CN115595354A - Method for visually and rapidly detecting nitrilase - Google Patents
Method for visually and rapidly detecting nitrilase Download PDFInfo
- Publication number
- CN115595354A CN115595354A CN202211335853.1A CN202211335853A CN115595354A CN 115595354 A CN115595354 A CN 115595354A CN 202211335853 A CN202211335853 A CN 202211335853A CN 115595354 A CN115595354 A CN 115595354A
- Authority
- CN
- China
- Prior art keywords
- photonic crystal
- nitrilase
- nitrile
- based polymer
- inverse opal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/29—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using visual detection
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Genetics & Genomics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
Abstract
The invention belongs to the technical field of detection, and relates to a method for visually and rapidly detecting nitrilase, which comprises the steps of mixing a functional monomer, a cross-linking agent and an initiator in a solvent to form a copolymer system, carrying out ultrasonic treatment to uniformly mix the copolymer system, obtaining a pre-polymerized liquid, and refrigerating the pre-polymerized liquid for later use; the functional monomer is a nitrile compound with double bonds; dripping a pre-polymerization liquid into gaps of the photonic crystal template, polymerizing under an ultraviolet lamp, after the polymerization is finished, etching to obtain inverse opal nitrile-based polymer photonic crystals, and washing the inverse opal nitrile-based polymer photonic crystals with deionized water to ensure that the inverse opal nitrile-based polymer photonic crystals are balanced in swelling and neutral; putting inverse opal nitrile-based polymer photonic crystals into an alkaline solution to obtain a mixed system; and (3) dripping nitrilase solutions with different concentrations into the mixed system, and utilizing nitrilase to catalyze and hydrolyze nitrile groups on the inverse opal nitrile-based polymer photonic crystal into carboxyl groups, so that the photonic crystal swells, and the lattice constant changes, thereby realizing the visual detection of the nitrilase.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for visually and rapidly detecting nitrilase.
Background
Nitrilase is an important biocatalyst, can hydrolyze nitrile compounds into corresponding carboxylic acid and ammonia under mild conditions, has no by-products, and has little environmental pollution. The preparation of fine chemicals by using nitrilase with selectivity has great application value. Since nitrilase has high stereoselectivity for hydrolyzing nitrile compounds, nitrilase can be used for hydrolyzing corresponding nitrile compounds to prepare carboxylic acids which are difficult to synthesize or have low yield under common chemical conditions. For example, dinitriles can be hydrolyzed enzymatically to give diacids. Iminodiacetic acid is an important raw material for synthesizing herbicides, can be obtained by chemical methods such as a chloroacetic acid method, a diethanolamine method and the like, but has serious pollution in the reaction process and high requirements on equipment. Researchers use nitrilase to catalyze iminodiacetonitrile to obtain iminodiacetic acid, the conversion rate reaches 72% by optimizing Alcaligenes faecalalis ZJB-09133 nitrilase, and then, some people perform immobilization research on Arthrobacter aureofaciens CYC705 nitrilase, and metal ions are added into a reaction system, so that 200mmol/L iminodiacetonitrile can be completely converted into iminodiacetic acid within 1h, and the reaction rate and the conversion rate are greatly improved. Nitrilases also have important applications in biodegradation and repair. Agricultural wastewater mostly contains pesticides or herbicides which take nitrile compounds as raw materials, and acetonitrile and acrylonitrile are important chemical raw materials, so that a large amount of nitrile compounds also exist in the industrial wastewater, which causes pollution to the nature to a certain extent, and soil remediation and water quality remediation can be carried out by using nitrilase. The bacterial colony capable of producing nitrilase is added into the waste water to make it secrete nitrilase, so that it can implement biodegradation, and can remove carcinogenic and teratogenic nitrile group compound, and said method possesses a certain continuity and cyclability.
Nowadays, more and more researchers are engaged in research work related to nitrilase, and how to synthesize and identify nitrilase is also becoming a hot topic. The researchers can obtain nitrilase by culturing and screening the bacterial colony and extracting the secretion of the bacterial colony. Meanwhile, in the aspect of detecting nitrilase, researchers mostly utilize high performance liquid chromatography to identify the nitrilase and detect the nitrilase activity, the detection method is complex, high in cost and high in requirements for instruments and equipment, secondary analysis is needed by means of software, and the method has great limitation in practical application, so that the establishment of a new simple and visual detection method is of great significance.
Disclosure of Invention
The invention aims to provide a method for visually and rapidly detecting nitrilase, and solves the problems of complexity and high cost of the conventional method for detecting nitrilase.
The invention is realized by the following technical scheme:
a method for visually and rapidly detecting nitrilase comprises the following steps:
step one, mixing a functional monomer, a cross-linking agent and an initiator in a solvent to form a copolymer system, performing ultrasonic treatment to uniformly mix the copolymer system to obtain a pre-polymerization solution, and refrigerating the pre-polymerization solution for later use;
the functional monomer is a nitrile compound with double bonds;
dripping pre-polymerization liquid into gaps of the photonic crystal template, polymerizing under an ultraviolet lamp, etching to obtain inverse opal nitrile-based polymer photonic crystals after polymerization is finished, and washing the inverse opal nitrile-based polymer photonic crystals with deionized water to ensure that the inverse opal nitrile-based polymer photonic crystals are balanced in swelling and neutral;
thirdly, preparing an alkaline solution, and putting the inverse opal nitrile polymer photonic crystal into the alkaline solution to obtain a mixed system;
and step four, dripping nitrilase solutions with different concentrations into the mixed system, oscillating and shaking up, and realizing the detection of nitrilase by observing the color change of the film and the movement of a reflection peak of the photonic crystal.
Further, in the step one, the nitrile compound with double bond is acrylonitrile or dichloroacrylonitrile.
Furthermore, in the first step, the mol ratio of the functional monomer to the cross-linking agent to the solvent is 5 (0.1-0.5) to 5.
Further, in the second step, the ordered units of the photonic crystal template are silica colloid particles.
Further, in the third step, the alkaline solution is a sodium carbonate or sodium bicarbonate solution with the mass fraction of 5% -15%.
Further, in the fourth step, the concentration of the nitrilase is 0.2U/mL to 10.0U/mL.
Further, in the first step, the cross-linking agent is ethylene glycol dimethacrylate or N, N-methylene bisacrylamide.
Further, in the step one, the solvent is absolute ethyl alcohol.
Further, in the first step, the initiator is 2-hydroxy-2-methyl propiophenone.
Further, in the second step, the preparation process of the inverse opal nitrile-based polymer photonic crystal specifically comprises the following steps:
putting the photonic crystal template into a culture dish with an inclined angle, and dropwise adding a prepolymerization liquid to the edge of the photonic crystal template to ensure that the prepolymerization liquid enters pores of the photonic crystal template;
covering the organic glass sheet on the photonic crystal template with the pre-polymerized liquid on the surface to enable the organic glass sheet, the pre-polymerized liquid and the photonic crystal template to present a sandwich structure, and irradiating the sandwich structure under an ultraviolet lamp to realize polymerization and solidification;
and adding hydrofluoric acid into the cured copolymer photonic crystal template to etch silicon dioxide in the photonic crystal template to obtain the inverse opal nitrile-based polymer photonic crystal.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention discloses a method for visually and rapidly detecting nitrilase, which comprises the steps of firstly preparing inverse opal nitrile-based polymer photonic crystals, and firstly placing the inverse opal nitrile-based polymer photonic crystals in alkaline solution to obtain an inverse opal nitrile-based polymer photonic crystal sensor under the alkaline condition required by hydrolysis of nitrile groups; after a solution to be detected of nitrilase is added, based on the optical self-expression of the photonic crystal, nitrile groups on the photonic crystal of the inverse opal nitrile-based polymer are catalyzed and hydrolyzed into carboxyl groups by the nitrilase, so that the photonic crystal of the inverse opal nitrile-based polymer is swelled, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. Compared with the traditional method, the method does not need a specially-assigned person for operation, is simple to operate, high in sensitivity, high in response speed, low in cost and portable, can perform field real-time visual detection without depending on other large instruments, and has the lowest detection limit of 0.2U/mL.
Drawings
FIG. 1 is a diffraction spectrum of 0.2U/mL nitrilase detected by the inverse opal nitrile based polymer photonic crystal sensor of example 1.
FIG. 2 is a diffraction spectrum of 0.4U/mL nitrilase detected by the inverse opal nitrile based polymer photonic crystal sensor of example 2.
FIG. 3 is a diffraction spectrum of the inverse opal nitrile based polymer photonic crystal sensor of example 3 detecting nitrilase at 0.6U/mL.
FIG. 4 is a diffraction spectrum of 0.8U/mL nitrilase detected by the inverse opal nitrile based polymer photonic crystal sensor of example 4.
FIG. 5 is a diffraction spectrum of the inverse opal nitrile based polymer photonic crystal sensor of example 5 for detecting 1.0U/mL nitrilase.
FIG. 6 is a diffraction spectrum of the inverse opal nitrile-based polymer photonic crystal sensor of example 6 detecting 0.6U/mL nitrilase in addition to an interfering solution of NaCl.
FIG. 7 is a diffraction spectrum of the nitrilase after inactivation at high temperature of 0.6U/mL detected by the inverse opal nitrile based polymer photonic crystal sensor of example 7.
FIG. 8 is a diffraction spectrum of the inverse opal nitrile-based polymer photonic crystal sensor of example 8 at 0.8U/mL nitrilase under acidic conditions.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description is made with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The components illustrated and described in the figures and embodiments of the present invention may be arranged and designed in a wide variety of different configurations, and accordingly, the detailed description of the embodiments of the present invention provided in the figures that follow is not intended to limit the scope of the invention, as claimed, but is merely representative of a selected embodiment of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the figures and embodiments of the present invention, belong to the scope of protection of the present invention.
It should be noted that: the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, element, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, element, method, article, or apparatus. Furthermore, the terms "horizontal" and "vertical" are based on the orientation and positional relationship of the devices or components shown in the drawings and are intended only for the purpose of better describing the invention, but do not require that the devices, components or apparatuses shown must have this particular orientation and therefore should not be construed as limiting the invention.
The invention aims to provide a method for visually and rapidly detecting nitrilase. The method comprises the following specific steps:
the method comprises the following steps: the functional monomer, the cross-linking agent and the initiator are mixed in a solvent according to a certain proportion to form a copolymer system, and then the copolymer system is subjected to ultrasonic treatment to be uniformly mixed to obtain a pre-polymerization solution which is refrigerated for standby.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
And taking out the whole sandwich, putting the whole sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and putting the inverse opal nitrile-based polymer photonic crystal on the organic glass sheet to obtain the inverse opal nitrile-based polymer photonic crystal. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step three: and preparing an alkaline solution with a certain concentration, and putting the inverse opal nitrile-based polymer photonic crystal into the solution to form a complete inverse opal nitrile-based polymer photonic crystal sensor system.
Step four: and dripping nitrilase solutions with different concentrations into the solution, oscillating and shaking up the solution, and observing the color change of the film and the movement of a photonic crystal reflection peak to realize the detection of the nitrilase.
In the first step, the functional monomer is a nitrile compound with double bonds; the functional monomer is acrylonitrile and dichloroacrylonitrile; the cross-linking agent is ethylene glycol dimethacrylate or N, N-methylene bisacrylamide; the solvent is absolute ethyl alcohol; the initiator is 2-hydroxy-2-methyl propiophenone.
In the first step, the mol ratio of the functional monomer, the cross-linking agent, the solvent and the photoinitiator is 5 (0.1-0.5) to 5, and the mol ratio of the photoinitiator is 0.2 mu L.
In the second step, the ordered units of the photonic crystal template are silica colloid particles.
In the third step, the alkaline solution is a sodium carbonate solution and a sodium bicarbonate solution with the mass fraction of 5-15%.
In the fourth step: the concentration of nitrilase was 0.2U/mL to 10.0U/mL.
The photonic crystal sensor is widely applied to the fields of biology and chemistry, can show macroscopic structural color change after meeting specific stimulation, and has the advantage of visual and visible 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 on an object to be detected. The nitrile group is introduced into the inverse opal nitrile-based polymer photonic crystal, and a new nitrilase detection method is explored and researched to meet the detection requirements of visual results, simplicity in operation and low price.
The features and properties of the present invention are further described in detail below with reference to examples.
Example 1
The experimental procedure for the detection of a nitrilase solution of 0.2U/mL is as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet rinsed and dried by ethanol on the photonic crystal template with the pre-polymerized liquid on the surface to enable the organic glass sheet, the pre-polymerized liquid and the photonic crystal template to be in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile polymer photonic crystal to ensure that the inverse opal nitrile polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1.0U/mL of nitrilase solution of 1mL, shaking uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 0.2U/mL, nitrile groups on the inverse opal nitrile-based polymer photonic crystal are hydrolyzed into carboxyl groups by the nitrilase, so that the inverse opal nitrile-based polymer photonic crystal is swelled in an alkaline solution, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. It can be observed from fig. 1 that the reflection peak of the sensor is shifted from 637nm initially to 657nm for a total shift of 20nm. At equilibrium the film also changed color from dark orange to red, thus enabling visual detection of 0.2U/mL nitrilase.
Example 2
The experimental procedure for testing the nitrilase solution at 0.4U/mL was as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 2.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 0.4U/mL, nitrile groups on the inverse opal nitrile-based polymer photonic crystal are hydrolyzed into carboxyl groups by the nitrilase, so that the inverse opal nitrile-based polymer photonic crystal is swelled in an alkaline solution, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. From fig. 2, it can be observed that the diffraction peak of the sensor is shifted from the initial 632nm to 652nm, and is shifted by 21nm altogether. At equilibrium the film also changed color from dark orange to red, thus enabling visual detection of 0.4U/mL nitrilase.
Example 3
The experimental procedure for testing the nitrilase solution at 0.6U/mL was as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile polymer photonic crystal to ensure that the inverse opal nitrile polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 3.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 0.6U/mL, nitrile groups on the inverse opal nitrile-based polymer photonic crystal are hydrolyzed into carboxyl groups by the nitrilase, so that the inverse opal nitrile-based polymer photonic crystal is swelled in an alkaline solution, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. It can be observed from FIG. 3 that the diffraction peak of the sensor is shifted from 633nm initially to 709nm for a total shift of 76nm. At equilibrium the film also changed color from dark orange to dark red, thus enabling visual detection of 0.6U/mL nitrilase.
Example 4
The experimental procedure for testing the nitrilase solution at 0.8U/mL was as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 4.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 0.8U/mL, nitrile groups on the inverse opal nitrile-based polymer photonic crystal are hydrolyzed into carboxyl groups by the nitrilase, so that the inverse opal nitrile-based polymer photonic crystal is swelled in an alkaline solution, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. It can be observed from fig. 4 that the diffraction peak of the sensor is shifted from the initial 628nm to 717nm for a total of 89nm. At equilibrium the film also changed in color from orange to deep red, thus enabling visual detection of 0.8U/mL nitrilase.
Example 5
The experimental procedure for detecting a 1.0U/mL nitrilase solution is as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 5.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 1.0U/mL, nitrile groups on the inverse opal nitrile-based polymer photonic crystal are hydrolyzed into carboxyl groups by the nitrilase, so that the inverse opal nitrile-based polymer photonic crystal is swelled in an alkaline solution, and the lattice constant is changed, thereby realizing the naked eye visual detection of the nitrilase. It can be observed from fig. 5 that the diffraction peak of the sensor has shifted from the initial 628nm to 719nm, for a total of 91nm. At equilibrium the film also changed color from orange to deep red, thus enabling visual detection of 1.0U/mL nitrilase.
Example 6
The experimental procedure for determining the effect on a 0.6U/mL nitrilase solution when NaCl was added as an interfering factor was as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet which is rinsed by ethanol and dried on the photonic crystal template with the pre-polymerized liquid on the surface, so that the organic glass sheet, the pre-polymerized liquid and the photonic crystal template are in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile polymer photonic crystal to ensure that the inverse opal nitrile polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 15% sodium carbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: 1mL of 3.0U/mL nitrilase solution was dropped into the solution and shaken, and then a Nacl interfering solution was added to observe the color of the film and record the presence or absence of a change in the diffraction peak of the photonic crystal as compared with example five.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects 0.6U/mL nitrilase, nacl interfering ions are added, the color of the photonic crystal film and the diffraction peak of the photonic crystal film are not obviously changed, and it can be observed from fig. 6 that the diffraction peak of the sensor is shifted from 635nm to 714nm, the total shift is 79nm, and the color is still changed from dark orange to dark red. Therefore, the photonic crystal sensor has strong anti-interference capability, the color of the photonic crystal sensor is not easily influenced by other external ions, and the practical application level can be achieved.
Example 7
The experimental procedure for detecting a nitrilase solution of 0.6U/mL after high temperature inactivation was as follows:
the method comprises the following steps: preparing a solution by taking acrylonitrile as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet rinsed and dried by ethanol on the photonic crystal template with the pre-polymerized liquid on the surface to enable the organic glass sheet, the pre-polymerized liquid and the photonic crystal template to be in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 10% sodium bicarbonate solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 3.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile polymer photonic crystal sensor detects a nitrilase solution inactivated at a high temperature of 0.6U/mL, the nitrilase cannot swell in an alkaline solution because the reaction temperature of the nitrilase is usually 30-50 ℃ and the nitrilase loses activity due to high-temperature treatment. From FIG. 7, it can be observed that the diffraction peak and the color of the sensor do not fluctuate, so we can also preliminarily predict the activity of nitrilase by this feature.
Example 8
The experimental procedure for testing a 0.8U/mL nitrilase solution under acidic conditions was as follows:
the method comprises the following steps: preparing a solution by taking dichloroacrylonitrile as a functional monomer, N, N-methylene bisacrylamide as a cross-linking agent and ethanol as a solvent according to a molar ratio of 5.
Step two: and putting the prepared photonic crystal template into a glass culture dish with an inclination angle of 15 degrees, dripping the prepared pre-polymerization liquid to the edge of the photonic crystal template, and making the pre-polymerization liquid slowly enter the pores of the photonic crystal template by utilizing the capillary action. And covering the organic glass sheet rinsed and dried by ethanol on the photonic crystal template with the pre-polymerized liquid on the surface to enable the organic glass sheet, the pre-polymerized liquid and the photonic crystal template to be in a sandwich state, clamping the sandwich by using tweezers, and wiping the redundant liquid on the edge by using filter paper. The sandwich was then placed in a clean glass petri dish and irradiated under uv light to effect polymerization curing.
Step three: taking out the whole sandwich, putting the sandwich into a plastic surface dish, adding hydrofluoric acid with the volume concentration of 3% into the plastic surface dish to etch away silicon dioxide in the photonic crystal template, throwing away the glass slide, and enabling the inverse opal nitrile-based polymer photonic crystal to be arranged on the organic glass sheet. And then deionized water is used for washing the inverse opal nitrile-based polymer photonic crystal to ensure that the inverse opal nitrile-based polymer photonic crystal reaches swelling balance and is neutral.
Step four: preparing 4mL of 5% hydrochloric acid solution, and putting the inverse opal nitrile-based polymer photonic crystal into the solution.
Step five: and (3) dripping 1mL of 4.0U/mL nitrilase solution into the solution, shaking the solution uniformly, observing the color change of the film, and recording the movement of a diffraction peak of the photonic crystal to realize the detection of the nitrilase.
And (4) conclusion: when the inverse opal nitrile-based polymer photonic crystal sensor detects a nitrilase solution of 0.8U/mL, the activity of the nitrilase rapidly decreases under an acidic condition, and almost no activity exists when the pH of the solution =5, so that the color and the diffraction peak of the sensor are almost unchanged. It can be observed from fig. 8 that the diffraction peak of the sensor is shifted from 630 to 633nm, which is the initial value, and is shifted by only 3nm. The color of the film does not change at equilibrium, and therefore the catalytic reaction at nitrilase generally needs to be carried out under alkaline conditions.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the embodiments of the invention without departing from the spirit and scope of the invention, which is to be covered by the claims.
Claims (10)
1. A method for visually and rapidly detecting nitrilase is characterized by comprising the following steps:
step one, mixing a functional monomer, a cross-linking agent and an initiator in a solvent to form a copolymer system, performing ultrasonic treatment to uniformly mix the copolymer system to obtain a pre-polymerization solution, and refrigerating the pre-polymerization solution for later use;
the functional monomer is a nitrile compound with double bonds;
secondly, dripping a pre-polymerization liquid into gaps of the photonic crystal template, polymerizing under an ultraviolet lamp, after the polymerization is finished, etching to obtain inverse opal nitrile-based polymer photonic crystals, and washing the inverse opal nitrile-based polymer photonic crystals with deionized water to enable the inverse opal nitrile-based polymer photonic crystals to be balanced in swelling and neutral;
thirdly, preparing an alkaline solution, and putting the inverse opal nitrile-based polymer photonic crystal into the alkaline solution to obtain a mixed system;
and step four, dripping nitrilase solutions with different concentrations into the mixed system, oscillating and shaking up, and realizing the detection of nitrilase by observing the color change of the film and the movement of a reflection peak of the photonic crystal.
2. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the step one, the nitrile compound with double bond is acrylonitrile or dichloroacrylonitrile.
3. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the first step, the molar ratio of the functional monomer to the cross-linking agent to the solvent is 5 (0.1-0.5) to 5.
4. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the second step, the ordered units of the photonic crystal template are silica colloidal particles.
5. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the third step, the alkaline solution is 5 to 15 mass percent sodium carbonate or sodium bicarbonate solution.
6. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the fourth step, the concentration of nitrilase is 0.2U/mL to 10.0U/mL.
7. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the first step, the cross-linking agent is ethylene glycol dimethacrylate or N, N-methylene bisacrylamide.
8. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the first step, the solvent is absolute ethyl alcohol.
9. The method for visually and rapidly detecting nitrilase of claim 1, wherein in the first step, the initiator is 2-hydroxy-2-methyl propiophenone.
10. The method for visually and rapidly detecting nitrilase according to claim 1, wherein in the second step, the preparation process of the inverse opal nitrile-based polymer photonic crystal specifically comprises the following steps:
putting the photonic crystal template into a culture dish with an inclined angle, and dropwise adding a prepolymerization liquid to the edge of the photonic crystal template to ensure that the prepolymerization liquid enters pores of the photonic crystal template;
covering the organic glass sheet on the photonic crystal template with the pre-polymerized liquid on the surface to enable the organic glass sheet, the pre-polymerized liquid and the photonic crystal template to be in a sandwich structure, and irradiating the sandwich structure under an ultraviolet lamp to realize polymerization and solidification;
and adding hydrofluoric acid into the cured copolymer photonic crystal template to etch silicon dioxide in the photonic crystal template to obtain the inverse opal nitrile-based polymer photonic crystal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211335853.1A CN115595354A (en) | 2022-10-28 | 2022-10-28 | Method for visually and rapidly detecting nitrilase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211335853.1A CN115595354A (en) | 2022-10-28 | 2022-10-28 | Method for visually and rapidly detecting nitrilase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115595354A true CN115595354A (en) | 2023-01-13 |
Family
ID=84851883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211335853.1A Pending CN115595354A (en) | 2022-10-28 | 2022-10-28 | Method for visually and rapidly detecting nitrilase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115595354A (en) |
-
2022
- 2022-10-28 CN CN202211335853.1A patent/CN115595354A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106497302B (en) | Photocatalytic self-cleaning parents' network marine anti-pollution coating and preparation method thereof | |
| Cantone et al. | Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods | |
| CN101215355B (en) | High oil absorption resin and synthetic method thereof | |
| US9316579B2 (en) | Method and apparatus for optical sensing using an optical sensor including a leaky mode waveguide | |
| Vitola et al. | PVDF membrane biofunctionalization by chemical grafting | |
| KR101114356B1 (en) | Porous structure for forming anti-fingerprint coating, method of forming anti-fingerprint coating, substrate comprising the anti-fingerprint coating formed by the method, and product comprising the substrate | |
| CN108484825B (en) | Reversible light-operated hydrophobic azobenzene fluorine-containing copolymer and membrane preparation method thereof | |
| CN106085173A (en) | A kind of functionally gradient composite construction abrasion-resistant clear super-hydrophobic coat and preparation method thereof | |
| CN102898833B (en) | Alignment film material, alignment film and preparation method of alignment film, and liquid display device | |
| Türünç et al. | Immobilization of α-amylase onto cyclic carbonate bearing hybrid material | |
| CN111621985A (en) | Preparation method of hydrophilic modified polyester non-woven fabric | |
| CN101864165A (en) | Organic-silicon-modified polyurethane sealant and processing technology thereof | |
| CN115595354A (en) | Method for visually and rapidly detecting nitrilase | |
| CN105153456A (en) | Photonic crystal material with humidity response allochroic characteristic and preparing method thereof | |
| CN106496464B (en) | A kind of amphipathic network of UV light anti-fouling type and preparation method thereof | |
| CN107664637A (en) | A kind of molecular imprinting photonic crystal detection card and application | |
| CN109825157A (en) | Reagent for the grease proofing diffusion of product surface | |
| CN107474254A (en) | The preparation and application of organic-inorganic hydrophilic hybrid integral material | |
| CN105670368A (en) | Method for preparing covalence immobilized antifouling enzyme thermo-sensitive self-polishing resin | |
| JP2004275064A (en) | Method for producing (meth)acrylic acid ester | |
| CN114775287A (en) | Modified regenerated fiber for waste wind power blades and preparation method thereof | |
| CN106701729A (en) | Immobilized enzyme taking polypeptide-modified amino resin as carrier and preparation method thereof | |
| CN111333794A (en) | Antibacterial polypropylene plastic and preparation method thereof | |
| CN110041543A (en) | A kind of corrosion resistant schemochrome hydrogel thin film and preparation method thereof | |
| US5077078A (en) | Surface modification of plastic optical fibers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |