Co-MOF nano-enzyme colorimetric detection method for L-Cys and application of Co-MOF nano-enzyme colorimetric detection method

Abstract

The invention relates to a method for colorimetric detection of L-Cys by Co-MOF nano-enzyme in the technical field of detection. The method comprises the following steps: S1, preparing a standard detection solution, namely fully and uniformly mixing a buffer solution, Co-MOF nano-enzyme with catalase simulation activity and no peroxidase and oxidase activity, H2O2, triphenylmethane dye and L-Cys standard solutions with different concentrations for reaction; s2, preparing a blank detection solution: replacing the L-Cys standard solution with distilled water, and preparing according to the same conditions. S3, drawing a standard curve: measuring the absorbance of the two solutions, establishing a quantitative relation between the absorbance and the L-Cys concentration, drawing the curve and obtaining a regression equation. S4, detecting a to-be-detected sample: replacing the L-Cys standard solution with the same amount of the to-be-detected sample solution, preparing a detection solution according to the same conditions, detecting the absorbance, and substituting the absorbance into the regression equation to obtain the concentration of the L-Cys. The method has the advantages of being easy and convenient to operate, low in cost and short in consumed time.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method and application of colorimetric detection of L-Cys using Co-MOF nanozymes. Background Technology

[0002] L-cysteine ​​(L-Cys) is an important substance with physiological functions, widely distributed in nature. In plants and animals, it is an amino acid used by various tissues and cells to defend against harmful substances and increase vitality. L-Cys possesses antioxidant, chelating, and flavoring properties and is widely used in the food industry. Examples include its use as a food flavoring and coloring agent, bread additive, antioxidant and preservative for oils and fats, and in preventing food browning.

[0003] Experimental studies have shown that low concentrations of L-Cys can protect nerves, while high concentrations may cause neuronal damage. Therefore, the amount of L-Cys added to different foods is subject to different strict limits, and its addition amount in food has become one of the important indicators for food safety control.

[0004] Currently, traditional methods for detecting L-Cys include potentiometric titration, fluorescence analysis, spectrophotometry, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). While these methods generally ensure effective detection of L-Cys, they suffer from drawbacks such as complex sample pretreatment, high technical requirements, high cost, and long detection time. These limitations restrict their application and make them unsuitable for the convenience, economy, and efficiency demands of rapid on-site detection. For example, HPLC utilizes the different distributions of L-Cys in the stationary and mobile phases to achieve separation via a chromatographic column. Then, an ultraviolet (UV) or fluorescence detector is used to detect the absorption or emission of L-Cys under specific colored light. By comparing with standard samples of known concentrations, the L-cysteine ​​content in the sample is determined. This method offers high separation efficiency, effectively separating L-Cys from other impurities in complex samples; it also boasts high sensitivity and a low detection limit, accurately determining trace amounts of L-Cys. However, the equipment is expensive and has high maintenance costs; it requires high pretreatment of samples, including complex extraction and purification steps, and requires specialized operators to perform the testing.

[0005] For example, potentiometric titration uses the change in potential to indicate the titration endpoint. During titration, as the titrant is added, the concentration of L-Cys in the solution changes continuously, causing a corresponding change in the electrode potential. When the stoichiometric point is reached, the potential changes, which is used to determine the titration endpoint and then calculate the L-Cys content. This method does not require the use of indicators, is unaffected by factors such as the color or turbidity of the sample solution, and has a wide range of applications, including colored or turbid solutions. The disadvantages are that the detection results are affected by the pH of the sample solution, the operation is relatively complex, it requires the use of instruments such as a potentiometric titrator, and the maintenance and calibration of the electrodes are quite important.

[0006] In addition, there are also methods for colorimetric detection of L-Cys using the oxidase activity of nanozymes. For example, patent publication number "CN117960117A" discloses a method for preparing a FeCoZn-NC nanozyme with biomimetic cascade capability and its application in the colorimetric detection of L-cysteine. Patent publication number "CN120774467A" discloses a method for preparing a manganese tetroxide / graphite nanozyme for sensitive detection of L-cysteine.

[0007] YangW et al. disclosed a method based on Co 2+ A method for synthesizing Co-MOF nanozymes by coordination self-assembly with 2-methylimidazole was discovered. The nanozymes exhibited catalase activity but lacked peroxidase and oxidase activity. Furthermore, the catalase activity was applied to the degradation of dyes.

[0008] However, there are currently no reports on the use of catalase activity based on Co-MOF nanozymes for L-Cys colorimetric detection. Summary of the Invention

[0009] The present invention aims to provide a method for colorimetric detection of L-Cys using Co-MOF nanozymes, so as to achieve rapid colorimetric detection of L-Cys.

[0010] Firstly, to achieve the above objectives, the present invention adopts the following technical solution: a method for colorimetric detection of L-Cys using Co-MOF nanozymes, comprising the following steps: S1. Preparation of standard detection solution: The buffer solution, Co-MOF nanozyme, H2O2, triphenylmethane dye, and L-Cys standard solutions of different concentrations are thoroughly mixed and reacted to obtain the standard detection solution; the Co-MOF nanozyme has catalase-mimicking activity but does not have peroxidase or oxidase activity; the triphenylmethane dye is fuchsin or crystal violet; S2. Preparation of blank detection solution: Using deionized water instead of the L-Cys standard solution in step S1, a blank detection solution is prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and L-Cys concentration, construct the standard curve, and obtain the regression equation; S4. Detection of L-Cys in the test sample: Using an equal volume of the test sample solution to replace the L-Cys standard solution in step S1, the test sample detection solution system is prepared according to step S1 under the same conditions; the absorbance of the test sample detection solution is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of L-Cys in the test sample.

[0011] The beneficial effects of this scheme are as follows: This scheme utilizes the catalase-like activity of Co-MOF nanozymes to catalyze the generation of H2O and O2 from H2O2, thereby degrading crystal violet or fuchsin. Furthermore, the inhibitory effect of L-Cys on this reaction slows down the degradation rate of crystal violet and fuchsin. By leveraging the direct proportionality between solution color and L-Cys concentration, rapid detection of L-Cys can be achieved.

[0012] Preferably, as an improvement, the pH value of the buffer solution is 6.5 to 7.5. More preferably, the pH value is 7.0.

[0013] Preferably, as an improvement, the ion concentration of the buffer solution is 0 M to 0.2 M. 0 M is preferred.

[0014] Preferably, as an improvement, the buffer solution is water.

[0015] Preferably, as an improvement, the final concentration of H2O2 in the test sample detection solution system is 0.5% to 0.7% (v / v). More preferably, the final concentration is 0.6% (v / v).

[0016] Preferably, as an improvement, the reaction temperature is 10°C to 20°C. More preferably, it is 15°C.

[0017] Preferably, as an improvement, when the triphenylmethane dye is crystal violet, the final concentrations of the Co-MOF nanozyme and crystal violet in the test sample detection solution system are 0.4–0.5 μg / mL and 0.008–0.012 mg / mL, respectively, the reaction time is 10–15 min, and the detection wavelength is 590 nm. More preferably, the final concentrations of the Co-MOF nanozyme and crystal violet in the test sample detection solution system are 0.45 μg / mL and 0.01 mg / mL, respectively, and the reaction time is 12 min.

[0018] Preferably, as an improvement, when the triphenylmethane dye is fuchsin, the final concentrations of the Co-MOF nanozyme and fuchsin in the test sample detection solution system are 0.08–0.10 μg / mL and 0.03–0.05 mg / mL, respectively, the reaction time is 8–15 min, and the detection wavelength is 546 nm. Preferably, the final concentrations of the Co-MOF nanozyme and fuchsin in the test sample detection solution system are 0.09 μg / mL and 0.04 mg / mL, respectively, and the reaction time is 10 min.

[0019] Secondly, the present invention provides the application of the above method in detecting L-Cys content.

[0020] Preferably, as an improvement, the detection of L-Cys content refers to the detection of L-Cys content in food, pharmaceutical or biological samples.

[0021] The beneficial effects of this invention are: 1. This study is the first to utilize the catalase-like activity of Co-MOF nanozymes to catalyze the degradation of triphenylmethane dyes (crystal violet / magenta) by H2O2, and achieves rapid and visual detection of their concentration by leveraging the inhibitory effect of L-Cys on this degradation reaction. The mechanism is clear and the selectivity is excellent. The entire detection process requires no complex instruments or cumbersome pretreatment; only the absorbance needs to be measured after mixing the reaction. The reaction time is short (8–15 minutes), making it suitable for rapid on-site detection.

[0022] 2. The Co-MOF nanozyme used is simple to synthesize and has good stability. The dyes and reagents such as H2O2 are all common and readily available materials, significantly reducing the detection cost. Furthermore, within the range of 0–0.005 mol / L, the absorbance shows a good linear relationship with the L-Cys concentration (R0). 2 The detection limits are as low as 1.5 mmol / L (crystal violet system) and 4.5 mmol / L (magenta system), meeting the needs of practical sample detection. Validated by spiked recovery experiments, the method achieved recoveries of 98.1%–106.2% in real-world samples such as milk, fruit juice, and functional beverages, with RSDs less than 5%, demonstrating good accuracy and repeatability. Furthermore, it maintains high selectivity for L-Cys detection even under conditions of coexistence of various sugars, amino acids, and ions, making it suitable for complex matrix samples (such as food and beverages). Attached Figure Description

[0023] Figure 1 SEM images of Co-MOF nanozymes; A: Co-MOF nanozymes at 2000x magnification; B: Co-MOF nanozymes at 5000x magnification.

[0024] Figure 2Figure A shows the activity verification diagrams for Co-MOF nanozymes; Figure B shows the activity verification diagram for CAT (sample 1: H2O2; sample 2: Co-MOF; sample 3: H2O2+Co-MOF); Figure C shows the activity verification diagram for OPD (sample 1: TMB; sample 2: TMB+ H2O2; sample 3: TMB+H2O2+Co-MOF); Figure C shows the activity verification diagram for OXD (sample 1: TMB; sample 2: Co-MOF; sample 3: TMB+ Co-MOF).

[0025] Figure 3 Figure A shows the feasibility verification results of the L-cysteine ​​detection method; Figure A is the control group of the crystal violet colorimetric sensing system (sample 1: Co-MOF; sample 2: Co-MOF + L-Cys; sample 3: H2O2 + crystal violet + L-Cys; sample 4: L-Cys; sample 5: H2O2 + crystal violet); Figure B is the experimental group of the crystal violet colorimetric sensing system (sample 1: Co-MOF + H2O2 + crystal violet + deionized water; sample 2: Co-MOF + H2O2 + crystal violet + 0.09M L-Cys; sample 3: Co-MOF + H2O2 + crystal violet + 0.05M L-Cys; sample 4: Co-MOF + H2O2 + crystal violet + 0.025M L-Cys). Figure C shows the control group of the magenta colorimetric sensing system (sample 1: Co-MOF; sample 2: Co-MOF + L-Cys; sample 3: H2O2 + magenta + L-Cys; sample 4: L-Cys; sample 5: H2O2 + magenta; Figure D shows the experimental group of the magenta colorimetric sensing system (sample 1: Co-MOF + H2O2 + magenta + deionized water; sample 2: Co-MOF + H2O2 + magenta + 0.09M L-Cys; sample 3: Co-MOF + H2O2 + magenta + 0.05M L-Cys; sample 4: Co-MOF + H2O2 + magenta + 0.025M L-Cys).

[0026] Figure 4 Figure A shows the optimization results of the buffer type; A is the crystal violet colorimetric sensor (blank group: Co-MOF+H2O2+crystal violet+deionized water; experimental group: Co-MOF+H2O2+crystal violet+0.1M L-Cys); B is the fuchsin colorimetric sensor system (blank group: Co-MOF+H2O2+fuchsin+deionized water; experimental group: Co-MOF+H2O2+fuchsin+0.1M L-Cys).

[0027] Figure 5Figure A shows the optimized pH results of the buffer solution; A represents the crystal violet colorimetric sensing system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1M L-Cys); B represents the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1M L-Cys).

[0028] Figure 6 Figure A shows the optimization results of the buffer ion concentration; A is the crystal violet colorimetric system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1M L-Cys); B is the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1M L-Cys).

[0029] Figure 7 Figure A shows the optimized concentration of Co-MOF nanozymes; A represents the crystal violet colorimetric sensing system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1M L-Cys); B represents the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1M L-Cys).

[0030] Figure 8 Figure A shows the optimized results of H2O2 concentration; A is the crystal violet colorimetric system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1M L-Cys); B is the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1M L-Cys).

[0031] Figure 9 Figure A shows the results of the triphenylmethane dye concentration optimization; A is the crystal violet colorimetric system (blank group: Co-MOF+H2O2+crystal violet+deionized water; experimental group: Co-MOF+H2O2+crystal violet+0.1M L-Cys); B is the fuchsin colorimetric sensing system (blank group: Co-MOF+H2O2+fuchsin+deionized water; experimental group: Co-MOF+H2O2+fuchsin+0.1M L-Cys).

[0032] Figure 10Figure A shows the optimized reaction temperature results; A represents the crystal violet colorimetric system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1M L-Cys); B represents the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1M L-Cys).

[0033] Figure 11 Figure A shows the optimized reaction time results; A represents the crystal violet colorimetric system (blank group: Co-MOF + H2O2 + crystal violet + deionized water; experimental group: Co-MOF + H2O2 + crystal violet + 0.1 mL - Cys); B represents the fuchsin colorimetric sensing system (blank group: Co-MOF + H2O2 + fuchsin + deionized water; experimental group: Co-MOF + H2O2 + fuchsin + 0.1 mL - Cys).

[0034] Figure 12 Figure 1 shows the sensitivity detection results; A and B belong to the crystal violet colorimetric sensing system, and C and D belong to the magenta colorimetric sensing system; Figures A and C: absorption spectra of L-Cys reaction solutions with different concentrations; Figures B and D: standard curves of absorbance of L-Cys reaction solutions with different concentrations.

[0035] Figure 13 Figure A shows the results of specificity analysis; Figure A: Results of specificity analysis with the addition of glucose, fructose, sucrose, glycine, L-tyrosine, L-valine and Ca. 2+ Bar chart for testing the anti-interference ability of the crystal violet colorimetric sensor system; Figure B: [Chart showing the effect of adding maltose, fructose, sucrose, L-leucine, L-phenylalanine, L-proline, and Na] + Bar chart showing the anti-interference capability test of the magenta colorimetric sensor system. Detailed Implementation

[0036] The following detailed description illustrates the specific implementation method: 1. Experimental Materials and Methods 1.1. Experimental Equipment and Reagents Table 1 shows the experimental equipment used in the experiment.

[0037] Table 2. Reagents used in the experiment

[0038] All experimental water used was deionized water. 2-Methylimidazole (2-MI) and cobalt nitrate hexahydrate (Co(NO3)2·6H2O) were used in methanol as solvent; Co-MOF nanozymes were used in anhydrous ethanol as solvent; 3,3',5,5'-tetramethylbenzidine (TMB) was used in dimethyl sulfoxide (DMSO) as solvent. All other reagents were used in deionized water as solvent.

[0039] 1.2 Test Methods 1.2.1 Synthesis of Co-MOF hydrogen peroxide mimic enzyme Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was prepared into a 40 mL solution with a concentration of 3.75 mg / mL using methanol. Simultaneously, 2-methylimidazole (2-MI) was prepared into a 10 mL solution with a concentration of 720 mg / mL using methanol. The two solutions were mixed thoroughly to initiate a coordination reaction. After 6 hours of reaction, the reaction solution was centrifuged at 5000 rpm for 5 min. The precipitate was then washed more than 3 times with anhydrous ethanol and dried in a vacuum drying oven at 60℃ for 3 hours. The resulting purple powder was the hydrogen peroxide mimic enzyme—Co-MOF nanozyme (Reference: Yang W, Zhu L, Xu W. Biologically inspired Co-MOF as catalase mimics with an unexpected ROS production ability for the efficient removal of organic dyes[J]. Journal of Environmental Chemical Engineering,2024,12(2):112358).

[0040] Unless otherwise specified, nanozymes in the following text refer to Co-MOF nanozymes.

[0041] 1.2.2 Characterization of Co-MOF hydrogen peroxide mimic enzyme The size and morphology of Co-MOF nanozymes were analyzed using scanning electron microscopy (SEM) to understand their microscopic characteristics.

[0042] 1.2.3 Investigation into the activity of Co-MOF hydrogen peroxide mimic enzyme Catalase (CAT) activity was investigated: The CAT activity was verified using Co-MOF nanozyme catalysis of H2O2. Specifically, 490 μL of deionized water (pH=7.0), 5 μL of H2O2 (stock solution), and 5 μL of Co-MOF nanozyme (45 μg / mL) were added to 1.5 mL centrifuge tubes, mixed thoroughly, and reacted at 15℃ for 10 min. The reaction phenomena in each centrifuge tube were then observed.

[0043] Peroxidase (POD) activity investigation: The POD activity of the TMB-H2O2 system was verified using Co-MOF nanozyme catalysis. Specifically, 485 μL of sodium dihydrogen phosphate buffer (25 mM, pH=7.0) was added to a 1.5 mL centrifuge tube, followed by 5 μL of H2O2 (stock solution), 5 μL of TMB (40 mM), and 5 μL of Co-MOF (9 μg / mL). After mixing thoroughly and reacting at 15℃ for 10 min, the absorbance of the reaction solution in the wavelength range of 500–800 nm was measured using a microplate reader, and the change in absorbance at 652 nm was observed.

[0044] Oxidase (OXD) activity investigation: The OXD activity of TMB was verified by using Co-MOF nanozyme catalysis. Specifically, 490 μL of sodium dihydrogen phosphate buffer (25 mM, pH=7.0) was added to a 1.5 mL centrifuge tube, followed by 5 μL of TMB (40 mM) and 5 μL of Co-MOF (45 μg / mL). After mixing thoroughly, the reaction was carried out at 15℃ for 10 min. The absorbance of the reaction solution in the wavelength range of 500–800 nm was measured using a microplate reader, and the change in absorbance at 652 nm was observed.

[0045] 1.2.4 Establishment of a rapid detection method for L-cysteine ​​based on hydrogen peroxide mimicry enzyme Co-MOF nanozymes exhibit excellent catalase activity, catalyzing the production of H2O and O2 from H2O2. This reaction can degrade crystal violet or fuchsin, both of which have absorption peaks at specific wavelengths (crystal violet: 590 nm, fuchsin: 546 nm). The addition of L-Cys inhibits the degradation of crystal violet or fuchsin, and changes in absorbance can be detected using a microplate reader. Visually, the concentration of L-Cys is directly proportional to the solution color; higher concentrations result in a deeper color. Based on this, a colorimetric sensor for L-Cys detection is established. The specific operation is as follows: Establishment of the crystal violet colorimetric sensing system: Based on the catalysis of the "crystal violet-H2O2" system by Co-MOF nanozyme, this colorimetric sensor was designed. Specifically, 5 μL of H2O2 (stock solution), 5 μL of crystal violet (0.001 g / mL), 5 μL of L-Cys at different concentrations (0.09 M / 0.05 M / 0.025 M), and 5 μL of Co-MOF nanozyme (45 μg / mL) were added to 480 μL of deionized water (pH=7.0), mixed thoroughly, and reacted at 10℃ for 10 min. The absorbance of the solution in the wavelength range of 400~700 nm was detected using a microplate reader, and the absorption spectrum was plotted. Establishment of the fuchsin colorimetric sensing system: A colorimetric sensor was designed using a Co-MOF nanozyme to catalyze a fuchsin-H2O2 system. 5 μL of H2O2 (stock solution), 10 μL of fuchsin (0.001 g / mL), 5 μL of L-Cys at different concentrations (0.09 M / 0.05 M / 0.025 M), and 5 μL of Co-MOF nanozyme (45 μg / mL) were added to 475 μL of deionized water (pH=7.0). After mixing thoroughly and reacting at 10℃ for 10 min, the absorbance of the solution in the wavelength range of 400–650 nm was measured using a microplate reader, and the absorption spectrum was plotted.

[0046] 1.2.5 Optimization of Reaction Conditions To optimize the performance of the L-Cys detection method, optimization work was carried out on several key influencing factors in the reaction system, building upon the original colorimetric sensing method. Optimizations included buffer type, buffer pH, buffer ion concentration, nanozyme concentration, substrate concentration, reaction time, and reaction temperature. Blank and parallel groups were included in all experiments. Specific procedures are as follows: Optimization of buffer type: (1) Crystal violet colorimetric sensing: 5 μL of H2O2 (stock solution), crystal violet (0.001 g / mL), Co-MOF nanozyme (45 μg / mL) and L-Cys (0.1 M) were added to 480 μL of different types of buffers (deionized water, pH=7.0; sodium dihydrogen phosphate solution, pH=7.0, 25 mM; sodium acetate solution, pH=7.0, 25 mM), mixed evenly, and a blank group was set up. After reacting at 10℃ for 10 min, the absorbance of the solution was detected at 590 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader. (2) Fuchsin colorimetric sensing: 5 μL H2O2 (stock solution), 10 μL fuchsin (0.001 g / mL), 5 μL Co-MOF nanozyme (45 μg / mL) and 5 μL L-Cys were added to 475 μL of different types of buffer solutions (deionized water, pH=7.0; sodium dihydrogen phosphate solution, pH=7.0, 25 mM; sodium acetate solution, pH=7.0, 25 mM), respectively, mixed thoroughly, and a blank group was set up. The reaction was carried out at 10℃ for 10 min, and the absorbance of the solution was detected at a wavelength of 546 nm using an ELISA reader. A bar graph of absorbance of the experimental group (A) and the blank group (A0) was plotted based on the experimental data. The difference in absorbance between the two groups (ΔA=A-A0) was compared. The buffer type with the largest difference was the optimal buffer type.

[0047] Optimization of buffer pH: The pH of deionized water was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 with 0.1 M dilute hydrochloric acid or dilute sodium hydroxide solution for later use. (1) Crystal violet colorimetric sensing: 5 μL of H2O2 (stock solution), Co-MOF nanozyme (45 μg / mL), crystal violet (0.001 g / mL) and L-Cys (0.1 M) were added to 480 μL of deionized water with different pH values, mixed evenly, and a blank group was set up. After reacting at 10℃ for 10 min, the absorbance of the solution was detected at 590 nm wavelength using an enzyme-linked immunosorbent assay reader. (2) Fuchsin colorimetric sensing: 5 μL H2O2 (stock solution), 10 μL fuchsin (0.001 g / mL), 5 μL Co-MOF nanozyme (45 μg / mL) and 5 μL L-Cys (0.1 M) were added to 475 μL of deionized water at different pH values ​​and mixed well. A blank group was set up. After reacting at 10℃ for 10 min, the absorbance of the solution was detected at 546 nm using an ELISA reader. A line graph was plotted based on the experimental data, and the absorbance difference between the experimental group (A) and the blank group (A0) was calculated (ΔA=A-A0). The largest difference was the optimal buffer pH.

[0048] Optimization of buffer ion concentration: Sodium chloride was dissolved in deionized water and prepared into deionized water with different salt ion concentrations of 0, 20, 40, 80, 100, 200 and 400 mM for later use. (1) Crystal violet colorimetric sensing: 5 μL H2O2 (stock solution), Co-MOF nanozyme (45 μg / mL), crystal violet (0.001 g / mL) and L-Cys (0.1 M) were mixed evenly with 480 μL of deionized water (pH=7.0) with different salt ion concentrations. A blank group was set up and reacted at 10℃ for 10 min. The absorbance of the solution was detected at a wavelength of 590 nm using an enzyme-linked immunosorbent assay reader. (2) Fuchsin colorimetric sensing: 5 μL of H2O2 (stock solution), 10 μL of fuchsin (0.001 g / mL), 5 μL of Co-MOF nanozyme (45 μg / mL) and L-Cys (0.1 M) were added to 475 μL of deionized water (pH=7.0) with different salt ion concentrations and mixed well. A blank group was set up. After reacting at 10℃ for 10 min, the absorbance of the solution was detected at a wavelength of 546 nm using an ELISA reader. A line graph was plotted based on the experimental data, and the absorbance difference between the experimental group (A) and the blank group (A0) was calculated (ΔA=A-A0). The largest difference was the optimal buffer ion concentration.

[0049] Optimization of nanozyme concentration: (1) Crystal violet colorimetric sensing: Five different Co-MOF nanozyme volume gradients of 1, 3, 4, 5 and 6 μL were set up. 5 μL H2O2 (stock solution), 5 μL crystal violet (0.001 g / mL), 5 μL L-Cys (0.1 M) and different volumes of Co-MOF nanozyme (45 μg / mL) were added to deionized water (pH=7.0; the pH changed with the nanozyme volume, and the total solution volume was kept at 500 μL) and mixed evenly. A blank group was set up and reacted at 10℃ for 10 min. The absorbance of the solution was detected at a wavelength of 590 nm using an ELISA reader.

[0050] (2) Fuchsin colorimetric sensing: Five different Co-MOF nanozyme volume gradients (0.5, 1, 2, 3, and 5 μL) were set up. 5 μL of H2O2 (stock solution) and L-Cys (0.1 M), 10 μL of fuchsin (0.001 g / mL) and different volumes of Co-MOF nanozyme (45 μg / mL) were added to deionized water (pH=7.0; the pH varied with the nanozyme volume, maintaining a total solution volume of 500 μL) and mixed well. A blank group was set up. After reacting at 10℃ for 10 min, the absorbance of the solution was detected at 546 nm using an ELISA reader. A line graph was plotted based on the experimental data, and the absorbance difference between the experimental group (A) and the blank group (A0) (ΔA=A-A0) was calculated. The largest difference was the optimal nanozyme concentration.

[0051] Optimization of H2O2 concentration: In the two colorimetric sensing systems of crystal violet and fuchsin, H2O2 with different volume gradients of 5, 10, 15, 20 and 25 μL was set, and the above different volumes of H2O2 were used in the two colorimetric systems. (1) Crystal violet colorimetric sensing: A certain volume of deionized water (pH=7.0; the volume of buffer solution changed with the volume of added H2O2, keeping the total volume of the reaction system at 500 μL) was added to a 1.5 mL centrifuge tube, along with 5 μL of crystal violet (0.001 g / mL), 5 μL of L-Cys (0.1 M), 5 μL of Co-MOF nanozyme (45 μg / mL) and different volumes of H2O2 (stock solution), and a blank group was set up. After mixing evenly, the mixture was reacted at 10℃ for 10 min. (2) Fuchsin colorimetric sensing: Add a certain volume of buffer solution (the volume of buffer solution varies with the volume of added H2O2, keeping the total volume of the reaction system at 500 μL), 10 μL of fuchsin (0.001 g / mL), 5 μL of L-Cys (0.1 M) and Co-MOF nanozyme (45 μg / mL) to a 1.5 mL centrifuge tube, add different volumes of H2O2 (stock solution) respectively, set up a blank group, mix well, and react at 10℃ for 10 min. Use an ELISA reader to detect the absorbance of the solution at the specified wavelength (crystal violet: 590 nm, fuchsin: 546 nm), draw a line graph based on the experimental data, and calculate the absorbance difference (ΔA=A-A0) between the experimental group (A) and the blank group (A0). The largest difference is the optimal H2O2 concentration.

[0052] Optimization of crystal violet or magenta colorimetric concentration: (1) Crystal violet colorimetric sensing: Five different volume gradients of crystal violet solution were set up: 2, 5, 7, 10 and 15 μL. A certain volume of deionized water (pH=7.0; the volume changed with the volume of crystal violet solution, keeping the total reaction volume at 500 μL), 5 μL of Co-MOF nanozyme (45 μg / mL), 5 μL of L-Cys (0.1 M) and 10 μL of H2O2 (stock solution) were added to a 1.5 mL centrifuge tube. Different volumes of crystal violet solution (0.001 g / mL) were added to each tube. A blank group was set up. After mixing, the mixture was reacted at 10℃ for 10 min.

[0053] (2) Fuchsin colorimetric sensing: Five different volume gradients of fuchsin solution were set up: 5, 10, 15, 20, and 25 μL. A certain volume of deionized water (pH=7.0; the volume varied with the volume of fuchsin solution, keeping the total reaction volume at 500 μL) was added to a 1.5 mL centrifuge tube. 1 μL of Co-MOF nanozyme (45 μg / mL), 5 μL of L-Cys (0.1 M), and 10 μL of H2O2 (stock solution) were added. Different volumes of fuchsin solution (0.001 g / mL) were added to each tube, and a blank group was set up. After mixing, the mixture was reacted at 10℃ for 10 min. The absorbance of the reaction solution at the specified wavelengths (crystal violet: 590 nm, fuchsin: 546 nm) was detected using an ELISA reader. A line graph was plotted based on the data, and the absorbance difference (ΔA=A-A0) between the experimental group (A) and the blank group (A0) was calculated. The largest difference was the optimal concentration of the chromogenic substrate.

[0054] Optimization of reaction time: Based on the optimization results above, the crystal violet and fuchsin colorimetric sensors were reacted at 10℃ within time ranges of 0–10 min and 0–12 min, respectively. The absorbance of the reaction solutions of the two colorimetric sensors at different reaction times was measured at specific wavelengths (crystal violet: 590 nm, fuchsin: 546 nm). Line graphs were plotted based on the data, and the absorbance difference (ΔA = A – A0) between the experimental group (A) and the blank group (A0) was calculated. The largest difference was considered the optimal reaction time.

[0055] Optimization of reaction temperature: Based on the optimization results, the optimal reaction temperature was selected. Five temperature gradients (15, 20, 25, 30, and 35 °C) were set using a metal bath to conduct experiments on the crystal violet and fuchsin colorimetric sensing systems at different temperatures. Using a microplate reader, the absorbance of the two colorimetric sensing reaction solutions was measured at specific wavelengths (crystal violet: 590 nm, fuchsin: 546 nm) at different reaction temperatures. A line graph was plotted based on the data, and the absorbance difference (ΔA = A - A0) between the experimental group (A) and the blank group (A0) was calculated. The maximum difference was identified as the optimal reaction temperature.

[0056] 1.2.6 Sensitivity Test In the optimized colorimetric sensors, 5 μL of L-Cys with a concentration range of 0–0.1 M was added to both crystal violet and magenta colorimetric sensors.

[0057] (1) Crystal violet colorimetric sensing: 5 μL of Co-MOF nanozyme (45 μg / mL), crystal violet (0.001 g / mL), L-Cys at different concentrations and 10 μL of H2O2 (stock solution) were added to 475 μL of deionized water, a blank group was set up, and the mixture was mixed and reacted at 15℃ for 10 min.

[0058] (2) Fuchs colorimetric sensing: 5 μL of L-Cys at different concentrations, 1 μL of Co-MOF nanozyme (45 μg / mL), 20 μL of fuchsin (0.001 g / mL), and 10 μL of H2O2 (stock solution) were added to 464 μL of deionized water (pH=7.0) to set up a blank group. The solutions were mixed and reacted at 15℃ for 12 min. The absorbance of the solution was measured using an ELISA reader within the specified wavelength range (crystal violet: 400~700 nm, fuchsin: 400~650 nm), and the spectral graph was plotted based on the experimental results. Similarly, based on optimized conditions, 5 μL of L-Cys at concentrations ranging from 0 to 0.1 M was added to both crystal violet and magenta colorimetric sensors. The detection results were analyzed and calculated to obtain the limits of detection (LOD) for L-Cys in both colorimetric sensor systems.

[0059] (1) Crystal violet colorimetric sensing: 5 μL Co-MOF nanozyme (45 μg / mL), crystal violet (0.001 g / mL), different concentrations of L-Cys and 10 μL H2O2 (stock solution) were added to 475 μL of deionized water (pH=7.0) to set up a blank group. The mixture was stirred and reacted at 15℃ for 10 min.

[0060] (2) Fuchs-based colorimetric sensing: 5 μL of L-Cys at different concentrations, 1 μL of Co-MOF nanozyme (45 μg / mL), 20 μL of fuchsin (0.001 g / mL), and 10 μL of H2O2 (stock solution) were added to 464 μL of deionized water (pH=7.0). A blank group was set up, and the solutions were mixed thoroughly and reacted at 15℃ for 12 min. The absorbance of the two colorimetric sensing reaction solutions was measured at specific wavelengths (crystal violet: 590 nm, fuchsin: 546 nm). A standard curve was plotted for different L-Cys concentrations versus absorbance, and the limit of detection (LOD) of the L-Cys detection method was calculated. LOD = 3σ / k (σ is the standard error of 20 blank absorbance samples) k (The slope of the standard curve).

[0061] 1.2.7 Specificity analysis Based on the optimization of conditions, the anti-interference ability of colorimetric sensors was evaluated by adding common interfering substances such as sugars, amino acids and ions to the colorimetric sensors. (1) Crystal violet colorimetric sensor: 5 μL of glucose, fructose, sucrose, glycine and calcium chloride at a concentration of 1 mol / L, 0.1 mol / L L-tyrosine and 0.5 mol / L L-valine as interfering substances were added to the colorimetric sensor, and the absorbance of the solution was detected at a wavelength of 590 nm using an enzyme-linked immunosorbent assay (ELISA) reader. (2) Fuchsin colorimetric sensor: 5 μL of maltose, fructose, sucrose and sodium chloride at a concentration of 1 mol / L, 0.1 mol / L L-leucine and L-phenylalanine and 0.5 mol / L L-proline as interfering substances were added to the colorimetric sensor, and the absorbance of the solution was detected at a wavelength of 546 nm using an ELISA reader.

[0062] Record the absorbance of the reaction solution for blank, L-Cys (0.1 mol / L), interfering substances, and both when crystal violet or magenta colorimetric sensors are present. Calculate the difference between the experimental group (A) and the blank group (A0) (ΔA=A-A0), plot a bar chart, and determine the magnitude of interference of the interfering substances on the two detection systems.

[0063] 1.2.8 Actual Sample Testing Based on condition optimization, the performance of crystal violet and magenta colorimetric sensors in detecting actual samples was evaluated by spiked recovery method. (1) Crystal violet colorimetric sensor: Pure milk (Yili) and mineral water (Wahaha) purchased from the school supermarket were used as the actual samples for crystal violet colorimetric sensor detection. Before detection, pure milk was diluted 50 times with deionized water; mineral water was used in its original state. The L-Cys content in the test sample was detected by the colorimetric sensor. (2) Magenta colorimetric sensor: Mixed fruit juice beverage (Nongfu Spring), functional beverage (Dongpeng Special Drink) and mineral water (Wahaha) purchased from the school were used as the actual samples for magenta colorimetric sensor detection. First, the mixed fruit juice beverage and functional beverage were diluted with deionized water to prepare test solutions of 12,000 times and 10,000 times respectively, while mineral water was used in its original state. The L-Cys content in the test solution was detected by the colorimetric sensor. The preparation of standard detection solutions and blank detection solutions should refer to section 1.2.6, Sensitivity Testing. Standard curves and regression equations should be plotted using Origin or other graphing software (the linear equation for the magenta colorimetric sensor system is y = 19.56845x + 0.20952 (R²)). 2 =0.992), the linear equation of the crystal violet colorimetric sensing system is y=39.14093x+0.11645 (R = 0.992). 2 =0.996); The detection process of L-Cys in the sample to be tested is the same as that of the experimental group.

[0064] 1.2.9 Data Analysis The data used in this paper are the average values ​​obtained from 3-4 sets of parallel experimental results. The difference between the control group and the experimental group was calculated based on the average value. Error bars were calculated using the STDEV function in Excel. Excel was used for initial data processing, and Origin was used to plot and export the processed data.

[0065] 2. Results Analysis and Discussion 2.1 Synthesis and Characterization of Co-MOF Hydrogen Peroxide Mimic Enzyme A hydrogen peroxide mimic enzyme—Co-MOF nanozyme—was synthesized using a coordination-driven self-assembly method, and its microstructure was observed using SEM. The results are as follows: Figure 1 -A and Figure 1 As shown in Figure B, at low magnification (2000x), Co-MOF nanozymes exhibit particles of varying sizes, without aggregation or uneven dispersion, with smooth surfaces and sharp edges. At high magnification (5000x), Co-MOF nanozymes display a more complete rhomboid dodecahedral geometry; similarly, their smooth surfaces, distinct edges, regular shapes, and relatively dispersed particle distribution are also visible.

[0066] 2.2 Activity of Co-MOF hydrogen peroxide mimic enzyme To understand the enzyme-like activities of Co-MOF nanozymes, this experiment was designed based on the catalytic characteristics of catalase (CAT), peroxidase (POD), and oxidase (OXD) activities. The experimental results are as follows: Experiment investigating catalase (CAT) activity: Experimental results are as follows Figure 2 As shown in Figure -A, when only H2O2 or Co-MOF nanozymes were present in the solution, no significant changes occurred. However, when H2O2 and Co-MOF nanozymes were placed in the same solution, a large number of bubbles were generated and adhered to the centrifuge tube wall.

[0067] Experiment investigating peroxidase (POD) activity: Experimental results are as follows Figure 2 As shown in -B, when only TMB is present in the solution, or when TMB and H2O2 coexist, or when TMB, H2O2 and Co-MOF nanozyme are present simultaneously, the solution does not show any obvious changes and there is no obvious absorption peak at 655 nm.

[0068] Experiment investigating the activity of oxidase (OXD): Experimental results are as follows Figure 2 As shown in -C, when only TMB or Co-MOF nanozymes are present in the solution, or when TMB and Co-MOF nanozymes coexist, the solution does not show any obvious changes, and there is no obvious absorption peak at 655 nm.

[0069] In summary, Co-MOF nanozymes did not exhibit catalytic activity in either the "H2O2-TMB" or "TMB" systems, indicating that Co-MOF nanozymes lack POD or OXD activity. In the CAT activity investigation experiment, when H2O2 and Co-MOF nanozymes were present simultaneously, a large number of bubbles were generated in the reaction solution, indicating that Co-MOF nanozymes catalyzed the production of O2 from H2O2. Therefore, this experiment fully demonstrates that Co-MOF nanozymes possess good catalase (CAT) activity. This conclusion is consistent with literature reports.

[38] 5 .

[0070] 2.3 Feasibility of L-cysteine ​​detection method In the activity investigation experiment, it was confirmed that the Co-MOF nanozyme possesses catalase (CAT) activity, capable of catalyzing the production of H2O and O2 from H2O2. This reaction can degrade crystal violet or fuchsin. Based on the CAT activity of Co-MOF nanozyme in degrading crystal violet or fuchsin and the inhibitory effect of L-Cys on degradation, this paper establishes a colorimetric detection method for L-Cys. When L-Cys is present in the colorimetric sensing system, the degradation rate of crystal violet or fuchsin decreases, and the degradation rate of the dye is related to the concentration of L-Cys. The higher the concentration of L-Cys, the slower the degradation rate and the deeper the color of the reaction solution. Based on the above principle, two colorimetric detection methods for L-Cys were established using crystal violet and fuchsin reaction systems, respectively. The specific experimental results are as follows: For the crystal violet colorimetric sensing system: the experimental results of the control group are as follows Figure 3 As shown in Figure A, the reaction solutions in centrifuge tubes 1, 2, and 4 showed no color change, and no obvious absorption peak at 590 nm. The reaction solutions in centrifuge tubes 3 and 5 were deep blue due to the addition of crystal violet, and showed obvious absorption peaks at 590 nm; the two absorbance curves almost overlapped. The experimental results for the experimental groups are as follows: Figure 3 As shown in Figure B, the crystal violet in centrifuge tube 1 (blank) was degraded, and the solution became almost colorless, with no obvious absorption peak at 590 nm. However, in the crystal violet colorimetric sensor, different concentrations of L-Cys (2: 0.09 M, 3: 0.05 M, 4: 0.025 M) were added, and the reaction solution showed obvious absorption at 590 nm to varying degrees. The higher the L-Cys concentration, the greater the absorbance at 590 nm.

[0071] For the magenta colorimetric sensor system: the experimental results of the control group are as follows Figure 3As shown in Figure C, the reaction solutions in centrifuge tubes 1, 2, and 4 showed no color change, and no obvious absorption peak at 546 nm. The reaction solutions in centrifuge tubes 3 and 5 were red due to the addition of fuchsin, and showed a clear absorption peak at 546 nm; the two absorbance curves were also very close. The experimental results for the experimental groups are as follows: Figure 3 As shown in Figure D, the fuchsin in centrifuge tube 1 (blank) degraded, resulting in a solution that was almost colorless, with a small absorption peak at 546 nm. However, in the fuchsin colorimetric sensor, the addition of different concentrations of L-Cys (2: 0.09 M, 3: 0.05 M, 4: 0.025 M) resulted in significant and varying degrees of absorption at 546 nm. The higher the L-Cys concentration, the greater the absorbance at 546 nm.

[0072] 2.4 Optimization of reaction conditions 2.4.1 Buffer Type The buffer solution, as the primary carrier in the entire reaction system, provides a stable environment for the reaction to occur. Therefore, it is necessary to optimize the type of buffer solution to ensure the stable progress of the reaction. Three commonly used buffer solutions were selected for this experiment: deionized water (pH=7.0), sodium dihydrogen phosphate (25 mM, pH=7.0), and sodium acetate (25 mM, pH=7.0). The results are as follows: Figure 4 As shown, in the two colorimetric sensing systems of crystal violet and magenta, the absorbance difference (ΔA) between the experimental group (A) and the blank group (A0) reaction solutions of different types of buffer solutions was compared to obtain ΔA. 去离子水 >ΔA 乙酸钠 >ΔA 磷酸二氢钠 Therefore, based on the experimental results, deionized water performed better than sodium acetate and sodium dihydrogen phosphate solutions. Thus, deionized water was chosen as the buffer solution for both crystal violet and fuchsin colorimetric sensing systems. This conclusion will be used in subsequent experiments.

[0073] 2.4.2 pH of the buffer solution A suitable buffer pH not only facilitates the catalytic reaction of nanozymes but also accelerates the rate of reactions between other substances. Therefore, the pH of the buffer solution in both colorimetric sensing systems was optimized, specifically the pH of the deionized water.

[0074] The optimization results of the pH of deionized water in the crystal violet colorimetric sensing system are as follows: Figure 5 As shown in Figure A, at a wavelength of 590 nm, when the pH of deionized water is less than 7.0, the absorbance difference (ΔA) between the blank group and the experimental group increases slowly with increasing pH; the difference reaches its maximum when the pH of deionized water is 7.0; when the pH is greater than 7.0, the difference decreases rapidly and tends to level off after 8.0.

[0075] In the magenta colorimetric sensing system, the pH optimization results for deionized water are as follows: Figure 5 As shown in Figure -B, at a wavelength of 546 nm, the absorbance difference (ΔA) between the experimental group and the control group showed only a slight increase and was not significant when the pH of deionized water was between 4.0 and 6.0. However, when pH > 6.0 and pH < 7.0, the difference increased rapidly; it reached its maximum at pH = 7.0; after pH > 7.0, the difference decreased rapidly; and within the pH range of 8.0 to 10.0, the difference gradually decreased.

[0076] In summary, in both the crystal violet and fuchsin colorimetric sensing systems, the absorbance difference (ΔA) between the experimental and control groups was largest when deionized water was at pH 7.0. Therefore, deionized water at pH 7.0 was chosen as the buffer solution in both the crystal violet and fuchsin colorimetric sensing reaction systems. This conclusion will be used in subsequent experiments.

[0077] 2.4.3 Buffer Ion Concentration To maximize the catalase activity of the Co-MOF nanozyme and ensure its catalytic activity, the salt ion concentration in the deionized water was optimized. The experimental results are as follows: Crystal Violet Colorimetric Sensor: Experimental results are as follows Figure 6 As shown in Figure A, at a wavelength of 590 nm, the absorbance difference (ΔA) between the experimental group and the control group is the largest when the salt ion concentration in the deionized water is 0. When the salt ion concentration is in the range of 0~100 mM, the absorbance difference (ΔA) decreases rapidly with the increase of salt ion concentration. When the salt ion concentration is greater than 100 mM, the absorbance difference gradually tends to level off.

[0078] Magenta colorimetric sensor: Experimental results are as follows Figure 6 As shown in Figure B, at a wavelength of 546 nm, the absorbance difference (ΔA) between the experimental group and the blank group was the largest when the salt ion concentration in the deionized water was 0; as the salt ion concentration in the buffer solution increased, the absorbance difference (ΔA) gradually decreased. Therefore, in the crystal violet and fuchsin colorimetric sensing system, the optimal buffer ion concentration is achieved when no salt ions are added to the deionized water. This conclusion was used in subsequent experiments.

[0079] 2.4.4 Hydrogen peroxide mimic enzyme concentration The concentration of Co-MOF nanozymes in the reaction system directly affects the catalytic reaction rate and the efficiency of the L-Cys detection method. To maximize the overall reaction rate and shorten the detection time, the concentration of Co-MOF nanozymes was optimized. The specific optimization results are as follows: Crystal Violet Colorimetric Sensor: Experimental results are as follows Figure 7As shown in Figure A, at a wavelength of 590 nm, when the volume of the Co-MOF nanozyme in the reaction system was 1–4 μL, the absorbance difference of the reaction solution increased rapidly; when the volume of the nanozyme was 4–5 μL, the increase in absorbance difference gradually slowed down; when the volume of the nanozyme was 5 μL, the absorbance difference between the experimental group and the blank group reached its maximum; when the volume of the nanozyme was 5–6 μL, the absorbance difference showed a decreasing trend.

[0080] Magenta colorimetric sensor: Experimental results are as follows Figure 7 As shown in Figure -B, at a wavelength of 546 nm, when the volume of Co-MOF nanozyme in the reaction system was between 0.5 and 2 μL, the absorbance difference between the experimental group and the blank group first increased and then decreased, reaching its maximum when the nanozyme volume was 1 μL. When the concentration of nanozyme in the system was greater than 2 μL, the absorbance difference of the reaction solution gradually decreased and tended to level off.

[0081] In summary, the optimal volume of Co-MOF nanozyme is 5 μL in crystal violet colorimetric sensing, and 1 μL in the fuchsin colorimetric system. This conclusion will be used in subsequent experiments.

[0082] 2.4.5 Substrate concentration The selection of the optimal substrate concentration involves optimizing the concentration of H2O2 and the concentration of dyes (crystal violet and magenta).

[0083] (1) Optimization of H2O2 concentration Crystal Violet Colorimetric Sensor: Experimental results are as follows Figure 8 As shown in Figure A, at a wavelength of 590 nm, when the volume of H2O2 in the reaction solution is in the range of 5 to 10 μL, the absorbance difference (ΔA) between the blank group and the experimental group increases slowly with the increase of the volume of H2O2; when the volume of H2O2 is 10 μL, the difference reaches its maximum; when the volume of H2O2 is in the range of 10 to 25 μL, the absorbance difference decreases slowly with the increase of the volume of H2O2 and tends to level off.

[0084] Magenta colorimetric sensor: Experimental results are as follows Figure 8 As shown in Figure B, at a wavelength of 546 nm, when the volume of H2O2 in the reaction solution is in the range of 5–10 μL, the absorbance difference (ΔA) between the blank group and the experimental group increases rapidly with the increase of the volume of H2O2. When the volume of H2O2 in the reaction solution is 10 μL, the absorbance difference (ΔA) reaches its maximum. When the volume of H2O2 is in the range of 10–25 μL, the absorbance difference gradually decreases with the increase of the volume of H2O2.

[0085] (2) Optimization of crystal violet dye concentration The results are as follows Figure 9As shown in Figure -A, at a wavelength of 590 nm, the absorbance difference (ΔA) between the experimental and blank groups in the crystal violet colorimetric sensing system did not show a significant difference with increasing crystal violet volume. When the volume of crystal violet in the reaction solution was 5 μL and 7 μL, the absorbance difference was similar, and the difference (ΔA) was more pronounced compared to other volumes of crystal violet. However, considering the convenience of experimental operation and the actual experimental phenomena, 5 μL of crystal violet dye was ultimately selected as the optimal substrate volume for this colorimetric sensing system. This conclusion was used in subsequent experiments.

[0086] (3) Optimization of the volume of magenta dye, the results are as follows: Figure 9 As shown in Figure -B, at a wavelength of 546 nm, when the volume of fuchsin in the fuchsin colorimetric sensing system is in the range of 5–10 μL, the absorbance difference (ΔA) between the experimental group and the blank group is not significantly different; as the volume of fuchsin exceeds 10 μL, the absorbance difference (ΔA) increases rapidly. When the volume of fuchsin in the reaction system is 20 μL, the absorbance difference reaches its maximum; when the volume of fuchsin exceeds 20 μL, the absorbance difference (ΔA) decreases.

[0087] In summary, the optimal H₂O₂ volume is 10 μL for both crystal violet and fuchsin colorimetric sensing reaction systems. The optimal substrate concentration is 5 μL for crystal violet colorimetric sensing and 20 μL for fuchsin colorimetric sensing. This conclusion will be applied to subsequent experiments.

[0088] 2.4.6 Reaction Temperature By exploring the optimal temperature of the reaction system, the most suitable working environment for the L-Cys detection method was found, and the reaction rate was accelerated, saving detection time. Experimental results are as follows: Figure 10 -A and Figure 10 As shown in Figure -B, at specific wavelengths (crystal violet: 590 nm, magenta: 546 nm), the absorbance difference between the two colorimetric sensing systems, crystal violet and magenta, exhibits roughly the same trend with temperature changes; that is, as the reaction temperature increases, the overall reaction slows down, and the absorbance difference (ΔA) decreases. In conclusion, the optimal reaction temperature for both crystal violet and magenta colorimetric sensing systems is 15 °C. This conclusion will be used in subsequent experiments.

[0089] 2.4.7 Reaction Time An appropriate reaction time facilitates the observation of detection results and improves detection accuracy; therefore, the reaction time was optimized. The experimental results are as follows: Crystal Violet Colorimetric Sensor: Experimental results are as follows Figure 11As shown in Figure -A, the reaction solution reacted at a wavelength of 590 nm within a time range of 0 to 10 min. The absorbance difference (ΔA) between the experimental group and the blank group gradually increased with the extension of time.

[0090] Magenta colorimetric sensor: Experimental results are as follows Figure 11 As shown in Figure B, the reaction solution reacted within a time range of 0 to 12 minutes at a wavelength of 546 nm. As the reaction time increased, the absorbance difference (ΔA) between the experimental group and the blank group also increased.

[0091] In summary, the absorbance difference (ΔA) between the crystal violet and magenta colorimetric sensing systems gradually increases with increasing reaction time. Based on the experimental results and actual performance, the optimal reaction time for the crystal violet colorimetric sensing system was determined to be 10 min, and for the magenta colorimetric sensing system, 12 min.

[0092] 2.5 Sensitivity Detection To improve the sensitivity and accuracy of the rapid L-Cys detection method, L-Cys at concentrations ranging from 0 to 0.1 M was added to two colorimetric sensing systems (crystal violet and fuchsin) after condition optimization. The absorbance of the reaction solutions in both systems was measured using a microplate reader. Absorption spectra of different concentrations of L-Cys with respect to absorbance were obtained in the wavelength ranges of 400–700 nm and 400–650 nm. Furthermore, nonlinear and linear relationships between different concentrations of L-Cys and absorbance were plotted at characteristic wavelengths (crystal violet: 590 nm, fuchsin: 546 nm). Specific experimental results are as follows: Experimental results are as follows Figure 12 -A and Figure 12 As shown in Figure -C, with the continuous increase of L-Cys concentration, the absorbance of the reaction solutions of the two colorimetric sensing systems at wavelengths of 590 nm (crystal violet) and 546 nm (magenta) gradually increases, and the higher the L-Cys concentration, the greater the absorbance.

[0093] When the L-Cys concentration in the crystal violet colorimetric sensing system reaches 0.1 M and the L-Cys concentration in the magenta colorimetric sensing system reaches 0.06 M, the absorbance gradually becomes more stable, such as... Figure 12 -B and Figure 12 -D is shown. Furthermore, in both colorimetric sensors, when the L-Cys concentration is in the range of 0~0.005 M, there is a good linear relationship between the L-Cys concentration and the absorbance.

[0094] In summary, the sensitivity detection results are as follows: ① The linear response range of the crystal violet colorimetric sensing system is 0~0.005 mol / L, and the linear equation is y=39.14093x+0.11645 (R0). 2 =0.996). Using the limit of detection formula (LOD=3σ / k), the limit of detection for L-Cys in this colorimetric sensing system is 1.5 mmol / L. ② The linear response range of the magenta colorimetric sensing system is 0~0.005 mol / L, and the linear equation is y=19.56845x+0.20952(R² / k). 2 =0.992), the limit of detection for L-Cys by this colorimetric sensing system is 4.5 mmol / L.

[0095] 2.6 Specificity Analysis To evaluate the anti-interference capability of the L-Cys rapid detection method and explore its specificity, the specific experimental results are as follows: Add 5 μL of glucose, fructose, sucrose, glycine, and Ca to the crystal violet colorimetric sensing system at a concentration of 1 mol / L. 2+ 0.1 mol / L L-tyrosine and 0.5 mol / L L-valine were used as interfering substances. The absorbance of the solution was measured at 590 nm using an ELISA reader. The results are as follows: Figure 13 As shown in Figure -A. By comparing the difference (ΔA) between the absorbance of each reaction solution and the absorbance of the blank, it can be seen that the above-mentioned interfering substances have little impact on the crystal violet colorimetric system.

[0096] Add 5 μL of 1 mol / L maltose, fructose, sucrose, and Na to the magenta color development system. + 0.1 mol / L L-leucine, L-phenylalanine, and 0.5 mol / L L-proline were used as interfering substances, and the absorbance of the solution was measured at 546 nm using an ELISA reader. The results are as follows: Figure 13 As shown in -B, by comparing the magnitude of the absorbance difference (ΔA), it can also be seen that the above-mentioned interfering substances have little impact on the magenta colorimetric sensing system.

[0097] Experiments have shown that the crystal violet and magenta colorimetric sensing systems have good selectivity and anti-interference ability for the detection of L-Cys.

[0098] 2.7 Detection of L-cysteine ​​in actual samples To investigate the application potential of the rapid L-Cys detection method based on hydrogen peroxide mimicry enzyme in practical sample detection, the method was applied in a real-world test. The specific procedure is detailed in section 2.5, Sensitivity Detection. The experimental results are as follows: Crystal violet colorimetric sensing: Pure milk and mineral water were used as actual samples for the crystal violet colorimetric sensing system. The detection results are shown in Table 3. The recovery rate of L-Cys in the actual samples ranged from 98.1% to 102.1%, and the relative standard deviation (RSD) ranged from 2.7% to 4.9%.

[0099] Magenta colorimetric sensing: Mixed fruit juice beverages, functional beverages, and mineral water were used as actual samples for the magenta colorimetric sensing system. The detection results are shown in Table 4. The recovery rate of L-Cys in the actual samples was 98.4%~100.2%, and the relative standard deviation (RSD) was 2.3%~4.5%.

[0100] In summary, crystal violet and magenta colorimetric sensors have good practical application potential for the detection of L-Cys.

[0101] Table 3. Actual sample test results (crystal violet colorimetric sensor)

[0102] Table 4. Actual sample test results (magenta colorimetric sensor)

[0103] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for colorimetric detection of L-Cys using Co-MOF nanozymes, characterized in that: Includes the following steps: S1. Preparation of standard detection solution: The buffer solution, Co-MOF nanozyme, H2O2, triphenylmethane dye, and L-Cys standard solutions of different concentrations are thoroughly mixed and reacted to obtain the standard detection solution; the Co-MOF nanozyme has catalase-mimicking activity but does not have peroxidase or oxidase activity; the triphenylmethane dye is fuchsin or crystal violet; S2. Preparation of blank detection solution: Using deionized water instead of the L-Cys standard solution in step S1, a blank detection solution is prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and L-Cys concentration, construct the standard curve, and obtain the regression equation; S4. Detection of L-Cys in the test sample: Using an equal volume of the test sample solution to replace the L-Cys standard solution in step S1, the test sample detection solution system is prepared according to step S1 under the same conditions; the absorbance of the test sample detection solution is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of L-Cys in the test sample.

2. The method for colorimetric detection of L-Cys using Co-MOF nanozymes according to claim 1, characterized in that: The pH value of the buffer solution is 6.5 to 7.

5.

3. The method for colorimetric detection of L-Cys using Co-MOF nanozymes according to claim 2, characterized in that: The ion concentration of the buffer solution is 0 M to 0.2 M.

4. The method for colorimetric detection of L-Cys using Co-MOF nanozymes according to claim 3, characterized in that: The buffer solution is water.

5. The method for colorimetric detection of L-Cys using Co-MOF nanozymes according to claim 4, characterized in that: The final concentration of H2O2 in the test solution system is 0.5% to 0.7% (v / v).

6. The method for colorimetric detection of L-Cys using Co-MOF nanozymes according to claim 5, characterized in that: The reaction temperature is 10℃~20℃.

7. A method for colorimetric detection of L-Cys using Co-MOF nanozymes according to any one of claims 1 to 6, characterized in that: When the triphenylmethane dye is crystal violet, the final concentrations of Co-MOF nanozyme and crystal violet in the test sample detection solution system are 0.4–0.5 μg / mL and 0.008–0.012 mg / mL, respectively, with a reaction time of 10–15 min and a detection wavelength of 590 nm; when the triphenylmethane dye is fuchsin, the final concentrations of Co-MOF nanozyme and fuchsin in the test sample detection solution system are 0.08–0.10 μg / mL and 0.03–0.05 mg / mL, respectively, with a reaction time of 8–15 min and a detection wavelength of 546 nm.

8. The application of the method according to claim 7 in the detection of L-Cys content.

9. The application according to claim 8, characterized in that: The detection of L-Cys content refers to the detection of L-Cys content in food, pharmaceutical, or biological samples.

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