CN112980827B - Immobilized glucose oxidase of metal organic framework material and preparation method and application thereof - Google Patents
Immobilized glucose oxidase of metal organic framework material and preparation method and application thereof Download PDFInfo
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- CN112980827B CN112980827B CN202110156009.1A CN202110156009A CN112980827B CN 112980827 B CN112980827 B CN 112980827B CN 202110156009 A CN202110156009 A CN 202110156009A CN 112980827 B CN112980827 B CN 112980827B
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- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 261
- 239000000463 material Substances 0.000 title claims abstract description 34
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 33
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 33
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 33
- 239000012621 metal-organic framework Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 38
- 239000008103 glucose Substances 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 14
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- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 32
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- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 18
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Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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)
-
- 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
Abstract
The invention provides a metal organic framework material immobilized glucose oxidase and a preparation method and application thereof, belonging to the technical field of material preparation; in the invention, glucose oxidase is introduced into a pore canal 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 the 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, a preparation method and application thereof.
Background
The enzyme is a biological macromolecular catalyst, and has the characteristics of substrate specificity, selectivity, green chemistry and the like, so that the enzyme is widely applied to the fields of medicine, food, chemical industry, agriculture and the like. However, the natural enzymes have low thermostability and operational stability, narrow optimal pH ranges, low tolerance to most organic solvents, difficult recovery and lack of reusability under operational 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 to organic ligands. In recent years, MOFs have the advantages of high specific surface area and pore volume, easiness in pore diameter adjustment, easiness in modification of metal cations or clusters and organic ligands, mild synthesis conditions and the like, and are considered to be a very promising enzyme immobilized support matrix. In addition, MOFs nodes and connectors provide a large number of anchor points for enzyme combination in the modes of coordination bonds, covalent bonds, hydrogen bonds, van der Waals forces and the like, so that leaching and denaturation of the enzyme during heating, dehydration and solvent change can be prevented, and the reusability of the catalyst is improved. However, enzyme @ MOF still has a disadvantage in substrate affinity and catalytic efficiency, and the time to induce 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 the 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 of a granular structure, and the grain size is 180-200 nm.
The invention also provides a preparation method of the immobilized glucose oxidase of the metal organic framework material, which comprises the following specific preparation steps:
weighing glucose oxidase GOx, uniformly mixing with polyvinylpyrrolidone PVP, adding 3-MPBA and 2-methylimidazole 2-MIM solution, uniformly mixing, adding zinc nitrate solution, fully mixing to obtain mixed solution, standing and culturing the mixed solution for reaction, centrifuging at 4 ℃, washing, and drying to obtain the immobilized glucose oxidase of the metal organic framework material, which is marked as 3-MPBA/GOx@ZIF-8.
Further, the final concentration of GOx in the mixed solution is 0.2-1.8mg/mL.
Further, the final concentration of GOx in the mixed solution was 1mg/mL.
Further, the PVP concentration in the mixed solution is 0.1-1.2mg/mL, preferably 0.8mg/mL.
Further, the final concentration of the 3-MPBA in the mixed solution is 0.1-1mg/mL, preferably 0.6mg/mL.
Further, the final concentration of the 2-MIM in the mixed solution is 64mM; the final concentration of zinc nitrate in the mixed solution was 16mM.
Further, the stationary culture time is 10min-24h, preferably 30min.
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 immobilization of enzymes is usually carried out by firstly synthesizing an immobilization material and then immobilizing the enzymes, so that the loss of enzyme activity is large; the invention innovatively selects a rapid one-step synthesis method of 3-mercaptophenylboronic acid, and glucose oxidase is introduced simultaneously in the MOFS forming process, thereby realizing immobilization. The immobilization of the enzyme and the preparation of the frame material are carried out simultaneously, so that the defects that the pore canal of the metal organic frame material is too small, the enzyme cannot enter the material, or the pore canal of the material is too large to play a role in immobilization are avoided, the temperature stability, the acid and alkali resistance and the storage stability of the enzyme are improved, the recycling rate is obviously improved, and the glucose degradation rate of glucose oxidase is also improved.
(2) According to the invention, after glucose oxidase solution and PVP are uniformly mixed, the mixture is sequentially and uniformly mixed with 3-mercaptophenylboronic acid, 2-methylimidazole and zinc nitrate solution, and immobilized enzyme 3-MPBA/GOx@ZIF-8 is successfully prepared under the conditions of normal temperature, water phase and mild conditions, 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 greatest extent by adjusting the PVP, the 3-mercaptophenylboronic acid and the enzyme, and the immobilization condition is optimized, when the concentration of the selected enzyme is 1mg/mL, the concentration of the 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 activity retention of the enzyme reach the optimal values of 97.32% and 86.15%, respectively.
(4) The invention examines the enzymology property of the immobilized enzyme 3-MPBA/GOx@ZIF-8, and discovers 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 glucose oxidase, so that the influence of 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 material are improved.
Drawings
FIG. 1 is a schematic diagram (a) of the synthesis of 3-MPBA/GOx@ZIF-8, a schematic diagram (b) of a different packaging system and a comparison diagram (c) of packaging efficiency.
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 N of 3-MPBA@ZIF-8 (a) and 3-MPBA/GOx@ZIF-8 (b) 2 Adsorption-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 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 enzymes.
FIG. 8 is a graph showing the effect of the addition amount of glucose oxidase on the encapsulation efficiency of the enzyme.
FIG. 9 is an SEM image of 3-MPBA/GOx@ZIF-8 at various 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 enzyme encapsulation efficiency 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 a SEM image and a photograph of suspensions of GOx@ZIF-8 and 3-MPBA/GOx@ZIF-8 at different incubation times.
FIG. 14 is a graph (a) showing the reaction of GOx@ZIF-8 and 3-MPBA/GOx@ZIF-8 with glucose and a graph (b) showing the rate of catalytic production of H2O 2.
FIG. 15 is a graph showing the effect of pH on the catalytic activity of free GOx and 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 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 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 recycling stability verification of the immobilized enzyme 3-MPBA/GOx@ZIF-8.
FIG. 21 is a graph (a) showing the selective detection of glucose by immobilized enzyme 3-MPBA/GOx@ZIF-8 and a graph (b) showing the visual detection of glucose at different concentrations.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
The immobilized enzyme obtained by the present invention was verified for its properties by the following means:
(1) Measurement of encapsulation efficiency of enzyme:
the assay was performed using a modified version of the Bradford protein assay kit. Specifically, 10. Mu.L of enzyme solution and supernatant after centrifugation are added into 100. Mu.L of working solution, and incubated for 10min at room temperature. Absorbance was measured in parallel at 595, 595 nm, and the protein concentration and further the encapsulation efficiency of glucose oxidase were determined from the linear regression equation of the protein standard curve.
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 measurement is carried out by adopting a colorimetric method, glucose is taken as a substrate, the product obtained by catalytic hydrolysis is hydrogen peroxide (H2O 2), indigo carmine is faded, and the characteristic absorption peak is arranged at 615 and nm, so that the colorimetric measurement can be directly carried out.
Preparing indigo carmine solution: a 0.0233 g indigo carmine sample was weighed, dissolved in a suitable amount of deionized water and then fixed to a volume of 50 mL to give a 1 mM indigo carmine solution.
0.2M acetic acid-sodium acetate (NaAc-HAc) solution: 2.46 g g sodium acetate solid is weighed into a beaker, fully dissolved with a proper amount of deionized water, added with 594 mu L of glacial acetic acid, and fixed to a volume of 200 mL.
0.2M glucose solution preparation: the sodium acetate solid, 0.9, g, was weighed into a beaker and dissolved with a suitable amount of deionized water to a volume of 25: 25 mL.
Measurement of enzyme Activity: 1mL glucose (0.2M) and 1mL enzyme were preheated in a 37℃water bath for 5 min. The two solutions were then mixed and reacted at 37℃for 10 min. Next, 3 mL of NaAc-HAc (0.2M, pH 5.2) buffer, 1.3 mL indigo carmine (1 mM) and the reaction solution were added to a 25 mL cuvette. Deionized water was added to the tube to a constant volume to the scale mark, reacted in boiling water for 13 min, and finally cooled with running water for 5min to terminate the reaction. The absorbance of the solution was measured at 615 nm using deionized water as a reference.
(3) Calculating enzyme activity:
definition of glucose oxidase Activity: catalyzing glucose to produce H per unit time (min) at 37 DEG C 2 O 2 Is used as a catalyst.
(4) Application of immobilized enzyme 3-MPBA/GOx@ZIF-8 in degrading glucose:
the immobilized enzyme 3-MPBA/GOx@ZIF-8 and cellulase are used for visually detecting glucose under the action of 3,3', 5' -tetramethyl benzidine (TMB): the visual detection of glucose was performed with a specially made round cotton piece. mu.L of 3-MPBA/GOx@ZIF-8 was added dropwise to the cotton sheets and dried completely at room temperature. Then, 15 μl of glucose, 10 mM TMB and HRP were mixed and added to the cotton sheets. 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 molecular weight of 8000, uniformly mixing, then adding 2-MIM with concentration of 160 mM and zinc nitrate with concentration of 2 mL of 40 mM, uniformly mixing 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, centrifuging the standing mixed solution 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 molecular weight of 8000, uniformly mixing, then adding 2-MIM with concentration of 160 mM and zinc nitrate with concentration of 2 mL of 40 mM, uniformly mixing so that 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, centrifuging at 8000 rpm for 5 minutes, washing, and vacuum drying to obtain 3-MPBA/GOx@ZIF-8.
As shown in FIG. 1a, due to 3-MPBA and Zn 2+ The strong complexation of 3-MPBA may accelerate 3-MPBA and Zn around GOx 2+ Nucleation cluster formation and triggering the encapsulation of these proteins by MOFs. The encapsulation effect of 3-MPBA and PVP on GOx is shown in FIGS. 1b and 1c, the amount of precipitate after PVP addition is significantly increased compared to that without PVP addition, and GOx encapsulation efficiencyThe improvement of 15.29 percent is obviously superior to the encapsulation experiment without PVP. The encapsulation efficiency of GOx and the amount of sediment were further increased by the encapsulation experiments with 3-MPBA, which showed a 12.23% increase compared to the encapsulation experiments without 3-MPBA, indicating that the increase of thiol groups could promote the formation of 3-MPB/GOx@ZIF-8.
The molecular structures of three samples of free GOx, metal organic framework material ZIF-8 monomer and immobilized enzyme 3-MPBA/GOx@ZIF-8 are detected by adopting a Fourier transform infrared spectrometer, and the measurement results are shown in figure 2. In the figure, a is the infrared spectrogram of free GOx (a), and it can be seen that the characteristic peak of GOx appears in 1400-1600 cm -1 (-CONH) and 3200-3530 cm -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 in 1580 cm -1 (c=n) and 1350-1500 cm -1 At (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). In addition, in the process of generating 3-MPBA/GOx@ZIF-8, the carboxyl groups and Zn on the enzyme 2+ Is coordinated by 1650 cm -1 Is 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, respectively 2 Adsorption-desorption isotherms and pore distribution curves. As can be seen from the graph, the curve intersection point is between 0.6 and 0.8, which indicates that the 3-MPBA@ZIF-8 and the 3-MPBA/GOx@ZIF-8 are mesoporous materials. In combination with the pore distribution curve, it can be concluded that the presence of pores may be due to stacking between materials. Meanwhile, the specific surface areas of the 3-MPBA@ZIF-8 and the 3-MPBA/GOx@ZIF-8 are 70.4218 m respectively 2 /g and 45.0515 m 2 And/g. The pore volumes of 3-MPBA@ZIF-8 and 3-MPBA/GOx@ZIF-8 were 0.241558 cm and 0.195621 cm d/g, respectively. The decrease in specific surface area and pore volume indicated that the added GOx blocked the remaining pores in 3-MPBA@ZIF-8, indicating that GOx was successfully embedded in a short period of 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 molecular weight of 8000, uniformly mixing, then adding 2-MIM with concentration of 160 mM and zinc nitrate with concentration of 2 mL of 40 mM, uniformly mixing 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, 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, centrifuging the standing mixed solution at 8000 rpm for 5 minutes, washing, and vacuum drying 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, both 3-MPBA@ZIF-8 and 3-MPBA/GOx@ZIF-8 are in the form of particles, the size is about 200nm, and MOFs show a particle size reduction after GOx addition, which also indicates that the introduction of glucose oxidase does not destroy the morphology of ZIF-8.
FIG. 5 is an LPSD of 3-MPBA@ZIF-8 and immobilized enzyme 3-MPBA/GOx@ZIF-8, wherein 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 were 234.6 nm and 183.4 nm, respectively, and the particle size of GOx@MOF was reduced after GOx addition, mainly due to the increase in 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 molecular weight of 8000, uniformly mixing, then adding 2-MIM with concentration of 160 mM and zinc nitrate with concentration of 2 mL of 40 mM, uniformly mixing so 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, the final concentration of 3-mercaptophenylboronic acid in the mixed solution is 1mg/mL, standing the mixed solution at room temperature for 24h, centrifuging the standing mixed solution at 8000 rpm for 5min, washing, and vacuum drying to obtain 3-MPBA/GOx@ZIF-8.
FIGS. 6a, 6b and 6c are thermogravimetric analyses of ZIF-8, 3-MPBA@ZIF-8 and immobilized enzyme 3-MPBA@ZIF-8, respectively. As shown in FIG. 6, ZIF-8 lost about 6% of its mass due to evaporation of water molecules in the sample from 25℃to 211℃and the second stage started around 357℃with a slow rate of weight loss due to self-decomposition of ZIF-8 with a weight loss rate of 25.79%. From the weight loss curves of 3-MPBA@ZIF-8 and 3-MPBA/GOx@ZIF-8, it can be seen that the first stage of weight loss is also below 102 ℃ due to 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 of 3-MPBA/GOx@ZIF-8 (305-800 ℃) presents a trend of first-to-last, total weight loss of about 84.50%, lost GOx weight of about 33.14% wt%, starting from 305-357 ℃, mass loss due to decomposition of 3-MPBA and GOx; at temperatures above 357 ℃, the slow drop in weight is mainly caused by continued collapse and decomposition of MOFs material portions. Analysis of three weight loss patterns of ZIF-8, 3-MPBA@ZIF-8 and immobilized enzyme 3-MPBA@ZIF-8 revealed that glucose oxidase was successfully immobilized in ZIF-8.
Example 4: immobilization condition optimization of 3-MPBA/GOx@ZIF-8
In this example, the effect of immobilization of 3-MPBA/GOx@ZIF-8 on its encapsulation efficiency and recovery of enzyme activity under different conditions was examined, respectively.
(1) Effect of PVP addition on immobilization:
PVP is a biocompatible macromolecule similar to protein, can be used as an encapsulating agent to form PVP/protein complex, and is beneficial to guiding amino acid to form 3-MPBA/GOx@ZIF-8 towards protein through hydrogen bond interaction, and the encapsulation effect of PVP on GOx is shown in figure 7. As can be seen from the figure, the amount of precipitation after PVP addition was significantly increased compared to that without PVP addition, and the encapsulation efficiency of GOx was improved by 15.29% significantly better than that of the encapsulation experiment without PVP addition. With increasing PVP concentration, the encapsulation efficiency of GOx is shown to be improved and then maintained unchanged, and the recovery rate of enzyme activity is improved and then reduced, which is probably due to the fact that 3-MPBA/GOx@ZIF-8 is seriously aggregated due to the fact that the PVP concentration is too high, and the reaction of 3-MPBA/GOx@ZIF-8 with a substrate is inhibited. At PVP concentration of 0.8mg/mL, the encapsulation efficiency and the recovery rate of enzyme activity were optimal, at which time the encapsulation efficiency was 96.18.+ -. 2.03% and the recovery rate of enzyme activity was 84.58.+ -. 2.20%.
(2) Effect of enzyme addition on immobilization:
in other steps, the influence of the amount of GOx on the formation of 3-MPBA/GOx@ZIF-8 was examined without changing the addition amount of GOx. As can be seen from fig. 8, the encapsulation efficiency of GOx and the recovery rate of enzyme activity were increased and then decreased with increasing concentration of GOx. When the GOx concentration is 1.0 mg/mL, the precipitation amount is maximum, and at this time, the GOx encapsulation rate 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 various glucose oxidase concentrations. As seen from the SEM image, as the concentration of GOx increased from 0.2mg/mL to 1.8mg/mL, the crystal size of 3-MPBA/GOx@ZIF-8 also decreased, indicating that the size and morphology of 3-MPBA/GOx@ZIF-8 can be controlled by adjusting the amount of GOx.
(3) Effect of the amount of 3-mercaptophenylboronic acid added and time on immobilization:
the effect of the addition amount of 3-mercaptophenylboronic acid on the formation of 3-MPBA/GOx@ZIF-8 was examined in other steps without changing the addition amount of 3-mercaptophenylboronic acid, and FIG. 10 is a graph showing the effect of the addition amount of 3-mercaptophenylboronic acid on the effect of the enzyme on the encapsulation efficiency.
As shown in FIG. 10, the increase in thiol groups was 12.23% compared to the encapsulation experiments without 3-MPBA, indicating that the increase in thiol groups can promote the formation of 3-MPB/GOx@ZIF-8. With increasing 3-MPBA usage, GOx encapsulation efficiency tends to increase and decrease. When the concentration of 3-MPBA was 0.6mg/mL, the encapsulation efficiency and the recovery rate of the enzyme activity reached the optimum levels, and at this time, the encapsulation efficiency was 96.98.+ -. 2.08% and the recovery rate of the enzyme activity was 85.71.+ -. 3.02%.
FIG. 11 is a graph showing the effect of immobilization time on the effect of enzyme encapsulation efficiency in the presence or absence of 3-mercaptophenylboronic acid, and FIG. 12 is a graph showing the effect of immobilization time on the effect of enzyme activity retention 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 completely encapsulated within 10-30min, the encapsulation efficiency reaches the highest value 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, with GOx@ZIF-8 still exhibiting 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 morphology 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 with the time. GOx@ZIF-8 without 3-MPBA required at least 12 hours to complete encapsulation, with an encapsulation efficiency of 93.83+ -2.30%. The reason for this may be that the presence of thiol groups interacts with metal ions changing the shape and size of the nanoparticle.
FIG. 13 is a SEM image and a photograph of suspensions of GOx@ZIF-8 and 3-MPBA/GOx@ZIF-8 at different incubation times. As can be seen from the figure, the GOx@ZIF-8 containing no 3-MPBA is flocculent, and the 3-MPBA/GOx@ZIF-8 is quite uniformly dispersed.
(4) Addition of 3-mercaptophenylboronic acid catalyzes the production of H from glucose 2 O 2 Effects of rate:
FIG. 14a is a schematic representation of the reactions of GOx@ZIF-8 and 3-MPBA/GOx@ZIF-8 with glucose, showing that glucose can rapidly aggregate near 3-MPBA/GOx@ZIF-8, resulting in a higher concentration of glucose near 3-MPBA/GOx@ZIF-8 than in the bulk solution, with a significant decrease in glucose enrichmentK m The apparent enzyme activity is increased. 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 to GOx@ZIF-8 as the glucose concentration around GOx@ZIF-8 decreases.
FIG. 14b shows the catalytic production of H by GOx@ZIF-8 and 3-MPBA/GOx@ZIF-8 2 O 2 Is a velocity profile of (a). As can be seen from the figure, at the same glucose concentration, 3-MPBA/GOx@ZIF-8 produced H 2 O 2 Is significantly faster than GOx@ZIF-8. This is because 3-MPBA/GOx@ZIF-8 can capture glucose substrates by virtue of the affinity of phenylboronic acid for 3-MPBA and glucose substrates, thereby improving substrate affinity and catalytic efficiency of 3-MPBA/GOx@ZIF-8.
Example 5: enzymatic Properties of immobilized enzyme 3-MPBA/GOx@ZIF-8
(1) Optimal catalytic reaction pH of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8:
the pH is an important factor influencing the enzyme activity, and the conformation of the enzyme is easy to be changed greatly by the change of the pH, so that the loss of the enzyme activity is caused; while the dissolution state and pH change of some substrates are also closely related. As shown in FIG. 15, the optimal reaction pH for both free and immobilized enzyme was 6.0, indicating that the conformation of GOx in 3-MPBA/GOx@ZIF-8 was not significantly changed. Compared with free GOx, 3-MPBA/GOx@ZIF-8 shows higher relative activity at pH values of 5.5-8.0. The relative activity of free GOx at pH 7.0 was only 77.28.+ -. 3.24%, while 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 characteristic can be used for medicine slow release and the like.
(2) Optimal catalytic reaction temperature of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8:
the temperature is another important factor affecting the catalytic reaction activity of the enzyme, and the invention explores the catalytic reaction activity of the free GOx and the immobilized enzyme 3-MPBA/GOx@ZIF-8 under different temperature systems. As shown in FIG. 16, the optimal reaction temperature of free GOx and 3-MPBA/GOx@ZIF-8 is 35 ℃, but the relative activity of 3-MPBA/GOx@ZIF-8 is still 47.15+ -2.95% at 70 ℃, and the relative activity of free GOx is only 23.31+ -2.78%, which is probably due to the structural rigidity enhancement brought by ZIF-8, and is more beneficial to the adaptation of 3-MPBA/GOx@ZIF-8 to temperature.
(3) Free GOx and pH stability of immobilized enzyme 3-MPBA/GOx@ZIF-8:
the present example examined the retention of the enzyme activity after the free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8 were cultured for 30min at pH 3.0-8.0. As shown in FIG. 17, free GOx is more stable at pH less than 5, because 3-MPBA/GOx@ZIF-8 will dissolve at pH less than 5; the 3-MPBA/GOx@ZIF-8 has higher activity at the pH of 5.5-8.0, because the different charges on the surface of the 3-MPBA/GOx@ZIF-8 have a certain buffer effect on the microenvironment. Therefore, after the ZIF-8 material is used for immobilizing the enzyme, the ZIF-8 pore canal framework provides a rigid shielding environment, so that adverse effects caused by an acid-base environment are 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 time required for the enzymatic reaction is long, and the reaction needs to be carried out at a higher temperature in order to increase the catalytic rate, which places high demands on the thermal stability of the enzyme. Although the enzymatic hydrolysis rate is fast at high temperatures, irreversible inactivation of the enzyme is easily caused, and the meaning of obtaining high yields is lost.
In this example, the relative enzyme activity of the free GOx and immobilized enzyme was measured after culturing at 20-70℃for 30min. As shown in fig. 18, when the temperature exceeds 40 ℃, the stability of free GOx rapidly decreases; at 50 ℃, the relative activity of 3-MPBA/GOx@ZIF-8 is 84.69+/-3.28% higher than that of free GOx by 53.60+/-4.00%; when the temperature reached 70℃the free GOx was almost deactivated and the relative activity of 3-MPBA/GOx@ZIF-8 was still 32.27.+ -. 3.02%. It can be seen that the high temperature environment damages the structure of GOx, resulting in irreversible deactivation, while 3-MPBA/GOx@ZIF-8 provides a rigid structure for GOx, improving thermal stability.
(5) Storage stability of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8:
the problems of storage conditions and stability of enzymes are the problems that must be dealt with in the industrial application of enzymes. In this example, free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8 were stored in a refrigerator at 4℃and their retained activity was measured over 30 days. As shown in FIG. 19, the relative activities of free GOx and 3-MPBA/GOx@ZIF-8 were measured after 30 days at 4deg.C and were 78.2.+ -. 3.09% and 65.73.+ -. 3.25%, respectively. 3-MPBA/GOx@ZIF-8 can be successfully stored at a certain temperature and time without losing activity, probably because the structure of ZIF-8 has a stronger protection effect on enzymes than free GOx.
(6) Reuse stability of immobilized enzyme 3-MPBA/GOx@ZIF-8:
as shown in FIG. 20, excluding a series of factors that disrupt the enzyme activity, such as centrifugation, in the experiment, the relative activity of 3-MPBA/GOx@ZIF-8 remained 71.47.+ -. 3.59% after 9 cycles with the first degradation enzyme activity of 100%. The 3-MPBA/GOx@ZIF-8 is simple to use, can be effectively recovered and can be reused, and the economic and green chemical requirements are met.
(7) Thermal inactivation kinetic parameters of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8:
table 1 shows the free GOx andthermal inactivation kinetics parameters of 3-MPBA/GOx@ZIF-8k d Half life periodt 1/2 And enthalpy changeΔ HAs a result.
TABLE 1 thermal inactivation kinetics parameters of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8
As can be seen from Table 1, with increasing temperature, free GOx and 3-MPBA/GOx@ZIF-8k d The value is gradually decreased and the temperature of the liquid,t 1/2 andΔHthe value gradually increases. At the same temperature, 3-MPBA/GOx@ZIF-8k d A value less than free GOxt 1/2 AndΔHthe values are the opposite. 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 shows that 3-MPBA/GOx@ZIF-8 has higher temperature resistance, which is consistent with the temperature stability test results. Inactivation energy of free GOx and 3-MPBA/GOx@ZIF-8E d ) 72.56.+ -. 2.37 kJ/mol and 84.02.+ -. 3.15 kJ/mol, respectively. 3-MPBA/GOx@ZIF-8 vs. free GOxE d Higher, indicating that it requires more energy to deactivate and is therefore more temperature stable.
(8) Kinetic constants of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8:
preparing glucose with a 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 activity of the free GOx and immobilized enzyme by using a Lineweaver-Burk methodK m The value of the sum of the values,k cat value Andk cat /K m Values.
TABLE 2 kinetic constants of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8
As can be seen from Table 2, 3-MPBA/GOx@ZIF-8 compared to free GOx and GOx@ZIF-8K m The value is greatly reduced, which indicates that 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 examinedk cat ) And catalytic efficiency%k cat /K m ). GOx@ZIF-8k cat The lower probability is due to the encapsulation of ZIF-8. It can be seen that with free GOx and GOx@ZIF-8k cat /K m Compared with the value, the catalytic efficiency of the 3-MPBA/GOx@ZIF-8 is obviously 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: visual detection of free GOx and immobilized enzyme 3-MPBA/GOx@ZIF-8
(1) Selectivity of immobilized enzyme 3-MPBA/GOx@ZIF-8 for glucose detection:
diabetes screening can be achieved by regular changes in blue on cotton flakes, and to avoid interference with other substances, the selectivity of 3-MPBA/GOx@ZIF-8 was identified, with interfering substances including galactose, trehalose, rhamnose, mannose, sorbose, ribose, arabinose, xylose, maltose and ascorbic acid at 10 mM. FIG. 21 (a) shows that at the same concentration, only the cotton piece containing glucose exhibited a distinct blue color, indicating that 3-MPBA/GOx@ZIF-8 had a specific color reaction sensitive to glucose. Based on the characteristics, the 3-MPBA/GOx@ZIF-8 can be well applied to blood glucose detection.
(2) Visual detection of glucose at different concentrations by immobilized enzyme 3-MPBA/GOx@ZIF-8:
the glucose concentration in the serum of diabetics was approximately 9-40 mM, with 0-20 mM being selected for visual detection. As shown in fig. 21 (b), the cotton piece showed a darkening color with increasing glucose concentration, and the lowest concentration of visually detected glucose was 2.0. 2.0 mM. The result shows that the visual detection of the glucose by the 3-MPBA/GOx@ZIF-8 has a large visual detection range, has good selectivity and simplicity, and can be applied to the fields of industry and medicine.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (4)
1. A method for preparing a glucose oxidase immobilized on a metal organic framework material, which is characterized by comprising the following steps:
weighing glucose oxidase GOx, uniformly mixing with polyvinylpyrrolidone PVP, adding 3-MPBA and 2-methylimidazole 2-MIM solution, uniformly mixing, adding zinc nitrate solution, fully mixing to obtain mixed solution, standing and culturing the mixed solution for reaction, centrifuging after the reaction is finished, washing, and drying to obtain immobilized glucose oxidase of a metal organic frame material, which is marked as 3-MPBA/GOx@ZIF-8;
the final concentration of GOx in the mixed solution is 1mg/mL; the concentration of PVP in the mixed solution is 0.8mg/mL; the final concentration of the 3-MPBA in the mixed solution is 0.6mg/mL;
the final concentration of the 2-MIM in the mixed solution is 64mM; the final concentration of the zinc nitrate in the mixed solution is 16mM;
the stationary culture time is 10min-24h.
2. The method for producing a glucose oxidase immobilized on a metal-organic framework material according to claim 1, wherein the stationary culture is performed for 30 minutes.
3. The immobilized glucose oxidase of a metal organic framework material prepared by the method of any one of claims 1-2, which is marked as 3-MPBA/GOx@ZIF-8, wherein the 3-MPBA/GOx@ZIF-8 is of a granular structure and has a particle size of 180-200 nm.
4. Use of the metal organic framework material immobilized glucose oxidase 3-MPBA/gox@zif-8 in the preparation of a visual detection glucose kit.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105136888A (en) * | 2015-08-07 | 2015-12-09 | 南京理工大学 | Graphene derivative based glucose oxidase electrode and preparation method thereof |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN109897846A (en) * | 2019-04-03 | 2019-06-18 | 辽宁大学 | A kind of immobilized glucose oxidase and its preparation method and application |
| CN111118111A (en) * | 2019-12-11 | 2020-05-08 | 山东农业大学 | Rapid detection method of glucose |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105136888A (en) * | 2015-08-07 | 2015-12-09 | 南京理工大学 | Graphene derivative based glucose oxidase electrode and preparation method thereof |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN109897846A (en) * | 2019-04-03 | 2019-06-18 | 辽宁大学 | A kind of immobilized glucose oxidase and its preparation method and application |
| CN111118111A (en) * | 2019-12-11 | 2020-05-08 | 山东农业大学 | Rapid detection method of glucose |
Non-Patent Citations (1)
| Title |
|---|
| A Convenient and Versatile Amino Acid-Boosted Biomimetic Strategy for Nondestructive Encapsulation of Biomacromolecules within Metal−Organic Framework;Chen Guosheng等;Angewandte Chemie International Edition;第58卷(第5期);第2页右栏第2段，第3页左栏第1段 * |
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