CN114717289B - Electronic medium reagent of lactic acid biosensor and application thereof - Google Patents

Electronic medium reagent of lactic acid biosensor and application thereof Download PDF

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CN114717289B
CN114717289B CN202210351871.2A CN202210351871A CN114717289B CN 114717289 B CN114717289 B CN 114717289B CN 202210351871 A CN202210351871 A CN 202210351871A CN 114717289 B CN114717289 B CN 114717289B
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enzyme solution
functionalized
immobilized enzyme
ferrocene
reagent
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CN114717289A (en
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吴克南
景振辉
陈明嘉
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Guangzhou Wondfo Biotech Co Ltd
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Abstract

The invention provides an electronic medium reagent, which comprises the following components in percentage by mass: 0.1 to 5 percent of functionalized electrocatalyst, 0.5 to 5 percent of ferrocene dicarboxaldehyde, 1 to 10 percent of hydrophilic compound, 0.5 to 5 percent of surfactant and the balance of water. The electron mediator provided by the invention can effectively avoid gradual loss of the electron mediator in the test process, simultaneously directly establish a connection between the electron mediator and the soluble enzyme liquid, efficiently transfer electrons, effectively promote the enzyme catalytic reaction rate, and realize the following advantages of H 2 O 2 The sensor has the advantages of being not easy to accumulate, large in test linear range, low in detection voltage (0.05-0.25V vs Ag/AgCl), short in response time of the sensor and longer in service life.

Description

Electronic medium reagent of lactic acid biosensor and application thereof
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to an electronic medium reagent of a lactic acid biosensor and application thereof.
Background
Lactic acid is a common analyte in the food, chemical and pharmaceutical industries, and has important significance for its accurate detection. The general principle of lactic acid sensor for detecting lactic acid concentration is: lactic acid and dissolved oxygen in the sample are catalyzed to hydrogen peroxide in the presence of a lactate oxidase. Hydrogen peroxide produces an electrical current signal at the working electrode that is linear over a range of lactic acid concentrations.
The traditional lactic acid sensor uses natural oxygen as an electron acceptor, so that the high-concentration lactic acid is difficult to detect when the content of dissolved oxygen in a sample is insufficient, and the test linear range is smaller; and fluctuations in the concentration of oxygen in the test liquid will also affect the response signal. In addition, hydrogen peroxide under the system needs a larger input voltage (0.4V-0.6V vs Ag/AgCl) on a metal electrode wire (generally a platinum wire) to generate an ideal current signal, and under the voltage, some endogenous reducing substances (ascorbic acid, uric acid and the like) and electrochemical active substances (acetaminophen, acetylsalicylic acid and the like) and the like of the reducing drugs are easily oxidized on the surface of the electrode to generate an interference current signal, so that the detection accuracy is reduced. While a slower electrode reaction rate results in H 2 O 2 Accumulation in the enzyme layer reduces the service life of the sensor.
For this problem of oxygen sensitivity and poor interference resistance, it is reported that an additional film layer is added to the electrode: the diffusion limiting outer membrane serves to enhance oxygen transmission and the anti-interference inner membrane serves to reduce diffusion of interfering substances. However, such a solution generally has poor film thickness uniformity and poor adhesion, requires a relatively complicated process, and reduces response sensitivity and prolongs response time.
There are also reports of schemes for adding electron mediators to the system, but generally, soluble small molecule electron mediators are easily lost gradually during the test process, and immobilization of the electron mediators is also an additional technical problem. For insoluble or slightly soluble electron mediators, it is difficult to directly connect with soluble enzyme solutions, and the transfer of electrons between the active center of the enzyme and the electrode surface may be hindered by the formation of a film of the insoluble electron mediator alone.
Disclosure of Invention
Based on this, the present invention aims to provide an electronic mediator reagent of a lactate biosensor and an application thereof; the electron mediator can efficiently transfer electrons and reduce detection voltage; the enzyme catalytic reaction rate is effectively improved, and the response time of the sensor is shortened; and H is 2 O 2 Is not easy to accumulate and prolongs the service life of the sensor.
The specific technical scheme is as follows.
An electronic mediator reagent comprising the following components in percentage by mass: 0.1 to 5 percent of functionalized electrocatalyst, 0.5 to 5 percent of ferrocene dicarboxaldehyde, 1 to 10 percent of hydrophilic compound, 0.5 to 5 percent of surfactant and the balance of water.
In some embodiments, the electron mediator reagent comprises the following components in percentage by mass: 0.5 to 2.5 percent of functionalized electrocatalyst, 1 to 3 percent of ferrocene dicarboxaldehyde, 5 to 10 percent of hydrophilic compound, 0.5 to 2.5 percent of surfactant and the balance of water.
In some of these embodiments, the mass ratio of the functionalized electrocatalyst to ferrocene dicarboxaldehyde is 1: (2.5-3.5).
In some of these embodiments, it is preferred that the mass ratio of the functionalized electrocatalyst to ferrocene dicarboxaldehyde is 1:3.
in some of these embodiments, the functionalized electrocatalyst is selected from the group consisting of carboxyiized or aminated FeOOH, fe 3 O 4 、Fe 2 O 3 And MnO 2 At least one of them.
In some of these embodiments, the functionalized electrocatalyst is a carboxylated or aminated FeOOH.
In some of these embodiments, the functionalized electrocatalyst is an aminated FeOOH.
In some of these embodiments, the functionalizing agent used in preparing the carboxyl or aminated FeOOH is an aminosilane or a carboxysilane.
In some of these embodiments, the aminosilane is 3-aminopropyl trimethoxysilane; the carboxyl silane is 3- (triethoxysilyl) propyl succinic anhydride.
In some of these embodiments, it is preferred that the functionalized electrocatalyst is 3-aminopropyl trimethoxysilane functionalized FeOOH.
In some of these embodiments, the hydrophilic compound is selected from at least one of diethylene glycol, propylene glycol, and glycerol.
In some of these embodiments, the hydrophilic compound is diethylene glycol.
In some of these embodiments, the surfactant is selected from at least one of polyethylene glycol trimethylnonyl ether, polyethylene glycol t-octylphenyl ether (Triton X-100), and polyethylene glycol dodecyl ether (Brij L4).
In some of these embodiments, the surfactant is polyethylene glycol dodecyl ether.
The invention also provides an immobilized enzyme solution, which contains the electronic medium reagent with the mass percent of 0.5-10%.
In some embodiments, the immobilized enzyme solution comprises the following components in percentage by mass: the electronic mediator comprises 0.5-10% of the electronic mediator, 1-10% of lactic acid oxidase, 1-5% of an enzyme protecting agent, 0.1-5% of a high polymer and the balance of Hepes buffer solution.
In some of these embodiments, the enzyme protecting agent is BSA.
In some of these embodiments, the high molecular polymer is selected from at least one of PVP (polyvinylpyrrolidone), PVA (polyvinyl alcohol) and PEI (polyethylenimine).
In some of these embodiments, the Hepes buffer has a pH of 5.0 to 6.5.
The invention also provides application of the electronic mediator reagent or the immobilized enzyme solution in preparing a lactic acid sensor.
The invention also provides a working electrode for lactic acid detection, and the preparation method of the working electrode comprises the following steps: and uniformly coating the immobilized enzyme solution on a metal wire, and drying to obtain the working electrode.
In some embodiments, the drying is performed at 35-50 ℃ for 1-3 hours; preferably, the drying is performed in a drying oven at 37 ℃ for 2 hours.
In some of these embodiments, the wire material is gold, platinum, silver; preferably the material of the wire is gold.
In some of these embodiments, the wire has a diameter of 0.5 mm to 1mm and a length of 4 mm to 6 mm; preferably, the wire has a diameter of 0.6 mm and a length of 5 mm.
The invention obtains an electron mediator reagent capable of efficiently transferring electrons between the active center of the lactic acid oxidase and the surface of an electrode through optimization, and the electron mediator reagent is prepared by compounding a functionalized electrocatalyst, ferrocene dicarboxaldehyde, a hydrophilic compound and a surfactant according to a proper proportion. The inventor finds that when the electronic mediator is used for lactic acid detection, ferrocene dicarboxaldehyde can serve as an electronic mediator and has the function of a crosslinking agent: the ferrocene dicarboxaldehyde can transfer electrons between an enzyme active center and the surface of an electrode, so that the linear detection range of the electrode is improved; in addition, the functional group-containing electrocatalyst-ferrocene dicarboxaldehyde-enzyme can be combined with amino or carboxyl on enzyme, so that the functional group-containing electrocatalyst-ferrocene dicarboxaldehyde-enzyme is not easy to run off in a test, and simultaneously plays a role of a cross-linking agent, so that the enzyme catalytic reaction rate is further improved, and the response time of the sensor is shortened. The inventors have also found that the mass ratio of the functionalized electrocatalyst to ferrocene dicarboxaldehyde in the electron mediator reagent has a relatively important effect on obtaining a larger test linearity range, and that a larger test linearity range can be obtained with a suitable mass ratio.
The electron mediator provided by the invention can effectively avoid gradual loss of the electron mediator in the test process, simultaneously directly establish a connection between the electron mediator and the enzyme solution, efficiently transfer electrons, effectively promote the enzyme catalytic reaction rate, and realize the following advantages of H 2 O 2 Is not easy to accumulate and has a test linearity rangeThe sensor has the advantages of large circumference, low detection voltage (0.05V-0.25V vs Ag/AgCl), short response time and longer service life.
Drawings
FIG. 1 is a graph showing the results of testing lactic acid solutions of different concentrations with respect to working electrodes obtained by preparing an immobilized enzyme solution in examples 1 to 3.
FIG. 2 is a graph showing the results of testing lactic acid solutions of different concentrations with working electrodes obtained by preparing the immobilized enzyme solutions of comparative examples 1 to 4.
FIG. 3 is a graph showing the results of monitoring the working electrode obtained by preparing the immobilized enzyme solutions of example 1 and comparative examples 1 to 3 for a continuous sensitivity of 30 d.
FIG. 4 is an i-t curve of the working electrode obtained by preparing the immobilized enzyme solution of example 1, in which an interfering substance is added to a chronoamperometric test solution.
FIG. 5 is an i-t curve of the working electrode obtained by preparing the immobilized enzyme solution of comparative example 1 in which an interfering substance is added to a chronoamperometric test solution.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
Example 1
An electron mediator reagent of this embodiment comprises the following components: 0.01g of functionalized FeOOH, 0.03g of ferrocene dicarboxaldehyde, 0.1g of diethylene glycol, 0.01g of Brij L4 and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: 0.02g of FeOOH and 0.5g of 3-aminopropyl trimethoxysilane are taken and added into 3g of toluene, the mixture is stirred for 2h at 60 ℃, solid precipitate is obtained by filtration, and after three times of washing with toluene, the solid precipitate is dried in air, and functionalized FeOOH is obtained for standby.
The preparation method of the electronic mediator reagent comprises the following steps: (a) the mass ratio is 1:3 taking functionalized FeOOH and ferrocene dicarboxaldehyde (0.01 g and 0.03g respectively), and dispersing in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The implementation also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Dissolving 0.06g of lactic acid oxidase in the above solution to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Example 2
An electron mediator reagent of this embodiment comprises the following components: 0.01g of functionalized FeOOH, 0.01g of ferrocene dicarboxaldehyde, 0.1g of diethylene glycol, 0.01g of Brij L4 and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: 0.02g of FeOOH and 0.5g of 3-aminopropyl trimethoxysilane are taken and added into 3g of toluene, the mixture is stirred for 2h at 60 ℃, solid precipitate is obtained by filtration, and after three times of washing with toluene, the solid precipitate is dried in air, and functionalized FeOOH is obtained for standby.
The preparation method of the electronic mediator reagent comprises the following steps: (a) the mass ratio is 1:1 taking functionalized FeOOH and ferrocene dicarboxaldehyde (0.01 g and 0.01g respectively), and dispersing in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The implementation also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Dissolving 0.06g of lactic acid oxidase in the above solution to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Example 3
An electron mediator reagent of this embodiment comprises the following components: 0.01g of functionalized FeOOH, 0.05g of ferrocene dicarboxaldehyde, 0.1g of diethylene glycol, 0.01g of Brij L4 and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: 0.02g of FeOOH and 0.5g of 3-aminopropyl trimethoxysilane are taken and added into 3g of toluene, the mixture is stirred for 2h at 60 ℃, solid precipitate is obtained by filtration, and after three times of washing with toluene, the solid precipitate is dried in air, and functionalized FeOOH is obtained for standby.
The preparation method of the electronic mediator reagent comprises the following steps: (a) the mass ratio is 1:5, dispersing the functionalized FeOOH and ferrocene dicarboxaldehyde (0.01 g and 0.05g respectively) in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The implementation also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Dissolving 0.06g of lactic acid oxidase in the above solution to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Comparative example 1
The comparative example provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) 0.06g of lactic acid oxidase was dissolved in the above solution to obtain an enzyme solution.
Comparative example 2
This comparative example provides an electron mediator reagent comprising the following components: ferrocene 0.03g, diethylene glycol 0.1g, brij L40.01g, water 1g.
The preparation method of the electronic mediator reagent comprises the following steps: (a) taking 0.03g of ferrocene, and dispersing the ferrocene in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The comparative example also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Dissolving 0.06g of lactic acid oxidase and 0.03g of 25wt% glutaraldehyde water solution in the solution, and uniformly stirring to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Comparative example 3
This comparative example provides an electron mediator reagent comprising the following components: 0.01g of functionalized FeOOH, 0.03g of ferrocene, 0.1g of diethylene glycol, 0.01g of Brij L4 and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: 0.02g of FeOOH and 0.5g of 3-aminopropyl trimethoxysilane are taken and added into 3g of toluene, the mixture is stirred for 2h at 60 ℃, solid precipitate is obtained by filtration, and after three times of washing with toluene, the solid precipitate is dried in air, and functionalized FeOOH is obtained for standby.
The preparation method of the electronic mediator reagent comprises the following steps: (a) the mass ratio is 1:3 taking functionalized FeOOH and ferrocene (0.01 g and 0.03g respectively) and dispersing in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The comparative example also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Adding 0.06g of lactic acid oxidase and 0.03g of 25wt% glutaraldehyde water solution into the solution, and uniformly stirring to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Comparative example 4
An electron mediator reagent of this comparative example comprises the following components: ferrocene dicarboxaldehyde 0.03g, diethylene glycol 0.1g, brij L4.01 g, water 1g.
The preparation method of the electronic mediator reagent comprises the following steps: (a) Taking 0.03g of ferrocene dicarboxaldehyde, and dispersing in 1g of ultrapure water; (b) And adding 0.1g of diethylene glycol and 0.01g of Brij L4 into the reagent successively, and stirring uniformly to obtain the electronic medium reagent.
The implementation also provides an immobilized enzyme solution, and the preparation method of the immobilized enzyme solution comprises the following steps:
(a) Adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min to dissolve to obtain PVP mother solution for later use;
(b) Adding 0.5g PVP mother liquor into 1.5g Hepes buffer solution, uniformly stirring, and adding 0.05g BSA;
(c) Dissolving 0.06g of lactic acid oxidase in the above solution to obtain an enzyme solution;
(d) And (c) dispersing 0.2g of the electronic mediator in the enzyme solution obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme solution.
Working electrodes were prepared using the immobilized enzyme solutions in examples 1 to 3 and comparative examples 1 to 4 described above, and a lactic acid solution was tested.
Preparing a working electrode: the immobilized enzyme solutions in examples 1 to 3 and comparative examples 1 to 4 were uniformly coated on a wire, and dried in a drying oven at 37℃for 2 hours to obtain a working electrode. The diameter of the metal wire is 0.6 mm, the length of the metal wire is 5mm, and the materials are selected from: gold, platinum, silver, gold being preferred.
And (3) selecting an Ag/AgCl reference electrode, a platinum wire counter electrode and the self-made working electrode to construct a three-electrode system, and testing corresponding response currents of lactic acid solutions with different concentrations by using a chronoamperometry. The results are shown in FIGS. 1 to 4 and Table 1.
Table 1 test of the obtained linear range, sensitivity and R 2 Summary of (2)
As can be seen from Table 1 and FIGS. 1-2, the electron mediator reagents of the present invention have a better linear range and higher sensitivity. The ferrocene dicarboxaldehyde used by the invention not only has the function of an electron mediator, but also can be used as a cross-linking agent to be covalently combined with the functionalized FeOOH, so that the linear range and the sensitivity of the sensor are further improved. The inventors have also found that the mass ratio of functionalized FeOOH to ferrocene dicarboxaldehyde has a relatively important effect on obtaining a large test linearity range when the mass ratio of functionalized FeOOH to ferrocene dicarboxaldehyde in the electron mediator reagent is 1: and 3, the effect is optimal.
As can be seen from comparative examples 1 and 2, the conventional electron mediator ferrocene (comparative example 2) can accelerate electron transfer to some extent, improving the linear range and sensitivity of the sensor. However, due to the presence of the functionalized FeOOH as an electrocatalyst (example 1), the improvement in the electrochemical reaction rate of the sensor can be directly performed, thereby further improving the detection performance of the sensor.
As is clear from comparative examples 1 and 3, the present invention can improve the linearity range and sensitivity of the electrode better than the system using conventional electron mediator and crosslinking agent, because ferrocene dicarboxaldehyde itself is used as a mediator for transferring electrons, and also acts as a crosslinking agent between enzyme and electrocatalyst to form a covalent system of enzyme-electron mediator-electrocatalyst, which can shorten the transfer path of electrons between the active center of enzyme and the surface of electrode.
As can be seen from comparative examples 1 and 4, the combination of ferrocene dicarboxaldehyde and functionalized FeOOH can effectively expand the linear range of detection and improve the sensitivity of detection.
FIG. 3 is a graph showing the results of continuous 30d monitoring of the sensitivity of the working electrode obtained by preparing the immobilized enzyme solutions of example 1 and comparative examples 1-3, which shows that the detection sensitivity is remarkably lost within 7d without the electron mediator and without the crosslinking agent (comparative example 1), and the stability is poor. When glutaraldehyde was added to the immobilized enzyme solution as a cross-linking agent (comparative examples 2, 3), although stability was improved to some extent as compared with comparative example 1, the stability was still significantly lower than in example 1, probably because ferrocene dicarboxaldehyde was milder to the active center of the enzyme as compared with glutaraldehyde.
FIGS. 4 and 5 are i-t curves of the addition of an interfering substance to a chronoamperometric test solution for working electrodes obtained by preparing immobilized enzyme solutions of example 1 and comparative example 1, respectively. The results show that in the continuous four rounds of testing, example 1 remained essentially consistent with respect to 5mM lactic acid response current, with negligible sensor interference from 0.1mM ascorbic acid and 0.1mM uric acid, whereas comparative example 1 did not. This is because the detection can be performed at a lower voltage using the electronic mediator of the present invention, thereby improving the anti-interference effect.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

1. An electronic mediator reagent, which is characterized by comprising the following components in percentage by mass: 0.1-5% of functionalized electrocatalyst, 0.5-5% of ferrocene dicarboxaldehyde, 1-10% of hydrophilic compound, 0.5-5% of surfactant and the balance of water;
the functionalized electrocatalyst is selected from at least one of carboxyl or aminated FeOOH;
the mass ratio of the functionalized electrocatalyst to ferrocene dicarboxaldehyde is 1: (2.5 to 3.5).
2. The electronic mediator reagent of claim 1, wherein the electronic mediator reagent comprises the following components in mass percent: 0.5-2.5% of functionalized electrocatalyst, 1-3% of ferrocene dicarboxaldehyde, 5-10% of hydrophilic compound, 0.5-2.5% of surfactant and the balance of water.
3. The electronic mediator reagent of claim 1, wherein the hydrophilic compound is selected from at least one of diethylene glycol, propylene glycol and glycerol.
4. The electron mediator reagent of claim 1, wherein the surfactant is selected from at least one of polyethylene glycol trimethylnonyl ether, polyethylene glycol t-octyl phenyl ether and polyethylene glycol dodecyl ether.
5. An immobilized enzyme solution, characterized in that the immobilized enzyme solution contains 0.5-10% by mass of the electronic mediator reagent according to any one of claims 1-4.
6. The immobilized enzyme solution of claim 5, wherein the immobilized enzyme solution comprises the following components in percentage by mass: the electron mediator reagent according to any one of claims 1 to 4, wherein the electron mediator reagent comprises 0.5 to 10%, lactate oxidase 1 to 10%, enzyme protectant 1 to 5%, polymer 0.1 to 5%, and Hepes buffer solution in balance.
7. The immobilized enzyme solution of claim 6, wherein the enzyme protectant is BSA.
8. The immobilized enzyme solution according to claim 6, wherein the high molecular polymer is at least one selected from polyvinylpyrrolidone, polyvinyl alcohol and polyethyleneimine.
9. The use of an electronic mediator reagent according to any one of claims 1 to 4 or an immobilized enzyme solution according to any one of claims 5 to 8 in the preparation of a lactate sensor.
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