A fluorescent probe and its preparation method and application in detecting sulfur dioxide derivatives

Abstract

The present invention relates to the technical field of small molecule fluorescent probes, and provides a fluorescent probe, a preparation method thereof, and an application in detecting sulfur dioxide derivatives. The present invention uses triphenylamine as a fluorescent platform to construct a fluorescent probe having the structure shown in ZR‑B‑I. The obtained fluorescent probe can respond to sulfur dioxide derivatives in a short time, achieve rapid detection, and has high sensitivity. The detection limit reaches the micromolar level. It also has the advantages of good selectivity, strong anti-interference ability, and fast response. It can quickly and efficiently identify sulfur dioxide derivatives by colorimetry and fluorescence. The present invention also provides a method for preparing the fluorescent probe described in the above scheme, which adopts a four-step synthesis method to synthesize the fluorescent probe having the structure shown in formula ZR‑B‑I. The synthesis process is simple and easy to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of organic small molecule fluorescent probes, in particular to a fluorescent probe and a preparation method thereof and application in detecting sulfur dioxide derivatives. Background Art

[0002] Food safety has always been one of the most pressing issues for the public. In the food processing industry, sulfur dioxide, as a food additive with numerous advantages, is widely used. Typically, sulfur dioxide is added to food in the form of sulfites such as potassium metabisulfite, sodium metabisulfite, sodium sulfite, sodium bisulfite, and sodium hyposulfite, or used in food processing via sulfur fumigation, providing color protection, preservation, bleaching, and antioxidant benefits. However, the misuse, excessive use, or even illegal use of sulfur dioxide can easily lead to excessive sulfur dioxide residues, which can harm consumers' physical and mental health. Therefore, developing a rapid, convenient, sensitive, and on-site detection method is of practical significance.

[0003] Currently, traditional methods for detecting SO2 content in food include iodine titration and chromatography. However, these traditional methods all have drawbacks to varying degrees, such as cumbersome sample pretreatment, the need for large-scale instrumentation, complex processes, and long detection times. In recent years, fluorescent probe analysis and detection methods have attracted widespread attention from researchers. These methods not only offer many unique advantages, such as good selectivity, high sensitivity, low cost, and ease of operation, but also enable non-destructive testing. Therefore, the application of fluorescent probes for the detection of SO2 in food holds great promise. Currently, fluorescent probes for detecting SO2 in food have achieved real-time dynamic monitoring of SO2 to a certain extent, but they still suffer from low sensitivity and poor anti-interference properties. Summary of the Invention

[0004] In light of this, the present invention provides a fluorescent probe, a preparation method thereof, and its use in detecting sulfur dioxide derivatives. The fluorescent probe provided by the present invention can rapidly and highly selectively identify sulfur dioxide derivatives through colorimetry and fluorescence, has high sensitivity, and exhibits strong anti-interference capabilities, making it suitable for practical sample detection and bioimaging research.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A fluorescent probe having the structure shown in formula ZR-BI:

[0007]

[0008] The present invention also provides a method for preparing the fluorescent probe described in the above scheme, comprising the following steps:

[0009] Mixing 4-triphenylamine borate, 4-bromo-2-hydroxybenzaldehyde, a first basic compound, a palladium catalyst, and an organic solvent to perform a Suzuki coupling reaction to obtain a compound with a structure shown in Formula 1;

[0010]

[0011] Mixing the compound of Formula 1, acrolein, a second basic compound, and an organic solvent to perform an addition reaction to obtain a compound of Formula 2;

[0012]

[0013] 2-methylbenzothiazole, methyl iodide and an organic solvent are mixed to carry out a quaternization reaction to obtain a compound with a structure shown in Formula 3;

[0014]

[0015] The compound with the structure represented by Formula 2, the compound with the structure represented by Formula 3 and an organic solvent are mixed and subjected to a condensation reaction to obtain a fluorescent probe with the structure represented by Formula ZR-BI.

[0016] Preferably, the molar ratio of 4-triphenylamine borate to 4-bromo-2-hydroxybenzaldehyde is 1:1.2 to 1:1.5;

[0017] The first alkaline compound is potassium carbonate; the molar ratio of 4-triphenylamine borate to potassium carbonate is 1:3 to 1:6;

[0018] The palladium catalyst is PdCl2(dppf); the molar ratio of the 4-triphenylamine borate to the palladium catalyst is 1:0.05 to 1:0.1;

[0019] The organic solvents used in the Suzuki coupling reaction are toluene and anhydrous ethanol, and the volume ratio of toluene to anhydrous ethanol is 2:1. The Suzuki coupling reaction is carried out under heating reflux conditions, and the reaction time is 5 to 6 hours.

[0020] Preferably, after the Suzuki coupling reaction is completed, the method further comprises cooling the obtained reaction solution and concentrating it, dissolving the obtained concentrate and then filtering it with diatomaceous earth and separating and purifying it with a silica gel column to obtain a compound with a structure represented by Formula 1; the eluent used for the silica gel column separation and purification is a mixed solvent of ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:200 to 1:20.

[0021] Preferably, the molar ratio of the compound of the structure represented by Formula 1 to acrolein is 1:2 to 1:3;

[0022] The second alkaline compound is potassium carbonate; the molar ratio of the compound represented by the structure of Formula 1 to potassium carbonate is 1:2 to 1:3;

[0023] The organic solvent used in the addition reaction is 1,4-dioxane; the addition reaction is carried out under heating reflux conditions, and the reaction time is 72 hours.

[0024] Preferably, after the addition reaction is completed, the method further comprises cooling the obtained reaction liquid and then extracting it, and subjecting the obtained extract product to silica gel column separation and purification to obtain a compound with a structure shown in Formula 2; the solvent used for the extraction is ethyl acetate and water; the elution method for the silica gel column separation and purification is gradient elution, and the eluent used is a mixed solvent of ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate and petroleum ether in the mixed solvent during the gradient elution changes from 1:200 to 1:10.

[0025] Preferably, the molar ratio of 2-methylbenzothiazole to methyl iodide is 1:1 to 1:1.2; the organic solvent used in the quaternization reaction is toluene; the quaternization reaction is carried out under heating reflux conditions, and the reaction time is 12 to 16 hours.

[0026] Preferably, the molar ratio of the compound represented by Formula 2 to the compound represented by Formula 3 is 1:1 to 1:1.2;

[0027] The organic solvents used in the condensation reaction are toluene and n-butanol, and the volume ratio of the toluene and n-butanol is 1:1. The condensation reaction is carried out under heating reflux conditions, and the reaction time is 12 to 16 hours.

[0028] Preferably, after the condensation reaction is completed, the obtained reaction solution is cooled and then concentrated and separated and purified by silica gel column to obtain a fluorescent probe having a structure shown in formula ZR-BI; the elution process of the silica gel column separation and purification includes a first stage and a second stage, the eluent of the first stage is a mixed solvent of dichloromethane and petroleum ether, and the volume ratio of dichloromethane and petroleum ether is 1:1; the elution method of the second stage is gradient elution, and the eluent used is a mixed solvent of methanol and dichloromethane. During the gradient elution, the volume ratio of methanol and dichloromethane in the mixed solvent changes from 1:200 to 1:10.

[0029] The present invention also provides the use of the fluorescent probe described in the above scheme in detecting sulfur dioxide derivatives.

[0030] The present invention provides a fluorescent probe having the structure represented by the formula ZR-BI. The fluorescent probe is constructed using triphenylamine as a fluorescent platform. The excellent planarity of triphenylamine enables it to exhibit high-intensity fluorescence characteristics, enabling rapid detection of sulfur dioxide derivatives in a short period of time. Furthermore, the fluorescent probe constructed in the present invention exhibits high sensitivity to sulfur dioxide derivatives, with a detection limit reaching the micromolar level. It also exhibits advantages such as good selectivity, strong anti-interference ability, and fast response, enabling rapid and efficient identification of sulfur dioxide derivatives through colorimetry and fluorescence.

[0031] The present invention also provides a method for preparing the fluorescent probe described in the above scheme. The present invention adopts a four-step synthesis method to synthesize the fluorescent probe having the structure shown in the formula ZR-BI. The synthesis process is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the H NMR spectrum of compound 1 (solvent is CDCl3);

[0033] Figure 2 is the carbon NMR spectrum of compound 1 (solvent is CDCl3);

[0034] Figure 3 is the H NMR spectrum of compound 2 (the solvent is deuterated DMSO);

[0035] Figure 4 is the carbon NMR spectrum of compound 2 (the solvent is deuterated DMSO);

[0036] Figure 5 is the H NMR spectrum of compound 3 (the solvent is deuterated DMSO);

[0037] Figure 6 is the carbon NMR spectrum of compound 3 (the solvent is deuterated DMSO);

[0038] Figure 7 This is the H NMR spectrum of probe ZR-BI (the solvent is deuterated DMSO);

[0039] Figure 8 This is the carbon NMR spectrum of the probe ZR-BI (the solvent is deuterated DMSO);

[0040] Figure 9 The fluorescence emission spectra of probe ZR-BI for identifying different concentrations of sulfur dioxide are as follows: the excitation wavelength is 354 nm, and the emission wavelength is 478 nm;

[0041] Figure 10 This is the fluorescence selectivity curve of probe ZR-BI for recognizing sulfur dioxide, with an excitation wavelength of 354 nm and an emission wavelength of 478 nm;

[0042] Figure 11 This is the fluorescence interference resistance diagram of probe ZR-BI in recognizing sulfur dioxide, with an excitation wavelength of 354 nm and an emission wavelength of 478 nm;

[0043] Figure 12 This is the minimum detection limit of probe ZR-BI for identifying sulfur dioxide, with an excitation wavelength of 354 nm and an emission wavelength of 478 nm;

[0044] Figure 13 This is the fluorescence kinetics of the probe ZR-BI recognizing sulfur dioxide, with an excitation wavelength of 354 nm and an emission wavelength of 478 nm;

[0045] Figure 14 This is the pH applicable range of probe ZR-BI for recognizing sulfur dioxide, with an excitation wavelength of 354 nm and an emission wavelength of 478 nm. DETAILED DESCRIPTION

[0046] The present invention provides a fluorescent probe having a structure shown in the formula ZR-BI:

[0047]

[0048] The present invention also provides a method for preparing the fluorescent probe described in the above scheme, comprising the following steps:

[0049] Mixing 4-triphenylamine borate, 4-bromo-2-hydroxybenzaldehyde, a first basic compound, a palladium catalyst, and an organic solvent to perform a Suzuki coupling reaction to obtain a compound with a structure shown in Formula 1 (denoted as Compound 1);

[0050]

[0051] The compound of the structure shown in Formula 1, acrolein, a second basic compound and an organic solvent are mixed and subjected to an addition reaction to obtain a compound of the structure shown in Formula 2 (denoted as Compound 2);

[0052]

[0053] 2-methylbenzothiazole, methyl iodide and an organic solvent are mixed to carry out a quaternization reaction to obtain a compound with a structure shown in Formula 3 (denoted as Compound 3);

[0054]

[0055] The compound with the structure represented by Formula 2, the compound with the structure represented by Formula 3 and an organic solvent are mixed and subjected to a condensation reaction to obtain a fluorescent probe with the structure represented by Formula ZR-BI.

[0056] The synthetic route of the present invention is as follows:

[0057]

[0058] The preparation method of the present invention is described in detail below.

[0059] 4-boric acid triphenylamine, 4-bromo-2-hydroxybenzaldehyde, a first basic compound, a palladium catalyst and an organic solvent are mixed to carry out a Suzuki coupling reaction to obtain a compound with a structure shown in Formula 1. In the present invention, the molar ratio of the 4-boric acid triphenylamine and 4-bromo-2-hydroxybenzaldehyde is preferably 1:1.2; the molar ratio of the 4-boric acid triphenylamine to the basic compound is preferably 1:3 to 1:6, more preferably 1:5; the basic compound is preferably potassium carbonate; the molar ratio of the 4-boric acid triphenylamine to the palladium catalyst is preferably 1:0.05 to 1:0.1, more preferably 1:0.1; the palladium catalyst is preferably PdCl2(dppf); the organic solvent used in the Suzuki coupling reaction is preferably toluene and anhydrous ethanol; the volume ratio of toluene and anhydrous ethanol is preferably 2:1; the Suzuki coupling reaction is preferably carried out under heating reflux conditions, the heating reflux temperature is preferably 60°C, the Suzuki coupling reaction time is preferably 5 to 6 hours, and the Suzuki coupling reaction is preferably carried out under nitrogen protection. In a specific embodiment of the present invention, it is preferred to first dissolve 4-triphenylamine borate and 4-bromo-2-hydroxybenzaldehyde in a mixed solvent of toluene and anhydrous ethanol, and then add the alkaline compound and palladium catalyst, followed by heating to reflux for reaction.

[0060] After the Suzuki coupling reaction is completed, the present invention preferably cools the obtained reaction solution and then concentrates it, dissolves the obtained concentrate and then sequentially filters it through diatomaceous earth and separates and purifies it through a silica gel column to obtain a compound with a structure represented by Formula 1; the elution mode of the silica gel column separation and purification is gradient elution, and the eluent used is a mixed solvent of ethyl acetate and petroleum ether. During the gradient elution, the volume ratio of ethyl acetate and petroleum ether in the mixed solvent is preferably changed from 1:200 to 1:20. The present invention has no special requirements for the specific gradient of the gradient elution, as long as it can achieve sufficient elution of the product; the present invention removes the solvent in the reaction solution by concentration; the solvent used to dissolve the concentrate is preferably dichloromethane.

[0061] After obtaining the compound of Formula 1, the present invention mixes the compound of Formula 1, acrolein, a second basic compound, and an organic solvent to perform an addition reaction to obtain a compound of Formula 2. In the present invention, the molar ratio of the compound of Formula 1 to acrolein is preferably 1:2 to 1:3, more preferably 1:2.5; the second basic compound is preferably potassium carbonate; the molar ratio of the compound of Formula 1 to potassium carbonate is preferably 1:2 to 1:3, more preferably 1:2.5; the solvent used in the addition reaction is preferably 1,4-dioxane; the addition reaction is carried out under heating and reflux conditions, the heating and reflux temperature is preferably 105°C, and the addition reaction time is preferably 72 hours.

[0062] After the addition reaction is completed, the present invention preferably cools the obtained reaction solution and extracts it, and the obtained extract is separated and purified by silica gel column to obtain a compound with a structure shown in Formula 2; the solvent used for the extraction is preferably ethyl acetate and water; the present invention has no special requirements for the ratio of ethyl acetate and water, as long as sufficient extraction can be achieved; the elution method for the silica gel column separation and purification is gradient elution, and the eluent used is preferably a mixed solvent of ethyl acetate and petroleum ether. During the gradient elution, the volume ratio of ethyl acetate and petroleum ether in the mixed solvent is preferably changed from 1:200 to 1:10. The present invention has no special requirements for the specific gradient of the gradient elution, as long as sufficient elution of the product can be achieved.

[0063] The present invention involves mixing 2-methylbenzothiazole, methyl iodide, and an organic solvent for a quaternization reaction to obtain a compound having the structure shown in Formula 3. In the present invention, the molar ratio of 2-methylbenzothiazole to methyl iodide is preferably 1:1 to 1:1.2, more preferably 1:1. The organic solvent used in the quaternization reaction is preferably toluene. The quaternization reaction is preferably carried out under heating and reflux conditions, the heating and reflux temperature is preferably 110°C, and the quaternization reaction time is preferably 12 to 16 hours. After the quaternization reaction is completed, the resulting reaction solution is preferably cooled and filtered, and the resulting filter cake is washed and dried to obtain the compound having the structure shown in Formula 2. The washing detergent is preferably glacial ethanol.

[0064] After obtaining the compound of the structure represented by Formula 2 and the compound of the structure represented by Formula 3, the present invention mixes the compound of the structure represented by Formula 2, the compound of the structure represented by Formula 3, and an organic solvent for a condensation reaction to obtain a fluorescent probe having a structure represented by Formula ZR-BI; in the present invention, the molar ratio of the compound of the structure represented by Formula 2 and the compound of the structure represented by Formula 3 is preferably 1:1 to 1:1.2, more preferably 1:1.1; the organic solvent used in the condensation reaction is preferably toluene and n-butanol, and the volume ratio of toluene and n-butanol is preferably 1:1; the condensation reaction is preferably carried out under heating reflux conditions, the heating reflux temperature is preferably 110°C, and the condensation reaction time is preferably 12 to 16 hours.

[0065] After the condensation reaction is completed, the present invention preferably cools the obtained reaction solution and then concentrates and separates and purifies it on a silica gel column to obtain a fluorescent probe having a structure represented by the formula ZR-BI; the elution process of the silica gel column separation and purification includes a first stage and a second stage, and the eluent of the first stage is preferably a mixed solvent of dichloromethane and petroleum ether, and the volume ratio of dichloromethane to petroleum ether is preferably 1:1; the eluent of the second stage is preferably a mixed solvent of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is preferably 1:200 to 1:10.

[0066] The present invention also provides the use of the fluorescent probe described in the above scheme in detecting sulfur dioxide derivatives; the detection is specifically quantitative detection; the sulfur dioxide derivative is preferably SO3 2- ; The fluorescence intensity of the fluorescent probe provided by the present invention increases with the increase of the concentration of sulfur dioxide derivatives; in a specific embodiment of the present invention, the fluorescent probe and the analyte are preferably added to the detection system for fluorescence detection, and the content of the sulfur dioxide derivative is determined according to the fluorescence intensity and the standard curve; the detection system is preferably PBS / DMSO, wherein the concentration of PBS is 10mM, and the volume ratio of PBS and DMSO is 8:2, hereinafter referred to as PBS / DMSO (10mM, v / v=8:2); the sulfur dioxide derivatives are prepared with PBS buffer; the concentration of the fluorescent probe in the detection system is preferably 10μM; the pH value of the detection system is preferably 7-10, and the standard curve is preferably a curve showing the relationship between the concentration of sulfur dioxide derivatives and the fluorescence intensity; in the present invention, the conditions for detecting sulfur dioxide or its derivatives using the fluorescent probe preferably include: an excitation wavelength of 354nm, a voltage of 400V, a detection temperature of 37°C, a slit of 10nm×10nm, and an emission wavelength of 478nm.

[0067] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. 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.

[0068] Example 1

[0069] In a 250 mL round-bottom flask, 4-triphenylamine borate (1.735 g, 6 mmol) and 4-bromo-2-hydroxybenzaldehyde (1.447 g, 7.2 mmol) were dissolved in a mixed solution of 60 mL of toluene and 30 mL of ethanol. K2CO3 (4.14 g, 30 mmol) and PdCl2(dppf) (0.439 g, 0.6 mmol) were added. Under a nitrogen atmosphere, the reaction was refluxed at 60°C for 5 h. After cooling to room temperature, the toluene and ethanol were removed by distillation under reduced pressure. The product was dissolved in dichloromethane and passed through diatomaceous earth to preliminarily remove impurities. The product was then separated and purified on a silica gel column using ethyl acetate:petroleum ether = 1:200 to 1:20 as eluent to obtain compound 1.

[0070] The NMR identification data of compound 1 are as follows: 1 H NMR(400MHz,Chloroform-d)δ11.10(s,1H),9.85(s,1H),7.54(d,J=8.1Hz,1H),7.47(d,J =8.7Hz,2H),7.29-7.23(m,4H),7.23-7.18(m,1H),7.17-7.09(m,6H),7.09-7.01(m,3H); 13 C NMR (101 MHz, Chloroform-d) δ 195.87, 162.16, 149.38, 148.95, 147.37, 134.20, 132.27, 129.57, 128.19, 125.16, 123.75, 122.87, 119.31, 118.32, 114.90. H NMR spectrum is shown in Figure 1 As shown, the carbon NMR spectrum is as follows Figure 2 shown.

[0071] Example 2

[0072] In a 100 mL round-bottom flask, compound 1 (200 mg, 0.55 mmol) and acrolein (91.33 μL, 1.37 mmol) were dissolved in 30 mL of 1,4-dioxane, and K2CO3 (189.26 mg, 1.37 mmol) was added. The reaction was refluxed at 105°C for 72 h. After cooling to room temperature, the mixture was extracted with EA and water. The extract was dehydrated by passing through anhydrous sodium sulfate and then purified by silica gel column with ethyl acetate: petroleum ether = 1:200 to 1:10 as eluent to obtain compound 2.

[0073] The NMR identification data of compound 2 are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.57(s,1H),7.77-7.53(m,3H),7.50-7.23(m,6H),7.06(m,9H),4.97(s,2H); 13 C NMR (101 MHz, DMSO-d6) δ 190.53, 155.88, 147.61, 146.79, 144.35, 140.79, 132.07, 130.84, 130.27, 129.69, 127.71, 124.58, 123.64, 122.42, 119.77, 119.24, 113.11, 62.85. H NMR spectrum is shown in Figure 1. Figure 3 As shown, the carbon NMR spectrum is as follows Figure 4 shown.

[0074] Example 3

[0075] 2-Methylbenzothiazole (2 g, 13.4 mmol) and methyl iodide (1.9 g, 13.39 mmol) were dissolved in 20 mL of toluene and reacted under reflux at 110°C for 16 h. After the reaction was completed, the reaction solution was cooled and filtered, and the filter cake was rinsed with ice-cold EtOH and dried to obtain compound 3.

[0076] The NMR identification data of compound 3 are as follows 1 H NMR (400MHz, DMSO-d6) δ8.46(dd,J=8.3,1.2Hz,1H),8.30(m,1H),7.90(m,1H),7.81(m,1H),4.21(s,3H),3.19(s,3H); 13 C NMR (101 MHz, DMSO-d6) δ 177.21, 141.56, 129.23, 128.68, 128.04, 124.50, 116.76, 36.28, 17.23. H NMR spectrum is shown in Figure 5 As shown, the carbon NMR spectrum is as follows Figure 6 shown.

[0077] Example 4

[0078] Compound 2 (100 mg, 0.25 mmol) and compound 3 (79.33 mg, 0.27 mmol) were dissolved in a mixed solvent of 10 mL of toluene and 10 mL of n-butanol, and the mixture was reacted at 110°C under reflux for 16 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the product was separated and purified on a silica gel column using dichloromethane: petroleum ether = 1:1 and then methanol: dichloromethane = 1:200-1:10 as eluent to obtain the target product ZR-BI.

[0079] The NMR identification data of ZR-BI are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=8.1Hz,1H),8.21(d,J=8.5Hz,1H),7.92(d,J=15.6Hz,1H),7.85(m,1H),7.76(m,1H),7.63(d,J=8. 3Hz,2H),7.48(s,1H),7.41-7.22(m,7H),7.19(s,1H),7.09(dd,J=17.2,7.8Hz,6H),6.98(d,J=8.3Hz,2H),5.28(s,2H),4.29(s,3H); 13 C NMR (101 MHz, DMSO-d6) δ 171.22, 155.36, 147.58, 146.76, 145.10, 143.55, 142.09, 135.68, 131.96, 129.73, 129.66, 129.41, 129.23, 128.33, 127.86, 127.65, 124.65, 124.26, 123.71, 122.29, 120.56, 119.88, 116.78, 112.84, 112.60, 64.48, 48.62, 36.33. Figure 7 As shown, the carbon NMR spectrum is as follows Figure 8 shown.

[0080] Example 5

[0081] Fluorescence Spectral Determination of Sulfur Dioxide Using Fluorescent Probe ZR-BI

[0082] (1) Preparation of fluorescent probe ZR-BI solution: The fluorescent probe ZR-BI was prepared into a stock solution using dimethyl sulfoxide (DMSO).

[0083] (2) Preparation of Na2SO3 solution: Dissolve Na2SO3 powder in PBS buffer to prepare a stock solution.

[0084] The detection system used was PBS / DMSO solution (10 mM, pH=8.1, v / v=8 / 2), and the concentration of the fluorescent probe in the control detection system was fixed at 10 μM.

[0085] In PBS / DMSO solution (10 mM, pH = 8.1, v / v = 8 / 2), 10 μM fluorescent probe ZR-BI responded to different final concentrations of Na2SO3 solution (0-1000 μM), and its fluorescence emission spectrum was measured under the conditions of excitation wavelength of 354 nm, voltage of 400 V, temperature of 37 ° C, and slit size of 10 nm × 10 nm (the following examples all adopt this detection condition and will not be described in detail). Figure 9 As shown. Figure 9 As can be seen, the fluorescence intensity at 478 nm gradually increases with increasing Na2SO3 concentration, so the fluorescent probe ZR-BI can be used as a fluorescent probe for the quantitative detection of sulfur dioxide. The fluorescent probe ZR-BI of the present invention has good solubility and detection sensitivity and has important application value in fluorescent probes, biological detection, and fluorescence imaging.

[0086] Example 6

[0087] Selectivity experiment of fluorescent probe ZR-BI

[0088] The fluorescent probe must be able to achieve single recognition of the detected species, so it is necessary to test the fluorescence selectivity of the probe ZR-BI to different active small molecules. The test solution for the fluorescence selectivity experiment of the probe ZR-BI is PBS / DMSO solution (10mM, pH=8.1, v / v=8 / 2), and the probe concentration is fixed at 10μM. Figure 10 As shown in the figure, under 354 nm excitation, the probe alone showed almost no fluorescence emission at 478 nm. However, after adding Na2SO3 (40 eq.), the fluorescence intensity at 478 nm was significantly enhanced. However, after adding other active species: CH3COONa, Al(NO3)3, NaBr, CaCO3, NaCl, NaClO, K2CO3, CuSO4·5H2O, NaF, FeCl3, KHCO3, NaHS, KI, MgSO4·7H2O, NaNO2, Na2S2SO3·5H2O, CaSO4·2H2O, ZnCl (the addition amount of the above active species was 40 eq.) and GSH, Cys, Hcy (the addition amount of GSH, Cys, and Hcy was 100 eq.), the fluorescence intensity at 478 nm of the solution was the same as that of the probe alone, with no significant enhancement. The above selectivity experimental results show that under 354nm excitation, probe ZR-BI has good selectivity for sulfur dioxide.

[0089] Example 7

[0090] Anti-interference experiment of fluorescent probe ZR-BI

[0091] To investigate the anti-interference ability of probe ZR-BI in response to sulfur dioxide in a complex environment, other active small molecules were tested using fluorescence emission spectroscopy. The test solution for the fluorescence interference experiment of probe ZR-BI was PBS / DMSO solution (10 mM, pH = 8.1, v / v = 8 / 2). Under 354nm excitation, the probe concentration was fixed at 10μM, and CH3COONa, Al(NO3)3, NaBr, CaCO3, NaCl, NaClO, K2CO3, CuSO4·5H2O, NaF, FeCl3, KHCO3, NaHS, KI, MgSO4·7H2O, NaNO2, Na2S2SO3·5H2O, CaSO4·2H2O, ZnCl (the addition amount of the above active species was 40eq.) and GSH, Cys, Hcy (the addition amount of GSH, Cys, Hcy was 100eq.) were added to the individual probe solutions, and then 40eq. of Na2SO3 was added to each solution. The fluorescence emission intensity of the solution at 478nm was detected. The results are shown in Figure 2. Figure 11 As shown by Figure 11 It can be seen that the fluorescence intensity of the solution at 478 nm is basically the same as the fluorescence intensity of the solution with Na2SO3 added alone at 478 nm, which shows that the probe ZR-BI has a strong anti-interference ability against other active small molecules when detecting sulfur dioxide.

[0092] Example 8

[0093] Minimum detection limit experiment of fluorescent probe ZR-BI

[0094] Fluorescent probes are required to have very high detection sensitivity, so the minimum detection limit is also an important indicator to measure the properties of the probe. Fluorescence emission spectroscopy is used to test the minimum detection limit of probe ZR-BI for sulfur dioxide. The experimental test solution for the minimum detection limit of probe ZR-BI is PBS / DMSO solution (10mM, pH=8.1, v / v=8 / 2). Under 354nm excitation, the concentration of probe ZR-BI is fixed at 10μM, the concentration of Na2SO3 in the solution is adjusted (0μM to 100μM), and the fluorescence intensity of the probe solution containing different concentrations of Na2SO3 at 478nm is measured. The results are as follows: Figure 12 As shown. Figure 12 It can be seen that the fluorescence intensity at 478 nm of the solution has a good linear relationship with the Na2SO3 concentration in the range of 0 to 90 μM (R 2=0.9916). According to IUPAC rules, the detection limit of probe ZR-BI for sulfur dioxide was calculated to be 0.1 μM using the detection limit formula (3σ / k). These detection limit experiments demonstrate that probe ZR-BI has high sensitivity for sulfur dioxide and can quantitatively detect extremely trace concentrations of sulfur dioxide.

[0095] Example 9

[0096] Fluorescence kinetics experiment of fluorescent probe ZR-BI

[0097] Response time is also an important indicator for judging the quality of fluorescent probes. Fluorescence emission spectroscopy was used to test the fluorescence kinetics of probe ZR-BI to sulfur dioxide. The experimental solution for probe ZR-BI fluorescence kinetics was PBS / DMSO solution (10mM, pH=8.1, v / v=8 / 2). Under 354nm excitation, the concentration of fixed probe ZR-BI was 10μM, and the change of 478nm fluorescence intensity of the solution without and with Na2SO3 (100μM) was measured over time. The results are as follows Figure 13 As shown, according to Figure 13 As can be seen, the fluorescence intensity at 478 nm of the Na2SO3 (100 μM) solution increases rapidly over time and reaches a plateau after 7 minutes. The fluorescence kinetics experimental results show that the probe ZR-BI responds quickly to sulfur dioxide and can achieve rapid detection.

[0098] Example 10

[0099] pH stability experiment of fluorescent probe ZR-BI

[0100] pH is also an important criterion for measuring the versatility of probes. Fluorescence spectroscopy was used to investigate the ability of probe ZR-BI to recognize sulfur dioxide in different pH environments. The pH test solution for probe ZR-BI was a PBS / DMSO solution (10mM, v / v=8 / 2) with a pH range of 1 to 14. The concentrations of probe and Na2SO3 were 10μM and 100μM, respectively. Figure 14 As shown, according to Figure 14 As can be seen, the fluorescence emission intensity of the probe alone at 478 nm remained unchanged within the pH range of 1 to 14. However, in the presence of Na₂SO₃, the fluorescence emission intensity at 478 nm of the test solution increased significantly within the pH range of 7 to 10. The pH test results demonstrate that the ZR-BI probe can detect sulfur dioxide under physiological conditions.

[0101] Example 11

[0102] Testing food samples

[0103] The food samples used were rock sugar, dried mango, and rock sugar kumquat. The test method was as follows: the food samples were appropriately chopped, and an appropriate amount was weighed and ultrasonically extracted with PBS buffer for 30 minutes. The sample was then filtered to prepare the test solution. The test system used was a PBS / DMSO solution (10 mM, pH = 8.1, v / v = 8 / 2). Each sample was divided into a control group and a test group. The experiment was repeated three times for each sample. The fluorescence signal of the sample was recorded at 478 nm, and the accuracy of the method was evaluated by spike recovery.

[0104] The test results are shown in Table 1.

[0105] Table 1 Food sample testing

[0106]

[0107] According to the data in Table 1, it can be seen that the fluorescent probe of the present invention is used to detect sulfur dioxide derivatives in food, and the detection results are highly accurate.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that It has the structure shown in formula ZR-BI: Formula ZR-B-I.

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: Mixing 4-triphenylamine borate, 4-bromo-2-hydroxybenzaldehyde, a first basic compound, a palladium catalyst, and an organic solvent to perform a Suzuki coupling reaction to obtain a compound with a structure shown in Formula 1; Formula 1; Mixing the compound of Formula 1, acrolein, a second basic compound, and an organic solvent to perform an addition reaction to obtain a compound of Formula 2; Formula 2; 2-methylbenzothiazole, methyl iodide and an organic solvent are mixed to carry out a quaternization reaction to obtain a compound with a structure shown in Formula 3; Formula 3; The compound with the structure represented by Formula 2, the compound with the structure represented by Formula 3 and an organic solvent are mixed and subjected to a condensation reaction to obtain a fluorescent probe with the structure represented by Formula ZR-BI.

3. The preparation method according to claim 2, characterized in that The molar ratio of the 4-triphenylamine borate to 4-bromo-2-hydroxybenzaldehyde is 1:1.2 to 1:1.5; The first alkaline compound is potassium carbonate; the molar ratio of 4-triphenylamine borate to potassium carbonate is 1:3 to 1:6 The palladium catalyst is PdCl2(dppf); the molar ratio of the 4-triphenylamine borate to the palladium catalyst is 1:0.05 to 1:0.1; The organic solvents used in the Suzuki coupling reaction are toluene and anhydrous ethanol, and the volume ratio of toluene to anhydrous ethanol is 2:

1. The Suzuki coupling reaction is carried out under heating reflux conditions, and the reaction time is 5 to 6 hours.

4. The preparation method according to claim 2, characterized in that After the Suzuki coupling reaction is completed, the method further includes cooling the obtained reaction solution and concentrating it, dissolving the obtained concentrate and then filtering it with diatomaceous earth and separating and purifying it with a silica gel column to obtain a compound with a structure represented by Formula 1; the elution method of the silica gel column separation and purification is gradient elution, and the eluent used is a mixed solvent of ethyl acetate and petroleum ether. During the gradient elution, the volume ratio of ethyl acetate and petroleum ether in the mixed solvent changes from 1:200 to 1:

20.

5. The preparation method according to claim 2, characterized in that The molar ratio of the compound represented by the formula 1 to acrolein is 1:2 to 1:3; The second alkaline compound is potassium carbonate; the molar ratio of the compound represented by the structure of Formula 1 to potassium carbonate is 1:2 to 1:3; The organic solvent used in the addition reaction is 1,4-dioxane; the addition reaction is carried out under heating reflux conditions, and the reaction time is 72 h.

6. The preparation method according to claim 2, characterized in that After the addition reaction is completed, the method further includes cooling the obtained reaction liquid and then extracting it, and subjecting the obtained extract product to silica gel column separation and purification to obtain a compound with a structure shown in Formula 2; the solvent used for the extraction is ethyl acetate and water; the elution method for the silica gel column separation and purification is gradient elution, and the eluent used is a mixed solvent of ethyl acetate and petroleum ether. During the gradient elution, the volume ratio of ethyl acetate and petroleum ether in the mixed solvent changes from 1:200 to 1:

10.

7. The preparation method according to claim 2, characterized in that The molar ratio of 2-methylbenzothiazole to methyl iodide is 1:1 to 1:1.2; the organic solvent used in the quaternization reaction is toluene; the quaternization reaction is carried out under heating reflux conditions, and the reaction time is 12 to 16 hours.

8. The preparation method according to claim 2, characterized in that The molar ratio of the compound represented by Formula 2 to the compound represented by Formula 3 is 1:1 to 1:1.2; The organic solvents used in the condensation reaction are toluene and n-butanol, and the volume ratio of toluene to n-butanol is 1:

1. The condensation reaction is carried out under heating reflux conditions, and the reaction time is 12 to 16 hours.

9. The preparation method according to claim 2, characterized in that After the condensation reaction is completed, the obtained reaction solution is cooled and then concentrated and separated and purified by silica gel column to obtain a fluorescent probe having a structure represented by formula ZR-BI; the elution process of the silica gel column separation and purification includes a first stage and a second stage, the eluent of the first stage is a mixed solvent of dichloromethane and petroleum ether, and the volume ratio of dichloromethane and petroleum ether is 1:1; the elution method of the second stage is gradient elution, and the eluent is a mixed solvent of methanol and dichloromethane. During the gradient elution, the volume ratio of methanol and dichloromethane changes from 1:200 to 1:

10.

10. Use of the fluorescent probe according to claim 1 in detecting sulfur dioxide derivatives; the sulfur dioxide derivative is SO3 2- .

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