Hydrogel molecularly imprinted inverse photonic crystal microspheres, preparation method and application thereof

Abstract

The present application relates to biomaterial technology, disclose a kind of hydrogel molecular imprinting anti-structure photonic crystal microspheres and its preparation method and application.The preparation method of the anti-structure photonic crystal microspheres includes the following steps: preparing positive structure photonic crystal microspheres by microfluidic method;Gibberellin is mixed as imprinting molecule with the mixed solution containing hydrogel and photoinitiator to form pre-gel mixture, the positive structure photonic crystal microspheres are soaked in the pre-gel mixture to obtain pre-gel system;After solidification I of the pre-gel system under ultraviolet irradiation, the solidified microspheres are obtained by stripping;The solidified microspheres are mixed with etching agent and reacted, washed.The photonic crystal microspheres can specifically and efficiently recognize and adsorb gibberellin, and the quantitative determination is completed by the shift value of reflection peak, and the detection sensitivity of gibberellin is high, and the detection condition requirement is low.

Description

Technical Field

[0001] This invention relates to biomaterials technology, specifically to a hydrogel molecularly imprinted reverse structure photonic crystal microsphere, its preparation method and application, and a method for detecting gibberellin. Background Technology

[0002] Gibberellins (GA) are tetracyclic, diterpenoid plant hormones synthesized through a complex pathway and widely distributed in angiosperms, gymnosperms, bacteria, and various fungi. Gibberellins are diverse, with different types exhibiting varying biological activities; gibberellin A3 (GA3) shows the highest activity. Gibberellins are among the most important plant growth regulators, crucial for many plant developmental processes, including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation, and flowering induction. Gibberellins are widely used in agricultural production; adjusting gibberellins to specific concentrations can improve crop yield and quality. However, excessive use of gibberellins in crop cultivation can lead to excessive gibberellin residues in crops, which can accumulate in the human body, causing chronic poisoning and harming human health, such as causing endocrine system disorders, chronic organ poisoning, and even cancer. Therefore, the detection and control of gibberellin content in plant growth environments and food is of great significance for food safety related to agricultural products.

[0003] Traditional methods for gibberellin detection mainly include high-performance liquid chromatography, immunoassay, and electrochemical methods. Although these methods have high sensitivity, they suffer from problems such as harsh reaction conditions, low detection efficiency, expensive detection equipment, and cumbersome operation. There is a need to find a high-throughput gibberellin detection platform and method that is fast, efficient, and accurate. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a hydrogel molecularly imprinted reverse structure photonic crystal microsphere, its preparation method and application, as well as a method for detecting gibberellin.

[0005] During their research, the inventors of this invention unexpectedly obtained a novel gibberellin detection platform by utilizing molecular imprinting technology and photonic crystals. They constructed a molecular imprinted photonic crystal that can specifically bind and capture gibberellin molecules and determine gibberellin content. The photonic crystal microspheres can specifically and efficiently identify gibberellin and complete quantitative determination by the shift value of the reflection peak. It has high detection sensitivity for gibberellin and low requirements for detection conditions.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for preparing hydrogel molecularly imprinted anti-structure photonic crystal microspheres, comprising the following steps:

[0007] (1) Prepared positive structure photonic crystal microspheres by microfluidic methods;

[0008] (2) Gibberellin is used as an imprinted molecule and mixed with a mixed solution containing hydrogel and photoinitiator to form a pregel mixture. The positive structure photonic crystal microspheres are immersed in the pregel mixture to obtain a pregel system.

[0009] (3) After curing the pregel system under ultraviolet irradiation, the cured microspheres are obtained by peeling.

[0010] (4) The solidified microspheres are mixed with an etchant for reaction and cleaning.

[0011] Preferably, the preparation process of the positive structure photonic crystal microspheres in step (1) includes: forming microfluidic droplets by using an aqueous dispersion containing silica nanoparticles as the inner phase and silicone oil as the outer phase, solidifying the microfluidic droplets to form preliminary microspheres, and calcining the preliminary microspheres.

[0012] Preferably, the microfluidic droplets are formed using a microfluidic device, wherein the flow rate of the internal phase in the microfluidic device is 0.4-0.6 mL / h, and the flow rate of the external phase is 4-6 mL / h.

[0013] Preferably, the average particle size of the silica nanoparticles is greater than 150 nm and less than 290 nm.

[0014] Preferably, the solvent used in the aqueous dispersion is ultrapure water, and the concentration of silica nanoparticles in the aqueous dispersion is 5-20 mg / mL.

[0015] Preferably, the curing process II includes: dispersing the microfluidic droplets in silicone oil and heating to remove water molecules.

[0016] Preferably, the heating temperature is 55-80°C.

[0017] Preferably, the calcination process includes: washing and drying the preliminary microspheres with n-hexane, and then calcining them at a temperature of 600-800℃ for 8-12 hours.

[0018] Preferably, the weight ratio of the positive structure photonic crystal microspheres, the gibberellin and the mixed solution in step (2) is 0.1-0.2:0.01-0.05:1.

[0019] Preferably, the solvent of the mixed solution is water; the hydrogel is selected from at least one of methacrylated gelatin, polyethylene glycol diacrylate, ethoxylated trimethylolpropane triacrylate and hyaluronic acid methacrylate; and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone.

[0020] Preferably, the amount of the hydrogel used is 50-500 mg relative to 1 mL of water, and the amount of the photoinitiator is 10-15 μL.

[0021] Preferably, the soaking conditions in step (2) include at least a time of 5-10 hours.

[0022] Preferably, the curing conditions in step (3) include at least: a UV wavelength of 380-405 nm and an irradiation intensity of 20-50 mW / cm². 2 The time is 1-2 minutes.

[0023] Preferably, the etchant used in step (4) is a hydrofluoric acid solution with a concentration of 3-6% by weight.

[0024] Preferably, the weight ratio of the positive structure photonic crystal microspheres to the etchant is 1:20-100.

[0025] Preferably, the reaction conditions include at least the following: a temperature of 20-30°C and a time of 5-8 hours.

[0026] Preferably, the cleaning solution used for cleaning is PBS buffer.

[0027] The second aspect of the present invention provides hydrogel molecularly imprinted reverse structure photonic crystal microspheres prepared by the above-described preparation method.

[0028] The third aspect of this invention provides the application of the above-described hydrogel molecularly imprinted reverse structure photonic crystal microspheres in the detection of gibberellins.

[0029] A fourth aspect of the present invention provides a method for detecting gibberellin, the method comprising the following steps:

[0030] S1. Gibberellin standard solutions of different concentrations were mixed with the above-mentioned hydrogel molecularly imprinted reverse structure photonic crystal microspheres for adsorption, and the shift of the reflection peak of the photonic crystal microspheres before and after adsorption was detected.

[0031] S2. Using the reflection peak displacement as the ordinate and the concentration of the gibberellin standard solution as the abscissa, establish the equation for the reflection peak displacement curve;

[0032] S3. The test solution is mixed with the photonic crystal microspheres for adsorption. The shift of the reflection peak of the photonic crystal microspheres before and after adsorption is detected. Then, the gibberellin content in the test solution is calculated according to the equation.

[0033] Preferably, the adsorption conditions include: a temperature of 0-40°C and a time of 25-35 min.

[0034] Preferably, the equation is y = 64.71 - 6.20 * x, where y is the reflection peak shift and x is the concentration of the gibberellin standard solution.

[0035] The beneficial effects of the present invention through the above technical solution are as follows:

[0036] The hydrogel molecularly imprinted reverse-structure photonic crystal microspheres provided by this invention, based on the specific recognition characteristics of molecular imprints, precisely bind with gibberellin molecules, causing fluctuations in the refractive index of the photonic crystal microspheres and corresponding changes in the characteristic reflection peaks, thereby enabling the detection of gibberellin concentration and improving the sensitivity and selectivity of gibberellin detection. The photonic crystal microspheres obtained by the preparation method provided by this invention combine hydrogels with imprinted molecular holes and photonic crystals as a carrier for gibberellin detection. Compared with other forms of carriers, they have advantages such as low angle dependence, large specific surface area, and rapid material exchange, enabling rapid, real-time, specific, and label-free detection of gibberellin.

[0037] The method for detecting gibberellin provided by this invention, compared with traditional gibberellin detection methods, does not require expensive instruments, has a simple and easy preparation method, and low requirements for reaction conditions. It improves the sensitivity and selectivity of detection and is a new type of rapid, efficient and accurate high-throughput gibberellin detection platform, which is suitable for promotion and application. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation method of hydrogel molecularly imprinted reverse structure photonic crystal microspheres provided by the present invention;

[0039] Figure 2 These are electron microscope images of the positive structure photonic crystal microsphere and the negative structure photonic crystal microsphere in Example 1, where a is the positive structure photonic crystal microsphere and b is the negative structure photonic crystal microsphere.

[0040] Figure 3 These are light micrographs of the normal structure photonic crystal microsphere and the anti-structure photonic crystal microsphere before and after adsorption of gibberellin molecules in Example 1. In this image, a is the normal structure photonic crystal microsphere, b is the anti-structure photonic crystal microsphere before adsorption, and c is the anti-structure photonic crystal microsphere after adsorption of gibberellin molecules.

[0041] Figure 4 This is a standard curve of gibberellin concentration detected by the anti-structure photonic crystal microspheres obtained in Example 1. Detailed Implementation

[0042] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0043] The first aspect of this invention provides a method for preparing hydrogel molecularly imprinted anti-structure photonic crystal microspheres, comprising the following steps:

[0044] (1) Prepared positive structure photonic crystal microspheres by microfluidic methods;

[0045] (2) Gibberellin is used as an imprinting molecule and mixed with a mixed solution containing hydrogel and photoinitiator to form a pregel mixture. The positive structure photonic crystal microspheres are immersed in the pregel mixture to obtain a pregel system.

[0046] (3) After curing the pregel system under ultraviolet irradiation, the cured microspheres are obtained by peeling.

[0047] (4) The solidified microspheres are mixed with an etchant for reaction and cleaning.

[0048] The hydrogel molecularly imprinted reverse-structure photonic crystal microspheres provided by this invention, based on the specific recognition characteristics of molecular imprints, precisely bind with gibberellin molecules, causing fluctuations in the refractive index of the photonic crystal microspheres and corresponding changes in the characteristic reflection peaks, thereby realizing the detection of gibberellin concentration and improving the sensitivity and selectivity of gibberellin detection. The photonic crystal microspheres obtained by the preparation method provided by this invention combine hydrogels with imprinted molecular holes and photonic crystals as a carrier for gibberellin detection. Compared with other forms of carriers, they have advantages such as low angle dependence, large specific surface area, and rapid material exchange, enabling rapid, real-time, specific, and label-free detection of gibberellin.

[0049] According to the present invention, positive structure photonic crystal microspheres are prepared using a microfluidic method, obtained by confined assembly within microfluidic emulsion droplets. Preferably, the preparation process of the positive structure photonic crystal microspheres in step (1) includes: forming microfluidic droplets by using an aqueous dispersion containing silica nanoparticles as the inner phase and silicone oil as the outer phase; solidifying the microfluidic droplets to form preliminary microspheres via a curing process II; and calcining the preliminary microspheres. The inventors have found that, under this preferred embodiment, it is beneficial to improve the structural stability of the positive structure photonic crystal microspheres.

[0050] In this invention, silica nanoparticles can be commercially available or prepared in-house. Exemplarily, silica nanoparticles are prepared using the Stober method, a physicochemical method specifically referring to the process of generating nano-silica particles by adding tetraethyl orthosilicate (TEOS) to ethanol and ammonia water.

[0051] According to the present invention, preferably, the microfluidic droplets are formed using a microfluidic device, wherein the flow rate of the inner phase in the microfluidic device is 0.4-0.6 mL / h, and the flow rate of the outer phase is 4-6 mL / h. The inventors have found that this preferred embodiment is beneficial for improving the preparation efficiency of positive structure photonic crystal microspheres and further enhancing the structural stability of the positive structure photonic crystal microspheres.

[0052] According to the present invention, preferably, the average particle size of the silica nanoparticles is greater than 150 nm and less than 290 nm, more preferably 180-250 nm. The inventors have found that, under this preferred embodiment, it is beneficial to improve the encapsulation effect of droplets on silica nanoparticles and promote the formation of positively structured photonic crystal microspheres.

[0053] According to the present invention, preferably, the solvent used in the aqueous dispersion is ultrapure water, and the concentration of silica nanoparticles in the aqueous dispersion is 5-20 mg / mL. The inventors have found that this preferred embodiment is beneficial for promoting the formation of positively structured photonic crystal microspheres.

[0054] According to the present invention, preferably, the curing II process includes: dispersing the microfluidic droplets in silicone oil and heating to remove water molecules. More preferably, the heating temperature is 55-80°C. The inventors have discovered that, under this preferred embodiment, silica nanoparticles can self-assemble into an ordered lattice during the water evaporation process in the droplets.

[0055] According to the present invention, preferably, the calcination process includes: washing and drying the preliminary microspheres with n-hexane, and then calcining them at a temperature of 600-800°C for 8-12 hours. The inventors have found that, under this preferred embodiment, closely packed positive-structure photonic crystal microspheres can be obtained, thereby improving the mechanical strength of the positive-structure photonic crystal microspheres.

[0056] According to the present invention, preferably, the weight ratio of the positive structure photonic crystal microspheres, the gibberellin, and the mixed solution in step (2) is 0.1-0.2:0.01-0.05:1. The inventors have found that, under this preferred embodiment, it is advantageous for gibberellin-imprinted molecules to be grafted onto the positive structure photonic crystal microspheres.

[0057] According to the present invention, preferably, the solvent of the mixed solution is water; the hydrogel is selected from at least one of gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), ethoxylated trimethylolpropane triacrylate (ETPTA), and hyaluronic acid methacrylate (HAMA); and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone (HMPP). The inventors have found that, under this preferred embodiment, it is beneficial to promote the swelling of the gibberellin-grafted positive-structure photonic crystal microsphere composite structure in the pre-gel mixture, thereby completely filling the void spaces between the silica nanoparticles.

[0058] According to the present invention, preferably, the amount of the hydrogel used is 50-500 mg relative to 1 mL of water, and the amount of the photoinitiator used is 10-15 μL. Exemplarily, relative to 1 mL of water, the amount of the hydrogel is 200-500 mg when it is GelMA, 50-100 μL when it is PEGDA, and 100-200 mg when it is HAMA.

[0059] According to the present invention, preferably, the soaking conditions in step (2) include at least a time of 5-10 hours. The inventors have found that, under this preferred embodiment, it is beneficial to promote the full swelling of the gibberellin-grafted positive-structure photonic crystal microsphere composite structure in the pregel mixture.

[0060] According to the present invention, preferably, the curing conditions in step (3) include at least: a UV wavelength of 380-405 nm and an irradiation intensity of 20-50 mW / cm². 2 The time is 1-2 minutes. The inventors have discovered that, under this preferred embodiment, it is beneficial to effectively polymerize the gibberellin-grafted positive structure photonic crystal microsphere composite structure after the voids between the silica nanoparticles are completely filled, thus forming a stable solidified microsphere structure.

[0061] According to the present invention, the etchant is a reagent capable of corroding silicon dioxide, such as hydrofluoric acid. Preferably, the etchant in step (4) is a hydrofluoric acid solution with a concentration of 3-6% by weight, so as to remove the gibberellin of the solidified microspheres by etching with hydrofluoric acid, forming a gibberellin molecular imprint, thereby achieving highly specific recognition of gibberellin.

[0062] According to the present invention, preferably, the weight ratio of the positive-structure photonic crystal microspheres to the etchant is 1:20-100. The inventors have found that, under this preferred embodiment, it is beneficial to improve the gibberellin sensitivity and selectivity of the hydrogel molecularly imprinted inverse-structure photonic crystal microspheres.

[0063] According to the present invention, preferably, the reaction conditions include at least the following: a temperature of 20-30°C and a time of 5-8 hours.

[0064] According to the present invention, preferably, the cleaning solution used is PBS buffer to remove imprinted molecules and purify the hydrogel molecularly imprinted inverse photonic crystal microspheres.

[0065] According to a particularly preferred embodiment of the present invention, see [link to embodiment]. Figure 1 The preparation methods of hydrogel molecularly imprinted reverse structure photonic crystal microspheres include:

[0066] (1) An aqueous dispersion containing silica nanoparticles with an average particle size greater than 150 nm and less than 290 nm (concentration of 5-20 mg / mL) is used as the inner phase and silicone oil as the outer phase. Microfluidic droplets are formed by using a microfluidic device with the inner phase flow rate of 0.4-0.6 mL / h and the outer phase flow rate of 4-6 mL / h. The microfluidic droplets are dispersed in silicone oil and heated to 55-80 °C to remove water molecules to form preliminary microspheres. The preliminary microspheres are washed with n-hexane, dried, and calcined at 600-800 °C for 8-12 h to obtain positive structure photonic crystal microspheres.

[0067] (2) Gibberellin is used as an imprinted molecule and mixed with a mixed solution containing hydrogel and photoinitiator (the amount of hydrogel is 50-500 mg and the amount of photoinitiator is 10-15 μL relative to 1 mL of water) to form a pregel mixture. The positive structure photonic crystal microspheres are immersed in the pregel mixture for 5-10 h to obtain a pregel system, wherein the weight ratio of positive structure photonic crystal microspheres, gibberellin and mixed solution is 0.1-0.2:0.01-0.05:1;

[0068] (3) Under UV wavelengths of 380-405nm and irradiation intensities of 20-50mW / cm² 2 Under ultraviolet irradiation, the pregel system was cured for 1-2 minutes and then peeled off to obtain cured microspheres;

[0069] (4) The solidified microspheres are mixed with a hydrofluoric acid solution (etchant) with a concentration of 3-6% by weight so that the weight ratio of positive structure photonic crystal microspheres to etchant is 1:20-100. The mixture is reacted at a temperature of 20-30℃ for 5-8 hours and then washed with PBS solution to remove the imprinted molecules.

[0070] The second aspect of the present invention provides hydrogel molecularly imprinted reverse structure photonic crystal microspheres prepared by the above-described preparation method.

[0071] Based on the characteristic of the hydrogel molecularly imprinted reverse structure photonic crystal microspheres provided by this invention that they can specifically recognize gibberellins, the detection of gibberellin concentration is achieved, and the sensitivity and selectivity of gibberellin detection are improved. A third aspect of this invention provides the application of the above-mentioned hydrogel molecularly imprinted reverse structure photonic crystal microspheres in the detection of gibberellins.

[0072] The application method for detecting gibberellin involves directly mixing the test solution with hydrogel molecularly imprinted reverse-structure photonic crystal microspheres for quantitative detection. The detection principle is as follows: gibberellin solutions of different concentration gradients are added to hydrogel molecularly imprinted reverse-structure photonic crystal microspheres. Due to the binding of gibberellin with the gibberellin imprint, the refractive index of the photonic crystal microspheres fluctuates, and its structural color and characteristic reflection peaks change accordingly. By measuring the characteristic reflection peaks of the photonic crystal microspheres, the initial concentration of gibberellin in the test solution can be quantitatively determined.

[0073] A fourth aspect of the present invention provides a method for detecting gibberellin, the method comprising the following steps:

[0074] S1. Gibberellin standard solutions of different concentrations were mixed with the above-mentioned hydrogel molecularly imprinted reverse structure photonic crystal microspheres for adsorption, and the shift of the reflection peak of the photonic crystal microspheres before and after adsorption was detected.

[0075] S2. Using the reflection peak displacement as the ordinate and the concentration of the gibberellin standard solution as the abscissa, establish the equation for the reflection peak displacement curve;

[0076] S3. The test solution is mixed with the photonic crystal microspheres for adsorption. The shift of the reflection peak of the photonic crystal microspheres before and after adsorption is detected. Then, the gibberellin content in the test solution is calculated according to the equation.

[0077] According to the present invention, preferably, the adsorption conditions include: a temperature of 0-40°C and a time of 25-35 min.

[0078] According to the present invention, preferably, the equation is y = 64.71 - 6.20 * x, where y is the reflection peak shift and x is the concentration of the gibberellin standard solution.

[0079] The present invention will be described in detail below through embodiments.

[0080] In the following examples, the scanning electron microscope was purchased from JEOL Ltd. (JSM-5610LV), the structural color of the particles was obtained using an optical microscope (Olympus Corporation, BX53 upright microscope), the microfluidic device was purchased from Baoding Lange Co., Ltd. (LSP02-1B), and the fiber optic spectrometer was purchased from Shanghai Fuxiang Optics Co., Ltd. (PG2000-Pro-EX). GA3 standard was purchased from Jiangxi Xinruifeng Biochemical Co., Ltd. (GA3 technical grade), and the aqueous dispersion of silica nanoparticles was purchased from Nanjing Caina Biotechnology Co., Ltd. (MS-06-101). Unless otherwise specified, all other reagents and raw materials were commercially available.

[0081] In the following examples, unless otherwise specified, room temperature refers to 25±5℃.

[0082] Example 1

[0083] (1) An aqueous dispersion of monodisperse silica nanoparticles with an average particle size of 250 nm and a concentration of 10 mg / mL was used as the inner phase, and 50 cs silicone oil was used as the oil phase. The two fluids were injected into a co-flow microfluidic device, and droplets were generated by controlling the inner phase flow rate at 0.5 mL / h and the outer phase flow rate at 5 mL / h. The droplets were collected in a container filled with excess 500 cs silicone oil. The droplet container was heated at 70 °C, allowing the silica nanoparticles to self-assemble into an ordered lattice during water evaporation in the droplets, resulting in preliminary microspheres. The preliminary microspheres were washed with excess n-hexane, repeated 3-5 times until residual silicone oil was removed. The preliminary microspheres were then air-dried and calcined at 800 °C for 10 h to obtain closely packed positive-structure photonic crystal microspheres. The microstructure of these positive-structure photonic crystal microspheres was observed using a scanning electron microscope as follows: Figure 2 As shown in Figure a, its structural color was observed under an optical microscope as follows: Figure 3 As shown in Figure a;

[0084] (2) Dissolve 50 mg of gibberellin template molecule and 200 mg of methacrylated gelatin (GelMA) in 900 μL of distilled water, then add 10 μL of 2-hydroxy-2-methyl-1-phenylpropanone (HMPP) and mix to prepare a pregel mixture. Soak 200 mg of positive structure photonic crystal microspheres obtained in step (1) in excess of the pregel mixture for 5 h to obtain a pregel system.

[0085] (3) The pregel system obtained in step (2) is subjected to irradiation at a wavelength of 405 nm and an intensity of 25 mW / cm. 2 Polymerize under ultraviolet irradiation for 2 minutes to completely solidify the system, and manually peel off the solidified microspheres.

[0086] (4) The solidified microspheres obtained in step (3) were mixed with an excess of 4% hydrofluoric acid solution, so that the weight ratio of the positive structure photonic crystal microspheres to the etchant was 1:50. The mixture was reacted at 25°C for 6 hours to etch away the silica particles in the solidified microspheres. Then, the imprinted gibberellin molecules were washed away with an excess of PBS buffer to obtain hydrogel molecularly imprinted inverse structure photonic crystal microspheres. The microstructure of these microspheres was observed by scanning electron microscopy as follows: Figure 2 As shown in Figure b, its structural color, as observed under an optical microscope, is as follows: Figure 3 As shown in Figure b, due to the change in the refractive index of the gel material, the structural color and reflection peak position of the anti-structure photonic crystal microspheres undergo a blue shift.

[0087] Example 2

[0088] (1) An aqueous dispersion of monodisperse silica nanoparticles with an average particle size of 200 nm and a concentration of 5 mg / mL was used as the inner phase, and 50 cs silicone oil was used as the oil phase. The two fluids were injected into a co-flow microfluidic device, and droplets were generated by controlling the flow rate of the inner phase to 0.4 mL / h and the flow rate of the outer phase to 4 mL / h. The droplets were collected in a container filled with excess 500 cs silicone oil. The droplet container was heated at 55 °C, so that the silica nanoparticles self-assembled into an ordered lattice during the evaporation of water in the droplets, and preliminary microspheres were obtained. The preliminary microspheres were washed with excess n-hexane, and the process was repeated 3-5 times until the residual silicone oil was washed away. The preliminary microspheres were then air-dried and calcined at 600 °C for 12 h to obtain closely packed positive structure photonic crystal microspheres.

[0089] (2) Dissolve 40 mg of gibberellin template molecule, 100 μL of polyethylene glycol diacrylate (PEGDA), and 10 mg of ethoxylated trimethylolpropane triacrylate (ETPTA) in 900 μL of distilled water, and then add 15 μL of 2-hydroxy-2-methyl-1-phenylpropanone (HMPP) to prepare a pregel mixture. Soak 150 mg of the positive structure photonic crystal microspheres obtained in step (1) in an excess of the pregel mixture for 8 h to obtain the pregel system.

[0090] (3) The pregel system obtained in step (2) is subjected to irradiation at a wavelength of 380 nm and an intensity of 50 mW / cm. 2 Polymerize under ultraviolet irradiation for 1 minute to completely solidify the system, and manually peel off the solidified microspheres.

[0091] (4) The solidified microspheres obtained in step (3) are mixed with an excess of hydrofluoric acid solution with a concentration of 3% by weight, so that the weight ratio of positive structure photonic crystal microspheres to etchant is 1:100. The mixture is reacted at 20°C for 8 hours to etch and remove silica particles from the solidified microspheres. Then, the imprinted gibberellin molecules are removed by rinsing with excess PBS buffer to obtain hydrogel molecular imprinted antistructure photonic crystal microspheres.

[0092] Example 3

[0093] (1) An aqueous dispersion of monodisperse silica nanoparticles with an average particle size of 180 nm and a concentration of 20 mg / mL was used as the inner phase, and 50 cs silicone oil was used as the oil phase. The two fluids were injected into a co-flow microfluidic device, and droplets were generated by controlling the flow rate of the inner phase to be 0.6 mL / h and the flow rate of the outer phase to be 6 mL / h. The droplets were collected in a container filled with excess 500 cs silicone oil. The droplet container was heated at 80 °C, so that the silica nanoparticles self-assembled into an ordered lattice during the evaporation of water in the droplets, and preliminary microspheres were obtained. The preliminary microspheres were washed with excess n-hexane, and the process was repeated 3-5 times until the residual silicone oil was washed away. The preliminary microspheres were then air-dried and calcined at 800 °C for 8 h to obtain closely packed positive structure photonic crystal microspheres.

[0094] (2) Dissolve 15 mg of template molecule gibberellin and 150 mg of hyaluronic acid methacrylate (HAMA) in 900 μL of distilled water, then add 10 μL of 2-hydroxy-2-methyl-1-phenylpropanone (HMPP) and mix to prepare a pregel mixture. Soak 110 mg of positive structure photonic crystal microspheres obtained in step (1) in excess of the pregel mixture for 10 h to obtain a pregel system.

[0095] (3) The pregel system obtained in step (2) is subjected to irradiation at a wavelength of 400 nm and an intensity of 20 mW / cm. 2 Polymerize under ultraviolet irradiation for 1.5 min to completely solidify the system, and manually peel off the solidified microspheres.

[0096] (4) The solidified microspheres obtained in step (3) are mixed with an excess of hydrofluoric acid solution with a concentration of 6% by weight, so that the weight ratio of positive structure photonic crystal microspheres to etchant is 1:20. The mixture is reacted at 30°C for 5 hours to etch and remove silica particles from the solidified microspheres. Then, the imprinted gibberellin molecules are removed by rinsing with excess PBS buffer to obtain hydrogel molecular imprinted antistructure photonic crystal microspheres.

[0097] Example 4

[0098] Hydrogel molecularly imprinted reverse-structure photonic crystal microspheres were prepared according to the method of Example 3, except that step (3) was replaced with:

[0099] (3) The pregel system obtained in step (2) is subjected to irradiation at a wavelength of 300 nm and an intensity of 20 mW / cm. 2 Polymerize under ultraviolet irradiation for 2 minutes to completely solidify the system, and then manually peel off the solidified microspheres.

[0100] Example 5

[0101] Hydrogel molecularly imprinted reverse-structure photonic crystal microspheres were prepared according to the method of Example 3, except that step (2) was replaced with:

[0102] (2) Dissolve 80 mg of gibberellin template molecule and 150 mg of hyaluronic acid methacrylate (HAMA) in 900 μL of distilled water, then add 10 μL of 2-hydroxy-2-methyl-1-phenylpropanone (HMPP) and mix to prepare a pregel mixture. Soak 240 mg of positive structure photonic crystal microspheres obtained in step (1) in excess of the pregel mixture for 10 h to obtain a pregel system.

[0103] Test Example 1

[0104] S1. Preparation of standard working solution: Weigh 0.35 mg of gibberellin standard (purchased from Shenzhen Antibiotechnology Co., Ltd., product number AT01GAAg) into a 100 mL volumetric flask, dissolve in PBS, dilute to the mark, and sonicate for 10 min to obtain the standard solution; pipette 10 mL of the above standard solution into a 100 mL volumetric flask, dilute to the mark with PBS to obtain a mixed intermediate solution, so as to obtain a concentration gradient of 10. -5 10 -6 10 -7 10 -8 10 -9 Gibberellin standard working solution at mol / L;

[0105] S2. The reflectance spectrum of the hydrogel molecularly imprinted reverse-structure photonic crystal microspheres prepared in Example 1 was measured using a fiber optic spectrometer. The position of the reflectance peak before adsorption was recorded as 605 nm. Then, 100 mg of the hydrogel molecularly imprinted reverse-structure photonic crystal microspheres prepared in Example 1 were added to 2 mL of gibberellin standard working solutions with different concentration gradients obtained in step S1. The microspheres were allowed to adsorb for 30 min at room temperature until saturation. The reverse-structure photonic crystal microspheres after adsorption of gibberellin were then removed (their structure and color were observed under an optical microscope as shown in the figure). Figure 3 As shown in Figure c, compared with before adsorption, the structural color and reflection peak position of the microspheres undergo a red shift. The reflection spectrum of the anti-structure photonic crystal microspheres after gibberellin adsorption was measured using a fiber optic spectrometer, and the reflection peak position after the reaction was recorded. A standard curve was plotted between the detected reflection peak shift value (the difference in characteristic peak positions before and after the reaction) and the gibberellin solution concentration, as shown in Figure c. Figure 4 As shown, the formula for obtaining the standard curve is y = 64.71 - 6.20 * x, where y is the reflection peak shift value and x is the negative logarithm of the concentration of the gibberellin standard solution.

[0106] S3. Add 100 mg of the hydrogel molecularly imprinted reverse structure photonic crystal microspheres prepared in Example 1 to 2 mL of the test solution I (using a concentration of 5.0 × 10⁻⁶).-4 A gibberellin standard working solution of mol / L was used as the test solution I). The mixture was thoroughly mixed and allowed to adsorb for 30 min at room temperature until saturation. The inverse-structure photonic crystal microspheres after gibberellin adsorption were then removed. The reflection spectrum of the inverse-structure photonic crystal microspheres after adsorption was measured using a fiber optic spectrometer, and the reflection peak position was recorded as 649 nm. The reflection peak shift value of the test solution was calculated. This shift value was substituted into the standard curve obtained in step S2, and the gibberellin content in the test solution was finally calculated to be 4.57 × 10⁻⁶. -4 M.

[0107] The detection limit for gibberellin by the hydrogel molecularly imprinted reverse structure photonic crystal microspheres obtained in Example 1 was 3.63 × 10⁻⁶. -11 M.

[0108] Comparative Example 1

[0109] Prepare the gibberellin standard working solution according to step S1 in Test Example 1. Detect the characteristic peak area of ​​the gibberellin standard using HPLC-MS-MS, and plot a standard curve comparing the characteristic peak area with the gibberellin concentration. Detect the characteristic peak area of ​​gibberellin in the test solution I using HPLC-MS-MS, and substitute this value into the standard curve to obtain a gibberellin content of 6.52 × 10⁻⁶. -4 M.

[0110] Compared with Comparative Example 1, the hydrogel molecularly imprinted reverse structure photonic crystal microspheres provided in Example 1 have better detection specificity, sensitivity and accuracy.

[0111] Comparative Example 2

[0112] Hydrogel molecularly imprinted reverse-structure photonic crystal microspheres were prepared according to the method in Example 3, except that the average particle size of the silica nanoparticles was 150 nm.

[0113] The theoretical value of the reflection peak of the inverse structure photonic crystal microspheres prepared in Comparative Example 2 is less than 390 nm, which reaches the critical value for visible light measurement. Therefore, the position of its reflection peak cannot be accurately measured, and thus it cannot be applied to the detection of gibberellin.

[0114] Comparative Example 3

[0115] Hydrogel molecularly imprinted reverse-structure photonic crystal microspheres were prepared according to the method of Example 3, except that the 6% by weight hydrofluoric acid solution in step (4) was replaced with a 6% by weight dilute sulfuric acid solution.

[0116] The dilute sulfuric acid solution used in Comparative Example 3 could not corrode the silica on the solidified microspheres obtained in step (3), thus failing to obtain the reverse-structure photonic crystal microspheres and failing to achieve specific recognition and adsorption of gibberellin.

[0117] Test Example 2

[0118] (1) Prepare the gibberellin standard working solution according to step S1 in Test Example 1;

[0119] (2) The reflectance spectra of the hydrogel molecularly imprinted reverse-structure photonic crystal microspheres prepared in Examples 2-5 and Comparative Examples 2-3 were measured using a fiber optic spectrometer. The position of the reflectance peak before adsorption was recorded. Then, 100 mg of the hydrogel molecularly imprinted reverse-structure photonic crystal microspheres prepared in Examples 2-5 and Comparative Examples 2-3 were added to 2 mL of gibberellin standard working solutions with different concentration gradients obtained in step S1. The microspheres were adsorbed at room temperature for 30 min until they were saturated. The reverse-structure photonic crystal microspheres after adsorption of gibberellin were removed. The reflectance spectra of the reverse-structure photonic crystal microspheres after adsorption of gibberellin were measured using a fiber optic spectrometer. The position of the reflectance peak after the reaction was recorded. The relationship between the detected reflectance peak shift value (the difference between the position of the characteristic peak before and after the reaction) and the concentration of the gibberellin solution was plotted into a standard curve. The formula of the standard curve is shown in Table 1, where y is the reflectance peak shift value and x is the negative logarithm of the concentration of the gibberellin standard solution.

[0120] (3) 100 mg of the hydrogel molecularly imprinted reverse structure photonic crystal microspheres prepared in Examples 2-5 and Comparative Examples 2-3 were added to 2 mL of test solution I (using a concentration of 5.0 × 10⁻⁶). -4 A gibberellin standard working solution of mol / L was used as the test solution I. The mixture was thoroughly mixed and allowed to adsorb for 30 min at room temperature until it was saturated. The reverse structure photonic crystal microspheres after adsorption of gibberellin were then removed. The reflection spectrum of the reverse structure photonic crystal microspheres after adsorption was measured using a fiber optic spectrometer, and the position of the reflection peak was recorded (the results are shown in Table 1). The reflection peak shift value of the test solution was calculated. The reflection peak shift value was substituted into the standard curve obtained in step S2, and the gibberellin content in the test solution I was finally calculated. The results are shown in Table 1.

[0121] The detection limits of gibberellin for the hydrogel molecularly imprinted reverse structure photonic crystal microspheres prepared in Examples 2-5 and Comparative Examples 2-3 are shown in Table 1.

[0122] Table 1

[0123]

[0124] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing hydrogel molecularly imprinted inverse structure photonic crystal microspheres, characterized in that, Includes the following steps: (1) A water dispersion containing silica nanoparticles is used as the inner phase and silicone oil is used as the outer phase to form microfluidic droplets. The microfluidic droplets are solidified in a solidification process II to form preliminary microspheres. The preliminary microspheres are calcined to obtain positive structure photonic crystal microspheres. The microfluidic droplets are formed using a microfluidic device. The flow rate of the inner phase in the microfluidic device is 0.4-0.6 mL / h, the flow rate of the outer phase is 4-6 mL / h, and the average particle size of the silica nanoparticles is 180-250 nm. (2) Gibberellin is used as an imprinting molecule and mixed with a mixed solution containing hydrogel and photoinitiator to form a pregel mixture. The positive structure photonic crystal microspheres are immersed in the pregel mixture to obtain a pregel system. The solvent of the mixed solution is water, the hydrogel is methacrylated gelatin, and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone. (3) After curing the pregel system under ultraviolet irradiation, the cured microspheres are obtained by peeling. (4) The solidified microspheres are mixed with an etchant for reaction and cleaning. The etchant is a hydrofluoric acid solution with a concentration of 3-6% by weight. The curing process II includes: dispersing the microfluidic droplets in silicone oil and heating to remove water molecules; the calcination process includes: washing and drying the preliminary microspheres with n-hexane and then calcining them at a temperature of 600-800℃ for 8-12 hours.

2. The preparation method according to claim 1, characterized in that, The solvent used in the aqueous dispersion is ultrapure water, and the concentration of silica nanoparticles in the aqueous dispersion is 5-20 mg / mL.

3. The preparation method according to claim 2, characterized in that, The heating temperature is 55-80℃.

4. The preparation method according to any one of claims 1 to 3, characterized in that, In step (2), the weight ratio of the positive structure photonic crystal microspheres, the gibberellin, and the mixed solution is 0.1-0.2:0.01-0.05:1; The amount of the hydrogel used relative to 1 mL of water is 50-500 mg, and the amount of the photoinitiator used is 10-15 µL.

5. The preparation method according to any one of claims 1 to 3, characterized in that, The soaking conditions described in step (2) include at least the following: a soaking time of 5-10 hours; The curing conditions described in step (3) include at least the following: UV wavelength of 380-405 nm and irradiation intensity of 20-50 mW / cm². 2 The time is 1-2 minutes.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The weight ratio of the positive structure photonic crystal microspheres to the etchant is 1:20-100; The reaction conditions include at least the following: temperature of 20-30℃ and time of 5-8h; The cleaning solution used for the cleaning process is PBS buffer.

7. Hydrogel molecularly imprinted reverse-structure photonic crystal microspheres prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the hydrogel molecularly imprinted reverse structure photonic crystal microspheres according to claim 7 in the detection of gibberellins.

9. A method for detecting gibberellin, characterized in that, The method includes the following steps: S1. Gibberellin standard solutions of different concentrations are mixed with the hydrogel molecularly imprinted reverse structure photonic crystal microspheres described in claim 7 for adsorption, and the shift of the reflection peak of the photonic crystal microspheres before and after adsorption is detected. S2. Using the reflection peak displacement as the ordinate and the concentration of the gibberellin standard solution as the abscissa, establish the equation for the reflection peak displacement curve; S3. The test solution is mixed with the photonic crystal microspheres for adsorption, and the shift of the reflection peak of the photonic crystal microspheres before and after adsorption is detected. Then, the content of gibberellin in the test solution is calculated according to the equation. The adsorption conditions include: a temperature of 0-40℃ and a time of 25-35 min; The equation is y = 64.71 - 6.20x, where y is the reflection peak shift and x is the concentration of the gibberellin standard solution.

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