Fluorescence determination method for free amino acids in tobacco leaves

Abstract

The invention relates to the technical field of chemical analysis and detection, in particular to a fluorescence determination method for free amino acid in tobacco leaves, based on the fluorescence characteristic of epinephrine-based zinc-doped carbon dots Zn / NA-CDs, free amino acid reacts with phthalic dicarboxaldehyde to generate a Schiff base product, and Zn / NA-CDs and Schiff base have hydrogen bond interaction and charge transfer effect, so that the content of free amino acid in tobacco leaves is determined. Therefore, the Schiff base has a fluorescence sensitization effect on the Zn / NA-CDs, and a free amino acid fluorescence detection method is established. According to the method, the detection limit can reach 0.01 mg / L, and the recovery rate is between 90.52% and 105.33%, so that the method also has high sensitivity to low-concentration free amino acid, can meet the requirement for accurately measuring trace amino acid in tobacco leaves, and has excellent accuracy and reliability. Meanwhile, the method further has the advantages of being easy and convenient to operate, low in cost, environmentally friendly, wide in applicability and the like, a new technical means is provided for tobacco leaf quality detection, and beneficial reference is provided for amino acid determination in other fields.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, in particular to a fluorescence determination method for free amino acids in tobacco leaves. Background Art

[0002] Free amino acids are amino acids that exist in a free state, unbound to protein molecules. As a key nitrogen-containing compound in tobacco, their presence and content are closely linked to tobacco leaf quality and are a key component of tobacco aroma. Throughout the tobacco growth cycle, from planting and curing to processing and ultimately smoking, amino acids undergo a series of complex chemical reactions with the reducing sugars in the tobacco. These reactions include both enzymatic and non-enzymatic browning reactions, forming heterocyclic compounds such as pyrazines, pyrroles, and furans, which significantly impact the smoking quality of the tobacco.

[0003] Monitoring the free amino acid content in tobacco is crucial for assessing leaf quality and monitoring its mold growth. During mold growth, protein hydrolysis causes changes in the total amount and type of free amino acids, which are crucial for determining whether a leaf is moldy. Therefore, developing an accurate, reliable, and cost-effective free amino acid assay is crucial for the tobacco industry.

[0004] Currently, methods used to determine amino acid content primarily include ion chromatography, liquid chromatography, gas chromatography, ion exchange chromatography, ninhydrin colorimetry, and amino acid analyzers. While these methods are capable of detecting amino acids to a certain extent, they lack selectivity, sensitivity, reliability, and ease of use. For example, some methods may require complex sample pretreatment and cumbersome testing procedures, which not only increases testing costs but also may affect accuracy and efficiency.

[0005] To overcome the limitations of existing technologies, researchers are exploring new detection methods. Carbon dots, a novel nanomaterial, have attracted considerable attention due to their excellent water solubility, low toxicity, biocompatibility, and fluorescent properties. In particular, when doped with transition metal atoms, their physicochemical properties undergo significant changes, expanding their potential for applications in sensing and detection.

[0006] In view of this, the present invention proposes a method for fluorescence determination of free amino acids in tobacco leaves using adrenaline zinc-doped carbon dots. Summary of the Invention

[0007] The present invention aims to provide a fluorescent method for the determination of free amino acids in tobacco leaves. This method, based on the novel fluorescent probe Zn / NA-CDs, achieves highly sensitive and accurate determination of free amino acids in tobacco leaves. Furthermore, this method offers advantages such as ease of operation, low cost, environmental friendliness, and wide applicability. It provides a new technical approach for tobacco leaf quality testing and offers a valuable reference for amino acid determination in other fields.

[0008] The present invention provides a fluorescence determination method for free amino acids in tobacco leaves. The method is based on the fluorescence characteristics of adrenaline zinc-doped carbon dots (Zn / NA-CDs). Free amino acids react with o-phthalaldehyde to generate Schiff base products. Hydrogen bond interactions and charge transfer occur between Zn / NA-CDs and the Schiff base, resulting in fluorescence sensitization of the Zn / NA-CDs by the Schiff base. Thus, a fluorescence detection method for free amino acids is established.

[0009] The adrenaline zinc-doped carbon dots Zn / NA-CDs prepared in the present invention have a large specific surface area and contain abundant carboxyl, hydroxyl and Zn 0 / Zn 2+ Among them, the carboxyl groups and Zn 2+ There are hydrogen bonds and coordination bonds between amino acid derivatives (Schiff bases) and -C=N. The carbon on the aromatic ring also has strong electron-withdrawing ability and can form Ar-H...O hydrogen bonds, which will lead to electron transfer between Zn / NA-CDs and amino acid derivative Schiff bases. The electron transfer caused by the strong molecular force enhances its fluorescence, thereby establishing a fluorescence detection method for free amino acids.

[0010] Studies have shown that the detection limit of this method can reach 0.01 mg / L, and the recovery rate is between 90.52% and 105.33%, indicating that this method has high sensitivity for low concentrations of free amino acids, can meet the needs of accurate determination of trace amino acids in tobacco leaves, and has excellent accuracy and reliability.

[0011] As a preferred embodiment of the present invention, the fluorescence determination method of free amino acids in tobacco leaves of the present invention specifically comprises the following steps:

[0012] First, add free amino acids (glycine Gly can be used as a model molecule for free amino acids), o-phthalaldehyde solution (OPA), and NaOH solution to the detection system and react for 5-10 minutes. Then, add Zn / NA-CDs solution and phosphate buffer solution and react for 5-10 minutes. The fluorescence intensity is measured, and a quantitative relationship between fluorescence intensity and free amino acid concentration is established. A standard curve is drawn to obtain a regression equation.

[0013] Then, a tobacco leaf extract was prepared, and o-phthalaldehyde solution and NaOH solution were added to the tobacco leaf extract, and the reaction was carried out for 5-10 minutes. Then, Zn / NA-CDs solution and phosphate buffer solution were added, and the reaction was carried out for 5-10 minutes. The fluorescence intensity was tested, and the fluorescence intensity was substituted into the regression equation to calculate the free amino acid content in the tobacco leaf sample.

[0014] As a preferred embodiment of the present invention, during the establishment of the standard curve or the sample testing process, the concentration of the free amino acid solution used is 2.5-250 mg / L;

[0015] The concentration of Zn / NA-CDs solution was 1 mg / mL, and the addition volume was 20-50 μL;

[0016] The concentration of o-phthalaldehyde solution is 5mmol / L, and the amount added is 50-100μL;

[0017] The concentration of NaOH solution is 1 mol / L, and the amount added is 50-100 μL.

[0018] As a preferred embodiment of the present technical solution, during the fluorescence intensity test, the excitation wavelength is 310 nm and the emission wavelength is 412 nm.

[0019] As a preferred embodiment of the present invention, the pH of the phosphate buffer solution is 5-7, and preferably 6.

[0020] As a preferred embodiment of the present invention, the method for preparing Zn / NA-CDs comprises the following steps:

[0021] S1. Add ethylenediamine and deionized water to ZnCl2, epinephrine and citric acid, and ultrasonicate for 20-30 minutes to obtain a mixture;

[0022] S2. The mixture is transferred to a high-temperature reactor for hydrothermal reaction, cooled to room temperature, and the obtained product is sequentially centrifuged, membrane filtered and freeze-dried to obtain Zn / NA-CDs.

[0023] In the preparation method of Zn / NA-CDs of the present invention, first, in step S1, ZnCl2, epinephrine, citric acid, and ethylenediamine are used as raw materials. These substances are relatively inexpensive and readily available, reducing preparation costs. Furthermore, epinephrine not only serves as a carbon source but also potentially imparts specific biological activity or functionality to the carbon dots, increasing the added value of the product. Ultrasonic treatment for 20-30 minutes effectively promotes the dispersion and initial reaction of the raw materials, laying the foundation for the subsequent high-temperature carbonization process. Secondly, in step S2, the mixture is transferred to a polytetrafluoroethylene high-temperature reactor and reacted in a muffle furnace, ensuring stable reaction at high temperatures, which is conducive to the formation and structural stability of the carbon dots. Finally, centrifugation and membrane filtration effectively remove large particulate impurities and unreacted raw materials from the reaction system, thereby improving the purity of the product. The application of freeze-drying technology avoids the potential for carbon dot agglomeration or structural damage caused by traditional drying methods, preserving the excellent dispersibility and stability of the carbon dots. These excellent dispersibility and stability make Zn / NA-CDs promising for broad application in fields such as sensors, imaging, and drug delivery.

[0024] In summary, the Zn / NA-CDs prepared in the present invention have excellent fluorescence properties, which is due to the combined action of epinephrine and citric acid, as well as the structural regulation during the high-temperature carbonization process.

[0025] As a preferred embodiment of the present technical solution, in step S1, the amounts of epinephrine, citric acid, ethylenediamine and deionized water required for every 0.05-0.10 g ZnCl2 are 25-30 mg, 2.0-2.5 g, 50-100 μL and 30-40 mL, respectively.

[0026] As a preferred embodiment of this technical solution, in step S2, the hydrothermal reaction is performed at a temperature of 180-220°C and a reaction time of 10-12 hours. Controlling the heating time to 10-12 hours ensures sufficient reaction while avoiding structural damage or performance degradation of the carbon dots that may result from prolonged heating.

[0027] As a preferred embodiment of the present technical solution, in step S2, during the centrifugal separation, the centrifugation is carried out at a rotation speed of 10,000-13,000 rpm for 15-20 minutes.

[0028] As a preferred embodiment of the present technical solution, in step S2, during the membrane filtration, the upper yellow liquid obtained by centrifugation is filtered using a 0.22 μm filter membrane.

[0029] The fluorescence determination method of free amino acids in tobacco leaves of the present invention has at least the following beneficial effects:

[0030] 1. The present invention is based on the fluorescence properties of the prepared adrenaline zinc-doped carbon dots (Zn / NA-CDs), and the fact that amino acids react with o-phthalaldehyde (OPA) to generate Schiff base products, which are derivatives of amino acids. The hydrogen bond interaction and charge transfer between Zn / NA-CDs and Schiff bases will cause the amino acid derivative Schiff base to produce fluorescence sensitization on Zn / NA-CDs, thus establishing a new method for amino acid fluorescence detection. This method is based on the novel fluorescent probe Zn / NA-CDs and achieves high sensitivity and high accuracy in the determination of free amino acids in tobacco leaves. At the same time, this method also has the advantages of simple operation, low cost, environmental friendliness and wide applicability, providing a new technical means for tobacco quality detection and a useful reference for the determination of amino acids in other fields.

[0031] 2. In the fluorescence determination method of free amino acids in tobacco leaves of the present invention, the detection limit of the method can reach 0.01 mg / L, indicating that the method has high sensitivity even for low concentrations of free amino acids and can meet the needs of accurate determination of trace amino acids in tobacco leaves; and the recovery rate ranges from 90.52% to 105.33%, indicating that the method has excellent accuracy and reliability in practical applications;

[0032] 3. The fluorescent determination method for free amino acids in tobacco leaves established by the present invention can detect a variety of free amino acids including histidine (His), valine (Val), alanine (Ala), leucine (Leu), aspartic acid (Asp), serine (Ser), cystine (Cys), lysine (Lys), and hydroxyproline (Hyp). Satisfactory results were obtained in the determination of free amino acids in tobacco leaves, and the detection is reliable, simple, and rapid.

[0033] 4. The fluorescent determination method for free amino acids in tobacco leaves established by the present invention simplifies the complex steps of traditional free amino acid determination, reduces the cumbersome operational procedures, and thus improves work efficiency. Due to the simplified processing steps, the time required for the entire determination process is greatly shortened, which is particularly important for the rapid detection and analysis of large numbers of samples. In addition, compared with other methods that require expensive reagents or complex equipment, the present invention is more economical in reagent consumption and equipment use, thereby reducing detection costs. The method is easy to master and operate, does not require highly specialized technicians, and avoids the use of expensive and complex large-scale instruments and equipment, reducing the requirements for personnel skills and equipment.

[0034] In summary, the fluorescent determination method for free amino acids in tobacco leaves developed in this paper not only has significant technical advantages but also demonstrates strong competitiveness in practical applications. The promotion and application of this method will help improve the efficiency and accuracy of tobacco leaf quality testing, providing strong support for the sustainable development of the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 TEM image of Zn / NA-CDs fluorescent carbon dots synthesized in an embodiment of the present invention;

[0037] Figure 2 Graph showing the emission spectra of Zn / NA-CDs at different excitation wavelengths (368 nm-408 nm) in an embodiment of the present invention;

[0038] Figure 3 The UV-visible absorption spectra of Gly and OPA under alkaline conditions and with the addition of Zn / NA-CDs in the examples of the present invention are shown;

[0039] Figure 4 : The fluorescence spectra of Gly+OPA and Zn / NA-CDs+Gly+OPA in the embodiment of the present invention;

[0040] Figure 5 This is a graph showing the linear fluorescence spectrum of Gly in an embodiment of the present invention;

[0041] Figure 6 is the linear regression equation of Gly in the embodiment of the present invention;

[0042] Figure 7 This is a diagram showing the effects of other amino acids on the fluorescent probe detection system;

[0043] Figure 8 This is a diagram showing the impact of coexisting substances in tobacco leaves on the fluorescent probe detection system. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0047] Example

[0048] 1. The preparation method of Zn / NA-CDs is as follows:

[0049] S1. Take 0.05 g ZnCl2, 25 mg epinephrine, 2.0 g citric acid and 50 μL ethylenediamine, add them to 30 mL deionized water, and sonicate for 20 min to obtain a mixture;

[0050] S2. The mixture was transferred to a polytetrafluoroethylene high-temperature reactor, placed in a muffle furnace and heated at 200°C for 10 hours. After naturally cooling to room temperature, the liquid in the reactor was poured out and centrifuged at 10,000 rpm for 20 minutes. The yellow liquid above was filtered with a 0.22 μm filter membrane and freeze-dried to obtain Zn / NA-CDs.

[0051] Figure 1 This is a transmission electron microscopy (TEM) image of the Zn / NA-CDs fluorescent carbon dots synthesized in this example. The image shows that the prepared Zn / NA-CDs are approximately spherical and evenly dispersed, with an average diameter of approximately 3.5 nm. The high-resolution transmission electron microscopy (HR-TEM) image in the inset shows a lattice spacing of approximately 0.22 nm, close to that of graphite.

[0052] 2. Determination of fluorescence quantum yield and evaluation of optical properties of Zn / NA-CDs

[0053] The fluorescence quantum yield of Zn / NA-CDs is usually determined using quinine sulfate (0.1 mol / L H2SO4 as solvent; Q R =0.54) as a reference substance and calculated according to the following formula:

[0054]

[0055] Where Q represents the quantum yield, I represents the fluorescence intensity, A represents the absorbance, and η represents the refractive index of the solvent (where η S 2 / η R 2 =1), the subscripts R and S represent the reference quinine sulfate and the sample carbon quantum dots, respectively.

[0056] Figure 2 Figure 2 is the emission spectrum of Zn / NA-CDs at different excitation wavelengths (368nm-408nm). It can be seen from the figure that Zn / NA-CDs exhibit excitation-dependent emission properties. As the excitation wavelength increases, the fluorescence intensity first increases and then decreases, which may be related to the surface state of Zn / NA-CDs.

[0057] Figure 3 Figure 3 is the UV-visible absorption spectrum of Gly and OPA under alkaline conditions and with the addition of Zn / NA-CDs. The UV-visible absorption spectrum shows a strong absorption peak at 281 nm, which is attributed to the n-π* transition of the C=O bond. The UV absorption is further enhanced after the addition of Zn / NA-CDs.

[0058] Figure 4 Fluorescence spectra revealed an emission peak at 460 nm under an excitation wavelength of 388 nm. The addition of Zn / NA-CDs to the system enhanced the fluorescence intensity. Using quinine sulfate as a reference, the fluorescence yield of Zn / NA-CDs was approximately 47.3%, demonstrating the influence of Zn doping on the luminescence properties of carbon dots. The structural properties of carbon dots and doped metal ions on surface trapped electrons can alter the ordered structure of the carbon rings, thereby improving the scavenging of electron-hole pairs by the newly generated emission traps.

[0059] 3. Preparation of standard working curve

[0060] In a 5 mL stoppered colorimetric tube, add 50 μL of 5 mmol / L OPA solution with a concentration range of 2.5-250 mg / L Gly and 100 μL of 1 mol / L NaOH solution. The reaction was carried out for 10 minutes. Then, 20 μL of 1 mg / mL Zn / NA-CDs solution and 500 μL of 30 mM pH 6.0 phosphate buffer solution were added. The volume was adjusted to 4 mL with pure water and the reaction was carried out for 10 minutes. The fluorescence intensity was measured at an excitation wavelength of 310 nm and an emission wavelength of 412 nm. A quantitative relationship between fluorescence intensity and Gly concentration was established, a standard curve was plotted, and a regression equation was obtained.

[0061] Figure 5 and Figure 6 They are the linear fluorescence spectrum and linear regression equation of Gly respectively; the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0062] Table 1 Linear equation, correlation coefficient, relative standard deviation, and linear range

[0063]

[0064] 4. Influence of other amino acids on the detection system

[0065] Substituting histidine (His), alanine (Ala), leucine (Leu), aspartic acid (Asp), and serine (Ser) for glycine (Gly), with other conditions remaining the same, the fluorescence intensity was measured. The results were as follows: Figure 7 As shown in the figure, it can be seen that histidine, alanine, leucine, aspartic acid and serine can also undergo reactions similar to Gly.

[0066] 5. Investigation of method specificity

[0067] Gly and other tobacco coexistents were mixed to investigate the specificity of the detection system. As shown in Figure 8, when the Gly concentration was 12.5 mg / L, 100-fold concentrations of Mg and 2+ , Ca 2+ , K + 、Na + NH4 + 、SO4 2- 、Cl - ; At the same time, add 5 times the concentration of chlorogenic acid, nicotine, carotene and lutein.

[0068] The research results show that inorganic ions and coexisting substances have almost no effect on the fluorescence system, so there is no interference in the determination results, further indicating that this method can be used for the determination of free amino acids.

[0069] Test example

[0070] Determination of free amino acids in tobacco leaves

[0071] 1. The preparation method of tobacco leaf extract is as follows:

[0072] Weigh 1.000 g of flue-cured tobacco sample, add 20 mL (0.1 mol / L) hydrochloric acid solution, place in an ultrasonic cleaner and ultrasonicate for 30 min (100 Hz), filter, and dilute the filtrate to a 50 mL volumetric flask to prepare a tobacco leaf extract.

[0073] 2. Determination of free amino acids in tobacco leaves:

[0074] In a 5 mL stoppered colorimetric tube, 1 mL of tobacco leaf extract, 50 μL of 5 mmol / L OPA solution, and 100 μL of 1 mol / L NaOH solution were added and reacted for 10 min. Then, 20 μL of 1 mg / mL Zn / NA-CDs solution and 500 μL of 30 mM pH 6.0 phosphate buffer solution were added and the volume was made up to 4 mL with pure water. The reaction was continued for 10 min. The fluorescence intensity was measured at an excitation wavelength of 310 nm and an emission wavelength of 412 nm. The fluorescence intensity was substituted into the regression equation in Table 1 to calculate the free amino acid content in the tobacco leaf sample as shown in Table 2. The sample spike recovery rate is shown in Table 2.

[0075] Table 2 Determination of amino acid content in tobacco leaf samples (n=6)

[0076]

[0077] As shown in Table 2, after adding different amounts of amino acid standards to tobacco leaf samples, the recovery rates were generally high, with most ranging from 93% to 108.2%. This indicates that the experimental method has high accuracy and reliability and can accurately determine the content of free amino acids in tobacco leaves. In addition, each sample was measured multiple times (n=6) in the experiment, and the relative standard deviation (RSD%) was calculated. The RSD% values ​​were all low, indicating that the experimental results were reproducible and the data were reliable. Therefore, the method of the present invention is effective, accurate, and reliable and can be used to determine the content of free amino acids in tobacco leaves.

[0078] In summary, the fluorescent determination method of free amino acids in tobacco leaves established by the present invention simplifies the complex steps of traditional determination of free amino acids, reduces cumbersome operating procedures, and thus improves work efficiency; due to the simplification of the processing steps, the time required for the entire determination process is greatly shortened, which is particularly important for rapid detection and analysis of a large number of samples; in addition, compared with other methods that require expensive reagents or complex equipment, the present invention is more economical in reagent consumption and equipment use, reducing detection costs; the method is easy to master and operate, does not require highly professional technicians, and avoids the use of expensive and complex large-scale instruments and equipment, reducing the requirements for personnel skills and equipment. Therefore, the fluorescent determination method of free amino acids in tobacco leaves established by the present invention not only has significant technical advantages, but also shows strong competitiveness in practical applications. The promotion and application of this method will help improve the efficiency and accuracy of tobacco leaf quality detection and provide strong support for the sustainable development of the tobacco industry.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the fluorescence determination of free amino acids in tobacco leaves, characterized in that: Based on the fluorescence properties of adrenaline-based zinc-doped carbon dots Zn / NA-CDs, free amino acids react with o-phthalaldehyde to generate Schiff base products. Due to the hydrogen bond interaction and charge transfer between Zn / NA-CDs and the Schiff base, the Schiff base produces a fluorescence sensitization effect on Zn / NA-CDs, and a fluorescence detection method for free amino acids is established.

2. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 1, characterized in that: The following steps are involved: Free amino acids, o-phthalaldehyde solution and NaOH solution were added to the detection system, and the reaction was continued for 5-10 minutes. Then, Zn / NA-CDs solution and phosphate buffer solution were added, and the reaction was continued for 5-10 minutes. The fluorescence intensity was tested, and a quantitative relationship between the fluorescence intensity and the free amino acid concentration was established. A standard curve was drawn to obtain a regression equation. Add o-phthalaldehyde solution and NaOH solution to the tobacco leaf extract, react for 5-10 minutes, then add Zn / NA-CDs solution and phosphate buffer solution, react for 5-10 minutes, test the fluorescence intensity, and substitute the fluorescence intensity into the regression equation to calculate the free amino acid content in the tobacco leaf sample.

3. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 2, characterized in that: During the establishment of the standard curve or sample testing, the concentration of the free amino acid solution used was 2.5-250 mg / L; The concentration of Zn / NA-CDs solution was 1 mg / mL, and the amount added was 20-50 μL; The concentration of o-phthalaldehyde solution is 5mmol / L, and the amount added is 50-100μL; The concentration of NaOH solution is 1 mol / L, and the amount added is 50-100 μL.

4. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 2, characterized in that: When the fluorescence intensity is tested, the excitation wavelength is 310 nm and the emission wavelength is 412 nm.

5. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 2, characterized in that: The pH of the phosphate buffer solution is 5-7.

6. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 2, characterized in that: The preparation method of the Zn / NA-CDs comprises the following steps: S1. Add ethylenediamine and deionized water to ZnCl2, epinephrine and citric acid, and perform ultrasonic treatment for 20-30 min to obtain a mixture; S2. The mixture is transferred to a high temperature reactor for hydrothermal reaction, cooled to room temperature, and the obtained product is sequentially centrifuged, membrane filtered and freeze-dried to obtain Zn / NA-CDs.

7. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 6, characterized in that: In step S1, the amounts of adrenaline, citric acid, ethylenediamine and deionized water required for every 0.05-0.10 g ZnCl2 are 25-30 mg, 2.0-2.5 g, 50-100 μL and 30-40 mL, respectively.

8. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 6, characterized in that: In step S2, during the hydrothermal reaction, the temperature is controlled to be 180-220° C. and the reaction time is 10-12 h.

9. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 6, characterized in that: In step S2, during the centrifugal separation, the centrifuge is performed at a speed of 10000-13000 rpm for 15-20 min.

10. The method for fluorescence determination of free amino acids in tobacco leaves according to claim 6, characterized in that: In step S2, during the membrane filtration, the upper yellow liquid obtained by centrifugal separation is filtered using a 0.22 μm filter membrane.

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