CN115494042A

CN115494042A - Method for detecting Hg by using 'off-on' type fluorescence sensor 2+ And glutathione production method

Info

Publication number: CN115494042A
Application number: CN202211333647.7A
Authority: CN
Inventors: 陈国庆; 李鑫; 吴亚敏; 马超群; 李磊; 朱纯; 辜姣; 高辉
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2022-12-20

Abstract

The invention discloses a method for detecting Hg by using an 'off-on' type fluorescence sensor ²⁺ And glutathione, belonging to the field of analysis and detection. The detection method comprises the following steps: 1) Preparing carbon dots by using polyethyleneimine and ammonium citrate as precursors; 2) Hg in carbon dots ²⁺ Hg is constructed based on the addition of (A) quenching fluorescence by dynamic quenching and photoinduced electron transfer ²⁺ Concentration and degree of fluorescence quenching and obtaining Hg in lake water samples ²⁺ The concentration of (c); 3) Glutathione was mixed with serum and added to the carbon dots and Hg ²⁺ System (2)In the method, a standard curve of the concentration of the glutathione and the fluorescence recovery degree is constructed, the concentration of the glutathione in the serum sample is obtained, and the detection limit is as low as 61.89nmol/L. The invention firstly uses the 'off-on' type fluorescence sensor to sequentially couple Hg ²⁺ And glutathione, and the method is simple to operate, safe, efficient and suitable for routine analysis.

Description

Hg detection by 'off-on' type fluorescence sensor 2+ And glutathione production method

Technical Field

The invention relates to a method for detecting Hg by using an 'off-on' type fluorescence sensor ²⁺ And glutathione, belonging to the field of analysis and detection.

Background

Glutathione (GSH), a type of biological thiol, is a tripeptide composed of glutamic acid, cysteine, and glycine condensed through peptide bonds. GSH contains free sulfydryl, is beneficial to inhibiting apoptosis, participates in constructing complex multidimensional protein synthesis, and plays an extremely important role in eliminating free radicals, superoxide, peroxide and the like in cells. Research has found that the generation of many diseases is related to the change of intracellular GSH levels, such as some immune diseases, digestive system diseases, cardiovascular diseases, aging diseases, alzheimer's disease, cancer, etc., so in the field of early clinical diagnosis of these diseases, it is very necessary to develop a simple and low-cost method for selectively and sensitively detecting glutathione in a biological sample. In addition, with the development of modern industry, heavy metal ion pollution is more serious. Therefore, it is necessary to develop a method which is efficient, sensitive, specific and can detect trace mercury ions.

In recent years, various analytical methods for detecting GSH have been developed, mainly including high performance liquid chromatography, electrochemical methods, chemiluminescence methods, and the like, which have problems of poor selectivity, high toxicity, low sensitivity, complicated operation, and the like, although having high accuracy and reliability. Therefore, there is an urgent need to find a simpler, faster and more sensitive method for detecting GSH.

Disclosure of Invention

The technical problem is as follows:

the currently developed various analytical methods for detecting glutathione mainly include high performance liquid chromatography, electrochemical methods, chemiluminescence methods, and the like, and these methods have problems of poor selectivity, high toxicity, low sensitivity, complicated operation, and the like. Meanwhile, few reports are reported for detecting glutathione in serum by using a fluorescence sensing technology, and the sensitivity and detection limit are not superior to those of the traditional method.

The technical scheme is as follows:

the invention provides a method for detecting Hg based on a 'switch-on' type fluorescence sensor ²⁺ And glutathione, comprising the steps of:

(1) Preparing a carbon dot solution by using polyethyleneimine and ammonium citrate as precursors through a hydrothermal method;

(2) Different concentrations of Hg are configured ²⁺ Standard solution of Hg ²⁺ Uniformly mixing the standard solution, the carbon dot solution, the buffer solution and the lake water sample to obtain a sample solution, incubating, and then carrying out fluorescence spectrum detection; by degree of fluorescence quenching (F) ₀ -F)/F ₀ And Hg ²⁺ Concentration of Standard solution construction of Hg ² A linear detection model; wherein, F ₀ Is Hg ²⁺ Fluorescence intensity at a concentration of 0;

(3) Mixing the lake water sample to be detected with the carbon dot solution and the buffer solution to obtain a lake water to be detected solution, measuring the fluorescence spectrum detection of the lake water to be detected solution, and detecting according to the Hg in the step (2) ² Obtaining Hg in lake water sample by linear detection model ²⁺ Concentration;

(4) Preparing glutathione standard solutions with different concentrations, and mixing the glutathione standard solutions with a serum sample to obtain a mixed solution; then adding the mixed solution into carbon dot solution and Hg ²⁺ Mixing the solution and the buffer solution uniformly to obtain a final reaction solution, incubating, and then carrying out fluorescence spectrum detection; degree of quenching by fluorescence (F '-F' ₀ )/F’ ₀ And glutathione Standard solution concentration construction of the troughA linear detection model of cystine; wherein, F' ₀ The fluorescence intensity is the concentration of glutathione is 0;

(5) Adding the serum sample to be detected into the carbon dot solution and Hg ²⁺ And (4) obtaining a serum solution to be detected in a mixed system of the solution and the buffer solution, measuring the fluorescence spectrum detection of the serum solution to be detected, and obtaining the concentration of the glutathione in the serum sample according to the glutathione linear detection model in the step (4).

In one embodiment of the present invention, the carbon dots in step (1) are prepared by the following steps: dispersing polyethyleneimine and ammonium citrate in deionized water, performing ultrasonic treatment to fully dissolve the mixture, performing hydrothermal reaction at 180-220 ℃, and after the reaction is finished, purifying and diluting to obtain a carbon dot solution.

In one embodiment of the invention, the mass ratio of polyethyleneimine to ammonium citrate is 1.

In one embodiment of the invention, the ratio of the total mass of polyethyleneimine and ammonium citrate to the amount of water used is 1g/20mL.

In one embodiment of the invention, the hydrothermal reaction is carried out for a period of time of 2 to 8 hours.

In one embodiment of the present invention, the hydrothermal reaction conditions may be specifically selected from 200 ℃ for 4h.

In one embodiment of the invention, the time of sonication is 6min.

In one embodiment of the present invention, the carbon dots are purified by the steps of: and (3) obtaining a crude carbon dot solution after the reaction is finished, centrifuging the crude carbon dot solution at 10000rpm for 10min, filtering the crude carbon dot solution through a 0.22 mu M microporous filter membrane to remove unreacted particles, and finally dialyzing and purifying the carbon dot for 36h by using a dialysis membrane with the molecular weight cutoff of 500Da to obtain the final carbon dot.

In one embodiment of the invention, the dilution factor after purification is 500-fold.

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

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

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

In one embodiment of the present invention, hg as described in step (2) ² The linear detection model is:

(F ₀ -F)/F ₀ ＝0.02913c(Hg ²⁺ ) +0.00622 wherein F ₀ And F each represents Hg ²⁺ Fluorescence intensity of the system at a concentration of 0 or other than 0, c (Hg) ²⁺ ) Is expressed in Hg ²⁺ The concentration of (2).

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.

in one embodiment of the present invention, the linear relationship in step (4) is:

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

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

in one embodiment of the present invention, the carbon dot solution, hg, in step (4) ²⁺ The volume ratio of the solution to the buffer is 1:1:1.

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

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

In one embodiment of the invention, hg is fixed in step (4) ²⁺ The concentration of the solution was 50. Mu. Mol/L.

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

has the beneficial effects that:

the invention applies to Hg by a 'switch-on' type fluorescence sensor ²⁺ And glutathione detection, the carbon dots are spherical or quasi-spherical in appearance and rich in functional groups on the surface, so that the water solubility of the carbon dots and Hg are improved ²⁺ The binding ability of (c);

the invention firstly uses the carbon dots synthesized by taking the polyethyleneimine and the ammonium citrate as raw materials as the fluorescence sensor to successively realize the Hg in the lake water ²⁺ And the quantitative detection of glutathione in serum, and the method is simple, rapid and safe to operate and is suitable for routine analysis. Hg in the process of the invention ²⁺ Interacts with the functional group on the surface of the carbon dot and generates electron transfer to cause fluorescence quenching, and has dynamic quenching function on Hg ²⁺ Has higher selectivity, and the sulfydryl and Hg in the glutathione molecule ²⁺ Has strong binding capacity, and then recovers fluorescence, and the sensor can be used for Hg in the same system ²⁺ And sequentially detecting the glutathione.

The invention detects Hg ²⁺ The linear interval of (a) is 0-25 mu mol/L, and the detection limit is as low as 22.45nmol/L; the linear interval for detecting the glutathione is 30-50 mu mol/L, and the detection limit is 61.89nmol/L, which has important significance for the fields of environmental monitoring and biomedicine.

Drawings

FIG. 1 shows the detection of Hg by an "off-on" type fluorescence sensor ²⁺ And a scheme for glutathione.

FIG. 2 shows the addition of different concentrations of Hg to the system of example 2 ²⁺ Fluorescence spectrum of time.

FIG. 3 shows the degree of fluorescence quenching and Hg in example 2 ²⁺ Concentration dependence.

FIG. 4 is a graph of fluorescence quenching and Hg concentration ranging from 0 to 25 μ M in example 2 ²⁺ Is linearly fitted to the curve.

FIG. 5 is a graph of the fluorescence spectra of the system of example 3 when glutathione was added at different concentrations.

FIG. 6 is a graph showing the relationship between the recovery degree of fluorescence and the glutathione concentration in example 3.

FIG. 7 is a linear fit of the degree of fluorescence recovery versus glutathione concentration in the range of 30-50. Mu.M in example 3.

FIG. 8 shows Hg detection in example 4 ²⁺ The test result of selectivity is shown.

FIG. 9 is a graph showing the results of the test for glutathione selectivity in example 5.

FIG. 10 shows Hg in detection of glutathione in example 6 ²⁺ Test results plot of concentration effect.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

EXAMPLE 1 preparation of Carbon Dot (CDs) solutions

0.5g of polyethyleneimine and 0.5g of ammonium citrate were weighed into a beaker, 20mL of deionized water was added, and sonication was performed for 6min to dissolve the mixture sufficiently, followed by reaction at 200 ℃ for 4h. The synthesized crude carbon dot solution is centrifuged at 10000rpm for 10min, then unreacted particles are removed by filtration through a 0.22 mu M microporous membrane, and finally the carbon dot is dialyzed and purified for 36h by a dialysis membrane with the molecular weight cutoff of 500Da to obtain the final carbon dot. The carbon dot solutions for subsequent experiments were based on this.

EXAMPLE 2 construction of Hg ²⁺ Linear measurement model of (2)

Preparing a sample solution: carbon dot solution (prepared by 500-fold dilution of the final carbon dot obtained in example 1), phosphate buffer (pH = 5), lake water sample, and Hg at concentrations of 0 (blank), 1. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu.M, and 50. Mu.M, respectively ²⁺ A standard solution;

mixing the four substances, and mixing the carbon dot solution, the phosphate buffer solution, the lake water sample and different concentrations of Hg ²⁺ The standard solutions had a volume of 0.4mL, then using deionized water to fix the volume to 4mL, standing and incubating for 3min to obtain different Hg ²⁺ Adding a standard lake aqueous solution with concentration and carrying out fluorescence spectrum detection, wherein the reaction temperature is 20 ℃;

fluorescence spectrum of the measurement system: scanning conditions are as follows: the excitation wavelength was 350nm, the emission wavelength scan range was 360-600nm, scanning was performed every 1nm, the slit width was set to 3.5nm/3.5nm (excitation slit/emission slit), and the fluorescence intensity peak F at 440nm was obtained (fig. 2); hg is added ²⁺ Performing fluorescence spectrum detection on the sample solution with the concentration of 0 to obtain the fluorescence intensity peak value F at 440nm ₀ The degree of fluorescence quenching (F) was recorded ₀ -F)/F ₀ ；

Plotting the degree of fluorescence quenching and Hg of the sample solution ²⁺ Concentration dependence, as shown in FIG. 3, degree of quenching and Hg ² ⁺ The fitted curve of (2) is shown in fig. 4, and can be seen from fig. 3 and 4: when Hg is contained ²⁺ The degree of fluorescence quenching and Hg of the solution at a concentration of 0-25 μ M ²⁺ The concentration is in a linear relationship, and the linear equation is (F) ₀ -F)/F ₀ ＝0.02913c(Hg ²⁺ ) +0.00622 with a correlation coefficient R ² =0.99967, detection limit 22.45nM.

Example 3 construction of Linear assay model for glutathione

Preparing a sample solution: carbon dot solution (500-fold dilution from final carbon dot), phosphate buffer (pH = 7.4), hg ²⁺ Standard solutions (50. Mu.M), serum samples and glutathione at concentrations of 0 (blank), 1. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, respectively;

firstly, carbon dot solution, phosphate buffer solution and Hg are added ²⁺ Uniformly mixing the standard solution, wherein the volumes of the three solutions are 0.4mL, adding glutathione serum solutions with different concentrations (the volumes of the glutathione and the serum are 0.4 mL), finally diluting to 4mL with deionized water, standing and incubating for 3min to obtain serum solutions with different glutathione concentrations and adding a standard, and performing fluorescence spectrum detection;

fluorescence spectrum of the measurement system: scanning conditions and detection of Hg ²⁺ Keeping the same, addingPerforming fluorescence spectrum detection on a sample solution with the glutathione concentration of 0 to obtain a fluorescence intensity peak value F at 440nm ₁ The peak value of the fluorescence intensity obtained by adding the sample solution of glutathione with other concentrations is F, and the fluorescence recovery degree (F-F) is recorded ₁ )/F ₁ As shown in fig. 5;

the fluorescence recovery degree of the sample solution is plotted as a function of the glutathione concentration, as shown in fig. 6, and the curve fitted with the recovery degree and glutathione is shown in fig. 7, as can be seen from fig. 6 and 7: when the concentration of glutathione is 30-50 mu M, the fluorescence recovery degree of the solution is in linear relation with the concentration of glutathione, and the linear equation is (F-F) ₁ )/F ₁ =0.07513c (GSH) -1.92279, correlation coefficient R ² =0.99437, detection limit 61.89nM.

Example 4 detection of Hg by probing the fluorescence "off" Process ²⁺ Selectivity of (2)

Referring to example 2, 12 common different metal ions (Cu) were selected ²⁺ 、Fe ³⁺ 、Fe ²⁺ 、Co ²⁺ 、Zn ²⁺ 、K ⁺ 、Na ⁺ 、Ca ²⁺ 、Mg ²⁺ 、Mn ²⁺ 、Cr ³⁺ 、Pb ²⁺ ) As an interfering substance, as shown in FIG. 8, F ₀ And F represent the fluorescence intensity before and after the addition of the metal ion, respectively. All cation concentrations were 60. Mu.M and all fluorescence measurements were performed under the same conditions.

According to the detection results, some interfering substances can quench fluorescence to some extent, but do not have a significant influence on the fluorescence intensity.

Example 5 exploration of the fluorescence "on" Process to detect glutathione Selectivity

Referring to example 3, CDs-Hg was investigated using 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) as interferents ²⁺ The selectivity of the system for detecting the glutathione is that the concentration of the glutathione and the concentration of the interferent are both 10 mu M and Hg ²⁺ The concentrations were all fixed at 100. Mu.M. The detection results are shown in fig. 9.

According to the image, glutathione and cysteine are corresponding to CDs-Hg ²⁺ The fluorescence of the system has obvious recovery effect, other substances basically cannot recover the fluorescence, and the content of glutathione in biological thiol (glutathione, cysteine and homocysteine) is more than 90 percent in serum or cells, so that the detection of the glutathione is not influenced by the existence of the cysteine.

Example 6 investigation of Hg in detection of glutathione ²⁺ Influence of concentration

With reference to example 3, hg was added at various concentrations (25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu.M, 50. Mu.M) ²⁺ When the standard solution was added to the carbo-dot solution and the concentration of glutathione was controlled to 1. Mu.M in order to maximize the sensitivity of detecting glutathione, the results were obtained as shown in FIG. 10 when Hg was added to the carbo-dot solution ²⁺ At a concentration of 50. Mu.M, the effect of recovering fluorescence was slightly remarkable, and therefore Hg was used in the detection of glutathione ²⁺ The concentration was fixed at 50. Mu.M.

Example 7 detection of Hg in lake Water Environment ²⁺

With reference to example 1, hg was measured at a concentration of 10, 15, 20. Mu.M ²⁺ The results are shown in Table 1.

Table 1 test results of example 7

Standard concentration (μ M)	Detection concentration (μ M)	Recovery (%)	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

Example 8 detection of glutathione in serum Environment

Referring to example 2, glutathione with the concentration of 30, 35 and 45 μ M was measured, and the detection results are shown in table 2, which indicates that the method is accurate and feasible, and can be applied to the detection of glutathione in the biomedical field.

Table 2 test results of example 8

Standard concentration (μ M)	Detect concentration (μ M)	Recovery (%)	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

Example 9 comparison of detection limits for glutathione detection by different methods

Referring to example 3, the detection limit obtained by the method is compared with other fluorescence detection methods, and the results are shown in table 3, and it is obvious from the data in the table that a lower detection limit can be obtained when carbon dots synthesized by using polyethyleneimine and ammonium citrate as raw materials are used for detecting glutathione, which proves that the carbon dots have high sensitivity for detecting glutathione.

Table 3 comparative results of example 9

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Comparative example 1 preparation of different carbon dots

Carbon point A:

0.6g of polyethyleneimine and 0.4g of ammonium citrate were weighed into a beaker, 20mL of deionized water was added, and sonication was performed for 6min to sufficiently dissolve the mixture, followed by reaction at 200 ℃ for 4h. And (3) centrifuging the synthesized crude carbon dot solution at 10000rpm for 10min, filtering by a 0.22 mu M microporous filter membrane to remove unreacted particles, and finally dialyzing and purifying the carbon dot by a dialysis membrane with the molecular weight cutoff of 500Da for 36h to obtain the carbon dot solution.

The carbon dot of example 1 and the carbon dot A of comparative example 1 are diluted 100 times and subjected to fluorescence spectrum detection under excitation light with the wavelength of 350nm, and the test results show that the fluorescence emission peak positions of the carbon dot and the carbon dot are consistent, but the fluorescence intensity of the carbon dot obtained in example 1 is higher than that of comparative example 1, which is related to the use amount of the raw materials of the carbon dot and the carbon dot A. Therefore, the carbon dot synthesis scheme in example 1 is preferable.

Carbon point B:

0.5g of polyethyleneimine and 0.5g of citric acid were weighed into a beaker, 20mL of deionized water was added, and sonication was performed for 6min to sufficiently dissolve the mixture, followed by reaction at 200 ℃ for 4h. And (3) centrifuging the synthesized crude carbon dot solution at 10000rpm for 10min, filtering by a 0.22 mu M microporous filter membrane to remove unreacted particles, and finally dialyzing and purifying the carbon dot by a dialysis membrane with the molecular weight cutoff of 500Da for 36h to obtain the carbon dot solution.

The carbon dots of example 1 and the carbon dots B of comparative example 1 are diluted 100 times and subjected to fluorescence spectrum detection under excitation light with the wavelength of 350nm, the fluorescence peak positions of the carbon dots and the excitation light are different, the fluorescence colors of the carbon dots and the excitation light are different, 0.4mL of each of the two carbon dots is taken out of a test tube, and 0.4mL of 100 mu M Hg is added into the test tube respectively ²⁺ And (3) diluting the solution to 4mL with deionized water, incubating for 3min, and detecting the solution by fluorescence spectrum, wherein the quenching rate of the carbon dot system obtained in the example 1 is higher than that of the carbon dot system obtained in the comparative example 1, and the quenching rate exceeds 90%.

Taking another two test tubes, respectively taking 0.4mL of each of the two carbon spots in the test tubes, and respectively adding 0.4mL of 100 mu M Hg ²⁺ And adding 0.4mL of 100 mu M glutathione solution into the two test tubes respectively, diluting the volume to 4mL by using deionized water, incubating for 3min, and then carrying out fluorescence spectrum detection, wherein the fluorescence recovery degree in the carbon dot system obtained in the example 1 is higher than that in the comparative example 1, and the recovery degree is close to 100%, namely the fluorescence is completely recovered. Therefore, the carbon dot synthesis scheme in example 1 is preferable.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Hg detection based on 'off-on' type fluorescence sensor ²⁺ And glutathione, characterized in that the method comprises the following steps:

(4) Preparing glutathione standard solutions with different concentrations, and mixing the glutathione standard solutions with a serum sample to obtain a mixed solution; then adding the mixed solution into carbon dot solution and Hg ²⁺ Uniformly mixing the solution and the buffer solution in a mixed system to obtain a final reaction solution, incubating, and then carrying out fluorescence spectrum detection; degree of quenching by fluorescence (F '-F' ₀ )/F’ ₀ Constructing a glutathione linear detection model according to the concentration of the glutathione standard solution; wherein, F' ₀ The fluorescence intensity of glutathione at a concentration of 0;

(5) Adding the serum sample to be detected into the carbon dot solution and Hg ²⁺ Obtaining serum to-be-detected liquid in a mixed system of the solution and the buffer solution, measuring the fluorescence spectrum detection of the serum to-be-detected liquid, and carrying outAnd (5) obtaining the concentration of glutathione in the serum sample according to the glutathione linear detection model in the step (4).

2. The method according to claim 1, wherein the carbon dots in step (1) are prepared by: dispersing polyethyleneimine and ammonium citrate in deionized water, performing ultrasonic treatment to fully dissolve the mixture, performing hydrothermal reaction at 180-220 ℃, and after the reaction is finished, purifying and diluting to obtain a carbon dot solution.

3. The method according to claim 2, wherein the mass ratio of polyethyleneimine to ammonium citrate is 1.

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

5. The method according to claim 1, wherein the fluorescence spectrum detection in steps (2) and (4) is performed under the following conditions: measuring a fluorescence spectrum by using a fluorescence spectrometer, wherein the widths of an excitation slit and an emission slit of the spectrometer are both 3.5nm, and the integration time is 0.1s; the excitation wavelength of the fluorescence spectrometer is 350nm, the emission wavelength range is 360nm-600nm, and the step length is 1nm.

6. The method of claim 1, wherein Hg in step (2) ²⁺ The volume ratio of the standard solution, the carbon dot solution, the buffer solution and the lake water sample is 1:1:1:1.

7. 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.

8. the method according to claim 1, wherein the volume ratio of the glutathione standard solution to the serum sample in the mixed solution in the step (4) is 1:1; carbon point solution, hg ²⁺ The volume ratio of the solution to the buffer is 1:1:1.

9. the method as claimed in claim 1, wherein in step (4) Hg is fixed ²⁺ The concentration of the solution was 50. Mu. Mol/L.

10. The method according to any one of claims 1 to 9, wherein in step (5), the serum sample to be tested, the carbon dot solution, hg, are added ²⁺ The volume ratio of the solution to the buffer is 1:1:1:1.