CN110865074A - Photonic crystal heavy metal sensor with immobilized enzyme and preparation method thereof - Google Patents
Photonic crystal heavy metal sensor with immobilized enzyme and preparation method thereof Download PDFInfo
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- CN110865074A CN110865074A CN201911149888.4A CN201911149888A CN110865074A CN 110865074 A CN110865074 A CN 110865074A CN 201911149888 A CN201911149888 A CN 201911149888A CN 110865074 A CN110865074 A CN 110865074A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01005—Urease (3.5.1.5)
Abstract
The invention discloses a photonic crystal heavy metal sensor with immobilized enzyme and a preparation method thereof, and the method comprises the following specific steps: firstly, preparing a prepolymerization solution containing a functional monomer, a cross-linking agent, ethanol and a photoinitiator, filling the prepolymerization solution into gaps of photonic crystal colloidal particles, and placing the photonic crystal colloidal particles under an ultraviolet lamp for polymerization; after polymerization is completed, etching by hydrofluoric acid to obtain the inverse protein structure photonic crystal sensor with pH responsiveness; inserting one side of the photonic crystal sensor, which is not provided with the photonic crystal organic glass sheet, into the polydopamine solution for placement; and taking out the sensor, and inserting one side with the polydopamine coating into a urease solution with a certain concentration to fix urease to obtain the photonic crystal heavy metal sensor with the immobilized enzyme. Compared with the traditional detection method, the inverse opal structure photonic crystal sensor with the immobilized enzyme prepared by the invention is simpler and more convenient to operate, better in stability, higher in sensitivity and faster in response speed.
Description
Technical Field
The invention belongs to the technical field of nano functional material sensors, and relates to a photonic crystal heavy metal sensor with immobilized enzyme and a preparation method thereof.
Background
With the development of society and economy, China has achieved very remarkable achievements in the industrial field, people's life is greatly improved, but negative effects brought by the growth of an industrial system are not negligible, and people's life and health are seriously affected by the discharge of industrial waste water, the random landfill of industrial garbage and the like. Among them, the harm of heavy metal pollution to people is one of the more common and serious pollution. Heavy metals are not strictly defined at home and abroad, but in the chemical field, the relative specific gravity is generally greater than or equal to 4.5g/cm3The metal element includes mercury (Hg), lead (Pb), copper (Cu), cadmium (Cr), etc.
Heavy metals are ubiquitous in mineral reserves, the atmosphere and water systems, and such metals or compounds are extracted industrially and utilized by processing. Heavy metal-containing wastewater or solid waste discharged by industry can permeate into a water layer through soil and finally enter a human body through direct drinking or a food chain, so that the human health is seriously harmed. Heavy metals destroy the normal metabolic activity of the human body by inhibiting the activity of enzymes after entering the human body. For example, mercuric ions and their compounds can interfere with endocrine functions, destroy normal kidney function by accumulating in the human body, cause central nervous disorders, severely cause shock, or die. Therefore, the establishment of a method for detecting heavy metals quickly, simply and efficiently is of great significance.
The existing methods for detecting trace heavy metals include an ultraviolet-visible light spectrophotometry method, a surface enhanced Raman spectroscopy method, an atomic absorption method and the like. These methods also generally have higher sensitivity and accuracy, but also have greater limitations: the analysis method depending on a large instrument is relatively expensive in cost, complicated in pretreatment steps, long in time consumption, needs special equipment and operators, and is difficult to meet the requirements of miniaturization, real-time and rapid field detection.
The photonic crystal is an ordered structure material formed by materials with different refractive indexes according to periodic arrangement, the photonic band gap is a unique characteristic of the photonic crystal, when visible light passes through the photonic crystal, light with the wavelength at the position of the photonic band gap is reflected, so that the bright color is displayed, and the self-expression characteristic can realize field visualization rapid identification and detection independent of other analytical instruments.
Compared with free enzyme, the immobilized enzyme can better maintain the catalytic activity, stability and tolerance, so the immobilized enzyme can be rapidly developed in the fields of biology, bioengineering, medicine, biochemical sensors and the like. The marine organism mussel can secrete a mucus which can be firmly adhered to the surfaces of films of various substrates, the main component of the mucus is mussel adhesive protein, and dopamine in the adhesive protein plays a decisive role in endowing the mussel adhesive protein with super-strong adhesive performance. Based on the sensor, the invention develops the sensor for visually detecting the heavy metal based on the immobilized enzyme. Alkaline ions generated by urea hydrolysis under the catalysis of immobilized urease can stimulate a photonic crystal sensor to generate visual response. When heavy metal ions exist in the system, the heavy metal can inhibit the catalytic action of urease, so that the pH change of the system is not obvious or even does not change, and the high-efficiency visual detection of the heavy metal is realized.
Disclosure of Invention
The invention relates to a photonic crystal heavy metal sensor with immobilized enzyme and a preparation method thereof, aiming at realizing efficient visual field semi-quantitative detection of heavy metal ions.
In order to achieve the purpose, the invention provides a preparation method of a photonic crystal heavy metal sensor with immobilized enzyme, which comprises the following steps:
firstly, preparing a prepolymerization solution containing a functional monomer, a cross-linking agent, ethanol and a photoinitiator, filling the prepolymerization solution into gaps of photonic crystal colloidal particles, and placing the photonic crystal colloidal particles under an ultraviolet lamp for polymerization; after polymerization is completed, etching by hydrofluoric acid to obtain the inverse protein structure photonic crystal sensor with pH responsiveness; preparing a polydopamine solution with a certain concentration, inserting one side of the photonic crystal sensor, which is not provided with the photonic crystal organic glass sheet, into the polydopamine solution, and placing the polydopamine solution for a period of time to form a polydopamine coating on the organic glass sheet; and then taking out the sensor, inserting one side with the polydopamine coating into a urease solution with a certain concentration to fix urease, and obtaining the photonic crystal heavy metal sensor with the immobilized enzyme.
The preparation method specifically comprises the following steps:
the method comprises the following steps: preparing an ethanol pre-polymerization solution containing a functional monomer, a cross-linking agent and an initiator, wherein the volume ratio of the functional monomer to the cross-linking agent to the solvent to the photoinitiator is (1-5: 0.1-1: 5-15): 0.1-0.5, shaking the pre-polymerization solution uniformly for dissolving, and refrigerating overnight;
step two: infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 3-6 hours, finally taking out the photonic crystal and etching by hydrofluoric acid to obtain a polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use;
step three: weighing dopamine, adding the dopamine into tris (hydroxymethyl amino) methane with the pH value of 8.5 to prepare polydopamine solution, wherein the concentration of the polydopamine is 10-40 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 3-5 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the urease into a urease solution with a certain concentration to fix the urease; and (3) taking out the sensor after standing for 1-4 hours, and washing away redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
In the second step, the concentration of the hydrofluoric acid is 1% -4%. The ordered unit of the photonic crystal is silica colloid particles, polystyrene particles, polyacrylamide particles or polymethyl methacrylate particles.
The photonic crystal heavy metal sensor with the immobilized enzyme is prepared according to the preparation method.
Compared with the prior art, the invention has the beneficial effects that:
the used poly-dopamine biomimetic bonding technology can complete the immobilization of free enzyme under mild conditions, thereby improving the catalytic activity, stability, tolerance and the like of the enzyme, being more beneficial to the occurrence of an enzyme catalysis process and not influencing the response performance of the polymer photonic crystal. Meanwhile, the whole detection system is repeatedly utilized due to the immobilization of the enzyme, so that the detection cost is greatly reduced. The contact area between the sensor and a system can be increased due to the characteristics of the porous intercommunicating structure, the high specific surface area and the like of the sensor, so that the mass transfer resistance can be reduced, and the identification response of the sensor is very facilitated. Therefore, compared with the traditional detection method, the photonic crystal sensor with the inverse opal structure and the immobilized enzyme has the advantages of simpler operation, better stability, higher sensitivity and higher response speed.
Drawings
FIG. 1 is a graph of the reflectance spectra of the sensor of example 1 before and after equilibration when placed in a blank solution.
FIG. 2 shows the sensor of example 1 placed to contain 5X10-10Reflection spectra before and after equilibrium in g/L lead ion solution.
FIG. 3 is a graph of the reflectance spectrum of the sensor of example 2 before and after equilibration when placed in a blank solution.
FIG. 4 shows the sensor of example 2 placed at 5X10-12Reflection spectra before and after equilibrium in g/L mercury ion solution.
FIG. 5 is a graph of the reflectance spectrum of the sensor of example 3 before and after equilibration when placed in a blank solution.
FIG. 6 shows the sensor of example 3 placed at 5X10-13Reflection spectra before and after equilibrium in g/L mercury ion solution.
FIG. 7 is a graph of the reflectance spectrum of the sensor of example 4 before and after equilibration when placed in a blank solution.
FIG. 8 shows the sensor of example 4 placed at 5X10-14Reflection spectra before and after equilibrium in g/L mercury ion solution.
FIG. 9 is a graph of the reflectance spectrum of the sensor of example 5 before and after equilibration when placed in a blank solution.
FIG. 10 shows an example of a 5 sensor set to contain 5x10-7Reflection spectra before and after equilibrium in g/L copper ion solution.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention include, but are not limited to, the scope shown in the following examples.
A photonic crystal heavy metal sensor with immobilized enzyme and a preparation method thereof comprise the following steps:
the method comprises the following steps: preparing an ethanol pre-polymerization solution containing a functional monomer, a cross-linking agent and an initiator, wherein the volume ratio of the functional monomer to the cross-linking agent to the solvent to the photoinitiator is (1-5) to (0.1-1) to (5-15): (0.1-0.5), then shaking the pre-polymerization solution to dissolve, and putting the pre-polymerization solution into a refrigerator for refrigeration overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 3-6 hours, finally taking out the photonic crystal, and etching by hydrofluoric acid, wherein the concentration of the hydrofluoric acid is 1% -4%, so that the polymer photonic crystal is obtained and put into water to be balanced for later use.
Step three: weighing dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution, wherein the concentration of the polydopamine is 10-40 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 3-5 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the urease into a urease solution with a certain concentration to fix the urease, wherein the concentration of the urease is 1mg/mL-10 mg/mL; and (3) taking out the sensor after standing for 1-4 hours, and washing away redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
In the first step, the functional monomer is methacrylic acid, acrylic acid or itaconic acid.
In the first step, the cross-linking agent is ethylene glycol dimethacrylate or N, N-methylene bisacrylamide; the solvent is ethanol, methanol, water or a mixture thereof; the initiator is 2-hydroxy-2-methyl propiophenone and azodiisobutyronitrile.
In the second step, the ordered units of the photonic crystals are silica colloid particles, polystyrene particles, polyacrylamide particles or polymethyl methacrylate particles.
Example 1
The method comprises the following steps: according to the weight ratio of 1: 0.1: 5: preparing a prepolymerization solution containing methacrylic acid, ethylene glycol dimethacrylate, ethanol and dihydroxyl dimethyl propiophenone at a volume ratio of 0.1, shaking the prepolymerization solution uniformly to dissolve, and putting the solution into a refrigerator for refrigeration overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 4 hours, finally taking out the photonic crystal, etching by hydrofluoric acid to obtain the polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use.
Step three: weighing 120mg of dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution with the concentration of 20 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 4 hours; then taking out the polydopamine, and washing the polydopamine with a Tris-HCl buffer solution to remove redundant polydopamine; then inserting the immobilized urease into 10mg/mL urease solution to immobilize the urease; and (3) taking out the sensor after standing for 3 hours, and washing away the redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
The photonic crystal heavy metal sensor with immobilized enzyme prepared in example 1 is used for a blank system (water) and 5x10-10The detection is carried out by g/L lead ion solution, and the detection steps and results are as follows.
Blank system
1 mL of deionized water was added to 9 mL of 200 mM urea solution, and then the photonic crystal sensor was placed in this solution system, and the color change of the sensor before and after equilibrium was observed and the reflectance spectrum before and after equilibrium was recorded by a spectrometer, i.e., FIG. 1.
When no heavy metal exists in the system, urease catalyzes urea hydrolysis, so that the pH value of the system is obviously changed, and it can be observed from figure 1 that the reflection peak position is shifted from initial 540 nm to 620 nm when the photonic crystal sensor is balanced, the reflection peak position is shifted by 80nm altogether, and macroscopically, the reflection peak position is changed from green to deep red.
-10Lead ion solution
1 mL of 5X10-9g/L of lead ions are added into 9 mL of 200 mM urea solution, and then the photonic crystal sensor is put into the solution system, wherein the lead ion concentration of the solution system is 5x10-10g/L, observing the color change of the sensor before and after the balance and recording the reflection spectrum before and after the balance by a spectrometer, namely figure 2.
When the sensor detects the concentration of 5x10-10And g/L lead ions generate an inhibiting effect on urease, so that the catalytic effect of the urease on urea is weakened, and the change of the pH value of the system is reduced. From fig. 2 it can be observed that the photonic crystal sensor has shifted from the initial 540 nm to 611 nm for a total of 71 nm. Compared with the blank system (FIG. 1, total shift of 80 nm), the shift is reduced by 9nm, the color at equilibrium is also changed from dark red to light red, and the 5x10 color pair is realized-10And (5) detecting the visual response of the g/L lead ions.
Example 2
The method comprises the following steps: according to the weight ratio of 1: 0.5: 5: preparing prepolymerization liquid containing acrylic acid, ethylene glycol dimethacrylate, ethanol and dihydroxy dimethyl propiophenone in a volume ratio of 0.1, shaking the prepolymerization liquid to dissolve, and refrigerating in a refrigerator overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 4 hours, finally taking out the photonic crystal, etching by hydrofluoric acid to obtain the polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use.
Step three: weighing 120mg of dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution with the concentration of 20 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 4 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the immobilized urease into 10mg/mL urease solution to immobilize the urease; and (3) taking out the sensor after standing for 3 hours, and washing away the redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
The photonic crystal heavy metal sensor with immobilized enzyme prepared in example 2 is used for a blank system (water) and 5x10-12g/L mercury ion solution, the detection steps and the results are as follows.
Blank system
1 mL of deionized water was added to 9 mL of 200 mM urea solution, and then the photonic crystal sensor was placed in this solution system, and the color change of the sensor before and after equilibrium was observed and the reflectance spectrum before and after equilibrium was recorded by a spectrometer, i.e., FIG. 3.
When no heavy metal exists in the system, urease catalyzes urea hydrolysis, so that the pH value of the system is obviously changed, and it can be observed from figure 3 that the reflection peak position is shifted from initial 544nm to 620 nm when the photonic crystal sensor is balanced, the reflection peak position is shifted by 76nm altogether, and the reflection peak position is changed from green to deep red macroscopically.
-12Mercury ion solution
1 mL of 5X10-11Adding g/L mercury ions into 9 mL of 200 mM urea solution, and putting the photonic crystal sensor into the solution system, wherein the mercury ion concentration of the solution system is 5x10-12g/L, the sensor color change before and after equilibrium is observed and the reflectance spectra before and after equilibrium are recorded by the spectrometer, i.e. fig. 4.
When the sensor detects the concentration of 5x10-12In g/L of mercury ions, the urease is inhibited by the mercury ions, so that the catalytic effect of the urease on urea is weakened, and the pH change of a system is reduced. It can be observed from fig. 3 that the photonic crystal sensor has shifted from the initial 544nm to 552 nm for a total of 8 nm. Compared to the blank system (FIG. 3, a total shift of 76 nm), the shift is reduced by 68 nm, the color at equilibrium also changes from dark red to green, thus achieving a color contrast of 5 × 10-12And (5) detecting the visual response of the g/L mercury ions.
Example 3
The method comprises the following steps: according to the weight ratio of 1: 1.0: 5: preparing prepolymerization liquid containing itaconic acid, ethylene glycol dimethacrylate, ethanol and dihydroxy dimethyl propiophenone at a volume ratio of 0.1, shaking the prepolymerization liquid to dissolve, and refrigerating in a refrigerator overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 4 hours, finally taking out the photonic crystal, etching by hydrofluoric acid to obtain the polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use.
Step three: weighing 120mg of dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution with the concentration of 20 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 4 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the immobilized urease into 10mg/mL urease solution to immobilize the urease; and (3) taking out the sensor after standing for 3 hours, and washing away the redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
The photonic crystal heavy metal sensor with immobilized enzyme prepared in example 3 is used for a blank system (water) and 5x10-13g/L mercury ion solution, the detection steps and the results are as follows.
Blank system
1 mL of deionized water was added to 9 mL of 200 mM urea solution, and then the photonic crystal sensor was placed in this solution system, and the color change of the sensor before and after equilibrium was observed and the reflectance spectrum before and after equilibrium was recorded by a spectrometer, i.e., FIG. 5.
When no heavy metal exists in the system, urease catalyzes urea hydrolysis, so that the pH value of the system is obviously changed, and it can be observed from figure 5 that the reflection peak position is shifted from initial 541nm to 630 nm when the photonic crystal sensor is balanced, the reflection peak position is shifted by 89nm altogether, and the reflection peak position is changed from green to deep red macroscopically.
-13Mercury ion solution
1 mL of 5X10-12Adding g/L mercury ions into 9 mL of 200 mM urea solution, and putting the photonic crystal sensor into the solutionIn the solution system, the mercury ion concentration of the solution system is 5x10-13g/L, the sensor color change before and after equilibrium is observed and the reflectance spectra before and after equilibrium are recorded by the spectrometer, i.e. fig. 6.
When the sensor detects the concentration of 5x10-13In g/L of mercury ions, the urease is inhibited by the mercury ions, so that the catalytic effect of the urease on urea is weakened, and the pH change of a system is reduced. It can be observed from FIG. 6 that the photonic crystal sensor has shifted from the initial 541nm to 615 nm for a total of 74 nm. Compared to the blank system (FIG. 5, total shift 89 nm), the shift was reduced by 15 nm, and the color at equilibrium also changed from dark red to light red, thus achieving a 5 × 10 color pair- 13And (5) detecting the visual response of the g/L mercury ions.
Example 4
The method comprises the following steps: according to the weight ratio of 1: 0.1: 5: preparing prepolymerization liquid containing itaconic acid, ethylene glycol dimethacrylate, ethanol and dihydroxy dimethyl propiophenone at a volume ratio of 0.1, shaking the prepolymerization liquid to dissolve, and refrigerating in a refrigerator overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 4 hours, finally taking out the photonic crystal, etching by hydrofluoric acid to obtain the polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use.
Step three: weighing 120mg of dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution with the concentration of 20 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 4 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the immobilized urease into 10mg/mL urease solution to immobilize the urease; and (3) taking out the sensor after standing for 3 hours, and washing away the redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
A photon with immobilized enzyme prepared in example 4 was usedA crystal heavy metal sensor for a blank system (water) and 5x10-14g/L mercury ion solution, the detection steps and the results are as follows.
Blank system
1 mL of deionized water was added to 9 mL of 200 mM urea solution, and then the photonic crystal sensor was placed in this solution system, and the color change of the sensor before and after equilibrium was observed and the reflectance spectrum before and after equilibrium was recorded by a spectrometer, i.e., FIG. 7.
When no heavy metal exists in the system, urease catalyzes urea hydrolysis, so that the pH value of the system is obviously changed, and it can be observed from figure 7 that the reflection peak position is shifted from initial 545nm to 632 nm when the photonic crystal sensor is balanced, the reflection peak position is shifted by 87nm altogether, and the reflection peak position is changed from green to deep red macroscopically.
-14Mercury ion solution
1 mL of 5X10-13Adding g/L mercury ions into 9 mL of 200 mM urea solution, and putting the photonic crystal sensor into the solution system, wherein the mercury ion concentration of the solution system is 5x10-14g/L, observing the color change of the sensor before and after the balance, and recording the reflection spectrum before and after the balance by a spectrometer, namely figure 8.
When the sensor detects the concentration of 5x10-14In g/L of mercury ions, the urease is inhibited by the mercury ions, so that the catalytic effect of the urease on urea is weakened, and the pH change of a system is reduced. It can be observed from FIG. 8 that the photonic crystal sensor has moved from the initial 545nm to 628 nm for a total of 83 nm. Compared to the blank system (FIG. 1, total shift 87 nm), the shift was reduced by 4nm and there was essentially no change in color at equilibrium, indicating that the mercury ion was 5X10-14And g/L is the lowest detection limit of the sensor.
Example 5
The method comprises the following steps: according to the weight ratio of 1: 0.1: 5: preparing prepolymerization liquid containing acrylic acid, ethylene glycol dimethacrylate, ethanol and dihydroxy dimethyl propiophenone in a volume ratio of 0.1, shaking the prepolymerization liquid to dissolve, and refrigerating in a refrigerator overnight.
Step two: and (3) infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 4 hours, finally taking out the photonic crystal, etching by hydrofluoric acid to obtain the polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use.
Step three: weighing 120mg of dopamine, adding the dopamine into a tris (hydroxymethyl amino) methane buffer solution with the pH value of 8.5 to prepare a polydopamine solution with the concentration of 20 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 4 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the immobilized urease into 10mg/mL urease solution to immobilize the urease; and (3) taking out the sensor after standing for 3 hours, and washing away the redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
The photonic crystal heavy metal sensor with immobilized enzyme prepared in example 5 is used for a blank system (water) and 5x10-7The detection is carried out by g/L copper ion solution, and the detection steps and results are as follows.
Blank system
1 mL of deionized water was added to 9 mL of 200 mM urea solution, and then the photonic crystal sensor was placed in this solution system, and the color change of the sensor before and after equilibrium was observed and the reflectance spectrum before and after equilibrium was recorded by a spectrometer, i.e., FIG. 9.
When no heavy metal exists in the system, urease catalyzes urea hydrolysis, so that the pH value of the system is obviously changed, and it can be observed from figure 9 that the reflection peak position is shifted from initial 545nm to 620 nm when the photonic crystal sensor is balanced, the reflection peak position is shifted by 75nm altogether, and macroscopically, the reflection peak position is changed from green to deep red.
-7Copper ion solution
1 mL of 5X10-6Adding copper ions in g/L into 9 mL of 200 mM urea solution, and putting the photonic crystal sensor into the solution system, wherein the copper ion concentration of the solution system is 5x10-7g/L, observing the color change of the sensor before and after balance and recording the color change by a spectrometerThe reflectance spectra before and after the equilibrium were recorded, i.e., FIG. 10.
When the sensor detects the concentration of 5x10-7In g/L of copper ions, the mercury ions have an inhibiting effect on urease, so that the catalytic effect of the urease on urea is weakened, and the pH change of a system is reduced. From FIG. 10, it can be observed that the photonic crystal sensor has shifted from the initial 545nm to 598 nm for a total of 53 nm. Compared to the blank system (FIG. 10, a total shift of 75 nm), the shift was reduced by 22 nm, and the color at equilibrium also changed from deep red to yellow, thus achieving a color contrast of 5X10- 7And g/L visual response detection of copper ions.
After the reaction is finished, putting the photonic crystal sensor into 2.5 mg/mL EDTA-disodium solution, and then washing with a large amount of water to realize the reactivation of urease; after activation, the sensor is placed into 1% acetic acid solution, the photonic crystal sensor can restore to the initial color, and the sensor can be reused after being placed into water for balance.
Claims (4)
1. A preparation method of a photonic crystal heavy metal sensor with immobilized enzyme is characterized by comprising the following steps:
firstly, preparing a prepolymerization solution containing a functional monomer, a cross-linking agent, ethanol and a photoinitiator, filling the prepolymerization solution into gaps of photonic crystal colloidal particles, and placing the photonic crystal colloidal particles under an ultraviolet lamp for polymerization; after polymerization is completed, etching by hydrofluoric acid to obtain the inverse protein structure photonic crystal sensor with pH responsiveness; preparing a polydopamine solution with a certain concentration, inserting one side of the photonic crystal sensor, which is not provided with the photonic crystal organic glass sheet, into the polydopamine solution, and placing the polydopamine solution for a period of time to form a polydopamine coating on the organic glass sheet; and then taking out the sensor, inserting one side with the polydopamine coating into a urease solution with a certain concentration to fix urease, and obtaining the photonic crystal heavy metal sensor with the immobilized enzyme.
2. The preparation method of the photonic crystal heavy metal sensor with the immobilized enzyme according to claim 1, wherein the method comprises the following steps:
the method specifically comprises the following steps:
the method comprises the following steps: preparing an ethanol pre-polymerization solution containing a functional monomer, a cross-linking agent and an initiator, wherein the volume ratio of the functional monomer to the cross-linking agent to the solvent to the photoinitiator is (1-5: 0.1-1: 5-15): 0.1-0.5, shaking the pre-polymerization solution uniformly for dissolving, and refrigerating overnight;
step two: infiltrating the pre-polymerized liquid obtained in the step one from the edge of the photonic crystal, covering an organic glass sheet on the photonic crystal when the photonic crystal is transparent, then putting the photonic crystal under an ultraviolet lamp for irradiating for 3-6 hours, finally taking out the photonic crystal and etching by hydrofluoric acid to obtain a polymer photonic crystal, and putting the polymer photonic crystal into water to balance for later use;
step three: weighing dopamine, adding the dopamine into tris (hydroxymethyl amino) methane with the pH value of 8.5 to prepare polydopamine solution, wherein the concentration of the polydopamine is 10-40 mg/mL, and inserting the polymer-carrying photonic crystal obtained in the second step into the polydopamine solution for 3-5 hours; then taking out the polydopamine, and washing the polydopamine with a tris (hydroxymethyl amino) methane buffer solution; then inserting the urease into a urease solution with a certain concentration to fix the urease; and (3) taking out the sensor after standing for 1-4 hours, and washing away redundant urease solution by using deionized water to prepare the photonic crystal sensor with the immobilized urease.
3. The preparation method of the photonic crystal heavy metal sensor with the immobilized enzyme according to claim 2, wherein the method comprises the following steps:
in the second step, the concentration of hydrofluoric acid is 1% -4%; the ordered unit of the photonic crystal is silica colloid particles, polystyrene particles, polyacrylamide particles or polymethyl methacrylate particles.
4. A photonic crystal heavy metal sensor with immobilized enzyme prepared by the preparation method of claims 1-3.
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