阅读说明：本技术 一种基于水相的光子晶体分子印迹水凝胶传感器及其制备方法和检测方法 (Photonic crystal molecularly imprinted hydrogel sensor based on water phase and preparation method and detection method thereof ) 是由 罗爱芹 刘宗坤 侯慧鹏 梁阿新 刘毅权 许立霖 王微 于 2021-04-23 设计创作，主要内容包括：本发明研究了一种基于水相的的光子晶体分子印迹水凝胶传感器及其制备方法和检测方法,属于分子印迹传感领域。本发明选择溶菌酶(Lysozyme,Lyz)作为研究对象,将分子印迹技术与光子晶体相结合,通过一种新的印迹方法和印迹体系,在水相中通过一种简单的方式制备了光子晶体分子印迹水凝胶传感器。首先采用改进的法制备了单分散二氧化硅微球,随后使用垂直自组装法制备光子晶体模板,聚合溶液在模板表面聚合成膜后,使用氢氟酸除去光子晶体模板,最后使用模板得到反蛋白石结构的光子晶体分子印迹水凝胶传感器。该水凝胶传感器具有良好的选择性和灵敏度,已经可以成功用于实际样品中溶菌酶的检测。(The invention researches a photonic crystal molecularly imprinted hydrogel sensor based on a water phase, and a preparation method and a detection method thereof, and belongs to the field of molecularly imprinted sensing. The invention selects Lysozyme (Lysozyme, Lyz) as a research object, combines the molecular imprinting technology with photonic crystal, and adopts a novel imprinting method and imprintingThe photonic crystal molecularly imprinted hydrogel sensor is prepared in a simple mode in a water phase. First of all by improved The method comprises the steps of preparing monodisperse silicon dioxide microspheres, preparing a photonic crystal template by using a vertical self-assembly method, polymerizing a polymerization solution on the surface of the template to form a film, removing the photonic crystal template by using hydrofluoric acid, and finally obtaining the photonic crystal molecularly imprinted hydrogel sensor with an inverse opal structure by using the template. The hydrogel sensor has good selectivity and sensitivity, and can be successfully used for detecting lysozyme in actual samples.)

1. The water phase-based photonic crystal molecularly imprinted polymer hydrogel sensor is characterized in that the hydrogel has an inverse opal photonic crystal structure, is a highly ordered porous material, has a photonic energy band and a band gap, has a large specific surface area, and can generate a Bragg diffraction phenomenon under the irradiation of light with a specific wavelength.

2. The photonic crystal molecularly imprinted hydrogel sensor of claim 1, wherein the template of the inverse opal structure is obtained by a vertical self-assembly method from silica microspheres with a size of 283nm, the microspheres are arranged closely and have a regular structure. The hydrogel is polymerized on the surface of the template and wraps the template, and the silica microspheres are removed by hydrofluoric acid to obtain the hydrogel with the inverse opal photonic crystal structure.

3. The photonic crystal molecularly imprinted hydrogel sensor according to claim 1, wherein the hydrogel film is composed of N, N' -methylenebisacrylamide as a cross-linking agent, acrylamide as a functional monomer, and a target protein. The acrylamide and the target protein are subjected to prepolymerization at low temperature, and the target protein is wrapped in the polymer when the cross-linking agent and the functional monomer are polymerized on the surface of the template, so that the specific recognition site is introduced into the hydrogel.

4. The photonic crystal molecularly imprinted hydrogel sensor of claim 1, wherein the synthesis of the hydrogel is completely carried out in an aqueous phase, so that the activity and structure of the target protein can be well protected.

5. A method for preparing the photonic crystal molecularly imprinted hydrogel sensor of claim 1, comprising the following steps

1) Synthesizing silica microspheres with the diameter of 283nm by using tetraethyl silicate, ammonia water, water and ethanol, washing the product by using ethanol, centrifuging and drying the product, and preparing a silica-ethanol dispersion liquid with the mass fraction of 2% from the dried product;

2) the glass slide was cut into two sizes of 1 mm. times.8 mm. times.76 mm and 1 mm. times.10 mm. times.30 mm, as a slide glass and a pressed sheet, respectively. Washing with deionized water in piranha solution (98% H)₂SO₄-30％H₂O₂7:3, v/v) for 24 hours, washing with deionized water and ethanol in sequence, and drying with nitrogen;

3) vertically inserting the glass slide into the dispersion liquid, and assembling a photonic crystal template with an opal structure on the surface of the glass by adopting a vertical self-assembly method; the self-assembly temperature is 35 ℃, and the time is 72 hours;

4) the target protein was dissolved in phosphate buffer (0.2mol/L, pH 6.4), and an amount of N, N' -methylenebisacrylamide and an amount of acrylamide were added. The mass ratio of the target protein to the N, N' -methylene bisacrylamide to the acrylamide is 1:2: 15-2: 3: 17; mixing for 5 minutes by using a vortex mixer, placing the mixture in a refrigerator at 4 ℃ for prepolymerization for 3 hours, introducing nitrogen for 10 minutes, and adding an ammonium persulfate solution with the mass fraction of 10% and a certain amount of tetramethylethylenediamine to initiate polymerization;

5) quickly injecting the initiated mixed solution into a sandwich structure consisting of a pressing sheet and a glass slide with a photonic crystal template, clamping by using a clamp, and standing for 1 hour;

6) and (3) placing the sandwich structure in deionized water until the gel falls off automatically. Soaking the silicon dioxide microsphere template for 20-30 minutes by using hydrofluoric acid with the mass fraction of 1 percent to remove the silicon dioxide microsphere template;

7) immersing the mixture into a mixed solution of 10% acetic acid and 10% sodium dodecyl sulfate at a volume ratio of 1:1 for 15 minutes to remove the target protein.

6. The method according to claim 5, wherein step 7) is followed by step 8) to prepare a control, and the non-imprinted polymer is prepared according to the methods of steps 1) to 7), without adding the target protein during the polymerization.

7. The preparation method according to claim 5, wherein the volume ratio of the 10% ammonium persulfate solution to the tetramethylethylenediamine in the step 4) is 4: 1.

8. the method according to claim 5, wherein the thickness of the sandwich structure used in step 5) is 1 mm.

9. The photonic crystal molecularly imprinted hydrogel sensor of claim 1, wherein the target protein used is lysozyme.

10. The method for detecting lysozyme by using the photonic crystal molecularly imprinted hydrogel sensor as claimed in claim 1, which is characterized by comprising the following steps:

1) the hydrogel was immersed in lysozyme standard solutions containing different concentration gradients, respectively. After incubation for 20min at room temperature, measuring the Bragg diffraction peak wavelength of the hydrogel by using an ultraviolet spectrophotometer, and drawing by using the variation of the wavelength and the concentration;

2) immersing the hydrogel into a lysozyme standard solution of 500 mu g/mL, measuring the Bragg diffraction peak wavelength of the hydrogel once every 2.5 minutes by using an ultraviolet spectrophotometer until the wavelength is not changed, and plotting the wavelength and the time;

3) and (3) selecting Bovine serum albumin (Bovine albumin), L-glutamic acid (L-Glu) and Escherichia coli holoprotein (The white Cell Proteins in Escherichia coli) as an interferent of lysozyme to determine The selectivity of The photonic crystal molecularly imprinted hydrogel membrane.

Technical Field

The invention belongs to the field of molecular imprinting sensing, and particularly relates to a photonic crystal molecular imprinting hydrogel sensor based on a water phase, and a preparation method and a detection method thereof.

Background

Lysozyme (Lyz) is a macromolecular protein with a molecular weight of about 14-15 kDa. As an alkaline enzyme, it can hydrolyze mucopolysaccharide in bacteria to crack the bacteria, and has certain antibacterial effect. Lysozyme is widely present in microorganisms, animal and plant tissues, egg white of birds and birds, tears of mammals, blood and milk, and about 13. mu.g of lysozyme is contained in cow's milk per 100 mL. Due to the special antibacterial property, the product can be used as a good additive in infant food and beverage. At present, some specific antigens are inoculated to dairy cows to improve the content of lysozyme in milk and further improve the quality of the milk.

At present, methods for detecting the content of lysozyme in food mainly comprise chromatography, spectrophotometry, capillary electrophoresis, enzyme-linked immunosorbent assay (ELISA), High Performance Liquid Chromatography (HPLC) and the like. Most of the methods have the advantages of high sensitivity and high accuracy. However, a complex pre-processing process is often required, resulting in low detection efficiency. In addition, each detection method has respective disadvantages, such as that ELISA method requires specific antibody, HPLC method is complex in operation and high in cost, and efficient detection is difficult. Therefore, a rapid lysozyme content detection method with low cost and high detection efficiency is urgently needed to be developed.

Photonic Crystals (PCs) are optical materials with Photonic energy bands and band gaps, which are formed by periodically arranging and arranging several materials with different refractive indexes in space, can control the motion mode of photons, and have an ordered structure similar to that of crystals. Molecular Imprinting Technique (MIT) is a technique for creating a polymer system with specific three-dimensional recognition and binding sites, i.e. by preparing a polymer with specific binding sites complementary to specific template molecules, specific recognition of a target is achieved. Has been applied to the separation and purification of certain substances in food. However, since the molecular imprinting only has selective adsorption and does not have sensing characteristics, after the molecular imprinting is used for separation, other instruments (such as a spectrophotometer) are needed to detect the adsorption result, and the detection efficiency is reduced. By combining the molecular imprinting technology with the photonic crystal technology, photonic crystal Molecularly imprinted hydrogels (MIPHs) with high selectivity and high sensing performance can be obtained. After the material is combined with the template molecules, the interplanar spacing and the refractive index of the material can be changed, so that the wavelength change of a Bragg diffraction peak of the material is caused, the change is macroscopically expressed as the change of the color of the material, and the sensing of a target substance is realized. Therefore, MIPH has the advantages of no need of other instruments, simple preparation process, fast response speed, and the like, and has been used for detection of some small molecules and ions.

Although the current methods for preparing MIPH and detection targets are diverse, the conventional method for preparing MIPH in an organic phase is difficult to be applied to the detection of specific protein molecules because proteins have a specific three-dimensional structure and are easily denatured. At present, a preparation method for directly synthesizing a photonic crystal molecularly imprinted hydrogel sensor for protein in a water phase by a one-step method is not reported.

Disclosure of Invention

The invention aims to provide a preparation method for directly synthesizing a photonic crystal molecularly imprinted hydrogel sensor aiming at protein in a water phase by a one-step method, and the method is used for quickly detecting lysozyme.

The purpose of the invention is mainly realized by the following technical means:

the invention relates to a photonic crystal molecularly imprinted hydrogel sensor, which has the following structure:

1) the substrate is selected from pure hydrophilized glass sheet, and a layer of silicon dioxide (SiO) is self-assembled on the surface of the glass sheet₂) Microspheres, forming a photonic crystal template (PC).

2) The surface of PC is covered with a layer of hydrophilized glass sheet, and a prepolymerization solution (containing phosphate buffer, functional monomer, crosslinking agent, Lyz) after adding an initiator is injected into the gap between PC and glass and polymerized.

3) Stripping the two glass sheets and removing SiO therein using hydrofluoric acid₂And (4) carrying out microsphere preparation to obtain the photonic crystal molecularly imprinted hydrogel (MIPH) with the inverse opal structure.

4) Finally, the template protein Lyz is removed by eluent to form Lyz-MIPH.

The method for preparing the photonic crystal molecularly imprinted hydrogel sensor for the protein in the water phase by the one-step method comprises the following specific steps:

1) a certain amount of ethanol, water and ammonia water are measured, the mixture is uniformly mixed and then placed in a water bath for stirring at a certain temperature, and then a certain amount of tetraethyl silicate and ethanol are measured, and the mixture is quickly added into the solution for reaction for 3 hours after uniform mixing. Centrifuging and drying after the reaction is finished to obtain 283nm silicon dioxide microspheres;

2) dispersing the microspheres in ethanol to form a silicon dioxide-ethanol dispersion liquid with a certain mass fraction;

3) the glass slide was cut into two sizes of 1 mm. times.8 mm. times.76 mm and 1 mm. times.10 mm. times.30 mm, as a slide glass and a pressed sheet, respectively. Washed in piranha solution (98% H)₂SO₄-30％H₂O₂7:3, v/v) for 24 hours, cleaning and drying;

4) vertically inserting the glass slide into the dispersion liquid, and assembling PC with an opal structure on the surface of the glass by adopting a vertical self-assembly method; the self-assembly temperature is 35 ℃, and the time is 72 hours;

5) weighing a certain amount of lysozyme, acrylamide and N, N' -methylene bisacrylamide, dissolving in a phosphate buffer solution with the pH value of 6.4, and performing prepolymerization for 2 h; adding a certain amount of 10 mass percent ammonium persulfate solution and tetramethylethylenediamine into the prepolymerization solution, then quickly dropping the solution into a gap between PC and glass with a certain height, and polymerizing for 1h at room temperature;

6) treating the glass and the hydrogel by using a hydrofluoric acid solution with a certain concentration to remove the glass substrate and the silicon dioxide microspheres;

7) washing the obtained hydrogel, and removing target protein Lyz in the hydrogel by using an eluent to obtain Lyz-MIPH;

8) as a control group, a non-imprinted polymer hydrogel (NIPH) was prepared according to the same procedure, except that the target protein Lyz was not added thereto, in the same manner as Lyz-MIPH;

specifically recognizing and detecting lysozyme based on a photonic crystal molecularly imprinted polymer hydrogel sensor:

9) the hydrogel was immersed in lysozyme standard solutions containing different concentration gradients, respectively. After incubation for 20min at room temperature, measuring the Bragg diffraction peak wavelength of the hydrogel by using an ultraviolet spectrophotometer, and drawing by using the variation of the wavelength and the concentration;

10) immersing the hydrogel into a lysozyme standard solution of 500 mu g/mL, measuring the Bragg diffraction peak wavelength of the hydrogel once every 2.5 minutes by using an ultraviolet spectrophotometer until the wavelength is not changed, and plotting the wavelength and the time;

11) and (3) selecting Bovine serum albumin (Bovine albumin), L-glutamic acid (L-Glu) and Escherichia coli holoprotein (The white Cell Proteins in Escherichia coli) as an interferent of lysozyme to determine The selectivity of The photonic crystal molecularly imprinted hydrogel membrane.

Further, in the step 1), the reaction temperature is 35 ℃; the volume of each substance in the mixed solution of the ethanol, the water and the ammonia water is 16.25mL, 24.75mL and 9mL respectively; the volumes of the tetraethyl silicate and ethanol in the mixture were 4.5mL and 45.5mL, respectively.

Further, in the step 2), the mass fraction of the silica-ethanol dispersion is 2%.

Further, in the step 5), the mass ratio of the lysozyme to the N, N' -methylene bisacrylamide to the acrylamide is 1:2: 15-2: 3: 17.

Further, in the step 5), the volume ratio of 10% by mass of ammonium persulfate solution to tetramethylethylenediamine is 4: 1.

further, in the step 6), the mass fraction of the hydrofluoric acid solution is 1%, and the treatment time is 30 min.

Further, in step 7), the eluent is 20mL of 10% (v/v) acetic acid: 10% sodium dodecyl sulfate, and the elution time is 72 h.

The invention has the beneficial effects that: the hydrogel sensor synthesized by the method has good stability and strength, can realize specific identification and adsorption of lysozyme in a complex sample, can show the change of gel color, has short response time, and has good linear relation between the wavelength of a Bragg diffraction peak and the concentration of the lysozyme.

According to the invention, the photonic crystal molecularly imprinted hydrogel sensor for lysozyme is directly synthesized in a water phase system, so that compared with the reported photonic crystal molecularly imprinted hydrogel, the photonic crystal molecularly imprinted hydrogel sensor can better keep the activity of protein and has better sensing performance; organic solvent is not needed in the preparation process, so that the environment can be effectively protected. In the invention, the sensor is directly prepared by adopting a one-step method, complex molecular imprinting preparation and treatment processes are not needed, and the method has the advantages of simple preparation, simple operation and the like. The hydrogel sensor has good selectivity and sensitivity, and can be successfully used for detecting lysozyme in actual samples.

Drawings

FIG. 1 is an SEM image of a photonic crystal template (PC)

FIG. 2 is an SEM image of a photonic crystal molecularly imprinted hydrogel sensor (Lyz-MIPH)

FIG. 3 is a linear relationship between Bragg diffraction peak wavelength and lysozyme concentration of photonic crystal molecularly imprinted hydrogel sensor (Lyz-MIPH)

FIG. 4 shows the relationship between Bragg diffraction peak wavelength and adsorption time of photonic crystal molecularly imprinted hydrogel sensor (Lyz-MIPH) and non-imprinted sensor (Lyz-NIPH)

FIG. 5 shows the Bragg diffraction peak variation of photonic crystal molecularly imprinted hydrogel sensor (Lyz-MIPH) and non-imprinted sensor (Lyz-NIPH) for different substances

Detailed Description

Example 1

1. The water phase-based photonic crystal molecularly imprinted polymer hydrogel is characterized in that the hydrogel has an inverse opal photonic crystal structure, is a highly ordered porous material, has a photonic energy band and a band gap, has a large specific surface area, and can generate a Bragg diffraction phenomenon under the irradiation of light with a specific wavelength.

2. The photonic crystal molecularly imprinted hydrogel film of claim 1, wherein the template of the inverse opal structure is obtained by a vertical self-assembly method from silica microspheres with a size of 283nm, the microspheres are arranged closely and have a regular structure. The hydrogel is polymerized on the surface of the template and wraps the template, and the silica microspheres are removed by hydrofluoric acid to obtain the hydrogel with the inverse opal photonic crystal structure.

3. The photonic crystal molecularly imprinted hydrogel film of claim 1, wherein the hydrogel film is composed of N, N' -methylenebisacrylamide as a cross-linking agent, acrylamide as a functional monomer, and a target protein. The acrylamide and the target protein are subjected to prepolymerization at low temperature, and the target protein is wrapped in the polymer when the cross-linking agent and the functional monomer are polymerized on the surface of the template, so that the specific recognition site is introduced into the hydrogel.

4. The photonic crystal molecularly imprinted hydrogel membrane of claim 1, wherein the synthesis of the hydrogel is completely carried out in the aqueous phase, so as to better protect the activity and structure of the target protein.

5. The photonic crystal molecularly imprinted hydrogel membrane of claim 1, wherein the target protein used is lysozyme.

Example 2

1) Synthesizing silica microspheres with the diameter of 283nm by using tetraethyl silicate, ammonia water, water and ethanol, washing the product by using ethanol, centrifuging and drying the product, and preparing a silica-ethanol dispersion liquid with the mass fraction of 2% from the dried product;

2) the glass slide was cut into two sizes of 1 mm. times.8 mm. times.76 mm and 1 mm. times.10 mm. times.30 mm, as a slide glass and a pressed sheet, respectively. Washing with deionized water, soaking in piranha solution (98% H2SO 4-30% H2O2, 7:3, v/v) for 24 hr, washing with deionized water and ethanol, and drying with nitrogen;

3) vertically inserting the glass slide into the dispersion liquid, and assembling a photonic crystal template with an opal structure on the surface of the glass by adopting a vertical self-assembly method; the self-assembly temperature is 35 ℃, and the time is 72 hours;

4) dissolving 23mg of target protein in 1.0mL of phosphate buffer (0.2mol/L, pH 6.4), adding 15mg of N, N' -methylene bisacrylamide and 174mg of acrylamide, mixing for 5 minutes by using a vortex mixer, placing in a refrigerator at 4 ℃ for prepolymerization for 3 hours, introducing nitrogen for 10 minutes, and adding 20. mu.L of a 10% ammonium persulfate solution by mass fraction and 2.5. mu.L of tetramethylethylenediamine to initiate polymerization;

5) quickly injecting the initiated mixed solution into a sandwich structure which is 1mm thick and consists of a tabletting and a glass slide with a photonic crystal template, clamping the sandwich structure by using a clamp, and standing for 1 hour;

6) and (3) placing the sandwich structure in deionized water until the gel falls off automatically. Soaking the silicon dioxide microsphere template for 30 minutes by using 1% hydrofluoric acid to remove the silicon dioxide microsphere template;

7) immersing the protein into a mixed solution of 10% acetic acid and 10% sodium dodecyl sulfate in a volume ratio of 1:1 for 15 minutes to remove the target protein;

8) a control group was prepared, and a non-imprinted polymer was prepared according to the same procedure without adding the target protein during the polymerization.

Example 3

The photonic crystal molecularly imprinted hydrogel sensor prepared based on example 2 was used for detection:

1) the hydrogel was immersed in lysozyme standard solutions containing different concentration gradients, respectively. After incubation for 20min at room temperature, measuring the Bragg diffraction peak wavelength of the hydrogel by using an ultraviolet spectrophotometer, and drawing by using the variation of the wavelength and the concentration;

2) immersing the hydrogel into a lysozyme standard solution of 500 mu g/mL, measuring the Bragg diffraction peak wavelength of the hydrogel once every 2.5 minutes by using an ultraviolet spectrophotometer until the wavelength is not changed, and plotting the wavelength and the time;

3) and (3) selecting Bovine serum albumin (Bovine albumin), L-glutamic acid (L-Glu) and an escherichia coli protein extract as an interferent of lysozyme to determine the selectivity of the photonic crystal molecularly imprinted hydrogel membrane.

As shown in FIG. 3, the wavelength of the Bragg diffraction peak and the concentration of Lyz are in good linear relation in the range of 100-500 μ g/mL, and the linear equation is that the Delta lambda is-4.5 +0.109C (R)²0.984), C is mass concentration of Lyz, and Δ λ is the amount of change in wavelength of Bragg diffraction peak. The sensor prepared by the working method has a good linear range and a lower detection limit, and can be applied to content detection of lysozyme in an actual sample.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.