CN112980827A - Metal organic framework material immobilized glucose oxidase and preparation method and application thereof - Google Patents
Metal organic framework material immobilized glucose oxidase and preparation method and application thereof Download PDFInfo
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- CN112980827A CN112980827A CN202110156009.1A CN202110156009A CN112980827A CN 112980827 A CN112980827 A CN 112980827A CN 202110156009 A CN202110156009 A CN 202110156009A CN 112980827 A CN112980827 A CN 112980827A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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Abstract
The invention provides a metal organic framework material immobilized glucose oxidase, a preparation method and application thereof, belonging to the technical field of material preparation; in the invention, a one-step immobilization method is adopted to introduce glucose oxidase into a pore channel structure of a metal organic framework material ZIF-8 formed by metal zinc ions and 2-methylimidazole, so that one-step rapid immobilization of the enzyme is realized; the obtained immobilized enzyme 3-MPBA/GOx @ ZIF-8 overcomes the defects of free glucose oxidase in aspects of pH stability, thermal stability, storage stability, urea tolerance and the like, and has good application in visual detection of glucose.
Description
Technical Field
The invention belongs to the technical field of material preparation, and particularly relates to a metal organic framework material immobilized glucose oxidase, and a preparation method and application thereof.
Background
The enzyme is a biological macromolecular catalyst, and is widely applied to the fields of medicine, food, chemical industry, agriculture and the like due to the characteristics of substrate specificity, selectivity, green chemistry and the like. However, natural enzymes have low thermostability and handling stability, narrow optimal pH range, low resistance to most organic solvents, difficult recovery and lack of reusability under handling conditions. Furthermore, enzymes generally perform best in homogeneous systems, which results in complex separation steps being required to remove the enzyme from the product mixture.
Metal Organic Frameworks (MOFs) are three-dimensional network structures with high crystallinity and porosity formed by coordination of metal cations or clusters with organic ligands. In recent years, MOFs are considered to be a promising enzyme immobilization support matrix due to their advantages of high specific surface area and pore volume, easy pore size adjustment, easy modification of metal cations or clusters and organic ligands, mild synthesis conditions, and the like. In addition, as the MOFs node and the connector provide a large number of anchor points for the combination of the enzyme through coordination bonds, covalent bonds, hydrogen bonds, van der Waals force and the like, the leaching and denaturation of the enzyme when being heated, dehydrated and changed in solvent can be prevented, and the reusability of the catalyst is improved. However, the enzyme @ MOF still has deficiencies in substrate affinity and catalytic efficiency, and the time for inducing the enzyme @ MOF in aqueous solution is long.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a metal organic framework material immobilized glucose oxidase, a preparation method and application thereof. In the invention, glucose oxidase (GOx for short) is introduced into a pore structure of a metal organic framework material ZIF-8 formed by metal zinc ions and 2-methylimidazole by adopting a one-step immobilization method, so that one-step rapid immobilization of the enzyme is realized; the obtained immobilized enzyme 3-MPBA/GOx @ ZIF-8 overcomes the defects of free glucose oxidase in aspects of pH stability, thermal stability, storage stability, urea tolerance and the like, and has good application in visual detection of glucose.
The invention firstly provides a metal organic framework material immobilized glucose oxidase which is marked as 3-MPBA/GOx @ ZIF-8, wherein the 3-MPBA/GOx @ ZIF-8 is in a granular structure, and the grain diameter is 180-200 nm.
The invention also provides a preparation method of the metal organic framework material immobilized glucose oxidase, which comprises the following specific preparation steps:
weighing glucose oxidase GOx, uniformly mixing the glucose oxidase GOx with polyvinylpyrrolidone PVP (polyvinylpyrrolidone), adding 3-MPBA and 2-methylimidazole 2-MIM (metal-imide-phosphate) solution, uniformly mixing, then adding zinc nitrate solution, fully mixing to obtain mixed solution, standing the mixed solution for culture reaction, centrifuging at 4 ℃ after the reaction is finished, washing and drying to obtain the immobilized glucose oxidase of the metal organic framework material, which is recorded as 3-MPBA/GOx @ ZIF-8.
Further, the final concentration of GOx in the mixed solution is 0.2-1.8 mg/mL.
Further, the final concentration of GOx in the mixed solution was 1 mg/mL.
Further, the concentration of PVP in the mixed solution is 0.1-1.2mg/mL, preferably 0.8 mg/mL.
Further, the final concentration of the 3-MPBA in the mixed solution is 0.1-1mg/mL, preferably 0.6 mg/mL.
Further, the final concentration of the 2-MIM in the mixed solution is 64 mM; the final concentration of the zinc nitrate in the mixed solution was 16 mM.
Further, the time of the static culture is 10min-24h, preferably 30 min.
The invention also provides application of the metal organic framework material immobilized glucose oxidase 3-MPBA/GOx @ ZIF-8 in visual detection of glucose.
Compared with the prior art, the invention has the beneficial effects that:
(1) at present, the enzyme immobilization usually comprises the steps of firstly synthesizing an immobilization material and then carrying out enzyme immobilization, so that the loss of enzyme activity is large; the invention innovatively selects a rapid one-step synthesis method of 3-mercaptophenylboronic acid, and the invention simultaneously introduces glucose oxidase in the MOFS forming process to realize immobilization. The defects that the pore channel of the metal organic framework material is too small, the enzyme cannot enter the material, or the pore channel of the material is too large and cannot play a role in immobilization are overcome by simultaneously immobilizing the enzyme and preparing the framework material, so that the temperature stability, the acid and alkali resistance and the storage stability of the enzyme are improved, the cyclic utilization rate is obviously improved, and the rate of degrading glucose by using glucose oxidase is improved.
(2) According to the invention, a glucose oxidase solution is uniformly mixed with PVP, and then sequentially and uniformly mixed with a 3-mercaptophenylboronic acid solution, 2-methylimidazole and a zinc nitrate solution, so that the immobilized enzyme 3-MPBA/GOx @ ZIF-8 is successfully prepared under the mild conditions of normal temperature and water phase, and compared with a packaging experiment without 3-MPBA, the packaging efficiency is improved by 12.23%.
(3) The immobilized enzyme is obtained to the maximum extent by adjusting the amounts of PVP, 3-mercaptophenylboronic acid and the enzyme, the immobilization condition is optimized, and when the concentration of the selected enzyme is 1mg/mL, the concentration of a polyvinylpyrrolidone solution is 0.8mg/mL, the concentration of the 3-mercaptophenylboronic acid is 0.6mg/mL, and the immobilization time is 30min, the encapsulation efficiency and the retention activity of the enzyme reach the optimal values of 97.32% and 86.15% respectively.
(4) The invention inspects the enzymological properties of the immobilized enzyme 3-MPBA/GOx @ ZIF-8, and finds that the stability and the like of the immobilized enzyme are obviously improved. The three-dimensional pore structure of the ZIF-8 material provides a rigid shielding environment for the glucose oxidase, so that the influence of an external adverse environment on the enzyme activity is effectively reduced, and the characteristics of thermal stability, acid and alkali resistance, storage stability, recycling stability and the like of the ZIF-8 material are improved.
Drawings
FIG. 1 is a schematic diagram of the synthesis of 3-MPBA/GOx @ ZIF-8 (a), a schematic diagram of different packaging systems (b) and a comparative diagram of packaging efficiency (c).
FIG. 2 is an infrared spectrum of free GOx (a), metal-organic framework material ZIF-8 (b), and immobilized enzyme 3-MPBA/GOx @ ZIF-8 (c).
FIG. 3 is a drawing showing N of 3-MPBA @ ZIF-8 (a) and 3-MPBA/GOx @ ZIF-8 (b)2Adsorption-desorption isotherms and pore distribution curves.
FIG. 4 is a TEM image of 3-MPBA @ ZIF-8 (a) and 3-MPBA/GOx @ ZIF-8 (b).
FIG. 5 is an LPSD map of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8, wherein a is 3-MPBA @ ZIF-8 and b is 3-MPBA/GOx @ ZIF-8.
FIG. 6 is a thermogravimetric analysis of ZIF-8 (a) and 3-MPBA @ ZIF-8 (b) and 3-MPBA/GOx @ ZIF-8 (c).
FIG. 7 is a graph showing the effect of PVP addition on the encapsulation efficiency of the enzyme.
FIG. 8 is a graph showing the effect of the amount of glucose oxidase added on the efficiency of encapsulation of the enzyme.
FIG. 9 is an SEM image of 3-MPBA/GOx @ ZIF-8 at different glucose oxidase concentrations.
FIG. 10 is a graph showing the effect of the amount of 3-mercaptophenylboronic acid added on the encapsulation efficiency of the enzyme.
FIG. 11 is a graph showing the effect of immobilization time on the efficiency of enzyme encapsulation in the presence or absence of 3-mercaptophenylboronic acid.
FIG. 12 is a graph showing the effect of immobilization time on enzyme activity retention in the presence or absence of 3-mercaptophenylboronic acid.
FIG. 13 is an SEM image of GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 at different incubation times and a photograph of a suspension thereof.
FIG. 14 is a graph showing the reaction of GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 with glucose (a) and the rate profile for catalytic generation of H2O2 (b).
FIG. 15 is a graph showing the effect of pH on the catalytic activity of free GOx and its immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 16 is a graph showing the effect of temperature on the catalytic activity of free GOx and its immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 17 is a graph showing the results of pH stability verification of free GOx and its immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 18 is a graph showing the results of thermal stability verification of free GOx and its immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 19 is a graph showing the results of the storage stability verification of free GOx and its immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 20 is a graph showing the results of the stability verification of the reuse of the immobilized enzyme 3-MPBA/GOx @ ZIF-8.
FIG. 21 is a graph showing the selective visual detection of glucose by the immobilized enzyme 3-MPBA/GOx @ ZIF-8 (a) and the visual detection of glucose at different concentrations (b).
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
The properties of the immobilized enzyme obtained by the invention are verified in the following way:
(1) enzyme encapsulation efficiency assay:
measured using a modified version of the Bradford protein detection kit. The specific method comprises adding 10 μ L enzyme solution and centrifuged supernatant into 100 μ L working solution, and incubating at room temperature for 10 min. And (3) parallelly measuring the absorbance at 595 nm, and calculating the protein concentration according to a linear regression equation of a protein standard curve, thereby further calculating the packaging efficiency of the glucose oxidase.
Encapsulation efficiency (%) = (amount of enzyme added-amount of enzyme in supernatant)/total amount of enzyme × 100%
(2) Determination of glucose oxidase activity:
the enzyme activity is measured by adopting a colorimetric method, glucose is taken as a substrate, a product obtained by catalytic hydrolysis is hydrogen peroxide (H2O 2), the indigo carmine is faded, a characteristic absorption peak exists at 615 nm, and the colorimetric determination can be directly carried out.
Preparing an indigo carmine solution: 0.0233 g of indigo carmine sample was weighed, dissolved in an appropriate amount of deionized water and made to volume of 50 mL to obtain a 1 mM indigo carmine solution.
Preparing 0.2M acetic acid-sodium acetate (NaAc-HAc) solution: 2.46 g of sodium acetate solid is weighed into a beaker, dissolved fully by using a proper amount of deionized water, and then 594 mu L of glacial acetic acid is added to the beaker, and the volume is adjusted to 200 mL.
Preparing a 0.2M glucose solution: 0.9 g of sodium acetate solid is weighed into a beaker, dissolved by a proper amount of deionized water and then added to 25 mL.
Determination of enzyme activity: 1mL of glucose (0.2M) and 1mL of enzyme were preheated in a water bath at 37 ℃ for 5 min. The two solutions were then mixed and reacted at 37 ℃ for 10 min. Then, 3 mL of NaAc-HAc (0.2M, pH 5.2) buffer, 1.3 mL of indigo carmine (1 mM) and the above reaction solution were added to a 25 mL colorimetric tube. Adding deionized water into the tube, adding water to a certain volume, reacting in boiling water for 13 min, and cooling with flowing water for 5min to terminate the reaction. The absorbance of the solution was measured at 615 nm against deionized water.
(3) Calculating enzyme activity:
definition of glucose oxidase activity: single-site (min) catalysis of glucose to produce H at 37 ℃2O2The amount of enzyme (c).
(4) The immobilized enzyme 3-MPBA/GOx @ ZIF-8 is used for degrading glucose:
the immobilized enzyme 3-MPBA/GOx @ ZIF-8 and the cellulase are used for visually detecting glucose under the action of 3,3',5,5' -Tetramethylbenzidine (TMB): visual detection of glucose was performed with a specially prepared round cotton piece. 50 μ L of 3-MPBA/GOx @ ZIF-8 was dropped into the cotton piece and completely dried at room temperature. Then, 15 μ L of different concentrations of glucose, 10 mM TMB and HRP were mixed and added to the cotton pad. The color of the cotton piece was recorded with a camera after 15 min at room temperature and normal pressure.
Example 1: preparation of metal organic framework material immobilized enzyme 3-MPBA/GOx @ ZIF-8
Preparation of 3-MPBA/GOx @ ZIF-8: dissolving 3-MPBA in 50 mu L of ethanol, adding 1mL of GOx and PVP with the molecular weight of 8000, mixing uniformly, then adding 2 mL of 2-MIM with the concentration of 160 mM and 2 mL of zinc nitrate solution with the concentration of 40 mM, mixing uniformly so that the final concentration of glucose oxidase in the mixed solution is 0.2mg/mL, the final concentration of PVP in the mixed solution is 0.1mg/mL, and the final concentration of 3-mercaptophenylboronic acid in the mixed solution is 0.1mg/mL, standing the mixed solution at room temperature for 10 minutes, then centrifuging the mixed solution after standing at 8000 rpm for 5 minutes, washing, and drying in vacuum to obtain 3-MPBA/GOx @ ZIF-8.
Preparation of 3-MPBA @ ZIF-8: dissolving 3-MPBA in 50 mu L of ethanol, adding 1mL of deionized water and PVP with the molecular weight of 8000, uniformly mixing, then adding 2 mL of 2-MIM with the concentration of 160 mM and 2 mL of zinc nitrate solution with the concentration of 40 mM, uniformly mixing to ensure that the final concentration of the PVP in the mixed solution is 0.1mg/mL and the final concentration of the 3-mercaptophenylboronic acid in the mixed solution is 0.1mg/mL, standing the mixed solution at room temperature for 10 minutes, then centrifuging the mixed solution after standing at 8000 rpm for 5 minutes, washing, and drying in vacuum to obtain the 3-MPBA/GOx @ ZIF-8.
As shown in FIG. 1a, due to 3-MPBA and Zn2+The strong coordination of 3-MPBA may accelerate the 3-MPBA and Zn around GOx2+Nucleate cluster formation and trigger encapsulation of these proteins by MOFs. The encapsulation effect of 3-MPBA and PVP on GOx is shown in figures 1b and 1c, compared with that of the encapsulation experiment without PVP, the precipitation amount is obviously increased after the PVP is added, the encapsulation efficiency of the GOx is improved by 15.29%, and the encapsulation experiment is obviously better than that without the PVP. The encapsulation experiment of 3-MPBA shows that the precipitation amount and the encapsulation efficiency of GOx are further increased by 12.23% compared with the encapsulation experiment without 3-MPBA, which indicates that the increase of sulfydryl can promote the formation of 3-MPB/GOx @ ZIF-8.
A Fourier transform infrared spectrometer is adopted to detect the molecular structures of three samples, namely free GOx, a metal organic framework material ZIF-8 monomer and an immobilized enzyme thereof, namely 3-MPBA/GOx @ ZIF-8, and the determination result is shown in figure 2. In the figure, a is the infrared spectrum of free GOx (a), and it can be seen that the characteristic peak of GOx appears at 1400-1600 cm-1(-CONH) and 3200--1(N-H). b is an infrared spectrogram of a metal-organic framework material ZIF-8, and a characteristic peak of the ZIF-8 appears at 1580 cm-1(C = N) and 1350--1Point (characteristic peak of imidazole ring). c is an infrared spectrogram of the immobilized enzyme 3-MPBA/GOx @ ZIF-8, the 3-MPBA/GOx @ ZIF-8 comprises peaks in a and b, and a characteristic peak 680 cm of the 3-MPBA appears-1(C-S). Furthermore, in the process of producing 3-MPBA/GOx @ ZIF-8, the carboxyl group on the enzyme is reacted with Zn2+The coordination of (2) appeared to be 1650 cm-1Indicating that GOx is not simply adsorbed but is embedded in ZIF-8.
FIGS. 3a and 3b are N of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8, respectively2Adsorption-desorption isotherms and pore distribution curves. As can be seen in the figure, the intersection of the curves is between 0.6 and 0.8, which indicates that 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 are mesoporous materials. In conjunction with the pore distribution curve, it can be concluded that the presence of pores is likely due to packing between materials. Meanwhile, the specific surface areas of the 3-MPBA @ ZIF-8 and the 3-MPBA/GOx @ ZIF-8 are 70.4218 m2G and 45.0515 m2(ii) in terms of/g. The pore volumes of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 were respectively carried out at 0.241558 cm and 0.195621 cm respectively. The decrease in specific surface area and pore volume indicated that the remaining pores in 3-MPBA @ ZIF-8 were blocked by the addition of GOx, indicating that GOx was successfully embedded in as little as 30 minutes.
Example 2: preparation of metal organic framework material immobilized enzyme 3-MPBA/GOx @ ZIF-8
Dissolving 3-MPBA in 50 mu L of ethanol, adding 1mL of GOx and PVP with the molecular weight of 8000, mixing uniformly, then adding 2 mL of 2-MIM with the concentration of 160 mM and 2 mL of zinc nitrate solution with the concentration of 40 mM, mixing uniformly so that the final concentration of GOx in the mixed solution is 1mg/mL, the final concentration of PVP in the mixed solution is 0.8mg/mL, and the final concentration of 3-mercaptophenylboronic acid in the mixed solution is 0.6mg/mL, standing the mixed solution at room temperature for 30 minutes, then centrifuging the mixed solution after standing at 8000 rpm for 5 minutes, washing, and drying in vacuum to obtain 3-MPBA/GOx @ ZIF-8.
FIGS. 4a and 4b are TEM images of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8, respectively. As can be seen by comparison, the 3-MPBA @ ZIF-8 and the 3-MPBA/GOx @ ZIF-8 are granular and have the size of about 200nm, and the phenomenon that the grain size of MOFs is reduced after the GOx is added also shows that the appearance of the ZIF-8 is not damaged by the introduction of the glucose oxidase.
FIG. 5 is an LPSD map of 3-MPBA @ ZIF-8 and immobilized enzyme 3-MPBA/GOx @ ZIF-8, in which a is 3-MPBA @ ZIF-8 and b is immobilized enzyme 3-MPBA/GOx @ ZIF-8. As can be seen from the figure, the average particle sizes of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 are 234.6 nm and 183.4 nm, respectively, and the particle size of GOx @ MOF decreases after the addition of GOx, which is mainly due to the increase of nucleation sites in GOx @ MOF.
Example 3: preparation of metal organic framework material immobilized enzyme 3-MPBA/GOx @ ZIF-8
Dissolving 3-MPBA in 50 mu L of ethanol, adding 1mL of GOx and PVP with the molecular weight of 8000, mixing uniformly, then adding 2 mL of 2-MIM with the concentration of 160 mM and 2 mL of zinc nitrate solution with the concentration of 40 mM, mixing uniformly to ensure that the final concentration of GOx in the mixed solution is 1.8mg/mL, the final concentration of PVP in the mixed solution is 1.2mg/mL, and the final concentration of 3-mercaptophenylboronic acid in the mixed solution is 1mg/mL, standing the mixed solution at room temperature for 24h, then centrifuging the mixed solution after standing at 8000 rpm for 5min, washing, and drying in vacuum to obtain 3-MPBA/GOx @ ZIF-8.
FIGS. 6a, 6b and 6c are thermogravimetric analysis plots of ZIF-8, 3-MPBA @ ZIF-8 and its immobilized enzyme, 3-MPBA @ ZIF-8, respectively. As shown in fig. 6, from 25 to 211 ℃, ZIF-8 lost about 6% of its mass due to the evaporation of water molecules in the sample, and the second stage started from about 357 ℃, the weight loss rate was slow, which was due to the decomposition of ZIF-8 itself, and the weight loss rate was 25.79%. As can be seen from the weight loss curves of 3-MPBA @ ZIF-8 and 3-MPBA/GOx @ ZIF-8, the first stage of weight loss is below 102 ℃ due to the evaporation of water molecules; in the 3-MPBA @ ZIF-8 curve, the second stage (277 ℃ -800 ℃) is mainly caused by the decomposition of 3-MPBA and ZIF-8, and the weight loss rate is about 51.36%. In contrast, the second phase of the weight loss curve for 3-MPBA/GOx @ ZIF-8 (305 ℃ C. -800 ℃ C.) exhibited a tendency of rapid first followed by slow, total weight loss of about 84.50%, loss of about 33.14% wt% GOx weight, beginning at 305 ℃ C. -357 ℃ C, mass loss due to decomposition of 3-MPBA and GOx; at temperatures above 357 ℃, the slow drop in weight is mainly due to the continued collapse and decomposition of the MOFs material parts. Three weight loss graphs of ZIF-8, 3-MPBA @ ZIF-8 and immobilized enzyme 3-MPBA @ ZIF-8 thereof are analyzed, so that the glucose oxidase is successfully immobilized in the ZIF-8.
Example 4: 3-MPBA/GOx @ ZIF-8 immobilization condition optimization
In this example, the influence of immobilization of 3-MPBA/GOx @ ZIF-8 under different conditions on the encapsulation efficiency and the recovery rate of enzyme activity was examined.
(1) Effect of PVP addition on immobilization:
PVP is a biocompatible macromolecule similar to protein, can be used as an encapsulating agent to form a PVP/protein compound, and is favorable for guiding amino acid inclined protein to form 3-MPBA/GOx @ ZIF-8 through hydrogen bond interaction, and the encapsulating effect of PVP on GOx is shown in figure 7. As can be seen from the figure, compared with the method without adding PVP, the amount of precipitation after adding PVP is significantly increased, and the encapsulation efficiency of GOx is improved by 15.29%, which is significantly better than that of the encapsulation experiment without adding PVP. Along with the increase of the concentration of PVP, the encapsulation efficiency of GOx is shown to be firstly improved and then kept unchanged, and the enzyme activity recovery rate is firstly improved and then reduced, which is probably because the serious aggregation of 3-MPBA/GOx @ ZIF-8 is caused by the overhigh concentration of PVP, and the reaction of the 3-MPBA/GOx @ ZIF-8 and a substrate is inhibited. At a PVP concentration of 0.8mg/mL, the encapsulation efficiency and the enzyme activity recovery rate are optimal, wherein the encapsulation efficiency is 96.18 +/-2.03%, and the enzyme activity recovery rate is 84.58 +/-2.20%.
(2) Influence of the amount of enzyme added on immobilization:
the influence of the amount of GOx on the formation of 3-MPBA/GOx @ ZIF-8 was examined by changing the amount of GOx added in other steps without changing. As can be seen from fig. 8, the encapsulation efficiency and enzyme activity recovery rate of GOx are increased and then decreased as the concentration of GOx is increased. When the concentration of GOx is 1.0 mg/mL, the precipitation amount is the largest, at the moment, the GOx encapsulation efficiency reaches 96.98 +/-2.57%, and the enzyme activity recovery rate is 85.87 +/-2.13%.
FIG. 9 is an SEM image of 3-MPBA/GOx @ ZIF-8 at different glucose oxidase concentrations. From the SEM image, as the concentration of GOx increased from 0.2mg/mL to 1.8mg/mL, the 3-MPBA/GOx @ ZIF-8 crystal size decreased, indicating that the size and morphology of the 3-MPBA/GOx @ ZIF-8 can be controlled by adjusting the amount of GOx.
(3) Influence of the amount and time of the 3-mercaptophenylboronic acid added on immobilization:
the influence of the addition amount of 3-mercaptophenylboronic acid on the formation of 3-MPBA/GOx @ ZIF-8 was examined by changing the addition amount of 3-mercaptophenylboronic acid without changing the other steps, and FIG. 10 is a result chart of the influence of the addition amount of 3-mercaptophenylboronic acid on the encapsulation efficiency effect of the enzyme.
As shown in FIG. 10, the improvement was 12.23% compared to the encapsulation experiment without 3-MPBA, indicating that the increase in thiol groups may promote the formation of 3-MPB/GOx @ ZIF-8. With the increase of the dosage of 3-MPBA, the encapsulation efficiency of GOx tends to increase first and then decrease. When the concentration of the 3-MPBA is 0.6mg/mL, the encapsulation efficiency and the enzyme activity recovery rate reach the optimal level, and the encapsulation efficiency and the enzyme activity recovery rate are 96.98 +/-2.08 percent and 85.71 +/-3.02 percent respectively.
FIG. 11 is a graph showing the effect of immobilization time on the efficiency of enzyme encapsulation in the presence or absence of 3-mercaptophenylboronic acid, and FIG. 12 is a graph showing the effect of immobilization time on the activity retention of the enzyme in the presence or absence of 3-mercaptophenylboronic acid. As can be seen by comparing the two graphs, the 3-MPBA/GOx @ ZIF-8 is gradually and completely encapsulated at 10-30min, the encapsulation efficiency reaches the highest value of 97.32 +/-2.82%, and the enzyme activity recovery rate is 86.15 +/-2.56%. 3-MPBA/GOx @ ZIF-8 exhibited a uniform granular structure at 30 minutes, and GOx @ ZIF-8 still exhibited an unassembled lamellar structure. When the immobilization time is 30 minutes to 24 hours, the encapsulation efficiency of the 3-MPBA/GOx @ ZIF-8 is kept stable, the size and the shape of the 3-MPBA/GOx @ ZIF-8 are kept unchanged, and the recovery rate of the enzyme activity of the 3-MPBA/GOx @ ZIF-8 is reduced along with the prolonging of the time. GOx @ ZIF-8 without the addition of 3-MPBA takes at least 12 hours to complete encapsulation, and the encapsulation efficiency is 93.83 +/-2.30%. The reason for this may be that the presence of thiol groups interacts with metal ions to change the shape and size of the nanoparticles.
FIG. 13 is an SEM image of GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 at different incubation times and a photograph of a suspension thereof. As can be seen from the figure, GOx @ ZIF-8 without 3-MPBA is in a flocculated state, and 3-MPBA/GOx @ ZIF-8 is dispersed very uniformly.
(4) Addition of 3-mercaptophenylboronic acid to catalyze the production of H from glucose2O2Influence of the velocity:
FIG. 14a is a schematic diagram of the reaction of GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 with glucose, and it can be seen that glucose can rapidly aggregate near 3-MPBA/GOx @ ZIF-8, resulting in a higher glucose concentration near 3-MPBA/GOx @ ZIF-8 than in bulk solution, and the glucose enrichment is significantly reducedK mResulting in an increase in apparent enzyme activity. However, in the absence of 3-MPBA, the interaction of glucose with GOx @ ZIF-8 is a dynamic equilibrium process: glucose first interacts with GOx @ ZIF-8 by contact and then reacts by diffusing glucose from the microenvironment into GOx @ ZIF-8 as the glucose concentration around GOx @ ZIF-8 decreases.
FIG. 14b is a schematic representation of GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 catalyzed generation of H2O2Velocity profile of (c). It can be seen from the figure that 3-MPBA/GOx @ ZIF-8 produces H at the same glucose concentration2O2The speed of (2) is obviously faster than GOx @ ZIF-8. This is because 3-MPBA/GOx @ ZIF-8 is responsible for the coupling of 3-MPBA and glucose substrates by phenylboronic acidThe affinity of (3-MPBA/GOx @ ZIF-8) can be used for capturing a glucose substrate, so that the substrate affinity and the catalytic efficiency of the 3-MPBA/GOx @ ZIF-8 are improved.
Example 5: enzymological properties of immobilized enzyme 3-MPBA/GOx @ ZIF-8
(1) The optimum catalytic reaction pH value of the free GOx and the immobilized enzyme 3-MPBA/GOx @ ZIF-8 is as follows:
the pH is an important factor influencing the enzyme activity, and the conformation of the enzyme is easy to change greatly by the change of the pH, so that the enzyme activity is lost; while the dissolution state and pH changes of some substrates are also closely related. As shown in FIG. 15, the optimal reaction pH for both the free and immobilized enzymes was 6.0, indicating that the conformation of GOx in 3-MPBA/GOx @ ZIF-8 did not change significantly. 3-MPBA/GOx @ ZIF-8 showed higher relative activity at pH 5.5-8.0 compared to free GOx. The relative activity of free GOx at pH 7.0 was only 77.28 + -3.24%, whereas the relative activity of 3-MPBA/GOx @ ZIF-8 was 88.30 + -2.76%; when the pH value is lower than 5.5, ZIF-8 can dissolve and release the enzyme contained in the ZIF-8, and the property can be used for the aspects of drug slow release and the like.
(2) The optimum catalytic reaction temperature of the free GOx and the immobilized enzyme 3-MPBA/GOx @ ZIF-8 is as follows:
the temperature is another important factor influencing the catalytic reaction activity of the enzyme, and the invention researches the catalytic reaction activity of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8 under different temperature systems. As shown in FIG. 16, the optimal reaction temperature for both free GOx and 3-MPBA/GOx @ ZIF-8 is 35 deg.C, but the relative activity of 3-MPBA/GOx @ ZIF-8 is still 47.15 + -2.95% at 70 deg.C, while the relative activity of free GOx is only 23.31 + -2.78%, which may be due to the increased structural rigidity brought by ZIF-8, which is more favorable for the temperature adaptation of 3-MPBA/GOx @ ZIF-8.
(3) pH stability of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8:
in this example, the enzyme activity retention conditions of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8 after culturing for 30min in a pH 3.0-8.0 system were examined. As shown in FIG. 17, free GOx was more stable at pH less than 5 because 3-MPBA/GOx @ ZIF-8 would dissolve at pH less than 5; the 3-MPBA/GOx @ ZIF-8 has higher activity when the pH is 5.5-8.0, because different charges on the surface of the 3-MPBA/GOx @ ZIF-8 have certain buffering effect on a microenvironment. Therefore, after the ZIF-8 material is immobilized with the enzyme, a rigid shielding environment is provided by the ZIF-8 pore channel framework, so that the adverse effect caused by an acid-base environment is reduced, and the pH stability of the enzyme is improved.
(4) Thermal stability of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8:
the enzymatic reaction requires a long time, and the reaction needs to be carried out at a high temperature in order to increase the catalytic rate, which puts high demands on the thermal stability of the enzyme. Although the enzymatic hydrolysis rate at high temperature is fast, irreversible inactivation of the enzyme is easily caused, thereby losing the meaning of obtaining high yield.
In this example, free GOx and immobilized enzyme thereof were cultured at 20-70 ℃ for 30min, and then the relative enzyme activity was measured. As shown in fig. 18, the stability of free GOx decreased rapidly at temperatures above 40 ℃; the relative activity of 3-MPBA/GOx @ ZIF-8 is 84.69 +/-3.28 percent higher than that of free GOx by 53.60 +/-4.00 percent at the temperature of 50 ℃; when the temperature reached 70 ℃, the free GOx was almost inactivated and the relative activity of 3-MPBA/GOx @ ZIF-8 was still 32.27. + -. 3.02%. Therefore, the high-temperature environment can damage the structure of GOx, and irreversible inactivation is caused, and 3-MPBA/GOx @ ZIF-8 provides a rigid structure for GOx and improves thermal stability.
(5) Storage stability of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8:
the problems such as storage conditions and stability of the enzyme are problems that must be dealt with in industrial application of the enzyme. In this example, free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8 were stored in a refrigerator at 4 ℃ and their retained activities were measured within 30 days. As shown in FIG. 19, the relative activities of free GOx and 3-MPBA/GOx @ ZIF-8 were measured to be 78.2. + -. 3.09% and 65.73. + -. 3.25% after 30 days at 4 ℃ respectively. 3-MPBA/GOx @ ZIF-8 was able to be successfully stored at a range of temperatures and times without loss of activity, probably because the structure of ZIF-8 protects the enzyme more strongly than free GOx.
(6) The recycling stability of the immobilized enzyme 3-MPBA/GOx @ ZIF-8 is as follows:
as shown in FIG. 20, a series of factors which destroy the enzyme activity such as centrifugation in the experiment are eliminated, and under the condition that the activity of the first degrading enzyme is 100%, the relative activity of 71.47 +/-3.59% of the 3-MPBA/GOx @ ZIF-8 is still maintained after 9 cycles. The 3-MPBA/GOx @ ZIF-8 is simple to use, can be effectively recycled and reused, and meets the requirements of economy and green chemistry.
(7) Thermal inactivation kinetic parameters of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8:
table 1 shows the thermal deactivation kinetics parameters of free GOx and 3-MPBA/GOx @ ZIF-8k dHalf life period oft 1/2And change in enthalpyΔ HAnd (6) obtaining the result.
TABLE 1 thermal deactivation kinetics parameters Table for free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8
As can be seen from Table 1, the ratio of free GOx to 3-MPBA/GOx @ ZIF-8 increases with increasing temperaturek dThe value is gradually decreased and the number of the second phase is gradually decreased,t 1/2andΔHthe value gradually increases. Of 3-MPBA/GOx @ ZIF-8 at the same temperaturek dA value less than free GOx, andt 1/2andΔHthe values are reversed. The results show that the higher the temperature, the less energy is required for enzyme inactivation, while 3-MPBA/GOx @ ZIF-8 requires more energy at the same temperature than free GOx. This indicates that 3-MPBA/GOx @ ZIF-8 has a higher temperature resistance, which is consistent with the temperature stability test results. Inactivation energy of free GOx and 3-MPBA/GOx @ ZIF-8: (E d) Respectively 72.56 +/-2.37 kJ/mol and 84.02 +/-3.15 kJ/mol. 3-MPBA/GOx @ ZIF-8 for free GOxE dHigher, indicating that its deactivation requires more energy and is therefore more temperature stable.
(8) Kinetic constants of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8:
preparing substrate glucose with concentration of 0-0.2M, performing catalytic reaction in a short time, measuring enzyme activities of free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8, and determining the enzyme activities by using a Lineweaver-Burk methodK mThe value of the one or more of,k cat valueAndk cat/K mthe value is obtained.
TABLE 2 kinetic constants tables for free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8
As can be seen from Table 2, 3-MPBA/GOx @ ZIF-8 is comparable to free GOx and GOx @ ZIF-8K mThe value is greatly reduced, which shows that the 3-MPBA/GOx @ ZIF-8 has better affinity with the substrate. In addition, the catalytic constants of free GOx, GOx @ ZIF-8 and 3-MPBA/GOx @ ZIF-8 were also examined ((R))k cat) And catalytic efficiency (k cat/K m). Of GOx @ ZIF-8k catThe lower may be due to the encapsulation of ZIF-8. It can be seen that the compounds are related to free GOx and GOx @ ZIF-8k cat/K mCompared with the value, the catalytic efficiency of the 3-MPBA/GOx @ ZIF-8 is remarkably improved. This further demonstrates that the introduction of 3-MPBA can significantly improve the affinity and catalytic efficiency of 3-MPBA/GOx @ ZIF-8 for the substrate.
Example 6: application test for visually detecting glucose by using free GOx and immobilized enzyme 3-MPBA/GOx @ ZIF-8
(1) The selectivity of immobilized enzyme 3-MPBA/GOx @ ZIF-8 on glucose detection:
diabetes screening can be achieved by regular changes in blue color on cotton sheets, and selectivity for 3-MPBA/GOx @ ZIF-8 was identified to avoid interference from other substances including 10 mM galactose, trehalose, rhamnose, mannose, sorbose, ribose, arabinose, xylose, maltose and ascorbic acid. FIG. 21 (a) shows that only the glucose-containing cotton piece exhibited a distinct blue color at the same concentration, indicating that 3-MPBA/GOx @ ZIF-8 has a sensitive specific color response to glucose. Based on the characteristics, the 3-MPBA/GOx @ ZIF-8 can be well applied to the detection of blood sugar.
(2) Visual detection of immobilized enzyme 3-MPBA/GOx @ ZIF-8 on glucose with different concentrations:
the concentration of glucose in the serum of diabetic patients is about 9-40 mM, and the concentration of glucose in the serum is selected from 0-20 mM for visual detection. As shown in FIG. 21 (b), the color of the cotton piece was deepened with the increase of the glucose concentration, and the minimum concentration of glucose was 2.0 mM in visual inspection. The result shows that the 3-MPBA/GOx @ ZIF-8 visual detection of glucose has a large visual detection range, good selectivity and simplicity, and can be applied to the industrial and medical fields.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. A preparation method of metal organic framework material immobilized glucose oxidase is characterized by comprising the following steps:
weighing glucose oxidase GOx, uniformly mixing the glucose oxidase GOx with polyvinylpyrrolidone PVP (polyvinylpyrrolidone), adding 3-MPBA and 2-methylimidazole 2-MIM (metal-imide-phosphate) solution, uniformly mixing, adding zinc nitrate solution, fully mixing to obtain mixed solution, standing the mixed solution for culture reaction, centrifuging, washing and drying after the reaction is finished to obtain the immobilized glucose oxidase of the metal organic framework material, and marking the immobilized glucose oxidase as 3-MPBA/GOx @ ZIF-8.
2. The method for preparing metal organic framework material immobilized glucose oxidase of claim 1, wherein the final concentration of GOx in the mixed solution is 0.2-1.8 mg/mL.
3. The method for preparing metal organic framework material immobilized glucose oxidase of claim 2, wherein the final concentration of GOx in the mixed solution is 1 mg/mL.
4. The preparation method of the metal-organic framework material immobilized glucose oxidase of claim 1, wherein the concentration of the PVP in the mixed solution is 0.1-1.2 mg/mL; the final concentration of the 3-MPBA in the mixed solution is 0.1-1 mg/mL.
5. The method for preparing metal organic framework material immobilized glucose oxidase of claim 4, wherein the concentration of PVP in the mixed solution is 0.8 mg/mL; the final concentration of the 3-MPBA in the mixed solution is 0.6 mg/mL.
6. The method for preparing metal-organic framework material immobilized glucose oxidase of claim 1, wherein the final concentration of 2-MIM in the mixed solution is 64 mM; the final concentration of the zinc nitrate in the mixed solution was 16 mM.
7. The preparation method of the metal-organic framework material immobilized glucose oxidase according to claim 1, wherein the standing culture time is 10min-24 h.
8. The method for preparing metal organic framework material immobilized glucose oxidase of claim 7, wherein the time of the static culture is 30 min.
9. The metal organic framework material immobilized glucose oxidase prepared by the method of any one of claims 1 to 8, which is 3-MPBA/GOx @ ZIF-8, wherein the 3-MPBA/GOx @ ZIF-8 is a granular structure and has a particle size of 180 to 200 nm.
10. The use of the metal organic framework material immobilized glucose oxidase 3-MPBA/GOx @ ZIF-8 of claim 9 for the visual detection of glucose.
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