Cyanine-like fluorescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a cyanine-like fluorescent probe as well as a synthesis method and application thereof, and belongs to the field of organic small molecule fluorescent probes. The molecular formula of the cyanine-like fluorescent probe is C22H17ClN2O4, and the cyanine-like fluorescent probe is marked as N-3-I; the novel cyanine-like fluorescent probe synthesis method provided by the invention is simple in operation steps and rapid in process, and the synthesized cyanine-like fluorescent probe can react with sulfite under very mild conditions to generate a strong sensing process with rapid response and high sensitivity; when sulfite exists in a test sample, the color of a fluorescent probe solution is obviously changed from original deep pink to colorless, real-time naked-eye monitoring of the content of the sulfite in the traditional Chinese medicine materials can be visually realized, and the monitoring level can reach a nanomole level; the cyanine fluorescent probe N-3-I can be applied to visual detection of sulfite and development of related detection test paper, and has important practical significance on safety evaluation of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of organic small molecule fluorescent probes, and specifically relates to a cyanine-like fluorescent probe and a synthesis method and application thereof. Background Art

[0002] Sulfite is a commonly used bleaching agent and preservative in the food industry. As a food additive, it plays the following roles in food: (1) preventing enzymatic and non-enzymatic browning of food, making the food look bright and white; (2) inhibiting the growth of microorganisms and thus extending the shelf life of food; (3) inhibiting the activity of oxidase in food, preventing oxidase from causing the destruction of nutrients and discoloration; (4) combining with colored substances in food and playing a bleaching role due to its reducing property.

[0003] However, as research progresses, the toxicity of sulfites has received increasing attention. A large number of studies have shown that abnormal levels of SO2 in food can cause harm to human health, and excessive sulfites entering the human circulation can cause renal dysfunction, cardiovascular disease, and respiratory diseases. The toxicity of sulfites is mainly manifested in the following aspects: (1) Sulfites can react with substances such as amino acids and proteins, and can also combine with various vitamins to break them into other products, thereby seriously destroying the nutrients in food and reducing the nutritional value of food. (2) Long-term exposure to low concentrations of sulfites can cause headaches, dizziness, and general fatigue. In severe cases, it can cause symptoms such as chronic pharyngitis, bronchitis, and loss of smell and taste. (3) If animals eat foods containing sulfites for a long time, they will develop adverse symptoms such as neuritis and bone marrow atrophy, which will hinder the growth of animals. (4) Sulfites can also cause cell mutations and induce oxidation of unsaturated fatty acids.

[0004] Therefore, the content of sulfite additives in food must be strictly tested and controlled. For example, the Codex Alimentarius Commission (CAC) stipulates that the sulfite content in dried fruits, vegetables and wine should be less than 100 mg / kg, 200 mg / kg and 250 mg / kg respectively. The US Food and Drug Administration (FDA) stipulates that HSO3 in food and beverages should not exceed 100 mg / kg, 200 mg / kg and 250 mg / kg respectively. - The content should be less than 10ppm (125μmol / L), and the "Sanitary Standard for the Use of Food Additives" (GB2760-2015) clearly stipulates the allowable amount of sulfites in various foods. That is, the amount of sulfites used in food shall not exceed 30mg / kg, and the Pharmacopoeia of the People's Republic of China (PRC) also stipulates that the sulfite content in traditional Chinese medicines shall not exceed 400mg / kg.

[0005] In view of some side effects of sulfite, it is urgent to develop a method for rapid quantitative detection of sulfite. Sulfite is an oxygen-containing salt, and the acid radical ion contained in its structure is sulfite (SO3 2- ), the sulfite detection is actually the detection of sulfite ions (SO3 2- ) for detection. Traditional methods for detecting sulfite mainly include ion chromatography, electrochemical method and capillary electrophoresis. Since these methods require complex pre-treatment steps, high-end instruments and equipment, complex experimental operations and long-term analysis, they cannot achieve the real-time monitoring needs of the target object, so they are subject to certain limitations in food detection applications. Photochemical sensors have developed rapidly in recent years and have received widespread attention due to their advantages such as low price, simple method, rapid response and large amount of information. The main principle is to use the change of the optical properties (absorption or emission) of the sensor unit as the output signal to realize the detection of the analyte.

[0006] Optical chemical sensors can be divided into fluorescent chemical sensors and colorimetric chemical sensors according to their signal detection. Fluorescent chemical sensors (fluorescent probes) mainly rely on fluorescent signals as a detection method, usually with fluorescence enhancement, quenching or changes in luminescent wavelength; colorimetric chemical sensors rely on changes in color tone, especially color enhancement type colorimetric sensors that can be detected by naked eye observation, which is convenient for practical application. At present, single fluorescent chemical sensors or colorimetric chemical sensors are increasingly used in the detection of biological molecules due to their good selectivity, high sensitivity, and fast response time.

[0007] In 2022, GONG W, ZHANG C, ZHANG X et al. published "Mitochondria-targetable colorimetric and far-red fluorescent sensor for rapid detection of SO2 derivatives in food samples and living cells" in "Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy". The probe AQCB is based on a rhodamine analog skeleton containing a 3-quinoline carboxaldehyde part and a 1-methylquinoline part, which produces a receptor-π-receptor structure that can quench fluorescence, that is, a new HSO3 - Colorimetric far-red fluorescent probe. Quinoline, as a cationic surfactant, can improve membrane permeability and biocompatibility. Increase the intermolecular reaction rate and shorten the reaction time. When HSO3 -AQCB undergoes a 1,4-nucleophilic addition reaction with the quinoline part to form an electron-rich enamine part, forming a donor-π-donor structure, and producing strong purple fluorescence. AQCB has good water solubility. In PBS, it is soluble in HSO3 - It has fast response (<15s), high sensitivity (LOD = 49nM) and selectivity. The probe has good cell membrane properties and can be successfully applied to the mitochondrial HSO3 of living cells. - Imaging. It can not only quantitatively detect HSO3 in food samples - , and can visually detect HSO3 in mitochondria - .

[0008]

[0009] In 2023, LI Y, WANG Y, LEI X and others reported in Food Chemistry "Development of a responsive probe for colorimetric and fluorescent detection of bisulfite infood and animal serum samples in 100% aqueous solution", in which the probe NPO has good water solubility, fast reaction, and a detection limit as low as 0.25μM, accompanied by a change in solution color from blue to yellow. The test strip experiment achieved HSO3 - In addition, it has also been successfully used for the detection of SO2 in the environment, as well as HSO3 in real food samples (sugar, liquor, dried edible fungi, etc.) and animal serum samples. - Detection.

[0010]

[0011] In 2021, Zhang C, Han L, Liu Q, et al. introduced "A colorimetric and far-red fluorescent probe for rapid detection of bisulfite / sulfite in full water-soluble based on biquinolinium and its applications" in Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. The probe DQ reacts with HSO3 via a 1,4 nucleophilic addition reaction. -The detection is highly selective, with the color ranging from colorless to purple-red in pure aqueous solution, and has significant far-red fluorescence enhancement. Probe DQ can be used to detect HSO3 on real samples such as paper strips, vermicelli, sugar, etc. - and HSO3 on live cell images - .

[0012]

[0013] Although the fluorescent probes disclosed in the above-mentioned prior art have the advantages of high sensitivity, good selectivity, and rapid detection, their application scenarios are wide, and their use scope includes the entire food and even biological and environmental safety fields. They cannot reflect excellent specificity and specificity when detecting Chinese medicinal materials. As the quality of life of the Chinese people improves, the food safety issues of Chinese medicinal materials have increasingly attracted people's attention. Therefore, it is urgent to study a fluorescent probe that is simple to operate, has real-time and rapid detection, and is targeted at the detection of sulfite in Chinese medicinal materials, so as to provide a strong technical guarantee for the self-monitoring and market supervision of Chinese medicinal materials manufacturers. Summary of the invention

[0014] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a cyanine-like fluorescent probe and its synthesis method and application. The cyanine-like fluorescent probe N-3-I can be applied to the visual detection of sulfite and the development of related test strips, which has important practical significance for the safety assessment of traditional Chinese medicine.

[0015] The technical solution of the present invention is as follows:

[0016] One of the purposes of the present invention is to provide a cyanine-like fluorescent probe, characterized in that the cyanine-like fluorescent probe is labeled as N-3-I, the solution color is dark pink, and the molecular formula is C 22 H 17 ClN2O4, chemical structure is shown in Ⅰ:

[0017]

[0018] A second object of the present invention is to provide a method for synthesizing a cyanine-like fluorescent probe, characterized in that it comprises the following steps:

[0019] (1) Cesium carbonate and 1,8-naphtholactam are dissolved in acetonitrile, iodomethane is added dropwise, and the mixture is stirred at room temperature for 3-5 hours under an argon atmosphere. The solvent is removed by rotary evaporation, and a bright yellow powder is obtained by separation and purification, namely, compound 1, whose structural formula is shown in II:

[0020]

[0021] (2) Compound 1 obtained in step 1 is dissolved in anhydrous tetrahydrofuran at 0°C, methylmagnesium chloride is added dropwise and stirred, and then refluxed in an oil bath at 50-70°C for 1-3h under argon atmosphere. After the reaction is completed, it is placed in an ice bath until the temperature drops to 0°C, a small amount of water is added to quench the reaction, and then perchloric acid is added to the reaction mixture and stirred thoroughly to obtain a yellow-green solution, which is added to ice water, vacuum filtered and dried to obtain a green powder, namely compound 2, whose structural formula is shown in III:

[0022]

[0023] (3) Compound 2 and 5-indolecarboxaldehyde were mixed in a round-bottom flask, and then a mixed solution of ethanol and acetonitrile was added to react overnight at room temperature in the dark. After the reaction was completed, the black precipitate was filtered and the reaction mixture was separated and purified by silica gel column chromatography to obtain a cyanine-like fluorescent probe N-3-I, whose structural formula is shown in IV:

[0024]

[0025] Furthermore, the step (1) is reacted as follows:

[0026]

[0027] Furthermore, in the step (1), the molar ratio of 1,8-naphtholactam, cesium carbonate and methyl iodide is 1:1.5:1.2.

[0028] Furthermore, the separation and purification in step (1) is carried out by column chromatography, wherein the chromatography eluent is a mixed solution of petroleum ether / ethyl acetate in a volume ratio of 23:1.

[0029] Furthermore, the step (2) is reacted as follows:

[0030]

[0031] Furthermore, in the step (2), the molar ratio of the compound 1 to methylmagnesium chloride is 1:4.5.

[0032] Furthermore, the step (3) is reacted as follows:

[0033]

[0034] Furthermore, in the step (3), the molar ratio of compound 2 to 5-indolecarboxaldehyde is 1:1.

[0035] Furthermore, the volume ratio of ethanol to acetonitrile in the ethanol-acetonitrile mixed solution in step (3) is 9:1.

[0036] Furthermore, the separation and purification in step (3) is performed by column chromatography, wherein the chromatography eluent is a mixed solution of dichloromethane / methanol in a volume ratio of 10:1.

[0037] The third object of the present invention is to provide a cyanine-like fluorescent probe for use in detecting the sulfite content of traditional Chinese medicine.

[0038] Furthermore, when sulfite is present in the Chinese medicinal material test sample, the cyanine-like fluorescent probe can respond quickly under very mild conditions, causing its solution to significantly change from the original deep pink to colorless.

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] 1. The present invention designs a cyanine-like fluorescent probe as a new sensor that can quickly and sensitively detect sulfite. The cyanine-like fluorescent probe has good selectivity and specificity for sulfite, can react with sulfite under very mild conditions, and the sulfite / bisulfite selectively reacts rapidly with the carbon-carbon double bond connecting the indole ring and the benzindole, destroying the large conjugated system of the probe and causing a blue shift in fluorescence. The probe can respond quickly within 1 minute, has high sensitivity (the detection limit can reach 49nM), good selectivity, and can effectively eliminate the interference of other food additives in the sample.

[0041] 2. Compared with the existing fluorescent probe technology, the anthocyanin fluorescent probe N-3-I disclosed in the present invention has a simple synthesis method, a fast synthesis process, simple operation steps, and is environmentally friendly. When sulfite is present in the test sample, the color of the fluorescent probe solution changes significantly from the original dark pink to colorless, which can intuitively realize the real-time "naked eye" monitoring of the sulfite content in traditional Chinese medicines, and the monitoring level can reach the nanomolar level. There are no special restrictions on the use of the anthocyanin fluorescent probe, and the detection can be completed at normal room temperature and other mild conditions, showing good practical applicability.

[0042] 3. The cyanine-like fluorescent probe N-3-I of the present invention can be applied to the safety assessment of sulfite in Chinese medicinal materials. The method has a small dosage, a fast detection time, and a high detection performance, and therefore has broad application prospects in the development of sulfite visualization detection and detection test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the cyanine-like fluorescent probe N-3-I synthesized in Example 1 of the present invention;

[0044] Figure 2The ultraviolet spectrum characteristic diagram (A) of the cyanine-like fluorescent probe N-3-I acting on Angelica dahurica, the fluorescence spectrum characteristic diagram (B), and the linear relationship diagram between the fluorescence intensity (488nm) and the Angelica dahurica concentration (C) of the present invention are shown in FIG. Figure 7 The excitation light for the fluorescence spectrum in B is 414 nm;

[0045] Figure 3 The ultraviolet spectrum characteristic diagram (A), fluorescence spectrum characteristic diagram (B), and linear relationship diagram (C) between fluorescence intensity (488nm) and licorice concentration of the cyanine-like fluorescent probe N-3-I in Example 2 of the present invention are shown in FIG. Figure 8 The excitation light for the fluorescence spectrum in B is 414 nm;

[0046] Figure 4 The ultraviolet spectrum characteristic diagram (A), fluorescence spectrum characteristic diagram (B), and linear relationship diagram (C) between fluorescence intensity (488nm) and American ginseng concentration of the cyanine-like fluorescent probe N-3-I in Example 2 of the present invention are shown in FIG. Fig. 9 The excitation light for the fluorescence spectrum in B is 414 nm;

[0047] Figure 5 The change multiple of the fluorescence intensity at 488 nm after the cyanine-like fluorescent probe N-3-I reacts with Na2SO3 in different solvents in the performance test of the present invention;

[0048] Figure 6 The ultraviolet spectrum characteristic diagram and fluorescence spectrum characteristic diagram of the reaction of the cyanine-like fluorescent probe N-3-I with 10 equivalents of Na2SO3 in different solvents in the performance test of the present invention are shown in FIG. Figure 3 A is the ultraviolet spectrum, Figure 3 B is the fluorescence spectrum (excitation light is 414nm), Figure 3 C is a bar graph of fluorescence intensity of different solvents at 488 nm;

[0049] Figure 7 The ultraviolet spectrum characteristic diagram (A), fluorescence spectrum characteristic diagram (B), and linear relationship diagram (C) between fluorescence intensity (488nm) and NaHSO3 concentration of the cyanine-like fluorescent probe N-3-I in the performance test of the present invention are shown in FIG. Figure 4 The excitation light for the fluorescence spectrum in B is 414 nm;

[0050] Figure 8 The ultraviolet spectrum characteristic diagram (A), fluorescence spectrum characteristic diagram (B), and linear relationship diagram (C) between fluorescence intensity (488nm) and Na2S2O4 concentration of the cyanine-like fluorescent probe N-3-I in the performance test of the present invention are shown, wherein Figure 8 The excitation light for the fluorescence spectrum in B is 414 nm;

[0051] Fig. 9 The ultraviolet spectrum characteristic diagram (A), fluorescence spectrum characteristic diagram (B), and linear relationship diagram (C) between fluorescence intensity (488nm) and Na2S2O5 concentration of the cyanine-like fluorescent probe N-3-I in the performance test of the present invention are shown in FIG. Figure 6 The excitation light for the fluorescence spectrum in B is 414 nm;

[0052] Fig.10 This is a bar graph showing the fluorescence intensity change multiples at 488 nm after the cyanine fluorescent probe N-3-I reacts with different sulfur-containing compounds (100 μM) in the performance test of the present invention. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with the accompanying drawings and preferred embodiments. The given embodiments are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources;

[0055] The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0056] The experimental methods in the following embodiments are conventional methods unless otherwise specified.

[0057] The drying solvents used in the following examples are all molecular sieves 4A (sodium-A molecular sieve) or molecular sieves 3A (potassium-A molecular sieve);

[0058] 1 H spectra were recorded on a JEOL ECZ600S (600 MHz) spectrometer using CDCl3 or CD3OD as solvent;

[0059] Upfield chemical shifts are reported in parts per million based on internal TMS (trimethylsilane) reference data;

[0060] The coupling constant (J) is expressed in Hertz (Hz), and the spin multiple states are expressed as s (singlet), d (doublet), t (triplet), and m (multiplet);

[0061] Column chromatography used thick-walled glass columns and silica gel (300-400 mesh); commercially available 0.25 mm silica gel plates were used for thin layer chromatography (TLC), and ultraviolet light was used for display;

[0062] The UV absorption spectra in the solutions were obtained using a Shimadzu UV-1900 UV-visible spectrophotometer;

[0063] Fluorescence spectra were measured using Spectrofluorometer FS5 fluorescence spectrometer;

[0064] Mass spectra were recorded on a ThermoFisher HPLC-MS / MS instrument.

[0065] Example 1

[0066] This embodiment provides a cyanine-like fluorescent probe, and the synthesis method thereof comprises the following steps:

[0067] S1. Dissolve cesium carbonate (0.48 g, 1.5 mmol) and 1,8-naphtholactam (0.17 g, 1 mmol) in 8 mL of acetonitrile, add iodomethane (0.124 mL, 2 mmol) dropwise, and stir at room temperature for 4 h under an argon atmosphere. Remove the solvent, separate and purify to obtain a bright yellow powder, namely compound 1. The chromatography eluent is a mixed solution of petroleum ether / ethyl acetate in a volume ratio of 23:1. The specific reaction process is as follows:

[0068]

[0069] S2. Compound 1 (0.182 g, 1 mmol) obtained in step 1 was dissolved in anhydrous THF at 0°C, methylmagnesium chloride (1.5 mL, 4.5 mmol) was added dropwise and stirred for 10 min. The mixture was refluxed in an oil bath at 60°C for 2 h under an argon atmosphere. After the reaction was completed, the mixture was placed in an ice bath at 0°C. When the temperature dropped to 0°C, a small amount of water (0.5 mL) was added to quench the reaction. Then perchloric acid (60%, 0.6 mL) was added to the reaction mixture and stirred sufficiently. The yellow-green solution was added to ice water (90 mL), vacuum filtered, and dried to obtain a green powder, namely compound 2. The specific reaction process is as follows:

[0070]

[0071] S3. Add the mixed compound 2 (1.5 mmol, 0.27 g) and 5-indolecarboxaldehyde (1.5 mmol, 0.217 g) into a round-bottom flask, and then add a mixed solution of ethanol and acetonitrile (the volume ratio of ethanol to acetonitrile is 9:1 respectively). Allow to react overnight at room temperature in the dark. After the reaction is completed, filter the black precipitate. Use silica gel column chromatography to separate and purify the reaction mixture. The chromatography eluent is a mixed solution of dichloromethane / methanol in a volume ratio of 10:1 to obtain a cyanine-like fluorescent probe N-3-I.

[0072]

[0073] S4. The cyanine fluorescent probe N-3-I was analyzed by nuclear magnetic resonance hydrogen spectrum. The spectrum is shown in the attached figure. Figure 1 shown.

[0074] Among them, this synthesis method can also adjust S1 to be stirred for 3 hours or 5 hours under argon atmosphere, and adjust S2 to be refluxed in a 50° C. oil bath for 3 hours or in a 70° C. oil bath for 1 hour according to actual operating conditions.

[0075] Example 2

[0076] This example uses the cyanine-like fluorescent probe in Example 1 to detect the sulfite content in actual samples of liquorice root, Angelica dahurica and American genseng, including the following steps:

[0077] S1. Select Chinese medicinal materials Angelica dahurica, Licorice, and American ginseng as samples, add water to the samples and place them at room temperature for 1 hour before taking the filtrate;

[0078] S2. Under an excitation light of 414 nm, measure the fluorescence spectra of different sample filtrates with different volumes (0-30 μL) added to the cyanine fluorescent probe N-3-I (10 μM), record the maximum fluorescence intensity at the peak, and draw a linear relationship graph based on the obtained data.

[0079] As attached Figure 2 As shown in B-4B, as the sample volume increases, the concentration of sulfite in the sample rises, and the N-3-I probe binds to sulfite, resulting in an increase in the fluorescence value at 488 nm.

[0080] In summary, the combination of N-3-I probe and sulfite can be used to determine whether the sulfite content in Chinese medicinal materials samples exceeds the standard. Therefore, the fluorescent probe N-3-I can be used to evaluate sulfite in Chinese medicinal materials samples.

[0081] Performance Testing

[0082] 1. Fluorescence spectrum test

[0083] Fluorescence spectroscopy tests were performed on the cyanine-like fluorescent probe N-3-I described in Example 1 and 100 μM Na2SO3 in solvents with different polarities and dielectric constants.

[0084] The cyanine probe N-3-I described in Example 1 was dissolved in dimethyl sulfoxide and placed in various solvents including petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, acetonitrile, ethyl acetate, ethanol, and methanol. The fluorescence spectrum after the reaction with the addition of 100 μM Na2SO3 and a bar graph based on the maximum fluorescence value at 488 nm were measured, wherein the conditions of the fluorescence spectrometer were: λex=414nm / λem=488nm, Step:1.00nm, Dwell:0.1s, Repeats:1, Bdw=4.

[0085] As attached Figure 5 As shown in the spectrum, the cyanine-like fluorescent probe N-3-I shows the largest emission peak at 488nm, among which in PBS solution, the fluorescence emission peak of probe N-3-I at 488nm is the highest; at the same time, after adding 100μM Na2SO3, the degree of change of probe N-3-I in PBS solution is obvious, so from the results of fluorescence spectrum, the detection effect of probe on Na2SO3 in PBS solution is better than that in other solvents.

[0086] 2. Ultraviolet absorption spectrum test

[0087] The ultraviolet absorption spectrum of the cyanine-like fluorescent probe N-3-I described in Example 1 was tested under different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5.

[0088] S1. Dissolve the cyanine fluorescent probe N-3-I described in Example 1 in dimethyl sulfoxide, and add PBS buffer solution to dilute it into a 10 μM probe buffer solution;

[0089] S2. Take several portions of the above probe buffer solution, add Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 solutions respectively to make their concentrations: 0-4μM, 0-7μM, 0-4μM, and 0-6μM, and then measure the ultraviolet absorption spectra of the cyanine fluorescent probe N-3-I after reacting with amines of different concentrations.

[0090] As attached Figure 6 A- Fig. 9 As shown in A, in the ultraviolet spectrum, the cyanine-like fluorescent probe N-3-I shows a maximum absorption value at 520nm. After reacting with Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 respectively, as the concentration of sulfite substances increases, the absorption peak of the probe at 520nm gradually decreases, and the color of the solution changes from the original dark pink to colorless, indicating that the probe N-3-I can more intuitively realize the "naked eye" detection of sulfite from a visual perspective.

[0091] 3. Fluorescence spectrum test

[0092] Fluorescence spectrum test of the cyanine-like fluorescent probe N-3-I described in Example 1 under different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5:

[0093] S1. Dissolve the cyanine fluorescent probe N-3-I described in Example 1 in dimethyl sulfoxide, and add PBS buffer solution to dilute it into a 10 μM probe buffer solution;

[0094] S2. Take several portions of the above-mentioned probe buffer solution, add Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 solutions respectively, so that their concentrations are: 0-4μM, 0-8μM, 0-3μM, and 0-4μM, respectively. Under the condition of excitation light of 414nm, measure the fluorescence spectra of the cyanine-like fluorescent probe N-3-I after reacting with different concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5, respectively. Among them, the conditions of the fluorescence spectrometer are: λex=414nm, Step:1.00nm, Dwell:0.1s, Repeats:1, Bdw=4.

[0095] like Figure 6 B- Fig. 9 As shown in Figure B, in the fluorescence spectrum, the behavior of the cyanine-like fluorescent probe probe N-3-I is consistent with the ultraviolet spectrum, showing similar sensitivity and selectivity. When the excitation light is 414nm, with the addition of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5, the fluorescence of the probe N-3-I at 488nm gradually increases.

[0096] from Figure 6 C- Fig. 9 As can be seen from Figure C, the fluorescence intensity of the cyanine-like fluorescent probe N-3-I has a good linear relationship with the concentrations of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 (the linear correlation coefficients are R 2 =0.9931, R 2 =0.9997, R 2 =0.9964, R 2 =0.9952), the surface probe N-3-I can be used for quantitative detection of sulfites. The detection limits for Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 can be calculated by the formula to be 1.182nM, 1.046nMμM, 0.422nM, and 0.495nM, respectively.

[0097] from Fig.10 It can be seen that the cyanine-like fluorescent probe N-3-I has a good detection effect on these four sulfites. Among them, the fluorescence change of probe N-3-I for tetravalent S sulfite is stronger and more significant, and the fluorescence intensity of Na2SO3, NaHSO3, Na2S2O4, and Na2S2O5 is enhanced by about 100 times, while the fluorescence intensity enhancement multiples for other sulfides are weak, which shows that the selectivity of probe N-3-I for sulfite is related to the valence of sulfite S.

[0098] 4. Spike recovery test

[0099] The cyanine-like fluorescent probe N-3-I described in Example 1 was used to perform a spike recovery test on actual samples using sulfur dioxide.

[0100] S1. Cut the samples of licorice, angelica dahurica and American ginseng into pieces and soak them at room temperature to obtain the filtrate. Under the excitation light of 414nm, add different volumes (0-30μL) of the supernatant of different Chinese medicinal materials samples to the buffer solution (10μM) of the cyanine fluorescent probe N-3-I to measure the fluorescence spectrum. After reacting for 2-3min at room temperature, measure the changes before and after.

[0101] S2, obtain the fluorescence value from the fluorescence spectrum of S1, substitute it into different standard curves to calculate the SO2 content, and then calculate the spike recovery rate according to the spike recovery formula. The spike recovery formula is as follows:

[0102] P (recovery rate) = (amount of spiked sample - amount of sample measured) / spiked amount;

[0103] The calculation results are shown in Table 1:

[0104] Table 1 Results of spike recovery experiments of probe N-3-I and different samples

[0105]

[0106] As can be seen from Table 1, the average recovery rate of the cyanine-like fluorescent probe N-3-I for spiked Chinese medicinal materials samples is 89%-103%, all within the good range, the relative standard deviation (RSD) is relatively low, and the repeatability is relatively good, indicating that the cyanine-like fluorescent probe N-3-I has a relatively high-quality detection ability in actual sample detection.

[0107] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A cyanine-like fluorescent probe, characterized in that: The cyanine-like fluorescent probe is labeled as N-3-I, and its solution color is dark pink, and its molecular formula is C 22 H 17 ClN2O4, chemical structure is shown in Ⅰ:

2. A method for synthesizing a cyanine-like fluorescent probe as claimed in claim 1, characterized in that: The following steps are included: (1) Cesium carbonate and 1,8-naphtholactam are dissolved in acetonitrile, iodomethane is added dropwise, and the mixture is stirred at room temperature for 3-5 hours under an argon atmosphere. The solvent is removed by rotary evaporation, and a bright yellow powder is obtained by separation and purification, namely, compound 1, whose structural formula is shown in II: (2) Compound 1 obtained in step 1 is dissolved in anhydrous tetrahydrofuran at 0°C, methylmagnesium chloride is added dropwise and stirred, and then refluxed in an oil bath at 50-70°C for 1-3h under argon atmosphere. After the reaction, it is placed in an ice bath until the temperature drops to 0°C, a small amount of water is added to quench the reaction, and then perchloric acid is added to the reaction to obtain a yellow-green solution, which is added to ice water, vacuum filtered and dried to obtain a green powder, namely compound 2, whose structural formula is shown in III: (3) Compound 2 and 5-indolecarboxaldehyde were mixed in a round-bottom flask, and a mixed solution of ethanol and acetonitrile was added. The mixture was reacted at room temperature in the dark overnight. After the reaction, the black precipitate was filtered and the reaction mixture was separated and purified by silica gel column chromatography to obtain a cyanine-like fluorescent probe N-3-I, whose structural formula is shown in IV:

3. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The molar ratio of 1,8-naphtholactam, cesium carbonate and methyl iodide in the step (1) is 1:1.5:1.

2.

4. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The separation and purification in step (1) is carried out by column chromatography, wherein the chromatography eluent is a mixed solution of petroleum ether / ethyl acetate in a volume ratio of 23:

1.

5. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The molar ratio of compound 1 to methylmagnesium chloride in step (2) is 1:4.

5.

6. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The molar ratio of the compound 2 to 5-indolecarboxaldehyde in the step (3) is 1:

1.

7. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The volume ratio of ethanol to acetonitrile in the ethanol-acetonitrile mixed solution in step (3) is 9:

1.

8. The method for synthesizing a cyanine-like fluorescent probe according to claim 2, characterized in that: The separation and purification in step (3) is carried out by column chromatography, wherein the chromatography eluent is a mixed solution of dichloromethane / methanol in a volume ratio of 10:

1.

9. Use of the cyanine-like fluorescent probe according to claim 1 in detecting the sulfite content of traditional Chinese medicine.

10. The use of a cyanine-like fluorescent probe according to claim 9, characterized in that: When sulfite is present in the Chinese medicinal material test sample, the cyanine-like fluorescent probe can respond quickly under very mild conditions, causing its solution to change significantly from the original deep pink to colorless.

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