Digital micro-fluidic chip integrated with coupling active membrane and preparation method and application thereof

Abstract

The invention discloses a digital micro-fluidic chip integrated with a coupling active membrane, the digital micro-fluidic chip comprises a chip top plate and a chip substrate, and the coupling active membrane is arranged on the lower surface of the chip top plate or the upper surface of the chip substrate; the coupling active film is prepared by carrying out secondary photocuring on a photocuring raw material containing a coupling active group. According to the prepared digital micro-fluidic chip, the coupling active group on the surface of the digital micro-fluidic chip can be subjected to covalent coupling reaction with functional groups such as amino, carboxyl, sulfydryl or aldehyde group in biomolecules, so that the connection efficiency and stability of the biomolecules can be greatly improved. The chip can effectively overcome the problems of complex system, high cost, difficult miniaturization and integration and the like caused by using magnetic beads as a solid medium. Besides, the processing process of the coupled active membrane is simple, convenient and rapid, has small influence on the performance of the DMF chip, has the potential of batch replication, and is expected to have very good application prospects in the fields of detection of biological molecules such as nucleic acid and protein, detection, separation and culture of microorganisms and cells and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chip design, and in particular to a digital microfluidic chip integrated with a coupled active membrane, and a preparation method and application thereof. Background Art

[0002] Digital microfluidics (DMF) is an emerging fluid manipulation technology. It is based on the principle of electrowetting-on-dielectric (EWOD) to manipulate droplet generation, movement, fusion, and splitting on a microelectrode array. It has the advantages of flexible operation and ease of automation, and has been increasingly widely used in biochemical testing, biomedical research, and other fields.

[0003] However, applications such as immunoassays often require solid matrices to immobilize biomolecules in order to achieve reactions, separations, cleaning and other operations. However, DMF technology has very high requirements for the strong hydrophobicity of the chip surface, making the stable connection of biomolecules very difficult. Therefore, current DMF chips generally use magnetic beads as solid matrices. However, the manipulation of magnetic beads must rely on complex peripheral mechanical and electrical equipment, including magnets, motors, and related control circuits for automation. This will not only greatly increase the complexity and cost of the equipment, introduce peripheral mechanical disturbances, but also pose great challenges to the miniaturization, integration and automation of DMF.

[0004] In order to overcome the adverse effects of magnetic beads, researchers have also tried many other methods to fix biomolecules on the surface of DMF chips, such as nonspecific adsorption, microcontact printing, and bioanalytical surfaces based on photolithography. However, these methods still have great limitations. For example, the coupling efficiency and stability of nonspecific adsorption are low, which is not conducive to reducing the detection limit of immunoassays; microcontact printing and photolithography processes are very complicated and easily affect the performance of the DMF chip itself.

[0005] Therefore, there is still an urgent need to develop DMF chips with good surface coupling activity, stable properties, and simple and stable biomolecule connection. Summary of the invention

[0006] In view of the above problems, the present invention discloses a digital microfluidic chip with an integrated coupling active membrane. By integrating the coupling active membrane with coupling active groups on the surface into the interior of the digital microfluidic chip, the coupling reaction can be directly carried out with the amino, carboxyl, thiol or aldehyde functional groups in the biomolecules to achieve covalent connection, thereby greatly improving the efficiency and stability of biomolecule connection. In addition, the chip processing process is simple and fast, and no peripheral machinery is required, which has a good application prospect.

[0007] The specific technical solutions are as follows:

[0008] A digital microfluidic chip with an integrated coupled active membrane comprises a chip top plate and a chip base;

[0009] The chip top plate includes an upper support layer, a ground electrode layer, and an upper hydrophobic layer from top to bottom;

[0010] The chip substrate comprises, from bottom to top, a patterned electrode layer and a lower hydrophobic layer;

[0011] The lower surface of the chip top plate or the upper surface of the chip substrate is also provided with a coupling active film;

[0012] The coupling active film is prepared by secondary photocuring of a photocurable raw material including coupling active groups.

[0013] The invention discloses a digital microfluidic chip integrated with a coupling active membrane. The coupling active membrane with coupling active groups is integrated inside the digital microfluidic chip by means of photocuring. The coupling active groups on the surface of the coupling active membrane can directly react with functional groups such as amino, carboxyl, thiol or aldehyde in biological molecules to achieve covalent connection, thereby greatly improving the efficiency and stability of biological molecule connection. The chip is used in the fields of biological molecule detection, microorganism and cell detection, separation, culture, nucleic acid aptamer screening, etc.

[0014] In the present invention, the coupling active membrane can be arranged on the lower surface of the chip top plate (i.e., the position of the upper hydrophobic layer) or the upper surface of the chip substrate (i.e., the position of the lower hydrophobic layer); the specific arrangement includes two kinds, one is to solidify the coupling active membrane to the lower surface of the chip top plate or the outer side of the upper surface of the chip substrate (protruding); the other is to integrate the coupling active membrane with the lower surface of the chip top plate or the upper surface of the chip substrate (flush). Regardless of the arrangement method, how to achieve stable and tight connection with the upper and lower hydrophobic layers with hydrophobicity without falling off is faced; only by solving this problem can the coupling active membrane be guaranteed to play the function of subsequent covalent connection with biological molecules. In addition, it was also found in the experiment that the coupling active membrane may also have problems such as swelling of the substance to be tested and poor coupling activity with the substance to be tested in the subsequent application process.

[0015] Based on the above practical problems, the inventors found after a large number of experiments that by using two photocuring steps when preparing the coupling active membrane and controlling the time of the two photocuring steps, it is possible to ensure that the coupling active membrane is stably and tightly connected to the hydrophobic upper and lower hydrophobic layers; and it is possible to ensure that the coupling active membrane has both high coupling activity and excellent chemical stability.

[0016] Specifically:

[0017] When the coupling active film is located on the lower surface of the chip top plate, specifically on the outer surface of the upper hydrophobic layer;

[0018] Alternatively, when the coupling active film is located on the upper surface of the chip substrate, specifically on the outer surface of the lower hydrophobic layer;

[0019] The secondary light curing is performed in which the first light curing is performed before demoulding, and the curing time is 30 to 50 seconds; the second light curing is performed after demoulding, and the curing time is 10 to 40 seconds.

[0020] In view of the above situation, the present invention also discloses a method for preparing the digital microfluidic chip with integrated coupling active membrane, adopting scheme A), comprising the following steps:

[0021] A-1) making a mold for preparing a coupling active membrane;

[0022] A-2) preparing a chip top plate or a chip base;

[0023] A-3) preparing a photocurable raw material according to the mass fraction, dropping the photocurable raw material onto the mold, covering the chip top plate or chip base, performing a primary photocuring for 30 to 50 seconds, then demoulding, and performing a secondary photocuring for 10 to 40 seconds;

[0024] A-4) Assembling the prepared chip substrate or chip top plate with the chip top plate or chip substrate with the coupled active membrane prepared in step A-3) to obtain a digital microfluidic chip integrated with the coupled active membrane.

[0025] It has been found through experiments that the time control of the two photocurings disclosed in the present invention is very critical. The shorter the first photocuring time is, the stronger the viscosity between the coupling-active photocuring material and the hydrophobic layer is. However, if the first photocuring time is too short, it is not conducive to the molding of the microstructure set on the surface of the coupling-active film, and if the first photocuring time is too long, it is not conducive to the fixation and demolding of the coupling-active film on the hydrophobic layer; when the second photocuring time is too long, the spontaneous fluorescence of the coupling-active film will be enhanced, and at the same time the coupling activity of the coupling-active photocuring material will be weakened, affecting the effect of coupling biological molecules; and when the secondary photocuring time is too short or even no secondary photocuring is performed, the curing performance of the coupling-active photocuring site is poor and is easily swollen, which is not conducive to the conduct of biological experiments.

[0026] When the above configuration is adopted, the thickness of the coupling active film is recorded as h, and the vertical distance between the upper hydrophobic layer of the chip top plate and the lower hydrophobic layer of the chip substrate is recorded as H;

[0027] Then it satisfies, 0 <h≤H / 3。

[0028] Experiments have found that excessive protrusion of the coupling active membrane will affect the normal driving of droplets by the digital microfluidic chip.

[0029] Preferred:

[0030] A microstructure is arranged on the surface of the coupling active membrane to increase the specific surface area of ​​the site, and the microstructure includes but is not limited to a microcolumn array and the like.

[0031] The number of the coupled active membranes is N, where N≥1.

[0032] Further preferred:

[0033] The microstructure is selected from a microcolumn array arranged in a ring, the microcolumn diameter d is selected from 20 to 200 μm, the ratio of the microcolumn height h1 to the microcolumn diameter d is selected from 0.2 to 1.5, and the ratio of the microcolumn spacing d1 to the microcolumn diameter d is selected from 1 to 3.

[0034] Experiments have found that the annular arrangement can ensure the uniform diffusion of the coupled active photocurable material on the mold; when the ratio of the microcolumn height h1 to the microcolumn diameter d is too large, or the ratio of the microcolumn array spacing d1 to the microcolumn diameter d is too small, incomplete demolding is likely to occur during the processing process, thereby affecting the quality of the microstructure; when the ratio of the microcolumn height h1 to the microcolumn diameter d is too small, or the ratio of the microcolumn array spacing d1 to the microcolumn diameter d is too large, the effect of increasing the specific surface area is not obvious.

[0035] When the coupling active membrane is located on the lower surface of the chip top plate, specifically integrated with the upper hydrophobic layer;

[0036] Alternatively, when the coupling active film is located on the upper surface of the chip substrate, specifically integrated with the lower hydrophobic layer;

[0037] The secondary photocuring is performed for 5 to 10 seconds before the first photocuring is performed in contact with the raw material for preparing the upper hydrophobic layer or the lower hydrophobic layer; the second photocuring is performed after the second photocuring is performed in contact with the raw material for preparing the upper hydrophobic layer or the lower hydrophobic layer, and the curing time is 30 to 60 seconds.

[0038] In view of the above situation, the present invention also discloses a method for preparing the digital microfluidic chip with integrated coupling active membrane;

[0039] When the coupling active membrane is integrated with the lower surface of the chip top plate, the preparation process adopts scheme B):

[0040] B-1) making a mold for preparing a coupling active membrane;

[0041] B-2) preparing a photocurable raw material by mass fraction; preparing a hydrophobic photocurable raw material for preparing an upper hydrophobic layer by mass fraction;

[0042] B-3) dropping the photocurable raw material onto the mold, performing a primary photocuring for 5 to 10 seconds, then placing the hydrophobic photocurable raw material on the mold, spreading the hydrophobic photocurable raw material into a thin layer by means of scraping or spin coating, and then covering with an upper support layer with a ground electrode layer, flattening, so that the hydrophobic photocurable raw material and the photocurable raw material for preparing the coupling active film form a thin layer, and then performing a secondary photocuring for 30 to 60 seconds; the hydrophobic photocurable raw material and the coupling active photocurable raw material are completely cured to form a chip top plate with a coupling active film;

[0043] B-4) Assembling the prepared chip substrate with the chip top plate with the coupled active membrane prepared in step B-3) to obtain a digital microfluidic chip with integrated coupled active membrane.

[0044] When the coupling active film is integrated with the upper surface of the chip substrate, the preparation process adopts scheme C):

[0045] C-1) making a mold for preparing a coupling active membrane;

[0046] C-2) preparing a photocurable raw material by mass fraction; preparing a hydrophobic photocurable raw material for preparing a lower hydrophobic layer by mass fraction;

[0047] C-3) dripping the photocurable material onto the mold, photocuring for 5 to 10 seconds, then placing the hydrophobic photocurable material on the mold, spreading the hydrophobic photocurable material into a thin layer by scraping or spin coating, then covering it with a patterned electrode with a dielectric layer, flattening it, so that the hydrophobic photocurable material and the photocurable material for preparing the coupling active film form a thin layer, and then performing secondary photocuring for 30 to 60 seconds; the hydrophobic photocurable material and the coupling active photocurable material are completely cured to form a chip substrate with a coupling active film;

[0048] C-4) Assembling the prepared chip top plate with the chip substrate with the coupled active membrane prepared in step C-3) to obtain a digital microfluidic chip with an integrated coupled active membrane.

[0049] The experiment found that when the initial curing time (i.e., one-time photocuring) of the photocurable raw material for preparing the coupling active film is too short, the fluidity is strong and it is easy to diffuse outward from the corresponding position of the mold, which is not conducive to controlling the size and range of the coupling active film; when the initial curing time is too long, the hardness of the coupling active film is too high, which is not conducive to subsequent molding with the hydrophobic photocurable material.

[0050] In the present invention, the photocurable raw material includes a photocurable material, a coupling agent and an initiator;

[0051] The photocurable material is selected from one or more monomers or oligomers containing photocurable functional group I;

[0052] The coupling agent is selected from one or more monomers or oligomers containing both a photocurable functional group II and a coupling active group;

[0053] The photocurable functional group I and the photocurable functional group II are independently selected from one or more of a thiol functional group, an alkenyl functional group, an acrylate functional group, a methacrylate functional group, a vinyl ether functional group, and an epoxy functional group;

[0054] The coupling active group is selected from one or more of an amino group, a carboxyl group, a thiol group, an ethylene oxide group, a glycidyl group, an N-hydroxysuccinimide ester (NHS), a maleimide group, an aldehyde group, and a sulfonyl chloride group.

[0055] Preferred:

[0056] The photocurable material is selected from one or more of polyethylene glycol acrylate, polyurethane acrylate, polysiloxane acrylate, perfluoropolyether acrylate, mercapto-containing polysiloxane, alkenyl-containing polysiloxane, hydroxypropyl acrylate, isobornyl acrylate, ethoxylated trimethylolpropane triacrylate, 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione, trimethylolpropane diallyl ether, pentaerythritol tetrakis(3-mercaptopropionic acid), pentaerythritol tetrakis(2-mercaptoacetic acid), triallyl isocyanurate, pentaerythritol triallyl ether, trimethylolpropane triacrylate, tris[2-(3-mercaptopropionic acid)ethyl]isocyanurate, and trimethylolpropane tris(3-mercaptopropionic acid);

[0057] The coupling agent is selected from one or more of glycidyl acrylate, glycidyl methacrylate, 3-mercaptopropane-N-hydroxysuccinimide ester, acrylate-N-hydroxysuccinimide ester, methacrylate-N-hydroxysuccinimide ester, N-(allyloxycarbonyloxy)succinimide, allyl glycidyl ether, 3-mercapto-1-propylamine, acrylic acid, mercaptopropionic acid, acrylamide, and pentaerythritol tetrakis-3-mercaptopropionate.

[0058] Preferably, in terms of mass percentage, the composition of the photocurable raw material includes:

[0059] Light-curing materials 45.0~94.5%;

[0060] Coupling agent 5-50%;

[0061] Initiator 0.5~5.0%.

[0062] The experiment found that when the coupling agent content is too low, the efficiency of the coupling active membrane connecting biological molecules is reduced, resulting in a decrease in detection sensitivity; when the coupling agent content is too high, the material curing performance will deteriorate, making the coupling active membrane difficult to process.

[0063] Further preferred:

[0064] When the photocurable material is selected from acrylate materials, the coupling agent is selected from one or more of glycidyl acrylate, glycidyl methacrylate, acrylate-N-hydroxysuccinimide ester, methacrylate-N-hydroxysuccinimide ester, and the mass ratio of the coupling agent is selected from 5 to 40%;

[0065] When the photocurable material is selected from thiol-olefin materials, the coupling agent is selected from one or more of 3-mercaptopropane-N-hydroxysuccinimide ester, N-(allyloxycarbonyloxy)succinimide, allyl glycidyl ether, acrylamide, and pentaerythritol tetrakis-3-mercaptopropionate, and the mass ratio of the coupling agent is selected from 20 to 50%.

[0066] There is no special requirement for the initiator, and it can be selected from conventional types in the art, such as one or more of benzil compounds, alkylphenone compounds, and acylphosphine oxides.

[0067] The hydrophobic photocuring raw material is selected from a photocuring aid with good hydrophobic properties, such as a fluorine-containing photocuring aid; or a photocuring material with good hydrophobic properties, such as 1H, 1H, 2H, 2H-perfluorodecyl acrylate, 2-(perfluorooctyl) ethyl methacrylate, 1H, 1H, 2H, 2H-perfluorodecyl mercaptan, allyl 1H, 1H-perfluorooctyl ether, polysiloxane acrylate oligomer, perfluoropolyether acrylate oligomer, mercapto polysiloxane oligomer, alkenyl polysiloxane oligomer or more.

[0068] In the present invention, the light source selected for the photocuring is ultraviolet light (such as 365nm), and the radiation intensity is 30 to 600mW / cm 2 .

[0069] In the present invention, the processing of the chip top plate and the chip base adopts conventional technical means in the art.

[0070] The upper hydrophobic layer in the chip top plate is a thin layer with hydrophobicity, a water contact angle greater than 105°, and a thickness selected from 0.1 to 50 μm;

[0071] The lower hydrophobic layer in the chip substrate is also a thin layer with hydrophobicity, a water contact angle greater than 105°, and a thickness selected from 0.1 to 50 μm.

[0072] The component of the hydrophobic thin layer is selected from one or more of Teflon, a hydrophobic modification agent, a hydrophobic photocuring agent, and a fluorine-containing silane coupling agent.

[0073] The patterned electrode layer in the chip substrate is a substrate with a plurality of electrodes processed on the surface, and the plurality of electrodes constitute an electrode pattern, and the patterned electrode layer can be connected to an external control circuit;

[0074] The substrate is selected from one or more of glass, silicon wafer, printed circuit board, plastic plate, rubber plate, photocurable resin plate;

[0075] The material of the plurality of electrodes is selected from one or more of conductive metal oxides, metals, conductive polymers, conductive inks, conductive inks, and graphene;

[0076] The chip substrate further comprises a dielectric layer, which is a separate thin layer and is located between the patterned electrode layer and the lower hydrophobic layer; or is combined with the lower hydrophobic layer to form a composite hydrophobic dielectric layer;

[0077] When the dielectric layer is a separate thin layer, the thin layer has a high dielectric constant, a relative dielectric constant selected from 2.5 to 10, and a thickness selected from 10 to 120 μm;

[0078] The material of the dielectric layer is selected from one or more of metal oxides, metal nitrides, photoresists, plastics, organic polymers, and photocurable materials;

[0079] Preferably, the material of the dielectric layer is selected from one or more of polyvinylidene fluoride (PVDF), polyimide (PI), polyparaxylene (Parylene C), aluminum oxide, silicon nitride, SU-8 photoresist, polydimethylsiloxane (PDMS), pentaerythritol tetrakis(3-mercaptopropionate), tris[2-(3-mercaptopropionate)ethyl]isocyanurate, trimethylolpropane tris(3-mercaptopropionate), pentaerythritol tetrakis(2-mercaptoacetic acid), 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione, and trimethylolpropane diallyl ether.

[0080] When the dielectric layer and the lower hydrophobic layer are combined into a composite hydrophobic dielectric layer, the raw material is a mixture of dielectric layer material and hydrophobic layer material.

[0081] The relative dielectric constant of the composite hydrophobic medium layer is selected from 2.5 to 10, the thickness is selected from 10 to 120 μm, and the water contact angle is greater than 105°.

[0082] The present invention also discloses the application of the digital microfluidic chip with integrated coupled active membrane in the fields of biological molecule detection, microorganism and cell detection, separation, culture, and nucleic acid aptamer screening.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] The present invention discloses a digital microfluidic chip with integrated coupling active membrane. By adopting a photocurable raw material with coupling activity and optimizing the photocuring process, a coupling active membrane can be easily constructed on the lower surface of the chip top plate or the upper surface of the chip substrate. These sites have coupling active groups directly on the surface, so that covalent coupling reactions can occur with functional groups such as amino, carboxyl, sulfhydryl or aldehyde groups in biological molecules without complicated surface modification, thereby greatly improving the efficiency and stability of biological molecule connection. The chip can be used to carry out biochemical tests and biomedical research, and can replace traditional solid matrices such as magnetic beads, thereby avoiding the introduction of complex peripheral mechanical and electrical equipment, making the digital microfluidic chip system easier to miniaturize, integrate and automate.

[0085] The coupled active photocurable raw materials can also be easily used to prepare microstructures such as microcolumn arrays, thereby greatly increasing the specific surface area and helping to improve the sensitivity and dynamic range of detection. In addition, the processing of the coupled active membrane is simple and fast, has little effect on the performance of the DMF chip, and has the potential for batch replication. Therefore, it will have very good application prospects in the detection of biological molecules such as nucleic acids and proteins, as well as the detection, separation, and cultivation of microorganisms and cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 This is a schematic diagram of the structure of a digital microfluidic chip with an integrated coupled active membrane prepared by the present invention. Figure 1 Figures A and B show two relative positions of the coupling active membrane and the chip surface, protruding (A) and flush (B).

[0087] 1-chip top plate, 2-chip substrate, 3-coupling active membrane, 4-upper support layer, 5-ground electrode layer, 6-upper hydrophobic layer, 7-lower hydrophobic layer, 8-medium layer, 9-patterned electrode layer, 91-electrode;

[0088] Figure 2 The double-layer mask used in the mold for preparing the coupling active membrane in Example 1, A is the first mask, and B is the second mask;

[0089] Figure 3 The photos are of the chip top plates with integrated coupling active membranes prepared in Example 1 (A), Comparative Example 1 (B), and Comparative Example 2 (C), respectively;

[0090] Figure 4 The schematic diagram is a principle diagram of H5N1 detection using the digital microfluidic chip with integrated coupled active membrane prepared in Example 1 of the present invention, in which:

[0091] 11-capture antibody, 12-BSA (bovine serum albumin), 13-H5N1, 14-detection antibody, 15-TMB chromogen substrate;

[0092] Figure 5 The results of H5N1 detection using the digital microfluidic chip with integrated coupled active membrane prepared in Example 1 of the present invention are shown in Figure A. In the figure, Figure A is a photo of the detection results of H5N1 antigen, and the concentrations of H5N1 antigen from left to right are 4000pg / mL, 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, and 0pg / mL, respectively; Figure B is a standard curve diagram drawn by extracting the color channel (RGB) information of different coupled active membranes in Figure A;

[0093] Figure 6 These are experimental fluorescent images of nucleic acid molecule coupling using the chip top plates with integrated coupling active membranes prepared in Example 1 (A), Comparative Example 4 (B), and Comparative Example 5 (C), respectively. Specific implementation methods

[0095] To further understand the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to these embodiments. Non-essential improvements and adjustments made by technicians in this field under the core guiding ideology of the present invention still fall within the scope of protection of the present invention.

[0096] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0097] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. The terms "comprise", "include", etc. used herein indicate the existence of the features, steps, operations and / or components, but do not exclude the existence or addition of one or more other features, steps, operations or components.

[0098] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner.

[0099] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the usual meanings understood by persons with ordinary skills in the field to which the present invention belongs. If the full text involves descriptions such as "first", "second", etc., the descriptions such as "first", "second", etc. are only used to distinguish similar objects, and cannot be understood as indicating or implying their relative importance, order of precedence, or implicitly indicating the number of technical features indicated. It should be understood that the data described by "first", "second", etc. can be interchangeable under appropriate circumstances.

[0100] Figure 1 The schematic diagram of the structure of the digital microfluidic chip with integrated coupling active membrane prepared by the present invention, in which A and B respectively show two relative positional relationships between the coupling active membrane and the chip surface, convex (A) and flush (B);

[0101] The digital microfluidic chip with integrated coupled active membrane disclosed in FIG. A comprises a chip top plate 1 and a chip substrate 2;

[0102] The chip top plate 1 includes an upper support layer 4, a ground electrode layer and an upper hydrophobic layer 6 from top to bottom, and the coupling active film 3 protrudes from the outer surface of the upper hydrophobic layer 6;

[0103] The chip substrate 2 includes a patterned electrode layer 9 , a dielectric layer 8 and a lower hydrophobic layer 7 from bottom to top, and a plurality of electrodes 91 are processed on the surface of the patterned electrode layer 9 .

[0104] The digital microfluidic chip with integrated coupling active membrane disclosed in FIG. B is similar to that in FIG. A, except that the coupling active membrane 3 and the upper hydrophobic layer 6 are integrated.

[0105] When performing the test, just drop the test liquid onto the coupled active membrane.

[0106] Figure 2 This is a double-layer mask used to make the mold for preparing the coupled active membrane in Example 1. In the figure, A is the first mask layer and B is the second mask layer. The two masks are exposed, developed, and dried in sequence to obtain a coupled active membrane mold, and the structure is shown in the figure on the far right.

[0107] Example 1: Chip with coupling active membrane protruding from the lower surface of the chip top plate

[0108] a) Coupling active film mold fabrication: Spin-coat photoresist (Microchem, SU-83050) on a clean and dry silicon wafer with a thickness of 20 μm and pre-bake; Figure 3 A) is placed on the photoresist, exposed for 12 seconds, and then baked; the photoresist is spin-coated again with a thickness of 70 μm and then baked; the second mask ( Figure 3B) Align with the first layer of photoresist pattern, expose for 15s, post-bake; finally develop and hard bake. Before using the SU-8 mold, modify it with 0.5wt% 1H,1H,2H,2H-perfluorodecyltriethoxysilane fluorine oil solution to facilitate subsequent demolding of polydimethylsiloxane (PDMS) mold. Prepare PDMS prepolymer and curing agent (Momentive, RTV-615) at a mass ratio of 10:1, mix well and remove bubbles, then take 20g and place it on the SU-8 mold, heat and cure to form a PDMS mold with a thickness of about 5mm.

[0109] b) Preparation of coupled active photocurable material: Calculated by mass fraction, a coupled active photocurable material containing 30% of N-(allyloxycarbonyloxy)succinimide, 51.5% of pentaerythritol tetrakis(3-mercaptopropionate), 17.5% of triallyl isocyanurate, and 1% of a photopolymerization initiator 2,4,6-trimethylbenzoyldiphenylphosphine oxide was prepared.

[0110] c) Chip top plate processing: The ITO glass was cleaned with anhydrous ethanol, blown dry, and then spin-coated with PTFE solution (dissolved in FC40 fluorinated liquid, concentration of 1wt%), and baked at 150°C for 30min to form a hydrophobic thin layer.

[0111] d) Processing of chip top plate with integrated coupling active membrane: drip the coupling active photocurable material in step b) onto the corresponding position of the PDMS mold in step a) and cover the chip top plate in step c), and then irradiate with ultraviolet light (365nm, 45mW / cm 2 ) and light-cured for 40 seconds; after demolding, the coupled active light-cured material was further cured by secondary light-irradiation for 30 seconds to form a chip top plate with an integrated coupled active film.

[0112] e) Chip substrate processing: The patterned electrode layer is printed on the flexible film of PI medium to form an FPC flexible circuit board, and the printed electrode side is pasted on the PMMA chip substrate with the PI flexible film as the dielectric layer. The dielectric layer is spin-coated with Teflon solution (dissolved in FC40, the concentration is 1%, and baked at 85°C for 30 minutes to form a lower hydrophobic layer.

[0113] f) Assembling the chip top plate of step d) and the chip base of step e) together to form a digital microfluidic chip with an integrated coupled active membrane.

[0114] Comparative Example 1

[0115] The preparation process is basically the same as that in Example 1, the only difference is that the time of the two photocuring in step d) is different, specifically, the time of the first photocuring is replaced by 60s, and the time of the second photocuring is replaced by 10s.

[0116] Comparative Example 2

[0117] The preparation process is basically the same as that in Example 1, the only difference being the time of the first photocuring in step d), specifically, the time of the first photocuring is replaced with 20 seconds.

[0118] Figure 3 The photographs of the chip top plate with integrated coupling active membrane prepared in Example 1, Comparative Examples 1 and 2 are respectively shown. By comparison, it is found that in the chip top plate with integrated coupling active membrane prepared in Example 1 (Figure A), the coupling active membrane is stably and tightly connected to the lower hydrophobic layer; while in the chip top plate with integrated coupling active membrane prepared in Comparative Example 1 (Figure B), the surface adhesion between the coupling active membrane and the lower hydrophobic layer is not strong, and the four sides are separated from the hydrophobic surface, which is related to the fact that the time of the first photocuring before demolding is too long; in the chip top plate with integrated coupling active membrane prepared in Comparative Example 2 (Figure C), the shape of the coupling active membrane is severely deformed, and the microcolumn array structure arranged thereon cannot be formed, which is related to the fact that the time of the first photocuring before demolding is too short and cannot be effectively cured.

[0119] Comparative Example 3

[0120] The preparation process is basically the same as that in Example 1, except that only one photocuring is used in step d), specifically:

[0121] The coupled active photocurable material in step b) is dripped onto the corresponding position of the PDMS mold in step a) and covers the chip top plate in step c), and then irradiated with ultraviolet light (365 nm, 45 mW / cm 2 ) and light-cured for 70 seconds, then demoulded.

[0122] This comparative example is prepared by only one-time photocuring, and the surface adhesion between the coupling active film and the lower hydrophobic layer in the prepared chip top plate with integrated coupling active film is not strong, which is similar to the situation in comparative example 1.

[0123] Comparative Example 4

[0124] The preparation process is basically the same as that in Example 1, except that only one photocuring is used in step d), specifically:

[0125] The coupled active photocurable material in step b) is dripped onto the corresponding position of the PDMS mold in step a) and covers the chip top plate in step c), and then irradiated with ultraviolet light (365 nm, 45 mW / cm 2 ) and light-cured for 40 seconds, and then demoulded.

[0126] Comparative Example 5

[0127] The preparation process is basically the same as that in Example 1, the only difference being the time of the second photocuring in step d), specifically, the second photocuring time is replaced with 50 seconds.

[0128] Example 2

[0129] The preparation process is basically the same as that in Example 1, the only difference is that the time of the two photocuring in step d) is different, specifically, the first photocuring time is replaced by 30s, and the second photocuring time is replaced by 40s.

[0130] It was observed that in the chip top plate with integrated coupling active membrane prepared in this embodiment, the coupling active membrane was stably and tightly connected to the lower hydrophobic layer, similar to that in Embodiment 1.

[0131] Example 3

[0132] The preparation process is basically the same as that in Example 1, the only difference is that the time of the two photocuring in step d) is different, specifically, the first photocuring time is replaced by 50s, and the second photocuring time is replaced by 10s.

[0133] It was observed that in the chip top plate with integrated coupling active membrane prepared in this embodiment, the coupling active membrane was stably and tightly connected to the lower hydrophobic layer, similar to that in Embodiment 1.

[0134] Example 4: Chip with coupling active membrane flush with the lower surface of the chip top plate

[0135] a) Configuration of coupled active light-curing material: Calculated by mass fraction, a coupled active light-curing material containing 7% of glycidyl methacrylate, 92% of polyethylene glycol acrylate, and 1% of a photopolymerization initiator 2,4,6-trimethylbenzoyldiphenylphosphine oxide was prepared.

[0136] b) Preparation of hydrophobic photocurable material: A photocurable hydrophobic material containing 99% of 2-(perfluorooctyl)ethyl methacrylate and 1% of a photopolymerization initiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was prepared according to the mass fraction.

[0137] c) Chip top plate processing: clean ITO glass is washed with anhydrous ethanol, blown dry and set aside; the coupled active photocurable material of step a) is dripped onto a flat mold and irradiated with ultraviolet light for 10 seconds to perform a photocuring process; then the hydrophobic photocurable material of step b) is placed on the mold and covered with ITO glass to form a thin layer of the hydrophobic photocurable material; finally, the ultraviolet light (365nm, 45mW / cm 2 ) and then subjected to secondary photocuring for 60 seconds, followed by demoulding to form a chip top plate with a coupling active film.

[0138] d) Chip substrate processing: According to the mass fraction, a composite hydrophobic dielectric layer material containing 10% fluorine-containing photocuring aid, 37.8% tetrakis (3-mercaptopropionic acid) pentaerythritol ester, 51.2% 1,3,5-triallyl-1,3,5-triazine-2,4,6 (1H, 3H, 5H)-trione, and 1% photopolymerization initiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is prepared. A chip substrate of PCB material is prepared, and a plurality of patterned electrodes are provided on the substrate; the patterned electrode layer is cleaned and dried with anhydrous ethanol, and the surface is covered with a composite hydrophobic dielectric layer material, and a release film is used to flatten it into a thin layer, and UV light is used to cure it for 60 seconds, and the release film is removed to form a composite hydrophobic dielectric layer.

[0139] e) Assembling the chip top plate of step c) and the chip base of step d) together to form a digital microfluidic chip with an integrated coupled active membrane.

[0140] Application Example 1: Detection of H5N1

[0141] First, a 2 μg / mL capture antibody (mouse anti-H5N1 hemagglutinin monoclonal antibody) solution was prepared with a buffer solution (1xPBS containing 0.05% Tween-20 and 1 mM MgCl2), and then added dropwise to the coupled active membrane of the chip top plate prepared in Example 1, and incubated for 4 hours at room temperature; then, the solution was rinsed several times with a 1xPBS solution to remove unattached capture antibody molecules, and then treated with a 5% BSA blocking solution for 30 minutes to block sites not bound to proteins to avoid nonspecific adsorption; the chip top plate was sealed with the chip substrate to form a complete chip.

[0142] H5N1 detection principle Figure 4 As shown. The specific operation process is as follows: by applying a driving voltage to the DMF chip, a droplet containing the H5N1 standard sample (recombinant H5N1 hemagglutinin) is moved from the sample area to the coupled active membrane with the capture antibody, and the droplet is removed after incubation for 45 minutes; then a droplet containing 1xPBS is driven to wash the coupled active membrane several times to remove the unconnected sample; then a 0.8μg / mL detection antibody (rabbit anti-H5N1 hemagglutinin polyclonal antibody connected to horseradish peroxidase) is driven to the coupled active membrane, and the droplet is removed after incubation for 20 minutes; a droplet containing 1xPBS is driven to wash several times to remove the unconnected detection antibody; then a standard TMB chromogen substrate droplet is driven to the coupled active membrane and incubated for 5 minutes. During this process, TMB will be catalyzed by horseradish peroxidase (HRP) to change color to form a blue cationic radical.

[0143] Figure 5are the test results. In the figure, A is a photo of the test results of H5N1 antigen. From left to right, the concentrations of H5N1 antigen are 4000pg / mL, 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, and 0pg / mL; Figure B is a standard curve diagram drawn by extracting the color channel (RGB) information of different coupled active membranes in Figure A.

[0144] It can be calculated from the standard curve that the detection limit of H5N1 antigen detected by the digital microfluidic chip with integrated coupled active membrane prepared in Example 1 is 147 pg / mL.

[0145] Application Example 2 Detection of Nucleic Acid Molecules

[0146] First, a 10 μM nucleic acid molecule (NH2-AGACCTTGGGCAGGTTCC-FAM) solution was prepared with a buffer solution (1xPBS), and then added dropwise to the coupled active membrane of the chip top plate prepared in Example 1 and Comparative Examples 4 to 5, and incubated for 4 hours at room temperature; then rinsed with 1xPBS solution several times to remove unconnected nucleic acid molecules.

[0147] Since one side of the nucleic acid molecule has an amino group, it can be covalently linked to the molecules on the surface of the coupled active membrane; the other side has a fluorescent FAM group that can emit green fluorescence under 494nm excitation light. Observe the coupled active membrane coupled with nucleic acid molecules under a fluorescence microscope, such as Figure 6 As shown. Figure A is an experimental fluorescence image of nucleic acid molecule coupling of the coupling active membrane in the chip top plate of the integrated coupling active membrane prepared in Example 1. Under the excitation light, uniform and strong green fluorescence was observed, indicating that the surface biological molecule coupling effect of the coupling active membrane prepared in this example is good, and further indicating that the surface coupling active functional group is highly active; and the coupling active membrane is not deformed or swollen after long-term incubation in the buffer solution, and the microcolumn array is complete and clear, indicating that the curing property is stable; Figure B is an experimental fluorescence image of nucleic acid molecule coupling of the coupling active membrane in the chip top plate of the integrated coupling active membrane prepared in Comparative Example 4. It is observed that the coupling active membrane has obvious swelling, which may be due to the insufficient time of secondary photocuring after demolding; Figure C is an experimental fluorescence image of nucleic acid molecule coupling of the coupling active membrane in the chip top plate of the integrated coupling active membrane prepared in Comparative Example 5. It is observed that the fluorescence intensity is significantly lower than that of Example 1, indicating that the coupling active membrane prepared in this comparative example has fewer biological molecules coupled, which may be due to the excessively long time of secondary photocuring after demolding, the coupling activity is destroyed, and the effect of coupling biological molecules is poor.

Claims

1. A digital microfluidic chip with an integrated coupled active membrane, comprising a chip top plate and a chip substrate, characterized in that: The chip top plate includes, from top to bottom, an upper support layer, a ground electrode layer, and an upper hydrophobic layer; The chip substrate comprises, from bottom to top, a patterned electrode layer and a lower hydrophobic layer; The lower surface of the chip top plate or the upper surface of the chip substrate is also provided with a coupling active film; The coupling active film is prepared by secondary photocuring of a photocurable raw material including coupling active groups.

2. The digital microfluidic chip with integrated coupled active membrane according to claim 1, characterized in that: The photocurable raw materials include photocurable materials, coupling agents and initiators; The photocurable material is selected from one or more monomers or oligomers containing photocurable functional group I; The coupling agent is selected from one or more monomers or oligomers containing both a photocurable functional group II and a coupling active group; The photocurable functional group I and the photocurable functional group II are independently selected from one or more of a thiol functional group, an alkenyl functional group, an acrylate functional group, a methacrylate functional group, a vinyl ether functional group, and an epoxy functional group; The coupling active group is selected from one or more of an amino group, a carboxyl group, a thiol group, an ethylene oxide group, a glycidyl group, an N-hydroxysuccinimide ester, a maleimide group, an aldehyde group, and a sulfonyl chloride group.

3. The digital microfluidic chip with integrated coupled active membrane according to claim 2, characterized in that: The photocurable material is selected from one or more of polyethylene glycol acrylate, polyurethane acrylate, polysiloxane acrylate, perfluoropolyether acrylate, mercapto-containing polysiloxane, alkenyl-containing polysiloxane, hydroxypropyl acrylate, isobornyl acrylate, ethoxylated trimethylolpropane triacrylate, 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione, trimethylolpropane diallyl ether, pentaerythritol tetrakis(3-mercaptopropionic acid), pentaerythritol tetrakis(2-mercaptoacetic acid), triallyl isocyanurate, pentaerythritol triallyl ether, trimethylolpropane triacrylate, tris[2-(3-mercaptopropionic acid)ethyl]isocyanurate, and trimethylolpropane tris(3-mercaptopropionic acid); The coupling agent is selected from one or more of glycidyl acrylate, glycidyl methacrylate, 3-mercaptopropane-N-hydroxysuccinimide ester, acrylate-N-hydroxysuccinimide ester, methacrylate-N-hydroxysuccinimide ester, N-(allyloxycarbonyloxy)succinimide, allyl glycidyl ether, 3-mercapto-1-propylamine, acrylic acid, mercaptopropionic acid, acrylamide, and pentaerythritol tetrakis-3-mercaptopropionate.

4. The digital microfluidic chip with integrated coupled active membrane according to claim 2, characterized in that: In terms of mass percentage, the composition of the photocurable raw material includes: Light-curing materials 45.0~94.5%; Coupling agent 5-50%; Initiator 0.5~5.0%; The initiator is selected from one or more of benzil compounds, alkylphenone compounds, and acylphosphine oxides.

5. The digital microfluidic chip with integrated coupled active membrane according to claim 1, characterized in that: When the coupling active film is located on the lower surface of the chip top plate, specifically on the outer surface of the upper hydrophobic layer; Alternatively, when the coupling active film is located on the upper surface of the chip substrate, specifically on the outer surface of the lower hydrophobic layer; The secondary light curing is performed before demoulding for a first time, and the curing time is 30 to 50 seconds; the second light curing is performed after demoulding for a second time, and the curing time is 10 to 40 seconds; The thickness of the coupling active film is recorded as h, and the vertical distance between the upper hydrophobic layer of the chip top plate and the lower hydrophobic layer of the chip substrate is recorded as H; Then it satisfies, 0 <h≤H / 3。 6. The digital microfluidic chip with integrated coupled active membrane according to claim 1, characterized in that: When the coupling active membrane is located on the lower surface of the chip top plate, specifically integrated with the upper hydrophobic layer; Alternatively, when the coupling active film is located on the upper surface of the chip substrate, specifically integrated with the lower hydrophobic layer; The secondary photocuring, the first photocuring is before contacting with the raw material for preparing the upper hydrophobic layer or the lower hydrophobic layer, the curing time is 5 to 10 seconds; The second photocuring is performed after contacting with the raw material for preparing the upper hydrophobic layer or the lower hydrophobic layer, and the curing time is 30 to 60 seconds.

7. The digital microfluidic chip with integrated coupled active membrane according to claim 1, characterized in that: A microstructure is arranged on the surface of the coupling active membrane; The microstructures include but are not limited to micropillar arrays; The number of the coupled active membranes is N, where N≥1.

8. A method for preparing a digital microfluidic chip with integrated coupled active membrane according to any one of claims 1 to 7, characterized in that: The steps include: When the coupling active film is located on the lower surface of the chip top plate or the upper surface of the chip substrate, the preparation process adopts scheme A): A-1) making a mold for preparing a coupling active membrane; A-2) preparing a chip top plate or a chip substrate; A-3) preparing a photocurable raw material according to the mass fraction, dropping the photocurable raw material onto the mold, covering the chip top plate or chip base, performing a primary photocuring for 30 to 50 seconds, then demoulding, and performing a secondary photocuring for 10 to 40 seconds; A-4) assembling the prepared chip substrate or chip top plate with the chip top plate or chip substrate with the coupled active membrane prepared in step A-3) to obtain a digital microfluidic chip with an integrated coupled active membrane; When the coupling active membrane is integrated with the lower surface of the chip top plate, the preparation process adopts scheme B): B-1) making a mold for preparing a coupling active membrane; B-2) preparing light-curing raw materials according to mass fraction; A hydrophobic photocurable raw material for preparing an upper hydrophobic layer is prepared according to mass fraction; B-3) dripping the photocurable raw material onto the mold, performing a primary photocuring for 5 to 10 seconds, placing the hydrophobic photocurable raw material on the mold, and covering it with an upper support layer with a ground electrode layer to form a thin layer, and performing a secondary photocuring for 30 to 60 seconds; B-4) assembling the prepared chip substrate with the chip top plate with the coupled active membrane prepared in step B-3) to obtain a digital microfluidic chip with an integrated coupled active membrane; When the coupling active film is integrated with the upper surface of the chip substrate, the preparation process adopts scheme C): C-1) making a mold for preparing a coupling active membrane; C-2) preparing light-curing raw materials according to mass fraction; A hydrophobic photocurable raw material for preparing a lower hydrophobic layer is prepared according to mass fraction; C-3) dropping the photocurable raw material onto the mold, performing a primary photocuring for 5 to 10 seconds, placing the hydrophobic photocurable raw material on the mold, and covering it with a patterned electrode with a selective dielectric layer to form a thin layer, and performing a secondary photocuring for 30 to 60 seconds; C-4) Assembling the prepared chip top plate with the chip substrate with the coupled active membrane prepared in step C-3) to obtain a digital microfluidic chip with an integrated coupled active membrane.

9. The method for preparing a digital microfluidic chip with integrated coupled active membrane according to claim 8, characterized in that: The upper hydrophobic layer and the lower hydrophobic layer are thin layers independently having hydrophobicity, with a water contact angle greater than 105° and a thickness selected from 0.1 to 50 μm; The patterned electrode layer is a substrate with a plurality of electrodes processed on the surface, and the plurality of electrodes constitute an electrode pattern, and the patterned electrode layer can be connected to an external control circuit; The substrate is selected from one or more of glass, silicon wafer, printed circuit board, plastic plate, rubber plate, photocurable resin plate; The material of the plurality of electrodes is selected from one or more of conductive metal oxides, metals, conductive polymers, conductive inks, conductive inks, and graphene; The chip substrate further comprises a dielectric layer, which is a separate thin layer and is located between the patterned electrode layer and the lower hydrophobic layer; or is combined with the lower hydrophobic layer to form a composite hydrophobic dielectric layer; When the dielectric layer is a separate thin layer, the thin layer has a high dielectric constant, a relative dielectric constant selected from 2.5 to 10, and a thickness selected from 10 to 120 μm; When the dielectric layer and the lower hydrophobic layer are combined into a composite hydrophobic dielectric layer, the relative dielectric constant of the composite hydrophobic dielectric layer is selected from 2.5 to 10, the thickness is selected from 10 to 120 μm, and the water contact angle is greater than 105°.

10. Use of the digital microfluidic chip with integrated coupled active membrane according to any one of claims 1 to 7 in the fields of biomolecule detection, microorganism and cell detection, separation, culture, and nucleic acid aptamer screening.

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