Preparation method and application of choline oxidase visual sensor based on SDBS modified copper-based nano enzyme

Abstract

The invention discloses a preparation method and application of a choline oxidase visual sensor based on SDBS modified copper-based nano enzyme. The preparation method comprises the following specific steps: synthesizing SDBSamp; the GO-coated Cu-CuFe2O4 nano enzyme can oxidize colorless TMB into blue ox-TMB, the ultraviolet absorption intensity of the GO-coated Cu-CuFe2O4 nano enzyme is increased, and ultraviolet colorimetric detection of H2O2 can be realized based on the reaction. In the presence of oxygen, ChOx can catalytically decompose Ch into betaine and H2O2, along with the increase of the concentration of Ch (ChOx) in the solution, the amount of generated H2O2 is increased, the content of generated. OH is increased, the ultraviolet absorption in the solution is enhanced, the blue color of the solution is deepened, and an inhibitor Pb < 2 + > added with ChOx inhibits the catalytic decomposition of Ch, so that the content of H2O2 generated in the solution is reduced, and the ultraviolet absorption intensity is reduced. On the basis, analysis and detection of H2O2 and Ch (ChOx) and screening of inhibitors of H2O2 and Ch (ChOx) are realized through an ultraviolet colorimetric method, and finally, a novel choline oxidase visual mobile phone sensing device is constructed and has relatively high application value.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a visual biosensor, in particular to a preparation method of a biosensor based on SDBS-modified copper-based nanozyme and its application in the detection of choline, choline oxidase and its inhibitors, and further studies a handheld visual mobile device, belonging to the technical fields of biomaterials and chemical biosensing technology. Background Art

[0002] Choline (Ch) is produced endogenously, but mainly comes from dietary intake. As a major component of some animals and plants, choline is essential in the production of important membrane phospholipids, assisting transmembrane signal transduction, and providing cell membrane structure. Human deficiency in choline can cause various health problems, such as fatty liver disease, fatty liver, and arteriosclerosis. In recent years, enzyme-based biosensors have emerged for the detection of choline. Choline can be catalytically decomposed into H2O2 by choline oxidase (ChOx) in the presence of oxygen, and the corresponding signal can be detected by electrochemical and optical methods. Given the impact of choline and choline oxidase on human health and their pathogenicity, their quantitative detection is of great significance in clinical analysis. Currently, common methods for detecting choline include fluorescence, ultraviolet, mass spectrometry, electrochemistry and other methods, but methods for the analysis and detection of choline oxidase are very rare, especially visual analysis and detection methods. Therefore, the development of a simple, rapid, and visual analysis and detection method is very popular.

[0003] In recent years, spinel ferrites (M = Mn, Fe, Co, Ni, Cu) have been widely used in high-density magnetic storage, drug delivery, environmental remediation, and catalysis due to their excellent optical, electrical, and magnetic properties. Recently, copper-based ferrite (CuFe2O4) has been proven to have the ability to catalytically oxidize H2O2, and the detection of H2O2 is established based on the peroxidase-like activity of the CuFe2O4 material. Although the research on the peroxidase-like activity of Cu-CuFe2O4 composites is relatively less, the research on CuFe2O4 and other composites as nanozymes has been relatively mature. Generally, after combining CuFe2O4 with other active components, corresponding synergistic effects will be produced to enhance its peroxidase-like activity. Therefore, it is a part worthy of attention to modify the surface of the CuFe2O4 material to enhance its peroxidase-like activity and accelerate the catalytic oxidation of H2O2.

[0004] The present invention relates to a preparation method and application of a visual sensor for choline oxidase based on SDBS-modified copper-based nanozyme. This method introduces Cu 0Enhance the peroxidase-like activity of CuFe2O4, enhance the conductivity of the material, accelerate the electron transfer rate, and then increase its affinity for the positively charged substrate 3′3′5′5′-tetramethylbenzidine (TMB) by introducing the anionic surfactant SDBS, thereby improving the peroxidase activity of Cu-CuFe2O4. Regarding the problem that the addition of the SDBS anionic surfactant covers the active sites of the SDBS&Cu-CuFe2O4 complex, leveraging the characteristics of GO having a large surface area and conductivity, GO and Cu-CuFe2O4 can be closely linked through electrostatic interactions and the formation of Fe-O-C chemical bonds to form a heterojunction and improve the peroxidase-like activity. In summary, the catalytic activity of CuFe2O4 is improved in three aspects. The modified SDBS-modified copper-based nanozyme (SDBS&GO@Cu-CuFe2O4) has good catalytic activity towards H2O2. In the presence of the chromogenic substrate TMB, by catalyzing H2O2 to generate ·OH, the generated ·OH can further oxidize the colorless TMB itself into blue ox-TMB, with an increase in its ultraviolet absorption intensity and the solution changing from colorless to blue. Based on the above reaction, the ultraviolet colorimetric detection of H2O2 can be achieved. In the presence of oxygen, ChOx can catalyze the decomposition of Ch into betaine and H2O2. As the concentration of Ch(ChOx) in the solution increases, the amount of H2O2 generated increases, the content of ·OH generated increases, the ultraviolet absorption in the solution enhances, and the blue color of the solution deepens. Adding the inhibitor Pb 2+ inhibits its catalytic decomposition of Ch, resulting in a decrease in the content of H2O2 generated in the solution and a decrease in the ultraviolet absorption intensity. Subsequently, through the capture of RGB signals, the design and production of a handheld visual mobile device are completed, which has strong convenience. So far, no method and its application for preparing a choline oxidase visual sensor using SDBS&GO@Cu-CuFe2O4 nanozyme have been found, and most of the analytical detections are limited to the analysis and detection of choline, with very few reports on the analysis and detection of choline oxidase, showing good innovation. Summary of the Invention

[0005] The problem to be solved by the invention is to provide a method for preparing a choline oxidase visual sensor based on SDBS&GO@Cu-CuFe2O4 nanozyme with high selectivity, visualization, low cost, and fast detection speed, as well as its application.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A method for preparing a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme and its application, the specific steps are as follows:

[0007] (1) Preparation of modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4)

[0008] Synthesis by one-step solvothermal method: 1.0 - 2.0 g of ferric nitrate nonahydrate (Fe(NO3)3·9H2O) and 0.1 - 0.5 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) were placed in 10 - 15 mL of ethylene glycol solution. After mixing evenly, 0.2 - 0.5 g of urea (CO(NH2)2) was added and dissolved, and ultrasonicated until a homogeneous solution was obtained. Then, 0.1 - 0.5 g of sodium dodecylbenzenesulfonate (SDBS), 0.1 - 0.5 g of two-dimensional GO material were added to the above mixed solution. After ultrasonicating for 10 - 30 min, it was transferred to a 20 - 50 mL autoclave, and the temperature was set at 150 - 200 °C for reaction for 15 - 20 h. After the reaction ended, the above mixed solution was centrifuged to obtain a precipitate, and the obtained precipitate was washed alternately with distilled water and ethanol 2 - 3 times. The above obtained precipitate was placed in a vacuum drying oven at 50 - 80 °C for drying for 5 - 10 h to obtain modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4) powder. The above dried material was added to 2 mL of distilled water and redispersed into a 2 mg / mL solution for standby.

[0009] (2) Preparation of ultraviolet colorimetric sensor

[0010] a. The 96-well quartz microplate was placed in an ultrasonic cleaner and washed 1 - 3 times with ethanol and distilled water in turn, ultrasonicated for 5 - 10 min, and then dried in air for standby.

[0011] b. 2.0 - 5.0 μL of SDBS&GO@Cu-CuFe2O4 (final concentration: 1.0 - 3.0 mg / mL), 1 - 5 μL of H2O2 (final concentration: 100 - 500 μM), and 1 - 5 μL of TMB (final concentration: 0.2 - 0.5 mM) were taken from the solution in the above step (1) and added to 60 - 90 μL of 10 mM acetic acid solution with pH 4.5 to maintain the volume of the solution at 100 μL. After the above solution was mixed evenly, it was placed at room temperature for 5 - 10 min, and then the ultraviolet absorption curve was measured with a microplate reader in the wavelength range of 500 - 800 nm. After the reaction was completed, it was photographed with a high-definition camera.

[0012] c. ChOx enzyme reaction system: The total volume of the solution reaction was 20 μL. 1 - 5 μL of Ch (final concentration: 100 - 500 μM) and 1 - 5 μL of ChOx (final concentration: 600 - 1000 U / L) were added to phosphate buffer (Na2HPO4 / NaH2PO4, 10 mM, pH 8.5) in turn. The above reaction solution was reacted at 38 °C for 20 - 60 min. Then, according to the above step 2b, the H2O2 was replaced with the above reaction solution, and other operations remained unchanged. The change in ultraviolet absorption intensity was measured with a microplate reader, and the wavelength range was set at 500 - 800 nm. After the reaction was completed, it was photographed with a high-definition camera.

[0013] Detection and analysis of H2O2:

[0014] Based on the above steps 2a and 2b, by changing the concentration of H2O2 in step 2b (final concentration: 0 - 400 μM) while keeping other steps unchanged, measure the change in ultraviolet absorption in the wavelength range of 500 - 800 nm and observe the change in solution color. Establish a standard H2O2 ultraviolet absorption curve with the maximum absorption wavelength at 652 nm. Based on this, the detection of the ultraviolet absorption intensity of H2O2 can be achieved, and the visual detection of H2O2 can be realized through a colorimetric chart.

[0015] Detection and analysis of Ch(ChOx):

[0016] Based on the experimental steps in the above step 2c, when the ChOx enzyme reacts, by changing the concentration of Ch in step 2c (final concentration: 0 - 500 μM), and when the ChOx enzyme reacts, by changing the concentration of ChOx in step 2c (final concentration: 0 - 2000 U / L), while keeping other steps unchanged, measure the change in ultraviolet absorption in the wavelength range of 500 - 800 nm and observe the change in solution color. Establish a Ch(ChOx) ultraviolet absorption curve with the maximum absorption wavelength at 652 nm. Based on this, the detection of the ultraviolet absorption intensity of Ch(ChOx) can be achieved, and the visual detection of Ch(ChOx) can be realized through a colorimetric chart.

[0017] Screening of inhibitor Pb 2+ :

[0018] Based on the experimental steps in the above step 2c, different concentrations of Pb 2+ (final concentration: 0 - 600 nM) are added during the ChOx enzyme reaction. After mixing, the reaction lasts for 20 - 60 s while keeping other steps unchanged. Measure the change in ultraviolet absorption in the wavelength range of 500 - 800 nm and observe the change in solution color. Establish a Pb 2+ ultraviolet absorption curve. Based on this, the screening of ChOx inhibitors can be achieved.

[0019] Principle of the invention: The present invention designs a preparation method and application of a visual sensor for choline oxidase based on SDBS-modified copper-based nanozyme. First, aiming at the problem of low peroxidase activity of copper-based ferrite (Cu-CuFe2O4), Cu 0GO accelerates the electron transfer rate and catalytic activity. The introduction of anionic surfactant SDBS increases the attraction to the substrate TMB and accelerates the progress of the oxidation reaction. Finally, the modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4) is synthesized by a one-step hydrothermal method. Secondly, the nanozyme synthesized above can catalyze the reduction of H2O2 to generate ·OH. In the presence of the chromogenic substrate TMB, ·OH further oxidizes colorless TMB into ox-TMB, resulting in an increase in ultraviolet absorption and the solution changing from colorless to blue. Then, with the help of Ch (ChOx) which can decompose to produce H2O2 under alkaline conditions, the detection of Ch (ChOx) can be indirectly achieved by ultraviolet colorimetric analysis. In a certain concentration range, as the concentration of Ch (ChOx) in the solution increases, the ultraviolet absorption intensity of the solution increases and the blue degree of the solution color gradually deepens. The inhibitor Pb of ChOx 2+ can inhibit Ch (ChOx) from generating H2O2, fix the concentration of Ch (ChOx) in the reaction solution. In a certain concentration range, the ultraviolet absorption intensity of the solution decreases as the concentration of Pb 2+ increases, and the solution color gradually becomes colorless. The experimental results and phenomena show that the changes in ultraviolet absorption intensity and solution color are linearly related to the concentrations of H2O2 and Ch (ChOx) within a certain linear range. Based on this, the detection and analysis of H2O2 and Ch (ChOx) can be realized. Its advantages are as follows:

[0020] (1) High catalytic activity. The modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4) has high peroxidase catalytic activity and has a rapid catalytic reduction effect on H2O2. This patent first uses this material with excellent catalytic performance for the preparation of a choline oxidase visual sensor, which has good innovation.

[0021] (2) Novel biosensing method and device. Currently, most analytical detections are limited to the analysis and detection of choline, and there are few reports on the analysis and detection of choline oxidase. And integrating this sensing method into a mobile phone device through the RGB channel has good innovation.

[0022] (3) High sensitivity. Based on the preparation and application of a choline oxidase ultraviolet colorimetric biosensor mediated by a modified copper-based ferrite nanozyme, using the changes in ultraviolet absorption intensity of ·OH oxidizing TMB and the changes in solution color, three linear equations are obtained: the linear correlation equation between absorbance and H2O2 is y = 0.18LogC H2O2 +0.27, R 2 = 0.9930, and the detection limit is 7.9 nM; the linear correlation equation between absorbance and Ch is y = 0.04LogC Ch +0.08, R 2= 0.9988, and the detection limit is 3.4 nM; the linear correlation equation between absorbance and ChOx is y = 0.06LogC ChOx + 0.01, R 2 = 0.9942, and the detection limit is 0.26 U / L; the IC 2+ of Pb 50 is 15.48 nM, indicating that the sensor can achieve highly sensitive detection of H2O2, Ch, ChOx, and Pb 2+ .

[0023] (4) The detection principle is simple and rapid. The present invention is based on the principle that SDBS&GO@Cu-CuFe2O4 catalyzes the reduction of H2O2 to ·OH, and then ·OH further oxidizes TMB to achieve the detection of H2O2, Ch, ChOx, and Pb 2+ . The entire catalytic reduction only takes 8 min, with a short detection time and obvious results.

[0024] (5) High selectivity. For the detection of H2O2, other control substances such as dopamine (DA), glucose (GLC), fructose (Fru), alkaline phosphatase (ALP), citric acid (CA), and uric acid (UA) have no interference with the system; for the detection of Ch, other control substances such as alanine (Ala), serine (Ser), arginine (Arg), glycine (Gly), uric acid (UA), and xanthine (XA) have no interference with the system; for the detection of ChOx, other control substances such as acetylcholinesterase (AChE), alkaline phosphatase (ALP), cholesterol oxidase (COD), bovine serum albumin (BSA), histone acetyltransferase (HAT), and glucose oxidase (GOx) have no interference with the system.

[0025] In summary, the present invention constructs a preparation method and application of a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme, and applies it to the analysis and detection of H2O2, Ch, ChOx, and Pb 2+ by ultraviolet colorimetric analysis, which has the advantages of high catalytic activity, high sensitivity, simple detection principle, excellent selectivity, etc., and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the peroxidase activity test and colorimetric diagram of different types of copper-based ferrite nanozymes in the present invention;

[0027] Figure 2 is the feasibility analysis and colorimetric diagram of the sensor of the present invention;

[0028] Figure 3 is the linear relationship and colorimetric diagram of the ultraviolet absorption intensity of the sensor of the present invention for different concentrations of H2O2;

[0029] Figure 4 The linear relationship and colorimetric diagram of the ultraviolet absorption intensity of the sensor of the present invention for different concentrations of Ch;

[0030] Figure 5 The linear relationship and colorimetric diagram of the ultraviolet absorption intensity of the sensor of the present invention for different concentrations of ChOx;

[0031] Figure 6 The curve relationship and colorimetric diagram of the ultraviolet absorption intensity of the sensor of the present invention for different concentrations of Pb 2+ ;

[0032] Figure 7 The selectivity experiment and colorimetric diagram of the sensor of the present invention for H2O2, Ch and ChOx;

[0033] Figure 8 The experimental diagram of the activity monitoring of ChOx by the mobile phone device of the present invention. Specific embodiments

[0034] The present invention will be further described in detail below with reference to the embodiments in the accompanying drawings.

[0035] Example 1 Preparation of modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4)

[0036] Synthesized by a one-step solvothermal method: 1.40 g of iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O) and 0.3 g of copper(II) nitrate trihydrate (Cu(NO3)2·3H2O) were placed in 13 mL of ethylene glycol solution, mixed evenly, and then urea (CO(NH2)2) was added and dissolved and ultrasonicated until a homogeneous solution was obtained. Then, 0.20 g of sodium dodecylbenzenesulfonate (SDBS) and 0.3 g of two-dimensional GO material were added to the above mixed solution. After ultrasonicating for 20 min, it was transferred to a 30 mL high-pressure reaction kettle, and the temperature was set at 200 °C and reacted for 18 h. After the reaction was completed, the above mixed solution was centrifuged to obtain a precipitate, and the obtained precipitate was washed alternately with distilled water and ethanol twice, and the obtained precipitate was placed in a vacuum drying oven at 50 °C and dried for 8 h to obtain a powder of modified copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4). The above dried material was added with 2 mL of distilled water and redispersed into a 2 mg / mL solution for standby.

[0037] Example 2 Preparation of ultraviolet colorimetric sensor

[0038] a. The 96-well quartz enzyme plate was placed in an ultrasonic instrument and washed twice with ethanol and distilled water in turn, ultrasonically for 5 min, and then dried in air for standby.

[0039] b. Take 5 μL of the solution 5 μL SDBS&GO@Cu-CuFe2O4 (final concentration: 1.0 mg / mL), 5 μL of H2O2 (final concentration 100 μM), and 5 μL of TMB (final concentration: 0.2 mM) in the above step (1) and add them to 85 μL of 10 mM acetic acid solution at pH 4.5, maintaining the volume of the solution at 100 μL. After mixing the above solution evenly, place it at room temperature for 8 min, and then use a microplate reader to measure its ultraviolet absorption curve in the wavelength range of 500-800 nm. After the reaction is completed, take a photo with a high-definition camera.

[0040] c. ChOx enzyme reaction system: The total volume of the solution reaction is 20 μL. Add 2 μL of Ch (final concentration 100 μM) and 2 μL of ChOx (final concentration 600 U / L) to the phosphate buffer (Na2HPO4 / NaH2PO4, 10 mM, pH 8.5) in sequence. React the above reaction solution at 38 °C for 30 min. Subsequently, follow the above step 2b, replace H2O2 with the above reaction solution, and keep other operations unchanged. Use a microplate reader to measure the change in ultraviolet absorption intensity, set the wavelength range to 500-800 nm, and take a photo with a high-definition camera after the reaction is completed.

[0041] According to the above Example 2b, different types of copper-based ferrite nanozymes were added to the reaction solution, namely Cu-CuFe2O4, SDBS&Cu-CuFe2O4, SDBS&GO@Cu-CuFe2O4. The other experimental steps in the above Examples 2a and 2b remained unchanged, and the catalytic activities of different types of copper-based ferrite nanozymes towards H2O2 were compared.

[0042] The results are as Figure 1 shown. It can be seen from curves 1 and 2 in the figure that when only TMB and TMB + H2O2 exist in the solution, their ultraviolet absorption is very weak and the solution is nearly colorless. When different types of modified copper-based ferrite nanozymes are added to the solution, with the deepening of the modification level, the catalytic activity of the nanozyme gradually increases. It can be seen from the figure that the absorbance values of curves 3-6 gradually increase, and the blue color degree of the solution deepens. The curve of the solution added with SDBS&GO@Cu-CuFe2O4 nanozyme has the largest absorbance value and the deepest blue color degree, proving that the catalytic activity of SDBS&GO@Cu-CuFe2O4 nanozyme is the best.

[0043] Detect the ultraviolet absorption curve of the above-mentioned sensor in the range of 500-800 nm. The results are as Figure 2As shown, from curves 1 and 2 in the figure, it can be observed that when only TMB or TMB + H2O2 exists in the solution, there is almost no ultraviolet absorption peak in the solution, and the solution shows a nearly colorless state. From curves 3 and 4 in the figure, it can be observed that when TMB or H2O2 is lacking in the solution, there is also almost no absorption peak in the solution, and the solution is also nearly colorless. When SDBS&GO@Cu-CuFe2O4 + TMB + H2O2 exists in the solution, there is an obvious ultraviolet absorption peak in curve 5, and the color of the solution turns blue, indicating that TMB is catalytically oxidized by ·OH generated by SDBS&GO@Cu-CuFe2O4 catalyzing H2O2 to form ox-TMB. By comparing curve 5 with other curves, it shows that the prepared sensor has a good response to H2O2 and can be applied to the analysis and detection of H2O2 and Ch (ChOx).

[0044] Example 3 Detection and Analysis of H2O2

[0045] Based on the steps in the above Examples 2a and 2b, change the concentration of H2O2 in Example 2b (final concentrations: 0, 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400 μM), and keep other steps unchanged. Based on this, the detection and analysis of H2O2 can be achieved. The results are as Figure 3 shown. There is a good linear relationship between the absorbance of the sensor to H2O2 and the concentration. The linear correlation equation is y = 0.18LogC H2O2 + 0.27, R 2 = 0.9930. The linear range is 0.02 - 100 μM, and the detection limit is 7.9 nM. It can be seen from Figure 3 this that within a certain concentration range, as the concentration of H2O2 increases, its ultraviolet absorption intensity gradually increases, and the solution gradually changes from colorless to blue.

[0046] Example 4 Detection and Analysis of Ch (ChOx) Activity

[0047] Based on the steps in the above Example 2, fix the concentration of ChOx at 600 U / L, change the concentration of Ch in Example 2c (final concentrations: 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500 μM), and keep other steps unchanged. Based on this, the detection and analysis of Ch can be achieved. The results are as Figure 4 shown. There is a good linear relationship between the absorbance of the sensor to Ch and the concentration. The linear correlation equation is y = 0.04LogC Ch + 0.08, R 2 = 0.9988. The linear range is 0.01 - 100 μM, and the detection limit is 3.4 nM. It can be seen fromFigure 4 It can be seen that when the concentration of ChOx is fixed and within a certain concentration range, as the concentration of added Ch increases, the ultraviolet absorption intensity of the solution gradually increases, indicating that the amount of H2O2 generated in the solution increases, and TMB in the solution is oxidized, causing the solution to gradually change from colorless to light blue.

[0048] Based on the steps in Example 2 above, the concentration of Ch is fixed at 100 μM, and the concentration of ChOx in Example 2c is changed (final concentration: 0, 0.5, 1, 2, 4, 6, 10, 20, 40, 60, 100, 200, 400, 600, 1000, 2000 U / L), and other steps remain unchanged. Based on this, the detection and analysis of ChOx can be achieved. The results are as Figure 5 shown. There is a good linear relationship between the absorbance of the sensor for ChOx and the concentration. The linear correlation equation is y = 0.06LogC ChOx + 0.01, R 2 = 0.9942, the linear range is 1 - 600 U / L, and the detection limit is 0.26 U / L. From Figure 5 it can be seen that when the amount of added Ch is fixed and within a certain range, as the concentration of added ChOx increases, the ultraviolet absorption intensity of the solution gradually increases, the amount of H2O2 generated in the solution increases, and TMB in the solution is oxidized, causing the solution to gradually change from colorless to light blue.

[0049] Example 5 Screening of ChOx Inhibitors

[0050] Based on the steps in Example 2 above, the concentration of Ch is fixed at 100 μM, and the concentration of ChOx is 600 U / L. Pb 2+ and ChOx are mixed and reacted for 40 s. The concentration of Pb 2+ is changed (final concentration: 0, 0.1, 0.2, 0.4, 1, 2, 4, 6, 10, 20, 40, 60, 100, 200, 400, 600 nM), and other steps are the same as above. Based on this, the screening of ChOx inhibitors can be achieved. The results are as Figure 6 shown. In the range of 0.1 - 600 nM, it can be seen from the figure that as the concentration of added Pb 2+ increases, the ultraviolet absorption intensity of the solution decreases, indicating that the presence of Pb 2+ inhibits the above enzyme reaction, the amount of H2O2 generated in the solution decreases, and the solution gradually changes from light blue to colorless. The above phenomenon indicates that Pb 2+ has an inhibitory effect on the ChOx enzyme reaction, and its IC 50 is 15.48 nM.

[0051] Example 6 Selective Detection

[0052] To verify the selectivity of the prepared sensor for the above-mentioned H2O2, Ch, and ChOx, the sensor was prepared according to Example 2 above, and the concentrations of the selective substances used were the same as those of the above three substances. The reagents used in the H2O2 selectivity experiment were dopamine (DA), glucose (GLC), fructose (Fru), alkaline phosphatase (ALP), citric acid (CA), and uric acid (UA), which had no interference with the system; the reagents used in the Ch selectivity experiment were alanine (Ala), serine (Ser), arginine (Arg), glycine (Gly), uric acid (UA), and xanthine (XA), which had no interference with the system; the reagents used in the ChOx selectivity experiment were acetylcholinesterase (AChE), alkaline phosphatase (ALP), cholesterol oxidase (COD), bovine serum albumin (BSA), histone acetyltransferase (HAT), and glucose oxidase (GOx). The results are as Figure 7 shown. It can be found that the prepared sensor has selectivity for the detected substances corresponding to the above-mentioned selected substances, and the added control substances are all colorless, indicating that the prepared sensor has good selectivity for the detected substances.

[0053] Example 7 Mobile Device Design and Application

[0054] There are few visualization methods for monitoring ChOx activity, and none have been seen in mobile devices so far. Based on the catalytic effect of SDBS&GO@Cu-CuFe2O4 on the TMB and H2O2 system, this patent collects the change of TMB color through the RGB channel in the image capture software, and establishes a linear relationship based on the G / R value and ChOx concentration, which can be used for the convenient visualization analysis of ChOx in actual samples. As Figure 8 shown in A, the chromaticity block in Example 4 was input into the mobile phone, and the chromaticity was extracted to establish a linear relationship between the G / R value and the logarithm of the ChOx concentration, y = 1.1268x + 0.2664, and the linear range was 1 - 600 U / L. As Figure 8 shown in B, taking SMMC-7721 hepatoma cells as an example, they were dispersed into a suspension of 10,000 cells / L, and the protein in the cell nucleus was extracted after lysis. It can be judged from the signal value output by the mobile phone that the cells contain a certain amount of ChOx. As the inhibitor Pb 2+ increases, the signal will be inhibited. It can be seen that this method and device can be used for the visual quantitative analysis of intracellular ChOx and have great application value.

[0055] 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 essence of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A preparation method and application of a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme, and its mechanism is as follows: First, an improved copper-based ferrite nanozyme (SDBS&GO@Cu-CuFe2O4) is synthesized by a one-step hydrothermal method, which can oxidize the chromogenic substrate TMB to colorless TMB. Then, with the help of Ch (ChOx) which can decompose to produce H2O2 under alkaline conditions, the detection of Ch (ChOx) can be indirectly achieved by ultraviolet colorimetric analysis. The ultraviolet absorption intensity and the change in solution color are linearly related to the concentration of H2O 2、 Ch (ChOx) within a certain linear range, and based on this, the detection and analysis of H2O 2、 Ch (ChOx) can be realized.

2. The preparation method and application of a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme according to claim 1, characterized in that: At present, most of the analysis and detection are limited to the analysis and detection of choline, and there are few reports on the analysis and detection of choline oxidase. This patent constructs a new type of visual sensor for choline oxidase based on ultraviolet colorimetry technology, which has good innovation.

3. The preparation method and application of a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme according to claims 1-2, characterized in that: Using this new type of sensor, the analysis and detection of H2O2, Ch, and ChOx have been realized, with good sensitivity and excellent selectivity. The detection limit of H2O2 is 7.9 nM, the detection limit of Ch is 3.4 nM, and the detection limit of ChOx is 0.26 U / L; for the inhibitory effect of Pb 2+ , the IC 50 is 15.48 nM.

4. The preparation method and application of a choline oxidase visual sensor based on SDBS-modified copper-based nanozyme according to claims 1-3, characterized in that: By using the RGB channels to extract colors and establishing a linear relationship between the G / R value and the logarithmic value of the ChOx concentration, this method and device can be used for the visual quantitative analysis of intracellular ChOx and have great application value.

Images