TWI755126B - Inverse opal hydrogel sensor for sensing food additives and method for making the same - Google Patents

Abstract

The invention discloses an inverse opal hydrogel sensor for sensing food additives and a preparation method thereof. The silicon dioxide nano-microspheres are synthesized by a sol-gel method and arranged into a single-layer photonic crystal array. After polymerizing a prepolymer containing the target molecule of L-tryptophan to be tested and a polymer monomer, and then removing the photonic crystal and the target molecule, an inverse opal hydrogel with holes imprinted by the L-Trp molecule was obtained. The thin film was measured so that the sensor could measure L-Trp concentrations ranging from 10 ^-3 to 10 ^-9 M. When the L-Trp target molecule solution was dropped onto the inverse opal hydrogel sensing film, the L-Trp molecular imprinted holes on the inverse opal hydrogel sensing film matched with the L-Trp target molecules, causing hydrogels The shrinkage or swelling of the inverse opal hole array leads to the change of the structural parameters of the inverse opal cavity array, which in turn causes the shift of the wavelength position of the Bragg diffraction peak. Through chromaticity analysis technology, L-Trp is predicted by the chromaticity coordinate position corresponding to its reflection spectrum. The concentration of the target molecule solution achieves the purpose of simple preparation, low cost, rapid detection and replacing traditional detection technology.

Description

Inverse opal hydrogel sensor for sensing food additives and method for making the same

The present invention relates to an inverse opal hydrogel sensor used for sensing food additives and a manufacturing method thereof, in particular to an inverse opal hydrogel sensing film with L-Trp molecularly imprinted holes, and the use of the inverse opal hydrogel sensing film. The opal hydrogel sensing film predicts the concentration of L-Trp, and can achieve the technology of simple preparation, low cost, rapid detection and replacement of traditional detection technology.

In recent years, food safety issues have received worldwide attention. Governments all over the world have set strict regulations on additives in food. There are many kinds of additives commonly found in food, such as preservatives, nutritional additives, color retention agents and bulking agents, etc., to improve the shelf life of food, reduce production costs and increase flavor. L-Tryptophan in nutritional additives is one of the essential amino acids in the human body. It is mainly used to maintain the balance of muscle mass in the human body. Physiological precursor for the synthesis of nicotinic acid. In addition, L-Trp also has positive effects on improving sleep quality and anti-depression. People can produce L-Trp by ingesting milk, bananas or cheese, etc. Due to the low content of L-Trp in vegetables, it is usually added to food and feed, as a nutritional supplement or as a pharmaceutical preparation [Refs 1, 2]. The content of L-Trp in some commercial soy sauces was between 6.67×10 ⁻⁴ and 1.28×10 ⁻³ M [Ref. 3]. Excessive intake of L-Trp can easily lead to abnormal metabolism and cause brain-related diseases. Studies have found that when L-Trp is not metabolized properly, toxic metabolites are produced in the brain and have been shown to be one of the causes of Alzheimer's, Parkinson's and schizophrenia [Ref. 4].

At present, the detection of L-tryptophan is mainly by capillary electrophoresis [Reference 5], fluorescence method [Reference 6], and high performance liquid chromatography [Reference 7]. However, these methods usually require the use of complex equipment, take a long time to pre-process the samples, and require high operator skills, so they cannot be simply and effectively performed for rapid detection. Therefore, it is necessary to develop a technology that can rapidly detect trace amounts of L-tryptophan in food.

Molecularly imprinted polymers (MIPs) are sensing technologies gradually formed from the selective interaction of biological mechanisms in nature, including the recognition of antibodies and antigens, and the transcription and translation mechanisms of nucleic acids. Among them, high selectivity, low cost, high sensitivity and wide application level are also the future development trend of quantitative analysis technology [Reference 8].

At present, molecular imprinting technology has been widely used. In 2018, Lian et al. [Reference 9] synthesized molecularly imprinted microspheres with high recognition function for chloramphenicol by precipitation polymerization in order to improve the selectivity and analytical sensitivity of solid phase extraction (SPE). (Chloramphenicol molecularly imprinted microspheres), and using MIP microspheres as solid-phase extraction adsorbents to purify seawater and quantitatively analyze chloramphenicol in seawater. In 2016, Chantada-Vázquez et al. [Reference 10] used the selectivity of MIP to prepare an imprinted fluorescent artificial receptor on the surface of quantum dots that can detect cocaine, and to evaluate urine by fluorescence monitoring. Cocaine content in liquid. In 2019, Zhang et al. [Reference 11] used the advantages of molecular imprinting and photonic crystals to successfully produce an illegal additive-ethyl anthranilate (Ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, ethyl anthranilate, which can be screened by the naked eye to detect color changes.) EA) of molecularly imprinted photonic crystals.

The existence of colors in nature is mainly caused by the combination of human physiological behavior and subjective feelings. The generation of color is mainly due to the interaction of absorption, reflection and diffraction of materials in the process of light of different wavelengths entering the eye [Reference 12]. 1917 British physicist Rayleigh [Ref. 13] observed the feathers of birds and the scales of insects and found that under the reflection of light, the stacking of different layers and structures, called photonic crystals, cause color changes. In the world of insects, color is even more important because they usually rely on structural color for courtship, defense and camouflage. For example, the striking spots and colors on the elytra of weevil [Ref. 14] are warning signs that they are not edible. Most of the colors in nature are derived from multilayer interference, thin film interference, grating diffraction and photonic crystals.

In order to confirm the patentability of the present invention and the non-infringement risk, the inventor searched the prior patents and made an analysis and comparison as follows.

The patent case for the method of forming a single-layer photonic crystal structure (Patent No. I414637) is to apply a voltage to the electrophoretic suspension and the upper and lower working electrodes to form an electric field by the electrophoretic self-assembly technology, so that the particles in the suspension interact with the electric field and the gravitational field Under the action, a single-layer photonic crystal structure is formed, and a photonic crystal with a single-layer non-compact structure arrangement is achieved by changing the shape of the ring electrode. Difference from the present invention: This method uses electrophoresis to fabricate a single-layer photonic crystal structure, which is different from that of the present invention using the Langmuir-Blodgett dip-pull method to fabricate a compact-structure photonic crystal array, so there is no risk of infringement.

The patent case of molecularly imprinted polymer particles capable of high-capacity adsorption of polyphenolic compounds (Patent Publication No. 200836826) is that polymer nanoparticles with special functional groups are added to the surface by surface modification. To make the detection of target molecular polyphenols (such as: black tea polyphenols, catechins, proanthocyanidins, etc.) with high selectivity, the polymer nanoparticles have two characteristics: (1) The size of the nanoscale is used to increase the Large adsorption surface area, (2) the surface has special specific functional groups, two characteristics can make the target molecule have high efficiency selectivity and adsorption capacity, which can be used for purification or extraction of polyphenolic compounds. Differences of the present invention: This research method utilizes emulsification polymerization to make molecularly imprinted polymer microspheres, and the present invention utilizes molecular imprinting to prepare hole-like structures The structure is different, so there is no risk of infringement.

The preparation method of molecular imprinting film, the preparation method of molecular imprinting film, molecular sensing electrode, molecular sensing electrode, molecular sensing system and its use patent case (Patent No. I561821), the fruiting body of fungi is freeze-dried After grinding into powder, add 10%~100% of the first solvent, and extract it at a temperature of 10℃~100℃ to obtain an extract, and use the molecular imprinting polymer technology to add the triterpenoid molecular imprinting polymer ( molecularly imprinted polymer) in the extract, and the triterpenoids in the extract are separated. The difference of the present invention: this research method uses methanol, chloroform and acetone to separate the triterpenoids adsorbed on the molecular imprinting polymer, and the present invention uses methacrylic acid and ethylene glycol dimethacrylate to prepare L-Trp molecularly imprinted polymers are different, so there is no risk of infringement.

The patent case for a method of forming a molecular imprinting polymer film on a plastic substrate (Patent No. I427111) is a method for forming a molecular imprinting polymer film on a plastic substrate. The 2,6- Diisopropofol, functional monomer, initiator and cross-linking agent are mixed and coated on the plastic substrate, 2,6-diisopropofol: functional monomer: cross-linking agent: initiator The molar ratio is between 1:4:30:0.17 and 1:4:30:0.85, and the reaction mixture is exposed under the exposure energy range of 16J/cm ² ~72J/cm ² , so that A cured film is formed on the plastic substrate. Then, the cured film is washed with methanol to complete the removal of target molecules, so as to form a molecular imprinted polymer film on the plastic substrate. Differences of the present invention: This research method uses 2,6-diisopropofol as the target molecule, which is different from the use of the L-Trp target molecule in the present invention, so there is no risk of infringement.

The patent case (Patent No. I421037) of the polymer selected as the molecular imprinting of tobacco-specific nitrosamines and the method for using the same is to detect, quantify and separate the nitrosamines in tobacco in the sample, and the solvent The material of the mixture containing tobacco is extracted, and the molecular imprinting polymer of TSNA and the functional monomer MAA and 2-hydroxyethyl methacrylate are combined with the MIP characteristic. (HEMA) and the copolymerization of a hydrophobic crosslinker (EDMA) with a free radical initiator, followed by removal of the target molecules in the MIP. Can be used to analyze and isolate TSNA from biological fluids. The present invention is in template molecules, polymeric materials designed to bind TSNA present in organic or aqueous systems and ultimately use these materials in analytical or preparative separations, in analytical sample pretreatment and in chemical sensors . The difference of the present invention: This research method uses TSNA as the target molecule and functional monomer MAA, HEMA and hydrophobic cross-linking agent (EDMA) to make a polymerization solution, and the present invention uses L-Trp target molecule and functional monomer MAA and cross-linking agent (EDMA). There are differences in the polymerization solution made from the linking agent (EGDMA), so there is no risk of infringement.

The patent case for a detector with photonic crystal structure (patent number I418775) is to use styrene microspheres with uniform particle size to form a neatly arranged structure by molecular self-assembly technology. Then, the molecular imprinting technique was used to use the sub-bisphenol A as the template molecule. Then, the molecular imprinted polymer material is infiltrated into the gaps of the regularly arranged styrene microspheres, and the styrene microspheres are removed with toluene to combine the molecular holes with the phenyl group, and then the bisphenol A template molecules are removed with methanol. The optical signal is generated by the photonic crystal structure, and the concentration of the target substance to be tested in the test sample can be quickly known by the relationship between the signal change and the concentration. Differences of the present invention: This research method uses styrene microspheres to make bisphenol A molecular recognition hole detector and the material of the present invention uses silica microspheres to make holes, so there is no risk of infringement.

Using Molecular Template Sensor to Identify Dopamine in Neurotransmitter In order to improve the identification, this layer of film can improve the identification ability of dopamine molecules. In many neurotransmitters, a certain voltage is used to increase the oxidation current of dopamine, and then the dopaminergic material and the conductive polymer monomer are mixed into a uniform by heating polymerization method. The solution is coated with the conductive polymer monomer to form a conductive polymer film, and finally the dopamine substance in the conductive polymer film is extracted after washing with a solvent. Differences of the present invention: This research method utilizes electrochemical method to detect target molecule dopamine and the present invention utilizes color recognition concept to make L-tryptophan sensor, so there is no risk of infringement.

Molecular imprinting material and its preparation method, magnetic molecular imprinting material and its preparation method patent case (Patent No. I643869), is to use simple and low-cost molecular imprinting material and magnetic molecular imprinting material to identify pancreatic regeneration protein in the test body. The target molecule pancreatic regeneration protein is added to the template mixture and mixed with the solvent. The target molecule in the mixture contains hydrophobic, hydrophilic, phenylcyclic amino acid amino acids, and the polymer is chitin, ethylene-vinyl alcohol copolymer, poly Poly(hydroxymethyl 3,4-ethylene dioxythiophene), poly(aniline-co-metanilic acid), polyphenylene sulfide poly (p-phenylene sulfide), polythiophene poly(thiophene) in any combination, the solvent is volatilized to form a solidified product, and finally the target molecules are removed to form a molecular imprinting material for recognizing pancreatic regeneration proteins. The magnetic molecular imprinting material The previous preparation method is the same as that of the molecular rubbing material, the difference is that the mixture is mixed with magnetic particles, and finally the target molecules in the magnetic molecular rubbing product are removed to form a magnetic molecular rubbing material for recognizing pancreatic regeneration protein. Differences of the present invention : This research method uses magnetic particles and polymers to make molecular imprint identification detectors, which is different from the L-tryptophan sensors made of methacrylic acid and ethylene glycol dimethacrylate of the present invention. No risk of infringement.

The patent case for the medical use of molecularly imprinted polymers (Patent Publication No. 201636029) is for the preparation of medicines for the treatment of liver cancer. Molecular imprinting can bind to telomeres in liver cancer cells, resulting in the inability of the chromosomal DNA of cells to replicate. Cell growth inhibitory effect. Provide chitin solution of template molecules adenine, thymine, guanine, telomeres, cytosine and other sequences, magnetic substances and optical substances, and control molecular rubbing polymers by magnetic ferric oxide (Fe ₃ O ₄ ) And the optical substance Zinc Oxide (ZnO) can give the molecular imprinting polymer optical properties to confirm the true location of the molecular imprinting polymer in the individual, and finally clean the molecular imprinting polymer precursor to remove the template molecule. Removal of the template molecule Molecularly imprinted polymers can be formed with sites for recognition by telomeres. Differences of the present invention: This research method utilizes the combination of magnetic particles and telomeres to form a medicine for inhibiting liver cancer cells, which is different from the useless magnetic particles of the present invention, so there is no risk of infringement.

The purpose of the present invention is to provide an inverse opal hydrogel sensor for sensing food additives and a preparation method thereof. The main method is to synthesize silica nanospheres by sol-gel method, and then use Langmuir-Blodgett (LB) deposition technique to arrange the silica nanospheres into a monolayer photonic crystal array. Based on the concept of discoloration of insect surface structure, the pre-polymer solution containing the target molecule of L-Trp (L-tryptophan) to be tested and macromolecular monomers is filled in the gap of the photonic crystal array. After polymerizing the prepolymer, and removing the photonic crystal and target molecules, an inverse opal hydrogel sensing film with holes imprinted by L-Trp molecules can be obtained. When the molecularly imprinted holes on the inverse opal hydrogel sensing film match with the target molecule, the inverse opal hydrogel sensing film will shrink or swell, resulting in changes in the structural parameters of the inverse opal, which in turn causes Bragg diffraction Shift of the peak wavelength position. Different colors will appear on the macroscopic view, which can be used to check the concentration change of the target molecule to be detected. Therefore, combining molecularly imprinted hydrogels and photonic crystals can endow hydrogel signals with self-display properties without the need for expensive facilities for detection.

In addition to exploring the stimulus response effect of inverse opal hydrogel sensing films with different pore sizes to different concentrations of L-Trp solution and its specific identification function, the present invention also uses chromaticity analysis technology to detect the displacement and color of reflected wave peaks. The change of the degree coordinate position was used to predict the trace concentration change of L-Trp. The photonic crystal hydrogel formed by the combination of photonic crystal and molecular imprinting technology has the potential to replace traditional detection technology due to simple preparation, low cost and rapid detection.

10: The first glass substrate

11:LB deposition tank

12: Second glass substrate

13: Prepolymer

14: Sandwich Structure

15: Hydrofluoric acid solution

16: Glacial acetic acid solution

20: Silica SiO ₂ Microsphere Crystal Array

21: Silica Microsphere Crystal Array Structure Template

30: Inverse Opal Hydrogel Film Containing L-tryptophan

30: Shiraishi Hydrogel Film

31: Inverse Opal Hydrogel Sensing Films with L-Trp Molecularly Imprinted Holes

Fig. 1 is a schematic diagram of the main characteristic steps of preparing the L-tryptophan inverse opal hydrogel sensing film according to the present invention picture.

2 is a schematic diagram of the detailed flow of the present invention for preparing an L-tryptophan inverse opal hydrogel sensing film; (a) a monolayer silicon dioxide crystal array is deposited on the surface of the hydrophilic treated first glass substrate; (b) Filling the prepolymer containing L-Trp target molecules in the gaps between the nanospheres to form a sandwich structure; (c) After initiating photopolymerization by ultraviolet light, the silica particles are removed to obtain a hole array containing L-Trp; (d) After removing excess L-tryptophan target molecule with glacial acetic acid, the inverse opal hydrogel is completed.

Figure 3 is a chemical structure diagram of (a) L-Trp; (b) L-Phe of the present invention.

FIG. 4 is a particle size distribution diagram of SiO ₂ microspheres with different particle sizes produced by the present invention with ammonia water: anhydrous ethanol ratio of (a) 1:3 and (b) 1:5 respectively.

Fig. 5 is the self-assembled arrangement of SiO ₂ nanospheres of different sizes made by the present invention: (a) 234nm; (b) 542nm opal SEM photo: (i) top view; (ii) is a cross-sectional view corresponding to (i) .

FIG. 6 is a SEM photograph of the inverse opal hydrogel with the pore size of (a) 202 nm; (b) 428 nm of the present invention. (i) Top view; (ii) is a sectional view corresponding to (i).

Fig. 7 is the detection curve diagram of the L-Trp hydrogel of the present invention; (a) 202nm; (b) the change of the inverse opal hydrogel with 428nm hole in the L-Trp solution (i) is the reflection spectrum; (ii) ) is the linear regression curve of the intensity (Intensity) of the reflection peak in (i) corresponding to the logarithm (L-Trp) of the concentration of L-Trp solution.

Figure 8 is the specificity test of the hydrogel containing L-Trp target molecule of the present invention; inverse opal hydrogel with (a) 202nm; (b) 428nm pores, dropwise (i) L-Trp; (ii) Reflection spectrum after L-Phe solution.

Fig. 9 is the corresponding (a) reflection spectrogram after the holes of the present invention are (i) 202nm and (ii) 428nm inverse opal hydrogels dropped into L-Trp solutions of different concentrations; (b) is a chromaticity diagram; (c) corresponds to (b) The corresponding chromaticity coordinate movement path diagram.

Fig. 10 is the corresponding (a) absorption equilibrium diagram and (b) reproduction of the hydrogel sensor with the pore size of (i) 202 nm and (ii) 428 nm of the present invention to the concentration of 10 ^-6 M L-Trp Sex Analysis Chart.

The invention mainly uses sol-gel method to synthesize silicon dioxide nanometer microspheres, and then uses Langmuir-Blodgett (LB) deposition technology to arrange the nanometer microspheres into a monolayer array. Through the combination of photonic crystal and molecular imprinting technology, an inverse opal hydrogel sensor capable of recognizing L-tryptophan was fabricated. When the inverse opal hydrogel with specific molecular imprint is matched with the target molecule, it will cause swelling or shrinkage of the hydrogel, resulting in the change of the Bragg diffraction peak wavelength. Therefore, the present invention also provides an innovative and practical colorimetric analysis technology to detect the trace concentration changes of L-tryptophan. In addition, the sensor also has excellent specific identification for L-Trp target molecules, and its concentration can be predicted from the shift of its reflection peak and the change of the chromaticity coordinate position. The present invention also explores the stimuli-response effect of inverse opal hydrogels with different pore sizes to L-Trp target molecule solutions with different concentrations.

The embodiment of the present invention is to synthesize silicon dioxide nano-microspheres by a sol-gel method, and then arrange the silicon dioxide nano-microspheres into a single-layer photonic crystal array by the Langmuir-Blodgett deposition technique. Based on the concept of discoloration of insect surface structure, the pre-polymer solution containing the target molecule of L-Trp (L-tryptophan) to be tested and macromolecular monomers is filled in the gap of the photonic crystal array. After polymerizing the prepolymer, and removing the photonic crystal and target molecules, an inverse opal hydrogel sensing film with holes imprinted by L-Trp molecules can be obtained. When the molecularly imprinted holes on the inverse opal hydrogel sensing film match with the target molecule, the inverse opal hydrogel sensing film will shrink or swell, resulting in changes in the structural parameters of the inverse opal, which in turn causes Bragg diffraction Shift of the peak wavelength position. The macro will appear in different colors, which can be used to inspect Changes in the concentration of the L-Trp target molecule to be detected. Therefore, combining molecularly imprinted hydrogels and photonic crystals can endow hydrogel signals with self-display properties without the need for expensive facilities for detection. In addition to exploring the stimulus response effect of inverse opal hydrogels with different pore sizes to different concentrations of L-Trp solution and its specific identification function, the present invention also uses chromaticity analysis technology to determine the displacement of reflected wave peaks and the position of chromaticity coordinates. to predict the trace concentration changes of L-Trp. The photonic crystal hydrogel formed by the combination of the photonic crystal and the molecular imprinting technology has the potential to replace the traditional detection technology due to the simple preparation, low cost and rapid detection.

Please refer to FIGS. 1 and 2 . In order to achieve the purpose of the present invention, the specific embodiment includes the steps of synthesizing silica nano-microspheres, preparing the silica monolayer opal template and preparing the biosensor. The foregoing steps are described in detail as follows.

The silicon dioxide nano-microsphere synthesis step (A) of the present invention is to prepare two kinds of monodisperse silicon dioxide microspheres with different particle sizes as photonic crystal templates by the sol-gel method [Reference 15]. Specifically, add 20-30 (preferably 25) mL of tetraethyl silicate (Tetraethl orthosilicate, TEOS; Sigma-Aldrich) and 4-5.5 (preferably 25) mL of starting agent in a 250 mL conical flask. It is 4.8) mL of absolute ethanol (Ethanol; Honeywell Riedel deHaen ^TM ), stir evenly at 250-350 (preferably 300) rpm for 20-40 (preferably 30) minutes, and then add 4-10 (preferably 7) mL of ammonia water (Aqua ammonia, NH ₄ OH; JT Baker), 1 to 5 (preferably 3) mL of deionized water (deionized water) and 20 to 30 (preferably 25) mL of anhydrous ethanol ( Ethanol; Honeywell Riedel de Haen ^™ ) catalyzed the reaction. After 18-30 (preferably 24) hours, the nano-scale silica microsphere suspension can be prepared. Then pass the synthesized nanoscale silica microsphere suspension through a tabletop centrifuge (Tabletop Centrifuges; Legend Mach 1.6-R, Thermo Scientific Sorvall) at 2~6 (preferably 4) ℃ at a speed of 5000 After centrifugation at ~7000 (preferably 6000) rpm for 15-45 (preferably 30) minutes, the supernatant was removed to obtain SiO ₂ microspheres. The SiO ₂ microspheres were washed with deionized water, and after several repeated centrifugation and washing, SiO ₂ microspheres with uniform particle size could be obtained. Silicon dioxide microspheres with different particle sizes can be prepared by controlling the ratio of ammonia water to absolute ethanol to be 1:3 (7mL:21mL) and 1:5 (7mL:35mL).

The preparation step (B) of the silica monolayer opal template of the present invention is to soak the first glass substrate 10 in 8-12 (preferably 10) wt% sodium hydroxide aqueous solution for 20-40 (preferably 30) Surface hydrophilic modification in minutes. The modified first glass substrate 10 is washed with deionized water and then dried for use. In order to evenly distribute the SiO ₂ microspheres on the liquid surface of the LB deposition tank 11, we take 1~3 (preferably 2) g of the SiO ₂ microspheres and add 1~3 (preferably 2) mL of anhydrous In a triangular conical flask of ethanol and 6-10 (preferably 8) mL of chloroform (Chloroform, Merck), ultrasonically vibrate for 5-15 (preferably 10) minutes to obtain a SiO ₂ microsphere suspension . Then, 150-250 (preferably 200) mL of deionized water was poured into the LB deposition tank 11, and the injection flow rate of the SiO ₂ microsphere suspension was controlled by the peristaltic pump to be 50-60 (preferably 54.4) μl/ min, the SiO ₂ microsphere particles are spread on the liquid surface of the LB deposition tank 11 . Then, insert the modified hydrophilic first glass substrate 10 under the liquid surface of the LB deposition tank 11, and then slowly transfer the SiO ₂ microsphere particles spread on the surface of the liquid surface to the surface of the hydrophilic first glass substrate 11 to form a single The _SiO2 microsphere crystal array 20 layered on the layer of SiO2, after the liquid volatilizes, a single-layer silicon dioxide _SiO2 microsphere crystal array structure template 21 attached to the surface of the first glass substrate 11 can be obtained, and the process is shown in FIG. 2(a) shown.

The biosensor preparation step (C) of the present invention includes the L-Trp prepolymerization solution preparation step (C1) and the L-Trp hydrogel preparation step (C2). The preparation step of L-Trp prepolymerization solution is to take 0.01~0.03 (preferably 0.02) g of L-tryptophan (L-Tryptophan, L-Trp; Sigma-Aldrich) and dissolve it in 0.5~1.5 (preferably 1 ) mL methanol (Mthanol; Honeywell Riedel de Haen ^TM ), 0.3-0.7 (preferably 0.5) mL of methacrylic acid (Methacrylic acid, MAA; Sigma-Aldrich) and 0.3-0.7 (preferably 0.5) mL of ethyl acetate Ethylene glycol dimethacrylate (Ethylene glycol dimethacrylate, EGDMA; Sigma-Aldrich), placed in a conical flask and evenly stirred, placed in a refrigerator at 2~6 (preferably 4) ℃ for 10~14 (preferably 12 ) after 1 hour, take out, add 0.005~0.015 (preferably 0.01) g of 2,2-azobisisobutyronitrile (2,2'-Azobis(2-methylpropionitrile), AIBN; UR) with a rotating speed of 150~350 ( Preferably, it is stirred at 300) rpm, and nitrogen gas is introduced at the same time to remove oxygen, and then the prepolymer solution containing the target molecular imprint of L-Trp can be obtained. In the L-Trp hydrogel preparation step, a single-layer SiO ₂ microsphere crystal array structure template 21 containing a single-layer SiO ₂ opal array is taken and a second glass substrate 12 is covered on its surface, and after the two ends are fixed with clips, The template 21 is immersed in the prepolymer 13 solution containing the L-Trp target molecule. The prepolymer 13 solution containing the L-Trp target molecule was infiltrated into the gap between the SiO ₂ microsphere particles and the first and second glass substrates 10/12 by capillary action. When the template 21 becomes transparent, it means that the prepolymer 13 has completely filled the gaps between the SiO ₂ microspheres of the template 21 , and the fabrication process is shown in FIG. 2( b ). Then, the sandwich structure 14 of the first and second glass substrates 10/12 coated with the prepolymer 13 is subjected to a photopolymerization reaction with ultraviolet light with a wavelength of 365 nm for 1.5-2.5 (preferably 2) hours. Finally, the sandwich structure 14 is immersed in a 2-4 (preferably 3)% hydrofluoric acid solution 15 for 1.5-2.5 (preferably 2) hours to remove the SiO ₂ microspheres. Due to the removal of SiO ₂ microspheres, the upper and lower layers of the first and second glass substrates 10/12 of the sandwich structure 14 are separated, and an inverse opal hydrogel film 30 containing L-tryptophan can be obtained. The process is shown in Fig. 2(c). Subsequently, the inverse opal hydrogel film 30 containing L-tryptophan is placed in a 0.5-1.5 (preferably 1)% glacial acetic acid solution 16 to remove the L-Trp target molecules in the inverse opal hydrogel film , an inverse opal hydrogel sensing film 31 with holes capable of recognizing the L-Trp target molecular imprint and having L-Trp molecular imprint holes can be obtained, and the process is shown in FIG. 2(d).

In order to verify the feasibility of the present invention, the sensing material produced by the present invention was analyzed, and the analysis was carried out by using a laser nanoparticle particle size analysis and a potential analyzer (Zetasizer; 3000HS, Malvern Instruments) to measure the polymerized particles. _SiO2 microsphere particle size and distribution. High resolution thermal field emission scanning electron microscope (High Resolution Thermal Field Emission Scanning Electron Microscope, HRFEG-SEM; JSM-7610F, JEOL) was used to observe the pore morphology of SiO ₂ array and inverse opal structure. Fluorescence Spectrophotometer (Fluorescence Spectrophotometer; F-7000; HITACHI) was used to analyze the spectral changes and reproducibility of molecularly imprinted hydrogels with different pore sizes in different concentrations of L-Trp solutions. To test the reproducibility of two inverse opal hydrogels with different pore sizes, we used 0.5-1.5 (preferably 1) wt% glacial acetic acid as the eluent, and after removing the target molecules, the solution was added dropwise to a concentration of 10 ⁻ The ⁶ M L-Trp solution was recombined with the pore positions on the hydrogel, and the above steps were repeated five times to observe the repeated use. The specificity and adsorption equilibrium characteristics of molecularly imprinted hydrogels with different pore sizes were analyzed by UV-Visible Spectrophotometers (UH5000; HITACHI). In order to test whether the two inverse opal hydrogels with different pore sizes only have specific recognition for L-Trp solution, we chose L-Phe (chemical structure, as shown in Figure 3) with a similar structure to L-Trp as the test. . We dropped different concentrations of L-Trp solution and L-Phe solution into two inverse opal hydrogels with different pore sizes, and observed the difference and the adsorption equilibrium characteristics of L-Trp solution at a fixed concentration of 10 ^-6 M . In addition, the changes of the chromaticity coordinate position and reflectance spectrum in the variable-angle multifunctional optical characteristics measuring system (MF-630; HMT) were used to explore the molecularly imprinted hydrogels with different pore sizes. Differences presented after dropping different concentrations of L-Trp solutions.

The analysis of the experimental example of the present invention includes the analysis of opal and inverse opal arrays and the analysis of biosensors.

The opal and inverse opal array analysis of the present invention includes particle size analysis of silica nanospheres, silica opal structure analysis and silica inverse opal structure analysis. Particle size analysis of silica nanospheres: In order to compare the response characteristics of different pore sizes to hydrogels, we aggregated two types of SiO ₂ microspheres with different particle sizes (the ratio of ammonia water to anhydrous ethanol was 1:3, respectively and 1:5). With the increase of anhydrous ethanol content, the particle size of _SiO2 nanospheres also increased. The average particle size and dispersion coefficient (Polydispersity Index, PDI) were measured by laser nanoparticle particle size analysis and potential analyzer to be 234nm, 542nm and 0.001, 0.010, respectively, showing that the polymerized SiO ₂ microspheres The diameter distribution is quite uniform, as shown in Fig. 4(a)(b). Silica opal structure analysis: SiO ₂ microspheres are uniformly dispersed on the liquid surface, and then the surface gas/liquid interface area is continuously reduced by pushing with baffles on both sides, and finally a tightly arranged monolayer array is formed. 5(a)(b)-(i) are the surface morphologies of silica opal arrays with particle sizes of 234 nm and 542 nm observed by SEM, respectively, and (ii) is the cross-sectional view corresponding to (i). Structural analysis of silica inverse opal: Figure 6(a)(b)-(i) shows the average diameter of pores observed by SEM after the silica opal arrays with particle sizes of 234 nm and 542 nm were etched by hydrofluoric acid. are the top views of 202 nm and 428 nm inverse opal hydrogels, and (ii) is the cross-sectional view corresponding to (i). It can be seen from Fig. 6 that the SiO ₂ microspheres have been completely removed after being etched by the hydrofluoric acid solution. The pores are tightly connected to form a porous hydrogel, and the inverse opal pore structure will not collapse due to the removal of nanospheres after etching. It can be seen from (a)(b)-(ii) that we successfully prepared A single-layer inverse opal hydrogel structure was obtained.

The biosensor analysis of the present invention includes the detection and analysis of molecularly imprinted hydrogels, the specificity analysis of molecularly imprinted hydrogels, the chromatic response of molecularly imprinted hydrogels to trace concentrations, and the response speed of molecularly imprinted hydrogels. reproducibility. Detection and analysis of molecularly imprinted hydrogels: Figures 7(a)(b)-(i) are the reflection spectra of molecularly imprinted hydrogels with different pore sizes under different concentrations of L-Trp solutions. When the L-Trp solution was dropped into the inverse opal hydrogel with holes of 202 nm and 428 nm, the reflection peaks appeared at 520 nm and 733 nm, respectively. And the intensity of the reflection peak also increases gradually with the increase of L-Trp concentration, and reaches the maximum value when the concentration is 10 ^-3 M. And the corresponding reflectance spectra of the two can be clearly distinguished when the L-Trp concentration is between 10 ^-7 M and 10 ^-3 M. However, when the concentration of L-Trp is 10 ^-8 M and 10 ^-9 M, the overlap of the reflection spectra is not easy to distinguish. By Bragg's theoretical formulae (1) and (2) [Ref. 16], the theoretical position of the reflection peak of the inverse opal hydrogel can be calculated: λ _max =1.633 D(n ² _neff -sin ² θ) ^1/2 ( 1); n ² _eff =0.26×n ² _prepolymer + 0.74×n ² _L-Trp (2). In formula (1), λ _max is the wavelength of the maximum reflection peak of the hydrogel, D is the pore size of the inverse opal hydrogel, n _eff is the effective refractive index of the hydrogel, and θ is the angle of incident light. In formula (2), n ² _prepolymer is the refractive index of the molecularly imprinted hydrogel (n = 1.43) [Ref. 17], and n _L-Trp is the refractive index of L-tryptophan (n = 1.33) [20] . From equations (1) and (2), it can be calculated that when the L-Trp solution is dropped into the hydrogel with holes of 202 nm and 428 nm, the corresponding theoretical positions of the reflection peaks are located at 448 nm and 948 nm, respectively. From Figure 7(a)(b)-(i), it is found that the actual positions of the reflection peaks of the inverse opal hydrogels with different pore sizes appear at 520 nm and 733 nm, respectively. The theoretical value of the reflection peak position is closer to the actual value for the smaller hole, but the difference is larger for the larger hole. This may be due to the more serious local deformation of the larger hole after the hydrofluoric acid solution etching (see Figure 6). In addition, we also found that L-Trp solutions with different concentrations were dropped into the hydrogel with smaller pores, and the reflection peak shifted to the position with higher wavelength. The main reason is that when the target molecule is captured by a specific hole point on the sensor, the two will combine together, resulting in the expansion of the inverse opal structure, thus showing an obvious red-shift phenomenon. Figures 7(a)(b)-(ii) are plots of the intensity of the reflection peak in (i) versus the log log(L-Trp) of the L-Trp solution concentration. The logarithm (log(L-Trp)) of the reflected light intensity and the concentration corresponding to the L-Trp concentration between 10 ^-7 M and 10 ^-3 M is close to a linear relationship, which shows that the sensor has a good effect on L -Trp's detection range is in between. In addition, the regression curve and the regression coefficient between the logarithm of the reflected light intensity and the concentration of the molecularly imprinted hydrogel with a hole of 202 nm are y=-119.67x+894 and R ² =0.9901, respectively; while the hole of 428 nm, its regression The curve and regression coefficient are y=-40.267x+315.8 and R ² =0.9813, respectively. The greater the slope of the regression curve, the greater the displacement, and the more obvious the change in structure color. This phenomenon is also found in the study of the volatilization of ethanol [Reference 19]. In addition, the higher the linear regression coefficient (R ² ) value, the better the reproducibility. Specificity analysis of molecularly imprinted hydrogels: In order to test the specificity of inverse opal hydrogel identification, we used L-Phenylalanine (L-Phenylalanine, L-Phe; Sigma-Aldrich), which is structurally similar to L-tryptophan. carry out testing. Figures 8(a)(b)-(i) are the spectral changes after dripping L-Trp solution with a concentration of 10 ^-3 to 10 ^-9 M to molecularly imprinted hydrogels with pores of 202 nm and 428 nm, respectively. We found that when the L-Trp solution was dropped into the molecularly imprinted hydrogel with a hole of 202 nm, the absorption peak intensity in the ultraviolet band increased with the increase of the L-Trp concentration. However, the absorption peaks of the hole with 428nm only have obvious changes in the concentration of L-Trp between 10 ^-3 and 10 ^-5 M, and the rest are corresponding to the concentration of L-Trp between 10 ^-6 and 10 ^-9 M The spectra almost overlap. Figure 8(a)(b)-(ii) are the ^spectral responses of molecularly imprinted hydrogels with holes of 202 nm and 428 nm when they were dropped into L- ^Phe solution. There is a clear identification function between the concentrations, and the other spectra in the concentration range of 10 ^-6 to 10 ^-9 M overlap each other. We found that hydrogels with smaller pores were more specific for L-Trp recognition than those with larger pores. This may be because the hole size of the inverse opal hydrogel sensor is much larger than the target molecule to be recognized, resulting in a decrease in the matching accuracy of the hole position in the sensor with the target molecule [Ref. 20]. Chromatic response of molecularly imprinted hydrogels to trace concentrations: Figures 9(a)-(i) and (ii) show the molecularly imprinted hydrogels with different concentrations of L-Trp solutions dropped into the molecularly imprinted hydrogels with holes of 202 nm and 428 nm, respectively. Rendered reflectance spectrum. When the concentration of dropwise Trp solution increased from 10 ^-9 M to 10 ^-3 M, the reflection peak positions moved from 413 nm to 531 nm and 540 nm to 602 nm, respectively. The displacement of the former (Δn=118nm) is significantly larger than that of the latter (Δn=62nm), indicating that the former has the potential to identify the changes of trace concentrations with the naked eye. Figure 9(b) and Table 1 show the changes in the chromaticity coordinates of the corresponding chromaticity coordinates after different concentrations of L-Trp solutions were dropped into the pores of the hydrogel. The chromaticity coordinates of the molecularly imprinted hydrogel with a hole of 202 nm will move from blue (0.1873, 0.1212) (L-Trp concentration = 10 ^-9 M) to green (0.234, 0.5957) (L-Trp Concentration = 10 ^-3 M) position. And the position of the chromaticity coordinates of the hole is 428nm from blue (0.1916, 0.0969) to red (0.3576, 0.3182). Obviously, the inverse opal hydrogel with pore size of 202nm is the best choice for detecting the trace concentration of L-Trp. Figure 9(c) is the movement path diagram of the chromaticity position corresponding to (b), (i) and (ii) prepare inverse opal hydrogels from 202 nm and 428 nm, respectively. It can be found that the former coordinate point position is located at The dark blue area, with the increase of the concentration of Trp solution (10 ^-9 M→10 ^-7 M→10 ^-5 M→10 ^-3 M), gradually moves towards the green direction, that is, from blue to green ((0.1873, 0.1212)→(0.2090,0.2655)→(0.2931,0.4138))→(0.2340,0.5957); the latter gradually moves from the original dark blue to the red direction ((0.1916,0.0969)→(0.2843,0.2484)→(0.2941 ,0.2353))→(0.3576,0.3182). Response speed and reproducibility of molecularly imprinted hydrogels: Figure 10(a) shows the results of response time testing of molecularly imprinted hydrogels with 202nm and 428nm holes using L-Trp at a concentration of 10 ^-6 M, respectively. It was found that the absorption values of the two were between 0.3 and 0.4 when they were dropped into L-Trp, but with the increase of the adsorption time, it was found that the time for reaching the adsorption equilibrium for the target molecule was shorter with the smaller pores, and the two were at 12 and reached saturation after 13 minutes. The interconnected structure of the pores in the inverse opal hydrogel is easy to form an effective recognition site with the target molecule, but the size of the pores of the hydrogel has no significant effect on the adsorption equilibrium time of the target molecule. Figure 10(b) shows that after repeated adsorption and elution of molecularly imprinted hydrogels with pores of 202 nm and 428 nm with L-Trp at a concentration of 10 ^-6 M, it was found that both exhibited good response characteristics and did not Because the efficiency is reduced due to multiple use, it has good reusability. The molecularly imprinted hydrogels with holes of 202 nm and 428 nm showed a significant decrease in light intensity after the 20th and 16th repeated elution, respectively, indicating the number of times the sensor can be used repeatedly. Figure 10 and Table 2 compare the sensor of the present invention with the technology commonly used to detect L-Trp at present. It is found that the sensor produced by the present invention has the fastest response time, and the detection range is indistinguishable from other technologies. .

Conclusion: The present invention arranges the polymerized silica nanospheres into a monolayer array by LB deposition. And through the combination of photonic crystal and molecular imprinting technology, an inverse opal hydrogel sensor that can rapidly detect the change of L-Trp trace concentration was successfully prepared. The research results show that the hydrogel with smaller pores has better specificity for L-Trp identification than the one with larger pores, and has better detection effect at the concentration of 10 ^-3 ~10 ^-7 M. It can reach adsorption equilibrium with the target molecule to be measured within 12 minutes. When different concentrations of L-Trp were dropped into the hydrogel with smaller holes, the displacement of the reflection peak was significantly larger than that with larger holes. And the movement path corresponding to its chromaticity coordinates will move from blue to green, showing that the inverse opal hydrogel has the characteristic of signal self-display, without the need to use more expensive instruments and facilities to detect specific target molecules. In addition, under the condition of similar detection concentration, the sensor has the fastest response time compared with other technologies. In addition, the sensor will not reduce the efficiency due to repeated use, and still has excellent reproducibility. The production and detection technology of the visualized inverse opal hydrogel provided by the present invention has excellent application prospects in industries such as biological detection, agriculture and medical rapid screening in the future.

The above descriptions are only feasible embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent implementation of other changes based on the content, features and spirits described in the following claims shall be Included in the patent scope of the present invention. The structural features of the present invention, which are specifically defined in the claim, are not found in similar articles, and are practical and progressive, and have met the requirements for a patent for invention. The legitimate rights and interests of the applicant.

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Claims (7)

An inverse opal hydrogel sensor for sensing food additives, comprising an inverse opal hydrogel sensing film for identifying L-Trp molecular imprints, the inverse opal hydrogel sensing film is prepolymerized The inverse opal hole array structure is a single-layer inverse opal hole array structure, which is molded by a single-layer SiO ₂ microsphere crystal array structure template, and the size of each inverse opal hole is 180~450nm. In order to match with the L-Trp molecular imprint, the inverse opal hydrogel sensing film is an inverse opal hydrogel sensing film with L-Trp molecular imprint holes, and the inverse opal hydrogel senses The measurable L-Trp concentration range of the film is 10 ^-3 to 10 ^-9 M; wherein, when the L-Trp target molecule solution is dropped into the inverse opal hydrogel sensing film, the inverse opal hydrogel sensing film When the L-Trp molecularly imprinted holes on the L-Trp match with the L-Trp target molecule, the hydrogel will shrink or swell, resulting in the change of the structural parameters of the inverse opal hole array, which in turn causes the shift of the wavelength position of the Bragg diffraction peak. The colorimetric analysis technique predicts the concentration of the L-Trp target molecule solution according to the chromaticity coordinate position corresponding to its reflection spectrum. The inverse opal hydrogel sensor for sensing food additives according to claim 1, wherein the size of each inverse opal hole of the inverse opal hole array structure is 202 nm. A method for producing an inverse opal hydrogel sensor for sensing food additives as described in claim 1, comprising: (A) Synthesis steps of silicon dioxide (SiO ₂ ) nano-microspheres: mix 20~30(25)mL of tetraethyl silicate and 4~5.5mL of absolute ethanol, stir evenly at 250~350rpm for 20~ After 40 minutes, 4-10 mL of ammonia water, 1-5 mL of deionized water and 20-30 mL of deionized ethanol were added to conduct the catalytic reaction for 18-30 hours to obtain a nano-scale silica microsphere suspension; The synthesized nanoscale silica microsphere suspension was centrifuged at 2~6°C for 15~45 minutes at 5000~7000rpm, and the supernatant was removed to obtain SiO ₂ microspheres; SiO was washed with deionized water. ₂ microspheres to obtain SiO ₂ microspheres with uniform particle size; (B) The preparation step of the silica monolayer opal template: the first glass substrate is immersed in 8-12wt% sodium hydroxide aqueous solution for 20-40 minutes to modify the surface hydrophilicity; the modified first glass substrate is Rinse with deionized water and dry it for later use; take 1~3g of SiO ₂ microspheres and mix with 1~3mL of absolute ethanol and 6~10mL of chloroform, and then ultrasonically vibrate for 5~15 minutes to obtain SiO ₂ microsphere suspension Then pour 150~250mL of deionized water into the deposition tank, and control the injection flow rate of the SiO ₂ microsphere suspension to 50~60 μl/min by the pump, so that the SiO ₂ microspheres spread on the liquid surface of the deposition tank; Then, insert the modified hydrophilic glass sheet under the liquid surface of the deposition tank, and then slowly transfer the SiO ₂ microspheres spread on the surface of the liquid surface to the surface of the hydrophilic glass to form a single-layer SiO ₂ microsphere crystal array; After the liquid is volatilized, a single-layer SiO ₂ microsphere crystal array structure template attached to the surface of the first glass substrate is obtained; and (C) The preparation steps of the hydrogel sensor include: (C1) Preparation steps of L-Trp prepolymer solution: take 0.01~0.03g of L-tryptophan and dissolve it in 0.5~1.5mL of methanol, 0.3~0.7mL of methacrylic acid and 0.3~0.7mL of ethylene glycol After the dimethacrylate is evenly mixed and stirred, it is placed in a refrigerator at 2~6°C for 10~14 hours and taken out, and 0.005~0.015g of 2,2-azobisisobutyronitrile is added to stir at a speed of 150~350rpm. nitrogen gas was introduced to remove oxygen to obtain a prepolymer solution containing the L-Trp target molecule; and (C2) Preparation steps of L-Trp hydrogel sensing film: cover a second glass substrate on the surface of the single-layer SiO ₂ microsphere crystal array structure template, and immerse the single-layer SiO ₂ microsphere crystal array structure template in a In the prepolymer solution of the L-Trp target molecule, the prepolymer solution containing the L-Trp target molecule was infiltrated into the gap between the SiO ₂ microspheres and the glass by capillary action; when the template became transparent, it indicated that the The prepolymer of the L-Trp target molecule has completely filled the gaps between the spheres of the SiO ₂ microspheres; then the template containing the prepolymer containing the L-Trp target molecule was photopolymerized with ultraviolet light for 1.5~2.5 hours ; Then soak the template in a 2-4% hydrofluoric acid solution for 1.5-2.5 hours to remove the SiO ₂ microspheres and separate the first glass substrate and the second glass substrate of the upper and lower layers of the template to obtain a L-tryptophan inverse opal hydrogel film; then put the inverse opal hydrogel film containing L-tryptophan in 0.5-1.5% glacial acetic acid solution to remove the inverse opal hydrogel film L-Trp target molecule is obtained, and an inverse opal hydrogel sensing film with L-Trp molecular imprinting holes and capable of recognizing L-Trp molecular imprinting is obtained. The method according to claim 3, wherein, in step (a), 25 mL of tetraethyl silicate and 4.8 mL of anhydrous ethanol are mixed, and after uniform stirring at 300 rpm for 30 minutes, 7 mL of ammonia water and 3 mL of ammonia are added. After 24 hours of catalytic reaction with deionized water and 25 mL of anhydrous ethanol, a nano-scale silica microsphere suspension was obtained; then the synthesized nano-scale silica microsphere suspension was passed through a centrifuge at 4 °C After centrifugation at 6000 rpm for 30 minutes, the supernatant was removed to obtain SiO ₂ microspheres; the SiO ₂ microspheres were washed with deionized water to obtain SiO ₂ microspheres with uniform particle size. The method according to claim 3, wherein the particle size of the SiO ₂ microspheres is between 234 and 542 nm. The method according to claim 3, wherein, in step (b), the first glass substrate is immersed in a 10wt% sodium hydroxide aqueous solution for 30 minutes to carry out surface hydrophilic modification; 2g of SiO ₂ microspheres are mixed Add 2 mL of absolute ethanol and 8 mL of chloroform, and then ultrasonically shake for 10 minutes to obtain the SiO ₂ microsphere suspension; then pour 200 mL of deionized water into the sedimentation tank, and control the SiO ₂ microsphere suspension by the pump. The injection flow was 54.4 μl/min, so that the SiO ₂ microspheres spread on the liquid surface of the deposition tank. The method according to claim 3, wherein, in step (c1), 0.02 g of L-tryptophan is dissolved in 1 mL of methanol, 0.5 mL of methacrylic acid and 0.5 mL of ethylene glycol dimethacrylate After evenly stirring, it was placed in a refrigerator at 4°C for 12 hours, taken out, and 0.01 g of 2,2-azobisisobutyronitrile was added to stir at a rotational speed of 300 rpm; and, in step (c2), the target containing L-Trp The template of the molecular prepolymer was subjected to photopolymerization for 2 hours with ultraviolet light with a wavelength of 365 nm; the template was then immersed in a 3% hydrofluoric acid solution for 2 hours to remove the SiO ₂ microspheres; The inverse opal hydrogel film of amino acid was placed in a 1% glacial acetic acid solution to remove the L-Trp target molecules in the inverse opal gel film, and a hydrogel sensor capable of recognizing the molecular imprint of L-Trp was obtained. .

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