Construction of lactic acid electrochemical sensor LDHBiNPs (at) MWCNTs / GCE and serum lactic acid detection method

Abstract

The invention provides a lactic acid electrochemical sensor LDHamp; the invention relates to construction of BiNPs (at) MWCNTs / GCE and a serum lactic acid detection method, and belongs to the technical field of biosensors, the method comprises the following steps: taking sodium citrate, Bi (NO3) 35H2O and NaBH4 as raw materials, synthesizing BiNPs through a multi-step reaction, compounding the BiNPs with MWCNTs, carrying out carboxyl activation and loading LDH, and finally modifying on a glassy carbon electrode to obtain the sensor. During detection, the sensor is placed in a PBS system containing 5mM of NAD, a serum sample is added, and the concentration of lactic acid is measured by electrochemical scanning according to current response change. According to the method, the detection linear range is 0-200 [mu] M, the detection limit is as low as 0.03 [mu] M, the detection difference of different batches of sensors is small, the method shows the advantages of high sensitivity and good stability, meanwhile, efficient and accurate detection of lactic acid in serum is achieved, and powerful support is provided for clinical diagnosis and disease research.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to the construction of a lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE and a serum lactate detection method. Background Art

[0002] Lactate Lac, as a key metabolite of muscle anaerobic glucose metabolism, plays an important role in human physiological and pathological processes. In a healthy human body, lactate is rapidly cleared at a rate of 320mmol / L / h, making the "basal" level of lactate in arterial and venous blood less than 1mmol / L. However, under certain pathological conditions, such as cardiogenic or endogenous shock, respiratory failure, liver disease, renal failure, and tissue hypoxia, lactate levels can increase significantly. In addition, cancer cells mainly rely on anaerobic glycolysis to produce energy, which makes high levels of lactate one of the hallmarks of cancer. Therefore, timely and accurate monitoring of lactate levels is crucial for doctors to make clinical diagnoses, assess the condition, and formulate treatment plans.

[0003] At present, there are various quantitative analysis methods for lactic acid, mainly including colorimetry, enzyme spectrophotometry, fluorescence, electrochemical method, proton nuclear magnetic resonance, enzyme fluorescence, etc. Among them, the colorimetric method is relatively simple to operate, but has low sensitivity and is easily interfered by sample color and impurities; although the enzyme spectrophotometry has high accuracy, it requires the use of expensive instruments and equipment, and the operation is complicated; the fluorescence method has high sensitivity, but has strict requirements on experimental conditions and is easily affected by environmental factors; the proton nuclear magnetic resonance method can provide rich structural information, but the equipment is expensive and the detection cost is high, which is not suitable for routine clinical detection.

[0004] Electrochemical analysis methods have attracted wide attention in the field of lactate detection due to their advantages such as fast signal reading speed, high sensitivity, low detection limit, simple handling, high cost-effectiveness, good stability and good repeatability. Among the many materials used to construct electrochemical sensors, multi-walled carbon nanotubes (MWCNTs) are widely used to promote electron transfer between electrodes and enzyme redox centers due to their small size, excellent electrochemical performance and high specific surface area. Compared with non-covalent enzyme adsorption, covalent coupling between MWCNTs and enzymes can provide long-lasting attachment, enhance stability, selectivity, accessibility and prevent enzyme leaching. In addition, carbon nanotubes also have NADH oxidase activity, and electrochemical sensors related to NADH have been studied many times and have shown excellent performance.

[0005] Although research on sensors based on carbon nanotubes has made some progress, the existing technology still has many shortcomings. On the one hand, the research on anchoring metal nanoparticles on carbon nanotubes to produce specific or stronger catalytic reactions is not in-depth enough, and the use of BiNPs and MWCNTs in combination for NADH or Lac detection has not been reported before. There is still a lot of room for optimization and performance improvement of related technical solutions. On the other hand, when detecting lactate in serum, existing lactate electrochemical sensors have problems such as insufficient detection limit, limited detection range, and greater interference from complex serum components, which makes it difficult to meet the clinical demand for high-sensitivity and high-accuracy detection. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE and a method for detecting serum lactate. By synthesizing composite nanomaterials, optimizing the construction process of the sensor, and combining specific detection methods and parameter settings, efficient and accurate detection of lactate in serum is achieved, providing strong support for clinical diagnosis and disease research.

[0007] To achieve the above object, the present invention provides the following solutions: A method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE comprises the following steps: S1, sodium citrate and Bi(NO3)3·5H2O are dissolved in a solvent to obtain a mixed solution, multi-walled carbon nanotubes MWCNTs are added to the mixed solution for mixing; then NaBH4 solution is added, and a chemical reduction reaction is carried out under stirring conditions to prepare a multi-walled carbon nanotube composite material BiNPs@MWCNTs loaded with bismuth nanoparticles; S2, dispersing the BiNPs@MWCNTs obtained in S1 in a buffer solution containing an activator, and performing chemical activation treatment to obtain carboxyl-activated BiNPs@MWCNTs; S3, mixing the carboxyl-activated BiNPs@MWCNTs obtained in S2 with lactate dehydrogenase LDH solutions of different concentrations, and reacting them under low temperature conditions; after the reaction is completed, centrifuging, washing and freeze-drying to obtain LDH&BiNPs@MWCNTs composite materials; S4. Pretreat the glassy carbon electrode GCE to ensure that the surface of the glassy carbon electrode is clean and has good conductivity; then resuspend the LDH&BiNPs@MWCNTs composite material obtained in S3 in water, and then take an appropriate amount of solution and drop it on the surface of the pretreated glassy carbon electrode to obtain the modified lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

[0008] Preferably, in S1, the process for preparing BiNPs@MWCNTs is: adding multi-walled carbon nanotubes MWCNTs to a mixed solution of sodium citrate and Bi(NO3)3·5H2O, and ultrasonically treating for 30 minutes; then adding NaBH4 solution to initiate a chemical reduction reaction, reacting for 8 hours and centrifuging at 12000 rpm for 5 minutes, washing 3 times, collecting the precipitate, and freeze-drying it to obtain a solid powder, and resuspending the solid powder in water to obtain a multi-walled carbon nanotube composite material BiNPs@MWCNTs loaded with bismuth nanoparticles.

[0009] Preferably, in S2, the carboxyl activation process of BiNPs@MWCNTs is as follows: the BiNPs@MWCNTs obtained in S1 are dispersed in 2-(N-morpholine)ethanesulfonic acid MES buffer containing 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and 50 mM N-hydroxysuccinimide NHS, reacted at room temperature for 1 hour, and after the reaction is completed, centrifuged at 10000 rpm for 5 minutes, washed 3 times, and resuspended in phosphate buffer PBS at pH 7.4 to obtain carboxyl-activated BiNPs@MWCNTs.

[0010] Preferably, in S3, the preparation process of the LDH&BiNPs@MWCNTs composite material is specifically as follows: adding 1~5 mg / ml lactate dehydrogenase LDH solution of different concentrations to the carboxyl-activated BiNPs@MWCNTs obtained in S2; using a rotary mixer to react at 4°C for 12 hours; after the reaction is completed, centrifuging at 10000 rpm, washing 3 times with PBS, and then freeze-drying to obtain the LDH&BiNPs@MWCNTs composite material.

[0011] Preferably, in S4, the process of pretreating the glassy carbon electrode GCE includes: grinding and polishing the glassy carbon electrode GCE with a diameter of 3 mm with 0.3 μm and 0.05 μm alumina powder in turn, then first cleaning it with ultrapure water, then using dilute nitric acid and ethanol in a 1:1 volume ratio, and finally using ultrapure water for final ultrasonic cleaning, and then drying it under high-purity N2 after cleaning.

[0012] Preferably, the preparation process of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is as follows: resuspend the LDH&BiNPs@MWCNTs solid powder obtained in S3 in 50 μL of water, take 5 μL and drop it on the surface of the pretreated glassy carbon electrode, and after natural drying, finally obtain the modified lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

[0013] The present invention also provides a method for detecting lactic acid in serum using a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE constructed by the above-mentioned lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE construction method, specifically: placing the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE in a liquid containing a substrate NAD + In a PBS reaction system, electrochemical scanning is performed to record a basic current response; a serum sample to be tested is added to the reaction system to allow lactic acid to react with the sensor; under set electrochemical conditions, an electrochemical scanning is performed on the reaction system to record changes in the current response; and the concentration of lactic acid in the serum is calculated based on the relationship between the change in the current response and the lactic acid concentration.

[0014] Preferably, the parameters of the electrochemical scan are set as follows: the scanning potential range is -0.4-0.8V, the scanning speed is 0.01V / s, and the NAD + The concentration is 5 mM; the working electrode of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is a glassy carbon electrode with a diameter of 3 mm.

[0015] Preferably, the detection method also includes evaluating the linear range and detection limit of lactic acid detection, wherein the linear range is 0-200 μM and the detection limit is 0.03 μM; and the detection method also includes optimizing the performance of the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE under different pH values ​​and different initial potential conditions to determine that the optimal working pH value is 7.4 and the optimal working initial potential is 0.6V.

[0016] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects: (1) The lactate electrochemical sensor constructed by the present invention exhibits excellent performance in serum lactate detection. By precisely controlling the electrochemical scanning parameters, such as the scanning potential range of -0.4-0.8V and the scanning speed of 0.01V / s, combined with 5mM NAD + The linear range of the sensor is 0-200 μM, and the detection limit is as low as 0.03 μM. It can accurately detect different concentrations of lactic acid in serum and meet the strict requirements of clinical diagnosis on detection accuracy and range.

[0017] (2) In the process of material synthesis and modification, the present invention uses sodium citrate, Bi(NO3)3·5H2O, and NaBH4 to synthesize BiNPs, which are then compounded with MWCNTs, activated by carboxyl groups, and loaded with LDH to form a composite material with stable performance. The synergistic effect between the materials enhances the stability and repeatability of the sensor. The differences in the detection of lactic acid in serum by sensors from different batches are small, ensuring the reliability of the detection results.

[0018] (3) The present invention is not only applicable to laboratory research, but also extremely valuable in clinical applications. By detecting serum lactate, it can provide doctors with key disease diagnosis and condition monitoring information. After optimizing the optimal working pH value of 7.4 and the initial potential of 0.6V, the sensor can work efficiently under conditions close to human physiological conditions, which is helpful for early diagnosis of diseases and formulation of treatment plans, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 This is a flow chart of a method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE of the present invention; Figure 2 The data graph provided in Example 1 of the present invention is about the detection effect of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE on different concentrations of lactate (Lac) and the influence of different lactate dehydrogenase (LDH) loading amounts on the sensor performance; wherein, Figure 2 A in the figure indicates that LDH&BiNPs@MWCNTs / GCE contains NAD + Cyclic voltammetry scanning results after adding different concentrations of Lac into PBS; Figure 2 B in the figure represents the chronoamperometric test results of Lac catalysis by BiNPs@MWCNTs modified GCE loaded with different concentrations of LDH; Figure 3 This is a data diagram showing the effect of different pH values ​​and initial potentials on the catalytic lactic acid effect of the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE provided in Example 2 of the present invention; wherein, Figure 3 A in the figure represents the scanning chronoamperometry (it) results of LDH&BiNPs@MWCNTs / GCE under different pH conditions; Figure 3B in the figure represents the scanning chronoamperometry (it) results of LDH&BiNPs@MWCNTs / GCE on lactic acid at different initial potentials; Figure 4 The method provided in Example 3 of the present invention is used to analyze the performance of the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE for lactic acid detection, including the relationship between the current and the lactic acid concentration, the relationship between the current difference and the lactic acid concentration, and the linear relationship diagram related to the detection limit; wherein, Figure 4 A in the figure indicates that LDH&BiNPs@MWCNTs / GCE was treated with 5 mM NAD + Different concentrations of lactic acid were added to PBS in sequence, and the scanning chronoamperometry (it) curve was plotted; Figure 4 B in the figure represents the relationship between the current difference (Δi) of the current corresponding to each lactic acid concentration obtained by the it curve in Example 3 and the initial current and the lactic acid concentration (c(Lac)); Figure 4 C in the equation represents the current difference (Δi) and the square root of lactate concentration (c(Lac)) 1 / 2 ) Linear relationship graph in the range of 0-200 μM; Figure 5 This is a performance test comparison chart for comparing BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE provided in Comparative Example 1 of the present invention; wherein, Figure 5 A in the figure is the cyclic voltammetry (CV) test diagram; Figure 5 B in the figure is the electrochemical impedance spectroscopy (EIS) test diagram; Figure 6 The comparative example 2 of the present invention is used to compare BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE in the presence or absence of NAD + The catalytic effect data of lactic acid (Lac) when it exists; Figure 6 A in the figure indicates that BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE are synthesized without NAD + Under the condition of , the current changes with time when 20 μM Lac is added each time; Figure 6 B in the figure indicates that BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE were treated with NAD + Afterwards, the current changes with time when 20 μM Lac was added each time; Figure 7 This is a graph comparing the detection effects of LDH&BiNPs@MWCNTs / GCE on different concentrations of lactic acid (Lac) in artificial serum and PBS buffer, provided in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] like Figure 1 As shown, the present invention provides a method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE, comprising the following steps: S1, sodium citrate and Bi(NO3)3·5H2O are dissolved in a solvent to obtain a mixed solution, multi-walled carbon nanotubes MWCNTs are added to the mixed solution for mixing; then NaBH4 solution is added, and a chemical reduction reaction is carried out under stirring conditions to prepare a multi-walled carbon nanotube composite material BiNPs@MWCNTs loaded with bismuth nanoparticles; S2, dispersing the BiNPs@MWCNTs obtained in S1 in a buffer solution containing an activator, and performing chemical activation treatment to obtain carboxyl-activated BiNPs@MWCNTs; S3, mixing the carboxyl-activated BiNPs@MWCNTs obtained in S2 with lactate dehydrogenase LDH solutions of different concentrations, and reacting them under low temperature conditions; after the reaction is completed, centrifuging, washing and freeze-drying to obtain LDH&BiNPs@MWCNTs composite materials; S4. Pretreat the glassy carbon electrode GCE to ensure that the surface of the glassy carbon electrode is clean and has good conductivity; then resuspend the LDH&BiNPs@MWCNTs composite material obtained in S3 in water, and then take an appropriate amount of solution and drop it on the surface of the pretreated glassy carbon electrode to obtain the modified lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

[0024] In S1, the process of preparing BiNPs@MWCNTs is as follows: weigh 8.82 mg of sodium citrate and dissolve it in 10 mL of ultrapure water. Weigh 12.13 mg of Bi(NO3)3·5H2O in a 2 mL centrifuge tube, add 100 μL of ultrapure water, and add an appropriate amount of dilute nitric acid until it is clear and transparent. Add it to the sodium citrate solution and mix it thoroughly. Weigh 5 mg of MWCNTs and add it to the above solution and ultrasonicate for 30 minutes. Weigh 11.35 mg of NaBH4 and dissolve it in 3 mL of ultrapure water. Under rapid stirring, slowly add NaBH4 to the above solution and react for 8 hours. Centrifuge at 12000 rpm for 5 minutes, wash 3 times, collect the precipitate, and freeze-dry to obtain a solid powder, namely BiNPs@MWCNTs. The obtained solid powder is resuspended in 200 μL of water for modification of glassy carbon electrodes.

[0025] In S2, the carboxyl activation process of BiNPs@MWCNTs is as follows: 50 μL of 7 mg / mL BiNPs@MWCNTs is added to 2 mL of MES buffer containing 100 mM EDC and 50 mM NHS, and reacted at room temperature for 1 h using a rotating mixer to obtain carboxyl-activated BiNPs@MWCNTs.

[0026] In S3, the preparation process of LDH&BiNPs@MWCNTs composite material is as follows: activated BiNPs@MWCNTs were resuspended in 1 mL of PBS at pH 7.4, 1 mL of LDH (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL) was added thereto, and the mixture was reacted at 4°C for 12 h using a rotary mixer. Subsequently, the mixture was centrifuged at 10,000 rpm and washed three times with PBS, and the precipitate was collected and freeze-dried to obtain a solid powder, namely LDH&BiNPs@MWCNTs.

[0027] In S4, the process of pretreating the glassy carbon electrode GCE includes: grinding and polishing the glassy carbon electrode GCE with a diameter of 3 mm with 0.3 μm and 0.05 μm alumina powder in turn, then first cleaning it with ultrapure water, then using dilute nitric acid and ethanol in a 1:1 volume ratio, and finally using ultrapure water for final ultrasonic cleaning, and then drying it under high-purity N2 after cleaning.

[0028] The preparation process of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is as follows: resuspend the LDH&BiNPs@MWCNTs solid powder obtained in S3 in 50 μL of water, take 5 μL and drop it on the pretreated glassy carbon electrode surface, and after natural drying, finally obtain the modified lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

[0029] In addition, the present invention also provides a method for detecting lactic acid in serum using a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE constructed by the above-mentioned lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE construction method, specifically: placing the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE in a liquid containing a substrate NAD + In a PBS reaction system, electrochemical scanning is performed to record a basic current response; a serum sample to be tested is added to the reaction system to allow lactic acid to react with the sensor; under set electrochemical conditions, an electrochemical scanning is performed on the reaction system to record changes in the current response; and the concentration of lactic acid in the serum is calculated based on the relationship between the change in the current response and the lactic acid concentration.

[0030] The parameters of the electrochemical scan were set as follows: the scanning potential range was -0.4-0.8 V, the scanning speed was 0.01 V / s, and the NAD + The concentration is 5mM; the working electrode of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is a glassy carbon electrode with a diameter of 3mm. The above detection method also includes evaluating the linear range and detection limit of lactate detection, the linear range is 0-200μM, and the detection limit is 0.03μM; and also includes optimizing the performance of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE under different pH values ​​and different initial potential conditions to determine that the optimal working pH value is 7.4 and the optimal working initial potential is 0.6V.

[0031] The features and properties of the present invention are further described in detail below with reference to specific examples. The process conditions and numerical values ​​in the following examples and comparative examples are exemplary and can be referred to as shown in the foregoing content.

[0032] Example 1 In this example, based on a 5 mL PBS reaction system, the substrate NAD was first added. + 5mM, then add different concentrations of Lac (25μM, 50μM, 100μM, 200μM), use LDH&BiNPs@MWCN / GCE to scan CV of the reaction solution containing different concentrations of Lac. Parameter setting: -0.4-0.8V, scanning speed 0.01V / s. Analyze the CV of different concentrations of Lac, and the results are as follows Figure 2As shown in A, an oxidation peak current appears at 0.15 V, and the peak current increases with the increase of Lac concentration, indicating that with the increase of Lac, the generated NADH increases, and the current response of LDH&BiNPs@MWCNTs / GCE to NADH increases, indicating that the Lac content can be represented by the current change, that is, LDH&BiNPs@MWCNTs / GCE can detect different concentrations of Lac.

[0033] LDH&BiNPs@MWCNTs / GCE synthesized with different concentrations of LDH (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL) were scanned in 5 mL of PBS. First, the scan was stabilized for 100 s, and then 5 mM NAD was added. + Scan for 100 s, then add 20 μM Lac each time, scan for 50 s, repeat 5 times, observe and record the current changes, and the results are as follows Figure 2 As shown in B, the changing trends of the current of different electrodes to Lac are different. The current response degree of GCE modified by BiNPs@MWCNTs loaded with 1 mg / mL, 2 mg / mL, and 3 mg / mL LDH to Lac increases with the increase of LDH concentration. The current response is the largest at 3 mg / mL. When it continues to increase to 4 mg / mL and 5 mg / mL, the current sensitivity does not continue to increase, indicating that 3 mg / mL LDH loaded with BiNPs@MWCNTs achieves the best synergistic effect of electron transfer and catalysis of Lac, and makes LDH&BiNPs@MWCNTs / GCE achieve the best response to Lac. Therefore, LDH&BiNPs@MWCNTs modified GCE synthesized with 3 mg / mL LDH is selected for the detection of Lac.

[0034] Example 2 In this example, LDH&BiNPs@MWCN / GCE was scanned under different pH (pH6, pH7.0, pH7.4, pH8.0) and different initial potentials (0.3V, 0.4V, 0.5V, 0.6V, 0.7V, 0.8V). First, it was scanned for 100s to stabilize, and then 5mM NAD was added. + Scan for 100 seconds, then add 20 μM Lac each time, scan for 50 seconds, analyze the current changes under different conditions, and select the optimal pH and potential for catalyzing Lac. The results are as follows Figure 3 See Figure 3A in the figure shows that at pH 6.0, the response of LDH&BiNPs@MWCNTs / GCE to Lac is relatively poor, which is much improved at pH 7.0. The current is the best at pH 7.4, and the response to Lac does not get better at pH 8.0. Therefore, pH 7.4 is selected as the optimal working pH for LDH&BiNPs@MWCNTs / GCE to detect Lac. In addition, the working potential of LDH&BiNPs@MWCNTs / GCE is optimized, and Lac is scanned at different initial potentials (0.3V, 0.4V, 0.5V, 0.6V, 0.7V, and 0.8V). The results are shown in Figure 1. Figure 3 As shown in B, from 0.3V to 0.6V, the current response of LDH&BiNPs@MWCNTs / GCE to Lac becomes stronger and stronger. When the potential is further increased to 0.7V and 0.8V, the response degree does not continue to increase, indicating that 0.6V is the working potential of LDH&BiNPs@MWCNTs / GCE to detect Lac.

[0035] Example 3 In this example, LDH & BiNPs @ MWCNTs / GCE was scanned in 5 mL of PBS. After scanning for a certain period of time, 5 mM NAD was added. + Scan for 100s, then add different concentrations of Lac (2μM, 10μM, 20μM, 50μM, 100μM) in turn, scan for 20s each time, observe and record the current changes. The Lac concentrations in the reaction solution are 0, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 400, 500μM, and the current difference i between the current corresponding to each concentration and the initial current is obtained according to the it curve. The relationship curve is drawn, and the linear relationship between c(Lac) and i, the detection range and detection limit are calculated. The results are shown in the figure. Figure 4 See Figure 4 A in, as the Lac concentration c(Lac) increases, the current i increases, and the relationship between the current difference (i) and the initial current and the Lac concentration (c(Lac)) is obtained, as shown in Figure 4 As shown in B. After calculation, i and (c(Lac)) 1 / 2 There is a linear relationship in the range of 0-200μM, i=0.01959(c(Lac)) 1 / 2 -0.001492, R 2 =0.9982, e.g. Figure 4 As shown in C, the detection limit LOD=0.03μM is calculated based on LOD=3σ / k. This further proves that the detection limit of LDH&BiNPs@MWCNTs / GCE for lactic acid is low.

[0036] Example 4 In this example, in order to further demonstrate the practical application effect of LDH & BiNPs @ MWCNTs / GCE, artificial serum was used as a simulated sample, a certain amount of Lac was added thereto, and LDH & BiNPs @ MWCNTs / GCE was used to detect the difference in Lac in serum detected by LDH & BiNPs @ MWCNTs / GCE of the same batch and different batches, and the practical application potential of LDH & BiNPs @ MWCNTs / GCE was evaluated. The results are shown in Table 1.

[0037] Table 1 Differences in detection of Lac in serum by lactate electrochemical sensors from the same batch and different batches ;

[0038] Referring to Table 1, the differences in the detection of Lac of 10μM and 100μM in serum by LDH&BiNPs@MWCNTs / GCE within the same batch were low, 3.6% and 2.79%, respectively, with an average difference of 3.19%. The differences between different batches of LDH&BiNPs@MWCNTs / GCE were slightly larger, 6.66% and 6.38% for 10μM and 100μM, respectively, with an average difference of 6.52%. Although the differences between different batches were large, they were still within 10%, indicating that LDH&BiNPs@MWCNTs / GCE has the potential for practical application in serum.

[0039] Comparative Example 1 This comparative example is based on the prior art. BiNPs@MWCNTs are dripped on the surface of GCE and dried naturally at room temperature for 12 hours to obtain BiNPs@MWCNTs / GCE. LDH&BiNPs@MWCNTs are dripped on the surface of GCE to obtain the modified electrode LDH&BiNPs@MWCNTs / GCE. CV and EIS are tested in an electrolyte of 5mM K3[Fe(CN)6]+5mM K4[Fe(CN)6]+0.1M KCl. The results are as follows: Figure 5 See Figure 5 As shown in A and B in the figure, compared with BiNPs@MWCNTs / GCE, the oxidation peak current of LDH&BiNPs@MWCNTs / GCE is significantly reduced, indicating that the loading of LDH reduces the conductivity of LDH&BiNPs@MWCNTs / GCE. The impedance value of the response is 4239.0Ω, which is larger than that of BiNPs@MWCNTs / GCE (85.6Ω) without LDH loading. This is because LDH hinders the transfer of electrons at the electrode interface, which further indicates that LDH is successfully loaded on BiNPs@MWCNTs.

[0040] Comparative Example 2 In this comparative example, BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE were used to scan it in 5 mL of PBS, first for 100 s to stabilize, and then in the absence of NAD + In the case of adding 20μM Lac each time, scanning for 50s, observing the current changes, and analyzing the catalytic effect of BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE on Lac. Use BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE to scan it in 5mL PBS, first scan for 100s to stabilize, then add 5mM NAD + Scan for 100 s, then add 20 μM Lac each time, scan for 50 s, observe the current change, compare and analyze the catalytic effects of BiNPs@MWCNTs / GCE and LDH&BiNPs@MWCNTs / GCE on Lac. The results are as follows Figure 6 See Figure 6 A, BiNPs@MWCNTs / GCE without NAD + In the case of each addition of Lac, the current remained basically unchanged. + In the case of , BiNPs@MWCNTs / GCE also has no current response to Lac, indicating that BiNPs@MWCNTs / GCE cannot electrocatalyze Lac. + When 20 μM Lac was added, there was no current change. + After that, Lac is added, the current increases, and as Lac increases, the current also increases. The results are as follows Figure 6 As shown in B, it is only in NAD + LDH&BiNPs@MWCNTs / GCE can respond to Lac only in the presence of NAD. This is determined by the catalytic properties of LDH. + As a cofactor, LDH can catalyze Lac to produce pyruvate.

[0041] Comparative Example 3 In this comparative example, artificial serum was used as a simulated sample, a certain amount of Lac was added thereto, and LDH&BiNPs@MWCNTs / GCE was used for detection, and PBS was used as a control. The results were analyzed based on the application effect of LDH&BiNPs@MWCNTs / GCE on the detection of Lac in serum. The results are as follows: Figure 7As shown in the data, LDH&BiNPs@MWCNTs / GCE has a good response to different concentrations of Lac, and the detection recovery rate can reach about 90%. At the same time, in serum with complex components, the performance of LDH&BiNPs@MWCNTs / GCE is the same as the response to Lac in PBS, which proves that LDH&BiNPs@MWCNTs / GCE can be used to detect lactate in serum.

[0042] Therefore, the above-mentioned lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE was constructed and the serum lactate detection method was adopted. By synthesizing composite nanomaterials, optimizing the sensor construction process, and combining specific detection methods and parameter settings, efficient and accurate detection of lactate in serum was achieved, providing strong support for clinical diagnosis and disease research.

[0043] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0044] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE, characterized in that: The following steps are involved: S1, sodium citrate and Bi(NO3)3·5H2O are dissolved in a solvent to obtain a mixed solution, multi-walled carbon nanotubes MWCNTs are added to the mixed solution for mixing; then NaBH4 solution is added, and a chemical reduction reaction is carried out under stirring conditions to prepare a multi-walled carbon nanotube composite material BiNPs@MWCNTs loaded with bismuth nanoparticles; S2, dispersing the BiNPs@MWCNTs obtained in S1 in a buffer solution containing an activator, and performing chemical activation treatment to obtain carboxyl-activated BiNPs@MWCNTs; S3, mixing the carboxyl-activated BiNPs@MWCNTs obtained in S2 with lactate dehydrogenase LDH solutions of different concentrations, and reacting them under low temperature conditions; after the reaction is completed, centrifuging, washing and freeze-drying to obtain LDH&BiNPs@MWCNTs composite materials; S4. Pretreat the glassy carbon electrode GCE to ensure that the surface of the glassy carbon electrode is clean and has good conductivity; then resuspend the LDH&BiNPs@MWCNTs composite material obtained in S3 in water, and then take an appropriate amount of solution and drop it on the surface of the pretreated glassy carbon electrode to obtain the modified lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

2. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 1, characterized in that: In S1, the process of preparing BiNPs@MWCNTs is as follows: multi-walled carbon nanotubes MWCNTs are added to a mixed solution of sodium citrate and Bi(NO3)3·5H2O, and ultrasonic treatment is performed for 30 minutes; then NaBH4 solution is added to initiate a chemical reduction reaction, the reaction is performed for 8 hours, and the mixture is centrifuged at 12000 rpm for 5 minutes, washed 3 times, the precipitate is collected, and freeze-dried to obtain a solid powder, and the solid powder is resuspended in water to obtain a multi-walled carbon nanotube composite material BiNPs@MWCNTs loaded with bismuth nanoparticles.

3. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 1, characterized in that: In S2, the carboxyl activation process of BiNPs@MWCNTs is as follows: the BiNPs@MWCNTs obtained in S1 are dispersed in 2-(N-morpholino)ethanesulfonic acid MES buffer containing 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and 50 mM N-hydroxysuccinimide NHS, reacted at room temperature for 1 hour, and after the reaction is completed, centrifuged at 10000 rpm for 5 minutes, washed 3 times, and resuspended in phosphate buffer PBS at pH 7.4 to obtain carboxyl-activated BiNPs@MWCNTs.

4. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 1, characterized in that: In S3, the preparation process of the LDH&BiNPs@MWCNTs composite material is as follows: adding lactate dehydrogenase LDH solution of different concentrations of 1~5 mg / ml to the carboxyl-activated BiNPs@MWCNTs obtained in S2; using a rotary mixer to react at 4°C for 12 hours; after the reaction, centrifuging at 10000 rpm, washing with PBS three times, and then freeze-drying to obtain the LDH&BiNPs@MWCNTs composite material.

5. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 1, characterized in that: In S4, the process of pretreating the glassy carbon electrode GCE includes: grinding and polishing the glassy carbon electrode GCE with a diameter of 3 mm with 0.3 μm and 0.05 μm alumina powder in turn, then first cleaning it with ultrapure water, then using dilute nitric acid and ethanol in a 1:1 volume ratio, and finally using ultrapure water for final ultrasonic cleaning, and then drying it under high-purity N2 after cleaning.

6. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 5, characterized in that: The preparation process of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is as follows: resuspend the LDH&BiNPs@MWCNTs solid powder obtained in S3 in 50 μL of water, take 5 μL and drop it on the pretreated glassy carbon electrode surface, and after natural drying, finally obtain the modified lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE.

7. A method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE as claimed in any one of claims 1 to 6, wherein the method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE for detecting lactic acid in serum is characterized in that: Specifically, the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE was placed in a solution containing substrate NAD + In the PBS reaction system, electrochemical scanning was performed to record the basic current response; The serum sample to be tested is added to the reaction system to make lactic acid react with the sensor; under the set electrochemical conditions, the reaction system is electrochemically scanned to record the change of the current response; and the concentration of lactic acid in the serum is calculated based on the relationship between the change of the current response and the lactic acid concentration.

8. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 7 for detecting lactic acid in serum using the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE, characterized in that: The parameters of the electrochemical scan were set as follows: the scanning potential range was -0.4-0.8 V, the scanning speed was 0.01 V / s, and the NAD + The concentration is 5 mM; the working electrode of the lactate electrochemical sensor LDH&BiNPs@MWCNTs / GCE is a glassy carbon electrode with a diameter of 3 mm.

9. The method for constructing a lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE according to claim 8, wherein the method for detecting lactic acid in serum using the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE is characterized in that: The detection method also includes evaluating the linear range and detection limit of lactic acid detection, wherein the linear range is 0-200 μM and the detection limit is 0.03 μM; and the detection method also includes optimizing the performance of the lactic acid electrochemical sensor LDH&BiNPs@MWCNTs / GCE under different pH values ​​and different initial potential conditions to determine that the optimal working pH value is 7.4 and the optimal working initial potential is 0.6V.

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