CN110938667A - Enzyme electrode, biosensor, preparation method and application thereof - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- 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
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90212—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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)
Abstract
The invention discloses an enzyme electrode and a biosensor based on a coenzyme and an electron carrier which respectively form conjugates with coenzyme-dependent enzymes. Firstly, enzyme and coenzyme or an electronic carrier are respectively crosslinked and combined, then the enzyme and the coenzyme or the electronic carrier are fixed on an electrode together, when the coenzyme-dependent enzyme catalyzes a substance to be detected, the coenzyme is changed into a reduction state from an oxidation state, then the reduction coenzyme is changed into the oxidation state through the electronic carrier, meanwhile, the electronic carrier is changed into the reduction state from the oxidation state, voltage is set according to the oxidation potential of the electronic carrier, and corresponding oxidation current is obtained, so that the detection of a substrate is realized. The biosensor can effectively avoid the operation that coenzyme is required to be added when the conventional sensor detects coenzyme-dependent enzyme as a catalyst to catalyze a substance to be detected, and can ensure that the detection is free of reagent; the immobilized coenzyme/electron carrier and enzyme conjugate has stable structure, is not easy to lose and can be used repeatedly; and has a higher response value and a more sensitive detection limit.
Description
Technical Field
The invention belongs to the field of bioelectrochemistry, and particularly relates to an enzyme electrode and a biosensor based on a coenzyme/electron carrier and enzyme conjugate, and a preparation method and application thereof.
Background
Coenzymes are utilized in nature as secondary substrates by many enzymes. According to incomplete statistics, many enzymes are coenzyme-dependent enzymes, which are required to act as hydrogen or electron transporters during the reaction. Taking the common coenzymes I and II as examples, NAD+Or NADP+Is reduced to its reduced form NADH or NADPH. The determination of NADH or NADPH produced during the reaction is the basis for the construction of this biosensor. However, there are many problems in the current biosensors. Firstly, the direct electrochemical oxidation of NAD (P) H on the electrode surface generally comprises the following process
NAD(P)H→NAD(P)H+·+e-(1)
NAD(P)H+·→NAD(P)·+H+(2)
NAD(P)·→NAD(P)++e- (3)
Wherein the first step of the reaction is a rate-controlling step in the whole reaction. Experiments have shown that the transfer of electrons between nad (p) H and bare electrodes requires very high overpotentials (typically above 1.0V), which often lead to electrode contamination, enzyme inactivation, coenzyme dimers, etc. A common solution is to introduce an electron carrier. An electron carrier is a chemical substance that participates in a redox reaction and can accelerate the overall reaction. An ideal electron carrier should have a lower redox potential, higher chemical stability and faster electron transfer rate. The oxidation principle of NAD (P) H with the participation of electron carriers is as follows:
NAD(P)H+Mox→NAD++Mred(4)
Mred→Mox+e-(5)
secondly, electrochemical oxidation of the coenzyme can also be achieved using NADH oxidase. Diaphorase is the most common NADH oxidase, and can be used in naphthoquinones, etcThe oxidation of NADH is first effected in the presence of an electron carrier. Compared with the method of directly introducing an electronic carrier, the addition of the diaphorase can obviously improve the reaction speed of the electrode surface catalysis. In 2003, Antiochia et al studied the reaction kinetics of diaphorase catalyzed coenzyme oxidation reaction by using methylaminophenol as an electron carrier, and found that the electrocatalytic oxidation rate of NADH of diaphorase is significantly increased in the presence of the electron carrier. Thus diaphorase and various dehydrogenases can construct biosensors. However, in such sensors, it is generally necessary to add an additional coenzyme, such as NAD+Or NADP+The coenzyme is expensive, and the single use of the coenzyme causes the increase of the analysis cost, and has poor stability, which is not beneficial to practical application. In addition, many documents report that coenzymes, electron carriers, etc. are co-immobilized on an electrode together with enzymes by crosslinking, embedding, etc., but such methods have low reaction speed, measurement insensitivity, and low response because the co-immobilization method is complicated, difficult to control, and limits the effective transfer of the above substances.
Disclosure of Invention
Aiming at the defects of the existing biosensor, the invention provides an enzyme electrode based on cross-linking combination of coenzyme-dependent enzyme and coenzyme/electronic carrier, a biosensor, a preparation method and application thereof. The enzyme electrode and the biosensor have the advantages of higher detection limit and sensitivity, convenient use, cost reduction and the like.
In order to solve the above problems, the present invention provides the following technical solutions:
according to one aspect of the present invention, there is provided an enzyme electrode comprising a base electrode having at least one of a first conjugate and a second conjugate immobilized on a surface thereof, the first conjugate being a conjugate of a coenzyme and a coenzyme-dependent enzyme, and the second conjugate being a conjugate of an electron carrier and an enzyme.
According to an embodiment of the invention, said at least one shall be understood as either or both. When present, the immobilization may be either separately immobilized or co-immobilized with the first cross-linked conjugate and the second cross-linked conjugate.
According to an embodiment of the invention, the coenzyme may comprise a reactive functional group.
According to an embodiment of the present invention, the electron carrier may comprise a reactive functional group.
According to an embodiment of the present invention, the coenzyme may be selected from at least one of coenzyme I, coenzyme II, riboflavin, thiamine, vitamin B6, vitamin B12, biotin, tetrahydrofolic acid, pantothenic acid, coenzyme a, coenzyme Q, lipoic acid, S-adenosylmethionine, glutathione, and nicotinamide mononucleotide, 1-benzyl 3-carbamoyl-pyridine, 1-methylnicotinamide, and the like.
According to an embodiment of the present invention, the coenzyme-dependent enzyme may be at least one selected from the group consisting of alcohol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, shikimate dehydrogenase, α -ketoglutarate dehydrogenase, glycerol dehydrogenase, 3-glyceraldehyde phosphate dehydrogenase, dihydrolipoamide dehydrogenase, choline dehydrogenase, carnitine 3-dehydrogenase, glutamic-pyruvic transaminase, glutamic-oxaloacetate transaminase, guanidinoacetate methyltransferase, histone methyltransferase, choline acetylase, phosphotransacetylase, lipoic acid acetyltransferase, acetyl CoA carboxylase, pyruvate carboxylase, phenylalanine carboxylase, 1, 3-propanediol oxidoreductase, 3-hydroxybutyrate dehydrogenase, lysine decarboxylase, pyruvate oxidase, transketolase, NADH-Q reductase, methylmalonyl-CoA mutase, glyoxal, glutamate dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, xanthate dehydrogenase, xylose dehydrogenase, xylitol dehydrogenase, ribose dehydrogenase, galactose dehydrogenase, sorbitol dehydrogenase, mannitol dehydrogenase, rhamnose dehydrogenase, and inositol dehydrogenase.
The enzyme bound to the electron carrier may also be selected from one or more of the above-mentioned coenzyme-dependent enzymes, and from other enzymes known in the art that require interaction with hydrogen or an electron carrier during the reaction.
According to an embodiment of the present invention, the electron carrier may be any other carrier having an electron transfer function known in the art, for example, at least one selected from phenothiazines such as thionins, flavins, naphthoquinones, neutral red, methyl viologen, benzyl viologen, methylene blue, methyl orange, methyl red, methyl green, pyocyanin, 1-carboxamide phenazine, and humus analogs such as anthraquinone-2, 6-disulfonic acid.
According to an embodiment of the present invention, the reactive functional groups are the same as or different from each other, and may be, for example, independently selected from at least one of carboxyl, maleimide, amino, epoxy, thiol, phenyl or aldehyde functional groups.
According to an embodiment of the present invention, the base electrode may be at least one of a gold electrode, a glassy carbon electrode, a magnetic electrode, a screen-printed electrode, conductive glass, a carbon paste electrode, a graphite electrode, a carbon felt, and a carbon cloth.
According to a second aspect of the present invention, there is provided a method for producing the enzyme electrode, comprising immobilizing at least one of the first conjugate and the second conjugate on a surface of a base electrode to obtain the enzyme electrode.
Further, the preparation method may include cross-linking and binding the coenzyme to the coenzyme-dependent enzyme to obtain a first cross-linked conjugate; and/or crosslinking and combining the electron carrier and the enzyme to obtain a second crosslinking combination.
According to an embodiment of the present invention, specifically, the preparation method comprises the steps of:
(1) modifying coenzyme with active functional groups to obtain a coenzyme compound, mixing the coenzyme compound with coenzyme-dependent enzyme, and performing crosslinking combination to obtain a first crosslinking combination of the coenzyme and the coenzyme-dependent enzyme;
(2) modifying the electronic carrier by using an active functional group, mixing with an enzyme, and carrying out cross-linking combination to obtain a second cross-linking combination of the enzyme and the electronic carrier;
(3) immobilizing at least one of the first cross-linked conjugate or the second cross-linked conjugate to a surface of a base electrode to form an enzyme electrode.
According to embodiments of the present invention, the coenzyme or the electron carrier may be modified at a site of the coenzyme or the electron carrier by a reaction known in the art, and an appropriate functional group or spacer may be introduced to generate a modified coenzyme or electron carrier. Preferably, the active functional group modified on the coenzyme or the electron carrier is at least one of a carboxyl, maleimide, amino, epoxy, mercapto, benzene ring or aldehyde functional group.
According to an embodiment of the present invention, the active functional group may be cross-linked to the coenzyme-dependent enzyme by at least one of coupling an amide group, such as a maleimide group, to a thiol group, Huisgen cycloaddition, biotin or streptavidin, or the like.
According to an embodiment of the present invention, the immobilization of the first cross-linked conjugate or the second cross-linked conjugate to the base electrode may be achieved by any method known in the art suitable for immobilizing an enzyme to an electrode, including, but not limited to, at least one of a physical adsorption method, an ion adsorption method, an entrapment method, a covalent coupling method, and a cross-linking method.
According to a third aspect of the present invention, there is also provided a biosensor comprising the enzyme electrode of the first aspect as a working electrode.
According to an embodiment of the invention, the biosensor further comprises a reference electrode and a counter electrode. According to an embodiment of the invention, the reference electrode may be selected from a saturated calomel electrode, a hydrogen electrode, a silver | silver chloride electrode or a mercury | mercury oxide electrode.
According to an embodiment of the present invention, the counter electrode may be selected from a platinum wire electrode or a carbon electrode.
According to a fourth aspect of the present invention there is also provided the use of a biosensor as defined in the third aspect for detecting a biological sample or substrate.
The biosensor according to the present invention, wherein the enzyme is first cross-linked to the coenzyme or the electron carrier, respectively. When the coenzyme is co-immobilized on the electrode, when the coenzyme-dependent enzyme catalyzes a substance to be detected, the coenzyme is changed from the oxidation state to the reduction state, then the coenzyme in the reduction state is changed into the oxidation state through the electronic carrier, meanwhile, the electronic carrier is changed from the oxidation state to the reduction state, voltage is set according to the oxidation potential of the electronic carrier, and corresponding oxidation current is obtained, so that the detection of the substrate is realized. When one of the cross-linked conjugates is fixed on the electrode, the corresponding current change is generated by the oxidation-reduction state change of the coenzyme or the oxidation-reduction state change of the electron carrier, so that the detection of the substrate is realized. In particular, the sample or substrate concentration may be determined by means of cyclic voltammetry and/or chronoamperometry.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention connects coenzyme dependent enzyme with coenzyme, connects electronic carrier with enzyme to make two enzyme combinations, which shortens the distance between coenzyme and enzyme and between electronic carrier and enzyme, improves the catalytic efficiency of coenzyme and enzyme and between electronic carrier and enzyme, reduces the mass transfer internal resistance, and can effectively improve current response, reduce detection limit and improve detection sensitivity after making biosensor.
(2) In the actual operation, the coenzyme and the electronic carrier can be recycled without being added continuously, and the cost is reduced. The sensor belongs to a non-reagent sensor, and the immobilized enzyme conjugate is not easy to run off and can be used repeatedly.
Drawings
FIG. 1 shows the preparation principle and process of enzyme electrode as working electrode in biosensor.
FIG. 2 is a representation of the UV-VIS absorption spectrum of glucose-6-phosphate dehydrogenase in combination with coenzyme NAD. In the experiment, NAD was converted to NADH using formate dehydrogenase. Curve 1 is glucose-6-phosphate dehydrogenase, curve 2 is a polyethylene glycol PEG and NAD complex, and curve 3 is glucose-6-phosphate dehydrogenase and NAD conjugate.
FIG. 3 is a representation of the UV-VIS absorption spectrum of the conjugate of diaphorase and the electron carrier benzyl viologen. In the experiment, benzyl viologen was converted in the oxidized state to the reduced state with sodium dithionate. Curve 1 is diaphorase, curve 2 is benzyl viologen and curve 3 is the conjugate of diaphorase and benzyl viologen.
FIG. 4 is a drawing showingThe diaphorase and benzyl viologen conjugate catalyze NADH to generate an electrogram. Wherein curve 1 is free diaphorase and benzyl viologen, and the current density reaches 26 muA/cm2Curve 2 shows the combination of diaphorase and benzyl viologen, and the current density produced is 38 μ A/cm2。
FIG. 5 is a graph of the current generated by glucose-6-phosphate dehydrogenase in combination with NAD to catalyze the production of glucose-6-phosphate. Where curve 1 is the current density produced by the free enzyme and curve 2 is the current density produced by the conjugate.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following embodiments are provided to further illustrate the technical solutions of the present invention. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1: biosensor based on cross-linked conjugates of coenzymes and enzymes
10mM NAD+Reacting with 10mM carboxyphenylboronic acid with shaking at room temperature for 2h, activating with 50mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50mM N-hydroxysuccinimide at room temperature for 1h, adjusting pH to 7.4, adding 10mM amino-and carboxyl-modified polyethylene glycol, and reacting at 37 ℃ for 2h to obtain carboxyl-modified coenzyme NAD+。
Subsequently, 50mM of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50mM of N-hydroxysuccinimide were added thereto, and the mixture was activated at room temperature for 1 hour, adjusted to pH 7.4, and then 100. mu.M of glucose-6-phosphate dehydrogenase was added thereto, and reacted at 37 ℃ for 2 hours to obtain a conjugate of glucose-6-phosphate dehydrogenase and coenzyme.
Glucose-6-phosphate dehydrogenase was dialyzed against the coenzyme conjugate at 4 ℃ overnight, and concentrated by ultrafiltration.
A porous carbon felt electrode of 0.5mm multiplied by 0.5mm is used as a substrate electrode, 200uL of 50M glucose-6-phosphate dehydrogenase and coenzyme conjugate is dripped on the electrode, and the electrode is naturally dried. An enzyme electrode is prepared by physical adsorption and is used as a working electrode in the biosensor.
As can be seen from FIG. 5, the current density generated by the coenzyme and coenzyme-dependent enzyme cross-linked conjugate is 2 to 3 times that of the same free enzyme, demonstrating that the conjugate can greatly improve the detection limit and sensitivity of the biosensor.
Example 2: biosensor based on electron carrier and enzyme conjugate
10mM 4,4' -bipyridine and 21mmol 4- (bromomethyl) phenylacetic acid are added into 30mL dimethylformamide, and after refluxing for 6 hours at 120 ℃, light yellow precipitate, namely carboxyl modified electron carrier-benzyl viologen is obtained. The precipitate was dried in vacuo for 12 hours and the powder was stored in a refrigerator at 4 ℃.
Dissolving carboxyl modified benzyl viologen in 0.2M phosphate buffer (pH 5.6) to prepare 20mM benzyl viologen solution, adding 1M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1M N-hydroxysuccinimide to activate at room temperature for 1h, adjusting the pH to 7.4, adding 200 mu M diaphorase, and reacting at 37 ℃ for 2h to obtain the conjugate of the diaphorase and the benzyl viologen.
Diaphorase and benzyl viologen were dialyzed overnight at 4 ℃ and concentrated by ultrafiltration.
Continuing to add 1M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1M N-hydroxysuccinimide to 200 μ M diaphorase and benzyl viologen conjugate, activating at room temperature for 1h, adjusting pH to 7.4, and adding 1mg of amino-modified magnetic nanoparticle Fe3O4-NH2Incubating at 37 ℃ for 2h, centrifuging and collecting precipitate, and recording the precipitate as Fe3O4An enzyme conjugate.
Mixing Fe3O4The enzyme conjugate was dissolved in 0.1M PBS (pH 7.4) at a concentration of 0.1 mg/mL.
Using magnetic glassy carbon electrode as substrate electrode, 10uL of 0.1mg/mL Fe3O4And dripping the enzyme conjugate onto a magnetic glassy carbon electrode, and naturally drying. An enzyme electrode is prepared by magnetic adsorption and is used as a working electrode in the biosensor.
Diaphorase and benzyl viologen conjugate catalysisThe NADH production current is shown in FIG. 4. As can be seen from FIG. 4, curve 1 represents the free diaphorase and benzyl viologen, resulting in a current density of only 26 μ A/cm2Left and right, and curve 2 shows that the combination of diaphorase and benzyl viologen generates current density of 38 μ A/cm2Therefore, the current value of the diaphorase and the electronic carrier benzyl viologen is greatly increased by chemical coupling, and the detection limit and the sensitivity of the biosensor can be greatly improved.
Example 3: biosensor based on coenzyme and enzyme conjugate
Activation of 10mM maleimide and carboxyl modified polyethylene glycol with 50mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50mM N-hydroxysuccinimide at room temperature for 1h, followed by 10mM NAD+Incubation at 37 ℃ for 2h (pH 7.4) to obtain the maleimide-modified coenzyme NAD+。
100. mu.M of lactate dehydrogenase with cysteine mutated on the surface thereof was mixed with 1mM of maleimide coenzyme NAD+Incubation at 4 ℃ for 12h, ultrafiltration to remove unbound NAD+To prepare a conjugate of lactate dehydrogenase and a coenzyme.
The chitosan was dissolved in 2.0mol/L acetic acid solution to prepare a 0.5% (w/v) chitosan solution, which was then adjusted to pH 5.0 with NaOH solution and stored in a refrigerator for further use. The gold electrode was pretreated and 1. mu.L of glutaraldehyde (2.5%) and 5. mu.L of the lactate dehydrogenase and coenzyme conjugate were successively added dropwise to the surface of the electrode. The complex of lactate dehydrogenase and coenzyme is fixed on a gold electrode through glutaraldehyde to prepare an enzyme electrode which is used as a working electrode in a biosensor.
Claims (10)
1. An enzyme electrode, characterized in that: the electrode comprises a base electrode, wherein at least one of a first cross-linked conjugate and a second cross-linked conjugate is fixed on the surface of the base electrode, the first cross-linked conjugate is a cross-linked conjugate formed by coenzyme and coenzyme-dependent enzyme, and the second cross-linked conjugate is a cross-linked conjugate formed by an electron carrier and the enzyme.
2. The enzyme electrode according to claim 1, characterized in that: the immobilization is either a separate immobilization or co-immobilization of the first and second cross-linked conjugates.
3. The enzyme electrode according to claim 1 or 2, characterized in that: the coenzyme or the electron carrier includes a reactive functional group.
Preferably, the reactive functional groups, which are the same or different from each other, are independently selected from at least one of carboxyl, maleimide, amino, epoxy, thiol, phenyl or aldehyde functional groups.
4. The enzyme electrode according to any one of claims 1 to 3, characterized in that: the coenzyme is at least one of coenzyme I, coenzyme II, riboflavin, thiamine, vitamin B6, vitamin B12, biotin, tetrahydrofolic acid, pantothenic acid, coenzyme A, coenzyme Q, lipoic acid, S-adenosylmethionine, glutathione, and artificial coenzymes such as nicotinamide mononucleotide, 1-benzyl 3-carbamoyl-pyridine, and 1-methylnicotinamide.
Preferably, the coenzyme-dependent enzyme or the enzyme bound to the electron carrier is independently selected from at least one of alcohol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, shikimate dehydrogenase, α -ketoglutarate dehydrogenase, glycerol dehydrogenase, 3-glyceraldehyde phosphate dehydrogenase, dihydrolipoamide dehydrogenase, choline dehydrogenase, carnitine 3-dehydrogenase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, guanidinoacetate methyltransferase, histone transmethylase, choline acetylase, phosphate transacetylase, lipoic acid acetyltransferase, acetyl CoA carboxylase, pyruvate carboxylase, phenylalanine carboxylase, 1, 3-propanediol oxidoreductase, 3-hydroxybutyrate dehydrogenase, lysine decarboxylase, pyruvate oxidase, transketolase, NADH-Q reductase, methylmalonyl-CoA mutase, glutamate dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, xanthate dehydrogenase, xylose dehydrogenase, xylitol dehydrogenase, galactose dehydrogenase, arabinose dehydrogenase, sorbitol dehydrogenase, rhamnose dehydrogenase, mannuronate dehydrogenase, and inositol dehydrogenase.
Preferably, the electron carrier is selected from at least one of thionine, flavin, naphthoquinone, neutral red, methyl viologen, benzyl viologen, methylene blue, methyl orange, methyl red, methyl green, pyocin, phenazine, and anthraquinone-2, 6-disulfonic acid.
5. The enzyme electrode according to any one of claims 1 to 4, characterized in that: the basic electrode is at least one of a gold electrode, a glassy carbon electrode, a magnetic electrode, a screen printing electrode, conductive glass, a carbon paste electrode, a graphite electrode, a carbon felt and carbon cloth.
6. The method for producing an enzyme electrode according to any one of claims 1 to 5, characterized in that: comprising immobilizing at least one of the first cross-linked conjugate and the second cross-linked conjugate on a surface of a base electrode to obtain the enzyme electrode;
for example, the preparation method may further include cross-linking and binding the coenzyme to the coenzyme-dependent enzyme to obtain a first cross-linked conjugate; and/or crosslinking and combining the electron carrier and the enzyme to obtain a second crosslinking combination.
7. The method of claim 6, wherein:
(1) modifying coenzyme with active functional groups to obtain a coenzyme compound, mixing the coenzyme compound with coenzyme-dependent enzyme, and performing crosslinking combination to obtain a first crosslinking combination of the coenzyme and the coenzyme-dependent enzyme;
(2) modifying the electronic carrier by using an active functional group, mixing with an enzyme, and carrying out cross-linking combination to obtain a second cross-linking combination of the enzyme and the electronic carrier;
(3) immobilizing at least one of the first cross-linked conjugate or the second cross-linked conjugate to a surface of a base electrode to form an enzyme electrode.
8. The method according to any one of claims 6-7, wherein: the fixing method is at least one of a physical adsorption method, an ion adsorption method, an embedding method, a covalent coupling method and a crosslinking method.
Preferably, the reactive functional groups are the same or different from each other, and may be independently selected from at least one of carboxyl, maleimide, amino, epoxy, thiol, phenyl or aldehyde functional groups, for example.
Preferably, the cross-linking of the active functional group of the coenzyme or the electron carrier with the enzyme is achieved by one of amide bond, maleimide coupling to thiol, Huisgen cycloaddition, biotin or streptavidin.
9. A biosensor, characterized by: the sensor has an enzyme electrode according to any one of claims 1 to 5 as a working electrode.
10. Use of the biosensor of claim 9 for detecting a biological sample or substrate.
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Cited By (2)
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| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
| CN115337962A (en) * | 2022-09-02 | 2022-11-15 | 四川大学华西医院 | Lipoic acid micelle enzyme-simulating catalyst and preparation method thereof |
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| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
| CN115337962A (en) * | 2022-09-02 | 2022-11-15 | 四川大学华西医院 | Lipoic acid micelle enzyme-simulating catalyst and preparation method thereof |
| CN115337962B (en) * | 2022-09-02 | 2023-09-15 | 四川大学华西医院 | Lipoic acid micelle enzyme-like catalyst and preparation method thereof |
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