Method for rapidly and selectively detecting mercury by dithiothreitol carbon dot gold nano-enzyme

Abstract

The invention discloses a method for rapidly and selectively detecting mercury by using dithiothreitol carbon dot gold nano-enzyme, which comprises the following steps: synthesizing dithiothreitol-carbon dots through a microwave method, synthesizing dithiothreitol carbon dot gold nano-enzyme through secondary reduction by using the carbon dots as a reducing agent through a solvent evaporation method, and selectively forming gold amalgam with Hg (II) by using the nano-enzyme. The dithiothreitol carbon dot gold nano-enzyme has negative charges on the surface and can be effectively combined with Hg (II) with positive charges, the activity of the oxidoid enzyme is remarkably enhanced by adding the Hg (II), 3, 3, 5, 5-tetramethylbenzidine with low Raman activity is catalyzed to be converted into blue oxidized TMB (oxTMB) with high Raman activity, the concentration of the Hg (II) and the Raman signal intensity of the oxTMB are in a linear relation, and the detection time is within 15 min; the method can be applied to selective detection and analysis of Hg (II), and has the characteristics of high sensitivity, strong anti-interference performance, simplicity and rapidness in operation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and specifically relates to a method for rapidly and selectively detecting Hg(II) by using dithiothreitol carbon dot-based nanoenzymes. Background Art

[0002] Mercury and its compounds easily accumulate in environmental soil and water, posing a great threat to life and health. In particular, mercury ions Hg(II), a water-soluble and stable divalent Hg(II), can cause kidney damage, genetic malformations and nerve damage through the enrichment of trace amounts of Hg(II). Therefore, a sensitive, specific and rapid method for detecting Hg(II) is urgently needed. At present, the determination methods of Hg(II) mainly include colorimetry, atomic absorption spectroscopy, atomic fluorescence spectroscopy, inductively coupled plasma mass spectrometry, chromatography and titration. Among them, surface enhanced Raman scattering (SERS) spectroscopy has attracted much attention due to its simple operation, rapidity and the absence of complex instruments and 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 through SERS, and metal ions usually need to be indirectly detected through Raman substrates. As a Raman substrate, gold nanoparticles (AuNPs) can interact with the detected substance and significantly amplify the Raman signal, which has a very broad application prospect in sensing technology. Carbon dots (CDs) are a type of zero-dimensional fluorescent carbon material with a size of less than 10 nm and rich oxygen-containing groups on the surface. They can be used as excellent electron donors and reducing agents. Carbon dots also have photoluminescence and upconversion fluorescence properties, which make them show broad application prospects in chemical sensing, bioimaging, photocatalysis and other fields. Summary of the invention

[0003] The present invention provides a method for rapid and selective detection of Hg(II) by using a dithiothreitol carbon dot-based nanozyme with high sensitivity and strong selectivity. The method uses DL-dithiothreitol, citric acid and ethylenediamine as precursors, synthesizes dithiothreitol-carbon dots (DTT-CDs) by a microwave method, and uses the carbon dots as a reducing agent to synthesize dithiothreitol carbon dot-based nanozymes (DTT-CDs / AuNPs-2) by a hydrothermal method. The nanozyme DTT-CDs / AuNPs-2 can form gold amalgam (Au@HgNPs) with Hg(II) and has oxidase-like activity. On the one hand, the oxidase-like activity catalyzes 3,3`,5,5`-tetramethylbenzidine (TMB) to obtain a blue product, oxidized TMB (oxTMB). On the other hand, the surface of the nanozyme DTT-CDs / AuNPs-2 is negatively charged and effectively combines with the positively charged Hg(II), and Hg(II) The addition of enhanced the oxidase-like activity of the nanozyme. When DTT-CDs / AuNPs-2 oxidized the substrate, the addition of Hg(II) accelerated the oxidation reaction. The Hg(II) concentration was linearly related to the Raman signal enhancement of the oxidation product, and the detection limit was 4.77μg / L.

[0004] The method for the rapid and selective detection of mercury by dithiothreitol carbon dot-based nanozyme of the present invention is as follows: (1) Dissolve 0.08-1.30 g DL-dithiothreitol, 1.80-2.30 g citric acid, and 20-70 μL ethylenediamine in 25-35 mL deionized water, mix by ultrasonication, react at 160-180 °C for 1-2 h in a microwave oven, cool to room temperature, and filter through a 0.22 μm filter membrane to obtain dithiothreitol-carbon dots DTT-CDs; (2) Add 20-50 μL of dithiothreitol-carbon dots, 100-300 μL of 1-2% chloroauric acid solution and 20-70 μL of 1% polyvinyl pyrrolidone solution into 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, and finally put into an ice bath in ice water to obtain dithiothreitol carbon dots-based nanozyme DTT-CDs / AuNPs-2; (3) The dithiothreitol carbon dot-based nanozymes were mixed with Hg(II) solutions of different concentrations and incubated at 20-30°C for 3-8 min. TMB solution was added and the volume was adjusted to 250 μL with deionized water. The reaction was continued for 2-7 min. -1 The Raman signal is measured at the position, the linear relationship between Hg(II) concentration and Raman signal intensity is determined, a standard curve is drawn, and a linear equation is obtained; The concentration of Hg(II) in the reaction system is 2.88-384.60 μg / L; (4) The sample solution containing Hg(II) was mixed with DTT-CDs / AuNPs-2 nanozyme, incubated at 20-30°C for 3-8 min, and then TMB solution was added to the volume and the reaction was continued for 2-7 min. -1 The Raman signal intensity is measured at the position, and the Raman signal intensity is substituted into the regression equation in step (3) to obtain the Hg(II) content in the sample; The amount of DTT-CDs / AuNPs-2 nanozyme added is 50-100 μL; the concentration of TMB solution is 5 mmol / L, and the amount of addition is 5-50 μL.

[0005] Advantages and technical effects of the present invention: 1. The present invention uses dithiothreitol-carbon dots as a reducing agent to prepare dithiothreitol-carbon dot-based nanozymes with excellent simulated oxidase activity and stability. When oxidizing the substrate TMB, with the addition of Hg(II), the negatively charged nanozyme on the surface combines with the positively charged Hg(II) through electrostatic interaction to form amalgam, which accelerates the oxidation reaction. The concentration of Hg(II) is linearly related to the Raman signal enhancement. The detection range of the method of the present invention is 2.88-384.60 μg / L, and the detection time is within 15 min. 2. The DTT-CDs / AuNPs-2 nanozyme synthesized in the present invention can specifically recognize Hg(II), is not affected by methylmercury, and has high selectivity. The actual sample detection results show that the spike recovery rate is in the range of 96.53% to 110.54%, and can be applied to the detection and analysis of Hg(II) in water samples. The method of the present invention has the characteristics of high sensitivity, strong specificity, simple operation, and rapidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 This is a transmission electron microscope (TEM) image of DTT-CDs / AuNPs-2 prepared in Example 1 of the present invention; Figure 2 Zeta potential diagram of DTT-CDs / AuNPs-2 and DTT-CDs / AuNPs-2+Hg(II) in Example 1 of the present invention; Figure 3 The UV-visible absorption spectra of DTT-CDs+TMB, DTT-CDs / AuNPs-2+TMB and DTT-CDs / AuNPs-2+TMB+ Hg(II) in Example 1 of the present invention are shown; Figure 4The Raman spectra of DTT-CDs+TMB, DTT-CDs / AuNPs-2+TMB and DTT-CDs / AuNPs-2+TMB+Hg(II) in Example 1 and the 1605 cm -1 Peak intensity comparison of positions; Figure 5 In Example 1, free radical scavengers such as isopropanol (OH) and tryptophan ( 1 O 2 ) and p-benzoquinone (•O 2- ) after UV-visible absorption spectrum; Figure 6 For Example 1 • 2- (left) and 1 O 2 (right) Electron spin resonance spectrum; Figure 7 The Raman spectra of DTT-CDs / AuNPs-2+TMB and different Hg(II) in Example 1 (left) and the corresponding linear graphs (right); Figure 8 The results of the influence of coexisting metal ions on the Hg(II) detection system in Example 1 are shown. DETAILED DESCRIPTION

[0007] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; Example 1: Determination of Hg(II) in river water 1. Dissolve 1.30 g DL-dithiothreitol, 2.30 g citric acid and 70 μL ethylenediamine in 35 mL deionized water, mix by ultrasonication, react at 180 °C for 1 h in a microwave digester, cool to room temperature, and filter with a 0.22 μm filter membrane to obtain DTT-CDs; 2. Add 50 μL of DTT-CDs, 300 μL of 2% chloroauric acid solution, and 70 μL of 1% polyvinyl pyrrolidone solution into 30 mL of deionized water, heat at 130°C for 10 min, then add 50 μL of DTT-CDs and 300 μL of 2% chloroauric acid solution, continue heating at 130°C for 15 min, and finally put into an ice bath in ice water to obtain DTT-CDs / AuNPs-2; the TEM image of DTT-CDs / AuNPs-2 is shown in the figure Figure 1 As shown, DTT-CDs / AuNPs-2 exhibited an irregular spherical shape.

[0008] In order to investigate the mechanism of action between dithiothreitol carbon dot-based nanozymes and Hg(II), the Zeta potential of 100 μL DTT-CDs / AuNPs-2 and 50 μL 1 mg / L Hg(II) was measured before and after mixing. The results are shown in Figure 2. Figure 2 As shown in the figure, the Zeta of DTT-CDs / AuNPs-2 and DTT-CDs / AuNPs-2+Hg(II) were -15.9 mV and 16.9 mV, respectively. The results showed that the surface of DTT-CDs / AuNPs-2 had a negative charge. After interacting with the positively charged Hg(II), the charge changed from negative to positive, indicating that DTT-CDs / AuNPs-2 could interact with Hg(II) through electrostatic interaction.

[0009] 3. Evaluation of oxidase-like activity and Raman activity of dithiothreitol carbon dot-based nanozymes (1) Study on oxidase-like activity: 100 μL of 5 mmol / L TMB solution and 100 μL of DTT-CDs / AuNPs-2 solution were added to a 5 mL polytetrafluoroethylene centrifuge tube, and then the volume was adjusted to 3 mL with deionized water and mixed. After standing for 7 minutes, the UV-visible absorption spectrum of the system was measured; at the same time, keeping other conditions unchanged, the UV-visible absorption spectra of TMB (100 μL 5 mmol / L) + DTT-CDs (100 μL), TMB (100 μL 5 mmol / L) + DTT-CDs / AuNPs-2 (100 μL) + Hg(II) (50 μL 1 mg / L) systems were measured. The results are as follows. Figure 3 As shown in the figure, analysis of the absorbance at 654 nm showed that the oxidase activity of DTT-CDs / AuNPs-2 was significantly enhanced after interaction with Hg(II), which was mainly due to the formation of gold amalgam (Au@HgNPs).

[0010] (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 in sequence. After incubation for 5 min, 10 μL of 5 mmol / L TMB solution was added, and then deionized water was added to make the volume 250 μL. The reaction was continued for 5 min. Finally, the Raman spectrum of the system was measured and the 1605 cm -1 At the same time, keeping other conditions unchanged, the Raman spectra of TMB (100 μL 5mmol / L) + DTT-CDs (100 μL), TMB (100 μL 5mmol / L) + DTT-CDs / AuNPs-2 (100 μL) + Hg(II) (50 μL 1 mg / L) systems were measured, and the Raman signal at 1605 cm -1 The Raman signal at Figure 4 As shown in Figure 2, the interaction between DTT-CDs / AuNPs-2 and Hg(II) can significantly enhance the Raman signal of the system, 1605cm -1 The Raman peak at the position is significantly enhanced, which is mainly due to the formation of gold amalgam (Au@HgNPs).

[0011] 4. Detection of free radicals In order to explore the changes of 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 O 2 ), superoxide radicals (•O 2- ) and hydroxyl radical (•OH) scavengers.

[0012] Take 100 μL DTT-CDs / AuNPs-2 and 100 μL 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 5 mmol / L TMB and 100 μL scavenger, continue incubation for 7 min, and finally measure the UV-visible absorption spectra of different systems. The results are shown in Figure 5 As shown in the figure, it can be seen that the maximum UV absorption of the experimental group without adding scavenger and the system with adding isopropanol did not change significantly, indicating that •OH is not the key free radical in the reaction system, while the UV-visible spectrum and maximum UV absorption of the system with adding tryptophan and p-benzoquinone changed significantly, indicating that •O 2- and 1 O 2 is the key free radical in the reaction system; at the same time, the study further used electron spin resonance spectrometer (EPR) to determine •O 2- and 1 O 2 , the results are shown in Figure 6 It can be seen from the figure that the interaction between DTT-CDs / AuNPs-2 and Hg(II) produced a higher concentration of •O 2- and 1 O 2 , thereby improving the oxidase-like activity of the nanozyme.

[0013] 5. Preparation of Hg(II) working curve 50 μL of DTT-CDs / AuNPs-2 and Hg(II) solution were added to a 96-well plate and incubated at 20°C for 8 min. 10 μL of 5 mmol / L TMB solution was added and the volume was fixed to 250 μL with deionized water so that the concentration of Hg(II) in the mixed solution was 2.88-384.60 μg / L. After reacting for 5 min, the system was measured at 1605 cm-1 The Raman signal at the position of Hg(II) was plotted with the Hg(II) concentration as the horizontal axis and the Raman signal as the vertical axis to obtain the regression equation. Figure 7 ; The regression equation, correlation coefficient, relative standard deviation, linear range, detection limit, etc. are shown in Table 1; Table 1 Linear equation, correlation coefficient, relative standard deviation, linear range, detection limit

[0014] 6. Determination of Hg(II) in river water samples (1) First, add 50 μL of DTT-CDs / AuNPs-2 to a 96-well plate, then add 50 μL of the Hg(II) river water sample to be tested, incubate at 20°C for 8 min, then add 10 μL of 5 mmol / L TMB, make the volume up to 250 μL, continue the reaction for 5 min, and measure the 1605 cm -1 Substituting the Raman signal at into the regression equation, the result is that Hg(II) is not detected; (2) Recovery and precision experiments Three Hg(II) standard solutions of different concentrations were added to the river water samples, and each concentration was measured three times in parallel. The spiked recovery was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2. The spiked recovery of Hg(II) was measured to be between 96.53% and 110.54%, and the RSD was between 1.77% and 4.05%, indicating that this method has good accuracy and precision.

[0015] Table 2 Sample spike recovery and RSD (n = 3)

[0016] 7. Selective examination of methods The Hg(II) 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 + , H 2 PO4 - , HCO 3 - 、SO 4 2-) and then added the mixture to the DTT-CDs / AuNPs-2+TMB system (same amount as above) and measured at 1605cm -1 The Raman signals at the positions were compared with those of DTT-CDs / AuNPs-2+TMB (blank group) and DTT-CDs / AuNPs-2+TMB+Hg(II) systems to evaluate the specificity of the detection method.

[0017] The results of the test are shown in Figure 8 As can be seen from the figure, 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 this method has good selectivity and anti-interference in determining Hg(II).

[0018] Example 2: Determination of Hg(II) in tap water 1. Dissolve 0.08 g DL-dithiothreitol, 2.0 g citric acid, and 30 μL ethylenediamine in 25 mL deionized water, mix by ultrasonication, react at 160 °C for 2 h in a microwave digester, cool to room temperature, and filter with a 0.22 μm filter membrane to obtain DTT-CDs. 2. Add 20 μL of DTT-CDs, 100 μL of 1% chloroauric acid solution, and 20 μL of 1% polyvinyl pyrrolidone solution into 20 mL of deionized water, heat at 80°C for 20 min, then add 20 μL of DTT-CDs and 100 μL of 1% chloroauric acid solution, continue heating at 80°C for 25 min, and finally put into an ice bath in ice water to obtain DTT-CDs / AuNPs-2; 3. The Hg(II) working curve is prepared in the same way as in Example 1; 4. Determination of Hg(II) in tap water Determination of Hg(II) in samples: As in Example 1, no Hg(II) was detected in tap water.

[0019] The determination method established by the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, easier operation, and no need for large-scale instruments and equipment, thus having great advantages in actual detection.

Claims

1. A method for rapid and selective detection of mercury by dithiothreitol carbon dot-based nanozymes, characterized in that: Here are the steps: (1) Dissolve 0.08-1.30 g DL-dithiothreitol, 1.80-2.30 g citric acid, and 20-70 μL ethylenediamine in 25-35 mL deionized water, mix by ultrasonication, react under microwave at 160-180 °C for 1-2 h, cool to room temperature, and filter through a 0.22 μm 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% polyvinyl pyrrolidone solution into 20-30 mL of deionized water, heat at 80-130°C for 10-20 min, 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, and then place in an ice bath in ice water to obtain dithiothreitol-carbon dot-based nanozyme; (3) The dithiothreitol carbon dot-based nanozymes were mixed with Hg(II) solutions of different concentrations and incubated at 20-30°C for 3-8 min. TMB solution was added and the volume was fixed with deionized water. After the reaction was continued for 2-7 min, the concentration of Hg(II) solution at 1605 cm -1 The Raman signal was measured at , the linear relationship between Hg(II) concentration and Raman signal intensity was determined, a standard curve was drawn, and a linear regression equation was obtained; (4) Mix the Hg(II) sample solution with the dithiothreitol carbon dot-based nanozyme, incubate at 20-30°C for 3-8 min, add TMB solution to the fixed volume, and continue the reaction for 2-7 min. -1 The Raman signal intensity is measured at the position 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 by dithiothreitol carbon dot-based nanozymes according to claim 1, characterized in that: The concentration of Hg(II) in the reaction system is 2.88-384.60 μg / L; the amount of dithiothreitol carbon dot-based nanozyme added is 50-100 μL; the concentration of TMB solution is 5 mmol / L, and the amount added is 5-50 μL.

Images