CN115595318A - Method for ZIF-8 in-situ immobilized enzyme with high enzyme activity and application thereof - Google Patents
Method for ZIF-8 in-situ immobilized enzyme with high enzyme activity and application thereof Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 73
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 73
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 28
- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 title claims abstract description 24
- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 18
- 230000000694 effects Effects 0.000 title abstract description 37
- 229940088598 enzyme Drugs 0.000 claims abstract description 72
- 102000012440 Acetylcholinesterase Human genes 0.000 claims abstract description 62
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 62
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 62
- 108010000659 Choline oxidase Proteins 0.000 claims abstract description 60
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 22
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 claims abstract description 14
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000004246 zinc acetate Substances 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 37
- 238000002835 absorbance Methods 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 30
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 claims description 18
- 229960004373 acetylcholine Drugs 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 239000002244 precipitate Substances 0.000 claims description 15
- 239000007974 sodium acetate buffer Substances 0.000 claims description 13
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 239000000575 pesticide Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 239000011701 zinc Substances 0.000 abstract description 34
- 230000003197 catalytic effect Effects 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 6
- 229910052751 metal Inorganic materials 0.000 abstract description 4
- 239000002184 metal Substances 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 238000006555 catalytic reaction Methods 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000006911 nucleation Effects 0.000 abstract description 2
- 238000010899 nucleation Methods 0.000 abstract description 2
- 238000010668 complexation reaction Methods 0.000 abstract 1
- 239000003987 organophosphate pesticide Substances 0.000 abstract 1
- 239000002131 composite material Substances 0.000 description 14
- 239000012498 ultrapure water Substances 0.000 description 12
- 239000012621 metal-organic framework Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 238000004064 recycling Methods 0.000 description 8
- 230000000717 retained effect Effects 0.000 description 8
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 239000011148 porous material Substances 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- QNRATNLHPGXHMA-XZHTYLCXSA-N (r)-(6-ethoxyquinolin-4-yl)-[(2s,4s,5r)-5-ethyl-1-azabicyclo[2.2.2]octan-2-yl]methanol;hydrochloride Chemical compound Cl.C([C@H]([C@H](C1)CC)C2)CN1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OCC)C=C21 QNRATNLHPGXHMA-XZHTYLCXSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910021536 Zeolite Inorganic materials 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000010457 zeolite Substances 0.000 description 2
- RVEJOWGVUQQIIZ-UHFFFAOYSA-N 1-hexyl-3-methylimidazolium Chemical compound CCCCCCN1C=C[N+](C)=C1 RVEJOWGVUQQIIZ-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000018791 negative regulation of catalytic activity Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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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
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Abstract
Hair brushBelongs to the technical field of environmental monitoring, and relates to a method for preparing ZIF-8 in-situ immobilized enzyme with high enzyme activity and application thereof. According to the invention, zinc acetate with weak metal activity and 2-methylimidazole are mixed in a water phase, and acetylcholinesterase, choline oxidase and horseradish peroxidase are added to complete enzyme immobilization. The method reduces the higher pH and free Zn of the 2-methylimidazole 2+ The enzyme activity is inhibited by the complexation, so that the immobilized enzyme retains 91% of the catalytic activity of the free enzyme, and the nucleation of the ZIF-8 is slow due to the weak metal activity of the zinc acetate, so that the enzyme can be embedded. The preparation method is simple and quick, has low cost, and realizes quick and sensitive colorimetric biosensing detection of the organophosphorus pesticide by utilizing the cascade catalytic reaction of three enzymes.
Description
Technical Field
The invention belongs to the technical field of environmental monitoring, and relates to an immobilization method for simultaneously embedding acetylcholinesterase (AChE), choline oxidase (CHO) and horseradish peroxidase (HRP) in ZIF-8 in situ and retaining higher enzyme activity, and application of the immobilization method in colorimetric sensing detection of Organophosphorus Pesticides (OPs).
Background
Organophosphorus Pesticides (OPs) are widely used in agricultural production due to their remarkable ability to control and destroy pests, however, excessive use and abuse of OPs cause serious residue problems, pose serious threats to water environment and human health, and have become one of major environmental problems of global concern. Therefore, the development of sensitive, rapid, efficient, and real-time detection methods for controlling food safety, protecting ecosystem, and preventing diseases is of great significance for monitoring OPs in the environment.
Conventional detection methods for OPs, including high performance liquid chromatography, gas chromatography and mass spectrometry, have the defects of expensive equipment, long time consumption, complicated sample preparation and purification steps and the like, and are not suitable for application in field and real-time rapid detection. In recent years, the colorimetric biosensing technology has the advantages of convenience in carrying, simplicity in instruments and equipment, low detection cost and the like, and more importantly, the colorimetric biosensing technology provides possibility for realizing automatic real-time online monitoring. Among the many established colorimetric biosensing methods, natural enzymes are ideal biorecognition elements and have higher efficiency, specificity and sensitivity than artificial catalysts, but the fragile characteristics of natural enzymes, such as extreme sensitivity to temperature and pH fluctuations, poor tolerance to most organic solvents and small molecule inhibitors, low recoverability, etc., limit the application of colorimetric sensing platforms with natural enzymes as recognition elements in OPs detection (Talanta, 2022,240, 123145).
Immobilization of the enzyme is key to improving the fragile nature of the enzymeOne of the strategies is that the immobilized enzyme technology can more effectively control the reaction process, improve the storage capacity of the enzyme and the stability under extreme operating conditions. In addition, the multi-stage structure of the immobilized enzyme makes the immobilized enzyme easier to separate, realizes recycling and reduces the production cost. Metal-organic frameworks (MOFs) are a class of porous materials, and compared with conventional inorganic porous materials, MOFs have the advantages of customizable pore canals, high specific surface area, unique nano/micro environment, good biocompatibility and the like, so that the MOFs become an excellent platform for immobilizing enzymes, are prevented from being influenced by severe environment, and are improved in recycling performance. In-situ immobilization strategies enzymes are mixed with precursors of the MOFs, and the enzymes are embedded inside the MOFs while the MOFs are formed. The strategy can embed the enzyme with the size which is much larger than the pore size of the MOFs in the MOFs, and improve the stability of the enzyme under adverse conditions. Zeolite imidazole framework-8 (Zeolite imidazole Frameworks-8, ZIF-8) is made of Zn 2+ The node is connected with 2-methylimidazole (HmIM) through Zn-N bond, and is widely applied to in-situ immobilization of enzyme due to simple synthetic method and mild reaction conditions. In previous studies, the enzyme was first mixed with HmIM followed by Zn addition 2+ Or enzymes with Zn 2+ The enzymes were first mixed and then immobilized in situ by the addition of HmIM, however, these methods retain only low catalytic activity for the enzymes in ZIF-8 after immobilization.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a simple and rapid AChE/CHO/HRP @ ZIF-8 preparation method which reserves higher enzyme activity, solves the problem of enzyme activity reduction when ZIF-8 is used for in-situ enzyme immobilization, is applied to colorimetric sensing detection of OPs, realizes sensitive and rapid detection of OPs, and overcomes the defects of poor stability and low recycling property of natural enzyme. In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a ZIF-8 in-situ immobilized enzyme preparation method, which comprises the steps of mixing a zinc acetate aqueous solution with a 2-methylimidazole aqueous solution, adding acetylcholinesterase, choline oxidase and horseradish peroxidase, and carrying out mixed reaction to obtain the product.
In the above technical solution, further, the concentration of the zinc acetate aqueous solution is 32mM, the concentration of the 2-methylimidazole aqueous solution is 640-1920mM, the mixing ratio of the zinc acetate aqueous solution to the 2-methylimidazole aqueous solution is 1, the mixing ratio of the acetylcholinesterase, the choline oxidase and the horseradish peroxidase is 1.
In the above technical solution, further, the method includes the following steps:
(1) Mixing a zinc acetate aqueous solution and a 2-methylimidazole aqueous solution, and standing for 4-16 minutes to obtain a mixed solution;
(2) Adding acetylcholinesterase, choline oxidase and horseradish peroxidase into the mixed solution obtained in the step (1), and stirring at room temperature for 0.5-2.5 hours to react to obtain a mixture;
(3) And after the reaction is finished, centrifugally washing the mixture, collecting the precipitate, and drying to obtain the ZIF-8 in-situ immobilized enzyme.
In the above technical scheme, further, the drying is vacuum drying at 20-30 ℃ for 6-12 hours.
The second aspect of the invention protects a ZIF-8 in-situ immobilized enzyme prepared by the method.
The third aspect of the invention protects the application of the ZIF-8 in-situ immobilized enzyme in the quantitative detection of Organophosphorus Pesticides (OPs).
In the above technical solution, further, the quantitative detection method comprises:
(1) Determination of the standard curve: preparing 2mg/mL ZIF-8 in-situ immobilized enzyme aqueous solution, respectively adding 200 mu L of OPs solution with different gradient concentrations of 0-100nM, incubating for 10 minutes, then adding 200 mu L of 0.2M acetylcholine and 400 mu L of 0.2M acetic acid-sodium acetate buffer solution, incubating for 5 minutes at 37 ℃, adding 200 mu L of 17.5mM TMB, and measuring the variation curve of the absorbance of the system along with the absorption wavelength after 5 minutes to obtain the standard curve of the variation of the absorbance of the system along with the OPs concentration at 652 nM;
(2) Determination of the concentration of OPs in the sample to be tested: replacing OPs with different gradient concentrations with a sample to be detected according to the method in the step (1), measuring the absorbance value of the sample to be detected at 652nm, and substituting the absorbance value into the standard curve to obtain the concentration of the OPs in the sample to be detected.
In the invention, ZIF-8 with the advantages of large specific surface area, excellent pore channel, mild synthesis conditions and the like is utilized, and zinc acetate (Zn (OAc) with weaker metal activity is adopted 2 ) And HmIM is used as a precursor, and the AChE, the CHO and the HRP are added after the HmIM and the HmIM are premixed to construct an AChE/CHO/HRP @ ZIF-8 composite material. Zn (OAc) 2 Mixed with HmIM, the pH of the mixed solution is rapidly changed to be close to neutral due to acid-base neutralization reaction, and free Zn is reduced due to coordination of HmIM 2+ At a concentration at which the addition of the enzyme reduces the higher pH of the HmIM and free Zn 2+ Inhibition of enzyme activity, and due to Zn (OAc) 2 The method has weaker metal activity, so that the nucleation speed of ZIF-8 is lower, and therefore, the method not only can complete the embedding of the enzyme, but also can ensure that the enzyme retains higher catalytic activity. In addition, the ZIF-8 protective layer not only increases the tolerance of AChE, CHO and HRP against extreme environments such as hydrolytic protease, high temperature and organic solvent, but also effectively improves the storage stability and the recycling property of the enzyme. In addition, by utilizing the cascade catalytic reaction of three enzymes fixed in ZIF-8, the substrate acetylcholine is hydrolyzed to generate hydroxyl radicals, and further, the chromogenic substrate 3,3', 5' -Tetramethylbenzidine (TMB) is subjected to chromogenic reaction, when OPs exists, the activity of AChE is inhibited, so that the generation of the hydroxyl radicals is inhibited, and the absorbance of the system is reduced. Therefore, the absorbance value of the system is linearly and inversely related to the concentration of OPs in a certain range, thereby providing a basis for the quantitative analysis of OPs.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the enzyme is embedded in a manner of avoiding direct contact between the enzyme and the precursor of the ZIF-8, so that the inhibition effect of the precursor on the enzyme activity is reduced, and the prepared AChE/CHO/HRP @ ZIF-8 composite material enables the enzyme to retain higher catalytic activity; the preparation method is simple and quick;
the AChE/CHO/HRP @ ZIF-8 composite material prepared by the invention not only increases the tolerance of natural enzyme to the extreme environment, but also improves the storage stability and the recycling property of the natural enzyme.
The AChE/CHO/HRP @ ZIF-8 composite material prepared by the invention is used for OPs colorimetric sensing detection, realizes rapid and sensitive detection of OPs, and has the advantages of wide linear range, high sensitivity and detection limit of 0.026nM.
Drawings
FIG. 1 is a schematic diagram of a preparation method of the AChE/CHO/HRP @ ZIF-8 composite material of the present invention;
FIG. 2 is a scanning electron micrograph of the AChE/CHO/HRP @ ZIF-8 composite obtained in example 1;
FIG. 3 is a comparison of the activity of the enzymes in the AChE/CHO/HRP @ ZIF-8 composite;
FIG. 4 is a comparison of the activity of the enzymes when AChE/CHO/HRP @ ZIF-8 composites were synthesized using different concentrations of 2-methylimidazole;
FIG. 5 is a comparison of enzyme activities at different time intervals after mixing of zinc acetate and 2-methylimidazole when adding the enzyme-synthesized AChE/CHO/HRP @ ZIF-8 composite material;
FIG. 6 is a comparison of enzyme activities obtained for AChE/CHO/HRP @ ZIF-8 composites at different mixing times;
FIG. 7 is the stability of the enzymes in AChE/CHO/HRP @ ZIF-8 composites;
FIG. 8 is the recycling of AChE/CHO/HRP @ ZIF-8 composite;
FIG. 9 is a schematic diagram of the mechanism of AChE/CHO/HRP @ ZIF-8 composite for OPs detection;
FIG. 10 is a standard curve of AChE/CHO/HRP @ ZIF-8 composite for OPs detection;
FIG. 11 is the optimal acetylcholine concentration for AChE/CHO/HRP @ ZIF-8 composite for OPs detection;
FIG. 12 is the optimal TMB concentration for AChE/CHO/HRP @ ZIF-8 composite for OPs detection;
FIG. 13 is the optimal pH for AChE/CHO/HRP @ ZIF-8 composite for OPs detection.
Detailed Description
The invention is further illustrated but is not in any way limited by the following specific examples. The raw materials used in the examples, acetylcholinesterase, choline oxidase and horseradish peroxidase, unless otherwise specified, were commercially available.
Example 1
(1)32mM Zn(OAc) 2 And 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume was determined in a 100mL volumetric flask at O25 ℃ and 13136.0mg of HmIM was weighed in a 100mL volumetric flask at 25 ℃.
(2) Mix 15mL 32mM Zn (OAc) 2 The solution was mixed with 15mL of 1600mM HmIM solution, allowed to stand for 10 minutes, and a mixture of 60U AChE, 120U CHO, and 1200U HRP was added thereto, followed by stirring at 25 ℃ for 1 hour. (FIG. 1)
(3) And centrifugally washing the obtained mixture with high-purity water for 3-5 times, collecting the precipitate, and drying the precipitate in vacuum at 25 ℃ for 6-12 hours to obtain the AChE/CHO/HRP @ ZIF-8 immobilized enzyme, wherein the scanning electron microscope picture of the immobilized enzyme is shown in figure 2, and the AChE/CHO/HRP @ ZIF-8 has a dispersed rhombic dodecahedron structure with the average diameter of about 500nm.
(4) And (3) enzyme activity determination: the AChE/CHO/HRP @ zif-8 obtained in step (3) and the immobilized enzyme equivalent amounts of AChE, CHO and HRP mixed free enzyme were added separately to a 0.2M acetate-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine. Reacting the mixed solution at 37 ℃, and measuring the absorbance value of the system at 652nm to obtain the relative activity value of the enzyme (figure 3); the absorbance value of the free enzyme was 0.847, and the absorbance value of the immobilized enzyme AChE/CHO/HRP @ ZIF-8 was 0.771.
Comparative example 1:
(1)32mM Zn(OAc) 2 and 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume is determined in a 100mL volumetric flask at O25 ℃, 13136.0mg HmIM25 ℃ is weighed and determined in a 100mL volumetric flask at O25 ℃.
(2) A mixture of 60U AChE, 120U CHO and 1200U HRP was added to 15mL 1600mM HmIM solution, followed by 15mL 32mM Zn (OAc) 2 The solution was stirred at 25 ℃ for 1 hour.
(3) The resulting mixture was washed with high-purity water by centrifugation 3-5 times, and the precipitate was collected and vacuum-dried at 25 ℃ for 6-12 hours.
(4) And (3) enzyme activity determination: the AChE/CHO/hrp @ zif-8 obtained in step (3) was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine, respectively, the mixture was reacted at 37 ℃, and the absorbance value of the system at 652nm was measured to be 0.593.
Comparative example 2:
(1)32mM Zn(OAc) 2 and 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume was determined in a 100mL volumetric flask at O25 ℃ and 13136.0mg of HmIM was weighed in a 100mL volumetric flask at 25 ℃.
(2) A mix of 60U AChE, 120U CHO and 1200U HRP was added to 15mL 32mM Zn (OAc) 2 To the solution, 15mL1600 mM HmIM solution was added, and the mixture was stirred at 25 ℃ for 1 hour.
(3) The resulting mixture was washed with high-purity water by centrifugation 3-5 times, and the precipitate was collected and vacuum-dried at 25 ℃ for 6-12 hours.
(4) And (3) enzyme activity determination: the AChE/CHO/hrp @ zif-8 obtained in step (3) was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine, respectively, the mixture was reacted at 37 ℃, and the absorbance value of the system at 652nm was measured to be 0.246.
As can be seen from FIG. 3, zn (OAc) is first introduced 2 The enzyme was added after mixing with HmIM in such a way that 91% of the activity of the free enzyme was retained, whereas the enzyme was added first to HmIM and Zn (OAc) respectively 2 Only 70% and 29% of the activity of the free enzyme was retained.
Comparative example 3
(1)32mM Zn(OAc) 2 And (640, 960, 1280, 1600, 1920) mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume is determined in a 100mL volumetric flask at O25 ℃, and 5254.4, 7881.6, 10508.8, 13136.0 and 15763.2mg HmIM are weighed respectively in a 100mL volumetric flask at 25 ℃.
(2) Mix 15mL 32mM Zn (OAc) 2 The solution was mixed with 15mL (640-1920) mM HmIM solution, allowed to stand for 10 minutes, and 60U AChE, 120U CHO and1200U HRP, stirring at 25 ℃ for 1 hour.
(3) And centrifugally washing the obtained mixture with high-purity water for 3-5 times, collecting the precipitate, and drying the precipitate in vacuum at 25 ℃ for 6-12 hours to obtain the AChE/CHO/HRP @ ZIF-8 immobilized enzyme.
(4) And (3) enzyme activity determination: the AChE/CHO/hrp @ zif-8 obtained in step (3) was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine, respectively, the mixture was reacted at 37 ℃, and the absorbance value of the system at 652nm was measured. (FIG. 4)
As can be seen from FIG. 4, the absorbance value of the system was maximized at a 2-methylimidazole concentration of 1600mM, and the enzyme activity under these conditions was maximally retained.
Comparative example 4
(1)32mM Zn(OAc) 2 And 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume was determined in a 100mL volumetric flask at O25 ℃ and 13136.0mg of HmIM was weighed in a 100mL volumetric flask at 25 ℃.
(2) Mix 15mL 32mM Zn (OAc) 2 The solution was mixed with a solution of 15mL1600 mM HmIM, allowed to stand (4, 7, 10, 13 and 16) for minutes, and 60U of AChE, 120U of CHO and 1200U of HRP were added, followed by stirring at 25 ℃ for 1 hour.
(3) And centrifugally washing the obtained mixture with high-purity water for 3-5 times, collecting the precipitate, and drying the precipitate in vacuum at 25 ℃ for 6-12 hours to obtain the AChE/CHO/HRP @ ZIF-8 immobilized enzyme.
(4) And (3) enzyme activity determination: the AChE/CHO/hrp @ zif-8 obtained in step (3) was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine, respectively, the mixture was reacted at 37 ℃, and the absorbance value of the system at 652nm was measured. (FIG. 5)
As can be seen from FIG. 5, the absorbance value of the enzyme system added when the precursor was mixed for 10 minutes was the largest, and the enzyme activity retention under this condition was the largest.
Comparative example 5
(1)32mM Zn(OAc) 2 And 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume is determined in a 100mL volumetric flask at O25 ℃, 13136.0mg HmIM is weighed respectively and determined in 10 at 25 DEG0mL volumetric flask.
(2) Mix 15mL 32mM Zn (OAc) 2 The solution was mixed with a solution of 15mL1600 mM HmIM, left to stand for 10 minutes, and 60U of AChE, 120U of CHO and 1200U of HRP were added thereto, and stirred (0.5, 1,2,3, 4) at 25 ℃ for hours.
(3) And centrifugally washing the obtained mixture with high-purity water for 3-5 times, collecting the precipitate, and drying the precipitate in vacuum at 25 ℃ for 6-12 hours to obtain the AChE/CHO/HRP @ ZIF-8 immobilized enzyme.
(4) And (3) enzyme activity determination: the AChE/CHO/hrp @ zif-8 obtained in step (3) was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine, the mixture was reacted at 37 ℃, and the absorbance value of the system at 652nm was measured. (FIG. 6)
As can be seen from FIG. 6, when the stirring time was 1 hour, the absorbance value of the system was maximized and the enzyme activity under this condition was maximally retained.
Example 2:
(1)32mM Zn(OAc) 2 and 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume is determined in a 100mL volumetric flask at O25 ℃, and 13136.0mg HmIM is weighed in a 100mL volumetric flask at 25 ℃.
(2) Mix 15mL 32mM Zn (OAc) 2 The solution was mixed with 15mL of 1600mM HmIM solution, allowed to stand for 10 minutes, and a mixed enzyme of 60U AChE, 120U CHO and 1200U HRP was added thereto, followed by stirring at 25 ℃ for 1 hour.
(3) The resulting mixture was washed with high-purity water by centrifugation 3-5 times, and the precipitate was collected and vacuum-dried at 25 ℃ for 6-12 hours.
(4) And (3) enzyme stability determination: the AChE/CHO/HRP @ ZIF-8 and the immobilized enzyme equal amount of AChE, mixed free enzyme of CHO and HRP were taken, incubated at 70 deg.C, 6M urea, l mg/mL trypsin, l wt% EDTA, and 50% organic solvent (DMSO and DMF) for 30 minutes and stored at 25 deg.C for different time, and the absorbance of AChE/CHO/HRP @ ZIF-8 and its free enzyme before and after treatment was measured by acetylcholine-TMB reaction system, and the relative catalytic activity was calculated (FIG. 7).
As can be seen from FIG. 7, the activity of AChE/CHO/HRP @ ZIF-8 can still be retained by more than 80% under the adverse environment of high temperature, urea, protease, EDTA and organic solvent, while the activity of free enzyme is only retained by less than 40%. After 30 days of storage at room temperature, AChE/CHO/hrp @ zif-8 still retained 82% activity, whereas the free enzyme had little activity.
Example 3:
(1)32mM Zn(OAc) 2 and 1600mM HmIM solution, 702.4mg of Zn (OAc) was weighed 2 ·2H 2 The volume was determined in a 100mL volumetric flask at O25 ℃ and 13136.0mg of HmIM was weighed in a 100mL volumetric flask at 25 ℃.
(2) 15mL of 32mM Zn (OAc) 2 The solution was mixed with 15mL of 1600mM HmIM solution, allowed to stand for 10 minutes, and a mixed enzyme of 60U AChE, 120U CHO and 1200U HRP was added thereto, followed by stirring at 25 ℃ for 1 hour.
(3) The resulting mixture was washed with high-purity water by centrifugation 3-5 times, and the precipitate was collected and vacuum-dried at 25 ℃ for 6-12 hours.
(4) Study of enzyme recyclability: to test the recycling of the prepared AChE/CHO/hrp @ zif-8, a concentration of AChE/CHO/hrp @ zif-8 was added to a 0.2M acetic acid-sodium acetate buffer system (pH = 4) containing 1.75mM TMB and 0.02M acetylcholine. After the mixture was reacted at 37 ℃ for 10 minutes, the absorbance of the system at 652nm was measured at room temperature. The mixture was centrifuged at 13000rpm for 10 minutes to collect the precipitate, and the next enzymatic reaction was performed, which was repeated 10 times, and the relative catalytic activity was calculated using the change in catalytic activity before and after the recycling of the acetylcholine-TMB reaction system (fig. 8).
As can be seen from FIG. 8, AChE/CHO/HRP @ ZIF-8 retained 65% of its activity after 10 cycles, whereas the free enzyme had little activity after 2 cycles.
Example 4
Quantitative detection of OPs: the immobilized enzyme AChE/CHO/HRP @ ZIF-8 prepared in example 1 was used to detect the OPs content.
(1) 2mg of the dried immobilized enzyme powder was weighed and dissolved in 1mL of ultrapure water, and after sufficient dissolution, 200. Mu.L of OPs (0 to 100 nM) at various concentrations was added, followed by incubation for 10 minutes, followed by addition of 200. Mu.L of 0.2M acetylcholine and 400. Mu.L of 0.2M acetic acid-sodium acetate buffer solution (pH = 4), incubation at 37 ℃ for 5 minutes, addition of 200. Mu.L of 17.5mM TMB, and after 5 minutes, the absorbance change curve of the system with respect to the absorption wavelength (550 to 750 nM) was measured. (detection mechanism is shown in figure 9) AChE, CHO and HRP are simultaneously fixed in ZIF-8 with a rhombic dodecahedron structure, the substrate acetylcholine is hydrolyzed by utilizing the cascade catalytic reaction of three enzymes, and finally the colorless TMB generates blue oxTMB, and an ultraviolet absorption peak is formed at the wavelength of 652 nm. In the presence of OPs, AChE activity is inhibited, so that hydrolysis of acetylcholine as a substrate is inhibited, and finally, the generated oxTMB is reduced, so that the color of the detection system is lightened, and the ultraviolet absorption peak intensity at the wavelength of 652nm is reduced.
(2) Drawing a standard working curve: in the step (5), the absorbance of the reaction system at 652nM is continuously reduced along with the increase of the concentration of OPs, the absorbance of the reaction system and the concentration of OPs have a good linear relation in the range of 0.781-50nM, and the linear correlation coefficient R 2 =0.996 (fig. 10), detection limit is 0.026nM.
(3) And (3) determining the content of OPs in the actual water sample: and preparing OPs solution with concentration of 5.00nM by using an actual water sample by using a standard recovery experiment method. And (3) applying the sample to the step (1) for detection, and calculating the concentration of OPs according to the detection result by using the standard curve obtained in the step (2). The results of the experiment determined that the OPs content was 5.13nM and the recovery was 102.60%. The relative standard deviation RSD was 3.72% (n = 5).
Comparative example 6
2mg of the dried immobilized enzyme powder was weighed and dissolved in 1mL of ultrapure water, and after sufficient dissolution, 200. Mu.L of OPs (0-100 nM) at various concentrations were added and incubated for 10 minutes, then 200. Mu.L of acetylcholine (0.025, 0.05,0.1,0.2,0.3,0.4 and 0.5M) at various concentrations and 400. Mu.L of 0.2M acetic acid-sodium acetate buffer solution (pH = 4) were added, incubation was carried out at 37 ℃ for 5 minutes, 200. Mu.L of 17.5mM TMB was added, and after 5 minutes, the absorbance value of the system at a wavelength of 652nM was measured (FIG. 11), and it was seen that the absorbance value of the system was the maximum when 0.2M acetylcholine was added.
Comparative example 7
2mg of the dried immobilized enzyme powder was weighed and dissolved in 1mL of ultrapure water, and after sufficient dissolution, 200. Mu.L of OPs (0-100 nM) at different concentrations were added, and the mixture was incubated for 10 minutes, then 200. Mu.L of 0.2M acetylcholine and 400. Mu.L of 0.2M acetic acid-sodium acetate buffer solution (pH = 4) were added, and incubated at 37 ℃ for 5 minutes, and 200. Mu.L of TMB (4.38, 8.75, 13.13, 17.50, 26.25, 35.00, 43.75 mM) at different concentrations were added, and after 5 minutes, the absorbance of the system at 652nM wavelength was measured (FIG. 12), and it was seen that when 17.50mM of TMB was added, the absorbance of the system was maximal.
Comparative example 8
After 2mg of the dried immobilized enzyme powder was weighed and dissolved in 1mL of ultrapure water, and sufficiently dissolved, 200. Mu.L of OPs (0-100 nM) at different concentrations was added, and the mixture was incubated for 10 minutes, followed by addition of 200. Mu.L of 0.2M acetylcholine and 400. Mu.L of 0.2M acetic acid-sodium acetate buffer solutions (pH =3,4,5,6 and 7) at different pH, incubation at 37 ℃ for 5 minutes, addition of 200. Mu.L of 17.5mM TMB, and measurement of the absorbance of the system at 652nM wavelength after 5 minutes (FIG. 13), and it was found that the absorbance of the system was the maximum when the pH of the buffer was 4.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention.
Claims (7)
1. A ZIF-8 in-situ immobilization method is characterized in that zinc acetate aqueous solution and 2-methylimidazole aqueous solution are mixed, and acetylcholinesterase, choline oxidase and horseradish peroxidase are added for mixed reaction to obtain the enzyme.
2. The method according to claim 1, wherein the concentration of the aqueous solution of zinc acetate is 32mM, the concentration of the aqueous solution of 2-methylimidazole is 640-1920mM, the mixing ratio of the aqueous solution of zinc acetate and the aqueous solution of 2-methylimidazole is 1.
3. The method according to claim 1, characterized in that it comprises the steps of:
(1) Mixing a zinc acetate aqueous solution and a 2-methylimidazole aqueous solution, and standing for 4-16 minutes to obtain a mixed solution;
(2) Adding acetylcholinesterase, choline oxidase and horseradish peroxidase into the mixed solution obtained in the step (1), and stirring at room temperature for 0.5-2.5 hours to react to obtain a mixture;
(3) And after the reaction is finished, centrifugally washing the mixture, collecting the precipitate, and drying to obtain the ZIF-8 in-situ immobilized enzyme.
4. The method according to claim 3, wherein the drying is vacuum drying at 20-30 ℃ for 6-12 hours.
5. A ZIF-8 immobilized in situ enzyme prepared by the method of any one of claims 1 to 4.
6. The use of the ZIF-8 in situ immobilized enzyme of claim 5 for the quantitative detection of Organophosphorus Pesticides (OPs).
7. The use according to claim 6, wherein the quantitative detection method is:
(1) Determination of the standard curve: preparing 2mg/ml ZIF-8 in-situ immobilized enzyme aqueous solution, respectively adding 200 mu L of OPs solution with different gradient concentrations of 0-100nM, incubating for 10 minutes, then adding 200 mu L of 0.2M acetylcholine and 400 mu L of 0.2M acetic acid-sodium acetate buffer solution, incubating for 5 minutes at 37 ℃, adding 200 mu L17.5mM TMB, and measuring the variation curve of the absorbance of the system along with the absorption wavelength after 5 minutes to obtain the standard curve of the variation of the absorbance of the system along with the OPs concentration at 652 nM;
(2) Determination of the concentration of OPs in the sample to be tested: replacing OPs with different gradient concentrations with a sample to be detected according to the method in the step (1), measuring the absorbance value of the sample to be detected at 652nm, and substituting the absorbance value into the standard curve to obtain the concentration of the OPs in the sample to be detected.
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| CN110724682A (en) * | 2019-07-22 | 2020-01-24 | 江苏科技大学 | Method for preparing immobilized enzyme from zeolite imidazole ester framework compound |
| CN112175931A (en) * | 2020-10-16 | 2021-01-05 | 安徽师范大学 | Immobilized carboxylesterase, and preparation method and application thereof |
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| CN104007104A (en) * | 2014-05-22 | 2014-08-27 | 上海交通大学 | Kit for rapidly detecting organophosphorus pesticide residues and application method thereof |
| CN110724682A (en) * | 2019-07-22 | 2020-01-24 | 江苏科技大学 | Method for preparing immobilized enzyme from zeolite imidazole ester framework compound |
| CN112175931A (en) * | 2020-10-16 | 2021-01-05 | 安徽师范大学 | Immobilized carboxylesterase, and preparation method and application thereof |
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