical column of the microscope 350. Alternatively, the optical modulation subsystem 330 may include a device that generates its own light (thus eliminating the need for a light source 332), such as an organic light-emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon (FLCOS) device, or a transmissive liquid crystal display (LCD). The optical modulation subsystem 330 may be, for example, a projector. Thus, the optical modulation subsystem 330 may be capable of emitting both structured and unstructured light. In certain embodiments, the imaging module 164 and / or the driving module 162 of system 150 may control the optical modulation subsystem 330.

[0246] In certain embodiments, the imaging device 194 further includes a microscope 350. In such embodiments, the nest 300 and the optical modulation subsystem 330 may be configured individually to be mounted on the microscope 350. The microscope 350 may be configured, for example, to be a standard research-grade optical microscope or fluorescence microscope. Thus, the nest 300 may be configured to be mounted on the stage 344 of the microscope 350, and / or the optical modulation subsystem 330 may be configured to be mounted on a port of the microscope 350. In other embodiments, the nest 300 and the optical modulation subsystem 330 described herein may be integral components of the microscope 350.

[0247] In certain embodiments, the microscope 350 may further include one or more detectors 348. In some embodiments, the detectors 348 are controlled by an imaging module 164. The detectors 348 may include eyepieces, charge-coupled devices (CCDs), cameras (e.g., digital cameras), or any combination thereof. If at least two detectors 348 are present, one detector may be, for example, a high-frame-rate camera, while the other detector may be a high-sensitivity camera. Furthermore, the microscope 350 receives light reflected and / or emitted from the microfluidic device 320, and reduces the amount of reflected and / or emitted light. The optical cascading can include an optical cascading canon configured to image the canon portion onto one or more detectors 348. The optical cascading can also include different tube lenses (not shown) for different detectors so that the final magnification at each detector can be different.

[0248] In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, a first light source 332 can be used to generate structured light (e.g., via an optical modulation subsystem 330), and a second light source 334 can be used to provide unstructured light. The first light source 332 can generate structured light for optically operated electrokinesis and / or fluorescence excitation, and the second light source 334 can provide bright-field illumination. In these embodiments, a motive module 164 can be used to control the first light source 332, and the imaging device The second light source 334 can be controlled using the module 164. The optical array of the microscope 350 may be configured to (1) receive structured light from the optical modulation subsystem 330, and when the device is held by the nest 300, focus the structured light on at least a first region within a microfluidic device, such as an optically actuated electrodynamic device; and (2) receive light reflected and / or emitted from the microfluidic device, and focus at least a portion of such reflected and / or emitted light on the detector 348. The optical array may be further configured to receive unstructured light from the second light source, and when the device is held by the nest 300, focus the unstructured light on at least a second region of the microfluidic device. In certain embodiments, the first and second regions of the microfluidic device may be overlapping regions. For example, the first region may be a subset of the second region. In other embodiments, the second light source 334 may, in addition or alternatively, include a laser which may have any preferred wavelength of light. The diagram of the optical system shown in Figure 3B is only a schematic, and the optical system may include further filters, notch filters, lenses, etc. If the second light source 334 includes one or more light sources for bright-field and / or fluorescence excitation and laser irradiation, the physical arrangement of the light sources may differ from that shown in Figure 3B, and the laser irradiation may be introduced at any suitable physical position within the optical system. The schematic positions of light source 334 and light source 332 / optical modulation subsystem 330 may also be interchanged.

[0249] Figure 3B shows a first light source 332 supplying light to the optical modulation subsystem 330, which provides structured light to the optical column of a microscope 350 (not shown) in system 355. A second light source 334 is shown providing unstructured light to the optical column via a beam splitter 336. The structured light from the optical modulation subsystem 330 and, as shown, the unstructured light from the second light source 334 travel together through the optical column from the beam splitter 336 to the second beam splitter (or dichroic filter 338, depending on the light provided by the optical modulation subsystem 330), where the light is reflected and descends through the objective lens 336 to the sample surface 342. The reflected and / or emitted light from the sample surface 342 then travels again through the objective lens 340, through the beam splitter and / or dichroic filter 338, and reaches the dichroic filter 346. Only a portion of the light that reaches the dichroic filter 346 is transmitted and reaches the detector 348.

[0250] In some embodiments, the second light source 334 emits blue light. Using 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 optical modulation 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 wavelengths longer than 495 nm. Such filtering of light from the optical modulation subsystem 330 is completed only if the light emitted from the optical modulation subsystem does not contain any wavelengths shorter than 495 nm (as shown). In practice, the optical modulation subsystem If the light coming from stem 330 includes wavelengths shorter than 495 nm (e.g., blue wavelengths), some of the light from the optical modulation subsystem passes through filter 346 and reaches detector 348. In such embodiments, filter 346 plays a role in changing the balance between the amount of light reaching detector 348 from the first light source 332 and the amount of light reaching detector 348 from the second light source 334. This can be beneficial if the first light source 332 is much more powerful than the second light source 334. In other embodiments, the second light source 334 may emit red light, and the dichroic filter 346 may filter out and exclude visible light other than red light (e.g., visible light with wavelengths shorter than 650 nm).

[0251] Coating solution and coating agent. Without intending to impose theoretical limitations, the maintenance of biological microorganisms (e.g., living cells) within microfluidic devices (e.g., DEP and / or EW microfluidic devices) may be facilitated if at least one or more inner surfaces of the microfluidic device are tuned or coated to present a layer of organic and / or hydrophilic molecules that provide the primary interface between the microfluidic device and the biological microorganisms maintained within it (i.e., the biological microorganisms exhibit increased viability, greater proliferation, and / or greater portability within the microfluidic device). In some embodiments, one or more inner surfaces of a microfluidic device (e.g., the inner surface of the electrode activation substrate of a DEP microfluidic device, the microfluidic cover, and / or the surface of the circuit material) may be treated or modified with coating solutions and / or coating agents to generate a desired layer of organic and / or hydrophilic molecules.

[0252] The coating may be applied before or after the introduction of the biological microobjects, or it may be introduced simultaneously with the biological microobjects. In some embodiments, the biological microobjects may be introduced into a fluid medium containing one or more coatings of the microfluidic device. In other embodiments, the inner surface of the microfluidic device (e.g., a DEP-configured microfluidic device) is treated or "primed" with a coating solution containing the coating before the biological microobjects are introduced into the microfluidic device.

[0253] In some embodiments, at least one surface of the microfluidic device includes a coating that provides a layer of organic and / or hydrophilic molecules suitable for maintaining and / or growing biological microorganisms (e.g., providing a conditioned surface as described later). In some embodiments, substantially all inner surfaces of the microfluidic device include the coating material. The coated inner surfaces may include the surfaces of flow regions (e.g., channels), chambers, isolation pens, or combinations thereof. In some embodiments, each of a plurality of isolation pens has at least one inner surface coated with the coating material. In other embodiments, each of a plurality of flow regions or channels has at least one inner surface coated with the coating material. In some embodiments, at least one inner surface of each of a plurality of isolation pens and each of a plurality of channels is coated with the coating material.

[0254] Coating agent / solution. Any conventional coating agent / coating solution can be used, but is not limited to serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof.

[0255] Polymer-based coating material. At least one inner surface may include a coating material containing a polymer. The polymer may be covalently or noncovalently bonded (or nonspecifically attached) to at least one surface. The polymer may have a wide variety of structural motifs, such as those found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein.

[0256] The polymer may include polymers containing an alkylene ether moiety. A wide range of alkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein. A non-limiting and exemplary class of alkylene ether-containing polymers is an amphiphilic nonionic block copolymer containing blocks of polyethylene oxide (PEO) subunits and polypropylene oxide (PPO) subunits located in different proportions and locations within the polymer chain. Pluronic® polymers (BASF) are of this type and are known in the art to be suitable for use in contact with living cells. The polymer has an average molecular weight M W The hydrophilicity can range from approximately 2000 Da to approximately 20 kDa. In some embodiments, the PEO-PPO block copolymer can have a hydrophilic-lipophilic balance (HLB) greater than approximately 10 (e.g., 12 to 18). Certain Pluronic® polymers useful for resulting in coated surfaces include Pluronic® L44, L64, P85, and F127 (including F127NF). Another class of alkylene ether-containing polymers includes polyethylene glycol (PEG M W <100,000 Da) or alternatively polyethylene oxide (PEO, M W (>100,000). In some embodiments, PEG is approximately 1000Da, 5000Da, 10,000Da, or 20,000Da. W It may have.

[0257] In other embodiments, the coating material may include a polymer containing a carboxylic acid moiety. The carboxylic acid subunit may be an alkyl, alkenyl, or aromatic moiety-containing subunit. A non-limiting example is polylactic acid (PLA). In other embodiments, the coating material may include a polymer containing a phosphate moiety either at the ends of the polymer backbone or as a pendant from the polymer backbone. In yet another embodiment, the coating material may include a polymer containing a sulfonic acid moiety. The sulfonic acid subunit may be an alkyl, alkenyl, or aromatic moiety-containing subunit. A non-limiting example is polystyrene sulfonic acid (PSSA) or polyanethol sulfonic acid. In yet another embodiment, the coating material may include a polymer containing an amine moiety. The polyamino polymer may include a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine.

[0258] In other embodiments, the coating material may comprise a polymer containing a sugar moiety. In non-limiting examples, polysaccharides such as xanthan gum or dextran may be suitable for forming materials that can reduce or prevent cell penetration in microfluidic devices. For example, a dextran polymer having a size of about 3 kDa may be used to provide a coating material to a surface within a microfluidic device.

[0259] In other embodiments, the coating material may include a polymer containing a nucleotide moiety, i.e., a nucleic acid, having a ribonucleotide moiety or a deoxyribonucleotide moiety, and capable of providing a polymer electrolyte surface. The nucleic acid may consist solely of a native nucleotide moiety, or may, but is not limited to, a non-native nucleotide moiety containing nucleic acid bases such as 7-deazaadenine, pentose, methylphosphonic acid, or a phosphorothioate moiety, ribose, or a phosphate moiety analogue.

[0260] In yet another embodiment, the coating material may comprise a polymer containing an amino acid moiety. The polymer containing the amino acid moiety may comprise a naturally occurring amino acid-containing polymer or a non-natural amino acid-containing polymer, either of which may comprise a peptide, polypeptide, or protein. In a non-limiting example, the protein used as the coating agent may be bovine serum albumin (BSA) and / or serum (or a combination of several different serums) containing albumin and / or one or more other similar proteins. The serum may be from any convenient source, but is not limited to, fetal bovine serum, sheep serum, goat serum, horse serum, etc. In the application, BSA in the coating solution is present at concentrations ranging from approximately 1 mg / mL to approximately 100 mg / mL, including 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or any value greater than or between these. In certain embodiments, serum in the coating solution may be present at concentrations ranging from approximately 20% (v / v) to approximately 50% v / v, including 25%, 30%, 35%, 40%, 45%, or any value greater than or between these. In some embodiments, BSA may be present as a coating agent in the coating solution at 5 mg / mL, while in other embodiments, BSA may be present as a coating agent in the coating solution at 70 mg / mL. In certain embodiments, serum is present as a coating agent in the coating solution at 30%. In some embodiments, extracellular matrix (ECM) proteins may be provided within the coating material to optimize cell adhesion to Foster cell growth. Examples of cellular matrix proteins that may be incorporated into the coating material include, but are not limited to, collagen, elastin, RGD-containing peptides (e.g., fibronectin), or laminin. In yet other embodiments, growth factors, cytokines, hormones, or other cellular signaling species may be provided within the coating material of the microfluidic device.

[0261] In some embodiments, the coating material may comprise a polymer containing two or more alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, saccharide moieties, nucleotide moieties, or amino acid moieties. In other embodiments, the prepared surface of the polymer may comprise a mixture of two or more polymers each having an alkylene oxide moiety, carboxylic acid moiety, sulfonic acid moiety, phosphate moiety, saccharide moiety, nucleotide moiety, and / or amino acid moieties, which may be incorporated into the coating material independently or simultaneously.

[0262] Furthermore, in embodiments in which a covalently modified surface is used in conjunction with a coating agent, the anions, cations, and / or amphoteric ions of the covalently modified surface may form ionic bonds with the charged portions of non-covalent coating agents (e.g., proteins in solution) present in the fluid culture medium (e.g., coating solution) within the enclosure.

[0263] Further details regarding appropriate coating and modification can be found in U.S. Patent Application Publication No. 15 / 135,707, filed on 22 April 2016, which is incorporated herein by reference in whole.

[0264] An additional system component for maintaining the viability of cells within an isolation pen for microfluidic devices. To promote the growth and / or proliferation of cell populations, additional components of the system may provide environmental conditions that promote the maintenance of functional cells. For example, such additional components may provide nutrients, cell growth signaling species, pH adjustment, gas exchange, temperature control, and removal of waste products from cells.

[0265] kit. A kit for providing a microfluidic device having at least one covalently modified surface configured to support the growth, survival and / or portability of living cells in various embodiments comprises a microfluidic device comprising a base, a cover and a microfluidic circuit material, an enclosure containing the microfluidic circuit material defining a fluid circuit, wherein at least one inner surface of the base, cover and microfluidic circuit material has a first covalent surface modification comprising a first binding group and a first portion which is a first surface contact portion or a first reactive portion; and at least one inner surface of the base, cover and microfluidic circuit material has a second covalent surface modification comprising a second binding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first binding group and the second binding group are different from each other, and / or the first part is different from the second part.

[0266] The first and second covalent surface modifications of the microfluidic device in the kit are represented by formulas XXX, V, VII, XXXI, VIII, and IX, respectively. [ka] It may have a structure independently selected from, where LG is -W-Si(OZ)2O- or -OP(O)2O-; L fm R is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0 or 1 bonding group CG; R x is the reactive part; W is O, S, or N, Z is a bond to an adjacent silicon atom or a bond to the surface; n is an integer from 3 to 21, L sm is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, 2, 3, or 4 bonding groups CG; and [ka] This is a surface. A kit containing any of the microfluidic devices described herein may be provided.

[0267] The kit is formula XII: RP-L-Surface contact part Formula XII A surface modification reagent may further comprise the following structure, where RP is the reaction pair portion; L is the linker; and the surface contact portion is the portion that provides improved contact properties to biological microorganisms. L is the linker, and L comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and comprises 0, 1, 2, or 3 bonding groups CG. The surface contact portion is as defined above. L and the surface contact portion may have any combination in the surface modification reagent. One embodiment In this embodiment, the surface contact portion may contain polyethylene glycol. In another embodiment, the surface contact portion may contain dextran. The reaction pair portion is configured to react with the reactive portion of the functionalized surface, respectively.

[0268] The kit is formula XXXIV: RP-L fm -R x2 Formula XXXIV A secondary functionalizing reagent having the structure of the formula, where RP is a reaction pair for reacting with the reactive part of formula XXX, formula V, or formula VII; and L fm R is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0 or 1 bonding group CG. x2 It is selected so as not to react with the reactive portion of the functionalized surface.

[0269] The kit may further contain other reagents to be used in the preparation of a microfluidic device having at least one covalently modified surface of formula VIII. A suitable reaction medium, buffer, or reaction accelerator may be provided in the kit. Auxiliary reagents and / or surface modification reagents and / or secondary functionalization reagents may be provided in separate containers.

[0270] Synthesis of the compound of formula IV. A method for synthesizing a compound having the structure of formula VI is provided, comprising the steps of reacting a compound of formula XIII with an azide ion and producing a compound of formula VI represented by formula 2 (wherein h is 1 to 19, n is 3 to 21, and R is H or C1-C6 alkyl). In one embodiment, n is an integer from 7 to 21.

[0271] formula 2 [ka] Azide ions may be provided as sodium azide or any other suitable source of azide ions. The reaction may be carried out in any suitable solvent such as acetonitrile or DMF. The reaction may be carried out at an ambient temperature which may range from about 15°C to about 30°C. In some embodiments, the ambient reaction temperature may range from about 20°C to about 30°C. In some embodiments, the reaction may be carried out at a temperature selected from 30°C, 40°C, 50°C, 60°C, or 70°C. The reaction may be carried out under an inert atmosphere.

[0272] In another embodiment, formula XIII: [ka] A compound having the structure of formula II is provided, where h is an integer from 1 to 19, and R is independently selected from the group consisting of H and C1-C6 alkyl groups. The compound of formula II may be a useful starting material for the synthesis of the compound having the structure of formula I. In one embodiment, h may be from 5 to 19. In other embodiments, h may be from 7 to 21, 8 to 21, 9 to 21, 10 to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to 21, or 16 to 21. In other embodiments, h may be from 7, 12, or 14. In one embodiment, h may be from 10, 12, or 14. In one embodiment, h may be from 12, or 14. In various embodiments, each of R may be from Me or Et.

[0273] Synthesis of the compound of formula XIII. A method for synthesizing a compound having the structure of formula XIII is provided, comprising the step of reacting an olefinic compound (compound 1) with a silane (compound 2) in the presence of a catalyst or initiator to produce a compound having the structure of formula XIII (see formula 3).

[0274] formula 3 [ka]

[0275] In compounds 1, 2, and XIII of formula 3, h is an integer from 1 to 19, and each of R is independently H or a C1-C6 alkyl group. In some embodiments, h is an integer from about 5 to 19. In some embodiments, R is a C1-C6 alkyl group.

[0276] In one embodiment, each of R may be selected from methyl, ethyl, and propyl. In another embodiment, each of R may be methyl. In various embodiments, h may be 7 to 19. In another embodiment, h may be 7, 12, or 14. In one embodiment, h may be 7.

[0277] In one embodiment, the catalyst can be any suitable hydrosilylation catalyst. The catalyst is a transition metal complex M(L) n The ligand may be a ligand, and M may be a metal such as Fe(O), Co(I), Rh(I), Ni(O), Pd(O), Pt(II), or Pt(O). In one embodiment, the metal of the hydrosilylation catalyst complex may be Co(I), Rh(I), Ni(O), Pt(II), or Pt(O). In yet another embodiment, the metal may be Rh(I), Pt(II), or Pt(O). The ligand may be any suitable ligand, selected to produce an electron-rich complex. Ligands may include halogens (e.g., chlorine); olefins, nitriles, siloxanes (including simple tetraalkyldivinylsiloxanes or restricted SILOP ligands); aromatic moieties 2,2'-bis( Examples of suitable hydrosilylation catalysts include diphenylphosphine)-sterically constrained biphenyls or binaphthyl ligands such as BINAP, BIPHEP, BINEPINE, or PHANEPHOS; 2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphine)butyl (DIOP); (some examples of suitable hydrosilylation catalysts, but not limited to, H2PtCl6·6H2O / iPrOH (Speyer catalyst); chloro(1,5-cyclooctadiene)Rh(1) dimer ([Rh(cod)Cl]2; tris(triphenylphosphine)-rhodium(I) Chloride; [PtCl2(NCR)2 (where R can be a cyclic amine such as N(alkyl)2, especially methyl or N-piperidinyl, Ph, Ch2Ph); Karlstedt catalyst Pt2{[(CH2=CH2)SiMe2]O}3); and bis(imino)pyridine iron dinitrogen complex ( et PDI)Fe(N2)]2(mu2-azide) is one example.

[0278] In one embodiment, the catalyst may be a platinum(0) catalyst. The platinum(0) catalyst is a Pt(0)-1,1,3,3-tetramethyl-1,3-divinyldisiloxane complex (compound 3): [ka] It is possible.

[0279] In other embodiments, an initiator may be used, which may be present in an amount ranging from about 0.5 equivalents to about 1.4 equivalents. In some embodiments, the initiator may be a trialkylborane.

[0280] The reaction may be carried out in any solvent capable of dissolving the olefinic compound (compound 1 in formula 2), and the solvent is not limited to, but includes DMF, benzene, tetrahydrofuran, toluene, and fluorinated or partially fluorinated solvents, particularly 1,3-bistrifluoromethylbenzene. In some embodiments, toluene or dimethylformamide (DMF) may be used.

[0281] The reaction can be carried out under an inert atmosphere, which may be argon or nitrogen gas. Typically, the inert atmosphere will exclude water vapor.

[0282] High temperatures can be used to accelerate the reaction, and the reaction may take place at temperatures ranging from about 60°C to about 110°C. In one embodiment, the reaction may take place at about 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, or about 110°C. The reaction may be completed at about 6 hours, 10 hours, 14 hours, 18 hours, 24 hours, 30 hours, 48 ​​hours, 60 hours, or at any point in between.

[0283] In another embodiment, formula IV: [ka] A method for synthesizing a compound having the structure of formula XIV: [ka] A method may be provided comprising the step of reacting a compound having the structure with an alcohol ROH (wherein h is an integer from 1 to 19; each of X is Cl; ROH is methyl alcohol, ethyl alcohol, or propyl alcohol) to produce a compound of formula I (wherein h is an integer from 1 to 19, and R is a C1-C3 alkyl group). In some embodiments, the base may be pyridine. In some embodiments, h may be an integer from 5 to 19. In various embodiments, h may be 7, 12, or 14. In yet another embodiment, R may be methyl.

[0284] Synthesis of the compound of formula L. A method for synthesizing a compound having the structure of formula L is provided, comprising the step of reacting an olefinic compound (compound 1) with a silane (compound 2) in the presence of a catalyst or initiator to produce a compound having the structure of formula L (see formula 1).

[0285] formula 4 [ka]

[0286] In compounds 101, 1032 and L of formula 4, n can be an integer from 13 to 25; each of Y can independently be a halo, OH, or OR, where the halo is Br, Cl, or F; and R is a C1-C6 alkyl group. In one embodiment, each of Y is Cl. In other embodiments, each of Y can be methoxy, ethoxy, or propoxy. In various embodiments, n can be 13, 15, 17, or 19. In one embodiment, n can be 13 or 15.

[0287] In one embodiment, the compound of formula L may be a compound having the structure of formula LI, which may be produced according to the method shown in formula 5. An olefinic compound (compound 1) may be reacted with a silane (compound 3) having three substituents OR, where R may be H or a C1 to C6 alkyl group. In one embodiment, each of R may be selected from methyl, ethyl, and propyl. In another embodiment, each of R may be methyl. In various embodiments, n may be 13, 15, 17, or 19. In one embodiment, n may be 13 or 15.

[0288] formula 5 [ka]

[0289] In other embodiments, the compound of formula L may be a compound having the structure of formula LII, which can be produced according to the method shown in formula 6.

[0290] formula 6 [ka]

[0291] The olefinic compound, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-nonacosafluorohexadeca-1-ene (compound 105), can be reacted with trialkoxysilane (compound 104) in the presence of a catalyst or initiator to produce a molecule of formula LII. The catalyst can be any suitable hydrosilylation catalyst.

[0292] Synthesis of the compound of formula VI. The compound of formula VI can be synthesized by various routes, one of which may involve reacting the compound of formula IV with a metal acetylide. [Examples]

[0293] Examples Systems and microfluidic devices: Manufactured by Berkeley Lights, Inc. An OptoSelect chip, a nanofluidic device controlled by an optical instrument. The instrument included a mounting base for the chip connected to a temperature controller; a pump and fluid medium adjustment components; and an optical array including a camera and a structured light source suitable for activating the phototransistor within the chip. The OptoSelect® chip is constructed using OptoElectroPositioning® (OEP®) technology, which provides OET force activated by the phototransistor. It included a substrate. Each chip had multiple NanoPen® chambers (or spacing). The isolation pen also contained multiple microfluidic channels that were fluidically connected. The volume of each isolation pen was approximately 1 × 10⁻⁶ 6 It was cubic μm.

[0294] Priming solution: 0.1% Pluronic(registered trademark) F127 ((Life Technologies(registered trademark) catalog number) A complete growth medium containing (p. 6866).

[0295] Preparation for culture: A microfluidic device with a modified surface was filled into the system and purged with 100% carbon dioxide at 15 psi for 5 minutes. Immediately after the carbon dioxide purge, the priming solution was perfused through the microfluidic device at 5 microliters / second for 8 minutes. Next, the culture medium was flowed through the microfluidic device at 5 microliters / second for 5 minutes.

[0296] Priming plan. 250 microliters of 100% carbon dioxide were flowed at a rate of 12 microliters / second. Following this, 250 microliters of PBS containing 0.1% Pluronic® F27 (Life Technologies® catalog number P6866) were flowed at a rate of 12 microliters / second. The flow rate was 100ml / second. The final priming step involved flowing 250 microliters of PBS at 12 microliters / second. The introduction of the culture medium was as follows:

[0297] The Kanryo Project. The perfusion method was one of the following two methods: The mixture was perfused at 0.01 microliters / second for 1.2 hours, then at 2 microliters / second for 64 seconds, and this was repeated. 2. The mixture was perfused at 0.02 microliters / second for 100 seconds; the flow was stopped for 500 seconds; then perfused at 2 microliters / second for 64 seconds; and this was repeated.

[0298] Example 1. Synthesis of (11-bromoundecyl)trimethoxysilane (compound 4). 1.26g (5.4 mmol) of 11-bromoundeca-1-ene (Sigma Aldrich) Soluble the mixture in 150 ml of dry toluene (Oakwood Products) in a reaction vessel flushed with argon and equipped with a reflux condenser. Add trimethoxysilane (1.77 g, 14.5 mmol, Sigma Aldrich catalog no. 282626) through the septum using a syringe. The reaction is carried out under argon purging. Next, 1.5 g of the hydrosilylation catalyst solution and (0.08 mmol) of the hydrosilylation catalyst platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex (compound 3, 0.1 M in poly(dimethylsiloxane), Sigma Aldrich, catalog no. 479527) are added via syringe under argon purging. Add to [the compound]. Next, the reaction is continued for 24 hours at a temperature of 80°C under an argon atmosphere to produce (11-bromoundecyl)trimethoxysilane (compound 4). The reactants are cooled to room temperature under argon, filtered, and the product is extracted in pentane. The solvent is removed by rotary evaporation under reduced pressure. The product is purified by vacuum distillation.

[0299] Example 2. Synthesis of (11-azidowndecyl)trimethoxysilane (compound 5). (13-11-azidowndecyltrimethoxysilane) was synthesized from 11-bromoundecyltrimethoxysilane (Gelest) by substituting the bromide with sodium azide. In a typical reaction, 4.00 g of 11-bromoundecyltrimethoxysilane (Gelest catalog number SIB1908.0) was added to a solution containing 2.00 g of sodium azide (Sigma-Aldrich) in 60 mL of dry dimethylformamide (Acros). The solution was stirred under nitrogen at room temperature for 24 hours. The solution was then filtered, and the filtrate was extracted with dry pentane (Acros). The crude 11-azidowndecyltrimethoxysilane product was subjected to rotary evaporation. Therefore, the sample was concentrated, purified by two consecutive vacuum distillations, and characterized using NMR and FTIR spectroscopy.

[0300] Example 3. Preparation of a functionalized surface of a silicon wafer. A silicon wafer (780 μm thick, 1 cm × 1 cm) was treated in an oxygen plasma cleaner (Nordson Asymtek) for 5 minutes using 100 W of power, a pressure of 240 mTorr, and an oxygen flow rate of 440 sccm. A separate foil box at the bottom of the vacuum reactor... As a source of water reactant in the mixture, magnesium sulfate heptahydrate (0.5g, Acros catalog number) is used. In the presence of (10034-99-8), plasma-treated silicon wafers were treated in a vacuum reactor with (11-azidondecyl)trimethoxysilane (compound 5, 300 microliters) in a foil boat at the bottom of the vacuum reactor. The chamber was then pumped to 750 mTorr using a vacuum pump and then sealed. The vacuum reactor was placed in an oven heated to 110°C for 24-48 hours. This introduced a modified surface to the wafer, and the functionalized surface was given by formula XV: [ka] It has the structure, where Z is a bond to an adjacent silicon atom bonded to the surface, or a bond to the surface. [ka] This is the surface. After cooling to room temperature and introducing argon into a vacuum chamber, the wafer was removed from the reactor. The wafer was rinsed with acetone and isopropanol and dried under a nitrogen stream. The introduction of the functionalized surface was confirmed by polarization analysis and contact angle measurement.

[0301] Example 4. Modification of microfluidic circuit materials. An example of a microfluidic circuit material is photopatternable silicone, which was used to define fluid circuits in microfluidic devices. Evidence of modification of this material was obtained. ITO wafers on which photopatternable silicone structures were incorporated were placed in an oxygen plasma cleaner (Nordson Asymtek) for 5 minutes using 100 W of power, a pressure of 240 mTorr, and an oxygen flow rate of 440 sccm. Plasma-cleaned photopatterned An ITO wafer was processed as described in Example 3 to introduce a modified surface of formula XV onto a photopatterned silicon. The FTIR ATR (attenuated total internal reflection) spectrum was measured using a ThermoFisher Nicolet iS50 spectrometer with a liquid nitrogen-cooled MCT detector. By pressing the light-patterned silicone, the Harrick Vari-GATR auxiliary equipment is used. Spectra were collected, and the surface of the germanium crystal was modified with a surface of formula XV using a force of 200 N. When the modified photopatternable silicone material was pressed against the germanium crystal, an FTIR ATR of only the modified photopatternable silicone was obtained. 250 scans were collected at a resolution of 4 cm⁻¹ and referenced to the background spectrum of the bare germanium crystal. The spectrum was then measured using an Omnic software equipped with an FTIR spectrometer. Visualization was performed using software.

[0302] As shown in Figure 4, the peak at approximately 2098 cm⁻¹(410) is due to azid asymmetric stretching. The peaks at 2924 cm⁻¹(414) and 2854 cm⁻¹(412) are due to CH stretching modes.

[0303] Note: In the following examples of introducing modified surfaces to microfluidic devices, contact angle and thickness measurements were performed on silicon wafers modified in the same way as the specific modified surface in the microfluidic device.

[0304] Example 5. Fabrication of a microfluidic device having a modified inner surface of formula XV. An OptoSelect device, having a first silicon electrode activated substrate and a second ITO substrate on opposing walls, and a photo-patterned silicone microfluidic circuit material separating the two substrates, was treated in an oxygen plasma cleaner (Nordson Asymtek) for 1 minute using 100 W of power, a pressure of 240 mTorr, and an oxygen flow rate of 440 sccm. (Bottom of vacuum reactor) As a water reagent source in a separate foil boat in the section, magnesium sulfate heptahydrate (0.5g) In the presence of Acros, 3-azidundine was found in a foil boat at the bottom of the vacuum reactor. A plasma-treated microfluidic device was treated with sil)trimethoxysilane (compound 5, 300 microliters) in a vacuum reactor. The chamber was then pumped to 750 mTorr using a vacuum pump and then sealed. The vacuum reactor was placed in an oven heated to 110°C for 24–48 hours. This introduced a functionalized surface to all opposing inner surfaces of the microfluidic device, and the functionalized surface was conditioned by formula XV: [ka] It has the structure, where Z is a bond to an adjacent silicon atom bonded to the surface, or a bond to the surface. [ka] This is the surface. After cooling to room temperature and introducing argon into the vacuum chamber, the microfluidic device was removed from the reactor. Next, the microfluidic device having the functionalized surface was treated with an alkynyl species to introduce the desired modified surface, as described later in Examples 6 and 7.

[0305] Example 6. Introduction of polyethylene glycol (PEG) modified surface (formula XVI) into a microfluidic device. Materials: Alkyne-modified PEG (j=MW approx. 5000 Da) (Compound 6) was purchased from JenKem and used in its original state. [ka] I purchased sodium ascorbate and copper sulfate pentahydrate from Sigma-Aldrich and obtained them as is. It was used in its original state. (3[Tris(3-hydroxypropyltriazolylmethyl)amine) THPTA rate-accelerating ligand (Glen Research) was used in its original state.

[0306] At least 250 microliters of an aqueous solution containing 3.3 mmol of alkyne-modified PEG, 50 mmol of copper sulfate, 55 mmol of THPTA ligand, and 100 mmol of sodium ascorbate were passed through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand, thereby reacting the microfluidic device, a product from Example 5 having a surface of formula XV as described above, with alkyne-modified PEG (compound 5). The reaction was allowed to proceed at room temperature for at least 1 hour. Next, formula XVI: [ka] (In the formula, Z is defined above for formula VIII, [ka] (This is the surface.) Microfluidic devices having PEG-modified surfaces were rinsed by passing at least 250 microliters of deionized water through the devices. After completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.4 nm (functionalized surface thickness) to 5 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 35° (surface of formula XVI).

[0307] Example 7. Introduction of a dextran-modified surface (formula XVII) into a microfluidic device. By flowing at least 250 microliters of an aqueous solution containing 1.66 mmol of DBCO-dextran through a microfluidic device having a surface-modified azide ligand after deposition, a microfluidic device, the product from Example 5, having a surface of formula XV as described above, is obtained, containing dibenzylcyclooctinyl (DBCO)-modified dextran, weight-average molecular weight 3000 Da (compound 7, Nanocs): [ka] The reaction was carried out at room temperature for at least one hour. Next, formula XVII (one of the two positional isomers is shown): [ka] (In the formula, Z is defined above for formula VIII, [ka] (This is the surface.) The microfluidic device having the modified surface was rinsed by passing at least 250 microliters of deionized water through the tip.

[0308] Example 8. Alternative introduction of polyethylene glycol (PEG) modified surface (formula XVIII) into a microfluidic device. The microfluidic device, a product from Example 5 having the surface of formula XV as described above, was treated with dibenzylcyclooctinyl (DBCO) modified PEG, weight-average molecular weight 5000 Da (compound 8, Broadpharm, catalog no. BP-22461), by passing at least 250 microliters of aqueous solution containing 1.33 mmol of DBCO-PEG through the microfluidic device having a surface-modified azide ligand after deposition. The reaction was allowed to proceed at 40°C for at least 1 hour. Next, the microfluidic device having the modified surface of formula XVIII was rinsed by passing at least 250 microliters of deionized water through the tip. One of the two positional isomers is shown. [ka]

[0309] Example 9. Introduction of a poly-L-glutamic acid (PGA) modified surface (formula XIX) into a microfluidic device. By passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of alkyne-modified PGA, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand and 5 mmol of sodium ascorbate through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand, the microfluidic device, which is the product from Example 5 and has a surface of formula XV as described above, is obtained by flowing the solution through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand. 15,000 Da) (Compound 8, Alamanda® polymer, catalog number AK-PLE100): [ka] The reaction was carried out at room temperature for at least 1 hour or at 40°C for at least 15 minutes. Next, the microfluidic device having the PGA modified surface of formula XIV was rinsed by flowing at least 250 microliters of deionized water through the device. After completion of the cyclization reaction introducing the modified surface, the thickness of the layer increased from 1.1 nm (functionalized surface thickness) to 5.2 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula X) to 17° (surface of formula XIX). [ka]

[0310] Example 10. Introduction of a covalently modified surface of a biotin-functionalized PEG surface (formula XX) into a microfluidic device. By flowing at least 250 microliters of an aqueous solution containing 31.33 mmol of compound 9, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand (formula XV), the microfluidic device, which is the product from Example 5 and has the surface of formula XV as described above, is obtained: biotin-functionalized alkynyl PEG (PEG is 1 kDA, compound 9, Nanocs, catalog no. PG2-AKBN-1k): [ka] The reaction was carried out at room temperature for at least one hour. Next, formula XX [ka] Microfluidic devices having biotinylated PEG-modified surfaces were rinsed by passing at least 250 microliters of deionized water through the devices. After completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.4 nm (functionalized surface thickness) to 5 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 39° (surface of formula XX).

[0311] Example 11. Introduction of a covalently modified surface of a photocleavable biotin-functionalized PEG surface (formula XXI) into a microfluidic device. At least 250 microliters of an aqueous solution containing 1.33 mmol of compound 10, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate were passed through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand, while the microfluidic device, the product from Example 5 having a surface of formula XV as described above, was reacted with biotin-functionalized photocleavable alkyne PEG3 (compound 10, Broadpharm, catalog no. BP-22677, containing a photocleavable nitro-substituted phenyl group as the linker L portion). The reaction was allowed to proceed at room temperature for at least 1 hour. Next, formula XX1: [ka] Microfluidic devices having biotinylated PEG-modified surfaces were rinsed by passing at least 250 microliters of deionized water through the devices. After completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.4 nm (functionalized surface thickness) to approximately 5 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 42° (surface of formula XXI).

[0312] Example 12. Introduction of a propiolic acid-modified surface (formula XXII) into a microfluidic device. By passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of propiolic acid, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through a microfluidic device having an 11-azidoneundecylsiloxy surface-modified ligand, the microfluidic device, which is the product from Example 5 and has a surface of formula XV as described above, is obtained by flowing the product through a microfluidic device having an 11-azidoneundecylsiloxy surface-modified ligand. The reaction was carried out at room temperature for at least 1 hour or less. Both reactions were carried out at 40°C for 15 minutes. Next, the microfluidic devices with the carboxylic acid-modified surface of formula XXII were rinsed by passing at least 250 microliters of deionized water through the devices. After the completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.1 nm (functionalized surface thickness) to 2 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 64° (surface of formula XXII). [ka]

[0313] Example 13. Introduction of an amine-modified surface (formula XXIII) into a microfluidic device. By passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of propargylamine, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand, the microfluidic device, which is the product from Example 5 and has a surface of formula XV as described above, is obtained by flowing the solution through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand. The reaction was carried out with (catalog number P50900-5G). The reaction was allowed to proceed at room temperature for at least 1 hour or at 40°C for at least 15 minutes. Next, the microfluidic device having the amine-modified surface of formula XXIII was rinsed by passing at least 250 microliters of deionized water through the device. [ka]

[0314] Example 14. Introduction of a PEG carboxylic acid-modified surface (formula XXIV) into a microfluidic device. The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was reacted with alkyne PEG acid (PEG(f=5000Da, compound 11) Nanocs, catalog no. PG2-AKCA-5k) by passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of compound 11, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through the microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand. The reaction was allowed to proceed at room temperature for at least 1 hour or at 40°C for at least 15 minutes. Next, the microfluidic device having a carboxylic acid-modified surface of formula XXIV was rinsed by passing at least 250 microliters of deionized water through the device. After completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.1 nm (functionalized surface thickness) to 5 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 48° (surface of formula XXIV). [ka]

[0315] Example 15. Introduction of a polylysine-modified surface (formula XXV) into a microfluidic device. The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was reacted with a poly(lysine hydrobromide) graft-(4-pentinamide, compound 12, PLKB100-g-AK20 Alamanda polymer, catalog no. PLKB100-g-AK20, 100 lysine repeating units, 20% alkynylated, MW 21,000 Da) by passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of compound 12, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through a microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand. The reaction was allowed to proceed at room temperature for at least 1 hour or at 40°C for at least 15 minutes. Next, the microfluidic device having the amine-modified surface of formula XXV was rinsed by passing at least 250 microliters of deionized water through the device. After completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.1 nm (functionalized surface thickness) to approximately 3 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 50° (surface of formula XXV). [ka]

[0316] Example 16. Introduction of a polyglutamate-modified surface (formula XXVI) into a microfluidic device. The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was reacted with poly(glutamic acid) graft-(N-propargyl), compound 13, Alamanda polymer, catalog no. PLE100-g-AK20, 20% alkynylated, 100 glutamic acid repeats, MW 15,000 Da) by passing at least 250 microliters of buffered saline solution (5.4 × PBS pH 7.4) containing 1.33 mmol of compound 13, 500 micromoles of copper sulfate, 550 micromoles of THPTA ligand, and 5 mmol of sodium ascorbate through the microfluidic device having an 11-azid-undecylsiloxy surface-modified ligand. The reaction was allowed to proceed at room temperature for at least 1 hour or at 40°C for at least 15 minutes. The microfluidic device having a carboxylic acid-modified surface of formula XXVI was then rinsed by passing at least 250 microliters of deionized water through the device. After the completion of the cyclization reaction that introduces the modified surface, the layer thickness changes from 1.1 nm (functionalized surface thickness) to approximately 3 nm. The thickness was increased to nm. Furthermore, the droplet water contact angle decreased from approximately 80° (on the functionalized surface of formula XV) to 54° (on the surface of formula XXVI). [ka]

[0317] Example 17. Introduction of a biotinylated polyethylene glycol (PEG) modified surface to a microfluidic device using a disulfide-cleavable linker (formula XXVII). The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was treated with dibenzylcyclooctinyl (DBCO)SS biotin-modified PEG3 (compound 14, Broadpharm, catalog no. BP-22453) by passing at least 250 microliters of an aqueous solution containing 1.33 micromoles of compound 14 through the microfluidic device having a surface-modified azide ligand after deposition. The reaction was allowed to proceed at 40°C for at least 1 hour. Next, the microfluidic device having the modified surface of formula XXVII was rinsed by passing at least 250 microliters of deionized water through the tip. One of the two positional isomers is shown. [ka] After the completion of the cyclization reaction introducing the modified surface, the layer thickness increased from 1.1 nm (functionalized surface thickness) to approximately 2 nm. Furthermore, the droplet-water contact angle decreased from approximately 80° (functionalized surface of formula XV) to 66° (surface of formula XXVII).

[0318] Example 18. Introduction of a PEG5 carboxylic acid modified surface (formula XXVIII) into a microfluidic device. The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was treated with dibenzylcyclooctinyl (DBCO)-PEG5-acid (compound 15, Broadpharm, catalog no. BP-22449) by passing at least 250 microliters of an aqueous solution containing 1.33 micromoles of compound 15 through the microfluidic device having a surface-modified azide ligand after deposition. The reaction was allowed to proceed at 40°C for at least 1 hour. The microfluidic device having the modified surface of formula XXVII was then rinsed by passing at least 250 microliters of deionized water through the tip. The contact angle was measured at 47° and the thickness was 17.8 angstroms, which were measured as described herein, respectively. One of the two positional isomers This is shown. [ka]

[0319] Example 19. Introduction of a PEG3-modified surface (formula XXIX) into a microfluidic device. A microfluidic device (Berkeley Lights, Inc.) having a first silicon electrode activation substrate and a second ITO substrate on opposing walls, and a photo-patterned silicon microfluidic circuit material separating the two substrates, was subjected to a power of 100W, a pressure of 240mTorr, and 440sccm. Using this oxygen flow rate, the oxygen plasma cleaner (Nordson Asymtek) was used for 1 minute. The reaction was carried out in the presence of magnesium sulfate heptahydrate (0.5 g, Acros) as the water reagent source in a separate foil boat at the bottom of the vacuum reactor. In this process, a plasma-treated microfluidic device was treated in a vacuum reactor with methoxytriethyleneoxypropyltrimethoxysilane (compound 16, Gelest catalog number SIM6493.4, 300 microliters). The chamber was then pumped to 750 mTorr using a vacuum pump and subsequently sealed. The vacuum reactor was placed in an oven heated to 110°C for 24–48 hours. This introduced a functionalized surface to all opposing inner surfaces of the microfluidic device, which is then treated with formula XXIX: [ka] It has the structure, where Z is a bond to an adjacent silicon atom bonded to the surface, or a bond to the surface. [ka] This is the surface. After cooling to room temperature and introducing argon into the vacuum chamber, the microfluidic device was removed from the reactor. The contact angle for this surface was measured to be 55°, and the average thickness was measured to be 10.2 angstroms.

[0320] Example 20. Preparation of a phosphonate-bonded surface (formula XXXVI). A silicon chip (780 μm thick, 1 cm × 1 cm) was pretreated as described above for Example 3, and then treated with octadecylphosphonic acid (compound 17, Sigma Aldrich catalog number 715166) in the same manner as in Example 19, to obtain formula XXXVI (wherein Z is adjacent to The bond is to the phosphorus atom in the bonding group LG, or on the surface. [ka] A covalently modified surface (with a bond to) was obtained. The contact angle was measured to be 110°. [ka]

[0321] Example 21. Introduction of streptavidin-modified surface (formula XXXVII or formula XXXVIII) into a microfluidic device. Method A. The microfluidic device, a product from Example 5 having a surface of formula XV as described above, was treated with dibenzylcyclooctinyl (DBCO) streptavidin (SAV) compound 18 (Nanocs, catalog no. SV1-DB-1, where there are 2 to 7 DBCOs for each molecule of SAV) by passing at least 250 microliters of an aqueous solution containing 2 micromoles of compound 18 through the microfluidic device having a surface-modified azide ligand after deposition. The reaction was allowed to proceed at room temperature for at least 1 hour. Next, the microfluidic device having the modified surface of formula XXVII was rinsed by passing at least 250 microliters of 1×PBS through the device. [ka]

[0322] Method B. The modified surface, a product of the microfluidic device of Example 17 having a surface of formula XXVII, was washed with water and dried by repeated flashing of gaseous carbon dioxide while heating the tip to 40°C. A solution of 2 micromoles of SAV in 1×PBS (ThermoFisher catalog no. 434301) was flowed into the microfluidic device and contacted with the biotinylated surface for 30 minutes. Excess SAV was removed by flowing 1×PBS through the microfluidic device to obtain a surface of formula XXXVIII: [ka] The surface was obtained.

[0323] Example 22. Introduction of a fibronectin surface (formula XXXIX) into a microfluidic device. Method A. Example 21, a microfluidic device having a modified surface of formula XXXVIII, which is a product of Method B, is charged with 46 nM biotinylated bovine fibronectin (randomly biotinylated, Cytoskeleton Inc., catalog no. FNR03A, FNR03) in 1 × PBS. -B) is treated with 50 microliters of solution, incubated at 37°C for 1 hour, and formula XXXXIX: [ka] The surface of fibronectin was obtained.

[0324] Modification in the presence of living cells. In one embodiment, living cells were introduced into a microfluidic device having a surface of formula XXXVIII, and streptavidin was presented in the fluid region of the microfluidic device. After the cells were taken up into an isolation pen, biotinylated fibronectin was introduced into PBS and incubated for 1 hour. Adhesion was observed.

[0325] Method B. A microfluidic device having the surface of formula XXXVII described above is treated with biotinylated fibronectin to introduce a fibronectin surface.

[0326] Method C. A biotinylated fibronectin stock was prepared in 2.3 micromoles in PBS, and a streptavidin stock was prepared in 19.2 micromoles in PBS. These two stocks were mixed in a fibronectin to streptavidin ratio of 1:1 to 1:2 and diluted in 1×PBS to a final concentration of at least 300 nanomoles. This solution was incubated at room temperature for 15 minutes to allow the binding of fibronectin and streptavidin to form a surface-modified reagent with a biotin / streptavidin binding group CG.

[0327] The modified surface, which is the product of the microfluidic device of Example 17 having a surface of formula XXVII, was washed with water and dried by repeated flashing of gaseous carbon dioxide while heating the tip to 40°C. The surface modification reagent was poured into a microfluidic device. The device was incubated at room temperature for at least 30 minutes to obtain the modified surface of formula XXXIX.

[0328] Further generalization. Furthermore, by flowing biotinylated proteins, peptides, small molecules, or recognition motifs through the same process, various bio-related molecules can be introduced to the modified surface of a microfluidic device by binding to either the surface of formula XXXVII or formula XXXVIII. For example, biotinylated laminin can be flowed into a microfluidic device prepared as described above, having a surface of formula XXXVII or XXXVIII, to create a modified surface (formula XL) with a laminin surface contact portion: [ka] Generates.

[0329] Example 23. Introduction of a mixed surface of formula XLI in various ratios. A silicon wafer was subjected to an oxygen plasma cleaner (Nordson Asymtek) for 1 minute using 100W of power, a pressure of 240mTorr, and an oxygen flow rate of 440sccm. Processed. In the presence of magnesium sulfate heptahydrate (0.5 g, Acros) as a water reagent source in a separate foil boat at the bottom of the vacuum reactor, In this case, 3-azidundecyl)trimethoxysilane (compound 5, prepared as described above) and methoxytriethyleneoxypropyltrimethoxysilane (Gelest Inc.) A plasma-treated microfluidic device was processed in a vacuum reactor with a mixture of log number SIM6493.4 (having a molecular weight similar to compound 5 in various proportions). The chamber was then pumped to 750 mTorr using a vacuum pump and then sealed. The vacuum reactor was placed in an oven heated to 110°C for 24–48 hours.

[0330] After cooling to room temperature and introducing argon into the vacuum chamber, the wafer was removed from the reactor. The wafer was rinsed with acetone and isopropanol and dried under a stream of nitrogen. The mixture is given by formula XLI (wherein x and y are 1 to 1 × 10⁻⁶). 8 Modified surfaces (which may exist in any x:y or y:x ratio) were evaluated for thickness, contact angle, and the presence of azides in FTIR of the surface. Individual wafers were modified with mixtures of 1% undecyl azide:99% methoxyPEG3; 10% undecyl azide:90% methoxyPEG3; 50% undecyl azide:50% methoxyPEG3; and 100% undecyl azide. [ka]

[0331] As shown in Figure 5A, the overlay FTIR trace clearly showed an amount of azide asymmetric stretching 510 decreasing at approximately 2098 cm⁻¹. Figure 5B shows an enlarged portion of the overlay trace at the location of azide asymmetric stretching for wafers having 10% of formula XV and 1% of formula XV, respectively. The relative amounts of azide were clearly distinguishable and correlated with the ratio of formula XV:formula XXIX used.

[0332] As shown in Table 2, when different ratios of the surface of formula XV:formula XXIX are present on the modified surface, the contact angle and thickness of the modified surface also differ. The data indicate that adhesion control was achieved by changing the material ratio during the chemical vapor deposition process. The change in contact angle also indicates that different performance was possible with these surface modifications of different ratios.

[0333] [Table 2]

[0334] Example 24. Introduction of a modified surface (formula XLII) having a mixture of a first surface modification containing PEG and a second surface modification of a block copolymer containing poly-L-lysine, using a combination of surface modification reagents. As described above, the microfluidic device, a product from Example 5 having a surface of formula XV, was treated with copper sulfate (excess), THPTA ligand, and sodium ascorbate in a solution containing 1.3 mmol of propargyl-PEG1-disulfide-PEG1-propargyl (compound 19, BroadPharm Inc. catalog no. BP-23283). The excess copper sulfate prevented disulfide cleavage by ascorbate during the reaction, and the reaction was carried out at 40°C for approximately 15 minutes (alternatively, it could be carried out at room temperature for approximately 1 hour). After the incubation period was complete, excess reagents and byproducts were removed by flushing with water. The inside of the microfluidic device was dried by flushing with carbon dioxide gas while heating the microfluidic device to 40°C, and the alkynyl R x2A surface was obtained that is a secondary functionalized surface having a portion.

[0335] Next, by treating the microfluidic device with a mixture of two surface modification reagents, Alkinyl R x2 Microfluidic devices having a secondary functionalized surface with a portion were further modified. The surface modification reagent was PEG-azide (5 kDa, Aldrich Chemicals, catalog number 6). 89475): A mixture of azide-PEG5k-block copolymer poly-l-lysine 100 (Alamanda polymer, MW 1600) was poured into a microfluidic device at a concentration of 1.3 mmol, where the ratio of azide-PEG to azide-PEG5k-b-PLL varied between 1:50000 and 50000:1 with copper sulfate (excess), THPTA ligand, and sodium ascorbate. The excess copper sulfate was absorbed by the ascorbate during the reaction. To prevent disulfide cleavage, the reaction was carried out at 40°C for approximately 15 minutes (alternatively, it could be carried out at room temperature for approximately 1 hour). After the incubation period was complete, excess reagents and by-products were removed by flushing with water. The inside of the microfluidic device was dried by flushing with carbon dioxide gas while heating the microfluidic device to 40°C to provide the microfluidic device with a mixture of the hydrophilic first surface modification, PEG5K, and the second surface modification, PEG5k-b-PLL, where the PLL block provided a positive charge (Equation XLII). The ratio of azide-PEG to azide-PEG5k-b-PLL can be even higher, for example, 10,000:1 or more, and it was demonstrated that adhesion was observed even with a very low percentage of the block copolymer poly-L-lysine surface contact portion. [ka]

[0336] The modified surface of formula XLII is 1 to 1 × 10 8 It is possible to have x and y existing in any value in the ratio x:y or y:x.

[0337] An alternative method of modification. By passing at least 250 microliters of an aqueous solution containing 1.0 mmol of DBCO-PEG through a microfluidic device having a surface of formula XV, the microfluidic device having a surface of formula XV may be modified with DBCO-PEG4-alkyne (compound 20, Conju-Probes, Inc. catalog no. CP-2039) instead of propargyl-PEG1-disulfide-PEG1-propargyl (compound 19). The reaction was allowed to proceed at 40°C for at least 1 hour. Next, the microfluidic device having a modified surface of formula XVIII can be rinsed by passing at least 250 microliters of deionized water through a tip and treated as described in the previous paragraph to provide the microfluidic device with a mixture of a hydrophilic first surface modification, PEG5K, and a second surface modification, PEG5k-b-PLL, where the PLL block provides a positive charge (formula XLIII), and the linker portion of the surface modification differs from that of formula XLII. [ka]

[0338] The modified surface of formula XLIII is 1 to 1 × 10 8 It is possible to have x and y existing in any value in the ratio x:y or y:x.

[0339] It is useful for culturing adherent cells. Either surface of formula XLII or XLIII was useful, but not limited to, for providing fixed points (e.g., clusters of positively charged poly-L-lysine provided within the block copolymer) for culturing adherent cells such as HeLa cells. HeLa cells were observed to flatten, grow, and proliferate during culture on either of these surfaces (data not shown).

[0340] Example 25. Introduction of a modified surface (formula XLIV) having a mixture of a first surface modification containing PEG and a second surface modification of poly-L-lysine, using a combination of surface modification reagents. As described above, the microfluidic device, a product from Example 5 having a surface of formula XV, was combined with copper sulfate (excess), THPTA ligand and sodium ascorbate, along with alkyne-poly-L-lysine HBr salt (100 units, Alamanda polymer) and alkyne-modified PEG (j=MW approx. 5000 Da, compound 6, JenKem Technologies) 1: The mixture was treated with a 1.33 mmol solution containing a 1 stoichiometric mixture. Excess copper sulfate prevented disulfide cleavage by ascorbate during the reaction, and the reaction was carried out at 40°C for approximately 15 minutes (alternatively, it could be carried out at room temperature for approximately 1 hour). After the incubation period was complete, excess reagents and by-products were removed by flushing with water. The inside of the microfluidic device was dried by flushing with carbon dioxide gas while heating the microfluidic device to 40°C to provide the microfluidic device with a mixture of the hydrophilic first surface modification, PEG5K, and the second surface modification, poly-L-lysine, which provides a positive charge (formula XLIV). [ka]

[0341] On the surface of formula XLIV, ( * ) is a linker protected by ownership, and x and y are 1 to 1 × 10 8 It can exist in any ratio of x:y or y:x for any value of x.

[0342] It is useful for culturing adherent cells. The surface of formula XLIV has been useful, but is not limited to, providing fixed points (e.g., clusters of positively charged poly-L-lysine provided within the block copolymer) for culturing adherent cells such as HeLa cells. HeLa cells were observed to flatten, grow, and proliferate during culture on any of these surfaces (data not shown).

[0343] Titration of surface modification 1:surface modification 2. The ratio of the first surface modification (PEG 5kDa) and the second surface modification (poly-L-lysine) in formula XLIV was modified to a ratio of 99PEG 5kDa:1 poly-L-lysine to adjust the number of points designed to promote adhesion. Using a 1% level charged second surface modification (poly-L-lysine), laser bubble initiation of displacement was performed, followed by dielectrophoresis of the cells. Removal was observed (data is not shown).

[0344] A microfluidic device having an inner surface modified with a first surface modification of PEG 5kDa and a second surface modification of poly-L-lysine with a percentage of approximately 0.00001% or 0.000001% is expected to enable adhesion of adherent cells (such as HeLa cells) while still allowing the extraction of cultured cells using dielectrophoretic force without laser-induced replacement.

[0345] Example 26. Introduction of a mixed surface using branched chain PEG linker (formula XLV). A modified surface having a first cleavable biotinylated surface modification combined with a second surface modification of hydrophilic PEG was introduced using a highly branched PEG alkyne moiety. The amount of biotinylated moiety present was controlled by adjusting the amount of biotinylated moiety relative to the hydrophilic surface contact portion. The modification was performed by bonding portions containing each of the surface contact portions to the branches of the highly branched PEG alkyne, while leaving sufficient alkyneylated moiety on the highly branched PEG to efficiently modify the surface of the microfluidic device. The procedure is described for a 1:1 ratio of biotin moiety to PEG carboxylic acid moiety, but experiments were also performed with 100% biotin moiety; 10% biotin to 90% PEG carboxylic acid; and 1% biotin moiety to 99% PEG carboxylic acid moiety. [ka]

[0346] 1.3 mmol 4-branch PEG (Creative PEGWorks catalog number PSB-495) A 1:1 ratio solution of 1.3 mmol of azido-disulfide-biotin (compound 20, BroadPharm catalog no. BP-22877) and azido-PEG6-carboxylic acid (BroadPharm catalog no. BP-20612) in aqueous solution was reacted with sodium ascorbate in the presence of 2 times excess copper sulfate, and a di-modified polybranched PEG was formed by incubation at room temperature for approximately 30 minutes. The solution of the di-modified polybranched PEG was introduced onto a silicon wafer from Example 3 having a surface of formula XV as described above, along with 2 times excess copper sulfate and further aliquots of sodium ascorbate. The remaining alkyne ligand of the polybranched PEG was reacted with the azido-reactive moiety on the surface of formula XV to produce a mixed-modified surface (formula XLV) having a biotin-reactive moiety and a surface-contact moiety of PEG-carboxylic acid.

[0347] Next, this mixed surface was further modified by adding a 1 micromolar solution of streptavidin (SAV) in PBS and incubated at room temperature for 15 minutes to produce a surface of formula XLVI, where the first surface contact portion is PEG-COOH and the second reactive portion is SAV. The sample was washed and the thickness of the modified surface was measured.

[0348] The thickness of the modified layer is shown in Table 3 and, as expected, varies depending on the amount of streptavidin bound to the available biotin surface contact portion.

[0349] [Table 3]

[0350] The results indicate that a modified surface was obtained having a combination of streptavidin-reactive moieties and PEG-COOH surface contact moieties. Further modification of streptavidin with biotinylated species such as biotin-fibronectin or any biotinylated moiety can yield a mixed surface of the first contact moiety (e.g., fibronectin) and the second contact moiety of PEG-COOH in any desired ratio.

[0351] Example 27. Introduction of regioselective surface modification (formula XLVII) of PEG5k in the first region of a microfluidic device and in poly-L-lysine within an isolation pen. Pre-prepared, dry, unprimed (e.g., not flushed with carbon dioxide gas) microfluidic devices having a surface of formula XV were treated with 1.0 to 3.3 mmol aqueous solutions of dibenzylcyclooctinyl (DBCO)-modified PEG, weight-average molecular weight 5000 Da (compound 8, Broadpharm, catalog no. BP-22461) by aspirating the solution through the device's microfluidic channels at a pressure slightly below atmospheric pressure. As a result, the channels were filled with the reagent solution. However, due to the low pressure of fluid introduction and the unprimed nature of the surface within the microfluidic device, D The BCO-modified PEG5kDa solution was not placed in the isolation pen leading to the microfluidic channel. After incubation at room temperature for 30 minutes, 80 microliters of water were passed through the channel and aspirated under reduced pressure, washing away any remaining reagent from the microfluidic device. The solution was further controlled to flow only through the microfluidic channel. Further flushing with 1 microliter / second of water at low pressure was continued for approximately 5 minutes. The surface-modified microfluidic channel was repeatedly flushed with carbon dioxide gas while the device was heated to 90°C.

[0352] Next, a dried microfluidic device having the first surface modification of PEG5K was primed with carbon dioxide as described above. Then, the isolation pen leading to the microfluidic channel was subjected to a 1:1 mixture of alkyne-poly-L-lysine HBr salt (100-mer units, Alamanda polymer) and alkyne-modified PEG (j=MW approx. 5000 Da, compound 6, JenKem Technologies) along with copper sulfate (excess), THPTA ligand, and sodium ascorbate. The microfluidic device was regioselectively modified by flowing a 1.33 micromolar solution containing a stoichiometric mixture. Excess copper sulfate prevented disulfide cleavage by ascorbate during the reaction, and the reaction was carried out at 40°C for approximately 15 minutes (alternatively, it could be carried out at room temperature for approximately 1 hour). After the incubation period was complete, excess reagents and byproducts were removed by flushing with water. The inside of the microfluidic device was dried by flushing with carbon dioxide gas while heating the microfluidic device to 40°C, providing the microfluidic device with the regioselective introduction of a first surface modification containing only PEG5K and a second regioselective surface modification containing poly-L-lysine within the microfluidic channel, which provides a positive charge to promote adhesion of biological cells, only in the isolation pen (formula XLV). Note the ability to adjust the ratio of reagents used to modify the surface of the isolation pen. The PEG-5K:poly-L-lysine ratio varied from 0:100 to 99.9999:0.0001%, and HeLa cell adhesion was observed within the isolation pen, while the migration of motile HeLa cells was inhibited simply by the presence of hydrophilic surfaces within the channel.

[0353] A microfluidic device having an inner surface modified with a first surface modification of PEG 5kDa and a second surface modification of poly-L-lysine with a percentage of approximately 0.00001% or 0.000001% is expected to enable adhesion of adherent cells (such as HeLa cells) while still allowing the extraction of cultured cells using dielectrophoretic force without laser-induced replacement.

[0354] Further generalization. Any type of surface modification reagent can be used to introduce a second surface modification within the isolation pen, and is not limited to poly-L-lysine.

[0355] Secondary passivation of microfluidic channels with a second surface modification only within the channel region. After the initial surface modification of the microfluidic channel described above, unreacted reactive moieties (e.g., azides) may still be present within the channel. While we do not wish to be constrained by theory, this can occur when the modifying reagent is bulky. Secondary passivation with a less sterically bulky surface modifying reagent may allow for the application of a second surface modification to the modified channel surface without modifying the isolation pen surface, by utilizing the remaining reactive moieties.

[0356] A microfluidic device with PEG5kDa surface modification introduced only into the microfluidic channel was rinsed with water only after surface introduction. A second treatment was performed with DBCO-PEG4-OH (Aldrich catalog number 761982) at a concentration of 1.3 micromoles in an aqueous solution, as described above. The procedure was carried out in the same manner as the first process. Because the microfluidic device was not primed, the second surface modification reagent did not enter the isolation pen, and therefore only the channels were further modified. After washing, drying and heating, followed by carbon dioxide priming, then regioselective modification of the isolation pen. The decorations are done as described above.

[0357] Example 28. Culture of OKT3 cells in a microfluidic device having a PEG-modified surface. material: OKT3 cells and mouse B lymphocyte hybridomas were obtained from the American Type Culture Collection (ATCC) (catalog ATCC® CRL-8001®) and provided as a suspension cell system. 2 × 10 5 Cells were seeded at a rate of 10⁴ viable cells / mL and maintained by incubation at 37°C in a 5% carbon dioxide gas environment. Cells were repotted every 2-3 days at a rate of 2 × 10⁴. 4cells / mL or 1 × 10⁶ 5 Cells were divided into 100 cells / mL. The cells were frozen in 5% dimethyl sulfoxide (DMSO) / 95% complete growth medium.

[0358] Culture medium: IMDM (Gibco, catalog 12440053) contains 20% fetal bovine serum (FBS). and 1% penicillin-streptomycin (10,000 U / mL) (Gibco, Catalogue) (G15140122) was added. Next, the complete medium was filtered through a 0.2 μm PES sterile membrane filter unit (Nalgene, 567-0020).

[0359] Priming and perforation procedures: This is as described above in the section on general experimental details.

[0360] Systems and microfluidic devices: As described above in the section on general experimental details, the isolation pen is approximately 7 x 10 5 It has a volume of cubic μm.

[0361] Modified microfluidic surface. The microfluidic device had a covalently bonded PEG-modified surface (formula XVI) prepared as described above in Example 6.

[0362] The OKT3 cell suspension in culture medium was introduced into the microfluidic device by passing the suspension through the fluid inlet and through the microfluidic channel. The cells were randomly filled into the isolation pen by stopping the flow, tilting the tip, and allowing gravity to enter the cells into the isolation pen.

[0363] After filling isolation pens with OKT3 cells, the cells were perfused with culture medium through the microfluidic channels of a nanofluidic chip for a period of three days. Figure 6A shows the growth of OKT3 cells on the PEG-modified surface of the isolation pen of the microfluidic device. The growth of OKT3 cells on the PEG surface was improved compared to the unmodified surface of a similar microfluidic device (data not shown).

[0364] Next, OKT3 cells were extracted from the isolation pen by OET. Figure 6B shows the degree of extraction from the isolation pen at the end of the 20-minute period, which demonstrates the excellent ability to extract proliferated OKT3 cells into the flow channel, an improvement over the ability to extract OKT3 cells from an unconditioned surface of a similar microfluidic device. Next, OKT3 cells were extracted from a microfluidic device (not shown).

[0365] Example 29: Culture and extraction of T lymphocytes on a dextran-modified microfluidic surface. material. CD3+ cells are manufactured by AllCells Inc. and are anti-CD3 / anti-C at a ratio of 1 bead / 1 cell. The mixture was mixed with D28 electromagnetic beads (Dynabeads®, Thermofisher Scientific, catalog number 11453D). The mixture was incubated in a 5% CO2 incubator at 37°C for 5 hours in the same culture medium as the culture experiment itself. After incubation, the T cell / bead mixture was resuspended for use.

[0366] Culture medium. RPMI-1640 (GIBCO®, ThermoFisher Scientific, Catalog) (Vocation number 11875-127), 10% FBS, 2% human AB serum (50 U / ml IL2; R&D Systems).

[0367] Priming procedure: This is as described above in the section on general experimental details.

[0368] Kanri-drain project: This is as described above in the section on general experimental details.

[0369] Systems and microfluidic devices: As described above in the section on general experimental details, the isolation pen is approximately 7 x 10 5 It has a volume of cubic μm.

[0370] Modified microfluidic surface. The microfluidic device had a covalently bonded dextran-modified surface prepared as described above in Example 7.

[0371] The T cell (and bead) suspension was introduced into the microfluidic device by passing the resuspension through the fluid inlet and through the microfluidic channel. The T cells / beads were randomly filled into the isolation pen by stopping the flow, tilting the tip, and allowing gravity to enter the growth chamber.

[0372] After filling isolation pens with T cells / beads, the culture medium was perfused through the microfluidic channels of a nanofluidic chip for a period of 4 days. Figure 7A shows T cell growth on the dextran-modified surface of the isolation pen of the microfluidic device. T cell growth on the dextran surface was improved compared to the unmodified surface of a similar microfluidic device (data not shown).

[0373] Next, T cells were extracted from the isolation pen by gravity (e.g., by tilting the microfluidic device). Figure 7B shows the degree of extraction from the isolation pen at the end of the 20-minute period, which demonstrates a superior ability to extract proliferated T cells into the flow channel, an improved ability compared to the ability to extract T cells from an unmodified surface of a similar microfluidic device (data not shown). Next, T cells were extracted from the microfluidic device (not shown).

[0374] In addition to any modifications shown above, many other variations and alternative configurations can be devised by those skilled in the art without departing from the spirit and scope of this specification. Thus, while the information above is specifically and in detail relating to what is currently considered the most practical and preferred embodiment, it will be apparent to those skilled in the art that many variations, including form, function, method of operation and use, can be made without departing from the principles and concepts described herein, though not limited thereto. Where used herein, examples and embodiments are intended to be illustrative in all respects and should not be construed as limiting. The term "process" is used herein, but the term refers to various parts of the described method. It should also be noted that this is merely used for that purpose, and is not intended to describe the starting or ending point of any part of the method, nor is it intended to be otherwise restrictive.

[0375] List of embodiments of the present disclosure 1. A microfluidic device comprising a base, a cover, and a microfluidic circuit material, wherein the enclosure includes a microfluidic circuit material defining a fluid circuit, wherein at least one inner surface of the base, cover, and microfluidic circuit material has a plurality of first covalent surface modifications each comprising a first bonding group and a first portion which is a first surface contact portion or a first reactive portion; and at least one inner surface of the base, cover, and microfluidic circuit material has a plurality of second covalent surface modifications each comprising a second bonding group and a second portion which is a second surface contact portion or a second reactive portion, wherein the first bonding group and the second bonding group are different from each other, and / or the first portion is different from the second portion.

[0376] 2. The first and second parts may be covalently bonded to a surface, respectively, via a bonding group LG independently selected from -W-Si(OZ)2O- and -OP(O)2O-, where W is O, S, or N, and Z is a bond to a silicon atom in the adjacent bonding group LG or a bond to the surface, as described in Embodiment 1.

[0377] 3. The microfluidic device according to Embodiment 1 or 2, wherein the first surface contact portion may comprise one or more alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyhydric alcohol, alkylene ether, polymer electrolyte, amino, carboxylic acid, phosphonic acid, sulfonic acid anion, carboxybetaine, sulfobetaine, sulfamic acid, amino acid portion, or cleavable portion; and / or the second surface contact portion may comprise one or more alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyhydric alcohol, alkylene ether, polymer electrolyte, amino, carboxylic acid, phosphonic acid, sulfonic acid anion, carboxybetaine, sulfobetaine, sulfamic acid, amino acid portion, or cleavable portion.

[0378] 4. The microfluidic device according to Embodiment 1 or 2, wherein the first surface contact portion may include a polyethylene glycol portion, a dextran portion, a protein portion, a polycarboxylic acid, a polylysine portion, or any combination thereof; and / or the second surface contact portion may include a polyethylene glycol portion, a dextran portion, a protein portion, a polycarboxylic acid, a polylysine portion, or any combination thereof.

[0379] 5. The microfluidic device according to any one of Embodiments 1 to 4, wherein the first reactive portion may be an alkyne portion, an azide portion, a carboxylic acid portion, an amine portion, an olefinic portion, a tetradinyl portion, a trans-cyclooctenyl portion, a thiol portion, a maleimide portion, a biotin portion, a streptavidin portion, a halide portion, a cyano portion, an isocyanate portion, an epoxide portion, a hydroxyamine portion, or a sulfonyl fluoride portion; and / or the second reactive portion may be an alkyne portion, an azide portion, a carboxylic acid portion, an amine portion, an olefinic portion, a tetradinyl portion, a trans-cyclooctenyl portion, a thiol portion, a maleimide portion, a biotin portion, a streptavidin portion, a halide portion, a cyano portion, an isocyanate portion, an epoxide portion, a hydroxyamine portion, or a sulfonyl fluoride portion.

[0380] 6. A microfluidic device according to any one of Embodiments 1 to 5, wherein the first covalent surface modification may include a linker, the linker comprising 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms; and / or the second covalent surface modification may include a linker, the linker comprising 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[0381] 7. The microfluidic device according to Embodiment 6, wherein the linker of the first covalent surface modification may further include one or two bonding group CG moieties; and / or the linker of the second covalent surface modification may further include one or two bonding group CG moieties.

[0382] 8. The first covalent surface modification is given by formulas XXX, V, VII, XXXI, VIII and IX: [ka] (wherein LG is -W-Si(OZ)2O- or -OP(O)2O-; L fm R is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and may further comprise 0 or 1 bonding group CG; R x is the reactive part; W is O, S, or N; Z is a bond to an adjacent silicon atom or a bond to the surface; n is an integer from 3 to 21; L sm This is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and may further co...

Claims

1. A microfluidic device comprising an enclosure having a base, a cover, and a microfluidic circuit material defining a fluid circuit inside, The base, the cover, and at least one inner surface of the microfluidic circuit material The first bonding group, and The first part is a first surface contact portion or a first reactive portion. It has a plurality of first covalent surface modifications, each having the following characteristics: The base, the cover, and at least one inner surface of the microfluidic circuit material The second bonding group, and The second part is a second surface contact portion or a second reactive portion. It has a plurality of second covalent surface modifications, each having the following characteristics: A microfluidic device in which the first bonding group and the second bonding group are different from each other, and / or the first part is different from the second part.

2. The first part and the second part are -W-Si(OZ) ２ O- and -OP(O) ２ The microfluidic device according to claim 1, wherein each of the O- is covalently bonded to the surface via a bonding group LG independently selected from O-, where W is O, S, or N, and Z is a bond to a silicon atom in an adjacent bonding group LG, or a bond to the surface.

3. The first surface contact portion comprises one or more alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyhydric alcohol, alkylene ether, polymer electrolyte, amino, carboxylic acid, phosphonic acid, sulfonic acid anion, carboxybetaine, sulfobetaine, sulfamic acid, amino acid portion, or cleavable portion; and / or The microfluidic device according to claim 1, wherein the second surface contact portion comprises one or more alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyhydric alcohol, alkylene ether, polymer electrolyte, amino, carboxylic acid, phosphonic acid, sulfonic acid anion, carboxybetaine, sulfobetaine, sulfamic acid, amino acid portion, or cleavable portion.

4. The first surface contact portion comprises a polyethylene glycol portion, a dextran portion, a proteinaceous portion, a polycarboxylic acid, a polylysine portion, or any combination thereof; and / or The microfluidic device according to claim 1, wherein the second surface contact portion comprises a polyethylene glycol portion, a dextran portion, a proteinaceous portion, a polycarboxylic acid, a polylysine portion, or any combination thereof.

5. The first reactive portion is an alkyne portion, an azide portion, a carboxylic acid portion, an amine portion, an olefinic portion, a tetradinyl portion, a trans-cyclooctenyl portion, a thiol portion, a maleimide portion, a biotin portion, a streptavidin portion, a halide portion, a cyano portion, an isocyanate portion, an epoxide portion, a hydroxyamine portion, or a sulfonyl fluoride portion; and / or The microfluidic device according to claim 1, wherein the second reactive portion is an alkyne portion, an azide portion, a carboxylic acid portion, an amine portion, an olefinic portion, a tetradinyl portion, a trans-cyclooctenyl portion, a thiol portion, a maleimide portion, a biotin portion, a streptavidin portion, a halide portion, a cyano portion, an isocyanate portion, an epoxide portion, a hydroxyamine portion, or a sulfonyl fluoride portion.

6. Each first covalent surface modification includes a linker, the linker comprising 1 to 200 nonaqueous atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. Contains elementary atoms; and / or The microfluidic device according to claim 1, wherein each second covalent surface modification comprises a linker, the linker comprising 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

7. The linker of the first covalent surface modification further comprises one or two bond group CG portions; and / or The microfluidic device according to claim 6, wherein the linker of the second covalent surface modification further comprises one or two bonding group CG portions.

8. The first covalent surface modification is represented by formulas XXX, V, VII, XXXI, VIII, and IX: 【Chemistry 1】 (In the formula, LG is -W-Si(OZ) ２ O- or -OP(O) ２ It is O-; L ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R ｘ However, it is the reactive part; W is O, S, or N; Z is either a bond to an adjacent silicon atom or a bond to the surface; n is an integer from 3 to 21; L ｓｍ a linker comprising 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, 2, or 3 bonding groups CG; and 【Chemistry 2】 (However, this is the aforementioned surface.) A microfluidic device according to claim 1, having a structure selected from the following.

9. LG is -W-Si(OZ) ２ The microfluidic device according to claim 8, wherein W is O, and W is O.

10. The microfluidic device according to claim 8, wherein n is between 7 and 21.

11. The reactive moiety R ｘ The microfluidic device according to claim 8, wherein the reactive moiety R is an alkyne, an azide, an amine, a carboxylic acid, biotin or streptavidin.

12. The second covalent surface modification is represented by formulas XXX', V', VII', XXX', VIII', and IX': 【Transformation 3】 (In the formula, LG' is -W'-Si(OZ') ２ O- or -OP(O) ２ It is O-; L' ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R' ｘ However, it is the reactive part; W' is O, S, or N; Z' is either a bond to an adjacent silicon atom or a bond to the surface; n' is an integer between 3 and 21; L' ｓｍ a linker comprising 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprising 0, 1, 2, or 3 bonding groups CG; and 【Chemistry 4】 (However, this is the aforementioned surface.) A microfluidic device according to claim 8, having a structure selected from the following.

13. LG' is -W'-Si(OZ') ２ The microfluidic device according to claim 12, wherein W' is O, and here W' is O.

14. The microfluidic device according to claim 12, wherein n' is from 7 to 21.

15. The reactive portion R' ｘ The microfluidic device according to claim 12, wherein the alkyne is an alkyne, an azide, an amine, a carboxylic acid, a biotin, or a streptavidin.

16. The microfluidic device according to claim 1, wherein the first part differs from the second part.

17. The microfluidic device according to claim 13, wherein the first covalent surface modification has a structure selected from formulas XXX, V and VII, and the second covalent surface modification has a structure selected from formulas XXXI', VIII' and IX'.

18. The microfluidic device according to claim 17, wherein the first covalent surface modification and the second covalent surface modification are located on a common inner surface of the base, the cover, and / or the microfluidic circuit material.

19. The microfluidic device according to claim 18, wherein the first and second covalent surface modifications are randomly distributed on the common inner surface.

20. The microfluidic device according to claim 18, wherein the common inner surface includes a first region having the first covalent surface modification and a second region having the second covalent surface modification, and the first region is adjacent to the second region.

21. The microfluidic device according to claim 18, wherein the common inner surface includes a plurality of first regions having the first covalent surface modification and a second region having the second covalent surface modification, and the plurality of first regions are separated from each other by the second regions.

22. The microfluidic device according to claim 13, wherein the first covalent surface modification has a structure selected from formulas XXXI, VIII, and IX, the second covalent surface modification has a structure selected from formulas XXXI', VIII', and IX', and the first covalent surface modification is different from the second covalent surface modification.

23. The first covalent surface modification ligand comprises the structure of formula X, and the second covalent surface modification ligand comprises the structure of formula XI: 【Transformation 5】 (In the formula, CG is a bonding group; and L is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. A microfluidic device according to claim 22, comprising the structure described above.

24. The microfluidic device according to claim 22, wherein the first covalent surface modification and the second covalent surface modification are located on a common inner surface of the base, the cover, and / or the microfluidic circuit material.

25. The microfluidic device according to claim 24, wherein the first and second covalent surface modifications are randomly distributed on the common inner surface.

26. The microfluidic device according to claim 24, wherein the common inner surface includes a first region having the first covalent surface modification and a second region having the second covalent surface modification, and the first region is adjacent to the second region.

27. The microfluidic device according to claim 24, wherein the common inner surface includes a plurality of first regions having the first covalent surface modification and a second region having the second covalent surface modification, and the plurality of first regions are separated from each other by the second regions.

28. The microfluidic device according to claim 24, wherein the common inner surface comprises two or more proteinaceous portions.

29. The microfluidic device according to claim 22, wherein the surface modification ligand for the first covalent surface modification comprises a first proteinaceous moiety, the surface modification ligand for the second covalent surface modification comprises a second proteinaceous moiety, and the first and second proteinaceous moieties are different.

30. The microfluidic device according to claim 12, wherein the first covalent surface modification has a structure selected from formulas XXX, V and VII, the second covalent surface modification has a structure selected from formulas XXX', V' and VII', and the first covalent surface modification differs from the second covalent surface modification in that the reactive portion of the first covalent surface modification does not react with the reactive portion of the second covalent surface modification.

31. The microfluidic device according to claim 30, wherein the first covalent surface modification and the second covalent surface modification are located on a common inner surface of the base, the cover, and / or the microfluidic circuit material.

32. The microfluidic device according to claim 31, wherein the common inner surface includes a first region having the first covalent surface modification and a second region having the second covalent surface modification, and the first region is adjacent to the second region.

33. The microfluidic device according to claim 31, wherein the common inner surface includes a plurality of first regions having the first covalent surface modification and a second region having the second covalent surface modification, and the plurality of first regions are separated from each other by the second regions.

34. The microfluidic device according to claim 1, wherein the fluid circuit includes a flow region and an isolation pen, the isolation pen includes a separation region and a connection region, the connection region includes a base opening to the flow region, and the separation region is fluidly connected to the flow region.

35. The microfluidic device according to claim 34, wherein at least one surface of the flow region is modified with the first covalent surface modification, and at least one surface of the isolation pen is modified with the second covalent surface modification.

36. The microfluidic device according to claim 35, wherein the second covalent surface modification comprises a surface contact portion configured to fix adherent cells.

37. The microfluidic device according to claim 35, wherein the first covalent surface modification comprises a surface contact portion configured to inhibit the migration of motile cells from the isolation pen.

38. The microfluidic device according to claim 34, wherein the flow region is fluid-connected to a fluid inlet and a fluid outlet and is configured to provide a flow of a first fluid culture medium.

39. The microfluidic device according to claim 34, wherein the isolation pen comprises a wall made of a microfluidic circuit material.

40. The microfluidic device according to claim 34, wherein the fluid circuit further comprises a plurality of isolation pens, each having at least one inner surface modified with the first and / or second covalent surface modification.

41. The microfluidic device according to claim 1, wherein the first covalent surface modification and / or the second covalent surface modification form a single layer.

42. The microfluidic device according to claim 1, wherein the inner surface of the base and / or the inner surface of the cover of the enclosure is made of glass, silicon, silicon oxide, hafnium oxide, indium tantalum oxide, or aluminum oxide.

43. The microfluidic device according to claim 1, wherein the inner surface of the microfluidic circuit material is made of polydimethylsiloxane (PDMS) or photopatternable silicone (PPS).

44. The microfluidic device according to claim 1, wherein substantially all of the inner surface of the enclosure is modified covalently.

45. The microfluidic device according to claim 1, wherein the base, the cover, and at least one inner surface of the microfluidic circuit material have a third covalent surface modification comprising a third bonding group and a third portion which is a third surface contact portion or a third reactive portion, wherein the third bonding group is different from each of the first and second bonding groups, and / or the third portion is different from each of the first and second portions.

46. The cover and / or base comprises a semiconductor substrate, according to claim 1, for the microcurrent Body device.

47. The microfluidic device according to claim 46, wherein the semiconductor substrate has a dielectrophoretic (DEP) configuration.

48. The microfluidic device according to claim 1, wherein the cover is an integral part of the microfluidic circuit material.

49. The first or second covalent surface modification is given by the following formula: 【Transformation 6】 【Transformation 7】 【Transformation 8】 A microfluidic device according to any one of claims 1 to 48, having one of the structures.

50. A method for forming a covalently modified surface on at least one inner surface of a microfluidic device comprising a base, a cover, and a microfluidic circuit material, the enclosure having the microfluidic circuit material defining a fluid circuit therein, The method involves bringing at least one of the inner surfaces into contact with the first and second modifying reagents; Reacting the first modifying reagent with a plurality of first nucleophilic moieties on at least one inner surface; Reacting the second modifying reagent with a plurality of second nucleophilic portions on at least one inner surface; To form at least one covalently modified surface comprising a first covalent surface modification comprising a first bonding group and a first portion which is a first surface contact portion or a first reactive portion, and a second covalent surface modification comprising a second bonding group and a second portion which is a second surface contact portion or a second reactive portion. A method comprising the first bonding group being different from the second bonding group, and / or the first portion being different from the second portion.

51. The method according to claim 50, wherein the reaction of the first modifying reagent with the at least one inner surface is performed simultaneously with the reaction of the second modifying reagent with the at least one inner surface of the microfluidic device.

52. The method according to claim 50, wherein the first modifying reagent is reacted with the at least one inner surface before or after the second modifying reagent is reacted with the at least one inner surface of the microfluidic device.

53. The method according to claim 50, wherein the first modifying reagent is reacted under conditions that allow the first modifying reagent to react with any available nucleophilic portion of the at least one inner surface, and the second modifying reagent is reacted under conditions that allow the second modifying reagent to react with any available nucleophilic portion of the at least one inner surface, thereby randomly arranging the first and second covalent surface modifications on the at least one inner surface of the microfluidic device.

54. The method according to claim 50, wherein the first modifying reagent is reacted under conditions that promote a reaction between the first modifying reagent and a nucleophilic moiety located within a first region of the at least one surface, and the second modifying reagent is reacted under conditions that promote a reaction between the second modifying reagent and a nucleophilic moiety located within a second region of the at least one surface, and the first region is adjacent to the first region.

55. The method according to claim 50, wherein the first modifying reagent is reacted under conditions that promote a reaction between the first modifying reagent and a nucleophilic moiety located in any of a plurality of first regions separated from each other on at least one surface, and the second modifying reagent is reacted under conditions that promote a reaction between the second modifying reagent and a nucleophilic moiety located in a second region, wherein the second region is adjacent to or surrounds each of the plurality of first regions.

56. The method according to claim 50, wherein the fluid circuit includes a flow region and an isolation pen having a separation region and a connection region, the connection region includes a base opening to the flow region, and the separation region is fluidly connected to the flow region.

57. The method according to claim 56, wherein the first modifying reagent reacts with a first nucleophilic portion located on the surface of the flow region to form a first covalent surface modification on the surface, and the second modifying reagent reacts with a second nucleophilic portion located on the surface of the isolation pen to form a second covalent surface modification on the surface.

58. The method according to claim 57, wherein the first covalent surface modification includes a first reactive portion, and the second covalent surface modification comprises a second reactive portion.

59. The method according to claim 58, wherein the first and second reactive portions do not react with each other.

60. The method according to claim 57, wherein the second covalent surface modification comprises a surface contact portion that is a support portion for adherent cells.

61. The method according to claim 57 or 60, wherein the first covalent surface modification comprises a surface contact portion configured to inhibit the migration of motile cells from the isolation pen.

62. The method according to claim 50, wherein forming the at least one covalently modified surface comprises forming a covalently modified surface on substantially all of the inner surfaces of the microfluidic device.

63. The first modifying reagent is given by the following formula: 【Chemistry 9】 (In the formula, V is -P(O)(OH) ２ or -Si(T) ２ It is W; W is -T, -SH, or -NH ２ The portion is configured to form a covalent bond with the at least one inner surface; T independently becomes OH, OC １～６ Alkyl or halo; R is C １～６ It is alkyl; L ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R ｘ However, it is the reactive part; n is an integer from 3 to 21, and L ｓｍ (However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0, 1, 2, or 3 bonding groups CG.) The method according to claim 50, having one of the structures.

64. W is OC １～６ The method according to claim 63, wherein the alkyl or halo is used.

65. The method according to claim 63 or 64, wherein n is from 7 to 21.

66. T is OC １～３ It is alkyl or halo, and / or R is C １～３ The method according to claim 63, wherein the alkyl group is used.

67. The aforementioned reactive portion R ｘ The method according to claim 63, wherein the alkyne is an alkyne, an azide, an amine, a carboxylic acid, biotin, or streptavidin.

68. The first modification reagent has the structure of formula I, formula III, or formula XXXII, and the surface modification ligand of the first modification reagent is formula X or formula XI: 【Chemistry 10】 (In the formula, CG is a bonding group; L is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms; L ｓｍ And the sum of L, if present, is 1 to 200 non-hydrogen atoms that do not contain atoms of CG; and The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof in the microfluidic device. The method according to claim 63, having the structure described above.

69. The method according to claim 68, wherein the surface contact portion of the first modifying reagent comprises polyethylene glycol, a dextran portion, a protein portion, a polycarboxylic acid, or a polylysine portion.

70. The second modifying reagent is given by the following formula: 【Chemistry 11】 (In the formula, V' is -P(O)(OH) ２ or -Si(T') ２ It is W'; W' is -T', -SH, or -NH ２ The portion is configured to form a covalent bond with the at least one inner surface; T' is independent of OH and OC. １～６ Alkyl or halo; R' is C １～６ It is alkyl; L' ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R' ｘ However, it is the reactive part; n is an integer from 3 to 21, and L' ｓｍ (However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0, 1, 2, or 3 bonding groups CG.) The method according to claim 50, having one of the structures.

71. W' is OC １～６ The method according to claim 70, wherein the alkyl or halo.

72. The method according to claim 70 or 71, wherein n' is from 7 to 21.

73. T' is OC １～３ It is alkyl or halo, and / or R' is C １～３ The method according to claim 70, wherein the alkyl group is used.

74. The reactive portion R' ｘ The method according to claim 70, wherein the alkyne is an alkyne, an azide, an amine, a carboxylic acid, biotin, or streptavidin.

75. The second modifying reagent has the structure of formula I', formula III', or formula XXXII', and the surface modifying ligand of the second modifying reagent is formula X or formula XI: 【Chemistry 12】 (In the formula, CG is a bonding group; L is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms; L ｓｍ And the sum of L, if present, is 1 to 200 non-hydrogen atoms that do not contain atoms of CG; and The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof in the microfluidic device. The method according to claim 70, having the structure described above.

76. The method according to claim 75, wherein the surface contact portion of the first modifying reagent comprises polyethylene glycol, a dextran portion, a protein portion, a polycarboxylic acid, or a polylysine portion.

77. The method according to claim 68 or 75, wherein the surface contact portion of the first modifying reagent and / or the second modifying reagent supports the proliferation of adherent cells and / or enables the removal of adherent cells cultured thereon.

78. The method according to claim 68 or 75, wherein the surface contact portion of the first modifying reagent and / or the second modifying reagent inhibits motile cells from entering a selected region within the microfluidic device.

79. The method according to claim 63, wherein the first modifying reagent has the structure of formula I, formula III, or formula XXXII, and the second modifying reagent has the structure of formula IV', formula VI', or formula XXXIII'.

80. The first modifying reagent is formula IV, formula VI, or formula XXXIII The method according to claim 63, wherein the second modifying reagent has the structure of formula I', formula III', or formula XXXII'.

81. The at least one covalently modified surface is given formula XXXIV RP-L ｆｍ -R ｘ２ Formula XXXIV By bringing it into contact with the secondary functionalization reagent; The secondary functionalizing reagent is reacted with the reactive portion of the first or second covalent surface modification of the at least one covalently modified surface to form a further modified surface. Furthermore, the formula includes, RP is a reaction pair portion for reacting with the reactive portion of formula XXXIII, formula XXXIII', formula IV, formula IV', formula VI, or formula VI'; R ｘ２ However, the reactive portion is selected so as not to react with the reactive portion of formula XXXIII, formula XXXIII', formula IV, formula IV', formula VI or formula VI'; and L ｆｍ The method according to claim 63, wherein the linker comprises 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG.

82. The method according to claim 63, further comprising contacting the at least one covalently modified surface with a surface modification reagent, and reacting the surface modification reagent with a reactive portion on the at least one covalently modified surface.

83. The surface modification reagent is defined by formula XII: RP-L-Surface Contacting Part Formula XII It has the structure, and in the formula, RP is the reaction pair; The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof; and The method according to claim 82, wherein L is a linker comprising 1 to 200 nonhydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and comprising 0 or 1 bonding group CG.

84. The method according to claim 50, wherein the formation of the at least one covalently modified surface is performed after the assembly of the microfluidic device.

85. The method according to claim 50, wherein the formation of the at least one covalently modified surface is performed before the assembly of the microfluidic device.

86. Forming one first modified surface of the base or cover before assembling the microfluidic device; Assembling the microfluidic device, comprising assembling one of the first covalently modified surfaces of the base or the cover together with the microfluidic circuit material and the unmodified surface of the cover or base; Forming a second modified surface on the unmodified surface of the assembled microfluidic device The method according to claim 50, further comprising:

87. The method according to claim 50, wherein the first nucleophilic portion is a hydroxide, an amino acid, or a thiol, and / or the second nucleophilic portion is a hydroxide, an amino acid, or a thiol.

88. The method according to claim 50, wherein the inner surface of the base and / or cover is a metal, a metal oxide, glass, a polymer, or any combination thereof.

89. The method according to claim 50, wherein the microfluidic circuit material is a polymer.

90. The method according to claim 89, wherein the microfluidic circuit material is polydimethoxysilane (PDMS) or photopatternable silicone (PPS).

91. The method according to claim 50, wherein contact comprises bringing at least one inner surface into contact with a liquid solution containing the first modifying reagent and / or the second modifying reagent.

92. The method according to claim 50, wherein contact comprises bringing at least one inner surface into contact with a gas phase containing the first modifying reagent and / or the second modifying reagent.

93. The method according to claim 92, wherein contact is provided by bringing the at least one inner surface into contact with the first and / or second modifying reagent in the gas phase in the presence of a controlled amount of water vapor.

94. The method according to claim 93, wherein magnesium sulfate heptahydrate provides the controlled amount of water vapor.

95. The method according to claim 92, wherein contact is made by bringing the at least one inner surface into contact with the first and / or second modifying reagent in the gas phase in an environment under a pressure reduced compared to atmospheric pressure.

96. The method according to claim 50, wherein each of the at least one inner surfaces is pre-treated to introduce an oxide portion.

97. The method according to claim 63, wherein n is 9, 14, or 16.

98. The method according to claim 63, wherein n is 9.

99. The method according to claim 70 or 97, wherein n' is equal to 9, 11, 14, 16, 18 or n+2.

100. The method according to claim 81 or 82, wherein the reaction of the at least one covalently modified surface with a surface modification reagent or a secondary functionalizing reagent is carried out by contacting the at least one covalently modified surface with a solution comprising the surface modification reagent or the secondary functionalizing reagent.

101. The method according to claim 50, wherein forming the at least one covalently modified surface comprises forming a monolayer having a first covalent surface modification and / or a second covalent surface modification.

102. The method according to claim 50, wherein forming the at least one covalently modified surface comprises covalently bonding two or more proteinaceous moieties to the at least one covalently modified surface.

103. The method according to claim 50, wherein the cover of the microfluidic device is an integral part of the microfluidic circuit material.

104. The method according to claim 50, wherein the cover or base of the microfluidic device has a DEP configuration.

105. A method for positionally forming different covalently modified surfaces within a microfluidic device, wherein the microfluidic device includes an enclosure comprising a base, a cover, and a microfluidic circuit material having a microfluidic circuit material defining a microfluidic circuit, the microfluidic circuit includes a flow region and an isolation pen, the isolation pen includes a separation region and a connection region, the connection region includes a base-end opening to the flow region, and the separation region is fluidly connected to the flow region, and the method is The first modifying reagent is flowed through the flow region under conditions such that the first modifying reagent does not enter the separation region of the isolation pen; The first modifying reagent is reacted with a nucleophilic moiety on at least one surface of the flow region. The first modified surface is formed within the flow region such that the first modified surface does not extend into the separation region of the isolation pen; The second modifying reagent is flowed through the flow region under conditions such that the second modifying reagent enters the separation region of the isolation pen; The second modifying reagent is reacted with a nucleophilic portion on at least one surface of the separation region of the isolation pen, thereby forming a second modified surface within the separation region of the isolation pen. Includes, A method wherein the first modifying reagent does not have the same structure as the second modifying reagent.

106. The method according to claim 105, wherein the conditions for flowing the first modifying reagent through the flow region include applying negative pressure to the flow region.

107. The method according to claim 106, wherein the flow of the first modifying reagent comprises flowing a solution containing the first modifying reagent through the flow region at a speed of about 10 mm / second or more.

108. The method according to claim 105, wherein the conditions for flowing the first modifying reagent through the flow region include applying positive pressure to the flow region.

109. The method according to claim 108, wherein the flow of the first modifying reagent is performed by flowing a solution containing the first modifying reagent through the flow region at a speed of about 2 mm / second or less.

110. The method according to claim 108 or 109, wherein flowing the first modifying reagent includes flowing a solution comprising the first modifying reagent through the flow region, and the solution comprises a surfactant.

111. The method according to claim 105, wherein the second modifying reagent does not substantially react with the portion on the surface of the flow region.

112. The first modifying reagent comprises a first connecting portion and a first modified portion having a first surface contact portion or a first reactive portion; and The second modifying reagent includes a second connecting portion and a second modifying portion having a second surface contact portion or a second reactive portion, The method according to claim 105, wherein the first connecting portion is different from the second connecting portion, and / or the first modifying portion is different from the second modifying portion.

113. The first modifying reagent is given by the following formula: 【Chemistry 13】 (In the formula, V is -P(O)(OH) ２ or -Si(T) ２ It is W; W is -T, -SH, or -NH ２ The portion is configured to form a covalent bond with the at least one surface of the flow region; T independently becomes OH, OC １～６ Alkyl or halo; R is C １～６ It is alkyl; L ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R ｘ However, it is the reactive part; n is an integer from 3 to 21; and L ｓｍ (However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0, 1, 2, or 3 bonding groups CG.) The method according to claim 105, having one of the structures.

114. W is OC １～６ The method according to claim 113, wherein the alkyl or halo is used.

115. The method according to claim 113, wherein n is from 7 to 21.

116. T is OC １～３ It is alkyl or halo, and / or R is C １～３ The method according to claim 113, wherein the alkyl group is used.

117. The aforementioned reactive portion R ｘ The method according to claim 113, wherein the alkyne is an alkyne, an azide, an amine, a carboxylic acid, biotin, or streptavidin.

118. The first modification reagent has the structure of formula I, formula III, or formula XXXII, and the surface modification ligand of the first modification reagent is formula X or formula XI: 【Chemistry 14】 (In the formula, CG is a bonding group; L is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms; L ｓｍ And the sum of L, if present, is 1 to 200 non-hydrogen atoms that do not contain atoms of CG; and The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof in the microfluidic device. The method according to claim 113, having the structure described above.

119. The method according to claim 118, wherein the surface contact portion comprises polyethylene glycol, a dextran portion, a protein portion, a polycarboxylic acid, or a polylysine portion.

120. The second modifying reagent is given by the following formula: 【Chemistry 15】 (In the formula, V' is -P(O)(OH) ２ or -Si(T') ２ It is W'; W' is -T', -SH, or -NH ２ The portion is configured to form a covalent bond with the at least one inner surface; T' is independent of OH and OC. １～６ Alkyl or halo; R' is C １～６ It is alkyl; L' ｆｍ However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG; R' ｘ However, it is the reactive part; n is an integer from 3 to 21, and L' ｓｍ (However, the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0, 1, 2, or 3 bonding groups CG.) The method according to claim 113, having one of the structures.

121. W' is OC １～６ The method according to claim 120, wherein the alkyl or halo is used.

122. The method according to claim 120, wherein n' is from 7 to 21.

123. T' is OC １～３ It is alkyl or halo, and / or R' is C １～３ Alkyl A method according to claim 120.

124. The reactive portion R' ｘ The method according to any one of claims 140 to 143, wherein the is an alkyne, azide, amine, carboxylic acid, biotin, or streptavidin.

125. The second modifying reagent has the structure of formula I', formula III', or formula XXXII', and the surface modifying ligand' of the second modifying reagent is formula X or formula XI: 【Chemistry 16】 (In the formula, CG is a bonding group; L is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms; L ｓｍ And the sum of L, if present, is 1 to 200 non-hydrogen atoms that do not contain atoms of CG; and The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof in the microfluidic device. The method according to claim 120, having the structure described above.

126. The method according to claim 125, wherein the surface contact portion of the second modifying reagent comprises polyethylene glycol, a dextran portion, a protein portion, a polycarboxylic acid, or a polylysine portion.

127. The method according to claim 125, wherein the surface contact portion of the second modifying reagent supports the proliferation of adherent cells and / or enables the removal of adherent cells cultured thereon.

128. The method according to claim 118, wherein the surface contact portion of the first modifying reagent inhibits motile cells from entering the flow region of the microfluidic device.

129. The method according to claim 120, wherein the first modifying reagent has the structure of formula I, formula III, or formula XXXII, and the second modifying reagent has the structure of formula IV', formula VI', or formula XXXIII'.

130. The first modifying reagent is formula IV, formula VI, or formula XXXIII The method according to claim 120, wherein the second modifying reagent has the structure of formula I', formula III', or formula XXXII'.

131. The method according to claim 105, wherein the second modified surface within the separation region of the isolation pen comprises a second covalent surface modification having the structure of formula XXX', formula V', or formula VII', respectively.

132. The second modified surface is given by formula XII RP-L-Surface Contacting Part Formula XII Contact with a surface modification reagent; The second covalent surface modification of the second modified surface is reacted with the surface modification reagent to form a further modified surface within the separation region of the isolation pen. Furthermore, the formula includes, RP is the reaction pair; The surface contact portion is configured to support cell growth, survival, portability, or any combination thereof; and The method according to claim 131, wherein L is a linker, and L comprises 1 to 200 nonhydrogen atoms selected from any combination of bonds or silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and further comprises 0 or 1 bonding group CG.

133. Contacting the second modified surface with the surface modification reagent of formula XII is The solution comprising the surface modification reagent is poured into the flow region; The surface modification reagent is diffused into the separation region of the isolation pen and brought into contact with the second modified surface. The method according to claim 132, comprising:

134. The method according to claim 132, wherein the first modified surface in the flow region comprises a first covalent surface modification having the structure of formula XXXI, formula VIII, or formula IX, respectively.

135. The second modified surface is given by formula XXXIV RP-L ｆｍ - Reactive part ２ Formula XXXIV By bringing it into contact with the secondary functionalization reagent; The secondary functionalizing reagent is reacted with the reactive portion of the second covalent surface modification of the second modified surface to form a further modified surface within the separation region of the isolation pen. Furthermore, the formula includes, RP is a reaction pair portion for reacting with the reactive portion of formula XXX, formula V, or formula VII; R ｘ２ However, the reactive portion is selected so as not to react with the reactive portion of the second modified surface; and L ｆｍ The method according to claim 131, wherein the linker comprises 1 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, and further comprises 0 or 1 bonding group CG.

136. Contacting the second modified surface with the secondary functionalizing reagent of formula XXXIV is performed. The solution comprising the secondary functionalizing reagent is poured into the flow region; The secondary functionalizing reagent is diffused into the separation region of the isolation pen and brought into contact with the second modified surface. The method according to claim 135, comprising:

137. The method according to claim 135 or 136, wherein the second covalent surface modification reacted with the secondary functionalizing reagent comprises one or two CGs.

138. The method according to claim 105, wherein the nucleophilic portion on the surface of the flow region is selected from hydroxides, aminos, and thiols; and / or the nucleophilic portion on the surface of the isolation pen is selected from hydroxides, aminos, and thiols.

139. The microfluidic circuit comprises a plurality of isolation pens, each of which is processed to form at least one second modified surface or further modified surfaces. The method according to claim 105.

140. The method according to claim 105, wherein the inner surface of the base and / or cover is a metal, a metal oxide, glass, a polymer, or any combination thereof.

141. The method according to claim 105, wherein the microfluidic circuit material is a polymer.

142. The method according to claim 141, wherein the microfluidic circuit material is polydimethoxysilane (PDMS) or photopatternable silicone (PPS).

143. The method according to claim 105, wherein the cover of the microfluidic device is an integral part of the microfluidic circuit material.

144. The method according to claim 131 or 134, wherein the first covalent surface modification forms a single layer on the at least one surface of the flow region, and / or the second covalent surface modification forms a single layer on the at least one surface of the separation region of the isolation pen.

145. The method according to claim 105, wherein forming the first modified surface and / or the second modified surface comprises introducing two or more proteinaceous moieties.

146. The method according to claim 105, wherein the cover or base of the microfluidic device has a DEP configuration.

147. The method according to claim 146, wherein the DEP configuration is operated optically.

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