Biosensing element for detecting new pollutants and method for making same

Abstract

The present application provides a biosensor element for detecting new pollutants and a manufacturing method thereof. It comprises a biosensor slide and a detection flow cell carrier. The biosensor slide is etched with a light path, and the light path is sequentially covered with a bottom light transmission core layer, an isolation layer, a top light transmission core layer and a cladding layer, forming a slide with a laminated light path structure. The surface of the slide is modified with a biological sensitive material. The detection flow cell carrier has a detection flow cell in the middle. During detection, the test sample and the detection reagent are mixed and injected into the detection flow cell; the biosensor slide is tightly pressed above the detection flow cell carrier, so that the mixed liquid in the detection flow cell contacts the biological sensitive material modified on the surface of the biosensor slide; laser is emitted from one end of the biosensor slide, forms an evanescent wave through the light path in the slide, excites the fluorescent substance in the labeled antibody captured by the slide, and finally achieves the purpose of detecting whether the test sample contains new pollutants and the concentration thereof through a detection instrument.

Description

Technical Field

[0001] The present invention relates to a biosensor element for detecting new pollutants and a manufacturing method thereof. The biosensor element can be widely used in instruments and equipment for detecting new pollutants. Background Art

[0002] Emerging pollutants refer to persistent organic pollutants, endocrine disruptors, and antibiotics, all of which are regulated by international conventions. Emerging pollutants are characterized by high toxicity at low concentrations in the environment, making them highly destructive to the ecological environment and harmful to human health.

[0003] Biosensor technology is a method for quantitatively detecting a target by utilizing the specific recognition ability of bioactive substances. It has broad application prospects in biomedical research, disease diagnosis, environmental monitoring, and food hygiene testing. There are many types of biosensor elements. For example, a planar waveguide-based biosensor developed by Professor A. Brandenburg and others at the Fraunhofer Institute in Germany [Schuderer J, et al. Development of a multichannel fluorescence affinity sensor system. Analytical Chemistry. 2000, 72(16): 3942-3948.] can be used to detect a variety of pollutants. However, its disadvantages are that it has a complex optical path and requires multiple detection cells for separate testing, resulting in overlapping instrument flow paths, a large instrument size, and high cost. Summary of the Invention

[0004] In view of the above reasons, an object of the present invention is to provide a biosensor element for detecting new pollutants based on optical principles and having a simple optical path.

[0005] Another object of the present invention is to provide a method for manufacturing the biosensor element.

[0006] To achieve the above-mentioned object, the present invention adopts the following technical solutions: a biosensor element for detecting new pollutants, comprising a biosensor glass slide with a surface modified with a biosensitive material and a detection flow cell carrier for performing a biochemical reaction;

[0007] The biosensing slide is a laser-induced fluorescence slide based on CMOS processing, which comprises a substrate made of a silicon dioxide wafer with a thickness of 1 mm±0.01 mm; a first Y-shaped branch structure is etched at the end of the top surface of the substrate, and a second Y-shaped branch structure is etched at the tail of each branch of the first Y-shaped branch structure, and the two branches of the second Y-shaped branch structure extend to the other end of the substrate, and the four branches of the two second Y-shaped branch structures serve as the light splitting areas of the biosensing element.

[0008] A bottom light transmission core layer with a thickness of 25 nm±2 nm is sputtered on the top surface of the substrate by chemical vapor deposition, and a separation layer with a thickness of 100 nm±5 nm is covered on the top of the bottom light transmission core layer; a top light transmission core layer with a thickness of 75 nm±2 nm is sputtered on the top of the separation layer corresponding to the light splitting area of the substrate; after the bottom light transmission core layer, the separation layer and the top light transmission core layer on both sides of the substrate are etched and removed, a cladding layer is covered on the entire substrate and the top light transmission core layer, and the thickness from the upper surface of the substrate layer to the upper surface of the cladding layer is 1300 nm±10 nm.

[0009] The detection flow cell carrier is made of organic glass material, and a detection flow cell with a depth of only 0.01 mm±0.002 mm is separated in the middle part.

[0010] During detection, the biosensing slide is tightly pressed above the detection flow cell carrier.

[0011] Preferably, the front section of the first Y-shaped branch structure and the second Y-shaped branch structure corresponds to a low refractive index difference area of the bottom light transmission core layer, forming single-mode transmission of light; the rear section of the second Y-shaped branch structure corresponds to a high refractive index difference area of the bottom light transmission core layer and the top light transmission core layer, forming multi-mode transmission of light.

[0012] Preferably, a plurality of grooves are formed on the cladding layer at the top of each branch of the second Y-shaped branch structure, and the depth of the grooves reaches the top light transmission core layer, so that the top light transmission core layer is exposed to form a detection window.

[0013] Preferably, the materials of the bottom light transmission core layer and the top light transmission core layer are Si3N4; the materials of the separation layer and the cladding layer are SiO2.

[0014] Preferably, a sample inlet and a sample outlet are respectively formed at both ends of the detection flow cell; the sample inlet and the sample outlet are in a tapered structure, and the size, shape and liquid delivery pipe joint of the detection instrument connected thereto are matched.

[0015] Preferably, an O-shaped groove is opened at the edge of the detection flow cell carrier, and an elastic sealing ring is arranged in the O-shaped groove; the thickness of the elastic sealing ring is greater than the depth of the O-shaped groove.

[0016] A method for manufacturing a biosensor element for detecting new pollutants, comprising the following steps:

[0017] S1, etching a light path on a substrate made of a silicon dioxide wafer;

[0018] A substrate made of a silicon dioxide wafer with a thickness of 1mm±0.01mm is selected; a first Y-shaped branch structure is etched at the end of the top surface of the substrate, a second Y-shaped branch structure is etched at the tail of each branch of the first Y-shaped branch structure, and the two branches of the second Y-shaped branch structure are extended to the other end of the substrate; the four branches of the two second Y-shaped branch structures serve as the light splitting area of the biosensor element;

[0019] S2, covering the bottom light transmission core layer, the isolation layer, the top light transmission core layer and the cladding layer on the substrate to form a biosensor glass with a stacked light path structure;

[0020] A bottom light transmission core layer with a thickness of 25nm±2nm is sputtered on the top surface of the substrate etched with the light path by chemical vapor deposition, and a isolation layer with a thickness of 100nm±5nm is covered on the top of the bottom light transmission core layer; a top light transmission core layer with a thickness of 75nm±2nm is sputtered on the top of the isolation layer corresponding to the light splitting area of the substrate;

[0021] After etching and removing the bottom light transmission core layer, the isolation layer and the top light transmission core layer on both sides of the substrate, a cladding layer is covered on the entire substrate and the top light transmission core layer, and the thickness from the top surface of the substrate layer to the top surface of the cladding layer is 1300nm±10nm;

[0022] A plurality of grooves are formed on the cladding layer at the top of each branch of the two second Y-shaped branch structures, and the depth of the grooves reaches the top light transmission core layer, so that the top light transmission core layer is exposed to form a detection window;

[0023] S3, modifying the surface of the biosensor glass with a stacked optical structure prepared in step S2 with a biological sensitive material;

[0024] S3.1, cleaning the surface of the biosensor glass with a stacked optical structure and hydroxylating;

[0025] The biosensor glass is immersed in a glass culture dish containing piraha solution, reacted at 120℃ for 60min, then taken out, the surface is cleaned with ultrapure water, dried with nitrogen at room temperature, and stored in a vacuum drying box for standby;

[0026] S3.2, silanization modification of the biosensor slide surface;

[0027] Immerse the clean hydroxylated glass slide in the silanizing agent solution and let it react at room temperature for 2 hours. Rinse both sides of the glass slide with anhydrous toluene, blow dry with nitrogen, and store in a desiccator.

[0028] S3.3, Surface coupling of the detection window of the biosensor slide;

[0029] Apply the coupling agent solution to the detection window of the silanized glass slide and let it react at room temperature for 1 hour. Remove the glass slide and rinse the upper and lower surfaces of the slide with anhydrous ethanol and ultrapure water respectively. Then blow dry with nitrogen and store in a desiccator.

[0030] S3.4. Immobilize the antigen coated with the new pollutant to be detected on the surface of the biosensor slide, so that the antigen coated with the new pollutant to be detected is covalently bonded to the detection window surface;

[0031] Place the chemically treated slide on a spotter and, according to the programmed procedure, drop 5 μL of 1 mg / mL coated antigen onto the detection window of the slide. Place the slide in a sealed environment at a humidity of 55%-75% and a temperature of 4°C for 12 hours. Wash the upper and lower surfaces of the slide with ultrapure water, blow dry with nitrogen, and store in a desiccator for later use.

[0032] S4. Make a detection flow cell

[0033] S4.1. Select a piece of plexiglass that has been milled to a smooth surface as the carrier for the flow cell. Open a flow cell with a depth of 0.01 mm ± 0.002 mm in the center of the cell.

[0034] S4.2. An O-shaped groove is formed around the edge of the detection flow cell carrier. An elastic sealing ring is installed in the O-shaped groove. The thickness of the elastic sealing ring is greater than the depth of the O-shaped groove.

[0035] S4.3. A sample inlet and a sample outlet are respectively provided at both ends of the bottom surface of the detection flow cell; the size and shape of the sample inlet and sample outlet match the infusion tube connector of the detection instrument connected thereto.

[0036] Preferably, the piraha solution in step S3.1 is a mixture of H2O2 and H2SO4 in a volume ratio of 1:3;

[0037] The silanizing agent solution in step S3.2 is a mixture of 2 mL of 3-mercaptopropyltrimethoxysilane solution dispersed in 100 mL of toluene solution;

[0038] The coupling agent solution in step S3.3 is a mixture formed by dissolving 25 mg of 4-maleimidobutyric acid-N-succinimide ester powder in 100 uL of N,N-dimethylformamide and then diluting the volume to 50 mL of anhydrous ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the structure of the biosensor element of the present invention;

[0040] Figure 2A This is a front view of the end portion of the biosensor element of the present invention;

[0041] Figure 2B for Figure 2A AA cross-section of

[0042] Figure 3 A top view of the biosensor element slide of the present invention;

[0043] Figure 4 Schematic diagram of biosensor glass slide modified with biosensitive materials according to the present invention;

[0044] Figure 5 is the relationship curve between the fluorescent dye concentration and the test signal;

[0045] Figure 6A This is a 2,4-D curve diagram of pesticides detected by the biosensor element of the present invention;

[0046] Figure 6B This is a curve diagram of the endocrine disruptor bisphenol A (BPA) detected by the biosensor element of the present invention;

[0047] Figure 6C This is a curve diagram of the antibiotic sulfamethazine (SM2) detected by the biosensor element of the present invention. DETAILED DESCRIPTION

[0048] The structure and features of the present invention are described in detail below with reference to the accompanying drawings and embodiments. It should be noted that various modifications can be made to the embodiments disclosed herein. Therefore, the embodiments disclosed in the specification should not be regarded as limiting the present invention, but are merely examples of embodiments, the purpose of which is to make the features of the present invention apparent.

[0049] The biosensor element for detecting new pollutants of the present invention is a sensor element based on optical principles, such as Figure 1 As shown, it consists of a biosensor slide 1 with biosensitive materials fixed on it and a detection flow cell carrier 2 for performing biochemical reactions. The biosensor slide 1 is tightly pressed on the detection flow cell carrier 2. The biosensor slide 1 is etched with an optical path, covered with a light transmission core layer, and its surface is modified with biosensitive materials for identifying new pollutants.

[0050] When conducting new pollutant detection, the test sample and the detection reagent are mixed and injected into the detection flow cell on the detection flow cell carrier 2, and come into contact with the biosensitive material modified on the surface of the biosensor slide 1; the laser is injected from one end of the biosensor slide, forming an evanescent wave through the light path inside the biosensor slide, exciting the fluorescent substance in the detection reagent, and then the detection instrument is used to detect whether the test sample contains new pollutants and their concentration.

[0051] like Figure 1 、 Figure 2A 、 Figure 2B As shown, the biosensor slide 1 of the present invention is a laser-induced fluorescence slide based on CMOS (Complementary Metal Oxide Processing), which includes a substrate 11 made of a silicon dioxide wafer with a thickness of 1 mm ± 0.01 mm. Figure 3 As shown, a first Y-shaped branch structure 111 is etched at the left end of the top surface of the substrate 11, and an incident mark 112 is etched at the incident end of the first Y-shaped branch structure to align the incident laser with the optical path of the glass slide. Second Y-shaped branch structures 113 are etched at the tails of the two branches of the first Y-shaped branch structure respectively, and the two branches 114 of the second Y-shaped branch structure 113 extend all the way to the right end of the substrate 11. In this way, the two branches of the two second Y-shaped branch structures, a total of four branches, serve as the spectroscopic area of ​​the biosensor element.

[0052] A bottom optical transmission core layer 12 with a thickness of 25nm±2nm is sputtered on the top surface of the substrate 11 by chemical vapor deposition. A spacer layer 13 with a thickness of 100nm±5nm is placed on top of the bottom optical transmission core layer. A top optical transmission core layer 14 with a thickness of 75nm±2nm is sputtered on top of the spacer layer, corresponding to the substrate light-split region. The bottom optical transmission core layer 12, spacer layer 13, and top optical transmission core layer 14 on both sides of the substrate are etched away (see Figure 2B ), narrowing the optical transmission channel and concentrating the energy. Then, a cladding layer 15 is applied to the entire substrate 11, with a thickness of 1300nm ± 10nm from the upper surface of the substrate layer to the upper surface of the cladding layer. The regions of the underlying optical transmission core layer 12 corresponding to the front sections of the first and second Y-shaped branch structures 111 and 113 are low-refractive-index-difference regions, which facilitate single-mode transmission of light. The regions of the underlying optical transmission core layer 12 and the top optical transmission core layer 14 corresponding to the rear sections of the second Y-shaped branch structures 113 are high-refractive-index-difference regions, which facilitate multi-mode transmission of light.

[0053] Several grooves 151 are spaced apart in the cladding layer 15 at the top of the branches 114 of each second Y-shaped branch structure 113. The grooves extend to the top of the optical transmission core layer 14, exposing the top optical transmission core layer as a detection window. When light propagates through different media, its refractive index differs. When laser light is incident from a denser medium to a less dense medium, total internal reflection occurs, generating an electromagnetic wave energy, an evanescent wave, on the surface of the less dense medium. When laser light is incident from the end of the glass slide's base layer, an evanescent wave forms on the surface of the glass slide's top optical transmission core layer in contact with the air. This wave becomes the energy that excites the fluorescent agent in the labeled antibody captured by the antigen-coated new contaminant being detected, fixed on the surface of the detection window, to produce fluorescence.

[0054] In a preferred embodiment of the present invention, the size of each detection window is 1.5 mm×0.8 mm±0.01 mm, and the center distance between adjacent detection windows in the same branch is 4 mm±0.01 mm.

[0055] In a preferred embodiment of the present invention, the bottom optical transmission core layer 12 and the top optical transmission core layer 14 are made of Si₃N₄, and the isolation layer 13 and the cladding layer 15 are made of SiO₂. Si₃N₄ and SiO₂ enable low-loss total reflection transmission of laser light within the multi-layered biosensor slide.

[0056] The present invention adopts the principle of immunoassay to detect new pollutants. Therefore, the present invention modifies the surface of the biosensor glass slide 1 with a biosensitive material, namely the new pollutant antigen to be detected. When detecting new pollutants, the present invention first mixes a detection reagent containing antibodies with the test sample. The antibody concentration in the detection reagent is much greater than the concentration of the new pollutant to be detected. A portion of the antibodies binds to the new pollutant to be detected. The detection reagent containing the remaining antibodies and the mixed liquid of the test sample are injected into the detection flow cell together. After contacting with the modified new pollutant antigen on the biosensor glass slide, a chemical reaction occurs. At the same time, a laser is injected from one end of the biosensor glass slide with a laminated optical structure. The laser is stably transmitted in the glass slide and forms an evanescent wave with relatively strong energy at the detection window, which excites the fluorescent dye in the labeled antibody captured by the new pollutant-coated antigen here, generating a fluorescent signal, so that the detection instrument connected to the biosensor element of the present invention can quickly and accurately detect the intensity of the fluorescence, and then determine whether there is a new pollutant and the concentration of the new pollutant.

[0057] The method of modifying the biosensitive material on the surface of the biosensor slide of the present invention is as follows:

[0058] (1) Cleaning and hydroxylating the surface of a biosensor glass slide having a laminated optical structure.

[0059] The biosensor slide was immersed in a glass culture dish containing piraha solution (H2O2:H2SO4=1:3, v / v), reacted at 120℃ for 60min, taken out, washed with ultrapure water, dried with nitrogen at room temperature, and stored in a vacuum drying oven for later use.

[0060] (2) Silanization modification of the surface of the biosensor slide.

[0061] Immerse the clean hydroxylated glass slide in a silanizing agent solution (2 mL of 3-mercaptopropyltrimethoxysilane solution dispersed in 100 mL of toluene solution), let it react at room temperature for 2 hours, take it out, rinse both sides of the glass slide with anhydrous toluene, then blow it dry with nitrogen and store it in a desiccating container.

[0062] (3) Surface coupling of the biosensor slide detection window.

[0063] The coupling agent solution (25 mg of 4-maleimidobutyric acid-N-succinimide ester powder was first dissolved in 100 μL of N,N-dimethylformamide and then diluted to 50 mL of anhydrous ethanol) was applied to the detection window of the silanized glass slide. The reaction was allowed to stand at room temperature for 1 hour. The glass slide was removed and the upper and lower surfaces were rinsed with anhydrous ethanol and ultrapure water respectively. Then, the slide was blown dry with nitrogen and stored in a desiccating container.

[0064] (4) The new pollutant-coated antigen to be detected is fixed on the surface of the biosensor slide, so that the new pollutant-coated antigen to be detected is covalently bonded to the detection window surface.

[0065] Place the slide that has undergone the chemical treatment on the spotter. According to the programmed procedure, add 5 μL of 1 mg / mL coated antigen to the detection window 151 of the slide. Place the slide in a sealed environment with a humidity of 55%-75% and a temperature of 4°C for 12 hours. Then, wash the upper and lower surfaces of the slide with ultrapure water, blow dry with nitrogen, and store in a desiccator for later use (see Figure 4 ).

[0066] In the process of modifying the surface of the biosensor slide with biosensitive materials, covalent coupling methods are used to link multiple layers of chemical substances to each other to form a strong covalent bond, so that the biosensor slide and biosensor element are stable and reusable, with strong specific reaction and weak nonspecific adsorption, and have good immune response performance.

[0067] like Figure 1As shown, the biosensor element of the present invention also includes a detection flow cell carrier 2. The detection flow cell carrier 2 is a component that ensures that the mixed solution of the test sample and the detection reagent flows in a plug-flow sequence, uniformly contacts the biosensor glass slide 1 modified with a biosensitive material, and reacts with it. The detection flow cell carrier 2 is made of organic glass material, and a very thin detection flow cell 21 with a depth of only 0.01mm±0.002mm is opened in the middle part thereof, and a sample inlet 211 and a sample outlet 212 are respectively opened at both ends of the detection flow cell. The test sample enters the detection flow cell through the sample inlet 211 and flows out of the detection flow cell from the sample outlet 212. After the biosensor glass slide 1 is pressed against the detection flow cell carrier 2, the present invention utilizes the side wall effect of the upper and lower planes of the detection flow cell 21 and the viscosity of the liquid to form a laminar flow state in the detection flow cell, so that the mixed solution of the test sample and the detection reagent flows in a plug-flow sequence, ensuring uniform contact of the mixed liquid in the detection flow cell with the biosensitive material modified on the glass slide surface and stable biochemical reaction.

[0068] An O-shaped groove 22 is provided on the edge of the detection flow cell carrier 2, and an elastic sealing ring 23 is provided in the O-shaped groove. The thickness of the sealing ring 23 is greater than the depth of the O-shaped groove 22. The purpose is to ensure the thickness of the detection flow cell when the biosensor glass slide 1 is pressed against the detection flow cell carrier 2, and to ensure that the liquid in the detection flow cell is in a laminar state, which is conducive to forming conditions for thin layer reaction, accelerating the renewal of the mixed liquid of the test sample and the detection reagent in contact with the biosensitive material modified on the glass slide surface, and promoting the immune response.

[0069] like Figure 1 As shown, the sample inlet 211 and the sample outlet 212 of the detection flow pool 21 connected to the external flow path are conical structures with a cone angle of 37 degrees. The purpose of this design is to be able to be connected to the infusion tube connector of the detection instrument to form a completely closed integrated structure to prevent leakage of reagents and carrier liquids, as well as the entry of external air.

[0070] In a preferred embodiment of the present invention, the detection flow cell 21 has planar dimensions of 15 mm x 60 mm ± 0.01 mm, a depth of 0.01 mm ± 0.002 mm, and a capacity of approximately 9 μL. A circular hole with a diameter of 0.8 mm ± 0.01 mm is located at each end of the detection flow cell, serving as the sample inlet and outlet. The thickness of the elastic sealing ring within the O-shaped groove is greater than the groove depth (0.1 mm), ensuring extensive contact between the base 11 of the biosensor slide 1 and the test liquid within the detection flow cell 21 during assembly, while maintaining a good seal.

[0071] The principle of detecting new pollutants in the present invention is: 1) Utilizing laser-induced biosensor slides. Light has different refractive indices when propagating through different media. When light waves are incident from a denser medium to a less dense medium, total internal reflection occurs, generating an electromagnetic wave energy known as an evanescent wave on the surface of the less dense medium. The laminated biosensor slides of the present invention utilize the difference in refractive indices between Si3N4 (silicon nitride) and SiO2 (silicon dioxide) to ensure stable transmission of incident laser light through the slide, generating a high-energy evanescent wave at the detection window. This wave excites the fluorescent dye in the labeled antibody captured by the new pollutant-coated antigen, generating a fluorescent signal. 2) A sample containing the new contaminant is mixed with an excess of a fluorescent dye-labeled antibody (a biological protein that can recognize the contaminant) reagent to react. Part of the antibody binds to the contaminant, and a portion of the antibody remains unreacted. The mixed liquid is input into a detection flow cell, and the mixed liquid contacts the detection window of the glass slide. The unreacted fluorescent dye-labeled antibody binds to the coated antigen modified on the glass slide. When the laser enters the optical channel of the glass slide, an evanescent wave is formed at the detection window, which excites the fluorescent substance in the labeled antibody captured by the glass slide, generating fluorescence. The concentration of the new contaminant in the sample is then calculated based on the strength of the fluorescence signal by a detection system connected to the biosensor element of the present invention, thereby achieving the purpose of detecting the new contaminant.

[0072] The embodiment of the present invention is to combine a biosensor glass slide modified with a biosensitive material by laser induction with a mixed liquid to be tested flowing in a detection flow cell, and assemble them into a biosensor element for sample detection. The excitation light used is red light with a wavelength of 635nm. The laser enters the glass slide from the incident mark set at the incident end of the biosensor glass slide, and the Y-shaped branch structure is used to evenly distribute the excitation light to 32 (4 columns, 8 in each column) detection windows. First, a Cy5.5 fluorescent dye solution (excluding the new pollutant sample to be tested) is input into the detection flow cell. The detection glass slide has a sensitive response to Cy5.5 fluorescent dye solutions of different concentrations, and the detection limit can reach 1nM (see Figure 5 ), which fully demonstrates the high sensitivity of the biosensor element of the present invention; then, the mixed solution of the Cy5.5 fluorescent dye-labeled antibody and the test sample is input into the detection flow cell. After the reaction of the labeled antibody with the new pollutant and the binding of the remaining labeled antibody with the detection window antigen, the concentration of the new pollutant in the sample can be quantitatively measured.

[0073] The biosensor element for new pollutant detection is installed on a trace toxic pollutant detection system (instrument) to detect a variety of new pollutants, and a standard curve for detection can be obtained, such as Figures 6A-6C shown. Figure 6A The curve in the middle is the detected pesticide 2,4-D, which is a toxic organic compound. Figure 6B The curve in the middle is the detected bisphenol A (BPA), which is an endocrine disruptor. Figure 6CThe curve in the figure is the detected sulfamethazine (SM2) which belongs to antibiotics. The above results can meet the requirements of detecting new pollutants in water. The sensing element can detect different new pollutant targets by changing the glass modified biosensitive material. The time required for completing one detection is less than 20 min.

[0074] The advantage of the present application is that the laser-induced fluorescence glass and the thin layer flow cell based on CMOS processing are closely combined to form a multi-channel multi-detection window biosensing element, the functions of the multi-detection cell and the multi-flow path complex system are integrated in one sensing element, the optical path is greatly simplified, the sample and reagent flow path is simplified, the instrument structure is simple, and the miniaturization and automation of the instrument are provided.

[0075] Currently, new pollutants are mainly detected by large instruments, and the mainly used instruments are gas chromatography-mass spectrometer and liquid chromatography-mass spectrometer. Such instruments are very expensive, generally with a price of 1-2 million yuan per unit, and it takes several hours to test one sample, and the test fee is 500-1000 yuan, and skilled professional technicians are needed to operate. Moreover, most of the instruments are imported. The instrument with the biosensing element as the core element has only 1 / 10 of the price of the above-mentioned instrument, and the time for testing one sample is about 20 min, and the detection cost is only 1 / 20 of the above-mentioned instrument, and general technicians can operate after training. Therefore, the present application combines the glass sheet with special optical structure and the detection flow cell carrier with laminar flow characteristics to form a biosensing element, and cooperates with a detection instrument to realize quantitative detection of various new pollutants, has the advantages of high sensitivity, wide applicability, low instrument and detection cost, and easy popularization. The present application can be widely applied to the determination of new pollutants in water environment monitoring and other fields.

[0076] Finally, it should be noted that the above-described embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A biosensor element for detecting new pollutants, characterized in that: It includes biosensing slides; The biosensor slide is a laser-induced fluorescence slide based on CMOS processing. It includes a substrate made of a silicon dioxide wafer with a thickness of 1 mm ± 0.01 mm. A first Y-shaped branch structure is etched at the end of the top surface of the substrate. A second Y-shaped branch structure is etched at the tail of each of the two branches of the first Y-shaped branch structure. The two branches of the second Y-shaped branch structure extend all the way to the other end of the substrate. The two branches of the two second Y-shaped branch structures, a total of four branches, serve as the spectroscopic area of ​​the biosensor element. A bottom light transmission core layer with a thickness of 25nm±2nm is sputtered on the top surface of the substrate by chemical vapor deposition, and a spacer layer with a thickness of 100nm±5nm is covered on the top of the bottom light transmission core layer; a top light transmission core layer is sputtered on the top of the spacer layer, corresponding to the light splitting area of ​​the substrate; after the bottom light transmission core layer, the spacer layer and the top light transmission core layer on both sides of the substrate are etched away, a cladding layer is covered on the entire substrate and the top light transmission core layer; The surface of the biosensor glass slide is modified with a biosensitive material; the thickness of the light transmission core layer on the top of the substrate is 75nm±2nm; the thickness from the upper surface of the substrate layer to the upper surface of the cladding layer is 1300nm±10nm; Also included is a detection flow cell carrier for performing biochemical reactions; The detection flow cell carrier is made of organic glass material, and a detection flow cell with a depth of only 0.01mm±0.002mm is opened in the middle; During detection, the biosensor glass slide is pressed tightly against the detection flow cell carrier; The material of the bottom light transmission core layer and the top light transmission core layer is Si3N4; The materials of the isolation layer and the coating layer are SiO2.

2. The biosensor element for detecting new pollutants according to claim 1, characterized in that: The bottom optical transmission core layer area corresponding to the front section of the first Y-type branch structure and the second Y-type branch structure is a low refractive index difference area, forming single-mode transmission of light; the bottom optical transmission core layer area corresponding to the rear section of the second Y-type branch structure and the top optical transmission core layer area are high refractive index difference areas, forming multi-mode transmission of light.

3. The biosensor element for detecting new pollutants according to claim 2, characterized in that: A plurality of grooves are opened at intervals on the cladding layer at the top of each branch of the second Y-shaped branch structure, and the depth of the grooves reaches the top light transmission core layer, exposing the top light transmission core layer to form a detection window.

4. The biosensor element for detecting new pollutants according to claim 3, characterized in that: A sample inlet and a sample outlet are respectively provided at both ends of the detection flow cell; The sample inlet and the sample outlet are of conical structure, and the size and shape thereof match the infusion pipe joint of the detection instrument connected thereto.

5. The biosensor element for detecting new pollutants according to claim 4, characterized in that: An O-shaped groove is provided on the edge of the detection flow cell carrier, and an elastic sealing ring is provided in the O-shaped groove; The thickness of the elastic sealing ring is greater than the depth of the O-shaped groove.

6. A method for manufacturing the biosensor element for detecting new pollutants according to any one of claims 1 to 5, characterized in that: It includes the following steps: S1, etching an optical path on a substrate made of silicon dioxide wafer; A substrate made of a silicon dioxide wafer with a thickness of 1 mm ± 0.01 mm was selected; a first Y-shaped branch structure was etched at the end of the top surface of the substrate, and a second Y-shaped branch structure was etched at the tail of each of the two branches of the first Y-shaped branch structure. The two branches of the second Y-shaped branch structure extended all the way to the other end of the substrate. The two branches of the two second Y-shaped branch structures, a total of four branches, served as the spectroscopic region of the biosensor element; S2. Covering the substrate with a bottom light transmission core layer, an isolation layer, a top light transmission core layer, and a cladding layer to form a biosensor glass slide having a laminated optical path structure; A bottom optical transmission core layer with a thickness of 25nm±2nm is sputtered on the top surface of the substrate etched with the optical path by chemical vapor deposition, and a spacer layer with a thickness of 100nm±5nm is covered on the top of the bottom optical transmission core layer; a top optical transmission core layer with a thickness of 75nm±2nm is sputtered on the top of the spacer layer, corresponding to the substrate light splitting area; After etching away the bottom light transmission core layer, the isolation layer, and the top light transmission core layer on both sides of the substrate, a cladding layer is covered on the entire substrate and the top light transmission core layer, with a thickness of 1300nm±10nm from the upper surface of the substrate layer to the upper surface of the cladding layer; A plurality of grooves are formed at intervals on the cladding layer at the top of each branch of the two second Y-shaped branch structures, with the depth of the grooves reaching the top light transmission core layer, exposing the top light transmission core layer to form a detection window; S3, modifying the surface of the biosensor slide produced in step S2 with a biosensitive material; S3.

1. Cleaning and hydroxylating the surface of the biosensor glass slide having the laminated optical structure; Immerse the biosensor slide in a glass culture dish filled with piraha solution, react at 120°C for 60 min, then take it out, wash the surface with ultrapure water, blow dry with nitrogen at room temperature, and store it in a vacuum drying oven for later use; S3.2, silanization modification of the biosensor slide surface; Immerse the clean hydroxylated glass slide in the silanizing agent solution and let it react at room temperature for 2 hours. Rinse both sides of the glass slide with anhydrous toluene, blow dry with nitrogen, and store in a desiccator. S3.3, Surface coupling of the detection window of the biosensor slide; Apply the coupling agent solution to the detection window of the silanized glass slide and let it react at room temperature for 1 hour. Remove the glass slide and rinse the upper and lower surfaces of the slide with anhydrous ethanol and ultrapure water respectively. Then blow dry with nitrogen and store in a desiccator. S3.

4. Immobilize the antigen coated with the new pollutant to be detected on the surface of the biosensor slide, so that the antigen coated with the new pollutant to be detected is covalently bonded to the surface of the detection window; Place the chemically treated slide on the spotter and, according to the programmed procedure, drop 5 μL of 1 mg / mL coated antigen onto the detection window of the slide. Place the slide in a sealed environment at a humidity of 55%-75% and a temperature of 4°C for 12 hours. Then, wash the upper and lower surfaces of the slide with ultrapure water, blow dry with nitrogen, and store in a desiccator for later use. S4. Make a detection flow cell S4.

1. Select a piece of plexiglass that has been milled to a smooth surface as the carrier for the flow cell. Open a flow cell with a depth of 0.01 mm ± 0.002 mm in the center of the cell. S4.

2. An O-shaped groove is formed around the edge of the detection flow cell carrier. An elastic sealing ring is installed in the O-shaped groove. The thickness of the elastic sealing ring is greater than the depth of the O-shaped groove. S4.

3. A sample inlet and a sample outlet are respectively provided at both ends of the bottom surface of the detection flow cell; the size and shape of the sample inlet and sample outlet match the infusion tube connector of the detection instrument connected thereto.

7. The method for fabricating a biosensor element for detecting new pollutants according to claim 6, wherein: The piraha solution in step S3.1 is a mixture of H2O2 and H2SO4 in a volume ratio of 1:3; The silanizing agent solution in step S3.2 is a mixture of 2 mL of 3-mercaptopropyltrimethoxysilane solution dispersed in 100 mL of toluene solution; The coupling agent solution in step S3.3 is a mixture formed by dissolving 25 mg of 4-maleimidobutyric acid-N-succinimide ester powder in 100 uL of N,N-dimethylformamide and then diluting the volume to 50 mL of anhydrous ethanol.

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