A fluorescence-enhanced microfluidic chip and a preparation method and application thereof

Abstract

The application discloses a fluorescence-enhanced microfluidic chip, a preparation method and application thereof. The chip comprises a fluorescence-enhanced substrate and a cover plate with a flow channel. The fluorescence-enhanced substrate comprises: a substrate; an adhesion layer arranged on the substrate; a noble metal particle layer arranged on the adhesion layer; or a polyelectrolyte layer arranged on the noble metal layer; or a substrate; an adhesion layer; a multi-gel network-noble metal particle layer formed by the adhesion layer, noble metal particles and a reducing natural polymer; and optionally, a polyelectrolyte layer arranged on the multi-gel network-noble metal particle layer. When the application is used for detection, the detection process of sample injection, reaction, enrichment, fluorescence enhancement of biomolecules, parallel analysis of multiple markers and the like can be integrated, the detection sensitivity and accuracy can be improved, and multi-target joint detection can be realized.

Description

TECHNICAL FIELD

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

[0002] Cancer is one of the main causes of global deaths at present, and early diagnosis is particularly important for patients to develop treatment methods and recover prognosis. For breast cancer, for example, the current main methods for diagnosis and evaluation of efficacy are imaging and histopathology. The former is easily influenced by subjective factors such as the experience of clinicians and cannot accurately evaluate the efficacy; the latter is accurate and is the gold standard, but cannot timely understand the sensitivity of chemotherapeutic drugs and is difficult to adjust the chemotherapy regimen in time, thus missing the best treatment opportunity. More importantly, because of the need for special equipment and personnel or sampling difficulties, these methods cannot achieve repeated detection. Therefore, it is particularly important to seek a new index that can timely reflect the efficacy and can be repeatedly detected to achieve the purpose of real-time efficacy monitoring of cancer.

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

[0004] Currently, the methods for detecting tumor markers in serum mainly include immunocolloidal gold test paper detection, colorimetric detection method, electrochemiluminescence detection, etc. The detection methods based on colloidal gold and colorimetric method have low sensitivity and cannot achieve simultaneous quantitative detection of multiple low-concentration lung cancer markers. The electrochemiluminescence method requires large instruments and professional personnel to manage detection, patients need to go to large hospitals and the detection price is expensive, which cannot achieve comprehensive screening, real-time monitoring of efficacy and recurrence of lung cancer. Microfluidic chips can precisely manipulate microscale sample solutions, integrate sample detection processes such as sample addition, mixing, enrichment, reaction, detection on the chip, and achieve sample-in and result-out without the need for professional personnel to operate, with the advantages of less sample and reagent consumption, fast detection speed, simple operation, multiple parallelism, etc.

[0005] Currently, microfluidics has been applied to many scenarios including allergen detection, myocardial marker detection, infectious pathogen detection, etc. However, the concentration of tumor markers is extremely low, the fluorescence background interference of biomolecules in blood, the fluorescence background interference of chip materials, the parallel detection of multiple markers, and the like, how to design and prepare a microfluidic chip to enhance the fluorescence signal of tumor markers in the blood of cancer patients while realizing real-time joint detection has important value. SUMMARY

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

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0008] In a first aspect of the present application, a fluorescence-enhanced substrate is provided, comprising:

[0009] a substrate;

[0010] an adhesion layer disposed on the substrate;

[0011] a noble metal particle layer disposed on the adhesion layer;

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

[0013] a substrate;

[0014] an adhesion layer disposed on the substrate, the adhesion layer further forming a multiple gel network-noble metal particle layer with noble metal particles and a reducing natural polymer;

[0015] and optionally, a polyelectrolyte layer disposed on the multiple gel network-noble metal particle layer.

[0016] In the present application, the adhesion layer is disposed on the substrate, which can improve the bonding strength of the noble metal particles and the substrate, the polyelectrolyte layer is disposed on the noble metal particle layer or the noble metal particles form a multiple gel network-noble metal particle layer, which can effectively regulate the distance between the fluorescently labeled biomolecules to be detected and the noble metal particles, avoid direct contact between the two resulting in fluorescence quenching, thereby effectively regulating the localized surface plasmon resonance effect of the noble metal particles to enhance the fluorescence signal of the molecules to be detected, and improving the detection sensitivity and accuracy.

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

[0018] In some embodiments of the present application, the material of the adhesion layer comprises an adhesive polymer, which comprises at least one of binding silane (main component: 3-(isobutyryloxy)propyl trimethoxysilane), dopamine, lysine, polyethyleneimine (PEI), polyacrylamide, fibrin, cyanoacrylate, chitosan, natural polysaccharide, and polyurethane. Such polymer can provide better adhesion, and has strong adsorption characteristics for substrates, noble metal particles, and biomolecules. The thickness of the adhesion layer is 5-50 nm.

[0019] In some embodiments of the present application, the noble metal particles in the noble metal particle layer comprise at least one of nano-gold, nano-silver, nano-copper, and nano-platinum; the average particle size of the noble metal particles is 20-200 nm; and the ultraviolet absorption range of the noble metal particles is 300-600 nm. The thickness of the noble metal layer is 30 nm-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 application, the material of the polyelectrolyte layer comprises at least one of a cationic polyelectrolyte, an anionic polyelectrolyte, and a semiconductor medium; the cationic polyelectrolyte comprises at least one of polyethyleneimine (PEI), polydiallyldimethylammonium chloride (PDADMAC), and polypropylene; the anionic polyelectrolyte comprises at least one of polystyrene sulfonate (PSS), polyacrylic acid, and polymethacrylic acid; the semiconductor medium comprises at least one of silicon dioxide and aluminum oxide; the thickness of the polyelectrolyte layer is 5-25 nm, which can be regulated by the number of layers and thickness of the cationic polyelectrolyte and / or the anionic polyelectrolyte and / or the semiconductor medium; the thickness of a single layer is 1-3 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 comprises cationic polyelectrolyte layers and anionic polyelectrolyte layers alternately stacked.

[0021] In some embodiments of the present application, the reducing natural polymer comprises at least one of hyaluronic acid (HA), methacrylated gelatin (GelMa), sodium alginate (SA), natural polysaccharide, and chitosan. Such natural polymer has strong reducing property, and can form a complex with noble metal cations, which is reduced to noble metal particles.

[0022] In some embodiments of the present application, the mass concentration ratio of the noble metal particles, the adhesive polymer and the reducing natural polymer in the multi-gel network-noble metal particle layer is 0.15-7: 10-60: 0.5-4. The thickness of the multi-gel network-noble 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 application, the fluorescence-enhanced substrate further comprises a biomolecule modification layer disposed on the polyelectrolyte layer or the multi-gel network-noble metal particle layer. The material of the biomolecule modification layer comprises a biological recognition molecule, which comprises at least one of a specific antigen, a specific antibody or a nucleic acid molecule ligand, such as at least one of 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, progastrin-releasing peptide, neuron-specific enolase, nuclear matrix protein-22, tartrate-resistant acid phosphatase 5b, tumor M2 pyruvate kinase, abnormal prothrombin, bone bridge protein, glycosaminoglycan 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 application, the fluorescence-enhanced substrate further comprises a BSA encapsulation to seal the non-specific adsorption sites.

[0025] In a second aspect of the present application, a preparation method of the fluorescence-enhanced substrate is provided, 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 adhesive 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 the adhesive polymer and the reducing natural polymer on the substrate, cross-linking and solidifying, and then reacting in a solution containing noble metal ions, and then photochemically reducing and chemically cross-linking to form a multi-gel network-noble metal particle layer; and optionally,

[0030] S2-1: placing the product of S1-1 in a polyelectrolyte solution to form a polyelectrolyte layer.

[0031] In some embodiments of the present application, 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 application, in S2, the colloidal noble metal solution is used to deposit noble metal particles on the adhesive layer; the mass concentration of the colloidal noble metal solution is 0.1-5 mg / mL.

[0033] In some embodiments of the present application, 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 application, the preparation method of the fluorescence-enhanced substrate further comprises repeating steps S3 or S2-1 multiple times, so as to form polyelectrolyte layers with different thicknesses, such as 1-10 times.

[0035] In some embodiments of the present application, in S1-1, the mixed solution further comprises a first crosslinking agent and a photoinitiator, the first crosslinking agent comprises at least one of N-hydroxymethyl acrylamide, N-hydroxy acrylamide, N,N-methylene bisacrylamide (Bis), N,N'-methylene bisacrylamide (MBA), and at least one of photoinitiators lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 1-hydroxycyclohexyl phenyl ketone (UV-184), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl benzophenone (I2959); the first crosslinking agent and the adhesive polymer perform 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 crosslinking reaction is performed at a temperature of 10°C-60°C for 10-120 s.

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

[0037] In some embodiments of the present application, in S1-1, the photocuring comprises ultraviolet light irradiation curing, and the ultraviolet light irradiation intensity is 70 μW / cm 2 ~ 1200 mW / 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 application, the preparation method of the fluorescence-enhanced substrate further comprises placing the S3 product or the S2-1 product in a biological recognition molecule solution to form a biological molecule modification layer; the mass concentration of the biological recognition molecule solution is 100ng / mL-500μg / mL.

[0039] In a third aspect of the present application, a fluorescence-enhanced microfluidic chip is provided, comprising the fluorescence-enhanced substrate and a cover plate sealingly fitted to the upper surface of the fluorescence-enhanced substrate; the cover plate has a flow channel; one end of the flow channel is provided with a sample adding area, and the sample adding area is connected to a detection area through the flow channel.

[0040] In some embodiments of the present application, the cover plate is provided with a sample adding area, a detection area and a waste liquid area connected in sequence through the flow channel.

[0041] In some embodiments of the present application, the sample adding area comprises a sample adding hole, a washing liquid adding hole and a reagent adding hole.

[0042] In some embodiments of the present application, the detection area comprises a plurality of detection holes arranged in parallel, such as 1-10 detection holes.

[0043] In some embodiments of the present application, the detection area corresponds to the detection reaction layer of the fluorescence-enhanced substrate.

[0044] In some embodiments of the present application, the waste liquid area comprises a liquid storage area and an outflow channel connected thereto.

[0045] In a fourth aspect of the present application, the fluorescence-enhanced substrate or the fluorescence-enhanced microfluidic chip is used for detecting the concentration of a target.

[0046] In some embodiments of the present application, the detection method comprises contacting the target to be detected with the fluorescence-enhanced substrate for detection.

[0047] In some embodiments of the present application, the detection method comprises detecting the fluorescence intensity, obtaining the fluorescence intensity and determining the concentration of the target.

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

[0049] In some embodiments of the present application, the detection method comprises at least one of an immunological sandwich method, an immunofluorescence quenching method, and an adsorption method.

[0050] The present application has the following advantages:

[0051] The present application provides a fluorescence-enhanced microfluidic chip for multi-tumor marker joint detection, a preparation method and an application. When detecting markers in blood samples of tumor patients, the microfluidic chip is integrated for sample injection, mixing, reaction, enrichment, fluorescence enhancement of biomolecules, and parallel analysis of multiple markers. The local surface plasmon resonance effect of noble metal-based materials is used to enhance the fluorescence signal of fluorescent molecule markers in complex blood samples, thereby providing a new solution for solving the problems of low concentration of tumor markers in blood samples, blood component interference, uneven mixing of trace sample solution and reagent solution, and complex detection process, which makes it difficult to realize high sensitivity, high accuracy, and multi-target instant joint detection of tumor markers. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The structure of the fluorescence-enhanced substrate of the present application is shown in the schematic diagram.

[0053] Figure 2 The structure of the fluorescence-enhanced microfluidic chip of the present application is shown in the schematic diagram.

[0054] Figure 3 The structure of the fluorescence-enhanced microfluidic chip of the present application is shown in the schematic diagram.

[0055] Figure 4 The structure of the fluorescence-enhanced microfluidic chip of the present application is shown in the schematic diagram.

[0056] Figure 5 The structure of the fluorescence-enhanced microfluidic chip of the present application is shown in the schematic diagram.

[0057] Figure 6 The structure of the fluorescence-enhanced substrate of the present application is shown in the schematic diagram.

[0058] Figure 7 The structure of the fluorescence-enhanced substrate of the present application is shown in the schematic diagram.

[0059] 001, fluorescence enhanced substrate; 002, multi-channel structure cover sheet; 003, inflow / outflow / communication pipeline; 101, adhesion layer-noble metal film layer-isolation layer-biomolecule composite film; 201, sample loading area; 202, sample / reagent / waste liquid inflow pipeline; 203, detection area; 204, reagent 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, reagent storage tank. DETAILED DESCRIPTION

[0060] The content of the present application is further described in detail below through specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0061] Figure 1 is a structural schematic diagram of the fluorescence enhanced substrate of the embodiment of the present application, which comprises, from bottom to top, a substrate, an adhesion layer, a noble metal layer, alternating layers of cationic polyelectrolyte and anionic polyelectrolyte, and a biomolecule modification layer.

[0062] Example 1

[0063] In this embodiment, a fluorescence enhanced substrate is prepared, and the specific process is as follows:

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

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

[0066] Comparative Example 1

[0067] A substrate was prepared in this comparative example, and the preparation method was the same as in Example 1. The difference from Example 1 was that no polyelectrolyte layer was provided.

[0068] Example 2

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

[0070] A 0.5 mL mixture of sodium alginate (1 wt%), acrylamide (30 wt%), BIS (0.15 wt%), and LAP (1 wt%) was uniformly coated onto a circular glass slide (55 mm diameter; 1.1 mm thickness) and exposed to ultraviolet light (405 nm, 1200 mW / cm²). 2 After a 10-second fixation reaction under certain conditions, the mixture was immersed in 10 mL of 0.5 wt% silver nitrate solution for 5 minutes to form a multi-network gel containing noble metal ions. Subsequently, it was subjected to ultraviolet light (405 nm, 1200 mW / cm²). 2 Irradiated for 300s under deionized water, washed with 10mL of 1wt% calcium chloride for 5min crosslinking reaction, washed again with deionized water, and then dried in an oven at 55℃ to obtain the fluorescence-enhanced substrate.

[0071] Example 3

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

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

[0074] Example 4

[0075] This embodiment describes the fabrication of a fluorescence-enhanced microfluidic chip, the specific process of which is as follows:

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

[0077] The fluorescence-enhanced microfluidic chip is shown in the perspective view as Figure 3 .

[0078] The photolithography pattern is designed using CoreIDRA software, and after the pattern is exported, it is cut into a file format required by a maskless lithography machine through MATLAB software; 500 μL of SU-8-2050 negative photoresist is uniformly coated on the surface of a 2-inch diameter silicon wafer and placed in a spin coater for multi-step coating, pre-baked at 65°C for 3 minutes, post-baked at 95°C for 6 minutes, exposed to light using a maskless lithography machine, post-baked at 95°C for 6 minutes, developed using a developer (model: AZAOOK), fixed using isopropyl alcohol, and post-baked at 150°C for 3 minutes; a PDMS solution is prepared by taking a quantitative ratio of 1:100, uniformly coated on the patterned silicon wafer, vacuum degassed, and post-baked in a 65°C oven, and after molding, the cover sheet with a multi-channel structure for multi-process detection of the sample is cut.

[0079] After the substrate for fluorescence enhancement of target molecules in a sample and the cover sheet with a multi-channel structure for multi-process detection of the sample are bonded together after being subjected to plasma treatment for 20 seconds, they are placed in a 75-degree oven for 2 hours, connected through the channel for sample inflow / outflow / communication, and a fluorescence-enhanced microfluidic chip for simultaneous detection of multiple tumor markers is prepared.

[0080] The physical diagram of the fluorescence-enhanced microfluidic chip for multi-tumor marker joint detection is shown in Figure 4 .

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

[0082] Example 5

[0083] In this embodiment, the fluorescence-enhanced microfluidic chip for multi-tumor marker joint detection prepared in Example 4 is used for cancer marker detection. The specific process is as follows:

[0084] 50 μL 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 of CEA-fitc antibody PBS (pH = 7.4) solution to be tested are respectively added into the fluorescence-enhanced microfluidic chip through the sample liquid addition hole, and the results are shown in Figure 5 .

[0085] As can be seen from Figure 5 , a good linearity is presented in the range of 1-500 ng / mL of CEA-fitc antibody.

[0086] Test Example 1

[0087] The test example uses an enzyme marker to test the ultraviolet absorption spectrum of the base in Example 1 and Example 2 in the range of 350-800 nm, and the results are shown in Figure 6 , Figure 7 .

[0088] As can be seen from Figure 6 , the ultraviolet absorption peak of the fluorescence-enhanced base in Example 1 is about 420 nm, and the absorbance peak is about 0.55.

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

[0090] Test Example 2

[0091] The test example uses an enzyme marker to test the fluorescence intensity of 1 μg / mL lung cancer marker FITC-labeled neuron-specific enolase (NES-FITC) in the base of Example 1 and Comparative Example 1 under 465 nm excitation light and 535 nm emission light, and the results show that the base of Comparative Example 1 has no isolation layer, and the fluorescence intensity of NES-FITC attenuates by 25%.

[0092] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A fluorescence enhancing substrate, characterized by: The fluorescence enhancement substrate comprises: a substrate; an adhesion layer disposed on the substrate, the adhesion layer further forming a multi-gel network-gold particle layer with gold particles and a reducing natural polymer; a biomolecule modification layer disposed on the multi-gel network-gold particle layer; wherein the material of the adhesion layer comprises an adhesive polymer; the preparation method of the multi-gel network-gold particle layer comprises the following steps: placing a mixed solution of the adhesive polymer, the reducing natural polymer, the first crosslinking agent, and the photoinitiator on the substrate, crosslinking and solidifying, then reacting in a solution containing gold ions, photochemically reducing, and then chemically crosslinking with the second crosslinking agent to form the multi-gel network-gold particle layer; the first crosslinking agent forms an adhesive first gel network with the adhesive polymer; the second crosslinking agent forms a reducing second gel network with the reducing natural polymer; the reducing natural polymer forms a complex with the gold ions to reduce them into gold particles; The adhesive polymer includes at least one of an affinity silane of (meth)acryloyloxy propyl trimethoxysilane, dopamine, lysine, polyethyleneimine, polyacrylamide, fibrin, cyanoacrylate, chitosan, a natural polysaccharide, and polyurethane as a main ingredient (3-methacryloyloxy)propyl trimethoxysilane, dopamine, lysine, polyethyleneimine, polyacrylamide, fibrin, cyanoacrylate, chitosan, a natural polysaccharide, and polyurethane the first crosslinking agent comprises at least one of N-hydroxymethyl acrylamide, N-hydroxy acrylamide, and N,N'-methylene bisacrylamide; the second crosslinking agent comprises at least one of calcium chloride, magnesium chloride, aluminum chloride, and iron chloride.

2. A fluorescence enhancing substrate, characterized by: The fluorescence enhancement substrate comprises: a substrate; an adhesion layer disposed on the substrate, the adhesion layer further forming a multi-gel network-gold particle layer with gold particles and a reducing natural polymer; a polyelectrolyte layer disposed on the multi-gel network-gold particle layer; a biomolecule modification layer disposed on the polyelectrolyte layer; wherein the material of the adhesion layer comprises an adhesive polymer; the preparation method of the multi-gel network-gold particle layer comprises the following steps: placing a mixed solution of the adhesive polymer, the reducing natural polymer, the first crosslinking agent, and the photoinitiator on the substrate, crosslinking and solidifying, then reacting in a solution containing gold ions, photochemically reducing, and then chemically crosslinking with the second crosslinking agent to form the multi-gel network-gold particle layer; the first crosslinking agent forms an adhesive first gel network with the adhesive polymer; the second crosslinking agent forms a reducing second gel network with the reducing natural polymer; the reducing natural polymer forms a complex with the gold ions to reduce them into gold particles; The adhesive polymer includes at least one of an affinity silane of (meth)acryloyloxy propyl trimethoxysilane, dopamine, lysine, polyethyleneimine, polyacrylamide, fibrin, cyanoacrylate, chitosan, a natural polysaccharide, and polyurethane as a main ingredient The adhesive polymer includes at least one of an affinity silane of (meth)acryloyloxy propyl trimethoxysilane, dopamine, lysine, polyethyleneimine, polyacrylamide, fibrin, cyanoacrylate, chitosan, a natural polysaccharide, and polyurethane as a main ingredient the first crosslinking agent comprises at least one of N-hydroxymethyl acrylamide, N-hydroxy acrylamide, and N,N'-methylene bisacrylamide; the second crosslinking agent comprises at least one of calcium chloride, magnesium chloride, aluminum chloride, and iron chloride.

3. The fluorescence enhanced substrate of claim 2, wherein: The material of the polyelectrolyte layer comprises at least one of a cationic polyelectrolyte, an anionic polyelectrolyte, and a semiconductor medium; the cationic polyelectrolyte comprises at least one of polyethyleneimine, polydimethyldiallylammonium chloride, and polypropylene; the anionic polyelectrolyte comprises at least one of polystyrene sulfonate, polyacrylic acid, and polymethacrylic acid; and the semiconductor medium comprises at least one of silicon dioxide and aluminum oxide.

4. The fluorescence enhanced substrate of claim 3, wherein: The thickness of the polyelectrolyte layer is 5-25 nm; and / or the polyelectrolyte layer comprises cationic polyelectrolyte layer and anionic polyelectrolyte layer alternately stacked.

5. The fluorescence enhanced substrate of claim 1 or 2, wherein: The reducing natural polymer comprises at least one of hyaluronic acid, methacrylanated gelatin, sodium alginate, natural polysaccharide and chitosan.

6. The fluorescence enhanced substrate of claim 1 or 2, wherein: In the multi-gel network-gold particle layer, the mass concentration ratio of the gold particle, the adhesive polymer and the reducing natural polymer is 0.15-7:10-60:0.5-4.

7. A method of producing the fluorescence-enhancing substrate according to any one of claims 2 to 4, characterized by: The method comprises the following steps: S1-1: preparing a multi-gel network-gold particle layer; and S2-1: placing the product of S1-1 in a polyelectrolyte solution to form a polyelectrolyte layer; S3-1: placing the product of S2-1 in a biological recognition molecule solution to form a biological molecule modified layer.

8. A fluorescence-enhanced microfluidic chip comprising the fluorescence-enhanced substrate according to any one of claims 1-6 and a cover plate sealingly fitted to the upper surface of the fluorescence-enhanced substrate; the cover plate has a flow channel thereon; one end of the flow channel is provided with a sample adding area and is connected to a detection area through the flow channel; and the detection area corresponds to the detection reaction layer of the fluorescence-enhanced substrate.

9. Use of the fluorescence-enhanced substrate according to any one of claims 1-6 or the fluorescence-enhanced microfluidic chip according to claim 8 in detecting target concentration.

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