Nitrate detection method and hydrogen sulfide detection method based on glutathione carbon quantum dots

Abstract

The invention provides a nitrite detection method and a hydrogen sulfide detection method based on glutathione carbon quantum dots, belongs to the technical field of fluorescent probe detection, and comprises detection of NO2 <-> in a CDs / Fe < 2 + > system and detection of H2S in a CDs / Fe < 3 + > system. Wherein the NO2 <-> detection method comprises the following steps: establishing a fluorescence intensity standard curve equation A, adding NO2 <-> into a CDs / Fe < 2 + > system to oxidize Fe < 2 + > into Fe < 3 + >, adsorbing positively charged Fe < 3 + > on the surface of negatively charged CDs by utilizing an electrostatic attraction effect, quenching the fluorescence of the CDs by virtue of a synergistic effect of static quenching and an inner filter effect, and then testing the fluorescence intensity. And substituting into a fluorescence intensity standard curve equation A, and calculating to obtain the concentration of NO2 <->. The H2S detection method comprises the following steps: establishing a fluorescence intensity standard curve equation B, adding H2S into a CDs / Fe < 3 + > system to reduce Fe < 3 + > into Fe < 2 + > so as to recover the fluorescence of CDs, then testing the fluorescence intensity, and substituting the fluorescence intensity into the fluorescence intensity standard curve equation B. The detection method provided by the invention is wide in linear range, low in detection limit and short in response time.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probe detection, and in particular to a nitrite detection method and a hydrogen sulfide detection method based on glutathione carbon quantum dots. Background Art

[0002] In the food industry, nitrite (NO 2- ) is often used as a food additive in meat products for antibacterial, preservative or color development. However, NO 2- It can react with biogenic amines in the human body to produce carcinogens and has been classified as a Class 2A carcinogen by the World Health Organization. In an era when food safety is receiving widespread attention, it is important to accurately detect NO in food and physiological systems. 2- In addition, accurate detection of hydrogen sulfide (H2S) in food and the environment is particularly important. 2 The content of H2S is also very important. For example, in the food industry, especially in the production of alcohol, yeast uses sulfur-containing amino acids (such as cysteine ​​and methionine) as a nitrogen source during fermentation. This metabolic process may produce hydrogen sulfide, which affects the flavor and quality of the alcohol. In the process of fresh meat spoilage, proteins and sulfur-containing amino acids are decomposed by bacteria metabolism to produce hydrogen sulfide and other by-products. This process not only leads to a decline in the quality of meat products, but also produces unpleasant odors and food safety problems. In addition, H2S is a kind of amino acid that contains sulfide, which is a kind of amino acid that contains sulfide. It is also very important to have a certain amount of H2S in the food industry. In the production of alcohol, yeast uses sulfur-containing amino acids (such as cysteine ​​and methionine) as a nitrogen source during fermentation. This metabolic process may produce hydrogen sulfide, which affects the flavor and quality of the alcohol. In the process of fresh meat spoilage, proteins and sulfur-containing amino acids are decomposed by bacteria metabolism to produce hydrogen sulfide and other by-products. This process not only leads to a decline in the quality of meat products, but also produces unpleasant odors and food safety problems. In addition, H2S is a kind of amino acid that contains sulfide, which is a kind of amino acid that contains sulfide. In addition, H2S is a kind of amino acid that contains sulfide. It is also very important to have a certain amount of H2S in the food industry. In the production of alcohol, yeast uses sulfur-containing amino acids (such as cysteine ​​and methionine) as a nitrogen source during fermentation. This metabolic process may produce hydrogen sulfide, which affects the flavor and quality of the alcohol. In the process of fresh meat spoilage, proteins and sulfur-containing amino acids are decomposed by bacteria metabolism to produce hydrogen sulfide and other by-products. In the process of fresh meat spoilage, proteins and sulfur-containing amino acids are decomposed by bacteria metabolism to produce hydrogen sulfide and other by-products. In this process, H2S 2 S is also a common industrial by-product. It can not only be generated naturally in water bodies, but may also enter the water environment through industrial emissions, thereby causing negative impacts on the ecological environment.

[0003] In the prior art, commonly used detection methods for nitrite include photometry, fluorescence, oscillographic polarography and chromatography, etc., and detection methods for hydrogen sulfide include iodine titration, fluorescence, methylene blue spectrophotometry, mercury salt titration, electrochemical method, gas chromatography and absorption spectroscopy, etc. Among them, fluorescence detection technology has high sensitivity, strong stability and simple operation, and has received widespread attention. The effect of fluorescence detection technology is mainly related to the biocompatibility of fluorescent probes, selectivity for detection objects and sensitivity. How to reasonably use the characteristics of different fluorescent probes to accurately detect a variety of substances and achieve a wider linear range, lower detection limit and shorter response time is one of the key topics that need to be studied urgently.

[0004] In view of this, it is necessary to design a fluorescence detection method with high selectivity, wide detection range, high sensitivity and rapid response to accurately test NO in food or environment. 2- and H 2 S. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present application provides a nitrite detection method and a hydrogen sulfide detection method based on glutathione carbon quantum dots (CDs), by rationally utilizing the characteristics of glutathione carbon quantum dots and Fe 2+ with NO 2- , Fe 3+ With H 2 S, and a fluorescent probe was used to respectively 2- and H 2 The detection of S is used to solve the technical problems of insufficient linear range, low detection limit and long response time caused by insufficient selectivity and sensitivity of fluorescent probes.

[0006] Among them, the present application utilizes the negatively charged surface of glutathione carbon quantum dots to make the positively charged Fe 3+ First, the carbon dots are adsorbed on the negatively charged surface through electrostatic attraction, and then the fluorescence of the carbon dots is quenched through static quenching. At the same time, the optimal excitation wavelength of the carbon quantum dots at 335nm is used to quench the fluorescence of the carbon dots. 3+ The ultraviolet absorption peak at 295nm partially overlaps, making Fe 3+ The fluorescence of the carbon dots can also be further quenched by the inner filter effect; the synergistic effect of static quenching and the inner filter effect is utilized to make the detection method provided in the present application more sensitive, with a wide linear range of detection, a low detection limit and a short response time.

[0007] In the first aspect, the present invention provides a method for detecting nitrite based on glutathione carbon quantum dots. 2+ CDs / Fe and glutathione carbon quantum dots 2+ Add NO into the system 2- , make NO 2- The CDs / Fe 2+ Fe in the system 2+ Oxidized to Fe 3+ , which in turn makes the positively charged Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of the glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of the glutathione carbon quantum dots at 335 nm, making Fe 3+ The fluorescence of the glutathione carbon quantum dots is further quenched by the inner filter effect to reduce the fluorescence intensity of the glutathione carbon quantum dots. 2- The linear relationship between the concentration of NO and the 2- concentration;

[0008] Wherein, the glutathione carbon quantum dots are 2- The detection range is 1-1000 μM, and the detection limit is 0.164 μM.

[0009] In some embodiments, the detection of nitrite comprises the following steps:

[0010] S1, glutathione carbon quantum dot solution and Fe 2+ Solution, NO 2- The solutions were mixed to prepare a standard solution with gradient concentrations. 2+ The solutions were mixed to prepare a blank solution.

[0011] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation A with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa.

[0012] S3, after the sample is removed from the impurities, it is added to the blank solution prepared in step S1, and the sample is incubated at room temperature to obtain the sample solution, and the fluorescence intensity of the sample solution at the optimal emission wavelength is measured under the optimal excitation wavelength, and the fluorescence intensity is substituted into the fluorescence intensity standard curve equation A to obtain NO 2- concentration.

[0013] In some embodiments, in step S1, the method for preparing the standard solution is specifically to prepare n identical glutathione carbon quantum dot solutions, and to add the same volume and the same concentration of Fe 2+ After incubation at room temperature for a period of time, the same volume of NO was added in increasing concentrations. 2- The solution was incubated at room temperature for a period of time to 2+ Oxidized to Fe 3+ , to obtain the standard solution, the Fe in the standard solution 2+ The concentration decreases in a gradient. Specifically, n≥10. The method for preparing the blank solution is specifically to mix the glutathione carbon quantum dot solution with Fe 2+ After the solutions were mixed, they were incubated at room temperature for a period of time to obtain the blank solution. 2+ The concentration and volume of the solution are similar to those of the standard solution containing Fe 2+ The solutions have the same concentration and volume.

[0014] In some embodiments, the glutathione carbon quantum dot solution is prepared by dissolving glutathione in ultrapure water, then placing it in a high pressure reactor, hydrothermally reacting it at 200-220°C for 3-16 hours, cooling it to room temperature, filtering it, and dialyzing it with ultrapure water for 10-14 hours. The concentration of the glutathione carbon quantum dot solution is 2-20 mg / mL. 2+ The concentration of the solution is 100-700 μM. The incubation time is 1-5 min. The impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

[0015] In some embodiments, in step S3, the sample to be tested is bacon, sausage, luncheon meat, ham, bacon or hot dog.

[0016] In the second aspect, the present invention provides a method for detecting hydrogen sulfide based on glutathione carbon quantum dots. 3+ CDs / Fe and glutathione carbon quantum dots 3+ Add H into the system 2 S, make H 2 S will be the CDs / Fe 3+ Fe in the system 3+ Reduction to Fe 2+ , by reducing Fe 3+ The content of CDs / Fe 3+ Fe in the system 3+ The fluorescence quenching effect of the glutathione carbon quantum dots is reduced to restore the fluorescence intensity of the glutathione carbon quantum dots, and finally the fluorescence intensity is compared with H 2 The concentration of S is calculated to be linear within the detectable range. 2 S concentration;

[0017] Wherein, the glutathione carbon quantum dots are 2 The detection range of S was 25-425 μM, and the detection limit was 0.114 μM.

[0018] The fluorescence quenching effect refers to Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of the glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of the glutathione carbon quantum dots at 335 nm, making Fe 3+ The fluorescence of the glutathione carbon quantum dots is further quenched by the inner filter effect.

[0019] In some embodiments, the detection of hydrogen sulfide comprises the following steps:

[0020] S1, glutathione carbon quantum dot solution and Fe 3+ Solution, Na 2 S solution to prepare a standard solution with gradient concentration. 3+ The solutions were mixed to prepare a blank solution.

[0021] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation B with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa.

[0022] S3, after the sample to be tested is removed from impurities, it is added to the blank solution prepared in step S1, and the solution to be tested is obtained after incubation at room temperature, and the fluorescence intensity of the solution to be tested at the optimal emission wavelength is measured under the optimal excitation wavelength, and the fluorescence intensity is substituted into the fluorescence intensity standard curve equation B to obtain H 2 The concentration of S.

[0023] In some embodiments, in step S1, the method for preparing the standard solution is specifically to prepare n identical glutathione carbon quantum dot solutions, and to add the same volume and the same concentration of Fe 3+ After incubation at room temperature for a period of time, the same volume of Na 2 S solution, and continue to incubate at room temperature for a period of time to obtain the standard solution. Specifically, n≥5. The method for preparing the blank solution is specifically to mix the glutathione carbon quantum dot solution with Fe 3+ After the solutions were mixed, they were incubated at room temperature for a period of time to obtain the blank solution. 3+ The concentration and volume of the solution are similar to those of the standard solution containing Fe 3+ The solutions have the same concentration and volume.

[0024] In some embodiments, the glutathione carbon quantum dot solution is prepared by dissolving glutathione in ultrapure water, placing in a high pressure reactor, hydrothermally reacting at 200-220°C for 3-16 hours, cooling to room temperature, filtering, and dialyzing with ultrapure water for 10-14 hours. The concentration of the glutathione carbon quantum dot solution is 2-20 mg / mL. 3+ The concentration of the solution is 100-700 μM. The incubation time is 1-5 min, and further, the incubation time is preferably 3 min. The impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

[0025] In some embodiments, in step S3, the sample to be tested is tap water, beer, wine or river water.

[0026] The beneficial effects of this application are as follows:

[0027] The present application provides a nitrite detection method and a hydrogen sulfide detection method based on glutathione carbon quantum dots (CDs), using a glutathione-protected carbon quantum dot as a fluorescent probe, and rationally utilizing the characteristics of the carbon quantum dot, as well as Fe 2+ with NO 2- , Fe 3+ With H 2 S redox reaction between CDs / Fe 2+ System for NO 2- Detection and in CDs / Fe 3+ System for H 2 Detection of S.

[0028] (1)Fe 3+ There are two effects between Fe and CDs: static quenching and inner filtration effect. These two effects cooperate with each other to make Fe 3+ The fluorescence of CDs can be fully quenched, so that the fluorescence intensity of the carbon dots increases with the Fe 3+ The inner filtration effect mainly changes with the concentration of Fe 3+ The ultraviolet absorption peak at 295 nm partially overlaps with the optimal excitation wavelength of the carbon dots at 335 nm.

[0029] (2)Fe 3+ There is also an electrostatic attraction between the positively charged Fe and CDs, which promotes the generation of static quenching. 3+ It first adsorbs on the surface of negatively charged carbon dots through electrostatic attraction to form a complex, which quenches the fluorescence of the carbon dots.

[0030] (3) The synergistic quenching effect of static quenching and inner filter effect not only avoids the difficulty in recovering the fluorescence of the probe due to quencher overload, but also makes Fe 3+ The high selectivity for glutathione carbon quantum dots significantly improves the detection sensitivity. The nitrite detection method and hydrogen sulfide detection method based on glutathione carbon quantum dots provided in this application have a wide linear range, a low detection limit and a short response time. 2- The linear range of the detection was 1-1000 μM, and the detection limit was 0.164 μM. 2 The linear range of S was 25-425 μM, and the detection limit was 0.114 μM.

[0031] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 Fluorescence spectrum of glutathione carbon quantum dots prepared for this application;

[0034] Figure 2 To detect NO in acetate buffer at different pH values 2 - The obtained fluorescence intensity change graph;

[0035] Figure 3 The fluorescence intensity of glutathione carbon quantum dots in Example 1 of the present application changes with NO 2- Fluorescence spectra of concentration changes;

[0036] Figure 4 NO in Example 1 of the present application 2- When the concentration of glutathione carbon quantum dots is in the range of 1-100 μM, the fluorescence intensity of NO 2- Linear relationship graph of concentration;

[0037] Figure 5 NO in Example 1 of the present application 2- When the concentration of glutathione carbon quantum dots is in the range of 100-1000 μM, the fluorescence intensity of NO 2- Linear relationship graph of concentration;

[0038] Figure 6 The fluorescence intensity curves obtained by testing Example 7 and Comparative Examples 1-2 are the fluorescence intensity curves of the glutathione carbon quantum dot solution and the fluorescence intensity curve of NO in Example 1. 2 - Comparison of the fluorescence intensity curves corresponding to the solution concentration of 600 μM;

[0039] Figure 7 The paper-based fluorescent sensor loaded with glutathione carbon quantum dots was used to detect NO in Examples 8-10. 2- Observed fluorescence changes; where Figure 7 (a) Figure 7 (c) Figure 7 (e) All the paper-based fluorescent sensors were added with Fe 2+ , images under UV light and natural light respectively after drying; Figure 7 (b) Figure 7 (d) Figure 7 (f) Fe was added to the surface of the paper-based fluorescence sensor. 2+ + bacon extract, Fe 2+ +Sausage extract, Fe 2+ + Images of luncheon meat extract after drying under UV light and natural light;

[0040] Figure 8 To detect H in acetate buffer at different pH values 2 Fluorescence intensity change diagram obtained by S;

[0041] Fig. 9 The fluorescence intensity of glutathione carbon quantum dots in Example 9 of the present application changes with H 2 Fluorescence spectra of S concentration changes;

[0042] Fig.10 H in Example 11 of the present application 2 When the concentration of S was in the range of 25-425 μM, the fluorescence intensity of glutathione carbon quantum dots was similar to that of H 2 Linear relationship diagram of S concentration;

[0043] Fig.11 The fluorescence intensity curve obtained by the test of Comparative Example 3-4 is the fluorescence intensity curve of the glutathione carbon quantum dot solution and the fluorescence intensity curve of Na in Example 11. 2 Comparison of the fluorescence intensity curves corresponding to the S solution when the concentration is 400 μM;

[0044] Fig.12 The paper-based fluorescence sensor loaded with glutathione carbon quantum dots was used to detect H in Examples 15-17. 2 S observed fluorescence change diagram; where, Fig.12 (a) The fluorescence change of the paper-based fluorescence sensor under ultraviolet light after the paper-based fluorescence sensor was sealed with fresh meat for 0h and 2h; Fig.12 (b) The fluorescence change of the paper-based fluorescence sensor under ultraviolet light after the paper-based fluorescence sensor was sealed with the spoiled meat for 0h and 2h;

[0045] Fig.13 This is a comparison chart of the selectivity of different ions towards glutathione carbon quantum dots. DETAILED DESCRIPTION

[0046] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0048] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0049] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0050] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0051] The effect of fluorescence detection technology is mainly related to the biocompatibility of fluorescent probes, selectivity for the detected object and sensitivity. How to rationally utilize the characteristics of different fluorescent probes to accurately detect a variety of substances and achieve a wider linear range, lower detection limit and shorter response time is one of the key topics that urgently need to be studied.

[0052] In order to solve the technical problems of insufficient linear range, low detection limit and long response time caused by insufficient selectivity and sensitivity of fluorescent probes, the present application provides a nitrite detection method and a hydrogen sulfide detection method based on glutathione carbon quantum dots, wherein the negatively charged surface of glutathione carbon quantum dots is used to make the positively charged Fe 3+First, it is adsorbed on the negatively charged carbon dot surface through electrostatic attraction to form a complex, which promotes Fe 3+ The fluorescence of the carbon dots was quenched by static quenching. At the same time, the optimal excitation wavelength of the carbon quantum dots at 335 nm was used to quench the fluorescence of the carbon dots. 3+ The ultraviolet absorption peak at 295nm partially overlaps, making Fe 3+ The fluorescence of the carbon dots can also be further quenched by the inner filter effect; the two effects of static quenching and inner filter effect work together to make the detection method provided in the present application more sensitive, with a wide linear range of detection, a low detection limit and a short response time.

[0053] In the first aspect, the present invention provides a method for detecting nitrite based on glutathione carbon quantum dots. 2+ CDs / Fe and glutathione carbon quantum dots 2+ Add NO into the system 2- , make NO 2- CDs / Fe 2+ Fe in the system 2+ Oxidized to Fe 3+ , positively charged Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of glutathione carbon quantum dots at 335 nm, making Fe 3+ The fluorescence of glutathione carbon quantum dots is further quenched by the inner filter effect to reduce the CDs / Fe 2+ The fluorescence intensity of the system was then compared with that of NO 2- The linear relationship of NO concentration within the detectable range was calculated. 2- concentration.

[0054] Among them, glutathione carbon quantum dots can effectively inhibit NO 2- The detection range is 1-1000 μM, and the detection limit is 0.164 μM.

[0055] Specifically, the detection of nitrite includes the following steps:

[0056] S1, glutathione carbon quantum dot solution and Fe 2+ Solution, NO 2- The solutions were mixed to prepare a standard solution with gradient concentrations. 2+ The solutions were mixed to prepare a blank solution.

[0057] In some embodiments, the preparation method of the glutathione carbon quantum dot solution is to dissolve the glutathione powder in ultrapure water, then put it into a high-pressure reactor, hydrothermally react at 200-220°C for 3-16 hours, cool to room temperature, filter, and dialyze with ultrapure water for 10-14 hours. The concentration of the prepared glutathione carbon quantum dot solution is 2-20 mg / mL. Furthermore, the hydrothermal reaction uses a polytetrafluoroethylene-lined high-pressure reactor.

[0058] The optimal excitation wavelength of glutathione carbon quantum dots is 335nm, and the optimal emission wavelength is 430nm. The carbon quantum dots have small particle size, good dispersibility, high storage stability, high salt resistance and UV resistance, strong and stable fluorescence emission, and are very effective for NO 2- and H 2 S has good selectivity, is not easily interfered by other ions, amino acids and small molecules, and has a negative charge on its surface.

[0059] In some embodiments, the method for preparing the standard solution is specifically to take n portions of the glutathione carbon quantum dot solution prepared in step S1, and add the same volume and the same concentration of Fe 2+ After the first incubation at room temperature, the same volume of NO was added in increasing concentrations. 2- The solution was incubated for the second time at room temperature to obtain a set of standard solutions.

[0060] Where n≥10, Fe 2+ The concentration of the solution is 100-700 μM, and the incubation time is 1-5 min. Preferably, the first incubation time is 1 min, the second incubation time is 5 min, and NO 2- The concentration gradient of the solution is 1μM, 2μM, 3μM, 4μM, 5μM, 10μM, 15μM, 20μM, 25μM, 30μM, 35μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 120μM, 140μM, 160μM, 180μM, 200μM, 250μM, 300μM, 350μM, 400μM, 450μM, 500μM, 600μM, 700μM, 800μM, 900μM, 1000μM.

[0061] In some embodiments, the blank solution is prepared by taking the glutathione carbon quantum dot solution prepared in step S1 and adding Fe 2+ Solution was incubated at room temperature for a period of time. 2+ The concentration and volume of the solution are similar to those of the standard solution with Fe added 2+The concentration and volume of the solution are the same. The incubation time is 1-5 minutes. Preferably, the incubation time is 1 minute.

[0062] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S2 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation A with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa.

[0063] S3, after the impurity treatment, the sample to be tested is added to the blank solution prepared in step S1, and the test solution is obtained after incubation at room temperature, and the fluorescence intensity of the test solution at the optimal emission wavelength is measured under the optimal excitation wavelength, and the fluorescence intensity is substituted into the fluorescence intensity standard curve equation A obtained in step S2 to obtain NO 2- concentration.

[0064] In some embodiments, the sample to be tested is bacon, sausage, luncheon meat, ham, bacon or hot dog, and the incubation time is 1-5 minutes. Preferably, the incubation time is 5 minutes.

[0065] In some embodiments, the impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

[0066] In the second aspect, the present invention provides a method for detecting hydrogen sulfide based on glutathione carbon quantum dots, wherein the method comprises: 3+ CDs / Fe and glutathione carbon quantum dots 3+ Add H into the system 2 S, H 2 SCDs / Fe 3+ Fe in the system 3+ Reduction to Fe 2+ , by reducing Fe 3+ The content of CDs / Fe 3+ Fe in the system 3+ The fluorescence quenching effect of glutathione carbon quantum dots was reduced to restore the fluorescence intensity of glutathione carbon quantum dots. Finally, the fluorescence intensity was compared with H 2 The linear relationship between S concentration and H was calculated within the detectable range. 2 S concentration. Glutathione carbon quantum dots on H 2 The detection range of S was 25-425 μM, and the detection limit was 0.114 μM.

[0067] Among them, the fluorescence quenching effect refers to Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of glutathione carbon quantum dots at 335 nm, making Fe3+ The fluorescence of glutathione carbon quantum dots is further quenched through the inner filter effect.

[0068] Specifically, the detection of hydrogen sulfide includes the following steps:

[0069] S1, glutathione carbon quantum dot solution and Fe 3+ Solution, Na 2 S solution to prepare a standard solution with gradient concentrations. 3+ The blank solution was prepared by mixing the solutions. 2 S is H 2 The source of S.

[0070] In some embodiments, the preparation method of glutathione carbon quantum dot solution is to dissolve glutathione powder in ultrapure water, then put it into a high-pressure reactor, hydrothermally react at 200-220°C for 3-16 hours, cool to room temperature, filter, and dialyze with ultrapure water for 10-14 hours. The concentration of the prepared glutathione carbon quantum dot solution is 2-20 mg / mL. Furthermore, the hydrothermal reaction uses a polytetrafluoroethylene-lined high-pressure reactor. The optimal excitation wavelength of glutathione carbon quantum dots is 335 nm, and the optimal emission wavelength is 430 nm.

[0071] In some embodiments, the method for preparing the standard solution is specifically to take n portions of the glutathione carbon quantum dot solution prepared in step S1, and add the same volume and the same concentration of Fe 3+ After the first incubation at room temperature, the same volume of Na 2 S solution, and continue to incubate for the second time at room temperature to obtain a set of standard solutions.

[0072] Where n≥5, Fe 3+ The concentration of the solution is 100-700 μM, and the incubation time is 1-5 min. Preferably, the first incubation time is 1 min, the second incubation time is 3 min, and Na 2 The concentration gradient of S solution was 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, and 425 μM.

[0073] In some embodiments, the blank solution is prepared by taking the glutathione carbon quantum dot solution prepared in step S1 and adding Fe 3+ Solution was incubated at room temperature for a period of time. 3+ The concentration and volume of the solution are similar to those of the standard solution with Fe added 3+The concentration and volume of the solution are the same. The incubation time is 1-5 minutes. Preferably, the incubation time is 1 minute.

[0074] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation B with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa.

[0075] S3, after removing impurities from the sample to be tested, add it to the blank solution prepared in step S1, incubate at room temperature to obtain the test solution, measure the fluorescence intensity of the test solution at the optimal emission wavelength under the optimal excitation wavelength, and substitute the fluorescence intensity into the fluorescence intensity standard curve equation B to obtain H 2 The concentration of S.

[0076] In some embodiments, the sample to be tested is tap water, beer, wine or river water, and the incubation time is 1-5 min. Preferably, the incubation time is 3 min.

[0077] In some embodiments, the impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

[0078] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0079] Example 1

[0080] Example 1 provides a nitrite detection method based on glutathione carbon quantum dots, comprising the following steps:

[0081] S1, prepare standard solutions with gradient concentrations and blank solutions respectively.

[0082] S11, 0.5 g of glutathione powder was dissolved in 120 mL of ultrapure water, and then placed in a polytetrafluoroethylene-lined high-pressure reactor, hydrothermally reacted at 220° C. for 3 h, cooled to room temperature, filtered, and dialyzed with ultrapure water for 10 h to obtain a glutathione carbon quantum dot solution with a concentration of 2.5 mg / mL.

[0083] The prepared glutathione carbon quantum dots were characterized by fluorescence, and the obtained fluorescence spectrum is shown in Figure 1 As shown, it can be seen that the optimal excitation wavelength of the glutathione carbon quantum dots prepared in step S1 is 335 nm, and the optimal emission wavelength is 430 nm.

[0084] Take 10 portions of 0.3 mL of the prepared glutathione carbon quantum dot solution and add 12 μL of 0.1 M Fe 2+ The solution was incubated at room temperature for 1 min, and then 12 μL of 0.1 M NO 2- The solution was diluted to 2 mL with acetate buffer with pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively, and incubated at room temperature for 5 min to obtain the test solution. Test and calculate the fluorescence intensity difference between the glutathione carbon quantum dot solution and the test solution, that is, the fluorescence intensity change value. Figure 2 The results show that NO was detected in acetate buffer at different pH values. 2- The fluorescence intensity change graph obtained shows that when the pH value of the acetate buffer solution is 3, the fluorescence intensity change value is the largest, indicating that glutathione carbon quantum dots can detect NO 2- The most suitable pH value is 3.

[0085] S12, take 34 portions (0.3 mL) of the glutathione carbon quantum dot solution prepared in step S11, and add 12 μL of 0.1 M Fe 2+ The solution was incubated at room temperature for 1 min. Then, 2, 4, 6, 8, and 10 μL of 1 mM NO were added to 5 of them. 2 - 7 solutions were added with 2, 3, 4, 5, 6, 7, and 8 μL of 10 mM NO 2 - solution, and 11 portions were added with 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, and 20 μL of 50 mM NO 2- 11 portions were added with 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, and 20 μL of 100 mM NO 2- Next, the above-mentioned NO 2- The solution was diluted to 2 mL with pH 3 acetate buffer to make NO 2- The concentrations were 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM and 1000 μM, respectively. Incubate at room temperature for 5 min to obtain a set of standard solutions.

[0086] S13, take 0.3 mL of the glutathione carbon quantum dot solution prepared in step S11, add 12 μL of 0.1 M Fe 2+ The solution was diluted to 2 mL with acetate buffer at pH 3 and incubated at room temperature for 1 min to obtain a blank solution.

[0087] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and obtaining the fluorescence intensity of the glutathione carbon quantum dots with NO 2- For concentration curves, please refer to Figure 3 As shown, it can be seen that the CDs / Fe 2+ Add NO to the system 2- After that, the fluorescence intensity of glutathione carbon quantum dots decreased, and NO 2- The higher the concentration, the lower the fluorescence intensity.

[0088] See also Figures 4 to 5 As shown, it is a linear relationship diagram of the fluorescence intensity standard curve A, and the corresponding fluorescence intensity standard curve equation is as follows:

[0089] (1)Y=-89.99X+8123.61,R 2 =0.999, linear range is 1-40μM;

[0090] (2)Y=-30.63X+5720.29,R 2 =0.993, linear range 40-100 μM;

[0091] (3)Y=-8.04X+3554.05,R 2 =0.997, linear range is 100-200 μM;

[0092] (4)Y=-1.98X+2317.36,R 2 =0.985, linear range is 200-600 μM;

[0093] (5)Y=-0.58X+1510.99,R 2 =0.995, linear range is 600-1000 μM;

[0094] Where Y is the fluorescence intensity and X is the NO 2- concentration, and R is the linear fitting constant.

[0095] Therefore, the standard curve equation is applicable to NO 2- The detection range is 1-1000μM. According to the calculation formula of the detection limit (LOD = 3σ / m), NO 2- The detection limit was 0.164 μM.

[0096] S3, add 20 g of bacon to 100 mL of deionized water and grind into a homogenate, then add 10 mL of 0.5 M NaOH solution and 10 mL of 0.42 M ZnSO 4 The solution was heated in a water bath at 60°C for 10 min, diluted to 250 mL, filtered to obtain a sample solution, and the impurity removal treatment was completed. Next, 0.312 mL of a blank solution was prepared according to the method provided in step S13, 12 μL of the sample solution after impurity removal was added to the blank solution, and the solution was diluted to 2 mL with an acetate buffer solution of pH=3, and incubated at room temperature for 5 min to obtain a test solution. The fluorescence intensity of the test solution at the optimal emission wavelength was measured at the optimal excitation wavelength, and the obtained fluorescence intensity was substituted into the fluorescence intensity standard curve equation obtained in step S2 to obtain the NO in the test solution. 2- concentration.

[0097] Embodiment 2-6

[0098] The difference between Example 2-6 and Example 1 is that in step S3, the samples to be tested are sausage, luncheon meat, ham, bacon, and hot dog, respectively, and the rest is substantially the same as Example 1 and will not be described in detail here.

[0099] Example 7

[0100] The difference between Example 7 and Example 1 is that in Example 7, NO is first added 2- , then add Fe 2+ Specifically, in Example 7, the glutathione carbon quantum dot solution was prepared by the same method as in Example 1. 0.3 mL of the glutathione carbon quantum dot solution was taken, and 12 μL of 0.1 M NO 2 - The solution was incubated at room temperature for 1 min, and then 12 μL of 0.1 M Fe 2 + The solution was diluted to 2 mL with acetate buffer at pH 3, incubated at room temperature for 5 min to obtain the test solution, and its fluorescence intensity was tested.

[0101] Comparative Example 1

[0102] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, only NO 2- , without adding Fe 2+ Specifically, in Comparative Example 1, a glutathione carbon quantum dot solution was prepared by the same method as in Example 1. 0.3 mL of the glutathione carbon quantum dot solution was taken, and 12 μL of 0.1 M NO 2 -The solution was diluted to 2 mL with acetate buffer at pH 3, incubated at room temperature for 5 min to obtain the test solution, and its fluorescence intensity was tested.

[0103] Comparative Example 2

[0104] The difference between Comparative Example 2 and Example 1 is that in Comparative Example 2, only Fe 2+ , do not add NO 2- Specifically, in Comparative Example 2, a glutathione carbon quantum dot solution was prepared by the same method as in Example 1. 0.3 mL of glutathione carbon quantum dot solution was taken, and 12 μL of 0.1 M Fe 2+ The solution was diluted to 2 mL with acetate buffer at pH 3, incubated at room temperature for 1 min to obtain the test solution, and its fluorescence intensity was tested.

[0105] See also Figure 6 As shown, the fluorescence intensity curves obtained by testing Example 7 and Comparative Examples 1-2 are compared with the fluorescence intensity curves of the glutathione carbon quantum dot solution and the fluorescence intensity curve of NO in Example 1. 2- Comparison of the fluorescence intensity curves when the solution concentration is 600 μM. It can be seen that only when there is Fe 2+ and NO 2- When the fluorescence intensity of glutathione carbon quantum dots is significantly reduced, the Fe 2+ with NO 2- The order of addition has almost no effect on the fluorescence intensity of glutathione carbon quantum dots.

[0106] This is because the optimal fluorescence excitation wavelength of the glutathione carbon quantum dots used in this application is 335nm, and the surface is negatively charged. 2+ and NO 2- When Fe 2+ +2H + +NO 2- =Fe 3+ +NO↑+H 2 O, NO 2- Rapidly convert Fe 2+ Oxidized to Fe 3+ , so that the Fe in the glutathione carbon quantum dot solution 3+ As the concentration increases, the positively charged Fe 3+ First, it is adsorbed on the negatively charged carbon dot surface through electrostatic attraction to form a complex, and then the fluorescence of the carbon dot is quenched by static quenching. 3+ The ultraviolet absorption peak at 295 nm and the optimal excitation wavelength of the carbon dots at 335 nm partially overlap, making Fe 3+The fluorescence of the carbon dots can also be further quenched by the inner filter effect, and finally the fluorescence intensity of the glutathione carbon quantum dots can be adjusted along with the NO in the system. 2- In addition, Fe 3+ The static quenching and inner filtration effects between glutathione carbon quantum dots work synergistically to greatly improve the sensitivity of detection.

[0107] Example 8

[0108] Example 8 provides a method for detecting nitrite based on glutathione carbon quantum dots, using a paper-based fluorescent sensor based on glutathione carbon quantum dots to detect nitrite in bacon, comprising the following steps:

[0109] S1, preparation of glutathione carbon quantum dot paper-based fluorescence sensor.

[0110] S11, 0.5 g of glutathione powder was dissolved in 120 mL of ultrapure water, and then placed in a polytetrafluoroethylene-lined high-pressure reactor, hydrothermally reacted at 220° C. for 3 h, cooled to room temperature, filtered, and dialyzed with ultrapure water for 10 h to obtain a glutathione carbon quantum dot solution with a concentration of 2.5 mg / mL.

[0111] S12, cut the filter paper into 2×1.5cm filter paper strips, soak them in 3M NaOH solution for 30min to activate the filter paper strips, then wash the filter paper strips with ultrapure water until neutral, and air dry them at room temperature. Subsequently, soak the activated filter paper strips in a weakly acidic 2.5% glutaraldehyde aqueous solution, shake at 25°C for 30min, wash them with ultrapure water after standing for a period of time, and air dry them at room temperature. Next, soak the filter paper strips in the glutathione carbon quantum dot solution prepared in step S11, shake them in a constant temperature shaker at 25°C for 2h, wash the filter paper strips with ultrapure water and place them at room temperature to air dry them naturally, and obtain a paper-based fluorescent sensor loaded with glutathione carbon quantum dots.

[0112] S2, add 20 g of bacon to 100 mL of deionized water and grind into a homogenate, then add 10 mL of 0.5 M NaOH solution and 10 mL of 0.42 M ZnSO 4 The solution was heated in a water bath at 60°C for 10 min, diluted to 250 mL, and filtered to obtain the test solution.

[0113] S3, adding 0.1 M Fe 2+ After the solution is naturally air-dried, the test solution obtained in step S2 is dropped on the filter paper strip, and naturally air-dried again, and the fluorescence change of the filter paper strip before and after the addition of the test solution is observed.

[0114] Examples 9-10

[0115] The difference between Examples 9-10 and Example 8 is that in step S2, the bacon is replaced by sausage and luncheon meat respectively. The rest is the same as Example 8 and will not be described again.

[0116] See also Figure 7 , which is a paper-based fluorescent sensor loaded with glutathione carbon quantum dots for detecting NO in Examples 8-10 2- Observed fluorescence change diagram. It can be seen that after the test solution is added to the surface of the paper-based fluorescence sensor loaded with glutathione carbon quantum dots, the fluorescence of the paper-based fluorescence sensor is significantly reduced, further proving that the detection of NO based on glutathione carbon quantum dots 2- feasibility.

[0117] The CDs-Fe 2+The nitrite detection method under the system was compared with 10 nitrite detection methods in the prior art, and the results are shown in the table below. (The source of control sample 1 is L. Gan, Q. Su, Z. Chen, X. Yang, Exploration of pH-responsive carbon dots for detecting nitrite and ascorbic acid, Applied Surface Science, 530 (2020) 147269.; the source of control sample 2 is W. Li, S. Huang, H. Wen, Y. Luo, J. Cheng, Z. Jia, P. Han, W. Xue, Fluorescent recognition and selective detection of nitrite ions with carbon quantum dots, Anal Bioanal Chem, 412 (2020) 993-1002.; the source of control sample 3 is J. Nam, IB Jung, B. Kim, SMLee, SEKim, KNLee, DSShin, A colorimetric hydrogel biosensor for rapid detection of nitrite ions, Sensors and Actuators B:Chemical,270(2018)112-118.; The source of control sample 4 is Y. Zhang, J. Nie, H. Wei, H. Xu, Q. Wang, Y. Cong, J. Tao, Y. Zhang, L. Chu, Y. Zhou, X. Wu, Electrochemical detection of nitrite ions using Ag / Cu / MWNT nanoclusterselectrodeposited on aglassy carbon electrode, Sensors and Actuators B:Chemical,258(2018)1107-1116.; The source of control sample 5 is KK Karali, L. Sygellou, C.D.Stalikas, Highly fluorescent N-doped carbon nanodots as an effective multi-probe quenching system for the determination of nitrite, nitrate and ferric ions in food matrices, Talanta, 189 (2018) 480-488.; The source of control sample 6 is L. Singh, N. Ranjan, Highly Selective and Sensitive Detection of Nitrite Ion by an Unusual Nitration of a Fluorescent Benzimidazole, Journal of the American Chemical Society, 145 (2023) 2745-2749.; The source of control sample 7 is X. Yang, M. Zhang, J. Xu, S. Wen, Y. Zhang, J. Zhang, Synthesis of fluorescent terbium-based metal-organic framework for quantitative detection of nitrite and ferric ions in water samples, Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 253 (2021) 119553.; The source of control sample 8 is M. Zan, L. Rao, H. Huang, W. Xie, D. Zhu, L. Li, X. Qie, S. S. Guo, X. Z. Zhao, W. Liu, W. F. Dong, A strong green fluorescent nanoprobe for highly sensitive and selective detection of nitrite ions based on phosphorus and nitrogen co-doped carbon quantum dots, Sensors and Actuators B: Chemical, 262 (2018) 555-561.; The source of control sample 9 is K. Chaiendoo, K. Ngamdee, W. Limbut, C.Saiyasombat,W.Busayaporn,S.Ittisanronnachai,V.Promarak,K.Promsuwan,P.Thavarungkul,P.Kanatharana,W.Ngeontae,Gold nanoparticle-based cascadereaction-triggered fluorogenicity for highly selective nitrite ion detection in forensic samples,Microchemical Journal, 168 (2021) 106470.; The source of control sample 10 is Y. Kong, Q. Cheng, Y. He, Y. Ge, J. Zhou, G. Song, A dual-modal fluorometric and colorimetric nanoprobe based on graphitic carbon nitrite quantum dots and Fe(II)-bathophenanthroline complex for detection of nitrite in sausage andwater, Food Chem,312(2020)126089.).

[0118]

[0119]

[0120] Comparing the data in the above table, it can be seen that compared with the control samples 1-10, the CDs-Fe 2+ The nitrite detection method under the system has at least one advantage in a wider linear range, a lower detection limit or a shorter response time. Therefore, the CDs-Fe 2+ The nitrite detection method under this system has a wide range of application and good detection sensitivity.

[0121] Embodiment 11

[0122] Example 11 provides a method for detecting hydrogen sulfide based on glutathione carbon quantum dots, comprising the following steps:

[0123] S1, prepare standard solutions with gradient concentrations and blank solutions respectively.

[0124] S11, 0.5 g of glutathione powder was dissolved in 120 mL of ultrapure water, and then placed in a polytetrafluoroethylene-lined high-pressure reactor, hydrothermally reacted at 220° C. for 3 h, cooled to room temperature, filtered, and dialyzed with ultrapure water for 10 h to obtain a glutathione carbon quantum dot solution with a concentration of 2.5 mg / mL.

[0125] Take 10 portions of 0.3 mL of the prepared glutathione carbon quantum dot solution and add 12 μL of 0.1 M Fe 3+ The solution was incubated at room temperature for 1 min, and then 8 μL of 0.1 M Na 2 S solution, dilute to 2 mL with acetate buffer with pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, respectively, and incubate at room temperature for 3 min to obtain the test solution. Test and calculate the fluorescence intensity difference between the glutathione carbon quantum dot solution and the test solution, that is, the fluorescence intensity change value. Figure 8 The results show that H 2 From the fluorescence intensity change graph obtained by S, we can see that when the pH value of the acetate buffer solution is 3, the fluorescence intensity change value is the largest, indicating that glutathione carbon quantum dots can detect H 2 The optimum pH value of S is 3.

[0126] S12, take 12 portions of 0.3 mL of the glutathione carbon quantum dot solution prepared in step S11, and add 12 μL of 0.1 M Fe 3+ After incubation at room temperature for 1 min, 0.5, 1, 2, 3, 4, 5, 6, 6.5, 7, 7.5, 8, and 8.5 μL of 0.1 M Na 2 S solution, and then use pH = 3 acetate buffer to make the volume 2mL, so that Na 2 The concentrations of S were 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, and 425 μM, respectively. After incubation at room temperature for 3 min, a set of standard solutions was obtained.

[0127] S13, take 0.3 mL of the glutathione carbon quantum dot solution prepared in step S11, add 12 μL of 0.1 M Fe 3+ The solution was diluted to 2 mL with acetate buffer at pH 3 and incubated at room temperature for 1 min to obtain a blank solution.

[0128] S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and obtaining the fluorescence intensity of the glutathione carbon quantum dots with NO 2-For concentration curves, please refer to Fig. 9 As shown, it can be seen that the CDs / Fe 3+ Add H into the system 2 After S, the fluorescence intensity of glutathione carbon quantum dots increased, and H 2 The higher the S concentration, the stronger the fluorescence intensity.

[0129] See also Fig.10 As shown, it is a linear relationship diagram of the fluorescence intensity standard curve B, and the corresponding fluorescence intensity standard curve equation is as follows:

[0130] (1)Y=6.15X+1780.43,R 2 =0.983, linear range 25-300 μM;

[0131] (2)Y=20.78X-2525.64,R 2 =0.996, linear range 300-425 μM;

[0132] Where Y is the fluorescence intensity and X is H 2 S is the concentration, and R is the linear fitting constant.

[0133] Therefore, the standard curve equation is applicable to H 2 The detection range of S is 25-425μM. According to the calculation formula of the detection limit (LOD = 3σ / m), H 2 The detection limit of S was 0.114 μM.

[0134] S3, take 8 μL of wine as the sample to be tested, centrifuge, filter, add to 0.308 mL of blank solution prepared according to the method provided in step S13, dilute to 2 mL with acetate buffer of pH=3, incubate at room temperature for 3 min to obtain the solution to be tested, measure the fluorescence intensity of the solution to be tested at the optimal emission wavelength under the optimal excitation wavelength, and substitute the obtained fluorescence intensity into the fluorescence intensity standard curve equation obtained in step S2 to obtain H in the solution to be tested. 2 S concentration.

[0135] Examples 12-14

[0136] The difference between Examples 12-14 and Example 11 is that in step S3, the samples to be tested are tap water, beer, and river water, respectively, and the rest is substantially the same as Example 11 and will not be described again.

[0137] Comparative Example 3

[0138] The difference between Comparative Example 3 and Example 11 is that in Comparative Example 3, only Na 2 S, without Fe 3+Specifically, in Comparative Example 3, a glutathione carbon quantum dot solution was prepared by the same method as in Example 11. 0.3 mL of glutathione carbon quantum dot solution was taken, and 8 μL of 0.1 M Na 2 S solution was diluted to 2 mL with acetate buffer at pH 3, incubated at room temperature for 3 min to obtain the test solution, and its fluorescence intensity was tested.

[0139] Comparative Example 4

[0140] The difference between Comparative Example 4 and Example 11 is that in Comparative Example 4, only Fe 3+ , without adding Na 2 Specifically, Comparative Example 4 adopts the same method as Example 11 to prepare a glutathione carbon quantum dot solution, take 0.3 mL of glutathione carbon quantum dot solution, add 12 μL of 0.1 M Fe 3+ The solution was diluted to 2 mL with acetate buffer at pH 3, incubated at room temperature for 1 min to obtain the test solution, and its fluorescence intensity was tested.

[0141] See also Fig.11 As shown, the fluorescence intensity curves obtained by the test of Comparative Example 3-4 and the fluorescence intensity curve of the glutathione carbon quantum dot solution and the Na 2 Comparison of the fluorescence intensity curves when the concentration of S solution is 400 μM. 2 S solution, the fluorescence intensity of the glutathione carbon quantum dots solution remained almost unchanged; only when Fe 3+ When the system contains Na 2 S and Fe 3+ When , the fluorescence intensity of glutathione carbon quantum dots solution increases.

[0142] This is because the Fe 3+ First, the fluorescence of carbon quantum dots is quenched to the maximum extent through the synergistic effect of static quenching and inner filter effect, and then the fluorescence of CDs / Fe 3+ Adding Na to the system 2 S, Na reacts in the system 2 S+2H + =H 2 S↑+2Na + and 2Fe 3+ +H 2 S=2Fe 2+ +S+2H + , H 2 S rapidly converts Fe 3+ Reduction to Fe 2+ , so that Fe 3+The concentration decreases, and the fluorescence of glutathione carbon quantum dots recovers rapidly. This synergistic quenching effect of static quenching and inner filter effect avoids the disadvantage that the fluorescence may be difficult to recover due to quencher overload, thereby reducing the detection sensitivity.

[0143] Embodiment 15

[0144] Example 15 provides a method for detecting hydrogen sulfide based on glutathione carbon quantum dots, using a paper-based fluorescent sensor based on glutathione carbon quantum dots to detect the freshness of chicken, comprising the following steps:

[0145] S1. Prepare a glutathione carbon quantum dot paper-based fluorescence sensor according to the same method as step S1 in Example 8.

[0146] S2, add 0.1M Fe 3+ The solution was naturally air-dried and then sealed with 20 g of fresh chicken, and the fluorescence changes of the filter paper strips were recorded at 0 h and 2 h.

[0147] S3, adding 0.1 M Fe 3+ The solution was naturally air-dried and then sealed with 20g of spoiled chicken, and the fluorescence changes of the filter paper strips were recorded at 0h and 2h. Among them, the spoiled chicken was obtained by storing fresh chicken at 30℃ for 48h.

[0148] Examples 16-17

[0149] The difference between Examples 16-17 and Example 15 is that chicken is replaced with fish and pork respectively. The rest is the same as Example 15 and will not be repeated here.

[0150] See also Fig.12 As shown, the paper-based fluorescence sensor loaded with glutathione carbon quantum dots is used to detect H in Examples 15-17. 2 Figure 2. The fluorescence change diagram observed by S. It can be seen that compared with fresh meat, the fluorescence enhancement of the paper-based fluorescence sensor after being sealed with the rotten meat for 2 hours is more obvious, which further proves that the detection of H based on glutathione carbon quantum dots can be realized. 2 The feasibility of S.

[0151] The CDs-Fe 3+The hydrogen sulfide detection method under the system is compared with 10 hydrogen sulfide detection methods in the prior art, and the results are shown in the table below. (Among them, the source of control sample 11 is JRXu, Y.Zheng, TZLiu, BXZhao, KMWang, A turn-on fluorescent probe for the detection of hydrogen sulfide and thiophenol in water and beer samples, Journal of Food Composition and Analysis, 128 (2024) 106038.; the source of control sample 12 is G.Liu, H.Ge, R.Yin, L.Yu, C.Sun, W.Dong, Z.Sun, KA Lamry, HM Marwani, S.Wang, Carbon dots tailored with a fluorophore for sensitive and selective detection of hydrogen sulfide based on a ratiometric fluorescence signal, Analytical Methods, 12 (2020) 1617-1623.; the source of control sample 13 is X.Jia, W.Li, Z.Guo, Z.Guo, Y.Li, P.Zhang, C.Wei, X.Li, An NBD-Based Mitochondrial Targeting Ratiometric Fluorescent Probe for Hydrogen Sulfide Detection, Chemistry Select, 4 (2019) 8671-8675.; The source of reference sample 14 is Q. Gong, Y. Lai, W. Lin, A dual-color ESIPT-based probe for simultaneous detection of hydrogen sulfide and hydrazine, Journal of materials chemistry. B, 12 (2024) 5150-5156.; The source of reference sample 15 is Q. Chen, P. Xing, Y. Xu, H. Li, S.Sun, A Selective FluorescentSensor for Fast Detection of Hydrogen Sulfide in Red Wine, Chinese Journal of Chemistry, 35 (2017) 477-482.; The source of control sample 16 is W.Chen, A.Pacheco, Y.Takano, JJDay, K.Hanaoka, M.Xian, A Single Fluorescent Probe to Visualize Hydrogen Sulfide and Hydrogen Polysulfides with Different Fluorescence Signals, Angewandte Chemie, 55(2016)9993-9996.; The source of control sample 17 is H.Wang, D.Yang, R.Tan, ZJZhou, R.Xu, JFZhang, Y.Zhou, A cyanine-based colorimetric and fluorescence probe for detection of hydrogen sulfide in vivo, Sensors and Actuators B: Chemical, 247 (2017) 883-888.; the source of control sample 18 is F. Kong, X. Wang, J. Bai, X. Li, C. Yang, Y. Li, K. Xu, B. Tang, A "double-locked" probe for the detection of hydrogen sulfide in aviscous system, Chem Commun (Camb), 57 (2021) 6604-6607.; the source of control sample 19 is J. Gao, Q. Li, C. Wang, H. Tan, Copper (II)-mediated fluorescence of lanthanide coordination polymers doped with carbon dots for ratiometric detection of hydrogen sulfide, Sensors and Actuators B: Chemical, 253 (2017) 27-33.; the source of control sample 20 is YLPak, J. Li, KCKo, G. Kim, JY Lee, J.Yoon, Mitochondria-Targeted Reaction-BasedFluorescent Probe for Hydrogen Sulfide, Anal Chem, 88 (2016) 5476-5481.).

[0152]

[0153]

[0154] Comparing the data in the above table, it can be seen that compared with the control samples 11-20, the CDs-Fe 3+ The hydrogen sulfide detection method under the system has at least one advantage in a wider linear range, a lower detection limit or a shorter response time. Therefore, the CDs-Fe 3+ The hydrogen sulfide detection method under this system has a wide range of applications and good detection sensitivity.

[0155] The selectivity of different ions for glutathione carbon quantum dots was tested by fluorescence method. Specifically, 1 portion of 0.3 mL of glutathione carbon quantum dot solution was taken as blank solution; 30 portions of 0.3 mL of glutathione carbon quantum dot solution were taken and 0.1 M Na + , K + , Ca 2+ Mg 2+ 、Zn 2+ , Cu 2+ 、Ni + 、Co 2+ , Mn 2+ , Fe 2+ Cr 3+ , Cl - Br - 、NO 2 - , HCO 3 - 、SO 3 2- 、SO 4 2- , S 2 O 3 2- , alanine, glutamic acid, lysine, phenylalanine, cysteine, glutathione, homocysteine, aspartic acid, citric acid, urea, Na 2 S and Fe 3+ Solution (where Na 2 S solution, Fe 3+The volume of the blank solution is 2 μL, and the rest of the substance is 12 μL) as the test solution. The blank solution and the test solution are respectively made up to 2 mL with acetate buffer at pH = 3, incubated at room temperature for 1 min, and then the fluorescence intensity value is recorded with a fluorescence spectrophotometer. The results are obtained by referring to Fig.13 As shown. It can be seen that Fe 3+ It has a strong selective fluorescence quenching effect on carbon dots, while Fe 2+ 、NO 2- and H 2 S has little effect on the fluorescence of carbon quantum dots, further proving that the application of Fe 2+ with NO 2- , Fe 3+ With H 2 The redox reaction between S and NO 2- and H 2 The feasibility of selective detection of S.

[0156] In summary, the present application provides a method for detecting nitrite and hydrogen sulfide based on glutathione carbon quantum dots. 2- Quickly transfer CDs / Fe 2+ Fe in the system 2+ Oxidized to Fe 3+ , Fe 3+ The fluorescence of glutathione carbon quantum dots was quenched to achieve NO in the range of 1-1000 μM. 2- The detection limit is 0.164μM; using H 2 S quickly converts CDs / Fe 3+ Fe in the system 3+ Reduction to Fe 2+ , so that the fluorescence of glutathione carbon quantum dots can be restored to achieve H 2 The detection limit of S was 0.114 μM.

[0157] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A nitrite detection method based on glutathione carbon quantum dots, characterized in that: Towards Fe 2+ CDs / Fe and glutathione carbon quantum dots 2+ Add NO into the system 2- , make NO 2- The CDs / Fe 2+ Fe in the system 2+ Oxidized to Fe 3+ , which in turn makes the positively charged Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of the glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of the glutathione carbon quantum dots at 335 nm, making Fe 3+ The fluorescence of the glutathione carbon quantum dots is further quenched by the inner filter effect to reduce the fluorescence intensity of the glutathione carbon quantum dots. 2- The linear relationship between the concentration of NO and the 2- concentration; Wherein, the glutathione carbon quantum dots are 2- The detection range is 1-1000 μM, and the detection limit is 0.164 μM.

2. The nitrite detection method based on glutathione carbon quantum dots according to claim 1, characterized in that: The detection of the nitrite radical comprises the following steps: S1, glutathione carbon quantum dot solution and Fe 2+ Solution, NO 2- The solutions were mixed to prepare a standard solution with gradient concentrations; the glutathione carbon quantum dot solution was mixed with Fe 2+ The solutions were mixed to prepare a blank solution; S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation A with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa; S3, after the sample is removed from the impurities, it is added to the blank solution prepared in step S1, and the sample is incubated at room temperature to obtain the sample solution, and the fluorescence intensity of the sample solution at the optimal emission wavelength is measured under the optimal excitation wavelength, and the fluorescence intensity is substituted into the fluorescence intensity standard curve equation A to obtain NO 2- concentration.

3. The nitrite detection method based on glutathione carbon quantum dots according to claim 2, characterized in that, In step S1, the method for preparing the standard solution is specifically to prepare n identical glutathione carbon quantum dot solutions, and to add the same volume and the same concentration of Fe 2+ After incubation at room temperature for a period of time, the same volume of NO was added in increasing concentrations. 2- The solution was incubated at room temperature for a period of time to 2+ Oxidized to Fe 3+ , to obtain the standard solution, the Fe in the standard solution 2+ The concentration decreases in a gradient; specifically, n≥10; the method for preparing the blank solution is specifically to mix the glutathione carbon quantum dot solution with Fe 2+ After the solutions were mixed, they were incubated at room temperature for a period of time to obtain the blank solution; Fe 2+ The concentration and volume of the solution are similar to those of the standard solution containing Fe 2+ The solutions have the same concentration and volume.

4. The nitrite detection method based on glutathione carbon quantum dots according to claim 3, characterized in that: The glutathione carbon quantum dot solution is prepared by dissolving glutathione in ultrapure water, then placing it in a high-pressure reactor, hydrothermally reacting it at 200-220° C. for 3-16 hours, cooling it to room temperature, filtering it, and dialyzing it with ultrapure water for 10-14 hours; the concentration of the glutathione carbon quantum dot solution is 2-20 mg / mL; the Fe 2+ The concentration of the solution is 100-700 μM; the incubation time is 1-5 min; and the impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

5. The nitrite detection method based on glutathione carbon quantum dots according to claim 4, characterized in that, In step S3, the sample to be tested is bacon, sausage, luncheon meat, ham, bacon or hot dog.

6. A method for detecting hydrogen sulfide based on glutathione carbon quantum dots, characterized in that: Towards Fe 3+ CDs / Fe and glutathione carbon quantum dots 3+ H2S is added to the system to make the CDs / Fe 3+ Fe in the system 3+ Reduction to Fe 2+ , by reducing Fe 3+ The content of CDs / Fe 3+ Fe in the system 3+ The fluorescence quenching effect of the glutathione carbon quantum dots is reduced to restore the fluorescence intensity of the glutathione carbon quantum dots, and finally the H2S concentration is calculated according to the linear relationship between the fluorescence intensity and the H2S concentration within the detectable range; The detection range of glutathione carbon quantum dots for H2S is 25-425 μM, and the detection limit is 0.114 μM; The fluorescence quenching effect refers to Fe 3+ It adsorbs to the negatively charged surface of glutathione carbon quantum dots through electrostatic action and quenches the fluorescence of the glutathione carbon quantum dots through static quenching. 3+ The ultraviolet absorption peak of Fe partially overlaps with the optimal excitation wavelength of the glutathione carbon quantum dots at 335 nm, making Fe 3+ The fluorescence of the glutathione carbon quantum dots is further quenched by the inner filter effect.

7. The method for detecting hydrogen sulfide based on glutathione carbon quantum dots according to claim 6, characterized in that: The detection of hydrogen sulfide comprises the following steps: S1, glutathione carbon quantum dot solution and Fe 3+ solution and Na2S solution to prepare a standard solution with gradient concentrations; the glutathione carbon quantum dot solution was mixed with Fe 3+ The solutions were mixed to prepare a blank solution; S2, measuring the fluorescence intensity of the standard solution and the blank solution prepared in step S1 at the optimal emission wavelength under the optimal excitation wavelength, and establishing a fluorescence intensity standard curve equation B with the fluorescence intensity as the ordinate and the concentration of the analyte as the abscissa; S3, after removing impurities from the sample to be tested, add it to the blank solution prepared in step S1, incubate at room temperature to obtain the test solution, measure the fluorescence intensity of the test solution at the optimal emission wavelength under the optimal excitation wavelength, and substitute the fluorescence intensity into the fluorescence intensity standard curve equation B to obtain the concentration of H2S.

8. The method for detecting hydrogen sulfide based on glutathione carbon quantum dots according to claim 7, characterized in that: In step S1, the method for preparing the standard solution is specifically to prepare n identical glutathione carbon quantum dot solutions, and to add the same volume and the same concentration of Fe 3+ The solution was incubated at room temperature for a period of time, and then Na2S solutions with the same volume and increasing concentrations were added respectively, and the standard solution was obtained by incubating at room temperature for a period of time; specifically, n≥5; the preparation method of the blank solution is specifically as follows: the glutathione carbon quantum dot solution is mixed with Fe 3+ After the solutions were mixed, they were incubated at room temperature for a period of time to obtain the blank solution; Fe 3+ The concentration and volume of the solution are similar to those of the standard solution containing Fe 3+ The solutions have the same concentration and volume.

9. The method for detecting hydrogen sulfide based on glutathione carbon quantum dots according to claim 8, characterized in that: The glutathione carbon quantum dot solution is prepared by dissolving glutathione in ultrapure water, placing the solution in a high pressure reactor, performing a hydrothermal reaction at 200-220° C. for 3-16 hours, cooling the solution to room temperature, filtering the solution, and dialyzing the solution with ultrapure water for 10-14 hours. The concentration of the glutathione carbon quantum dot solution is 2-20 mg / mL. 3+ The concentration of the solution is 100-700 μM; the incubation time is 1-5 min, and further, the incubation time is preferably 3 min; the impurity removal treatment includes one or more of grinding, water bath heating, dilution, centrifugation, and filtration.

10. The method for detecting hydrogen sulfide based on glutathione carbon quantum dots according to claim 9, characterized in that: In step S3, the sample to be tested is tap water, beer, wine or river water.

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