The application is a divisional application with the title of 'a coenzyme factor compound, an enzyme electrode, an enzyme sensor, a preparation method and an application thereof' of application No. 2020101521251, 3/6/2020.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a coenzyme factor compound, an enzyme electrode, an enzyme sensor, a preparation method and application thereof. The invention firstly uses chemical means to make coenzyme factor small molecule NAD+Chemically modifying the inactive site; chemically modifying NAD by chemical means+Covalently linked with chitosan carrier to obtain NAD+Chitosan complex (coenzyme factor complex). On the basis, an enzyme electrode is prepared by the following steps: modifying a carbon nano tube on the surface of a substrate electrode, and using the carbon nano tube as a substrate material for detecting NADH; electrodeposition of ABTS on electrodes, use of ABTS as an electron mediator to achieve NAD+In-situ regeneration; dropwise prepared NAD+Chitosan complex, effecting small molecule NAD+Fixing; and (3) connecting dehydrogenase to the chitosan carrier through glutaraldehyde crosslinking to obtain the dehydrogenase electrode. Utilizing the NAD+The immobilization and regeneration methods are respectively combined with different dehydrogenases, so that various dehydrogenase electrodes/biosensors can be prepared, and the detection of various substrates such as malic acid, glucose, lactic acid and the like can be realized. Thus, immobilization and in situ regeneration of NAD in the present invention+And a method ofThe electrode preparation technology can be widely applied to the preparation process of the dehydrogenase electrode/sensor, so that the dehydrogenase electrode can be recycled, and an effective technical approach is provided for constructing the dehydrogenase electrode/biosensing.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, a coenzyme factor complex is provided, wherein the coenzyme factor complex is obtained by complexing a coenzyme factor and a loading material.
It should be noted that the complex means that the coenzyme factor and the carrier are attached to, bound to, integrated with, or linked to each other. Thus, they are not physically separate components, but rather can be compounded together as a single component (covalently or ionically bonded complex).
The coenzyme factor may be a natural coenzyme or an artificial coenzyme. "coenzyme" or "redox cofactor" refers to redox equivalents (e.g., hydride (H) ions) that can act as an enzymatic transfer-) A redox equivalent of the enzymatic transfer from a substrate (e.g., a target analyte) to an enzyme to a coenzyme. As used herein, "redox equivalents" refers to concepts commonly used in redox chemistry, which are well known to those skilled in the art.
In particular, it relates to the transfer of electrons from a substrate of a coenzyme-dependent enzyme (i.e., the target analyte) to the coenzyme or from the coenzyme to an electrode or an electron of an indicator reagent. Examples of coenzymes include, but are not limited to, NAD, NADP, PQQ, thio-NAD, thio-NADP, and the like.
In some cases, the coenzyme is an artificial coenzyme. Examples of artificial coenzymes include, but are not limited to, artificial NAD (P)/NAD (P) H compounds that are chemical derivatives of natural NAD/NADH or natural NADP/NADPH.
Specifically, the artificial coenzyme can be chemically modified at the inactive site of the natural coenzyme, so that the coenzyme carries a chemical group for fixation under the condition of not influencing the activity and the function of the coenzyme factor, and the coenzyme is further fixed.
In some cases, can be rightModifying amino on NAD adenine to obtain artificial coenzyme NAD+A compound is provided.
The load material may be a polymer carrier, and examples of the load material include, but are not limited to, chitosan, agarose, sodium alginate, polyethylene glycol, and the like; preferably, the chitosan is a high molecular water-soluble polysaccharide with free amino, has good film forming property and can be used as a carrier of coenzyme factors; more preferably, the chitosan is medium viscosity chitosan (200-400 mPa.s).
In some cases, the coenzyme factor complex of the invention may be NAD+-chitosan complex, said NAD+Chitosan complex by para-NAD+Modifying amino on adenine to obtain artificial NAD+Coenzyme, then covalently linked with chitosan to prepare the coenzyme, thereby actually realizing NAD+Immobilization of (2).
In particular, the NAD+The preparation method of the chitosan complex comprises the following steps:
1) to NAD+Adding iodoacetic acid into the aqueous solution, and heating to react to obtain n 1-carboxymethyl-NAD+;
2) At n 1-carboxymethyl-NAD+Adding a sodium thiosulfate solution into the aqueous solution, adjusting the pH to be alkaline, heating to react to obtain n 1-carboxymethyl-NADH, and simultaneously carrying out Dimroth rearrangement under a strong alkali condition to obtain n 6-carboxymethyl-NADH;
3) adding formaldehyde for heating reaction to obtain c 6-carboxymethyl NAD+。
4) Adjusting the pH of the reaction system to be neutral, adding N-hydroxysuccinimide (NHS) and (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC), adding a chitosan solution, and heating for reaction to obtain chitosan-NAD+And (c) a complex.
In some cases, in the step 1), the heating reaction is preferably water bath heating, and the water bath reaction condition is that the reaction is carried out for 0.5-3 h at the temperature of 60-80 ℃, and preferably for 1h at the temperature of 70 ℃;
in some cases, in the step 2), the pH is adjusted to be strong alkaline, preferably 10-11, the heating reaction is preferably heating in a water bath, and the water bath reaction condition is that the reaction is carried out for 0.5-3 h at the temperature of 60-80 ℃, preferably for 1h at the temperature of 70 ℃;
in some cases, in the step 3), the heating reaction is preferably water bath heating, and the water bath reaction condition is that the reaction is carried out for 0.5-3 h at the temperature of 60-80 ℃, and preferably for 1h at the temperature of 70 ℃;
in some cases, in the step 4), the heating reaction is preferably water bath heating, and the water bath reaction condition is that the reaction is carried out for 0.5-3 h at the temperature of 60-80 ℃, and preferably for 1h at the temperature of 70 ℃.
In a second aspect of the invention, there is provided the use of the above-described coenzyme factor complex in an enzyme electrode and/or in the preparation of an enzyme electrode.
In a third aspect of the present invention, there is provided an enzyme electrode comprising:
a base electrode, and a base material carried by the base electrode.
The substrate electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material is a carbon material including, but not limited to: the electrode material comprises activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel and Carbon Nanotubes (CNTs), and preferably the carbon nanotubes have large specific surface area and small internal resistance, so that the analysis performance of the chemically modified electrode can be remarkably improved, and the electrode material has good conductivity and chemical stability.
The carbon nano tube not only comprises a multi-wall carbon nano tube and a single-wall carbon nano tube, but also comprises a functionalized carbon nano tube modified by amination, carboxylation and the like.
In some cases, the enzyme electrode comprises: a GCE electrode, and CNTs loaded on the GCE electrode. Wherein the loading can be carried out by means of drop coating.
In some cases, the enzyme electrode comprises:
the substrate electrode is loaded with a substrate material, a medium is deposited on the surface of the substrate material, and the surface of the medium is coated with a coenzyme factor compound and an enzyme.
The substrate electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material is a carbon material including, but not limited to: the electrode material comprises activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel and Carbon Nanotubes (CNTs), and preferably the carbon nanotubes have large specific surface area and small internal resistance, so that the analysis performance of the chemically modified electrode can be remarkably improved, and the electrode material has good conductivity and chemical stability.
The carbon nano tube not only comprises a multi-wall carbon nano tube and a single-wall carbon nano tube, but also comprises a functionalized carbon nano tube modified by amination, carboxylation and the like.
In the present invention, the "mediator" refers to a compound that increases the reactivity of a reduced coenzyme obtained by reaction with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
The mediator can be any chemical species (typically electrochemically active) that can participate in a reaction scheme involving an analyte, a coenzyme-dependent enzyme, a coenzyme, and reaction products thereof to produce a detectable electrochemically active reaction product. In general, participation of the mediator in the reaction involves a change in its oxidation state upon interaction with any of the analyte, the coenzyme-dependent enzyme, the coenzyme, or a species that is a reaction product of one of these (e.g., the coenzyme reacts to a different oxidation state). The media may also be stable in its oxidized form, may optionally exhibit reversible redox electrochemistry, may exhibit good solubility in aqueous solutions, and may react rapidly to produce electrochemically active reaction products.
Examples of such mediators include, but are not limited to, 2,2' -diaza bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), azo compounds or azo precursors, benzoquinone, prussian blue, nitrosoaniline or nitrosoaniline-based precursors, thiazine or thiazine derivatives, transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, osmium derivatives, quinones or quinone derivatives, phenazine or phenazine-based precursors, and combinations of phenazine derivatives and ruthenium hexamine chloride, and derivatives thereof.
In some cases, when the coenzyme is NAD/NADH, the mediator may be 2,2' -diaza-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). In the present invention, carbon nanotubes are mixed with ABTS+The carbon nano tube is modified on the surface of a GCE electrode, and can realize the catalytic oxidation of NADH (nicotinamide adenine dinucleotide), ABTS (adenosine triphosphate), and+electron transfer can be increased. Simultaneous ABTS+Oxidation reduction occurs with NADH, the 4-position of pyridine ring in NADH and ABTS+Radical cation reaction, ABTS+The hydrogen ion is converted into ABTS, NADH is oxidized into NAD+Effecting NAD+And (4) in-situ regeneration. After the above process, ABTS loses electrons, and ABTS is generated at the positive electrode of the electrode+And entering the next cycle.
In the present invention, the "enzyme" refers specifically to a coenzyme-dependent enzyme, and the "coenzyme-dependent enzyme" refers to an enzyme requiring an organic or inorganic cofactor called a coenzyme for catalytic activity.
In some cases, the coenzyme-dependent enzyme can be a dehydrogenase. As used herein, "dehydrogenase" refers to a compound capable of passing a hydride (H) as a redox equivalent (redox equivalent)-) A protein or polypeptide that is transferred to a receptor molecule to catalyze the oxidation of a substrate. Examples of dehydrogenases include, but are not limited to, glucose dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, L-amino acid dehydrogenase, malate dehydrogenase, sorbitol dehydrogenase, or the like, especially nad (p)/nad (p) H-dependent dehydrogenase.
In a fourth aspect of the present invention, there is provided the above-mentioned enzyme electrode preparation method, which is not particularly limited, but the enzyme electrode may be formed by being applied on one surface of a base electrode or by being coated in the form of a film using the following method: such as drop coating, electrodeposition, sputtering, e-beam, thermal deposition, spin coating, screen printing, ink jet printing, doctor blading or gravure printing.
In some cases, the method of making comprises:
1) dripping a substrate material on the surface of the substrate electrode;
2) depositing a medium on the substrate electrode loaded with the substrate material prepared in the step 1) by adopting an electrochemical deposition method;
3) dripping a coenzyme factor compound on the material prepared in the step 2);
4) loading enzyme on the material prepared in the step 3).
In the step 1), the step (A) is carried out,
in some cases, the base electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material is a carbon material including, but not limited to: the electrode material comprises activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel and Carbon Nanotubes (CNTs), and preferably the carbon nanotubes have large specific surface area and small internal resistance, so that the analysis performance of the chemically modified electrode can be remarkably improved, and the electrode material has good conductivity and chemical stability.
The carbon nano tube not only comprises a multi-wall carbon nano tube and a single-wall carbon nano tube, but also comprises a functionalized carbon nano tube modified by amination, carboxylation and the like.
In the step 2), the step (c) is carried out,
"mediator" refers to a compound that increases the reactivity of a reduced coenzyme obtained by reaction with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
The mediator can be any chemical species (typically electrochemically active) that can participate in a reaction scheme involving an analyte, a coenzyme-dependent enzyme, a coenzyme, and reaction products thereof to produce a detectable electrochemically active reaction product. In general, participation of the mediator in the reaction involves a change in its oxidation state (e.g., reduction) upon interaction with any of the analyte, the coenzyme-dependent enzyme, the coenzyme, or a species that is a reaction product of one of these (e.g., the coenzyme reacts to a different oxidation state). A variety of media exhibit suitable electrochemical behavior. The media may also be stable in its oxidized form, may optionally exhibit reversible redox electrochemistry, may exhibit good solubility in aqueous solutions, and may react rapidly to produce electrochemically active reaction products.
Examples of such mediators include, but are not limited to, ABTS, azo compounds or azo precursors, benzoquinone, prussian blue, nitrosoaniline or nitrosoaniline-based precursors, thiazine or thiazine derivatives, transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, osmium derivatives, quinone or quinone derivatives, phenazine or phenazine-based precursors, and combinations of phenazine derivatives and ruthenium hexammine chloride, and derivatives thereof.
In some cases, when the coenzyme factor is NAD/NADH, the mediator may be 2,2' -diaza-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).
In the step 3), the step (c),
in some cases, a coenzyme factor complex, the coenzyme factor complex being complexed from a coenzyme factor and a support material.
It should be noted that the complex means that the coenzyme factor and the carrier are attached to, bound to, integrated with, or linked to each other. Thus, they are not physically separate components, but rather can be compounded together as a single component (covalently or ionically bonded complex).
The coenzyme factor may be a natural coenzyme or an artificial coenzyme. As used herein, "coenzyme" or "redox cofactor" refers to redox equivalents (e.g., hydride (H) ions) that can act as an enzymatic transfer-) A redox equivalent of the enzymatic transfer from a substrate (e.g., a target analyte) to an enzyme to a coenzyme. "Redox equivalents" refers to concepts commonly used in redox chemistry, which are well known to those skilled in the art.
In particular, it relates to the transfer of electrons from a substrate of a coenzyme-dependent enzyme (i.e., the target analyte) to the coenzyme or from the coenzyme to an electrode or an electron of an indicator reagent. Examples of coenzymes include, but are not limited to, NAD, NADP, PQQ, thio-NAD, thio-NADP, and the like.
In some cases, the coenzyme is an artificial coenzyme. Examples of artificial coenzymes include, but are not limited to, artificial NAD (P)/NAD (P) H compounds that are chemical derivatives of natural NAD/NADH or natural NADP/NADPH.
Specifically, the artificial coenzyme can be chemically modified at the inactive site of the natural coenzyme, so that the coenzyme carries a chemical group for fixation under the condition of not influencing the activity and the function of the coenzyme factor, and the coenzyme is further fixed.
In some cases, the amino group on NAD adenine may be modified to obtain an artificial coenzyme NAD compound.
In some cases, the support material may be a polymeric carrier, examples of which include, but are not limited to, chitosan, agarose, sodium alginate, polyethylene glycol, and the like; preferably, the chitosan is a high molecular water-soluble polysaccharide with free amino, has good film forming property and can be used as a carrier of coenzyme factors; more preferably, the chitosan is medium viscosity chitosan (200-400 mPa.s).
In some cases, the coenzyme factor complex of the invention may be NAD+-chitosan complex, said NAD+-chitosan complex, said NAD+Chitosan complex by para-NAD+Modifying amino on adenine to obtain artificial NAD+Coenzyme, then covalently linked with chitosan to prepare the coenzyme, thereby actually realizing NAD+Immobilization of (2).
In the step 4), the step of mixing the raw materials,
in some cases, the "enzyme" refers specifically to a coenzyme-dependent enzyme, and "coenzyme-dependent enzyme" refers to an enzyme that requires an organic or inorganic cofactor, called a coenzyme, for catalytic activity.
In some cases, the coenzyme-dependent enzyme can be a dehydrogenase. As used herein, "dehydrogenase" refers to a compound capable of passing a hydride (H) as a redox equivalent (redox equivalent)-) Transfer to an acceptor molecule to catalyze substrate oxygenA methylated protein or polypeptide. Examples of dehydrogenases include, but are not limited to, glucose dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, L-amino acid dehydrogenase, malate dehydrogenase, sorbitol dehydrogenase, or the like, especially nad (p)/nad (p) H-dependent dehydrogenase.
It should be noted that, in the step 4), the enzyme is loaded on the material prepared in the step 3), and the cross-linking effect of the glutaraldehyde on the enzyme can be realized. Specifically, when the loading material of the coenzyme factor compound in the step 3) is chitosan, the free amino group of the chitosan and one free aldehyde on glutaraldehyde are covalently connected to synthesize a Schiff base structure under the action of glutaraldehyde, and the other free aldehyde of the glutaraldehyde is connected with an enzyme to realize the immobilization of the enzyme.
In a fifth aspect of the present invention, there is provided a method for regenerating a coenzyme factor, the method comprising:
1) dripping a substrate material on the surface of the substrate electrode;
2) depositing a medium on the substrate electrode loaded with the substrate material prepared in the step 1) by adopting an electrochemical deposition method.
In the step 1), the step (A) is carried out,
in some cases, the base electrode includes, but is not limited to: a Glassy Carbon (GCE) electrode, a gold electrode, a graphite electrode, a carbon paste electrode, preferably GCE. The GCE has good mechanical stability, light stability and high conductivity.
The substrate material is a carbon material including, but not limited to: the electrode material comprises activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogel and Carbon Nanotubes (CNTs), and preferably the carbon nanotubes have large specific surface area and small internal resistance, so that the analysis performance of the chemically modified electrode can be remarkably improved, and the electrode material has good conductivity and chemical stability.
The carbon nano tube not only comprises a multi-wall carbon nano tube and a single-wall carbon nano tube, but also comprises a functionalized carbon nano tube modified by amination, carboxylation and the like.
In the step 2), the step (c) is carried out,
"mediator" refers to a compound that increases the reactivity of a reduced coenzyme obtained by reaction with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
The mediator can be any chemical species (typically electrochemically active) that can participate in a reaction scheme involving an analyte, a coenzyme-dependent enzyme, a coenzyme, and reaction products thereof to produce a detectable electrochemically active reaction product. In general, participation of the mediator in the reaction involves a change in its oxidation state (e.g., reduction) upon interaction with any of the analyte, the coenzyme-dependent enzyme, the coenzyme, or a species that is a reaction product of one of these (e.g., the coenzyme reacts to a different oxidation state). A variety of media exhibit suitable electrochemical behavior. The media may also be stable in its oxidized form, may optionally exhibit reversible redox electrochemistry, may exhibit good solubility in aqueous solutions, and may react rapidly to produce electrochemically active reaction products.
Examples of such mediators include, but are not limited to, ABTS, azo compounds or azo precursors, benzoquinone, prussian blue, nitrosoaniline or nitrosoaniline-based precursors, thiazine or thiazine derivatives, transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, osmium derivatives, quinone or quinone derivatives, phenazine or phenazine-based precursors, and combinations of phenazine derivatives and ruthenium hexammine chloride, and derivatives thereof.
In some cases, when the coenzyme factor is NAD, the mediator may be 2,2' -biazobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). In the present invention, carbon nanotubes are mixed with ABTS+The carbon nano tube is modified on the surface of a GCE electrode, and can realize the catalytic oxidation of NADH (nicotinamide adenine dinucleotide), ABTS (adenosine triphosphate), and+electron transfer can be increased. Simultaneous ABTS+Oxidation reduction occurs with NADH, the 4-position of pyridine ring in NADH and ABTS+Radical cation reaction, ABTS+The hydrogen ion is converted into ABTS, NADH is oxidized into NAD+Effecting NAD+And (4) in-situ regeneration. After the above process, ABTS loses electrons, and ABTS is generated at the positive electrode of the electrode+And entering the next cycle.
In a sixth aspect of the present invention, there is provided a use of the above-mentioned method for regenerating a coenzyme factor in an enzyme electrode and/or an enzyme sensor.
In a seventh aspect of the present invention, there is provided the use of the above-described coenzyme factor complex and/or enzyme electrode for the production of an enzyme sensor.
In an eighth aspect of the present invention, there is provided an enzyme sensor comprising at least two electrodes comprising at least the above-described coenzyme factor complex and/or the above-described enzyme electrode. The enzyme sensor has the advantages of high detection sensitivity, high detection repeatability and long-term storage stability.
In some cases, the enzyme sensor comprises two or three electrodes, and accordingly, the enzyme sensor is a two-electrode or three-electrode enzyme sensor.
In some cases, a sensor consisting of two electrodes (i.e., a two-electrode enzyme sensor), the electrodes being a working electrode and a counter electrode; wherein the working electrode is the coenzyme factor complex and/or the enzyme electrode.
In some cases, a sensor consisting of three electrodes (i.e., a three-electrode enzyme sensor), the electrodes being a working electrode, a counter electrode, and a reference electrode; wherein the working electrode is the coenzyme factor complex and/or the enzyme electrode.
In some cases, in a three-electrode enzyme sensor, the counter electrode is a platinum electrode; the reference electrode is an Ag/AgCl electrode.
In a ninth aspect of the invention, there is provided a method of electrochemically measuring the concentration or presence of a target analyte to be measured, the method comprising: and (3) contacting the enzyme electrode and/or the enzyme sensor with a liquid sample having or suspected of having a target analyte to be detected, measuring the response current intensity of the target analyte to be detected, and analyzing the concentration or the existence of the target analyte.
In some cases, the target analyte includes, but is not limited to, amino acids, glucose, ethanol, glycerol, lactic acid, malic acid, pyruvic acid, sorbitol, triglycerides, and uric acid.
In a tenth aspect of the present invention, there is provided a malate dehydrogenase electrode comprising: the GCE electrode is loaded with carbon nanotubes, ABTS is deposited on the surface of the carbon nanotubes, and chitosan-NAD is loaded on the surface of the ABTS+Complexes and malate dehydrogenase.
In some cases, the method of making a malate dehydrogenase electrode comprises:
1) dropping carbon nanotubes on the surface of the GCE electrode;
2) depositing ABTS on the substrate electrode loaded with the substrate material prepared in the step 1) by adopting an electrochemical deposition method;
3) dripping chitosan-NAD (nicotinamide adenine dinucleotide) on the material prepared in the step 2)+A complex;
4) loading malate dehydrogenase on the material prepared in the step 3).
In some cases, the method of making a malate dehydrogenase electrode comprises:
the carbon nanotube dispersion (0.1%) was drop-coated onto the working surface of the GCE electrode, and dried to obtain CNTs/GCE.
And (2) soaking the CNTs/GCE into the ABTS deposition storage solution, and performing Cyclic Voltammetry (CV) scanning for different times within a potential range of-200 mV to 600mV at a scanning rate of 50mV/s, wherein the electrode is ABTS/CNTs/GCE. ABTS cation free radicals deposited on the surface of the electrode can participate in NADH oxidation process to realize NAD+And (4) regenerating.
Wherein the ABTS deposition stock solution comprises: 2.5mmol/L FeCl3、2.5mmol/L K3Fe(CN)6200mmol/L HCl and 1mmol/L ABTS.
NAD prepared by dropwise adding on ABTS/CNTs/GCE surface+-chitosan complex, dried, electrode CTS-NAD+/ABTS/CNTs/GCE。
Wherein, NAD+-the preparation process of the chitosan complex comprises:
taken 5mL of NAD+Adding 1mL of iodoacetic acid (1mg/mL) into the aqueous solution (1mg/mL), and reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NAD+。
② in the above NAD+Adding 1mL of sodium thiosulfate solution (1.3mmol/L) into the aqueous solution, adjusting the pH value to 11, reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NADH, and simultaneously carrying out Dimroth rearrangement under a strong alkali condition to obtain n 6-carboxymethyl NADH.
③ adding 1mL of formaldehyde into the mixture to react for 1h in 70 ℃ water bath to obtain c 6-carboxymethyl NAD+。
Fourthly, after the pH value of the reaction system is adjusted to be neutral, 10mg of NHS and 10mg of EDC are added, 10mL of chitosan solution (0.1%) is added, and the mixture is reacted in water bath at 70 ℃ for 1h to obtain the chitosan-NAD+And (c) a complex.
Converting CTS-NAD+Immersing the/ABTS/CNTs/GCE working surface in 25% glutaraldehyde aqueous solution, taking out after 1h, and fully cleaning with deionized water. Subsequently, the working surface of the electrode is immersed in a solution of malate dehydrogenase (240U/mL), taken out after 1h, and sufficiently washed to take out the loosely bound enzyme molecules, the electrode being MDH/CTS-NAD+/ABTS/CNTs/GCE。
In some cases, the malate dehydrogenase sensor comprises: the malic dehydrogenase electrode is used as the working electrode; the counter electrode is a platinum electrode; the reference electrode was an Ag/AgCl electrode.
In an eleventh aspect of the present invention, there is provided a glucose dehydrogenase electrode comprising: the GCE electrode is loaded with carbon nanotubes, ABTS is deposited on the surface of the carbon nanotubes, and chitosan-NAD is loaded on the surface of the ABTS+A complex and glucose dehydrogenase.
In some cases, the method of making a glucose dehydrogenase electrode comprises:
1) dropping carbon nanotubes on the surface of the GCE electrode;
2) depositing ABTS on the substrate electrode loaded with the substrate material prepared in the step 1) by adopting an electrochemical deposition method;
3) dripping chitosan-NAD (nicotinamide adenine dinucleotide) on the material prepared in the step 2)+A complex;
4) loading glucose dehydrogenase on the material prepared in the step 3).
In some cases, the method of making a glucose dehydrogenase electrode comprises:
the carbon nanotube dispersion (0.1%) was drop-coated onto the working surface of the GCE electrode, and dried to obtain CNTs/GCE.
And (2) soaking the CNTs/GCE into the ABTS deposition storage solution, and performing Cyclic Voltammetry (CV) scanning for different times within a potential range of-200 mV to 600mV at a scanning rate of 50mV/s, wherein the electrode is ABTS/CNTs/GCE. ABTS cation free radicals deposited on the surface of the electrode can participate in NADH oxidation process to realize NAD+And (4) regenerating.
Wherein the ABTS deposition stock solution comprises: 2.5mmol/L FeCl3、2.5mmol/L K3Fe(CN)6200mmol/L HCl and 1mmol/L ABTS.
NAD prepared by dropwise adding on ABTS/CNTs/GCE surface+-chitosan complex, dried, electrode CTS-NAD+/ABTS/CNTs/GCE。
Wherein, NAD+-the preparation process of the chitosan complex comprises:
taken 5mL of NAD+Adding 1mL of iodoacetic acid (1mg/mL) into the aqueous solution (1mg/mL), and reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NAD+。
② in the above NAD+Adding 1mL of sodium thiosulfate solution (1.3mmol/L) into the aqueous solution, adjusting the pH value to 11, reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NADH, and simultaneously carrying out Dimroth rearrangement under a strong alkali condition to obtain n 6-carboxymethyl NADH.
③ adding 1mL of formaldehyde into the mixture to react for 1h in 70 ℃ water bath to obtain c 6-carboxymethyl NAD+。
Fourthly, after the pH value of the reaction system is adjusted to be neutral, 10mg of NHS and 10mg of EDC are added, 10mL of chitosan solution (0.1%) is added, and the mixture is reacted in water bath at 70 ℃ for 1h to obtain the chitosan-NAD+And (c) a complex.
Converting CTS-NAD+Immersing the/ABTS/CNTs/GCE working surface in 25% glutaraldehyde aqueous solution, taking out after 1h, and fully cleaning with deionized water. The working surface of the electrode was then immersed in a solution of glucose dehydrogenase (240U/mL) for 1h, removed, washed thoroughly to remove the loosely bound enzyme molecules, and the electrode was MDH/CTS-NAD+/ABTS/CNTs/GCE。
In some cases, the glucose dehydrogenase sensor comprises: a working electrode, a counter electrode and a reference electrode, wherein the glucose dehydrogenase electrode is used as the working electrode; the counter electrode is a platinum electrode; the reference electrode was an Ag/AgCl electrode.
In a twelfth aspect of the present invention, there is provided a lactate dehydrogenase electrode comprising: the GCE electrode is loaded with carbon nanotubes, ABTS is deposited on the surface of the carbon nanotubes, and the surface of the ABTS comprises chitosan-NAD+A complex and lactate dehydrogenase.
In some cases, the lactate dehydrogenase electrode is prepared by a method comprising:
1) dropping carbon nanotubes on the surface of the GCE electrode;
2) depositing ABTS on the substrate electrode loaded with the substrate material prepared in the step 1) by adopting an electrochemical deposition method;
3) dripping chitosan-NAD (nicotinamide adenine dinucleotide) on the material prepared in the step 2)+A complex;
4) loading lactate dehydrogenase on the material prepared in the step 3).
In some cases, the lactate dehydrogenase electrode is prepared by a method comprising:
the carbon nanotube dispersion (0.1%) was drop-coated onto the working surface of the GCE electrode, and dried to obtain CNTs/GCE.
And (2) soaking the CNTs/GCE into the ABTS deposition storage solution, and performing Cyclic Voltammetry (CV) scanning for different times within a potential range of-200 mV to 600mV at a scanning rate of 50mV/s, wherein the electrode is ABTS/CNTs/GCE. ABTS cation free radicals deposited on the surface of the electrode can participate in NADH oxidation process to realize NAD+And (4) regenerating.
Wherein the ABTS deposition stock solution comprises: 2.5mmol/L FeCl3、2.5mmol/L K3Fe(CN)6200mmol/L HCl and 1mmol/L ABTS.
NAD prepared by dropwise adding on ABTS/CNTs/GCE surface+-chitosan complex, dried, electrode CTS-NAD+/ABTS/CNTs/GCE。
Wherein, NAD+-the preparation process of the chitosan complex comprises:
taken 5mL of NAD+Adding 1mL of iodoacetic acid (1mg/mL) into the aqueous solution (1mg/mL), and reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NAD+。
② in the above NAD+Adding 1mL of sodium thiosulfate solution (1.3mmol/L) into the aqueous solution, adjusting the pH value to 11, reacting in a water bath at 70 ℃ for 1h to obtain n 1-carboxymethyl-NADH, and simultaneously carrying out Dimroth rearrangement under a strong alkali condition to obtain n 6-carboxymethyl NADH.
③ adding 1mL of formaldehyde into the mixture to react for 1h in 70 ℃ water bath to obtain c 6-carboxymethyl NAD+。
Fourthly, after the pH value of the reaction system is adjusted to be neutral, 10mg of NHS and 10mg of EDC are added, 10mL of chitosan solution (0.1%) is added, and the mixture is reacted in water bath at 70 ℃ for 1h to obtain the chitosan-NAD+And (c) a complex.
Converting CTS-NAD+Immersing the/ABTS/CNTs/GCE working surface in 25% glutaraldehyde aqueous solution, taking out after 1h, and fully cleaning with deionized water. The working surface of the electrode was then immersed in a solution of lactate dehydrogenase (240U/mL) for 1h, removed, and thoroughly washed to remove the loosely bound enzyme molecules, the electrode being MDH/CTS-NAD+/ABTS/CNTs/GCE。
In some cases, the lactate dehydrogenase sensor comprises: a working electrode, a counter electrode and a reference electrode, wherein the lactate dehydrogenase electrode is used as the working electrode; the counter electrode is a platinum electrode; the reference electrode was an Ag/AgCl electrode.
The invention has the beneficial technical effects that:
(1) the present invention uses simple NAD+Modification technology for preparing chitosan-NAD+The complex (coenzyme factor complex) and is used for the preparation of dehydrogenase electrodes. The complex is to NAD+The amino group on adenine is modified without affecting NAD+The exertion of active function effectively improves the immobilized NAD+Activity of (2). On one hand, the coenzyme factor compound is dripped on the surface of an electrode to realize NAD+Immobilization on the surface of the electrode, on the other hand, providing a chitosan binding site on the surface of the electrode for dehydrogenase, so that the coenzyme factor complex is on the coenzymeThe field of the dependent enzyme electrode/biosensor has wide application value.
(2) The invention uses ABTS as an electron mediator to realize NAD (NAD) through electron transfer of an electrode+And (4) regenerating. In situ regeneration of NAD by electrochemical means+Not only is convenient and fast, but also can avoid the influence of byproducts.
(3) The present invention utilizes NAD+The immobilized and in-situ regeneration technology prepares the dehydrogenase electrode which can be repeatedly used. In addition, compared with the traditional dehydrogenase electrode, the electrode has higher detection sensitivity, higher detection repeatability and better storage stability, thereby having good practical application value.