An ultrahigh-sensitivity photonic crystal chip, a preparation method thereof and application thereof in detection of CGRP

Abstract

The application discloses an ultrahigh-sensitivity photonic crystal chip, a preparation method thereof and application of the photonic crystal chip in detection of CGRP. The photonic crystal biological chip comprises a photonic crystal structure and an immune detection system; the immune detection system is fixed on the photonic crystal structure, the immune detection system contains antibodies of a biological detection marker; and the photonic crystal structure is formed by self-assembly of photonic crystals on a base material. For detection of CGRP, a target object which is easy to degrade and has an ultralow concentration, the application provides a quick and simple detection method and an optical detection platform. Compared with ELISA detection which needs several hours of detection time, the detection platform of the application is simple to operate and quick in detection (only 10 minutes are needed).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of material science and biology, and relates to a marker detection chip and a detection method, in particular to a photonic crystal chip with ultra-high sensitivity and a preparation method thereof and application thereof in detection of CGRP. BACKGROUND

[0002] Migraine is a disabling primary headache characterized by recurrent attacks of unilateral pulsating moderate to severe pain that is aggravated by movement. During migraine attacks, patients are often bedridden and unable to fulfill social and life obligations. In clinical practice, atypical migraine is often similar to other types of headache (such as tension-type headache), and it is difficult to make a definitive differential diagnosis based on the symptoms of the patient. Calcitonin gene-related peptide (CGRP) is a small molecule polypeptide that is rapidly degraded after release into the plasma with a half-life of about 10 minutes. It is considered a reliable biomarker of 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 migraine from tension-type headache. Its levels increase during migraine attacks, while remaining unchanged during tension-type headache.

[0003] Currently, the enzyme-linked immunosorbent assay (ELISA) method is considered the gold standard for detecting CGRP, with better sensitivity and reproducibility. However, this method also has the disadvantages of long analysis time, cumbersome operation and the need for professional knowledge, which severely limits its application in clinical practice, especially in the case of rapidly degrading and extremely low concentration 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 is both fast and highly sensitive in a short period of time. SUMMARY

[0004] For the detection of target substances such as calcitonin gene-related peptide (CGRP) that are easily degraded and have ultra-low concentrations, the present application provides a photonic crystal biological chip and a preparation method and detection method thereof.

[0005] The technical solutions provided by the present application are as follows:

[0006] A photonic crystal biological chip, the photonic crystal biological chip comprising a photonic crystal structure and an immunodetection system; the immunodetection system is fixed on the photonic crystal structure, and the immunodetection system contains antibodies of a biological detection marker;

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

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

[0009] According to an embodiment of the present application, the biological detection marker is a small molecule polypeptide such as CGRP which is easily degradable and in ultra-low concentration. Preferably, the immune detection system contains antibodies of CGRP. Preferably, the ultra-low concentration refers to a concentration of 0.1-1000 pg / mL, for example 0.5 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL.

[0010] According to an embodiment of the present application, 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 application, the particle size of the monodisperse latex spheres is 150-320 nm, for example 180-280 nm, and is exemplarily 180 nm, 190 nm, 200 nm, 205 nm, 215 nm, 220 nm, 230 nm, 250 nm, 260 nm, 280 nm.

[0012] According to an embodiment of the present application, 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., and are preferably poly(methyl methacrylate-acrylic acid-styrene) latex spheres.

[0013] According to an embodiment of the present application, the surface of the monodisperse latex spheres is provided with -COOH functional groups.

[0014] According to a preferred embodiment of the present application, 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., and are preferably carboxyl-modified poly(methyl methacrylate-acrylic acid-styrene) latex spheres.

[0015] According to an embodiment of the present application, the photonic crystals are provided by a photonic crystal ink.

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

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

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

[0019] According to an embodiment of the present application, the humectant can be at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerol, sorbitol, and the like, for example, ethylene glycol.

[0020] According to an embodiment of the present application, the mass concentration of the humectant in the photonic crystal ink is 5-10%, for example, 6-9%, and exemplarily 7%, 8%.

[0021] According to an embodiment of the present application, the wetting agent can be at least one selected from the group consisting of BYK-3400, BYK-3455, BYK-151, BKY-154, and the like, for example, BYK-3400.

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

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

[0024] According to an exemplary embodiment of the present application, 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, 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 an embodiment of the present application, the pattern of the photonic crystal structure is not particularly limited, 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 application, the photonic crystal structure is a water-resistant photonic crystal structure.

[0028] According to an embodiment of the present application, the photonic crystal structure can further comprise a substrate.

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

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

[0031] According to a preferred embodiment of the present application, the photonic crystal structure is prepared by a method comprising the following steps: inkjet printing by photonic crystal ink, self-assembly of the photonic crystal structure on a substrate and heat sintering, wherein the heat sintering is performed at a temperature of 100-150°C; and 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, a humectant, a wetting agent and a solvent; wherein the photonic crystals are monodisperse latex spheres.

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

[0035] The humectant 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 crystals is 5-30%, the mass concentration of the humectant is 5-10%, and the mass concentration of the wetting agent is 0.5-5‰.

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

[0039] According to an embodiment of the present application, the immune detection system is selected from an immune competition detection system.

[0040] The present application also provides a preparation method of the above photonic crystal biochip, which comprises: fixing an immune detection system on the photonic crystal structure to form the photonic crystal biochip.

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

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

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

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

[0045] (3) fixing the immune detection system on the photonic crystals in the photonic crystal structure through an amide reaction, so that the immune detection system is fixed on the photonic crystals in the photonic crystal structure, and the photonic crystal biochip is obtained.

[0046] According to an embodiment of the present application, 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 used in concentrations known in the art, for example, the concentration of EDC is 30-50 mg / mL, for example, 40 mg / mL; for example, the concentration of NHS is 4-50 mg / mL, for example, 40 mg / mL.

[0047] The present application also provides a use of the above-mentioned photonic crystal biochip in detecting CGRP.

[0048] The present application 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 application, the method for detecting CGRP comprises the following steps:

[0050] a) contacting standard immune detection systems with different concentrations with the photonic crystal biochip, determining the fluorescence value, and constructing a standard curve of the immune detection system;

[0051] b) contacting a sample to be tested with the photonic crystal biochip, determining 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 application, the volume of the sample to be tested is 10 μL-50 μL.

[0053] According to an embodiment of the present application, the standard curve is, for example, as shown in the following formula: Figure 3

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

[0055] According to an embodiment of the present application, the contacting can be carried out under conditions known in the art, for example, at room temperature, and the contacting time is 5-15 min, for example, 10 min.​

[0056] According to an embodiment of the present application, the fluorescence detection can be performed by using a device known in the art, for example, a portable fluorescence detector is used to test the ice and read data.

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

[0058] Preferably, in step b), the concentration of the target CGRP detected 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 application further provides a fluorescence detection device, which comprises at least the photonic crystal biochip.

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

[0061] According to an embodiment of the present application, the fluorescence detection device preferably comprises a CGRP detection kit, which comprises the photonic crystal biochip.

[0062] According to an embodiment of the present application, 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 application, the diluent is selected from at least one of deionized water and PBS buffer.

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

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

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

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

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

[0069] According to an embodiment of the present application, the sampling tube and the washing dropper can be prepared from materials known in the art, which are not specifically limited in the present application.

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

[0071] The present application also provides a detection method of the above-mentioned fluorescence detection device, which comprises collecting a sample to be detected into a sampling tube, optionally diluting the sample to be detected with a diluent or without dilution, then adding dropwise to a photonic crystal biochip, waiting for at least 10 min, then washing the photonic crystal biochip with a washing 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 application, the sample to be detected is selected from saliva.

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

[0074] The present application has the following beneficial effects:

[0075] 1. For the detection of CGRP, a target substance which is prone to degradation and has an ultra-low concentration, the present application provides a rapid and simple detection method and an optical detection platform.

[0076] 2. Compared with ELISA detection which requires several hours of detection time, the detection platform of the present application is simple to operate and rapid in detection (only 10 min is required), and can quickly assist clinicians in diagnosing migraine.

[0077] 3. The present application provides rapid and high-sensitivity detection for CGRP detection, which can quickly distinguish migraine from other tension-type headaches, which has important guiding significance for doctors in treatment. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 For the gain effect of the photonic crystal biochip.

[0079] Figure 2 Flow chart for detecting CGRP by competition method.

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

[0081] The present application also provides a preparation method of the above-mentioned photonic crystal ink, which comprises the following steps: mixing photonic crystals, a humectant and a wetting agent to prepare the photonic crystal ink; the photonic crystals have the meanings as described above.

[0082] Preferably, the photonic crystal is monodisperse latex spheres.

[0083] According to an embodiment of the present application, the photonic crystal, the humectant and the wetting agent are matched in mass ratio as shown above.

[0084] According to an embodiment of the present application, the humectant and the wetting agent are selected as shown above.

[0085] The present application also provides a method for preparing the photonic crystal structure as described above, comprising the step of self-assembling the photonic crystal on the substrate to form the photonic crystal structure.

[0086] Preferably, the method for preparing the photonic crystal structure comprises the step of self-assembling the photonic crystal ink as described above on the substrate by inkjet printing to form the photonic crystal structure.

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

[0088] According to an embodiment of the present application, 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 application, the method for preparing the photonic crystal structure further comprises heat sintering the obtained photonic crystal structure. The photonic crystal is better solidified on the PET substrate after heat sintering, achieving water resistance.

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

[0091] The technical solutions of the present application will be further described in detail below with reference to specific examples. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[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 biochip: wherein the printable polystyrene ink was prepared by mixing carboxyl-modified polystyrene microspheres (290 nm) with ethylene glycol and surfactant BYK3455 at a mass / volume ratio of 95:4:1, and the specific preparation process was as follows:

[0095] (1) The photonic crystal structure (including a 3x 3 dot array in the microarray, and the diameter d of a single dot in the dot array was 600 μm; each 3x 3 dot array was 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 10 min to sinter the photonic crystal structure and fix it on the PET substrate.

[0097] (3) 30 μL of a mixed solution of EDC (40 mg / mL) and NHS (40 mg / mL) was added to each detection unit, and the mixture was allowed to stand for 10 min to completely activate the carboxyl terminals on the surface of the photonic crystal.

[0098] (4) 10 μL of anti-CGRP antibody (0.58 μg / mL) was added to each test microarray, and the reaction was allowed to proceed for 2 h.

[0099] (5) The unreacted carboxyl groups on the surface of the photonic crystal were blocked using 1% BSA buffer + 0.005% sodium casein, and the blocking was allowed to proceed for 30 min, followed by washing with deionized water for 1 min to remove excess blocking solution, thereby obtaining 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 of photonic crystal chip (d = 600 μm; 3x 3) on Cy5 fluorescence gain:

[0103] The photonic crystal biochip prepared in Preparation Example 1 was taken, and Cy5-CGRP solution (0.5 μL, 7.9 μg / mL) was added to the photonic crystal dot array and the pure PET substrate on the chip, respectively. After drying, the fluorescence intensity value was read using a portable fluorescence tester.

[0104] The average value of the fluorescence intensity value of Cy5 on the photonic crystal lattice is 3445; the average value of the fluorescence intensity value of Cy5 on the pure PET substrate is 121. The fluorescence gain multiple of the photonic crystal can be obtained by calculating the average value of the fluorescence intensity value of Cy5 on the photonic crystal / the average value of the fluorescence intensity value of Cy5 on the pure PET substrate, which is 28.4 times. The gain effect of the photonic crystal biochip is shown in Figure 1 .

[0105] Example 2

[0106] A standard curve for detecting the CGRP marker of the photonic crystal biochip in Preparation Example 1 is established, and a competitive detection process is used, and the principle is shown in Figure 2 .

[0107] (1) The CGRP antigen standard is 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 different concentrations of the to-be-detected solution;

[0108] (2) 30 μL of the to-be-detected solution of the above different concentrations is added dropwise to a detection unit of a photonic crystal chip, and reacted at room temperature and in the dark for 10 min. Then, the reaction is terminated by washing with deionized water.

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

[0110] Example 3

[0111] The detection method of CGRP in the to-be-detected clinical sample is as follows:

[0112] 1) Collect a saliva sample as a to-be-detected clinical sample, and dilute the collected saliva sample 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 a PBS solution (pH=7.2-7.4) to obtain a to-be-detected sample.

[0113] 2) Take the photonic crystal biochip of Preparation Example 1, and add 30 μL of the to-be-detected sample of step 1) to the detection unit in the chip of Preparation Example 1, terminate the reaction after reacting for 10 min by washing with deionized water, and read the fluorescence intensity value A using a portable fluorescence tester.

[0114] 3) The fluorescence intensity value obtained in step 2) is compared with the standard curve Figure 3 , and the corresponding CGRP concentration is read, and after conversion, the real concentration of CGRP in the saliva sample is obtained, denoted as p PC .

[0115] The saliva samples of 65 patients in the clinic are tested, and the above steps 1)-3) are repeated, and the detection results corresponding to the samples to be tested are recorded in Table 1.

[0116] Table 1: Detection results of CGRP content in clinical saliva samples

[0117]

[0118]

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

Claims

1. A photonic crystal biochip, characterized by, 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 a biological detection marker; The photonic crystal structure is formed by self-assembly of photonic crystals on a substrate; the photonic crystal structure is provided by photonic crystal ink; the photonic crystal structure is formed by self-assembly of the photonic crystal ink on a substrate through inkjet printing; the photonic crystal ink comprises photonic crystals, a humectant and a wetting agent; The photonic crystals in the photonic crystal structure are monodisperse latex spheres; the monodisperse latex spheres are selected from at least one of poly(methyl methacrylate-acrylic acid-styrene) latex spheres, silica microspheres and polystyrene microspheres; the surface of the monodisperse latex spheres is provided with -COOH functional groups; The immune detection system is fixed on the photonic crystal structure through a coupling reaction; The biological detection marker is a small molecule polypeptide which is easily degradable and has an ultra-low concentration; the immune detection system contains antibodies of calcitonin gene-related peptide; the ultra-low concentration refers to a concentration of 0.1-1000 pg / mL.

2. The photonic crystal biochip of claim 1, wherein, The photonic crystal structure is a periodic regular arrangement formed by self-assembly of the photonic crystals on a substrate.

3. The photonic crystal biochip of claim 1, wherein, The particle size of the monodisperse latex spheres is 150-320 nm.

4. The photonic crystal biochip of claim 1, wherein, In the photonic crystal ink, the mass concentration of the photonic crystals is 5-30%; The humectant is selected from at least one of ethylene glycol, propylene glycol, glycerol and sorbitol; In the photonic crystal ink, the mass concentration of the humectant is 5-10%; The wetting agent is selected from at least one of BYK-3400, BYK-3455, BYK-151 and BKY-154; In the photonic crystal ink, the mass concentration of the wetting agent is 0.5-5 ‰; The photonic crystal ink contains a solvent.

5. The photonic crystal biochip of claim 1, wherein, The photonic crystal structure is a water-resistant photonic crystal structure; The photonic crystal structure further comprises a substrate; The photonic crystal structure has a fluorescence gain effect; The immune detection system is selected from an immune competition detection system.

6. The method for preparing a photonic crystal biochip according to any one of claims 1 to 5, wherein, It comprises: The immune detection system is fixed on the photonic crystal structure to form the photonic crystal biochip; The immune detection system is fixed on the photonic crystal structure through a coupling reaction.

7. The production method according to claim 6, characterized by, The preparation method of the photonic crystal biochip specifically comprises the following steps: (1) preparing a photonic crystal structure; (2) activating the photonic crystals in the photonic crystal structure to expose a large number of -COOH functional groups on the surface of the photonic crystals; (3) fixing the immune detection system on the photonic crystals in the photonic crystal structure through an amide reaction to obtain the photonic crystal biochip.

8. Use of the photonic crystal biochip according to any one of claims 1-5 in detection of CGRP.

9. A method for detecting CGRP, characterized by, The detection method comprises: contacting a sample to be detected with the photonic crystal biochip according to any one of claims 1-5, and recognizing the target CGRP through fluorescence detection.

10. The detection method according to claim 9, characterized in that, The detection method comprises the following steps: a) contacting the photonic crystal biochip with a standard immune detection system at different concentrations, determining the fluorescence value, and constructing a standard curve of the immune detection system; b) contacting the sample to be detected with the photonic crystal biochip, determining the fluorescence value, and calculating the content of the target CGRP in the sample to be detected according to the standard curve.

11. The detection method according to claim 10, characterized in that, The volume of the sample to be detected is 10 μL to 50 μL; The sample to be detected is saliva; In step b), after the contacting, rinsing is further performed; In step b), the lower limit of the concentration of the target CGRP detected is at least 0.01 pg / mL.

12. A fluorescence detection device, which comprises at least the photonic crystal biochip according to any one of claims 1 to 5.

13. The use of the fluorescence detection device according to claim 12 in the detection of CGRP.

14. The detection method of the fluorescence detection device according to claim 12, which comprises collecting the sample to be detected into a sampling tube, optionally diluting the sample to be detected with a diluent or without dilution, then dropping onto the detection photonic crystal biochip, waiting for at least 10 min, rinsing the photonic crystal biochip with a rinsing solution, and placing the photonic crystal biochip in a portable fluorescence detection instrument to read the fluorescence value.

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