Lactate oxidase portable mobile phone sensing method based on composite MXene thin-layer material and application of lactate oxidase portable mobile phone sensing method

Abstract

The invention discloses a lactate oxidase portable mobile phone sensing method based on a composite MXene thin-layer material and an application of the lactate oxidase portable mobile phone sensing method. Firstly, a Cu0. 9Zn0. 1S (at) MXene thin-layer material is synthesized and can catalyze H2O2 into hydroxyl free radicals, the generated hydroxyl free radicals react with a colorless 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) solution to generate blue oxidation-state 3, 3 ', 5, 5'-tetramethyl benzidine (oxTMB), the oxTMB can have an obvious ultraviolet absorption peak at 652nm, and a novel visual analysis method is constructed by taking the peak as signal output; besides, lactate oxidase can catalyze lactic acid to generate pyruvic acid and H2O2, a new method is used for LA or LOX analysis by taking H2O2 as a relation, H2O2 generated by the reaction can participate in the oxidation reaction of TMB, an obvious ultraviolet absorption peak can appear at 652nm, and the inhibitor Fe (III) can inhibit the reaction. On the basis, a portable mobile phone sensing method which is rapid, simple and low in cost is constructed, and the method is successfully applied to sensitive detection of LOX in HEK293S cells and has scientific significance.

Description

Technical Field

[0001] The present invention relates to an ultraviolet colorimetric visualization sensor, in particular to the construction of a portable mobile phone sensing method using a composite MXene thin film material and its application in the analysis and detection of lactic acid and lactate oxidase, and finally applied to the analysis of intracellular lactate oxidase activity, belonging to the fields of functional biomaterials and biosensing technology. Background Art

[0002] Lactic acid (LA, 2-hydroxypropionic acid) plays multiple biological functions. On the one hand, it can remove old and dull cells, protect the skin from sunburn by controlling moisture to make the skin work properly and increasing sensitivity to sunlight. On the other hand, abnormal lactic acid levels can cause various physiological diseases. When the lactic acid content in the human body is too high, it will lead to a decrease in blood pressure, reduce the oxygen supply in human tissues, and cause serious lung diseases, sepsis, congestive heart failure and anemia, etc. Too low lactic acid content will lead to liver diseases, uremia, etc. Therefore, the detection and analysis of lactic acid concentration levels are of great help for early disease diagnosis and subsequent treatment. There are currently many lactic acid detection methods, such as voltammetry, chromatography, fluorescence method and chemiluminescence method, etc., but they have disadvantages such as complex assembly, high cost and easy oxidation. In view of this, it is extremely necessary to develop a simple, low-cost and visually detectable analysis method. In addition, lactate oxidase (LOX) can specifically catalyze lactic acid to produce pyruvate and hydrogen peroxide (H2O2). Its activity is highly related to abnormal lactic acid metabolism, such as diabetes and lactic acidosis syndrome. Understanding LOX can further explore the pathogenesis of these diseases and develop new treatment methods for these diseases, which has very important clinical value.

[0003] Natural enzymes are highly specific biocatalysts that can selectively catalyze specific biological reactions. However, most natural enzymes are globular proteins or RNA ribozyme structures. After natural enzymes are exposed to extreme pH values and high temperatures, they usually lose their catalytic activity and have the disadvantages of high cost, easy variability and easy inactivation, which limit their practical applications. Nanozymes are inspired by natural enzymes and nanomaterials. The advantages of nanozymes such as low cost, excellent stability, controllable morphology and size to regulate catalytic activity have attracted wide attention, and significant progress has been made in the applications in the fields of biosensing, biotherapy and environment. Copper sulfide nanoparticles have excellent structural and surface properties. After functionalization, copper sulfide nanomaterials have excellent conductivity and catalytic properties, can promote the electron transfer ability between the sensor and biomolecules and better participate in catalytic reactions to achieve signal output, which expands its application in biosensing methods and has good application prospects.

[0004] The present invention designs a portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin film material. The Cu used in this method0.9 Zn 0.1 Copper ions in the CoZnS@MXene thin film material react with H2O2 in the Fenton reaction to generate hydroxyl radicals, which turn the colorless 3,3',5,5'-tetramethylbenzidine (TMB) solution into the blue oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB). oxTMB will exhibit an obvious ultraviolet absorption peak at 652 nm, thus constructing a new visual analysis and detection method through the signal output of H2O2. And the lactate-lactate oxidase system can produce a small amount of H2O2, so the above method can be indirectly used to detect lactate and lactate oxidase. With the changes in the concentrations of lactate and lactate oxidase, both the absorbance and the color of TMB show certain changes. By accurately analyzing the changes in lactate and lactate oxidase through the amount of change in absorbance, a visual analysis sensing method for detecting lactate and lactate oxidase is constructed. Furthermore, a portable mobile phone sensing method is developed through the RGB channel, which has a low cost and is convenient for on-site detection, providing new ideas for the related clinical diagnosis and drug development of lactate and lactate oxidase. Currently, based on Co 0.9 Zn 0.1 S@MXene thin film material to achieve the screening of lactate oxidase and its inhibitors has not been reported, especially the portable mobile phone sensing method for lactate oxidase, which has great scientific value and development space. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a portable mobile phone sensing method and application for lactate oxidase based on a composite MXene thin film material with good specificity, high sensitivity, fast detection speed, accurate and reliable results, low cost, and visualization.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A portable mobile phone sensing method and application for lactate oxidase based on a composite MXene thin film material, and the specific steps are as follows:

[0007] (1) Preparation of Co 0.9 Zn 0.1 S@MXene thin film material

[0008] a. Synthesis of MXene

[0009] Prepare 20 - 40 mL of 9 - 18 M HCl solution, mix it with 2 - 4 g of LiF powder, and stir magnetically at room temperature for 30 - 40 min to fully dissolve the mixture. Then, slowly add 1 - 3 g of Ti3AlC2 to the mixed solution, avoiding intense heat release. After complete addition, stir magnetically at a low speed at room temperature for 42 - 48 h, then take out the solution and centrifuge and wash it multiple times at 4000 - 5000 rpm until the pH of the supernatant is > 6. Then, take 40 - 50 mL of dimethyl sulfoxide (DMSO) and mix and shake it with the preliminarily treated MXene, stir at room temperature for 10 - 12 h, then dry the solution, add deionized water and sonicate for 25 - 30 min, and then centrifuge at 3500 - 4000 rpm for 20 - 30 min to obtain the supernatant, and perform freeze - drying to obtain the MXene solid.

[0010] b.Cu 0.9 Zn 0.1 Synthesis of S@MXene thin - layer material

[0011] Copper(II) chloride dihydrate (0.1 - 0.2 mmol, 17.1 - 34.2 mg), sodium citrate (0.05 - 0.068 mmol, 20 - 27.2 mg) and zinc acetate dihydrate (0.1 - 0.2 mmol, 30 - 60 mg), 80 - 100 mg of MXene are dissolved in 70 - 90 mL of deionized water and stirred at room temperature for 5 min. Then, 10 - 20 mL of sodium sulfide solution (Na2S, 0.02 - 0.04 M) is added to the light blue mixed solution, and the color of the mixture changes to dark brown. Then, after magnetic stirring at room temperature for 3 - 10 min, the mixed solution is transferred to a water bath at 80 - 90 °C and heated and stirred for 10 - 15 min. Subsequently, the prepared dark green mixed solution is cooled in an ice - water environment, then centrifuged at 6000 - 9000 rpm for 20 - 30 min. The centrifuged sample is 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. That is, Cu 0.9 Zn 0.1 S@MXene thin - layer material, and finally it is dispersed in 0.1% chitosan solution for standby.

[0012] (2) Construction of visual analysis sensing method

[0013] a. Put a 96 - well quartz microplate into an ultrasonic cleaner and wash it 2 - 3 times with ethanol and distilled water respectively, sonicate for 10 - 15 min, and then dry it for standby.

[0014] b. Take 1 - 2 μL of 1 - 2 mM H2O2 solution, 1 - 2 mg / mL, 20 - 25 μL of Cu 0.9 Zn 0.1The S@MXene thin film material and 10 - 20 mM 5 - 10 μL TMB were successively added to 20 - 25 μL of acetic acid buffer solution (0.1 M, pH = 4.0) and 40 - 50 μL of ultrapure water. Subsequently, they were fully mixed and placed at room temperature for 20 - 30 min, and the ultraviolet detection of H2O2 was completed in the wavelength range of 400 - 800 nm.

[0015] c. Take 1 - 2 μL of 40 - 50 mM lactic acid solution (final concentration: 2.5 - 3 mM), 1 - 2 μL of 4000 - 5000 U / L lactate oxidase (final concentration: 400 - 500 U / L), and 10 - 15 μL of PBS (100 mM, pH = 7.4), and add ultrapure water to make the total volume 20 - 25 μL. After fully mixing, wrap it with tin foil and place it in a water bath at 35 - 37 °C for reaction for 25 - 30 min, marked as solution 2.

[0016] d. Take 1 - 2 mg / mL 20 - 25 μL of Cu 0.9 Zn 0.1 The S@MXene thin film material, 8 - 10 μL of the reaction solution of lactic acid - lactate oxidase, and 10 - 20 mM 5 - 10 μL TMB were successively added to 20 - 25 μL of acetic acid buffer solution (0.1 M, pH = 4.0) and ultrapure water to make the total volume 100 - 120 μL. Subsequently, they were fully mixed and placed at room temperature for 20 - 30 min, marked as solution 3.

[0017] e. Take 1 - 2 μL of 4000 - 5000 U / L lactate oxidase (final concentration: 400 - 500 U / L, calculated based on a volume of 20 μL) and 1 - 2 μL of 10 - 20 mM Fe(III) (final concentration: 1 - 2 mM, calculated based on a volume of 20 μL), mix them thoroughly by shaking, wrap them with tin foil, and place them in a water bath at 35 - 37 °C for reaction for 25 - 30 min. Then add 1 - 2 mg / mL 20 - 25 μL of Cu 0.9 Zn 0.1 The S@MXene thin film material, 1 - 2 μL of 40 - 50 mM lactic acid solution (final concentration: 2.5 - 3 mM), 10 - 15 μL of PBS (100 mM, pH = 7.4), and ultrapure water were added to make the total volume 20 - 25 μL. After fully mixing, wrap it with tin foil and place it in a water bath at 35 - 37 °C for reaction for 25 - 30 min.

[0018] In the above step (2b), by changing the concentration of H2O2 (final concentrations: 0, 0.1, 0.3, 0.5, 0.8, 1, 2, 5, 10, 20, 50, 80, 100, 200, 300, 400, 500, 600 μM) while keeping other steps unchanged, the detection of H2O2 at different concentrations can be achieved.

[0019] In the above step (2c), by changing the LA concentration (final concentration: 0, 0.01, 0.05, 0.2, 0.5, 1, 6, 10, 20, 60, 150, 300, 600, 1500, 3000, 6000, 10000 μM) while keeping other steps unchanged, the detection of LA at different concentrations can be achieved.

[0020] In the above step (2d), by changing the LOX concentration (final concentration: 0, 0.1, 0.3, 0.6, 1, 2, 6, 10, 20, 50, 100, 200, 500, 1000, 1500, 2000 U / L) while keeping other steps unchanged, the detection of LOX at different concentrations can be achieved.

[0021] In the above step (2e), by changing the concentration of the inhibitor Fe(III) (final concentration: 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1500, 3000, 8000 μM) while keeping other steps unchanged, the detection of the lactate oxidase inhibitor Fe(III) at different concentrations can be achieved.

[0022] (3) Portable mobile phone sensing method and its analytical application in HEK293S cells

[0023] Prepare a mobile phone with color capture software. Based on the results in (2), input the TMB color change card into the mobile phone, and complete the signal output in the way of G / R through the RGB channel. Establish a linear relationship between the G / R value and the lactate oxidase concentration. The concentration of lactate oxidase in the sample can be calculated through the linear equation. Generally, the kidney contains a certain amount of lactate oxidase. In this patent, HEK293S cells are used as the research object. After extracting the protein in the cells, the monitoring of trace lactate oxidase activity in the cells is completed.

[0024] Principle of the invention: The present invention is a portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin film material. First, a Cu 0.9 Zn 0.1S@MXene thin-layer material, whose dispersibility in homogeneous solution is increased by chitosan, can catalyze H2O2 into hydroxyl radicals. The generated hydroxyl radicals react with the colorless 3,3',5,5'-tetramethylbenzidine (TMB) solution to form the blue oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB). oxTMB will show an obvious ultraviolet absorption peak at 652 nm. Based on this, a new type of visual analysis method was constructed; in addition, lactate oxidase can catalyze lactate to produce pyruvate and H2O2. Using H2O2 as a link, the new method was used for LA or LOX analysis. The generated H2O2 can participate in the oxidation reaction of TMB and will show an obvious ultraviolet absorption peak at 652 nm, while the inhibitor Fe(III) can inhibit this reaction. Based on this, a rapid, simple and low-cost visual analysis method was constructed for the sensitive detection of H2O2, LA or LOX, and finally a portable mobile phone sensing method was constructed to complete the activity monitoring of LOX in HEK293S cells.

[0025] Compared with the prior art, the advantages of the present invention are as follows: The present invention constructs a portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin-layer material. First, Cu 0.9 Zn 0.1 S@MXene material was synthesized by a water bath method. In this material, MXene and zinc ions can effectively promote the electron transfer rate, improve the carrier transport ability, and accelerate the transfer of electrons from the surface of copper sulfide nanoparticles to H2O2 in the solution, and can catalyze the decomposition of H2O2 to produce hydroxyl radicals. Secondly, this material does not directly oxidize TMB, so the ultraviolet colorimetric method can be used to detect different concentrations of LA and LOX. Obviously, within a certain concentration range, the greater the concentration of LA, the more H2O2 is produced, and the more obvious the ultraviolet absorption peak; similarly, the greater the concentration of LOX, the more obvious the ultraviolet absorption peak. The experimental results show that the size of the ultraviolet absorption peak has 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:

[0026] (1) High catalytic activity. Cu 0.9 Zn 0.1 S@MXene thin-layer material has excellent conductivity and a large specific surface area, which can better increase the electron transfer rate and catalytic performance of the reaction, and has high catalytic activity for H2O2.

[0027] (2) High sensitivity. The present invention is the first to be based on Cu 0.9 Zn 0.1The MXene thin film material has developed a portable mobile phone sensing method and application for lactate oxidase, and three linear equations are obtained respectively: The linear correlation equation between the UV response and the H2O2 concentration is y = 0.1083lgC H2O2 + 0.0444, R 2 = 0.9929, and the detection limit is 0.052 μM; The linear correlation equation between the UV response and the LA concentration is y = 0.0216lgC LA + 0.0140, R 2 = 0.9915, and the detection limit is 0.0034 μM; The linear correlation equation between the UV response and the LOX concentration is y = 0.0252lgC LOX + 0.0128, R 2 = 0.9985, and the detection limit is 0.092 U / L; It shows that the sensor can achieve highly sensitive detection of H2O2, LA and LOX.

[0028] (3) High specificity. For the detection of LA: Other control substances, such as uric acid (UA), glucose, caffeine, ascorbic acid (AA), citric acid (CA), dopamine (DA), glucose (GLU) and sucrose (SUC), have no interference on the system; For the detection of LOX: Acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase) and lysozyme (LZM), papain and trypsin have no interference on the system.

[0029] (4) Portable mobile phone. It can realize on-site analysis and detection, with simple equipment and low cost. It can detect LOX with less materials and reagents, and finally explore the activity monitoring of LOX in HEK293S cells.

[0030] In summary, the present invention constructs a portable mobile phone sensing method and application for lactate oxidase based on a composite MXene thin film material, which has the advantages of high sensitivity, good selectivity, simple operation, fast analysis, easy operation, etc. It can detect low concentrations of LA and LOX, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the UV colorimetric analysis diagram of the feasibility of the LA-LOX biological pathway by the sensor of the present invention;

[0032] Figure 2 It is the linear relationship and colorimetric diagram of the absorbance of the sensor of the present invention to different concentrations of H2O2 versus the logarithm of the concentration;

[0033] Figure 3The linear relationship between the absorbance of the sensor of the present invention for different concentrations of LA and the logarithm of the concentration, and the colorimetric diagram;

[0034] Figure 4 The linear relationship between the absorbance of the sensor of the present invention for different concentrations of LOX and the logarithm of the concentration, and the colorimetric diagram;

[0035] Figure 5 The linear relationship between the absorbance of the sensor of the present invention for different concentrations of Fe(III) and the logarithm of the concentration, and the colorimetric diagram;

[0036] Figure 6 The specific experiment diagram of the sensor of the present invention for LA;

[0037] Figure 7 The specific experiment diagram of the sensor of the present invention for LOX;

[0038] Figure 8 The comparison experiment diagram of the portable mobile phone and the large instrument test of the present invention;

[0039] Figure 9 The experimental diagram of the portable mobile phone of the present invention for monitoring the intracellular LOX activity. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0041] Example 1 Cu 0.9 Zn 0.1 Preparation of Cu

[0042] a. Synthesis of MXene

[0043] Prepare 40 mL of 9 M HCl solution, mix it with 3.2 g of LiF powder, magnetically stir the reaction at room temperature for 30 min to fully dissolve the mixture, and then slowly add 2 g of Ti3AlC2 to the mixed solution to avoid violent heat release. After complete addition, magnetically stir at low speed at room temperature for 48 h, then take out the solution and centrifuge and wash it multiple times at 4000 rpm until the pH of the supernatant > 6. Then take 50 mL of dimethyl sulfoxide (DMSO) and mix it with the preliminarily treated MXene and shake it, stir at room temperature for 12 h, then dry the solution, add deionized water and ultrasonicate for 30 min, and then centrifuge at 3500 rpm for 30 min to obtain the supernatant, and perform freeze-drying to obtain the MXene solid.

[0044] b. Synthesis of Cu 0.9 Zn 0.1 S@MXene thin layer material

[0045] Copper(II) chloride 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 MXene 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 dark 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 three times with absolute ethanol and deionized water respectively, and then dried in an oven at 50 °C for 4 h. That is, Cu 0.9 Zn 0.1 S@MXene thin-layer material was obtained. It was dispersed in 0.1% chitosan solution and labeled as Solution 1 for standby.

[0046] Example 2 Construction of Visual Analysis Sensing Method

[0047] a. The 96-well quartz microplate was placed in an ultrasonic cleaner and washed twice with ethanol and distilled water respectively, ultrasonically treated for 10 min, and then left to dry for standby.

[0048] b. 2 μL of 1 mM H2O2 solution, 20 μL of 2 mg / mL Cu 0.9 Zn 0.1 S@MXene thin-layer material, and 5 μL of 20 mM TMB were successively added to 25 μL of acetate buffer (0.1 M, pH = 4.0) and 48 μL of ultrapure water. Subsequently, they were thoroughly mixed and placed at room temperature for 20 min. The ultraviolet detection of H2O2 was completed in the wavelength range of 400 - 800 nm.

[0049] c. 1.2 μL of 50 mM lactic acid solution (final concentration: 3 mM), 2 μL of 5000 U / L lactate oxidase (final concentration: 500 U / L), 10 μL of PBS (100 mM, pH = 7.4), and 6.8 μL of ultrapure water were thoroughly mixed, then wrapped with tin foil and placed in a 37 °C water bath for reaction for 30 min, and labeled as Solution 2.

[0050] d. 20 μL of 1 mg / mL Cu 0.9 Zn 0.1 S@MXene thin-layer material, 20 μL of the reaction solution of lactic acid - lactate oxidase, and 5 μL of 20 mM TMB were successively added to 25 μL of acetate buffer (0.1 M, pH 4.0) and 30 μL of ultrapure water. Subsequently, they were thoroughly mixed and placed at room temperature for 20 min, and labeled as Solution 3.

[0051] e. Take 2 μL of 5000 U / L lactate oxidase (final concentration: 500 U / L, calculated based on a 20 μL volume), mix it thoroughly with 2 μL of 20 mM Fe(III) (final concentration: 2 mM, calculated based on a 20 μL volume). Wrap it with tin foil and place it in a 37 °C water bath for reaction for 30 min. Then add 20 μL of 1.0 mg / mL Cu 0.9 Zn 0.1 S@MXene thin film material, 1.2 μL of 50 mM lactic acid solution (final concentration: 3 mM), 10 μL of PBS (100 mM, pH = 7.4), and ultrapure water to make the total volume 20 μL. After thorough mixing, wrap it with tin foil and place it in a 37 °C water bath for reaction for 30 min.

[0052] Prepare the sensor according to the above steps. Mix 5 μL of 20 mM TMB with 95 μL of ultrapure water, and label it as solution 4; mix 5 μL of 20 mM TMB, 20 μL of 1 mg / mL Cu 0.9 Zn 0.1 S@MXene solution with 25 μL of acetate buffer (0.1 M, pH 4.0) and 50 μL of ultrapure water, and label it as solution 5; mix 5 μL of 20 mM TMB, 20 μL of the reaction solution of lactic acid - lactate oxidase with 25 μL of acetate buffer (0.1 M, pH 4.0) and 50 μL of ultrapure water, and label it as solution 6; mix 20 μL of 1 mg / mL Cu 0.9 Zn 0.1 S@MXene solution, 20 μL of the reaction solution of lactic acid - lactate oxidase with 25 μL of acetate buffer (0.1 M, pH 4.0) and 35 μL of ultrapure water, and label it as solution 7. Then use a microplate reader to measure the ultraviolet absorption curve, with the wavelength range of 400 - 800 nm.

[0053] The results are as Figure 1 shown. Solutions 1, 2, and 4 only contain Cu 0.9 Zn 0.1 S@MXene, the reaction solution of lactic acid - lactate oxidase, and TMB respectively, and the solutions are colorless with extremely low absorbance; solutions 5, 6, and 7 contain TMB and Cu 0.9 Zn 0.1 S@MXene, TMB, the reaction solution of lactic acid - lactate oxidase, and Cu 0.9 Zn 0.1 S@MXene and the reaction solution of lactic acid - lactate oxidase respectively. Only curve 6 shows a slightly pale blue color and a weak absorbance. Solution 3 is Cu 0.9 Zn 0.1The mixture of S@MXene, the reaction solution of lactic acid - lactate oxidase, and TMB shows a lighter blue color than Solution 6, and its absorbance is also greater than that of Solution 6. Based on this, it shows that this sensing method has a good response to lactic acid - lactate oxidase and can be used for the analysis and detection of LA and LOX.

[0054] Based on the steps in Examples 1 and 2a, by changing the concentration of H2O2 in Example 2b (final concentrations: 0, 0.1, 0.3, 0.5, 0.8, 1, 2, 5, 10, 20, 50, 80, 100, 200, 300, 400, 500, 600 μM), with other steps unchanged, the detection of H2O2 can be achieved. The results are as Figure 2 shown. The intensity of the ultraviolet absorption peak of the sensor shows a good linear relationship with the logarithm of the H2O2 concentration. As the H2O2 concentration increases, the absorbance value gradually increases and the blue color gradually deepens. The linear correlation equation between the intensity of the ultraviolet absorption peak of the sensor and the H2O2 concentration is y = 0.1083lgC H2O2 + 0.0444, R 2 = 0.9929, the linear range is 0.3 - 400 μM, and the detection limit is 0.052 μM.

[0055] Analysis and detection of LA and LOX in Example 3

[0056] Based on the steps in Examples 1 and 2, by changing the LA concentration in Example 2c (final concentrations: 0, 0.01, 0.05, 0.2, 0.5, 1, 6, 10, 20, 60, 150, 300, 600, 1500, 3000, 6000, 10000 μM), with other steps unchanged, the detection of LA can be achieved. The results are as Figure 3 shown. The intensity of the ultraviolet absorption peak of the sensor shows a good linear relationship with the logarithm of the LA concentration. As the LA concentration increases, the blue color gradually deepens. The linear correlation equation between the intensity of the ultraviolet absorption peak of the sensor and the LA concentration is y = 0.0216lgC LA + 0.0140, R 2 = 0.9915, the linear range is 0.2 - 3000 μM, and the detection limit is 0.0034 μM.

[0057] Based on the above steps in Examples 1 and 2, by changing the LOX concentration in Example 2c (final concentrations: 0, 0.1, 0.3, 0.6, 1, 2, 6, 10, 20, 50, 100, 200, 500, 1000, 1500, 2000 U / L), the detection of LOX can be achieved. The results are as Figure 4As shown, the intensity of the ultraviolet absorption peak of the sensor shows a good linear relationship with the logarithm of the LOX concentration, and as the LOX concentration increases, the blue color gradually deepens. The linear correlation equation between the ultraviolet absorption peak intensity of the sensor and the LOX concentration is y = 0.0252lgC LOX + 0.0128, R 2 = 0.9985, the linear range is 0.3 - 1000 U / L, and the detection limit is 0.092 U / L. It shows that the sensor has achieved highly sensitive detection of LA and LOX.

[0058] Example 4 Detection of LOX inhibitor Fe(III)

[0059] Based on the steps in Examples 1 and 2, by changing Example 2e, different concentrations of Fe(III) solutions (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1500, 3000, 8000 μM) were mixed with 2 μL of 5000 U / L lactate oxidase (final concentration: 500 U / L), reacted in a 37 °C water bath for 30 min, and then 1.0 mg / mL 20 μL of Cu 0.9 Zn 0.1 S@MXene solution, 6 μL of 50 mM lactic acid (final concentration: 3 mM), and 5 μL of 20 mM TMB were successively added to 25 μL of acetate buffer (0.1 M pH 4.0) and ultrapure water, with a total volume of 100 μL. Under other unchanged conditions, the ultraviolet absorption peak was detected by an enzyme-linked immunosorbent assay reader. The results are as Figure 5 shown. As the Fe(III) concentration increases, the blue color continuously fades, indicating that the inhibitory effect of Fe(III) on LOX activity is stronger. The half-inhibitory concentration of Fe(III) is 18.6 μM.

[0060] Example 5 Specific detection

[0061] To verify the selectivity of the sensor for LA, according to the sensor preparation steps in Examples 1 and 2 above, uric acid (UA), glucose, caffeine, ascorbic acid (AA), citric acid (CA), dopamine (DA), glucose (GLU), and sucrose (SUC) with the same concentration as LA were respectively added to participate in the reaction; acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP), exonuclease I (Exo I), pyrophosphatase (PPase), and lysozyme (LZM), papain, and trypsin with the same concentration as LOX were added to participate in the reaction. The results are as Figure 6 , Figure 7 shown. It can be seen that the sensor has good selectivity for LA and LOX.

[0062] Example 6 Portable Mobile Phone Sensing

[0063] To verify its potential for on-site analysis, this patent is based on the colorimetric chart in Example 3, integrates it into the mobile phone through color capture software, and outputs signals in the form of G / R. As Figure 8 shown in A, the G / R value has a linear relationship with the logarithm of the LOX concentration, y = 0.9609x + 1.8038, R 2 = 0.988, and the linear range is 0.3 - 1000 U / L. Subsequently, LOX concentrations of 0, 1, 10, and 100 U / L were taken, and monitored using a large instrument (microplate reader) and the mobile phone device respectively. As Figure 8 shown in B, the detection results of the two methods are not very different, but the mobile phone device is more portable and easily forms network data, which is in line with the goal of the development of intelligent healthcare.

[0064] Subsequently, this patent uses HEK293S cells as a cell model to discuss the activity of lactate oxidase in these cells. To stimulate the enzyme activity in the cells, the cells were cultured in a solution containing 1 mM LA for 2 h, then centrifuged to collect these cells for lysis, and then the proteins in the cells were extracted. It was found from Figure 9 A that as the number of cells increases, the content of lactate oxidase increases and the signal shows an increasing phenomenon. Later, normal cells were used as a comparison. As Figure 9 shown in B, the content of lactate oxidase in HEK293S cells is lower than that in normal cells. It can be seen that the lactate oxidase in cancer cells shows abnormal conditions, which is likely to lead to the occurrence of hyperlactacidemia and more likely to cause other complications. This is beneficial for the subsequent exploration of disease mechanisms and helps to reveal the occurrence and development process of cancer.

[0065] 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 this technical field who make changes, modifications, additions, or substitutions within the substantial scope of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A portable mobile phone sensing method and application of lactate oxidase based on composite MXene thin film materials, the mechanism of which is as follows: The copper ions in the Cu 0.9 Zn 0.1 S@MXene thin film material react with H2O2 in the Fenton reaction, and the generated hydroxyl radicals turn the colorless 3,3',5,5'-tetramethylbenzidine (TMB) solution into the blue oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB). oxTMB will show an obvious ultraviolet absorption peak at 652 nm, thus constructing a new visual analysis and detection method through the signal output of H2O2; while the lactate-lactate oxidase system can produce a small amount of H2O2, so the above method can be indirectly used to detect lactate and lactate oxidase. With the change of the concentration of lactate and lactate oxidase, both the absorbance and the color of TMB show a certain degree of change. By accurately analyzing the change of lactate and lactate oxidase through the amount of change in absorbance, a visual analysis sensing method for detecting lactate and lactate oxidase is constructed. At present, there has been no report on the case of realizing the screening of lactate oxidase and its inhibitors based on the Cu 0.9 Zn 0.1 S@MXene thin film material, which has great scientific value and development space.

2. A portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin film material according to claim 1, characterized in that: The present invention combines Cu 0.9 Zn 0.1 S@MXene, an ultraviolet colorimetric visualization method, and the LOX-catalyzed LA reaction for the first time to achieve sensitive analytical detection of LA and LOX.

3. The portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin film material according to claims 1-2, characterized in that: Set the wavelength at 400 - 800 nm, and use an enzyme - linked immunosorbent assay reader to analyze and detect different concentrations of LA and LOX. This method has good sensitivity and specificity. The detection limit of LA is 0.0034 μM, and the detection limit of LOX is 0.092 U / L. The inhibitor IC 50 = 18.6 μM.

4. A portable mobile phone sensing method and application of lactate oxidase based on a composite MXene thin film material according to claims 1-3, characterized in that: Integrate the data into the mobile phone through color capture software, and output the signal in the G / R mode. Compared with large-scale enzyme-linked immunosorbent assay (ELISA) instruments, the detection results of the mobile phone are not much different, but the mobile phone device is more portable and easier to form network data, which is in line with the goal of the development of intelligent healthcare. Using HEK293S cells as a cell model, discuss the activity of lactate oxidase in these cells and normal cells. Lactate oxidase shows low expression in cancer cells, providing new ideas for revealing the development law of cancer.

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