A "off-on" type fluorescence sensor for detecting Hg 2+ and glutathione methods

Abstract

The present invention discloses a method for detecting Hg with an "off-on" type fluorescence sensor. 2+ The method for detecting Hg in carbon dots is a method for detecting Hg in carbon dots. The method includes: 1) preparing carbon dots with polyethyleneimine and ammonium citrate as precursors; 2) detecting Hg in carbon dots. 2+ The addition of Hg 2+ The standard curve of Hg concentration and fluorescence quenching degree was obtained and the Hg 2+ 3) Glutathione was mixed with serum and then added to carbon dots and Hg 2+ In the system, a standard curve of glutathione concentration and fluorescence recovery degree was constructed to obtain the concentration of glutathione in serum samples, with a detection limit as low as 61.89 nmol / L. This invention is the first to use an "off-on" type fluorescence sensor to detect Hg 2+ It can quantitatively detect two substances, GSH and glutathione, with simple operation, safe and efficient, and suitable for routine analysis.

Description

Technical Field

[0001] The present invention relates to a method for detecting Hg by using an "off-on" type fluorescence sensor. 2+ The invention discloses a method for determining the amount of glutathione and glutathione, and belongs to the field of analysis and detection. Background Art

[0002] Glutathione (GSH), a type of biothiol, is a tripeptide formed by the condensation of glutamic acid, cysteine, and glycine through peptide bonds. GSH contains free sulfhydryl groups, which help inhibit cell apoptosis and participate in the complex, multidimensional synthesis of proteins. It plays a crucial role in scavenging free radicals, superoxides, and peroxides within cells. Studies have linked many diseases to changes in intracellular GSH levels, including immune disorders, digestive system diseases, cardiovascular diseases, aging disorders, Alzheimer's disease, and cancer. Therefore, the development of simple, low-cost methods for the selective and sensitive detection of glutathione in biological samples is crucial for the early clinical diagnosis of these diseases. Furthermore, with the development of modern industry, heavy metal ion pollution has become increasingly serious. Therefore, the development of an efficient, sensitive, and specific method for the detection of trace amounts of mercury ions is essential.

[0003] In recent years, various analytical methods have been developed for detecting GSH, including high-performance liquid chromatography, electrochemical methods, and chemiluminescence methods. Although these methods offer high accuracy and reliability, they suffer from poor selectivity, high toxicity, low sensitivity, and complex procedures. Therefore, there is an urgent need to find a simpler, faster, and more sensitive method for detecting GSH. Summary of the Invention

[0004] Technical issues:

[0005] Currently developed analytical methods for glutathione detection primarily include high-performance liquid chromatography, electrochemical methods, and chemiluminescence methods. These methods suffer from poor selectivity, high toxicity, low sensitivity, and complex procedures. Furthermore, reports on the use of fluorescence sensing technology for the detection of glutathione in serum are limited, and their sensitivity and detection limits are inferior to those of traditional methods.

[0006] Technical solution:

[0007] The present invention provides a method for detecting Hg based on an "off-on" type fluorescence sensor. 2+ and glutathione, comprising the steps of:

[0008] (1) A carbon dot solution was prepared by a hydrothermal method using polyethyleneimine and ammonium citrate as precursors;

[0009] (2) Configure different concentrations of Hg 2+ Standard solution, Hg 2+The standard solution, carbon dot solution, buffer solution, and lake water sample were mixed evenly to obtain the sample solution, incubated, and then the fluorescence spectrum was detected. The fluorescence quenching degree (F0-F) / F0 and Hg 2+ The concentration of the standard solution is used to construct Hg 2 Linear detection model; where F0 is Hg 2+ Fluorescence intensity when the concentration is 0;

[0010] (3) Mixing the lake water sample to be tested with the carbon dot solution and the buffer solution to obtain the lake water test solution, measuring the fluorescence spectrum of the lake water test solution, and 2 Linear detection model to obtain Hg in lake water samples 2+ concentration;

[0011] (4) Prepare glutathione standard solutions of different concentrations, mix the glutathione standard solutions with serum samples to obtain mixed solutions; then add the mixed solutions to the carbon dot solution, Hg 2+ The solution and the buffer solution are mixed to obtain a final reaction solution, incubated, and then fluorescence spectrum detection is performed; a glutathione linear detection model is constructed by the fluorescence quenching degree (F'-F'0) / F'0 and the concentration of the glutathione standard solution; wherein F'0 is the fluorescence intensity when the glutathione concentration is 0;

[0012] (5) Add the serum sample to be tested into the carbon dot solution, Hg 2+ The serum test solution is obtained from a mixed system of the solution and the buffer solution, and the fluorescence spectrum of the serum test solution is measured, and the concentration of glutathione in the serum sample is obtained according to the glutathione linear detection model in step (4).

[0013] In one embodiment of the present invention, the preparation process of carbon dots in step (1) is as follows: polyethyleneimine and ammonium citrate are dispersed in deionized water, ultrasonic treatment is performed to fully dissolve the mixture, and then a hydrothermal reaction is carried out at 180-220°C. After the reaction is completed, purification and dilution are performed to obtain a carbon dot solution.

[0014] In one embodiment of the present invention, the mass ratio of polyethyleneimine to ammonium citrate is 1:1.

[0015] In one embodiment of the present invention, the ratio of the total mass of polyethyleneimine and ammonium citrate to water is 1 g / 20 mL.

[0016] In one embodiment of the present invention, the hydrothermal reaction time is 2-8 hours.

[0017] In one embodiment of the present invention, the hydrothermal reaction conditions can be specifically selected to be 200° C. for 4 hours.

[0018] In one embodiment of the present invention, the ultrasonic treatment time is 6 minutes.

[0019] In one embodiment of the present invention, the purification steps of carbon dots are as follows: after the reaction is completed, a crude carbon dot solution is obtained, the crude carbon dot solution is centrifuged at 10,000 rpm for 10 minutes, and then filtered through a 0.22 μM microporous filter membrane to remove unreacted particles, and finally the carbon dots are dialyzed and purified for 36 hours using a dialysis membrane with a molecular weight cutoff of 500 Da to obtain the final carbon dots.

[0020] In one embodiment of the present invention, the dilution factor after purification is 500 times.

[0021] In one embodiment of the present invention, the conditions for fluorescence spectrum detection in steps (2) and (4) are: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit and emission slit widths of the spectrometer are both 3.5 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 350 nm, the emission wavelength range is 360 nm-600 nm, and the step size is 1 nm.

[0022] In one embodiment of the present invention, in step (2), Hg 2+ The volume ratio of standard solution, carbon dot solution, buffer solution, and lake water sample was 1:1:1:1.

[0023] In one embodiment of the present invention, the buffer in step (2) is a phosphate buffer (pH=7.4).

[0024] In one embodiment of the present invention, the Hg 2 The linear detection model is:

[0025] (F0-F) / F0=0.02913c(Hg 2+ )+0.00622, where F0 and F represent Hg 2+ The fluorescence intensity of the system when the concentration is 0 or not, c(Hg 2+ ) represents Hg 2+ concentration.

[0026] In one embodiment of the present invention, in step (3), the volume ratio of the lake water sample to be tested, the carbon dot solution, and the buffer solution is 1:1:1.

[0027] In one embodiment of the present invention, the linear relationship described in step (4) is:

[0028] (F'-F'0) / F'0=0.07513c(GSH)-1.92279, wherein F'0 and F' represent the fluorescence intensity of the system when the glutathione concentration is 0 or not, respectively, and c(GSH) represents the concentration of glutathione.

[0029] In one embodiment of the present invention, the volume ratio of the glutathione standard solution to the serum sample in the mixed solution in step (4) is 1:1.

[0030] In one embodiment of the present invention, in step (4), the carbon dot solution, Hg 2+ The volume ratio of the solution and the buffer solution is 1:1:1.

[0031] In one embodiment of the present invention, the buffer in step (4) is a phosphate buffer (pH=7.4).

[0032] In one embodiment of the present invention, the volume ratio of the mixed solution to the carbon dot solution in step (4) is 2:1.

[0033] In one embodiment of the present invention, in step (4), Hg is fixed 2+ The concentration of the solution was 50 μmol / L.

[0034] In one embodiment of the present invention, in step (5), the serum sample to be tested, the carbon dot solution, the Hg 2+ The volume ratio of solution and buffer is 1:1:1:1.

[0035] Beneficial effects:

[0036] The present invention is applied to Hg by an "off-on" type fluorescence sensor 2+ The carbon dots are spherical or quasi-spherical in appearance and have rich functional groups on the surface, which improves the water solubility of the carbon dots and their affinity with Hg 2+ The ability to combine;

[0037] This invention is the first to use polyethyleneimine and ammonium citrate as raw materials to synthesize carbon dots as fluorescent sensors to achieve the detection of Hg in lake water. 2+ The quantitative detection of glutathione in serum is simple, rapid and safe, and suitable for routine analysis. 2+ It interacts with the surface functional groups of carbon dots and causes electron transfer, which leads to fluorescence quenching. It also has a dynamic quenching effect. 2+ It has high selectivity, the sulfhydryl group in glutathione molecule reacts with Hg 2+ It has a strong binding ability, which then restores the fluorescence. This sensor can detect Hg in the same system. 2+ and glutathione were tested successively.

[0038] The present invention detects Hg 2+The linear range of the detection of glutathione is 0-25μmol / L, and the detection limit is as low as 22.45nmol / L; the linear range of the detection of glutathione is 30-50μmol / L, and the detection limit is 61.89nmol / L, which is of great significance for environmental monitoring and biomedical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 To detect Hg using an off-on fluorescence sensor 2+ and glutathione flow chart.

[0040] Figure 2 Add different concentrations of Hg to the system in Example 2 2+ Fluorescence spectra of .

[0041] Figure 3 The fluorescence quenching degree and Hg 2+ Concentration relationship curve.

[0042] Figure 4 The fluorescence quenching degree and Hg concentration range of 0-25 μM in Example 2 are 2+ The linear fitting curve of .

[0043] Figure 5 The fluorescence spectra when different concentrations of glutathione were added to the system in Example 3 are shown.

[0044] Figure 6 This is the relationship curve between the fluorescence recovery degree and the glutathione concentration in Example 3.

[0045] Figure 7 : is the linear fitting curve of the fluorescence recovery degree in Example 3 and the glutathione concentration range of 30-50 μM.

[0046] Figure 8 For the detection of Hg in Example 4 2+ Optional test result graph.

[0047] Figure 9 This is a graph showing the test results for detecting glutathione selectivity in Example 5.

[0048] Figure 10 Hg is used for the detection of glutathione in Example 6 2+ Graph of test results for the effect of concentration. DETAILED DESCRIPTION

[0049] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0050] Example 1 Preparation of Carbon Dots (CDs) Solution

[0051] Weigh 0.5g of polyethyleneimine and 0.5g of ammonium citrate into a beaker, add 20mL of deionized water, and sonicate for 6 minutes to fully dissolve the mixture. Then, react at 200°C for 4 hours. The resulting crude carbon dot solution was centrifuged at 10,000rpm for 10 minutes, then filtered through a 0.22μM microporous filter to remove unreacted particles. Finally, the carbon dots were dialyzed for 36 hours using a 500Da molecular weight cutoff membrane to obtain the final carbon dots. This carbon dot solution was used in all subsequent experiments.

[0052] Example 2 Construction of Hg 2+ Linear determination model

[0053] Prepare sample solution: carbon dot solution (prepared by diluting the final carbon dots obtained in Example 1 500 times), phosphate buffer (pH = 5), lake water sample and Hg at concentrations of 0 (blank control), 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM. 2+ Standard solution;

[0054] The four substances were mixed, and the carbon dot solution, phosphate buffer, lake water sample and different concentrations of Hg 2+ The volume of the standard solution was 0.4 mL, and then it was diluted to 4 mL with deionized water and incubated for 3 minutes to obtain different Hg 2+ The concentration of spiked lake water solution was tested by fluorescence spectroscopy, and the reaction temperature was 20℃;

[0055] Fluorescence spectrum of the measurement system: Scanning conditions: excitation wavelength of 350nm, emission wavelength scanning range of 360-600nm, scanning every 1nm, slit width of 3.5nm / 3.5nm (excitation slit / emission slit), the fluorescence intensity peak F( Figure 2 ); add Hg 2+ The sample solution with a concentration of 0 was subjected to fluorescence spectrum detection to obtain the fluorescence intensity peak F0 at 440 nm, and the degree of fluorescence quenching (F0-F) / F0 was recorded;

[0056] Plot the fluorescence quenching degree of the sample solution versus Hg 2+ The concentration relationship curve, such as Figure 3 As shown, the quenching degree and Hg 2 + The fitting curve of Figure 4 ,from Figure 3 、 Figure 4 It can be seen that when Hg 2+When the concentration is 0-25μM, the degree of fluorescence quenching of the solution is similar to that of Hg 2+ The concentration is linearly related, and the linear equation is (F0-F) / F0=0.02913c(Hg 2+ )+0.00622, the correlation coefficient is R 2 =0.99967, and the detection limit was 22.45 nM.

[0057] Example 3 Construction of a linear determination model for glutathione

[0058] Prepare sample solution: carbon dot solution (500 times diluted from the final carbon dot), phosphate buffer (pH = 7.4), Hg 2+ Standard solution (50 μM), serum samples, and standard solutions of glutathione with concentrations of 0 (blank control), 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, and 80 μM, respectively;

[0059] First, the carbon dot solution, phosphate buffer and Hg 2+ The standard solutions were mixed evenly, with the volume of all three being 0.4 mL. Then, different concentrations of glutathione serum solutions were added (the volume of glutathione and serum was 0.4 mL). Finally, the volume was adjusted to 4 mL with deionized water and incubated for 3 minutes to obtain serum solutions spiked with different glutathione concentrations and subjected to fluorescence spectroscopy detection.

[0060] Fluorescence spectrum of the measurement system: scanning conditions and detection of Hg 2+ Keeping the same, add the sample solution with glutathione concentration of 0 to perform fluorescence spectrum detection, and obtain the fluorescence intensity peak F1 at 440nm. Add the sample solution with other concentrations of glutathione to obtain the fluorescence intensity peak F, and record the fluorescence recovery degree (F-F1) / F1, such as Figure 5 As shown;

[0061] Draw a curve showing the relationship between the fluorescence recovery degree of the sample solution and the glutathione concentration, such as Figure 6 The fitting curves of recovery degree and glutathione are shown in Figure 7 ,from Figure 6 、 Figure 7 It can be seen that when the glutathione concentration is 30-50 μM, the fluorescence recovery degree of the solution is linearly related to the glutathione concentration, the linear equation is (F-F1) / F1=0.07513c(GSH)-1.92279, and the correlation coefficient is R 2 =0.99437, and the detection limit was 61.89 nM.

[0062] Example 4: Detection of Hg by Fluorescence "Off" Process 2+ Selectivity

[0063] Referring to Example 2, 12 common different metal ions (Cu 2+ 、Fe 3+ 、Fe 2+ 、Co 2+ 、Zn 2+ , K + 、Na + , Ca 2+ Mg 2+ 、Mn 2+ Cr 3+ , Pb 2+ ) as interfering substances, such as Figure 8 As shown, F0 and F represent the fluorescence intensity before and after the addition of metal ions, respectively. All cation concentrations were 60 μM, and all fluorescence measurements were performed under the same conditions.

[0064] According to the test results, some interfering substances can quench fluorescence to a certain extent, but have no obvious effect on the fluorescence intensity.

[0065] Example 5: Exploring the Selectivity of Glutathione Detection by the Fluorescence "On" Process

[0066] Referring to Example 3, 16 common amino acids (L-cysteine, D-phenylalanine, L-alanine, glycine, L-glutamic acid, DL-methionine, L-arginine, L-tyrosine, L-leucine, L-proline, DL-tryptophan, DL-serine, L-threonine, L-aspartic acid, L-valine, L-histidine) were used as interfering substances to study the CDs-Hg 2+ The selectivity of the system was tested for glutathione. The concentrations of glutathione and interfering substances were both 10 μM. 2+ The concentration was fixed at 100 μM. Figure 9 shown.

[0067] According to the image, glutathione and cysteine ​​have a great influence on the activity of CDs-Hg. 2+ The fluorescence of the system has a significant recovery effect, and other substances basically cannot restore the fluorescence. Considering that the content of glutathione in biological thiols (glutathione, cysteine, homocysteine) is above 90% in both serum and cells, the presence of cysteine ​​does not affect the detection of glutathione.

[0068] Example 6: Hg in the detection of glutathione 2+ Effect of concentration

[0069] Referring to Example 3, different concentrations (25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM) of Hg 2+The standard solution was added to the carbon dot solution. In order to maximize the sensitivity of glutathione detection, the concentration of glutathione was controlled to 1 μM. The measured results were as follows: Figure 10 As shown, when Hg 2+ When the concentration is 50 μM, the fluorescence recovery effect is slightly obvious. Therefore, Hg 2+ The concentration was fixed at 50 μM.

[0070] Example 7 Detection of Hg in Lake Water Environment 2+

[0071] Referring to Example 1, Hg concentrations of 10, 15, and 20 μM were measured. 2+ , the test results are shown in Table 1.

[0072] Table 1 Test results of Example 7

[0073] Spike concentration (μM) Detection concentration (μM) Recovery rate (%) Relative standard deviation (%), n = 3 10 9.32 93.2 0.97 15 15.53 103.5 0.76 20 20.02 100.1 0.54

[0074] Example 8 Detection of glutathione in serum environment

[0075] Referring to Example 2, glutathione at concentrations of 30, 35, and 45 μM was measured. The test results are shown in Table 2. The test results are relatively satisfactory, indicating that this method is accurate and feasible and can be applied to the detection of glutathione in the biomedical field.

[0076] Table 2 Test results of Example 8

[0077] Spike concentration (μM) Detection concentration (μM) Recovery rate (%) Relative standard deviation (%), n = 3 30 29.94 99.8 1.78 35 34.69 99.1 1.93 45 46.21 102.7 1.91

[0078] Example 9 Comparison of detection limits of glutathione by different methods

[0079] Referring to Example 3, the detection limits obtained by this method were compared with those of other fluorescence detection methods. The results are shown in Table 3. As can be clearly seen from the data in the table, the carbon dots synthesized using polyethyleneimine and ammonium citrate as raw materials can achieve a lower detection limit for glutathione detection, demonstrating the high sensitivity of these carbon dots for glutathione detection.

[0080] Table 3 Comparative results of Example 9

[0081]

[0082]

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[0091] [9] C. Liang,

[0092] Comparative Example 1 Preparation of different carbon dots

[0093] Carbon dot A:

[0094] 0.6 g of polyethyleneimine and 0.4 g of ammonium citrate were weighed into a beaker, and 20 mL of deionized water was added. The mixture was sonicated for 6 minutes to fully dissolve, and then reacted at 200°C for 4 hours. The resulting crude carbon dot solution was centrifuged at 10,000 rpm for 10 minutes, then filtered through a 0.22 μM microporous filter to remove unreacted particles. Finally, the carbon dots were dialyzed for 36 hours using a 500 Da molecular weight cutoff membrane to obtain a carbon dot solution.

[0095] Fluorescence spectra of the carbon dots from Example 1 and carbon dots A from Comparative Example 1, diluted 100-fold under 350 nm excitation light, revealed that their fluorescence emission peaks were aligned, but the fluorescence intensity of the carbon dots from Example 1 was higher than that from Comparative Example 1, which is related to the raw material dosages used. Therefore, the carbon dot synthesis scheme in Example 1 is preferred.

[0096] Carbon dot B:

[0097] 0.5 g of polyethyleneimine and 0.5 g of citric acid were weighed into a beaker, added to 20 mL of deionized water, and sonicated for 6 minutes to fully dissolve the mixture. The mixture was then reacted at 200°C for 4 hours. The resulting crude carbon dot solution was centrifuged at 10,000 rpm for 10 minutes, then filtered through a 0.22 μM microporous membrane to remove unreacted particles. Finally, the carbon dots were dialyzed for 36 hours using a 500 Da molecular weight cutoff membrane to obtain a carbon dot solution.

[0098] The carbon dots of Example 1 and the carbon dots B of Comparative Example 1 were diluted 100 times and fluorescence spectra were detected under an excitation light with a wavelength of 350 nm. The fluorescence peak positions and fluorescence colors of the two carbon dots were different. 0.4 mL of each carbon dot was taken in a test tube, and 0.4 mL of 100 μM Hg was added to each of them. 2+The solution was diluted to 4 mL with deionized water and incubated for 3 min before fluorescence spectrum detection. The fluorescence quenching rate of the carbon dot system obtained in Example 1 was higher than that in Comparative Example 1, and the quenching rate exceeded 90%.

[0099] Take two test tubes, take 0.4 mL of each of the two carbon dots into the test tube, and add 0.4 mL of 100 μM Hg 2+ The solution was then added to each of the two test tubes, with 0.4 mL of a 100 μM glutathione solution added. The volume was then adjusted to 4 mL with deionized water. After incubation for 3 minutes, fluorescence spectroscopy was performed. The degree of fluorescence recovery in the carbon dot system obtained in Example 1 was higher than that in Comparative Example 1, and the degree of recovery was close to 100%, indicating complete fluorescence recovery. Therefore, the carbon dot synthesis scheme in Example 1 is more desirable.

[0100] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for detecting Hg based on an "off-on" fluorescence sensor 2+ and glutathione, characterized in that, The following steps are involved: (1) Carbon dot solution was prepared by hydrothermal method using polyethyleneimine and ammonium citrate as precursors; (2) Configuring different concentrations of Hg 2+ Standard solution, Hg 2+ The standard solution, carbon dot solution, buffer solution, and lake water sample were mixed evenly to obtain the sample solution, incubated, and then fluorescence spectrum detection was performed; the fluorescence quenching degree (F0-F) / F0 and Hg 2+ The concentration of the standard solution is used to construct Hg 2+ Linear detection model; where F0 is Hg 2+ Fluorescence intensity when the concentration is 0, F is Hg 2+ Fluorescence intensity when the concentration is not 0; (3) Mix the lake water sample to be tested with the carbon dot solution and buffer solution to obtain the lake water test solution, measure the fluorescence spectrum of the lake water test solution, and calculate the Hg 2+ Linear detection model to obtain Hg in lake water samples 2+ concentration; (4) Prepare glutathione standard solutions of different concentrations, mix the glutathione standard solutions with serum samples to obtain mixed solutions; then add the mixed solutions to the carbon dot solution, Hg 2+ The solution and the buffer solution are mixed to obtain a final reaction solution, incubated, and then fluorescence spectrum detection is performed; a glutathione linear detection model is constructed by the fluorescence quenching degree (F'-F'0) / F'0 and the concentration of the glutathione standard solution; wherein F'0 is the fluorescence intensity when the glutathione concentration is 0, and F' is the fluorescence intensity when the glutathione concentration is not 0; (5) Add the serum sample to be tested into the carbon dot solution, Hg 2+ solution, and a mixed system of a buffer solution to obtain a serum test solution, measure the fluorescence spectrum of the serum test solution, and obtain the concentration of glutathione in the serum sample according to the glutathione linear detection model in step (4); The preparation process of carbon dots in step (1) is as follows: polyethyleneimine and ammonium citrate are dispersed in deionized water, ultrasonic treatment is performed to fully dissolve the mixture, and then a hydrothermal reaction is carried out at 180-220°C. After the reaction is completed, the mixture is purified and diluted to obtain a carbon dot solution; The mass ratio of polyethyleneimine to ammonium citrate is 1:

1.

2. The method according to claim 1, characterized in that The total mass ratio of polyethyleneimine and ammonium citrate to water is 1 g / 20 mL.

3. The method according to claim 1, characterized in that The conditions for fluorescence spectrum detection in steps (2) and (4) are as follows: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit and emission slit widths of the spectrometer are both 3.5 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 350 nm, the emission wavelength range is 360 nm-600 nm, and the step size is 1 nm.

4. The method according to claim 1, wherein Hg in step (2) 2+ The volume ratio of standard solution, carbon dot solution, buffer solution, and lake water sample was 1:1:1:

1.

5. The method according to claim 1, wherein In step (3), the volume ratio of the lake water sample to be tested, the carbon dot solution, and the buffer solution is 1:1:

1.

6. The method according to claim 1, characterized in that In step (4), the volume ratio of glutathione standard solution to serum sample in the mixed solution is 1:1; carbon dot solution, Hg 2+ The volume ratio of the solution and the buffer solution is 1:1:

1.

7. The method according to claim 1, characterized in that Hg in step (4) 2+ The concentration of the solution was 50 μmol / L.

8. The method according to any one of claims 1 to 7, characterized in that In step (5), the serum sample to be tested, carbon dot solution, Hg 2+ The volume ratio of solution and buffer is 1:1:1:1.

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