Three-dimensional gel photonic crystal, preparation method thereof and application of three-dimensional gel photonic crystal in glycocholic acid detection

Abstract

The invention discloses a three-dimensional gel photonic crystal. The three-dimensional gel photonic crystal is prepared by the following steps: preparing polyvinyl alcohol three-dimensional PCH, acrylated glycocholic acid and an acrylated glycocholic acid antibody, and preparing glycocholic acid antigen-antibody cross-linked PCH through a photopolymerization reaction. The PCH prepared by the invention can be used for detecting glycocholic acid on the basis of a competitive immunoassay principle, free glycocholic acid molecules in a sample and covalently loaded glycocholic acid molecules in hydrogel compete to be combined with covalently loaded glycocholic acid antibodies, so that the initial antigen-antibody crosslinking degree in the hydrogel is reduced to cause the volume change of the hydrogel; further, the structural color change and diffraction wavelength migration of the PCH are caused, so that the visual semi-quantitative and optical quantitative analysis of the glycocholic acid is realized. The PCH-based sensor is simple and convenient to use, can meet the requirement of instant detection, and has the advantages of high sensitivity, strong specificity and reusability, the actual application potential of the sensor is also proved by an artificial serum standard recovery experiment, and the PCH-based sensor has a very good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of sensors and immunoassay, and particularly relates to a three-dimensional gel photonic crystal (PCH), a preparation method thereof and application thereof in detection of glycocholic acid. BACKGROUND

[0002] Liver disease is one of the major diseases of human beings, which has affected the health of millions of people in the past few decades. Liver diseases such as alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), hepatitis C virus (HCV) and hepatitis B virus (HBV) infection, hepatocellular carcinoma (HCC) and cirrhosis, etc. pose a great threat to human health. Early diagnosis and precise medical analysis of liver disease are of great significance for the prevention and treatment of liver disease.

[0003] In recent years, glycocholic acid has become a new type of liver disease biomarker due to its strong specificity and high sensitivity. When liver function is impaired, glycocholic acid cannot be effectively absorbed by hepatocytes, resulting in a significant increase in the content of glycocholic acid in the blood. Previous studies have shown that the content of glycocholic acid in the blood and urine of patients with intrahepatic cholestasis during pregnancy, cirrhosis, viral hepatitis and liver cancer is significantly increased, and the content of glycocholic acid is positively correlated with the degree of hepatocyte damage, and is not affected by jaundice, biliary system diseases and other non-liver diseases. Studies have shown that the glycocholic acid level of cirrhosis patients is about 3-100 times higher than that of healthy people. Based on its specificity and high sensitivity, glycocholic acid has great advantages as a liver disease biomarker. The detection of glycocholic acid in blood and urine can accurately reflect the degree of hepatocyte damage, providing an important basis for the diagnosis, treatment and prognosis analysis of liver disease. The detection methods of glycocholic acid mainly include instrumental analysis and immunoassay. Instrumental analysis includes thin layer scanning quantitative method, thin layer fluorescence method and chromatography method, etc. Although instrumental analysis has high accuracy, the instrument equipment is expensive and complex, the processing process is tedious and time-consuming, the cost is high, and the personnel technology is required. Immunoassay includes radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, lateral flow immunoassay (LFIA) and fluorescence polarization immunoassay (FPIA), etc. However, the current established immunoassay still has the disadvantages of radioactive substance hazards, tedious detection steps, long detection time, high requirements for instrument equipment and operating personnel, etc. It is of great significance to develop a new type of glycocholic acid content instant detection method which is convenient and fast.

[0004] Photonic crystal is a periodic and highly ordered structure assembled by monodisperse colloidal particles (micron / sub-micron level), which has photonic band gap characteristics. Due to the existence of photonic band gap, photonic crystal exhibits unique structural color and diffraction wavelength. Its diffraction wavelength is mainly related to its average refractive index (n) and lattice spacing (d) and follows the Bragg formula (mλ=2ndsinθ, where m is the diffraction order, λ is the diffraction wavelength, n is the average refractive index of the photonic crystal, d is the lattice spacing of the photonic crystal, and θ is the angle of incident light). Combining responsive hydrogel with photonic crystal template can obtain photonic crystal hydrogel (PCH), which can respond to external stimuli (such as temperature, pH, analyte concentration, etc.) by changing its n or d, thereby changing its structural color and shifting its diffraction wavelength, realizing sensing function. The change of structural color of PCH can be directly observed by naked eye, and the change of diffraction wavelength can be quantitatively determined by fiber spectrometer, making it stand out in biological and chemical visualization sensing. So far, no sensor and corresponding detection method for real-time detection of glycocholic acid content by using the above PCH have been found. SUMMARY

[0005] The present application provides a three-dimensional gel photonic crystal, which can be used as a sensor based on the principle of competitive immunoassay and for visual qualitative and spectral quantitative detection of liver biomarker glycocholic acid.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] A three-dimensional gel photonic crystal (PCH) is obtained by the following steps:

[0008] S1, acrylate glycocholic acid to obtain acrylated glycocholic acid;

[0009] S2, acrylate glycocholic acid antibody to obtain acrylated glycocholic acid antibody;

[0010] S3, dissolve polyvinyl alcohol in a solvent to form a sol, mix the obtained sol with a nanometer microsphere suspension and repeatedly heat and shake until a strong structural color is obtained to obtain a photonic crystal sol, and then inject the photonic crystal sol into a mold to form a polyvinyl alcohol PCH by freezing and thawing;

[0011] S4, mix monomer, crosslinking agent, photoinitiator, acrylated antigen obtained in step S1 and acrylated antibody obtained in step S2 to obtain a pre-polymer solution, and then add the polyvinyl alcohol PCH in step S3 to perform photopolymerization reaction. After the reaction is completed, purify and heat in water bath to remove polyvinyl alcohol to obtain the three-dimensional PCH.

[0012] The amount of the cross-linking agent in the prepolymer solution is 0.10-0.30 wt %; the mass ratio of the acrylated antibody to the acrylated glycocholic acid antigen is 1-60:1.

[0013] The principle and process of this invention for detecting glycocholic acid using three-dimensional PCH are as follows: the prepared PCH is immersed in a sample solution containing glycocholic acid. Free glycocholic acid molecules in the solution compete with covalently loaded glycocholic acid in the hydrogel for binding to the covalently loaded glycocholic acid antibody, reducing the initial antigen-antibody crosslinking in the hydrogel and causing the hydrogel to expand. This, in turn, causes a change in the structural color of the PCH and a shift in the diffraction wavelength. The higher the glycocholic acid content in the sample, the more pronounced the structural color change of the PCH and the greater the shift in the diffraction wavelength.

[0014] Preferably, step S1 includes the following steps:

[0015] S11, reacting glycocholic acid with an amination reagent, wherein the amination reagent is tert-butyl (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate, to generate terminally aminated glycocholic acid -NH2;

[0016] S12, glycocholic acid-NH2 and N-acryloyloxysuccinimide react to obtain acrylated glycocholic acid in a molar ratio of 1:1 to 1:1.5.

[0017] More preferably, the amination reaction temperature in step S11 is room temperature, and the crude reaction product is purified by silica gel column chromatography, with the mobile phase being a DCM / MeOH mixed solution (DCM / MeOH = 10:1 to 1:1) containing 1 v / v% ammonia water.

[0018] Preferably, in step S2, N-acryloyloxysuccinimide reacts with the primary amino group of the lysine residue in the glycocholic acid antibody molecule to form an amide bond to obtain the acrylated antibody; the molar ratio of N-acryloyloxysuccinimide to the glycocholic acid antibody molecule is 1:5 to 1:15.

[0019] More preferably, the glycocholic acid antibody in step S2 is selected from a monoclonal antibody, a polyclonal antibody, a nanobody or a single-chain antibody.

[0020] Preferably, the molecular weight of the polyvinyl alcohol in step S3 is 10,800-108,000 Da; and the mass concentration of the polyvinyl alcohol sol is 5-15 wt %.

[0021] Preferably, the nanosphere material used in step S3 is selected from one or more of silicon dioxide, polystyrene, polymethyl methacrylate, titanium dioxide, iron oxide, gold, and silver. Specifically, the iron oxide can be ferroferric oxide Fe3O4.

[0022] More preferably, the particle size of the nanospheres used in step S3 is 50-200 nm; the mass concentration of the nanosphere suspension is 5-30%.

[0023] Preferably, the mass ratio of the polyvinyl alcohol sol to the nanosphere suspension in step S3 is 3:1-1:3.

[0024] More preferably, the solvent used in step S3 is any one or more of water, dimethyl sulfoxide, dimethyl formamide, ethylene glycol, and glycerol.

[0025] Preferably, the monomer of the hydrogel pre-polymer solution in step S4 is one or more of acrylamide, methyl methacrylate, polyhydroxyethyl methacrylate, cellulose acetate butyrate, siloxane methacrylate, fluorosilicon methacrylate, N-vinyl pyrrolidone, glycidyl methacrylate, or ethylene glycol dimethacrylate.

[0026] More preferably, the crosslinking agent of the hydrogel pre-polymer solution in step S4 is one or more of N,N'-methylene bisacrylamide, polyethylene glycol diacrylate, divinylbenzene, ethylene glycol dimethacrylate, 1,6-hexanediol diacrylate, and polyethylene glycol dimethacrylate.

[0027] More preferably, the freeze-thawing in step S4 is freezing at -20℃ for 6-18 hours and thawing at room temperature for 12-36 hours.

[0028] Preferably, the photopolymerization reaction in step S4 is performed using ultraviolet light irradiation, the wavelength of the ultraviolet light is 365 nm, the power is 3-16 W, and the polymerization time is 5-20 minutes.

[0029] The application also provides the use of the three-dimensional PCH as an immune analysis sensor in the detection of glycocholic acid.

[0030] Further, the PCH sensor is immersed in a sample solution containing glycocholic acid, the free glycocholic acid molecules in the solution and the covalently loaded glycocholic acid in the hydrogel compete to bind to the covalently loaded glycocholic acid antibody, which reduces the initial antigen-antibody cross-linking degree in the hydrogel, causing the volume of the hydrogel to expand, resulting in changes in the structural color of the PCH and a shift in the diffraction wavelength. The visual qualitative analysis of glycocholic acid can be achieved by observing the structural color change of the PCH with the naked eye or a mobile phone APP, and the optical quantitative analysis of glycocholic acid can be achieved by measuring the diffraction peak wavelength change of the PCH using a portable fiber-optic spectrometer. The higher the content of glycocholic acid in the solution, the more obvious the structural color change of the PCH, and the greater the change in the diffraction peak wavelength.

[0031] Compared with the prior art, the application has the following advantages:

[0032] The application is based on the principle of competitive immunoassay and the principle of structural color and diffraction wavelength change of photonic crystal, and a three-dimensional PCH immunological analysis sensor with distinct structural color containing antigen-antibody complex as a reversible crosslinking agent is prepared, the sensor can realize visual qualitative and spectral quantitative detection of glycocholic acid, is simple to use, can meet the needs of point-of-care testing (POCT), has the advantages of high sensitivity, strong specificity, reusability, and has practical application potential. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Preparation process of the PCH immunological analysis sensor;

[0034] Figure 2 Scanning electron microscope image of polystyrene microspheres;

[0035] Figure 3 Scanning electron microscope image of PCH after drying;

[0036] Figure 4 Reflection spectrum of 3D-PCH prepared by using different mass concentrations of Bis;

[0037] Figure 5 Reflection spectrum of 3D-PCH prepared by using different mass concentrations of acrylated glycocholic acid monoclonal antibody;

[0038] Figure 6 Reflection spectrum of PCH in different concentrations of glycocholic acid solution;

[0039] Figure 7 Standard curve of the wavelength shift value of the diffraction peak of PCH in different concentrations of glycocholic acid solution and the concentration;

[0040] Figure 8 Wavelength shift value of the diffraction peak of PCH in 100 μg / mL glycocholic acid and its structural analogues;

[0041] Figure 9 Reusability of PCH. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be described clearly and completely below by combining the examples and comparative examples of the present application. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0043] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0044] Example 1 Synthesis of polystyrene microspheres

[0045] like Figure 1 As shown, 50 mg of sodium bicarbonate and 38 mL of ultrapure water were added to a round-bottom flask. 17 mL of styrene and 500 mg of divinylbenzene were placed in a light-shielded constant pressure funnel. The stirring rate was set to 100 rpm, and nitrogen deoxygenation was performed for 10 minutes. 500 mg of sodium dihexyl sulfosuccinate was dissolved in 1 mL of ultrapure water and added to the round-bottom flask. Deoxygenation was continued for 10 minutes. The stirring rate was increased to 400 rpm, the temperature was raised to 50°C, styrene and divinylbenzene were added to the constant pressure funnel, and the addition rate was controlled at 0.1 mL / s. After the addition of the ingredients, 900 mg of sodium allylhydroxypropyl sulfonate was added. After the temperature was raised to 70°C, 230 mg of ammonium persulfate solution dissolved in 1 mL of water was added. After reacting under nitrogen for 2 hours, 300 mg of sodium allylhydroxypropyl sulfonate and 150 mg of ammonium persulfate solution dissolved in 1 mL of water were added, and the reaction was continued for 1 hour. After cooling to 50°C, the resulting polystyrene microsphere suspension was filtered through glass wool and dialyzed for 14 days using a dialysis bag (MW cutoff ~14,000Da). The purified polystyrene microsphere suspension exhibited a bright iridescent color. A small amount of the polystyrene microsphere suspension was dropped onto a glass slide, dried, sprayed with gold, and observed under a scanning electron microscope. Figure 2 As shown, the polystyrene microspheres self-assembled into a regular hexagonal structure with uniform particle size.

[0046] Example 2 Synthesis of Polyvinyl Alcohol Gel Photonic Crystals (PCH)

[0047] like Figure 1 As shown, 100 μL of 100 nm diameter monodisperse polystyrene colloidal microspheres (16 wt%) were added to 100 μL of a 10 wt% polyvinyl alcohol-DMSO sol. After adding 0.1 g of ion exchange resin, the suspension was heated until a distinct structural color was produced. The sol was then poured into a mold, frozen at -20°C for 12 hours, and then thawed at room temperature for 24 hours to obtain physically cross-linked PCH.

[0048] Example 3 Preparation of three-dimensional PCH containing glycocholic acid antigen-antibody crosslinking

[0049] like Figure 1 As shown,

[0050] (1) Synthesis of acrylated glycocholic acid: 100 mg of glycocholic acid, 88 mg of tert-butyl (14-amino-3,6,9,12-tetraoxatetradecyl) carbamate, 36 mg of N-hydroxy-7-azabenzotriazole, 50 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 100 μL of triethylamine were added in 2 mL of N,N-dimethylformamide (DMF) and stirred overnight at room temperature; after 2 h of stirring after adding 1 mL of trifluoroacetic acid in the product, N2 was blown for 10 min, and 10 mL of saturated NaHCO3 solution was added to obtain the crude product. The crude product was purified by silica gel column chromatography using a DCM / MeOH mixed solution containing 1 v / v% ammonia water as the mobile phase (DCM / MeOH = 10:1-1:1). The purified glycocholic acid-NH2 was obtained as a yellow-brown viscous liquid after drying. 1 HNMR (DMSO-d6) and mass spectrometry characterization. Glycocholic acid-NH2 was dissolved in 100 μL of DMSO, and 1.2 equivalents of N-acryloyloxy succinimide were added to react at room temperature in the dark. Sampling points were taken every 1 h for ninhydrin color development until the reaction stopped when it was colorless. The resulting product was stored at 4°C in the dark for later use.

[0051] (2) Synthesis of acrylated glycocholic acid monoclonal antibody: glycocholic acid monoclonal antibody (mAbs) was diluted to 1 mg / mL with 0.1 M NaHCO3 (pH 8.3) solution (the antibody was derived from the existing glycocholic acid hybridoma cell strain (CG-706, preservation number CGMCC No. 15292, but the present application is not limited thereto, and antibodies with the above-mentioned effects are all applicable), which was purified by mouse ascites method, and 15 equivalents of N-acryloyloxy succinimide DMSO solution were added thereto to react at room temperature in the dark for 2 h. The purified reaction solution was dialyzed in PBS (MW cutoff ~ 14,000 Da) at 4°C, and the solution was changed every 4 h, and dialysis was performed for 1-2 days. The purified product was ultrafiltrated and concentrated to 6 mg / mL for later use.

[0052] (3) Synthesis of PCH containing antigen-antibody cross-linking: 50 μg of acrylated glycocholic acid monoclonal antibody, 1 μg of acrylated glycocholic acid, 5 mg of acrylamide, 0.1 mg of N,N'-methylene bisacrylamide (Bis), and 0.3 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone were dissolved in 50 μL of PBS to prepare a prepolymer solution. The physical cross-linking hydrogel obtained in Example 2 was soaked in the prepolymer solution for 8 hours, taken out, and placed in the same mold as in Example 2. After injecting the excess prepolymer solution into the mold, polymerization was performed under a UV lamp (365 nm, power 3-16 W) for 10 min. After the polymerization was completed, the hydrogel was taken out and washed in a PBS buffer at 55°C for 5 min to remove polyvinyl alcohol, thereby obtaining PCH containing antigen-antibody cross-linking. Figure 3 ), and then immersed in PBS for equilibration.

[0053] Comparative Example 1

[0054] The present comparative example is similar to the synthetic procedure of step (3) in Example 3, except that no acrydite antigen and acrydite antibody were added. Different amounts of crosslinker N,N'-methylenebisacrylamide (Bis) (0.05 mg, 0.075 mg, 0.10 mg, 0.125 mg, 0.15 mg, respectively) were dissolved in 50 μL PBS together with 5 mg acrylamide and 0.3 μL 2-hydroxy-2-methyl-1-phenyl-1-propanone to prepare pre-polymer solutions with different concentrations of Bis, the concentrations of Bis in the pre-polymer solutions were 0.10, 0.15, 0.20, 0.25, 0.30 wt%, respectively. The physical crosslinked hydrogel obtained in Example 2 was immersed in the pre-polymer solution for 8 hours, and then taken out and placed in the same mold as in Example 2. Excess pre-polymer solution was injected into the mold, and then polymerized under a UV lamp (365 nm, power 3-16 W) for 10 minutes. After polymerization, the hydrogel was taken out and washed in PBS buffer at 55 °C for 5 minutes to remove the polyvinyl alcohol, and then three-dimensional PCHs with different crosslinking degrees were obtained. After equilibration in PBS, the diffraction wavelengths were measured.

[0055] The amount of N,N'-methylenebisacrylamide (Bis) used affects the crosslinking degree of the three-dimensional PCH. A suitable crosslinking degree is conducive to maintaining a moderate mechanical property and elasticity of the three-dimensional PCH, so that it has better optical responsiveness in the visible light band and produces a wider diffraction wavelength shift. As shown in FIG. 2, as the amount of Bis used increases, the crosslinking degree of the three-dimensional PCH increases, and the diffraction wavelength correspondingly blueshifts. Although a low-crosslinking-degree three-dimensional PCH prepared using a low amount of Bis has a wider shift in the visible light band when the antigen-antibody crosslinking is subsequently increased, the mechanical property of the three-dimensional PCH prepared using less than 0.20 wt% Bis is too poor, and it is extremely easy to break in actual detection. Therefore, to meet the basic mechanical property requirements of the PCH and to improve the optical responsiveness as much as possible, 0.10 mg (0.20 wt%) Bis is selected as the optimal amount of crosslinking agent. Figure 4 Comparative Example 2

[0056]

[0057] ​The comparative example is the same as the synthesis process of step (3) in Example 3, except that different amounts of acrylated glycocholic acid antibody (0, 20, 30, 40, 50, 60 μg) are respectively dissolved in 50 μl PBS with 5 mg acrylamide, 0.3 μL 2-hydroxy-2-methyl-1-phenyl-1-propanone and 0.10 mg N,N'-methylene bisacrylamide, 1 μg acrylated glycocholic acid to prepare a pre-polymer solution. Then, the physical cross-linked hydrogel obtained in Example 2 is soaked in the pre-polymer solution for 8 hours, taken out, placed in the same mold as in Example 2, and the excess pre-polymer solution is injected into the mold and polymerized under a UV lamp (365 nm, power 3-16 W) for 10 minutes. After polymerization, the hydrogel is taken out, washed in PBS buffer at 55°C for 5 minutes to remove the polyvinyl alcohol, and PCH with different amounts of glycocholic acid antibody is obtained. After equilibration in PBS, the diffraction wavelength is measured.

[0058] As shown in Figure 5 , due to the addition of acrylated glycocholic acid and acrylated glycocholic acid monoclonal antibody, the cross-linking degree of the double-crosslinked three-dimensional PCH is improved, and the diffraction wavelength of the three-dimensional PCH significantly blue shifts within the range of 0-30 μg with the increase of the amount of antibody; with further increase of the amount of antibody, the blue shift value of the diffraction wavelength of the obtained three-dimensional PCH gradually slows down, and when the amount of antibody reaches 50 μg, the change value of the PCH diffraction wavelength is not obvious. Since the prepared three-dimensional PCH will be used for glycocholic acid detection based on the competition method, the PCH with the smallest diffraction wavelength is the best choice because the PCH diffraction wavelength red shifts with the increase of the concentration of glycocholic acid during the detection process. At the same time, considering the high cost of the antibody and the economic consideration, 50 μg is selected as the optimal amount of acrylated antibody.

[0059] Performance study of glycocholic acid detection of three-dimensional PCH prepared in Example 4

[0060] (1) Preparation of different concentrations of glycocholic acid solutions: 10 mg of glycocholic acid was weighed and dissolved in PBS to make up to 50 mL to obtain a 200 μg / mL glycocholic acid solution, which was then diluted to 10, 20, 40, 60, 80, 100, 120 μg / mL gradient concentration solutions.

[0061] (2) Preparation of glycocholic acid analog solution: 100 μg / mL PBS solutions of deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and tauroursodeoxycholic acid (TUDCA) were prepared respectively.

[0062] (3) Preparation of artificial serum spiked samples: The artificial serum was diluted 100 times with PBS and used to prepare 20, 40, 60 μg / mL glycocholic acid solutions.

[0063] (4) Glycocholic acid detection step: The initial diffraction wavelength of PCH prepared in Example 3 was recorded by the fiber spectrometer. Then PCH was placed in a series of gradient concentrations of glycocholic acid solution, respectively, and the diffraction peak wavelength value of PCH was detected after 1 h, and the displacement value of the diffraction wavelength before and after the response was calculated. The glycocholic acid concentration and the displacement value of the diffraction wavelength were plotted and linearly fitted to obtain the standard curve for glycocholic acid detection.

[0064] Linear fitting curve (standard curve): y = ax + b

[0065] Where: y is the displacement value of the diffraction wavelength of PCH after responding to different concentrations of glycocholic acid solution, x is the concentration of glycocholic acid, a is the slope of the fitting curve, and b is the intercept of the fitting curve.

[0066] In this test example, the position of the diffraction wavelength of PCH in different concentrations of glycocholic acid solution was detected by the fiber spectrometer, and the relationship between the diffraction wavelength and the displacement value of the diffraction wavelength and the concentration of glycocholic acid solution was obtained. From Figure 6 It can be seen that as the concentration of glycocholic acid solution increases, the structural color of PCH changes significantly, from blue in 0 μg / mL solution to orange-red in 120 μg / mL solution, which can be observed qualitatively by naked eye; at the same time, the red shift of PCH diffraction wavelength is also gradually obvious, from 508 nm when the concentration is 0 μg / mL to 593 nm when the concentration is 120 μg / mL, and the displacement value of the diffraction peak wavelength is 85 nm; when the concentration of glycocholic acid is greater than 120 μg / mL, the red shift of the diffraction wavelength gradually becomes smaller, indicating that PCH has reached a response balance with glycocholic acid. Linear fitting Figure 7 ) was performed on the data of the relationship between the displacement value of the diffraction wavelength and the concentration of glycocholic acid, and the fitting equation was obtained: y = 0.756x - 4.882, and it was known that the linear detection range of PCH was 10 μg-120 μg, which could realize spectral quantitative detection.

[0067] (5) Specificity: The initial diffraction wavelength of PCH prepared in Example 3 was recorded by the fiber spectrometer. PCH was placed in glycocholic acid and its structural analog solution, the test conditions were kept unchanged, and PCH was taken out after 1 h, the diffraction wavelength of PCH in different solutions was detected, and the displacement value of the diffraction wavelength before and after the response was calculated.

[0068] A column chart was drawn using the displacement value of the diffraction wavelength of PCH in 100 μg / mL glycocholic acid and its structural analog solution Figure 8 It can be seen from the figure that PCH shows good selectivity to glycocholic acid, which indicates that the prepared PCH has good specific detection ability for glycocholic acid. This is due to the high binding affinity and specific selectivity between antigen and antibody in the immunoassay method, and also indicates that the prepared PCH can well avoid the non-specific binding of other molecules.

[0069] (6) Reusability: The PCH prepared in Example 3 was immersed in 100 μg / mL glycocholic acid solution for 1 h after recording the initial diffraction wavelength, and then the diffraction wavelength was measured again after taking out. The PCH was immersed in 50 mL of PBS and washed on a shaker for 8 h, and then the diffraction wavelength was recorded again. The cycle test was repeated 4 times, and the results are shown in Table 1. It can be seen that the PCH has almost no change in glycocholic acid response performance after four cycles, which proves the reusability of the PCH. Figure 9

[0070] (7) Spiked recovery experiment: Compared with PBS, the composition of real human serum is much more complex. In order to further study the actual clinical application potential of PCH, the detection performance of PCH in artificial serum was tested in this embodiment. The initial diffraction wavelength value of the PCH prepared in Example 3 was recorded by the fiber spectrometer. The PCH was placed in artificial serum spiked samples with different concentrations, and the diffraction wavelength shift value of the PCH was recorded after incubation at 37°C for 1 h. According to the standard curve obtained in step (4) of this embodiment, the spiked recovery rate of glycocholic acid was calculated, and the results are shown in Table 1. The results show that the PCH has excellent anti-interference ability and can be used for the detection of glycocholic acid content in actual serum samples.

[0071] Table 1 Spiked recovery experiment results of PCH for detecting glycocholic acid (GCA)

[0072]

[0073] Obviously, the above embodiments of the present application are only examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the specific embodiments of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.​

Claims

1. A three-dimensional gel photonic crystal, characterized in that: The following steps are included: S1, performing acrylation on glycocholic acid to obtain acrylated glycocholic acid; S2, acrylating the glycocholic acid antibody to obtain the acrylated glycocholic acid antibody; S3, dissolving polyvinyl alcohol in a solvent to form a sol, mixing the obtained sol with the nanosphere suspension and repeatedly heating and shaking until a strong structural color is obtained to obtain a photonic crystal sol, and then shaping the above photonic crystal sol and forming polyvinyl alcohol PCH by freeze-thawing; S4. Mixing the monomer, cross-linking agent, photoinitiator, acrylated glycocholic acid antigen obtained in step S1, and acrylated glycocholic acid antibody obtained in step S2 to obtain a prepolymer solution, which is then added to the polyvinyl alcohol (PCH) prepared in step S3 to carry out a photopolymerization reaction. After the reaction is completed, the polyvinyl alcohol is removed by purification and water bath heating to obtain the three-dimensional PCH; the amount of cross-linking agent used in the prepolymer solution is 0.10-0.30 wt%; and the mass ratio of acrylated antibody to acrylated glycocholic acid antigen is 1-60:

1.

2. The three-dimensional gel photonic crystal according to claim 1, characterized in that: Step S1 includes the following steps: S11, reacting glycocholic acid with an amination reagent to generate terminally aminated glycocholic acid -NH2; S12, glycocholic acid-NH2 reacts with N-acryloyloxysuccinimide to obtain acrylated glycocholic acid.

3. The three-dimensional gel photonic crystal according to claim 1, characterized in that: In step S2, N-acryloyloxysuccinimide is reacted with the primary amino group of the lysine residue in the glycocholic acid antibody molecule to form an amide bond to obtain the acrylated glycocholic acid antibody.

4. The three-dimensional gel photonic crystal according to claim 1, characterized in that: The antibody in step S2 is selected from a monoclonal antibody, a polyclonal antibody, a nanobody or a single-chain antibody.

5. The three-dimensional gel photonic crystal according to claim 1, characterized in that: The nanosphere material used in step S3 is selected from one or more of silicon dioxide, polystyrene, polymethyl methacrylate, titanium dioxide, iron oxide, gold, and silver.

6. The three-dimensional gel photonic crystal according to claim 1 or 5, characterized in that: The particle size of the nanospheres used in step S3 is 50 nm to 200 nm; the mass concentration of the nanosphere suspension is 5% to 30%.

7. The three-dimensional gel photonic crystal according to claim 1, characterized in that: The solvent selected in step S3 is any one or more of water, dimethyl sulfoxide, dimethylformamide, ethylene glycol, and glycerol.

8. The three-dimensional gel photonic crystal according to claim 1, characterized in that: The monomers in step S4 are one or more of acrylamide, methyl methacrylate, polyhydroxyethyl methacrylate, cellulose acetate butyrate, silicone methacrylate, fluorosilicone methacrylate, N-vinyl pyrrolidone, glycidyl methacrylate or ethylene glycol dimethacrylate.

9. The three-dimensional gel photonic crystal according to claim 1, characterized in that: The cross-linking agent in step S4 is one or more of N,N'-methylenebisacrylamide, polyethylene glycol diacrylate, divinylbenzene, ethylene glycol dimethacrylate, 1,6-hexanediol diacrylate, and polyethylene glycol dimethacrylate.

10. Use of the three-dimensional gel photonic crystal according to claim 1 as an immunoassay sensor in the detection of glycocholic acid.

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