Dual detection of viscosity and HSO 3 - Fluorescent probe and preparation method thereof

Abstract

The present invention belongs to the technical field of ion detection, and specifically relates to a dual detection method for viscosity and HSO3 ‑ The preparation method of the fluorescent probe comprises: dissolving 2-methylquinoline and iodoethane in acetonitrile to obtain product 1; dissolving product 1 and malononitrile in anhydrous ethanol, and adding sodium ethoxide to obtain product 2; dissolving product 2 and quinoline-3-carboxaldehyde in acetonitrile, and adding piperidine to obtain product 3; dissolving product 3 and methyl trifluoromethanesulfonate in dichloromethane, and obtaining fluorescent probe QC-QNM under nitrogen protection; and dissolving the fluorescent probe QC-QNM in DMSO to prepare a mother solution. The fluorescent probe of the present invention can react with HSO3 under high viscosity conditions. ‑ Response, viscosity and HSO3 ‑ Dual detection, simple synthesis route, simple detection equipment and methods, can be used in biological systems HSO3 ‑ Qualitative and quantitative detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of ion detection, and specifically relates to a dual detection method for viscosity and HSO3 - The invention relates to a method for preparing a fluorescent probe. Background Art

[0002] SO2 is an environmental pollutant mainly due to two hydrates (SO3 2- / HSO3 - ). HSO3 - It is widely used in food and medicine because of its antioxidant, antiseptic and bleaching effects. However, excessive intake of HSO3 by the human body - It can cause oxidative damage to the human body, which can lead to respiratory diseases, cardiovascular diseases, asthma, lung cancer, etc. At the same time, intracellular viscosity is involved in many cellular processes and plays a very important role in the physiological activities of cells, such as the transport of substances, the transduction of signals, and the interaction between biological substances. Moreover, abnormal intracellular viscosity is closely related to diseases such as Alzheimer's disease, diabetes, and cancer. At the same time, HSO3, as an antioxidant, - It can regulate the redox state and protect cells from oxidative stress. This shows that cell viscosity is related to HSO3 - However, it is possible to achieve simultaneous dual detection of HSO3 - There is no report on fluorescent probes for level and viscosity changes, which will be detrimental to the in-depth study of HSO3 - The relationship between level and viscosity changes and related diseases. Fluorescent probes have good selectivity, high sensitivity, wide detection range, fast response time, and no damage to samples during the detection process. They show great potential in the rapid detection of food and in real-time in situ imaging of cells, tissues and living bodies. Therefore, it is necessary to develop a method that can simultaneously detect HSO3 - Fluorescent probes of level and viscosity changes are crucial. Summary of the invention

[0003] In view of the problems existing in the existing fluorescent probe detection technology, the present invention provides a method for detecting viscosity and HSO3 - The invention discloses a method for preparing a dual-detection fluorescent probe. The method has simple synthesis steps, high yield of target products, fast response speed, good selectivity, high sensitivity and low detection limit.

[0004] To solve the above technical problems, the present invention is achieved as follows:

[0005] A dual detection viscosity and HSO3 - The method for preparing a fluorescent probe comprises the following steps:

[0006] (1) 2-Methylquinoline and iodoethane were dissolved in acetonitrile, the resulting mixture was heated and stirred, and after the reaction was completed, the mixture was cooled to room temperature, filtered, and vacuum dried to obtain product 1;

[0007] (2) dissolving the product 1 obtained in step (1) and malononitrile in anhydrous ethanol, slowly adding sodium ethoxide at 0°C, pouring into ice water after the reaction at room temperature, adjusting the pH to weak alkalinity, filtering, and vacuum drying to obtain product 2;

[0008] (3) dissolving the product 2 obtained in step (2) and quinoline-3-carboxaldehyde in acetonitrile, adding piperidine, and heating the resulting mixture with stirring. After the reaction is completed, cooling to room temperature, filtering, and vacuum drying are performed to obtain product 3;

[0009] (4) The product 3 obtained in step (3) and methyl trifluoromethanesulfonate are dissolved in dichloromethane, and the resulting mixture is stirred at room temperature. After the reaction is completed, the precipitated solid is separated, washed and purified with dichloromethane, and vacuum dried to obtain the target product, the fluorescent probe QC-QNM.

[0010] Furthermore, in the step (1), the molar ratio of 2-methylquinoline to ethyl iodide is 1:1.

[0011] Furthermore, in the step (2), the molar ratio of the product 1 to malononitrile is 1:4.

[0012] Furthermore, in the step (3), the molar ratio of the product 2 to quinoline-3-carboxaldehyde is 1:1.

[0013] Furthermore, in the step (4), the molar ratio of the product 3 to methyl trifluoromethanesulfonate is 1:1.

[0014] Furthermore, in step (4), the structure of the fluorescent probe QC-QNM is as follows:

[0015]

[0016] Furthermore, in the step (1), 2-methylquinoline and iodoethane are dissolved in acetonitrile, and the resulting mixture is heated to 80° C. and stirred for 12 hours under N2 protection conditions.

[0017] Furthermore, in step (2), the obtained mixture is placed in an ice water bath.

[0018] Sodium ethoxide was added, and the mixture was stirred at room temperature for 8 hours. In step (3), the mixture was placed under N2 protection, heated to 80°C and stirred for 12 hours.

[0019] Furthermore, in the step (4), the obtained mixture is placed under N2 protection and stirred at room temperature for 12 hours.

[0020] The above dual detection viscosity and HSO3 - The fluorescent probe prepared by the fluorescent probe preparation method has a specific reaction process as follows:

[0021]

[0022] The present invention uses 2-methylquinoline, iodoethane, malononitrile, quinoline-3-carboxaldehyde and methyl trifluoromethanesulfonate to prepare the required fluorescent probe. Under pure water conditions, on the one hand, when bisulfite is present, no significant fluorescence enhancement phenomenon occurs with the increase of bisulfite concentration. On the other hand, under high viscosity conditions, the fluorescent probe also does not undergo significant changes in fluorescence intensity. However, under certain viscosity conditions, in the presence of HSO3 - When present, the fluorescent probe will show a significant fluorescence enhancement phenomenon with the increase of bisulfite concentration, and has dual detection viscosity and HSO3 - Compared with some existing detection technologies, the fluorescent chemical probe in the present invention has low cost investment, simple synthesis route, convenient post-processing, and can realize dual detection of viscosity and HSO3 - , which enhances the accuracy of the detection process and has potential application value, especially in biological systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be described in detail below through specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the definition of the attached claims. Unless otherwise specified, the various reagents and materials used in the present invention can be purchased from the market.

[0024] Figure 1 This is the H NMR spectrum of the prepared fluorescent probe QC-QNM;

[0025] Figure 2 This is the NMR C spectrum of the prepared fluorescent probe QC-QNM;

[0026] Figure 3 Fluorescent probe QC-QNM and HSO3 in solutions with different viscosities - Response fluorescence emission spectra;

[0027] Figure 4 Fluorescent probe QC-QNM and HSO3- The fitting curve corresponding to the fluorescence intensity as the solution viscosity changes and the function graph corresponding to the curve;

[0028] Figure 5 The fluorescent probe QC-QNM was used in the presence of 80% glycerol and HSO3 - Fluorescence emission spectra with increasing concentrations;

[0029] Figure 6 For the fluorescent probe QC-QNM with HSO3 - The fitting curve corresponding to the fluorescence intensity of the concentration change and the function graph corresponding to the curve;

[0030] Figure 7 The selectivity diagram of the fluorescent probe QC-QNM to various analytes in the presence of 80% glycerol;

[0031] Figure 8 The fluorescence probe QC-QNM was used to detect HSO3 in the presence of 80% glycerol. - Time scan of the response.

[0032] The concentration of added ions was 1×10 -3 mol / L, 425nm as excitation wavelength, 590nm as emission wavelength. (Note: The probe concentration is 10μM, and the response time is 15s) DETAILED DESCRIPTION

[0033] Example 1

[0034] Dual detection of viscosity and HSO3 - The method for preparing a fluorescent probe comprises the following steps:

[0035] (1) Preparation of products 1-3.

[0036] 2-Methylquinoline (5 mmol) and iodoethane (5 mmol) were added to a round-bottom flask containing 15 mL of acetonitrile. The mixture was heated to reflux for 12 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered, and dried in vacuo to synthesize product 1.

[0037] The product 1 (3 mmol) and malononitrile (12 mmol) were dissolved in 30 mL of anhydrous ethanol, and sodium ethoxide (6 mmol) was slowly added in an ice bath. The mixture was stirred at room temperature for 8 h. After the reaction, the mixture was poured into ice water, the pH was adjusted to weak alkalinity, filtered, and vacuum dried to synthesize the product 2.

[0038] Product 2 (1 mmol) and quinoline-3-carboxaldehyde (1 mmol) were dissolved in 10 mL of dry acetonitrile and 2 drops of piperidine. The mixture was heated to reflux for 12 h under N2 protection. After the reaction was completed, the precipitated solid was separated to synthesize product 3.

[0039] (2) Using product 3, a probe is synthesized.

[0040] The product 3 (0.5 mmol) and methyl trifluoromethanesulfonate (1 mmol) were weighed and added to a round-bottom flask containing 10 mL of dichloromethane, and the mixture was reacted at room temperature for 8 h under N2 protection. After cooling to room temperature, the mixture was filtered and washed with ice dichloromethane, and then dried in vacuum to obtain the fluorescent probe QC-QNM.

[0041] QC-QNM (1 mmol) was weighed and dissolved in DMSO to prepare a 1 mM stock solution.

[0042] QC-QNM+HSO3 - Viscosity test:

[0043] Take 10 5 mL sample bottles, add glycerol and PBS mixed solution (glycerol content: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%), continue to add 20 μL of QC-QNM solution prepared by the fluorescent probe obtained in Example 1 (the concentration of the fluorescent probe is 10 μM), and then add 1×10 -3 28 μL of 1 mol / L sodium bisulfite solution was added, stirred at room temperature for 1 min, and the fluorescence intensity of these samples was measured at 425 nm as the excitation wavelength. The fluorescence intensity emission spectra of 10 samples were obtained. Figure 3 The results showed that the fluorescence intensity of the fluorescent probe gradually increased with the increase of bisulfite concentration. Figure 3 The fluorescence intensity Log I f (I f The change value of fluorescence emission intensity can be used to make the corresponding Log Ƞ (Ƞ: solution system viscosity) fitting function curve and the function graph corresponding to the curve (y=ax+b, a=0.5711, b=2.645, R 2 =0.9908) Figure 4 .

[0044] QC-QNM in 80% glycerol to HSO3 - Detection test:

[0045] Take 8 5 mL sample bottles, add 20 μL of the fluorescent probe solution QC-QNM obtained in Example 1 (the concentration of the fluorescent probe is 10 μM), and then add different concentrations (2×10 -3 mol / L,4×10 -3 mol / L,6×10 - 3 mol / L,8×10 -3 mol / L,10×10 -3 mol / L,12×10 -3 mol / L,14×10 -3 mol / L) of HSO3 - The solution was stirred at room temperature for 1 min, and the concentrations of the above samples were measured at 425 nm as the excitation wavelength to obtain the fluorescence emission wavelength change spectra of the 8 samples. Figure 5 The results show that the fluorescence intensity of the fluorescent probe is - The concentration of Figure 5 The fluorescence intensity change value can be used to make the corresponding fitted function curve and the function graph corresponding to the curve (y=ax+b, a=165.35, b=413.57, R 2 =0.9956) Figure 6 .

[0046] Other analyte comparison tests:

[0047] Take 18 5 mL sample bottles, add 20 μL of the solution QC-QNM prepared by the fluorescent probe obtained in Example 1 (the concentration of the fluorescent probe is 10 μM), and then add 1×10 -2 mol / L other analytes and HSO3 - Take 28 μL of each sample and add it to the other 20 sample bottles. Sample No. 1 is the blank sample. Then measure the fluorescence emission intensity of the 18 samples at 425 nm as excitation and 590 nm as emission wavelength. The results are shown in Figure 7 The results showed that the other analytes had no significant effect on the intensity of the prepared fluorescent probe. - The response time is 1 min for the samples and 10 min for the other samples.

[0048] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A dual detection viscosity and HSO3 - A method for preparing a fluorescent probe, characterized in that: The following steps are involved: (1) Dissolve 2-methylquinoline and iodoethane in acetonitrile, heat and stir the resulting mixture, cool to room temperature after the reaction, filter, and vacuum dry to obtain product 1; the structural formula of product 1 is: (2) The product 1 obtained in step (1) and malononitrile are dissolved in anhydrous ethanol, sodium ethoxide is slowly added at 0°C, and after the reaction at room temperature is completed, the mixture is poured into ice water, the pH is adjusted to weak alkalinity, filtered, and vacuum dried to obtain product 2; the structural formula of product 2 is: (3) dissolving the product 2 obtained in step (2) and quinoline-3-carboxaldehyde in acetonitrile, adding piperidine, and heating the resulting mixture with stirring. After the reaction is completed, cooling to room temperature, filtering, and vacuum drying are performed to obtain product 3; the structural formula of product 3 is: (4) The product 3 obtained in step (3) and methyl trifluoromethanesulfonate are dissolved in dichloromethane, and the resulting mixture is stirred at room temperature. After the reaction is completed, the precipitated solid is separated, washed and purified with dichloromethane, and vacuum dried to obtain the target product, fluorescent probe QC-QNM; the structural formula of the fluorescent probe QC-QNM is as follows:

2. According to claim 1, the double detection viscosity and HSO3 - The method for preparing a fluorescent probe is characterized by: In the step (1), the molar ratio of 2-methylquinoline to ethyl iodide is 1:

1.

3. According to claim 2, the double detection viscosity and HSO3 - A method for preparing a fluorescent probe, characterized in that: In the step (2), the molar ratio of the product 1 to malononitrile is 1:

4.

4. According to claim 3, the double detection viscosity and HSO3 - A method for preparing a fluorescent probe, characterized in that: In the step (3), the molar ratio of product 2 to quinoline-3-carboxaldehyde is 1:

1.

5. According to claim 4, the double detection viscosity and HSO3 - A method for preparing a fluorescent probe, characterized in that: In the step (4), the molar ratio of product 3 to methyl trifluoromethanesulfonate is 1:

1.

6. Dual detection of viscosity and HSO3 according to claim 5 - The method for preparing a fluorescent probe is characterized by: In the step (1), 2-methylquinoline and iodoethane are dissolved in acetonitrile, and the resulting mixture is heated to 80° C. and stirred for 12 hours under N2 protection.

7. Dual detection of viscosity and HSO3 according to claim 6 - A method for preparing a fluorescent probe, characterized in that: In the step (2), the obtained mixture is placed in an ice-water bath, sodium ethoxide is added, and the mixture is stirred at room temperature for 8 hours; in the step (3), the obtained mixture is placed under N2 protection, the temperature is raised to 80°C, and stirred for 12 hours.

8. Dual detection of viscosity and HSO3 according to claim 7 - A method for preparing a fluorescent probe, characterized in that: In the step (4), the obtained mixture is placed under N2 protection and stirred at room temperature for 12 hours.

9. A dual detection viscosity and HSO3 as claimed in any one of claims 1 to 8 - The fluorescent probe is obtained by the fluorescent probe preparation method.

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