Method for detecting vitamin B1 and glyphosate by nano enzyme base
By preparing Cu2O/Mn@TCPP nanosheets, the activity of vitamin B1 was enhanced to improve its peroxidase-like activity and glyphosate inhibitory effect, thus solving the problems of low sensitivity and susceptibility to interference in the detection of vitamin B1 and glyphosate, and achieving rapid detection with high sensitivity and specificity.
Patent Information
- Application Number
- CN202311726520.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-04-05
- Filing Date
- 2023-12-15
- Publication Date
- 2026-01-16
AI Technical Summary
In the existing technologies, the detection methods for vitamin B1 and glyphosate have the disadvantages of low sensitivity, susceptibility to interference, and inability to achieve rapid on-site detection. Instrumental analysis methods are costly and complex, while nanozymes have low activity and are susceptible to interference.
Cu2O/Mn@TCPP nanosheets were prepared using Cu2O cubes as templates and manganese ions as donors via a hydrothermal method under UV irradiation. Cu2O/Mn@TCPP exhibited peroxidase-like activity under neutral conditions. The affinity for the substrate was enhanced by the addition of vitamin B1, generating hydroxyl radicals. A detection method was established, and glyphosate inhibited the enzyme-catalyzed oxidation reaction.
It achieves highly sensitive detection of vitamin B1 and glyphosate under neutral conditions, with detection limits of 0.20 mg/kg and 0.025 mg/kg, respectively. It features simple operation, high specificity, and no interference from other substances.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of chemical analysis detection, in particular to a method for detecting vitamin B1 and glyphosate based on nano-enzyme. BACKGROUND
[0002] Vitamin B1, also known as thiamine or antineuritic factor, is a B vitamin formed by the combination of a pyrimidine ring and a thiazole ring. Although its content in the body is very small, it plays an important role in the growth, metabolism and development of the human body, and the determination of vitamin B1 in food is of great significance. At present, the methods for determining vitamin B1 mainly include high performance liquid chromatography, fluorescence method, chemiluminescence method and spectrophotometry. The spectrophotometry is simple to operate, and the instrument used is cheap and easy to popularize. However, the spectrophotometry is rarely reported for the detection of vitamin B1 due to its serious interference. Glyphosate, chemical name N-(phosphonomethyl) glycine, is a white solid with strong polarity, which is an organic acid and a water-soluble herbicide insoluble in organic solvents. Glyphosate has strong systemic conductance and broad-spectrum killing effect, and is widely used in many countries in the world, which pollutes water resources, damages plants and threatens human and animal health. Therefore, the detection of glyphosate is of great significance. Glyphosate lacks chromophoric and fluorescent groups, and has strong binding ability with organic matter in plants, making it difficult to analyze directly. The detection methods of glyphosate include ion chromatography, gas chromatography, high performance liquid chromatography and high performance liquid chromatography-tandem mass spectrometry.
[0003] Instrument analysis detection method has great advantages in detection sensitivity and accuracy, but due to the limitations of expensive equipment requirements, complex sample pretreatment process and long detection period, instrument analysis detection method cannot be used for on-site and rapid detection of vitamin B1 and glyphosate. Nano-enzyme refers to a nano material with natural enzyme catalytic activity, which has low cost, easy storage and adjustable enzyme activity, and is widely used in the field of detection. However, compared with natural enzymes, nano-enzymes also have low enzyme activity, are easily disturbed during detection and have low detection sensitivity. SUMMARY
[0004] In view of the deficiencies of the prior art, the present application provides a kind of nanometer enzyme-based detection method of vitamin B1 and glyphosate, which uses Cu2O cube as template and manganese ion as donor to prepare Cu2O / Mn under ultraviolet irradiation, and hydrothermal method is used to prepare two-dimensional Mn-tetra (4-carboxyphenyl) porphyrin (TCPP) nanosheet (Cu2O / Mn@TCPP).Cu2O / Mn@TCPP shows very weak pseudo-peroxidase activity under neutral conditions, while the addition of vitamin B1 (VB1) significantly enhances the pseudo-peroxidase activity, thanks to the fact that VB1 enhances the affinity of Cu2O / Mn@TCPP and substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating more hydroxyl radicals (·OH).In the presence of hydrogen peroxide, colorless TMB is oxidized to produce a blue solution, and the presence of glyphosate inhibits the catalytic oxidation reaction of the enzyme, thanks to the interaction between glyphosate and vitamin B1, which inhibits the affinity of VB1 and TMB.The pseudo-peroxidase activity of Cu2O / Mn@TCPP increases linearly with the increase of VB1 concentration, while the absorbance decreases linearly with the increase of glyphosate concentration, thus establishing a new method for detecting VB1 and glyphosate under neutral conditions in nanometer enzyme, with detection limits of 0.20 mg / kg and 0.025 mg / kg respectively, and other vitamins and pesticides do not interfere with this reaction, so the detection method has specificity.The method has the characteristics of simple operation, high sensitivity and rapidness.
[0005] The nanometer enzyme-based detection method of vitamin B1 and glyphosate according to the present application comprises the following steps:
[0006] (1) Add Cu2O / Mn@TCPP nanometer enzyme to the vitamin B1 (VB1) standard solution, incubate for 5 min, add 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, and dilute with pH 7.2 glycine-NaOH buffer solution to prepare a VB1 solution with a concentration range of 0.8-80 mg / L, shake well, stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, establish the quantitative relationship between absorbance and VB1 concentration, draw the standard curve, and obtain the regression equation;
[0007] (2) Add Cu2O / Mn@TCPP nanometer enzyme, 3,3',5,5'-tetramethylbenzidine (TMB), H2O2 and VB1 to the glyphosate standard solution, and dilute with pH 7.2 glycine-NaOH buffer solution to prepare a glyphosate solution with a concentration range of 0.05-60 mg / L, shake well, stand for 5-10 min, centrifuge, take the supernatant and measure the absorbance at 654 nm wavelength, establish the quantitative relationship between absorbance and glyphosate concentration, draw the standard curve, and obtain the regression equation;
[0008] (3) Extract and purify glyphosate in the sample to obtain a sample determination solution, add Cu2O / Mn@TCPP nanoscale enzyme, TMB and H2O2 to the sample determination solution, and then add a pH 7.2 glycine-NaOH buffer solution to make up the volume, shake well, stand for 5-10 min, centrifuge, take the supernatant, and then measure the absorbance at a wavelength of 654 nm, and then put the absorbance into the regression equation in step (1) to obtain the content of VB1 in the sample.
[0009] (4) Extract and purify VB1 in the sample to obtain a sample determination solution, add Cu2O / Mn@TCPP nanoscale enzyme, TMB, H2O2 and VB1 to the sample determination solution, and then add a pH 7.2 glycine-NaOH buffer solution to make up the volume, shake well, stand for 5-10 min, centrifuge, take the supernatant, and then measure the absorbance at a wavelength of 654 nm, and then put the absorbance into the regression equation in step (2) to obtain the content of glyphosate in the sample.
[0010] The Cu2O / Mn@TCPP nanoscale enzyme is prepared as follows:
[0011] a. Preparation of Cu2O nanoparticles: 10-15 mL of NaOH (2 mol / L), 100-120 mL of CuCl2 (0.01 mol / L) and 4-5 g of polyvinylpyrrolidone (PVP) are stirred and uniformly mixed for 30-40 min, then 0.5-0.7 mol / L ascorbic acid (AA) is added, and stirring is carried out in a 55°C water bath for 5-6 h. After washing with deionized water, vacuum drying is performed to obtain the product.
[0012] b. Preparation of Cu2O / Mn nanoparticles: 30-40 mg of Cu2O prepared in step (a) is added to a mixture of 10-15 mL of deionized water and 10-15 mL of ethanol, and then 10-15 mL of MnCl2 (0.1 mol / L) solution is added. Ultrasonic treatment is performed for 20-30 min, and then stirring is carried out under ultraviolet light for 1-1.5 h to obtain the product.
[0013] c. Preparation of Cu2O / Mn@TCPP nanoscale enzyme: 10-15 mL of tetra(4-carboxyphenyl)porphyrin (TCPP, 0.025 mmol / L in DMF) is added to the Cu2O / Mn solution prepared in step (b), and stirring is carried out in a 60°C water bath for 6-8 h. Centrifugation is performed, and then washing is carried out with ethanol and deionized water. Vacuum drying is performed to obtain the product.
[0014] The sample determination solution is processed according to a relevant standard method.
[0015] The concentration of Cu2O / Mn@TCPP nanoenzyme is 1 mg / mL, the added amount is 100-150 μL; the concentration of TMB is 50 mmol / L, the added amount is 50-100 μL; the concentration of H2O2 is 50 mmol / L, the amount used is 50-100 μL; the concentration of VB1 is 80 mg / L, the added amount is 50-100 μL.
[0016] Centrifugation is treated at 8000-10000 r / min for 10-15 min.
[0017] The advantages and technical effects of the present application are as follows:
[0018] 1. The Cu2O / Mn@TCPP synthesized in the present application has weak peroxidase-like activity under neutral conditions, and the addition of VB1 significantly enhances the peroxidase-like activity, thanks to the fact that VB1 enhances the affinity of Cu2O / Mn@TCPP and substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is verified by the Michaelis equation, and in the presence of hydrogen peroxide, more hydroxyl radicals (·OH) are generated, which is verified by the ·OH capture test, the blue solution is generated by the oxidation of colorless TMB by the nanoenzyme, and the catalytic oxidation reaction of the enzyme is inhibited in the presence of glyphosate, thanks to the interaction between glyphosate and vitamin B1, which inhibits the affinity of VB1 and TMB; the absorbance linearly increases with the increase of the concentration of VB1, and the absorbance linearly decreases with the increase of the concentration of glyphosate, thus a new method for detecting VB1 and glyphosate under neutral conditions of the nanoenzyme is established.
[0019] 2. The detection method for VB1 and glyphosate established in the present application has high detection sensitivity, and the detection limits of VB1 and glyphosate are 0.20 mg / kg and 0.01 mg / kg respectively, and the coexisting VB2, VB3, VB6, VB 12 and other pesticides do not interfere with the determination, and the method has good selectivity.
[0020] 3. The detection method for VB1 and glyphosate established in the present application can independently detect VB1 and glyphosate, and when glyphosate is detected, VB1 is used as a sensitizer of the enzyme detection system to improve the sensitivity and specificity of glyphosate detection. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 The TEM image of Cu2O synthesized in Example 1 (left image) and the TEM image of Cu2O / Mn@TCPP (right image).
[0022] Figure 2UV-Vis absorption spectra of Cu2O / Mn@TCPP oxidizing TMB+H2O2, TMB+H2O2+VB1 and TMB+H2O2+VB1+glyphosate (Gly) in Example 1.
[0023] Figure 3 Michaelis-Menten kinetic curves of Cu2O / Mn@TCPP oxidizing TMB in Example 1.
[0024] Figure 4 Michaelis-Menten kinetic curves of Cu2O / Mn@TCPP oxidizing TMB+VB1 in Example 1.
[0025] Figure 5 Hydroxyl radical (·OH) trapping diagrams of Cu2O / Mn@TCPP oxidizing TMB+H2O2 and TMB+H2O2+VB1 systems in Example 1.
[0026] Figure 6 Linear UV-Vis absorption spectra (left) and regression equation (right) of Cu2O / Mn@TCPP+TMB+H2O2 system detecting VB1 in Example 1.
[0027] Figure 7 Linear UV-Vis absorption spectra (left) and regression equation (right) of Cu2O / Mn@TCPP+TMB+H2O2+VB1 system detecting Gly in Example 1.
[0028] Figure 8 Results of the influence of coexisting substances on VB1 in Example 1.
[0029] Figure 9 Results of the influence of coexisting substances on pesticides in Example 1. DETAILED DESCRIPTION
[0030] The technical solutions of the present application will be further described in detail below in combination with specific examples, but the protection scope of the present application is not limited to this.
[0031] Example 1: Determination of VB1 in milk powder samples and Gly in hanging ear coffee samples
[0032] 1. Preparation of Cu2O nanoparticles: 10 mL of NaOH (2 mol / L), 100 mL of CuCl2 (0.01 mol / L) and 4 g of polyvinylpyrrolidone (PVP) were stirred and uniformly mixed for 30 min, then 0.6 mol / L ascorbic acid (AA) was added, and the mixture was stirred in a 55°C water bath for 5 h. After washing with deionized water, vacuum drying was performed to obtain the Cu2O nanoparticles. Figure 1 TEM image of Cu2O prepared, from which it can be seen that the prepared Cu2O nanoparticles have a cubic structure and are uniformly dispersed.
[0033] 2. Cu2O / Mn nanoparticle preparation: Take 35 mg of Cu2O prepared in step 1, add 15 mL of deionized water and 15 mL of ethanol mixture, then add 15 mL of MnCl2(0.1 mol / L) solution, ultrasonic treatment for 30 min, then irradiate under ultraviolet lamp for 1 h with stirring, and obtain.
[0034] 3. Cu2O / Mn@TCPP nanoscale enzyme preparation: Add 10 mL of tetrakis(4-carboxyphenyl) porphyrin (TCPP, 0.025 mmol / L DMF solution) to the Cu2O / Mn solution prepared in step 2, stir in a 60°C water bath for 6-8 h, centrifuge, wash with ethanol and deionized water alternately, and vacuum dry to obtain. Figure 2 To prepare the TEM image of Cu2O / Mn@TCPP, it can be seen from the figure that the prepared Cu2O / Mn@TCPP retains the structure of Cu2O, and clear crystal lattice fringes are observed, with a fringe spacing of 0.34 nm corresponding to the (111) crystal plane CeO2.
[0035] 4. Cu2O / Mn@TCPP nanoscale enzyme peroxidase activity evaluation: Take 100 μL of TMB with a concentration of 50 mmol / L, add 100 μL of Cu2O / Mn@TCPP with a concentration of 1 mg / mL and 100 μL of H2O2 with a concentration of 50 mmol / L, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, and measure the absorbance at 655 nm with a UV-visible spectrophotometer; At the same time, incubate 100 μL of Cu2O / Mn@TCPP with a concentration of 1 mg / mL and 100 μL of VB1 aqueous solution with a concentration of 1 mg / mL for 5 min, then add 100 μL of TMB with a concentration of 50 mmol / L and 100 μL of H2O2 with a concentration of 50 mmol / L, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, and measure the absorbance at 654 nm with a UV-visible spectrophotometer; Incubate 100 μL of Cu2O / Mn@TCPP with a concentration of 1 mg / mL and 100 μL of VB1 aqueous solution with a concentration of 1 mg / mL for 5 min, then add 100 μL of 5 mg / mL Gly, 100 μL of TMB with a concentration of 50 mmol / L and 100 μL of H2O2 with a concentration of 50 mmol / L, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, and measure the absorbance at 654 nm with a UV-visible spectrophotometer. The results are as follows Figure 2 , in Figure 2In this study, Cu2O / Mn@TCPP oxidized TMB showed weak peroxidase activity under neutral conditions. When VB1 was added, the absorbance of the system was significantly improved. However, when Gly was added, the absorbance of the system was inhibited, and the absorbance decreased. Meanwhile, the Michaelis-Menten catalytic kinetic parameters were determined (Table 1). Figure 3 , Figure 4 The Michaelis constant K m before and after the addition of VB1 was 1.242 mM -1 and 0.924 mM -1 , respectively, and the reaction rate constant was 1.47 x 10 -8 M s -1 and 2.39 x 10 -8 M s -1 , respectively, indicating that the addition of VB1 greatly enhanced the affinity and reaction rate of Cu2O / Mn@TCPP nanoszyme and substrate.
[0036] Table 1 Michaelis-Menten catalytic kinetic parameters
[0037] Enzymes Substrate K m (mM -1 )]]> V max (10 -8 M·s -1 )]]> Cu-Mn-TCPP TMB 1.242 1.47 [Cu-Mn-TCPP + VB1] TMB 0.924 2.39
[0038] 5. Hydroxyl radical (·OH) capture test: methylene blue (MB) was used as a ·OH capture agent, and the change in absorbance when mixed with nanoszyme and H2O2 was detected. Specifically, 200 μL of 1 mg / mL MB solution was mixed with 50 mmol / L H2O2 100 μL and 100 μL of 1 mg / mL Cu2O / Mn@TCPP in a pH 7.2 glycine-NaOH buffer solution, and incubated in the dark at room temperature for 1 h. The absorbance was measured at 664 nm, and the absorbance was negatively correlated with the production of ·OH. At the same time, the fluorescence intensity was determined by adding VB1 under the same experimental conditions, and the results are shown in Figure 5 . The results showed that the absorbance of the system decreased after the addition of VB1, indicating that more ·OH was produced. However, the production of ·OH was inhibited after the addition of Gly.
[0039] 6. VB1 working curve preparation: 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanoszyme and VB1 standard solution with a concentration of 0.8-80 mg / L were added to a 5 mL stoppered cuvette, incubated for 5 min, and then 100 μL of TMB with a concentration of 50 mmol / L and 100 μL of H2O2 with a concentration of 50 mmol / L were added. The pH 7.2 glycine-NaOH buffer solution was added to 4 mL, and the reaction was allowed to proceed for 10 min. The absorbance was measured at 655 nm using a UV-visible spectrophotometer, the VB1 concentration was taken as the abscissa, and the absorbance A was taken as the ordinate. The standard curve was drawn, and the regression equation is shown in Figure 6 . The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.
[0040] 7、Gly working curve: in 5 mL stoppered cuvette, add 100 μL 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme and 100 μL 1 mg / mL VB1 solution, incubate for 5 min, then add 0.05-60 mg / L Gly standard solution, 50 mmol / L TMB and 50 mmol / L H2O2 100 μL, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, measure absorbance at 655 nm by UV-visible spectrophotometer, Gly concentration is the abscissa, absorbance A is the ordinate, draw the standard curve, get the regression equation, see Figure 7 ; regression equation, correlation coefficient, relative standard deviation, linear range, etc. see Table 2.
[0041] Table 2 Linear equation, correlation coefficient, relative standard deviation, linear range
[0042]
[0043]
[0044] 7、Method specificity investigation: VB1 and other vitamins are mixed, the influence of coexisting vitamins on VB1 in the above detection system is detected, VB1 concentration is 10 mg / kg, the above interfering substance concentration is 50 mg / kg, Figure 8 is the influence result of coexisting vitamins (VB2, VB3, VB6, VB 12 , VD, and Vc) on VB1, from the figure, it can be seen that Cu2O / Mn@TCPP detection system has good selectivity, VB1 has obvious promotion of oxidation reaction, and other substances almost have no effect, the method has good selectivity for determination of VB1; the selectivity of determination of Gly is also investigated, Gly and other possible coexisting substances are mixed, the influence of coexisting substances on the detection system is detected, Gly concentration is 10 mg / g, the above interfering substance concentration is 5 mg / g, Figure 9 is the influence result of coexisting substances profenofos, phorate, Rogor, chlorpyrifos, phoxim, isocarbophos, acetamiprid, paraquat on glyphosate, from the figure, it can be seen that only glyphosate has obvious inhibitory effect on the catalytic activity of nanometer enzyme, and other substances almost have no inhibitory effect, the method has good selectivity.
[0045] 8、Determination of VB1 in milk powder samples
[0046] (1) Sample treatment: accurately weigh 2 g of milk powder sample (accurate to 0.01 g), place it in a conical flask, add 10 mL of 0.01 mol / L HCl solution, shake well, place it in a boiling water bath for 30 min, take it out and cool it to room temperature, then adjust it to pH 4.5-5.0 (to precipitate the protein) with 1.0 mol / L NaOH solution, filter, and place the filtrate in a 25 mL volumetric flask, add 5.00 mL of 100 g / L EDTA (to mask Fe 3+ , Al 3+ , Cu 2+ ), and then dilute to 25 mL with water, which is the test solution.
[0047] (2) Determination of VB1 in milk powder sample: in a 5 mL colorimetric tube with a stopper, add 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme, 2 mL of the above test solution, incubate for 5 min, then add 100 μL of 50 mmol / L TMB and 50 mmol / L H2O2, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, measure the absorbance at 655 nm with a UV-visible spectrophotometer, and calculate the content of VB1 as 1.25 mg / kg by substituting into the regression equation.
[0048] 9. Determination of Gly in a hanging ear coffee sample
[0049] ① Extraction of Gly
[0050] Accurately weigh 1.00 g of hanging ear coffee sample (accurate to 0.001 g) and place it in a 50 mL polyethylene centrifuge tube with a stopper, add 1 mL of 1 mol / L NaOH and 30 mL of deionized water, ultrasonically treat for 15 min, centrifuge at 8000 rpm for 5 min, and transfer the supernatant to another centrifuge tube to obtain a brown extract;
[0051] ② Purification
[0052] This purification process is treated by two times of precipitation, and the specific steps are as follows: take 2 mL of the brown extract, add 0.4 g of ZnSO4, mix well, add 400 μL of 1 mol / L NaOH, the solution becomes turbid and a precipitate is formed, vortex for 40 s, centrifuge at 4000 rpm for 5 min, and the lower layer is a brown precipitate, and the upper layer solution is clear and transparent, transfer the supernatant to another centrifuge tube, add 200 μL of 1 mol / L NaOH, vortex for 40 s, centrifuge at 4000 rpm for 5 min, and take the supernatant to obtain the sample purification solution;
[0053] ③ Determination of Gly in a hanging ear coffee sample
[0054] In a 5 mL colorimetric tube, 100 μg / mL Fe3O4 / Mo / P nanoscale enzyme 100 μL, sample purification solution 2 mL, 10 mmol / L TMB 50 μL, 40 mmol / L H2O2 250 μL, dilute to 4 mL with pH 2.0 acetic acid-sodium acetate buffer, irradiate with infrared light at 808 nm wavelength for 10 min, separate Fe3O4 / Mo / P nanoscale enzyme with a magnet, pour the solution into a 1 cm cuvette, and measure the absorbance at 654 nm wavelength, substitute into the regression equation of step (4), and the sample glyphosate is not detected;
[0055] (8) Recovery and precision experiment: 3 different concentrations of glyphosate standard solution were added to the hanging ear coffee sample respectively; each concentration was determined in triplicate, the recovery rate was calculated, and the relative standard deviation RSD was calculated, the results were shown in Table 2; the measured recovery rate of glyphosate was 98.2% to 103.5%, and the RSD was 1.81% to 3.20%, the method had good accuracy and precision;
[0056] Example 2: Determination of VB1 in spirulina samples and Gly in tea samples
[0057] 1. Preparation of Cu2O nanoparticles: same as example 1.
[0058] 2. Preparation of Cu2O / Mn nanoparticles: same as example 1.
[0059] 3. Preparation of Cu2O / Mn@TCPP nanoscale enzyme: same as example 1.
[0060] 4. Preparation of VB1 working curve: same as example 1.
[0061] 5. Preparation of Gly working curve: same as example 1.
[0062] 6. Determination of VB1 in spirulina samples
[0063] (1) Sample treatment: 50 tablets were ground and mixed, 2 g (accurate to 0.001 g) was weighed into a 50 mL centrifuge tube with a stopper, 30 mL of 0.2 mol / L HCl was added, vortexed for 30 s, and placed in a constant temperature drying oven at 121 ℃ for 30 min. After cooling to below 40 ℃, it was taken out and shaken several times; adjust the pH value to about 4.5 with 2.0 mol / L sodium acetate solution, add 2.0 mL of mixed enzyme solution (papain (500,000 activity units / g), amylase (37,000 activity units / g)), shake well, and place in a 37 ℃ incubator overnight; dilute to 50 mL with water, filter with filter paper, and the filtrate is the test solution.
[0064] (2) Determination of VB1 in spirulina samples: 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme, 2 mL of the above test solution, 100 μL of 50 mmol / L TMB and 100 μL of 50 mmol / L H2O2 were added in a 5 mL colorimetric tube with a stopper, and incubated for 5 min, and then 4 mL of pH 7.2 glycine-NaOH buffer solution was added, and reacted for 10 min. The absorbance was measured at 655 nm by ultraviolet-visible spectrophotometer, and the content of VB1 was calculated as 30.5 mg / kg by substituting the regression equation.
[0065] 7. Determination of Gly content in tea leaves
[0066] (1) Extraction: The tea leaf sample was respectively placed in a dry pulverizer, crushed through a 40 mesh sieve, and 1.00 g (accurate to 0.001 g) of the sample was accurately weighed in a 50 mL centrifuge tube, 20 mL of ultrapure water was added, and oscillated thoroughly for 2 min, and ultrasonic extraction was performed for 30 min. After extraction, centrifugation was performed at 5000 r / min for 5 min, and the supernatant was taken for purification.
[0067] (2) Purification: The solid phase extraction column was activated with 3 mL of methanol and 3 mL of ultrapure water, respectively, and 5 mL of the supernatant was passed through the solid phase extraction column, and the effluent was collected to obtain the test solution.
[0068] (3) Determination of Gly content in tea leaves: 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme and 100 μL of 1 mg / mL VB1 solution were added in a 5 mL colorimetric tube with a stopper, incubated for 5 min, and then 2 mL of the above test solution, 100 μL of 50 mmol / L TMB and 100 μL of 50 mmol / L H2O2 were added, and 4 mL of pH 7.2 glycine-NaOH buffer solution was added, and reacted for 10 min. The absorbance was measured at 655 nm by ultraviolet-visible spectrophotometer, and the Gly content was 0.32 mg / kg.
[0069] Example 3: Determination of VB1 in Anshen Bumao granules and Gly in water samples
[0070] 1. Preparation of Cu2O nanoparticles: same as Example 1.
[0071] 2. Preparation of Cu2O / Mn nanoparticles: same as Example 1.
[0072] 3. Preparation of Cu2O / Mn@TCPP nanometer enzyme: same as Example 1.
[0073] 4. Preparation of VB1 working curve: same as Example 1.
[0074] 5. Preparation of Gly working curve: same as Example 1.
[0075] 6. Determination of VB1 in Anshen Bnao Granules sample
[0076] (1) Sample treatment: Take 10 bags of the product, grind finely, weigh about 0.5 g (accurate to 0.001 g), place in a 50 mL volumetric flask, add about 30 mL of ultrapure water, tightly cap, ultrasonic to dissolve, cool, dilute to the mark with water, shake well, filter, take the filtrate, and obtain the test solution.
[0077] (2) Determination of VB1 in Anshen Bnao Granules sample: In a 5 mL colorimetric tube with a stopper, add 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme, 0.1 mL of the above test solution, incubate for 5 min, then add 100 μL of TMB with a concentration of 50 mmol / L and 100 μL of H2O2 with a concentration of 50 mmol / L, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, measure the absorbance at 655 nm with a UV-visible spectrophotometer, substitute into the regression equation, and calculate the content of VB1 as 5.40 mg / g.
[0078] 7. Determination of Gly content in water sample: In a 5 mL colorimetric tube with a stopper, add 100 μL of 1 mg / mL Cu2O / Mn@TCPP nanometer enzyme and 100 μL of 1 mg / mL VB1 solution, incubate for 5 min, then add 2 mL of water sample filtered with a 0.45 μm filter membrane, 100 μL of TMB with a concentration of 50 mmol / L and 100 μL of H2O2 with a concentration of 50 mmol / L, add pH 7.2 glycine-NaOH buffer solution to 4 mL, react for 10 min, measure the absorbance at 655 nm with a UV-visible spectrophotometer, and Gly is not detected.
[0079] Recovery rate and precision experiment of the sample detected in the above examples: In the samples detected in Examples 1-3, add 2 different concentrations of VB1 and 3 different concentrations of Gly standard solution respectively; each concentration is determined in triplicate, the standard addition recovery rate is calculated, and the relative standard deviation RSD is calculated, the results are shown in Table 3; the standard addition recovery rate of VB1 is measured as 92.7% to 98.4%, and the RSD is 2.98% to 5.21%, the standard addition recovery rate of Gly is 91.3% to 105.5%, and the RSD is 1.81% to 5.90%, this method has good accuracy and precision.
[0080] Table 3 Standard addition recovery rate and RSD of VB1 in sample (n = 3)
[0081]
[0082] Table 4 Standard addition recovery rate and RSD of Gly in sample (n = 3)
[0083]
[0084] The established determination method of VB1 and glyphosate has the advantages of less processing steps, short processing time, low processing cost, simple operation, and no need for large-scale instruments and equipment, and has strong advantages in actual detection.
Claims
1. A method for detecting vitamin B1 and glyphosate based on nanoscale enzyme, characterized in that, The method comprises the following steps: (1) adding Cu2O / Mn@TCPP nanoscale enzyme into a vitamin B1 (VB1) standard solution, incubating for 5 min, adding 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, and using a pH 7.2 glycine-NaOH buffer solution to make up the volume, to prepare a VB1 solution with a concentration range of 0.8-80 mg / L, shaking, standing for 5-10 min, centrifuging, taking the supernatant to measure the absorbance at a wavelength of 654 nm, establishing a quantitative relationship between the absorbance and the concentration of VB1, drawing a standard curve, and obtaining a regression equation; (2) adding Cu2O / Mn@TCPP nanoscale enzyme, 3,3',5,5'-tetramethylbenzidine (TMB), H2O2 and VB1 into a glyphosate standard solution, and using a pH 7.2 glycine-NaOH buffer solution to make up the volume, to prepare a glyphosate solution with a concentration range of 0.05-60 mg / L, shaking, standing for 5-10 min, centrifuging, taking the supernatant to measure the absorbance at a wavelength of 654 nm, establishing a quantitative relationship between the absorbance and the concentration of glyphosate, drawing a standard curve, and obtaining a regression equation; (3) extracting and purifying glyphosate in a sample to obtain a sample determination solution, adding Cu2O / Mn@TCPP nanoscale enzyme, TMB and H2O2 into the sample determination solution, using a pH 7.2 glycine-NaOH buffer solution to make up the volume, shaking, standing for 5-10 min, centrifuging, taking the supernatant to measure the absorbance at a wavelength of 654 nm, and substituting the absorbance into the regression equation in step (1) to obtain the content of VB1 in the sample. (4) extracting and purifying VB1 in a sample to obtain a sample determination solution, adding Cu2O / Mn@TCPP nanoscale enzyme, TMB, H2O2 and VB1 into the sample determination solution, using a pH 7.2 glycine-NaOH buffer solution to make up the volume, shaking, standing for 5-10 min, centrifuging, taking the supernatant to measure the absorbance at a wavelength of 654 nm, and substituting the absorbance into the regression equation in step (2) to obtain the content of glyphosate in the sample. The Cu2O / Mn@TCPP nanoscale enzyme is prepared as follows: a. Cu2O nanoparticle preparation: 10-15 mL of NaOH (2 mol / L), 100-120 mL of CuCl2 (0.01 mol / L) and 4-5 g of polyvinylpyrrolidone (PVP) are stirred and uniformly mixed for 30-40 min, then 0.5-0.7 mol / L ascorbic acid (AA) is added, stirring in a 55°C water bath for 5-6 h, washing with deionized water, and vacuum drying to obtain the product. b. Cu2O / Mn nanoparticle preparation: 30-40 mg of Cu2O prepared in step (a) is added into a mixture of 10-15 mL of deionized water and 10-15 mL of ethanol, and then 10-15 mL of MnCl2 (0.1 mol / L) solution is added, ultrasonic treatment is performed for 20-30 min, and then stirring is performed under ultraviolet light for 1-1.5 h to obtain the product. c. Preparation of Cu2O / Mn@TCPP nanoszyme: 10-15 mL of tetrakis(4-carboxyphenyl) porphyrin (TCPP, 0.025 mmol / L DMF solution) was added to the Cu2O / Mn solution prepared in step (b), stirred in a 60°C water bath for 6-8 h, centrifuged, washed with ethanol and deionized water alternately, and vacuum dried to obtain the product.
2. The method of claim 1, wherein: The sample determination solution is processed according to a relevant standard method.
3. The method of claim 1, wherein: The concentration of Cu2O / Ce-TCPP nanoszyme is 1 mg / mL, the added amount is 100-150 μL; the concentration of TMB is 50 mmol / L, the added amount is 50-100 μL; the concentration of H2O2 is 50 mmol / L, the amount used is 50-100 μL; the concentration of VB1 is 80 mg / L, the added amount is 50-100 μL.
4. The method of claim 2, wherein: Centrifugation is performed at 8000-10000 r / min for 10-15 min.