CN-116808962-B - Hydrogel molecular imprinting inverse photonic crystal microsphere and preparation method and application thereof

CN116808962BCN 116808962 BCN116808962 BCN 116808962BCN-116808962-B

Abstract

The invention relates to a biological material technology, and discloses a hydrogel molecular imprinting inverse structure photon crystal microsphere, a preparation method and application thereof. The preparation method of the anti-structure photonic crystal microsphere comprises the following steps of preparing a positive structure photonic crystal microsphere through a microfluidic method, mixing gibberellin serving as a imprinting molecule with a mixed solution containing hydrogel and a photoinitiator to form a pre-gel mixture, soaking the positive structure photonic crystal microsphere in the pre-gel mixture to obtain a pre-gel system, solidifying the pre-gel system under ultraviolet irradiation, stripping to obtain a solidified microsphere, and mixing the solidified microsphere with an etchant for reaction and cleaning. The photonic crystal microsphere can specifically and efficiently identify and adsorb gibberellin, and the quantitative determination is completed through the offset value of the reflection peak, so that the detection sensitivity of gibberellin is high, and the detection condition requirement is low.

Inventors

HUANG HE
LI XIANG
YANG QIAOYI
WANG YUETONG
Shi Tiankong
YE CHAO
NIE ZHIKUI
Zhao Danshan

Assignees

南京师范大学
江西新瑞丰生化股份有限公司

Dates

Publication Date: 20260512
Application Date: 20230530

Claims (9)

1. The preparation method of the hydrogel molecular imprinting inverse structure photonic crystal microsphere is characterized by comprising the following steps of: (1) Forming microfluidic liquid drops by taking aqueous dispersion containing silica nanoparticles as an internal phase and silicone oil as an external phase, forming preliminary microspheres by solidifying the microfluidic liquid drops and calcining the preliminary microspheres to obtain photonic crystal microspheres with positive structures, wherein the microfluidic liquid drops are formed by adopting a microfluidic device, the flow rate of the internal phase in the microfluidic device is 0.4-0.6mL/h, the flow rate of the external phase is 4-6mL/h, and the average particle size of the silica nanoparticles is 180-250nm; (2) Mixing gibberellin serving as a imprinting molecule with a mixed solution containing hydrogel and a photoinitiator to form a pregelatinized mixture, and soaking the positive-structure photonic crystal microsphere in the pregelatinized mixture to obtain a pregelatinized system, wherein the solvent of the mixed solution is water, the hydrogel is methacrylic gelatin, and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropion; (3) Curing the pre-gel system under ultraviolet irradiation, and stripping to obtain cured microspheres; (4) Mixing the solidified microspheres with an etchant for reaction and cleaning, wherein the etchant adopts hydrofluoric acid solution with the concentration of 3-6 wt%; The curing process of the II comprises dispersing the microfluidic liquid drops in silicone oil, heating to remove water molecules, and the calcining process comprises washing and drying the primary microspheres by normal hexane and calcining for 8-12h at 600-800 ℃.
2. The method according to claim 1, wherein the solvent used in the aqueous dispersion is ultrapure water, and the concentration of the silica nanoparticles in the aqueous dispersion is 5-20mg/mL.
3. The method of claim 2, wherein the heating is at a temperature of 55-80 ℃.
4. The method according to any one of claims 1 to 3, wherein the weight ratio of the positive-structure photonic crystal microsphere, the gibberellin, and the mixed solution in step (2) is 0.1-0.2:0.01-0.05:1; the hydrogel is used in an amount of 50-500mg and the photoinitiator is used in an amount of 10-15 μl relative to 1mL of water.
5. The method according to any one of claims 1 to 3, wherein the conditions for soaking in step (2) include at least a time period of 5 to 10 hours; the curing condition in the step (3) at least comprises the UV wavelength of 380-405nm, the irradiation intensity of 20-50mW/cm 2 and the time of 1-2min.
6. A method of preparation according to any one of claims 1 to 3 wherein the weight ratio of positive-structure photonic crystal microspheres to etchant is 1:20-100; the reaction conditions at least comprise 20-30 ℃ and 5-8 hours; the cleaning solution is PBS buffer solution.
7. A hydrogel molecularly imprinted inverse photonic crystal microsphere prepared by the preparation method according to any one of claims 1 to 6.
8. The use of the hydrogel molecularly imprinted inverse photonic crystal microsphere of claim 7 in detecting gibberellin.
9. A method for detecting gibberellin, the method comprising the steps of: S1, respectively mixing gibberellin standard solutions with different concentrations with the hydrogel molecular imprinting inverse structure photonic crystal microsphere of claim 7 for adsorption, and detecting the reflection peak displacement of the photonic crystal microsphere before and after adsorption; s2, establishing an equation of a reflection peak displacement curve by taking the reflection peak displacement as an ordinate and the concentration of the gibberellin standard solution as an abscissa; S3, mixing the solution to be detected with the photonic crystal microsphere for adsorption, detecting the reflection peak displacement of the photonic crystal microsphere before and after adsorption, and then calculating the content of gibberellin in the solution to be detected according to the equation; The adsorption conditions comprise temperature of 0-40deg.C and time of 25-35min; The equation is y= 64.71-6.20x, wherein y is the reflection peak displacement, and x is the concentration of gibberellin standard solution.

Description

Hydrogel molecular imprinting inverse photonic crystal microsphere and preparation method and application thereof Technical Field The invention relates to a biological material technology, in particular to a hydrogel molecular imprinting inverse structure photonic crystal microsphere, a preparation method and application thereof, and a gibberellin detection method. Background Gibberellin (GA) is a tetracyclic, diterpene phytohormone that is biosynthesized by complex pathways and is widely distributed in angiosperms, gymnosperms, bacteria and various fungi. Gibberellins are abundant in species and different gibberellins have different biological activities, wherein gibberellin A3 (GA 3) has the highest activity. Gibberellins are one of the most important plant growth regulators and are critical to many developmental processes in plants, including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation, and induction of flowering. Gibberellin is used in agricultural production, and the yield and quality of crops can be improved by adjusting gibberellin to a certain concentration. However, if excessive gibberellin is used in cultivating crops, the gibberellin content of the crops may exceed the standard, and the crops are easy to accumulate in human body, so that chronic poisoning is caused, and human health is endangered, such as disorder of endocrine system of human body, chronic organ poisoning even cancer is caused. Therefore, the gibberellin content in plant growth environment and food is detected and controlled, and the method has important significance for food safety related to crops. The traditional gibberellin detection method mainly comprises a high-performance liquid chromatography method, an immunoassay method, an electrochemical method and the like, and the method has the problems of severe reaction conditions, low detection efficiency, expensive detection equipment, complex operation process and the like although the sensitivity is high, so that a rapid, efficient and accurate-result high-throughput gibberellin detection platform and method are required to be found. Disclosure of Invention The invention aims to overcome the problems in the prior art and provides a hydrogel molecular imprinting reverse structure photonic crystal microsphere, a preparation method and application thereof, and a method for detecting gibberellin. In the research process, the inventor of the invention uses a molecular imprinting technology and a photonic crystal to unexpectedly obtain a novel gibberellin detection platform, and constructs a molecular imprinting photonic crystal capable of specifically combining and capturing gibberellin molecules and measuring gibberellin content. In order to achieve the above object, a first aspect of the present invention provides a method for preparing hydrogel molecularly imprinted inverse photonic crystal microspheres, comprising the following steps: (1) Preparing a photonic crystal microsphere with a positive structure by a microfluidic method; (2) Mixing gibberellin serving as a imprinting molecule with a mixed solution containing hydrogel and a photoinitiator to form a pregel mixture, and soaking the positive-structure photonic crystal microsphere in the pregel mixture to obtain a pregel system; (3) Curing the pre-gel system under ultraviolet irradiation, and stripping to obtain cured microspheres; (4) Mixing the solidified microsphere with an etchant for reaction and cleaning. Preferably, the preparation process of the positive structure photonic crystal microsphere in the step (1) comprises the steps of forming microfluidic liquid drops by taking an aqueous dispersion liquid containing silica nanoparticles as an inner phase and silicone oil as an outer phase, forming preliminary microspheres by curing the microfluidic liquid drops II, and calcining the preliminary microspheres. Preferably, the microfluidic droplet is formed using a microfluidic device in which the flow rate of the inner phase is 0.4-0.6mL/h and the flow rate of the outer phase is 4-6mL/h. Preferably, the silica nanoparticles have an average particle diameter greater than 150nm and less than 290nm. Preferably, the solvent adopted by the aqueous dispersion is ultrapure water, and the concentration of the silica nanoparticles in the aqueous dispersion is 5-20mg/mL. Preferably, the curing II process comprises dispersing the microfluidic droplets in silicone oil and heating to remove water molecules. Preferably, the temperature of the heating is 55-80 ℃. Preferably, the calcination process comprises washing the primary microspheres with n-hexane, drying, and calcining at 600-800 ℃ for 8-12h. Preferably, in the step (2), the weight ratio of the positive structure photonic crystal microsphere to the gibberellin to the mixed solution is 0.1-0.2:0.01-0.05:1. Preferably, the solvent of the mixed solution is water, the hydrogel is at least one selected from methacrylic gelatin, polyet