Super-high-sensitivity photonic crystal chip, preparation method thereof and application of super-high-sensitivity photonic crystal chip in CGRP detection

Abstract

The invention discloses a photonic crystal chip with ultrahigh sensitivity, a preparation method of the photonic crystal chip and application of the photonic crystal chip in detection of CGRP. The photonic crystal biochip comprises a photonic crystal structure and an immunodetection system, the immunodetection system is fixed on the photonic crystal structure, and the immunodetection system contains an antibody of a biological detection marker; the photonic crystal structure is formed by self-assembly of photonic crystals on a base material. The invention provides a rapid, simple and convenient detection method and an optical detection platform aiming at the detection of target objects which are easy to degrade and ultralow in concentration, such as CGRP. Compared with ELISA (enzyme-linked immunosorbent assay) detection which needs several hours, the detection platform disclosed by the invention is simple and convenient to operate and rapid in detection (only 10 minutes).

Description

Technical Field

[0001] The present invention belongs to the field of materials science and biology, and relates to a marker detection chip and a detection method, and in particular to an ultra-high sensitivity photonic crystal chip and a preparation method thereof and an application in detecting CGRP. Background Art

[0002] Migraine is a disabling primary headache characterized by recurrent unilateral throbbing moderate to severe pain that worsens with movement. During a migraine attack, patients are often bedridden and unable to fulfill social and life obligations. In clinical practice, atypical migraines often mimic other types of headaches (such as tension-type headaches), and it is difficult to make a definitive differential diagnosis based on the patient's symptoms alone. Calcitonin gene-related peptide (CGRP) is a small molecule polypeptide that is rapidly degraded after release into plasma, with a half-life of approximately 10 minutes. It is considered a reliable biomarker for migraine and is closely related to the pathogenesis of the disease. Notably, studies have shown that it has the potential to serve as an objective marker to distinguish between migraine and tension-type headaches. Its levels increase during migraine attacks and remain unchanged during tension-type headaches.

[0003] Currently, the enzyme-linked immunosorbent assay (ELISA) method is considered the gold standard for detecting CGRP, with better sensitivity and repeatability. However, this method also has the disadvantages of long analysis time, cumbersome operation and professional knowledge, which seriously limits its application in clinical practice, especially in the case of rapid degradation and extremely low concentration of biomarkers, the existing detection methods cannot meet the needs of these markers in clinical practice. Therefore, there is an urgent need to develop a detection method that can be both rapid and highly sensitive in a short time. Summary of the invention

[0004] Aiming at the detection of easily degradable and ultra-low concentration target substances such as calcitonin gene-related peptide (CGRP), the present invention provides a photonic crystal biochip and a preparation method and a detection method thereof.

[0005] The technical solution provided by the present invention is as follows:

[0006] A photonic crystal biochip, comprising a photonic crystal structure and an immune detection system; the immune detection system is fixed on the photonic crystal structure, and the immune detection system contains antibodies of biological detection markers;

[0007] The photonic crystal structure is formed by self-assembly of photonic crystals on a substrate.

[0008] According to an embodiment of the present invention, the immunodetection system is fixed on the photonic crystal structure through a coupling reaction.

[0009] According to an embodiment of the present invention, the biodetection marker is a small molecule polypeptide that is easily degradable and has an ultra-low concentration, such as CGRP. Preferably, the immunoassay system contains an antibody to CGRP. Preferably, the ultra-low concentration refers to a concentration of 0.1-1000pg / mL, such as 0.5pg / mL, 50pg / mL, 100pg / mL, 200pg / mL, 500pg / mL.

[0010] According to an embodiment of the present invention, the photonic crystal structure is a periodic regular arrangement formed by self-assembly of the photonic crystals on a substrate. Preferably, the photonic crystals are monodisperse latex spheres.

[0011] According to an embodiment of the present invention, the particle size of the monodisperse latex spheres is 150-320 nm, for example, 180-280 nm, exemplified by 180 nm, 190 nm, 200 nm, 205 nm, 215 nm, 220 nm, 230 nm, 250 nm, 260 nm, and 280 nm.

[0012] According to an embodiment of the present invention, the monodisperse latex spheres can be selected from at least one of poly(methyl methacrylate-acrylic acid-styrene) latex spheres, silica microspheres, polystyrene microspheres, etc., preferably poly(methyl methacrylate-acrylic acid-styrene) latex spheres.

[0013] According to an embodiment of the present invention, the surface of the monodisperse latex spheres carries -COOH functional groups.

[0014] According to a preferred embodiment of the present invention, the monodisperse latex spheres can be selected from at least one of carboxyl-modified poly(methyl methacrylate-acrylic acid-styrene) latex spheres, carboxyl-modified silica microspheres, carboxyl-modified polystyrene microspheres, etc., preferably carboxyl-modified poly(methyl methacrylate-acrylic acid-styrene) latex spheres.

[0015] According to an embodiment of the present invention, the photonic crystal is provided by photonic crystal ink.

[0016] According to an embodiment of the present invention, the photonic crystal structure is formed by self-assembly on a substrate by inkjet printing of the photonic crystal ink.

[0017] According to an embodiment of the present invention, the photonic crystal ink comprises photonic crystals, a humectant and a wetting agent.

[0018] According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the photonic crystals is 5-30%, preferably 10-25%, more preferably 10-15%, for example 10%, 11%, 12%, 13%, 14%, 15%.

[0019] According to an embodiment of the present invention, the humectant may be at least one selected from ethylene glycol, propylene glycol, glycerol, sorbitol, etc., for example, ethylene glycol.

[0020] According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the moisturizing agent is 5-10%, such as 6-9%, and exemplified by 7% or 8%.

[0021] According to an embodiment of the present invention, the wetting agent may be selected from at least one of BYK-3400, BYK-3455, BYK-151, BKY-154, etc., for example, BYK-3400.

[0022] According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the wetting agent is 0.5-5‰, such as 1-3‰.

[0023] According to an embodiment of the present invention, the photonic crystal ink contains a solvent. Preferably, the solvent is water.

[0024] According to an exemplary embodiment of the present invention, the photonic crystal ink comprises monodisperse poly(methyl methacrylate-acrylic acid-styrene) latex spheres, ethylene glycol and BYK-3400;

[0025] The particle size of the monodisperse poly(methyl methacrylate-acrylic acid-styrene) latex spheres is 150-320 nm, and the mass concentration of the monodisperse poly(methyl methacrylate-acrylic acid-styrene) latex spheres is 5-30%; the mass concentration of the ethylene glycol is 5-10%, and the mass concentration of the BYK-3400 is 0.5-5‰.

[0026] According to the embodiment of the present invention, there is no particular limitation on the pattern of the photonic crystal structure, for example, the pattern can be designed. As an example, the photonic crystal structure can be a dot array.

[0027] According to an embodiment of the present invention, the photonic crystal structure is a water-resistant photonic crystal structure.

[0028] According to an embodiment of the present invention, the photonic crystal structure may further include a substrate.

[0029] According to an embodiment of the present invention, the substrate can be glass (the glass can be ordinary glass, quartz glass or organic glass, etc.), a metal sheet / film (aluminum foil or copper sheet, etc.), a plastic sheet / film (PET sheet / film, polystyrene sheet / film, polymethacrylic sheet / film, polypropylene sheet / film, or polyvinyl chloride sheet / film, etc.), etc.

[0030] According to an embodiment of the present invention, the contact angle of the substrate is greater than 60°.

[0031] According to a preferred embodiment of the present invention, the photonic crystal structure is prepared by a method comprising the following steps: inkjet printing of photonic crystal ink, self-assembly on a substrate to form a photonic crystal structure and thermal sintering, wherein the thermal sintering temperature is 100-150° C.; the substrate is a PET film;

[0032] The photonic crystal structure is a periodic regular arrangement formed by self-assembly of photonic crystals on a substrate;

[0033] The photonic crystal ink is composed of photonic crystals, moisturizing agent, wetting agent and solvent; wherein the photonic crystals are monodisperse latex spheres;

[0034] The particle size of the monodisperse latex sphere is 150-320 nm; the monodisperse latex sphere is selected from at least one of poly(methyl methacrylate-acrylic acid-styrene) latex sphere, silica microsphere, and polystyrene microsphere; the surface of the monodisperse latex sphere has a -COOH functional group;

[0035] The moisturizing agent is selected from at least one of ethylene glycol, propylene glycol, glycerol and sorbitol;

[0036] The wetting agent is selected from at least one of BYK-3400, BYK-3455, BYK-151, and BKY-154;

[0037] In the photonic crystal ink, the mass concentration of the photonic crystal is 5-30%, the mass concentration of the moisturizing agent is 5-10%, and the mass concentration of the wetting agent is 0.5-5‰.

[0038] According to an embodiment of the present invention, the photonic crystal structure has a fluorescence gain effect, for example, the fluorescence gain effect can be 1 to 100 times, for example, 10 times or 50 times.

[0039] According to an embodiment of the present invention, the immunoassay system is selected from an immunocompetitive assay system.

[0040] The present invention also provides a method for preparing the above-mentioned photonic crystal biochip, which comprises: fixing an immune detection system on the photonic crystal structure to form the photonic crystal biochip.

[0041] Preferably, the immunoassay system and the photonic crystal structure have the meanings as described above. Preferably, the immunoassay system is fixed on the photonic crystal structure through a coupling reaction to form the photonic crystal biochip.

[0042] According to a preferred embodiment of the present invention, the method for preparing the photonic crystal biochip specifically comprises the following steps:

[0043] (1) preparing a photonic crystal structure;

[0044] (2) activating the photonic crystal in the photonic crystal structure so that a large number of -COOH functional groups are exposed on the surface of the photonic crystal;

[0045] (3) The immunoassay system and the photonic crystal are subjected to an amide reaction, and the immunoassay system is fixed on the photonic crystal on the photonic crystal structure to obtain the photonic crystal biochip.

[0046] According to an embodiment of the present invention, the reagents used in the amide reaction are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Preferably, EDC and NHS can be selected at concentrations known in the art, such as 30-50 mg / mL for EDC, and 40 mg / mL for example; and 4-50 mg / mL for NHS, and 40 mg / mL for example.

[0047] The present invention also provides an application of the photonic crystal biochip in detecting CGRP.

[0048] The present invention also provides a method for detecting CGRP, which comprises: contacting a sample to be tested with the above-mentioned photonic crystal biochip, and identifying the target CGRP through fluorescence detection.

[0049] According to an embodiment of the present invention, the detection method comprises the following steps:

[0050] a) contacting the photonic crystal biochip with a standard immunoassay system of different concentrations, measuring the fluorescence value, and constructing a standard curve of the immunoassay system;

[0051] b) contacting the sample to be tested with the photonic crystal biochip, measuring the fluorescence value, and calculating the content of the target CGRP in the sample to be tested according to the standard curve.

[0052] According to an embodiment of the present invention, the volume of the sample to be tested is 10 μL to 50 μL.

[0053] According to an embodiment of the present invention, the standard curve is, for example, Figure 3 as shown in .

[0054] According to an embodiment of the present invention, the sample to be tested is saliva. Preferably, the sample to be tested can be diluted according to actual conditions. Further, deionized water, PBS buffer, etc. can be used for dilution.

[0055] According to an embodiment of the present invention, the contacting can be carried out under conditions known in the art, such as at room temperature, and the contacting time is 5 to 15 minutes, such as 10 minutes.

[0056] According to an embodiment of the present invention, the fluorescence detection can be performed using equipment known in the art, such as using a portable fluorescence tester to test ice reading data.

[0057] According to an embodiment of the present invention, in step b), rinsing is further performed after contacting. Preferably, deionized water is used for rinsing, and the number of rinsing is not limited, for example, 3 to 5 times.

[0058] Preferably, in step b), the lower limit of the concentration of the detected target CGRP is at least 0.01 pg / mL, for example, 0.1-1000 pg / mL, for example, 0.5 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL.

[0059] The present invention also provides a fluorescence detection device, which at least comprises the above-mentioned photonic crystal biochip.

[0060] According to an embodiment of the present invention, the fluorescence detection device is a portable detection device.

[0061] According to an embodiment of the present invention, the fluorescence detection device preferably includes a CGRP detection kit, and the CGRP detection kit includes the above-mentioned photonic crystal biochip.

[0062] According to an embodiment of the present invention, the CGRP detection kit further comprises a sampling tube, a cleaning solution, a detection probe and a storage medium. Further, the CGRP detection kit further comprises a diluent, a blocking agent and a pipette.

[0063] According to an embodiment of the present invention, the diluent is selected from at least one of deionized water and PBS buffer.

[0064] According to an embodiment of the present invention, the detection probe is selected from a standard of CGRP labeled with a fluorescent molecule.

[0065] According to an embodiment of the present invention, the fluorescent molecule is, for example, Cy5.

[0066] According to an embodiment of the present invention, the storage medium is selected from a sealed heat-pressed light-proof bag.

[0067] According to an embodiment of the present invention, the cleaning solution is selected from at least one of water and PBS buffer.

[0068] According to an embodiment of the present invention, the blocking agent is selected from at least one of sodium caseinate, BSA (eg, 1% BSA), and goat serum.

[0069] According to the embodiment of the present invention, the sampling tube and the washing dropper can be prepared by using materials known in the art, and this application does not make any specific limitation.

[0070] The present invention also provides an application of the fluorescence detection device in detecting CGRP.

[0071] The present invention also provides a detection method for the above-mentioned fluorescence detection device, which comprises collecting the sample to be tested into a sampling tube, optionally diluting the sample to be tested with a diluent or not, then dripping it onto a detection photonic crystal biochip, waiting for at least 10 minutes, rinsing the photonic crystal biochip with a cleaning solution, and placing the photonic crystal biochip in a portable fluorescence detection instrument to read the fluorescence value.

[0072] According to an embodiment of the present invention, the sample to be tested is selected from saliva.

[0073] According to an embodiment of the present invention, after the fluorescence value is measured, the content of the target CGRP in the sample to be tested is optionally calculated according to a standard curve.

[0074] Beneficial effects of the present invention:

[0075] 1. For the detection of CGRP, a target that is easily degraded and has ultra-low concentration, the present invention provides a rapid and simple detection method and optical detection platform.

[0076] 2. Compared with the ELISA test which takes several hours, the detection platform of the present invention is simple and easy to operate, and the detection is fast (only 10 minutes), which can quickly assist clinicians in diagnosing migraine.

[0077] 3. The present invention provides a rapid and highly sensitive detection for CGRP detection, which can quickly distinguish migraine from other tension-type headaches, which has important guiding significance for doctors in treating and prescribing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Gain effect for photonic crystal biochip.

[0079] Figure 2 Flow chart for the competition assay of CGRP.

[0080] Figure 3 This is the standard curve of the photonic crystal biochip prepared in Example 2 (taking CGRP as an example). DETAILED DESCRIPTION

[0081] The present invention also provides a method for preparing the above-mentioned photonic crystal ink, comprising the following steps: mixing photonic crystals, a moisturizing agent and a wetting agent to prepare the photonic crystal ink; the photonic crystals have the meaning as described above.

[0082] Preferably, the photonic crystals are monodisperse latex spheres.

[0083] According to an embodiment of the present invention, the photonic crystal, moisturizing agent and wetting agent have a mass ratio as shown above.

[0084] According to an embodiment of the present invention, the moisturizing agent and the humectant have the selections as shown above.

[0085] The present invention also provides a method for preparing the above-mentioned photonic crystal structure, comprising the following steps: forming the photonic crystal structure by self-assembly of photonic crystals on the substrate.

[0086] Preferably, the method for preparing the photonic crystal structure comprises the following steps: forming the photonic crystal structure by self-assembly on the substrate by inkjet printing the photonic crystal ink.

[0087] According to an embodiment of the present invention, the photonic crystal has the meaning as described above. Preferably, the photonic crystal is provided by the above-mentioned photonic crystal ink.

[0088] According to an embodiment of the present invention, the substrate has the selection as shown above. Preferably, the substrate is a PET film; more preferably, the contact angle of the PET film is 70°.

[0089] According to an embodiment of the present invention, the method for preparing the photonic crystal structure further comprises thermally sintering the obtained photonic crystal structure. After thermal sintering, the photonic crystal can be better solidified on the PET substrate to achieve water resistance.

[0090] According to an embodiment of the present invention, the conditions of the thermal sintering can be adjusted according to the particle size of the photonic crystal. For example, the temperature of the thermal sintering is 100-150° C., preferably 110-130° C., for example 120° C. For example, the time of the thermal sintering is 10-20 min.

[0091] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0092] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0093] Preparation Example 1

[0094] Preparation of photonic crystal biochips: The printable polystyrene ink is made of carboxyl-modified polystyrene microspheres (290 nm), mixed with ethylene glycol and surfactant BYK3455 in a mass / volume ratio of 95:4:1. The specific preparation process is as follows:

[0095] (1) The photonic crystal structure (the microarray includes a 3x 3 dot array, and the diameter of a single dot in the dot array is d = 600 μm; each 3x 3 dot array is a "detection unit" described below) was printed on a polyethylene terephthalate (PET) substrate using a Sonoplot Microplotter II. The volume of each droplet was about 400 pL.

[0096] (2) The printed photonic crystal structure was naturally dried at room temperature (25°C) and then placed in an oven at 110°C for sintering for 10 min to fix the photonic crystal structure on the PET substrate.

[0097] (3) Add 30 μL of a mixed solution of EDC (40 mg / mL) and NHS (40 mg / mL) to each detection unit and let it stand for 10 min to completely activate the carboxyl termini on the surface of the photonic crystal.

[0098] (4) Add 10 μL of anti-CGRP antibody (0.58 μg / mL) to each test microarray and react for 2 h.

[0099] (5) Use 1% BSA buffer + 0.005% sodium caseinate to block the unreacted carboxyl groups on the surface of the photonic crystal for 30 minutes, and then rinse with deionized water for 1 minute to remove excess blocking solution to obtain a photonic crystal biochip.

[0100] All the above steps were carried out at room temperature (25°C). The prepared photonic crystal biochip was stored at 4°C.

[0101] Example 1

[0102] Test photonic crystal chip (d = 600 μm; 3 x 3) for Cy5 fluorescence gain:

[0103] Take the photonic crystal biochip of Preparation Example 1, add Cy5-CGRP solution (0.5 μL, 7.9 μg / mL) to the photonic crystal lattice and pure PET substrate in the chip respectively, and after drying, use a portable fluorescence tester to read the fluorescence intensity value.

[0104] The average fluorescence intensity of Cy5 on the photonic crystal lattice is 3445; the average fluorescence intensity of Cy5 on the pure PET substrate is 121. By calculating the average fluorescence intensity of Cy5 on the photonic crystal / the average fluorescence intensity of Cy5 on the pure PET substrate, the fluorescence gain of the photonic crystal is 28.4 times. Figure 1 shown.

[0105] Example 2

[0106] A standard curve for detecting CGRP markers using the photonic crystal biochip in Preparation Example 1 was established, and a competitive detection process was used. The principle is as follows: Figure 2 As shown:

[0107] (1) The CGRP antigen standard was diluted to different concentrations (0.05 pg / mL-1000 pg / mL, specifically 0.05 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL), and mixed with Cy5-CGRP (7.9 μg / mL) at a ratio of 8:2 to obtain test solutions of different concentrations;

[0108] (2) Take 30 μL of the above-mentioned test solutions of different concentrations and add them dropwise to the detection unit of a photonic crystal chip, and react for 10 minutes at room temperature and in the dark. Then rinse with deionized water to terminate the reaction.

[0109] (3) Use a portable fluorescence tester to detect the fluorescence intensity and read the fluorescence intensity of the detection unit corresponding to the different test solutions. The standard curve of the photonic crystal biochip for detecting CGRP is shown in Figure 3 As shown by Figure 3 It can be seen that the detection limit of the photonic crystal biochip prepared by the present invention can be as low as 0.05 pg / mL.

[0110] Example 3

[0111] The detection method of CGRP in the clinical samples to be tested is as follows:

[0112] 1) Collect saliva samples as clinical samples to be tested, dilute the collected saliva samples to 1 / 10 of the original concentration, mix with Cy5-CGRP (7.9 μg / mL) at a volume ratio of 8:2, and then dilute with PBS solution (pH=7.2-7.4) to obtain the sample to be tested.

[0113] 2) Take the photonic crystal biochip of Preparation Example 1, and drop 30 μL of the sample to be tested in step 1) into the detection unit in the chip of Preparation Example 1. After reacting for 10 minutes, rinse with deionized water to terminate the reaction, and use a portable fluorescence tester to read the fluorescence intensity value A.

[0114] 3) Compare the fluorescence intensity value obtained in step 2) with Figure 3 , read the corresponding CGRP concentration, and then convert it to get the true concentration of CGRP in the saliva sample, recorded as ρ PC .

[0115] Saliva samples of 65 clinical patients were tested by repeating the above steps 1)-3) respectively, and the test results of the corresponding samples were recorded in Table 1.

[0116] Table 1 Statistics of the test results of CGRP content in clinical saliva samples

[0117]

[0118]

[0119] The above is a description of the exemplary embodiments of the present invention. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A photonic crystal biochip, characterized in that: The photonic crystal biochip comprises a photonic crystal structure and an immune detection system; the immune detection system is fixed on the photonic crystal structure, and the immune detection system contains antibodies of biological detection markers; The photonic crystal structure is formed by self-assembly of photonic crystals on a substrate.

2. The photonic crystal biochip according to claim 1, characterized in that: The immunodetection system is fixed on the photonic crystal structure through a coupling reaction. Preferably, the biomarker is a small molecule polypeptide that is easily degradable and has an ultra-low concentration. Preferably, the immunoassay system contains an antibody to CGRP. Preferably, the ultra-low concentration refers to a concentration of 0.1-1000 pg / mL. Preferably, the photonic crystal structure is a periodic regular arrangement formed by self-assembly of the photonic crystals on a substrate. Preferably, the photonic crystals are monodisperse latex spheres. Preferably, the particle size of the monodisperse latex spheres is 150-320 nm. Preferably, the monodisperse latex spheres are selected from at least one of poly(methyl methacrylate-acrylic acid-styrene) latex spheres, silica microspheres, and polystyrene microspheres. Preferably, the surface of the monodisperse latex spheres has -COOH functional groups.

3. The photonic crystal biochip according to claim 1 or 2, characterized in that: The photonic crystal is provided by photonic crystal ink. Preferably, the photonic crystal structure is formed by self-assembly on a substrate by inkjet printing of the photonic crystal ink. Preferably, the photonic crystal ink comprises photonic crystals, a moisturizing agent and a wetting agent. Preferably, in the photonic crystal ink, the mass concentration of the photonic crystals is 5-30%. Preferably, the humectant is selected from at least one of ethylene glycol, propylene glycol, glycerol and sorbitol. Preferably, in the photonic crystal ink, the mass concentration of the moisturizing agent is 5-10%. Preferably, the wetting agent is selected from at least one of BYK-3400, BYK-3455, BYK-151, and BKY-154. Preferably, in the photonic crystal ink, the mass concentration of the wetting agent is 0.5-5‰.

4. The photonic crystal biochip according to any one of claims 1 to 3, characterized in that: The photonic crystal ink contains a solvent. Preferably, the photonic crystal structure is a water-resistant photonic crystal structure. Preferably, the photonic crystal structure further comprises a substrate. Preferably, the photonic crystal structure has a fluorescence gain effect. Preferably, the immunoassay system is selected from an immunocompetitive assay system.

5. The method for preparing the photonic crystal biochip according to any one of claims 1 to 4, comprising: The immune detection system is fixed on the photonic crystal structure to form the photonic crystal biochip. Preferably, the immunoassay system is fixed on the photonic crystal structure through a coupling reaction to form the photonic crystal biochip. According to a preferred embodiment of the present invention, the method for preparing the photonic crystal biochip specifically comprises the following steps: (1) preparing a photonic crystal structure; (2) activating the photonic crystal in the photonic crystal structure so that a large number of -COOH functional groups are exposed on the surface of the photonic crystal; (3) The immunoassay system and the photonic crystal are subjected to an amide reaction, and the immunoassay system is fixed on the photonic crystal on the photonic crystal structure to obtain the photonic crystal biochip.

6. Use of the photonic crystal biochip according to any one of claims 1 to 4 in detecting CGRP.

7. A method for detecting CGRP, comprising: The sample to be tested is contacted with the photonic crystal biochip according to any one of claims 1 to 4, and the target CGRP is identified through fluorescence detection. Preferably, the detection method comprises the following steps: a) contacting the photonic crystal biochip with a standard immunoassay system of different concentrations, measuring the fluorescence value, and constructing a standard curve of the immunoassay system; b) contacting the sample to be tested with the photonic crystal biochip, measuring the fluorescence value, and calculating the content of the target CGRP in the sample to be tested according to the standard curve. Preferably, the volume of the sample to be tested is 10 μL to 50 μL. Preferably, the sample to be tested is saliva. Preferably, in step b), rinsing is performed after contacting. Preferably, in step b), the lower limit of the concentration of the target CGRP detected is at least 0.01 pg / mL.

8. A fluorescence detection device, comprising at least the photonic crystal biochip according to any one of claims 1 to 4.

9. Use of the fluorescence detection device according to claim 8 in detecting CGRP.

10. The detection method of the fluorescence detection device according to claim 8, the detection method comprising collecting the sample to be tested into a sampling tube, optionally diluting the sample to be tested with a diluent or not, then dripping it onto the detection photonic crystal biochip, waiting for at least 10 minutes, rinsing the photonic crystal biochip with a cleaning solution, and placing the photonic crystal biochip in a portable fluorescence detection instrument to read the fluorescence value.

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