Preparation method of microfluidic chip and application of microfluidic chip in chemiluminescence detection

Abstract

The application provides a preparation method of a microfluidic chip and application of the microfluidic chip in chemiluminescence detection, and the application is characterized in that: a flow channel is subjected to hydrophilic treatment, the flow channel and a sample inlet are sealed, the bottom of a chip disc is sealed twice after the sealing is completed, then a composition composed of a solid oil phase mixture and a liquid oil phase mixture is added into a micro-pool, a stable semi-solid / high-viscosity state is formed at 2-8 DEG C through a specific proportion of the solid oil phase mixture and the liquid oil phase mixture composite system, which is like a "physical fence" and effectively blocks the diffusion of water phase molecules, and the problem of hole channeling in long-term refrigerated storage is solved. The microfluidic chip prepared by the application has excellent storage stability, excellent biocompatibility, ideal controllable phase change behavior and optimized interface dynamics, and meanwhile, the process is friendly and the cost is controllable, so the application has wide application prospects in chemiluminescence detection.

Description

Technical Field

[0001] This invention relates to the technical field of molecular detection, specifically to a method for preparing a microfluidic chip and its application in chemiluminescence detection. Background Technology

[0002] Microfluidic chip technology has been widely used in the field of in vitro diagnostics due to its advantages such as integration, automation, and low reagent consumption. In order to achieve multi-step continuous reactions, multiple independent aqueous reaction chambers are often set on the chip and physically separated by an oil phase (usually fluorinated oil or silicone oil) to form a "water-in-oil" structure.

[0003] However, current microfluidic chips have the following problems: (1) Poor storage stability: Under the refrigerated storage conditions of 2-8°C for microfluidic chip products, common oil phases (such as some silicone oil and mineral oil mixtures) may undergo changes in properties, viscosity changes, or instability at the interface with the aqueous phase, leading to mutual permeation (pore crossing) of adjacent aqueous phase components, causing premature degradation of reagents or cross-contamination, resulting in chip failure. (2) Significant interference with the reaction: Some oil phase components may dissolve or adsorb active ingredients (such as enzymes, antibodies, magnetic beads) in the aqueous phase, or release inhibitors into the aqueous phase, affecting the accuracy and sensitivity of the final detection results; (3) Poor phase control: During the reaction heating process, the oil phase failed to form a uniform and clear molten state to encapsulate the water phase, but instead formed an emulsion, which caused the "water-in-oil" droplets in different chambers to merge with each other, thus disrupting the compartmentalization of the reaction. (4) Insufficient interface performance: The surface tension of the oil phase is mismatched, which makes it difficult for the sample or magnetic beads to penetrate the oil-water interface efficiently, resulting in low sample entry efficiency, incomplete magnetic bead transfer or slow dispersion, which affects reaction kinetics and uniformity. (5) Liquid mixing problem: When multiple reagents are present in the chip channel at the same time, they are easily mixed due to diffusion, capillary action or external vibration, resulting in detection failure. (6) Complexity of production process: Traditional filling and sealing processes are complicated and difficult to achieve high-throughput, high-efficiency automated production, and the sealing reliability is poor.

[0004] Therefore, there is an urgent need for a microfluidic chip structure that can effectively isolate multiple reagents and ensure the long-term stability of the reagents, as well as a simple and reliable manufacturing process. Summary of the Invention

[0005] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a method for preparing a microfluidic chip and its application in chemiluminescence detection.

[0006] The first object of the present invention is to provide a composition.

[0007] A second objective of this invention is to provide a reagent card.

[0008] A third objective of this invention is to provide a microfluidic system.

[0009] The fourth objective of this invention is to provide a microfluidic chip.

[0010] A fifth object of the present invention is to provide the application of the above-described compositions, reagent cards, microfluidic systems and / or microfluidic chips in chemiluminescence detection.

[0011] The sixth objective of this invention is to provide a method for fabricating the microfluidic chip described above.

[0012] The seventh objective of this invention is to provide a detection method for the microfluidic chip described above.

[0013] This invention claims protection for the following: A composition comprising a solid oil phase mixture and a liquid oil phase mixture; The solid oil phase mixture includes solid grease, liquid grease, and surfactants; The liquid oil phase mixture includes liquid oils and surfactants; The liquid oil and surfactant in the liquid oil phase mixture are the same in type and amount as those in the solid oil phase mixture; The solid fats include one or more of petrolatum, beeswax, solid paraffin, stearic acid, palmitic acid, and fatty acid glycerides; The liquid grease includes one or more of hexadecane, silicone oil, mineral oil, and liquid paraffin; The surfactants include one or more of Span 80, Span 85, Brij 92, polydimethylsiloxane copolyol, silicone surfactants, oleyl alcohol, lauryl alcohol, and isopropyl palmitate.

[0014] Preferably, in the solid oil phase mixture, the mass ratio of the solid oil to the liquid oil is 1:(2-9), and the mass of the surfactant is 0.001-0.1% of the sum of the masses of the solid oil and the liquid oil.

[0015] More preferably, the mass ratio of the solid oil to the liquid oil is 1:(3-6), and the mass of the surfactant is 0.01-0.05% of the sum of the masses of the solid oil and the liquid oil.

[0016] Preferably, in the liquid oil phase mixture, the mass of the surfactant is 0.001 to 0.1% of the mass of the liquid oil.

[0017] More preferably, the mass of the surfactant is 0.01 to 0.05% of the mass of the liquid oil.

[0018] A reagent card comprising a plurality of sequentially connected microcells, each microcell having a first chamber, a second chamber and a third chamber, wherein the first chamber, the second chamber and the third chamber are connected in sequence, and the second chamber is filled with the aforementioned composition.

[0019] A microfluidic system comprising sequentially connected injection ports, microchannels, and one or more reagent cards.

[0020] A microfluidic chip comprising one or more of the above-mentioned microfluidic systems.

[0021] The application of the above-mentioned compositions, reagent cards, microfluidic systems and / or microfluidic chips in chemiluminescence detection.

[0022] Preferably, the chemiluminescence detection includes the detection of inflammatory markers, thyroid dysfunction markers, and / or myocardial infarction markers.

[0023] More preferably, the inflammatory markers include CRP, PCT, and SAA; The markers of thyroid dysfunction include TSH, FT3, and FT4; The myocardial infarction markers include cTnI, CKMB, and MYO.

[0024] The above-described method for fabricating a microfluidic chip includes the following steps: S1. A microfluidic system is formed by injection molding, wherein the sample inlet, microchannel and reagent card in the microfluidic system are all connected on both the top and bottom surfaces; S2. All microchannels are hydrophilically treated. After the hydrophilic treatment, the microchannels and injection ports are sealed by heat using a heat-sealing film. S3. Use heat-sealing film to heat-seal the entire bottom of the chip pad; S4. Except for the microcells that need to be photometered, use an adhesive to bond the opaque sheet to the rest of the bottom of the chip disk, and then heat seal it. S5. Add the above composition to the second chamber of each microcell, and add the reaction reagent to the third chamber of each microcell; S6. Use a heat-sealing film to heat-seal the top of the chip disk.

[0025] Preferably, in step S1, the hydrophilic treatment method is plasma treatment or coating with a hydrophilic coating.

[0026] Preferably, in steps S1 and S2, the material of the heat-sealing film includes PMMA, PC, PS, COP, or PP.

[0027] Preferably, in step S2, the conditions for heating and sealing are a temperature of 100–200°C, a time of 1–20 s, and a pressure of 10–100 kgf.

[0028] More preferably, in step S2, the conditions for heating and sealing are a temperature of 145–150°C, a time of 5–6 s, and a pressure of 45–50 kgf.

[0029] Preferably, in step S3, the conditions for heating and sealing are a temperature of 100–200°C, a time of 1–20 s, and a pressure of 10–100 kgf.

[0030] More preferably, in step S3, the conditions for heating and sealing are a temperature of 148–150°C, a time of 5.5–6.5 s, and a pressure of 56–60 kgf.

[0031] Preferably, in step S4, the conditions for heating and sealing are a temperature of 100–150°C, a time of 1–15 s, and a pressure of 10–100 kgf.

[0032] More preferably, in step S4, the conditions for heating and sealing are a temperature of 125–130°C, a time of 4–4.8 s, and a pressure of 48–52 kgf.

[0033] Preferably, in step S5, the amount of the solid oil phase added is 30-100 μL per microcell, the amount of the liquid oil phase added is 3-20 μL per microcell, and the amount of the reaction reagent added is 30-150 μL per microcell.

[0034] More preferably, in step S5, the amount of the solid oil phase added is 50 μL per microcell, the amount of the liquid oil phase added is 5 μL per microcell, and the amount of the reaction reagent added is 30 μL per microcell.

[0035] Preferably, in step S6, the conditions for heating and sealing are a temperature of 100–200°C, a time of 1–20 s, and a pressure of 10–100 kgf.

[0036] More preferably, in step S6, the conditions for heating and sealing are a temperature of 150–152°C, a time of 4.6–5.5 s, and a pressure of 62–65 kgf.

[0037] The above-mentioned detection method for microfluidic chips includes the following steps: The sample to be tested is added into the injection port. The sample to be tested flows into the first chamber of the first microcell through the microchannel. Then the bottom of the entire microfluidic chip is heated to melt the composition filled in the second chamber of each microcell. The melted composition forms a connecting channel, so that the first chamber, second chamber and third chamber in the same microcell are interconnected. Adjacent microcells are blocked by an oil phase. In the first microcell, the sample to be tested in the first chamber is mixed with the reaction reagent with magnetic beads in the third chamber, and a reaction occurs. After the reaction is complete, the reaction product in the first microcell is moved to the next microcell by a magnetic mechanism, where it mixes with the reagent in that microcell and reacts, and so on. After all the reactions in the microcells have been completed, the chemiluminescence signal of the final product in the photometric microcell of the microfluidic chip is acquired and detected using a photometric mechanism.

[0038] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for fabricating a microfluidic chip and its application in chemiluminescence detection, which has the following advantages: (1) Excellent storage stability: Through the composite system of solid oil phase mixture and liquid oil phase mixture, a stable semi-solid / high viscosity state is formed at 2-8℃, which is like a "physical fence" to effectively block the diffusion of water phase molecules and solve the problem of pores in long-term cold storage.

[0039] (2) Excellent biocompatibility: The composite system of solid oil phase mixture and liquid oil phase mixture has stable chemical properties and does not adsorb or damage biomolecules such as proteins and enzymes. The extremely low concentration of mild surfactant further reduces the adverse effects of oil phase on biological interfaces.

[0040] (3) Ideal controllable phase change behavior: At the reaction temperature (e.g., 37°C), the solid oil phase mixture melts and the whole system becomes a low viscosity liquid, which can smoothly wrap the water phase to form independent droplets. Moreover, due to the high uniformity of the formulation, emulsification is avoided, and accidental mixing of different reaction zones is prevented.

[0041] (4) Optimized interfacial kinetics: The addition of surfactants significantly reduced the surface tension of the oil phase, making it easier to wet the aqueous phase. This brings two major benefits: first, it allows serum / plasma samples to quickly penetrate the oil layer and enter the lower aqueous phase reagent; second, it reduces the resistance when magnetic beads carrying reaction products pass through the oil layer from the upper aqueous phase to the lower aqueous phase, and allows them to disperse quickly and evenly in the lower aqueous phase, ensuring the uniformity and efficiency of subsequent reactions.

[0042] (5) Process-friendly and cost-controllable: The raw materials used are all common and low-cost chemical reagents. The preparation and spotting processes are simple and suitable for large-scale production. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the microfluidic chip in Example 1.

[0044] Figure 2 This is a schematic diagram of the microfluidic chip in Example 2.

[0045] Figure 3 This is a schematic diagram of the microfluidic chip in Example 3.

[0046] Figure 4 This is a schematic diagram of the microfluidic chip in Example 4.

[0047] Figure 5 The correlation between the microfluidic chip and the Beckman reagent kit of this invention in detecting the results of three inflammatory markers (CRP, PCT, SAA) is shown.

[0048] Figure 6 The correlation between the microfluidic chip of this invention and the Beckman reagent kit for detecting three thyroid function tests (TSH, FT3, FT4) is shown.

[0049] Figure 7 The correlation between the microfluidic chip and the Beckman reagent kit of this invention for detecting the results of three myocardial infarction parameters (cTnI, CKMB, MYO) is shown.

[0050] In the attached diagram: 1. Body; 2. Inlet port; 3. Reaction port; 4. Gas injection port; 6. Inlet channel; 7. Drive channel; 701. First channel; 702. Second channel; 16. Positioning part; 18. Reagent card; 1801. First microcell; 1802. Second microcell; 1803. Third microcell; 1804. Fourth microcell; 1805. Fifth microcell; 19. Channel. Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0052] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0053] Example 1: A method for fabricating a microfluidic chip This embodiment provides a method for fabricating a microfluidic chip, specifically including the following steps: 1. Microfluidic chip structure like Figure 1As shown, a square chip disk (both top and bottom unsealed) is injection molded using PMMA. The chip disk includes a body 1, an injection port 2 (diameter 6 mm, height 5 mm), a flow channel 19 (width 0.6 mm), and a reagent card 18. The injection port 2, the flow channel 19, and the reagent card 18 are connected in sequence to form a microfluidic system. The reagent card 18 includes five microcells connected in series, namely the first microcell 1801, the second microcell 1802, the third microcell 1803, the fourth microcell 1804, and the fifth microcell 1805, which are respectively a magnetic bead chamber, a labeled antibody chamber, a washing chamber, a washing chamber, and a luminescent substrate reaction chamber; each microcell has a first chamber, a second chamber, and a third chamber arranged in the direction from liquid inlet to liquid outlet, and each chamber is 7 mm long, 4 mm wide, and 4 mm high. The first chamber of the first microcell 1801 is connected to the flow channel 19. The first chamber of the other microcells is connected to the previous microcell. The second chamber is used to fill the encapsulation oil (solid oil phase and liquid oil phase). The third chamber is used to fill the reaction reagents: the third chamber of the magnetic bead chamber (first microcell 1801) is filled with magnetic microparticle reagents, the third chamber of the labeled antibody chamber (second microcell 1802) is filled with enzyme reagents, the third chamber of the washing chamber (third microcell 1803 and fourth microcell 1804) is filled with washing solution, and the third chamber of the luminescent substrate chamber (fifth microcell 1805) is filled with luminescent substrate solution.

[0054] The sample inlet 2, flow channel 19, and reagent card 18 are all structures that are open on both the top and bottom.

[0055] 2. Detection principle of microfluidic chips The encapsulation oil is solid at room temperature. When heated, it transforms from solid to liquid. Initially, the three chambers within the same microcell are not interconnected due to the encapsulation oil. During testing, the sample to be tested flows from the injection port 2 into the first chamber of the first microcell 1801 through the flow channel 19. Then, the bottom of the entire microfluidic chip is heated, causing the encapsulation oil to become liquid. The three chambers within the same microcell then connect, while adjacent microcells are separated by an oil phase barrier. In the first microcell 1801, the sample to be tested in the first chamber comes into contact with and mixes with the reaction reagent in the third chamber, and a reaction occurs. After the reaction is completed, the reaction product (with magnetic particles) in the first microcell 1801 is moved to the next microcell by a magnetic mechanism, where it mixes with the reaction reagent in the microcell and reacts, and so on. After all the reactions in the microcells have been completed, the chemiluminescence signal of the final product in the fifth microcell 1805 is collected and detected using a photometric device.

[0056] 3. Fabrication process of microfluidic chips (1) Hydrophilic treatment and preliminary sealing of the flow channel To ensure smooth sample introduction, the flow channel 19 is hydrophilically treated by plasma treatment or by coating with a hydrophilic anti-fog nano-coating ATF202, using a brush coating method.

[0057] After the hydrophilic treatment, a PMMA film with a thickness of 50 μm was used to seal the flow channel 19 and the injection port 2 by a heat sealing process. The specific conditions of the heat sealing process were: temperature 150℃, time 5 s, and pressure 45 kgf.

[0058] (2) The bottom of the chip disk is sealed. The bottom of the chip disk is sealed twice to provide a rapidly heating substrate: First sealing: A 50 μm thick PMMA film was used to seal the entire bottom of the chip pad using a heat sealing process. The specific conditions for the heat sealing process were: temperature 148℃, time 6.5 s, and pressure 56 kgf. Second sealing: Use an adhesive (double-sided tape or hot melt adhesive) to bond the aluminum foil sheet to the bottom of the chip disk after the first sealing (except for the part of the light-emitting substrate reaction chamber, i.e., the fifth microcell 1805), and then bond it together by a heat sealing process. The specific conditions of the heat sealing process are: temperature 125℃, time 4.8 s, and pressure 48 kgf.

[0059] (3) Liquid filling Solid oil phase: Weigh 1.0 g of solid paraffin and 3.0 g of liquid paraffin and place them in a glass bottle. Heat and stir in a 65°C water bath until the solid paraffin is completely dissolved to obtain a clear mixture. Add 2.0 mg of surfactant Span85 (0.05% of the total mass of the clear mixture) to the clear mixture and mix thoroughly for 30 min to obtain a homogeneous solid oil phase; this solid oil phase serves as an isolation agent.

[0060] Liquid oil phase: 3.0 g of liquid paraffin, and 1.5 mg of Span 85 (0.05% of the mass of liquid paraffin) was added to the liquid paraffin; this liquid oil phase serves to moisturize and prevent reagent evaporation.

[0061] Add 50 μL of solid oil phase liquid (used after heating and melting) to the second chamber of each microcell on the chip disk. After the solid oil phase solidifies, add 5 μL of liquid oil phase to the second chamber of each microcell.

[0062] Add the micro-dropletized magnetic microparticle reagent, enzyme reagent, washing solution, and luminescent substrate solution to the magnetic bead chamber, labeled antibody chamber, washing chamber, and the third chamber of the luminescent substrate reaction chamber in the reagent card, respectively. The volume of liquid added to each well is 30 μL.

[0063] Adding two oil phases can physically isolate the magnetic microparticle reagent, enzyme reagent, washing solution and luminescent substrate solution from each other, preventing them from evaporating and mixing, and generating uniform and stable water-in-oil droplets.

[0064] (4) Final sealing of the top of the chip disk After liquid filling is completed, a PMMA film with a thickness of 50 μm is used to achieve a tight seal between the PMMA film and the top of the chip disk through a heat sealing process. This completely seals all liquids and structures in a sterile, evaporation-proof environment, resulting in the finished microfluidic chip. The specific conditions for the heat sealing process are: temperature 152℃, time 5.5 s, and pressure 62 kgf.

[0065] Example 2: A method for fabricating a microfluidic chip This embodiment provides a method for fabricating a microfluidic chip according to Embodiment 1, the difference being that the finished microfluidic chip integrates multiple microfluidic systems (…). Figure 2 ).

[0066] Example 3: A method for fabricating a microfluidic chip This embodiment provides a method for fabricating a microfluidic chip according to Embodiment 1, the difference being that the structure of the microfluidic chip in step 1 is as follows: like Figure 3 As shown, a circular chip disk (unsealed at both ends) is injection molded from PMMA, with a diameter of 8 cm. The chip disk includes a body 1, an inlet port 2 (6 mm in diameter, 5 mm in height), a flow channel 19 (0.6 mm in width), and three reagent cards 18. The inlet port 2, the flow channel 19, and the reagent cards 18 are connected sequentially. The three reagent cards 18 are evenly distributed around the circumference of the body 1, and the inlet port 2 is located at the center of the body 1. The body 1 has a triangular region, and the three vertices of the triangular region correspond to the liquid inlets of the three reagent cards.

[0067] Each reagent card 18 includes five sequentially connected arc-shaped microcells, namely the first microcell 1801, the second microcell 1802, the third microcell 1803, the fourth microcell 1804, and the fifth microcell 1805, which are respectively a magnetic bead chamber, a labeled antibody chamber, a washing chamber, a washing chamber, and a luminescent substrate reaction chamber; each microcell has a first chamber, a second chamber, and a third chamber arranged in the direction from liquid inlet to liquid outlet, and each chamber is 7 mm long, 4 mm wide, and 4 mm high. The first chamber of the first microcell 1801 is connected to the flow channel 19, and the first chamber of the other microcells is connected to the previous microcell. The second chamber is used to fill the encapsulation oil (solid oil phase and liquid oil phase), and the third chamber is used to fill the reaction reagents: the third chamber of the magnetic bead chamber (first microcell 1801) is filled with magnetic microparticle reagents, the third chamber of the labeled antibody chamber (second microcell 1802) is filled with enzyme reagents, the third chamber of the washing chamber (third microcell 1803 and fourth microcell 1804) is filled with washing solution, and the third chamber of the luminescent substrate chamber (fifth microcell 1805) is filled with luminescent substrate solution. The sample inlet 2, the flow channel 19, and the reagent card 18 are all structures that are open on both the top and bottom, and the sample inlet 2 and the flow channel 19 are located in the triangular planar area of ​​the body 1.

[0068] In step “(1) Hydrophilic treatment and preliminary sealing of the flow channel”, the triangular planar area of ​​the chip disk is sealed by a heat sealing process, and the remaining steps are the same as in Example 1.

[0069] Example 4: A method for fabricating a microfluidic chip This embodiment provides a method for fabricating a microfluidic chip according to Embodiment 1, the difference being that the microfluidic chip structure in step 1 is as follows: like Figure 4 As shown, the microfluidic chip is configured according to Chinese Utility Model Patent CN223717180U (both top and bottom are unsealed). The chip disk has a diameter of 8 cm and includes a body 1, an inlet port 2 (diameter 6 mm, height 5 mm), a flow channel 19 (width 0.6 mm), a reaction port 3, and three reagent cards 18. The inlet port 2, flow channel 19, reaction port 3, and reagent cards 18 are connected sequentially. The flow channel 19 includes an inlet flow channel 6, a drive flow channel 7, and a third flow channel (not shown in the figure, used to connect the reaction port 3 and the reagent cards 18). The three reagent cards 18 are evenly distributed around the circumference of the body 1. The body 1 has a triangular planar region, and the three vertices of the triangular planar region correspond to the liquid inlets of the three reagent cards. The microfluidic chip also includes other components as described in Chinese Utility Model Patent CN223717180U.

[0070] The sample inlet 2, flow channel, reaction port 3, and reagent card 18 are all structures that are open on both the top and bottom. The sample inlet 2, flow channel, and reaction port 3 are located within the triangular planar area of ​​the body 1.

[0071] Each reagent card 18 includes five sequentially connected arc-shaped microcells: microcell 1801, microcell 1802, microcell 1803, microcell 1804, and microcell 1805, which are respectively a magnetic bead chamber, a labeled antibody chamber, a washing chamber, a washing chamber, and a luminescent substrate reaction chamber. Within each microcell, following the direction from liquid inlet to outlet, there are sequentially arranged first, second, and third chambers. Each chamber is 7 mm long, 4 mm wide, and 4 mm high. The first chamber of microcell 1801 is connected to the reaction well 3. The first chambers of the other microcells are connected to the preceding microcell. The second chamber is used to fill the encapsulation oil (solid oil phase and liquid oil phase), and the third chamber is used to fill the reaction reagents (sequentially filled with magnetic microparticle reagent, enzyme reagent, washing solution, or luminescent substrate solution).

[0072] In step “(1) Hydrophilic treatment and preliminary sealing of the flow channel”, the triangular planar area of ​​the chip disk is sealed by a heat sealing process, and the remaining steps are the same as in Example 1.

[0073] Example 5: A method for fabricating a microfluidic chip This embodiment provides a method for preparing a microfluidic chip according to Embodiment 1, the difference being that in “(1) hydrophilic treatment of the flow channel and preliminary sealing”, the specific conditions for the heating sealing process are: temperature 145℃, time 6 s, pressure 50 kgf; In “(2) Sealing the bottom of the chip disk”, the specific conditions for the heat sealing process during the first sealing are: temperature 150℃, time 5.5 s, and pressure 60 kgf; The specific conditions for the heat sealing process during the second sealing are: temperature 130℃, time 4 s, and pressure 52 kgf. In “(4) Final sealing of the top of the chip disk”, the specific conditions for the heat sealing process are: temperature 150℃, time 4.6s, and pressure 65 kgf.

[0074] Example 6: Microfluidic chip detection of three inflammatory markers (CRP, PCT, SAA) I. Experimental Methods 1. Reagent preparation CRP magnetic microparticle preservation buffer: 50 mM HEPES, 10 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.1% Proclin-300 (v / v). CRP-labeled antibody preservation buffer: 50 mM Tris-HCl, 20 mM NaCl, 0.02% Tween-20 (v / v), 1% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.05% zinc chloride (w / v); CRP antibody: purchased from Phytobio, catalog number CRP-REAB-G1-012 or CRP-Ab6#; PCT magnetic microparticle preservation buffer: 50 mM HEPES, 10 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.2% Proclin-300 (v / v). PCT enzyme-labeled antibody preservation buffer: 100 mM Tris-HCl, 10 mM NaCl, 0.01% Tween-20 (v / v), 2% BSA (w / v), 0.1% Proclin-300 (v / v), 0.05% BIT-10 (w / v), 0.1% magnesium chloride (w / v), 0.01% zinc chloride (w / v); PCT antibody: purchased from Boyue Biotechnology, catalog number PCT301 or PCT302; SAA magnetic microparticle preservation buffer: 100 mM Tris-HCl, 10 mM NaCl, 2% BSA (w / v), 0.1% Proclin-300 (v / v). SAA enzyme-labeled antibody preservation buffer: 100 mM Tris-HCl, 100 mM NaCl, 0.02% Tween-20 (v / v), 3% BSA (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.1% zinc chloride (w / v). SAA antibody: purchased from Qiancheng Biotechnology, catalog number MC00301 or MC00302; Coupling buffer: Weigh 10 g MES into a beaker, add 1 L of purified water, stir and mix well, then adjust the pH to 6.0 with NaOH solution and store at 4℃ for later use; Washing solution: 10 mM PBS buffer, 0.1% Tween-20 (v / v); Luminescent substrate solution: APS-5 chemiluminescent substrate solution.

[0075] 2. Preparation of magnetic microparticle reagents (1) Microsphere activation: Add 40 μL of 100 mg / mL magnetic microparticle suspension to 1 mL of 50 mM MES coupling buffer (pH 6.0), then add 1 mg EDC and 10 mg NHS for activation treatment for 20 min, magnetic adsorption treatment for 10 min, discard the supernatant, resuspend the precipitate with coupling buffer to obtain a 4 mg / mL activated microsphere solution; (2) Protein coupling: Add 0.2 mg of antibody (CRP antibody, PCT antibody or SAA antibody) to 4 mg / mL activated microsphere solution, mix at room temperature for 2 h, magnetically treat for 10 min, discard the supernatant, block the precipitate with 50 mM PBS solution containing 5 mg BSA for 60 min, stir and mix, magnetically treat for 10 min, and collect the precipitate and store it in 40 mL of the corresponding magnetic microparticle storage buffer solution, which is the magnetic microparticle reagent.

[0076] 3. Preparation of enzyme reagents (1) Antibody activation: Add 20 μg of Sulfo-SMCC to 0.2 mg of antibody (CRP antibody, PCT antibody or SAA antibody), react for 4 h to obtain solution A; (2) ALP thiol modification: 70 μg of SATA was added to 0.6 mg ALP, the reaction was carried out for 40 min, and then 10 μL of 0.5 M hydroxylamine hydrochloride solution was added for deacylation treatment for 2 h to obtain solution B; (3) Mix solution A and solution B to obtain ALP-labeled antibody solution. Add the antibody solution to 40 mL of the corresponding enzyme-labeled antibody storage buffer to obtain the enzyme reagent.

[0077] 4. Testing According to Example 4, a microfluidic chip was prepared, and the magnetic microparticle reagents, enzyme reagents, washing solutions and luminescent substrate solutions prepared in steps 1 to 3 corresponding to CRP, PCT and SAA were respectively added to the third chamber of the magnetic bead chamber, labeled antibody chamber, washing chamber and luminescent substrate reaction chamber of the microfluidic chip.

[0078] Precision experiment: One normal human serum sample and one CRP / PCT / SAA abnormal human serum sample were selected, and each sample was tested 10 times. The detection method was as follows: 100 μL of sample was added to the sample inlet 2 of the microfluidic chip, and then the microfluidic chip was placed into the microdroplet detection bioanalysis system (Chinese Utility Model Patent CN216864170U) for detection.

[0079] Sensitivity experiment: CRP, PCT, and SAA reference samples were selected, and 60 repeated tests were performed using the microfluidic chip of this invention according to the above detection method, obtaining 60 test data points. The normality of the test data and the limit of detection (LOD) were calculated according to the requirements of CLSI EP17-A2. When the test data conformed to a normal distribution, the LOD was calculated using a parametric method; when the test data did not conform to a normal distribution, the LOD was calculated using a non-parametric method and verified.

[0080] Correlation experiment: Another 87 clinical samples were selected and tested using the microfluidic chip of the present invention according to the above detection method. CRP, PCT and SAA were also detected using the Beckman reagent kit to evaluate the correlation between the two.

[0081] II. Experimental Results The results of the precision experiment are shown in Table 1. According to the results in Table 1, the CV of CRP, PCT and SAA are all <10%, indicating that the microfluidic chip of the present invention has good detection precision.

[0082] Table 1 Precision Experiment Results

[0083] The sensitivity test results are shown in Table 2. The results show that the limits of detection of the microfluidic chip of the present invention for the three inflammatory markers CRP, PCT and SAA are 0.007 μg / mL, 0.05 μg / mL and 1 μg / mL, respectively, which have good sensitivity.

[0084] Table 2 Sensitivity Experiment Results

[0085]

[0086] The results of the correlation experiment are shown in Table 3 and Figure 5 As shown, the results indicate that the correlation R 2 A value greater than 0.9 indicates that the microfluidic chip of the present invention has good consistency with the detection method of the Beckman reagent kit.

[0087] Table 3. Results of the correlation experiment

[0088]

[0089]

[0090] Example 7: Microfluidic chip detection of three functional indicators (TSH, FT3, FT4) I. Experimental Methods 1. Reagent preparation FT3 magnetic microparticle preservation buffer: 50 mM HEPES, 100 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 5% glycerol (v / v), 0.2% Proclin-300 (v / v). FT3 enzyme-labeled antibody preservation buffer: 50 mM Tris-HCl, 50 mM NaCl, 0.02% Tween-20 (v / v), 0.5% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.05% zinc chloride (w / v); FT3 antibody: purchased from Qiancheng Biotechnology, catalog number MC09601 or MC09645; FT4 magnetic microparticle preservation buffer: 100 mM HEPES, 50 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.2% Proclin-300 (v / v). FT4 enzyme-labeled antibody preservation buffer: 100 mM Tris-HCl, 50 mM NaCl, 0.02% Tween-20 (v / v), 0.5% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.05% BIT-10 (v / v), 0.1% magnesium chloride (w / v), 0.01% zinc chloride (w / v); FT4 antibody: purchased from Qiancheng Biotechnology, catalog number MC09701 or MC09753S; TSH magnetic microparticle preservation buffer: 100 mM Tris-HCl, 50 mM NaCl, 0.05% SDS (w / v), 1% BSA (w / v), 0.5% skim milk powder (w / v), 0.1% Proclin-300 (v / v). TSH enzyme-labeled antibody preservation buffer: 100 mM Tris-HCl, 150 mM NaCl, 0.02% Tween-20 (v / v), 3% BSA (w / v), 0.05% glycine (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.1% zinc chloride (w / v); TSH antibody: purchased from Qiancheng Biotechnology, catalog number MC04101 or MC04102; Coupling buffer: Weigh 10 g MES into a beaker, add 1 L of purified water, stir and mix well, then adjust the pH to 6.0 with NaOH solution and store at 4℃ for later use; Washing solution: 10 mM PBS buffer, 0.1% Tween-20 (v / v); Luminescent substrate solution: APS-5 chemiluminescent substrate solution.

[0091] 2. Preparation of magnetic microparticle reagents and enzyme reagents The magnetic microparticle reagent and enzyme reagent were prepared according to steps 2 and 3 in Example 3.

[0092] 3. Testing According to Example 4, a microfluidic chip was prepared, and the magnetic microparticle reagents, enzyme reagents, washing solutions and luminescent substrate solutions corresponding to FT3, FT4 and TSH prepared in steps 1 to 2 were added to the third chamber of the magnetic bead chamber, labeled antibody chamber, washing chamber and luminescent substrate reaction chamber of the microfluidic chip, respectively.

[0093] Precision experiment: One normal human serum sample and one TSH / FT3 / FT4 abnormal human serum sample were selected, and each sample was tested 10 times. The detection method was as follows: 100 μL of sample was added to the sample inlet 2 of the microfluidic chip, and then the microfluidic chip was placed into the microdroplet detection bioanalysis system (Chinese Utility Model Patent CN216864170U) for detection.

[0094] Sensitivity experiment: FT3, FT4, and TSH reference samples were selected, and 60 repeated tests were performed using the microfluidic chip of this invention according to the above detection method, obtaining 60 test data points. The test data were then subjected to normality testing and limit of detection (LOD) calculation according to the requirements of CLSI EP17-A2. When the test data conformed to a normal distribution, the LOD was calculated using a parametric method; when the test data did not conform to a normal distribution, the LOD was calculated using a non-parametric method and verified.

[0095] Correlation experiment: Another 140 clinical samples were selected and tested using the microfluidic chip of the present invention according to the above detection method. At the same time, FT3, FT4 and TSH were detected using the Beckman reagent kit to evaluate the correlation between the two.

[0096] II. Experimental Results The results of the precision experiment are shown in Table 4. According to the results in Table 4, the CV of TSH, FT3 and FT4 are all <10%, indicating that the microfluidic chip of the present invention has good detection precision.

[0097] Table 4 Precision Experiment Results

[0098] The sensitivity test results are shown in Table 5. The results show that the microfluidic chip of the present invention has the lowest detection limits of 0.0045 μIU / mL, 0.81 pg / mL and 0.2433 ng / dL for the three methylformamide parameters TSH, FT3 and FT4, respectively, which are good.

[0099] Table 5 Sensitivity Experiment Results

[0100]

[0101] The results of the correlation experiment are shown in Table 6 and Figure 6 As shown, the results indicate that the correlation R 2 A value greater than 0.9 indicates that the microfluidic chip of the present invention has good consistency with the detection method of the Beckman reagent kit.

[0102] Table 6. Results of the correlation experiment

[0103]

[0104]

[0105] Example 8: Microfluidic chip detection of three myocardial infarction markers (cTnI, CKMB, MYO) I. Experimental Methods 1. Reagent preparation cTnI magnetic microparticle preservation buffer: 50 mM HEPES, 10 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.1% Proclin-300 (v / v). cTnI enzyme-labeled antibody preservation buffer: 50 mM Tris-HCl, 50 mM NaCl, 0.02% Tween-20 (v / v), 0.5% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.05% zinc chloride (w / v); cTnI antibody: purchased from Qiancheng Biotechnology, catalog number MC00501 or MC00502; CKMB magnetic microparticle preservation buffer: 50 mM HEPES, 10 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.1% Proclin-300 (v / v). CKMB enzyme-labeled antibody preservation buffer: 50 mM Tris-HCl, 50 mM NaCl, 0.02% Tween-20 (v / v), 0.5% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.05% zinc chloride (w / v); CKMB antibody: purchased from Qiancheng Biotechnology, catalog number MC00703 or MC00704; MYO magnetic microparticle preservation buffer: 50 mM HEPES, 10 mM NaCl, 0.05% Tween-20 (v / v), 2% BSA (w / v), 0.1% Proclin-300 (v / v). MYO enzyme-labeled antibody preservation buffer: 50 mM Tris-HCl, 50 mM NaCl, 0.02% Tween-20 (v / v), 0.5% BSA (w / v), 0.5% sodium caseinate (w / v), 0.1% Proclin-300 (v / v), 0.1% magnesium chloride (w / v), 0.05% zinc chloride (w / v); MYO antibody: purchased from Qiancheng Biotechnology, catalog number MC00201 or MC00202; Coupling buffer: Weigh 10 g MES into a beaker, add 1 L of purified water, stir and mix well, then adjust the pH to 6.0 with NaOH solution and store at 4℃ for later use; Washing solution: 10 mM PBS buffer, 0.1% Tween-20 (v / v); Luminescent substrate solution: APS-5 chemiluminescent substrate solution.

[0106] 2. Preparation of magnetic microparticle reagents and enzyme reagents The magnetic microparticle reagent and enzyme reagent were prepared according to steps 2 and 3 in Example 3.

[0107] 3. Testing According to Example 4, a microfluidic chip was prepared, and the magnetic microparticle reagents, enzyme reagents, washing solutions and luminescent substrate solutions corresponding to cTnI, CKMB and MYO prepared in steps 1 to 2 were added to the third chamber of the magnetic bead chamber, labeled antibody chamber, washing chamber and luminescent substrate reaction chamber of the microfluidic chip, respectively.

[0108] Precision experiment: One normal human serum sample and one abnormal cTnI / CKMB / MYO human serum sample were selected. Each sample was tested 10 times. The detection method was as follows: 100 μL of sample was added to the sample inlet 2 of the microfluidic chip, and then the microfluidic chip was placed in the microdroplet detection bioanalysis system (Chinese Utility Model Patent CN216864170U) for detection.

[0109] Sensitivity experiment: Using cTnI / CKMB / MYO reference samples, the microfluidic chip of this invention was used to perform 60 repeated tests according to the above detection method, obtaining 60 test data points. The test data were then subjected to normality testing and limit of detection (LOD) calculation according to the requirements of CLSI EP17-A2. When the test data conformed to a normal distribution, the LOD was calculated using a parametric method; when the test data did not conform to a normal distribution, the LOD was calculated using a non-parametric method and verified.

[0110] Correlation experiment: Another 76 clinical samples were selected and detected using the microfluidic chip of the present invention according to the above detection method. At the same time, cTnI, CKMB and MYO were detected using the Beckman reagent kit to evaluate the correlation between the two.

[0111] II. Experimental Results The results of the precision experiment are shown in Table 7. According to the results in Table 7, the CV of cTnI, CKMB and MYO are all <10%, indicating that the microfluidic chip of the present invention has good detection precision.

[0112] Table 7 Precision Experiment Results

[0113] The sensitivity test results are shown in Table 8. The results show that the minimum detection limits of the microfluidic chip of the present invention for the three myocardial infarction markers cTnI, CKMB and MYO are 2 pg / mL, 0.11 ng / mL and 1 ng / mL, respectively, which have good sensitivity.

[0114] Table 8 Sensitivity Experiment Results

[0115]

[0116] The results of the correlation experiment are shown in Table 9 and Figure 7 As shown, the results indicate that the correlation R 2 A value greater than 0.9 indicates that the microfluidic chip of the present invention has good consistency with the detection method of the Beckman reagent kit.

[0117] Table 9. Results of the correlation experiment

[0118]

[0119] Comparative Example 1 I. Experimental Methods The microfluidic chip was prepared according to Example 4, except that in "4. Liquid filling", pure silicone oil was added to the second chamber of each microcell on the chip disk to obtain microfluidic chip A; The microfluidic chip prepared in Example 4 is used as microfluidic chip B.

[0120] Storage stability test: Microfluidic chip A and microfluidic chip B were stored in a 4°C refrigerator for 28 days. Samples were taken weekly, and the presence of pores was observed using dye-labeled reaction reagents.

[0121] Storage and moisture retention test: 20 microfluidic chips A and 20 microfluidic chips B were stored in a 4°C refrigerator for 28 days. They were weighed weekly, and the weights were compared to determine whether the reaction reagents had evaporated.

[0122] Phase observation: Observe the reaction process on a constant temperature stage at 37℃.

[0123] Interference test: Using microfluidic chip A and microfluidic chip B, the three inflammatory reagents were prepared according to Example 3 and filled into the third chamber of each microcell on microfluidic chip A and microfluidic chip B respectively. Then, the samples with abnormal inflammatory values ​​were tested repeatedly. By comparing the repeatability test data of microfluidic chip A and microfluidic chip B, the mean deviation and CV of the test were calculated to determine whether microfluidic chip A and microfluidic chip B would interfere with the sample test.

[0124] II. Experimental Results Storage stability tests showed that no dye migration was observed in microfluidic chip B within 28 days, while observable dye migration began to appear in microfluidic chip A on day 14, indicating that the microfluidic chip prepared in Example 4 has good storage stability.

[0125] The results of the storage and hydration test are shown in Table 10. The results show that no significant weight change was observed in microfluidic chip B within 28 days, while observable weight change began to appear in microfluidic chip A on day 7. This indicates that the microfluidic chip prepared in Example 4 has a good storage and hydration effect.

[0126] Table 10 Storage and Moisture Retention Test Results of Microfluidic Chip A

[0127] Table 11 Storage and Moisture Retention Test Results of Microfluidic Chip B

[0128] Phase observation results show that microfluidic chip B completely melts into a clear liquid within 2 minutes, clearly encapsulates the aqueous phase droplets, and does not emulsify. The magnetic beads can quickly pass through the oil layer and disperse uniformly in the next aqueous phase.

[0129] The results of the reaction interference test are shown in Table 12. The results show that the repeatability test deviation of microfluidic chip B is no greater than 5%, the CV is no greater than 10%, and it has no significant interference with the reaction.

[0130] Table 12 Results of Reaction Interference Test

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that, The composition comprises a solid oil phase mixture and a liquid oil phase mixture; The solid oil phase mixture includes solid grease, liquid grease, and surfactants; The liquid oil phase mixture includes liquid oils and surfactants; The liquid oil and surfactant in the liquid oil phase mixture are the same in type and amount as those in the solid oil phase mixture; The solid fats include one or more of petrolatum, beeswax, solid paraffin, stearic acid, palmitic acid, and fatty acid glycerides; The liquid grease includes one or more of hexadecane, silicone oil, mineral oil, and liquid paraffin; The surfactant includes one or more of Span 80, Span 85, Brij 92, polydimethylsiloxane copolyol, silicone surfactant, oleyl alcohol, lauryl alcohol, and isopropyl palmitate.

2. The composition according to claim 1, characterized in that, In the solid oil phase mixture, the mass ratio of the solid oil to the liquid oil is 1:(2-9), and the mass of the surfactant is 0.001-0.1% of the sum of the masses of the solid oil and the liquid oil.

3. The composition according to claim 1, characterized in that, In the liquid oil phase mixture, the mass of the surfactant is 0.001 to 0.1% of the mass of the liquid oil.

4. A reagent card, the reagent card comprising a plurality of sequentially connected microcells, each microcell having a first chamber, a second chamber, and a third chamber, wherein the first chamber, the second chamber, and the third chamber are connected sequentially, characterized in that, The second chamber is filled with the composition according to any one of claims 1 to 3.

5. A microfluidic system, characterized in that, The microfluidic system comprises sequentially connected injection ports, microchannels, and one or more reagent cards as described in claim 4.

6. A microfluidic chip, characterized in that, It includes one or more of the microfluidic systems described in claim 5.

7. The application of the composition according to any one of claims 1 to 3, the reagent card according to claim 4, the microfluidic system according to claim 5, and / or the microfluidic chip according to claim 6 in chemiluminescence detection.

8. The method for fabricating the microfluidic chip according to claim 6, characterized in that, The preparation method includes the following steps: S1. A microfluidic system is formed by injection molding, wherein the sample inlet, microchannel and reagent card in the microfluidic system are all connected on both the top and bottom surfaces; S2. All microchannels are hydrophilically treated. After the hydrophilic treatment, the microchannels and injection ports are sealed by heat using a heat-sealing film. S3. Use heat-sealing film to heat-seal the entire bottom of the chip pad; S4. Except for the microcells that need to be photometered, use an adhesive to bond the opaque sheet to the rest of the bottom of the chip disk, and then heat seal it. S5. After heating and melting the solid oil phase mixture according to any one of claims 1 to 3, add it to the second chamber of each micro-pool. After the solid oil phase mixture solidifies, add the liquid oil phase mixture according to any one of claims 1 to 3. Add the reaction reagent into the third chamber of each microcell; S6. Use a heat-sealing film to heat-seal the top of the chip disk.

9. The preparation method according to claim 8, characterized in that, In steps S2, S3, S5 and S6, the conditions for heating and sealing are a temperature of 100 to 200°C, a time of 1 to 20 seconds, and a pressure of 10 to 100 kgf. In step S4, the conditions for heating and sealing are a temperature of 100-150°C, a time of 1-15 seconds, and a pressure of 10-100 kgf.

10. The detection method for the microfluidic chip according to claim 6, characterized in that, Includes the following steps: The sample to be tested is added into the injection port. The sample to be tested flows into the first chamber of the first microcell through the microchannel. Then the bottom of the entire microfluidic chip is heated to melt the composition filled in the second chamber of each microcell. The melted composition forms a connecting channel, so that the first chamber, second chamber and third chamber in the same microcell are interconnected. Adjacent microcells are blocked by an oil phase. In the first microcell, the sample to be tested in the first chamber is mixed with the reaction reagent with magnetic beads in the third chamber, and a reaction occurs. After the reaction is complete, the reaction product in the first microcell is moved to the next microcell by a magnetic mechanism, where it mixes with the reagent in that microcell and reacts, and so on. After all the reactions in the microcells have been completed, the chemiluminescence signal of the final product in the photometric microcell of the microfluidic chip is acquired and detected using a photometric mechanism.

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