Fluorescence-enhanced micro-fluidic chip as well as preparation method and application thereof

Abstract

The invention discloses a fluorescence-enhanced micro-fluidic chip and a preparation method and application thereof, the chip comprises a fluorescence-enhanced substrate and a cover plate with a circulation pipeline, the fluorescence-enhanced substrate comprises: a substrate; the adhesion layer is arranged on the substrate; a noble metal particle layer provided on the adhesive layer; the polyelectrolyte layer is arranged on the noble metal layer; or, a substrate; the adhesion layer, the noble metal particles and the reducing natural polymer form a multi-gel network-noble metal particle layer, and the multi-gel network-noble metal particle layer is arranged on the substrate; and optionally, a polyelectrolyte layer, the polyelectrolyte layer being disposed on the multi-gel network-noble metal particle layer. When the device is used for detection, the detection processes such as sample introduction, reaction, enrichment, biomolecular fluorescence enhancement and parallel analysis of multiple markers can be effectively integrated, the detection sensitivity and accuracy are improved, and meanwhile multi-target joint detection is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field, and in particular to a fluorescence enhanced microfluidic chip and a preparation method and application thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide. Early diagnosis is particularly important for the formulation of treatment methods and prognosis recovery for patients. For example, for breast cancer, imaging and histopathology are currently the main methods for diagnosis and evaluation of efficacy. The former is easily affected by subjective factors such as the experience of clinical physicians and cannot accurately evaluate the efficacy; although the latter is highly accurate and is the gold standard, it cannot timely understand the sensitivity of chemotherapy drugs, making it difficult to adjust chemotherapy regimens in a timely manner and miss the best treatment opportunity. More importantly, these methods cannot achieve repeated testing because they require special equipment and personnel or are difficult to sample. Therefore, it is particularly important to seek new indicators that can respond to the efficacy in a timely manner and can be repeatedly tested to achieve the purpose of real-time cancer efficacy monitoring.

[0003] Antigen antibodies (such as CEA, NSE, CYFRA21-1 and SCC), nucleic acids (ctDNA, circRNA, etc.) in serum are extremely valuable early markers for diagnosing lung cancer. Detection of such markers is a promising liquid biopsy method that can be measured repeatedly. However, the concentration of tumor markers in the serum of cancer patients, especially those with early cancer, is extremely low and the matrix environment is complex. It is extremely challenging to specifically and sensitively identify tumor markers clinically related to cancer diagnosis and to perform specific quantitative analysis on them. Studies have shown that the combined determination of multiple tumor markers can improve diagnostic sensitivity and accuracy, but how to quantitatively analyze multiple markers from the blood of cancer patients with high sensitivity and specificity is an urgent problem to be solved.

[0004] At present, the detection methods of tumor markers in serum mainly include immunocolloidal gold test strips, colorimetric detection methods, electrochemiluminescence detection and other methods. The detection sensitivity based on colloidal gold and colorimetric methods is low and it is impossible to achieve simultaneous quantitative detection of multi-indicator low-concentration lung cancer markers. The electrochemiluminescence method requires large instruments and professionals to manage the detection. Patients need to go to larger hospitals and the detection is expensive. It is impossible to achieve comprehensive screening of lung cancer, real-time monitoring of efficacy and recurrence. Microfluidic chips can realize precise manipulation of trace sample solutions at the micro-nano scale, and can integrate the sample detection processes such as sample addition, mixing, enrichment, reaction, and detection on the chip. At the same time, it does not require professional personnel to operate to realize sample input and result output. It has the advantages of low sample reagent consumption, fast detection speed, simple operation, and multiple parallelization.

[0005] Currently, microfluidics has been applied to many scenarios including allergen detection, myocardial marker detection, infectious pathogen detection, etc. However, there are problems such as extremely low tumor marker concentration, fluorescence background interference of biological molecules in the blood, fluorescence background interference of chip materials, and parallel detection of multiple markers. How to design and prepare microfluidic chips to enhance the fluorescence signal of tumor markers in the blood of cancer patients and realize instant joint detection is of great value. Summary of the invention

[0006] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention aims to provide a fluorescence enhanced microfluidic chip and a preparation method and application thereof.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a fluorescence enhancement substrate, comprising:

[0009] substrate;

[0010] an adhesive layer disposed on the substrate;

[0011] A noble metal particle layer, wherein the noble metal particle layer is disposed on the adhesion layer;

[0012] a polyelectrolyte layer, wherein the polyelectrolyte layer is disposed on the noble metal layer; or

[0013] substrate;

[0014] An adhesive layer, the adhesive layer is disposed on the substrate, and the adhesive layer also forms a multi-gel network-noble metal particle layer with the noble metal particles and the reducing natural polymer;

[0015] And optionally, a polyelectrolyte layer, wherein the polyelectrolyte layer is disposed on the multigel network-noble metal particle layer.

[0016] In the present invention, an adhesion layer is provided on the substrate to improve the bonding strength between the noble metal particles and the substrate, and a polyelectrolyte layer is provided on the noble metal particle layer or the noble metal particles are formed into a multi-gel network-noble metal particle layer. When the fluorescence enhancement substrate is used for detection, the distance between the fluorescently labeled biological molecules to be detected and the noble metal particles can be effectively regulated to avoid fluorescence quenching caused by direct contact between the two, thereby effectively regulating the local surface plasma resonance effect of the noble metal particles to enhance the fluorescence signal of the molecules to be detected, thereby improving the detection sensitivity and accuracy.

[0017] In some embodiments of the present invention, the material of the substrate includes at least one of glass, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polystyrene (PS), and medical tape; and the thickness of the substrate is 1.5 to 2.5 mm.

[0018] In some embodiments of the present invention, the material of the adhesive layer includes an adhesive polymer, and the adhesive polymer includes at least one of a binding silane (mainly composed of 3-(isobutyleneoxy)propyltrimethoxysilane), dopamine, lysine, polyethyleneimine (PEI), polyacrylamide, fibrin, cyanoacrylate, chitosan, natural polysaccharide, and polyurethane. Such polymers can provide good adhesion and have strong adsorption properties for substrates, noble metal particles, and biomolecules. The thickness of the adhesive layer is 5 to 50 nm.

[0019] In some embodiments of the present invention, the noble metal particles in the noble metal particle layer include at least one of nano-gold, nano-silver, nano-copper, and nano-platinum; the average particle size of the noble metal particles is 20 to 200 nm; the ultraviolet absorption range of the noble metal particles is 300 to 600 nm. The thickness of the noble metal layer is 30 nm to 200 μm, such as 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 μm, 100 μm, etc.

[0020] In some embodiments of the present invention, the material of the polyelectrolyte layer includes at least one of a cationic polyelectrolyte, an anionic polyelectrolyte, and a semiconductor medium; the cationic polyelectrolyte includes at least one of polyethyleneimine (PEI), polydimethyl ammonium chloride (PDADMAC), and polypropylene; the anionic polyelectrolyte includes at least one of polystyrene sulfonate (PSS), polyacrylic acid, and polymethacrylic acid; the semiconductor medium includes at least one of silicon dioxide and aluminum oxide; the thickness of the polyelectrolyte layer is 5 to 25 nm, which can be regulated by the number and thickness of layers formed by cationic polyelectrolytes and / or anionic polyelectrolytes and / or semiconductor mediums; the thickness of a single layer is 1 to 3 nm, such as 1.2 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2.0 nm, 2.5 nm, etc. The polyelectrolyte layer includes cationic polyelectrolyte layers and anionic polyelectrolyte layers alternately stacked.

[0021] In some embodiments of the present invention, the reducing natural polymer is at least one of hyaluronic acid (HA), methacrylated gelatin (GelMa), sodium alginate (SA), natural polysaccharides, and chitosan; this type of natural polymer has strong reducing properties and can form complexes with precious metal cations to reduce them to precious metal particles.

[0022] In some embodiments of the present invention, in the multi-gel network-precious metal particle layer, the mass concentration ratio of the precious metal particles, the adhesive polymer and the reducing natural polymer is 0.15-7: 10-60: 0.5-4. The thickness of the multi-gel network-precious metal particle layer is 0.04 μm-1 mm, such as 0.1 μm, 0.3 μm, 0.5 μm, 0.8 μm.

[0023] In some embodiments of the present invention, the fluorescence enhancement substrate further comprises a biomolecule modification layer, which is disposed on the polyelectrolyte layer or the multi-gel network-noble metal particle layer. The material of the biomolecule modification layer comprises a biorecognition molecule, which comprises at least one of a specific antigen, a specific antibody or a nucleic acid molecule ligand, such as carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen 125, carbohydrate antigen 15-3, carbohydrate antigen, carbohydrate antigen 72-4, carbohydrate antigen 24-2, carbohydrate antigen 50, prostate specific antigen, squamous cell carcinoma antigen, cytokeratin 19 fragment, gastrin releasing peptide precursor, neuron specific enolase, nuclear matrix protein-22, tartrate-resistant acid phosphatase 5b, tumor M2 type pyruvate kinase, abnormal prothrombin, osteopontin, phosphatidylinositol proteoglycan 3, Golgi protein 73, metalloproteinase 1, human gastric cancer MG7 antigen, pepsinogen, lectin DC-SIGN, lectin DC-SIGNR, and human chromogranin A.

[0024] In some embodiments of the present invention, the fluorescence enhancement substrate further comprises BSA encapsulation to block non-specific adsorption sites.

[0025] The second aspect of the present invention provides a method for preparing the fluorescence enhancement substrate, comprising the following steps:

[0026] S1: placing the substrate in an adhesive polymer solution to form an adhesive layer;

[0027] S2: depositing noble metal particles on the adhesion layer to form a noble metal particle layer;

[0028] S3: placing the product of S2 in a polyelectrolyte solution to form a polyelectrolyte layer; or,

[0029] S1-1: placing a mixed solution of an adhesive polymer and a reducing natural polymer on a substrate, crosslinking and curing, and then placing it in a solution containing precious metal ions for reaction, photochemically reducing, and chemically crosslinking to form a multi-gel network-precious metal particle layer; and optionally,

[0030] S2-1: The product of S1-1 is placed in a polyelectrolyte solution to form a polyelectrolyte layer.

[0031] In some embodiments of the present invention, the mass concentration of the adhesive polymer solution is 0.5-60%, such as 1.0%, 1.5%, 2.0%, 5.0%, 10%, 20%, 30%, 40%, 50%, etc.

[0032] In some embodiments of the present invention, in S2, a colloidal precious metal solution is used to deposit the precious metal particles on the adhesion layer; the mass concentration of the colloidal precious metal solution is 0.1-5 mg / mL.

[0033] In some embodiments of the present invention, the mass concentration of the polyelectrolyte solution is 0.5-5.0%, such as 1.0%, 1.5%, 2.0%, etc.

[0034] In some embodiments of the present invention, the method for preparing the fluorescence enhancement substrate further comprises repeating step S3 or S2-1 multiple times to form polyelectrolyte layers with different thicknesses, such as repeating 1 to 10 times.

[0035] In some embodiments of the present invention, in S1-1, the mixed solution further includes a first crosslinking agent and a photoinitiator, wherein the first crosslinking agent includes at least one of N-hydroxymethyl acrylamide, N-hydroxy acrylamide, N,N-methylenebisacrylamide (Bis), and N,N'-methylenebisacrylamide (MBA) and at least one of photoinitiators phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), 1-hydroxycyclohexyl phenyl ketone (UV-184), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylbenzoyl benzoyl (I2959); the first crosslinking agent and the adhesive polymer undergo a crosslinking reaction to form an adhesive first gel network; the mass concentration ratio of the first crosslinking agent, the photoinitiator, and the adhesive polymer is 0.15-0.75: 0.1-0.5: 10-60. The temperature of the crosslinking reaction is 10°C-60°C, and the time is 10-120s.

[0036] In some embodiments of the present invention, in S1-1, the mixed solution further includes a second crosslinking agent, the second crosslinking agent includes a metal salt, such as at least one of calcium chloride, magnesium chloride, aluminum chloride, ferric chloride, and sodium hydroxide; the second crosslinking agent and the reducing natural polymer undergo a crosslinking reaction to form a reducing second gel network; the mass concentration ratio of the second crosslinking agent to the reducing natural polymer is 0.1 to 10: 0.5 to 4, such as 0.5 to 2: 0.5 to 4. The temperature of the crosslinking reaction is 10°C to 60°C, and the time is 60 to 600s.

[0037] In some embodiments of the present invention, in S1-1, the light curing includes ultraviolet light curing, and the ultraviolet light intensity is 70 μW / cm 2 ~1200mW / cm2 The irradiation time is 10s~1000s, such as 20s~800s, 30s~600s, 50s, 80s, 100s, 120s, 200s, 300s, 400s, 500s, etc.

[0038] In some embodiments of the present invention, the method for preparing the fluorescence enhancement substrate further includes placing the S3 product or the S2-1 product in a biorecognition molecule solution to form a biorecognition molecule modification layer; the mass concentration of the biorecognition molecule solution is 100 ng / mL to 500 μg / mL.

[0039] The third aspect of the present invention provides a fluorescence-enhanced microfluidic chip, comprising the fluorescence-enhanced substrate and a cover sheet sealed on the upper surface of the fluorescence-enhanced substrate; the cover sheet has a flow channel; a sample addition area is provided at one end of the flow channel, and is connected to the detection area via the flow channel.

[0040] In some embodiments of the present invention, the cover sheet is provided with a sample addition area, a detection area, and a waste liquid area which are sequentially connected by a flow pipe.

[0041] In some embodiments of the present invention, the sample adding area includes independently provided sample adding holes for the sample to be tested, washing solution adding holes, and reagent adding holes.

[0042] In some embodiments of the present invention, the detection area includes a plurality of detection holes arranged in parallel, such as 1 to 10 detection holes.

[0043] In some embodiments of the present invention, the detection area corresponds to the detection reaction layer of the fluorescence enhancement substrate.

[0044] In some embodiments of the present invention, the waste liquid area includes a liquid storage area and an outflow pipe connected thereto.

[0045] The fourth aspect of the present invention provides an application of the fluorescence enhancement substrate or the fluorescence enhancement microfluidic chip in detecting target concentration.

[0046] In some embodiments of the present invention, the detection method includes contacting the target to be detected with the fluorescence enhancement substrate for detection.

[0047] In some embodiments of the present invention, the detection method includes performing fluorescence intensity detection, obtaining fluorescence intensity, and determining target concentration.

[0048] In some embodiments of the present invention, the target includes at least one of a small molecule, a nucleic acid, a protein, and a cell.

[0049] In some embodiments of the present invention, the detection method comprises at least one of an immunosandwich method, an immunofluorescence decay method, and an adsorption method.

[0050] The beneficial effects of the present invention are:

[0051] The present invention provides a fluorescence-enhanced microfluidic chip for joint detection of multiple tumor markers, a preparation method and an application thereof, which integrates sampling, mixing, reaction, enrichment, biomolecule fluorescence enhancement, parallel analysis of multiple markers and the like in the detection of markers in blood samples of tumor patients on a microfluidic chip, and simultaneously utilizes the local surface plasmon resonance effect of precious metal-based materials to enhance the fluorescence signals of fluorescent molecule-labeled targets in complex blood samples, thereby providing a new solution to solve the problems of extremely low tumor marker concentrations in blood samples, interference from blood components, uneven mixing of trace sample solutions and reagent solutions, and complex detection procedures, which make it difficult to perform high-sensitivity, high-accuracy, and real-time joint detection of multiple targets for tumor markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the structure of the fluorescence enhancement substrate according to an embodiment of the present invention.

[0053] Figure 2 This is a schematic diagram of the structure of the fluorescence enhanced microfluidic chip according to Example 4 of the present invention.

[0054] Figure 3 This is a schematic diagram of the three-dimensional structure of the fluorescence enhanced microfluidic chip according to Example 4 of the present invention.

[0055] Figure 4 This is a physical picture of the fluorescence enhanced microfluidic chip according to Example 4 of the present invention.

[0056] Figure 5 This is a graph showing the test results of the fluorescence enhanced microfluidic chip used for the tumor marker CEA-FITC in Example 4 of the present invention.

[0057] Figure 6 This is the ultraviolet absorption spectrum of the fluorescence enhancement substrate in Example 1 of the present invention.

[0058] Figure 7 This is the ultraviolet absorption spectrum of the fluorescence enhancement substrate in Example 2 of the present invention.

[0059] Figure numerals: 001, fluorescence enhanced substrate; 002, multi-channel structure cover; 003, inflow / outflow / connecting pipeline; 101, adhesion layer-precious metal base film layer-isolation layer-biomolecule composite membrane; 201, sample loading area; 202, sample / reagent / waste liquid inflow pipeline; 203, detection area; 204, liquid storage area; 205, waste liquid outflow pipeline; 2011, sample liquid loading hole; 2012, washing liquid loading hole; 2013, reagent loading hole; 2021, sample liquid inflow pipeline; 2022, washing liquid inflow pipeline; 2023, reagent inflow pipeline; 2031, detection hole; 2041, liquid storage tank. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0061] Figure 1 It is a schematic structural diagram of a fluorescence enhancement substrate according to an embodiment of the present invention, which includes, from bottom to top, a substrate, an adhesion layer, a noble metal layer, alternatingly stacked cationic polyelectrolyte layers and anionic polyelectrolyte layers, and a biomolecule modification layer.

[0062] Example 1

[0063] This embodiment prepares a fluorescence enhancement substrate, and the specific process is as follows:

[0064] S1: 90 mg of silver nitrate was dissolved in 500 mL of deionized water and then boiled, followed by adding 10 mL of 1% sodium citrate to react for 1 hour to obtain a colloidal silver solution, the precipitate was centrifuged, washed with deionized water, and resuspended in deionized water to obtain a 1 mg / mL nano-colloidal silver solution;

[0065] S2: The pre-cleaned glass slide was plasma treated for 20 seconds and then immersed in 50mL 1wt% PEI solution for 20 minutes, then washed with deionized water for 3 times, 1mL of colloidal silver solution (1mg / mL) with an average diameter of about 40nm and an ultraviolet absorption peak of about 420nm was deposited on the glass slide and the unbound nanosilver was thoroughly washed away to obtain a silver film layer with a thickness of about 40nm; the glass slide with the nanosilver film deposited was then immersed in 50mL 1wt% PEI cationic polyelectrolyte solution, deionized water washing and 50mL 1wt% PSS anionic polyelectrolyte solution for 0 to 7 times, wherein each PEI-PSS layer was about 1.4nm, and the 7 layers were about 13nm in total; and the nanosilver film layer was retained only in the required area by template chemical etching to obtain a noble metal-multilayer polyelectrolyte composite membrane to obtain a fluorescence enhancement substrate.

[0066] Comparative Example 1

[0067] In this comparative example, a substrate is prepared. The preparation method is referred to Example 1, but the difference from Example 1 is that no polyelectrolyte layer is provided.

[0068] Example 2

[0069] This embodiment prepares a fluorescence enhancement substrate, and the specific process is as follows:

[0070] 0.5 mL of a mixed solution of sodium alginate (1 wt%), acrylamide (30 wt%), BIS (0.15 wt%), and LAP (1 wt%) was evenly coated on a round glass slide (55 mm diameter; 1.1 mm thickness) and exposed to ultraviolet light (405 nm, 1200 mW / cm 2 ) for 10 s, and then immersed in 10 mL of silver nitrate solution (0.5 wt%) for 5 min to form a multi-network gel containing noble metal ions. 2 ) for 300 s, washed with deionized water, and then cross-linked with 10 mL of 1 wt % calcium chloride for 5 min, then washed with deionized water, and dried in an oven at 55°C to obtain a fluorescence enhancement substrate.

[0071] Example 3

[0072] This embodiment prepares a fluorescence enhancement substrate, and the specific process is as follows:

[0073] The substrate prepared in Example 1 was immersed in 1 mL of 1000 ng / mL CEA antigen solution in PBS (pH=7.4) for 1 hour, then washed with deionized water to modify the biomolecule recognition layer to obtain a fluorescence enhancement substrate with a noble metal-multilayer polyelectrolyte-biomolecule composite membrane.

[0074] Example 4

[0075] This embodiment prepares a fluorescence enhanced microfluidic chip, and the specific process is as follows:

[0076] like Figure 2As shown, the fluorescence enhancement microfluidic chip of this embodiment includes a substrate (001) for fluorescence enhancement of target molecules in a sample, a multi-channel structure cover (002) for multi-process detection of samples, and a sample / reagent / waste liquid inlet / outlet / connecting pipeline (003). Its structure sequentially includes a sample addition area (201), a sample / reagent / waste liquid inlet pipeline (202), a detection area (203), a liquid storage area (204), and a waste liquid outflow pipeline (205) which are sequentially connected. The sample adding area is provided with a sample liquid adding hole (2011), a washing liquid adding hole (2012) and a reagent adding hole (2013); the sample or reagent inflow pipeline is provided with a sample liquid inflow pipeline (2021), a washing liquid inflow pipeline (2022) and a reagent inflow pipeline (2023); the detection area (204) is provided with a plurality of parallel detection holes (2031), and the sample liquid adding hole (2011) is connected to the detection hole (2031) through the sample liquid inflow pipeline (2021). The reagent loading hole (2013) is connected to the detection hole (2031) through a reagent inflow pipe (2023); the detection area (203) on the fluorescence enhancement substrate (001) is provided with an adhesion layer-precious metal-multilayer polyelectrolyte-biomolecule composite membrane (101) for fluorescence enhancement of target molecules in a sample, and the detection hole (2031) is placed on the adhesion layer-precious metal-polyelectrolyte-biomolecule composite membrane (101); the liquid storage area (204) is provided with a plurality of liquid storage tanks (2041).

[0077] A three-dimensional image of a fluorescence-enhanced microfluidic chip Figure 3 shown.

[0078] CoreIDRA software was used to design the photolithography graphics. After the graphics were exported, they were cut into the file format required by the maskless lithography machine through MATLAB software. 500 μL of SU-8-2050 photolithography negative resist was spread on the surface of a 2-inch diameter silicon wafer and placed in a spin coater for multi-step coating. It was pre-baked at 65°C for 3 minutes and post-baked at 95°C for 6 minutes. After exposure to light with a maskless lithography machine, it was baked at 95°C for 6 minutes. Subsequently, it was developed with a developer (model: AZAOOK) and fixed with isopropanol, and then placed on a 150°C baking table for hard baking for 3 minutes. A quantitative 1:100 ratio of PDMS solution was prepared, and the solution was spread and stirred and poured on a patterned silicon wafer. After vacuum degassing, it was placed in a 65°C oven for baking. After molding, it was cut to obtain a multi-channel structure cover sheet for multi-process detection of samples.

[0079] Subsequently, the substrate for fluorescence enhancement of target molecules in the sample and the multi-channel structure cover for multi-process detection of the sample were bonded together after plasma treatment for 20 seconds, and then placed in an oven at 75 degrees for 2 hours. After being connected with the sample inlet / outlet / connecting pipes, a fluorescence enhanced microfluidic chip for multi-tumor marker joint detection was prepared.

[0080] The actual picture of the fluorescence enhanced microfluidic chip for multi-tumor marker joint detection is as follows Figure 4 shown.

[0081] The workflow of the fluorescence enhanced microfluidic chip for joint detection of multiple tumor markers is as follows: when testing multiple tumor markers based on the double antibody sandwich method, the sample liquid to be tested enters the sample liquid inflow pipe (2021) through the sample addition hole (2011), and then flows to each detection hole (2031) in the detection area, and the target in the sample binds to the multiple specific recognition molecules in the adhesion layer-precious metal-polyelectrolyte-biomolecule composite membrane (101) on the substrate (001) in the detection hole, and the waste liquid flows out of the chip through the liquid storage area (204) and the waste liquid outflow channel (205); then the washing liquid enters the washing liquid inflow pipe (2022) through the washing liquid sample addition hole (2012), and then flows to each detection hole (2031) in the detection area. The detection hole (2031) is washed to remove the unbound components in the detection hole (2031), and the waste liquid flows out of the chip through the liquid storage area (204) and the waste liquid outflow channel (205); finally, the detection fluorescent reagent enters the reagent inflow pipe (2023) through the reagent sample addition hole (2023), and then flows to each detection hole (2031) in the detection area, specifically binds to the adsorbed sample target to be detected in the detection hole, and the waste liquid flows out of the chip through the liquid storage area (204) and the waste liquid outflow channel (205); the biological molecule-target to be detected-biofluorescent molecule in the detection hole (2031) is tested for fluorescence intensity under excitation light, and the concentration of the target to be detected is obtained through the relationship between the fluorescence intensity and the concentration of the target to be detected. When testing multiple tumor markers based on the direct method, the surface of the isolation layer of the substrate (001) used for fluorescence enhancement of the target molecule in the sample is not modified with specific antigen antibodies or nucleic acid molecule ligands, and other conditions remain unchanged.

[0082] Example 5

[0083] This example uses the fluorescence-enhanced microfluidic chip for joint detection of multiple tumor markers prepared in Example 4 to detect cancer markers. The specific process is as follows:

[0084] 50 μL of PBS (pH=7.4) test solutions of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL CEA-fitc antibody were respectively passed through the sample solution injection hole of the fluorescence enhanced microfluidic chip for testing. The results are shown in FIG. Figure 5 shown.

[0085] from Figure 5 It can be seen that the CEA-fitc antibody exhibits good linearity within the range of 1 to 500 ng / mL.

[0086] Test Example 1

[0087] This test example uses an ELISA instrument to test the ultraviolet absorption spectra of the substrates in Example 1 and Example 2 in the range of 350 to 800 nm. The results are as follows: Figure 6 , Figure 7 shown.

[0088] from Figure 6 It can be seen that the ultraviolet absorption peak of the fluorescence enhancement substrate in Example 1 is around 420 nm, and the peak absorbance is around 0.55.

[0089] from Figure 7 It can be seen that the ultraviolet absorption of the fluorescence enhanced substrate in Example 2 is at 480 nm, the absorbance peak is around 1.75, the in-situ grown silver nanoparticles are larger in size, higher in density and more stable, and the construction of the noble metal-polyelectrolyte-biomolecule composite membrane is simpler.

[0090] Test Example 2

[0091] In this test example, the fluorescence intensity of 1 μg / mL lung cancer marker FITC-labeled neuron-specific enolase (NES-FITC) in the substrates of Example 1 and Comparative Example 1 was tested using a microplate reader under 465 nm excitation light and 535 nm emission light. The results showed that the substrate of Comparative Example 1 had no isolation layer and the fluorescence intensity of NES-FITC was attenuated by 25%.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A fluorescence enhancement substrate, characterized in that: include: substrate; an adhesive layer disposed on the substrate; A noble metal particle layer, wherein the noble metal particle layer is disposed on the adhesion layer; a polyelectrolyte layer, wherein the polyelectrolyte layer is disposed on the noble metal layer; or substrate; An adhesive layer, the adhesive layer is disposed on the substrate, and the adhesive layer also forms a multi-gel network-noble metal particle layer with the noble metal particles and the reducing natural polymer; And optionally, a polyelectrolyte layer, wherein the polyelectrolyte layer is disposed on the multigel network-noble metal particle layer.

2. The fluorescence enhancement substrate according to claim 1, characterized in that: The material of the adhesive layer includes an adhesive polymer, and the adhesive polymer includes at least one of affinity silane, dopamine, lysine, polyacrylamide, fibrin, cyanoacrylate, chitosan, natural polysaccharide, and polyurethane.

3. The fluorescence enhancement substrate according to claim 1, characterized in that: The material of the polyelectrolyte layer includes at least one of a cationic polyelectrolyte, an anionic polyelectrolyte, and a semiconductor medium; the cationic polyelectrolyte includes at least one of polyethyleneimine, polydimethyl ammonium chloride, and polypropylene; the anionic polyelectrolyte includes at least one of polystyrene sulfonate, polyacrylic acid, and polymethacrylic acid; and the semiconductor medium includes at least one of silicon dioxide and aluminum oxide.

4. The fluorescence enhancement substrate according to claim 3, characterized in that: The thickness of the polyelectrolyte layer is 5 to 25 nm; and / or the polyelectrolyte layer comprises cationic polyelectrolyte layers and anionic polyelectrolyte layers alternately stacked.

5. The fluorescence enhancement substrate according to claim 1, characterized in that: The reducing natural high molecular weight hyaluronic acid, methacrylic anhydride gelatin, sodium alginate, natural polysaccharide, chitosan is at least one kind.

6. The fluorescence enhancement substrate according to claim 1, characterized in that: In the multi-gel network-precious metal particle layer, the mass concentration ratio of the precious metal particles, the adhesive polymer and the reducing natural polymer is 0.15-7:10-60:0.5-4.

7. The fluorescence enhancement substrate according to claim 1, characterized in that: The fluorescence enhancement substrate further comprises a biomolecule modification layer, and the biomolecule modification layer is arranged on the polyelectrolyte layer or the multi-gel network-noble metal particle layer.

8. A method for preparing a fluorescence enhancement substrate according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1: placing the substrate in an adhesive polymer solution to form an adhesive layer; S2: depositing noble metal particles on the adhesion layer to form a noble metal particle layer; S3: placing the product of S2 in a polyelectrolyte solution to form a polyelectrolyte layer; or, S1-1: placing a mixed solution of an adhesive polymer and a reducing natural polymer on a substrate, crosslinking and curing, and then placing it in a solution containing precious metal ions for reaction, photochemically reducing, and chemically crosslinking to form a multi-gel network-precious metal particle layer; and optionally, S2-1: The product of S1-1 is placed in a polyelectrolyte solution to form a polyelectrolyte layer.

9. A fluorescence-enhanced microfluidic chip, comprising the fluorescence-enhanced substrate according to any one of claims 1 to 7, and a cover sheet sealed and fitted on the upper surface of the fluorescence-enhanced substrate; the cover sheet has a flow channel; a sample addition area is provided at one end of the flow channel, and is connected to a detection area via the flow channel.

10. Use of the fluorescence enhancement substrate according to any one of claims 1 to 7 or the fluorescence enhancement microfluidic chip according to claim 9 in detecting target concentration.

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