CN113218891A - Method for colorimetric detection of NADH (nicotinamide adenine dinucleotide) based on cascade reaction of NADH-imitating oxidase and biological enzyme - Google Patents
Method for colorimetric detection of NADH (nicotinamide adenine dinucleotide) based on cascade reaction of NADH-imitating oxidase and biological enzyme Download PDFInfo
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 title description 84
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Abstract
The invention provides a method for colorimetric detection of NADH based on a cascade reaction of an NADH-like oxidase and a biological enzyme. Co-MoS2The nanoparticles have NADH oxidase activity, can catalyze NADH oxidation to generate hydrogen peroxide, can develop color after HRP and TMB are added, and estimate the NADH content in the sample by measuring the light absorption value at 652nm by using an enzyme-labeling instrument. The invention has wide development prospect in the aspects of biochemical analysis, nano enzyme-like catalysis, clinical medicine detection and the like, and has good practical application value.
Description
Technical Field
The invention relates to the technical field of nano materials, biological catalysis and analysis detection, in particular to a method for colorimetric detection of NADH based on a cascade reaction of an NADH-like oxidase and a biological enzyme.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Nicotinamide adenine dinucleotide phosphate (NADH), also known as reduced coenzyme I, is Nicotinamide Adenine Dinucleotide (NAD)+) Reduced state of NADH with NAD+Is an important coenzyme factor participating in a plurality of oxidation-reduction reactions in organisms. Under aerobic conditions, NADH produced via glycolysis and the tricarboxylic acid cycle can produce large amounts of ATP via oxidative phosphate reactions. The amount of NADH is directly related to the amount of ATP producedIn turn, the more NADH that each cell contains, the more energy is produced. The more energy-requiring organ, the higher the amount of NADH it contains (or requires). NADH molecules are control markers in the energy-producing chain in mitochondria. An increase in NADH levels indicates the occurrence of a metabolic imbalance. The traditional measurement method is ultraviolet spectroscopy, which is based on the change of the characteristic absorption peak of NADH at 340nm, but the method has low sensitivity, large interference and large sample size, which makes the measurement of NADH impractical under the limited sample size. AmpliteTMThe colorimetry can detect NADH as low as 300nM after incubating the solution to be detected for 1 hour by monitoring the increase of the absorbance of the NADH solution at 575 +/-5 nM, and the sensitivity is improved but the time is longer. The other method utilizes ethanol to generate acetaldehyde by oxidation under the action of Alcohol Dehydrogenase (ADH), and NAD is generated in the reaction process+Is reduced to NADH; the WST-8 is reduced by the generated NADH under the action of an electronic coupling reagent 1-Methoxy-5-methylphenazinium Methyl Sulfate to generate orange yellow formazan which has the maximum absorption peak at about 450nm and detects the linear range of the NADH from 0.25 mu M to 10 mu M.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for colorimetric detection of NADH based on a cascade reaction of Nicotinamide Adenine Dinucleotide (NADH) oxidase and biological enzyme. The method is simple and feasible, simple and convenient to synthesize and low in cost, and the prepared Co-MoS2The nanoparticles have NADH oxidase activity and can catalyze NADH oxidation to generate H2O2For colorimetric detection of NADH, Co-MoS in samples2The nano particles can be recycled, have strong specificity to NADH and have good selectivity.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, the invention provides Co-MoS2Use of a nanoparticle for the preparation of a product having NADH oxidase activity.
Such as a catalyst, for the catalytic oxidation of NADH.
Having NADH oxidase activityCo-MoS of2The nanoparticles can catalyze NADH to oxidize to generate hydrogen peroxide, can be developed after a cascade reaction is carried out after horseradish peroxidase (HRP) and 3 ', 5, 5' -Tetramethylbenzidine (TMB) are added, and the absorbance value at 652nm is measured by a microplate reader to evaluate the NADH content in a sample, so that Co-MoS2The nanoparticles can be used for detecting NADH, and producing H2O2。
In an embodiment of the invention, Co-MoS having NADH oxidase activity2The nanoparticles can be prepared by a hydrothermal method.
The preparation method comprises the following steps: and (3) dropwise adding a divalent cobalt salt solution into a mixed aqueous solution of sulfide and molybdate to react, and drying after the reaction is finished.
In an embodiment of the invention, the molar ratio of molybdate to divalent cobalt salt is 3 to 4: 1;
in some embodiments of the invention, the sulfide salt is selected from sodium sulfide and potassium sulfide, and the molybdate is selected from sodium molybdate, potassium molybdate, and ammonium molybdate; the divalent cobalt salt is selected from cobalt chloride, cobalt nitrate and cobalt sulfate.
In some embodiments of the invention, the reaction conditions are: the reaction temperature is 160-200 ℃; the reaction time is 18-36 h.
In some embodiments of the invention, the drying is vacuum heating drying, the drying temperature is 50-60 ℃, and the drying time is 4-8 h.
In some embodiments of the invention, the hydrothermal reaction is followed by a centrifugal wash and the centrifuged precipitate is subjected to at least one wash.
In some embodiments of the invention, the Co-MoS2The preparation method of the nano-particles comprises the following steps: adding cobalt chloride dropwise into a mixed solution containing a sodium sulfide aqueous solution and a sodium molybdate aqueous solution, stirring at room temperature, transferring into a high-pressure reaction kettle, sealing the reaction kettle, then placing the reaction kettle into a thermostat, heating to 160 ℃, reacting for 24 hours, naturally cooling after the reaction is finished, washing with deionized water and ethanol to be neutral, and removing soluble substances. Drying the final product in a vacuum drying oven at 60 ℃ for 6h to obtain Co-MoS2And (3) nanoparticles. The inventionThe room temperature refers to the temperature of the indoor environment, and is generally 15-30 ℃ unless otherwise specified.
In an embodiment of the present invention, the Co-MoS2The diameter of the nano-particles is 400-500 nm.
In a second aspect of the invention, the invention provides Co-MoS2Use of nanoparticles for the detection of NADH.
In an embodiment of the invention, colorimetric detection of NADH is based on Co-MoS2The nanoparticles are realized by simulating a cascade reaction of NADH oxidase and HRP. The invention discovers that Co-MoS is utilized by experimental research2The good NADH oxidase activity of the nano-particles can catalyze NADH to generate hydrogen peroxide, and the absorbance of a characteristic absorption peak is generated at 652nm by combining HRP catalytic oxidation TMB, so that the NADH content can be evaluated by the process.
In a third aspect of the invention, the invention provides Co-MoS2Nanoparticles in oxidizing NADH or in producing H2O2The use of (1).
Co-MoS2The nano-particles have good activity of imitating NADH oxidase and can catalyze and oxidize NADH into NAD+Hydrogen peroxide is generated.
In a fourth aspect of the present invention, there is provided a kit for detecting NADH, which comprises Co-MoS2And (3) nanoparticles.
In addition, the kit also contains acetate buffer, HRP and TMB. The acetate buffer has a pH of 4-4.5.
In a fifth aspect of the invention, the invention provides a colorimetric sensor for the detection of NADH comprising at least Co-MoS2And (3) nanoparticles.
In a sixth aspect of the invention, the invention provides a catalyst for the catalytic oxidation of NADH comprising Co-MoS2And (3) nanoparticles.
In a seventh aspect of the present invention, the present invention provides a method for colorimetric detection of NADH using Co-MoS2Nanoparticles or using the kit described above.
In some embodiments of the invention, the ratio isThe method for detecting NADH comprises the following steps: mixing Co-MoS2The nanoparticles are added into acetate buffer solution of a sample to be detected (for example, the sample to be detected can be dissolved in the acetate buffer solution) for reaction, then centrifugation is carried out, supernate is taken and added into the acetate buffer solution containing HRP and TMB (for example, the detection is carried out by using the kit, the HRP and TMB can be dissolved in the acetate buffer solution), and the NADH content is evaluated by detecting the absorbance at 652 nm.
In some embodiments of the invention, the visible absorption spectrum is recorded with a microplate reader in the range of 500-800 nm. Further using the absorbance value at 652nm to draw a standard working curve of NADH, wherein the linear range is 0.5-800 mu M, and y is 0.00087x +0.0736 (R)2＝0.997)。
In some embodiments of the invention, the measurement may also be performed in a relatively simple manner in the form of a 96-well microtiter plate, and the NADH content may be assessed by measuring the absorbance of the sample at 652nm using a microplate reader.
In an embodiment of the invention, the acetate buffer has a pH of 4 to 4.5.
In an embodiment of the invention, the reaction temperature is 25-30 ℃.
In an embodiment of the invention, Co-MoS2The concentration ratio of the nanoparticles, TMB and HRP is 50-100 μ g/ml: 0.5 mM: 15 ng/ml.
In an eighth aspect of the invention, there is provided a method of catalyzing the oxidation of NADH to NAD+The method of (1), which comprises reacting with Co-MoS2The nanoparticles are catalysts.
In some embodiments of the invention, a method of catalyzing oxidation of NADH to NAD + comprises: mixing Co-MoS2The nanoparticles were added to acetate buffer containing NADH, and then the reaction was performed.
In an embodiment of the invention, the acetate buffer has a pH of 4 to 4.5.
In an embodiment of the invention, Co-MoS2The addition concentration ratio of the nano particles to NADH is 50-100 mu g/ml: 0.25 mM.
In an embodiment of the invention, the reaction temperature is 25-30 ℃.
In a ninth aspect of the invention, the invention provides a process for producing H2O2Method with Co-MoS2Production of H by oxidation of NADH with nanoparticles as catalyst2O2。
In some embodiments of the invention, the method comprises: mixing Co-MoS2Adding the nanoparticles into acetate buffer solution containing NADH for reaction, centrifuging, taking supernatant, and adding acetate buffer solution containing HRP and TMB for reaction.
In an embodiment of the invention, the acetate buffer has a pH of 4 to 4.5.
In an embodiment of the invention, Co-MoS2The concentration ratio of the nanoparticles, NADH, TMB and HRP is 50-100 mug/ml: 0.5 mM: 15 ng/ml.
In some embodiments of the invention, the concentration of HRP is 10-15 ng/ml.
In an embodiment of the invention, the reaction temperature is 25-30 ℃.
Compared with the prior art, the invention has the advantages that:
the invention is based on Co-MoS2The colorimetric detection of NADH is realized by the enzyme-like activity of the nano particles, the material synthesis is simple and convenient, and the cost is low; the invention estimates the content of NADH by detecting the absorbance at 652nm, and has small interference and high sensitivity. Furthermore, the Co-MoS of the invention2The nano particles can be recycled, have strong specificity, have wide development prospect in the aspects of biochemical analysis, nano enzyme-like catalysis, clinical medicine detection and the like, and have good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows Co-MoS of example 1 of the present invention2Scanning electron micrographs of nanoparticles;
FIG. 2 shows the present inventionWhile Co-MoS in EXAMPLE 22A graph of the effect of the NADH oxidase activity of the nanoparticles;
FIG. 3 is a standard curve of NADH concentration provided in example 3 of the present invention;
FIG. 4 shows Co-MoS in example 4 of the present invention2Nanoparticle catalysis of NADH to produce H2O2Effect graphs;
FIG. 5 shows Co-MoS in example 5 of the present invention2Measuring an NADH effect graph by the nanoparticles;
FIG. 6 is a graph showing the standard operation of the quantitative determination of NADH in example 5 of the present invention;
FIG. 7 shows Co-MoS in example 6 of the present invention2A recycling effect graph of the nano particles;
FIG. 8 is a graph showing the specific effects of NADH detection in example 7 of the present invention;
FIG. 9 shows Co-MoS2Schematic diagram of nanoparticle detection of NADH.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
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. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1
Co-MoS with NADH oxidase activity2And (4) preparing nanoparticles.
The reaction mixture was heated at room temperature to room temperature in a reactor containing an aqueous solution of sodium sulfide (1.53mM,10mL) and an aqueous solution of sodium molybdate (0.25mM,15mL) is added with cobalt chloride (0.21mM,5mL) drop by drop, stirred at room temperature, then transferred into a high-pressure reaction kettle, the reaction kettle is sealed, then placed into a thermostat and heated to 160 ℃, reacted for 24 hours, after the reaction is finished, naturally cooled, washed to be neutral by deionized water and ethanol, and soluble substances are removed. Drying the final product in a vacuum drying oven at 60 ℃ for 6h to obtain Co-MoS2And (3) nanoparticles. Mixing Co-MoS2The nanoparticles were formulated into a 1.0mg/mL solution for use. FIG. 1 shows Co-MoS observed by a scanning electron microscope2SEM imaging of nanoparticles. Co-MoS2The particle size of the nano-particles is 400-500 nm.
Example 2
Co-MoS2And (3) verification of the activity of the NADH-imitating oxidase of the nanoparticles:
experiment system a: the catalytic reaction system contained acetate buffer (pH 4.5,100mM), Co-MoS obtained in example 1 above2(100. mu.g/mL) and NADH (0.25mM) were mixed, and the absorbance after 30 minutes of reaction at room temperature was measured, and the absorbance spectrum in the range of 200-450nm was recorded by means of a UV-visible spectrophotometer.
Control experiment b: the acetate buffer (pH 4.5,100mM) and NADH (0.25mM) were contained in the catalyst system, and the absorbance of NADH itself was measured under the same conditions as in the above-described experimental system.
Control experiment c: the catalytic reaction system contained acetate buffer (pH 4.5,100mM), Co-MoS obtained in example 1 above2(100. mu.g/mL) and NADH (0.25mM) were mixed, and the absorbance at the beginning of the reaction at room temperature was measured, and the absorbance spectrum in the range of 200-450nm was recorded by means of a UV-visible spectrophotometer.
Control experiment d: the catalytic reaction system only contains Co-MoS2The absorbance of the material itself was measured under the same conditions as in the above-mentioned experimental system.
As shown in FIG. 2, A, the absorption peak of the experimental system a at 340nm is gradual and the increase of the absorption peak at 260nm can be observed, which shows that Co-MoS2Has the catalytic activity of imitating NADH oxidase and catalyzes the oxidation of NADH into NAD+(ii) a Control experiment b at 340nm andan absorption peak is respectively arranged at 260nm, and the Co-MoS is judged by mainly observing the change of the absorption peak at 340nm2The simulated NADH oxidase activity of (1); the control experiment c shows a decrease in the absorption peak at 340nm, indicating the addition of Co-MoS2Then the reaction system is influenced; control experiment d had no significant absorption peak at 340 nm.
As shown in FIG. 2, B, NADH has absorption peaks at 260nm and 340nm, NAD+And an obvious absorption peak is only formed at 260nm, and the change of the absorption spectrum of the experimental system a after 30min of reaction is verified.
Example 3
Co-MoS2And (3) quantitatively detecting the activity of the NADH-like oxidase of the nanoparticles:
the experimental system is as follows: the Co-MoS obtained in example 1 above was used2(50. mu.g/mL) was added to acetate buffers (pH 4.5,100mM) containing different concentrations of NADH (0.1-1mM), reacted at room temperature for 30min, centrifuged at 10000rpm for 3 min, the supernatant was taken, the change of absorbance at 340nm with the NADH concentration was recorded by UV-visible spectrophotometer (FIG. 3), and Co-MoS was calculated according to Lambert beer's law2The relative enzyme activity of NADH oxidase of (1) was 0.1331U/mg.
Example 4
Co-MoS2Nanoparticle catalysis of NADH to produce H2O2
The catalytic reaction system contained the Co-MoS obtained in example 1 above2(100. mu.g/mL), NADH (0.25mM) and acetate buffer (pH 4.5,100mM) in 1250. mu.L, reacted at room temperature for 30min, centrifuged at 10000rpm for 3 min, and 250. mu.L of the supernatant was added to each of four centrifuge tubes (FIG. 4).
Experiment system a: the catalytic reaction system contained the supernatant (250. mu.L) obtained in example 4, TMB (0.5mM), H2O2(0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
Control system b: the catalytic reaction system comprises TMB (0.5mM) and H2O2(0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
Control system c: the catalytic reaction system contained the supernatant (250. mu.L) obtained in example 4, TMB (0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
Control system d: the catalytic reaction system contained the supernatant (250. mu.L) obtained in example 4, TMB (0.5mM) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum thereof in the range of 500-800nm was recorded by a microplate reader.
Control system e: the catalytic reaction system contained the supernatant (250. mu.L) obtained in example 4, TMB (0.5mM), H2O2(0.5mM) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
Control system f: the catalytic reaction system comprises TMB (0.5mM) and H2O2(0.5mM) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
Control system g: the catalytic reaction system contained TMB (0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded by a microplate reader.
Control system h: the catalytic reaction system comprises H2O2(0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), and after 10 minutes of reaction at 25 ℃, the visible absorption spectrum in the range of 500-800nm was recorded using a microplate reader.
As shown in FIG. 4, the absorbance value of the experimental system a at 652nm is higher than that of the control systems b and c, while the control systems d, e, f, g and h have no absorption peak at 652nm, which indicates that the absorption peak at 652nm of the control system c is due to the fact that the supernatant contains hydrogen peroxide and is further catalyzed by HRPThe Co-MoS obtained in example 1 was explained by oxidizing TMB by decomposing the generated hydroxyl radical2The nanoparticles do oxidize NADH to produce hydrogen peroxide.
Example 5
Using Co-MoS2Detecting NADH (nicotinamide adenine dinucleotide) by the enzyme activity of the nanoparticles in a colorimetric manner:
the experimental system contained Co-MoS including the Co obtained in example 12(100. mu.g/mL), NADH (0-800. mu.M), acetate buffer (pH 4.5,100mM), reacted at room temperature for 30min, centrifuged at 10000rpm for 3 min, 500. mu.L of the supernatant was added with TMB (0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), reacted at room temperature for 10min, and then the visible absorption spectrum in the range of 500-800nm was recorded with a microplate reader (FIG. 5). Further using the absorbance at 652nm to plot the standard working curve of NADH, as shown in FIG. 6, the linear range is 0.5-800. mu.M, y is 0.00087x +0.0736 (R)2＝0.997)。
Example 6
Co-MoS2And (3) carrying out colorimetric detection on the recyclable property of NADH by the nanoparticles:
the catalytic reaction system contained the Co-MoS obtained in example 1 above2(100. mu.g/mL), NADH (0-800. mu.M), acetate buffer (pH 4.5,100mM), reacted at room temperature for 30min, centrifuged at 10000rpm/min for 3 min, and 500. mu.L of the supernatant was added with TMB (0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), reacted at room temperature for 10min, and the absorbance at 652nm was recorded with a microplate reader and defined as 100% of the enzyme activity at this time. The centrifuged material was collected for the first time, NADH (0.25mM) and acetate buffer (pH 4.5,100mM) were added in this order, and the reaction was carried out at room temperature for 30min, and the subsequent steps described above were repeated. The 2 nd, 3 rd, 4 th, 5 th and 6 th recovery are carried out in sequence according to the steps, and the light absorption value at 652nm is recorded by a microplate reader.
As shown in FIG. 7, Co-MoS increases with the number of cycles2The enzyme-imitating activity of the nano-particles is still over 70 percent, so the nano-particles can be recycled when being applied to colorimetric detection of NADH.
Example 7
Co-MoS2And (3) carrying out colorimetric detection on specificity of NADH by the nanoparticles:
the catalytic reaction system contained the Co-MoS obtained in example 1 above2(100. mu.g/mL), each substrate (0.25mM), acetate buffer (pH 4.5,100mM), reacted at room temperature for 30min, centrifuged at 10000rpm for 3 min, and 500. mu.L of the supernatant was added with TMB (0.5mM), HRP (15ng/mL) and acetate buffer (pH 4.5,100mM), reacted at room temperature for 10min, and the absorbance at 652nm was recorded with a microplate reader. As shown in FIG. 8, the method has good selectivity for NADH.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1.Co-MoS2Use of a nanoparticle for the preparation of a product having NADH oxidase activity.
2.Co-MoS2Use of nanoparticles for the detection of NADH.
3.Co-MoS2Nanoparticles in oxidizing NADH or in producing H2O2The use of (1).
4. A kit for detecting NADH comprising Co-MoS2And (3) nanoparticles.
5. The kit of claim 4, further comprising acetate buffer, HRP and TMB.
6. A colorimetric sensor for detecting NADH, at leastComprising Co-MoS2And (3) nanoparticles.
7. A catalyst for the catalytic oxidation of NADH comprising Co-MoS2And (3) nanoparticles.
8. A method for colorimetric detection of NADH, characterized in that Co-MoS is used2Nanoparticles or use of a kit according to claim 4 or 5;
preferably, the method comprises: mixing Co-MoS2And adding the nanoparticles into an acetate buffer solution of a sample to be detected for reaction, centrifuging, taking supernate, adding the acetate buffer solution containing HRP and TMB, and detecting the absorbance at 652nm to evaluate the content of NADH.
9. Catalyzing oxidation of NADH into NAD+The method of (1), which comprises reacting with Co-MoS2The nano particles are used as catalysts:
preferably, the method comprises: mixing Co-MoS2The nanoparticles were added to acetate buffer containing NADH, and then the reaction was performed.
10. Production of H2O2Method with Co-MoS2Production of H by oxidation of NADH with nanoparticles as catalyst2O2；
Preferably, the method comprises: mixing Co-MoS2Adding the nanoparticles into acetate buffer solution containing NADH for reaction, centrifuging, taking supernatant, and adding acetate buffer solution containing HRP and TMB for reaction.
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