CN114717289A - 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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CN114717289A
CN114717289A CN202210351871.2A CN202210351871A CN114717289A CN 114717289 A CN114717289 A CN 114717289A CN 202210351871 A CN202210351871 A CN 202210351871A CN 114717289 A CN114717289 A CN 114717289A
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reagent
electronic
functionalized
electrocatalyst
ferrocene
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CN114717289B (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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    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
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    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
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Abstract

The invention provides an electronic medium reagent, which comprises the following components in percentage by mass: 0.1-5% of functionalized electrocatalyst, 0.5-5% of ferrocene diformaldehyde, 1-10% of hydrophilic compound and surface activity0.5 to 5 percent of sex agent and the balance of water. The electron mediator reagent provided by the invention can effectively avoid the gradual loss of the electron mediator in the test process, simultaneously directly establishes the relation between the electron mediator and the soluble enzyme solution, can efficiently transfer electrons, effectively improves the enzyme catalysis reaction rate, and has H2O2The sensor is not easy to accumulate, and has the advantages of large linear range of test, low detection voltage (0.05V-0.25V vs Ag/AgCl), short response time of the sensor and longer 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 accurate detection of the lactic acid. The general principle of lactate sensors for detecting lactate concentration is: the lactic acid and dissolved oxygen in the sample are catalyzed to generate hydrogen peroxide in the presence of lactate oxidase. The hydrogen peroxide produces a current signal at the working electrode that is linear with lactate concentration over a range.
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 dissolved oxygen content in a sample is insufficient, and the linear range of the detection is small; and fluctuations in the oxygen concentration in the test fluid will also affect the response signal. In addition, hydrogen peroxide in 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, electrochemical active substances such as some endogenous reducing substances (ascorbic acid, uric acid and the like) and reducing drugs (acetaminophen, acetylsalicylic acid and the like) are also easily oxidized on the surface of an electrode to generate an interference current signal, so that the detection accuracy is reduced. While slower electrode reaction rates may result in H2O2And the enzyme layer is accumulated, so that the service life of the sensor is shortened.
In response to the problem of poor oxygen sensitivity and interference resistance, it has been reported that an additional film layer is added to the electrode: the outer diffusion limiting film is used for improving the oxygen transmission rate and the inner interference resisting film is used for reducing the diffusion of interference substances. However, such a similar scheme generally has poor film thickness uniformity and poor adhesion, requires a complicated process, and decreases 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 easy to gradually lose in the test process, and the immobilization of the electron mediators also becomes an additional technical problem. The insoluble or slightly soluble electron mediator is difficult to directly establish connection with soluble enzyme liquid, and the film formation of the insoluble electron mediator alone can block the transfer of electrons between the enzyme activity center and the electrode surface.
Disclosure of Invention
Based on the above, the invention aims to provide an electronic mediator reagent of a lactate biosensor and application thereof; the electron mediator reagent can transfer electrons efficiently and reduce detection voltage; the enzyme catalysis reaction rate is effectively improved, and the response time of the sensor is shortened; and H2O2The accumulation is not easy, and the service life of the sensor is prolonged.
The specific technical scheme is as follows.
An electronic medium reagent comprises the following components in percentage by mass: 0.1-5% of functionalized electrocatalyst, 0.5-5% of ferrocene diformaldehyde, 1-10% of hydrophilic compound, 0.5-5% of surfactant and the balance of water.
In some embodiments, the electronic media reagent comprises the following components in percentage by mass: 0.5-2.5% of functionalized electrocatalyst, 1-3% of ferrocene diformaldehyde, 5-10% of hydrophilic compound, 0.5-2.5% 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 carboxyl or aminated FeOOH, Fe3O4、Fe2O3And MnO2At least one of (1).
In some of these embodiments, the functionalized electrocatalyst is a carboxyl or aminated FeOOH.
In some of these embodiments, the functionalized electrocatalyst is aminated FeOOH.
In some of these embodiments, the functionalizing agent used in preparing the carboxy or aminated FeOOH is an aminosilane or a carboxysilane.
In some of these embodiments, the aminosilane is 3-aminopropyltrimethoxysilane; the carboxyl silane is 3- (triethoxysilyl) propyl succinic anhydride.
In some of these embodiments, it is preferred that the functionalized electrocatalyst is 3-aminopropyltrimethoxysilane 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 liquid which contains the electronic medium reagent with the mass percentage content of 0.5-10%.
In some embodiments, the immobilized enzyme liquid comprises the following components in percentage by mass: 0.5-10% of the electronic medium reagent, 1-10% of lactate oxidase, 1-5% of an enzyme protective agent, 0.1-5% of a high molecular 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 (polyethyleneimine).
In some embodiments, the pH value of the Hepes buffer is 5.0-6.5.
The invention also provides application of the electronic medium reagent or the immobilized enzyme liquid in preparation of a lactic acid sensor.
The invention also provides a working electrode for detecting lactic acid, and the preparation method of the working electrode comprises the following steps: and (3) uniformly coating the immobilized enzyme liquid on a metal wire, and drying to obtain the working electrode.
In some embodiments, the drying is performed for 1 to 3 hours at 35 to 50 ℃; preferably, the drying is drying in a drying oven at 37 ℃ for 2 h.
In some of these embodiments, the material of the wire 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 electronic mediator reagent which can efficiently transfer electrons between the active center of the lactate oxidase and the surface of an electrode by optimization, and the electronic mediator reagent is prepared by compounding a functionalized electrocatalyst, ferrocene diformaldehyde, a hydrophilic compound and a surfactant in a proper proportion. The inventor finds that when the electron mediator is used for detecting lactic acid, ferrocene dicarboxaldehyde can be used as an electron mediator and has the function of a cross-linking agent: ferrocene diformaldehyde can transfer electrons between an enzyme activity center and the surface of an electrode, so that the linear range of electrode detection is improved; in addition, the compound can be combined with amino or carboxyl on enzyme, is not easy to run off in the test, and simultaneously plays the role of a cross-linking agent to form a complex of a functionalized electrocatalyst, namely ferrocene diformaldehyde and enzyme, so that the catalytic reaction rate of the enzyme is further improved, and the response time of the sensor is shortened. The inventor also finds that the mass ratio of the functionalized electrocatalyst to the ferrocene dicarboxaldehyde in the electronic mediator reagent has a relatively important influence on obtaining a larger test linear range, and a larger test linear range can be obtained by a proper mass ratio.
The electron mediator reagent provided by the invention can effectively avoid the gradual loss of the electron mediator in the test process, simultaneously directly establishes the relation between the electron mediator and the enzyme solution, can efficiently transfer electrons, effectively improves the enzyme catalysis reaction rate, and has H2O2The sensor is not easy to accumulate, and has the advantages of large linear range of test, low detection voltage (0.05V-0.25V vs Ag/AgCl), short response time of the sensor and longer service life.
Drawings
FIG. 1 is a graph showing the results of tests on working electrodes with different concentrations of lactic acid solutions, obtained by preparing immobilized enzyme liquids in examples 1 to 3.
FIG. 2 is a graph showing the results of tests on working electrodes prepared from immobilized enzyme solutions in comparative examples 1 to 4, with respect to lactic acid solutions of different concentrations.
FIG. 3 is a graph showing the results of monitoring the sensitivity of working electrodes obtained by preparing immobilized enzyme liquids in example 1 and comparative examples 1 to 3 for 30 days.
FIG. 4 is an i-t curve of a working electrode obtained by preparation of an immobilized enzyme liquid in example 1, to which an interfering substance was added in a test liquid by a chronoamperometry.
FIG. 5 is an i-t curve of the working electrode obtained by preparing the immobilized enzyme liquid of comparative example 1, with an interfering substance added to the test liquid by chronoamperometry.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals 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 non-exclusive inclusions. For example, a process, method, apparatus, article, or apparatus that comprises a list of steps is not limited to only those steps or modules recited, but may alternatively include other steps not recited, or may alternatively include other steps inherent to such process, method, article, or apparatus.
Example 1
The embodiment provides an electronic medium reagent, which comprises the following components: 0.01g of functionalized FeOOH, 0.03g of ferrocene dialdehyde, 0.1g of diethylene glycol, 40.01g of Brij Lj and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: adding 0.02g FeOOH and 0.5g 3-aminopropyltrimethoxysilane into 3g toluene, stirring for 2h at 60 ℃, filtering to obtain a solid precipitate, washing with toluene for three times, and drying in the air to obtain functionalized FeOOH for later use.
The preparation method of the electronic mediator reagent comprises the following steps: (a) according to the mass ratio of 1: 3 dispersing functional FeOOH and ferrocene dicarboxaldehyde (0.01 g and 0.03g respectively) in 1g of ultrapure water; (b) and adding 0.1g of diethylene glycol and Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The implementation also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid comprises the following steps:
(a) adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min, and dissolving to obtain PVP mother liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) dissolving 0.06g of lactate oxidase in the solution to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Example 2
The embodiment provides an electronic medium reagent, which comprises the following components: 0.01g of functionalized FeOOH, 0.01g of ferrocene dicarboxaldehyde, 0.1g of diethylene glycol, Brij L40.01g and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: adding 0.02g FeOOH and 0.5g 3-aminopropyltrimethoxysilane into 3g toluene, stirring for 2h at 60 ℃, filtering to obtain a solid precipitate, washing with toluene for three times, and drying in the air to obtain functionalized FeOOH for later use.
The preparation method of the electronic mediator reagent comprises the following steps: (a) according to the mass ratio of 1: 1 dispersing functional FeOOH and ferrocene dicarboxaldehyde (0.01 g and 0.01g respectively) in 1g of ultrapure water; (b) and adding 0.1g of diethylene glycol and Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The implementation also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid comprises the following steps:
(a) adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min, and dissolving to obtain PVP mother liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) dissolving 0.06g of lactate oxidase in the solution to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Example 3
The embodiment provides an electronic medium reagent, which comprises the following components: 0.01g of functionalized FeOOH, 0.05g of ferrocene dicarboxaldehyde, 0.1g of diethylene glycol, Brij L40.01g and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: adding 0.02g FeOOH and 0.5g 3-aminopropyltrimethoxysilane into 3g toluene, stirring for 2h at 60 ℃, filtering to obtain a solid precipitate, washing with toluene for three times, and drying in the air to obtain functionalized FeOOH for later use.
The preparation method of the electronic mediator reagent comprises the following steps: (a) according to the mass ratio of 1: 5 dispersing 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 Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The implementation also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid comprises the following steps:
(a) adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min, and dissolving to obtain PVP mother liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) dissolving 0.06g of lactate oxidase in the solution to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Comparative example 1
The comparative example provides an immobilized enzyme liquid, which is prepared by the following steps:
(a) adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min, and dissolving to obtain PVP mother liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) 0.06g of lactate oxidase was dissolved in the above solution to obtain an enzyme solution.
Comparative example 2
This comparative example provides an electron mediator agent comprising the following components: 0.03g of ferrocene, 0.1g of diethylene glycol, Brij L40.01g and 1g of water.
The preparation method of the electronic mediator reagent comprises the following steps: (a) taking 0.03g of ferrocene, and dispersing in 1g of ultrapure water; (b) and adding 0.1g of diethylene glycol and Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The comparative example also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid 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 liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) dissolving 0.06g of lactate oxidase and 0.03g of 25 wt% glutaraldehyde aqueous solution in the solution, and uniformly stirring to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Comparative example 3
This comparative example provides an electron mediator agent comprising the following components: 0.01g of functionalized FeOOH, 0.03g of ferrocene, 0.1g of diethylene glycol, Brij L40.01g and 1g of water.
The preparation method of the functionalized FeOOH comprises the following steps: adding 0.02g FeOOH and 0.5g 3-aminopropyltrimethoxysilane into 3g toluene, stirring for 2h at 60 ℃, filtering to obtain a solid precipitate, washing with toluene for three times, and drying in the air to obtain functionalized FeOOH for later use.
The preparation method of the electronic mediator reagent comprises the following steps: (a) according to the mass ratio of 1: 3 dispersing functionalized FeOOH and ferrocene (0.01 g and 0.03g respectively) in 1g of ultrapure water; (b) and adding 0.1g of diethylene glycol and Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The comparative example also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid comprises the following steps:
(a) adding 0.04g of PVP into 1.96g of ultrapure water, stirring at 90 ℃ for 30min, and dissolving to obtain PVP mother liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) adding 0.06g of lactate oxidase and 0.03g of 25 wt% glutaraldehyde aqueous solution into the solution, and uniformly stirring to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Comparative example 4
The comparative example is an electron mediator agent comprising the following components: 0.03g of ferrocene diformaldehyde, 0.1g of diethylene glycol, 40.01g of Brij Lj and 1g of water.
The preparation method of the electronic mediator reagent comprises the following steps: (a) taking 0.03g of ferrocene dimethyl aldehyde by mass, and dispersing in 1g of ultrapure water; (b) and adding 0.1g of diethylene glycol and Brij L40.01g into the reagents in sequence, and uniformly stirring to obtain the electron mediator reagent.
The implementation also provides an immobilized enzyme liquid, and the preparation method of the immobilized enzyme liquid 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 liquor for later use;
(b) adding 0.5g of PVP mother liquor into 1.5g of Hepes buffer solution, stirring uniformly, and adding 0.05g of BSA;
(c) dissolving 0.06g of lactate oxidase in the solution to obtain an enzyme solution;
(d) and (c) dispersing 0.2g of the electronic mediator reagent in the enzyme liquid obtained in the step (c), and uniformly stirring to obtain the immobilized enzyme liquid.
Working electrodes were prepared using the immobilized enzyme liquids in examples 1 to 3 and comparative examples 1 to 4 described above, and tests were performed using a lactic acid solution.
Preparing a working electrode: the immobilized enzyme liquids in examples 1 to 3 and comparative examples 1 to 4 were uniformly coated on a metal 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 metal wire can be made of the following materials: gold, platinum, silver, preferably gold.
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 the corresponding response currents of the lactic acid solutions with different concentrations by using a chronoamperometry. The results are shown in FIGS. 1 to 4 and Table 1.
TABLE 1 Linear Range, sensitivity and R obtained by testing2Summary of (1)
Figure BDA0003580906540000071
Figure BDA0003580906540000081
As can be seen from Table 1 and FIGS. 1-2, the electron mediator reagent of the present invention has a better linear range and higher sensitivity. The ferrocene diformaldehyde used in the invention not only has the function of an electronic mediator, but also can be covalently combined with functionalized FeOOH as a cross-linking agent, thereby further improving the linear range and the sensitivity of the sensor. The inventor also found that the mass ratio of the functionalized FeOOH to the ferrocene dicarboxaldehyde has a more important influence on obtaining a larger linear range of the test, when the mass ratio of the functionalized FeOOH to the ferrocene dicarboxaldehyde in the electronic medium reagent is 1: and 3, the effect is optimal.
Comparing example 1 and comparative example 2, it can be seen that the conventional electron mediator ferrocene (comparative example 2) can accelerate electron transfer to a certain extent, and improve the linear range and sensitivity of the sensor. However, due to the presence of the functionalized FeOOH as an electrocatalyst (example 1), the electrochemical reaction rate of the sensor can be directly improved, so that the detection performance of the sensor is further improved.
Comparing example 1 and comparative example 3, it can be seen that the linear range and sensitivity of the electrode can be better improved by using ferrocene diformaldehyde with both functions of an electronic mediator and a crosslinking agent compared with a system using a conventional electronic mediator and a crosslinking agent, which is probably because ferrocene diformaldehyde itself is used as a medium for transferring electrons and has a crosslinking effect between the enzyme and the electrocatalyst to form a covalent system of the enzyme-electronic mediator-electrocatalyst, and the transfer path of electrons between the enzyme activity center and the electrode surface can be shortened.
As can be seen from comparative example 1 and comparative example 4, the combined use 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 monitoring the sensitivity of the working electrode obtained by preparing the immobilized enzyme liquid of example 1 and comparative examples 1 to 3 for 30d continuously, and the results show that the detection sensitivity is remarkably lost within 7d and the stability is poor without the electron mediator and without the crosslinking agent (comparative example 1). When glutaraldehyde is added to the immobilized enzyme solution as a cross-linking agent (comparative examples 2 and 3), although the stability is improved to some extent compared with that of comparative example 1, the stability is still obviously lower than that of example 1, probably because ferrocene diformaldehyde is milder to the active center of the enzyme compared with glutaraldehyde.
FIGS. 4 and 5 are i-t curves of working electrodes obtained by preparation of immobilized enzyme liquids of example 1 and comparative example 1, respectively, for adding an interfering substance to a chronoamperometric test solution. The results show that in the four successive tests, the response current of example 1 to 5mM of lactic acid is substantially consistent, the interfering effect of 0.1mM of ascorbic acid and 0.1mM of uric acid on the sensor is negligible, while comparative example 1 has no such effect. This is because the use of the electronic mediator reagent of the present invention allows detection at lower voltages, thereby enhancing interference rejection.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (14)

1. The electronic medium reagent is characterized by comprising the following components in percentage by mass: 0.1-5% of functionalized electrocatalyst, 0.5-5% of ferrocene diformaldehyde, 1-10% of hydrophilic compound, 0.5-5% of surfactant and the balance of water.
2. The electronic media reagent of claim 1, wherein the electronic media reagent comprises the following components in percentage by mass: 0.5-2.5% of functionalized electrocatalyst, 1-3% of ferrocene diformaldehyde, 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 mass ratio of the functionalized electrocatalyst to ferrocene dicarboxaldehyde is from 1: (2.5-3.5).
4. The electronic mediator agent of claim 1, wherein the functionalized electrocatalyst is selected from the group consisting of carboxyl or aminated FeOOH, Fe3O4、Fe2O3And MnO2At least one of (1).
5. The electronic mediator reagent of claim 4, wherein the functionalized electrocatalyst is a carboxyl group or an aminated FeOOH.
6. The electronic mediator agent of claim 5, wherein the functionalizing agent used in the preparation of the carboxy or aminated FeOOH is an aminosilane or a carboxysilane.
7. The electronic mediating agent of claim 6, wherein the aminosilane is 3-aminopropyltrimethoxysilane; the carboxyl silane is 3- (triethoxysilyl) propyl succinic anhydride.
8. The electronic mediator agent of claim 1, wherein the hydrophilic compound is selected from at least one of the group consisting of diethylene glycol, propylene glycol, and glycerol.
9. The electronic mediator agent of claim 1, wherein the surfactant is selected from at least one of polyethylene glycol trimethyl nonyl ether, polyethylene glycol t-octyl phenyl ether, and polyethylene glycol dodecyl ether.
10. An immobilized enzyme liquid, which contains the electronic mediator reagent according to any one of claims 1 to 9 in an amount of 0.5 to 10% by mass.
11. The immobilized enzyme liquid of claim 10, comprising the following components in percentage by mass: the electronic vector reagent of any one of claims 1 to 9, wherein the reagent comprises 0.5 to 10 percent of lactate oxidase, 1 to 10 percent of enzyme protective agent, 0.1 to 5 percent of high molecular polymer, and the balance of Hepes buffer solution.
12. The immobilized enzyme liquid of claim 11, wherein the enzyme protecting agent is BSA.
13. The immobilized enzyme liquid of claim 11, wherein the high molecular polymer is at least one selected from polyvinylpyrrolidone, polyvinyl alcohol, and polyethyleneimine.
14. Use of the electronic mediator reagent according to any one of claims 1 to 9 or the immobilized enzyme liquid according to any one of claims 10 to 13 in the preparation of a lactate sensor.
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