Preparation method of long-wavelength-emission fluorescent probe capable of rapidly detecting bisulfite and preparation method of long-wavelength-emission fluorescent probe

Abstract

The invention belongs to the technical field of ion detection, and particularly relates to a preparation method of a long-wavelength emission fluorescent probe for rapidly detecting bisulfite, which comprises the following steps: dissolving isophorone and malononitrile in ethanol to obtain a product 1; 3-bromoquinoline and 5-formyl-2-thiopheneboronic acid are dissolved in a mixed solvent of dry methanol and dry toluene, and a product 2 is obtained; dissolving the product 2 in dichloromethane, and adding methyl trifluoromethanesulfonate to obtain a product 3; and dissolving the product 1 and the product 3 in ethanol, and adding piperidine to obtain the required fluorescent probe LSQ. Dissolving the obtained fluorescent probe LSQ in DMSO to prepare mother liquor A; a target product is obtained. The fluorescent probe provided by the invention has the advantages of high-selectivity rapid response to bisulfite, long-wavelength emission, no organic solvent detection, simple synthesis route and simple and convenient detection equipment and method, and can be used for qualitative and quantitative detection of bisulfite in food and biological systems.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of ion detection, and particularly relates to a long-wavelength emission rapid detection sulfite fluorescent probe and a preparation method thereof. BACKGROUND

[0002] Sulfur dioxide derivatives have the effects of disinfection, oxidation resistance and bleaching, and are widely used in the fields of food and industry. If too much sulfur dioxide is ingested, it will cause different degrees of damage to the human body. Therefore, it is urgent to develop a high-selectivity and high-sensitivity, rapid-response sulfite (HSO3 - ) fluorescent probe, which has important scientific significance and application prospect for portable detection of HSO3 - .

[0003] The fluorescence detection requires relatively simple instruments, has high selectivity and sensitivity, wide detection range, rapid response time, no sample damage in the detection process, little harm to cells, and can realize real-time in-situ imaging of cells, tissues and living bodies. SUMMARY

[0004] In view of the problems existing in the prior art fluorescence imaging technology, the application provides a long-wavelength emission rapid detection sulfite fluorescent probe which can respond to HSO3 - , and a preparation method thereof. The method has the advantages of simple synthesis steps, high yield, rapid response speed, good selectivity, high sensitivity and low detection limit.

[0005] To solve the above technical problems, the application is implemented as follows: The preparation method of the long-wavelength emission rapid detection sulfite fluorescent probe comprises the following steps: (1) isophorone and malononitrile are dissolved in dry anhydrous ethanol, a piperidine catalyst is added, and the mixture is stirred and reacted at an elevated temperature for 8 hours, then cooled to room temperature, filtered, and vacuum dried to obtain product 1; (2) 3-bromoquinoline and 5-aldehyde-2-thiophene boronic acid are dissolved in a mixed solvent of dry methanol and dry toluene, palladium dichloride and cesium carbonate are added, the mixture is stirred at an elevated temperature, poured into water, filtered, and vacuum dried to obtain product 2; (3) product 2 synthesized in step (2) is dissolved in dry dichloromethane, methyl trifluoromethanesulfonate is added, and the mixture is precipitated at room temperature, filtered, and vacuum dried to obtain product 3; (4) product 1 synthesized in step (1) and product 3 synthesized in step (3) are dissolved in ethanol, piperidine is added, the mixture is stirred at an elevated temperature, most of the ethanol is removed after the reaction is completed, and vacuum drying is performed to obtain the required fluorescent probe LSQ; (5) the fluorescent probe LSQ obtained in step (4) is dissolved in DMSO to prepare mother liquor A; and the rapid detection sulfite fluorescent probe is obtained.

[0006] Further, in the step (1), the molar ratio of isophorone and malononitrile is 1:1.

[0007] Further, in the step (2), the molar ratio of 3-bromoquinoline and 5-formyl-2-thiophene boronic acid is 1:1.

[0008] Further, in the step (4), the molar ratio of product 1 and product 3 is 1:1.

[0009]

[0010] Further, in the step (1), isophorone and malononitrile are dissolved in dry anhydrous ethanol, a few drops of piperidine are added, and the temperature is raised to 60 DEG C and stirred for 8 hours.

[0011] Further, in the step (2), 3-bromoquinoline and 5-formyl-2-thiophene boronic acid are dissolved in a mixed solvent of dry methanol and dry toluene, and the temperature is raised to 90 DEG C and stirred for 12 hours under N2 protection.

[0012] Further, in the step (5), the fluorescent probe LSQ obtained in the step (4) is dissolved in DMSO to prepare a 1 mM stock solution.

[0013] The fluorescent probe obtained by the preparation method of the long-wavelength emission fluorescent probe for rapidly detecting bisulfite has the following structure: .

[0014] The application further provides application of the fluorescent probe LSQ in detection of bisulfite.

[0015] The preparation method of the long-wavelength emission fluorescent probe for rapidly detecting bisulfite has the following specific reaction process: , The fluorescent probe is prepared by reacting isophorone, malononitrile, 3-bromoquinoline and 5-formyl-2-thiophene boronic acid, and the fluorescent probe has a significant fluorescence enhancement phenomenon with the increase of the concentration of bisulfite under the condition of HEPES / DMSO (5 / 5 v:v pH=7.4) in the presence of bisulfite, and the fluorescent probe has rapid response, high selectivity and high sensitivity in detection of bisulfite. Compared with some existing detection technologies, the fluorescent chemical probe in the application has the advantages of low cost, simple synthesis route, convenient post-processing, and can directly realize rapid and specific recognition of bisulfite in a HEPES / DMSO (5 / 5 v:v pH=7.4) system, and has potential application value in food and biological systems. BRIEF DESCRIPTION OF DRAWINGS

[0016] The present application will be described in detail below with specific embodiments. These embodiments are provided so that the present application will be thorough and complete, and will fully convey the scope of the application to those skilled in the art. As used throughout the specification and claims, the terms "comprising" or "including" are to be construed as open-ended terms, meaning that "comprising" or "including" will be given its broadest interpretation to include all and any subsequent appended claims. The description that follows is intended to be a best mode of carrying out the present application, and is not intended to limit the scope of the present application. The scope of the present application is defined by the appended claims. Unless otherwise specified, all reagents and materials used in the present application are commercially available.

[0017] Figure 1 NMR spectrum of the prepared fluorescent probe LSQ; Figure 2 Fluorescent intensity of the fluorescent probe LSQ as a function of pH value in the presence and absence of bisulfite; Figure 3 Fluorescent emission spectrum of the fluorescent probe LSQ at different bisulfite concentrations; Figure 4 Fitting curve of the fluorescent intensity of the fluorescent probe LSQ as a function of bisulfite concentration and the function graph corresponding to the curve; Figure 5 Selectivity and competition of the fluorescent probe LSQ for other analytes; Figure 6 Time scan of the response of the fluorescent probe LSQ to bisulfite.

[0018] The concentration of the added ions was 2 x 10 -2 mol / L, 590 nm was the excitation wavelength, and 820 nm was the emission wavelength. DETAILED DESCRIPTION

[0019] Example 1

[0020] The preparation method of the long-wavelength emission rapid detection bisulfite fluorescent probe includes the following steps: (1) Preparation of product 1, product 2 and product 3 Isophorone (70 mmol) and malononitrile (70 mmol) were added to a round-bottom flask containing 20 mL of dry anhydrous ethanol, and a piperidine catalyst was added dropwise. The mixture was stirred under heating reflux for 8 hours, cooled to room temperature, and filtered under suction to obtain the desired product, which was dried under vacuum to synthesize product 1.

[0021] 3-Bromoquinoline (22 mmol) and 5-aldehyde-2-thiopheneboronic acid (22 mmol) were dissolved in methanol (10 mL) and toluene (15 mL), and palladium dichloride (0.45 mmol) and cesium carbonate (22 mmol) were added. The mixture was heated to 90 °C and stirred under reflux for 12 hours. The mixture was then poured into water, filtered, and dried under vacuum to obtain product 2.

[0022] Product 2 was dissolved in dry dichloromethane (78 mmol), methyl trifluoromethanesulfonate (40 mmol) was added, and the product was precipitated at room temperature. The precipitate was then filtered and dried under vacuum to synthesize product 3.

[0023] (2) Preparation of fluorescent probe LSQ Product 1 (1 mmol) and product 3 (1 mmol) were weighed and added to a round-bottom flask containing 15 mL of ethanol. Piperidine was added, and the mixture was refluxed for 4 h. After cooling to room temperature, the mixture was filtered and dried under vacuum to obtain the probe LSQ.

[0024] Weigh out 1 mmol of LSQ and dissolve it in DMSO to prepare a 1 mM stock solution. This is a fluorescent probe for rapid detection of bisulfite using long-wavelength emission.

[0025] Detection test for sulfite ions: Take nine 5 mL sample vials and add 20 μL of the fluorescent probe solution (10 μM concentration) prepared in Example 1 to each vial. Then, add [HSO3] to each of the nine sample vials. - ]=0(a)1×10 -5 mol / L (b), 2×10 -5 mol / L(c), 3×10 -5 mol / L (d), 4×10 -5 mol / L(e), 5×10 -5 mol / L (f), 6×10 -5 mol / L (g), 7×10 -5 mol / L (h), 8×10 -5 A mol / L (i) solution of bisulfite was stirred at room temperature for 1 s, and the fluorescence intensity of these samples was measured at an excitation wavelength of 590 nm. The fluorescence intensity emission spectrum changes of the nine samples are shown in the figure. Figure 3 The results showed that the fluorescence intensity of the fluorescent probe gradually increased with the gradual increase of bisulfite ion concentration. Based on... Figure 3 The fluorescence intensity changes can be used to plot the corresponding fitted function curve and the corresponding function graph (y=ax+b, a=35.44122, b=28.27689, R). 2 =0.99) SeeFigure 4 .

[0026] Other analyte comparison tests: Take 23 5 mL sample vials and add 20 μL of the fluorescent probe solution LSQ prepared in Example 1 (the concentration of the fluorescent probe is 10 μM). Then, add 2 × 10⁻⁶ μL of the solution to each vial. -2 Other analytes and HSO3 mol / L - 20 μL of each sample was added to one of the 22 sample vials, with sample 1 serving as the blank. The fluorescence emission intensity of each of the 22 samples was then measured at an excitation wavelength of 590 nm and an emission wavelength of 820 nm. The results are shown below. Figure 5 The results showed that the other analytes had no significant effect on the intensity of the prepared fluorescent probe. (Note: HSO3) - The response time is 1 second, while the response time for other substances is 5 minutes. The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims fall within the protection scope of the present invention.

Claims

1. A method for preparing a long-wavelength emission fluorescent probe for rapid detection of bisulfite, characterized in that, Includes the following steps: (1) Dissolve isophorone and malononitrile in dry anhydrous ethanol, add piperidine catalyst, heat and stir for 8 hours, cool to room temperature, filter and dry under vacuum to obtain product 1; (2) Dissolve 3-bromoquinoline and 5-aldehyde-2-thiopheneboronic acid in a mixed solvent of methanol and toluene, add palladium dichloride and cesium carbonate, heat and stir, pour into water and filter, and dry under vacuum to obtain product 2; (3) Dissolve the product 2 synthesized in step (2) in dry dichloromethane, add methyl trifluoromethanesulfonate, precipitate at room temperature, filter, and dry under vacuum to obtain product 3; (4) Dissolve product 1 synthesized in step (1) and product 3 synthesized in step (3) in ethanol, add piperidine, heat and stir the mixture, remove most of the ethanol after the reaction is complete, and dry under vacuum to obtain the desired fluorescent probe LSQ. (5) Dissolve the fluorescent probe LSQ obtained in step (4) in DMSO to prepare mother liquor A; thus, the target product, a fluorescent probe for rapid detection of bisulfite by long-wavelength emission, is obtained.

2. The method for preparing a long-wavelength emission rapid detection fluorescent probe for bisulfite ions according to claim 1, characterized in that: In step (1), the molar ratio of isophorone to malononitrile is 1:

1.

3. The method for preparing the long-wavelength emission rapid detection fluorescent probe for bisulfite ions according to claim 2, characterized in that: In step (2), the molar ratio of 3-bromoquinoline and 5-aldehyde-2-thiopheneboronic acid is 1:

1.

4. The method for preparing a fluorescent probe for rapid detection of bisulfite using long-wavelength emission according to claim 3, characterized in that: In step (4), the molar ratio of product 1 to product 3 is 1:

1.

5. The method for preparing the long-wavelength emission rapid detection fluorescent probe for bisulfite ions according to claim 4, characterized in that: In step (1), isophorone and malononitrile are dissolved in dry anhydrous ethanol, piperidine catalyst is added, the temperature is raised to 60 °C and stirred for 8 hours, cooled to room temperature, filtered, and vacuum dried to obtain product 1.

6. The method for preparing the long-wavelength emission rapid detection fluorescent probe for bisulfite ions according to claim 5, characterized in that: In step (2), 3-bromoquinoline and 5-aldehyde-2-thiophene boric acid are dissolved in a mixed solvent of methanol and toluene, and then heated to 90 °C and stirred for 12 hours under N2 protection.

7. The method for preparing the long-wavelength emission rapid detection fluorescent probe for bisulfite ions according to claim 6, characterized in that: In step (5), the fluorescent probe LSQ obtained in step (4) is dissolved in DMSO to prepare a 1 mM stock solution.

8. A fluorescent probe prepared using the method for rapid detection of bisulfite using long-wavelength emission as described in any one of claims 1 to 7, has the following structure: 。 9. The application of the fluorescent probe LSQ as described in claim 8 in the detection of bisulfite.

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