Two-channel fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a dual-channel fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of chemical analysis and detection. The preparation method of the two-channel fluorescent probe comprises the following steps: mixing a compound 1, methylene blue acylating chloride, alkali and an organic solvent, and reacting to obtain the novel two-channel fluorescent probe for accurate detection of HClO. The probe has a function of detecting HClO in two spectrum channels (blue and red), and the two channels have relatively high spectrum separation degrees and can independently respond to HClO, so that background noise interference in an actual detection process is remarkably reduced. In addition, the fluorescent probe shows excellent selectivity, short response time and low cytotoxicity, and due to the characteristics, the fluorescent probe becomes 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 an important impact on a range of physiological and pathological reactions within biological systems. In the immune system, HClO is produced by neutrophils and, as an effective bactericidal agent, can penetrate the cell walls of pathogens and disrupt their metabolic processes, thereby rapidly eliminating invading bacteria, viruses, and other microorganisms; the above processes are essential for protecting the body from infection. In addition, HClO is involved in regulating inflammatory responses, affecting intracellular signaling pathways, participating in the activation of immune cells and the release of inflammatory mediators, thereby playing a regulatory role in maintaining tissue homeostasis and promoting wound healing. However, the production and clearance of HClO must be balanced. 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. With the in-depth study of disease mechanisms, it has been found that abnormal HClO concentrations are associated with a range of pathological conditions, including inflammation, liver damage, and even cancer. Although great progress has been made in the above research, there is still uncertainty about the specific function and significance of HClO, and further research is needed to clarify its mechanism and significance. Therefore, it is imperative to develop an accurate and rapid method for detecting HClO. This approach will help reveal their potential for therapeutic intervention, providing valuable insights into their biological significance and potential therapeutic applications.

[0003] Compared with traditional methods, fluorescent probes provide a more sophisticated and direct means of observation for the detection of active molecules in biological systems with their real-time monitoring capabilities, excellent biocompatibility, and non-invasive detection characteristics. To date, numerous fluorescent probes have been developed for the detection of HClO, using a variety of fluorophores such as fluorescein, coumarin, rhodamine, and benzothiazole. Despite significant progress, existing probes still face many challenges in in vivo imaging, such as insufficient tissue penetration and limited application range. In addition, most probes only monitor changes in HClO concentration within a single detection channel and are susceptible to interference from complex environmental factors and photobleaching, limiting their application in actual 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 purpose of the present invention is to provide a dual-channel fluorescent probe and a preparation method and application thereof, so as to solve the technical problem that the detection method for monitoring the concentration change of HClO in a single channel in the prior art is easily interfered by environmental factors and photobleaching, and its application in actual imaging experiments is limited.

[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 acyl chloride in an organic solvent, reacting under the action of a base, and obtaining a dual-channel fluorescent probe;

[0010] The structural formula of the compound 1 is

[0011] The structural formula of the methylene blue acyl chloride 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 the 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 to 100° C., and the reaction time is 2 to 12 hours.

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

[0018] Beneficial effects of the present invention:

[0019] 1) A new dual-channel fluorescent probe was constructed using methylene blue and benzothiazole as fluorophores and dimethylaminothiocarbamate as the specific recognition group of HClO. The probe uses N,N-dimethylthiocarbamate as the recognition group. Since the donors in methylene blue and benzothiazole are protected by the recognition group, the intramolecular charge transfer and excited-state intramolecular proton transfer processes in the system are inhibited, so the probe molecule has almost no fluorescence in the blue light and near-infrared regions. When HClO molecules interact with the probe, a nucleophilic reaction occurs first, and at the same time, the cleavage of the intramolecular self-eliminating group is triggered, thereby releasing the benzothiazole fluorophore with strong blue light emission and the methylene blue fluorophore with near-infrared emission. Qualitative detection and quantitative analysis of HClO dual channels (blue and red channels) are achieved.

[0020] 2) A new dual-channel fluorescent probe for accurate detection of HClO was obtained through molecular design. The probe provides dual detection capabilities and improves sensitivity by synergistically amplifying 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 mitigate 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 detection concentration of the probe molecule for HClO is 1.0-40.0μmol / L, and the lowest detection limit is 18.7nM (blue channel). Good selectivity and low cytotoxicity make it an ideal tool for detecting HClO.

[0021] 3) The fluorescent probe of the present invention has good cell membrane permeability and can perform confocal imaging of endogenous and exogenous HClO in living HeLa cells. Therefore, the probe molecule can be used for the detection of HClO in aqueous solutions and complex biological systems, and is a promising molecular tool for studying the function and synergy 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 nuclear magnetic resonance spectrum of the dual-channel fluorescent probe of Example 1;

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

[0025] Figure 4The figure is a linear relationship diagram of the fluorescence intensity of the fluorescent probe of Example 1 at 471nm and 681nm and the HClO concentration; the illustrations are linear fitting diagrams of the fluorescence intensity of the fluorescent probe of Example 1 at 471nm and 681nm and (0.0-10.0μmol / L) HClO; wherein, the left is 471nm and the right is 681nm;

[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 of Example 1 detecting exogenous HClO in HeLa cells, wherein a1-a4 are respectively the bright field image, 633nm excited red channel fluorescence image, 405nm excited blue channel fluorescence image and superposition image of HeLa cells cultured with the fluorescent probe; b1-b4 are respectively the bright field image, 633nm excited red fluorescence channel fluorescence image, 405nm 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 added; c1-c4 are the bright field images, red fluorescence channel fluorescence images excited by 633nm, blue fluorescence channel fluorescence images excited by 405nm, and superimposed images of HeLa cells first pre-incubated with HClO (20.0μmol / L) and then cultured with fluorescent probes, respectively; d1-d4 are the bright field images, red fluorescence channel fluorescence images excited by 633nm, blue fluorescence channel fluorescence images excited by 405nm, and superimposed images of HeLa cells first pre-incubated with HClO (40.0μmol / L) and then cultured with fluorescent probes, respectively;

[0028] Figure 7 The fluorescence imaging images of the fluorescent probe of Example 1 detecting endogenous HClO in HeLa cells, wherein a1-a4 are respectively 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 respectively 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 respectively 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 acyl chloride in an organic solvent, reacting under the action of a base, and obtaining a dual-channel fluorescent probe;

[0032] The structural formula of the compound 1 is

[0033] The structural formula of the methylene blue acyl chloride 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 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, further preferably dichloromethane or ethanol, 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 to 100° C., preferably 40 to 80° C., more preferably 60° C., and the reaction time is 2 to 12 h, preferably 4 to 10 h, more preferably 5 to 8 h, and more preferably 7 h.

[0044] The present invention also provides an application of a dual-channel fluorescent probe in the preparation of a 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

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

[0053]

[0054] The nuclear magnetic resonance hydrogen spectrum of the dual-channel fluorescent probe obtained in this example 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] 1.0mmol compound 1, 1.2mmol methylene blue acyl chloride, 0.8mmol potassium carbonate and 2mmol DMAP were placed in 30mL dichloromethane and reacted at 25°C for 5h. After the reaction, the reaction solution was added to 20mL water, extracted twice with dichloromethane, and washed twice with saturated brine. The combined organic layer was dried over anhydrous sodium sulfate for 15min and concentrated under reduced pressure at -0.1MPa for 10min 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 accurate 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 embodiment is 302.5 mg, and the yield is 46.2%. The structural formula of the new dual-channel fluorescent probe for accurate detection of HClO is:

[0061]

[0062] Example 3

[0063] 1.0mmol compound 1, 1.0mmol methylene blue chloride, 1.0mmol triethylamine and 2mmol DMAP were placed in 25mL dichloromethane and reacted at 25°C for 6h. After the reaction, the reaction solution was added to 18mL water, extracted with dichloromethane 4 times, and washed with saturated brine twice. The combined organic layer was dried over anhydrous sodium sulfate for 15min and concentrated under reduced pressure at -0.1MPa for 10min 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 accurate 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 embodiment is 250.2 mg, and the yield is 38.2%. The structural formula of the new dual-channel fluorescent probe for accurate detection of HClO is:

[0067]

[0068] 1) Spectral determination of HClO by dual-channel fluorescence probe

[0069] In order to study the in vitro response of the probe to HClO, the spectral response of the probe to HClO was first studied. 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. The stock solution was added to a phosphate buffer solution (containing acetonitrile with a volume fraction of 20%) with a concentration of 20 mmol / L and a pH value of 7.4 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 (the concentrations were 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, and 40.0 μmol / L) were placed in the above probe solution, mixed evenly, 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 about 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), and the 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 suitability 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, 5B show that in the presence of various analytes including HClO (e.g. Ca 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, a significant fluorescence signal was observed only in the presence of HClO, which confirmed that the probe was highly selective. In addition, competition experiments further confirmed that the probe had excellent anti-interference ability for the detection of HClO. Even in the presence of background interfering substances, the probe was still able to maintain a high sensitivity response to HClO ( Figure 5 C, 5D). These comprehensive experimental results show that the probe is not only highly selective for HClO, but also can maintain excellent anti-interference performance in complex biological samples. These characteristics provide a solid scientific basis for the application of the probe 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 the HeLa cells with 10.0 μmol / L probe solution in the spectral measurement, the bright field image, 633 nm excitation fluorescence image (red channel), 405 nm excitation fluorescence image (blue channel) and superimposed image are shown as follows: Figure 6 As shown in . As can be seen from Figures a1 to a4, cells incubated only with the probe have weak fluorescence in the blue and red channels. After HeLa cells were incubated with different concentrations of HClO (10.0, 20.0, 40.0 μmol / L) for 30 minutes, they were co-incubated with a probe solution with 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 a1 to a4. Figure 6 As shown in b1 to d1 in FIG. As shown in FIG. b1 to d1, after adding 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 probe

[0076] We tested the effectiveness of the probe in monitoring endogenous HClO in HeLa cells. After HeLa cells were incubated with 10 μmol / L of the 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 in Figure 2. 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 a low background fluorescence. Figure 7Figures b1 to b4 show the bright field images, 633nm excitation fluorescence images (red channel), 405nm excitation fluorescence images (blue channel), and superimposed images of HeLa cells pre-stimulated with LPS / PMA and then incubated with 10μmol / L probe solution in the spectral measurement. The results show that the endogenous HClO produced by LPS / PMA pre-stimulation significantly increases the intracellular fluorescence after probe incubation. c1 to c4 are the bright field images, 633nm excitation fluorescence images (red channel), 405nm excitation fluorescence images (blue channel), and superimposed images of cells pre-treated with NAC, HeLa cells pre-stimulated with LPS / PMA, and then incubated with 10μmol / L probe solution. The results show that the fluorescence in both channels of cells pre-treated with NAC is significantly reduced after subsequent LPS / PMA stimulation. This phenomenon confirms that the detected fluorescence signal is indeed caused by the probe's response to endogenous HClO. These observations not only verify the specificity of the probe, but also prove that it can accurately report HClO levels, which is crucial for evaluating the performance of the probe in biological environments.

[0077] It can be seen from the above embodiments that the present invention provides a dual-channel fluorescent probe and 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 for monitoring the concentration change of HClO 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 principle of the present invention. These improvements and modifications should also be regarded as 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, characterized in that: 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 acyl chloride is 3. The method for preparing a dual-channel fluorescent probe according to claim 2, characterized in that: The organic solvent includes one or more of dichloromethane, ethanol, acetonitrile, tetrahydrofuran and N,N-dimethylformamide.

4. A method for preparing a dual-channel fluorescent probe according to claim 2 or 3, characterized in that: 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, characterized in that: 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 a dual-channel fluorescent probe according to claim 1 in preparing a HClO detection product.

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