A dual-channel fluorescent probe and its preparation method and application

Abstract

The present invention provides a dual-channel fluorescent probe, a preparation method and application thereof, and relates to the field of chemical analysis and detection technology. The preparation method of the dual-channel fluorescent probe of the present invention comprises the following steps: compound 1, methylene blue chloride, a base and an organic solvent are mixed and reacted to obtain a novel dual-channel fluorescent probe for accurate detection of HClO. The probe has the function of detecting HClO in two spectral channels (blue and red). The two channels have high spectral separation and can respond to HClO independently, thereby significantly reducing background noise interference in the actual detection process. In addition, the fluorescent probe exhibits excellent selectivity, fast response time and low cytotoxicity, which make it an ideal tool for detecting the concentration of HClO in living cells.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a dual-channel fluorescent probe and a preparation method and application thereof. Background Art

[0002] As a key member of the ROS family, HClO has a significant impact on a range of physiological and pathological responses within biological systems. In the immune system, HClO is produced by neutrophils and acts as a potent bactericidal agent, capable of penetrating the cell walls of pathogens and disrupting their metabolic processes, thereby rapidly eliminating invading bacteria, viruses, and other microorganisms. These processes are crucial for protecting the body from infection. HClO also regulates inflammatory responses, influencing intracellular signaling pathways, contributing to immune cell activation and the release of inflammatory mediators, thereby playing a regulatory role in maintaining tissue homeostasis and promoting wound healing. However, a balance must be maintained between the production and clearance of HClO. Excessive HClO can lead to oxidative stress, which can damage cellular components, including lipids, proteins, and nucleic acids, thereby affecting cell function and tissue structure. As disease mechanisms deepen, abnormal HClO concentrations have been linked to a range of pathological conditions, including inflammation, liver damage, and even cancer. Despite significant progress in this research, the specific functions and significance of HClO remain uncertain, and further research is needed to elucidate its mechanisms and significance. Therefore, the development of accurate and rapid methods for the detection of HClO is imperative. This approach will help reveal their potential for therapeutic intervention, providing valuable insights into their biological significance and potential therapeutic applications.

[0003] Compared to traditional methods, fluorescent probes, with their real-time monitoring capabilities, excellent biocompatibility, and non-invasive detection characteristics, provide a more precise and direct means of observing active molecules in biological systems. To date, numerous fluorescent probes have been developed for the detection of HClO, employing a variety of fluorophores such as fluorescein, coumarin, rhodamine, and benzothiazole. Despite significant progress, existing probes still face numerous challenges in in vivo imaging, such as insufficient tissue penetration and limited application. Furthermore, most probes monitor HClO concentration changes within a single detection channel and are susceptible to interference from complex environmental factors and photobleaching, limiting their application in practical imaging experiments.

[0004] Therefore, developing a dual-channel fluorescent probe that can be cross-calibrated in two different and non-overlapping channels to achieve accurate imaging of HClO in vivo is a challenging task. Summary of the Invention

[0005] The present invention aims to provide a dual-channel fluorescent probe, its preparation method, and application, to address the technical problem that existing detection methods for monitoring HClO concentration changes in a single channel are susceptible to interference from environmental factors and photobleaching, limiting their application in actual imaging experiments.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a dual-channel fluorescent probe, the structural formula of the dual-channel fluorescent probe is:

[0008]

[0009] The present invention provides a method for preparing a dual-channel fluorescent probe, comprising mixing compound 1 and methylene blue chloride in an organic solvent, and reacting the mixture under the action of a base to obtain a dual-channel fluorescent probe;

[0010] The structural formula of the compound 1 is

[0011] The structural formula of the methylene blue chlorination is

[0012] Furthermore, the organic solvent includes one or more of dichloromethane, ethanol, acetonitrile, tetrahydrofuran and N,N-dimethylformamide.

[0013] Furthermore, the base includes one or more of sodium carbonate, potassium carbonate, triethylamine, sodium hydroxide and ammonium acetate.

[0014] Furthermore, the molar ratio of the compound 1, methylene blue acyl chloride and base is 1:1-1.5:0.8-1.2;

[0015] The molar volume ratio of the compound 1 to the organic solvent is 1 mol: 20-40 mL.

[0016] Furthermore, the reaction temperature is 25-100° C., and the reaction time is 2-12 hours.

[0017] The present invention also provides an application of a dual-channel fluorescent probe in the preparation of an HClO detection product.

[0018] Beneficial effects of the present invention:

[0019] 1) A novel dual-channel fluorescent probe was constructed using methylene blue and benzothiazole as fluorophores and dimethylaminothiocarbamate as a specific recognition group for HClO. The probe uses N,N-dimethylthiocarbamate as the recognition group. Because the donors in methylene blue and benzothiazole are protected by the recognition group, intramolecular charge transfer and excited-state intramolecular proton transfer in the system are inhibited, resulting in almost no fluorescence in the blue and near-infrared regions. When HClO molecules interact with the probe, a nucleophilic reaction occurs first, simultaneously triggering the cleavage of the intramolecular self-eliminating group, releasing the benzothiazole fluorophore with strong blue emission and the methylene blue fluorophore emitting in the near-infrared. This enables qualitative detection and quantitative analysis of HClO in two channels (blue and red).

[0020] 2) Through molecular design, a new dual-channel fluorescent probe for the precise detection of HClO was obtained. The probe provides dual detection capabilities and improves sensitivity by synergistically amplifying the signals in the blue and red channels. The two channels have significant spectral separation characteristics, allowing independent responses to HClO, thereby effectively minimizing background interference. In addition, different fluorescence channels help to alleviate errors caused by changes in sample preparation and measurement conditions, giving the probe greater application flexibility. At the same time, it also has the advantages of high selectivity, ultra-low detection limit and rapid response. The probe molecule detects HClO at a concentration of 1.0-40.0 μmol / L, with a minimum detection limit of 18.7 nM (blue channel). Its good selectivity and low cytotoxicity make it an ideal tool for detecting HClO.

[0021] 3) The fluorescent probe of this invention has good cell membrane permeability, enabling confocal imaging of endogenous and exogenous HClO in living HeLa cells. Therefore, the probe molecule can be used to detect HClO in aqueous solutions and complex biological systems, representing a promising molecular tool for studying the functions and synergistic effects of HClO in physiological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the dual-channel fluorescent probe of Example 1;

[0023] Figure 2 is the carbon NMR spectrum of the dual-channel fluorescent probe of Example 1;

[0024] Figure 3 The fluorescence spectra of the fluorescent probe of Example 1 after reacting with HClO at different concentrations;

[0025] Figure 4The figure shows the linear relationship between the fluorescence intensity of the fluorescent probe of Example 1 at 471 nm and 681 nm and the HClO concentration; the insets show the linear fitting graphs of the fluorescence intensity of the fluorescent probe of Example 1 at 471 nm and 681 nm and (0.0-10.0 μmol / L) HClO; wherein, the left one is 471 nm and the right one is 681 nm;

[0026] Figure 5 Schematic diagram of the selectivity and competitiveness of the fluorescent probe of Example 1 to HClO at 471 nm and 681 nm;

[0027] Figure 6 The fluorescence imaging diagram of the fluorescent probe in Example 1 detecting exogenous HClO in HeLa cells, wherein a1 to a4 are respectively the bright field image, 633 nm excited red channel fluorescence image, 405 nm excited blue channel fluorescence image and superposition image of HeLa cells cultured with the fluorescent probe; b1 to b4 are respectively the bright field image, 633 nm excited red fluorescence channel fluorescence image, 405 nm excited blue fluorescence channel fluorescence image and superposition image of HeLa cells pre-incubated with HClO (10.0 μmol / L) and then cultured with the fluorescent probe. Figure 5: Addition of images; c1-c4 are the bright field images, fluorescence images of the red fluorescence channel excited at 633 nm, fluorescence images of the blue fluorescence channel excited at 405 nm, and overlay images of HeLa cells pre-incubated with HClO (20.0 μmol / L) and then cultured with the fluorescent probe, respectively; d1-d4 are the bright field images, fluorescence images of the red fluorescence channel excited at 633 nm, fluorescence images of the blue fluorescence channel excited at 405 nm, and overlay images of HeLa cells pre-incubated with HClO (40.0 μmol / L) and then cultured with the fluorescent probe, respectively.

[0028] Figure 7 These are fluorescence imaging images of the fluorescent probe of Example 1 detecting endogenous HClO in HeLa cells, wherein a1-a4 are the bright field image, red fluorescence channel fluorescence image, blue fluorescence channel fluorescence image, and overlay image of HeLa cells cultured only with the fluorescent probe; b1-b3 are the bright field image, red fluorescence channel fluorescence image, blue fluorescence channel fluorescence image, and overlay image of HeLa cells first stimulated with LPS and PMA and then incubated with the fluorescent probe; c1-c3 are the bright field image, red fluorescence channel fluorescence image, blue fluorescence channel fluorescence image, and overlay image of HeLa cells first pre-incubated with NAC, then stimulated with LPS and PMA, and then incubated with the fluorescent probe. DETAILED DESCRIPTION

[0029] The present invention provides a dual-channel fluorescent probe, the structural formula of the dual-channel fluorescent probe is:

[0030]

[0031] The present invention provides a method for preparing a dual-channel fluorescent probe, comprising mixing compound 1 and methylene blue chloride in an organic solvent, and reacting the mixture under the action of a base to obtain a dual-channel fluorescent probe;

[0032] The structural formula of the compound 1 is

[0033] The structural formula of the methylene blue chlorination is

[0034] In the present invention, after the reaction is completed, the obtained product is preferably sequentially extracted, washed, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a dual-channel fluorescent probe.

[0035] In the present invention, the extraction reagent is preferably dichloromethane, and the number of extractions is preferably 2 to 4 times, more preferably 3 times.

[0036] In the present invention, the washing agent is preferably saturated saline, and the number of washing times is preferably 2 to 4 times, more preferably 3 times.

[0037] In the present invention, the dry reagent is preferably anhydrous sodium sulfate.

[0038] In the present invention, in the silica gel column chromatography purification, the eluent is preferably a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 1 to 8:1, more preferably 2 to 6:1, and even more preferably 3:1.

[0039] In the present invention, the organic solvent includes one or more of dichloromethane, ethanol, acetonitrile, tetrahydrofuran and N,N-dimethylformamide, preferably one or more of dichloromethane, ethanol, acetonitrile and tetrahydrofuran, more preferably dichloromethane or ethanol, and more preferably dichloromethane.

[0040] In the present invention, the base includes one or more of sodium carbonate, potassium carbonate, triethylamine, sodium hydroxide and ammonium acetate, preferably one or more of sodium carbonate, potassium carbonate, triethylamine and sodium hydroxide, further preferably one or more of sodium carbonate, potassium carbonate and triethylamine, more preferably sodium carbonate.

[0041] In the present invention, the molar ratio of the compound 1, methylene blue acyl chloride and the base is 1:1-1.5:0.8-1.2, preferably 1:1.2-1.4:0.9-1.1, and more preferably 1:1.3:1;

[0042] The molar volume ratio of the compound 1 to the organic solvent is 1 mol:20-40 mL, preferably 1 mol:25-35 mL, and more preferably 1 mol:30 mL.

[0043] In the present invention, the reaction temperature is 25-100°C, preferably 40-80°C, more preferably 60°C, and the reaction time is 2-12h, preferably 4-10h, more preferably 5-8h, and more preferably 7h.

[0044] The present invention also provides an application of a dual-channel fluorescent probe in the preparation of an HClO detection product.

[0045] In the present invention, a fluorescent probe is used to prepare a product for qualitatively and quantitatively detecting HClO in an acetonitrile / PBS mixed solution (the volume ratio of acetonitrile to PBS is 2:8) or in a biological system, and exogenous and endogenous HClO in cells is successfully detected.

[0046] In the present invention, the dual-channel fluorescent probe is dissolved in a PBS buffer solution of acetonitrile, and HClO in the PBS solution is quantitatively detected. In the PBS solution, the volume ratio of water to acetonitrile is preferably 6-9:4-1, and more preferably 8:2.

[0047] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] 1.0 mmol of compound 1, 1.2 mmol of methylene blue acyl chloride, 0.8 mmol of anhydrous sodium carbonate, and 2 mmol of DMAP were placed in 30 mL of dichloromethane and reacted at 30°C for 8 hours. After the reaction, the reaction solution was added to 15 mL of water, extracted three times with dichloromethane, and washed three times with saturated brine. The combined organic layer was dried over anhydrous sodium sulfate for 15 minutes and concentrated under reduced pressure at -0.1 MPa for 10 minutes to remove the solvent. The resulting residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate, volume ratio 3:1) to obtain a green solid product, the dual-channel fluorescent probe.

[0050] The structural formula of compound 1 in this example is:

[0051]

[0052] The mass of the dual-channel fluorescent probe in this embodiment is 325.5 mg, and the yield is 49.6%. The structural formula of the dual-channel fluorescent probe for accurate HClO detection is:

[0053]

[0054] The nuclear magnetic resonance hydrogen spectrum of the dual-channel fluorescent probe obtained in this embodiment is as follows Figure 1 As shown: 1H NMR (600MHz, DMSO) δ8.27(dd,J=7.8,1.4Hz,1H),8.16(d,J=7.9Hz,1H),8.08(d,J=8.1Hz,1H),7.57(dd,J=11.0,4.1Hz,2H),7.49(t,J=7.6H z, 2H), 7.35 (d, J = 7.8Hz, 2H), 6.70 (d, J = 2.6Hz, 2H), 6.66 (d, J = 8.6Hz, 2H), 5.18 (t, J = 17.9Hz, 2H), 3.46 (s, 3H), 3.38 (s, 3H), 2.89 (s, 12H).

[0055] The carbon NMR spectrum of the dual-channel fluorescent probe obtained in this example is as follows: Figure 2 As shown: 13 C NMR(151MHz,DMSO)δ183.97,161.77,153.17,152.01,148.82,148.50,131.85,1 26.98,126.81,126.53,125.52,122.78,122.00,110.70,109.56,62.23,40.02.

[0056] Example 2

[0057] 1.0 mmol of compound 1, 1.2 mmol of methylene blue acyl chloride, 0.8 mmol of potassium carbonate, and 2 mmol of DMAP were placed in 30 mL of dichloromethane and reacted at 25°C for 5 h. After the reaction, the reaction solution was added to 20 mL of water, extracted twice with dichloromethane, and washed twice with saturated brine. The combined organic layer was dried over anhydrous sodium sulfate for 15 min and concentrated under reduced pressure at -0.1 MPa for 10 min to remove the solvent. The residue was purified by silica gel column chromatography (volume ratio of petroleum ether and ethyl acetate was 3:1) to obtain a green solid product - a new dual-channel fluorescent probe for the precise detection of HClO.

[0058] The structural formula of compound 1 in this example is:

[0059]

[0060] The mass of the dual-channel fluorescent probe in this example is 302.5 mg, and the yield is 46.2%. The structural formula of the new dual-channel fluorescent probe for accurate HClO detection is:

[0061]

[0062] Example 3

[0063] 1.0 mmol of compound 1, 1.0 mmol of methylene blue chloride, 1.0 mmol of triethylamine, and 2 mmol of DMAP were placed in 25 mL of dichloromethane and reacted at 25 ° C for 6 hours. After the reaction, the reaction solution was added to 18 mL of water, extracted with dichloromethane 4 times, and washed twice with saturated brine. The combined organic layer was dried over anhydrous sodium sulfate for 15 minutes and concentrated under reduced pressure at -0.1 MPa for 10 minutes to remove the solvent. The residue was purified by silica gel column chromatography (volume ratio of petroleum ether and ethyl acetate was 3:1) to obtain a green solid product - a new dual-channel fluorescent probe for the precise detection of HClO.

[0064] The structural formula of compound 1 in this example is:

[0065]

[0066] The mass of the dual-channel fluorescent probe in this example is 250.2 mg, and the yield is 38.2%. The structural formula of the new dual-channel fluorescent probe for accurate HClO detection is:

[0067]

[0068] 1) Spectral determination of HClO using a dual-channel fluorescent probe

[0069] To investigate the probe's in vitro response to HClO, the probe's spectral response to HClO was first investigated. The fluorescent probe prepared in Example 1 was dissolved in acetonitrile to obtain a stock solution with a fluorescent probe concentration of 1 mmol / L. This stock solution was then added to a 20 mmol / L, pH 7.4 phosphate buffer solution (containing 20% ​​by volume acetonitrile) to obtain a probe solution with a fluorescent probe concentration of 10 μmol / L. In a 3 mL quartz test tube, different concentrations of HClO (0.0 μmol / L, 1.0 μmol / L, 2.0 μmol / L, 3.0 μmol / L, 4.0 μmol / L, 5.0 μmol / L, 6.0 μmol / L, 7.0 μmol / L, 8.0 μmol / L, 9.0 μmol / L, 10.0 μmol / L, 15.0 μmol / L, 20.0 μmol / L, 30.0 μmol / L, 40.0 μmol / L) were placed in the above probe solution, mixed well, and then tested.

[0070] The fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of HClO are shown in FIG. Figure 3 As shown by Figure 3It can be seen that when different concentrations of HClO were added to the probe solution, two obvious fluorescence emission bands were observed at 471nm and 681nm. At a concentration of 40.0μmol / L HClO, the fluorescence intensities of these two emission bands were enhanced by approximately 29 times and 11 times, respectively. In addition, there was a good linear relationship between the fluorescence intensity at 471nm and 681nm and the HClO concentration (0.0-10.0μmol / L), with a correlation coefficient (R 2 ) were 0.9976 and 0.9932, respectively. The detection limits were 18.7 nM in the blue fluorescence channel and 45.0 nM in the red fluorescence channel, indicating that the probe has high sensitivity to HClO in both channels.

[0071] 2) Selectivity and competitiveness test of fluorescent probes for HClO

[0072] To evaluate the applicability of the probe for imaging in complex in vivo environments, a series of experiments were performed to test its reactivity to multiple analytes and its specific response to HClO in the presence of these analytes. Figure 5 A and B in Figure 2 show that the 2+ , K + , Zn 2+ , Cu 2+ , Hg 2+ , Na + , Al 3+ , Cl - , I - , Br - , HSO3 - , CO3 2- , PO4 3- , NO3 - , Cys, Hcy, GSH, H2O2, OH - , ONOO - , O2 - ) After incubation, significant fluorescence signals were observed only in the presence of HClO, confirming the high selectivity of the probe. In addition, competition experiments further confirmed that the probe has excellent anti-interference ability for the detection of HClO. Even in the presence of background interfering substances, the probe can still maintain a high sensitivity response to HClO ( Figure 5 C and D in Figure 1). These combined experimental results demonstrate that the probe is not only highly selective for HClO but also maintains excellent anti-interference properties in complex biological samples. These properties provide a solid scientific basis for the probe's application in future in vivo studies.

[0073] 3) Cellular imaging test of fluorescent probes for exogenous HClO

[0074] The density is 1×10 5 After incubating HeLa cells with 10.0 μmol / L probe solution in the spectral measurement, the bright field image, 633 nm excited fluorescence image (red channel), 405 nm excited fluorescence image (blue channel) and superimposed image are shown as follows: Figure 6 As shown in . Figure 6 As can be seen from a1 to a4 in the figure, cells incubated only with the probe showed weak fluorescence in the blue and red channels. HeLa cells were incubated with different concentrations of HClO (10.0, 20.0, 40.0 μmol / L) for 30 min and then co-incubated with a probe solution at a concentration of 10.0 μmol / L. The bright field image, 633 nm excitation fluorescence image (red channel), 405 nm excitation fluorescence image (blue channel) and superimposed image are shown in Figures 1 and 2. Figure 6 As shown by b1 to d1 in . Figure 6 As can be seen from b1 to d1 in the figure, after the addition of HClO, the fluorescence intensity of the two channels is significantly enhanced, and this enhancement increases with the increase of HClO concentration, indicating that the reaction between the probe and HClO is concentration-dependent.

[0075] 4) Cellular imaging test of endogenous HClO using fluorescent probes

[0076] We tested the effectiveness of the probe in monitoring endogenous HClO in HeLa cells. After incubating HeLa cells with 10 μmol / L probe solution, the bright field image, 633 nm excitation fluorescence image (red channel), 405 nm excitation fluorescence image (blue channel) and overlay image were shown as follows: Figure 7 As shown in a1 to a4 in the figure, there is no obvious fluorescence signal in the two channels, indicating that the probe has low background fluorescence. Figure 7Figures b1 to b4 show brightfield images, fluorescence images excited at 633 nm (red channel), fluorescence images excited at 405 nm (blue channel), and overlay images of HeLa cells pre-stimulated with LPS / PMA and then incubated with a 10 μmol / L probe solution in the spectroscopic assay. The results demonstrate that LPS / PMA pre-stimulation induces endogenous HClO production, leading to a significant increase in intracellular fluorescence after probe incubation. Figures c1 to c4 show brightfield images, fluorescence images excited at 633 nm (red channel), fluorescence images excited at 405 nm (blue channel), and overlay images of HeLa cells pre-treated with NAC and then incubated with a 10 μmol / L probe solution. The results show that, following LPS / PMA stimulation, fluorescence in both channels decreases significantly in cells pre-treated with NAC. This phenomenon confirms that the detected fluorescence signal is indeed caused by the probe's response to endogenous HClO. These observations not only validate the probe's specificity but also demonstrate its ability to accurately report HClO levels, which is crucial for evaluating probe performance in biological environments.

[0077] As can be seen from the above embodiments, the present invention provides a dual-channel fluorescent probe, its preparation method and application. According to the aforementioned series of tests, the dual-channel fluorescent probe prepared by the present invention has high sensitivity to HClO in both channels, and has high selectivity, excellent anti-interference ability and concentration dependence. It can be seen that the present invention solves the technical problem that the detection method of monitoring HClO concentration changes in a single channel in the prior art is easily affected by environmental factors and photobleaching, and its application in actual imaging experiments is limited. Therefore, the present invention is of great significance.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A dual-channel fluorescent probe, characterized in that: The structural formula of the dual-channel fluorescent probe is:

2. The method for preparing a dual-channel fluorescent probe according to claim 1, wherein: Compound 1 and methylene blue acyl chloride are mixed in an organic solvent and reacted under the action of a base to obtain a dual-channel fluorescent probe; The structural formula of the compound 1 is The structural formula of the methylene blue chloride is 3. The method for preparing a dual-channel fluorescent probe according to claim 2, wherein: The organic solvent includes one or more of dichloromethane, ethanol, acetonitrile, tetrahydrofuran and N,N-dimethylformamide.

4. The method for preparing a dual-channel fluorescent probe according to claim 2 or 3, wherein: The base includes one or more of sodium carbonate, potassium carbonate, triethylamine, sodium hydroxide and ammonium acetate.

5. The method for preparing a dual-channel fluorescent probe according to claim 4, wherein: The molar ratio of the compound 1, methylene blue acyl chloride and the base is 1:1-1.5:0.8-1.2; The molar volume ratio of the compound 1 to the organic solvent is 1 mol: 20-40 mL.

6. The method for preparing a dual-channel fluorescent probe according to claim 2 or 5, characterized in that: The reaction temperature is 25-100° C., and the reaction time is 2-12 hours.

7. Use of the dual-channel fluorescent probe according to claim 1 in preparing a HClO detection product.

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