A method for visualizing rapid detection of nitrilase

Abstract

The application belongs to the technical field of detection and relates to a method for visual rapid detection of nitrilase. Functional monomers, cross-linking agents and initiators are mixed in a solvent to form a copolymer system, which is then ultrasonically treated to make the mixture uniform, so as to obtain a prepolymer solution, which is stored in cold storage for standby use. The functional monomers are double-bonded nitrile compounds. The prepolymer solution is dropped into the gap of a photonic crystal template and polymerized under a UV lamp. After polymerization is completed, the inverse opal nitrile-based polymer photonic crystal is obtained through etching, and then the inverse opal nitrile-based polymer photonic crystal is washed with deionized water so as to reach swelling equilibrium and be neutral. The inverse opal nitrile-based polymer photonic crystal is placed in an alkaline solution to obtain a mixed system. Nitrilase solutions with different concentrations are dropped into the mixed system. The nitrilase makes the nitrile groups on the inverse opal nitrile-based polymer photonic crystal catalytically hydrolyzed into carboxyl groups, so that the photonic crystal swells and the lattice constant changes, thereby realizing visual detection of the nitrilase.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a method for visual rapid detection of nitrilase. BACKGROUND

[0002] Nitrilase is an important biological catalyst, which can hydrolyze nitrile compounds into corresponding carboxylic acid and ammonia under mild conditions, and has no by-products and little environmental pollution. Using nitrilase with selectivity to prepare fine chemicals has great application value. Because nitrilase has high stereoselectivity in hydrolysis of nitrile compounds, the corresponding nitrile compounds can be hydrolyzed by nitrilase to prepare carboxylic acids which are difficult to synthesize or have low yield under general chemical conditions. For example, diacid can be obtained by enzyme hydrolysis of dinitrile. Iminodiacetic acid is an important raw material for synthesizing herbicides, which can be obtained by chemical methods such as chloroacetic acid method and diethanolamine method, but the reaction process is seriously polluted and requires high equipment. Researchers use nitrilase to catalyze imino diethyl nitrile to obtain imino diacetic acid, and optimize Alcaligenes faecalis ZJB-09133 nitrilase to make the conversion rate reach 72%. Later, someone immobilized Arthrobacter aurescens CYC705 nitrilase and added metal ions to the reaction system, so that 200 mmol / L of imino diethyl nitrile can be completely converted into imino diacetic acid in 1 h, greatly improving the reaction rate and conversion rate. Nitrilase also has important applications in biodegradation and remediation. Agricultural wastewater often contains pesticides or herbicides made of nitrile compounds, and acetonitrile and acrylonitrile are important chemical raw materials, so there are a large amount of nitrile compounds in industrial wastewater, which pollutes the environment to some extent. Nitrilase can be used for soil remediation and water quality remediation. Adding bacteria that can produce nitrilase to wastewater can make them secrete nitrilase, so as to realize biodegradation and remove carcinogenic and teratogenic nitrile compounds, and the method has certain sustainability and recyclability.

[0003] Nowadays, more and more researchers are engaged in the research work of nitrilase, and how to synthesize and identify nitrilase has gradually become a hot topic. Researchers can obtain nitrilase by culturing and screening bacteria and then extracting their secretions. At the same time, researchers often use high-performance liquid chromatography to identify and detect the activity of nitrilase. This detection method is relatively complex, has high cost, requires instruments and equipment, and needs secondary analysis by software, so it has great limitations in practical application. Therefore, it is of great significance to establish a new simple and intuitive detection method. SUMMARY

[0004] The application aims to provide a method for visualizing and rapidly detecting nitrilase, and solves the problems of complex and high cost of the existing method for detecting nitrilase.

[0005] The application is realized by the following technical scheme:

[0006] The application aims to provide a method for visualizing and rapidly detecting nitrilase, and solves the problems of complex and high cost of the existing method for detecting nitrilase.

[0007] Step one, mix the functional monomer, crosslinking agent and initiator in a solvent to form a copolymer system, and then perform ultrasonic treatment to make the mixture uniform, so as to obtain a prepolymer solution, which is stored in cold storage for standby;

[0008] The functional monomer is a nitrile compound with a double bond.

[0009] Step two, drop the prepolymer solution into the gap of the photonic crystal template, and polymerize under an ultraviolet lamp, and then perform etching to obtain inverse opal nitrile-based polymer photonic crystals, and then wash the inverse opal nitrile-based polymer photonic crystals with deionized water to make them reach a swelling equilibrium and be neutral.

[0010] Step three, configure an alkaline solution, and put the inverse opal nitrile-based polymer photonic crystals into the alkaline solution to obtain a mixed system.

[0011] Step four, drop nitrilase solutions with different concentrations into the mixed system, shake well, and realize the detection of nitrilase by observing the color change of the film and the movement of the reflection peak of the photonic crystal.

[0012] Further, in step one, the nitrile compound with a double bond is acrylonitrile or dichloropropenyl nitrile.

[0013] Further, in step one, the molar ratio of the functional monomer, the crosslinking agent and the solvent is 5:(0.1-0.5):5.

[0014] Further, in step two, the ordered unit of the photonic crystal template is silica colloidal particles.

[0015] Further, in step three, the alkaline solution is a sodium carbonate or sodium bicarbonate solution with a mass fraction of 5%-15%.

[0016] Further, in step four, the concentration of the nitrilase is 0.2U / mL-10.0U / mL.

[0017] Further, in step one, the crosslinking agent is ethylene glycol dimethacrylate or N,N-methylene bisacrylamide.

[0018] Further, in step one, the solvent is anhydrous ethanol.

[0019] Further, in step one, the initiator is 2-hydroxy-2-methylbenzophenone.

[0020] Further, in step two, the preparation process of the inverse opal nitrile-based polymer photonic crystal is specifically as follows:

[0021] Put the photonic crystal template into a culture dish with an inclined angle, and drop the prepolymer liquid to the edge of the photonic crystal template, so that the prepolymer liquid enters the pores of the photonic crystal template;

[0022] Then, cover the photonic crystal template with the prepolymer liquid on the surface with an organic glass sheet, so that the organic glass sheet, the prepolymer liquid and the photonic crystal template form a "sandwich" structure, and then irradiate the "sandwich" structure under a UV lamp to realize polymerization and curing;

[0023] Add hydrofluoric acid to the cured copolymer photonic crystal template to etch off the silicon dioxide in the photonic crystal template, and obtain the inverse opal nitrile-based polymer photonic crystal.

[0024] Compared with the prior art, the present application has the following beneficial technical effects:

[0025] The application discloses a method for visual rapid detection of nitrilase, and first, an inverse opal nitrile-based polymer photonic crystal is prepared, and then the inverse opal nitrile-based polymer photonic crystal is placed in an alkaline solution, because the hydrolysis of a nitrile group requires an alkaline condition, to obtain an inverse opal nitrile-based polymer photonic crystal sensor; when a nitrilase solution to be detected is added, based on the optical self-expression of the photonic crystal, the nitrilase is used to catalyze the hydrolysis of the nitrile group on the inverse opal nitrile-based polymer photonic crystal into a carboxyl group, so that the inverse opal nitrile-based polymer photonic crystal is swollen, the lattice constant is changed, and the "naked eye" visual detection of the nitrilase is realized. Compared with a traditional method, the present application does not need special operation, is simple to operate, has high sensitivity, fast response speed, is cheap and portable, and can be used for on-site real-time visual detection without depending on other large instruments, and the minimum detection limit can reach 0.2 U / mL. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a diffraction spectrum diagram of the inverse opal nitrile-based polymer photonic crystal sensor of Example 1 for detecting 0.2 U / mL nitrilase.

[0027] Figure 2 Figure 2 is a diffraction spectrum diagram of the inverse opal nitrile-based polymer photonic crystal sensor of Example 2 for detecting 0.4 U / mL nitrilase.

[0028] Figure 3 Figure 3 is a diffraction spectrum diagram of the inverse opal nitrile-based polymer photonic crystal sensor of Example 3 for detecting 0.6 U / mL nitrilase.

[0029] Figure 4is the diffraction spectrum of the inverse opal nitrile-based polymeric photonic crystal sensor of Example 4 detecting 0.8 U / mL of nitrilase.

[0030] Figure 5 is the diffraction spectrum of the inverse opal nitrile-based polymeric photonic crystal sensor of Example 5 detecting 1.0 U / mL of nitrilase.

[0031] Figure 6 is the diffraction spectrum of the inverse opal nitrile-based polymeric photonic crystal sensor of Example 6 detecting 0.6 U / mL of nitrilase with the addition of interference solution NaCl.

[0032] Figure 7 is the diffraction spectrum of the inverse opal nitrile-based polymeric photonic crystal sensor of Example 7 detecting 0.6 U / mL of nitrilase after high temperature inactivation.

[0033] Figure 8 is the diffraction spectrum of the inverse opal nitrile-based polymeric photonic crystal sensor of Example 8 detecting 0.8 U / mL of nitrilase under acidic conditions. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made in combination with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application, i.e., the described examples are only a part of the examples of the present application, but not all the examples.

[0035] The components described and shown in the accompanying drawings and examples of the present application can be arranged and designed in various different configurations, therefore, the detailed description of the examples of the present application provided in the following accompanying drawings is not intended to limit the scope of the claimed present application, but only to represent a selected embodiment of the present application. Based on the accompanying drawings and examples of the present application, all other examples obtained by those skilled in the art without creative efforts belong to the protection scope of the present application.

[0036] It should be noted that the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, element, method, article or device including a list of elements does not only include those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, element, method, article or device. In addition, the terms "horizontal", "vertical" are based on the orientation and position relationship of the devices or components shown in the drawings, and are only used to better describe the present application, and are not required to have the specific orientation of the shown devices, components or equipment, and therefore cannot be understood as a limitation of the present application.

[0037] The application aims to provide a method for visualizing rapid detection of nitrilase.

[0038] Step one: functional monomer, crosslinking agent and initiator are mixed in solvent according to certain proportion to form copolymer system, and then ultrasonic treatment is conducted to make them uniformly mixed to obtain pre-polymer liquid, which is stored in cold storage for standby.

[0039] Step two: the prepared photonic crystal template is placed in a glass culture dish with an inclination angle of 15°, the prepared pre-polymer liquid is added to the edge of the photonic crystal template, and the pre-polymer liquid is slowly introduced into the pores of the photonic crystal template by capillary action. Then an organic glass sheet washed with ethanol and dried is covered on the photonic crystal template with pre-polymer liquid on the surface, so that the organic glass sheet, pre-polymer liquid and photonic crystal template present a "sandwich" state. The "sandwich" is clamped with tweezers, and the excess liquid at the edge is wiped dry with filter paper. Then the "sandwich" is placed in a clean glass culture dish and irradiated under a UV lamp to realize polymerization and curing.

[0040] The "sandwich" is taken out as a whole and placed in a plastic surface dish, 3% hydrofluoric acid by volume concentration is added to etch off the silica in the photonic crystal template, and the glass slide is discarded. The inverse opal nitrile-based polymer photonic crystal is obtained on the organic glass sheet. Then the inverse opal nitrile-based polymer photonic crystal is washed with deionized water to make it reach swelling equilibrium and be neutral.

[0041] Step three: a certain concentration of alkaline solution is configured, and the inverse opal nitrile-based polymer photonic crystal is placed in the solution to form a complete inverse opal nitrile-based polymer photonic crystal sensor system.

[0042] Step four: different concentrations of nitrilase solution are added dropwise and shaken, and the detection of nitrilase is realized by observing the color change of the film and the movement of the photonic crystal reflection peak.

[0043] In step one, the functional monomer is a double-bonded nitrile compound; the functional monomer is acrylonitrile or dichloropropenyl nitrile; the crosslinking agent is ethylene glycol dimethacrylate or N, N-methylene bisacrylamide; the solvent used is anhydrous ethanol; and the initiator used is 2-hydroxy-2-methylbenzene propionone.

[0044] In step one, the molar ratio of the functional monomer, the crosslinking agent, the solvent and the photoinitiator is 5:(0.1-0.5):5, and the photoinitiator is 0.2 μL.

[0045] In step two, the ordered unit of the photonic crystal template is silica colloidal particles.

[0046] In step three, the alkaline solution is a sodium carbonate and sodium bicarbonate solution with a mass fraction of 5%-15%.

[0047] In step four: the concentration of nitrilase is 0.2 U / mL-10.0 U / mL.

[0048] The photonic crystal sensor is widely used in biological and chemical fields, and the sensor can exhibit macroscopic structural color change after encountering specific stimulation, and has the advantages of intuitive and visual detection results. The inverse opal nitrile-based polymer photonic crystal sensor prepared by the photonic crystal template has good flexibility and high porosity, and has high detection rate for the measured substance. The nitrile group is introduced into the inverse opal nitrile-based polymer photonic crystal, and a new nitrilase detection method is explored to meet the detection requirements of intuitive results, simple operation and low price.

[0049] The features and performances of the present application are further described in detail in combination with the following examples.

[0050] Example 1

[0051] The experimental steps for detecting 0.2 U / mL nitrilase solution are as follows:

[0052] Step one: acrylonitrile is used as a functional monomer, ethylene glycol dimethacrylate is used as a crosslinking agent, and ethanol is used as a solvent to prepare a solution with a molar ratio of 5:0.1:5, then 2 μL of dihydroxydimethylbenzophenone is added as a photoinitiator to obtain the required prepolymer solution, which is mixed by ultrasonic treatment for 1 h, and then refrigerated for standby.

[0053] Step two: the prepared photonic crystal template is placed in a glass culture dish with an inclination angle of 15°, and the prepared prepolymer solution is added to the edge of the photonic crystal template, and the prepolymer solution slowly enters the pores of the photonic crystal template by capillary action. Then an organic glass sheet that has been washed with ethanol and dried is covered on the photonic crystal template with prepolymer solution on the surface, so that the organic glass sheet, prepolymer solution and photonic crystal template form a "sandwich" state, and the "sandwich" is clamped with tweezers, and the excess liquid at the edge is wiped dry with filter paper. Then the "sandwich" is placed in a clean glass culture dish and irradiated under a UV lamp to achieve polymerization and curing.

[0054] Step three: the "sandwich" is taken out as a whole and placed in a plastic surface dish, 3% hydrofluoric acid is added to etch off the silicon dioxide in the photonic crystal template, and the glass slide is discarded, and the inverse opal nitrile-based polymer photonic crystal is obtained on the organic glass sheet. Then the inverse opal nitrile-based polymer photonic crystal is washed with deionized water to achieve swelling equilibrium and neutrality.

[0055] Step four: 4 mL of 5% sodium carbonate solution is prepared, and the inverse opal nitrile-based polymer photonic crystal is placed in the solution.

[0056] Step five: 1 mL of 1.0 U / mL nitrilase solution was dropped into the oscillation and shaken, the color change of the film was observed and the shift of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0057] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 0.2 U / mL nitrilase solution. Because nitrilase hydrolyzes the nitrile group on the inverse opal nitrile-based polymer photonic crystal to carboxyl group, it swells in alkaline solution, and the lattice constant changes, thereby realizing the "naked eye" visual detection of nitrilase. From the experimental results, it can be seen that the reflection peak of the sensor moves from the initial 637 nm to 657 nm, a total of 20 nm. The color of the film also changes from deep orange to red when it is balanced, so it can achieve visual detection of 0.2 U / mL nitrilase. Figure 1 The reflection peak of the sensor can be observed to move from the initial 637 nm to 657 nm, a total of 20 nm. The color of the film also changes from deep orange to red when it is balanced, so it can achieve visual detection of 0.2 U / mL nitrilase.

[0058] Example 2

[0059] The experimental steps for detecting 0.4 U / mL nitrilase solution are as follows:

[0060] Step one: acrylonitrile was used as the functional monomer, ethylene glycol dimethacrylate was used as the crosslinking agent, and ethanol was used as the solvent to prepare a solution with a molar ratio of 5:0.2:5. Then 2 μL of dihydroxydimethylbenzophenone was added as a photoinitiator to obtain the required prepolymer solution. The solution was mixed thoroughly by ultrasonic treatment for 1 h and then refrigerated for later use.

[0061] Step two: The prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°. The prepared prepolymer solution was added to the edge of the photonic crystal template, and the capillary action was used to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then an organic glass sheet that had been rinsed with ethanol and dried was placed on the photonic crystal template with the prepolymer solution on the surface, so that the organic glass sheet, the prepolymer solution, and the photonic crystal template formed a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to achieve polymerization and curing.

[0062] Step three: The "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was left on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0063] Step four: 4 mL of 5% sodium carbonate solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0064] Step five: 1 mL of 2.0 U / mL nitrilase solution was dropped into the oscillation and shaken, the color change of the film was observed and the shift of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0065] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 0.4 U / mL nitrilase solution. Because nitrilase hydrolyzes the nitrile group on the inverse opal nitrile-based polymer photonic crystal to carboxyl group, it swells in alkaline solution, and the lattice constant changes, thus realizing the "naked eye" visual detection of nitrilase. From the experimental results, it can be seen that the diffraction peak of the sensor moves from the initial 632 nm to 652 nm, a total of 21 nm. The color of the film also changes from dark orange to red when it is balanced, so it can achieve visual detection of 0.4 U / mL nitrilase. Figure 2 The diffraction peak of the sensor can be observed to move from the initial 632 nm to 652 nm, a total of 21 nm. The color of the film also changes from dark orange to red when it is balanced, so it can achieve visual detection of 0.4 U / mL nitrilase.

[0066] Example 3

[0067] The experimental steps for detecting 0.6 U / mL nitrilase solution are as follows:

[0068] Step one: acrylonitrile was used as the functional monomer, ethylene glycol dimethacrylate was used as the crosslinking agent, and ethanol was used as the solvent to prepare a solution with a molar ratio of 5:0.3:5. Then 2 μL of dihydroxydimethylbenzophenone was added as a photoinitiator to obtain the required prepolymer solution. After ultrasonic treatment for 1 h, it was mixed thoroughly and then refrigerated for later use.

[0069] Step two: The prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°. The prepared prepolymer solution was added to the edge of the photonic crystal template, and the capillary action was used to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then an organic glass sheet that had been rinsed with ethanol and dried was placed on the photonic crystal template with the prepolymer solution on the surface, so that the organic glass sheet, the prepolymer solution, and the photonic crystal template formed a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to realize polymerization and curing.

[0070] Step three: The "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was left on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0071] Step four: 4 mL of 5% sodium carbonate solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0072] Step five: 1 mL of 3.0 U / mL nitrilase solution was dropped into the oscillation and shaken, the color change of the film was observed and the movement of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0073] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 0.6 U / mL nitrilase solution. The nitrile group on the inverse opal nitrile-based polymer photonic crystal is hydrolyzed to carboxyl group by nitrilase, which causes the swelling of the inverse opal nitrile-based polymer photonic crystal in alkaline solution, and the change of the lattice constant, so as to realize the "naked eye" visual detection of nitrilase. Figure 3 It can be observed that the diffraction peak of the sensor moves from the initial 633 nm to 709 nm, a total of 76 nm. The color of the film also changes from deep orange to deep red at equilibrium, so that the visual detection of 0.6 U / mL nitrilase can be achieved.

[0074] Example 4

[0075] The experimental steps for detecting 0.8 U / mL nitrilase solution are as follows:

[0076] Step one: acrylonitrile was used as a functional monomer, ethylene glycol dimethacrylate was used as a crosslinking agent, and ethanol was used as a solvent to prepare a solution with a molar ratio of 5:0.4:5. Then 2 μL of dihydroxydimethylbenzophenone was added as a photoinitiator to obtain the required prepolymer solution. The solution was mixed thoroughly by ultrasonic treatment for 1 h and then stored in the refrigerator for later use.

[0077] Step two: the prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°. The prepared prepolymer solution was added to the edge of the photonic crystal template, and the capillary action was used to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then an organic glass sheet that had been rinsed with ethanol and dried was placed on the photonic crystal template with the prepolymer solution on the surface, so that the organic glass sheet, the prepolymer solution, and the photonic crystal template formed a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to realize polymerization and curing.

[0078] Step three: the "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was left on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0079] Step four: 4 mL of 5% sodium carbonate solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0080] Step five: 1 mL of 4.0 U / mL nitrilase solution was dropped into the oscillation and shaken, the color change of the film was observed and the shift of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0081] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 0.8 U / mL nitrilase solution. The nitrile group on the inverse opal nitrile-based polymer photonic crystal is hydrolyzed to carboxyl group by nitrilase, which causes the swelling of the inverse opal nitrile-based polymer photonic crystal in alkaline solution, and the change of the lattice constant. In this way, the "naked eye" visual detection of nitrilase is realized. The diffraction peak of the sensor can be observed to move from the initial 628 nm to 717 nm, a total of 89 nm. The color of the film also changes from orange to dark red when it is balanced, so the visual detection of 0.8 U / mL nitrilase can be achieved. Figure 4

[0082] Example 5

[0083] The experimental steps for detecting 1.0 U / mL nitrilase solution are as follows:

[0084] Step one: acrylonitrile was used as the functional monomer, ethylene glycol dimethacrylate was used as the crosslinking agent, and ethanol was used as the solvent to prepare a solution with a molar ratio of 5:0.5:5. Then 2 μL of dihydroxydimethylbenzophenone was added as a photoinitiator to obtain the pre-polymer solution required for the experiment. After ultrasonic treatment for 1 h, it was mixed thoroughly and then refrigerated for later use.

[0085] Step two: the prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°, and the pre-polymer solution prepared was added to the edge of the photonic crystal template. The pre-polymer solution slowly entered the pores of the photonic crystal template by capillary action. Then an organic glass sheet that had been rinsed with ethanol and dried was covered on the photonic crystal template with pre-polymer solution on the surface, so that the organic glass sheet, pre-polymer solution, and photonic crystal template formed a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to realize polymerization and curing.

[0086] Step three: the "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was left on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0087] Step four: 4 mL of 5% sodium carbonate solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0088] ​Step five: 1 mL of 5.0 U / mL nitrilase solution was dropped into the oscillation and shaken, the color change of the film was observed and the shift of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0089] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 1.0 U / mL nitrilase solution. The nitrile group on the inverse opal nitrile-based polymer photonic crystal is hydrolyzed to carboxyl group by nitrilase, which causes the swelling of the inverse opal nitrile-based polymer photonic crystal in alkaline solution, and the change of the lattice constant. In this way, the "naked eye" visual detection of nitrilase is realized. The diffraction peak of the sensor can be observed to move from the initial 628 nm to 719 nm, a total of 91 nm. The color of the film also changes from orange to deep red when it is balanced, so the visual detection of 1.0 U / mL nitrilase can be achieved. Figure 5

[0090] Example 6

[0091] The experimental steps for detecting the influence of 0.6 U / mL nitrilase solution when NaCl is added as an interference factor are as follows:

[0092] Step one: acrylonitrile was used as a functional monomer, ethylene glycol dimethacrylate was used as a crosslinking agent, and ethanol was used as a solvent to prepare a solution with a molar ratio of 5:0.3:5. Then 2 μL of dihydroxy dimethyl benzophenone was added as a photoinitiator to obtain the required prepolymer solution. The solution was mixed thoroughly by ultrasonic treatment for 1 h and then refrigerated for later use.

[0093] Step two: the prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°. The prepared prepolymer solution was added to the edge of the photonic crystal template, and the capillary action was used to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then an organic glass sheet that had been rinsed with ethanol and dried was placed on the photonic crystal template with the prepolymer solution on the surface, so that the organic glass sheet, the prepolymer solution, and the photonic crystal template formed a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to realize polymerization and curing.

[0094] Step three: the "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was left on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0095] Step four: 4 mL of 15% sodium carbonate solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0096] ​Step five: 1 mL of 3.0 U / mL nitrilase solution was dropped into the oscillation and shaken, then Nacl interference solution was added, and the color of the film was observed and the changes of the diffraction peak of the photonic crystal were recorded.

[0097] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor did not change significantly in color and diffraction peak when detecting 0.6 U / mL nitrilase and adding Nacl interference ions, and the color of the photonic crystal film did not change significantly. Figure 6 It can be observed that the diffraction peak of the sensor moves from the initial 635 nm to 714 nm, a total of 79 nm, and the color changes from deep orange to deep red. Therefore, it can be concluded that the photonic crystal sensor has strong anti-interference ability and its color is not easily affected by other external ions, which can achieve the level of practical application.

[0098] Example 7

[0099] The experimental steps for detecting 0.6 U / mL nitrilase solution after high temperature inactivation are as follows:

[0100] Step one: acrylonitrile was used as a functional monomer, dimethyl acrylate was used as a crosslinking agent, and ethanol was used as a solvent to prepare a solution with a molar ratio of 5:0.3:5. 2 μL of dihydroxy dimethyl benzophenone was added as a photoinitiator to obtain the required prepolymer solution. After ultrasonic treatment for 1 h, it was mixed thoroughly and stored in the refrigerator for later use.

[0101] Step two: Put the prepared photonic crystal template into a glass culture dish with an inclination angle of 15°, and add the prepared prepolymer solution to the edge of the photonic crystal template. Use capillary action to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then cover the photonic crystal template with a piece of organic glass that has been rinsed with ethanol and dried, so that the organic glass, prepolymer, and photonic crystal template form a "sandwich". Use tweezers to clamp the "sandwich" and wipe off the excess liquid with filter paper. Then place the "sandwich" in a clean glass culture dish and irradiate it under a UV lamp to achieve polymerization and curing.

[0102] Step three: Take out the "sandwich" as a whole and place it in a plastic surface dish. Add 3% hydrogen fluoride acid to etch away the silicon dioxide in the photonic crystal template. Discard the glass slide and the inverse opal nitrile-based polymer photonic crystal is formed on the organic glass sheet. Rinse the inverse opal nitrile-based polymer photonic crystal with deionized water to achieve swelling equilibrium and neutrality.

[0103] Step four: Prepare 4 mL of 10% sodium bicarbonate solution and place the inverse opal nitrile-based polymer photonic crystal in the solution.

[0104] Step five: 1 mL of 3.0 U / mL nitrilase solution was added dropwise into the oscillation and shaken, the color change of the film was observed and the movement of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0105] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor can detect 0.6 U / mL of nitrilase solution after high temperature inactivation. Since the reaction temperature of nitrilase is usually 30-50°C, high temperature treatment will cause nitrilase to lose activity, so it cannot be swollen in alkaline solution. The diffraction peak and color of the sensor do not fluctuate, so we can also preliminarily judge the activity of nitrilase by this feature. Figure 7 The diffraction peak and color of the sensor do not fluctuate, so we can also preliminarily judge the activity of nitrilase by this feature.

[0106] Example 8

[0107] The experimental steps for detecting 0.8 U / mL nitrilase solution under acidic conditions are as follows:

[0108] Step one: A solution was prepared with dichloroallyl nitrile as the functional monomer, N, N-methylene bisacrylamide as the crosslinking agent, and ethanol as the solvent in a molar ratio of 5:0.4:5. Then 2 μL of dihydroxydimethylbenzophenone was added as a photoinitiator to obtain the required prepolymer solution. The solution was mixed thoroughly by ultrasonic treatment for 1 h and then refrigerated for later use.

[0109] Step two: The prepared photonic crystal template was placed in a glass culture dish with an inclination angle of 15°. The prepared prepolymer solution was added to the edge of the photonic crystal template. The capillary action was used to slowly fill the prepolymer solution into the pores of the photonic crystal template. Then an organic glass sheet that had been rinsed with ethanol and dried was placed on the photonic crystal template with the prepolymer solution on the surface, forming a "sandwich" state. The "sandwich" was clamped with tweezers, and the excess liquid at the edge was wiped off with filter paper. Then the "sandwich" was placed in a clean glass culture dish and irradiated under a UV lamp to achieve polymerization and curing.

[0110] Step three: The "sandwich" was taken out as a whole and placed in a plastic surface dish. Hydrofluoric acid with a volume concentration of 3% was added to etch away the silicon dioxide in the photonic crystal template. The glass slide was discarded, and the inverse opal nitrile-based polymer photonic crystal was obtained on the organic glass sheet. The inverse opal nitrile-based polymer photonic crystal was then rinsed with deionized water to achieve swelling equilibrium and neutrality.

[0111] Step four: 4 mL of 5% hydrochloric acid solution was prepared, and the inverse opal nitrile-based polymer photonic crystal was placed in the solution.

[0112] Step five: 1 mL of 4.0 U / mL nitrilase solution was added dropwise into the oscillation and shaken, the color change of the film was observed and the movement of the photonic crystal diffraction peak was recorded to realize the detection of nitrilase.

[0113] Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor has little change in color and diffraction peak when detecting 0.8 U / mL nitrile hydratase solution, because the activity of nitrile hydratase will rapidly decrease under acidic conditions, and there is almost no activity at pH = 5. Therefore, the color and diffraction peak of the sensor have little change. The diffraction peak of the sensor can be observed to move from the initial 630 to 633 nm, only 3 nm. The color of the film does not change at equilibrium, so the catalytic reaction of nitrile hydratase usually needs to be carried out under alkaline conditions. Figure 8 Conclusion: The inverse opal nitrile-based polymer photonic crystal sensor has little change in color and diffraction peak when detecting 0.8 U / mL nitrile hydratase solution, because the activity of nitrile hydratase will rapidly decrease under acidic conditions, and there is almost no activity at pH = 5. Therefore, the color and diffraction peak of the sensor have little change. The diffraction peak of the sensor can be observed to move from the initial 630 to 633 nm, only 3 nm. The color of the film does not change at equilibrium, so the catalytic reaction of nitrile hydratase usually needs to be carried out under alkaline conditions.

[0114] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limiting it, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that: the specific embodiments of the present application can still be modified or replaced by the equivalent, without departing from the spirit and scope of the present application, any modification or equivalent replacement, which should be covered within the protection scope of the claims of the present application.

Claims

1. A method for rapid visual detection of nitrilase, characterized in that: The following steps are involved: Step 1: Mix the functional monomer, cross-linking agent and initiator in a solvent to form a copolymer system, then perform ultrasonic treatment to mix them evenly to obtain a prepolymer solution, which is refrigerated for later use; The functional monomer is a nitrile compound with a double bond; the nitrile compound with a double bond is acrylonitrile or dichloroacrylonitrile; Step 2: dripping prepolymer solution into the gap of the photonic crystal template, polymerizing under ultraviolet light, and after the polymerization is completed, etching to obtain an inverse opal nitrile-based polymer photonic crystal, and then rinsing the inverse opal nitrile-based polymer photonic crystal with deionized water to make it reach swelling equilibrium and become neutral; Step 3: preparing an alkaline solution, placing the inverse opal nitrile-based polymer photonic crystal into the alkaline solution to obtain a mixed system; Step 4: Add nitrilase solutions of different concentrations dropwise into the mixed system, shake the system to evenly distribute the mixture, and detect the nitrilase by observing the color change of the film and the shift of the reflection peak of the photonic crystal.

2. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 1, the molar ratio of the functional monomer, the cross-linking agent, and the solvent is 5:(0.1-0.5):

5.

3. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 2, the ordered units of the photonic crystal template are silica colloidal particles.

4. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 3, the alkaline solution is a sodium carbonate or sodium bicarbonate solution with a mass fraction of 5%-15%.

5. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 4, the concentration of nitrilase is 0.2 U / mL-10.0 U / mL.

6. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 1, the cross-linking agent is ethylene glycol dimethacrylate or N,N-methylenebisacrylamide.

7. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 1, the solvent is anhydrous ethanol.

8. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 1, the initiator is 2-hydroxy-2-methylpropiophenone.

9. The method for rapid visual detection of nitrilase according to claim 1, wherein: In step 2, the preparation process of the inverse opal nitrile-based polymer photonic crystal is specifically as follows: The photonic crystal template is placed in a culture dish with an inclined angle, and a prepolymer solution is dripped onto the edge of the photonic crystal template so that the prepolymer solution enters the pores of the photonic crystal template; Then, a plexiglass sheet is covered on the photonic crystal template with the prepolymer liquid on the surface, so that the plexiglass sheet, the prepolymer liquid, and the photonic crystal template form a "sandwich" structure, and then the "sandwich" structure is irradiated under ultraviolet light to achieve polymerization and curing; Hydrofluoric acid is added to the solidified copolymer photonic crystal template to etch away the silicon dioxide in the photonic crystal template to obtain an inverse opal nitrile-based polymer photonic crystal.

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