CN111518862B - Preparation method of lactate oxidase bioelectrode - Google Patents
Preparation method of lactate oxidase bioelectrode Download PDFInfo
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- CN111518862B CN111518862B CN202010517752.0A CN202010517752A CN111518862B CN 111518862 B CN111518862 B CN 111518862B CN 202010517752 A CN202010517752 A CN 202010517752A CN 111518862 B CN111518862 B CN 111518862B
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- 108010073450 Lactate 2-monooxygenase Proteins 0.000 title claims abstract description 87
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 41
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 27
- 229910017604 nitric acid Inorganic materials 0.000 claims description 27
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- 238000002484 cyclic voltammetry Methods 0.000 claims description 9
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- 238000012258 culturing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 241000235013 Yarrowia Species 0.000 claims description 5
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- 238000000605 extraction Methods 0.000 claims description 5
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- 239000008101 lactose Substances 0.000 claims description 5
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- 239000012141 concentrate Substances 0.000 description 15
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 9
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 8
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- 229920000656 polylysine Polymers 0.000 description 8
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 5
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- 238000001514 detection method Methods 0.000 description 5
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- 235000014655 lactic acid Nutrition 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
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- 239000000969 carrier Substances 0.000 description 4
- 239000007772 electrode material Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 101150070593 lox gene Proteins 0.000 description 3
- 229940107700 pyruvic acid Drugs 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
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- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 2
- UQSQSQZYBQSBJZ-UHFFFAOYSA-N fluorosulfonic acid Chemical compound OS(F)(=O)=O UQSQSQZYBQSBJZ-UHFFFAOYSA-N 0.000 description 2
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- 208000010444 Acidosis Diseases 0.000 description 1
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
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- 230000007950 acidosis Effects 0.000 description 1
- 208000026545 acidosis disease Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the technical field of bioengineering, and particularly provides a preparation method of a lactate oxidase bioelectrode. The first aspect of the invention provides a preparation method of a lactate oxidase bioelectrode, which comprises the following steps: electrochemical oxidation treatment of the Pb electrode; the enzyme is immobilized by using an immobilization carrier. The invention provides a preparation method of a lactate oxidase bioelectrode, which solves the problems of low immobilization rate of lactate oxidase and poor biocompatibility of immobilized materials by electrochemical oxidation treatment of a Pb electrode and a method for preparing the bioelectrode by immobilizing specific enzyme solution; realizes the high efficiency and repeatability of the lactate oxidase bioelectrode, and is an enzyme immobilization technology with high immobilization efficiency and good biocompatibility.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly provides a preparation method of a lactate oxidase bioelectrode.
Background
Lactate oxidase is a flavoprotein, uses oxygen as a substrate, uses FMN and FAD as cofactors, and can directly convert lactic acid into pyruvic acid. FMN and FAD are firmly combined with enzyme protein, and no cofactor is required to be added from an external source. The production of pyruvic acid by using lactate oxidase is one of the important means for the industrial production of pyruvic acid. In addition, lactic acid oxidase is of great significance for the detection of lactic acid in humans because lactic acid acidosis symptoms occur when large amounts of lactic acid are present in humans and cannot be metabolized.
Lactate oxidase, as a normal temperature enzyme, has poor stability, is easy to inactivate and can not be reused, and the product is mixed after the reaction, so that the purification is difficult, and the wider application in detection is difficult, therefore, an optimized immobilization mode is needed to improve the availability of the lactate oxidase.
At present, the commonly used immobilization methods of the lactate oxidase include a cross-linking method, a covalent bonding method, a microencapsulation method, a grid method, a physical adsorption method, a crystallization method, an ion bonding method and the like, but the enzyme reaction activity is reduced to a certain extent and the enzyme electron transfer is blocked in the application of a bioelectrode, so that the development of an optimized immobilization method of the lactate oxidase is necessary.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a method for preparing a lactate oxidase bioelectrode, comprising the steps of:
(1) electrochemical oxidation treatment of the Pb electrode;
(2) the enzyme is immobilized by using an immobilization carrier.
As a preferable aspect of the present invention, the electrochemical oxidation treatment of the Pb electrode includes the steps of:
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;
a2: and scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment.
As a preferred technical scheme, the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%.
As a preferred technical scheme of the invention, the immobilized carrier is selected from any one or combination of more of glutaraldehyde, chitosan, polylysine and perfluorosulfonic acid (Nafion); preferably chitosan.
As a preferred technical scheme, the volume concentration of the chitosan is 0.25-1.50%, and the chitosan is stored at the temperature of 3-6 ℃.
As a preferred technical scheme of the invention, the immobilization mode of immobilizing the enzyme by utilizing the immobilization carrier comprises the following steps:
c1: dropwise adding enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at 3-6 ℃ until the electrode is dried;
c2: and D, dropwise adding the immobilized carrier, covering and titrating the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the immobilized carrier in the dark at the temperature of 3-6 ℃ until the immobilized carrier is dried.
As a preferred technical scheme of the invention, the volume ratio of the enzyme solution to the immobilized carrier is 1: (0.8 to 1.2).
As a preferable technical scheme of the invention, the preparation method of the enzyme solution comprises the following steps:
b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;
b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;
b3: constructing a gene engineering bacterium E.coli BL21 containing LOX gene by using a host bacterium E.coli BL21, inoculating the constructed LOX strain into an LB shake flask, culturing at 34-39 ℃ for 2.5-4.5 h, adding 50mM lactose solution, culturing at 18-22 ℃ for 22-26 h, and expressing the lactate oxidase gene;
b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.
The second aspect of the present invention provides a bioelectrode prepared according to the method for preparing a lactate oxidase bioelectrode.
In a third aspect, the present invention provides a lactate oxidase biosensor comprising the bioelectrode.
Has the advantages that: the invention provides a preparation method of a lactate oxidase bioelectrode, which solves the problems of low immobilization rate of lactate oxidase and poor biocompatibility of immobilized materials by electrochemical oxidation treatment of a Pb electrode and a method for preparing the bioelectrode by immobilizing specific enzyme solution; realizes the high efficiency and repeatability of the lactate oxidase bioelectrode, and is an enzyme immobilization technology with high immobilization efficiency and good biocompatibility.
Drawings
FIG. 1 is an SDS-PAGE pattern of lactate oxidase;
FIG. 2 is a graph showing the influence of different immobilization carriers on the enzyme activity of immobilized lactate oxidase;
FIG. 3 is a graph showing the comparison of immobilization rates of different immobilization carriers;
FIG. 4 is a graph showing comparative immobilization curves for different curing modes;
FIG. 5: comparing the tolerance curves of the enzyme at different temperatures and pH values;
FIG. 6 is a graph showing the reproducibility of lactate oxidase electrodes.
Detailed Description
The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto.
The words "preferred", "more preferred", and the like, in the present invention refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range from "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10. Exemplary subranges of the range 1 to 10 include, but are not limited to, 1 to 6.1, 3.5 to 7.8, 5.5 to 10, and the like.
The invention mainly provides a preparation method of the lactate oxidase bioelectrode, which is used for solving the problems of low lactate oxidase bioelectrode fixing efficiency and poor sensitivity caused by poor stability, easy inactivation and incapability of being reused of lactate oxidase and improving the high efficiency and reusability of the lactate oxidase bioelectrode.
The first aspect of the invention provides a preparation method of a lactate oxidase bioelectrode, which comprises the following steps:
(1) electrochemical oxidation treatment of the Pb electrode;
(2) the enzyme is immobilized by using an immobilization carrier.
In one embodiment, the electrochemical oxidation treatment of the Pb electrode includes the steps of:
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;
a2: and scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment.
In one embodiment, the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%; preferably, the mass concentration of the nitric acid is 9-11%; the mass concentration of the potassium dichromate is 2.1-2.8%; more preferably, the mass concentration of nitric acid is 10%; the mass concentration of potassium dichromate is 2.6%.
In one embodiment, the mass ratio of the nitric acid to the potassium dichromate is 0.6 to 1; preferably 0.7 to 0.9; more preferably 0.82.
In one embodiment, the immobilization support is selected from the group consisting of glutaraldehyde, chitosan, polylysine, a combination of any one or more of perfluorosulfonic acids (Nafion); preferably chitosan; further preferably, the volume concentration of the chitosan is 0.25-1.5%; further preferably, the volume concentration of the chitosan is 0.5-1.25%; further preferably, the volume concentration of the chitosan is 0.75-1.25%; further preferably, the volume concentration of the chitosan is 1.0-1.25%; more preferably, the chitosan is stored at 3-6 ℃.
In one embodiment, the immobilization of the enzyme by the immobilization carrier is performed by:
c1: dropwise adding the enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at the temperature of 3-6 ℃ until the electrode is dried;
c2: and D, covering and dropwise adding the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the electrode at 3-6 ℃ in a dark place until the electrode is dried.
In another embodiment, the immobilization of the enzyme by the immobilization carrier is performed by: and (3) mixing the enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in the dark until the mixture is dried.
In one embodiment, the volume ratio of the enzyme solution to the immobilization carrier is 1: (0.8 to 1.2); preferably, the volume ratio of the enzyme solution to the immobilization carrier is 1: (0.9 to 1.1); more preferably, the volume ratio of the enzyme solution to the immobilization carrier is 1: 1.
in one embodiment, the amount of the enzyme solution in the step C1 is 0.8-1.2U; preferably 0.9-1.1U; more preferably 1.0U.
In one embodiment, the method of preparing an enzyme solution comprises:
b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;
b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;
b3: constructing a gene engineering bacterium E.coliBL21(DE3-pET-LOX) containing LOX genes by using a host bacterium E.colibl21 (DE3), inoculating the constructed LOX strain into an LB shake flask, culturing for 2.5-4.5 h at 34-39 ℃, adding 50mM lactose solution, culturing for 22-26 h at 18-22 ℃, and expressing a lactate oxidase gene;
b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.
Preferably, the preparation method of the enzyme solution comprises:
b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;
b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;
b3: constructing a gene engineering bacterium E.coliBL21 (recorded as DE3-pET-LOX) containing LOX gene by using a host bacterium E.colibl21 (recorded as DE3), inoculating the constructed LOX strain into an LB shake flask, culturing for 3.5h at 37 ℃, adding 25 mu L of 50mM lactose solution, and culturing for 24h at 20 ℃ to express a lactate oxidase gene;
b4: and D, adsorbing the substance obtained in the step B3 by using a nickel ion column, and concentrating by using an ultrafiltration tube to obtain the LOX protease liquid.
The second aspect of the present invention provides a bioelectrode prepared according to the method for preparing a lactate oxidase bioelectrode.
In a third aspect, the present invention provides a lactate oxidase biosensor comprising the bioelectrode.
Example 1
Example 1 of the present invention provides lactate oxidase, which is prepared by the following method:
b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;
b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;
b3: a gene engineering bacterium E.coliBL21(DE3-pET-LOX) containing LOX gene is constructed by using a host bacterium E.colibl21 (DE3), the constructed LOX strain is inoculated into an LB shake flask, cultured for 3.5h at 37 ℃, then added with 25 mu L of 50mM lactose solution, cultured for 24h at 20 ℃, and the lactate oxidase gene is expressed. Carrying out ultrasonic crushing, wherein the supernatant obtained by crushing is crushed supernatant, and the obtained precipitate is crushed precipitate;
b4: and D, adsorbing the substance obtained in the step B3 for 10Min by using a nickel ion column, releasing after adsorption to obtain a penetrating liquid, eluting by using imidazole solutions with the concentrations of 50mM, 100 mM, 200 mM and 250mM respectively to obtain eluates with corresponding concentrations, and concentrating by using an ultrafiltration tube to obtain LOX protease liquid, namely obtaining the lactate oxidase concentrated solution.
Example 2
Example 2 of the present invention provides a bioelectrode material, the preparation method of which is as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) the enzyme is immobilized by utilizing an immobilization carrier, and the immobilization mode is as follows:
mixing 1.0U of enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in a dark place until the mixture is dried; the volume ratio of the enzyme solution to the immobilized carrier is 1: 1;
the enzyme solution was the lactate oxidase concentrate prepared in example 1;
the immobilized carrier is glutaraldehyde, and the volume concentration of the glutaraldehyde is 0.25%, 0.5%, 0.75%, 1% and 1.25%, respectively.
Example 3
Example 3 of the present invention provides a bioelectrode material, the preparation method of which is as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) the enzyme is immobilized by utilizing an immobilization carrier, and the immobilization mode is as follows:
mixing 1.0U of enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in a dark place until the mixture is dried; the volume ratio of the enzyme solution to the immobilized carrier is 1: 1;
the enzyme solution was the lactate oxidase concentrate prepared in example 1;
the immobilized carrier is chitosan, the volume concentration of the chitosan is 0.25%, 0.5%, 0.75%, 1% and 1.25%, which is purchased from Shanghai Yuan leaf Biotech Co.
Example 4
Example 4 of the present invention provides a bioelectrode material, which is prepared as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) the enzyme is immobilized by utilizing an immobilization carrier, and the immobilization mode is as follows:
mixing 1.0U of enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in a dark place until the mixture is dried; the volume ratio of the enzyme solution to the immobilized carrier is 1: 1;
the enzyme solution was the lactate oxidase concentrate prepared in example 1;
the immobilization carrier is Nafion, the volume concentration of Nafion is 0.25%, 0.5%, 0.75%, 1%, 1.25%, which is purchased from Shanghai Yuan Ye Biotech Co., Ltd.
Example 5
Example 5 of the present invention provides a bioelectrode material, which is prepared as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) the enzyme is immobilized by utilizing an immobilization carrier, and the immobilization mode is as follows:
mixing 1.0U of enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in a dark place until the mixture is dried; the volume ratio of the enzyme solution to the immobilized carrier is 1: 1;
the enzyme solution was the lactate oxidase concentrate prepared in example 1;
the immobilized carrier is polylysine, the volume concentration of the polylysine is 0.25 percent, 0.5 percent, 0.75 percent, 1 percent and 1.25 percent respectively, and the immobilized carrier is purchased from Shanghai Yuan leaf Biotech Co.
Example 6
Example 6 of the present invention provides a bioelectrode material, which is prepared as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) the enzyme is immobilized by utilizing an immobilization carrier, and the immobilization mode is as follows:
c1: dropwise adding 1.0U of enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode at 4 ℃ in a dark place until the electrode is dried;
c2: covering and dripping the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the immobilized carrier in the dark at the temperature of 4 ℃ until the immobilized carrier is dried; the volume ratio of the enzyme solution to the immobilized carrier is 1: 1;
the enzyme solution was the lactate oxidase concentrate prepared in example 1;
the immobilized carrier is chitosan, the volume concentration of the chitosan is 1.25%, and the immobilized carrier is purchased from Shanghai-sourced leaf Biotechnology Co.
Example 7
Example 7 of the present invention provides a bioelectrode material, which is prepared as follows:
(1) electrochemical oxidation treatment of the Pb electrode;
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment; the mass concentration of the nitric acid is 10 percent; the mass concentration of the potassium dichromate is 2.6 percent; the mass ratio of the nitric acid to the potassium dichromate is 0.82;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
(2) dropwise adding 1.0U of enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode at 4 ℃ in a dark place until the electrode is dried; the enzyme solution was the lactate oxidase concentrate prepared in example 1.
Performance evaluation
1. SDS-PAGE detection of lactate oxidase: SDS-PAGE detection of different fractions obtained in the lactate oxidase preparation process of example 1 is carried out, the detection results are shown in FIG. 1,
2. biocompatibility testing of different immobilization carriers: taking the electrode materials of the embodiments 2-5, then washing the electrode surface with distilled water, and testing the enzyme activity; the results of the experiment are shown in FIG. 2, in which the activity of the lactate oxidase concentrate obtained in example 1 is defined as 100%; 1 represents polylysine, 2 represents glutaraldehyde, 3 represents chitosan, and 4 represents Nafion; the experiment results show that the chitosan and the polylysine have good fixing effect on the lactate oxidase.
3. Immobilization rate experiments for different immobilization carriers: taking the electrode material obtained in example 7, the lactate oxidase electrode corresponding to the volume concentration of 1.25% of chitosan in example 3, and the lactate oxidase electrode corresponding to the volume concentration of 0.25% of polylysine in example 5, soaking in a PBS buffer solution, keeping the room temperature at 25 ℃, taking out the lactate oxidase electrode at fixed intervals, testing the activity of the lactate oxidase electrode, and detecting the activity of the lactate oxidase electrode, wherein the activity of the lactate oxidase concentrate obtained in example 1 is 100%, and the immobilization rate is calculated according to the ratio of the residual activities, and the test results are shown in fig. 3, wherein 1 represents the test result of 1.25% of chitosan, 2 represents the test result of 0.25% of polylysine, and 3 represents the test result of example 7.
4. Immobilization rate experiments for different curing modes: taking the electrode material obtained in the example 7, the lactate oxidase electrode corresponding to the chitosan volume concentration of 1.25% in the example 3, and the lactate oxidase electrode obtained in the example 6, soaking in a PBS buffer solution, keeping the room temperature at 25 ℃, taking out the lactate oxidase electrode at fixed intervals, testing the activity of the lactate oxidase electrode, and detecting the enzyme activity, wherein the enzyme activity of the lactate oxidase concentrate obtained in the example 1 is 100%, the immobilization rate is calculated according to the ratio of the residual enzyme activity, and the test results are shown in fig. 3, wherein 1 represents the test result of 1.25% of chitosan in the example 3, 2 represents the test result of the example 7, and 3 represents the test result of the example 6.
5. Enzyme tolerance test at different temperatures and pH:
(1) pH tolerance test of lactate oxidase: taking the lactate oxidase electrode obtained in example 6 and the lactate oxidase concentrate obtained in example 1, keeping the temperature for 2h at 37 ℃ and pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively, sampling and measuring the residual relative activity of the lactate oxidase at pH 6.525 ℃, defining the activity of the untreated lactate oxidase to be 100%, and testing results are shown in the left graph of fig. 5, wherein 1 represents the lactate oxidase electrode obtained in example 6, and 2 represents the lactate oxidase concentrate obtained in example 1; the experimental result shows that the lactate oxidase electrode provided by the invention improves the tolerance of the lactate oxidase to pH;
(2) temperature tolerance test of lactate oxidase: taking the lactate oxidase electrode obtained in example 6 and the lactate oxidase concentrate obtained in example 1, placing the lactate oxidase electrode and the lactate oxidase concentrate obtained in example 1 in water bath pots at different temperatures (15 ℃, 25 ℃, 35 ℃, 45 ℃ and 55 ℃) for heat preservation for 2 hours, then sampling and measuring the residual relative activity of the lactate oxidase at the pH of 6.525 ℃, wherein the enzyme activity of the lactate oxidase concentrate obtained according to example 1 is 100%, and the relative activity is shown in the right graph of fig. 5, 1 represents the lactate oxidase electrode obtained in example 6, and 2 represents the lactate oxidase concentrate obtained in example 1; the experimental result shows that the lactate oxidase electrode provided by the invention improves the temperature tolerance of the lactate oxidase.
6. Repeatability test of lactate oxidase electrode:
(1) the lactate oxidase electrode obtained in the embodiment 6 is accessed to an electrochemical workstation to scan an IT image, 0.1mM lactate solution is added after the baseline is stable, and the response current of the lactate oxidase electrode is detected;
(2) the experiment in the step (1) is repeated, the response current value of each time is recorded, the relative current response is calculated according to the first response current of 100%, the repeatability is detected, the experimental result is shown in figure 6, and the experimental result shows that the lactate oxidase electrode provided by the invention effectively improves the immobilization rate and the use repetition frequency of the lactate oxidase biosensor electrode, and has important guiding significance for the development and application of subsequent biosensors.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (4)
1. A preparation method of a lactate oxidase bioelectrode is characterized by comprising the following steps:
(1) electrochemical oxidation treatment of the Pb electrode;
(2) immobilizing the enzyme by using an immobilized carrier;
the immobilized carrier is chitosan; the volume concentration of the chitosan is 1.00-1.25%, and the chitosan is stored at the temperature of 3-6 ℃;
the immobilization method for immobilizing the enzyme by using the immobilization carrier comprises the following steps:
c1: dropwise adding enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at 3-6 ℃ until the electrode is dried;
c2: dropwise adding the immobilized carrier, covering and titrating the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the immobilized carrier in the dark at the temperature of 3-6 ℃ until the immobilized carrier is dried;
the electrochemical oxidation treatment of the Pb electrode comprises the following steps:
a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;
a2: scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment;
the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%;
the volume ratio of the enzyme solution to the immobilized carrier is 1: (0.8 to 1.2).
2. The method for preparing lactate oxidase bioelectrode according to claim 1, wherein the method for preparing the enzyme solution comprises:
b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;
b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;
b3: constructing a gene engineering bacterium E.coliBL21 containing LOX genes by using a host bacterium E.coliBL21, inoculating the constructed LOX strain into an LB shake flask, culturing at 34-39 ℃ for 2.5-4.5 h, adding 50mM lactose solution, culturing at 18-22 ℃ for 22-26 h, and expressing a lactate oxidase gene;
b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.
3. A bioelectrode obtained by the production method of a lactate oxidase bioelectrode according to any one of claims 1 to 2.
4. A lactate oxidase biosensor comprising the bioelectrode of claim 3.
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