A microfluidic chip and detection method for tumor marker detection

Abstract

The present invention discloses a microfluidic chip and a detection method for tumor marker detection, the chip comprises a reaction layer substrate and a microfluidic sample layer located thereon, the reaction layer substrate comprises a glass slide substrate and a graphene oxide quantum dot nanomaterial substrate assembled on the glass slide substrate, the graphene oxide quantum dot nanomaterial substrate is printed with a strip detection band and a quality control band; the bottom of the microfluidic sample layer is provided with a plurality of microfluidic grooves, the plurality of microfluidic grooves and the plurality of strip detection bands and quality control bands are arranged vertically to form a plurality of reaction dot arrays, the microfluidic sample layer is also provided with an inflow hole and an outflow hole that are connected up and down, and the two ends of the microfluidic groove are respectively connected to the inflow hole and the outflow hole. The chip and the detection method disclosed in the present invention can detect a variety of different tumor markers at the same time, and have the advantages of rapid, stable, and on-site high-throughput detection.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chips, and in particular to a microfluidic chip and a detection method for tumor marker detection. Background Art

[0002] In recent years, various cancers have seriously affected human health. If the tumor is discovered early, early treatment and intervention can greatly improve the patient's survival period and survival rate. In the early stage of tumor onset, the concentration of certain biological molecules in human serum begins to change. For example, CEA, CA125, CA199, and CA153 in the serum of breast cancer patients are important markers of breast cancer and have an important relationship with the diagnosis and prognosis of breast cancer. They are breast cancer-related antigens, present in breast cancer cells, and can be released into the blood. Detection of CEA, CA125, CA199, and CA153 levels in the blood can be used to detect breast cancer. The detection of tumor markers in blood or tissue provides important information for early diagnosis, clinical monitoring, disease treatment, and rehabilitation monitoring of cancer patients.

[0003] Due to the increasing demand for various disease control, sensitive, low-cost, and instant detection methods have greatly attracted the interest of relevant researchers. Traditional tumor marker detection methods mainly include enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence analysis, mass spectrometry, etc. However, these methods cannot meet the requirements of instant detection. The traditional detection method, ELISA kit, consumes a large amount of samples to complete the detection of multiple biomarkers by testing one by one. The electrochemical method has high sensitivity, but its high-throughput detection capability is limited.

[0004] In recent years, nanomaterial integrated biosensors have played an important role in biomolecule detection due to their special interactions with bioactive molecules, cells and organisms. Recently, carbon-based nanomaterials have attracted the attention of researchers. Some carbon-based luminescent nanomaterials have excellent photoelectric properties and good biocompatibility. Due to their single-atom layered structure, they have unique physical adsorption, luminescence properties, low toxicity, stability, and easy preparation. There are a large number of oxygen-containing groups on some carbon-based nanomaterials, making them more compatible in polar solvents (i.e., water). Therefore, nanomaterials combined with technologies such as microfluidic chips have broad application prospects in improving the sensitivity of protein detection, improving the reliability of protein detection, and high throughput. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a microfluidic chip and a detection method for tumor marker detection, so as to achieve the purpose of simultaneously detecting multiple different tumor markers with rapid, stable and on-site high-throughput detection.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A microfluidic chip for tumor marker detection comprises a reaction layer substrate and a microfluidic sample loading layer thereon, wherein the reaction layer substrate comprises a glass slide substrate and a graphene oxide quantum dot nanomaterial substrate assembled on the glass slide substrate, and a strip detection band and a quality control band are printed on the graphene oxide quantum dot nanomaterial substrate; a plurality of microfluidic grooves are arranged at the bottom of the microfluidic sample loading layer, and the plurality of microfluidic grooves and the plurality of strip detection bands and quality control bands are arranged vertically to form a plurality of reaction dot arrays; an inflow hole and an outflow hole which are connected vertically are also provided on the microfluidic sample loading layer, and the two ends of the microfluidic groove are respectively connected to the inflow hole and the outflow hole.

[0008] In the above scheme, a plurality of the strip-shaped detection bands and quality control bands are periodically laid on the graphene oxide quantum dot nanomaterial substrate to form a probe area.

[0009] In the above scheme, the plurality of strip-shaped detection bands and quality control bands are evenly arranged and arranged in parallel and at intervals, and the plurality of microfluidic channel grooves are evenly arranged perpendicular to the plurality of strip-shaped detection bands and quality control bands.

[0010] In the above solution, the material of the glass slide substrate is calcium silicate, sodium silicate or silicon dioxide.

[0011] In the above scheme, the graphene oxide quantum dot nanomaterial substrate is obtained by reacting the glass slide substrate with the graphene oxide quantum dot nanomaterial, followed by washing and drying.

[0012] In the above scheme, the strip detection band and the quality control band are biological molecules and graphene oxide quantum dot nanomaterial substrates physically adsorbed on the glass slide substrate through functional groups or π-π bonds.

[0013] A method for detecting tumor markers, using the above-mentioned microfluidic chip for detecting tumor markers, comprises the following process:

[0014] (1) Inject different samples to be tested from multiple inlet holes respectively, and incubate for 15-30 minutes to allow the antigens to be tested in the samples to be tested to fully bind to the tumor marker detection antibodies in the strip detection bands and quality control bands on the reaction layer substrate. After the incubation is completed, remove the excess samples to be tested from the outlet holes. At this time, the antigens to be tested are specifically bound to the tumor marker detection antibodies and fixed on the reaction layer substrate;

[0015] (2) removing the slide in DPBS containing 0.5-5% BSA, and washing away the antigen to be tested that is not adsorbed on the reaction layer substrate with 0.5-5% BSA;

[0016] (3) Spread the mixed detection antibody solution on the reaction layer substrate and incubate for 10-30 minutes;

[0017] (4) Rinse with BSA at a mass concentration of 0.5-5%, and then spread the APC-streptavidin conjugate on the reaction layer substrate. At this time, the APC-streptavidin conjugate specifically binds to the tumor marker detection antibody labeled with biotin and is fixed on the reaction layer substrate, and then the tumor marker is fluorescently detected.

[0018] Through the above technical solution, the microfluidic chip and detection method for tumor marker detection provided by the present invention have the following beneficial effects:

[0019] The self-assembled graphene oxide quantum dot nanomaterial substrate of the present invention is integrated with a detection chip of microfluidic technology. The microfluidic sample loading layer includes a plurality of microfluidic grooves uniformly arranged perpendicularly to a plurality of strip detection bands and quality control bands, and a plurality of biochemical or chemical indexes of the sample can be detected simultaneously. When the chip is in use, only the outflow hole and the inflow hole are connected to the outside world, and most of the reaction processes are completed in a closed microfluidic system, which effectively reduces the interference of the external environment on the reaction and detection process. In addition, the chip has a simple structure and is easy to integrate and combine with supporting automation equipment to realize automated detection.

[0020] The chip can detect a variety of different tumor markers at the same time, and has the functions of rapid, stable, and on-site high-throughput detection, making on-site sample detection more convenient, simple, and rapid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0022] Figure 1 This is a schematic diagram of the overall structure of a microfluidic chip for tumor marker detection disclosed in an embodiment of the present invention;

[0023] Figure 2 A schematic diagram of the reaction layer substrate structure disclosed in an embodiment of the present invention;

[0024] Figure 3 A schematic diagram of a microfluidic sample loading layer disclosed in an embodiment of the present invention;

[0025] Figure 4 It is a standard curve diagram of an embodiment of the present invention.

[0026] In the figure, 1. reaction layer substrate; 2. microfluidic sample loading layer; 3. glass slide substrate; 4. graphene oxide quantum dot nanomaterial substrate; 5. detection belt; 6. quality control belt; 7. inflow hole; 8. outflow hole; 9. microfluidic groove. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0028] The present invention provides a microfluidic chip for tumor marker detection, such as Figure 1 As shown, it includes a reaction layer substrate 1 and a microfluidic sample loading layer 2 located thereon. Figure 2 As shown, the reaction layer substrate 1 includes a glass slide substrate 3 and a graphene oxide quantum dot nanomaterial substrate 4 assembled on the glass slide substrate 3, and a strip detection band 5 and a quality control band 6 are printed on the graphene oxide quantum dot nanomaterial substrate 4; Figure 3 As shown, a plurality of microchannel grooves 9 are provided at the bottom of the microchannel loading layer 2, and the plurality of microchannel grooves 9 are vertically arranged with the plurality of strip detection bands 5 and the quality control bands 6 to form a plurality of reaction arrays. The microchannel loading layer 2 is also provided with an inlet hole 7 and an outlet hole 8 which pass through from top to bottom, and the two ends of the microchannel groove 9 are connected with the inlet hole 7 and the outlet hole 8 respectively.

[0029] A plurality of strip-shaped detection bands 5 and quality control bands 6 are periodically laid flat on the graphene oxide quantum dot nanomaterial substrate 4 to form a probe area. The plurality of strip-shaped detection bands 5 and quality control bands 6 are evenly arranged and arranged in parallel and spaced apart, and the plurality of microfluidic grooves 9 are evenly arranged perpendicular to the plurality of strip-shaped detection bands 5 and quality control bands 6.

[0030] The material of the slide glass substrate 3 is calcium silicate, sodium silicate or silicon dioxide.

[0031] The graphene oxide quantum dot nanomaterial substrate 4 is obtained by reacting the glass slide substrate 3 with the graphene oxide quantum dot nanomaterial, followed by washing and drying.

[0032] The plurality of strip detection bands 5 and quality control bands 6 are strip detection bands 5 and quality control bands 6 patterns printed on the graphene oxide quantum dot nanomaterial substrate 4 using a straight-line microfluidic chip. The strip detection bands 5 and quality control bands 6 are biomolecules and the graphene oxide quantum dot nanomaterial substrate 4 are physically adsorbed on the glass slide substrate 3 through functional groups or π-π bonds.

[0033] The microchannel sample loading layer 2 is made of polydimethylsiloxane material by casting or injection molding. The detection chip can detect 60 samples from different sources, and each sample can detect 50 antibodies at the same time, thus greatly improving the detection efficiency.

[0034] In order to verify the uniformity of the strip detection band 5 and the quality control band 6, one end of the short side of the glass slide substrate 3 fixed with the strip detection band 5 and the quality control band 6 is immersed in the goat anti-mouse fluorescent secondary antibody solution, incubated for 10 minutes, and then rinsed with DPBS containing 1% BSA mass concentration, and then the entire glass slide substrate 3 fixed with the barcode of the strip detection band 5 and the quality control band 6 is placed in the following solutions 100% DPBS, 50% DPBS, DI Water for cleaning, reducing the detection background and improving the detection sensitivity. Finally, the glass slide substrate 3 fixed with the strip detection band 5 and the quality control band 6 is placed in a glass slide centrifuge to spin dry for 20-30 seconds, and finally the fluorescence signal is observed with a microarray gene scanner for standby use. At this time, the microfluidic sample layer 2 and the processed reaction layer substrate 1 fixed with the strip detection band 5 and the quality control band 6 are bonded by self-weight, so that a complete and usable chip system is prepared.

[0035] A method for detecting tumor markers, using the above-mentioned microfluidic chip for detecting tumor markers, comprises the following process:

[0036] (1) 1uL of the sample to be tested of different concentrations is injected from the multiple inflow holes 7 respectively, and incubated for 20 minutes to allow the antigen to be tested in the sample to be tested to fully bind with the tumor marker detection antibody on the strip detection band 5 and the quality control band 6 on the reaction layer substrate 1. After the incubation is completed, the excess sample to be tested is drained from the outflow hole 8. At this time, the antigen to be tested is specifically bound to the tumor marker detection antibody and fixed on the reaction layer substrate 1;

[0037] (2) removing the slide in DPBS containing 1% BSA by mass concentration, and washing away the antigen to be tested that is not adsorbed on the reaction layer substrate 1 with 1% BSA by mass concentration;

[0038] (3) Use a pipette to spread the mixed detection antibody solution on the reaction layer substrate 1 and incubate for 20 minutes;

[0039] (4) Rinse with BSA at a mass concentration of 1%, then use a pipette to take 200ul of APC-streptavidin conjugate and spread it on the reaction layer substrate 1. At this time, the APC-streptavidin conjugate is specifically bound to the biotin-labeled tumor marker detection antibody and fixed on the reaction layer substrate 1, and then the tumor marker is fluorescently detected.

[0040] (5) The intensity of the optical signal was detected by GenePix 4400 microarray scanner (the detection wavelength was 635 nm), and the detection results were given. The total detection time was 30 min. Each standard was measured three times using three microfluidic chip system detection holes, and the average of the results was taken to draw a standard curve.

[0041] Method for drawing standard curve: dilute recombinant proteins CEA and AFP in gradients, i.e. 50000pg / ml, 5000pg / ml, 500pg / ml, 50pg / ml, 5pg / ml; dilute recombinant proteins CA125, CA199 and CA153 in gradients, i.e. 1000u / ml, 100u / ml, 10u / ml, 1u / ml, 0.1u / ml. Perform the following operations for each concentration of recombinant protein: take 2μL of recombinant protein standard with a pipette, inject it into the detection sample chip structure of the chip system through the injection hole of the chip, perform the above operations, and the obtained standard curve is shown in Figure 4 .

[0042] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for using a microfluidic chip for tumor marker detection for non-diagnostic and / or therapeutic purposes, characterized in that: The microfluidic chip comprises a reaction layer substrate and a microfluidic sample loading layer thereon, wherein the reaction layer substrate comprises a glass slide substrate and a graphene oxide quantum dot nanomaterial substrate assembled on the glass slide substrate, and a plurality of strip detection bands and quality control bands are printed on the graphene oxide quantum dot nanomaterial substrate; a plurality of microfluidic grooves are arranged vertically with the plurality of strip detection bands and quality control bands at the bottom of the microfluidic sample loading layer to form a plurality of reaction dot arrays, and an inflow hole and an outflow hole which are connected up and down are also provided on the microfluidic sample loading layer, and the two ends of the microfluidic groove are connected with the inflow hole and the outflow hole respectively; the graphene oxide quantum dot nanomaterial substrate is obtained by reacting the glass slide substrate with the graphene oxide quantum dot nanomaterial, then washing and drying; the strip detection band and the quality control band are formed by physical adsorption of biological molecules and the graphene oxide quantum dot nanomaterial substrate on the glass slide substrate through functional groups or π-π bonds; The method for using the microfluidic chip comprises the following process: (1) Different samples to be tested are injected from multiple inlet holes respectively and incubated for 15-30 minutes to allow the antigens to be tested in the samples to be fully combined with the tumor marker detection antibodies in the strip detection bands and quality control bands on the reaction layer substrate. After the incubation is completed, the excess samples to be tested are taken out from the outlet holes. At this time, the antigens to be tested are specifically combined with the tumor marker detection antibodies and fixed on the reaction layer substrate; (2) Remove the slide in DPBS containing 0.5-5% BSA, and rinse the antigen that is not adsorbed on the reaction layer substrate with 0.5-5% BSA; (3) Spread the mixed detection antibody solution on the reaction layer substrate and incubate for 10-30 minutes; (4) Rinse with BSA at a mass concentration of 0.5-5%, and then spread the APC-streptavidin conjugate on the reaction layer substrate. At this time, the APC-streptavidin conjugate specifically binds to the tumor marker detection antibody labeled with biotin and is fixed on the reaction layer substrate, and then the tumor marker is fluorescently detected.

2. The method for using the microfluidic chip for tumor marker detection for non-diagnostic and / or therapeutic purposes according to claim 1, characterized in that: A plurality of the strip-shaped detection bands and quality control bands are periodically laid on the graphene oxide quantum dot nanomaterial substrate to form a probe area.

3. The method for using the microfluidic chip for tumor marker detection for non-diagnostic and / or therapeutic purposes according to claim 1, characterized in that: The plurality of strip-shaped detection bands and quality control bands are evenly arranged and arranged in parallel and at intervals, and the plurality of microfluidic channel grooves are evenly arranged perpendicular to the plurality of strip-shaped detection bands and quality control bands.

4. The method for using the microfluidic chip for tumor marker detection for non-diagnostic and / or therapeutic purposes according to claim 1, characterized in that: The material of the glass slide substrate is calcium silicate, sodium silicate or silicon dioxide.

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