Preparation and application of fluorescent probe PCH for detecting HSO3 <-> based on hemicyanine

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to preparation and application of a fluorescent probe PCH for detecting HSO3 <-> based on hemicyanine. Experimental results show that the fluorescent probe PCH shows specific fluorescent response to HSO3 <->, and is high in selectivity and strong in anti-interference capability. In the HSO3 <-> detection process, the color of the solution is changed from purple to yellow, and visual detection can be carried out. Compared with the prior art, the method has the advantages that the reaction process is mild, the probe performance is excellent, and the method has a wide application prospect in the aspect of HSO3 <-> detection.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and particularly relates to the preparation and application of a fluorescent probe PCH based on merocyanine for detecting HSO3 - . Background Art

[0002] Bisulfite is a common additive and plays an important role in the processing and production of food, beverages and pharmaceuticals due to its excellent antibacterial and antioxidant properties. As a preservative, HSO3 - can prevent or delay the deterioration of food due to oxidation and extend the storage life of food. However, excessive HSO3 - can cause damage to individual tissues, and its dosage in food needs to be strictly controlled. As a reducing agent, HSO3 - has a wide range of applications in industries such as dyes, papermaking, leather making and chemical synthesis, such as bleaching cotton fabric organic substrates, treating chromium-containing wastewater, and being used as an electroplating additive. In addition, bisulfite, as a reactive sulfur species, participates in many physiological processes. Toxicological studies have shown that low concentrations of HSO3 - are beneficial to vasodilation and can play a role in relieving inflammation and antioxidation. While high concentrations of HSO3 - can cause irreversible damage such as cell necrosis and lead to a series of diseases, such as atherosclerosis, essential hypertension, hypoxic pulmonary hypertension, etc. Therefore, it is of great significance to study a method that can efficiently detect HSO3 - .

[0003] Currently, the conventional analytical techniques for detecting bisulfite include chromatography, chemiluminescence measurement, electrochemistry, spectrophotometry and phosphorescence determination, etc. These methods have certain defects, such as complex operation, high cost, and inability to be directly used for in vivo detection. In recent years, the fluorescent probe technology has formed unique advantages due to its simple operation, low cost, high sensitivity and ability to perform visual detection. So far, researchers have developed many fluorescent probes for specific detection of bisulfite. Bisulfite reacts with specific groups to achieve the purpose of detection. Common reaction mechanisms include deprotection reaction of levulinic acid groups, nucleophilic addition reaction with aldehydes, hydrogen bonds, and nucleophilic addition reaction of carbon-carbon double bonds, etc.

[0004] Based on the merocyanine structure, a reactive fluorescent probe PCH for detecting HSO3 - was designed and synthesized in this paper. Experiments show that the recognition and detection of HSO3 - by the PCH probe can be completed within 5 minutes, and the detection limit is as low as 2.47 nM, providing a convenient and fast method for the detection of HSO3 - . Summary of the Invention

[0005] The object of the present invention is to provide a preparation method and application of a fluorescent probe based on a merocyanine structure for detecting HSO3 -

[0006] The technical solution to achieve the object of the present invention is as follows:

[0007] A fluorescent probe PCH based on merocyanine for detecting HSO3 - The structure of this material is as shown in the attached specification Figure 1 .

[0008] A preparation method of a fluorescent probe PCH based on merocyanine for detecting HSO3 - in the present invention includes the following steps:

[0009] A preparation method of a fluorescent probe PCH based on merocyanine for detecting HSO3 - in the present invention includes the following steps: Take 1.22 g of salicylaldehyde and 1.27 ml of ethyl acetoacetate and place them in a flask, add 20 ml of absolute ethanol and 330 μL of piperidine, and react at 78 °C for 4 hours. After filtration, wash the obtained solid with absolute ethanol and dry it to obtain compound 1; Weigh 0.33 g of 3 - diethylaminophenol and 0.296 g of phthalic anhydride, place them in a flask, add 50 ml of toluene, and reflux at 110 °C for 4 hours. Cool the mixture to 50 - 60 °C, then add 50 ml of NaOH solution, and react at 90 °C for 6 hours. Pour the reaction mixture into 200 ml of ice water, add concentrated hydrochloric acid for acidification and let it stand for 2 hours. After the solid precipitates, filter it, wash it with absolute ethanol and dry it, and then recrystallize to obtain compound 2; Weigh 188 mg of compound 1 and 313 mg of compound 2 and place them together in a flask, add 3 ml of methanesulfonic acid, heat and reflux at 90 °C for 12 hours. After the reaction is completed, add 25 ml of saturated brine. After the solid precipitates, filter it, wash it 3 times with 100 ml of ice water, and then use CH2Cl2 / CH3OH with a volume ratio of 100∶1 as the eluent for silica gel column chromatography purification. After rotary evaporation, a dark red solid is obtained, which is the final product fluorescent probe PCH with a yield of 67%; The structure of compound 1 is as follows: The structure of compound 2 is as follows:

[0010] The fluorescent probe PCH based on merocyanine described in the present invention can be used for detecting HSO 3- .

[0011] Compared with the prior art, the significant advantages of the present invention are: (1) The present invention has synthesized a fluorescent probe for detecting HSO 3- ​The merocyanine-based fluorescent probe PCH for ions has the advantages of strong selectivity and low detection limit. (2) The raw materials selected in the present invention have low cost, simple synthesis method, mild reaction conditions, high yield, few post-treatment steps, and are easy to realize large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Structure of the fluorescent probe PCH for detecting HSO3 -

[0013] Figure 2 1H NMR spectrum of Compound 1 (CDCl3, 400 MHz) 1 (CDCl3, 400 MHz)

[0014] Figure 3 1H NMR spectrum of Compound 2 (CDCl3, 400 MHz) 1 (CDCl3, 400 MHz)

[0015] Figure 4 1H NMR spectrum of the fluorescent probe PCH (CDCl3, 400 MHz) 1 (CDCl3, 400 MHz)

[0016] Figure 5 13C NMR spectrum of the fluorescent probe PCH (CDCl3, 101 MHz) 13 (CDCl3, 101 MHz)

[0017] Figure 6 ESI-MS spectrum of the fluorescent probe PCH

[0018] Figure 7 ESI-MS spectrum of the fluorescent probe PCH after binding with HSO3 -

[0019] Figure 8 Synthesis route of the fluorescent probe PCH

[0020] Figure 9 (A) UV-Vis absorption spectra of the probe PCH with various different anions added (B) Changes in the UV-Vis absorption spectra of the probe PCH in the presence of different concentrations of HSO3 - (C) Fluorescence spectra of PCH with different anions and cations added (D) Competitive analysis in the presence of various anions (1: F - ; 2: Cl - ; 3: Br - ; 4: I - ; 5: NO2 - ; 6: NO3 - ; 7: S2O3 2- ; 8: ClO4 - ; 9: CO3 2- ​​; 10: H2PO4 - ; 11: HCO3 - ; 12::SO4 2- ; 13: SO3 2- ; 14: CYS; 15: GSH; 16: HCY; 17: HSO3 - )

[0021] Figure 10 The fluorescence spectra of PCH added with different metal cations and HSO3 -

[0022] Figure 11 The fluorescence spectra of PCH (1 mM) added with HSO3 - changing with time

[0023] Figure 12 The fluorescence spectra of PCH in solutions with different concentrations of HSO3 - Inset: The fluorescence intensity values of PCH in the presence of different equivalents of HSO3 - at 580 nm

[0024] Figure 13 The linear correlation curve of fluorescence intensity vs. the concentration of HSO3 - in a solution containing 1 mM probe PCH

[0025] Figure 14 In vivo fluorescence images of zebrafish treated with PCH and PCH-HSO3 - . (A) Fluorescence image, (B) Bright-field image, (C) Overlay image Detailed implementation manners

[0026] Example 1 Synthesis and structural characterization of the fluorescent probe PCH

[0027] Example 2 Selective response ability of the fluorescent probe PCH to HSO3 -

[0028] Example 3 Response time of the fluorescent probe PCH to HSO3 -

[0029] Example 4 Minimum detection limit of the fluorescent probe PCH to HSO3 -

[0030] Example 5 Application of the fluorescent probe PCH in in vivo fluorescence imaging

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples.

[0032] Example 1

[0033] ​​​​Synthesis and Structural Characterization of Fluorescent Probe PCH

[0034] Take 1.22 g of salicylaldehyde and 1.27 ml of ethyl acetoacetate and place them in a flask. Add 20 ml of absolute ethanol and 330 μL of piperidine, and react at 78 °C for 4 hours. After filtration, wash the obtained solid with absolute ethanol and dry it to obtain Compound 1; weigh 0.33 g of 3 - diethylaminophenol and 0.296 g of phthalic anhydride, place them in a flask, add 50 ml of toluene, and reflux at 110 °C for 4 hours. Cool the mixture to 50 - 60 °C, then add 50 ml of NaOH solution, and react at 90 °C for 6 hours. Pour the reaction mixture into 200 ml of ice water, add concentrated hydrochloric acid for acidification and let it stand for 2 hours. After the solid precipitates, filter it, wash it with absolute ethanol and dry it, and then recrystallize it to obtain Compound 2; weigh 188 mg of Compound 1 and 313 mg of Compound 2 and place them together in a flask. Add 3 ml of methanesulfonic acid, heat and reflux at 90 °C for 12 hours. After the reaction is completed, add 25 ml of saturated brine. After the solid precipitates, filter it, wash it 3 times with 100 ml of ice water, and then use CH2Cl2 / CH3OH with a volume ratio of 100∶1 as the eluent for silica gel column chromatography purification. After rotary evaporation, a dark red solid is obtained, which is the final product fluorescent probe PCH with a yield of 67%; the structure of Compound 1 is as follows: The structure of Compound 2 is as follows:

[0035] For Compound 1 and Compound 2 1 The 1H NMR spectra are shown in Appendix Figure 2 , 3 of the specification.

[0036] For the fluorescent probe PCH 1 1H NMR spectra, 13 13C NMR spectra and ESI - MS spectra are shown in Appendix Figure 4 , 5, 6 of the specification. The ESI - MS spectrum of the fluorescent probe PCH after binding with HSO3 - is shown in Appendix Figure 7 of the specification.

[0037] The specific synthesis route of the fluorescent probe PCH is shown in Appendix Figure 8 of the specification.

[0038] Example 2

[0039] The selective response ability of the fluorescent probe PCH to HSO3 -

[0040] Add the fluorescent probe PCH to the PBS aqueous solution, and respectively add in the probe solution except for HSO3 - ​For other cationic and anionic solutions, it can be found that obvious absorption peaks appear at 360 nm and 570 nm in the UV-visible absorption spectra of the samples. When HSO3 - is added, due to its nucleophilic addition reaction with PCH, the conjugated structure of the probe is destroyed, causing the absorption peak at 570 nm to shift towards shorter wavelengths and form a new absorption peak at 470 nm (Appendix Figure 9 A).

[0041] The UV titration experiment better verified this phenomenon. As the concentration of HSO3 - in the sample gradually increased, the intensity of the absorption peak at 570 nm gradually decreased while the intensity of the absorption peak at 470 nm gradually increased (Appendix Figure 9 B). At the same time, the fluorescence spectrum showed that PCH itself did not emit fluorescence. When PCH contacted with other anions and cations, the emitted fluorescence was very weak and could be ignored. Only when HSO3 - was added did strong fluorescence occur (Appendix Figure 9 C). The above analysis indicated that PCH could produce a specific response to HSO3 - . In addition, with the addition of the HSO3 - ionic solution, the color of the probe solution quickly changed from purple to yellow, and the yellow color gradually deepened over time. However, when other anions, biothiols, or cations were added, the solution color did not change. This phenomenon indicated that the response process of PCH to HSO3 - could be visualized.

[0042] The fluorescence spectral ion competition experiment was used to exclude the influence of coexisting anions on the probe. When the analyte sample did not contain HSO3 - , the fluorescence intensity of all samples was very weak. After adding HSO3 - , its fluorescence intensity increased significantly. Compared with the sample only added with HSO3 - , the fluorescence intensity was not much different (Appendix Figure 9 D). This showed that in the presence of other anions, PCH could still specifically detect HSO3 - .

[0043] The influence of the presence of common metal cations on the fluorescence probe PCH was further studied. Different metal cation solutions were added to the sample containing HSO3 - , and fluorescence spectral tests were conducted. The experimental results showed that after adding cations, the fluorescence intensity of the sample did not change significantly compared with that of the sample only added with HSO3 - (Appendix Figure 10 ). This indicated that the fluorescence probe PCH could still detect HSO3 -The response is hardly affected and it has strong anti-interference ability.

[0044] Example 3

[0045] The response time of the fluorescent probe PCH to HSO3 -

[0046] Take 60 μL of the probe stock solution and add it to 3 mL of PBS buffer solution. Immediately after adding 60 μL of HSO3 - solution, fluorescence spectrum test was carried out immediately. The results showed that the fluorescence intensity of the system gradually increased over time and gradually stabilized at the maximum value after 240 s (attached Figure 11 ). This indicates that the fluorescent probe PCH can achieve rapid detection of HSO3 - .

[0047] Example 4

[0048] The lowest detection limit of the fluorescent probe PCH to HSO3 -

[0049] To further prove the practical application ability of the probe, the lowest detection limit of the fluorescent probe PCH was calculated by fluorescence titration. The fluorescence titration results showed that as the titration equivalent of HSO3 - increased, the fluorescence intensity of the probe gradually increased. When the titration equivalent reached 100, the fluorescence intensity gradually stabilized and reached the highest value ( Figure 12 ). The lowest detection limit of the probe was calculated according to the results of fluorescence titration. First, the fluorescence intensity of the blank sample was measured, and it was measured 10 times and the standard deviation of its fluorescence intensity at 580 nm was calculated. Then, a curve was made based on the fluorescence intensity of the sample at 580 nm and the concentration of HSO3 - to obtain its slope ( Figure 13 ). The detection limit was calculated according to the following formula: Finally, the detection limit was obtained as 2.47 nM.

[0050] Example 5

[0051] Application of the fluorescent probe PCH in fluorescence imaging in animals

[0052] The feasibility of using the fluorescent probe PCH for in vivo monitoring of HSO3 - in animals was evaluated using zebrafish as a model. Attached Figure 14 shows the in vivo fluorescence imaging of zebrafish. From left to right are the fluorescence image, bright field image and merged image. From the first row of images (attached Figure 14 A1, C1), it can be seen that after the zebrafish was incubated with 20 μM of the fluorescent probe PCH solution at 27 °C for 1 h, almost no fluorescence was observed. The second row of images (attached​​Figure 14 A2, C2) It can be observed that after incubating with the fluorescent probe PCH for 1 h and then incubating with a 40 μM NaHSO3 solution for 1 h, bright yellow fluorescent signals are shown in the zebrafish body. It can be concluded that the fluorescent probe PCH has good biocompatibility and can be used to trace the distribution of HSO3 - in the animal body.

Claims

1. A fluorescence probe PCH based on merocyanine for detecting HSO3 - with the following structure 2. A preparation method of a fluorescent probe PCH based on merocyanine for detecting HSO3 - as described in claim 1, comprising the following steps: Take 1.22 g of salicylaldehyde and 1.27 ml of ethyl acetoacetate and place them in a flask. Add 20 ml of absolute ethanol and 330 μL of piperidine and react at 78 °C for four hours. After filtration, wash the obtained solid with absolute ethanol and then dry it to obtain Compound 1; weigh 0.33 g of 3-diethylaminophenol and 0.296 g of phthalic anhydride and place them in a flask. Add 50 ml of toluene and reflux at 110 °C for four hours. Cool the mixture to 50 - 60 °C, then add 50 ml of NaOH solution and react at 90 °C for six hours. Pour the reaction mixture into 200 ml of ice water, add concentrated hydrochloric acid for acidification and let it stand for 2 hours. After the solid precipitates, filter it, wash it with absolute ethanol, dry it and then recrystallize to obtain Compound 2; weigh 188 mg of Compound 1 and 313 mg of Compound 2 and place them together in a flask. Add 3 ml of methanesulfonic acid and heat under reflux at 90 °C for 12 hours. After the reaction is completed, add 25 ml of saturated brine. After the solid precipitates, filter it, wash it 3 times with 100 ml of ice water, and then use CH2Cl2 / CH3OH with a volume ratio of 100∶1 as the eluent for silica gel column chromatography purification. After rotary evaporation, a dark red solid is obtained, which is the final product, the fluorescent probe PCH; the structure of Compound 1 is as follows: The structure of Compound 2 is as follows:

3. Use of a fluorescent probe PCH based on a phthalocyanine for detecting HSO3 - , characterized in that: The phthalocyanine-based fluorescent probe PCH described in claim 1 can be used to detect HSO3 in an aqueous phase - .

Images