A self-pumping microfluidic chip for quantitative detection of whole blood biomarkers

Abstract

The present invention discloses a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers, which relates to the technical field of dark-field microscopy detection of tumor markers. The chip comprises a glass substrate and a cover plate, wherein an inlet, a functional microchannel and an outlet are provided on the cover plate, and the functional microchannel comprises a mixing microchannel, a reaction chamber, a separation microchannel, a detection area, a first Tesla microvalve, a pressing chamber and a second Tesla microvalve connected in sequence; a magnetic bead accumulation area is connected to the separation microchannel, and a magnet placement area is provided next to the magnetic bead accumulation area; and a liquid storage tank is provided on the first inlet. The self-pumping microfluidic chip provided by the present invention has stable properties, good detection performance, and can realize the precise transportation of liquids. It will get rid of the problems of cumbersome operation and additional bulky instruments of traditional detection, and can accurately perform process judgment and quantitative detection of biomarkers in human whole blood samples.

Description

Technical Field

[0001] The present invention relates to the technical field of dark-field microscope detection of tumor markers, and in particular to a self-pumping microfluidic chip for quantitatively detecting whole blood biomarkers. Background Art

[0002] In clinical sample testing, highly sensitive and accurate quantitative detection of biomarkers is crucial for early screening and monitoring. Highly sensitive, portable, and rapid tests have become a research hotspot. Existing conventional detection methods, such as enzyme-linked immunosorbent assay (ELISA) and PCR, have drawbacks such as complex plasma and blood cell separation, cumbersome manual procedures, and low sensitivity when testing whole blood samples. Both ELISA and PCR require complex pretreatment procedures and sophisticated instrumentation, requiring only specialized personnel to operate, significantly limiting their practical application.

[0003] Microfluidic chips compensate for the above-mentioned shortcomings of traditional detection methods in terms of microscale and functional integration. At the same time, microfluidic chips reduce reagent and sample consumption during the detection process, improve detection sensitivity, and provide a more efficient platform for the detection of cancer markers in complex real samples. However, the reported microfluidic chips for tumor markers cannot integrate blood separation and detection. They usually require multiple steps, such as additional microfluidic injection pumps and connectors and large test systems, to achieve biomarker detection, which greatly increases the cost of the entire microfluidic system and limits the application scenarios. Although some pump-free miniaturized microfluidic systems have been reported, these pump-free microfluidic chips have deficiencies in flow control and microvalve manufacturing, which is also an inevitable problem in the design of some soft microfluidic systems. In addition, such microfluidic sensor chips cannot meet the portable requirements for markers in whole blood samples. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers, so as to solve the problems of existing whole blood sample separation, cumbersome detection operation, and excessively large and non-integrated system.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers is provided, comprising a glass substrate and a cover plate, which is arranged on the glass substrate; an inlet, a functional microchannel and an outlet are arranged on the cover plate, which are connected in sequence; the inlet and the outlet are both through holes, and the inlet includes a first inlet, a second inlet and a third inlet; the functional microchannel is a groove, and the functional microchannel is arranged at the bottom of the cover plate close to the glass substrate, and the functional microchannel includes a mixing microchannel, a reaction chamber, a separation microchannel, a detection area, a first Tesla microvalve, a pressing chamber and a second Tesla microvalve, which are connected in sequence, the mixing microchannel is connected to the inlet, and the second Tesla microvalve is connected to the outlet; a magnetic bead accumulation area is connected to the separation microchannel, and a magnet placement area is arranged next to the magnetic bead accumulation area; a liquid reservoir is arranged on the first inlet, and a filter membrane is arranged at the bottom of the liquid reservoir; the glass substrate surface of the detection area is provided with a positive charge.

[0006] The beneficial effects of the present invention are as follows: the chip of the present invention mainly includes a glass substrate, a filter device and a functional microchannel. The glass substrate is used to fix the functional microchannel to form a closed microcavity; the filter device is used to separate the blood cells from the serum in the whole blood sample. The filter device of the present invention is a liquid reservoir and a filter membrane, which can accommodate the whole blood sample containing biomarkers, and the bottom of the filter paper is facing the first sample inlet of the lower cover plate, and the whole blood sample containing biomarkers flows into the first sample inlet through the filter paper; the functional microchannel is used for the mixing, reaction, detection and other functions of tumor markers, wherein the mixing microchannel is used to mix the plasma from the first sample inlet and the detection probe from the second sample inlet, and then flow into the reaction chamber, so that the aptamer of the detection probe specifically recognizes the antigen, thereby connecting the detection probe to the magnetic The gold nanoparticles on the surface of the beads are released into the plasma, and the detection probe is then separated into two parts. The magnet accumulates the separated magnetic beads in the magnetic bead accumulation area, and the fluorescence intensity of the magnetic beads is measured using a fluorescence microscope. The glass surface of the detection area is covered with cations, so that the gold nanoparticles connected to the antigen passing through are adsorbed here, and the quantitative detection of biomarkers is achieved by the number of green spots presented in the dark field microscope; the first Tesla valve and the second Tesla valve are one-way control valves, which can realize the forward rapid passage of liquid and prevent the reverse backflow of liquid; the pressing chamber is pressed by the finger to form a negative pressure inside the microfluidic chip, and the negative pressure is used to realize the transport flow of liquid.

[0007] The microfluidic chip of the present invention has a simple structure, is easy to operate, and does not pollute samples. Combined with dark-field microscope imaging detection technology, it can accurately perform process judgment and quantitative detection of prostate markers or tumor markers in human whole blood samples.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Furthermore, the positive charge on the surface of the glass substrate in the detection area is obtained by the following method: the surface of the glass substrate in the detection area is immersed in a piranha solution, then rinsed and dried with nitrogen, and then immersed in an alcohol solution containing 3-aminopropyltriethoxysilane, and then rinsed and dried with nitrogen, and heated to obtain a glass substrate surface modified with a positive charge.

[0010] Furthermore, the glass substrate surface of the detection area was immersed in piranha solution at 60° C. for 30 min.

[0011] Furthermore, the piranha solution is a mixture of concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3.

[0012] Further, rinse with deionized water.

[0013] Further, the film was immersed in an alcohol solution containing 3-aminopropyltriethoxysilane for 3 hours.

[0014] Furthermore, the concentration of 3-aminopropyltriethoxysilane in the alcohol solution is 10 wt %.

[0015] Furthermore, the mixing microchannel includes a first microchannel, a second microchannel and a third microchannel that are connected in sequence, the first microchannel is connected to the injection port, the second microchannel is S-shaped, and the third microchannel is connected to the reaction chamber.

[0016] The beneficial effect of adopting the above-mentioned further technical solution is that the liquid passing through the inlet is stratified due to the laminar flow, the liquid flow is slowed down through the S-shaped pipe, and the mutual exchange between particles in the liquid is increased, thereby achieving full mixing and integration of the two liquids, plasma and detection probe.

[0017] Furthermore, the cover plate is made of polydimethylsiloxane.

[0018] The beneficial effects of adopting the above-mentioned further technical solution are: the cover is made of polydimethylsiloxane, which is easy to observe: high transparency, no spontaneous fluorescence will not interfere with observation, good biocompatibility, and will not affect the experiment; low cost; easy to process: PDMS does not require high curing temperature, and the softness and hardness can be adjusted according to the proportion and temperature. It can be wearable and microfluidic. Cutting and punching can be done manually, and it is easy to modify.

[0019] Furthermore, the material of the liquid storage tank is polydimethylsiloxane.

[0020] Furthermore, the liquid storage tank has an inlet and an outlet, the pore size of the inlet is smaller than the pore size of the outlet, a flow channel is formed from the inlet to the outlet, and the outlet is provided with a filter membrane.

[0021] The beneficial effects of adopting the above-mentioned further technical solution are: the cover plate and the liquid reservoir are bonded due to the interaction between PDMS to avoid leakage. The liquid reservoir is small at the top and large at the bottom so that the filter membrane can be embedded in the cover plate, similar to a sink hole, so that the filter membrane and the cover plate form a seal and will not cause leakage.

[0022] Furthermore, the filter membrane is a rapid filter paper.

[0023] Furthermore, the depths of the reaction chamber, the detection area, and the pressing chamber are all greater than the depths of the mixing microchannel, the separation microchannel, the first Tesla microvalve, or the second Tesla microvalve.

[0024] Furthermore, the depth of the pressing cavity>the depth of the detection area>the depth of the reaction cavity.

[0025] Furthermore, the dimensions of the cover plate are 75×25×7 mm.

[0026] Furthermore, the mixing microchannel has a width of 500 μm and a depth of 100 μm.

[0027] Furthermore, the reaction chamber has a diameter of 6 mm and a depth of 0.8 mm.

[0028] Furthermore, the separation microchannel has a width of 500 μm and a depth of 100 μm.

[0029] Furthermore, the detection area has a diameter of 4 mm and a depth of 1.6 mm.

[0030] Furthermore, the arc length of the first Tesla microvalve and the second Tesla microvalve are both 1.41 mm; the radius is both 1.35 mm, and the depth is 100 μm.

[0031] Furthermore, the pressing cavity has a diameter of 13 mm and a depth of 4 mm.

[0032] Furthermore, the inner diameter of the outlet of the liquid reservoir is 6 mm, the inner diameter of the inlet is 4 mm, and the outer diameter of the liquid reservoir is 9 mm.

[0033] The present invention also provides a method for preparing the above-mentioned self-pumping microfluidic chip for quantitatively detecting whole blood biomarkers, comprising the following steps:

[0034] (1) Preparation of cover plate mold by photolithography: The chip is drawn using AutoCAD, a plastic mask is prepared, SU-8 photoresist is coated on the surface of the silicon wafer, and then the photolithography process is completed through pre-baking, exposure, heat curing and development to form a photoresist pattern. A frame is then set around the silicon wafer to obtain the cover plate mold;

[0035] (2) Preparation of the cover plate: According to the depth of the functional microchannel, acrylic plates of different heights are attached to the cover plate mold obtained in step (1), and then a mixed solution of PDMS prepolymer and curing agent is added. After removing bubbles, the mixture is heated and cured. Then, the cover plate mold is removed, and the sample inlet and sample outlet of the cover plate are punched into through holes to prepare the cover plate;

[0036] (3) Preparation of the liquid reservoir: The inner mold and outer mold of the liquid reservoir are made by laser engraving, and then the inner mold and the outer mold are clamped to form a cavity, and a mixed solution of PDMS prepolymer and curing agent is added. After removing bubbles, the mixture is heated and cured, and then the inner mold and the outer mold are removed to obtain the liquid reservoir;

[0037] (4) The cover plate prepared in step (2) and the glass substrate are connected by oxygen plasma bonding, and then the liquid reservoir prepared in step (3) and the first sample inlet of the cover plate are also connected by oxygen plasma bonding to prepare a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers.

[0038] Furthermore, the oxygen plasma bonding in step (4) includes the following steps: performing oxygen plasma surface treatment on the surfaces to be connected, then aligning and laminating them, heating them, and completing the oxygen plasma bonding process.

[0039] Furthermore, the surface was treated with oxygen plasma in a surface plasma cleaning machine for 40 seconds.

[0040] Further, the mixture was heated at 75°C for 10 min.

[0041] The present invention also provides a method for quantitatively detecting whole blood biomarkers, which uses the above-mentioned microfluidic chip to perform quantitative detection of whole blood biomarkers.

[0042] Furthermore, the method for quantitatively detecting biomarkers in whole blood comprises the following steps:

[0043] S1: inject the detection probe solution into the second injection port and inject the blood containing biomarkers into the reservoir;

[0044] S2: By pressing the pressing chamber, the blood containing biomarkers is filtered and mixed with the detection probe through the mixing microchannel to form a mixed solution, which enters the reaction chamber and reacts for 20 minutes;

[0045] S3: Press the pressing chamber again to allow the reaction mixture to flow into the magnetic bead accumulation area and the detection area respectively, and then pour deionized water from the third inlet and flush by pressing the pressing chamber;

[0046] S4: The magnetic bead accumulation area is observed with a fluorescence microscope, and the detection area is observed with a dark-field microscope. The target object is detected by counting.

[0047] Furthermore, in S1, the biomarker is a tumor marker.

[0048] Furthermore, in step S1, the volume ratio of the detection probe solution, the blood containing the biomarker, and the deionized water in S3 is 1:1:2.

[0049] Furthermore, in step S1, the concentration of the detection probe solution is 500 μg / mL.

[0050] Furthermore, the detection probe is an assembly of silica magnetic beads modified with fluorescent aptamers and gold nanoparticles modified with nucleic acid aptamers. The fluorescent aptamers and nucleic acid aptamers can be base-complementarily paired, and the nucleic acid aptamers and biomarkers can be base-complementarily paired.

[0051] Furthermore, the detection probe is an assembly of silica magnetic beads modified with fluorescent aptamers and gold nanoparticles modified with nucleic acid aptamers. The fluorescent aptamer and the nucleic acid aptamer can base-complementarily pair with each other, and the nucleic acid aptamer and the biomarker can specifically recognize and capture each other.

[0052] Furthermore, the nucleic acid aptamer is labeled with carboxyfluorescein to obtain a fluorescent aptamer.

[0053] Furthermore, the detection probe was prepared by the following method:

[0054] S11: adding the activated SH-DNA solution to the AuNPs solution, incubating for the first time, adding the NaCl solution in portions, centrifuging, adding to the BSA solution, incubating for the second time, washing, centrifuging to obtain the aptamer-modified AuNPs, and resuspending in PBS buffer to prepare reaction solution 1;

[0055] S12: At room temperature in the dark, the silica magnetic bead solution and the fluorescent aptamer solution are mixed and shaken, then washed and magnetically separated, and resuspended in PBS buffer to obtain a fluorescent aptamer-modified silica magnetic bead solution, and the reaction solution 1 prepared in step S11 is added to assemble and wash to obtain a composite nanomaterial, i.e., a detection probe.

[0056] Furthermore, in step S11, the AuNPs solution is prepared by sodium citrate reduction method.

[0057] Furthermore, in step S11, the AuNPs solution is pre-treated by centrifugation to remove sodium citrate.

[0058] Furthermore, in step S11, the sequence of SH-DNA is 5′-TTGATGGCGAGCTTTAAT-SH-3′.

[0059] Furthermore, in step S11, the activation method is: adding TCEP solution to the SH-DNA solution and reacting in the dark for 30 minutes.

[0060] Furthermore, the volume ratio of the SH-DNA solution to the TCEP solution was 10:1.

[0061] Furthermore, the concentration of the SH-DNA solution was 10 mmol / L.

[0062] Furthermore, the concentration of the TCEP solution was 10 mmol / L.

[0063] Furthermore, in step S11 , the volume ratio of the SH-DNA solution to the AuNPs solution is 1:500.

[0064] Furthermore, in step S11 , the concentration of the SH-DNA solution is 10 μmol / L.

[0065] Furthermore, in step S11, the concentration of the AuNPs solution is 2.5 nmol / L.

[0066] Furthermore, in step S11, the first incubation is carried out at 25° C. for 150 min.

[0067] Furthermore, in step S11, the concentration of the NaCl solution is 1 mol / L.

[0068] Furthermore, in step S11, NaCl solution is added in portions until the concentration of NaCl in the system reaches 0.15 mol / L.

[0069] Furthermore, in step S11, the concentration of the BSA solution is 1 wt%.

[0070] Furthermore, in step S11, the second incubation is performed for 30 minutes.

[0071] Furthermore, in step S11, the volume ratio of the AuNPs solution to the BSA solution is 10:1.

[0072] Furthermore, in step S11, the concentration of the reaction solution 1 is 2.5 nmol / L.

[0073] Furthermore, in step S12, the fluorescent aptamer is 5′-FAM-ATTAAAGCTCGCCATCAAATAGC-Biotion-3′.

[0074] The beneficial effects of adopting the above further technical solution are: the role of the fluorescent group is to determine whether there is a gold nanoparticle connection. After the gold nanoparticles are connected, the fluorescence will be quenched, and when the gold nanoparticles fall, the fluorescence will recover. There is fluorescence resonance energy transfer between the fluorescent group and the gold nanoparticles.

[0075] Furthermore, in step S12, the volume ratio of the fluorescent aptamer solution, the silica magnetic bead solution and the reaction solution 1 is 150:1000:350.

[0076] Furthermore, in step S12, the concentration of the fluorescent aptamer solution is 100 μmol / L.

[0077] Furthermore, in step S12, the concentration of the silica magnetic bead solution is 500 μg / mL.

[0078] Furthermore, in step S12, the cell was washed 5 times with PBS buffer.

[0079] Furthermore, in step S3, the rinsing is repeated three times.

[0080] The present invention has the following beneficial effects:

[0081] The present invention utilizes the base complementary pairing principle of silica magnetic beads and gold nanoparticle surface aptamers to assemble the two together, quench the fluorescence of the silica magnetic beads, and obtain an immune detection probe with a core-satellite structure. During the test, the blood to be tested is added to the liquid reservoir, the detection probe is added to the second injection port, and then the pressing chamber is driven by pressing the finger to complete the blood filtration. The filtered blood and the detection probe are fully mixed through the mixing microchannel and flow into the reaction chamber. After 20 minutes of reaction, the biomarkers in the plasma competitively bind to the gold nanoparticles through the specific binding between the aptamer on the surface of the gold nanoparticles and the antigen, and the gold nanoparticles will be freed from the surface of the magnetic beads into the plasma, and the magnetic beads will resume fluorescence; the finger presses the pressing chamber again. Since a magnet is set next to the magnetic bead accumulation area, the fluorescent magnetic beads stay in the magnetic bead accumulation area and are observed under a fluorescence microscope. The changes in spectral intensity under different concentrations of biomarkers are observed, and the gold nanoparticles bound to the biomarkers flow into the detection area. The surface of the functional area has a positive charge. The gold nanoparticles are adsorbed in the functional area through positive and negative charges. The excellent optical properties of the gold nanoparticles themselves are used to observe green spots under a dark field microscope, and the detection of biomarkers is achieved by counting.

[0082] 1. The method of the present invention separates the serum from the blood by pressing the finger, and accurately transports it and the detection probe into the reaction chamber. After the reaction is complete, the magnetic beads and gold nanoparticles are transported to the corresponding detection area, and the separated gold nanoparticles are mathematically counted by fluorescence and dark-field microscopy. This method does not require the addition of signal molecules, which can avoid contamination of reagents or test objects. At the same time, the requirements for cost and use are greatly reduced. Compared with existing methods, it can effectively improve the sensitivity and reliability of detection.

[0083] 2. The self-pumping microfluidic chip provided by the present invention has stable properties, good detection performance, and can achieve precise transportation of liquids. It will get rid of the problems of cumbersome operation and additional bulky instruments of traditional detection. Because of its simple manufacturing structure, convenient operation, and no pollution to samples, combined with dark-field microscopy imaging detection technology, it can accurately perform process judgment and quantitative detection of prostate markers or tumor markers in human whole blood samples.

[0084] 3. The present invention adopts Tesla microvalve as a damper to prevent rapid backflow of liquid and realizes precise transportation of liquid through finger drive; the self-pumping microfluidic chip designed by the present invention can realize a detection system integrating separation, identification and detection through a filtering device; the detection method designed by the present invention can realize dual-modal detection to improve the accuracy and correctness of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Schematic diagram of a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers;

[0086] Figure 2 for Figure 1 A top view of the cover;

[0087] Figure 3 is a schematic diagram of the liquid reservoir;

[0088] Figure 4 A partial diagram of the first Tesla microvalve and the second Tesla microvalve;

[0089] Figure 5 For the test results.

[0090] 1. Glass substrate; 2. Cover plate; 3. First sample inlet; 4. Second sample inlet; 5. Third sample inlet; 6. First microchannel; 7. Second microchannel; 8. Third microchannel; 9. Reaction chamber; 10. Separation microchannel; 11. Detection area; 12. First Tesla microvalve; 13. Pressing chamber; 14. Second Tesla microvalve; 15. Sample outlet; 16. Magnetic bead accumulation area; 17. Magnet placement area; 18. Liquid reservoir; 19. Filter membrane. DETAILED DESCRIPTION

[0091] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not intended to limit the scope of the invention. In the embodiments, if specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0092] Combine Figure 1-4A self-pumping microfluidic chip for quantitative detection of whole blood biomarkers comprises a glass substrate 1 and a cover plate 2, the cover plate 2 being arranged on the glass substrate 1; the cover plate 2 being provided with an inlet, a functional microchannel and an outlet 15 which are sequentially connected, the inlet and the outlet 15 being through holes, the inlet comprising a first inlet 3, a second inlet and a third inlet 5; the functional microchannel being a groove, the functional microchannel being arranged at the bottom of the cover plate 2 close to the glass substrate 1; the functional microchannel comprising a mixing microchannel, a reaction microchannel, a mixing microchannel and a reaction microchannel which are sequentially connected The response chamber 9, the separation microchannel 10, the detection area 11, the first Tesla microvalve 12, the pressing chamber 13 and the second Tesla microvalve 14; the mixing microchannel is connected to the sample inlet, and the second Tesla microvalve 14 is connected to the sample outlet 15; the separation microchannel 10 is connected to the magnetic bead accumulation area 16, and the magnetic bead accumulation area 16 is provided next to the magnet placement area 17; the first sample inlet 3 is provided with a liquid reservoir 18, and the bottom of the liquid reservoir 18 is provided with a filter membrane 19; the surface of the glass substrate 1 of the detection area 11 is provided with a positive charge.

[0093] The chip of the present invention mainly includes a glass substrate 1, a filter device and a functional microchannel. The glass substrate 1 is used to fix the functional microchannel to form a closed microcavity; the filter device is used to separate blood cells from serum in a whole blood sample. The filter device of the present invention is a liquid reservoir 18 and a filter membrane 19, which can accommodate a whole blood sample containing a biomarker, and the bottom of the filter paper is directly opposite to the first sample inlet 3 of the lower cover 2. The whole blood sample containing the biomarker flows into the first sample inlet 3 through the filter paper; the functional microchannel is used for mixing, reacting, detecting and other functions of tumor markers, wherein the mixing microchannel is used to mix the plasma from the first sample inlet 3 and the detection probe from the second sample inlet 4, and then flow into the reaction chamber 9, so that the aptamer of the detection probe specifically recognizes the antigen, thereby connecting the detection probe. The gold nanoparticles on the surface of the magnetic beads are released into the plasma, and the detection probe is then separated into two parts. The magnet accumulates the separated magnetic beads in the magnetic bead accumulation area 16, and the fluorescence intensity of the magnetic beads is measured using a fluorescence microscope. The glass surface of the detection area 11 is covered with cations, so that the gold nanoparticles connected to the antigen passing through are adsorbed here, and the quantitative detection of biomarkers is achieved by the number of green spots presented in the dark field microscope; the first Tesla valve and the second Tesla valve are one-way control valves, which can realize the forward rapid passage of the liquid and prevent the liquid from flowing back in the reverse direction; the pressing cavity 13 is pressed by the finger to form a negative pressure inside the microfluidic chip, and the negative pressure is used to realize the transport flow of the liquid. The positive charge on the surface of the glass substrate in the detection area was obtained by the following method: the glass substrate surface in the detection area was immersed in a 60°C piranha solution (a mixture of concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3) for 30 minutes, then rinsed with deionized water and blown dry with nitrogen, and then immersed in an alcohol solution containing 10wt% 3-aminopropyltriethoxysilane for 3 hours, and continued to rinse with deionized water, blown dry with nitrogen, and heated to obtain a glass substrate surface modified with a positive charge.

[0094] The microfluidic chip of the present invention has a simple structure, is easy to operate, and does not pollute samples. Combined with dark-field microscope imaging detection technology, it can accurately perform process judgment and quantitative detection of prostate markers or tumor markers in human whole blood samples.

[0095] The mixing microchannel includes a first microchannel 6 , a second microchannel 7 and a third microchannel 8 which are connected in sequence. The first microchannel 6 is connected to the injection port, the second microchannel 7 is S-shaped, and the third microchannel 8 is connected to the reaction chamber 9 .

[0096] The liquid passing through the injection port is stratified due to the laminar flow, which slows down the liquid flow through the S-shaped pipe and increases the mutual exchange between particles in the liquid, thereby achieving full mixing and integration of the two liquids, plasma and detection probe.

[0097] Cover plate 2 is made of polydimethylsiloxane. This material facilitates observation: high transparency, no autofluorescence that interferes with observation, good biocompatibility, and no impact on experiments. It is also low-cost and easy to process: PDMS has low curing temperature requirements, and its hardness can be adjusted based on the ratio and temperature. It can be soft for wearables and hard for microfluidics. Cutting and punching can be done manually, making modifications simple.

[0098] The reservoir 18 is made of polydimethylsiloxane and has an inlet and an outlet. The inlet has a smaller pore size than the outlet, forming a flow channel from the inlet to the outlet. The outlet is provided with a filter membrane 19. The filter membrane is a fast filter paper. The cover and the reservoir are bonded due to the interaction of PDMS to prevent leakage. The reservoir is smaller at the top and larger at the bottom so that the filter membrane can be embedded in the cover, similar to a countersunk hole, so that the filter membrane and the cover form a seal and prevent leakage.

[0099] The depth of the reaction chamber, detection area, and pressing chamber is greater than the depth of the mixing microchannel, separation microchannel, first Tesla microvalve, or second Tesla microvalve.

[0100] The depth of the pressing cavity > the depth of the detection area > the depth of the reaction cavity. This setting allows for smooth pressing.

[0101] The dimensions of the cover plate are 75×25×7mm; the width of the mixing microchannel is 500μm and the depth is 100μm; the diameter of the reaction chamber is 6mm and the depth is 0.8mm; the width of the separation microchannel is 500μm and the depth is 100μm; the diameter of the detection area is 4mm and the depth is 1.6mm; the arc length of the first Tesla microvalve and the first Tesla microvalve is 1.41mm; the radius is 1.32mm and the depth is 100μm; the diameter of the pressing chamber is 13mm and the depth is 4mm; the inner aperture of the liquid reservoir outlet is 6mm, the inner aperture of the inlet is 4mm, and the outer diameter of the liquid reservoir is 9mm.

[0102] A method for preparing a self-pumping microfluidic chip for quantitatively detecting whole blood biomarkers comprises the following steps:

[0103] (1) Preparation of cover plate mold by photolithography: The chip is drawn using AutoCAD, a plastic mask is prepared, SU-8 photoresist is coated on the surface of the silicon wafer, and then the photolithography process is completed through pre-baking, exposure, heat curing and development to form a photoresist pattern. A frame is then set around the silicon wafer to obtain the cover plate mold;

[0104] (2) Preparation of cover plate 2: According to the depth of the functional microchannel, acrylic plates of different heights are attached to the cover plate mold obtained in step (1), and then a mixed solution of PDMS prepolymer and curing agent (volume ratio of 10:1) is added. After removing bubbles, the mixture is heated and cured. Then, the cover plate mold is removed, and the sample inlet and sample outlet of the cover plate are punched into through holes to obtain cover plate 2;

[0105] (3) Preparation of the liquid reservoir 18: Laser engraving is used to make the inner mold and outer mold of the liquid reservoir 18, and then the inner mold and the outer mold are clamped to form a cavity, and a mixed solution of PDMS prepolymer and curing agent (volume ratio of 10:1) is added. After removing bubbles, the mixture is heated and cured, and then the inner mold and the outer mold are removed to obtain the liquid reservoir 18;

[0106] (4) The cover plate 2 and the glass substrate 1 prepared in step (2) are connected by oxygen plasma bonding (the surfaces to be bonded are surface treated with oxygen plasma in a surface plasma cleaning machine for 40 seconds, then aligned and bonded, appropriate pressure is applied to make them fit tightly, and heated at 75°C for 10 minutes), and then the liquid reservoir 18 prepared in step (3) and the first sample inlet 3 of the cover plate 2 are also connected by oxygen plasma bonding to prepare a self-pumping microfluidic chip for quantitative detection of whole blood biomarkers.

[0107] A method for quantitatively detecting whole blood biomarkers uses the above-mentioned self-pump microfluidic chip for quantitatively detecting whole blood biomarkers to quantitatively detect whole blood biomarkers.

[0108] A method for quantitatively detecting biomarkers in whole blood comprises the following steps:

[0109] S11: AuNPs solution was prepared by sodium citrate reduction method. The AuNPs solution was centrifuged 3 times at 5000 rpm for 15 min to remove as much excess sodium citrate as possible. 10 μL of 10 mmol / L TCEP (tris(2-carboxyethyl)phosphine) solution (solvent: PBS) was added to 100 μL of 10 mmol / L SH-DNA solution. The reaction was carried out in the dark for 30 min to ensure that the disulfide bonds were completely broken to obtain activated SH-DNA. The activated SH-DNA solution (10 μmol / L, 20 μL, sequence: 5′-TTGATGGCGAGCTTTAAT-SH-3′) was added to the AuNPs solution (2.5 nmol / L, 1 mL) and incubated at 25°C for 15 min. 0 min, slowly add NaCl solution (1 mol / L) at 30 min intervals until the concentration of NaCl in the solution reaches 0.15 mol / L, centrifuge at 5000 rpm for 3 min, repeat three times to remove excess aptamer, add to BSA solution (1 wt%, 100 μL), block the inactivated site and incubate for 30 min, centrifuge and wash twice to remove unbound bovine serum albumin to obtain aptamer-modified AuNPs, and resuspend in PBS buffer to prepare reaction solution 1 (2.5 nmol / L);

[0110] S12: Under room temperature and dark conditions, the fluorescent aptamer (150 μL, 100 μmol / L, 5′-FAM-ATTAAAGCTCGCCATCAAATAGC-Biotion-3′) was added to silica magnetic beads (1 mL, 500 μg / mL), mixed and shaken at 37°C for 2 h, then washed and magnetically separated, and then resuspended in PBS to obtain fluorescent aptamer-modified silica magnetic beads (1 mL, 500 μg / mL). 350 μL of reaction solution 1 prepared in step S11 was added, heated at 95°C for 1 min, and naturally cooled to room temperature. Assembly was carried out according to the base complementary pairing principle between the aptamers, and washed 5 times with PBS buffer to remove uncoupled AuNPs to obtain a composite nanomaterial, i.e., a detection probe.

[0111] S1: Inject the detection probe solution (solvent: PBS buffer, concentration: 500 μg / mL) into the second injection port 4, and inject the blood containing the biomarker (prostate antigen PSA) into the reservoir 18; wherein the volume of the detection probe solution and the biomarker is 10 μL each;

[0112] S2: By pressing the pressing chamber 13, the blood containing biomarkers is filtered and mixed with the detection probe through the mixing microchannel to form a mixed solution, which enters the reaction chamber 9 and reacts for 20 minutes;

[0113] S3: Press the pressing chamber 13 again to allow the reaction mixture to flow into the magnetic bead accumulation area 16 and the detection area 11 respectively. Add 20 μL of deionized water to the injection port 5. Press the pressing chamber to transport the washing water to each reaction functional area for rinsing. Repeat the rinsing three times.

[0114] S4: The magnetic bead accumulation area 16 is observed with a fluorescence microscope, and the detection area 11 is observed with a dark field microscope, and the target object is detected by counting.

[0115] The same method was used to test different samples. The test results are shown in Figure 4 (The illustration shows the corresponding relationship between the logarithm of concentration and the number of green light scattering points, where N and N0 represent the number of green light scattering points in the experimental group and the blank control group, respectively).

[0116] Depend on Figure 5 It can be seen that as the PSA concentration in the blood (0-1μg / mL) increases, the number of green light scattering points generated by gold nanoparticles in the detection area increases accordingly. The functional relationship diagram corresponding to different PSA concentrations was also drawn. The results showed that in the concentration range of 1pg / mL-100ng / mL / , N-N0 and the logarithm of PSA concentration (log C PSA) There was a positive correlation, and the detection limit was 0.05 pg / mL.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A self-pumping microfluidic chip for quantitative detection of whole blood biomarkers, characterized in that: The invention comprises a glass substrate (1) and a cover plate (2), wherein the cover plate (2) is arranged on the glass substrate (1); the cover plate (2) is provided with an injection port, a functional micro-channel and an injection port (15) which are connected in sequence; the injection port and the injection port are both through holes, and the injection port comprises a first injection port (3), a second injection port (4) and a third injection port (5); the functional micro-channel is a groove, and the functional micro-channel is arranged at the bottom of the cover plate (2) close to the glass substrate (1); the functional micro-channel comprises a mixing micro-channel, a reaction chamber (9), a separation micro-channel (10), a detection micro-channel (11) which are connected in sequence The invention relates to a detection area (11), a first Tesla microvalve (12), a pressing chamber (13) and a second Tesla microvalve (14); the mixing microchannel is connected to the injection port, and the second Tesla microvalve (14) is connected to the sample outlet (15); the separation microchannel (10) is connected to a magnetic bead accumulation area (16), and a magnet placement area (17) is provided next to the magnetic bead accumulation area (16); the first injection port (3) is provided with a liquid storage tank (18), and the bottom of the liquid storage tank (18) is provided with a filter membrane (19); the surface of the glass substrate (1) of the detection area (11) is provided with positive charges.

2. The self-pumping microfluidic chip for quantitative detection of whole blood biomarkers according to claim 1, characterized in that: The mixing microchannel comprises a first microchannel (6), a second microchannel (7) and a third microchannel (8) which are connected in sequence, wherein the first microchannel (6) is connected to the injection port, the second microchannel (7) is S-shaped, and the third microchannel (8) is connected to the reaction chamber (9).

3. The self-pumping microfluidic chip for quantitative detection of whole blood biomarkers according to claim 1, characterized in that: The material of the cover plate (2) is polydimethylsiloxane.

4. The self-pumping microfluidic chip for quantitative detection of whole blood biomarkers according to claim 1, characterized in that: The liquid storage tank (18) has an inlet and an outlet, the aperture of the inlet is smaller than the aperture of the outlet, a flow channel is formed from the inlet to the outlet, and the outlet is provided with a filter membrane (19).

5. The self-pumping microfluidic chip for quantitative detection of whole blood biomarkers according to claim 1, characterized in that: The depths of the reaction chamber, the detection area, and the pressing chamber are all greater than the depths of the mixing microchannel, the separation microchannel, the first Tesla microvalve, or the second Tesla microvalve.

6. A method for quantitatively detecting biomarkers in whole blood, characterized in that: The self-pump microfluidic chip for quantitatively detecting whole blood biomarkers according to any one of claims 1 to 4 is used to quantitatively detect whole blood biomarkers.

7. The method for quantitative detection of whole blood biomarkers according to claim 6, characterized in that: The following steps are included in sequence: S1: injecting the detection probe solution into the second injection port (4) and injecting the blood containing the biomarker into the reservoir (18); S2: By pressing the pressing chamber (13), the blood containing the biomarker is filtered and mixed with the detection probe solution through the mixing microchannel to form a mixed solution, which enters the reaction chamber (9) and reacts for 20 minutes; S3: Press the pressing chamber (13) again to allow the reaction mixture to flow into the magnetic bead accumulation area (16) and the detection area (11), and then add deionized water to the third injection port (5) and flush by pressing the pressing chamber (13); S4: The magnetic bead accumulation area (16) is observed with a fluorescence microscope, and the detection area (11) is observed with a dark field microscope, and the target object is detected by counting.

8. The method for quantitative detection of whole blood biomarkers according to claim 7, characterized in that: In step S1, the biomarker is a tumor marker.

9. The method for quantitative detection of whole blood biomarkers according to claim 7, characterized in that: In step S1, the detection probe is an assembly of silica magnetic beads modified with fluorescent aptamers and gold nanoparticles modified with nucleic acid aptamers. The fluorescent aptamers and nucleic acid aptamers can be base-complementarily paired, and the nucleic acid aptamers and biomarkers can be specifically recognized and captured.

10. The method for quantitative detection of whole blood biomarkers according to claim 7, characterized in that: In step S1, the detection probe is prepared by the following method: S11: adding the activated SH-DNA solution to the AuNPs solution, incubating for the first time, adding the NaCl solution in portions, centrifuging, adding to the BSA solution, incubating for the second time, washing, centrifuging to obtain the nucleic acid aptamer-modified AuNPs, and resuspending in PBS buffer to prepare reaction solution 1; S12: At room temperature in the dark, the silica magnetic bead solution and the fluorescent aptamer solution are mixed and shaken, then washed and magnetically separated, and resuspended in PBS buffer to obtain a fluorescent aptamer-modified silica magnetic bead solution, and the reaction solution 1 prepared in step S11 is added to assemble and wash to obtain a composite nanomaterial, i.e., a detection probe.

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