Accurate quantitative analysis method for lactic acid and lactate oxidase based on Amperometric i-t technology and application

Abstract

The invention designs an accurate quantitative analysis method for lactic acid and lactate oxidase based on an Amperometric i-t technology, and the method comprises the following steps: firstly, synthesizing Cu0. 9Zn0. 1S-coated black phosphorus nanosheet nano-enzyme which has excellent dispersity, excellent adhesion and better catalytic performance; secondly, lactic acid oxidase (LOX) can oxidize LA to generate pyruvic acid and H2O2, and analysis and detection of lactic acid and lactic acid oxidase can be realized by utilizing electrochemical response of the material to H2O2. Therefore, the Amperometric i-t technology is used, the law of current change along with time change under different concentrations of LA and LOX is explored through the mutual relation between the current and the time, and a real-time dynamic analysis LA and LOX accurate quantitative analysis detection method is developed. In conclusion, the accurate quantitative analysis method based on the Amperometric i-t Curve electrochemical technology is constructed, and the method can be well applied to activity analysis of the lactate oxidase in the liver cancer cells and has good scientific significance and social benefits.

Description

Technical Field

[0001] The present invention relates to an analysis method mediated by Amperometric IT technology and its application, in particular to a quantitative analysis method based on Amperometric IT technology and its application in the analysis and detection of lactate and lactate oxidase, which is ultimately applied to the analysis of lactate oxidase activity in normal cells and liver cancer cells, belonging to the field of functional biomaterials and biosensor technology. Background Art

[0002] Lactate is a metabolite produced by glucose or glycogen in the blood under anaerobic or hypoxic conditions. During strenuous exercise, large amounts of lactate are produced. Lactate that cannot be broken down quickly accumulates in the body and can cause harm. Monitoring lactate levels can also reflect fatigue. Lactate levels can be used to monitor an athlete's exercise intensity and metabolic status, allowing adjustments to be made for more effective training. Therefore, lactate detection is of great significance in fields such as medicine and sports. The most commonly used analytical methods for measuring lactate include high-performance liquid chromatography, fluorescence, colorimetry, nuclear magnetic resonance spectroscopy, and chemiluminescence. These methods all have static characteristics and are not suitable for dynamic analysis, meaning they cannot track changes in lactate levels in real time. Electrochemical methods stand out due to their unique advantages. While there are numerous electrochemical analysis methods with widely varying sensitivity, amperometry is capable of capturing minute changes or effects in real time. Currently, no literature has been found using this technique to develop electrochemical lactate sensors.

[0003] The foundation of Amperometric it Curve real-time dynamic electrochemical technology lies in the modification of the electrochemical substrate electrode, which requires high electron transfer rates, excellent catalytic performance, and excellent anti-shedding properties. With the rapid development of nanotechnology, nanozymes have replaced natural enzymes due to their advantages, such as resistance to loss of catalytic activity under extreme pH and high temperature conditions, low cost, and easy storage. Nanozymes are mainly divided into carbon-based nanozymes, metal-based nanozymes, metal oxide-based nanozymes, metal sulfide nanozymes, and nanozymes with metal frameworks. Copper sulfide, as an important p-type semiconductor nanomaterial, possesses excellent structure and surface properties and is widely used in biosensors, batteries, optoelectronic devices, and other fields. Common methods for synthesizing copper sulfide nanoparticles include solvothermal, microwave-assisted synthesis, template-directed synthesis, and cation exchange. To date, there are many reports on copper sulfide nanoparticles, but few reports on doped copper sulfide nanozymes. In particular, reports on copper sulfide nanozymes with excellent dispersibility and catalytic performance and their application in the analysis and detection of lactate and lactate oxidase are extremely rare and hold great potential.

[0004] The present invention designs a precise quantitative analysis method and application of lactic acid and lactate oxidase based on Amperometric i-t technology. In this method, zinc ions are first doped into copper sulfide. Due to the presence of zinc ions, the carrier transport rate is accelerated. Then, by compounding with black phosphorus nanosheet materials, the electron transport rate is further increased. This nanozyme has good peroxidase activity, but its dispersion performance in water is poor. Therefore, in order to better apply this nanozyme to Amperometric i-t Curve electrochemical technology, 0.1% octylphenoxypolyethoxyethanol (Triton X-100) is added in this patent to improve its dispersibility and electrode adhesion. Secondly, lactate oxidase (LOX) can oxidize LA to generate pyruvate and H2O2. The electrochemical response of this material to H2O2 can be used to analyze and detect lactic acid and lactate oxidase. Therefore, this patent uses Amperometric i-t Curve electrochemical technology to explore the law of current change with time under different concentrations of LA and LOX through the mutual relationship between current and time, and develops a precise quantitative analysis and detection method for LA and LOX with real-time dynamic analysis, and finally applies it to the analysis and detection of target substances in liver cancer cells. In summary, the present invention constructs a precise quantitative analysis method based on Amperometric i-t technology, which can be well applied to the dynamic analysis of lactic acid and lactate oxidase, especially in tumor cells, solves the bottleneck problem of static analysis, and has good scientific significance and social benefits. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a precise quantitative analysis method and application of lactic acid and lactate oxidase based on Amperometric i-t technology with good specificity, high sensitivity, fast detection speed, accurate and reliable results, and low cost.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A precise quantitative analysis method and application of lactic acid and lactate oxidase based on Amperometric i-t technology, the specific steps are as follows:

[0007] (1) Preparation of nanozyme Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheets

[0008] a. Synthesis of Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheet Nanozyme

[0009] Copper(II) chloride dihydrate (0.1 - 0.2 mmol, 17.1 - 34.2 mg), sodium citrate (0.05 - 0.068 mmol, 20.0 - 27.2 mg), zinc acetate dihydrate (0.1 - 0.2 mmol, 30 - 60 mg), and 100 mg of black phosphorus nanosheets were dissolved in 90 mL of deionized water and stirred at room temperature for 3 - 5 min. Then, 10 - 20 mL of sodium sulfide solution (Na2S, 0.02 - 0.04 M) was added to the light blue mixed solution, and the color of the mixture changed to dark brown. After that, the mixed solution was magnetically stirred at room temperature for 3 - 10 min and then transferred to a water bath at 80 - 90 °C and heated with stirring for 10 - 15 min. Subsequently, the prepared dark green mixed solution was cooled in an ice water environment and then centrifuged at 6000 - 9000 rpm for 20 - 30 min. The centrifuged sample was washed 2 - 4 times with absolute ethanol and deionized water respectively and then dried in an oven at 45 - 50 °C for 4 - 6 h. Thus, Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme was obtained;

[0010] b. Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme with good dispersibility and adhesiveness

[0011] Weigh 1 - 2 mg of Triton X - 100 and prepare a Triton X - 100 solution with a mass fraction of 0.05 - 0.1%, and store it at 4 °C for later use. Then weigh 1 - 2 mg of Cu 0.9 Zn 0.1 S@black phosphorus nanosheets and add them to 1 - 2 mL of the Triton X - 100 solution. After ultrasonic treatment for 25 - 30 min, a uniformly dispersed 1 - 2 mg / mL Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme solution was obtained and reserved for use.

[0012] (2) Precise quantitative analysis method based on Amperometric i - t Curve electrochemical technology

[0013] a. The glassy carbon electrode (GCE, diameter 3 mm) was polished on suede with aluminum oxide powder of particle sizes 0.3 μm and 0.05 μm for 0.5 - 5 min in sequence. After polishing, the electrode was placed in an ultrasonic cleaner and ultrasonically cleaned with ultrapure water for 1 - 5 min, and then dried with N2, denoted as GCE;

[0014] b. Take 8 - 10 μL of the Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme solution from step (1c) above and drop it onto the GCE electrode. After air - drying at room temperature, it was reserved for use, denoted as Cu 0.9Zn 0.1 S@Black Phosphorus Nanosheets / GCE.

[0015] c. Place the electrode from step (2b) above into 1 - 2 mL of PBS solution (0.1 M, pH = 7.0), add 5 - 10 μL of 100 - 200 mM H2O2 (final concentration: 0.5 - 1 mM), and perform detection using Amperometric i - t Curve electrochemical technique with the voltage set at -0.5 V.

[0016] d. Lactate oxidase catalytic reaction: The total volume is 18 - 20 μL, including 1 - 2 μL of 40 - 50 mM lactate solution (final concentration: 2.5 - 3 mM), 1 - 2 μL of 4000 - 5000 U / L lactate oxidase (final concentration: 400 - 500 U / L), and 8 - 10 μL of PBS (100 mM, pH = 7.4) and ultrapure water. After thorough mixing, wrap it with tin foil and place it in a water bath at 35 - 37 °C for reaction for 25 - 30 min; place the electrode from step (2b) above into 1 - 2 mL of PBS solution (0.1 M, pH = 7.0), add 3 - 5 μL of the lactate oxidase catalytic reaction solution, and perform detection using Amperometric i - t Curve electrochemical technique with the voltage set at -0.5 V.

[0017] In step (2c) above, by changing the H2O2 concentration (final concentration: 0, 0.01, 0.1, 0.3, 0.5, 1, 3, 5, 10, 20, 50, 80, 100, 200, 500, 800, 1000 μM) while keeping other steps unchanged, quantitative analysis of H2O2 at different concentrations can be achieved.

[0018] In step (2d) above, by changing the LA concentration (final concentration: 0, 0.001, 0.005, 0.01, 0.03, 0.1, 0.3, 1, 4, 10, 35, 100, 300, 800, 1500, 3000, 6000, 10000 μM) while keeping other steps unchanged, detection of LA at different concentrations can be achieved; by changing the LOX concentration (final concentration: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 30, 60, 200, 500, 1000, 2000 U / L) while keeping other steps unchanged, detection of LOX at different concentrations can be achieved; by adding different concentrations of the inhibitor Fe(III) (final concentration: 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 800, 2000, 4000, 8000 μM) while keeping other steps unchanged, screening of LOX inhibitors can be achieved.

[0019] Using the above electrochemical precise quantitative analysis method and its application for detecting lactic acid and lactate oxidase, in order to achieve its dynamic real-time monitoring, the Amperometric i-t technique is adopted, the potential is set to -0.5V, and Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme is used for the electrochemical catalysis of H2O2, and a series of current signal magnitudes corresponding to different concentrations of LA are obtained in the electrolyte solution containing the LOX catalytic reaction solution, a quantitative relationship between the current response and LA is established, and according to the quantitative relationship between the two, the content of LA in the sample to be measured is determined. Similarly, a series of current signal magnitudes corresponding to different concentrations of LOX are obtained in the electrolyte solution containing the LA reaction solution, a quantitative relationship between the current response and LOX is established, and according to the quantitative relationship between the two, the content of LOX in the sample to be measured is determined.

[0020] Development principle: The present invention is a precise quantitative analysis method and application for lactic acid and lactate oxidase based on the Amperometric i-t technique. First, lactate oxidase can oxidize lactic acid to generate an unstable complex, and the complex will be oxidized to generate H2O2; second, Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme has excellent catalytic performance. As the concentration of LA or LOX increases, more H2O2 is generated, the current response is greater, and with the change of time, dynamic monitoring can be realized. Its final current value shows a linear relationship with the logarithm of the concentrations of LA and LOX within a certain range, realizing the detection of LA and LOX. Its advantages are as follows:

[0021] (1) Excellent Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme. It has good dispersibility, adhesiveness and catalytic performance, is an ideal electrode modification material, and has high catalytic activity for H2O2, laying a foundation for the dynamic analysis and detection of LA and LOX in this patent.

[0022] (2) Real-time dynamic analysis. The present invention is based on Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme to prepare an electrochemical sensor. By using LOX to catalyze LA to generate H2O2, the dynamic real-time analysis of the generation or consumption of LA can be realized through the Amperometric i-t Curve electrochemical technique, which is helpful for the monitoring of lactic acid changes in the field of physical exercise and the development of clinical test methods for biochemical indicators.

[0023] (3) High sensitivity. The precise quantitative analysis method for lactic acid and lactate oxidase based on the Amperometric i-t Curve electrochemical technology of the present invention obtains three linear equations; the correlation equation between the current response and the logarithm of the H2O2 concentration is y = -11.81lgC H2O2 -6.095, R 2 = 0.9927, and the detection limit is 0.0033 μM; the correlation equation between the current response and the logarithm of the LA concentration is y = -0.0372lgC LA -0.0617, R 2 = 0.9951, and the detection limit is 0.00026 μM; the correlation equation between the current response and the logarithm of the LOX concentration is y = -0.0457lgC LOX -0.0732, R 2 = 0.9919, and the detection limit is 0.0067 U / L; it shows that the sensor can achieve high-sensitivity detection of LA and LOX.

[0024] (4) High specificity. For the detection of LA, other control substances, such as urea, glucose, caffeine, ascorbic acid (AA), acetaminophen (AP), and glutathione (GSH), have no interference on the system; for the detection of LOX, other control substances, such as acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase), and lysozyme (LZM), have a negligible current response, achieving specific detection of LOX.

[0025] (5) Strong applicability. The present invention can achieve high-sensitivity detection of LA and LOX by consuming only a small amount of materials and reagents, and is finally applied to the analysis and detection of target substances in liver cancer cells, and the results are statistically significant.

[0026] In summary, the present invention constructs a precise quantitative analysis method and application for lactic acid and lactate oxidase based on the Amperometric i-t technology, which has the advantages of high sensitivity, good selectivity, simple operation, rapid analysis, and easy operation. It can detect low-concentration LOX in liver cancer cells and has good application prospects. Brief Description of the Drawings

[0027] Figure 1 It is the electrochemical response diagram of the sensor of the present invention to H2O2;

[0028] Figure 2 It is the linear relationship diagram between the current response of the sensor of the present invention to different concentrations of H2O2 and the logarithm of the concentration;

[0029] Figure 3Feasibility experiment diagram for the analysis and detection of LA and LOX by the sensor of the present invention;

[0030] Figure 4 Linear relationship diagram of the current response of the sensor of the present invention to LA versus the logarithm of concentration;

[0031] Figure 5 Linear relationship diagram of the current response of the sensor of the present invention to LOX versus the logarithm of concentration;

[0032] Figure 6 Linear relationship diagram of the current response of the sensor of the present invention to Fe(III) versus the logarithm of concentration;

[0033] Figure 7 Specificity experiment diagram of the sensor of the present invention for LA and LOX.

[0034] Figure 8 Detection experiment diagram of the LOX activity in liver cancer cells by the sensor of the present invention. Detailed Description of the Invention

[0035] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings.

[0036] Example 1: Preparation of nanozyme Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheets

[0037] a. Synthesis of Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheet Nanozyme

[0038] Copper dichloride dihydrate (0.2 mmol, 34.2 mg), sodium citrate (0.068 mmol, 20 mg), zinc acetate dihydrate (0.2 mmol, 60 mg), and 100 mg of black phosphorus nanosheets were dissolved in 90 mL of deionized water and stirred at room temperature for 5 min. Then, 10 mL of sodium sulfide solution (Na2S, 0.02 M) was added to the light blue mixed solution, and the color of the mixture changed to black-brown. After magnetic stirring at room temperature for 5 min, the mixed solution was transferred to a 90 °C water bath and heated with stirring for 15 min. Subsequently, the prepared dark green mixed solution was cooled in an ice water environment, and then centrifuged at 9000 rpm for 30 min. The centrifuged sample was washed 3 times with absolute ethanol and deionized water respectively, and then dried in an oven at 50 °C for 4 h to obtain Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheet Nanozyme;

[0039] b. Cu 0.9 Zn 0.1 S@Black Phosphorus Nanosheet Nanozyme with Good Dispersibility and Viscosity

[0040] Weigh 2 mg of Triton X-100 and dissolve it in 2 mL of 0.005 M HCl solution. After stirring well to dissolve, adjust the pH value to 7.0 with 0.1 mol / L NaOH solution to prepare a 0.1% (mass fraction) Triton X-100 solution, and store it at 4 °C for later use. Then weigh 1 mg of Cu 0.9 Zn 0.1 S@black phosphorus nanosheets and add them to 1 mL of the Triton X-100 solution. After ultrasonication for 30 min, a uniformly dispersed 1 mg / mL Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme solution is obtained for later use.

[0041] Example 2 Precise quantitative analysis method based on Amperometric i-t Curve electrochemical technology

[0042] a. Polish a glassy carbon electrode (GCE, diameter 3 mm) successively with 0.3 μm and 0.05 μm alumina powder on suede for 1 min. After polishing, place the electrode in an ultrasonic cleaner and ultrasonically clean it with ultrapure water for 1 min, then dry it with N2, denoted as GCE;

[0043] b. Take 10 μL of the Triton X-100 solution and drop it onto the GCE electrode. After drying at room temperature, it is ready for use, denoted as Triton X-100 / GCE;

[0044] c. Drop 10 μL of the Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme solution from step (1c) onto the GCE electrode. After drying at room temperature, it is ready for use, denoted as Cu 0.9 Zn 0.1 S@black phosphorus nanosheet / GCE.

[0045] Detect the electrochemical response of the above-mentioned electrode to a PBS (0.1 M, pH 7.0) electrolyte solution, and add 1 mM H2O2. As Figure 1 , it can be seen that the prepared sensor shows an obvious electrochemical response to H2O2 compared with the other two electrodes. Change the H2O2 concentration (final concentrations: 0, 0.01, 0.1, 0.3, 0.5, 1, 3, 5, 10, 20, 50, 80, 100, 200, 500, 800, 1000 μM), and keep other steps unchanged. As Figure 2 shown, there is a good linear relationship between the logarithm of the H2O2 concentration and the current signal value. The correlation equation of the current response to the logarithm of the H2O2 concentration is y = -11.81lgC H2O2 - 6.095, R 2= 0.9927, and the detection limit was 0.0033 μM. Thus, it can be concluded that the sensor can achieve quantitative analysis of H2O2 at different concentrations.

[0046] Feasibility Experiment of Example 3

[0047] According to the sensor preparation steps of the above Examples 1 and 2, a reaction solution with a total volume of 20 μL, including LA (final concentration: 3000 μM), LOX (500 U / L), and PBS (0.1 M, pH = 7.4), was reacted in a water bath at 37 °C for 30 min, and then the electrochemical response detection of Cu 0.9 Zn 0.1 S@black phosphorus nanosheets / GCE was carried out. The results are as Figure 3 shown in A. It can be seen that Cu 0.9 Zn 0.1 S@black phosphorus nanosheets / GCE has a good electrochemical response to LA and can be applied to the detection of LA.

[0048] According to the sensor preparation steps of the above Examples 1 and 2, a reaction solution with a total volume of 20 μL, including LA (final concentration: 3 mM), LOX (0, 500 U / L), and PBS (0.1 M, pH = 7.4), was reacted in a water bath at 37 °C for 30 min, and then the electrochemical response detection of Cu 0.9 Zn 0.1 S@black phosphorus nanosheets / GCE was carried out. The results are as Figure 3 shown in B. It can be seen that Cu 0.9 Zn 0.1 S@black phosphorus nanosheets / GCE has a good electrochemical response to LOX and can be applied to the detection of LOX.

[0049] Detection of LA, LOX and Its Inhibitor Fe(III) in Example 4

[0050] According to the responses of LA in the above Examples 1, 2 and Example 3, by changing the concentration of LA (final concentration: 0, 0.001, 0.005, 0.01, 0.03, 0.1, 0.3, 1, 4, 10, 35, 100, 300, 800, 1500, 3000, 6000, 10000 μM), the response of the reaction solution was detected using the sensor. The results are as Figure 4 shown. The current response of the sensor to LA shows a good linear relationship with the logarithm of its concentration. The linear correlation equation of the current response of the sensor corresponding to the logarithm of LA concentration is: y = -0.0372lgC LA - 0.0617, R 2 = 0.9951, and the linear range is 0.001 - 3000 μM, and the detection limit is 0.00026 μM, indicating that the sensor has achieved highly sensitive detection of LA.

[0051] According to the responses of LOX in the above Examples 1, 2, and 3, by changing the concentration of LOX (final concentrations: 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 30, 60, 200, 500, 1000, 2000 U / L), the responses of the reaction solution were detected using the sensor. The results are as Figure 5 shown. The current response of the sensor to LOX shows a good linear relationship with the logarithm of its concentration. The linear correlation equation of the current response of the sensor corresponding to the logarithm of the LOX concentration is: y = -0.0457lgC LOX - 0.0732, R 2 = 0.9919. The linear range is 0.05 - 500 U / L, and the detection limit is 0.0067 U / L, indicating that the sensor has achieved highly sensitive detection of LOX.

[0052] Based on the steps of the above Examples 1 and 2, different concentrations of Fe(III) (final concentrations: 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 800, 2000, 4000, 8000 μM) were mixed with 2 μL of 5000 U / L LOX (final concentration: 500 U / L) and PBS (0.1 M, pH = 7.4), incubated in a 37 °C water bath for 30 min, and then added to 1.2 μL of 50 mM lactic acid (final concentration: 3 mM), PBS (0.1 M, pH = 7.4), and ultrapure water. The total volume was 20 μL. After incubation in a 37 °C water bath for 30 min, 5 μL of the mixed solution was taken and added to 1 mL of PBS (0.1 M, pH = 7.0) electrolyte solution to test the inhibitory effect of Fe(III) on LOX. The results are as Figure 6 shown. As Fe(III) was added, the current response became smaller and smaller, indicating that the inhibitory effect of Fe(III) on the activity of LOX was stronger. The half - inhibitory concentration of Fe(III) was 12.4 μM.

[0053] Example 5 Specific Detection of LA and LOX

[0054] To verify the specificity of the sensor for LA, according to the sensor preparation steps of the above Examples 1 and 2, urea (Urea), glucose (Glucose), caffeine (Caffeine), ascorbic acid (AA), acetaminophen (AP), and glutathione (GSH) with the same concentration as LA were added respectively to detect the specificity of the sensor for LA. The results are as Figure 7 shown in A. It can be seen that compared with LA, other small - molecule substances had no current response, indicating that the sensor has good specificity for the detection of LA.

[0055] To verify the specificity of the sensor for LOX, according to the sensor preparation steps of Examples 1 and 2 above, acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase), and lysozyme (LZM) with the same concentration as LOX were added respectively to detect the selectivity of the sensor for LOX. The results are as Figure 7 shown in B. It can be seen that compared with LOX, other small molecule substances have no current response, indicating that the sensor has good specificity for the detection of LOX.

[0056] Example 6 Detection of LOX activity in liver cancer cells.

[0057] Using the snu449 cell line as a template, after subculture, in 1 mL of snu449 cell suspension (containing 5 million cells), the nuclear proteins of liver cancer cells were extracted using a kit, and LA (final concentration: 3 mM) was added. After incubation for 3 h and 12 h, the LOX activity was monitored. As Figure 8 shown, using normal laboratory cells as a comparison, Sample 1 and Sample 2 are normal cells after 3 h of incubation, Sample 3 and Sample 4 are liver cancer cells after 12 h of incubation. The signal of Sample 2 is stronger than that of Sample 1, indicating that the more lactic acid, the stronger the LOX activity and the more H2O2 is produced. The signal of Sample 4 is stronger than that of Sample 2, indicating that LOX is highly expressed in liver cancer cells. These results are statistically significant with P < 0.05. Generally speaking, excessive free radicals can cause great harm to the human body and lead to certain diseases. Therefore, the exploration of the activity of intracellular LOX can help understand the mechanism of disease occurrence and provide a new idea.

[0058] It should also be noted that the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Those skilled in the relevant art of the present technology field who make changes, modifications, additions, or substitutions within the scope of the essence of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. The precise quantitative analysis method and application of lactic acid and lactate oxidase based on Amperometric i-t technology, the mechanism is as follows: Using the above-mentioned electrochemical precise quantitative analysis method and application for detecting lactic acid and lactate oxidase, in order to achieve its dynamic real-time monitoring, Amperometric i-t Curve electrochemical technology is adopted, the potential is set to -0.5V, and Cu 0.9 Zn 0.1 The electrochemical catalysis of S@black phosphorus nanosheet nanozyme on H2O2 is used to obtain the magnitude of current signals corresponding to a series of different concentrations of LA in the electrolyte solution containing the LOX catalytic reaction solution, establish the quantitative relationship between the current response and LA, and determine the content of LA in the sample to be tested according to the quantitative relationship between the two. Similarly, the magnitude of current signals corresponding to a series of different concentrations of LOX is obtained in the electrolyte solution containing the LA reaction solution, establish the quantitative relationship between the current response and LOX, and determine the content of LOX in the sample to be tested according to the quantitative relationship between the two, which is helpful for the development of clinical test methods for biochemical indicators in liver cancer cells.

2. The precise quantitative analysis method and application of lactic acid and lactate oxidase based on the Amperometric i-t technology as described in claim 1, characterized in that: The present invention synthesizes Cu 0.9 Zn 0.1 S@black phosphorus nanosheet nanozyme with good dispersibility, adhesion and catalytic performance. The electron transfer rate is increased by zinc ion doping, the specific surface area is increased by black phosphorus nanosheets and the electron transfer rate is improved secondly, and the adhesion of electrode surface modification is increased by introducing 0.1% Triton X-100, laying a foundation for the use of Amperometric i-t Curve technology.

3. The precise quantitative analysis method and application of lactic acid and lactate oxidase based on Amperometric i-t technology as described in claims 1-2, characterized in that: For the first time, the current-time technique, the enzyme-catalyzed consumption reaction of LA, and the Cu 0.9 Zn 0.1 ZnS@black phosphorus nanosheet nanozyme are combined to construct a dynamic real-time electrochemical precise quantitative analysis method for LA and LOX.

4. The accurate quantitative analysis method and application of lactic acid and lactate oxidase based on the Amperometric i-t technique according to claims 1-3, characterized in that: Analysis and detection of different concentrations of LA and LOX were carried out using the Amperometric i-t Curve. The detection limits of LA and LOX were 0.00026 μM and 0.0067 U / L, respectively, and the IC 50 = 12.4 μM.

5. The precise quantitative analysis method and application of lactic acid and lactate oxidase based on the Amperometric i-t technology according to claims 1-3, characterized in that: The quantitative analysis of lactate oxidase in cells was completed using the method of this patent. After incubating the cells with excessive lactate, compared with normal cells, the expression level of lactate oxidase in liver cancer cells was higher, and more H2O2 was produced, which is beneficial to further explore the disease mechanism.

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