A method for preparing a microfluidic chip

Abstract

The invention discloses a preparation method of a microfluidic chip, comprising the following steps: after super-hydrophilic modification of a chip surface material, spraying a layer of sealing liquid on the surface of the microfluidic chip for surface sealing, and then drying the chip; covering a super-hydrophobic reagent on a groove microstructure, super-hydrophobic modification of the surface of the groove microstructure in the chip, and then drying the chip to obtain a super-hydrophobic pipeline; adding a fluorescent microsphere marker to a mixing pipeline in a chip substrate, and then drying, so that the fluorescent microsphere marker is dried in the chip and coated on a second micron column; coating a capture antibody reagent on a carrier film, and then drying, so that the capture antibody labeled with polystyrene microspheres is fixed on the carrier film, and the carrier film coated with the capture antibody reagent is placed on a detection pipeline of a chip substrate; placing absorbent paper in a waste liquid tank, and assembling and bonding a chip cover plate and a chip substrate to obtain the microfluidic chip.

Description

[0001] This application is a divisional application of the invention patent application with application date of December 13, 2018, application number 2018115239244, and invention name “A microfluidic chip, preparation method and detection method thereof”. Technical Field

[0002] The present invention belongs to the technical field of in vitro diagnosis of medical devices, and in particular relates to a method for preparing a microfluidic chip for point-of-care diagnosis (POCT). Background Art

[0003] Point of Care Testing (POCT) is to conduct rapid on-site diagnosis of patients with the help of miniaturized or moderate desktop equipment or reagents. POCT testing products are gradually being accepted and promoted by the market due to its simple and convenient operation, easy maintenance of the testing system, the ability to perform testing anytime and anywhere, and low testing costs.

[0004] In the prior art, POCT detection products used for disease diagnosis can be roughly divided into two categories according to detection methodology or product materials: chromatographic POCT detection technology and microfluidic POCT detection technology.

[0005] Chromatographic POCT detection technology has a history of more than 30 years, and the leading POCT detection products on the market are currently developed based on chromatographic technology. Although POCT products developed based on chromatographic technology are mature and have low technical barriers, their structure is not closed, which makes the detection susceptible to interference from the external environment, and the sample flow is uncontrolled, resulting in large differences between test individuals, which affects the detection sensitivity and repeatability, and cannot meet the detection performance requirements of laboratory doctors for clinical diagnosis.

[0006] Microfluidic chip POCT detection technology is a rapid detection technology developed in the past decade. Due to the characteristics of microfluidic technology such as sample flow control, chip closure, tiny pipelines, and strong controllability, the detection sensitivity and detection repeatability of microfluidic chip POCT detection technology have been improved to varying degrees compared with the chromatography POCT detection technology. Therefore, microfluidic chip POCT detection technology is increasingly favored by the market.

[0007] In the prior art, microfluidic chip POCT detection products can be roughly divided into two categories according to the different sample driving methods: one is a passive microfluidic detection chip, which drives the flow of samples in the chip by providing capillary force through tiny pipes in the chip. Although the passive microfluidic chip POCT has a simple detection method and low requirements for the detection system, the controllability of the entire sample detection reaction is not strong, and the reagent reaction is highly random. The reagent reaction chamber contained in the passive microfluidic chip is pre-filled with dry reaction reagents, and the dried reagents are redissolved after the sample flows into the reagent chamber. The redissolution process is random, and the sample redissolution volume and redissolution process are uncontrollable, resulting in poor detection repeatability. Its detection repeatability is not significantly improved compared with the chromatography POCT detection technology.

[0008] Another type of microfluidic chip POCT detection product is the active microfluidic detection chip, which drives the flow of samples in the chip through external forces such as air pressure, mechanical force, centrifugal force, and electrodynamics. The active microfluidic chip POCT accurately controls the flow and mixing reaction process of the test samples and reagents, thereby greatly improving the repeatability and sensitivity of reagent detection. However, the active microfluidic chip POCT that integrates magnetic particle detection technology has the main defect that it can only perform single-item detection, and cannot perform multiple-item detection simultaneously in a single-channel microfluidic chip, because the magnetic particles coated with different antibodies cannot be separated by the magnetic field. The active microfluidic chip POCT that integrates the pressure valve needs to integrate an elastic film in the chip, which makes the entire chip packaging process extremely complicated, resulting in low chip manufacturing yield. Summary of the invention

[0009] In view of this, in order to overcome the defects in the prior art, one of the objects of the present invention is to provide a method for preparing a microfluidic chip, and the prepared microfluidic chip can realize on-site rapid, accurate, highly sensitive and quantitative detection of multiple items.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A microfluidic chip includes a chip substrate and a chip cover, wherein the chip cover is provided with a sample addition port for adding a test sample, and the chip substrate is provided with a reaction chamber located below the sample addition port, a mixing channel connected to the reaction chamber, a detection channel connected to the mixing channel, and a waste liquid tank in sequence, wherein the mixing channel is coated with a fluorescent microsphere labeling reagent for providing a fluorescent detection signal, and the detection channel is loaded with a carrier film, and the carrier film is coated with at least one capture antibody reagent. In some embodiments, the depth of the detection channel is 50 μm-1000 μm; the depth of the waste liquid tank is 0.2 mm-2 mm.

[0012] When the microfluidic chip is used, the sample addition port can be connected to a driving device, which can be driven by air pressure, hydraulic pressure or electric power. The driving device provides a driving force for the added test sample to flow back and forth in the pipeline of the chip, so that the sample can fully react with the reagent in the pipeline.

[0013] According to some preferred aspects of the present invention, a super-hydrophobic pipe is also provided between the mixing pipe and the detection pipe. The super-hydrophobic pipe comprises a groove microstructure and a first micron column of a micron structure, and the surface of the groove microstructure has a super-hydrophobic reagent. The super-hydrophobic pipe can serve as a transition function for the reaction between the detection sample and the reagent to form a reaction complex and the reaction complex to flow to the detection pipe. The depth of each groove in the groove microstructure is 20-500μm and the width is 50-4000μm; the diameter of the first micron column is 20-500μm and the height is 20-500μm; the super-hydrophobic reagent is fluorosilane dissolved in an electronic fluoride liquid.

[0014] The superhydrophobic channel is composed of a groove microstructure, a first micron column and a superhydrophobic reagent covering the surface of the groove microstructure. The superhydrophobic surface formed by the superhydrophobic reagent and the groove microstructure can realize a passive flow blocking valve. When the test sample is driven to flow to the superhydrophobic surface, it can effectively prevent the liquid from continuing to flow. The liquid will remain in the groove and will not spread. The driving force must be increased to drive the liquid to continue to flow forward, thereby further enhancing the controllability of the liquid flow.

[0015] According to some preferred aspects of the present invention, a second micron column with a micron structure is arranged in the mixing channel, and the fluorescent microsphere labeling reagent is coated on the second micron column. The second micron column has a diameter of 10-500 μm and a height of 10-500 μm.

[0016] The mixing pipe is connected to the reaction chamber and can provide a space for the test sample to flow back and forth. The test sample is re-dissolved in the coated fluorescent microsphere labeling reagent and mixed evenly with it, so that a liquid homogeneous immune reaction occurs to form a reaction complex. After the test sample enters the mixing pipe, the fluorescent microsphere labeling reagent is first re-dissolved, then the test sample and the fluorescent microsphere labeling reagent are mixed, and finally the test sample and the fluorescent microsphere labeling reagent are incubated for reaction, wherein the re-dissolution and mixing basically occur in the mixing pipe, and the incubation mainly occurs in the reaction chamber. That is, the reaction chamber can store the test sample and provide a place for the test sample and the reagent to react.

[0017] More preferably, the fluorescent microsphere labeling reagent is a detection antibody labeled with fluorescent microspheres.

[0018] More preferably, the capture antibody reagent is a capture antibody labeled with nano-polystyrene microspheres.

[0019] According to some preferred aspects of the present invention, a transition duct is provided between the mixing duct and the reaction chamber, and a third micron column and a boss of a micron structure are provided in the transition duct. The third micron column and the boss in the transition duct can further mix the liquid flowing between the reaction chamber and the mixing channel, so that the reaction between the test sample and the reagent is more sufficient. The diameter of the third micron column is 20-500μm, and the height is 20-500μm; the height of the boss is 20-1000μm, and the width is 100-4000μm.

[0020] The carrier membrane is a nitrocellulose membrane. If only one capture antibody reagent is coated in the carrier membrane, the capture antibody reagent can be randomly dispersed in the carrier membrane; if two or more capture antibody reagents are coated in the carrier membrane, the carrier membrane needs to be divided into several areas, and different capture antibody reagents are coated in different areas. In this way, in the final detection, the fluorescent signals generated by the complexes corresponding to different areas can be distinguished.

[0021] The present invention also provides a method for preparing the microfluidic chip as described above, which specifically comprises the following steps:

[0022] Step 1) Preparation of fluorescent microsphere labeling reagent: labeling the purified detection antibody raw material using time-resolved fluorescent microsphere analysis, and collecting the fluorescent microsphere markers as the fluorescent microsphere labeling reagent;

[0023] Step 2) Preparation of capture antibody reagent: diluting the purified capture antibody raw material with a diluent, and marking the diluted capture antibody raw material on nano-polystyrene microspheres to prepare a capture antibody reagent;

[0024] Step 3) Super-hydrophilic modification of chip surface materials: Super-hydrophilic modification of chip surface materials by vacuum plasma bombardment or atmospheric plasma bombardment;

[0025] Step 4) Spraying and drying of the blocking solution: After the super-hydrophilic modification of the chip surface in step 3) is completed, a layer of blocking solution is sprayed on the surface of the microfluidic chip for surface blocking, and then the chip is dried;

[0026] Step 5) Preparation of super-hydrophobic channels: After the chip is dried in step 4), a precision spotter and a super-hydrophobic reagent are used to perform super-hydrophobic modification on the surface of a specific channel in the chip, i.e., the surface of the groove microstructure, and then the chip is dried to obtain a super-hydrophobic channel;

[0027] Step 6) Drying of the fluorescent microsphere labeling reagent: After the chip is dried in step 5), the fluorescent microsphere labeling material collected in step 1) is added to the mixing channel in the chip substrate, and then dried, so that the fluorescent microsphere labeling material is dried in the chip and coated on the second micron column;

[0028] Step 7) Fixation of capture antibody: After the chip is dried in step 6), the capture antibody reagent prepared in step 2) is coated on the carrier membrane using a precision spotter, and then dried to fix the capture antibody labeled with polystyrene microspheres on the carrier membrane, and the carrier membrane coated with the capture antibody reagent is placed on the detection channel of the chip substrate;

[0029] If only one capture antibody reagent is coated in the carrier membrane, the capture antibody reagent can be dispersed in the carrier membrane at will; if two or more capture antibody reagents are coated in the carrier membrane, the carrier membrane needs to be divided into several areas, and different capture antibody reagents are coated in different areas, so that in the final detection, the fluorescent signals generated by the complexes corresponding to different areas can be distinguished;

[0030] Step 8) Microfluidic chip packaging: Put absorbent paper in the waste liquid tank, and assemble and bond the chip cover plate and the chip substrate to obtain a microfluidic chip. The chip substrate and the cover plate are bonded together by pressure glue, ultrasound, laser, etc. to form a closed bedside diagnosis microfluidic chip.

[0031] Preferably, the blocking solution comprises 0.05%-0.5% BSA, 0.01%-0.5% Tween 20 and 10mM-200mM PBS; the diluent is 10mM PBS. BSA is bovine serum albumin, preferably the mass ratio of which to the total volume of the blocking solution is 0.1%; Tween 20 is a surfactant, preferably the volume ratio of which to the total volume of the blocking solution is 0.01%; PBS is a buffer solution, preferably the concentration of which is 10mM.

[0032] The present invention also provides a detection method of the microfluidic chip as described above, which specifically comprises the following steps:

[0033] Step A. Adding and mixing: adding samples with a pipette, sucking the test sample into the sample loading hole of the above-mentioned bonded microfluidic chip, then placing the loaded microfluidic chip into the card slot of the detection instrument, combining the sample loading hole of the microfluidic chip with the gas path driving device of the instrument, controlling the gas path driving device to generate alternating positive pressure and negative pressure in the sample loading hole, driving the test sample to flow back and forth in the reaction chamber and the mixing channel in the chip, and uniformly mixing the fluorescent microsphere labeling reagent in the mixing channel while re-dissolving it, and stopping the generation of positive air pressure or negative air pressure after the mixing is completed, and incubating the mixed solution to cause an immune complex reaction to form an antibody / antigen complex labeled with fluorescent microspheres;

[0034] Step B. Capturing the antibody / antigen complex: After completing step A, the air path driving device generates a high positive air pressure in the sample loading hole, drives the mixed sample containing the antibody / antigen complex to flow through the super-hydrophobic pipe, and then reduces the high positive air pressure to a low positive air pressure, drives the mixed sample to flow into the carrier membrane in the chip detection pipe, and at the same time, the capture antibody reagent coated in the carrier membrane reacts with the antibody / antigen complex in the mixed sample to form a capture antibody-antigen-labeled antibody complex, and other substances that are not captured flow into the waste liquid tank with the liquid and are absorbed by the absorbent paper;

[0035] Step C. Data reading: After step B is completed, the optical device of the detection instrument reads the fluorescence intensity on the carrier membrane in the microfluidic chip, calculates the capture antibody-antigen-labeled antibody concentration and generates the detection result.

[0036] The magnitude and time of the positive pressure, negative pressure, high positive pressure and low positive pressure generated by the driving device in the above detection steps are set according to actual needs.

[0037] Due to the implementation of the above technical solutions, the microfluidic chip of the present invention has the following advantages compared with the prior art: the preparation method of the microfluidic chip of the present invention, the prepared microfluidic chip has a reaction chamber with a fixed reaction volume and a mixing channel, combined with an active driving mode, drives the detection sample and reagent to flow in the channel, and accurately controls the re-dissolution and mixing process of the sample and the reagent, thereby achieving strict controllability of the reagent re-dissolution volume and reaction time, greatly improving the detection repeatability; the super-hydrophobic channel designed for the microfluidic chip forms a passive flow blocking valve, the packaging is simple, the chip production process is greatly simplified, and the chip yield is improved; the microfluidic chip is coated with more than one capture antibody in different areas of the carrier membrane to achieve multi-project joint detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 is a stereoscopic diagram of the microfluidic chip in the preferred embodiment 1 of the present invention;

[0040] Figure 2 A stacked diagram of a microfluidic chip according to a preferred embodiment 1 of the present invention;

[0041] Figure 3 for Figure 2 Enlarged view of the middle I part;

[0042] Figure 4 for Figure 2 Enlarged view of middle part II;

[0043] In the accompanying drawings: chip cover plate-1, chip substrate-2, sample loading port-3, reaction chamber-4, transition channel-5, third micron column-51, boss-52, mixing channel-6, second micron column-61, super hydrophobic channel-7, first micron column-71, groove microstructure-72, detection channel-8, carrier membrane-9, waste liquid tank-10, absorbent paper-11. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0045] It should be noted that the terms "first" and "second" in this article are intended to facilitate the distinction between multiple objects and have no limiting effect. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or equipment.

[0046] Example 1 Microfluidic Chip

[0047] like Figure 1-4 As shown, a microfluidic chip of this embodiment includes a chip substrate 2 and a chip cover plate 1, a sample adding port 3 for adding a test sample is provided on the chip cover plate 1, a reaction chamber 4 located below the sample adding port 3, a mixing pipe 6 connected to the reaction chamber 4, a detection pipe 8 connected to the mixing pipe 6, and a waste liquid tank 10 are provided on the chip substrate 2 in sequence, and a blotting paper 11 matched therewith is loaded in the waste liquid tank 10. In this embodiment, the depth of the detection pipe 8 is 200 μm, and the depth of the waste liquid tank 10 is 1 mm; in other embodiments, the depth of the detection pipe 8 ranges from 50 μm to 1000 μm, and the depth of the waste liquid tank 10 ranges from 0.2 mm to 2 mm.

[0048] The mixing channel 6 is coated with a fluorescent microsphere labeling reagent for providing a fluorescent detection signal, and the detection channel 8 is loaded with a carrier membrane 9, and at least one capture antibody reagent is coated on the carrier membrane 9. Among them, the fluorescent microsphere labeling reagent is a detection antibody labeled with fluorescent microspheres, and the capture antibody reagent is a capture antibody labeled with nano polystyrene microspheres.

[0049] The mixing pipe 6 is connected to the reaction chamber 4 and can provide a space for the test sample to flow back and forth. The test sample is re-dissolved in the coated fluorescent microsphere labeling reagent and mixed evenly with it, so that a liquid homogeneous immune reaction occurs to form a reaction complex. After the test sample enters the mixing pipe 6, the fluorescent microsphere labeling reagent is first re-dissolved, then the test sample and the fluorescent microsphere labeling reagent are mixed, and finally the test sample and the fluorescent microsphere labeling reagent are incubated for reaction, wherein the re-dissolution and mixing basically occur in the mixing pipe 6, and the incubation mainly occurs in the reaction chamber 4. The reaction chamber 4 can store the test sample and provide a reaction place for the test sample and the reagent.

[0050] like Figure 2-3 As shown, in this embodiment, a super-hydrophobic pipe 7 is also provided between the mixing pipe 6 and the detection pipe 8. The super-hydrophobic pipe 7 includes a groove microstructure 72 and a first micron column 71 of a micron structure, and the surface of the groove microstructure 72 has a super-hydrophobic reagent. The super-hydrophobic pipe 7 can serve as a transition function for the detection sample to react with the reagent to form a reaction complex and the reaction complex to flow to the detection pipe 8. In this embodiment, the depth of each groove in the groove microstructure 72 is 50μm and the width is 4mm; the diameter of the first micron column 71 is 100μm and the height is 100μm; the super-hydrophobic reagent is fluorosilane dissolved in an electronic fluoride liquid. In other embodiments, the depth of each groove in the groove microstructure 72 ranges from 20-500μm and the width ranges from 50-4000μm; the diameter of the first micron column 71 ranges from 20-500μm and the height ranges from 20-500μm.

[0051] The super-hydrophobic surface formed by the super-hydrophobic reagent and the groove microstructure 72 can realize a passive flow blocking valve. When the test sample is driven to flow to the super-hydrophobic surface, it can effectively block the liquid from continuing to flow. The liquid will remain in the groove and will not spread. The driving force must be increased to drive the liquid to continue to flow forward, thereby further enhancing the controllability of the liquid flow.

[0052] like Figure 2-3 As shown, a second micron column 61 with a micron structure is provided in the mixing pipe 6, and the fluorescent microsphere labeling reagent is coated on the second micron column 61. In this embodiment, the diameter of the second micron column 61 is 200 μm and the height is 200 μm. In other embodiments, the diameter of the second micron column 61 ranges from 10 to 500 μm and the height ranges from 10 to 500 μm.

[0053] like Figure 2 and 4As shown, in this embodiment, a transition pipe 5 is provided between the mixing pipe 6 and the reaction chamber 4, and a third micron column 51 and a boss 52 of a micron structure are provided in the transition pipe 5. The third micron column 51 and the boss 52 in the transition pipe 5 can further mix the liquid flowing between the reaction chamber 4 and the mixing channel 6, so that the reaction between the test sample and the reagent is more sufficient. The diameter of the third micron column 51 is 200μm and the height is 200μm; the height of the boss 52 is 200μm and the width is 200μm. In other embodiments, the diameter of the third micron column 51 ranges from 20-500μm and the height ranges from 20-500μm; the height of the boss 52 ranges from 20-1000μm and the width ranges from 100-4000μm.

[0054] The carrier membrane 9 in this embodiment is a nitrocellulose membrane. If only one capture antibody reagent is coated in the carrier membrane 9, the capture antibody reagent can be randomly dispersed in the carrier membrane 9; if two or more capture antibody reagents are coated in the carrier membrane 9, the carrier membrane 9 needs to be divided into several areas, and different capture antibody reagents are coated in different areas. In this way, in the final detection, the fluorescent signals generated by the complexes corresponding to different areas can be accurately distinguished.

[0055] When the microfluidic chip in this embodiment is used, the sample addition port can be connected to a driving device, which can be driven by air pressure, hydraulic pressure or electric power. The driving device provides a driving force for the added test sample to flow back and forth in the pipeline of the chip, so that the test sample can fully react with the reagent in the pipeline.

[0056] The microfluidic chip in this embodiment is designed with a reaction chamber and a mixing channel with a fixed reaction volume, and an active driving method is used to drive the detection samples and reagents to flow in the channel, and accurately control the re-dissolution and mixing process of the samples and reagents, so as to achieve strict control of the reagent re-dissolution volume and reaction time, greatly improving the detection repeatability; a super-hydrophobic channel is designed to form a passive flow blocking valve, which is simple to encapsulate, greatly simplifies the chip production process, and improves the chip yield; different areas in the carrier membrane can be coated with more than one capture antibody to achieve multi-project joint detection.

[0057] Example 2 Preparation method of microfluidic chip

[0058] This embodiment provides a method for preparing the microfluidic chip based on the embodiment 1, which specifically comprises the following steps:

[0059] Step 1) Preparation of fluorescent microsphere labeling reagent

[0060] The purified detection antibody raw material is labeled by time-resolved fluorescent microsphere analysis, and the fluorescent microsphere marker is collected to obtain the fluorescent microsphere labeling reagent.

[0061] Step 2) Preparation of capture antibody reagent

[0062] The purified capture antibody raw material is diluted with a diluent, and the diluted capture antibody raw material is marked on nano polystyrene microspheres to prepare a capture antibody reagent.

[0063] The diluent in this example is 10 mM PBS.

[0064] Step 3) Super-hydrophilic modification of chip surface materials

[0065] The chip surface material is modified to be super-hydrophilic by vacuum plasma bombardment or atmospheric plasma bombardment.

[0066] Step 4) Spraying and drying of sealing liquid

[0067] After the super-hydrophilic modification of the chip surface in step 3) is completed, a layer of blocking liquid is sprayed on the surface of the microfluidic chip for surface blocking, and then the chip is dried.

[0068] The blocking solution in this embodiment includes 0.1% BSA, 0.01% Tween 20 and 10 mM PBS, wherein BSA is bovine serum albumin, Tween 20 is a surfactant, and PBS is a buffer.

[0069] Step 5) Preparation of super hydrophobic pipe

[0070] After the chip in step 4) is dried, a precision spotter and a superhydrophobic reagent are used to perform superhydrophobic modification on the surface of a specific channel in the chip, i.e., the surface of the groove microstructure, and then the chip is dried to obtain a superhydrophobic channel.

[0071] Step 6) Drying of fluorescent microsphere labeling reagent

[0072] After the chip is dried in step 5), the fluorescent microsphere markers collected in step 1) are added to the mixing channel in the chip substrate and then dried to dry the fluorescent microsphere markers in the chip and coat the second micron column.

[0073] Step 7) Immobilization of capture antibody

[0074] After the chip is dried in step 6), the capture antibody reagent prepared in step 2) is coated on the carrier membrane using a precision spotter, and then dried to fix the capture antibody labeled with polystyrene microspheres on the carrier membrane, and the carrier membrane coated with the capture antibody reagent is placed on the detection channel of the chip substrate.

[0075] If only one capture antibody reagent is coated in the carrier membrane, the capture antibody reagent can be dispersed freely in the carrier membrane; if two or more capture antibody reagents are coated in the carrier membrane, the carrier membrane needs to be divided into several areas, and different capture antibody reagents are coated in different areas, so that in the final detection, the fluorescent signals generated by the complexes corresponding to different areas can be distinguished.

[0076] Step 8) Microfluidic chip packaging

[0077] Put absorbent paper in the waste liquid tank, and assemble and bond the chip cover plate and the chip substrate to obtain a microfluidic chip.

[0078] The chip substrate and the cover plate are bonded together by means of pressure glue, ultrasound, laser, etc. to form a closed microfluidic chip for bedside diagnosis.

[0079] Example 3 Detection method of microfluidic chip

[0080] This embodiment provides a detection method based on the microfluidic chip described in Example 1, which specifically includes the following steps:

[0081] Step A. Add sample and mix well

[0082] The sample is added by a pipette, and the test sample is sucked into the sample loading hole of the above-mentioned bonded microfluidic chip, and then the microfluidic chip with the added sample is placed in the card slot of the detection instrument. The sample loading hole of the microfluidic chip is combined with the gas path driving device of the instrument, and the gas path driving device is controlled to generate alternating positive pressure and negative pressure in the sample loading hole, driving the test sample to flow back and forth in the reaction chamber and the mixing channel in the chip, and the fluorescent microsphere labeling reagent in the mixing channel is evenly mixed while being redissolved. After the mixing is completed, the positive air pressure or negative air pressure is stopped, and the mixed solution is incubated to cause an immune complex reaction to form an antibody / antigen complex labeled with fluorescent microspheres.

[0083] Step B. Capture of Antibody / Antigen Complex

[0084] After step A is completed, the gas path driving device generates a high positive air pressure in the sample loading hole, drives the mixed sample containing the antibody / antigen complex to flow through the super-hydrophobic pipe, and then reduces the high positive air pressure to a low positive air pressure, drives the mixed sample to flow into the carrier membrane in the chip detection pipe, and at the same time, the capture antibody reagent coated in the carrier membrane reacts with the antibody / antigen complex in the mixed sample to form a capture antibody-antigen-labeled antibody complex, and other substances that are not captured flow into the waste liquid tank with the liquid and are absorbed by the absorbent paper.

[0085] Step C. Data reading

[0086] After step B is completed, the optical device of the detection instrument reads the fluorescence intensity on the microfluidic chip detection channel, calculates the capture antibody-antigen-labeled antibody concentration and generates the detection result.

[0087] The magnitude and time of the positive air pressure, negative air pressure, high positive air pressure and low positive air pressure generated by the driving device in the above detection steps are set according to actual needs.

[0088] Example 4 Detection of procalcitonin (PCT) by microfluidic chip

[0089] (I) Antibody labeling

[0090] A. Fluorescent microspheres labeled with detection antibodies

[0091] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0092] 2. Take 90ul of 10mM PBS buffer, add 10ul of 300nm fluorescent microspheres, and shake to mix.

[0093] 3. Take 5ul of EDC activation solution and add it to the buffer solution containing fluorescent microspheres prepared in step 2, shake and mix, and place it on a shaker for 15min (room temperature or 20°C, 250rpm).

[0094] 4. Prepare 100 μl labeled antibody: Use 10 mM PBS buffer to prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with a final concentration of 0.5 mg / ml.

[0095] 5. Place the activated fluorescent microsphere solution prepared in step 3 in a centrifuge, centrifuge at 15,000 rpm for 15 minutes, and discard the supernatant.

[0096] 6. Add the 0.5 mg / ml PCT antibody and rabbit IgG antibody solution prepared in step 4 to the activated fluorescent microsphere solution obtained after centrifugation in step 5, and shake to mix. Ultrasonicate for 2-3 minutes, shake and mix, and incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0097] 7. Take 20 μl of blocking solution (1% bovine serum albumin) and add it to the solution obtained in step 6. Place it on a shaker for blocking for 2 hours. The blocking conditions are: room temperature, 250 rpm.

[0098] 8. Place the solution obtained in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0099] 9. Add 500ul 10mM PBS buffer to the substance obtained in step 8 to redissolve the fluorescent microspheres. After vortexing and mixing, place in a centrifuge at 15000rpm for 15min and discard the supernatant.

[0100] 10. Add 200ul of microsphere preservation solution (0.1% BSA, 5% sucrose, 10mM PBS) to the substance obtained in step 9, shake and mix well, sonicate for 3min, and store at 2-8°C.

[0101] B. Polystyrene microspheres labeled with capture antibodies

[0102] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0103] 2. Take 90ul of 10mM PBS buffer, add 10ul of 150nm polystyrene microspheres, and shake to mix.

[0104] 3. Take 5ul of EDC activation solution and add it to the buffer solution with polystyrene microspheres prepared in step 2, shake and mix well, and place it on a shaker for 15min (room temperature or 20°C, 250rpm).

[0105] 4. Prepare 100 μl capture antibody: Use 10 mM PBS buffer to prepare PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solution with a final concentration of 0.5 mg / ml.

[0106] 5. Place the activated polystyrene microsphere solution prepared in step 3 in a centrifuge, centrifuge at 15,000 rpm for 15 minutes, and discard the supernatant.

[0107] 6. Add the 0.5 mg / ml PCT antibody and goat anti-rabbit IgG antibody solution prepared in step 4 to the substance obtained in step 5 and shake to mix. Ultrasonicate for 2-3 minutes, shake and mix, and incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0108] 7. Take 20 μl of blocking solution (1% bovine serum albumin) and add it to the substance prepared in step 6. Place it on a shaker for blocking for 2 hours. The blocking conditions are: room temperature, 250 rpm.

[0109] 8. Place the substance prepared in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0110] 9. Add 500 μl 10 mM PBS buffer to the material prepared in step 8 to redissolve the polystyrene microspheres. After vortexing and mixing, place in a centrifuge at 15,000 rpm for 15 min and discard the supernatant.

[0111] 10. Add 200ul 10mM PBS to the material prepared in step 9, shake to mix and sonicate for 3min, and store at 2-8℃.

[0112] (II) Microfluidic chip assembly

[0113] 1. Use vacuum plasma bombardment or atmospheric plasma bombardment to modify the surface material of the chip substrate to be super-hydrophilic.

[0114] 2. After the super-hydrophilic modification of the chip surface is completed, a layer of blocking solution is sprayed on the surface of the microfluidic chip for surface blocking, and then the chip is dried. The blocking solution contains 0.1% BSA, 0.01% Tween 20 and 10m MPBS.

[0115] 3. After the chip in step 2 is dried, a precision spotter and a superhydrophobic reagent are used to perform superhydrophobic modification on the surface of a specific channel in the chip, i.e., the surface of the groove microstructure, and then the chip is dried to obtain a superhydrophobic channel.

[0116] 4. After the chip in step 3 is dried, the PCT monoclonal antibody and rabbit IgG antibody labeled with fluorescent microspheres collected in step (i) are added to the mixing channel in the chip substrate, and then dried to dry the fluorescent microsphere markers in the mixing channel of the chip.

[0117] 5. After the chip in step 4 is dried, the PCT capture antibody reagent and goat anti-rabbit IgG antibody reagent labeled with polystyrene microspheres prepared in step (a) are coated on the carrier membrane using a precision spotter, and then dried to fix the capture antibody labeled with polystyrene microspheres on the carrier membrane, and the carrier membrane coated with the capture antibody reagent is placed on the detection channel of the chip substrate.

[0118] 6. Place absorbent paper in the waste liquid tank, assemble the chip cover and chip substrate and bond them with pressure glue to obtain a microfluidic chip.

[0119] (III) Sample testing

[0120] 1. Pipette the serum sample containing PCT antigen, draw 150 μL of the test sample into the sample well of the above-mentioned bonded microfluidic chip, then place the loaded microfluidic chip into the card slot of the detection instrument, the sample well of the microfluidic chip is combined with the gas path driving device of the instrument, the gas path driving device is controlled to generate alternating positive pressure and negative pressure in the sample well, drive the test sample to flow back and forth in the reaction chamber and mixing channel in the chip, and evenly mix the fluorescent microsphere-labeled PCT monoclonal antibody and rabbit IgG antibody reagent in the mixing channel while re-dissolving them. After the mixing is completed, stop generating positive or negative air pressure, and incubate the mixed solution to cause an immune complex reaction to form a PCT monoclonal antibody / PCT antigen complex labeled with fluorescent microspheres.

[0121] 2. After step 1 is completed, the gas path driving device generates a high positive air pressure in the sample loading hole, drives the mixed sample containing the PCT antibody / PCT antigen complex to flow through the super-hydrophobic pipe, and then reduces the high positive air pressure to a low positive air pressure, drives the mixed sample to flow into the carrier membrane in the chip detection pipe, and at the same time, the polystyrene-labeled PCT capture antibody reagent and the goat anti-rabbit IgG antibody reagent coated in the carrier membrane react with the PCT labeled antibody / PCT antigen complex and the rabbit IgG antibody in the mixed sample to form a PCT capture antibody-PCT antigen-PCT labeled antibody complex and a rabbit IgG-goat anti-rabbit IgG antibody complex. Other substances that are not captured flow into the waste liquid tank with the liquid and are absorbed by the absorbent paper.

[0122] 3. After step 2 is completed, the optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-sheep anti-rabbit IgG antibody complex on the microfluidic chip detection channel, and obtains the fluorescence intensity ratio (T / C value). The concentration of PCT capture antibody-PCT antigen-PCT labeled antibody is calculated through the calibration curve and the detection result is generated.

[0123] In the above detection steps, the magnitudes of the positive pressure, negative pressure, high positive pressure, low positive pressure generated by the driving device and the driving time are set according to actual needs.

[0124] Comparative Example 1

[0125] This comparative example provides a traditional passive bedside diagnostic microfluidic chip. The microfluidic chip provided in this comparative example is basically the same as Example 1, except that there is no superhydrophobic pipe and carrier membrane in this comparative example. The microfluidic chip in this comparative example includes a chip substrate and a chip cover plate, and a sample addition port for adding a test sample is provided on the chip cover plate, and a reaction chamber connected to the sample addition port, a detection pipe connected to the reaction chamber, and a waste liquid tank connected to the detection pipe are provided on the chip substrate. In this comparative example, a fluorescent microsphere labeling reagent for providing a fluorescent detection signal is coated in the reaction chamber, and a capture antibody reagent labeled with polystyrene is coated in the detection pipe.

[0126] (I) Antibody labeling

[0127] A. Fluorescent microspheres labeled with detection antibodies

[0128] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0129] 2. Take 90ul of 10mM PBS buffer, add 10ul of 300nm fluorescent microspheres, and shake to mix.

[0130] 3. Take 5ul of EDC activation solution and add it to the buffer solution containing fluorescent microspheres prepared in step 2, shake and mix, and place it on a shaker for 15min (room temperature or 20°C, 250rpm).

[0131] 4. Prepare 100 μl labeled antibody: Use 10 mM PBS buffer to prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with a final concentration of 0.5 mg / ml.

[0132] 5. Place the activated fluorescent microsphere solution prepared in step 3 in a centrifuge, centrifuge at 15,000 rpm for 15 minutes, and discard the supernatant.

[0133] 6. Add the 0.5 mg / ml PCT antibody and rabbit IgG antibody solution prepared in step 4 to the activated fluorescent microsphere solution obtained after centrifugation in step 5, and shake to mix. Ultrasonicate for 2-3 minutes, shake and mix, and incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0134] 7. Take 20 μl of blocking solution (1% bovine serum albumin) and add it to the solution obtained in step 6. Place it on a shaker for blocking for 2 hours. The blocking conditions are: room temperature, 250 rpm.

[0135] 8. Place the solution obtained in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0136] 9. Add 500ul 10mM PBS buffer to the substance obtained in step 8 to redissolve the fluorescent microspheres. After vortexing and mixing, place in a centrifuge at 15000rpm for 15min and discard the supernatant.

[0137] 10. Add 200ul of microsphere preservation solution (0.1% BSA, 5% sucrose, 10mM PBS) to the substance obtained in step 9, shake and mix well, sonicate for 3min, and store at 2-8°C.

[0138] B. Polystyrene microspheres labeled with capture antibodies

[0139] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0140] 2. Take 90ul of 10mM PBS buffer, add 10ul of 150nm polystyrene microspheres, and shake to mix.

[0141] 3. Take 5ul of EDC activation solution and add it to the buffer solution with polystyrene microspheres prepared in step 2, shake and mix well, and place it on a shaker for 15min (room temperature or 20°C, 250rpm).

[0142] 4. Prepare 100 μl capture antibody: Use 10 mM PBS buffer to prepare PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solution with a final concentration of 0.5 mg / ml.

[0143] 5. Place the activated polystyrene microsphere solution prepared in step 3 in a centrifuge, centrifuge at 15,000 rpm for 15 minutes, and discard the supernatant.

[0144] 6. Add the 0.5 mg / ml PCT antibody and goat anti-rabbit IgG antibody solution prepared in step 4 to the substance obtained in step 5 and shake to mix. Ultrasonicate for 2-3 minutes, shake and mix, and incubate on a shaker for 2 hours. Incubation conditions: room temperature, 250 rpm.

[0145] 7. Take 20 μl of blocking solution (1% bovine serum albumin) and add it to the substance prepared in step 6. Place it on a shaker for blocking for 2 hours. The blocking conditions are: room temperature, 250 rpm.

[0146] 8. Place the substance prepared in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0147] 9. Add 500 μl 10 mM PBS buffer to the material prepared in step 8 to redissolve the polystyrene microspheres. After vortexing and mixing, place in a centrifuge at 15,000 rpm for 15 min and discard the supernatant.

[0148] 10. Add 200ul 10mM PBS to the material prepared in step 9, shake to mix and sonicate for 3min, and store at 2-8℃.

[0149] (II) Microfluidic chip assembly

[0150] 1. Use vacuum plasma bombardment or atmospheric plasma bombardment to modify the surface material of the chip substrate to be super-hydrophilic.

[0151] 2. After the super-hydrophilic modification of the chip surface is completed, a layer of blocking solution is sprayed on the surface of the microfluidic chip for surface blocking, and then the chip is dried. The blocking solution contains 0.1% BSA, 0.01% Tween 20 and 10m MPBS.

[0152] 3. After the chip in step 2 is dried, the PCT monoclonal antibody and rabbit IgG antibody labeled with fluorescent microspheres collected in step (a) are added to the reaction chamber in the chip substrate, and then dried to dry the fluorescent microsphere markers in the reaction chamber in the chip.

[0153] 4. After the chip in step 3 is dried, the polystyrene-labeled PCT capture antibody and goat anti-rabbit IgG antibody reagent prepared in step (a) are coated on the micrometer column detection channel in the chip substrate using a precision spotter, and then dried to fix the capture antibody labeled with polystyrene microspheres on the micrometer column detection channel.

[0154] 5. Assemble the chip cover plate and the chip substrate and bond them by pressure adhesive to obtain a microfluidic chip.

[0155] (III) Sample testing

[0156] 1. Use a pipette to add serum samples containing PCT antigen, draw 150 μL of the test sample into the sample loading hole of the above-mentioned bonded microfluidic chip, then place the loaded microfluidic chip into the card slot of the detection instrument, and the test sample flows into the reaction chamber by capillary suction, and the fluorescent microspheres labeled with PCT monoclonal antibody and rabbit IgG antibody reagent in the reaction chamber are re-dissolved and reacted with them to form a fluorescent microsphere-labeled PCT monoclonal antibody / PCT antigen complex.

[0157] 2. Then, the mixed sample containing the PCT antibody / PCT antigen complex is self-driven by capillary force into the detection channel of the chip. At the same time, the PCT capture antibody and goat anti-rabbit IgG antibody labeled with polystyrene microspheres coated in the channel react with the PCT antibody / PCT antigen complex and rabbit IgG antibody in the sample to form a PCT capture antibody-PCT antigen-PCT labeled antibody complex and a rabbit IgG-goat anti-rabbit IgG antibody complex. Other substances that are not captured flow into the waste liquid tank with the liquid.

[0158] 3. After step 2 is completed, the optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-sheep anti-rabbit IgG antibody complex on the microfluidic chip detection channel, and obtains the fluorescence intensity ratio (T / C value). The concentration of PCT capture antibody-PCT antigen-PCT labeled antibody is calculated through the calibration curve and the detection result is generated.

[0159] Example 5 Results and Analysis

[0160] The microfluidic chips prepared in Example 4 and Comparative Example 1 were used to test the same test sample with a PCT concentration of 0.05 ng / mL 10 times, and the performance test and analysis comparison are shown in the following table:

[0161] Table 1 Test results comparison table

[0162]

[0163] The experimental results in Table 1 show that when the same test sample is tested, the active microfluidic chip in Example 1 and the corresponding detection method in Example 4 are compared with the passive microfluidic chip and its detection method in Comparative Example 1. The detection results of the active microfluidic chip in Example 1 can be seen from the T / C value that the detection sensitivity is significantly improved, and the detection repeatability can be greatly improved from the detection coefficient of variation.

[0164] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a microfluidic chip, characterized in that: The microfluidic chip comprises a chip substrate and a chip cover plate, wherein the chip cover plate is provided with a sample addition port for adding a test sample, and the chip substrate is provided with a reaction chamber, a mixing channel, a super-hydrophobic channel, a test channel and a waste liquid tank in sequence, wherein the reaction chamber is located below the sample addition port; The preparation method comprises the following steps: After the chip surface material is modified to be super-hydrophilic, a layer of blocking liquid is sprayed on the surface of the microfluidic chip to seal the surface, and then the chip is dried; The super-hydrophobic pipeline is composed of a groove microstructure, a first micron column, and a super-hydrophobic agent covering the surface of the groove microstructure; the super-hydrophobic agent is covered on the groove microstructure, the surface of the groove microstructure in the chip is super-hydrophobic modified, and then the chip is dried to obtain the super-hydrophobic pipeline; A second micron column with a micron structure is arranged in the mixing channel; the fluorescent microsphere marker is added into the mixing channel in the chip substrate, and then dried, so that the fluorescent microsphere marker is dried in the chip and coated on the second micron column; The capture antibody reagent is coated on the carrier membrane, and then dried to fix the capture antibody labeled with polystyrene microspheres on the carrier membrane, and the carrier membrane coated with the capture antibody reagent is placed on the detection channel of the chip substrate; Putting absorbent paper in the waste liquid tank, and assembling and bonding the chip cover plate and the chip substrate to obtain the microfluidic chip; The sample adding port is connected to a driving device, and the driving device is used to provide a driving force for the test sample to flow back and forth in the pipeline of the chip, so that the test sample and the reagent in the pipeline can fully react; A transition pipe is provided between the mixing pipe and the reaction chamber, and a third micron column and a boss with a micron structure are provided in the transition pipe; The super-hydrophobic reagent and the super-hydrophobic surface formed by the groove microstructure are used to realize a passive flow blocking valve; the mixing pipeline is connected to the reaction chamber to provide a space for the test sample to flow back and forth; the reaction chamber is used to store the test sample and provide a reaction place for the test sample and the reagent.

2. The preparation method according to claim 1, characterized in that: The driving mode of the driving device is pneumatic, hydraulic or electric.

3. The preparation method according to claim 1, characterized in that: The preparation of the fluorescent microsphere marker is carried out according to the following steps: using fluorescent microspheres to label the detection antibody raw material, and collecting the fluorescent microsphere marker to obtain the fluorescent microsphere labeling reagent.

4. The preparation method according to claim 1, characterized in that: The capture antibody reagent is prepared according to the following steps: the capture antibody raw material is diluted with a diluent, and the diluted capture antibody raw material is marked on nano polystyrene microspheres to prepare the capture antibody reagent.

5. The preparation method according to claim 1, characterized in that: The super hydrophobic agent is fluorosilane dissolved in electronic fluoride liquid.

6. The preparation method according to claim 1, characterized in that: The detection channel is loaded with a carrier membrane, and the carrier membrane is coated with at least one capture antibody reagent.

7. The preparation method according to claim 6, characterized in that: The carrier membrane is coated with two or more capture antibody reagents, and the carrier membrane is correspondingly divided into a plurality of regions, and different capture antibody reagents are coated in different regions.

8. The preparation method according to claim 1, characterized in that The blocking solution contains 0.05%-0.5% BSA, 0.01%-0.5% Tween 20 and 10mM-200mM PBS.

9. The preparation method according to any one of claims 1 to 8, characterized in that: The chip substrate and the chip cover are bonded together by means of pressure glue, ultrasound or laser to form the closed microfluidic chip for bedside diagnosis.

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