Bedside diagnosis micro-fluidic chip

Abstract

The invention discloses a bedside diagnosis micro-fluidic chip, which comprises a chip substrate, and the chip substrate is provided with a reaction tank, a buffer solution tank, a uniform mixing pipeline communicated with the reaction tank, a detection pipeline communicated with the buffer solution tank and the uniform mixing pipeline, and a waste liquid tank communicated with the detection pipeline. The reaction tank is used for storing a detection sample and providing a reaction place for the detection sample and a reagent; a super-hydrophobic pipeline located between the detection pipeline and the uniform mixing pipeline and / or between the detection pipeline and the buffer solution pool is arranged on the chip substrate, and the super-hydrophobic pipeline is composed of a groove microstructure and a super-hydrophobic reagent covering the surface of the groove microstructure; a super-hydrophobic surface formed by the super-hydrophobic reagent and the groove microstructure is used for realizing passive flow blocking. According to the bedside diagnosis micro-fluidic chip, the designed super-hydrophobic pipeline forms a passive flow blocking valve, the packaging is simple, the chip production process is greatly simplified, and the chip yield is improved.

Description

[0001] This application is a divisional application of the invention patent application with application date of December 13, 2018, application number 201811523063X, and invention name “Bedside diagnostic microfluidic chip and its preparation method and detection method”. Technical Field

[0002] The present invention belongs to the technical field of in vitro diagnosis of medical devices, and in particular relates to 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 bedside diagnostic microfluidic chip that can achieve rapid, accurate, highly sensitive and multi-item quantitative detection on site.

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

[0011] A bedside diagnostic microfluidic chip comprises a chip substrate and a chip cover plate, wherein the chip cover plate is provided with a first sample addition port for adding a test sample and a second sample addition port for adding a buffer solution, the chip substrate is provided with a reaction pool connected to the first sample addition port, a buffer solution pool connected to the second sample addition port, a mixing pipeline interconnected with the reaction pool, a detection pipeline connected to the buffer solution pool and the mixing pipeline, and a waste liquid pool connected to the detection pipeline, the mixing pipeline is coated with a fluorescent microsphere labeling reagent for providing a fluorescent detection signal, and the detection pipeline is coated with at least one capture antibody reagent.

[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 and buffer 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] In some embodiments, the chip substrate is provided with a first sample loading slot located below the first sample loading port, a first sample loading channel connecting the first sample loading slot and the reaction pool, a second sample loading slot located below the second sample loading port, a second sample loading channel connecting the second sample loading slot and the buffer pool, and a transition pipeline is also provided between the buffer pool and the detection pipeline.

[0014] The reaction pool can store test samples and provide a reaction site for test samples and reagents; the buffer pool can store buffer and realize the washing function of immune reaction.

[0015] According to some preferred aspects of the present invention, one end of the detection pipeline is connected to the waste liquid pool, and the other end of the detection pipeline is connected to a super-hydrophobic pipeline, and the super-hydrophobic pipeline includes a first super-hydrophobic pipeline arranged between the detection pipeline and the mixing pipeline and a second super-hydrophobic pipeline arranged between the detection pipeline and the buffer pool. A Y-shaped structure is formed between the first super-hydrophobic pipeline, the second super-hydrophobic pipeline and the detection pipeline, and the sample enters the detection pipeline through the first super-hydrophobic pipeline, and the buffer enters the detection pipeline through the second super-hydrophobic pipeline.

[0016] More preferably, the super-hydrophobic channel comprises a groove microstructure, the surface of the groove microstructure has a super-hydrophobic agent, each groove in the groove microstructure has a depth of 50-200 μm and a width of 100-2000 μm, and the super-hydrophobic agent is fluorosilane dissolved in electronic fluoride liquid.

[0017] The superhydrophobic channel is composed of a groove microstructure 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 or buffer solution 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.

[0018] According to some preferred aspects of the present invention, a first micron column with a micron structure is arranged in the mixing channel, and the fluorescent microsphere labeling reagent is coated on the first micron column. The first micron column has a diameter of 20-500 μm and a height of 10-2000 μm.

[0019] The mixing pipe is connected to the reaction pool 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 pool.

[0020] More preferably, the fluorescent microsphere labeling reagent is a detection antibody labeled with fluorescent microspheres.

[0021] According to some preferred aspects of the present invention, a second micron column with a micron structure is arranged in the detection channel, and the capture antibody reagent is coated on the second micron column. The second micron column has a diameter of 10-200 μm and a height of 10-200 μm.

[0022] If there is only one capture antibody reagent coated in the detection channel, the capture antibody reagent can be randomly dispersed in the detection channel; if there are two or more capture antibody reagents coated in the detection channel, the detection channel 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.

[0023] More preferably, the capture antibody reagent is a capture antibody labeled with nano-polystyrene microspheres.

[0024] The present invention also provides a method for preparing the above-mentioned bedside diagnosis microfluidic chip, which specifically comprises the following steps:

[0025] 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;

[0026] 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;

[0027] Step 3) Super-hydrophilic modification of chip surface materials: Super-hydrophilic modification of chip surface materials by vacuum plasma bombardment or atmospheric plasma bombardment;

[0028] 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;

[0029] 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;

[0030] 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 to dry the fluorescent microsphere labeling material in the chip;

[0031] 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 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 second micrometer column;

[0032] If only one capture antibody reagent is coated in the detection channel, the capture antibody reagent can be randomly dispersed in the detection channel; if two or more capture antibody reagents are coated in the detection channel, the detection channel 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;

[0033] Step 8) Microfluidic chip packaging: Assemble and bond the chip cover plate and the chip substrate to obtain a bedside diagnosis 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.

[0034] Preferably, the blocking solution includes 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.

[0035] The present invention also provides a detection method for the bedside diagnosis microfluidic chip as described above, which specifically comprises the following steps:

[0036] Step A. Adding and mixing: Adding samples with a pipette, respectively sucking the test sample and the buffer solution into the first and second sample wells of the above-mentioned bonded microfluidic chip, then placing the loaded microfluidic chip into the card slot of the detection instrument, combining the first and second sample wells of the microfluidic chip with the gas path driving device of the instrument, controlling the gas path driving device to generate alternating positive and negative pressures in the first sample well, driving the test sample to flow back and forth in the reaction pool and the mixing channel in the chip, and uniformly mixing the fluorescent microsphere labeling reagent in the mixing channel while redissolving it, and stopping the generation of positive 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;

[0037] Step B. Capturing the antibody / antigen complex: After completing step A, the gas path driving device generates a high positive air pressure in the first sample loading hole, drives the mixed sample containing the antibody / antigen complex to flow through the first 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 detection pipe of the chip, and at the same time, the capture antibody reagent coated in the pipe 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 pool along with the liquid;

[0038] Step C. Buffer washing: After step B is completed, the gas path driving device generates a high positive air pressure in the second sample loading hole, drives the buffer to flow through the second super-hydrophobic pipe, and then reduces the high positive air pressure to a low positive air pressure, drives the buffer to flow into the detection pipe and waste liquid pool of the chip, washes the detection pipe, and improves the detection sensitivity and detection specificity; preferably, the buffer comprises 1% BSA, 0.5% Tween20, and 10mM PBS;

[0039] Step D. Data reading: After step C 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.

[0040] 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.

[0041] Due to the implementation of the above technical solutions, the bedside diagnostic microfluidic chip of the present invention has the following advantages compared with the prior art:

[0042] 1. The bedside diagnostic microfluidic chip of the present invention is designed with a reaction pool and a mixing pipeline with a fixed reaction volume. Combined with an active driving method, it drives the test sample and reagent to flow in the pipeline, accurately controls the re-dissolution and mixing process of the sample and reagent, thereby achieving strict controllability of the reagent re-dissolution volume and reaction time, greatly improving the repeatability of the test;

[0043] 2. The super-hydrophobic pipeline designed in the bedside diagnosis microfluidic chip of the present invention forms a passive flow blocking valve, which has simple packaging, greatly simplifies the chip production process, and improves chip production;

[0044] 3. The bedside diagnostic microfluidic chip of the present invention is designed with a detection channel with a micron column, in which more than one capture antibody can be coated to achieve multi-item joint detection;

[0045] 4. The bedside diagnostic microfluidic chip of the present invention has a buffer washing structural design to wash the immune reaction, thereby further improving the detection sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] 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.

[0047] Figure 1 It is a stereoscopic diagram of the bedside diagnosis microfluidic chip in the preferred embodiment 1 of the present invention;

[0048] Figure 2 A three-dimensional diagram of a chip substrate in a bedside diagnosis microfluidic chip according to a preferred embodiment 1 of the present invention;

[0049] Figure 3 for Figure 2 Enlarged view of the middle part I;

[0050] In the accompanying drawings: chip cover plate-1, chip substrate-2, first sample loading port-31, second sample loading port-32, first sample loading slot-41, second sample loading slot-42, first sample loading channel-51, second sample loading channel-52, reaction pool-61, buffer pool-62, mixing pipe-71, transition pipe-72, first super-hydrophobic pipe-81, second super-hydrophobic pipe-82, first micron column-91, second micron column-92, groove microstructure-10, detection pipe-11, waste liquid pool-12. DETAILED DESCRIPTION

[0051] 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.

[0052] 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.

[0053] Example 1 Bedside Diagnosis Microfluidic Chip

[0054] like Figure 1-3 As shown, a bedside diagnostic microfluidic chip of this embodiment includes a chip substrate 2 and a chip cover 1, the chip cover 1 is provided with a first sample addition port 31 for adding a test sample and a second sample addition port 32 for adding a buffer solution, the chip substrate 2 is provided with a reaction pool 61 connected to the first sample addition port 31, a buffer pool 62 connected to the second sample addition port 32, a mixing pipe 71 interconnected with the reaction pool 61, a detection pipe 11 connected to the buffer pool 62 and the mixing pipe 71, and a waste liquid pool 12 connected to the detection pipe 11, the mixing pipe 71 is coated with a fluorescent microsphere labeling reagent for providing a fluorescent detection signal, and the detection pipe 11 is coated with at least one capture antibody reagent. Among them, the fluorescent microsphere labeling reagent is a detection antibody labeled with a fluorescent microsphere, and the capture antibody reagent is a capture antibody labeled with a nano polystyrene microsphere.

[0055] The mixing pipe 71 is connected to the reaction pool 61 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 71, 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 71, and the incubation mainly occurs in the reaction pool 61.

[0056] like Figure 2 As shown, in this embodiment, in order to better control the reaction volume, a first sample loading slot 41 located below the first sample loading port 31, a first sample loading channel 51 connecting the first sample loading slot 41 and the reaction pool 61, and a second sample loading slot 42 located below the second sample loading port 32, a second sample loading channel 52 connecting the second sample loading slot 42 and the buffer pool 62 are provided on the chip substrate 2, and a transition pipe 72 is also provided between the buffer pool 62 and the detection pipe 11.

[0057] The reaction pool 61 can store the test sample and provide a reaction site for the test sample and the reagent; the buffer pool 62 can store the buffer and realize the washing function of the immune reaction.

[0058] like Figure 2 As shown, in this embodiment, one end of the detection pipe 11 is connected to the waste liquid pool 12, and the other end of the detection pipe 11 is connected to a super-hydrophobic pipe, which includes a first super-hydrophobic pipe 81 disposed between the detection pipe 11 and the mixing pipe 71 and a second super-hydrophobic pipe 82 disposed between the detection pipe 11 and the transition pipe 72. A Y-shaped structure is formed between the first super-hydrophobic pipe 81, the second super-hydrophobic pipe 82 and the detection pipe 11, and the sample enters the detection pipe 11 through the first super-hydrophobic pipe 81, and the buffer enters the detection pipe 11 through the second super-hydrophobic pipe 82.

[0059] like Figure 2-3 As shown, the super-hydrophobic channel includes a groove microstructure 10, and the surface of the groove microstructure 10 has a super-hydrophobic agent. In this embodiment, the depth of each groove in the groove microstructure 10 is 50 μm and the width is 100 μm; the super-hydrophobic agent is fluorosilane dissolved in an electronic fluoride liquid. In other embodiments, the depth of each groove in the groove microstructure 10 ranges from 50 to 200 μm and the width ranges from 100 to 2000 μm.

[0060] The superhydrophobic surface formed by the superhydrophobic reagent and the groove microstructure 10 can realize a passive flow blocking valve. When the test sample or buffer solution is driven by the driving force and flows 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.

[0061] like Figure 3 As shown, a first micron column 91 with a micron structure is provided in the mixing pipe 71, and a fluorescent microsphere labeling reagent is coated on the first micron column 91; a second micron column 92 with a micron structure is provided in the detection pipe 11, and a capture antibody reagent is coated on the second micron column 92. The diameter of the first micron column 91 is 200 μm and the height is 200 μm; the diameter of the second micron column 92 is 100 μm and the height is 200 μm. The density of the first micron column 91 is sparser than that of the second micron column 92, and can be set according to actual needs. In other embodiments, the diameter range of the first micron column 91 is 20-500 μm, and the height range is 10-2000 μm; the diameter range of the second micron column 92 is 10-200 μm, and the height range is 10-200 μm.

[0062] If only one capture antibody reagent is coated in the detection channel 11, the capture antibody reagent can be randomly dispersed in the detection channel 11; if two or more capture antibody reagents are coated in the detection channel 11, the detection channel 11 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.

[0063] 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 and buffer 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.

[0064] The bedside diagnostic microfluidic chip in this embodiment is designed with a reaction pool and a mixing channel with a fixed reaction volume, and combined with an active driving method, it drives the detection sample and reagent to flow in the channel, accurately controls the re-dissolution and mixing process of the sample and reagent, thereby achieving 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 package, greatly simplifies the chip production process, and improves the chip yield; a detection channel with a micron column is designed, and different areas in the detection channel can be coated with more than one capture antibody to achieve multi-project joint detection; the structural design with buffer washing can wash the immune reaction, further improving the detection sensitivity and detection specificity.

[0065] Example 2 Preparation method of bedside diagnosis microfluidic chip

[0066] This embodiment provides a method for preparing a bedside diagnostic microfluidic chip based on the embodiment 1, which specifically comprises the following steps:

[0067] Step 1) Preparation of fluorescent microsphere labeling reagent

[0068] 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.

[0069] Step 2) Preparation of capture antibody reagent

[0070] The purified capture antibody raw material is diluted with a diluent, and the diluted capture antibody raw material is labeled on the nano polystyrene microspheres to prepare a capture antibody reagent. The diluent in this embodiment is 10 mM PBS.

[0071] Step 3) Super-hydrophilic modification of chip surface materials

[0072] The chip surface material is modified to be super-hydrophilic by vacuum plasma bombardment or atmospheric plasma bombardment.

[0073] Step 4) Spraying and drying of sealing liquid

[0074] 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.

[0075] 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.

[0076] Step 5) Preparation of super hydrophobic pipe

[0077] 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.

[0078] Step 6) Drying of fluorescent microsphere labeling reagent

[0079] 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.

[0080] Step 7) Immobilization of capture antibody

[0081] After the chip is dried in step 6), the capture antibody reagent prepared in step 2) is 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 second micrometer column.

[0082] If only one capture antibody reagent is coated in the detection channel, then the capture antibody reagent can be dispersed at will in the detection channel; if two or more capture antibody reagents are coated in the detection channel, the detection channel 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.

[0083] Step 8) Microfluidic chip packaging

[0084] The chip cover plate and the chip substrate are assembled and bonded to obtain a bedside diagnosis microfluidic chip.

[0085] 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.

[0086] Example 3 Detection method of bedside diagnosis microfluidic chip

[0087] This embodiment provides a detection method based on the bedside diagnosis microfluidic chip described in Example 1, which specifically includes the following steps:

[0088] Step A. Add sample and mix well

[0089] The sample is added by a pipette, and the test sample and the buffer solution are respectively sucked into the first sample loading hole and the second sample loading hole of the above-mentioned bonded microfluidic chip, and then the microfluidic chip after the sample loading is placed in the card slot of the detection instrument. The first sample loading hole and the second sample loading hole of the microfluidic chip are 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 first sample loading hole, driving the test sample to flow back and forth in the reaction pool and the mixing channel in the chip, and uniformly mixing the fluorescent microsphere labeling reagent in the mixing channel while redissolving it. 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.

[0090] Step B. Capture of Antibody / Antigen Complex

[0091] After step A is completed, the gas path driving device generates a high positive air pressure in the first sample loading hole, drives the mixed sample containing the antibody / antigen complex to flow through the first 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 detection pipe of the chip, and at the same time, the capture antibody reagent coated in the pipe 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 pool with the liquid.

[0092] Step C. Buffer Wash

[0093] After step B is completed, the gas circuit driving device generates a high positive air pressure in the second sample addition hole, and after driving the buffer solution to flow through the second super-hydrophobic pipe, the high positive air pressure is reduced to a low positive air pressure, and the buffer solution is driven to flow into the detection pipe and waste liquid pool of the chip, and the detection pipe is washed to improve the detection sensitivity and detection specificity. The buffer solution in this embodiment includes 1% BSA, 0.5% Tween20 and 10mM PBS.

[0094] Step D. Data reading

[0095] After step C 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.

[0096] 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.

[0097] Example 4: Determination of procalcitonin (PCT) by bedside diagnosis microfluidic chip

[0098] (I) Antibody labeling

[0099] A. Fluorescent microsphere labeled detection antibody

[0100] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0101] 2. Take 90ul of 10mM PBS buffer, add 10ul of 300nm fluorescent microspheres, and shake to mix.

[0102] 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).

[0103] 4. Prepare 100ul labeled antibody: Use 10mM PBS buffer to prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with a final concentration of 0.5mg / ml.

[0104] 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.

[0105] 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.

[0106] 7. Take 20ul 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, 250rpm.

[0107] 8. Place the solution obtained in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0108] 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.

[0109] 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 3 minutes, and store at 2-8°C.

[0110] B. Polystyrene microspheres labeled with capture antibodies

[0111] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0112] 2. Take 90ul of 10mM PBS buffer, add 10ul of 150nm polystyrene microspheres, and shake to mix.

[0113] 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).

[0114] 4. Prepare 100ul capture antibody: Use 10mM PBS buffer to prepare PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solution with a final concentration of 0.5mg / ml.

[0115] 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.

[0116] 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.

[0117] 7. Take 20ul 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, 250rpm.

[0118] 8. Place the substance prepared in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0119] 9. Add 500ul 10mM PBS buffer to the material prepared in step 8 to redissolve the polystyrene microspheres. After vortexing and mixing, place in a centrifuge at 15000rpm for 15min and discard the supernatant.

[0120] 10. Add 200ul 10mM PBS to the material prepared in step 9, shake and mix well, sonicate for 3min, and store at 2-8℃.

[0121] (II) Microfluidic chip assembly

[0122] 1. Use vacuum plasma bombardment or atmospheric plasma bombardment to modify the surface material of the chip substrate to be super-hydrophilic.

[0123] 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 10mM PBS.

[0124] 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.

[0125] 4. After the chip in step 3 is dried, the PCT monoclonal antibody and rabbit IgG antibody labeled with fluorescent microspheres collected in step (a) are added to the mixing channel in the chip substrate, and then dried to dry the fluorescent microsphere markers on the first micron column of the chip mixing channel.

[0126] 5. After the chip in step 4 is dried, the polystyrene-labeled PCT capture antibody and goat anti-rabbit IgG antibody reagent prepared in step (i) 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 second micrometer column.

[0127] 6. Assemble the chip cover plate and the chip substrate and bond them by pressure adhesive to obtain a bedside diagnostic microfluidic chip.

[0128] (III) Sample testing

[0129] 1. Use a pipette to add serum samples containing PCT antigens, and take 100 μL of the test sample and 100 μL of buffer solution and add them to the first and second sample wells of the above-mentioned bonded microfluidic chip, respectively. Then place the loaded microfluidic chip into the card slot of the detection instrument. The first and second sample wells of the microfluidic chip are 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 first sample well, driving the test sample to flow back and forth in the reaction pool and mixing channel in the chip, and uniformly 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 pressure or negative pressure, and incubate the mixed solution to cause an immune complex reaction to form a fluorescent microsphere-labeled PCT monoclonal antibody / PCT antigen complex.

[0130] 2. After step 1 is completed, the gas path driving device generates a high positive air pressure in the first sample loading hole, drives the mixed sample containing the PCT antibody / PCT antigen complex to flow through the first super-hydrophobic pipe, and then reduces the high positive air pressure to a low positive air pressure to drive the mixed sample to flow into the detection pipe of the chip. At the same time, the PCT capture antibody and goat anti-rabbit IgG antibody labeled with polystyrene microspheres coated in the pipe react with the PCT antibody / PCT antigen complex and 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 pool with the liquid.

[0131] 3. After step 2 is completed, the gas path driving device generates a high positive air pressure in the second sample loading hole. After driving the buffer solution to flow through the second super-hydrophobic pipe, the high positive air pressure is reduced to a low positive air pressure, and the buffer solution is driven to flow into the detection pipe and waste liquid pool of the chip to wash the detection pipe and improve the detection sensitivity and specificity.

[0132] 4. After step 3 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.

[0133] 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.

[0134] Comparative Example 1

[0135] 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 buffer pool, buffer injection port, etc. that matches the superhydrophobic pipe and buffer in this comparative example. The microfluidic chip in this embodiment includes a chip substrate and a chip cover plate. The chip cover plate is provided with an injection port for adding a test sample. The chip substrate is provided with a reaction pool connected to the injection port, a detection pipeline connected to the reaction pool, and a waste liquid pool connected to the detection pipeline. In this comparative example, a fluorescent microsphere labeling reagent for providing a fluorescent detection signal is coated in the reaction pool, and a polystyrene-labeled capture antibody reagent is coated in the detection pipeline.

[0136] (I) Antibody labeling

[0137] A. Fluorescent microsphere labeled detection antibody

[0138] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0139] 2. Take 90ul of 10mM PBS buffer, add 10ul of 300nm fluorescent microspheres, and shake to mix.

[0140] 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).

[0141] 4. Prepare 100ul labeled antibody: Use 10mM PBS buffer to prepare PCT detection monoclonal antibody and rabbit IgG antibody solution with a final concentration of 0.5mg / ml.

[0142] 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.

[0143] 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.

[0144] 7. Take 20ul 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, 250rpm.

[0145] 8. Place the solution obtained in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0146] 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.

[0147] 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 3 minutes, and store at 2-8°C.

[0148] B. Polystyrene microspheres labeled with capture antibodies

[0149] 1. Prepare 20 mg / ml EDC, 10 mM PBS solution.

[0150] 2. Take 90ul of 10mM PBS buffer, add 10ul of 150nm polystyrene microspheres, and shake to mix.

[0151] 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).

[0152] 4. Prepare 100ul capture antibody: Use 10mM PBS buffer to prepare PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solution with a final concentration of 0.5mg / ml.

[0153] 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.

[0154] 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.

[0155] 7. Take 20ul 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, 250rpm.

[0156] 8. Place the substance prepared in step 7 in a centrifuge, centrifuge at 15000 rpm for 15 min, and discard the supernatant.

[0157] 9. Add 500ul 10mM PBS buffer to the material prepared in step 8 to redissolve the polystyrene microspheres. After vortexing and mixing, place in a centrifuge at 15000rpm for 15min and discard the supernatant.

[0158] 10. Add 200ul 10mM PBS to the material prepared in step 9, shake and mix well, sonicate for 3min, and store at 2-8℃.

[0159] (II) Microfluidic chip assembly

[0160] 1. Use vacuum plasma bombardment or atmospheric plasma bombardment to modify the surface material of the chip substrate to be super-hydrophilic.

[0161] 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 10mM PBS.

[0162] 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 pool in the chip substrate, and then dried to dry the fluorescent microsphere markers in the reaction pool of the chip.

[0163] 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.

[0164] 5. Assemble the chip cover plate and the chip substrate and bond them by pressure adhesive to obtain a bedside diagnostic microfluidic chip.

[0165] (III) Sample testing

[0166] 1. Use a pipette to add serum samples containing PCT antigen, draw 100 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 pool by capillary suction, and the fluorescent microspheres labeled with PCT monoclonal antibody and rabbit IgG antibody reagent in the reaction pool are re-dissolved and reacted with them to form a fluorescent microsphere-labeled PCT monoclonal antibody / PCT antigen complex.

[0167] 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 pool with the liquid.

[0168] 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.

[0169] Example 5 Results and Analysis

[0170] 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:

[0171] Table 1 Test results comparison table

[0172]

[0173] 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.

[0174] 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 bedside diagnostic microfluidic chip, comprising a chip substrate, characterized in that: The chip substrate is provided with a reaction pool, a buffer pool, a mixing pipe connected to the reaction pool, a detection pipe connected to the buffer pool and the mixing pipe, and a waste liquid pool connected to the detection pipe. The reaction pool is used to store the test sample and provide a reaction place for the test sample and the reagent; The chip substrate is provided with a super-hydrophobic channel, which is composed of a groove microstructure and a super-hydrophobic agent covering the surface of the groove microstructure. The super-hydrophobic agent and the super-hydrophobic surface formed by the groove microstructure are used to achieve passive flow blocking.

2. The bedside diagnostic microfluidic chip according to claim 1, characterized in that: The depth of each groove in the groove microstructure is 50-200 μm and the width is 100-2000 μm; the super hydrophobic agent is fluorosilane dissolved in electronic fluoride liquid.

3. The bedside diagnostic microfluidic chip according to claim 1, characterized in that: The super-hydrophobic pipe includes a first super-hydrophobic pipe arranged between the detection pipe and the mixing pipe and a second super-hydrophobic pipe arranged between the detection pipe and the buffer pool; a Y-shaped structure is formed between the first super-hydrophobic pipe, the second super-hydrophobic pipe and the detection pipe.

4. The bedside diagnostic microfluidic chip according to claim 1, characterized in that: The chip cover comprises a first sample adding port for adding a test sample and a second sample adding port for adding a buffer solution. The reaction pool is connected to the first sample adding port, and the buffer solution pool is connected to the second sample adding port.

5. The bedside diagnostic microfluidic chip according to claim 4, characterized in that: When the bedside diagnostic microfluidic chip is in use, the sample addition port is connected to a driving device, which is used to provide a driving force for the added test sample and buffer to flow back and forth in the pipeline of the chip, so that the sample and the reagent in the pipeline can fully react.

6. The bedside diagnostic microfluidic chip according to claim 1, characterized in that: The mixing channel is coated with a fluorescent microsphere labeling reagent for providing a fluorescent detection signal, and the fluorescent microsphere labeling reagent is a detection antibody labeled with a fluorescent microsphere.

7. The bedside diagnostic microfluidic chip according to claim 6, characterized in that: A first micron column with a micron structure is arranged in the mixing channel, and the fluorescent microsphere labeling reagent is coated on the first micron column; the diameter of the first micron column is 20-500 μm, and the height is 10-2000 μm.

8. The bedside diagnostic microfluidic chip according to claim 1, characterized in that: At least one capture antibody reagent is coated in the detection channel, and the capture antibody reagent is a capture antibody labeled with polystyrene microspheres.

9. The bedside diagnostic microfluidic chip according to claim 8, characterized in that: The detection channel is coated with two or more capture antibody reagents. The detection channel is divided into several areas, and different capture antibody reagents are coated in different areas.

10. The bedside diagnostic microfluidic chip according to claim 8, characterized in that: A second micron column with a micron structure is arranged in the detection channel, and the capture antibody reagent is coated on the second micron column; the diameter of the second micron column is 10-200 μm, and the height is 10-200 μm.

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