A multi-sample flux multi-index detection device based on digital droplet microfluidics and application thereof

Abstract

The application discloses a kind of based on digital droplet microfluidic multi-sample flux multi-index detection device and application thereof, belong to electromechanical device, microfluidic chip and immunoprotein analysis field.The device includes droplet microfluidic equipment and immunoprotein analysis chip component, droplet microfluidic equipment is used to drive multiple droplets, mainly divided into three major systems: power supply system, computer operating system and chip clamp system, immunoprotein analysis chip component includes biological analysis substrate and microarray chip.The present application drives micro liquid sample by digital droplet microfluidic technology, realizes the multi-index detection of automated immunoprotein analysis chip.The detection device of the present application has a series of advantages such as rapidity, independence, low consumption, automation, function integration and multi-index detection, and is expected to become the most fruitful rapid diagnostic device for improving tumor diagnosis positive rate.

Description

Technical Field

[0001] This invention belongs to the fields of electromechanical equipment, microfluidic chips and immune protein analysis technology, specifically relating to a multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) and its application. Background Technology

[0002] Traditional cytokine detection typically uses a single indicator, making it prone to false negatives and false positives. Multi-indicator detection technology, based on the chemiluminescence detection principle of a double-antibody sandwich method, coats a solid matrix with multiple capture antibodies to capture corresponding markers in the sample. These markers are then combined with detection antibodies labeled with fluorescent groups, and the fluorescence signal is read by a fluorescence scanner for qualitative and quantitative detection, reducing the incidence of false positives and false negatives. However, this technology suffers from drawbacks such as frequent liquid blockage due to excessively long or narrow channels in the chip, lengthy sample incubation and immunoassay chip preparation times, and difficulty in automation, thus failing to reduce manual labor requirements.

[0003] Digital droplet microfluidics (DMF) is a droplet technology based on the control of individual discrete droplets moving in a plane. It can control liquids without the risk of clogging, is applicable to various biological liquid phases, and features low sample consumption, fast reaction, high parallelism, and automation capabilities. It does not require components such as microchannels, micropumps, and microvalves, is simple to construct, and can be dynamically configured. However, research on combining DMF technology with immunoprotein analysis to automate multi-parameter detection and solve problems such as long sample incubation time and liquid clogging within the channels has not yet been reported. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) and its application.

[0005] The objective of this invention is achieved through the following means:

[0006] This invention provides a multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF). By combining a digital droplet microfluidic driving chip with an immune protein multi-index detection chip, the preparation of the multi-index detection chip is automated. Its sensitivity is comparable to that of traditional multi-index detection, but it does not have the problem of liquid blockage in the channel and effectively reduces the time for sample incubation and chip preparation.

[0007] A multi-sample throughput and multi-index detection device based on digital droplet microfluidics is disclosed. The device includes a droplet microfluidic device and an immunoprotein analysis chip assembly. The droplet microfluidic device is used to drive multiple droplets and mainly includes three systems: a power supply system, a computer operating system, and a chip clamping system. The immunoprotein analysis chip assembly includes a bioanalytical substrate and a microarray chip. The microarray chip is used to assist in obtaining multiple different capture antibody barcode arrays on the bioanalytical substrate. The bioanalytical substrate (bioanalytical chip) with multiple different capture antibody barcode arrays is used to detect and analyze target molecules in the sample. The chip clamping system mainly includes fixing bolts and clamps. The bioanalytical substrate with multiple different capture antibody barcode arrays, spacers, and chrome plates are stacked sequentially from top to bottom and fixed by the chip clamping system.

[0008] The bioanalytical substrate is a silica glass slide coated from the glass surface to the surface with indium tin oxide (ITO), perfluorooctyltrimethoxysilane, and a hydrophobic material, including Teflon and polylysine.

[0009] Furthermore, the microarray chip is a stripe array chip.

[0010] Furthermore, the strip array is a parallel array of 10-100 strips with a specific width and spacing, wherein the specific width is 10-200µm and the spacing between each strip is 10-200µm.

[0011] Furthermore, the chip is made of polydimethylsiloxane (PDMS).

[0012] Furthermore, the chromium plate multi-sample throughput chip can achieve 10 throughputs per cycle.

[0013] Furthermore, the microarray chip is a PDMS microarray chip, and its fabrication method mainly includes the following steps:

[0014] 1) Mixing: Weigh RTV 615 type PDMS prepolymer A and B in a ratio of 10:1, mix and stir evenly, then place in a vacuum dryer and vacuum for 20-60 minutes to remove air bubbles;

[0015] 2) Pour the adhesive: Pour the obtained RTV 615 mixed adhesive onto the chip template, remove any residual air bubbles, place the template in a vacuum dryer and evacuate for 2-10 minutes, continue to remove any residual air bubbles, and let it stand for 5-10 minutes.

[0016] 3) Curing: Bake in an oven at 80℃ for 40 minutes to fully cure the RTV 615 mixed adhesive. After cooling to room temperature, cut it to the appropriate size using a scalpel and punch holes with a hole puncher to obtain the PDMS chip.

[0017] 4) Cleaning: Immerse the PDMS chip in anhydrous ethanol and sonicate for 5-30 minutes. Clean it once with deionized water and sonicate for 5-30 minutes. Blow it dry and bake it in an oven at 80℃ for 3-10 minutes. Cool it to room temperature.

[0018] Furthermore, the method for preparing the chip template mainly includes the following steps:

[0019] 1) Mask design: Use computer-aided design software to draw the mask design drawing and print the mask;

[0020] 2) Remove the negative photoresist from the refrigerator and allow it to return to room temperature before use; determine the experimental conditions according to the required thickness of the structure. The photoresist required for this experiment is SU8-3035 with a thickness of approximately 30 μm.

[0021] 3) Cleaning the glass slide: Immerse the glass slide in anhydrous ethanol and ultrasonically clean for 5-30 minutes. Rinse once with deionized water and then ultrasonically clean for 5-30 minutes. Blow dry and bake on a hot plate at 120℃ for 5-20 minutes. Cool to room temperature.

[0022] 4) Spin coating of photoresist: Place the glass slide in a plasma cleaner and treat it with oxygen plasma for 5-20 minutes. Pour SU8-3035 photoresist onto the glass slide to cover 4 / 5 of the area. Remove air bubbles from the photoresist and then spin coat at 2500 rpm for 60 seconds.

[0023] 5) Pre-baking: Bake at 95℃ for 5-15 minutes on a hot plate to remove solvent from the adhesive layer, improve the adhesion between the photoresist and the silicon wafer and the mechanical abrasion resistance of the adhesive film, and cool to room temperature;

[0024] 6) Exposure: Place the glass slide on the stage of the UV exposure machine. The side of the mask with the printed pattern should be bonded to the SU8-3035 photoresist coating. Use another glass slide to flatten the mask to ensure a tight bond. Expose at a UV intensity of 10.0 mW / cm². 2 Expose for 30 seconds under the specified conditions (5 seconds / time * 6 times, with a 10-second interval between times) to transfer the pattern onto the photoresist layer through exposure;

[0025] 7) Post-baking: Bake on a hot plate at 95℃ for 3-5 minutes until the pattern appears, then cool to room temperature;

[0026] 8) Development: Use ethyl lactate to remove the uncured SU8-3035 photoresist and develop; after development, wash off the ethyl lactate with isopropanol; after washing, rinse off the isopropanol residue with ddH2O, blow dry and examine under a microscope.

[0027] 9) Hardening: Bake at 165℃ for 20-40 minutes on a hot plate to evaporate the solvent and ensure the photoresist adheres firmly to the silicon wafer. Cool to room temperature.

[0028] 10) Surface treatment: Trimethylchlorosilane is used to hydrophobize the surface to form a hydrophobic film, which facilitates the peeling of the cured PDMS from the template without damaging the template structure.

[0029] 11) Cofferdam: Surround the glass sheet with plastic and secure it with high-temperature resistant tape to prevent leakage of fluid PDMS.

[0030] Furthermore, the preparation method of the bioanalytical substrate mainly includes the following steps:

[0031] 1) Preparation of 0.4% perfluorooctyltrimethoxysilane solution: Mix anhydrous ethanol and deionized water in a ratio of 9:1 to prepare a 90% ethanol solution; add 20 μL of 37% hydrochloric acid and 40 μL of perfluorooctyltrimethoxysilane stock solution to every 10 mL of 90% ethanol solution to prepare the solution. After protecting from light and mixing thoroughly for at least 5 hours, use within 24 hours.

[0032] 2) Cleaning indium tin oxide (ITO) glass: Immerse the ITO glass in RCA solution (28% ammonia, 30% hydrogen peroxide and ddH2O in a ratio of 1:1:6) and ultrasonically clean for 10-30 minutes. After rinsing with deionized water once, ultrasonically clean for 5-10 minutes, blow dry, bake in an oven at 120℃ for 10-30 minutes, and cool to room temperature.

[0033] 3) Mark the non-ITO side: Use a multimeter to measure the resistance of the two sides of the ITO. Mark the side that shows "1" as the non-ITO side; the other side is the ITO side and will be coated with subsequent materials.

[0034] 4) Coating adhesive layer: Place the obtained ITO glass in a plasma cleaner and treat it with oxygen plasma for 5-30 min, immerse it in 0.4% perfluorooctyltrimethoxysilane solution and let it stand for 10-30 min, then immediately bake it with a hot plate at 120℃ for 20-40 min, and cool it to room temperature.

[0035] 5) Coating with a hydrophobic layer:

[0036] ① Dilute Teflon-AF 1600 (stock solution: FC-40 ratio is 1:5); spin coat 1% Teflon-AF 1600 at 2000 rpm for 60 seconds, bake at 165℃ for 10-15 minutes on a hot plate, then increase the temperature to 330℃ for 10-15 minutes, cool to room temperature, and mark "TF-ITO" on the non-ITO side.

[0037] ② Spin coat 1% Cytop at 2000 rpm for 60 seconds, bake at 180°C on a hot plate for 15-20 minutes, cool to room temperature, and mark “TF-ITO” on the non-ITO side; or spin coat 1% FluoroPel PFC1601V-FS at 2000 rpm for 60 seconds, bake at 180°C on a hot plate for 15-20 minutes, cool to room temperature, and mark “TF-ITO” on the non-ITO side.

[0038] Furthermore, the preparation method of the chromium plate multi-sample throughput chip mainly includes the following steps:

[0039] 1. Chromium plate patterning: The commercial chromium plate used in this experiment is a 120nm thick layer of metallic chromium plated on glass, and the surface of the chromium is covered with a layer of AZ1505 positive photoresist.

[0040] 1) Mask design: Use computer-aided design software to draw the mask design drawing and print the mask;

[0041] 2) Exposure: Place the commercial chrome plate on the stage of the UV exposure machine. The side of the mask with the printed pattern is bonded to the AZ1505 coating on the chrome plate. A piece of glass is used to flatten the mask to ensure a tight bond. Exposure is performed at a UV intensity of 10.0 mW / cm². 2 Under the condition of exposure for 5 seconds, that is, W = PT = 50w;

[0042] 3) Development: Immerse the obtained chromium plate in AZ-MIF-300 developer for at least 1 minute until the AZ1505 photoresist in the ultraviolet (UV) irradiated area dissolves and the pattern is clearly visible. Rinse three times with deionized water for a total time of at least 2 minutes, and then blow dry with an air gun.

[0043] 4) Etching: Immerse the obtained chromium plate in cerium ammonium nitrate acetic acid etching solution (20% cerium ammonium nitrate, 1% acetic acid, deionized water as solvent) until the chromium not covered by AZ1505 photoresist is completely dissolved, rinse three times with deionized water, and blow dry with an air gun.

[0044] 5) Remove photoresist coating: Immerse the patterned chromium plate in N-methylpyrrolidone (NMP) for 2-10 minutes, wipe the entire chromium plate surface with a lint-free cloth soaked in acetone, immerse again in NMP for 1-5 minutes to completely remove the AZ1505 photoresist coating on the patterned surface, rinse three times with deionized water, blow dry with an air gun, bake at 120℃ for 10 minutes, and cool to room temperature;

[0045] 6) Protect the pins: Protect the pins on both the left and right ends of the chip with high-temperature resistant tape;

[0046] 7) Coating with insulating layer: The obtained patterned chromium plate is placed in a plasma cleaner and treated with oxygen plasma for 5-30 min. SU8-3010 is poured onto the chromium plate chip to cover all electrodes. Spin coating is performed at 4000 rpm for 60 s. The plate is then baked at 95°C for 5-10 min, cooled to room temperature, and then subjected to a light intensity of 10.0 mW / cm². 2 Expose for 30 seconds under the following conditions (5 seconds / time * 6 times, with a 10-second interval between two times), bake at 95℃ on a hot plate for 5-10 minutes, and then cool to room temperature;

[0047] 8) Coating with a hydrophobic layer: Spin-coat 1% Teflon-AF 1600 at 2000 rpm for 60 seconds, bake at 165°C on a hot plate for 20-40 minutes, and then cool to room temperature;

[0048] 9) Expose pins: Remove high-temperature tape.

[0049] In another aspect, the present invention provides an application of the above-mentioned multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF), the device being used to simultaneously capture, detect and analyze different cytokines in a variety of different substances.

[0050] Furthermore, the multi-sample throughput multi-index detection includes the analysis of 1-32 throughputs and 1-200 indicators.

[0051] Furthermore, the various substances include, but are not limited to, cell culture medium, serum, and standard proteins.

[0052] Furthermore, the cells include, but are not limited to, M1 type mononuclear macrophages and OSCC cells.

[0053] Furthermore, the serum includes, but is not limited to, normal individuals, patients with immune system diseases, and patients with other diseases involving abnormal cytokine secretion.

[0054] Furthermore, the analysis includes qualitative or quantitative detection of various substances, correlation analysis, or immunophenotyping analysis.

[0055] The specific steps of capture, detection, and analysis based on digital droplet microfluidics-based multi-throughput, multi-parameter detection technology are as follows:

[0056] Take out the bioanalytical substrate coated with multiple capture antibodies to form an antibody barcode array, align the antibody-coated side with the lower electrode of the DMF chromium plate multi-sample throughput chip and fix it with a clamp, then sequentially introduce the sample to be tested, the anti-target protein antibody connected to ligand A and the receptor B-fluorescein for incubation, remove the clamp and perform detection on the bioanalytical substrate.

[0057] The advantages of this invention over the prior art are as follows:

[0058] 1. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention is applicable to a variety of biological liquid phases, and can control the liquid while avoiding the risk of blockage.

[0059] 2. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention can process each reagent individually without the need for complex pipelines or micro-valve networks.

[0060] 3. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention has low sample consumption but is compatible with a wide range of solution volumes, making it very suitable for the detection and analysis of liquid samples.

[0061] 4. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention has high parallelism and can realize the simultaneous detection of multiple or single research objects with multiple indicators.

[0062] 5. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention has a high degree of automation capability to achieve dynamic configuration.

[0063] 6. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) of the present invention uses a chromium plate multi-throughput chip that is highly compatible with microlithography technology, is simple to manufacture, and can be reused to reduce costs.

[0064] 7. The microarray chip of the present invention is simple to manufacture, uses inexpensive materials, requires a small amount of antibody, and can be widely applied to high-throughput, multi-index immune protein analysis.

[0065] 8. The surface of the bioanalytical substrate of the present invention is a hydrophobic Teflon material. The interaction between proteins and the contact surface usually increases with the increase of surface hydrophobicity and proteins usually do not desorb. Therefore, this chip has a better protein adsorption effect and is more conducive to protein analysis. Attached Figure Description

[0066] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0067] Figure 1 The diagram (A) and the fixture structure (B) of the multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF) in Embodiment 1 of the present invention are shown below. In the diagram, 1: computer system, 2: power supply system, 3: video acquisition, 4: fixture device, 5: control panel, 6: connecting device, 7: fixing bolt, 8: clamping plate, 9: immune protein analysis chip, 10: spacer, 11: droplet, 12: chrome plate multi-sample throughput chip.

[0068] Figure 2 This is a schematic diagram of the bioanalytical substrate structure in Example 1, where 1: glass slide, 2: indium tin oxide, 3: perfluorooctyltrimethoxysilane, and 4: Teflon (Teflon-AF 1600).

[0069] Figure 3 The diagram shows the connection of the microarray chip in Example 1 and the diagram of the PDMS microarray chip set in a specific area, where 1: capture antibody entry and exit pore.

[0070] Figure 4 The diagram shows the connection method (A) and the structural diagram (B) of the chromium plate multi-sample throughput chip in Example 1, where 1: glass plate, 2: chromium plate, 3: photoresist (SUS-3010), and 4: Teflon (Teflon-AF 1600).

[0071] Figure 5 This is a schematic diagram and partial detailed structural diagram of the assembly of the bioanalytical chip coated with capture antibodies, spacer, and chromium plate multi-sample throughput chip in the multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF) in Example 1.

[0072] Figure 6 This is a schematic diagram of the preset test area of ​​the multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF) for population cell extracellular cytokine analysis in Example 1, wherein the transparent square area is the signal detection area.

[0073] Figure 7 This is a scanned image showing the results of the extracellular cytokine analysis experiment of the M1 type monocyte / macrophage population in Example 1. In the image, 0 / 0-4 represents culture medium used to stimulate monocytes / macrophages with 0 ng / ml lipopolysaccharide (LPS) for 0-4 hours; 0 / 4-8 represents culture medium used to stimulate monocytes / macrophages with 0 ng / ml LPS for 4-8 hours; 0 / 8-12, 0 / 12-16, and 0 / 16-24 have the same meaning; 10 / 0-4 represents culture medium used to stimulate monocytes / macrophages with 0 ng / ml LPS for 4-8 hours. The culture medium for stimulating monocytes and macrophages with 10 ng / ml lipopolysaccharide (LPS) for 0-4 hours, 10 / 4-8, 10 / 8-12, 10 / 12-16, and 10 / 16-24 have the same meaning as above; 100 / 0-4 indicates that the culture medium for stimulating monocytes and macrophages with 100 ng / ml lipopolysaccharide (LPS) for 0-4 hours, 100 / 4-8, 100 / 8-12, 100 / 12-16, and 100 / 16-24 have the same meaning as above.

[0074] Figure 8 This is a statistical analysis of the fluorescence intensity results of various signals in the extracellular cytokine analysis experiment of the M1 type mononuclear macrophage population in Example 1.

[0075] Figure 9 This is a scanned image of the standard curve experimental results for the protein standard in Example 2.

[0076] Figure 10 This is a graph showing the experimental results of the standard curve of the protein standard in Example 2.

[0077] Figure 11 This is a scan of the fluorescence signal interference experiment results for the protein standard in Example 2.

[0078] Figure 12 This is a graph showing the results of the fluorescence signal interference experiment of the protein standard in Example 2.

[0079] Figure 13 This is a scanned image showing the results of the extracellular cytokine analysis experiment of the OSCC cell population in Example 3.

[0080] Figure 14 This is a statistical analysis of the fluorescence intensity results of various signals in the OSCC cell population extracellular cytokine analysis experiment in Example 3. Detailed Implementation

[0081] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0082] All experimental reagents used in this invention are commercially available, as shown in Table 1.

[0083] Table 1: List of reagents used in the examples

[0084]

[0085]

[0086]

[0087] Example 1

[0088] This embodiment provides a multi-sample throughput and multi-index detection device based on digital droplet microfluidics (DMF), such as... Figures 1 to 6 As shown, this involves the fabrication of a chip based on digital droplet microfluidics (DMF) for multi-sample throughput and multi-index detection technology, and its application in the analysis of extracellular cytokines in M1 type mononuclear macrophages to understand the functional state of immune cells and their response to pathogen stimuli, such as... Figure 1 As shown.

[0089] This embodiment of the multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF) includes a droplet microfluidic device and an immunoprotein analysis chip assembly. The droplet microfluidic device is used to drive multiple droplets and mainly includes three systems: a power supply system, a computer operating system, and a chip clamping system. The immunoprotein analysis chip assembly includes a bioanalytical substrate and a microarray chip. The microarray chip is used to assist in obtaining multiple different capture antibody barcode arrays on the bioanalytical substrate. The bioanalytical substrate with multiple different capture antibody barcode arrays is used to detect and analyze target molecules in the sample. The chip clamping system mainly includes fixing bolts and clamps. The bioanalytical substrate with multiple different capture antibody barcode arrays, spacers, and chrome plates are stacked sequentially from top to bottom and fixed by the chip clamping system.

[0090] The biological analysis substrate is a silica glass (TF-ITO slide) coated from the glass surface to the top with indium tin oxide (ITO), perfluorooctyltrimethoxysilane, and Teflon AF-1600, respectively. Figure 2 As shown, it is used to incubate antibodies, form antibody arrays and proteins, and detect and analyze target molecules in samples;

[0091] The microarray chip consists of 20 parallel PDMS (polydimethylsiloxane) stripes, each 100µm in spacing and width, modified with trimethylchlorosilane. It provides a set of independent detection channels to assist in obtaining barcode arrays with various capture antibodies on a bioanalytical substrate, such as... Figure 3 As shown, 20 types of antibodies can be incubated each time, and the types of antibodies can be varied and selected according to the cells.

[0092] The chromium plate multi-sample throughput chip is used for automated control of liquid flow, avoiding liquid blockage in the channel, and can achieve a throughput of 10 samples per cycle. Figure 4 As shown.

[0093] The aforementioned multi-sample throughput multi-index detection device based on digital droplet microfluidics (DMF) can complete the detection of extracellular cytokines in a population of cells within 3 hours. The overall structural diagram and detailed structural diagram of the assembled multi-sample throughput chip, which includes a bioanalytical chip coated with capture antibodies, spacers, and a chromium plate, are shown below. Figure 5 As shown, the preset test area is as follows: Figure 6 The white area in the middle is shown.

[0094] The fabrication method of the PDMS microarray chip is as follows:

[0095] 1. Template fabrication using soft photolithography: This step involves etching geometric patterns onto a photoresist layer using exposure and development, followed by etching to transfer the pattern from the mask onto a substrate. The substrate used in this invention is a square glass with a side length of 10cm. The PDMS microarray chip specifications are as follows: a strip array of 20 parallel stripes, each with a spacing and width of 100µm.

[0096] 1) Mask design: Use computer-aided design software to draw mask design drawings and print masks.

[0097] 2) Remove the negative photoresist from the refrigerator and allow it to return to room temperature before use; determine the experimental conditions according to the required structure thickness. The photoresist used in this experiment is SU8-3035 with a thickness of 30μm.

[0098] 3) Cleaning the glass slide: Immerse the glass slide in anhydrous ethanol for more than 10 minutes, rinse it once with deionized water for more than 10 minutes, blow it dry with an air gun, bake it on a hot plate at 120°C for 10 minutes, and cool it to room temperature.

[0099] 4) Spin coating of photoresist: Place the glass slide in a plasma cleaner and treat it with oxygen plasma for 10 minutes. Pour SU8-3035 photoresist onto the glass slide until it covers about 4 / 5 of the area. Remove air bubbles from the photoresist with a pipette and then spin coat (2500 rpm, 60 seconds).

[0100] 5) Pre-baking: Bake at 95℃ for 15 minutes on a hot plate to remove solvent from the photoresist layer, improve the adhesion between the photoresist and the silicon wafer, and enhance the mechanical abrasion resistance of the photoresist film. Cool to room temperature.

[0101] 6) Exposure: Place the glass slide on the stage of the UV exposure machine. The side of the mask with the printed pattern should be bonded to the SU8-3035 photoresist coating. Use another glass slide to flatten the mask to ensure a tight bond. Expose at a UV intensity of 10.0 mW / cm². 2 Expose for 30 seconds under the specified conditions (5 seconds / time * 6 times, with a 10-second interval between times) to transfer the pattern onto the photoresist layer through exposure.

[0102] 7) Post-baking: Bake on a hot plate at 95℃ for 3-5 minutes until the pattern appears, then cool to room temperature.

[0103] 8) Development: Use ethyl lactate to remove the uncured SU8-3035 photoresist and develop; after development, wash off the ethyl lactate with isopropanol; after washing, rinse off the isopropanol residue with ddH2O, blow dry with an air gun and examine under a microscope.

[0104] 9) Hardening: Bake at 165℃ for 30 minutes on a hot plate to allow the solvent to evaporate so that the photoresist adheres firmly to the silicon wafer, and then cool to room temperature.

[0105] 10) Surface treatment: Trimethylchlorosilane is used to hydrophobize the surface to form a hydrophobic film, which facilitates the peeling of the cured PDMS from the template without damaging the template structure.

[0106] 11) Cofferdam: Surround the glass sheet with plastic and secure it with high-temperature resistant tape to prevent leakage of fluid PDMS.

[0107] 2. Chip infusion template:

[0108] 1) Mixing the adhesive: Weigh RTV 615 type A adhesive and B adhesive in a ratio of 10:1, stir evenly with a glass rod, place in a vacuum desiccator and vacuum for about 30 minutes, and take it out when the air bubbles are basically removed;

[0109] 2) Pour the adhesive: Pour the RTV 615 mixed adhesive onto the chip template, remove any remaining air bubbles with a suction bulb, place the template in a vacuum dryer and evacuate for about 5 minutes, then remove it and use the suction bulb again to remove any remaining air bubbles, and let it stand for 5 minutes.

[0110] 3) Curing: Bake in an oven at 80℃ for 40 minutes to fully cure the RTV 615 mixed adhesive. After cooling to room temperature, cut it to the appropriate size with a scalpel and punch holes as needed.

[0111] 4) Cleaning: Immerse the PDMS chip in anhydrous ethanol and sonicate for 10 minutes, clean it once with deionized water and sonicate for 10 minutes, blow it dry with an air gun, bake it in an oven at 80°C for 5 minutes, and cool it to room temperature.

[0112] The preparation method of the bioanalytical substrate is as follows:

[0113] 1. Preparation of TF-ITO glass slides:

[0114] 1) Preparation of 0.4% perfluorooctyltrimethoxysilane solution: Mix anhydrous ethanol and deionized water in a ratio of 9:1 to prepare a 90% ethanol solution; add 20 μL of 37% hydrochloric acid and 40 μL of perfluorooctyltrimethoxysilane stock solution to every 10 mL of 90% ethanol solution to prepare the solution. After mixing continuously for at least 5 h in the dark, use within 24 h.

[0115] 2) Cleaning indium tin oxide (ITO) glass: Immerse the ITO in RCA solution (28% ammonia, 30% hydrogen peroxide and ddH2O in a ratio of 1:1:6) and ultrasonically clean for 20 minutes. After rinsing with deionized water once, ultrasonically clean for 10 minutes, blow dry with an air gun, bake in an oven at 120℃ for 15 minutes, and cool to room temperature.

[0116] 3) Mark the non-ITO side: Use a multimeter to measure the resistance of the two sides of the ITO. Mark the side that shows "1" as the non-ITO side; the other side is the ITO side and will be covered with subsequent materials.

[0117] 4) Coating adhesive layer: The obtained ITO glass was placed in a plasma cleaner for oxygen plasma treatment for 10 min, immersed in 0.4% perfluorooctyltrimethoxysilane solution and left to stand for 20 min, and then immediately baked at 120℃ on a hot plate for 30 min, and cooled to room temperature.

[0118] 5) Coating with a hydrophobic layer: Dilute Teflon-AF 1600 (stock solution: FC-40 ratio of 1:5); spin-coat 1% Teflon (spin speed 2000 rpm, duration 60 s), heat to 165℃ for 15 min on a hot plate, then heat to 330℃ for 15 min, cool to room temperature, and mark “TF-ITO” on the non-ITO side.

[0119] 2. Antibody coating: forming a bioanalytical substrate with an antibody barcode array.

[0120] 1) Incubation: Attach the TF-ITO slide and PDMS chip; using a pipette, add 3 μL of capture antibody (stock solution and DPBS solution ratio 1:3) to each of the 20 channels from the beginning of the channel. Use a vacuum pump to aspirate from the channel outlet to fill the entire channel with antibody. Incubate at 37°C for 1 hour, then aspirate completely. The channel starting position is as follows: Figure 3 As shown in section A01-20, the channel exit location is as follows: Figure 3 As shown in Figures 01-20 at the B-terminus, the corresponding capture antibodies are: anti-CCL2, CCL3, CCL4, IL-1b, IL-6, IL-8, IL-10, IP-10, TNF-a, and GM-CSF purified capture antibodies.

[0121] 2) Blocking: Prepare a 2% BSA solution, pass the solution through to wash once, block at room temperature for 20 minutes, and then aspirate.

[0122] 3) Cleaning: Prepare a 50% DPBS solution (the ratio of DPBS solution to deionized water is 1:1); after removing the PDMS chip, clean the TF-ITO slide with the antibody barcode array in sequence with DPBS, 50% DPBS solution and deionized water, spin dry the slide and store it for later use (use within 1 week).

[0123] The preparation method of the multi-sample throughput chip (chrome plate) is as follows:

[0124] 1. Chromium plate patterning: The commercial chromium plate used in this experiment is a 120nm thick layer of metallic chromium plated on glass, and the surface of the chromium is covered with a layer of AZ1505 positive photoresist.

[0125] 1) Mask design: Use computer-aided design software to draw mask design drawings and print masks.

[0126] 2) Exposure: Place the chromium plate on the stage of the UV exposure machine, and attach the mask with the AZ1505 coating on the chromium plate. Use a piece of glass to flatten the mask to ensure a tight fit. Expose for 5 seconds under a light intensity of 10.0mW / cm2, i.e., W=PT=50w.

[0127] 3) Development: Immerse the chromium plate in AZ-MIF-300 developer for at least 1 minute until the AZ1505 photoresist in the ultraviolet (UV) irradiated area dissolves and the pattern is clearly visible. Rinse three times with deionized water for a total time of at least 2 minutes, and then blow dry with an air gun.

[0128] 4) Etching: The chromium plate is immersed in cerium ammonium nitrate acetic acid etching solution (20% cerium ammonium nitrate, 1% acetic acid, and deionized water as solvent) for about 1 minute until the chromium not covered by AZ1505 photoresist is completely dissolved. Rinse three times with deionized water and blow dry with an air gun. This completes the replication of the design pattern to the chromium layer.

[0129] 5) Remove photoresist coating: Immerse the patterned chromium plate in N-methylpyrrolidone (NMP) for 2 minutes, wipe the entire chromium plate surface with a lint-free cloth soaked in acetone, immerse again in NMP for 1 minute to completely remove the AZ1505 photoresist coating on the patterned surface, rinse three times with deionized water, blow dry with an air gun, bake at 120°C for 10 minutes, and cool to room temperature.

[0130] 6) Protect the pins: Use high-temperature resistant tape to protect the pins on both sides of the chip to prepare for the next step of spin coating the insulating layer and hydrophobic layer.

[0131] 2. Preparation of the insulating and hydrophobic layers of the chromium plate: The insulating layer used in this experiment is SU8-3010 negative photoresist, and the hydrophobic layer is 1% Teflon-AF 1600.

[0132] 1) Remove SU8-3010 from the refrigerator and allow it to return to room temperature before use.

[0133] 2) Coating with insulating layer: The patterned chromium plate was placed in a plasma cleaner and treated with oxygen plasma for 10 min. SU8-3010 was poured onto the chip to cover all electrodes, and then spin-coated (3000 rpm, 60 s). It was then baked on a hot plate at 95°C for 10 min, cooled to room temperature, and then coated under light at an intensity of 10.0 mW / cm². 2Expose for 30 seconds (5 seconds per exposure * 6 exposures, with a 10-second interval between exposures) under the specified conditions, bake at 95°C for 5 minutes on a hot plate, and then cool to room temperature.

[0134] 3) Coating with a hydrophobic layer: Spin-coat with 1% Teflon-AF 1600 (2000 rpm, 60 s), bake at 165°C on a hot plate for 30 min, and cool to room temperature.

[0135] 4) Exposed pins: Remove the high-temperature tape.

[0136] The process of analyzing extracellular cytokines in M1 type monocytes and macrophages is as follows:

[0137] 1. Collection of conditioned culture medium for M1 type mononuclear macrophages:

[0138] 1) Preparation of complete culture medium: Filter PAN serum through a 0.22um filter membrane and mix well; prepare complete culture medium according to the ratio of 79% RPMI medium, 20% FBS and 1% penicillin-streptomycin, mix well and set aside.

[0139] 2) Culture U937: Culture U937 in complete culture medium until the cell density reaches 80%.

[0140] 3) PMA differentiation of U937 into MΦ: Take U937 cells and mix well, transfer to a centrifuge tube and centrifuge (1000 rpm, 5 min), discard the supernatant, add 1 ml of complete culture medium, disperse and mix the cells; count the cells, and dilute with complete culture medium to a cell density of 1*102. 6 cells / ml; add an appropriate amount of 1 mg / ml PMA to the cell suspension to make the concentration 50 ng / ml, mix well, put into a culture dish / well plate, and place in a 37℃ 5% CO2 cell culture incubator for 48 h of differentiation, then replace with PMA-free complete medium and continue incubation for 24 h.

[0141] 4) LPS stimulation of MΦ: Dilute 1 mg / ml LPS to concentrations of 10 ng / ml and 100 ng / ml using FBS-free RPMI medium; add medium containing 0 ng / ml, 10 ng / ml and 100 ng / ml LPS to MΦ culture dishes / well plates respectively, and collect conditioned culture solutions for 0-4hrs, 4-8hrs, 8-12hrs, 12-16hrs and 16-24hrs, aliquot and store at -40℃ for later use.

[0142] 2. Detection of extracellular cytokines in M1 type monocytes and macrophages (DMF platform): 0.1% Tetronic 90R4 solution was used as solvent; the incubation conditions were room temperature, protected from light, and humidified; during sample addition and incubation, the droplets were driven to the pre-set position, and the waste droplets were driven to the rightmost waste liquid pool and aspirated with lint-free paper.

[0143] 1) Capture protein: Add M1 type mononuclear macrophage conditioned culture medium droplets and incubate for 30 min;

[0144] 2) Add detection antibodies (biotin): Prepare 0.1% Tetronic 90R4 solution and biotin solution (the ratio of biotin stock solution to 0.1% Tetronic 90R4 solution is 1:199); add biotin and incubate for 30 min; the biotin are biotin against CCL2, CCL3, CCL4, IL-1b, IL-6, IL-8, IL-10, IP-10, TNF-α, and GM-CSF respectively.

[0145] 3) Add the fluorescent dye Streptavidin-APC: Prepare Streptavidin-APC (APC stock solution and 0.1% Tetronic 90R4 solution in a ratio of 1:99); add Streptavidin-APC droplets and incubate for 15 min.

[0146] 4) Cleaning: Prepare 50% DPBS droplets (DPBS droplets and deionized water droplets in a 1:1 ratio), and add DPBS droplets, 50% DPBS droplets, and deionized water droplets in sequence for cleaning.

[0147] 5) Scanning: The slides were scanned and analyzed using a four-color laser chip scanner (Genepix+4300A).

[0148] 6) Data processing and statistical analysis: See the results of this embodiment. Figure 7-8 . Figure 7 The results of the slide scan show extracellular cytokine signals in the population of cells. Figure 8 for Figure 7 Statistical analysis of fluorescence intensity results for various signals.

[0149] Example 2

[0150] This embodiment uses the digital droplet microfluidic (DMF) based multi-sample throughput multi-index detection device from Example 1 for the detection of protein standards to complete the plotting of standard curves. It employs the PDMS microarray channel chip and chromium plate multi-sample throughput chip prepared in Example 1; specifically, it includes the following steps:

[0151] Protein standard detection (DMF platform): Tetronic 90R4 solution was used as solvent; incubation conditions were room temperature, protected from light, and humidified; during sample addition and incubation, droplets were driven to a pre-set position, and waste droplets were driven to the rightmost waste pool and removed with lint-free paper.

[0152] 1) Sample Preparation: Each standard (CCL2, CCL3, CCL4, IL-1a, IL-1b, IL-8, IL-10, IP-10, TNF-a, GM-CSF) contains 0.1% Tetronic 90R4. Standard Curve Experiment - Sample Preparation: Mix the original concentrations of CCL2, CCL3, CCL4, GM-CSF (1 μg / ml) and IL-1b (250 ng / ml) with the original concentrations of IL-1a, IL-8, IL-10 (150 ng / ml), TNF-a (15 ng / ml), and IP-10 (70 ng / ml) in a corresponding ratio to achieve their initial concentrations. Then, serially dilute the initial concentrations of the protein standards 5-fold. The initial concentrations of all proteins, except IP-10 (35 ng / ml) and TNF-a (10 ng / ml), are 100 ng / ml. Interference Experiment Sample Preparation: Dilute each original concentration standard to 10 ng / ml.

[0153] 2) Incubate the protein: Add protein standard droplets and incubate for 30 minutes.

[0154] 3) Add detection antibodies (biotin): Prepare 0.1% Tetronic 90R4 solution and biotin solution (the ratio of biotin stock solution to 0.1% Tetronic 90R4 solution is 1:199); add biotin and incubate for 30 min; the biotin are biotin against CCL2, CCL3, CCL4, IL-1a, IL-1b, IL-8, IL-10, IP-10, TNF-a, and GM-CSF respectively.

[0155] 4) Add the fluorescent dye Streptavidin-APC: Prepare Streptavidin-APC (APC stock solution and 0.1% Tetronic 90R4 solution in a ratio of 1:99); add Streptavidin-APC droplets and incubate for 15 min.

[0156] 5) Cleaning: Prepare 50% DPBS droplets (DPBS droplets and deionized water droplets in a 1:1 ratio), and add DPBS droplets, 50% DPBS droplets, and deionized water droplets in sequence for cleaning.

[0157] 6) Scanning: The slides were scanned and analyzed using a four-color laser chip scanner (Genepix+4300A).

[0158] 7) Data processing and statistical analysis: See the results of this embodiment. Figure 9-12 . Figure 9 This is a scanned image of the experimental results of the standard curve of protein standards, showing the fluorescence signal of the protein standards. Figure 10 for Figure 9Standard curves for each protein standard. Figure 11 This is a scan of the experimental results showing interference from the fluorescence signal of the protein standard, where the fluorescence signal of the protein standard is visible. Figure 12 for Figure 11 Analysis results of fluorescence signal interference experiment between protein standards.

[0159] Example 3

[0160] This embodiment uses the digital droplet microfluidic (DMF) based multi-sample throughput multi-index detection device from Example 1 for the analysis of extracellular cytokines in OSCC cells, to understand the response of OSCC cells to hydrogen (H2) stimulation. The PDMS microarray channel chip and chromium plate multi-sample throughput chip prepared in Example 1 are used; the specific steps include:

[0161] Antibody coating: forming a bioanalytical substrate with an antibody barcode array.

[0162] 1) Incubation: Attach the TF-ITO slide and PDMS chip; using a pipette, add 3 μL of capture antibody (stock solution and DPBS solution ratio 1:3) to each of the 20 channels from the beginning of the channel. Use a vacuum pump to aspirate from the channel outlet to fill the entire channel with antibody. Incubate at 37°C for 1 hour, then aspirate completely. The channel starting position is as follows: Figure 3 As shown in section A01-20, the channel exit location is as follows: Figure 3 As shown in Figures 01-20 at the B-terminus, the corresponding capture antibodies are: anti-CCL2, CCL4, IL-4, IL-6, IL-8, IL-10, IP-10, TNF-α, GM-CSF, MMP-9, VEGF, CD9, and CD63 purified capture antibodies.

[0163] 2) Blocking: Prepare a 2% BSA solution, pass the solution through to wash once, block at room temperature for 20 minutes, and then aspirate.

[0164] 3) Cleaning: Prepare a 50% DPBS solution (the ratio of DPBS solution to deionized water is 1:1); after removing the PDMS chip, clean the TF-ITO slide with the antibody barcode array in sequence with DPBS, 50% DPBS solution and deionized water, spin dry the slide and store it for later use (use within 1 week).

[0165] The procedure for analyzing extracellular cytokines in OSCC cells is as follows:

[0166] 1. Collection of OSCC cell conditioned culture medium:

[0167] 1) Preparation of complete culture medium: Filter PAN serum through a 0.22um filter membrane and mix well; prepare complete culture medium according to the ratio of 79% RPMI medium, 20% FBS and 1% penicillin-streptomycin, mix well and set aside.

[0168] 2) Culture OSCC cells: Culture OSCC cells in complete culture medium until the cell density reaches 80%.

[0169] 3) Digestion of OSCC cells: Remove OSCC cells from a 37℃ 5% CO2 cell culture incubator, aspirate the complete culture medium, and wash twice with DPBS; add 500 μL of trypsin and incubate for 3 min until the cells become round; gently pipette to mix, then transfer to a centrifuge tube and centrifuge (1000 rpm, 5 min), discard the supernatant, add 1 ml of FBS-free RPMI medium, agitate and mix the cells; count the cells, and dilute with complete culture medium to a cell density of 5*102. 5 Add cells / ml to the wells of a 96-well plate, 200 μL / well, and incubate at 37°C in a 5% CO2 cell culture incubator for 30 min.

[0170] 4) H2 stimulation of OSCC cells: Select one well of cells for normal incubation, and use the Xiaoxinqing portable hydrogen and oxygen meter (D-HO-001) to introduce hydrogen gas (20ml / min) into the other well of cells, and collect the conditioned culture medium for 48h, aliquot it, and store it at -40℃ for later use.

[0171] 2. OSCC cell extracellular cytokine detection (DMF platform): 0.1% Tetronic 90R4 solution was used as solvent; the incubation conditions were room temperature, protected from light, and humidified; during sample addition and incubation, the droplets were driven to the pre-set position, and the waste droplets were driven to the rightmost waste liquid pool and aspirated with lint-free paper.

[0172] 1) Capture protein: Add OSCC cell conditioned medium droplets and incubate for 30 min;

[0173] 2) Add detection antibodies (biotin): Prepare 0.1% Tetronic 90R4 solution and biotin solution (the ratio of biotin stock solution to 0.1% Tetronic 90R4 solution is 1:199); add biotin and incubate for 30 min; the biotin are biotin against CCL2, CCL4, IL-4, IL-6, IL-8, IL-10, IP-10, TNF-α, GM-CSF, MMP-9, VEGF, CD9, and CD63, respectively.

[0174] 3) Add the fluorescent dye Streptavidin-APC: Prepare Streptavidin-APC (APC stock solution and 0.1% Tetronic 90R4 solution in a ratio of 1:99); add Streptavidin-APC droplets and incubate for 15 min.

[0175] 4) Cleaning: Prepare 50% DPBS droplets (DPBS droplets and deionized water droplets in a 1:1 ratio), and add DPBS droplets, 50% DPBS droplets, and deionized water droplets in sequence for cleaning.

[0176] 5) Scanning: The slides were scanned and analyzed using a four-color laser chip scanner (Genepix+4300A).

[0177] 6) Data processing and statistical analysis: See the results of this embodiment. Figure 13-14 . Figure 13 The results of the slide scan show extracellular cytokine signals in the population of cells. Figure 14 for Figure 13 Statistical analysis of fluorescence intensity results for various signals.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-sample throughput and multi-index detection device based on digital droplet microfluidics, characterized in that, The device includes a droplet microfluidic device and an immunoprotein analysis chip assembly. The droplet microfluidic device, used to drive the droplets, mainly comprises three systems: a power supply system, a computer operating system, and a chip clamping system. The immunoprotein analysis chip assembly mainly comprises a bioanalytical substrate and a microarray chip. The microarray chip is used to assist in obtaining barcode arrays with various capture antibodies on the bioanalytical substrate. The bioanalytical substrate with various capture antibody barcode arrays is used to detect and analyze target molecules in samples. The chip clamping system mainly includes fixing bolts and clamps. Bioanalytical substrates with various capture antibody barcode arrays, spacers, and chrome plates are stacked sequentially and fixed by a chip clamping system. The bioanalytical substrate is a silica glass slide coated from the glass surface to the surface with indium tin oxide (ITO), perfluorooctyltrimethoxysilane and a hydrophobic material, including Teflon and polylysine. The microarray chip is a strip array chip; the strip array consists of 10-100 parallel stripes with a specific width and spacing, the specific width being 10-200µm and the spacing between each strip being 10-200µm.

2. The multi-sample throughput and multi-index detection device based on digital droplet microfluidics according to claim 1, characterized in that, The microarray chip is a PDMS microarray chip.

3. The application of the multi-sample throughput and multi-index detection device based on digital droplet microfluidics as described in any one of claims 1-2, characterized in that, The device is used to simultaneously capture, detect and analyze cytokines and exosomes in a variety of different samples; the multi-sample throughput and multi-index include 1-32 throughputs and 1-200 indices, respectively.

4. The application of the multi-sample throughput and multi-index detection device based on digital droplet microfluidics according to claim 3, characterized in that, The various samples mentioned include, but are not limited to, cell culture media, serum, and protein standards.

5. The application of the multi-sample throughput and multi-index detection device based on digital droplet microfluidics according to claim 3, characterized in that, The applications include qualitative or quantitative detection of various substances to be detected, correlation analysis, or immunophenotyping analysis.

6. The application of the multi-sample throughput and multi-index detection device based on digital droplet microfluidics according to claim 3, characterized in that, The specific steps of the detection process are as follows: Take out the bioanalytical substrate with multiple different capture antibody barcode arrays, align the side coated with the capture antibody with the chromium plate multi-sample throughput chip and fix it with the chip clamping system, sequentially introduce the sample to be tested, the anti-target protein antibody linked to ligand A and the receptor B-fluorescein for incubation, remove the clamp and perform detection on the bioanalytical substrate.

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