A fluorescent probe for simultaneous detection of bosonicine and sulfur dioxide derivatives, its preparation method and application

Abstract

This invention belongs to the field of organic fluorescent probe molecules, specifically referring to a fluorescent probe for the simultaneous detection of Bosein and sulfur dioxide derivatives, its preparation method, and its applications, addressing the technological gap in fluorescent probe detection of Bosein. The proposed fluorescent probe uses a triacrylonitrile furan as the Michael reaction site and introduces a catechol structure as an electron-donating group, enabling the probe to exhibit intramolecular charge transfer effects. Simultaneously, it can achieve ratiowise detection of sulfur dioxide derivatives at 595 nm and 336 nm. Furthermore, the catechol structure can also serve as a binding site for the boron-containing compound tavaborone. Because the hydroxyl group of Bosein has a stronger binding affinity for TVB, it can lead to the dissociation of TVB from the probe, thereby restoring the probe's fluorescence. The detection limits of this probe for Bosein and sulfur dioxide derivatives are 1.2 μM and 6.8 nM, respectively. The successful detection of Bosein by this probe provides design ideas for the future development of specific detection of some important substances that lack UV-Vis absorption and fluorescence, and offers reference for the design of other similar molecules and the detection of target substances.

Description

Technical Field

[0001] This invention belongs to the field of organic fluorescent probe molecules, and specifically refers to a fluorescent probe for simultaneously detecting bosonicine and sulfur dioxide derivatives, its preparation method, and its application. Background Technology

[0002] Pro-Xylane is a xylose derivative with anti-aging activity. Its active ingredient, chemically named hydroxypropyltetrahydropyranotriol, was originally derived from a plant called beech and developed by L'Oréal's research team for use in their products. Its main functions include promoting collagen synthesis, improving damaged skin, preventing moisture loss, and anti-aging. Pro-Xylane influences the formation of GAGs (glycosaminoglycans), an extracellular matrix that plays a crucial role in maintaining moisture in loose collagen proteins. Its hyaluronic acid content has high viscosity and can adhere to joint surfaces, providing lubrication and protection. Currently, many cosmetics on the market claim to contain Pro-Xylane, but few explicitly state the amount. Furthermore, the complex production process of Pro-Xylane, requiring meticulous extraction and purification, has led to a mixed market for raw materials and inconsistent product quality. Currently, there is no unified testing standard; therefore, a rapid method for detecting Pro-Xylane is urgently needed.

[0003] Currently, common methods for detecting Bosein include high-performance liquid chromatography (HPLC) with an evaporative light scattering detector.

[103] Methods such as ion chromatography with electrochemical detectors are available. However, evaporative light scattering (ELS) detectors detect concentration by evaporating the mobile phase into a gas and detecting the amount of light scattered in the gas. Therefore, substances with lower volatility than the mobile phase in the sample are also detected, making specific detection of Bosein impossible. Furthermore, the response value of ELS detectors shows an exponential relationship with the amount of substance, not the commonly used linear relationship. Liquid chromatography-mass spectrometry (LC-MS) generally only detects low concentrations of substances; for large amounts of added Bosein, dilution is required, which increases error. These methods all require large instruments, are complex to operate, and cannot perform in-situ sample detection. Therefore, developing fluorescence methods for Bosein detection is of great significance.

[0004] Sulfur dioxide has a bleaching effect and is therefore commonly used in the bleaching of cosmetic raw materials. However, excessive exposure to sulfur dioxide and its derivatives can lead to respiratory, nervous, cardiovascular, and inflammatory diseases. Currently, many fluorescent probes for detecting sulfur dioxide derivatives individually have been reported, such as the application of a fluorescent probe for detecting sulfur dioxide derivatives disclosed in patent CN115855899A. However, probes for simultaneously detecting Bosein and sulfur dioxide derivatives have not been reported. Therefore, developing a detection method capable of simultaneously detecting Bosein and sulfur dioxide derivatives is of great value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a fluorescent probe for the simultaneous detection of Bosein and sulfur dioxide derivatives, along with its preparation method and application. This fluorescent probe can simultaneously detect both sulfur dioxide derivatives and Bosein.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A fluorescent probe for the simultaneous detection of bosonic acid and sulfur dioxide derivatives, wherein the fluorescent probe has the structural formula shown in formula (I):

[0008]

[0009] The method for preparing the fluorescent probe for simultaneous detection of Bosein and sulfur dioxide derivatives comprises the following steps:

[0010] (1) Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile: Malonitrile, 3-hydroxy-3-methyl-2-butanone and sodium ethoxide were dissolved in solvent I, and after reflux reaction I and recrystallization purification, 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile was obtained;

[0011] (2) The 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile, 3,4-dihydroxybenzaldehyde and piperidine obtained in step (1) were added to solvent II. After reflux reaction II, the fluorescent probe was obtained after solvent removal and column chromatography separation.

[0012] In step (1), the molar ratio of malononitrile to 3-hydroxy-3-methyl-2-butanone is (3-4.5):1.

[0013] In step (1), the temperature of reflux reaction I is 60-85℃ and the time is 12-16h.

[0014] In step (2), the molar ratio of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonium to 3,4-dihydroxybenzaldehyde is 1:(1.2-1.5).

[0015] In step (1), solvent I is anhydrous ethanol; in step (2), solvent II is any one of anhydrous acetonitrile, methanol, and DMF.

[0016] In step (2), the temperature of reflux reaction II is 60-85℃ and the time is 2-6h.

[0017] In step (2), a silica gel column is used for column chromatography, and the eluent is V.二氯甲烷 :V 甲醇 =150:1.

[0018] The above-mentioned fluorescent probes are used in the detection of Bosein.

[0019] The application of the fluorescent probes described above in the detection of sulfur dioxide derivatives.

[0020] The beneficial effects of this invention are:

[0021] (1) This invention proposes a novel fluorescent probe, which is prepared by reacting malononitrile and 3-hydroxy-3-methyl-2-butanone to prepare the intermediate product 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile, followed by further reaction of the intermediate product with 3,4-dihydroxybenzaldehyde. This probe fills the gap in the fluorescence detection of Bosein and can simultaneously detect Bosein and sulfur dioxide derivatives. The successful detection of Bosein by this probe provides design ideas for the future development of specific detection of some important substances that do not have UV-Vis absorption and fluorescence, and provides reference for the design of other similar molecules and the detection of target substances.

[0022] (2) The fluorescent probe of this invention is based on the sensing mechanism of the ICT effect, and a small molecule fluorescent probe capable of simultaneously detecting Bosein and sulfur dioxide derivatives is designed. Using tricyano furan (TCF) as the Michael reaction site, a catechol structure is introduced as an electron-donating group to enable the probe to exhibit an intramolecular charge transfer (ICT) effect. Simultaneously, ratiometric detection of sulfur dioxide derivatives can be achieved at 595 nm and 336 nm, with a detection limit of 6.8 nM for sulfur dioxide derivatives by absorption spectroscopy. Even in the presence of common monosaccharides such as xylose, fructose, and glucose, the probe still exhibits good selectivity for Bosein. Furthermore, the catechol structure can also serve as a binding site for the boron-containing compound tavaborole (TVB). Since the hydroxyl group of Bosein has a stronger binding affinity to TVB, it can lead to the dissociation of TVB from the probe, thereby restoring the probe's fluorescence. Ratiometric detection of Bosein is achieved by comparing the absorbance ratio at 595 nm to 510 nm, with a detection limit of 1.20 μM for Bosein by absorption spectroscopy. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram illustrating the detection principle of the fluorescent probe for detecting bosonic acid and sulfur dioxide derivatives according to the present invention.

[0025] Figure 2 The images show the UV-Vis absorption and fluorescence emission spectra of the fluorescent probe of the present invention, wherein (A) the absorption spectra of fluorescent probes at different concentrations in PBS buffer (10mM, pH=7.4); and (B) the 3D fluorescence emission spectrum of the fluorescent probe (10μM) in PBS buffer, with integration time: 0.5s, cumulative detection intensity: 10, emission step size: 2nm, and slit size: 5 / 5nm.

[0026] Figure 3 The images show the absorption spectra of the fluorescent probe (10 μM) of the present invention in solutions of different pH values, with the inset showing photographs of the fluorescent probe (10 μM) in solutions of different pH values ​​under sunlight.

[0027] Figure 4 The diagram shows the structure of the fluorescent probe (10 μM) of the present invention in solutions with different pH values.

[0028] Figure 5 The UV-Vis absorption spectrum (A) and fluorescence spectrum (B) of the fluorescent probe (20 μM) in TVB solutions of different concentrations are shown. In (B), the excitation wavelength is 465 nm, the slit size is 5 / 5 nm, the integration time is 0.5 s, the cumulative detection intensity is 10, and the emission step size is 2 nm.

[0029] Figure 6 The UV-Vis absorption spectrum and fluorescence emission spectrum of the fluorescent probe after adding sulfur dioxide derivative are shown. (A) is the UV-Vis absorption spectrum of the fluorescent probe (10 μM) with sodium bisulfite added gradually; (B) is the fluorescence spectrum with an excitation wavelength of 335 nm; (C) is the fluorescence spectrum with an excitation wavelength of 595 nm. In the fluorescence test, the slit size was 5 / 5 nm, the integration time was 0.5 s, the cumulative intensity was 10, and the emission step size was 2 nm.

[0030] Figure 7 The absorption spectra of fluorescent probes with different interfering substances are shown below. (A) shows the absorption spectra of fluorescent probe (20 μM) with different interfering substances; (B) shows the absorbance at 595 nm of fluorescent probe (20 μM) with the relevant interfering substance (200 μM) added. Where 1: blank; 2: fluorescent probe + 200 μM TVB; 3: fluorescent probe + F - ; 4: Fluorescent probe + Cl - ;5: Fluorescent probe + Br - 6: Fluorescent probe + I -7: Fluorescent probe + CO3 2- ;8: Fluorescent probe + AcO - ;9: Fluorescent probe + SO4 2- ;10: Fluorescent probe + NO2 - ;11: Fluorescent probe + NO3 - ; 12: Fluorescent probe + Cys; 13: Fluorescent probe + GSH; 14: Fluorescent probe + HPO4 2- ;15: Fluorescent probe + K + ;16: Fluorescent probe + Ca 2+ ;17: Fluorescent probe + Mg 2+ ;18: Fluorescent probe + Al 3+ ;19: Fluorescent probe + Zn 2+ ;20: fluorescent probe + Cu 2+ ;21: fluorescent probe + NaHSO3 (20μM).

[0031] Figure 8 The absorption spectra of the fluorescent probe with added sugars are shown in Figure 1. (A) shows the absorption spectra of the fluorescent probe (10 μM) with different interfering sugars; (B) shows the absorbance at 595 nm of the fluorescent probe (10 μM) with added interfering sugars (500 μM), where 1: blank; 2: fluorescent probe + 100 μM TVB; 3: fluorescent probe + D-glucose; 4: fluorescent probe + D-xylose; 5: fluorescent probe + D-fructose; 6: fluorescent probe + xylitol.

[0032] Figure 9 (A) The reaction of fluorescent probe (10 μM) with different concentrations of sulfur dioxide derivatives (0-100 μM) 336 The graph shows the relationship between the value and the concentration of sulfur dioxide derivatives, where A 336 (A) Absorbance at 336 nm after the addition of sulfur dioxide derivatives; (B) A value of the reaction between fluorescent probe (10 μM) and sulfur dioxide derivatives of different concentrations (0-100 μM). 595 The graph shows the relationship between the value and the concentration of sulfur dioxide derivatives, where A 595 (c) Absorbance at 595 nm after the addition of sulfur dioxide derivatives; A 336 / A 595 The relationship between the value and the concentration of sulfur dioxide derivatives.

[0033] Figure 10(A) Absorption spectra of fluorescent probe (10 μM), TVB (100 μM), and Bosein (200 μL, 30%, m / m) before and after addition to PBS buffer (pH = 7.4); (B) Absorbance of the fluorescent probe (10 μM) and TVB (100 μM) system at 510 nm before and after addition of Bosein (200 μL, 30%, m / m) over time; (C) Absorbance of the fluorescent probe (10 μM) and TVB (100 μM) system at 595 nm before and after addition of Bosein (200 μL, 30%, m / m) over time.

[0034] Figure 11 (A) UV-Vis absorption spectra of probe (20 μM) with TVB (50 μM) and the gradual addition of different concentrations of bosonicine. Inset: from left to right: 20 μM probe, 20 μM probe with 50 μM TVB, 20 μM probe with 50 μM TVB and then 100-500 μL bosonicine; (B) A. Reaction of fluorescent probe with bosonicine after the addition of TVB. 595 / A 510 The relationship between the value and the concentration of bosonicine (30%, m / m) (A) 595 : The absorbance value at 595nm after adding bosonic to the probe; A 510 (The absorbance value at 510 nm after adding bosonic to the probe).

[0035] Figure 12 High-resolution mass spectra of the probe before (A) and after (B) reaction with TVB.

[0036] Figure 13 High-resolution mass spectra of the probe before (A) and after (B) reaction with Bosonic after the addition of TVB.

[0037] Figure 14 This is a high-resolution mass spectrum of the probe after reacting with a sulfur dioxide derivative.

[0038] Figure 15 This is a high-resolution mass spectrometry diagram showing possible fragmentation modes after the probe reacts with sulfur dioxide derivatives. Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] The materials used in this invention are: 3,4-dihydroxybenzaldehyde, ≥98%, Anhui Zesheng Technology Co., Ltd.; malononitrile, ≥98%, Anhui Zesheng Technology Co., Ltd.; 3-hydroxy-3-methyl-2-butanone, ≥97%, Anhui Zesheng Technology Co., Ltd.; tavaborone, ≥98%, Anhui Zesheng Technology Co., Ltd.; 1,4-dioxane, chromatographically pure, Anhui Zesheng Technology Co., Ltd.; acetonitrile, chromatographically pure, Anhui Zesheng Technology Co., Ltd.; methanol, chromatographically pure, Anhui Zesheng Technology Co., Ltd.; D-(+)-xylose, ≥98%, Shanghai Maclean Biochemical Technology Co., Ltd.; xylitol, ≥98%, Shanghai Aladdin Biochemical Technology Co., Ltd.; D-fructose, ≥99%, Shanghai Maclean Biochemical Technology Co., Ltd.; D-anhydrous glucose, ≥99%, Shanghai Maclean Biochemical Technology Co., Ltd.

[0041] Example 1

[0042] The synthetic route of the fluorescent probe for simultaneous detection of bosonic acid and sulfur dioxide derivatives in this embodiment is as follows:

[0043]

[0044] The preparation method of the fluorescent probe for simultaneous detection of Bosein and sulfur dioxide derivatives in this embodiment includes the following steps:

[0045] (1) Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile: A mixture of malononitrile (3 g, 45 mmol), 3-hydroxy-3-methyl-2-butanone (1.53 g, 15 mmol), and sodium ethoxide (1.156 g, 17 mmol) was dissolved in anhydrous ethanol (15 mL), and the mixture was refluxed at 80 °C for 14 h. The crude product was purified by recrystallization from ethanol and used directly in the next step. 2.1 g of yellow solid was obtained, yield: 70.3%.

[0046] (2) Synthesis of fluorescent probe: A mixture of 3,4-dihydroxybenzaldehyde (249.6 mg, 1.81 mmol) and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile (300 mg, 1.51 mmol) was refluxed at 80 °C for 2 h in the presence of piperidine (120 μL, 1.2 mmol) and 20 mL anhydrous acetonitrile. The reaction progress was monitored by thin-layer chromatography. The reaction was stopped after confirming that the reactants had reacted completely. The reaction mixture was then allowed to cool to room temperature. The reaction solution was evaporated to dryness using a rotary evaporator. The reactants were then dissolved in dichloromethane and methanol, and an appropriate amount of 30-40 mesh silica gel powder was added and stirred until homogeneous. The solution was purified by column chromatography using dichloromethane:dichloromethane = 150:1 as the mobile phase. The resulting solution was evaporated to dryness and then dried in a vacuum drying oven to obtain a black solid (0.16 g, yield 63.69%). 1 H NMR (600MHz, Methanol-d4) δ9.69(s,1H),7.81(d,J=16.2Hz,1H),7.29(t,J=6.2Hz,2H),7.20(dd,J =8.3,2.2Hz,1H),6.94(d,J=18.4Hz,1H),6.90(d,J=8.0Hz,1H),6.85(d,J=8.2Hz,1H),1.80(s,6H). 13 CNMR(151MHz,Methanol-d4)δ191.64,176.90,175.71,152.34,148.87,146.63,129.46,126.68,124.97,115.71,114.84, 114.14,113.99,111.53,111.22,110.74,97.42,48.02,47.88,47.74,47.60,47.46,47.31,47.17,24.83.HR-MS(ESI):m / z calcd for[C 18 H 13 N3O3] + =319.0957,found:([C 18 H 13 [N3O3]+Na) + =342.0856.

[0047] Example 2

[0048] The preparation method of the fluorescent probe for simultaneous detection of Bosein and sulfur dioxide derivatives in this embodiment includes the following steps:

[0049] (1) Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile: A mixture of malononitrile (4 g, 60 mmol), 3-hydroxy-3-methyl-2-butanone (1.53 g, 15 mmol), and sodium ethoxide (1.156 g, 17 mmol) was dissolved in anhydrous ethanol (15 mL), and the mixture was refluxed at 60 °C for 16 h. The crude product was purified by recrystallization from ethanol and used directly in the next step.

[0050] (2) Synthesis of the fluorescent probe: A mixture of 3,4-dihydroxybenzaldehyde (313 mg, 2.27 mmol) and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile (300 mg, 1.51 mmol) was refluxed at 60 °C for 6 h in the presence of piperidine (120 μL, 1.2 mmol) and 20 mL of methanol. The reaction progress was monitored by thin-layer chromatography. The reaction was stopped after confirming that the reactants had reacted completely. The reaction mixture was then allowed to cool to room temperature. The reaction solution was evaporated to dryness using a rotary evaporator. The reactants were then dissolved in dichloromethane and methanol, and an appropriate amount of 30-40 mesh silica gel powder was added and stirred until homogeneous. The solution was purified by column chromatography using dichloromethane:dichloromethane = 150:1 as the mobile phase. The resulting solution was evaporated to dryness and then dried in a vacuum drying oven to obtain a black solid. 1 H NMR (600MHz, Methanol-d4) δ9.69(s,1H),7.81(d,J=16.2Hz,1H),7.29(t,J=6.2Hz,2H),7.20(dd,J =8.3,2.2Hz,1H),6.94(d,J=18.4Hz,1H),6.90(d,J=8.0Hz,1H),6.85(d,J=8.2Hz,1H),1.80(s,6H). 13 C NMR(151MHz,Methanol-d4)δ191.64,176.90,175.71,152.34,148.87,146.63,129.46,126.68,124.97,115.71,114.84,1 14.14,113.99,111.53,111.22,110.74,97.42,48.02,47.88,47.74,47.60,47.46,47.31,47.17,24.83.HR-MS(ESI):m / z calcd for[C 18 H 13 N3O3] + =319.0957,found:([C 18 H 13 [N3O3]+Na) +=342.0856.

[0051] Example 3

[0052] The preparation method of the fluorescent probe for simultaneous detection of Bosein and sulfur dioxide derivatives in this embodiment includes the following steps:

[0053] (1) Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile: A mixture of malononitrile (3 g, 45 mmol), 3-hydroxy-3-methyl-2-butanone (1.02 g, 10 mmol), and sodium ethoxide (1.156 g, 17 mmol) was dissolved in anhydrous ethanol (15 mL), and the mixture was refluxed at 85 °C for 12 h. The crude product was purified by recrystallization from ethanol and used directly in the next step.

[0054] (2) Synthesis of the fluorescent probe: A mixture of 3,4-dihydroxybenzaldehyde (281.3 mg, 2.04 mmol) and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonium (300 mg, 1.51 mmol) was refluxed at 85 °C for 4 h in the presence of piperidine (120 μL, 1.2 mmol) and 20 mL anhydrous acetonitrile. The reaction progress was monitored by thin-layer chromatography. The reaction was stopped after confirming that the reactants had reacted completely. The reaction mixture was then allowed to cool to room temperature. The reaction solution was evaporated to dryness using a rotary evaporator. The reactants were then dissolved in dichloromethane and methanol, and an appropriate amount of 30-40 mesh silica gel powder was added and stirred until homogeneous. The solution was purified by column chromatography using dichloromethane:dichloromethane = 150:1 as the mobile phase. The resulting solution was evaporated to dryness and then dried in a vacuum drying oven to obtain a black solid. 1 H NMR (600MHz, Methanol-d4) δ9.69(s,1H),7.81(d,J=16.2Hz,1H),7.29(t,J=6.2Hz,2H),7.20(dd,J =8.3,2.2Hz,1H),6.94(d,J=18.4Hz,1H),6.90(d,J=8.0Hz,1H),6.85(d,J=8.2Hz,1H),1.80(s,6H). 13C NMR(151MHz,Methanol-d4)δ191.64,176.90,175.71,152.34,148.87,146.63,129.46,126.68,124.97,115.71,114.84,1 14.14,113.99,111.53,111.22,110.74,97.42,48.02,47.88,47.74,47.60,47.46,47.31,47.17,24.83.HR-MS(ESI):m / z calcd for[C 18 H 13 N3O3] + =319.0957,found:([C 18 H 13 [N3O3]+Na) + =342.0856.

[0055] The detection principle diagram of the fluorescent probe prepared in this invention for detecting bosonic acid and sulfur dioxide derivatives is shown below. Figure 1 As shown. Specifically, since Bosein lacks a conjugated structure, it does not absorb in the ultraviolet and visible regions, making it undetectable using ultraviolet-visible methods. Furthermore, Bosein lacks a clear fluorescent recognition site, preventing direct detection by fluorescence methods. Based on these issues, a probe for detecting Bosein using a chemical derivatization method was designed. First, the probe's own hydroxyl group binds to TVB (tavaborone), a substance with strong hydroxyl affinity, to form a complex. Then, due to the stronger binding affinity between Bosein and TVB, TVB dissociates from the probe and binds to Bosein, thus restoring the probe's absorption and fluorescence. To design a fluorescent probe that rapidly responds to sulfur dioxide derivatives, a carbon-carbon double bond was chosen as the nucleophilic addition reaction site. The probe still uses classic electron-pulling groups connected by carbon-carbon double bonds, a design that allows for a significant ICT (intramolecular charge transfer) effect. 3,4-Dihydroxybenzaldehyde and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile are linked together via a carbon-carbon double bond to form a long-wavelength emission fluorescent probe. The strong electron-withdrawing effect of the nitrile group causes the negative charge center of the entire molecule to face the nitrile group, while the phenolic hydroxyl side is relatively positively charged, thus forming a typical "D-π-A" structure, which helps the probe emit fluorescence.

[0056] Test case

[0057] I. Solution Preparation

[0058] Preparation of fluorescent probe stock solution: Weigh 2.5 mg of the fluorescent probe (M = 319.0957 g / mol) solid prepared in Example 1 of this invention, dissolve it in 4 mL of chromatographically pure dimethyl sulfoxide (DMSO) to make a stock solution with a concentration of 2 mM, and store it at room temperature in the dark for subsequent experiments.

[0059] Preparation of Boseine solution: Weigh 1.2 g of solid Boseine and dissolve it in 4 mL of ultrapure water to obtain a final Boseine solution with a mass concentration of 0.3 g / mL, i.e., a 30% Boseine aqueous solution. Store in a refrigerator at 4°C, protected from light and air, for subsequent experiments.

[0060] Preparation of TVB (tavarborone) stock solution: Weigh 3.0 mg of tavarborone solid and dissolve it in 10 mL of chromatographically pure dimethyl sulfoxide (DMSO) to make a 2 mM stock solution. Store it at room temperature in the dark for subsequent experiments.

[0061] Preparation of PBS buffer (10mM, pH=7.4): Weigh the required amounts of NaCl, KCl, Na₂HPO₄·12H₂O, and KH₂PO₄. Dissolve NaCl in 400mL of ultrapure water, then add the other three salts. Measure the pH using a calibrated pH meter, and adjust the pH of the solution to 7.4 with dilute hydrochloric acid and sodium hydroxide solution. Transfer the solution to a volumetric flask and bring the volume to 500mL for later use.

[0062] Preparation of sulfur dioxide derivative solution: Since there is currently no stable release agent for sulfur dioxide, an aqueous solution of sodium bisulfite was used as the standard test solution. Solid NaHSO3 was dissolved in ultrapure water to concentrations of 20 mM and 2 mM.

[0063] Preparation of liquids used in the potential interference experiment: During the interference testing of potential substances, solutions involving anions, cations, amino acids, etc., were prepared using a specific mass of salt dissolved in deionized water to a concentration of 20 mM, and stored at 4°C for later use. In the test, the cation corresponding to all potentially interfering anions was sodium ion. The anion corresponding to all potentially interfering cations was chloride ion.

[0064] II. Performance Testing of Fluorescent Probes

[0065] (1) Ultraviolet-Vis absorption spectroscopy test

[0066] Test conditions for UV-Vis absorption spectroscopy: absorption wavelength range: 300nm to 800nm, scan step size: 2nm.

[0067] UV-Vis absorption spectroscopy method: Add 10 μL of probe stock solution and the required concentrations of sodium bisulfite and Bosein aqueous solution (final probe concentration 10 μM) to a series of 1 cm thick quartz cuvettes, and then add the corresponding volume of PBS buffer (pH = 7.4, 10 mM) to maintain a total solution volume of 2 mL. Then perform the measurements using a UV-Vis spectrophotometer.

[0068] (2) Fluorescence spectroscopy test

[0069] Fluorescence spectroscopy test conditions: excitation wavelength 600nm, emission wavelength scanning range: 610nm to 900nm; integration time: 0.5s; cumulative intensity: 10; emission step size: 2nm; slit width: 5nm / 5nm.

[0070] Fluorescence emission spectroscopy assay method: Add 10 μL of probe stock solution and the required concentrations of sodium bisulfite aqueous solution and Bosein aqueous solution (final probe concentration 10 μM) to a series of 1 cm thick quartz cuvettes, and add the corresponding volume of PBS buffer (pH = 7.4, 10 mM) to maintain the total solution volume at 2 mL. After waiting 5 minutes, perform the assay using a fluorescence spectrometer.

[0071] (3) pH test for fluorescent probe detection

[0072] PBS buffer solution (pH 1 to 12) and 10 μL of fluorescent probe stock solution were added sequentially to a series of cuvettes, bringing the total volume to 2 mL, with a final probe concentration of 10 M. All test solutions were measured using a UV-Vis absorption spectrometer.

[0073] (4) Investigation of the reaction time of fluorescent probes to bosonic acid

[0074] Add PBS buffer solution (10 mM, pH = 7.4), 10 μL of probe stock solution, and 50 μL of TVB stock solution sequentially to a cuvette, and perform the test in absorption spectroscopy measurement mode. When the absorption spectrum of the solution reaches stability, quickly add 100 L of 30% Bosein solution, and monitor the change in absorbance every 1 min.

[0075] (5) Detection limit test of fluorescent probe

[0076] Based on the absorption spectral titration results, the ratio of absorbance (at 595 nm and 510 nm) after reacting the test solutions of probe (20 μM) and TVB (50 μM) with aqueous solutions of different concentrations of Bosein was plotted on the ordinate, and the aqueous solutions of different concentrations of Bosein were plotted on the abscissa. Then, a linear relationship between the absorbance ratio and the concentration of the aqueous solution of Bosein was fitted. The limit of detection (LOD) for Bosein concentration by absorption spectroscopy was calculated using the formula LOD = 3σ / k (where σ is the standard deviation of ten blank solutions and k is the slope of the fitted linear equation). Similarly, based on the absorption spectral titration results, the absorbance (at 336 nm and 595 nm) after reacting the test solution of probe (10 μM) with sulfur dioxide derivatives of different concentrations was plotted on the ordinate, and the sulfur dioxide derivatives of different concentrations were plotted on the abscissa. Then, a linear relationship between the absorbance ratio and the concentration of the sulfur dioxide derivative was fitted. The limit of detection (LOD) for sulfur dioxide derivatives by spectrophotometry was calculated using the formula LOD = 3σ / k (where σ is the standard deviation of ten blank solutions and k is the slope of the fitted linear equation). Furthermore, a graph was plotted with the ratio of the absorbance (at 336 nm to 595 nm) of the analyte after reacting the probe (10 μM) with different concentrations of sulfur dioxide derivatives as the ordinate and the reaction of different concentrations of sulfur dioxide derivatives as the abscissa. The LOD was calculated using the formula LOD = 3R / C (where R is the difference between the maximum and minimum values ​​in n independent measurements and C is the range coefficient). The lowest detection limit for sulfur dioxide derivatives by absorbance method is calculated by means of n (where n is the number of measurements).

[0077] III. Test Results and Analysis

[0078] (1) Investigation of the final concentration of fluorescent probe in spectral performance testing

[0079] Figure 2 (A) shows the absorption spectra of fluorescent probes at different concentrations in PBS buffer (10 mM, pH = 7.4). As can be seen from the figure, with increasing probe concentration, the fluorescent probe exhibits a shoulder peak at 465 nm and a maximum absorption peak at 595 nm. These two absorption peaks correspond to the single ionization structure of catechol and the structure of the probe itself, respectively, with strong absorption at the long wavelength of 595 nm. Since both excessively low and excessively high probe concentrations affect the detection of bosonic acid, too low a concentration results in an indistinct main absorption peak, while too high a concentration leads to minimal absorption change. Therefore, a final concentration of 10 μM for the fluorescent probe is recommended for more significant absorption changes. Unless otherwise specified, all subsequent experiments used a 10 μM fluorescent probe. Figure 2(B) shows the 3D fluorescence emission spectrum of the fluorescent probe (10 μM) in PBS buffer. As can be seen from the figure, the probe exhibits the highest emission intensity when excited at 595 nm. Therefore, 595 nm was chosen as the excitation wavelength for subsequent tests, with a corresponding emission wavelength of 635 nm. Since the excitation wavelength is also 470 nm, to ensure more accurate subsequent tests and minimize potential interference, 595 nm was ultimately selected as the excitation wavelength, with a corresponding emission wavelength of 635 nm. This emission wavelength reduces interference from background fluorescence.

[0080] (2) Effect of pH on fluorescent probes

[0081] PBS buffer solution (pH 1 to 12) and 10 μL of fluorescent probe stock solution were added sequentially to a series of cuvettes, bringing the total volume to 2 mL, with a final probe concentration of 10 μM. All test solutions were measured using UV-Vis absorption spectroscopy. The absorption spectra of the fluorescent probe in different pH buffer solutions are shown below. Figure 3 As shown, when the pH of the buffer solution increases from 1 to 5, the absorbance of the probe does not change significantly at the short wavelength of 425 nm, and the absorbance at the long wavelength approaches 0. However, when the pH is between 6 and 12, the absorbance of the probe decreases sharply at the short wavelength of 425 nm, while it increases sharply at the long wavelength of 595 nm. This is because the phenolic hydroxyl group on the probe structure ionizes into a hydrogen ion under alkaline conditions, resulting in a stronger charge transfer system within the probe molecule, thus causing the absorption wavelength to become longer. Figure 4 As shown, the probe's color changed significantly with increasing pH, as indicated by the following pattern. Figure 3 The illustration shows that this is consistent with the changes in the probe's absorption spectrum. At pH 7, the absorption intensities of long-wavelength and short-wavelength absorption are essentially the same, showing no change; at pH 8, only long-wavelength absorption is observed. In summary, when the pH of the buffer solution is between 7 and 8, the probe absorbs at both 425 nm (short-wavelength) and 595 nm (long-wavelength), which facilitates subsequent dual-wavelength ratio detection. Therefore, pH = 7.4 was chosen as the condition for subsequent experiments.

[0082] (3) Investigation into the concentration of TVB added

[0083] The detection of Bosein by fluorescent probes requires the addition of TVB solution to complex the fluorescent probe. Therefore, the interaction between the fluorescent probe and TVB was investigated using absorption and fluorescence spectra. 20 μM fluorescent probe stock solution and different concentrations of TVB solution were added to a series of cuvettes, along with corresponding volumes of PBS buffer (pH = 7.4, 10 mM), maintaining a total solution volume of 2 mL. The absorption and fluorescence spectra were then measured. Figure 5(A) It can be seen that as the concentration of the TVB solution gradually increases, the absorption at 595 nm gradually decreases, while the absorption wavelength at 505 nm gradually increases, forming an isoabsorption point at 550 nm. The absorption spectrum preliminarily proves that the fluorescent probe complexes with TVB to form a new substance. When the concentration of the TVB solution increases from 40 μM to 60 μM, the long-wavelength absorption decreases significantly; and the color of the system becomes more pronounced (transitioning from cyan to red). Therefore, considering the significant color difference and high long-wavelength absorption during subsequent investigations, a TVB solution concentration of 50 μM was chosen. Figure 5 (B) It can be seen that, when excited at 465 nm, the system produces a distinct fluorescence emission peak at 640 nm. The fluorescence intensity decreases with increasing TVB solution concentration, which is due to the complexation between the probe and TVB. Under sunlight, the solution color gradually transitions from cyan to wine red. Under a 365 nm UV lamp, the solution color does not change significantly.

[0084] In summary, the fluorescent probe formed the expected complex with the TVB solution. Subsequent testing was conducted using a 20 μM fluorescent probe and a 50 μM TVB solution, and the reaction between the fluorescent probe and the TVB solution and Bosein was investigated by observing changes in the absorption spectrum.

[0085] (4) Exploration of the feasibility of fluorescent probes for detecting sulfur dioxide derivatives

[0086] 10 μL of fluorescent probe stock solution and sodium bisulfite aqueous solution of the required concentration were added to a series of cuvettes, along with an appropriate volume of PBS buffer (pH = 7.4, 10 mM), maintaining a total solution volume of 2 mL. The feasibility was investigated by analyzing the changes in the UV-Vis absorption and fluorescence emission spectra of the fluorescent probe before and after the addition of the sulfur dioxide derivative. The results are as follows: Figure 6 As shown. Figure 6 As shown in (A), with the addition of the sulfur dioxide derivative, the absorption peak of the fluorescent probe at 595 nm gradually disappeared, and a new strong absorption peak gradually appeared at 335 nm. This indicates that the fluorescent probe reacted chemically with the sulfur dioxide derivative to generate a new substance. The above experiments suggest that a Michael addition reaction may have occurred between the fluorescent probe and the sulfur dioxide derivative, causing a change in the conjugated structure of the probe. (Fluorescence emission...) Figure 6 (B) and Figure 6 (C) shows that with the addition of sulfur dioxide derivatives, the emission peak at 445 nm gradually increases, but the fluorescence intensity is very weak. The emission peak at 650 nm gradually weakens. This indicates that the probe produces a new fluorescent substance after reacting with the sulfur dioxide derivative. These experiments preliminarily verify that the probe can achieve colorimetric and fluorescence detection of sulfur dioxide derivatives.

[0087] (5) Investigation of the selectivity of fluorescent probes for bosonic acid and sulfur dioxide derivatives

[0088] Since real samples often contain various anions and cations, and the molecules of monosaccharides and disaccharides also contain many hydroxyl groups, and the principle of fluorescent probe detection of bosonic also involves the coordination of hydroxyl groups, monosaccharides and disaccharides also need to be added when examining the selectivity of fluorescent probes.

[0089] a. Different interfering ions

[0090] A fluorescent probe (20 μM) and 10 equivalent volumes of interfering ion solution (containing F) were added to a series of cuvettes. - Cl - , Br - I - CO3 2- AcO - SO4 2- NO2 - NO3 - Cys, GSH, HPO4 2- K + Ca 2+ Mg 2+ Al 3+ Zn 2+ Cu 2+ The absorbance at 595 nm was compared with that of a blank fluorescent probe solution (20 μM) and a 20 μM sulfur dioxide derivative solution. Figure 7 As shown in (A), the absorbance of the fluorescent probe (20 μM) decreased significantly after the addition of TVB, indicating that the probe complexed with TVB. The absorption of the fluorescent probe at 595 nm decreased significantly after the addition of sodium bisulfite solution, indicating that the fluorescent probe underwent an addition reaction with the sulfur dioxide derivative. Figure 7 As shown in (B), apart from TVB and sodium bisulfite, the absorbance of the fluorescent probe did not change significantly in the presence of other interfering substances, indicating that the detection properties of the fluorescent probe are stable. The absorbance decreased by 2.2 times after the addition of sodium bisulfite, demonstrating that the fluorescent probe, which relies on absorbance for detection, has high anti-interference capability.

[0091] b. Interfering with carbohydrates

[0092] The selectivity of the fluorescent probe for four common sugars was tested. 10 μM of the fluorescent probe, 50 equivalents of interfering sugar solutions, and 100 μM of TVB solution were added to a series of cuvettes, and the absorbance at 595 nm was compared with that of the blank solution. Figure 8As shown in (A), the absorbance of the solution reaches its maximum at 595 nm when only the fluorescent probe is present. After adding TVB and interfering sugar solutions, the absorbance of the solution at 595 nm decreases significantly, and the absorbance values ​​remain approximately the same. Figure 8 As shown in (B). Therefore, the addition of interfering sugars does not affect the binding of the fluorescent probe to TVB.

[0093] (6) Investigation of the working curves of fluorescent probes on sulfur dioxide derivatives

[0094] Based on the above experimental data, the working curves of the reaction between the system and different concentrations of sulfur dioxide derivatives were further investigated. 10 μM fluorescent probe stock solution and 0-100 μM sodium bisulfite aqueous solution were added to a series of cuvettes, along with corresponding volumes of PBS buffer (pH = 7.4, 10 mM), maintaining a total solution volume of 2 mL. Since the probe fluorescence is weak, absorbance was used to quantitatively detect the sulfur dioxide derivatives. With the addition of sulfur dioxide derivatives, the absorbance of the fluorescent probe at 595 nm gradually decreased, while the absorbance at 336 nm gradually increased, such as... Figure 6 As shown, when the concentration of sulfur dioxide derivatives is between 0-100 μM, the absorbance values ​​at 595 nm and 336 nm both show a good linear relationship with the concentration of sulfur dioxide derivatives, as shown in the figure. Figure 9 As shown in (A) and 9(B), the working curve corresponding to the absorbance at 595 nm is y = -0.00415x + 0.418, R 2 =0.9684. Using the formula for calculating the limit of detection (LOD): LOD = 3σ / k (where σ is the standard deviation of ten blank solutions, and k is the slope of the fitted linear equation), the lowest detection limit for the absorption spectroscopy method for detecting sulfur dioxide derivatives is calculated to be 1.45 μM. The working curve corresponding to the absorbance at 336 nm is y = 0.0023x + 0.0087, R... 2 =0.9711. Using the formula for calculating the limit of detection (LOD): LOD = 3σ / k (σ is the standard deviation of ten blank solutions, and k is the slope of the fitted linear equation), the lowest limit of detection for sulfur dioxide derivatives by absorption spectroscopy is calculated to be 2.62 μM.

[0095] Meanwhile, the working curve corresponding to the ratio of absorbance at 595 nm to that at 336 nm in the probe system is as follows: R 2 =0.9966, such as Figure 9 As shown in (C). According to the formula LOD = 3R / C (where R is the difference between the maximum and minimum values ​​in n independent measurements, and C is the range coefficient), The lowest detection limit for sulfur dioxide derivatives by absorbance method was calculated to be 6.8 nM (where n is the number of measurements).

[0096] The above results indicate that the probe prepared in this invention can be used for the detection of trace amounts of sulfur dioxide derivatives.

[0097] (7) Investigation of the reaction time of fluorescent probes to bosonic acid

[0098] Since time sensitivity is often required in actual sample detection processes, the reaction time between the probe and Bosein is particularly important. To investigate the reaction time between the probe and Bosein, 100 μM TVB was added to a 10 μM probe solution, and the wavelength was continuously scanned in the 300 nm–800 nm range for 1 min. Then, a 30% (w / w) aqueous solution of Bosein was rapidly added. The absorption spectra before and after adding the probe (10 μM), TVB (100 μM), and Bosein (200 μL, 30%, m / m) to PBS buffer (pH = 7.4) are shown below. Figure 10 As shown in (A). The absorbance of the system was recorded every 1 minute. Within 1 minute after the addition of Bosonic, the absorbance of the probe at 510 nm decreased rapidly by 0.03 ( ). Figure 10 B), the absorbance at 595 nm remains essentially unchanged. Figure 10 C). The experimental data above demonstrate that the probe can achieve real-time and rapid detection of Bosein, and has the potential to become a colorimetric probe for detecting Bosein.

[0099] (8) Investigation of the working curve of the fluorescent probe to bosonic acid

[0100] Considering the degree of noticeable color change, a probe concentration of 20 μM and a TVB solution concentration of 50 μM were ultimately chosen as the subsequent testing conditions. The specific solution preparation involved adding 20 μM fluorescent probe stock solution and 50 μM TVB solution to a series of cuvettes, followed by the rapid addition of different concentrations of Bosein aqueous solution (100-500 μL), and then adding corresponding volumes of PBS buffer (pH = 7.4, 10 mM) to maintain a total solution volume of 2 mL. Figure 11 As shown in (A), when TVB solution was added to the probe, the absorbance of the overall solution at 595 nm decreased significantly, while the absorbance at 510 nm increased significantly. Subsequently, with increasing concentrations of the added Bosein aqueous solution, the absorbance of the overall solution at 595 nm increased significantly, while the absorbance at 510 nm decreased slightly. Figure 11 (A) The illustration clearly shows the probe color changing from cyan to red and then back to cyan. The working curve for probe detection of Bosein is derived by plotting the absorbance ratio at 595 nm and 510 nm on the ordinate and the concentration of the Bosein aqueous solution on the abscissa. For example... Figure 11 As shown in (B), a good linear relationship is observed when the concentration of bosonic acid is between 100 and 500 μL, and the corresponding working curve is y = 6.639 × 10⁻⁶.-4 x+1.565,R 2 =0.9604. Using the formula for calculating the limit of detection (LOD): LOD = 3σ / k (where σ is the standard deviation of ten blank solutions and k is the slope of the fitted linear equation), the lowest detection limit for the absorption spectroscopy method for detecting Bosein is calculated to be 1.20 μM. Experimental results show that the prepared probe can be used for the detection of trace amounts of Bosein.

[0101] (9) Investigation into the reaction mechanism of fluorescent probes with bosine and sulfur dioxide derivatives

[0102] The probe was designed based on the Michael addition reaction for detecting sulfur dioxide derivatives, and it contains two hydroxyl groups, allowing it to complex with TVB. Subsequently, because Boseine contains more hydroxyl groups and has a stronger binding affinity to TVB, the addition of Boseine disrupts the probe-TVB complex, releasing the probe and restoring its original color and fluorescence. To determine whether the probe undergoes an addition reaction with the sulfur dioxide derivative and whether it complexes with TVB, high-resolution mass spectra before and after the reactions of the probe with the sulfur dioxide derivative and Boseine were analyzed.

[0103] Figure 12 The images show high-resolution mass spectra before (A) and after (B) the reaction of the probe with TVB. As can be seen from the figures, the mass-to-charge ratio of the probe before the reaction with TVB is 342.0856 (its theoretical value is 319.0957). This mass-to-charge ratio corresponds to the mass-to-charge ratio of the probe molecule to Na. + Combining (its theoretical value is 342.0850), it can also be seen from the figure that the probe molecule interacts with H. + The mass-to-charge ratio of the combined product was 320.1039 (the theoretical value is 320.1030). After the probe reacted with TVB, the predicted mass-to-charge ratio of the product was found to be 452.1212 (the theoretical value is 459.1737). This provides preliminary verification of the proposed reaction mechanism.

[0104] Figure 13 The images show high-resolution mass spectra of the probe before (A) and after (B) reaction with Bosein following the addition of TVB. As can be seen from the images, when Bosein is added after TVB to the probe, the molecular ion peak of the probe is found in the mass spectrum, indicating a mass-to-charge ratio of 320.1039 (the probe is added with H+). + ) and 342.0857 (probe plus Na) + The molecular ion peaks of TVB Bosoxane, namely, mass-to-charge ratios of 325.1243 (TVB Bosoxane) and 343.1561 (TVB Bosoxane plus NH4), were also observed. + This indicates that TVB binds to Bosonic, releasing probe molecules and validating the probe design.

[0105] The mechanism of the reaction between the probe and sulfur dioxide derivatives was analyzed by high-resolution mass spectrometry, such as... Figure 14 As shown. The product molecules resulting from the reaction of the probe with the sulfur dioxide derivative may be unstable, and no corresponding molecular ion peak is observed in the mass spectrum. However, product L, which is fragmented by electron bombardment after the reaction, was found in the mass spectrum, and its structure is shown below. Figure 15 As shown. The theoretical molecular ion peak of the cleavage is 274.0511, but it was actually found to be 274.2759. Therefore, due to the strong electron-withdrawing ability of the nitrile group, the product after the addition of the probe with the sulfur dioxide derivative is cleaved by electron bombardment to give product L.

[0106] In summary, the mechanism of probe detection of Bosein and sulfur dioxide derivatives was verified by high-resolution mass spectrometry, which is consistent with the expected design.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a fluorescent probe in the detection of bosonic acid, characterized in that, The structure of the fluorescent probe is as shown in formula (Ⅰ): 。

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