Preparation and application of pump-free microfluidic chip for thrombin detection

Abstract

The application discloses a kind of preparation methods of pump-free microfluidic chip, comprising the following steps;Step S1, surface modification of gold-plated substrate;Step S2, the production of pump-free microfluidic chip;Step S3, the production of pump-free microfluidic chip internal drive liquid flow's finger pressure pump;Step S4, embedding and sealing of gold-plated substrate with chip.The application also provides the technical scheme of the application of the pump-free microfluidic chip prepared by the aforementioned pump-free microfluidic chip preparation method in thrombin detection.The pump-free microfluidic chip designed by the application has low manufacturing cost, does not need professional operation, and the detection operation process is simple.It realizes high sensitivity, short detection time and low detection lower limit.

Description

TECHNICAL FIELD

[0001] The present application relates to a surface-enhanced Raman spectroscopy detection method, in particular to a preparation of a pump-free microfluidic chip based on SERS and its application in thrombin detection. BACKGROUND

[0002] Surface-enhanced Raman scattering (SERS) is a powerful vibrational spectroscopy technique that allows for the high-sensitivity detection of low concentrations of target analytes by amplifying the electromagnetic field generated by the local surface plasmon excitation. In 1974, the Raman spectrum of pyridine on rough silver was first observed, however, the researchers at that time did not realize that these spectra were caused by any unusual, enhanced or new phenomenon. Since SERS was formally discovered in 1977, numerous researchers have shown great interest in it and carried out extensive research. The SERS enhancement factor is the product of the electromagnetic enhancement mechanism and the chemical enhancement mechanism. For a highly optimized surface, it can be close to about 1010-1011. The emergence of SERS makes Raman a viable option for biosensing, as it can use relatively simple laboratory equipment or even portable equipment on site to detect Raman spectra of a large number of biological molecules. SERS has high sensitivity, can extract different spectra from structurally and functionally similar molecules, and saves expensive reagents or time-consuming sample preparation steps related to other technologies such as polymerase chain reaction (PCR).

[0003] Thrombin is a serine protease with multiple functions, which can regulate various cell functions, including homeostasis, platelet activation, tissue regeneration, inflammation and cancer progression, etc. Thrombin is the main effector protease of the coagulation cascade, and once activated, it will produce a clot by converting fibrinogen into fibrin. Not only can it play a role in promoting coagulation activity, but it also plays a role in coordinating cell pro-inflammatory responses in cells. Overexpression of thrombin can induce inflammatory cytokines, adhesion molecules, angiogenic factors and matrix-degrading proteases, thereby inducing tumor cell metastasis, proliferation and angiogenesis processes. In the coagulation process related to malignant tumors, the concentration of thrombin can reach micromolar levels. On the other hand, studies have shown that even in the blood of patients with diseases related to abnormal coagulation, the content of thrombin has only reached high picomolar levels. Therefore, given its above characteristics, thrombin is likely to be a new diagnostic factor and a clinically valuable cancer marker, and its concentration in the blood is an indispensable biomarker in tumor diagnosis.

[0004] In 1971, two Swiss scientists, Engvall and Perlmann, developed the enzyme-linked immunosorbent assay (ELISA) by modifying the radioimmunoassay (RIA) and used this new method to measure IgG levels in rabbit serum. ELISA is a heterogeneous immunoassay technique for detecting soluble antigens and specific antibodies, and various types of ELISA have been developed to improve the specificity of detection based on the specificity of the structure and properties of the substance to be detected. One of the types of ELISA is the "sandwich structure" of the double antibody sandwich technique. In this modified method, a specific antibody is first adsorbed on a plate, then it is reacted with a test sample containing an antigen, and then an enzyme-labeled specific antibody is added, followed by the addition of an enzyme substrate. The "antigen" in the test sample is "captured" and fixed on the sensitized plate, and then it itself can fix the enzyme-labeled antibody. This method has high sensitivity and specificity for detecting high molecular weight antigens.

[0005] Aptamers are specific DNA or RNA strands selected from a huge library of nucleic acids using the "systematic evolution of ligands by exponential enrichment" (SELEX) process to evolve ligands and can bind to target molecules with extremely high specificity and affinity. The specific process of the SELEX scheme is as follows: a random nucleic acid library is incubated with target molecules, and unbound molecules are separated from bound molecules. The bound nucleic acids are eluted, amplified by PCR, and provide a rich nucleic acid library for the next cycle. For each target molecule, 6 to 12 consecutive cycles are performed, and the final enriched library is cloned and sequenced. Unlike antibody production methods that rely on inducing the animal immune system, the SELEX process can produce aptamers for non-immunogenic and toxic targets, which would not be possible to obtain by the immune system alone, so aptamers are also called "chemical antibodies". Compared with antibodies or enzymes, which are essentially proteins, aptamers are much smaller and less complex, have high chemical stability, are easy to manufacture, and are stable during storage. Given the above advantages, aptamers have gradually become a good alternative to antibodies for target molecule recognition. SUMMARY

[0006] The purpose of the present application is to solve the problem that the detection of thrombin in blood is not easy due to low content, in order to improve the convenience of instrument operation, and to alleviate the dependence on large and precise instruments and professional operation in clinical application. A pump-free microfluidic chip based on SERS is developed, which is simple to operate, low in manufacturing cost, short in detection time, low in detection lower limit, and high in sensitivity.

[0007] To achieve the purpose of the present application, the present application proposes the following technical solutions:

[0008] A preparation method of a pump-free microfluidic chip, comprising the following steps;

[0009] Step S1, surface modification of a gold-plated substrate;

[0010] In step S1, the surface modification method of the gold-plated substrate is:

[0011] The gold-plated substrate treated by the hydrophilic treatment is immersed in a 1 mL 70 nM thrombin DNA aptamer TBA15 solution for modifying TBA15 aptamer thereon;

[0012] Then it is placed in a vacuum pump for continuing vacuumizing for 20 minutes to remove the gas in the aptamer solution;

[0013] After the treatment, the aptamer solution in which the gold-plated substrate is immersed is placed in a honeycomb oscillator for oscillation for 24 hours;

[0014] After the oscillation, the treated gold-plated substrate is taken out from the aptamer solution by using tweezers and is immersed in a centrifuge tube in which 1 mL 2 mM 6-MCH solution is placed for 2 hours;

[0015] After the immersion, they are cleaned by using deionized water and alcohol respectively for multiple times, and are used after being naturally air-dried.

[0016] The sequence of the TBA15 DNA aptamer is: 5'-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3';

[0017] Step S2, fabrication of the pump-free microfluidic chip;

[0018] The design and fabrication method of the pump-free microfluidic chip is that the design drawing of the pump-free microfluidic chip is drawn on the software Auto CAD2018, the drawn pattern is printed on a transparent film after being completed, and the pattern is solidified on a silicon membrane plate by using an ultraviolet photoetching machine. After being mixed well according to the mass ratio of polydimethylsiloxane (PDMS) to curing agent of 10:1, the PDMS is poured on the silicon template for solidification, and the PDMS chip is obtained by mold turning. The length of the chip is 45 mm, the width is 15 mm, and the height is 10 mm.

[0019] Step S3, fabrication of a finger pressure pump for driving liquid flow in the pump-free microfluidic chip;

[0020] The fabrication method of the finger pressure pump for driving liquid flow in the chip is that before pouring the PDMS on the silicon template in which the chip pattern is solidified, a cylindrical copper block with a height of 8 mm and a radius of 5 mm is placed at a designated position on the silicon template as a reserved area of the finger pressure pump, and then the PDMS and the curing agent mixed well according to the mass ratio of 10:1 are poured.

[0021] After the PDMS liquid surface is stable, the culture dish is placed on a heating plate at 75 DEG C and heated and timely blown away the bubbles generated due to heating by using an ear ball, the culture dish is covered with a cover and transferred to a oven at 75 DEG C for heating for 2 hours to solidify the PDMS, and the pump-free microfluidic chip made of PDMS is obtained after heating.

[0022] Step S4, embedding of the gold-plated substrate and sealing of the chip.

[0023] The embedding of the gold-plated substrate and the sealing of the chip are as follows: the PDMS chip containing a microchannel surface is placed above and is placed in a plasma etching machine together with a glass slide, first vacuumizing in the machine for 70 seconds, and then performing oxygen plasma oxidation treatment by ultraviolet irradiation for 50 seconds; after the treatment, the two are taken out from the plasma etching machine, and the gold-plated substrate treated by hydrophilic treatment and surface modification is quickly placed in the hexagonal chamber arranged in the PDMS chip; after being placed, the glass slide is sealed, and thus a pump-free microfluidic chip made of PDMS is obtained.

[0024] The principle of quickly placing the gold-plated substrate before the PDMS chip and the glass slide are sealed is as follows: the treatment of the PDMS chip and the glass slide in the plasma etching machine is to open the silicon-oxygen bond on the surface of the two, but the opened chemical bond will be closed again at normal temperature and pressure, and if the gold-plated substrate is not placed in the chip in time, the PDMS chip and the glass slide cannot be tightly combined, and liquid leakage will occur after the reagent is introduced. Therefore, the gold-plated substrate is placed in the PDMS chip within 5 seconds, and then the PDMS chip and the glass slide are sealed, so that the chemical bond is recombined after the two are contacted, and thus irreversible sealing is formed, avoiding the liquid leakage in the experiment.

[0025] The hydrophilic treatment method of the gold-plated substrate is as follows: the gold-plated substrate is placed in a plasma etching machine, first vacuumizing in the machine for about 70 seconds, and then performing oxygen plasma oxidation treatment by ultraviolet irradiation for 5 minutes, and thus a hydrophilic substrate surface is obtained.

[0026] Preferably, two flow limiting valves with a height of 50 μm are arranged in the pump-free microfluidic chip, so as to reduce the flow speed of thrombin and thrombin detection probes in the chip, and the thrombin and the thrombin detection probes can fully react with each other before flowing to the gold-plated substrate treated by hydrophilic treatment and surface modification, so as to increase the immunocomplexes possibly formed on the surface of the gold-plated substrate.

[0027] The application further provides a technical scheme of the application of the pump-free microfluidic chip prepared by the preparation method of the pump-free microfluidic chip to thrombin detection.

[0028] The technical scheme of the application of the pump-free microfluidic chip in thrombin detection is specifically to detect by using a Raman detection method, and the specific steps include:

[0029] First, add thrombin from the liquid inlet by using a pipette, then simultaneously introduce gold nanoparticles modified with the aptamer TBA29 and the Raman reporter molecule MGITC as a thrombin detection probe, under the action of the serpentine channel, the thrombin antigen fully reacts with the detection probe, thereby forming an immunocomplex in the sandwich structure on the gold-plated substrate incubated with the TBA15 aptamer;

[0030] After the reagents are introduced, the chip is left to stand for a period of time, and then PBS buffer is introduced from the liquid inlet to wash the chip several times to wash away the thrombin and thrombin detection probe that are not combined with the TBA15 aptamer on the gold-plated substrate;

[0031] Finally, the immunocomplex formed on the gold-plated substrate is subjected to Raman detection following the principle of controlling variables.

[0032] The Raman characteristic peak of the thrombin detection probe labeled with the Raman reporter molecule MGITC is shifted to 1614 cm -1 , and the thrombin is qualitatively and quantitatively detected by detecting the peak intensity at 1614 cm -1 . The Raman spectrum is collected by a LabRam HREvolution system, the power of the helium-neon laser is 10 mW, the selected laser wavelength is 633 nm, the Raman shift range for detection is set to 700-1700 cm -1 , the integration time is 10 seconds, and the integration number is 3. The concentrations of the thrombin to be tested are 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM and 10 nM.

[0033] The preparation method of the thrombin detection probe is as follows: first, mix 1 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) with 50 μL of 100 μM DNA aptamer TBA29 of thrombin and incubate at room temperature for 6 hours to generate free thiol groups;

[0034] Then, add 100 μL of 1 μM TBA29 aptamer solution to 1 mL of gold nanoparticle solution, and then add 2.5 M NaCl solution and 0.5 M PBS buffer, so that the final concentrations of the two are 0.1 M and 10 mM, respectively;

[0035] After calculation, the required volumes of the added NaCl solution and PBS buffer are 46.8 μL and 23.4 μL, respectively;

[0036] The solution is incubated in a dry thermostat at 50°C for 48 hours for further aging, and 7.8 μL of NaCl solution is added in the TBA29 aptamer solution at an average of six times within 48 hours, namely, every 7.8 μL is added;

[0037] 4 μL of 10 mM MgCl2 solution is added in the TBA29 aptamer solution two hours before the end of the 48-hour incubation; -5 M Raman reporter molecule MGITC;

[0038] Finally, the mixed solution is placed in a differential centrifuge and centrifuged at a speed of 5500 rpm for 15 minutes, and the supernatant is removed with a pipette to remove free aptamer and MGITC not combined with gold nanoparticles, and the precipitate is suspended in PBS buffer for use,

[0039] wherein,

[0040] TBA29 DNA aptamer sequence: 5'-dithiol-TTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'.

[0041] The synthesis method of the gold nanoparticles is as follows: 49 mL of deionized water and 500 μL of 1% mass fraction of tetrachloroauric acid solution are added in a clean three-necked flask and placed in a heating jacket on a constant temperature magnetic stirrer for heating; after boiling, 500 μL of 1% mass fraction of sodium citrate solution is added with a pipette, and after the liquid color changes from transparent to purple red, the synthesized gold nanoparticles are matured by continuing to heat for 1 hour, so that gold nanoparticles with a particle size of about 40 nm are synthesized.

[0042] Advantages and beneficial effects of the present application:

[0043] 1. The pump-free microfluidic chip designed in the present application has low manufacturing cost and does not need to be operated by professionals, and the detection operation process is simple.

[0044] 2. The SERS detection realizes high sensitivity, short detection time and low detection lower limit. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0046] Figure 1 is a schematic diagram of the pump-free microfluidic chip of the present application;

[0047] Figure 2is the manufacturing and assembling schematic diagram of the pump-free microfluidic chip of the present application;

[0048] Figure 3 is the working schematic diagram of the internal flow limiting valve of the pump-free microfluidic chip of the present application;

[0049] Figure 4 is the schematic diagram of the formation of the immune complex on the gold-plated substrate;

[0050] Figure 5 is the thrombin concentration gradient Raman spectrum and its corresponding linear regression curve diagram. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings.

[0052] Figure 1 is the schematic diagram of the pump-free microfluidic chip of the present application. It can be seen that the pump-free microfluidic chip is mainly composed of three parts, which are a PDMS chip body, a gold-plated substrate and a glass slide.

[0053] Figure 2 is the manufacturing and assembling schematic diagram of the pump-free microfluidic chip of the present application. The preparation and assembling method is carried out in the following steps in turn:

[0054] Step S1, surface modification of the gold-plated substrate;

[0055] In step S1, the surface modification method of the gold-plated substrate is:

[0056] The gold-plated substrate treated by hydrophilic treatment is immersed in 1 mL of 70 nM thrombin DNA aptamer TBA15 solution for modifying TBA15 aptamer thereon;

[0057] Then it is placed in a vacuum pump to continue vacuuming for 20 minutes to remove the gas in the aptamer solution;

[0058] After treatment, the aptamer solution in which the gold-plated substrate is immersed is placed in a honeycomb oscillator to oscillate for 24 hours;

[0059] After oscillation, the treated gold-plated substrate is taken out of the aptamer solution with tweezers and is immersed in a centrifuge tube containing 1 mL of 2 mM 6-MCH solution for 2 hours;

[0060] After immersion, they are washed with deionized water and alcohol several times, and are used after natural air drying.

[0061] TBA15 DNA aptamer sequence: 5'-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3';

[0062] Step S2, fabrication of the pump-free microfluidic chip;

[0063] The design is performed on software Auto CAD2018, and after completion, the drawn pattern is printed on a transparent film sheet, and then the pattern is solidified on a silicon film plate using an ultraviolet photoetching machine. Before pouring PDMS on the silicon template solidified with the chip pattern, a cylindrical copper block with a height of 8 mm and a radius of 5 mm is placed on the designated position thereon as a reserved area of the finger pump, and then PDMS mixed with a curing agent in a mass ratio of 10:1 is poured. After the PDMS liquid surface is stable, the culture dish is placed on a heating plate at 75°C for heating and timely blowing away the bubbles generated due to heating with an ear bulb. After all the bubbles disappear, the culture dish is covered with a cover and transferred to a 75°C oven for heating for 2 hours to solidify the PDMS. After heating, the pump-free chip made of PDMS is obtained.

[0064] The gold-plated substrate is placed in a plasma etching machine, vacuum treatment is performed in the machine for about 70 seconds, and then oxygen plasma oxidation treatment is performed by ultraviolet irradiation for 5 minutes. The hydrophilic surface of the substrate is obtained after being taken out.

[0065] Step S2, fabrication of the pump-free microfluidic chip;

[0066] The design and fabrication method of the pump-free microfluidic chip is as follows: the design of the pump-free microfluidic chip is performed on software Auto CAD2018, and after completion, the drawn pattern is printed on a transparent film sheet, and then the pattern is solidified on a silicon film plate using an ultraviolet photoetching machine. After mixing polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 10:1, pouring the mixture on the silicon template for solidification, and obtaining the PDMS chip by inverse molding, the chip length is 45 mm, the width is 15 mm, and the height is 10 mm.

[0067] Step S3, fabrication of the finger pump for driving liquid flow in the pump-free microfluidic chip;

[0068] The fabrication method of the finger pump for driving liquid flow in the chip is as follows: before pouring PDMS on the silicon template solidified with the chip pattern, a cylindrical copper block with a height of 8 mm and a radius of 5 mm is placed on the designated position thereon as a reserved area of the finger pump, and then PDMS mixed with a curing agent in a mass ratio of 10:1 is poured.

[0069] After the PDMS liquid surface is stable, the culture dish is placed on a heating plate at 75℃ and heated and the air bubbles generated due to heating are timely blown away by an ear ball, the culture dish is covered with a cover after the air bubbles are completely disappeared, and then is transferred to an oven at 75℃ to heat for 2 hours to solidify the PDMS, and the pump-free microfluidic chip made of PDMS is obtained after heating.

[0070] Step S4, embedding of the gold-plated substrate and sealing of the chip.

[0071] The embedding of the gold-plated substrate and the sealing method of the chip is as follows: the PDMS chip containing a microchannel surface is placed above and is placed in a plasma etching machine together with a glass slide, first vacuumizing in the machine for 70 seconds, and then performing oxygen plasma oxidation treatment by ultraviolet irradiation for 50 seconds; after the treatment is completed, the two are taken out from the plasma etching machine, and the gold-plated substrate treated by hydrophilic treatment and surface modification is quickly placed in the hexagonal chamber arranged in the PDMS chip; after being placed, the glass slide is sealed, and thus a pump-free microfluidic chip made of PDMS is obtained.

[0072] The principle of quickly placing the gold-plated substrate before the PDMS chip and the glass slide are sealed is as follows: the treatment of the PDMS chip and the glass slide in the plasma etching machine is to open the silicon-oxygen bond on the surface of the two, but the opened chemical bond will be closed again at normal temperature and pressure, and if the gold-plated substrate cannot be placed in the chip in time, the PDMS chip and the glass slide cannot be tightly combined, and liquid leakage will occur after the reagent is introduced. Therefore, the gold-plated substrate is placed in the PDMS chip within 5 seconds, and then the PDMS chip and the glass slide are sealed, so that the chemical bond is recombined after the two are contacted, thereby forming an irreversible seal to avoid liquid leakage during the experiment.

[0073] Figure 3 is a schematic diagram of the operation of the internal flow limiting valve of the pump-free microfluidic chip of the present application. Two flow limiting valves with a height of 50 μm are arranged in the pump-free microfluidic chip to reduce the flow speed of thrombin and thrombin detection probes in the chip, so that they can fully react with each other before flowing to the gold-plated substrate treated by hydrophilic treatment and surface modification, thereby increasing the immune complex that can be formed on the surface of the gold-plated substrate.

[0074] The use method of the pump-free microfluidic chip of the present application is as follows: when the reagent adding step is performed each time, the index finger is first pressed on the finger pressure pump to form a pressure difference in the chip, at the same time, the reagent is added at the liquid inlet, and after the addition is completed, the pressure applied on the finger pressure pump by the index finger is released to drive the flow of the liquid in the microchannel.

[0075] Figure 4 is a schematic diagram of the formation of an immune complex on a gold-plated substrate, and the preparation process of a thrombin detection probe is performed in the following steps:

[0076] One μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) was mixed with 50 μL of 100 μM of the DNA aptamer TBA29 and incubated at room temperature for 6 hours to generate free thiols. Then 100 μL of 1 μM TBA29 aptamer solution was added to 1 mL of gold nanoparticle solution, followed by the addition of 2.5 M NaCl solution and 0.5 M PBS buffer to a final concentration of 0.1 M and 10 mM, respectively. The volume of NaCl solution and PBS buffer needed to be added was calculated to be 46.8 μL and 23.4 μL, respectively. The solution was incubated in a dry incubator at 50 °C for 48 hours for further aging. Since the aging of DNA aptamer with NaCl solution is a gradual process, the NaCl solution was added to the TBA29 aptamer solution in six equal portions over 48 hours, i.e. 7.8 μL each time. Two hours before the end of the 48 hours incubation, 4 μL of 10 -5 M Raman reporter MGITC was added. Finally, the mixed solution was centrifuged in a microfuge at 5500 rpm for 15 minutes, and the supernatant was removed with a pipette to remove free aptamer and MGITC that were not bound to the gold nanoparticles. The pellet was suspended in PBS buffer for use.

[0077] The experiment performed in the pumpless microfluidic chip was carried out in the following order:

[0078] The thrombin was first added from the inlet with a pipette, followed by the simultaneous introduction of gold nanoparticles modified with the aptamer TBA29 and the Raman reporter MGITC as the detection probe for thrombin. Under the action of the serpentine channel, the thrombin antigen reacted fully with its detection probe, forming a sandwich immunocomplex on the gold-plated substrate incubated with the TBA15 aptamer. After the reagents were introduced, the chip was left to react for a period of time, and then PBS buffer was introduced from the inlet to wash the chip several times to remove the thrombin and thrombin detection probe that were not bound to the TBA15 aptamer on the gold-plated substrate. In this way, a sandwich immunocomplex was formed on the gold-plated substrate.

[0079] Figure 5 is the Raman spectrum of the thrombin concentration gradient and its corresponding linear regression curve.

[0080] The Raman spectra were collected by LabRam HR Evolution system. Firstly, the laser spot was moved to the hexagonal detection area on which the gold-plated substrate was carried, then the 50x objective lens of microscope was used and the fine focus screw was adjusted to focus the laser spot on the gold-plated substrate. The power of the helium-neon laser was 10 mW, the selected laser wavelength was 633 nm, the Raman shift range for detection was set to 700-1700 cm -1 , the integration time was 10 seconds, and the integration number was 3. After the experimental data were collected, the LabSpec 5.0 software was used to remove the baseline and suppress the background noise. Each spectrum was the average value of five sampling points of the antigen at the concentration. The Raman shift was taken as the horizontal coordinate, and the Raman spectrum signal intensity was taken as the vertical coordinate, and the thrombin quantitative analysis was carried out by observing the change of the SERS intensity of the peak at 1614 cm -1 . The detection range of thrombin was between 0.01-10 nM, and the lower limit of detection could reach 0.01 nM. In addition, the Raman intensity at 1614 cm -1 of the thrombin at different concentrations was selected to draw a linear regression curve, and the linear fitting degree reached 0.97354. It can be seen that there is a good linear relationship between the concentration of thrombin and the SERS peak intensity.

[0081] The above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Application of a pump-free microfluidic chip in thrombin detection, characterized in that, The detection is carried out by using a Raman detection method, and the specific steps include: First, use a pipette to add thrombin from the liquid inlet, then simultaneously introduce gold nanoparticles modified with the aptamer TBA29 and the Raman reporter molecule MGITC as the thrombin detection probe, under the action of the serpentine channel, the thrombin antigen fully reacts with its detection probe, thereby forming a sandwich structure of the immune complex on the gold-plated substrate incubated with the TBA15 aptamer; After the reagents are introduced, the chip is left to stand for a period of time, and then PBS buffer is introduced from the liquid inlet to wash the chip several times to wash away the thrombin and thrombin detection probe that is not combined with the TBA15 aptamer on the gold-plated substrate; Finally, follow the principle of controlling variables, and perform Raman detection on the immune complex formed on the gold-plated substrate; The preparation method of the thrombin detection probe is as follows: first, mix 1 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) with 50 μL of 100 μM DNA aptamer TBA29 of thrombin and incubate at room temperature for 6 hours to generate free thiol groups; Then add 100 μL of 1 μM TBA29 aptamer solution to 1 mL of gold nanoparticle solution, followed by the addition of 2.5 M NaCl solution and 0.5 M PBS buffer, so that the final concentrations of the two are 0.1 M and 10 mM, respectively; According to the calculation, the required volumes of NaCl solution and PBS buffer to be added are 46.8 μL and 23.4 μL, respectively; The solution is incubated in a dry thermostat at 50°C for 48 hours for further aging, and the NaCl solution is added to the TBA29 aptamer solution at an average of six times within 48 hours, i.e. 7.8 μL each time; Four hours before the end of the 48 hours of incubation, 4 μL of 10 -5 Raman reporter molecule of M, MGITC; Finally, centrifuge the mixed solution in a differential centrifuge at a speed of 5500 rpm for 15 minutes, and remove the supernatant with a pipette to remove the free aptamer and MGITC that are not combined with the gold nanoparticles, and suspend the precipitate in PBS buffer for use, Wherein, The sequence of the TBA29 DNA aptamer is: 5'-dithiol-TTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'; The preparation method of the pump-free microfluidic chip includes the following steps: Step S1, surface modification of the gold-plated substrate; After hydrophilic treatment, immerse the gold-plated substrate in 1 mL of 70 nM DNA aptamer TBA15 solution of thrombin to modify the TBA15 aptamer thereon; Then put it into a vacuum pump to continue vacuuming for 20 minutes to remove the gas in the aptamer solution; After treatment, immerse the aptamer solution with the gold-plated substrate in a honeycomb oscillator for 24 hours; After the oscillation is completed, use tweezers to take out the treated gold-plated substrate from the aptamer solution and place them in a centrifuge tube containing 1 mL of 2 mM 6-MCH solution for 2 hours; After soaking, they are washed with deionized water and alcohol several times, and then used after natural drying; TBA15 DNA aptamer sequence: 5'-dithiol-TTTTTTTTTTGGTTGGTGTGGTTGG-3'; Step S2, the preparation of pump-free microfluidic chip; The drawn pattern is printed on the transparent film sheet, and the pattern is solidified on the silicon membrane plate by using the ultraviolet photoetching machine; After mixing the polydimethylsiloxane (PDMS) and the curing agent, pour them on the silicon template for solidification, and get the PDMS chip by inverse molding; Step S3, the preparation of the finger pressure pump for driving the liquid flow in the pump-free microfluidic chip; Step S4, embedding of the gold-plated substrate and sealing of the chip.

2. The application of a pump-free microfluidic chip in thrombin detection according to claim 1, wherein, The hydrophilic treatment method of the gold-plated substrate is as follows: the gold-plated substrate is placed in a plasma etching machine, vacuum treatment is first carried out in the machine for about 70 seconds, and then oxygen plasma oxidation treatment is carried out by ultraviolet irradiation for 5 minutes, and the hydrophilic surface of the substrate is obtained after taking out.

3. The use of a pump-free microfluidic chip in thrombin detection according to claim 1, characterized in that, In step S3, the preparation method of the finger pressure pump for driving the liquid flow in the chip is as follows: before pouring the PDMS on the silicon template on which the chip pattern is solidified, a cylindrical copper block with a height of 8 mm and a radius of 5 mm is placed at a specified position on the silicon template as a reserved area of the finger pressure pump, and then the mixed PDMS and curing agent are poured into the reserved area according to the mass ratio of 10:1 of the PDMS to the curing agent. After the PDMS liquid surface is stable, the culture dish is placed on a heating plate at 75℃ and heated in time with an ear bulb to blow away the bubbles generated by heating, and after the bubbles disappear, the culture dish is covered with a cover and transferred to a 75℃ oven for 2 hours to solidify the PDMS, and the pump-free microfluidic chip made of PDMS is obtained after heating.

4. The application of a pump-free microfluidic chip in thrombin detection according to claim 3, characterized in that, Two flow limiting valves with a height of 50 μm are arranged in the pump-free microfluidic chip to reduce the flow speed of thrombin and thrombin detection probe in the chip, so that they can fully react with each other before flowing to the gold-plated substrate treated by hydrophilic treatment and surface modification, thereby increasing the immune complexes that may be formed on the surface of the gold-plated substrate.

5. The use of a pump-free microfluidic chip in thrombin detection according to claim 1, characterized in that, In step S4, the embedding of the gold-plated substrate and the sealing method of the chip are as follows: the PDMS chip containing one side of the microchannel is placed on the upper side and placed in a plasma etching machine together with a glass slide, vacuum treatment is first carried out in the machine for 70 seconds, and then oxygen plasma oxidation treatment is carried out by ultraviolet irradiation for 50 seconds; after the treatment is completed, the two are taken out from the plasma etching machine, and the gold-plated substrate treated by hydrophilic treatment and surface modification is quickly placed in the hexagonal chamber arranged in the PDMS chip; after placing, the glass slide is sealed with it, and thus a pump-free microfluidic chip made of PDMS is obtained.

6. The use of a pump-free microfluidic chip in thrombin detection according to claim 1, characterized in that, The synthesis method of the gold nanoparticles is as follows: 49 mL of deionized water and 500 μL of 1% mass fraction of tetrachloroauric acid solution are added into a cleaned three-necked flask, and the flask is placed in a heating jacket of a constant-temperature magnetic stirrer for heating; after boiling, 500 μL of 1% mass fraction of sodium citrate solution is added by using a syringe, and after the liquid color completely changes from transparent to purple red, the synthesized gold nanoparticles are matured by continuing to heat for 1 hour, so that the gold nanoparticles with a particle size of about 40 nm are synthesized.

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