A portable hepatitis biomarker detection kit and a method for detecting hepatitis biomarkers.

Abstract

This invention belongs to the field of biochemical detection and discloses a portable hepatitis biomarker detection kit. The kit contains a hydrogel; the hydrogel is obtained by dispersing hollow manganese dioxide in water to obtain an H-MnO2 dispersion, mixing the H-MnO2 dispersion with sodium alginate solution to obtain a mixed solution, and then adding a calcium salt solution to obtain the hydrogel. The hollow manganese dioxide is obtained by etching manganese Prussian blue analog nanoparticles with sodium hydroxide. The manganese Prussian blue analog nanoparticles are prepared from potassium ferricyanide, polyvinylpyrrolidone, and manganese salts. This invention utilizes the color change of hollow manganese dioxide in relation to TMB to construct a method for detecting hepatitis biomarkers using a smartphone to capture signals. This kit reduces the detection cost of hepatitis biomarkers, does not require large instruments, and is highly portable and widely applicable, possessing great potential as a novel point-of-care testing device.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry detection, and relates to a portable hepatitis marker detection kit and a preparation method thereof, and a method for detecting hepatitis markers based on the kit. BACKGROUND

[0002] Liver is the place of metabolism of many drugs, and unreasonable drug use and excessive pressure can cause liver damage. Hepatitis refers to inflammation of the liver, which is usually caused by viruses, drugs, alcohol, and autoimmune factors. Acetaminophen is a commonly used fever-reducing drug, and in recent years, cases of liver damage caused by non-standard use of fever-reducing drugs have been increasing. Transaminases are an important class of liver enzymes, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which generally have high concentrations in the liver. When liver cells are damaged or inflamed, transaminases are released into the blood, causing an increase in the concentration of AST and ALT in the serum. In addition, liver damage can cause an increase in the concentration of alkaline phosphatase (ALP), indicating that the liver has some diseases or damage, such as bile duct obstruction, liver abscess, primary biliary cholangitis, hepatitis, cirrhosis, etc. Therefore, it is of great significance to simultaneously detect the above three hepatitis markers.

[0003] Currently, the methods for detecting transaminases and alkaline phosphatase mainly include colorimetry, fluorescence, chemiluminescence, enzyme kinetics, and electrochemistry. For example, Chinese Patent Application CN115656290A discloses a preparation method of a biosensor chip for rapid diagnosis of hepatitis. The method greatly improves the loading capacity of active enzyme molecules and electrochemical sensing performance by preparing a porous Prussian blue@metal oxide core-shell structure with ultra-high specific surface area. The detection chip preparation process is simple and controllable, and the biosensor based on the chip can realize rapid identification and simultaneous detection of alanine aminotransferase and aspartate aminotransferase in real serum within 1 min, greatly improving the diagnosis efficiency of hepatitis disease.

[0004] Although the above detection methods have good detection efficiency, there are still some deficiencies: the instruments and equipment relied on by the detection methods are relatively expensive and not suitable for remote areas with scarce resources; the detection methods are limited to laboratories, and the scope of use is limited, making it difficult to further expand commercialization. SUMMARY

[0005] The purpose of the present application is to provide a portable hepatitis marker detection kit.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The portable hepatitis marker detection kit contains a hydrogel; the hydrogel is obtained by dispersing hollow manganese dioxide (H-MnO2) in water to obtain a H-MnO2 dispersion, mixing the H-MnO2 dispersion with a sodium alginate solution to obtain a mixed solution, and adding a calcium salt solution to obtain the hydrogel; the hollow manganese dioxide (H-MnO2) is obtained by etching manganese prussian blue analog nanoparticles (Mn-PBA) with sodium hydroxide; the manganese prussian blue analog nanoparticles are prepared from potassium ferricyanide and polyvinylpyrrolidone, and manganese salt as raw materials.

[0008] Another object of the present application is to provide a preparation method of a portable hepatitis marker detection kit, comprising the following steps:

[0009] Step (1), preparation of hollow manganese dioxide (H-MnO2): dissolve potassium ferricyanide and polyvinylpyrrolidone in water to prepare solution A, dissolve manganese salt in the same volume of water to prepare solution B, slowly add solution B to solution A, incubate at room temperature for 10-24 h, centrifuge and dry to obtain manganese prussian blue analog nanoparticles; disperse the manganese prussian blue analog nanoparticles in a solvent, ultrasonicate to disperse the nanoparticles uniformly, add sodium hydroxide solution, stir at 25-60°C for 0.1-4 h, centrifuge and dry to obtain hollow manganese dioxide (H-MnO2);

[0010] Step (2), preparation of a portable hepatitis marker detection kit: weigh 100-350 mg of sodium alginate and dissolve it in 10 mL of NaAc-HAc buffer solution to obtain a sodium alginate solution; disperse H-MnO2 in water to obtain a H-MnO2 dispersion with a concentration of 1 mg / mL, add 100-250 μL of the H-MnO2 dispersion to the sodium alginate solution, stir to obtain a mixed solution; take 200 μL of the mixed solution, add it to a small dish, add 20 μL of a calcium salt solution, mix thoroughly to obtain a hydrogel, which is the portable hepatitis marker detection kit.

[0011] In step (1), the amount-of-substance ratio of potassium ferricyanide to manganese salt is 1:1-1:3, preferably 1:1.5; the amount ratio of potassium ferricyanide to polyvinylpyrrolidone is 1 mmol:3 g; after adding solution B to solution A, the concentration of potassium ferricyanide is 1-100 mM.

[0012] The manganese salt is any one of MnCl2, MnSO4, and Mn(NO3)2.

[0013] Disperse 20-200 mg of manganese prussian blue analog nanoparticles in 10-40 mL of solvent; use 40 mL of sodium hydroxide solution for every 20-200 mg of manganese prussian blue analog nanoparticles.

[0014] Preferably, 100 mg of manganese prussian blue analogue nanoparticles are dispersed in 20 mL of solvent; 40 mL of sodium hydroxide solution is used per 100 mg of manganese prussian blue analogue nanoparticles.

[0015] The solvent is one of anhydrous ethanol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO).

[0016] Specifically, the manganese prussian blue analogue nanoparticles are dispersed in the solvent, and ultrasonic dispersion is performed for 3 minutes to uniformly disperse the nanoparticles.

[0017] The concentration of NaOH in the sodium hydroxide solution is 0.01-1 M, preferably 0.05-1 M, and more preferably 0.1 M.

[0018] In step (2), the preparation method of the sodium alginate solution is as follows: 100-350 mg of sodium alginate is dissolved in 10 mL of NaAc-HAc buffer solution, and stirring is performed at room temperature for 12-24 h to obtain a sodium alginate solution.

[0019] The sodium alginate is crosslinked with calcium ions to form a hydrogel, and the hydrogel generally has a porous structure, which is beneficial to the diffusion of the substrate. By controlling the amount of sodium alginate and NaAc-HAc buffer solution, the present application avoids the situation that "the hydrogel formed by high-concentration sodium alginate has a very small pore size, which is not conducive to the diffusion of the substrate, and experiments show that the color change rate is slow and uneven when the concentration of sodium alginate is high; when the concentration is low, the sodium alginate does not have the property of forming a gel", in addition, the NaAc-HAc buffer solution provides an optimal pH environment for H-MnO2.

[0020] The preparation method of the mixed solution is as follows: 100-250 μL of H-MnO2 dispersion liquid is added to the sodium alginate solution, and stirring is performed for 0.5-4 h to obtain a mixed solution.

[0021] The concentration of the NaAc-HAc buffer solution is 0.025-0.2 M, and the pH of the NaAc-HAc buffer solution is 4-6, preferably 5.5.

[0022] The concentration of the calcium salt in the calcium salt solution is 20 mg / mL.

[0023] The calcium salt is one of CaCl2 and Ca(NO3)2.

[0024] The capacity of the small dish is 250-300 μL. Specifically, the small dish can use a centrifuge tube cover.

[0025] As a further preferred technical solution for the preparation method of the portable hepatitis biomarker detection kit of the present invention, the portable hepatitis biomarker detection kit is sealed and stored at 4°C.

[0026] Another object of the present invention is to provide the application of the portable hepatitis biomarker detection kit in the detection of hepatitis biomarkers.

[0027] The hepatitis markers mentioned are aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP).

[0028] Another object of the present invention is to provide a method for detecting hepatitis markers, comprising the following steps:

[0029] Step (a) Plotting the transaminase standard curve: Construct an incubation system with different concentrations of aspartate aminotransferase (AST), malate dehydrogenase (MDH), and substrate 1, and incubate; take the incubated solution and add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish an AST standard curve with AST concentration as the x-axis and gray value as the y-axis; Construct an incubation system with different concentrations of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and substrate 2, and incubate; take the incubated solution and add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish an ALT standard curve with ALT concentration as the x-axis and gray value as the y-axis;

[0030] Plotting the alkaline phosphatase standard curve: Construct an incubation system with different concentrations of alkaline phosphatase and catalytic substrate 3, and incubate; take the incubated solution, add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish the alkaline phosphatase standard curve with alkaline phosphatase concentration as the x-axis and gray value as the y-axis.

[0031] Step (b), Sample Analysis: Take a serum sample and dilute it;

[0032] The diluted serum sample was used to construct a detection system with malate dehydrogenase (MDH) and catalytic substrate 1, and incubated. The incubated solution was added to the portable hepatitis marker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were substituted into the aspartate aminotransferase standard curve to obtain the aspartate aminotransferase content in the serum sample.

[0033] The diluted serum sample was used to construct a detection system with lactate dehydrogenase (LDH) and catalytic substrate 2, and incubated. The incubated solution was added to the portable hepatitis marker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were substituted into the alanine aminotransferase standard curve to obtain the alanine aminotransferase content in the serum sample.

[0034] The diluted serum sample was used to construct a detection system with catalytic substrate 3 and incubated. The incubated solution was then added to the portable hepatitis biomarker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were then substituted into the alkaline phosphatase standard curve to obtain the alkaline phosphatase content in the serum sample.

[0035] In step (a), the catalytic substrate 1 of AST includes aspartic acid, α-ketoglutarate, and NADH.

[0036] In the incubation system, the final concentrations of aspartic acid, α-ketoglutarate, and NADH were 600 mM, 15 mM, and 1 mM, respectively; the final concentration of MDH was 300–1000 U / L; and the final concentration of aspartate aminotransferase was 5–170 U / L. The pH of the incubation system was 7.4.

[0037] The incubation temperature was 37°C and the incubation time was 30 minutes.

[0038] Specifically, the method for plotting the aspartate aminotransferase (AST) standard curve is as follows:

[0039] Preparation of substrate solution 1: Dissolve 7.986 g aspartic acid and 0.219 g α-ketoglutarate in 100 mL of 0.2 M sodium hydroxide solution, heat and stir to promote dissolution, adjust the pH to about 7.4, cool and add 0.663 g NADH to obtain catalytic substrate solution 1;

[0040] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), and sequentially add 50 μL of substrate solution 1, 50 μL of MDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to achieve a final MDH concentration of 600 U / L in the incubation solution, and 100 μL of AST solution (prepared with 10 mM Tris-hydrochloric acid buffer) to achieve final AST concentrations of 5, 10, 30, 50, 70, 90, 110, 130, 150, and 170 U / L in the incubation solution, respectively. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the portable hepatitis biomarker detection kit. Distribute 20 μL of TMB solution (20 mM, prepared with dimethyl sulfoxide) at more than 5 points, let stand for 20 minutes, and place the color-changing kit in a sealed box to avoid the influence of unstable external light on the color. Use a smartphone to take a picture to capture the color intensity of the kit; use the Color software... Picker processes photos to obtain their RGB values, and then calculates grayscale values ​​from these RGB values.

[0041] A standard curve for aspartate aminotransferase (AST) was established with AST concentration on the x-axis and grayscale value on the y-axis.

[0042] The catalytic substrates of alanine aminotransferase include alanine, α-ketoglutarate, and NADH.

[0043] In the incubation system, the final concentrations of alanine, α-ketoglutarate, and NADH were 400 mM, 15 mM, and 1 mM, respectively; the final concentration of LDH was 300–1000 U / L; and the final concentration of alanine aminotransferase was 5–180 U / L. The pH of the incubation system was 7.4.

[0044] The incubation temperature was 37°C and the incubation time was 30 minutes.

[0045] Specifically, the method for plotting the alanine aminotransferase (ALT) standard curve is as follows:

[0046] Preparation of substrate solution 2: Dissolve 5.844 g alanine and 0.219 g α-ketoglutarate in 100 mL of 0.2 M sodium hydroxide solution, heat and stir to promote dissolution, adjust the pH to about 7.4, cool and add 0.663 g NADH to obtain substrate solution 2;

[0047] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), and add 50 μL of substrate solution 2, 50 μL of LDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make a final concentration of 800 U / L, and 100 μL of ALT solution (prepared with 10 mM Tris-hydrochloric acid buffer) of different concentrations. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the portable hepatitis biomarker detection kit. Distribute 20 μL of TMB solution (20 mM, prepared with dimethyl sulfoxide) at more than 5 points. Let it stand for 20 minutes. Place the color-changing kit in a sealed box, take a picture with a smartphone to capture the color intensity of the kit, and process the photo with Color Picker software to obtain the RGB values ​​of the photo. Calculate the grayscale value from the RGB values.

[0048] A standard curve for alanine aminotransferase (ALT) was established with ALT concentration as the x-axis and grayscale value as the y-axis.

[0049] The catalytic substrate 3 of alkaline phosphatase is ascorbate phosphate (AA2P).

[0050] In the incubation system, the final concentration of ascorbic acid phosphate was 200 mM, the final concentration of alkaline phosphatase was 5–110 U / L, and the pH of the incubation system was 8.

[0051] The incubation temperature was 37°C and the incubation time was 30 minutes.

[0052] Specifically, the method for plotting the alkaline phosphatase standard curve is as follows:

[0053] Take 300 μL of Tris-hydrochloric acid buffer (0.01 M, pH 8), and add 100 μL of AA2P solution (prepared with water) to make the AA2P concentration in the incubation solution 200 mM. Add 100 μL of ALP solution of different concentrations (prepared with 10 mM Tris-hydrochloric acid buffer) to make the ALP concentrations in the incubation solution 5, 10, 30, 50, 70, 90, and 110 U / L, respectively. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the portable hepatitis biomarker detection kit. Distribute 20 μL of TMB (20 mM, prepared with dimethyl sulfoxide) at more than 5 points. Let it stand for 20 minutes. Place the color-changing kit in a sealed box, take a picture with a smartphone to capture the color intensity of the kit, process the picture with the software Color Picker to obtain the RGB value of the picture, and calculate the gray value from the RGB value.

[0054] A standard curve for alkaline phosphatase was established with alkaline phosphatase concentration as the x-axis and grayscale values ​​as the y-axis.

[0055] The formula for calculating the grayscale value is: Gray = (0.299*R) + (0.587*G) + (0.114*B).

[0056] In step (b), when detecting aspartate aminotransferase, the final concentrations of aspartic acid, α-ketoglutarate, and NADH in the detection system are 600 mM, 15 mM, and 1 mM, respectively, and the final concentration of MDH is 300–1000 U / L; the pH of the detection system is 7.4.

[0057] When detecting alanine aminotransferase (ALT), the final concentrations of alanine, α-ketoglutarate, and NADH in the detection system were 400 mM, 15 mM, and 1 mM, respectively, and the final concentration of LDH was 300–1000 U / L; the pH of the detection system was 7.4.

[0058] When detecting alkaline phosphatase, the final concentration of ascorbate phosphate (AA2P) in the detection system is 200 mM; the pH of the detection system is 8.

[0059] Three hepatitis biomarkers were detected using a portable hepatitis biomarker detection kit. The linear range for AST was 5–170 U / L, for ALT it was 5–180 U / L, and for alkaline phosphatase it was 5–110 U / L. The overall recovery rate was between 96.5% and 113%.

[0060] The working principle of this invention's kit: H-MnO2 possesses oxidase-like activity, catalyzing the conversion of colorless TMB to blue oxTMB. ALT catalyzes the transfer reaction between α-ketoglutarate and alanine, generating pyruvate and glutamate; under the action of LDH, pyruvate can react with NADH to produce lactic acid and NAD. + Similarly, AST catalyzes the reaction of aspartic acid and α-ketoglutarate to produce glutamic acid and oxaloacetic acid; under the action of MDH, oxaloacetic acid and NADH react to produce malic acid and NAD. + Both of the above reactions involve the use of NADH. Different concentrations of transaminase consume different amounts of NADH per unit time. The remaining NADH can inhibit the activity of oxidase-like enzymes. The concentration of transaminase can be indirectly reflected by the reaction of the TMB chromogenic agent with the oxidase-like enzyme. Alkaline phosphatase can catalyze the production of ascorbic acid (AA) from AA2P. AA can inhibit the activity of oxidase-like enzymes, reducing the oxidation of the chromogenic agent TMB by the oxidase-like enzymes.

[0061] The kit described in this invention is also applicable to the detection of other small molecules with reducing properties, such as glutathione, cysteine, dopamine, gallic acid, and acetaminophen.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0063] This invention uses polyvinylpyrrolidone as a morphology inducer to promote the reaction of potassium ferricyanide and manganese salt to generate a cubic manganese Prussian blue analog. Then, using the manganese Prussian blue analog as a template and sodium hydroxide as an etchant, hollow manganese dioxide with oxidase-like activity is prepared. This hollow manganese dioxide exhibits extremely high enzyme-like activity and demonstrates excellent optical sensing capabilities. Utilizing the gel-forming properties of sodium alginate, hollow manganese dioxide is encapsulated in a sodium alginate hydrogel to prepare a portable hydrogel kit that can be stored long-term at 4°C.

[0064] This invention utilizes the color change of hollow manganese dioxide in response to the chromogenic reagent TMB to construct a method for detecting hepatitis biomarkers using a smartphone to capture signals. Both NADH and ascorbic acid (AA) inhibit the activity of oxidase-like enzymes, and NADH and AA can serve as substrates for the catalysis of hepatitis biomarkers. Based on this principle, typical hepatitis biomarkers, AST, ALT, and ALP, were detected.

[0065] The hydrogel reagent kit of this invention reduces the detection cost of hepatitis biomarkers, does not require large instruments, and is highly portable and widely applicable, greatly expanding the application scenarios for hepatitis biomarker detection and showing great potential as a novel point-of-care testing (POCT) device. Attached Figure Description

[0066] Figure 1 XPS plots for Mn-PBA and H-MnO2.

[0067] Figure 2 This is a TEM image of H-MnO2.

[0068] Figure 3 It exhibits enzyme-like activity similar to H-MnO2.

[0069] Figure 4 Enzyme-like activities of H-MnO2 generated by etching with different concentrations of NaOH.

[0070] Figure 5 The effect of different pH values ​​on the enzyme-like activity of H-MnO2.

[0071] Figure 6 This is the standard curve for AST.

[0072] Figure 7 This is the standard curve for ALT.

[0073] Figure 8 This is the standard curve for ALP.

[0074] Figure 9 This is the result of the selectivity test of the kit. Detailed Implementation

[0075] Example 1

[0076] Preparation of hollow manganese dioxide (H-MnO2): Solution A was prepared by dissolving 1.0 mmol of anhydrous potassium ferricyanide and 3.0 g of polyvinylpyrrolidone in 50 mL of water, and solution B was prepared by dissolving 1.5 mmol of MnCl2·4H2O in 50 mL of water. Solution B was slowly added to solution A and incubated at room temperature for 24 h. After centrifugation and drying, manganese Prussian blue analog nanoparticles (Mn-PBA) were obtained. 100 mg of manganese Prussian blue analog nanoparticles were used as a template and dispersed in 20 mL of anhydrous ethanol. The nanoparticles were sonicated for 3 min to ensure complete dispersion. The dispersion of manganese Prussian blue analog nanoparticles was brownish-yellow. 40 mL of 0.1 M NaOH solution was added, and the solution gradually changed from dark brown to black. The mixture was stirred at 50 °C for 1 h, centrifuged at 4000 rpm for 5 min, and the precipitate was collected. The precipitate was washed with deionized water until the pH of the filtrate was about 7, then washed with anhydrous ethanol and dried under vacuum to obtain black H-MnO2.

[0077] Figure 1 The image shows the X-ray electron spectrum (XPS) of H-MnO2. After etching with sodium hydroxide, the iron peak of Mn-PBA disappeared, proving that H-MnO2 does not contain iron.

[0078] Figure 2 The projection electron microscope (TEM) shows the H-MnO2 product etched with 0.1M sodium hydroxide. After etching with sodium hydroxide, H-MnO2 exhibits a hollow cubic structure with a side length of approximately 400 nm.

[0079] TMB+H-MnO2 mixed solution, TMB+Mn-PBA mixed solution and control solution were prepared respectively, and the absorption spectra of the solutions in the range of 500-800 nm were measured using a UV-Vis absorption spectrophotometer (UV3600). Figure 3 ).

[0080] TMB + H-MnO2 mixture: Disperse H-MnO2 in water to prepare a 1 mg / mL H-MnO2 dispersion; take 970 μL of NaAc-HAc (0.1 M, pH 5.5) buffer solution, add 10 μL of H-MnO2 dispersion and 20 μL of TMB (20 mM, prepared by dimethyl sulfoxide, the same below) in sequence, mix well, and react at room temperature for 3 min. The color of the mixture quickly changes from colorless to blue.

[0081] TMB+Mn-PBA mixture: Disperse Mn-PBA in water to prepare a Mn-PBA dispersion with a concentration of 1 mg / mL; take 970 μL of NaAc-HAc (0.1 M, pH 5.5) buffer solution, add 10 μL of Mn-PBA dispersion and 20 μL of TMB (20 mM) in sequence, mix well, and react at room temperature for 3 min. The color change of the mixture is not obvious.

[0082] Control solution: Take 980 μL of NaAc-HAc (0.1M, pH 5.5) buffer solution, add 20 μL of TMB (20mM), mix well, react at room temperature for 3 min, the color of the mixture does not change, and remains colorless.

[0083] Depend on Figure 3 It can be seen that H-MnO2 can oxidize TMB and produce a strong absorption peak at 650 nm, while Mn-PBA produces a weaker absorption peak, proving that the hollow manganese dioxide produced by etching with NaOH solution has significant oxidase-like activity.

[0084] Example 2

[0085] Properties of H-MnO2 oxidase

[0086] Following the preparation method of Example 1, only the concentration of NaOH solution was adjusted to 0.01M, 0.05M, 0.5M, and 1M, respectively, to obtain H-MnO2 etching products of different concentrations of sodium hydroxide.

[0087] Different concentrations of NaOH etching product H-MnO2 were dispersed in water to prepare a 1 mg / mL H-MnO2 dispersion. 970 μL of NaAc-HAc (0.1 M, pH 5.5) buffer solution was taken, and 10 μL of H-MnO2 dispersion and 20 μL of TMB (20 mM) were added sequentially. The mixture was stirred evenly and reacted at room temperature for 3 min. The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (UV3600).

[0088] Using the H-MnO2 that produces the maximum absorbance in the mixed solution as the relative activity of 100%, calculate the relative activity of H-MnO2 etched by different concentrations of NaOH:

[0089] Relative activity = (Absorbance of the mixed solution ÷ Absorbance of the mixed solution with the highest absorbance) × 100%

[0090] like Figure 4As shown, the H-MnO2 nanoenzymes obtained by etching manganese-based Prussian blue analog nanoparticles with different concentrations of NaOH all exhibited oxidase activity, but the oxidase-like activities varied considerably. Hollow manganese dioxide etched with lower concentrations of NaOH likely had a smaller specific surface area, thus exhibiting only weaker oxidase-like activity. Hollow manganese dioxide etched with a 0.1M NaOH solution showed the best oxidase-like activity. When the NaOH concentration reached 1M, the activity decreased, possibly because the high concentration of NaOH disrupted the hollow structure. Therefore, 0.1M NaOH was chosen as the etching agent for the preparation of H-MnO2.

[0091] Example 3

[0092] The H-MnO2 prepared in Example 1 was dispersed in water to prepare an H-MnO2 dispersion with a concentration of 1 mg / mL. 970 μL of NaAc-HAc buffer solution at different pH values ​​(0.1 M, pH values ​​2, 3, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8) were taken, and 10 μL of the H-MnO2 dispersion and 20 μL of TMB (20 mM) were added sequentially. The mixtures were mixed thoroughly and reacted at room temperature for 3 min. The color of the mixtures rapidly changed from colorless to blue, and the color deepened. The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (UV3600). The relative activity of H-MnO2 in systems at different pH values ​​was calculated according to Example 2.

[0093] See results Figure 5 The results show that H-MnO2 exhibits oxidase activity in systems with different pH values, but the oxidase-like activities vary considerably. H-MnO2 demonstrates good oxidase activity at pH 4–6; particularly, it exhibits the best enzyme-like activity at pH 5.5. Therefore, it is preferable to use H-MnO2 in an environment with a pH of 5.5.

[0094] Example 4

[0095] The preparation of the hydrogel kit includes the following steps: 150 mg of sodium alginate is dissolved in 10 mL of NaAc-Hac solution (0.1 M, pH 5.5) and stirred at room temperature for 12 h to obtain a sodium alginate solution; H-MnO2 prepared in Example 1 is dispersed in water to prepare an H-MnO2 dispersion with a concentration of 1 mg / mL; 150 μL of the 1 mg / mL H-MnO2 dispersion is added to the sodium alginate solution and stirred for 4 hours to obtain a mixture; 200 μL of the mixture is added to a 1.5 mL centrifuge tube cap, and 20 μL of a 20 mg / mL CaCl2 solution is added to the centrifuge tube cap. The mixture is thoroughly mixed and waited for about 10 min to obtain a translucent hydrogel, which is the hydrogel kit. When the centrifuge tube cap is turned down, the hydrogel will not drip.

[0096] Example 5

[0097] Plotting the standard curve for AST involves the following steps:

[0098] Preparation of substrate solution 1: Dissolve 7.986 g of aspartic acid and 0.219 g of α-ketoglutarate in 100 mL of 0.2 M sodium hydroxide solution, heat and stir to promote dissolution, adjust the pH to about 7.4, cool and add 0.663 g of NADH to obtain substrate solution 1, and store it in a refrigerator at 4 °C.

[0099] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), and sequentially add 50 μL of substrate solution 1, 50 μL of MDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration of MDH in the incubation solution 600 U / L, and 100 μL of AST solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentrations of AST in the incubation solution 5, 10, 30, 50, 70, 90, 110, 130, 150, and 170 U / L, respectively. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the above incubation solution and add it to the hydrogel kit prepared in Example 4, and then disperse 20 μL at more than 5 points. For TMB (20mM), three parallel groups were prepared for each different concentration of AST. After standing for 20 minutes, the color-changing reagent kit was placed in a sealed box. Using a smartphone (iPhone 11), with the lens 11cm away from the reagent kit, a photo was taken to capture the color intensity of the reagent kit. The photo was processed using the software ColorPicker (downloadable from the App Store) to obtain the RGB values ​​of the photo. The gray value (Gray) was calculated from the RGB values: Gray = (0.299*R) + (0.587*G) + (0.114*B).

[0100] A standard curve for aspartate aminotransferase (AST) was established with AST concentration on the x-axis and grayscale values ​​on the y-axis. The results are as follows: Figure 6 As shown, the gray value of the kit decreases with increasing AST concentration, and there is a good linear relationship between AST concentration and the gray value of the kit, with a linear range of 5–170 U / L.

[0101] Example 6

[0102] Plotting the standard curve for ALT involves the following steps:

[0103] Preparation of substrate solution 2: Dissolve 5.844 g alanine and 0.219 g α-ketoglutarate in 100 mL of 0.2 M sodium hydroxide solution, heat and stir to promote dissolution, adjust the pH to about 7.4, cool and add 0.663 g NADH to obtain substrate solution 2, and store at 4 °C.

[0104] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), add 50 μL of substrate solution 2, 50 μL of LDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make a final concentration of 800 U / L, and 100 μL of ALT solution of different concentrations (prepared with 10 mM Tris-hydrochloric acid buffer). Incubate at 37°C for 30 minutes to obtain the incubation solution. The subsequent detection steps are the same as in Example 5, except that AST is replaced with ALT. Specifically, take 45 μL of the above incubation solution and add it to the hydrogel kit prepared in Example 4. Then, disperse 20 μL of TMB (20 mM). Prepare three parallel groups for each group of different ALT concentrations. Let stand for 20 minutes. Place the color-changing kit in a sealed box. Use a smartphone (iPhone 11) with the lens 11 cm away from the kit to take a picture and capture the color intensity of the kit. Use the software Color Picker to process the photo and obtain its RGB values. Then, calculate the grayscale value from the RGB values: Gray = (0.299*R) + (0.587*G) + (0.114*B).

[0105] A standard curve for alanine aminotransferase (ALT) was constructed with ALT concentration on the x-axis and grayscale values ​​on the y-axis. The results are as follows: Figure 7 As shown, the gray value of the kit decreases with increasing ALT concentration, indicating a good linear relationship between ALT concentration and kit gray value, with a linear range of 5–180 U / L.

[0106] Example 7

[0107] The standard curve plotting for ALP includes the following steps:

[0108] Take 300 μL of Tris-hydrochloric acid buffer (0.01 M, pH 8), and add 100 μL of AA2P solution (prepared with water) to make the AA2P concentration in the incubation solution 200 mM. Add 100 μL of ALP solution of different concentrations (prepared with 10 mM Tris-hydrochloric acid buffer) to make the ALP concentrations in the incubation solution 5, 10, 30, 50, 70, 90, and 110 U / L, respectively. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the above incubation solution and add it to the kit prepared in Example 4. Then, disperse 20 μL of TMB (20 mM) and prepare three parallel groups for each group of different ALP concentrations. Let it stand for 20 minutes. Place the color-changing kit in a sealed box and take a picture using a smartphone (iPhone 11) with the lens 11 cm away from the kit to capture the color intensity of the kit. Use the software Color Picker to process the photo and obtain the RGB values ​​of the photo. Calculate the grayscale value from the RGB values: Gray = (0.299*R) + (0.587*G) + (0.114*B).

[0109] A standard curve for alkaline phosphatase was constructed with alkaline phosphatase concentration on the x-axis and grayscale values ​​on the y-axis. The results are as follows: Figure 8 The results show that as the ALP concentration increases, the gray value of the kit increases, and there is a good linear relationship between the ALP concentration and the gray value of the kit, with a linear range of 5–110 U / L.

[0110] Example 8

[0111] Selective experiments were performed on the kit, including:

[0112] AST control group, ALT control group and ALP control group, AST experimental group, ALT experimental group and ALP experimental group were set up respectively.

[0113] AST control group: Take 300 μL of Tris-hydrochloric acid buffer (0.1M, pH 7.4), add 50 μL of substrate solution 1 (same as in Example 5), 50 μL of MDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration of MDH in the incubation solution 600 U / L, and 100 μL of AST solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration of AST in the incubation solution 50 U / L. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the hydrogel kit prepared in Example 4. Then, add 20 μL of TMB (20 mM) at more than 5 points. Let it stand for 20 minutes. Place the color-changing kit in a sealed box and obtain the gray value according to the method in Example 5.

[0114] AST experimental group: Following the preparation method of the AST control group incubation solution, the following interfering substances were added simultaneously: Amino acids (final concentration 10 μM): cysteine ​​(Cys), histidine (His), alanine (Ala), phenylalanine (Phe), lysine (Lys), tryptophan (Try), arginine (Arg), glutamic acid (Glu); Small molecule compounds (final concentration 10 μM): glutathione (GSH), ascorbic acid (AA), H2O2; Proteins (final concentration 20 μg / mL): glucose oxidase (GOx), bovine serum albumin (BSA); Ions (final concentration 5 mM): Ca... 2+ Na + K + HCO3 - HPO4 2- Take 45 μL of incubation solution and add it to the hydrogel kit prepared in Example 4. Then, add 20 μL of TMB (20 mM) at more than 5 points, let it stand for 20 min, place the color-changing kit in a sealed box, and obtain the gray value according to the method in Example 5.

[0115] ALT control group: Take 300 μL of Tris-hydrochloric acid buffer (0.1M, pH 7.4), add 50 μL of substrate solution 2 (same as in Example 6), 50 μL of LDH solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration 800 U / L, and 100 μL of ALT solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration in the incubation solution 50 U / L. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the hydrogel kit prepared in Example 4. Then, disperse 20 μL of TMB (20 mM) and let it stand for 20 minutes. Place the color-changing kit in a sealed box and obtain the gray value according to the method in Example 6.

[0116] ALT experimental group: Following the preparation method of the ALT control group incubation solution, the following interfering substances were added simultaneously: Amino acids (final concentration 10 μM): cysteine ​​(Cys), histidine (His), alanine (Ala), phenylalanine (Phe), lysine (Lys), tryptophan (Try), arginine (Arg), glutamic acid (Glu); Small molecule compounds (final concentration 10 μM): glutathione (GSH), ascorbic acid (AA), H2O2; Proteins (final concentration 20 μg / mL): glucose oxidase (GOx), bovine serum albumin (BSA); Ions (final concentration 5 mM): Ca... 2+ Na + K + HCO3 - HPO4 2-Take 45 μL of incubation solution and add it to the hydrogel kit prepared in Example 4. Then, add 20 μL of TMB (20 mM) at more than 5 points, let it stand for 20 min, place the color-changing kit in a sealed box, and obtain the gray value according to the method in Example 6.

[0117] ALP control group: Take 300 μL of Tris-hydrochloric acid buffer (0.01 M, pH 8), add 100 μL of AA2P solution (prepared with water) to make the final concentration of AA2P in the incubation solution 200 mM, and 100 μL of ALP solution (prepared with 10 mM Tris-hydrochloric acid buffer) to make the final concentration of ALP in the incubation solution 50 U / L. Incubate at 37°C for 30 minutes to obtain the incubation solution; take 45 μL of the incubation solution and add it to the kit prepared in Example 4, then disperse 20 μL of TMB (20 mM), let stand for 20 min, place the color-changing kit in a sealed box, and obtain the gray value according to the method in Example 7.

[0118] ALP experimental group: Following the preparation method of the ALP control group incubation solution, the following interfering substances were added simultaneously: Amino acids (final concentration 10 μM): cysteine ​​(Cys), histidine (His), alanine (Ala), phenylalanine (Phe), lysine (Lys), tryptophan (Try), arginine (Arg), glutamic acid (Glu); Small molecule compounds (final concentration 10 μM): glutathione (GSH), ascorbic acid (AA), H2O2; Proteins (final concentration 20 μg / mL): glucose oxidase (GOx), bovine serum albumin (BSA); Ions (final concentration 5 mM): Ca... 2+ Na + K + HCO3 - HPO4 2- Take 45 μL of incubation solution and add it to the hydrogel kit prepared in Example 4. Then, add 20 μL of TMB (20 mM) at more than 5 points, let it stand for 20 min, place the color-changing kit in a sealed box, and obtain the gray value according to the method in Example 6.

[0119] The gray values ​​obtained from the experimental group and the control group were compared separately, and the results are as follows: Figure 9 As shown, the grayscale value did not change much when hepatitis markers and interfering substances coexisted, indicating that these interfering substances had little interference with the kit, which is attributed to the specificity of the enzyme cascade reaction.

[0120] Example 9

[0121] The kit prepared in Example 4 was used to test real samples, including the following steps:

[0122] The serum sample was diluted to a volume fraction of 5% with phosphate buffer solution (pH=7.4) at a volume ratio of 1:19. AST, ALT and ALP standards were added to the diluted serum sample according to Table 1 for spiking tests.

[0123] The method for detecting AST levels in diluted serum samples is as described in Example 5:

[0124] Preparation of substrate solution 1: Same as in Example 5;

[0125] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), add 50 μL of substrate solution 1 and 50 μL of MDH solution (prepared from 10 mM Tris-hydrochloric acid buffer) to make the final concentration of MDH in the incubation solution 600 U / L, and 100 μL of diluted serum sample. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the hydrogel kit prepared in Example 4. Then, add 20 μL of TMB (20 mM) at more than 5 points and let it stand for 20 minutes. Place the color-changing kit in a sealed box, take a picture using a smartphone (iPhone 11) with the lens 11 cm away from the kit, capture the color intensity of the kit, and obtain the gray value according to the method in Example 5. Substitute the gray value into the aspartate aminotransferase standard curve to obtain the AST content in the diluted serum sample.

[0126] The method for detecting ALT levels in diluted serum samples is as described in Example 6:

[0127] Preparation of substrate solution 2: Same as in Example 6;

[0128] Take 300 μL of Tris-hydrochloric acid buffer (0.1 M, pH 7.4), add 50 μL of substrate solution 2 and 50 μL of LDH solution (prepared from 10 mM Tris-hydrochloric acid buffer) to make a final concentration of 800 U / L, and 100 μL of diluted serum sample. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the hydrogel kit prepared in Example 4. Then, disperse 20 μL of TMB (20 mM) and let it stand for 20 minutes. Place the color-changing kit in a sealed box. Use a smartphone (iPhone 11) with the lens 11 cm away from the kit to take a picture and capture the color intensity of the kit. Obtain the gray value according to the method in Example 6. Substitute the gray value into the ALT standard curve to obtain the ALT content in the diluted serum sample.

[0129] The method for detecting ALP levels in diluted serum samples is as described in Example 7:

[0130] Take 300 μL of Tris-hydrochloric acid buffer (0.01 M, pH 8), add 100 μL of AA2P solution (prepared with water) to make the AA2P concentration in the incubation solution reach a final concentration of 200 mM, and add 100 μL of diluted serum sample. Incubate at 37°C for 30 minutes to obtain the incubation solution. Take 45 μL of the incubation solution and add it to the kit prepared in Example 4. Then, disperse 20 μL of TMB (20 mM) and let it stand for 20 minutes. Place the color-changing kit in a sealed box, and take a picture using a smartphone (iPhone 11) with the lens 11 cm away from the kit to capture the color intensity of the kit. Obtain the gray value according to the method in Example 7. Substitute the gray value into the alkaline phosphatase standard curve to obtain the ALP content in the diluted serum sample.

[0131] Table 1. Detection results of clinical serum samples using the method of the present invention and commercial reagent kits.

[0132]

[0133] As shown in Table 1, the portable hepatitis biomarker detection kit of the present invention provides accurate and reliable results for the determination of hepatitis biomarkers in clinical samples. Specifically, the recovery rates of AST are 95.3%–105.4%, ALT is 97.3%–107.9%, and ALP is 97.1%–102.4%. Furthermore, the measurement error between the portable hepatitis biomarker detection kit of the present invention and commercial kits is less than 6%. Therefore, the portable hepatitis biomarker detection kit of the present invention has great commercial application potential.

Claims

1. A portable hepatitis biomarker detection kit, characterized in that: The test kit contains a hydrogel; the hydrogel is prepared by dispersing hollow manganese dioxide in water to obtain a hollow manganese dioxide dispersion, mixing the hollow manganese dioxide dispersion with sodium alginate solution to obtain a mixed solution, and adding calcium salt solution to obtain the hydrogel. The hollow manganese dioxide is obtained by etching manganese Prussian blue analog nanoparticles with sodium hydroxide, comprising: dissolving potassium ferricyanide and polyvinylpyrrolidone in water to prepare solution A, dissolving manganese salt in an equal volume of water to prepare solution B, slowly adding solution B to solution A, incubating at room temperature for 10-24 hours, centrifuging and drying to obtain manganese Prussian blue analog nanoparticles; dispersing 20-200 mg of manganese Prussian blue analog nanoparticles in 10-40 mL of solvent, sonicating to ensure uniform dispersion of nanoparticles, using 40 mL of sodium hydroxide solution for every 20-200 mg of manganese Prussian blue analog nanoparticles, wherein the concentration of NaOH in the sodium hydroxide solution is 0.05-1 M, stirring at 25-60 °C for 0.1-4 hours, centrifuging and drying to obtain hollow manganese dioxide; the preparation method of the sodium alginate solution is as follows: weighing 100-350 mg of sodium alginate and dissolving it in 10 mL Sodium alginate solution was obtained by stirring the NaAc-HAc buffer solution at room temperature for 12–24 h; the pH of the NaAc-HAc buffer solution was 4–6.

2. A method for preparing a portable hepatitis biomarker detection kit as described in claim 1, characterized in that: Includes the following steps: Step (1) Preparation of hollow manganese dioxide: Dissolve potassium ferricyanide and polyvinylpyrrolidone in water to prepare solution A, dissolve manganese salt in the same volume of water to prepare solution B, slowly add solution B to solution A, incubate at room temperature for 10-24 hours, centrifuge and dry to obtain manganese Prussian blue analog nanoparticles; disperse 20-200 mg of manganese Prussian blue analog nanoparticles in 10-40 mL of solvent, sonicate to make the nanoparticles uniformly dispersed, use 40 mL of sodium hydroxide solution for every 20-200 mg of manganese Prussian blue analog nanoparticles, the concentration of NaOH in the sodium hydroxide solution is 0.05-1 M, stir at 25-60℃ for 0.1-4 hours, centrifuge and dry to obtain hollow manganese dioxide; Step (2): Preparation of portable hepatitis biomarker detection kit: Weigh 100-350 mg of sodium alginate and dissolve it in 10 mL of NaAc-HAc buffer solution with pH 4-6 to obtain sodium alginate solution; disperse hollow manganese dioxide in water to obtain hollow manganese dioxide dispersion with a concentration of 1 mg / mL; add 100-250 mL of hollow manganese dioxide dispersion to sodium alginate solution and stir to obtain a mixture; take 200 mL of the mixture and add it to a small dish, add 20 mL of calcium salt solution with a concentration of 20 mg / mL, mix thoroughly to obtain hydrogel, which is the portable hepatitis biomarker detection kit.

3. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (1), the molar ratio of potassium ferricyanide to manganese salt is 1:1 to 1:3; the molar ratio of potassium ferricyanide to polyvinylpyrrolidone is 1 mmol: 3 g; after solution B is added to solution A, the concentration of potassium ferricyanide is 1 to 100 mM.

4. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (1), the manganese salt is any one of MnCl2, MnSO4, and Mn(NO3)2.

5. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (1), 100 mg of manganese Prussian blue analog nanoparticles were dispersed in 20 mL of solvent; 40 mL of sodium hydroxide solution was used for every 100 mg of manganese Prussian blue analog nanoparticles.

6. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (1), the solvent is one of anhydrous ethanol, N,N-dimethylformamide, and dimethyl sulfoxide.

7. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (1), the concentration of NaOH in the sodium hydroxide solution is 0.1M.

8. The method for preparing the portable hepatitis biomarker detection kit according to claim 2, characterized in that: In step (2), the pH of the NaAc-HAc buffer solution is 5.5; The calcium salt mentioned is one of CaCl2 and Ca(NO3)2.

9. The application of the hydrogel according to claim 1 in the preparation of a portable hepatitis biomarker detection kit, wherein the application comprises the following steps: Step (a) Plotting the transaminase standard curve: Construct an incubation system with different concentrations of aspartate aminotransferase (AST), malate dehydrogenase (MDH), and substrate 1, and incubate; take the incubated solution and add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish an AST standard curve with AST concentration as the x-axis and gray value as the y-axis; Construct an incubation system with different concentrations of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and substrate 2, and incubate; take the incubated solution and add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish an ALT standard curve with ALT concentration as the x-axis and gray value as the y-axis; Plotting the alkaline phosphatase standard curve: Construct an incubation system with different concentrations of alkaline phosphatase and catalytic substrate 3, and incubate; take the incubated solution, add it to the portable hepatitis biomarker detection kit, add TMB for color development, take a picture, obtain the RGB values ​​of the picture, calculate the gray value from the RGB values, and establish the alkaline phosphatase standard curve with alkaline phosphatase concentration as the x-axis and gray value as the y-axis. Step (b), Sample Analysis: Take a serum sample and dilute it; The diluted serum sample was used to construct a detection system with malate dehydrogenase and catalytic substrate 1 and incubated. The incubated solution was added to the portable hepatitis marker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were substituted into the aspartate aminotransferase standard curve to obtain the aspartate aminotransferase content in the serum sample. The diluted serum sample was used to construct a detection system with lactate dehydrogenase and catalytic substrate 2 and incubated. The incubated solution was then added to the portable hepatitis marker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were then substituted into the alanine aminotransferase standard curve to obtain the alanine aminotransferase content in the serum sample. The diluted serum sample was used to construct a detection system with catalytic substrate 3 and incubated. The incubated solution was then added to the portable hepatitis biomarker detection kit, TMB was added for color development, and gray values ​​were obtained according to step (a). The gray values ​​were then substituted into the alkaline phosphatase standard curve to obtain the alkaline phosphatase content in the serum sample.

10. The application according to claim 9, characterized in that: In step (a), the catalytic substrate 1 of AST includes aspartic acid, α-ketoglutarate, and NADH; In the incubation system, the final concentrations of aspartic acid, α-ketoglutarate, and NADH were 600 mM, 15 mM, and 1 mM, respectively; the final concentration of MDH was 300–1000 U / L; and the final concentration of aspartate aminotransferase was 5–170 U / L. The pH of the incubation system was 7.

4. The catalytic substrates of alanine aminotransferase (ALT) include alanine, α-ketoglutarate, and NADH. In the incubation system, the final concentrations of alanine, α-ketoglutarate, and NADH were 400 mM, 15 mM, and 1 mM, respectively; the final concentration of LDH was 300–1000 U / L; and the final concentration of alanine aminotransferase was 5–180 U / L. The pH of the incubation system was 7.

4. The catalytic substrate 3 of alkaline phosphatase is ascorbate phosphate; In the incubation system, the final concentration of ascorbic acid phosphate was 200 mM, the final concentration of alkaline phosphatase was 5–110 U / L, and the pH of the incubation system was 8. In step (b), when detecting aspartate aminotransferase, the final concentrations of aspartate, α-ketoglutarate, and NADH in the detection system are 600 mM, 15 mM, and 1 mM, respectively, and the final concentration of MDH is 300–1000 U / L; the pH of the detection system is 7.

4. When detecting alanine aminotransferase (ALT), the final concentrations of alanine, α-ketoglutarate, and NADH in the detection system were 400 mM, 15 mM, and 1 mM, respectively, and the final concentration of LDH was 300–1000 U / L; the pH of the detection system was 7.

4. When detecting alkaline phosphatase, the final concentration of ascorbate phosphate in the detection system is 200 mM; the pH of the detection system is 8.

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