Method for detecting methyl mercury based on gold nano-enzyme peroxidase

A technology of nano-enzyme peroxidase and methylmercury, which is applied in the direction of nano-carbon, nano-technology, nano-optics, etc., can solve the problems of unstable method, poor reproducibility, and easy aggregation, so as to avoid uneven color and carry Convenience, Analytical Sensitive Effects

Pending Publication Date: 2022-06-07

KUNMING UNIV OF SCI & TECH +1

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Problems solved by technology

Unlike inorganic mercury, the oxidation/reduction of methylmercury has not been fully studied so far in the development of portable nanosensors. Colorimetric determination method, but the method is uns...

Method used

4. recovery rate and precision experiment: in sample extraction liquid, add respectively the methylmercury standard solution of 2 different concentrations; Each concentration is measured in parallel 3 times, calculates standard addition recovery rate, and calculates relative standard deviation RSD , the results are shown in Table 2; the spiked recovery rate of methylmercury was measured at 97.4% to 104.1%, and the RSD was at 2.45% to 4.12%. Thi...

Abstract

According to the method, serotonin carbon dots and glucose are used as a reducing agent and a stabilizing agent to prepare carbon dot gold nano-enzyme, the carbon dot gold nano-enzyme has the characteristics and reducibility of quasi-peroxidase nano-enzyme and can catalyze reduction of methyl mercury, and due to the fact that the absolute electronegativity of Hg is smaller than that of Au, the carbon dot gold nano-enzyme can catalyze reduction of methyl mercury. Electrons transferred from Au-Hg amalgam are much easier than electrons transferred from Au, so that the catalytic activity of the nano-enzyme is enhanced after methyl mercury is introduced, TMB can be quickly oxidized into a blue oxidation product oxTMB by H2O2, on the basis of the principle, methyl mercury is quickly detected, a test strip capable of being used for on-site quick detection can be prepared, detection of the test strip is completed within 10 minutes, and the detection efficiency is greatly improved. The limit of quantitation of methyl mercury reaches 0.5 g/kg. The method has the characteristics of high specificity, convenience in carrying, rapidness, low cost, sensitivity in analysis and the like.

Application Domain

Material analysis by observing effect on chemical indicatorNanooptics +2

Technology Topic

MethylmercurySerotonin +9

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Examples

Experimental program(2)

Example Embodiment

[0030] Example 1: Rapid detection of methylmercury in fish by carbon dot gold nanozyme

[0031] (1) Weigh 2.0g of serotonin and 1.0g of glucose and dissolve them in 20mL of ultrapure water, ultrasonically mix evenly, transfer the solution to a polytetrafluoroethylene-lined hydrothermal reactor, and heat it at a constant temperature of 200 °C for 6 hours to react. After completion, it was naturally cooled to room temperature to obtain a brown solution; the brown solution was removed with a 0.22 μm filter membrane to remove large particles of impurities, and then centrifuged at 8000 r/min, and the supernatant was dried in vacuum at 60 °C to obtain serotonin carbon dots;

[0032] (2) Dissolve 12 mg of serotonin carbon dots in 40 mL of ultrapure water, heat the oil bath to 100°C, and add HAuCl 4 , HAuCl 4 The concentration in the mixed solution was 10 mg/mL, stirred for 60 minutes in the dark, cooled to room temperature, centrifuged at 8000 r/min, and the supernatant was dried to obtain carbon dot gold nanozymes (AuNPs/CDs);

[0033] (3) Preparation of working curve of methylmercury: add 20µL of 0.1mg/mL AuNPs/CDs nanozyme, methylmercury standard solution and 20µL of pH4, 0.2mol/L phosphate buffer solution to the microtiter plate, reaction 5 minutes, followed by the addition of 100 mmol/L H 2 O 220μL, 50mmol/L of TMB 20μL, react at room temperature for 10 minutes, and measure the absorbance at 652nm wavelength, where the concentration of methylmercury in the microtiter plate is in the range of 0.5-100μg/L, with the methylmercury concentration as the abscissa, The absorbance is the ordinate, and the absorption spectrum is shown in figure 1 , 2 , the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are obtained in Table 1. image 3;

[0034] Table 1 Linear equation, correlation coefficient, relative standard deviation, linear range

[0035] ;

[0036] (4) Determination of methylmercury in grass carp samples

[0037] Extraction of grass carp samples: Weigh 10g (accurate to 0.001g) grass carp, pipette 50mL of 5mol/L hydrochloric acid solution into a 100mL plastic centrifuge tube with a lid, extract in an ultrasonic water bath for 60 minutes at room temperature, and centrifuge at 8000 r/min at 4°C For 15 minutes, take out the supernatant, slowly add aqueous ammonia solution (made by mixing ammonia water and water at a volume ratio of 1:1) in a cold water bath, adjust the pH of the sample solution to 6, and add water to 50 mL to obtain a sample extract;

[0038] Sample extraction: extract the sample extract with 20 mL of dichloromethane by shaking twice, shake for 10 minutes each time, let stand for 10 minutes, collect and combine the dichloromethane extract into a 50 mL colorimetric tube, and accurately add 2 mL of back extraction with a graduated pipette The solution (aqueous solution containing 1% cysteine and 0.8% ammonium acetate) was extracted, shaken for 5 minutes and then left to stand for 10 minutes, and the upper aqueous solution was drawn to obtain the sample solution to be tested;

[0039] Take 50µL of the sample solution to be tested, add 20µL of 0.1mg/mL AuNPs/CDs nanozyme and 20µL of pH4, 0.2mol/L phosphate buffer solution, react for 5 minutes, and then add 100mmol/L H 2 O 2 20 μL of 20 μL, 50 mmol/L of TMB, react at room temperature for 10 minutes, measure the absorbance at a wavelength of 652 nm, and substitute it into the regression equation of step (3), the calculated concentration of methylmercury in grass carp is 3.01 μg/kg;

[0040] ④Recovery rate and precision experiment: Add 2 methylmercury standard solutions of different concentrations to the sample extract respectively; each concentration is measured 3 times in parallel, calculate the standard addition recovery rate, and calculate the relative standard deviation RSD, the results are shown in Table 2; the measured recovery of methyl mercury is 97.4% to 104.1%, and the RSD is 2.45% to 4.12%. This method has good accuracy and precision;

[0041] Table 2 Sample recovery and RSD of methylmercury spiked (n = 3)

[0042] ;

[0043] (5) Method specificity investigation: replace methylmercury with other substances, and detect whether other interfering substances have an impact on the detection of methylmercury in the above detection system. The concentration of methylmercury is 10 μg/L, Figure 4 For other interfering substances (AgCl, CoCl 2 , CdCl 2 , PbCl 2 , Cr(NO 3 ) 3 , FeCl 3 , NiCl 2 , HgCl 2 and Hg (NO 3 ) 2 etc.) interference results on the detection system, the concentration of the above interference substances is 100μg/L, it can be seen from the figure that the methylmercury colorimetric probe system has good selection specificity.

[0044] (6) Determination of methylmercury in grass carp samples with test strips

[0045] Test strip preparation:

[0046] a. The test strip includes a polyvinyl chloride (PVC) bottom plate and a nitrocellulose membrane. First, the nitrocellulose (NC) membrane was immersed in a phosphate buffer solution with a pH of 4 and incubated for 30 minutes, dried at 35 °C for 8 hours, and then, Immerse the NC film in 0.05mg/mL carbon dot gold nanozyme solution, take it out, and let it dry naturally;

[0047] b. Cut the dried NC film into strips of uniform size as control lines and test lines, and paste them on polyvinyl chloride (PVC) boards ( Figure 5 ), will contain 60mmol/L TMB and 100mmol/L H 2 O 2 The 1μg/L methylmercury standard solution of 2 O 2 Step (4) The sample to be tested is dropped on the test line, and it is found that the color of the test line turns blue, indicating that the methylmercury content in the sample exceeds 1 μg/kg ( Image 6 ).

Example Embodiment

[0048] Example 2: Rapid detection of methylmercury in environmental water samples by carbon dot gold nanozyme

[0049] (1) Weigh 3.0g of serotonin and 2.0g of glucose and dissolve them in 30mL of ultrapure water, ultrasonically mix evenly, transfer the solution to a polytetrafluoroethylene-lined hydrothermal reactor, and heat it at a constant temperature of 200 °C for 6 hours to react. After completion, it was naturally cooled to room temperature to obtain a brown solution; the brown solution was used to remove large particles of impurities with a 0.22 μm filter membrane, and then centrifuged at 8000 r/min, and the supernatant was vacuum-dried at 60 °C to obtain serotonin carbon dots;

[0050] (2) Dissolve 15 mg of serotonin carbon dots in 50 mL of ultrapure water, heat the oil bath to 100 °C, and add HAuCl 4 , HAuCl 4 The concentration in the mixed solution was 12 mg/mL, and the mixture was stirred for 60 minutes in the dark, cooled to room temperature, centrifuged at 8000 r/min, and the supernatant was dried to obtain the carbon dot gold nanozyme;

[0051] (3) The working curve of methyl mercury is made the same as in Example 1;

[0052] (4) Determination of methylmercury in environmental water samples

[0053] Environmental water sample pretreatment: 10.0mL water sample, add 0.20mL concentrated nitric acid, and leave it for 20 minutes to obtain the assay solution;

[0054] Take 50µL of the sample solution to be tested, add 20µL of 0.08mg/mL AuNPs/CDs nanozyme and 20µL of pH4, 0.2mol/L phosphate buffer solution, react for 5 minutes, and then add 100mmol/L H 2 O 2 20 μL of 20 μL, 50 mmol/L TMB, react at room temperature for 10 minutes, measure the absorbance at a wavelength of 652 nm, and substitute it into the regression equation of step (3), the methylmercury concentration in the environmental water sample was not detected;

[0055] (5) Determination of methylmercury in environmental water samples with test strips

[0056] a. The test strip includes a polyvinyl chloride (PVC) bottom plate and a nitrocellulose membrane. First, the nitrocellulose (NC) membrane was immersed in a phosphate buffer solution with a pH of 4 and incubated for 30 minutes, dried at 40 °C for 5 hours, and then, Immerse the NC film in 0.05mg/mL carbon dot gold nanozyme solution, take it out, and let it dry naturally;

[0057] b. Cut the dried NC film into strips of uniform size and use them as control lines and test lines, and paste them on polyvinyl chloride (PVC) boards respectively. 2 O 2 The 1μg/L methylmercury standard solution was added dropwise to the control line, and the solution containing 45mmol/L TMB and 80mmol/L H 2 O 2 (4) The water sample drops on the test line, and the test line of the test strip does not change color, indicating that the concentration of methylmercury in the sample is less than 1 μg/L.

[0058] The method for rapidly detecting methylmercury by carbon dot gold nanozyme established in the present invention utilizes the inherent catalytic and simulated enzyme properties of carbon dot gold nanometer and the reducibility of carbon dot, and adopts photometric method and test strip to detect organic mercury. The detection method has the following characteristics: High sensitivity, fast, easy to operate and so on.

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Classification and recommendation of technical efficacy words

Avoid uneven color
high sensitivity

Array substrate, manufacturing method of array substrate and display panel of array substrate

ActiveCN104091806Aavoid uneven colorImprove pass rate

Owner:BOE TECH GRP CO LTD +1

Preserved-fruit sulphuring method and preserved-fruit sulphuring device

InactiveCN106359789AUniform sulfur fumigationAvoid uneven color

Owner:徐州龙龙食品有限公司

Method for detecting methyl mercury based on gold nano-enzyme peroxidase

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