A method for rapid and selective detection of mercury by dithiothreitol carbon dots-based nanosensor

Abstract

The application discloses a method for rapidly and selectively detecting mercury by using dithiothreitol carbon dot-based nanoszyme, wherein dithiothreitol-carbon dots are synthesized by a microwave method, the carbon dots are used as a reducing agent, and dithiothreitol carbon dot-based nanoszyme is synthesized by a solvent evaporation method for secondary reduction; the nanoszyme can selectively form gold amalgam with Hg(II); the dithiothreitol carbon dot-based nanoszyme has negative charges on the surface and can be effectively combined with positively charged Hg(II); the addition of Hg(II) can significantly enhance the activity of the oxidase-like enzyme, and catalyze 3,3', 5,5'-tetramethylbenzidine with low Raman activity to be converted into blue oxidized TMB (oxTMB) with high Raman activity; the concentration of Hg(II) has a linear relationship with the Raman signal intensity of oxTMB, and the detection time is within 15 min; the method can be applied to the selective detection and analysis of Hg(II) and has the characteristics of high sensitivity, strong anti-interference, simple operation, rapidness and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical analysis and detection, and particularly relates to a method for rapidly and selectively detecting Hg(II) by using dithiothreitol carbon dot-based nanoszyme. BACKGROUND

[0002] Mercury and its compounds are easily accumulated in the environment soil and water, which poses a great threat to the health of life. In particular, mercury ion Hg(II), a water-soluble and stable divalent Hg(II), can cause kidney damage, genetic deformity and nerve damage through the enrichment of trace Hg(II). Therefore, there is an urgent need for a sensitive, specific and rapid Hg(II) detection method. At present, the determination methods of Hg(II) mainly include colorimetric method, atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry, chromatography and titration method, among which surface-enhanced Raman scattering (SERS) spectroscopy is of great concern due to its simple operation, rapidness and the absence of complex instrument equipment. SERS spectroscopy is a powerful analytical tool that can be used for rapid and instant detection of metal ions. Due to the small Raman scattering cross section of metal ions and the absence of vibrational modes, it is difficult to directly detect metal ions by SERS, and usually metal ions are indirectly detected by Raman substrates. Gold nanoparticles (AuNPs) as a kind of Raman substrate can interact with the detected substances and significantly amplify the Raman signal, and have very broad application prospects in sensing technology. Carbon dots (CDs) are a kind of zero-dimensional fluorescent carbon material with a size of less than 10 nm and a surface containing rich oxygen groups, which can be used as an excellent electron donor and reducing agent. Carbon dots also have photoluminescence and upconversion fluorescence properties, which make them show broad application prospects in the fields of chemical sensing, biological imaging, photocatalysis and the like. SUMMARY

[0003] The application provides a method for rapidly and selectively detecting Hg (II) by using a dithiothreitol carbon dot-based nanoscale enzyme, which has high sensitivity and selectivity, and the method is as follows: DL-dithiothreitol, citric acid and ethylenediamine are used as precursors, dithiothreitol-carbon dots (DTT-CDs) are synthesized by a microwave method, the carbon dots are used as a reducing agent, and dithiothreitol carbon dot-based nanoscale enzyme (DTT-CDs / AuNPs-2) is synthesized by a hydrothermal method. The nanoscale enzyme DTT-CDs / AuNPs-2 can form gold amalgam (Au@HgNPs) with Hg (II) and has oxidase-like activity. On one hand, the oxidase-like activity catalyzes 3,3',5,5'-tetramethylbenzidine (TMB) to obtain blue product oxidized TMB (oxTMB), and on the other hand, the nanoscale enzyme DTT-CDs / AuNPs-2 surface has negative charges and effectively combines with positively charged Hg (II). The addition of Hg (II) enhances the oxidase-like activity of the nanoscale enzyme, and when the DTT-CDs / AuNPs-2 oxidizes the substrate, the addition of Hg (II) accelerates the oxidation reaction. The concentration of Hg (II) has a linear relationship with the Raman signal enhancement of the oxidation product, and the detection limit is 4.77 μg / L.

[0004] The method for rapidly and selectively detecting mercury by using the dithiothreitol carbon dot-based nanoscale enzyme is as follows:

[0005] (1) 0.08-1.30 g of DL-dithiothreitol, 1.80-2.30 g of citric acid and 20-70 μL of ethylenediamine are dissolved in 25-35 mL of deionized water, ultrasonically mixed, reacted at 160-180 ℃ under microwave for 1-2 h, cooled to room temperature, filtered through a 0.22 µm filter membrane, and dithiothreitol-carbon dots DTT-CDs are obtained;

[0006] (2) 20-50 μL of dithiothreitol-carbon dots, 100-300 μL of a 1-2% chloroauric acid solution and 20-70 μL of a 1% polyvinylpyrrolidone solution are added to 20-30 mL of deionized water, heated at 80-130 ℃ for 10-20 min, then 20-50 μL of dithiothreitol-carbon dots and 100-300 μL of a 1-2% chloroauric acid solution are added, and the mixture is continuously heated at 80-130 ℃ for 10-30 min, and finally the mixture is placed in ice water for ice bath to obtain dithiothreitol carbon dot-based nanoscale enzyme DTT-CDs / AuNPs-2;

[0007] (3) The dithiothreitol carbon dot-based nanoscale enzyme is mixed with different concentrations of Hg (II) solution, incubated at 20-30 ℃ for 3-8 min, then TMB solution is added and diluted to 250 μL with deionized water, and the reaction is continued for 2-7 min, and then the Raman signal of the oxidation product is detected at 1605 cm -1The linear relationship between the concentration of Hg(II) and the Raman signal intensity is determined by determining the Raman signal, a standard curve is drawn, and a linear equation is obtained;

[0008] The concentration of Hg(II) in the reaction system is 2.88-384.60 μg / L;

[0009] (4) The sample liquid containing Hg(II) to be measured is mixed with the DTT-CDs / AuNPs-2 nanoenzyme, and after being incubated at 20-30 DEG C for 3-8 min, the TMB solution is added to constant volume, and the reaction is continued for 2-7 min, and the Raman signal intensity at 1605 cm -1 The Raman signal intensity is determined at 1605 cm

[0010] The addition amount of the DTT-CDs / AuNPs-2 nanoenzyme is 50-100 μL; the concentration of the TMB solution is 5 mmol / L, and the addition amount is 5-50 μL.

[0011] The advantages and technical effects of the present application are as follows:

[0012] 1. The dithiothreitol-carbon dots are used as a reducing agent to prepare dithiothreitol-carbon dot-based nanoenzymes with excellent simulated oxidase activity and stability, and when the oxidized substrate TMB is oxidized, the negatively charged nanoenzymes are combined with the positively charged Hg(II) through electrostatic interaction and form gold amalgam, which accelerates the oxidation reaction, and the concentration of Hg(II) is linearly related to the Raman signal enhancement, the detection range of the method is 2.88-384.60 μg / L, and the detection time is within 15 min;

[0013] 2. The DTT-CDs / AuNPs-2 nanoenzyme synthesized in the present application can specifically recognize Hg(II) and is not affected by methyl mercury, has high selectivity, and the actual sample detection results show that the standard addition recovery rate is in the range of 96.53% to 110.54%, and the method can be applied to the detection and analysis of Hg(II) in water samples, and the method has the characteristics of high sensitivity, strong specificity, simple operation, rapidness and the like. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The transmission electron microscope (TEM) image of the DTT-CDs / AuNPs-2 prepared in Example 1 of the present application;

[0015] Figure 2 The Zeta potential diagram of the DTT-CDs / AuNPs-2 and the DTT-CDs / AuNPs-2+Hg(II) in Example 1 of the present application;

[0016] Figure 3 UV-Vis absorption spectra of DTT-CDs+TMB, DTT-CDs / AuNPs-2+TMB and DTT-CDs / AuNPs-2+TMB+Hg(II) in Example 1;

[0017] Figure 4 Raman spectra of DTT-CDs+TMB, DTT-CDs / AuNPs-2+TMB and DTT-CDs / AuNPs-2+TMB+Hg(II) in Example 1 and the peak intensity contrast at 1605 cm -1

[0018] Figure 5 UV-Vis absorption spectra of DTT-CDs / AuNPs-2+TMB system in Example 1 after adding free radical scavengers isopropanol (·OH), tryptophan (·O 1 2) and p-benzoquinone (·O 2- 2) in Example 1;

[0019] Figure 6 ESR spectra of ·O 2- 2) (left) and O 1 2) (right) in Example 1;

[0020] Figure 7 Raman spectra (left) and corresponding linear graph (right) of DTT-CDs / AuNPs-2+TMB with different Hg(II) in Example 1;

[0021] Figure 8 Effect of coexisting metal ions on Hg(II) detection system in Example 1. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be described in further detail below in combination with specific examples, but the protection scope of the present application is not limited to this;

[0023] Example 1: Determination of Hg(II) in river water

[0024] 1. Dissolve 1.30 g of DL-dithiothreitol, 2.30 g of citric acid and 70 μL of ethylenediamine in 35 mL of deionized water, mix uniformly by ultrasonic, then react in a microwave digestion instrument at 180°C for 1 h, cool to room temperature, filter through a 0.22 µm filter membrane to obtain DTT-CDs;

[0025] ​2、Into 30 mL deionized water, 50 μL DTT-CDs, 300 μL of 2% mass concentration chloroauric acid solution, 70 μL of 1% mass concentration polyvinylpyrrolidone solution were added, heated at 130°C for 10 min, then 50 μL DTT-CDs and 300 μL of 2% mass concentration chloroauric acid solution were added, and heated at 130°C for 15 min, and finally placed in ice water for ice bath, to obtain DTT-CDs / AuNPs-2; the TEM image of DTT-CDs / AuNPs-2 is shown in Figure 1 , and DTT-CDs / AuNPs-2 presents irregular spherical shape.

[0026] To investigate the mechanism of the interaction between dithiothreitol carbon dots gold nanoszyme and Hg(II), the Zeta potentials of 100 μL DTT-CDs / AuNPs-2 before and after mixing with 50 μL of 1 mg / L Hg(II) were measured, and the results are shown in Figure 2 , the Zeta potentials of DTT-CDs / AuNPs-2 and DTT-CDs / AuNPs-2+Hg(II) are -15.9 mV and 16.9 mV, respectively, and the results show that DTT-CDs / AuNPs-2 has a negative charge on the surface, and the charge changes from negative to positive after interacting with positively charged Hg(II), indicating that DTT-CDs / AuNPs-2 can interact with Hg(II) through electrostatic interaction.

[0027] 3, Evaluation of the oxidase-like activity and Raman activity of dithiothreitol carbon dots gold nanoszyme

[0028] (1) Oxidase-like activity research: 100 μL of 5 mmol / L TMB solution and 100 μL of DTT-CDs / AuNPs-2 solution were added into a 5 mL polytetrafluoroethylene centrifuge tube in turn, then deionized water was added to 3 mL, and mixed, and after standing for 7 min, the ultraviolet-visible absorption spectrum of the system was measured; at the same time, while keeping other conditions unchanged, the ultraviolet-visible absorption spectrum of TMB (100 μL of 5 mmol / L) + DTT-CDs (100 μL), TMB (100 μL of 5 mmol / L) + DTT-CDs / AuNPs-2 (100 μL) + Hg(II) (50 μL of 1 mg / L) system was measured. The results are shown in Figure 3 , as can be seen from the absorbance at 654 nm, the oxidase-like activity of DTT-CDs / AuNPs-2 is significantly improved after interacting with Hg(II), which is mainly due to the formation of gold amalgam (Au@HgNPs).

[0029] (2) Raman activity evaluation: In a 96-well plate, 50 μL of DTT-CDs / AuNPs-2 and 20 μL of 1 mg / L Hg(II) solution were added sequentially. After incubation for 5 min, 10 μL of 5 mmol / L TMB solution was added, and then deionized water was added to bring the volume to 250 μL. The reaction was allowed to proceed for 5 min, and finally the Raman spectrum of the system was measured. The 1605 cm⁻¹ spectrum was analyzed. -1 The Raman signal at 1605 cm⁻¹ was measured. Simultaneously, keeping other conditions unchanged, the Raman spectra of the systems TMB (100 μL 5 mmol / L) + DTT-CDs (100 μL) and TMB (100 μL 5 mmol / L) + DTT-CDs / AuNPs-2 (100 μL) + Hg(II) (50 μL 1 mg / L) were determined, and the Raman spectra at 1605 cm⁻¹ were analyzed. -1 The Raman signal at the location. The results are as follows: Figure 4 As shown, the interaction between DTT-CDs / AuNPs-2 and Hg(II) significantly enhances the Raman signal of the system, reaching 1605 cm⁻¹. -1 The Raman peak at the location was significantly enhanced, mainly due to the formation of gold amalgam (Au@HgNPs).

[0030] 4. Detection of free radicals

[0031] To investigate the changes in free radicals during the interaction between DTT-CDs / AuNPs-2 and Hg(II), tryptophan (TRP), p-benzoquinone, and isopropanol were used as singlet oxygen (…). 1 O2), superoxide radicals (•O) 2- It is a scavenger of hydroxyl radicals (•OH).

[0032] Take 100 μL of DTT-CDs / AuNPs-2 and 100 μL of Hg(II) (1 mg / L), dilute to 3 mL with deionized water, mix thoroughly, incubate at room temperature for 8 min, then add 100 μL of 5 mmol / L TMB and 100 μL of scavenging agent, and continue incubation for 7 min. Finally, measure the UV-Vis absorption spectra of different systems. The results are as follows: Figure 5 As shown in the figure, the maximum UV absorption of the experimental group without scavenger and the system with isopropanol did not change significantly, indicating that •OH is not a key free radical in the reaction system. However, the UV-Vis spectra and maximum UV absorption of the system with tryptophan and p-benzoquinone showed significant changes, indicating that •OH is not a key free radical in the reaction system. 2- and 1 O2 is the key free radical in the reaction system; furthermore, the study used electron spin resonance spectroscopy (EPR) to determine •O 2- and 1 O2, results see Figure 6As can be seen from the figure, DTT-CDs / AuNPs-2 and Hg(II) interact to produce higher concentrations of •O 2- and 1 O2, thereby improving the oxidaselike activity of the nanoscale enzyme.

[0033] 5. Preparation of Hg(II) working curve

[0034] In a 96-well plate, 50 μL of DTT-CDs / AuNPs-2 and Hg(II) solution was added, incubated at 20°C for 8 min, 10 μL of 5 mmol / L TMB solution was added, and deionized water was added to a constant volume of 250 μL, so that the concentration of Hg(II) in the mixed solution was 2.88-384.60 μg / L, and after 5 min of reaction, the Raman signal of the system at 1605 cm -1 was measured. The standard curve was plotted with Hg(II) concentration as the abscissa and the Raman signal as the ordinate, and the regression equation was obtained, as shown in Figure 7 ; the regression equation, correlation coefficient, relative standard deviation, linear range, and detection limit are shown in Table 1;

[0035] Table 1 Linear equation, correlation coefficient, relative standard deviation, linear range, and detection limit

[0036]

[0037] 6. Determination of Hg(II) in river water samples

[0038] (1) In a 96-well plate, 50 μL of DTT-CDs / AuNPs-2 was first added, followed by 50 μL of Hg(II) river water sample to be tested, incubated at 20°C for 8 min, then 10 μL of 5 mmol / L TMB was added, and the volume was made up to 250 μL, and the reaction was continued for 5 min, after which the Raman signal at 1605 cm -1 was measured, and the result was that Hg(II) was not detected;

[0039] (2) Recovery and precision experiments

[0040] Three different concentrations of Hg(II) standard solution were added to the river water sample, each concentration was determined in triplicate, the recovery rate was calculated, and the relative standard deviation RSD was calculated, and the results are shown in Table 2. The measured recovery rate of Hg(II) was 96.53%-110.54%, and the RSD was 1.77%-4.05%, indicating that the method has good accuracy and precision.

[0041] Table 2 Sample recovery rate and RSD (n = 3)

[0042]

[0043] 7. Selective investigation of the method

[0044] Hg(II) containing solution (final concentration of 20 μg / L) and other ion solutions (final concentration of 200 μg / L, Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cu 2+ , Fe 3+ , Mg 2+ , Mn 2+ , Ni 2+ , Mg 2+ , MeHg + , H2PO4 - , HCO3 - , SO4 2- ) were mixed respectively, and the mixed solution was added to the DTT-CDs / AuNPs-2+TMB (same amount as above) system, and the Raman signal at 1605 cm -1 was measured. The Raman signal of the DTT-CDs / AuNPs-2+TMB (blank group) and the DTT-CDs / AuNPs-2+TMB+Hg(II) system was compared, and the specificity of the detection method was evaluated.

[0045] The determination results are shown in Figure 8 From the figure, it can be seen that Hg(II) can significantly enhance the Raman signal of the DTT-CDs / AuNPs-2+TMB system, and the addition of other ions has no obvious effect on the system, and the concentration of other ions is 10 times that of Hg(II). The above results show that the method for determining Hg(II) has good selectivity and anti-interference.

[0046] Example 2: Determination of Hg(II) in tap water

[0047] 1. 0.08 g of DL-dithiothreitol, 2.0 g of citric acid, 30 μL of ethylenediamine were dissolved in 25 mL of deionized water, and after ultrasonic mixing, it was reacted in a microwave digestion instrument at 160°C for 2 h, cooled to room temperature, and filtered through a 0.22 µm filter membrane to obtain DTT-CDs;

[0048] 2. 20 μL of DTT-CDs, 100 μL of 1% mass concentration of chloroauric acid solution, 20 μL of 1% mass concentration of polyvinylpyrrolidone solution were added to 20 mL of deionized water, and after heating at 80°C for 20 min, 20 μL of DTT-CDs and 100 μL of 1% mass concentration of chloroauric acid solution were added, and the mixture was heated at 80°C for 25 min. Finally, it was placed in ice water for ice bath to obtain DTT-CDs / AuNPs-2.

[0049] 3. Preparation of Hg(II) working curve, same as Example 1;

[0050] 4. Determination of Hg(II) in tap water

[0051] Determination of Hg(II) in sample: same as Example 1, no Hg(II) detected in tap water.

[0052] The determination method established by the application has the advantages of less processing steps, short processing time, low processing cost, simple operation, and no need for large-scale instrument equipment, and has strong advantages in actual detection.

Claims

1. A method for rapid and selective detection of mercury using dithiothreitol carbon dot nanozymes, characterized in that, The steps are as follows: (1) Dissolve 0.08-1.30g DL-dithiothreitol, 1.80-2.30g citric acid, and 20-70μL ethylenediamine in 25-35mL deionized water, mix by sonication, react in microwave at 160-180℃ for 1-2h, cool to room temperature, filter through a 0.22µm filter membrane to obtain dithiothreitol-carbon dots; (2) Add 20-50 μL of dithiothreitol-carbon dots, 100-300 μL of 1-2% chloroauric acid solution, and 20-70 μL of 1% polyvinylpyrrolidone solution to 20-30 mL of deionized water. Heat at 80-130 °C for 10-20 min, then add 20-50 μL of dithiothreitol-carbon dots and 100-300 μL of 1-2% chloroauric acid solution. Continue heating at 80-130 °C for 10-30 min, then place in an ice bath to obtain dithiothreitol carbon dot nanozyme. (3) Mix dithiothreitol carbon dot nanozyme with Hg(II) solutions of different concentrations, incubate at 20-30℃ for 3-8 min, add TMB solution and dilute with deionized water, continue the reaction for 2-7 min, and then incubate at 1605 cm⁻¹. -1 Raman signals were measured at the site to determine the linear relationship between Hg(II) concentration and Raman signal intensity, a standard curve was plotted, and the linear regression equation was obtained. (4) Mix the Hg(II) sample solution with dithiothreitol carbon dot nanozyme, incubate at 20-30℃ for 3-8 min, add TMB solution to make up the volume, and continue the reaction for 2-7 min. Then, at 1605 cm⁻¹... -1 The Raman signal intensity was measured and substituted into the regression equation in step (3) to obtain the Hg(II) content in the sample.

2. The method for rapid and selective detection of mercury using dithiothreitol carbon dot nanozymes according to claim 1, characterized in that: The concentration of Hg(II) in the reaction system was 2.88-384.60 μg / L; the amount of dithiothreitol carbon dot fund nanozyme added was 50-100 μL; the concentration of TMB solution was 5 mmol / L, and the amount added was 5-50 μL.

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