Method for fluorescence detection of viscosity and hydrogen peroxide based on intramolecular charge transfer mechanism probe

Abstract

The invention discloses a method for simultaneously detecting viscosity and hydrogen peroxide (H2O2) by using a molecular fluorescent probe based on an intramolecular charge transfer (ICT) mechanism. The method comprises the following steps: firstly carrying out hydroformylation on strong electron donor triphenylamine, then introducing a boronic acid pinacol ester group into a strong electron acceptor quinoline structure as a response site of H2O2, and then connecting the donor and the acceptor through freely rotatable carbon-carbon double bonds through aldol condensation reaction to form the molecular probe with a stable ICT structure. H2O2 is subjected to specific oxidation, boronic acid pinacol ester groups are eliminated, intramolecular charge transfer is reduced, emission wavelength blue shift is caused, fluorescence is enhanced, and therefore H2O2 detection is achieved; free rotation of double bonds can be inhibited by increasing the viscosity of the system, the radiative transition number of excited state electrons is increased, red color is enhanced, and therefore the viscosity level is detected. The fluorescent probe has the advantages of high speed, strong specificity and high sensitivity in response to viscosity and H2O2, and rapid qualitative and quantitative analysis on viscosity and H2O2 can be realized by utilizing fluorescence change.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for simultaneously detecting viscosity and hydrogen peroxide based on intramolecular charge transfer mechanism of molecular fluorescence probe, characterized by involving a high sensitivity and specificity fluorescence probe suitable for simultaneous fluorescence detection of viscosity and hydrogen peroxide. BACKGROUND

[0002] Mitochondrial dysfunction has become a key element in the pathogenesis of neurodegenerative diseases. As one of the key parameters for evaluating mitochondrial function, mitochondrial matrix viscosity regulates the interaction and transport of biological macromolecules and chemical signals in living cells, which has an impact on biological functions. Abnormal viscosity changes can lead to changes in mitochondrial network structure, and further affect the diffusion of metabolites. Another key parameter is the generation of reactive oxygen species (ROS) and oxidative stress signaling in mitochondria. ROS is a class of highly reactive oxygen-containing molecules with biological molecules, which plays an important role in many physiological and pathological processes. Hydrogen peroxide (H2O2), as the main component of ROS, is mainly produced in mitochondria, which is used to regulate cell proliferation, host defense, immune response and signaling pathways. Abnormal H2O2 levels can regulate mitochondrial fission and protein aggregation, trigger ultrastructural changes such as reversible swelling of mitochondria and disruption of cristae structure, and further lead to mitochondrial dysfunction and various serious diseases, including cancer, diabetes and neurodegenerative diseases. In order to further analyze the key role of viscosity and H2O2 in mitochondrial dysfunction and the physiological effects of abnormal changes, therefore, it is urgent to develop a new method for rapid, real-time and high-sensitivity simultaneous detection of viscosity and H2O2.

[0003] Traditional methods for detecting viscosity include capillary method and falling ball method, and traditional methods for detecting H2O2 include colorimetric method, gas chromatography and high performance liquid chromatography. These methods have been proven to be practical, but they have the disadvantages of low sensitivity, complex operation, the need for expensive detection equipment and professional personnel, and the inability to achieve real-time non-invasive in vivo detection. In recent years, fluorescence detection method may be suitable for dynamic monitoring of biomolecules and microenvironment parameters in living systems due to its high sensitivity, high selectivity, non-invasive detection and real-time analysis. At present, a variety of excellent fluorescent probes for single biological imaging viscosity and H2O2 have been developed. However, the single probe has the defect of being unable to realize the synchronous detection of viscosity and H2O2. If multiple fluorescent probes are used in the same system, signal crosstalk and other problems will usually lead to detection failure. Therefore, by designing a dual-response fluorescent probe with different imaging channels, the simultaneous detection of viscosity and H2O2 can be realized. However, the dual-response fluorescent probes reported at present generally have a small Stokes shift and a short wavelength excitation / emission, which are easily interfered by the biological autofluorescence in the living system, thus limiting the development of the probes in biological imaging applications. The present patent connects the electron donor and acceptor through a freely rotatable carbon-carbon double bond by means of hydroxy aldehyde condensation reaction, forms a D-π-A type fluorescent probe (TQB) with intramolecular charge transfer (ICT) mechanism, and realizes the high selectivity and rapid detection of target substances by using specific molecular rotor mechanism and specific recognition of H2O2. In the application of biological imaging, the probe TQB realizes the synchronous fluorescence imaging of viscosity and H2O2 in zebrafish in vivo. SUMMARY

[0004] The present application aims at the deficiencies of high operation cost, low sensitivity and the inability to realize in-situ real-time detection of the existing viscosity and H2O2 detection methods, and a novel fluorescent probe with simple preparation method is designed and synthesized, which can distinguish the viscosity and H2O2 conditions in the imaging life system by using different colors of fluorescence signals, and provides a fluorescence imaging method with the advantages of rapidness, high sensitivity and selectivity.

[0005] The present application provides a method for simultaneously detecting viscosity and H2O2 by using a ICT mechanism-based molecular fluorescent probe. The method first performs aldehyde group on strong electron donor triphenylamine, then introduces boronic acid pinacol group as a response site of H2O2 in a strong electron acceptor quinoline structure, and then connects the donor and acceptor through a freely rotatable carbon-carbon double bond through the aldol condensation reaction, to form a molecular probe with stable ICT structure. When H2O2 exists, it will be specifically oxidized and eliminated to reduce the degree of intramolecular charge transfer, resulting in blue shift of emission wavelength and enhanced fluorescence, thereby realizing the detection of H2O2; the increase of the viscosity of the system will inhibit the free rotation of the double bond, enhance the number of excited state electron radiation transition, and cause the red fluorescence to enhance, thereby realizing the detection of the viscosity level. The probe has fast response speed, strong specificity and high sensitivity to viscosity and H2O2, and the rapid qualitative or quantitative analysis of viscosity and H2O2 can be realized by using fluorescence change. The chemical structural formula of the fluorescent probe TQB is as follows:

[0006]

[0007] The synthesis method of the fluorescent probe TQB comprises the following steps:

[0008] 1) Synthesis of 4-diphenylaminobenzaldehyde (compound 1): dissolve an appropriate amount of triphenylamine in N,N-dimethylformamide, add phosphorus oxychloride at 0°C, and stir at 90°C for 3 hours. After the reaction is cooled to room temperature, the light white powder compound 1 is obtained after purification treatment.

[0009] 2) Synthesis of 4-methyl-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl] quinolin-1-ium (compound 2): dissolve an appropriate amount of 4-methylquinoline and 4-(bromomethyl)phenylboronic acid pinacol in toluene, and reflux the mixed system at 110°C for 12 hours. After the reaction is completed, the mixed system is cooled to room temperature, and the gray powder compound 2 is obtained after purification treatment.

[0010] 3) Synthesis of fluorescent probe (E)-4-[4-(diphenylamino)styryl]-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl] quinolin-1-ium (TQB): dissolve an appropriate amount of potassium tert-butoxide and compound 2 in anhydrous DMF, and stir at room temperature for 0.5 hours under nitrogen protection. Then, compound 1 is added, and the reaction is stirred at room temperature for 12 hours under nitrogen protection. After the reaction is completed, the product is treated by quenching, extraction and drying. Then, the compound TQB in the form of yellow powder is obtained after the product is purified by silica gel column chromatography, and is stored at-20°C.

[0011] The fluorescent probe TQB described in the application can be applied in the simultaneous fluorescence detection of viscosity and H2O2, and the specific application method is as follows: without special instructions, the probe TQB is usually dissolved in a dimethyl sulfoxide phosphate buffer solution (PBS) (pH = 7.4, volume ratio 1:9) as a test solution for analysis and detection at room temperature.

[0012] The specific characteristics of the fluorescent probe TQB for simultaneously detecting viscosity and H2O2 in the application are as follows: after the probe molecule is dissolved in the test solution, it is added to a system with different viscosity levels. With the increase of the viscosity level, the probe shows red fluorescence enhancement at 683 nm wavelength under 540 nm excitation wavelength, and the fluorescence intensity is enhanced by 42 times. After H2O2 is added to the test solution of the probe TQB, the probe shows obvious yellow fluorescence enhancement at 597 nm wavelength under 405 nm excitation wavelength, and the fluorescence intensity is enhanced by 33 times, the Stokes shift is 192 nm, and the detection limit is 219 nM. Therefore, the detection of different excitation and emission signals for specific analysis targets is realized. The fluorescent probe TQB can simultaneously distinguish and detect viscosity and H2O2 under the same detection conditions, and has no obvious fluorescence response to other interfering substances such as metal ions, halogen ions and other bioactive species. In addition, the probe can still maintain good fluorescence stability and selectivity to target analytes in the physiological pH range, which lays a foundation for further biological imaging application of the probe. Therefore, the detection method of the fluorescent probe disclosed in the application has high sensitivity and strong specificity, does not need professional operators and large instruments, and can be used for the qualitative and semi-quantitative analysis of viscosity and H2O2 by naked eye, and can be used for the rapid detection of viscosity and H2O2 in the field environment and in vivo BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The nuclear magnetic resonance spectrum and high resolution mass spectrum of the fluorescent probe TQB in the application.

[0014] Figure 2 The response mechanism of the fluorescent probe TQB in the application to viscosity and H2O2.

[0015] Figure 3 The ultraviolet absorption and fluorescence emission spectrum of the fluorescent probe TQB in the application for detecting viscosity and H2O2.

[0016] Figure 4 The time dependence and selectivity results of the fluorescent response of the fluorescent probe TQB in the application to viscosity and H2O2.

[0017] Figure 5 It is the test of cytotoxicity of the fluorescent probe TQB with different concentrations in the application.

[0018] Figure 6Fig. 1 is an image of the application of the fluorescent probe TQB to viscosity and H2O2 fluorescence imaging in zebrafish in vivo. DETAILED DESCRIPTION

[0019] The application is further described in conjunction with the accompanying drawings.

[0020] Example 1: The synthesis route of the probe TQB described in the application is shown in the following figure:

[0021]

[0022] (1) Synthesis of compound 1

[0023] Triphenylamine (5 g, 20.5 mmol) was dissolved in N,N-dimethylformamide (DMF, 30 mL) at 0°C. Phosphorus oxychloride (POCl3, 7.5 mL, 81.5 mmol) was slowly added to the system dropwise under ice bath. The reaction solution was continuously stirred at 0°C for 30 minutes, and then transferred to room temperature to stir until the solution showed a red-brown color. After the color changed, the reaction system was warmed to 90°C and stirred for 3 hours. The reaction solution was slowly poured into a beaker containing ice water, while stirring the ice water mixture (note: careful and slow operation is required to prevent overflow). After neutralization to neutral with sodium bicarbonate solution (0.5 M), extraction was performed using ethyl acetate. Then the organic solvent was removed by evaporation under reduced pressure, and the residue was washed with deionized water and filtered to obtain 5.5 g of light white powder, which was compound 1, with a yield of 89.9%. 1 H NMR (600 MHz, DMSO-d6) δ 9.77 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 7.7 Hz, 4H), 7.22 (dd, J = 24.3, 7.6 Hz, 6H), 6.89 (d, J = 8.5 Hz, 2H).

[0024] (2) Synthesis of compound 2

[0025] 4-Methylquinoline (0.3 g, 2.1 mmol) and 4-(bromomethyl)phenylboronic acid pinacol ester (0.9 g, 3.2 mmol) were dissolved in 15 mL of toluene under nitrogen protection, and the mixture was refluxed at 110°C for 12 hours. After the reaction was completed, the generated gray powder-like solid was collected by suction filtration, washed with toluene, and vacuum dried to obtain compound 2 (0.6 g, yield 65.1%). 1H NMR (600 MHz, DMSO-d6) δ 9.61 (d, J = 5.8 Hz, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.21 - 8.13 (m, 2H), 8.01 (t, J = 7.7 Hz, 1H), 7.66 (d, J = 7.6 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 6.35 (s, 2H), 3.05 (s, 3H), 1.26 (s, 12H). HR-MS (m / z): C 23 H 27 BNO2 + , calculated [M] + : 360.2129, found: 360.2129.

[0026] (3) Synthesis of probe TQB

[0027] Potassium tert-butoxide (0.14 g, 1.2 mmol) and compound 2 (0.36 g, 1.0 mmol) were dissolved in 15 mL of anhydrous DMF, and the mixture was stirred at room temperature for 0.5 h under nitrogen protection. Then compound 1 (0.27 g, 1.0 mmol) was added, and the mixture was stirred at room temperature for 12 h under nitrogen protection after ultrasonic degassing. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, 30 mL of brine was added to quench the reaction, and the compound was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and then removed by a rotary evaporator. Then, the product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 3:1) as the eluent to obtain compound TQB (0.30 g) in the form of yellow powder with a yield of 48.5%, which was stored at -20 °C. 1 H NMR (600 MHz, DMSO-d6) δ 9.54 (s, 2H), 8.57 (s, 2H), 8.10 (d, J = 23.7 Hz, 3H), 7.91 (s, 2H), 7.78 (d, J = 7.7 Hz, 2H), 7.54 - 7.06 (m, 10H), 6.97 (d, J = 8.5 Hz, 3H), 6.51 (s, 2H), 6.25 (s, 2H), 1.25 (d, J = 16.1 Hz, 12H). HR-MS (m / z): C 42 H 40 BN2O2 + , calculated [M] + : 615.3171, found: 615.3177.

[0028] Example 2: Mechanism analysis and theoretical calculation of the viscosity and H2O2 response of the probe TQB according to the present application Figure 2). The viscosity increase of the system inhibits the rotation of the intramolecular double bond, enhances the number of radiative transitions of the excited state electron, and enhances the red fluorescence. H2O2 can specifically oxidize and eliminate the pinacol borate group, reduce the degree of intramolecular charge transfer, cause the emission wavelength to blue shift, and enhance the fluorescence. In the results of density functional theory calculation, the energy gap and the oscillator strength of the probe molecule after reaction with H2O2 are increased, verifying the blue shift of the emission wavelength of the probe and the increase of the fluorescence intensity.

[0029] Example 3: Application of the probe TQB described in the present application for detecting viscosity and H2O2 in vitro environment Figure 3 First, different volumes of PBS solution were added to the glycerol solution to dilute the solution to obtain a viscosity level in the range of 5.1 cp-1410 cp, and the probe test solution with a concentration of 10 μM was prepared using the solution. The samples were tested by ultraviolet spectrophotometer and fluorescence spectrometer. The results show that with the increase of the viscosity of the system, the ultraviolet absorption intensity and the fluorescence emission intensity of the probe are increased (excitation wavelength: 540 nm, emission wavelength: 683 nm). In the study of H2O2 detection, different volumes of H2O2 were added to the probe test solution (10% DMSO in PBS solution) with a concentration of 10 μM to prepare H2O2 samples to be tested with a concentration range of 0-70 μM. The results of ultraviolet absorption and fluorescence emission spectra show that with the increase of the concentration of H2O2, the fluorescence of the probe at 597 nm wavelength is significantly enhanced (excitation wavelength: 405 nm), and the detection limit is 219 nM. Therefore, the fluorescent probe TQB disclosed in the present application can realize high-sensitivity detection of both.

[0030] Example 4: Time-dependent spectrum and fluorescence stability of the probe TQB described in the present application Figure 4Probe test solutions with a concentration of 10 μM were prepared using glycerol and PBS, respectively. Fluorescence emission results showed that the probe fluorescence intensity peaked within 8 minutes under high viscosity conditions and remained stable for a long time. Similarly, the H2O2 response process based on the chemical reaction mechanism of the probe was completed within 20 minutes and maintained excellent fluorescence stability thereafter. Then, the pH stability of the probe fluorescence response was studied by preparing test solutions with different pH values. In the viscosity fluorescence detection study, the probe maintained high stability over a wide pH range of 3-10. Furthermore, in the H2O2 fluorescence detection application, the probe maintained good fluorescence stability under neutral and weakly alkaline conditions. Different organs and tissues in living systems have different pH values, and the multi-pH adaptability of probes lays the foundation for their biofluorescence imaging applications. In addition, living systems have complex compositions and numerous interfering substances; therefore, probes need to possess excellent fluorescence stability and selectivity in the presence of many interfering substances. Interfering substances, such as cations, halide ions, and other bioactive species (concentration 100 μM), were added to the aforementioned probe test solutions, and then the probe solutions were subjected to spectral analysis. The results show that the probe maintains a good response to the fluorescence detection of viscosity and H2O2, exhibiting good fluorescence stability. Furthermore, when the target analyte is absent or at a low level, the probe's fluorescence intensity is also low, demonstrating good selectivity for the detection of viscosity and H2O2. Therefore, the fluorescent probe TQB disclosed in this invention exhibits good stability and selectivity in the detection of viscosity and H2O2.

[0031] Example 5: Cytotoxicity test of the probe TQB described in this invention. A prerequisite for the application of fluorescent probes in biological imaging is that they must not have significant toxic side effects on organisms. Therefore, in this example, the cytotoxicity of the probe TQB was investigated using the MTT assay. HeLa cells were passaged into sterilized plates and cultured in an incubator for 12 hours. Afterward, the culture medium was removed. A cell culture medium without probe solution served as the blank control group, while cell culture media supplemented with 1 μM, 2 μM, 3 μM, 5 μM, 10 μM, and 20 μM probe solution served as the experimental groups. Cell culture was continued. Cell viability was then calculated using the MTT assay. Figure 5 As shown, after culturing cells in a medium containing 10 μM probe solution for 24 hours, the cell survival rate remained above 80%. This result verifies that low-dose probe TQB has minimal toxicity to cells and has the potential for application in biological imaging.

[0032] Example 5: Application of the probe TQB described in this invention to fluorescence imaging of viscosity and endogenous and exogenous H2O2 in live zebrafish (Figure 6). Zebrafish, as the second largest vertebrate model, has a genome similarity of up to 87% with humans. In addition, early zebrafish embryos are transparent, develop rapidly, and are easy to study using biofluorescence imaging. The specific operation steps are as follows: Five-day-old zebrafish were incubated with culture media containing H2O2, phorbol ester (PMA, which stimulates the body to produce endogenous H2O2), and nystatin (Nys, which acts as an ion carrier to induce an increase in body viscosity). Then, a zebrafish culture medium containing 10 μM probe solution was prepared, and the zebrafish were incubated at 30°C. After washing off the culture medium, fluorescence imaging was performed using a fluorescence microscope (excitation wavelengths of 385 nm and 525 nm, respectively), and fluorescence signals were collected from the green and red channels, respectively. A weak green fluorescence signal was detected in the zebrafish that had only been incubated with the probe, which may be due to background fluorescence and trace amounts of reactive oxygen species in the zebrafish. In the presence of both exogenous and endogenous H2O2, imaging results showed significant fluorescence enhancement in the green channel of zebrafish. A disease model established under Nys stimulation increased the viscosity of the zebrafish, resulting in a corresponding significant enhancement of red fluorescence in the red channel. These results validate that the probe TQB of this invention is suitable for fluorescence imaging of in vivo zebrafish viscosity and H2O2.

[0033] The fluorescent probe proposed in this invention, based on an ICT mechanism, for the simultaneous fluorescence detection of viscosity and H2O2, achieves the simultaneous detection of viscosity and H2O2 by generating different fluorescent substances under the same conditions using a single probe, thereby emitting red and green fluorescence at a specific excitation wavelength. Furthermore, it successfully enables fluorescence imaging of viscosity and H2O2 in live zebrafish using independent channels. It is hoped that the content of this patent will provide insights for the design and development of future dual-response fluorescent probes.

Claims

1. A method for detecting viscosity and hydrogen peroxide using a molecular probe based on an intramolecular charge transfer mechanism, characterized in that, The chemical structural formula of the fluorescent probe is shown below, and its chemical molecular formula is: C 42 H 40 BN2O2 + .

2. The preparation of the fluorescent probe according to claim 1, characterized in that, The specific synthesis method of the fluorescent probe is as follows: (1) Synthesis of Compound 1 Triphenylamine (5 g, 20.5 mmol) was dissolved in N,N-dimethylformamide (DMF, 30 mL) at 0 °C. Phosphorus oxychloride (POCl3, 7.5 mL, 81.5 mmol) was slowly added dropwise to the system while maintaining an ice bath. The reaction mixture was stirred continuously at 0 °C for 30 minutes, then transferred to room temperature and stirred until the solution turned reddish-brown. After the color change, the reaction mixture was heated to 90 °C and stirred for 3 hours. The reaction mixture was slowly poured into a beaker containing ice water while stirring the ice-water mixture (Note: handle carefully and slowly to prevent overflow). After neutralization with sodium bicarbonate solution (0.5 M), extraction was performed using ethyl acetate. The organic solvent was then removed by evaporation under reduced pressure. The residue was washed with deionized water and filtered to give 5.5 g of pale white powder, which was compound 1, with a yield of 89.9%. (2) Synthesis of compound 2 4-Methylquinoline (0.3 g, 2.1 mmol) and pinacol ester of 4-(bromomethyl)phenylboronic acid (0.9 g, 3.2 mmol) were dissolved in 15 mL of toluene. The mixture was refluxed at 110 °C for 12 hours under nitrogen protection. After the reaction was completed, the resulting gray powdery solid was collected by filtration, washed with toluene, and dried under vacuum to obtain compound 2 (0.6 g, yield 65.1%). (3) Synthesis of probe TQB Potassium tert-butoxide (0.14 g, 1.2 mmol) and compound 2 (0.36 g, 1.0 mmol) were dissolved in 15 mL of anhydrous DMF. The mixture was stirred at room temperature for 0.5 h under nitrogen protection. Then, compound 1 (0.27 g, 1.0 mmol) was added, and after sonication to degas the mixture, it was stirred at room temperature for 12 h under nitrogen protection. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, 30 mL of brine was added to quench the reaction. The compound was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and removed by rotary evaporation. Subsequently, the product was purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixture (3:1 v / v) as the eluent to give a yellow powder, compound TQB (0.30 g), with a yield of 48.5%, which was stored at -20 °C.

3. The application of the probe TQB according to claim 1, characterized in that, The probe TQB enables quantitative analysis of viscosity and H2O2 levels in vitro, and dynamic monitoring of viscosity and H2O2 in vivo via fluorescence imaging.

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