AIE fluorescent probe for detecting CEES as well as preparation method and application of AIE fluorescent probe

Abstract

The invention relates to the technical field of chemical detection, in particular to an AIE fluorescent probe for detecting CEES and a preparation method and application of the AIE fluorescent probe. The invention also provides a composition and a detection system containing the fluorescent probe, an auxiliary reagent for enhancing the aggregation of the probe is added, the probe and CEES are subjected to a specific reaction, and the unreacted probe is oxidized into a product with stronger aggregation capability under the action of H2O2, so that the aggregation-induced emission (AIE) effect is remarkably amplified, and an obvious fluorescence difference signal is formed. By adding an aggregation amplification mechanism of an auxiliary reagent, the rapid and visual high-sensitivity detection of CEES is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical detection, in particular to an AIE fluorescent probe for detecting CEES, and a preparation method and application thereof. BACKGROUND

[0002] 2-chloroethyl ethyl sulfide (CEES for short) is a sulfur-containing organic compound containing a chloroethyl and an ethyl sulfide structure in the molecule, and has strong alkylation activity. CEES can react with various compounds containing sulfhydryl, hydroxyl or amine groups to generate corresponding addition products. Due to its similar reaction characteristics to thioether chemicals, relative safety and ease of operation, CEES is often used as a safe simulation of thioether chemical reaction mechanism and detection method.

[0003] Currently, the detection methods for CEES and similar compounds mainly include gas chromatography-mass spectrometry, ion mobility spectrometry, infrared spectroscopy, electrochemical method, surface-enhanced Raman spectroscopy, chemical coloration method and fluorescent probe method, etc. Among various detection methods for CEES, fluorescent probes stand out due to their high cost-effectiveness, simple operation and good selectivity.

[0004] Aggregation-induced emission (AIE) probes have weak fluorescence when fully dissolved in good solvents, but show significant fluorescence when aggregated due to intramolecular rotation restriction in poor solvents. By taking advantage of the difference in aggregation behavior between the probe and its reaction product, the fluorescence can be switched from "off-on" or "on-off", thereby used for target analyte detection. In recent years, various AIE fluorescent probes have been developed for detection purposes. These probes are usually prepared by coupling a tetraphenyl ethylene (TPE) unit with a target recognition moiety. When the probe reacts with the target, the product shows a significantly different molecular polarity from the parent probe, thereby generating fluorescence signal enhancement through AIE effect.

[0005] However, in some detection systems, the aggregation difference between the probe and its reaction product may not be sufficient, making it difficult for the probe to effectively detect the target analyte through AIE effect. This suggests the need to design AIE probes that can form aggregation tendencies that are significantly distinguishable compared to the parent probe, in order to achieve more efficient and sensitive detection methods. SUMMARY

[0006] To achieve the above purpose, the present application first provides an AIE fluorescent probe for detecting CEES, the structure of the fluorescent probe is as follows: 。

[0007] The present application also provides a composition for detecting CEES, comprising the AIE fluorescent probe described above.

[0008] In some embodiments, the composition further comprises an auxiliary agent capable of increasing the aggregation of the AIE fluorescent probe.

[0009] In some embodiments, the auxiliary agent comprises hydrogen peroxide, butenedioic acid, N-phenylmaleimide or 2-methyl-2-propen-1-ol.

[0010] The present application also provides a detection reagent combination for detecting CEES, comprising the AIE fluorescent probe described above.

[0011] In some embodiments, the detection system reagent combination further comprises an auxiliary agent capable of increasing the aggregation of the fluorescent probe described above.

[0012] In some embodiments, the auxiliary agent in the detection system reagent combination comprises hydrogen peroxide, butenedioic acid, N-phenylmaleimide or 2-methyl-2-propen-1-ol.

[0013] The present application also provides a method for detecting CEES using the fluorescent probe, composition or detection reagent combination described above, comprising the following steps: S1: Preparation of the reaction system: After mixing the AIE fluorescent probe with the sample to be tested, sodium bicarbonate buffer is added to adjust the pH, the concentration of the AIE fluorescent probe is 20 μM, and after thorough mixing, the reaction is heated at 80°C for 5 minutes; S2: Preparation of the detection system: Take the reaction solution of the reaction system after the reaction is completed, dilute with water and acetonitrile, the ratio of the reaction solution to water and acetonitrile is 1:9, and add an auxiliary agent with a volume fraction of 1%, thoroughly mix and stand for about 1 minute; the volume fraction of water in the detection system is 60%; S3: Excite the detection system at an excitation wavelength of 336 nm, and record the fluorescence intensity at an emission wavelength of 470 nm; S4: When the fluorescence intensity of the reaction system is lower than that of the detection system, it is determined that the CEES exists in the sample to be tested.

[0014] In some embodiments, the auxiliary agent in the method comprises hydrogen peroxide, butenedioic acid, N-phenylmaleimide or 2-methyl-2-propen-1-ol.

[0015] Compared with the prior art, the present application has at least the following beneficial effects: The application provides a CEES detection method based on a TPE-SH fluorescent probe. First, TPE-SH can specifically react with CEES to generate TPE-CEES, but the difference in aggregation between the two is small, which is not enough to cause a significant fluorescent response, so the application introduces TPE-SH into the system to oxidize the unreacted TPE-SH to generate a disulfide bond connected product TPE-S-S-TPE. The oxidation product has a stronger molecular aggregation tendency than TPE-SH and TPE-CEES, thereby significantly amplifying the aggregation-induced emission (AIE) effect, forming an observable fluorescent difference signal. The method has the advantages of high sensitivity, simple operation and fast detection speed, and is suitable for rapid identification and visual detection of CEES. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a high-resolution mass spectrum of TPE-SH of the application; Figure 2 is a 1 H NMR (400 MHz) spectrum of TPE-SH of the application; Figure 3 is a 13 C NMR (101 MHz) spectrum of TPE-SH of the application; Figure 4 is a mass spectrum of the reaction product of TPE-SH and CEES of the application; Figure 5 is a comparison chart of AIE fluorescent probes (TPE-SH) and TPE-SH / CEES AIE fluorescence of the application; Figure 6 (A) is a fluorescence intensity chart (emission wavelength: 470 nm, excitation wavelength: 336 nm) of TPE-SH / H2O2 and TPE-SH / CEES / H2O2 samples at different water fractions (fw); (B) is a fluorescence color photo of the corresponding samples at different water fractions (fw) under a 365 nm ultraviolet lamp; Figure 7 is a fluorescence intensity chart of TPE-SH / H2O2 and TPE-SH / CEES / H2O2 at different oxidation times (emission wavelength: 470 nm, excitation wavelength: 336 nm); Figure 8 (A) is a fluorescence spectrum chart of a TPE-SH (20 μM) detection solution with H2O2 as an auxiliary reagent at different CEES concentrations (0-500 μM); (B) is a chart of the relationship between the emission intensity at 470 nm in the detection solution and the CEES concentration (excitation wavelength: 336 nm); Figure 9To compare the AIE fluorescence of TPE-SH and TPE-SH / CEES under different auxiliary reagents, the auxiliary reagents are: Figure 9 In the middle, (A) is MA: butenedioic acid; (B) is PMI: N-phenylmaleimide; (C) is 3-BnA: 1-amino-3-butene; Figure 9 (D)MAA: 2-Methyl-2-propen-1-ol. Detailed Implementation

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0018] Materials and Methods: The compound (4-(bromomethyl)phenyl)ethylene-1,1,2-triyl)triphenyl was purchased from Bidex Pharmaceuticals. All other reagents were purchased from Shanghai Titan Technology Co., Ltd. Proton nuclear magnetic resonance (NMR) 1 H NMR) and carbon nuclear magnetic resonance (H NMR) 13 C10 NMR spectra were recorded at 600 MHz using a Varian NMR System 600 spectrometer. Electrospray ionization mass spectrometry (ESI-MS) was obtained using an Anglient 6470 Q-TOF mass spectrometer. Fluorescence spectroscopy measurements were performed using a Hitachi F-4600 fluorescence spectrophotometer (equipped with a 1 cm path length quartz cuvette, room temperature) or a Tecan Spark microplate multifunction reader. UV-Vis absorption spectra were also obtained using a Tecan Spark microplate multifunction reader. Visual inspection was performed using a handheld UV lamp. Column chromatography was performed on silica gel (300-400 mesh).

[0019] Example 1: Design and preparation of AIE fluorescent probes for detecting CEES Commercially available (4-(bromomethyl)phenyl)ethylene-1,1,2-triyl)triphenyl (0.532 g, 1.25 mmol), thiourea (0.195 g, 2.5 mmol), and ethanol (50 mL) were mixed and heated at 70 °C for 8 hours. After cooling, the solvent was evaporated under vacuum to obtain a white solid, which was used for subsequent reactions. Subsequently, sodium hydroxide solution was added to the white residue, and the resulting solution was refluxed under a nitrogen atmosphere for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, and the pH of the reaction solution was adjusted to 2 with 6 M hydrochloric acid. A large amount of solid precipitate was formed, which was filtered and washed three times with water. The collected solid was dissolved in 60 mL of ethyl acetate, and the solution was rinsed with anhydrous... The sample was dried overnight. After filtration, the solvent was evaporated under reduced pressure. The residue was then purified by rapid silica gel column chromatography using a 1:1 (v / v) mixture of petroleum ether and ethyl acetate (Rf = 0.14) as eluent to give the probe compound (TPE-SH) of this application as an off-white solid (yield: 47.0%). High-resolution mass spectrometry, m / z: 377.1362 (calculated [MH]-, 377.1369). 1 ¹H NMR (400 MHz, acetonitrile-d³) δ: 7.07 (dtd, J = 6.6, 5.1, 2.8 Hz, 10H), 7.03 (d, J = 2.0 Hz, 1H), 7.02–6.96 (m, 6H), 6.96–6.91 (m, 2H), 3.60 (d, J = 7.7 Hz, 2H), 1.97 (t, J = 7.7 Hz, 1H). 13 C NMR (101 MHz, acetonitrile-d3) δ: 143.39, 130.74, 130.50, 127.46, 127.42, 127.38, 127.12, 126.18, 27.33. The synthetic chemical formula is shown in Formula 1.

[0020] Formula 1: Through high-resolution mass spectrometry, 1 HNMR and 13 CNMR confirmed the presence of the probe TPE-SH ( Figures 1-3 ).

[0021] Example 2: Confirmation of the reaction products TPE-SH was initially treated with excess CEES (10 equivalents) at 80°C for 5 min in a mixture of ethanol and bicarbonate-hydroxide buffer (2:1 v / v, pH = 9.5). The reaction solution was then analyzed by mass spectrometry. Figure 4 As shown, in positive EI mode, a significant peak was observed at 489.4, corresponding to the calculated molecular weight of the TPE-CEES product (489.2, [M+H]). + C 31 H 31 S2 + This indicates that TPE-SH and CEES underwent alkylation. Simultaneously, no significant fluorescence change was observed in the reaction solution. According to the AIE mechanism, AIE molecules exhibit high fluorescence emission when they have poor solubility in solvents and aggregate. Therefore, it is necessary to select a suitable solvent system that allows TPE-SH and TPE-CEES to exhibit different aggregation characteristics.

[0022] Example 3 Direct detection of CEES by probe TPE-SH Reagent preparation: The probe (TPE-SH) synthesized in Example 1 was dissolved in anhydrous acetonitrile to prepare a stock solution (10 mM). Similarly, CEES was dissolved in anhydrous ethanol to prepare a stock solution (10 mM), and a sodium bicarbonate solution with a concentration of 20 mM was prepared using water.

[0023] Reaction group: 20 μL of the TPE-SH stock solution and 200 μL of the CEES stock solution were sequentially added to a mixture containing ethanol (450 μL), water (230 μL), and 100 μL of the 20 mM sodium bicarbonate solution. The resulting solution was then reacted at 80°C for 5 minutes.

[0024] Control group: 20 μL of the TPE-SH stock solution was added to a mixture containing ethanol (650 μL), water (230 μL), and 100 μL of the 20 mM sodium bicarbonate solution. The resulting solution was then reacted at 80°C for 5 minutes.

[0025] 100 μL of the reaction solution from the reaction group and the control group were taken and added to 900 μL of a water-acetonitrile mixture with proportions of 50%, 60%, 70%, 80%, and 90% to prepare two groups of 1 mL solutions with different water fractions for fluorescence analysis.

[0026] The results of the fluorescence analysis (fluorescence spectrometer, fluorescence measurement on the solution in a 1 cm cuvette, parameter settings: excitation wavelength: 336.0 nm) are shown in Figure 5 The results show that there is no distinguishable difference between the sample containing TPE-SH and CEES and the sample containing only the probe at any water fraction. This indicates that the difference in aggregation tendency between TPE-SH and TPE-CEES is insufficient.

[0027] Therefore, the present application amplifies the aggregation difference by adding an auxiliary reagent, thereby enabling TPE-SH to detect CEES.

[0028] Example 4 Detection of CEES by TPE-SH probe assisted by H2O2 In this example, hydrogen peroxide (H2O2) was chosen as the auxiliary reagent because of its high efficiency, strong specificity, and ease of handling. The mechanism of oxidizing TPE-SH to TPE-S-S-TPE is shown in Formula 2. The mechanism of oxidizing TPE-SH to TPE-S-S-TPE is shown in Formula 2.

[0029] Formula 2: The specific solution preparation method is as follows: ​Reaction solutions: A 10 mM TPE-SH probe solution was prepared using acetonitrile, and a 10 mM CEES solution was prepared using ethanol. Using a 65% ethanol-water solution as the reaction system, water, 20 mM NaKCO3 solution, and ethanol were added sequentially, mixed thoroughly, and then the acetonitrile probe solution (10 mM) and the ethanol solution of CEES (10 mM) were added. Specific amounts are shown in Table 1. The reaction was carried out in an 80℃ metal bath for 5 minutes, and then cooled to room temperature.

[0030] Table 1 H2O2-assisted probe reaction system

[0031] Using water-acetonitrile ratios of 40%, 50%, 60%, and 70% as detection systems, the solutions were mixed thoroughly and then added to the above reaction solutions. After further mixing, H₂O₂ (30% by volume) was added (specific amounts are shown in Table 2). The reactions were carried out at room temperature for 2 minutes. A set of 1 mL solutions with different water fractions were then prepared for fluorescence analysis.

[0032] Table 2 H2O2 Auxiliary Probe Detection System

[0033] The results of fluorescence analysis (excitation wavelength: 336.0 nm) are as follows: Figure 6 As shown. According to Figure 6 , The solution showed no fluorescence as the water fraction (fw) increased from 40% to 60%, but significant fluorescence emission was observed at a water fraction of 70%. This finding is consistent with the absence of... Results obtained from TPE-SH / CEES samples ( Figure 5 The consistency indicates that aggregation-induced emission behavior is not affected by... Impact. In contrast, when fw is 60%, at Significant emission was observed in solution, with TPE-SH or The samples showed significant differences compared to the others. This indicates that... This promotes the formation of TPE-SS-TPE in solution, thereby enhancing the aggregation tendency. Therefore, CEES can be detected by amplifying the aggregation difference between TPE-SH samples containing and without the target analyte using auxiliary reagents. Based on these results, this application selects a solution with an fw of 60% as the optimal detection system for CEES. In this system, the probe TPE-SH in... It exhibits significant fluorescence emission in the presence of [something], while [something else]... In the presence of [a specific substance], the reaction product of TPE-SH and CEES exhibited negligible fluorescence emission. Furthermore, under UV light (365 nm), the [response product] showed negligible fluorescence emission compared to the control sample. AIE fluorescence was visible to the naked eye in the (fw=60% solution), and was consistent with that of the test sample. A stark contrast ( Figure 6 (Right figure). The control sample emitted a bright blue light under 365 nm UV light, while the test sample did not emit light. These observations are consistent with fluorescence emission measurements.

[0034] Example 5: Use Response time of the TPE-SH detection system with auxiliary reagents to CEES Reaction solution: Using 65% ethanol-water solution as the reaction system, water, NaKCO3 solution (20 mM) and ethanol were added in sequence, mixed well, and then acetonitrile solution of probe (10 mM) and ethanol solution of CEES (10 mM) were added (the reaction system is the same as in Table 1 above). The reaction was carried out in a metal bath at 80℃ for 5 minutes and then cooled to room temperature.

[0035] Prepare 20 portions of a mixed solution of 555 μL water and 335 μL acetonitrile, then add 100 μL of the reaction solution prepared in this example to each portion, mix well, and then add... The reactions were carried out at room temperature for 0, 20, 40, 60, 80, 100, 120, 140, 160, and 180 seconds, respectively.

[0036] After the reaction, fluorescence analysis was performed on the above 20 samples (parameters set as follows: excitation wavelength: 336.0 nm, slit width: excitation end -5 nm; emission end -5 nm). The results are as follows. Figure 7 As shown, After adding the TPE-SH sample, a distinct fluorescence peak appeared at 470 nm, indicating that TPE-SH was... Rapid oxidation leads to a more aggregated product. The fluorescence intensity remains stable over time, indicating that the oxidation process can be completed quickly.

[0037] Example 6 Using The TPE-SH detection system for auxiliary reagents showed AIE fluorescence response to different concentrations of CEES. Reaction solution: Using 65% ethanol-water solution as the reaction system, water, NaKCO3 solution (20 mM) and ethanol were added sequentially, mixed well, and then acetonitrile solution (10 mM) of probe and CEES ethanol solution (10 mM) were added to prepare reaction solutions with CEES concentrations of 0 μM, 20 μM, 50 μM, 100 μM, 150 μM and 200 μM respectively. The reaction was carried out in a metal bath at 80℃ for 5 minutes and then cooled to room temperature.

[0038] Prepare six aliquots of a mixture of 555 μL water and 335 μL acetonitrile. Then, add 100 μL of the probe and different concentrations of CEES to each aliquot, mix well, and add 10 μL of 30% H2O2 to each aliquot. React at room temperature for 1 minute. After the reaction, use a multi-functional microplate reader (Tecan Spark) to detect the fluorescence of the samples (parameters set as follows: excitation wavelength: 336.0 nm, slit width = 20 nm).

[0039] The effect was further evaluated by detecting CEES at different concentrations (excitation wavelength: 336 nm). To assist in the performance testing of the TPE-SH system. For example... Figure 8 As shown, the fluorescence intensity decreased in a dose-dependent manner, indicating that TPE-SH can be used for semi-quantitative analysis of CEES. Significant fluorescence responses were still observed when the CEES concentration in the detection system was as low as 5 μM, demonstrating that the TPE-SH detection system has acceptable sensitivity.

[0040] Example 7 Other auxiliary reagents to assist TPE-SH probe detection of CEES Reagent preparation: Prepare a 10 mM TPE-SH probe solution with acetonitrile, a 10 mM CEES solution with ethanol, and 10 mM butenadic acid stock solutions, N-phenylmaleimide stock solutions, 1-amino-3-butene stock solutions, and 2-methyl-2-propen-1-ol stock solutions with dioxane.

[0041] Reaction system: A 65% ethanol-water solution was used as the reaction system. Water, NaKCO3 solution (20 mM), and ethanol were added sequentially, mixed well, and then the acetonitrile solution of the probe (10 mM) and the ethanol solution of CEES (10 mM) were added. The specific amounts are shown in Table 1. The reaction was carried out in a metal bath at 80℃ for 5 minutes and then cooled to room temperature.

[0042] Colorimetric system: Prepare eight aliquots of a mixture of 555 μL water and 335 μL acetonitrile. Add 100 μL of the reaction solution from the above reaction system to each aliquot, mix well, and then add 10 μL of butenic acid stock solution, N-phenylmaleimide stock solution, 1-amino-3-butene stock solution, and 2-methyl-2-propen-1-ol stock solution respectively. React at room temperature for 1 minute each, and then measure the fluorescence signal. (Parameters set as follows: EX: 336.0 nm, slit width = 20 nm).

[0043] Table 3. Auxiliary reagents that can react with TPE-SH

[0044] The application conceives that as long as an auxiliary reagent capable of reacting with TPE-SH is found, and a product different from the aggregation behavior of TPE-CEES is generated, the fluorescence phenomenon of the reaction group and the blank group can be different, thereby realizing the detection of CEES. Therefore, four reagents containing double bonds (see Table 3) were tested, and by addition reaction with TPE-SH, a product more prone to aggregation was generated, thereby producing fluorescence and realizing the detection of CEES (results see Figure 9 ).

[0045] In summary, the application provides a TPE-SH-based fluorescence probe system, which realizes high sensitivity and high selectivity detection of CEES by introducing auxiliary reaction and high-pH reaction medium. First, TPE-SH can specifically react with CEES to generate TPE-CEES, but the difference in aggregation between the two is small, which is not enough to cause a significant fluorescence response. Therefore, the application introduces to make the unreacted TPE-SH oxidize to generate a disulfide bond connected product TPE-S-S-TPE. The oxidation product has a stronger molecular aggregation tendency than TPE-SH and TPE-CEES, thereby significantly amplifying the aggregation-induced emission (AIE) effect, forming an observable fluorescence difference signal. Therefore, the application realizes rapid and visual detection of CEES by auxiliary aggregation amplification mechanism, and the TPE-SH probe has the advantages of response sensitivity, simple operation and wide applicability.

[0046] The above is a preferred embodiment of the application. It should be noted that for those skilled in the art, without departing from the principles of the application, several improvements and refinements can be made, which should also be considered within the scope of protection of the application.

Claims

1. An AIE fluorescent probe for detecting CEES, characterized in that, The structural formula of the fluorescent probe is: 。 2. A composition for detecting CEES, characterized in that, Includes the AIE fluorescent probe as described in claim 1.

3. The composition according to claim 2, characterized in that, It also includes auxiliary reagents that can increase the aggregation of the AIE fluorescent probe.

4. The composition according to claim 3, characterized in that, The auxiliary reagents include hydrogen peroxide, butenedioic acid, N-phenylmaleimide, or 2-methyl-2-propen-1-ol.

5. A reagent combination for detecting CEES, characterized in that, Includes the AIE fluorescent probe as described in claim 1.

6. The reagent combination of the detection system according to claim 5, characterized in that, It also includes auxiliary reagents that can increase the aggregation of the fluorescent probe of claim 1.

7. The detection reagent combination according to claim 6, characterized in that, The auxiliary reagents include hydrogen peroxide, butenedioic acid, N-phenylmaleimide, or 2-methyl-2-propen-1-ol.

8. A method for detecting CEES using the fluorescent probe, composition, or combination of detection reagents as described in claim 1, 2, or 5, characterized in that, Includes the following steps: S1: Preparation of the reaction system: After mixing the AIE fluorescent probe with the sample to be tested, sodium bicarbonate buffer is added to adjust the pH. The concentration of the AIE fluorescent probe is 20 μM. After thorough mixing, the mixture is heated at 80°C for 5 minutes. S2: Preparation of the detection system: Take the reaction solution from the reaction system after the reaction is completed, dilute it with water and acetonitrile, the ratio of reaction solution to water and acetonitrile is 1:9, add 1% (v / v) of auxiliary reagent, mix thoroughly and let stand for about 1 minute; the volume fraction of water in the detection system is 60%; S3: Excite the detection system at an excitation wavelength of 336 nm and record the fluorescence intensity at an emission wavelength of 470 nm; S4: When the fluorescence intensity of the reaction system is lower than that of the detection system, it is determined that the CEES is present in the sample to be tested.

9. The method according to claim 8, characterized in that, The auxiliary reagents include hydrogen peroxide, butenedioic acid, N-phenylmaleimide, or 2-methyl-2-propen-1-ol.

Images