Indole quinolyl fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, particularly discloses an indolyl quinolyl fluorescent probe as well as a preparation method and application thereof, and synthesizes a novel closed indolyl quinolyl fluorescent probe for hydrazine detection. The probe has the excellent characteristics of quick response, excellent selectivity, high sensitivity, low cytotoxicity and the like, and can realize quick imaging in in-vitro experiments, so that the probe becomes an ideal tool for monitoring in-vivo and in-vitro hydrazine concentration.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to an indolequinoline-based fluorescent probe, its preparation method, and its application. Background Technology

[0002] Hydrazine (hydrazine) is a simple molecule, first synthesized in 1875. Although hydrazine plays a vital role in our society's development, its pollution, including leaks during energy production and industrial use, causes serious damage to ecosystems and impacts human health, raising deep concerns from a sustainable development perspective. Many countries have established permissible thresholds for hydrazine in water. Therefore, developing novel qualitative and quantitative detection methods for hydrazine is imperative.

[0003] Compared to traditional analytical methods, fluorescence detection has garnered significant attention in the analytical community due to its advantages in hydrazine detection, including ultra-high sensitivity, excellent selectivity, and ease of operation. Since the advent of the first fluorescent probe for hydrazine detection in 2011, scientists have made substantial progress in this field through continuous research. Most fluorescent probes are designed based on the strong nucleophilicity of hydrazine, a property that allows them to react with multiple recognition sites, thereby "turning on" or "turning off" the fluorescence signal. Among the many hydrazine detection recognition sites, cyanovinyl, as a strong electron-withdrawing group, reacts with hydrazine to form a Schiff base, becoming one of the key recognition sites.

[0004] In recent years, various fluorescent dyes containing cyanoethylene, including phenothiazines, coumarins, anthrones, carbazoles, naphthalenes, and nopidones, have been widely used to construct specific fluorescent probes for the accurate detection of hydrazine.

[0005] There is limited research on the application of hydrazine detection in tumor imaging. If hydrazine is absorbed by tumor tissue or accumulates in large quantities in cancer patients through drug metabolism, a significant decrease in its concentration will synergistically enhance hypoxia, an inherent characteristic of solid tumors. This may increase tumor drug resistance, promote tumor angiogenesis, and make tumors more aggressive.

[0006] Although significant progress has been made in hydrazine detection research, its application in biomedical imaging, particularly in tumor diagnosis, is relatively limited yet of great value. Therefore, developing novel fluorescent probes capable of real-time in vitro and in vivo detection is crucial for a deeper understanding of the biological effects of hydrazine and related disease mechanisms.

[0007] In summary, the development of a near-infrared fluorescent probe with rapid response, high selectivity, high sensitivity, and good biocompatibility for the specific detection and imaging of hydrazine in the environment and in organisms remains a key scientific problem that urgently needs to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide an indolequinoline-based fluorescent probe, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] An indolequinoline-based fluorescent probe for detecting hydrazine has the following specific structural formula;

[0011] ;

[0012] Indolequinoline-based fluorescent probes, specifically 2-(4-(6-methyl-6H-indole[2,3-b]quinoline-9-yl)benzyl)malonitrile, are used in the preparation of hydrazine detection reagents and in in vitro and in vivo hydrazine imaging.

[0013] The fluorescent probe was reacted with hydrazine or a hydrazine-containing substance, and the change in fluorescence emission intensity at 615 nm was measured at an excitation wavelength of 413 nm.

[0014] A method for preparing an indolequinoline-based fluorescent probe, comprising the following steps;

[0015] S1. Synthesize 2-((5-bromo-1-methyl-1H-indol-3-yl)methyl)aniline;

[0016] S2. Synthesize 9-bromo-6-methyl-6H-indole[2,3-b]quinoline;

[0017] S3. Synthesize 4-(6-methyl-6H-indol[2,3-b]quinolin-9-yl)benzaldehyde;

[0018] S4. Synthesize the target probe compound;

[0019] The chemical formula is as follows;

[0020]

[0021] Preferably, the synthesis of 2-((5-bromo-1-methyl-1H-indole-3-yl)methyl)aniline in S1 is carried out by adding trifluoroacetic acid (2.37 mmol, 0.27 g) dropwise to a solution of 1,2-dichloroethane (40 mL) containing 2-aminophenylmethanol (9.5 mmol, 1.17 g) and 5-bromo-1-methyl-1H-indole (7.9 mmol, 1.65 g) under nitrogen protection. The mixture is then placed in an oil bath and heated at 50°C with stirring for 12 hours. When the reaction is completed as monitored by thin-layer chromatography, a saturated sodium carbonate aqueous solution is added, and the mixture is extracted three times with dichloromethane. The organic layers are combined, dried with anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture is purified by silica gel column chromatography to obtain the product.

[0022] Preferably, step S2 involves slowly adding iodophenyl diacetic acid (6.0 mmol, 1.93 g) to a solution of hexafluoroisopropanol (40 mL) containing the compound (5.0 mmol, 1.57 g) prepared in S1 at room temperature. The mixture is then stirred for 2 hours. When the reaction is complete as monitored by thin-layer chromatography, 20 mL of water is added, and the mixture is extracted with ethyl acetate. The organic layers are combined, dried with anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture is purified by silica gel column chromatography to obtain 9-bromo-6-methyl-6H-indole[2,3-b]quinoline.

[0023] Preferably, step S3 involves dissolving 9-bromo-6-methyl-6H-indole[2,3-b]quinoline (1 mmol, 0.31 g), 4-formylphenylboronic acid (1.2 mmol, 0.18 g), potassium carbonate (3 mmol, 0.41 g), and tetra(triphenylphosphine)palladium (10% mol, 0.116 g) in tetrahydrofuran (10 ml) under nitrogen protection. The mixture is heated to 55°C and stirred for 12 hours. After the reaction is completed as monitored by thin-layer chromatography, the solvent is removed under reduced pressure. The product is purified by silica gel column chromatography to obtain 4-(6-methyl-6H-indole[2,3-b]quinoline-9-yl)benzaldehyde.

[0024] Preferably, step S4 involves adding 0.5 mmol (0.168 g) of 4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzaldehyde and 1.0 mmol (0.066 g) of malononitrile to a 50 mL three-necked round-bottom flask. After evacuation, the flask is purged three times with nitrogen. Then, piperidine (1 mmol, 0.085 g), glacial acetic acid (1.5 mmol, 0.090 g), and ethanol (10 mL) are added to the flask. The mixture is refluxed overnight at 85 °C under nitrogen protection. After cooling to room temperature, the probe 2-(4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzyl)malononitrile is collected and washed three times with petroleum ether.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The indolequinoline-based fluorescent probe provided by this invention exhibits extremely high sensitivity, with its fluorescence intensity showing a good linear relationship with hydrazine concentration in the range of 0-12 μM, and a detection limit (LOD) as low as 0.68 μM. This excellent sensitivity enables it to meet the detection requirements for trace amounts of hydrazine in environmental samples and biological organisms.

[0027] Meanwhile, probe 1 exhibits high specificity for the recognition of hydrazine. Experiments show that various common anions and cations (such as Cl⁻, Br⁻, Na⁺, K⁺, Zn²⁺, Cu²⁺, etc.) and biothiols and amino acids (such as Cys, Gly, Leu, etc.) do not significantly interfere with the fluorescence signal at 615 nm, ensuring accuracy and reliability when used in complex systems.

[0028] Meanwhile, the reaction kinetics between the probe and hydrazine are rapid, and the fluorescence quenching reaction can reach equilibrium within 2 minutes, enabling rapid real-time detection of hydrazine.

[0029] MTT cytotoxicity assays showed that the probe did not exhibit significant toxicity to normal breast cells (MCF-10A) or breast cancer cells (MCF-7) at the experimental concentrations, and the cell survival rate remained above 77%, demonstrating its good biocompatibility and laying the foundation for its application in vivo.

[0030] This probe was successfully applied to hydrazine imaging in a live tumor model. After intravenous injection, the probe rapidly accumulates at the tumor site and exhibits a sensitive "off" response to intratumorally injected hydrazine, with significantly suppressed fluorescence signal for a long duration (over 24 hours) and a high signal-to-background ratio (NTTR). This demonstrates the potential of this type of probe for visualizing and monitoring hydrazine at the in vivo level, providing a powerful tool for studying the role of hydrazine in physiological and pathological processes.

[0031] High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) clearly confirmed that the reaction mechanism between the probe and hydrazine is based on a nucleophilic addition-elimination reaction of cyanoethylene, ultimately forming a hydrazone compound. This disrupts the intramolecular charge transfer (ICT) process, leading to fluorescence quenching. This well-defined mechanism provides a solid theoretical basis for the rational design and performance optimization of the probe.

[0032] In summary, the fluorescent probe provided by this invention integrates high sensitivity, high selectivity, rapid response, and good biological applicability, and has broad application prospects. Attached Figure Description

[0033] Figure 1 This is a spectral analysis detection diagram of probe 1 after the addition of hydrazine in an embodiment of the present invention;

[0034] Figure 2 This is a comparison of the fluorescence intensity of probe 1 of the present invention at 615 nm with that of different analytical hydrazines;

[0035] Figure 3 This embodiment of the invention shows the linear relationship between the fluorescence intensity of probe 1 at 615 nm and the equivalent amount of hydrazine (N2H4) when incubated at 25°C for 2 minutes.

[0036] Figure 4 This is a high-resolution mass spectrum of probe 1 in an embodiment of the present invention;

[0037] Figure 5 This is a high-resolution mass spectrum of probe 1 after reacting with hydrazine (N2H4) in an embodiment of the present invention;

[0038] Figure 6 This is the hydrogen nuclear magnetic resonance (¹H NMR) spectrum of probe 1 after reacting with hydrazine (N₂H₄) in an embodiment of the present invention;

[0039] Figure 7 This is the carbon-13 nuclear magnetic resonance (¹³CNMR) spectrum of probe 1 after reacting with hydrazine (N2H4) in an embodiment of the present invention.

[0040] Figure 8 This is a diagram illustrating the sensing mechanism of probe 1 for hydrazine according to Scheme 2 of this invention.

[0041] Figure 9 This is a compatibility evaluation diagram in MCF-10 and MCF-7 cells according to an embodiment of the present invention;

[0042] Figure 10 This is a region of interest (ROI) analysis diagram of the signal-to-background ratio according to an embodiment of the present invention;

[0043] Figure 11 This is a schematic diagram illustrating the real-time recording of fluorescence images of each nude mouse using an IVIS imaging system, as an embodiment of the present invention. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1:

[0046] Please see Figures 1 to 11 As shown

[0047] A method for preparing an indolequinoline-based fluorescent probe includes the following steps;

[0048] Step 1: Synthesis of 2-((5-bromo-1-methyl-1H-indol-3-yl)methyl)aniline

[0049] Under nitrogen protection, trifluoroacetic acid (2.37 mmol, 0.27 g) was added dropwise to a solution of 1,2-dichloroethane (40 mL) containing (2-aminophenyl)methanol (9.5 mmol, 1.17 g) and 5-bromo-1-methyl-1H-indole (7.9 mmol, 1.65 g). The mixture was then heated in an oil bath at 50°C with stirring for 12 hours. When the reaction was complete as monitored by thin-layer chromatography, a saturated aqueous sodium carbonate solution was added, and the mixture was extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by silica gel column chromatography to give the product (1.488 g, 60% yield). 1H NMR (400MHz, CDCl3) δ7.72 (d, J = 1.8 Hz, 1H), 7.32 (dd, J = 8.7 Hz, 1H), 7.17-7.2 (m, 3H), 6.81-6.68 (m, 1H), 6.72 (d, J = 7.7 Hz, 1H), 6.70 (s, 1H), 3.94 (s, 2H), 3.68 (s, 3H).

[0050] Step 2; Synthesis of 9-bromo-6-methyl-6H-indole[2,3-b]quinoline

[0051] At room temperature, iodophenyl diacetic acid (6.0 mmol, 1.93 g) was slowly added to a solution of hexafluoroisopropanol (40 mL) containing the product from step one (5.0 mmol, 1.57 g), and the mixture was stirred for 2 hours. When the reaction was complete as monitored by thin-layer chromatography, 20 mL of water was added, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by silica gel column chromatography to give 9-bromo-6-methyl-6H-indole[2,3-b]quinoline (1.038 g, 67% yield). 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H), 8.27 (d, J = 2.0 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.0, 1H), 7.72-7.76 (m, 1H), 7.68 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.46-7.50 (m, 1H), 7.31 (d, J = 8.8 Hz, 1H), 3.98 (s, 3H).

[0052] Step 3; Synthesis of 4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzaldehyde

[0053] Under nitrogen protection, the product from step two (1 mmol, 0.31 g), 4-formylphenylboronic acid (1.2 mmol, 0.18 g), potassium carbonate (3 mmol, 0.41 g), and tetra(triphenylphosphine)palladium (10% mol, 0.116 g) were dissolved in tetrahydrofuran (10 mL). The mixture was heated to 55 °C and stirred for 12 hours. After the reaction was completed as monitored by thin-layer chromatography, the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography to give 4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzaldehyde (0.2386 g, 71% yield). 1H NMR (400 MHz, CDCl3) δ 10.07 (s, 1H), 8.72 (s, 1H), 8.36 (d, J = 1.6 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.97-8.00 (m, 3H), 7.84-7.86 (m, 2H), 7.71-7.77 (m, 2H), 7.43-7.49 (m, 2H), 3.98 (s, 3H).

[0054] Step 4; Synthesis of 2-(4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzylmethyl)malononitrile

[0055] 4-(6-methyl-6H-indol[2,3-b]quinoline-9-yl)benzaldehyde (0.5 mmol, 0.168 g) and malononitrile (1.0 mmol, 0.066 g) were added to a 50 mL three-necked round-bottom flask. The flask was evacuated and then purged three times with nitrogen. Piperidine (1 mmol, 0.085 g), glacial acetic acid (1.5 mmol, 0.090 g), and ethanol (10 mL) were then added to the flask. The mixture was refluxed overnight at 85 °C under nitrogen protection. After cooling to room temperature, probe 1 was collected and washed three times with petroleum ether, with a yield of 68% (130.6 mg). 1H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.44 (d, J = 1.6 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.03-8.05 (m, 3H), 7.88-7.90 (m, 3H), 7.73-7.81 (m, 2H), 7.47-7.55 (m, 2H), 4.05 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 159.32, 153.23, 147.93, 147.20, 143.51, 131.77, 130.96, 129.49, 129.38, 128.79, 128.06,127.90 ,127.79,127.49,124.40,123.54,121.44,120.37,118.02,114.24,113.16,109.56,81.45,28.11. HRMS (ESI) m / z, calcdfor C26H16N4, ([M + H]+): 385.1448, found 385.1816.

[0056] The experimental steps of this invention are as follows;

[0057] Sensitivity measurement

[0058] To investigate the optical response of probe 1 to hydrazine detection, UV-Vis absorption and fluorescence spectroscopy were first performed in DMSO solution. Probe 1 showed strong absorption at 410 nm. Figure 1 A). When hydrazine is added, the UV-Vis absorption at 410 nm decreases. Notably, the color change from yellow to colorless is very pronounced. In the fluorescence spectrum, probe 1 exhibits strong red fluorescence emission at 615 nm (λex = 413 nm, Slit: 5 nm / 5 nm). Figure 1B). The larger Stokes shift is attributed to intramolecular charge transfer processes. With the addition of hydrazine, the fluorescence intensity peak at 615 nm gradually weakens. When hydrazine is added to 15 μM, this peak disappears, and other substances do not cause significant changes in the fluorescence intensity peak at 615 nm. Figure 1 C).

[0059] Furthermore, within the 0-12 μM range, the fluorescence intensity at 615 nm showed a good linear relationship with the hydrazine concentration. For example... Figure 3 As shown, the ratio of fluorescence intensity to hydrazine concentration and fluorescent probe intensity exhibits a good linear relationship (R² = 0.995, standard curve equation Y = -3966.44x + 5078.73). Theoretical calculations (LOD = 3δ / s, where δ is the standard deviation of probe fluorescence intensity and s is the slope of the equation) yielded a detection limit of 0.68 μM for probe 1. This low detection limit meets the requirements for hydrazine concentration detection in both environmental and biological environments. Subsequently, the fluorescence characteristics of probe 1 within 0 to 3 minutes after reacting with hydrazine were investigated.

[0060] like Figure 1 As shown in Figure D, the fluorescence intensity of probe 1 at 615 nm is negatively correlated with the reaction time of hydrazine. Upon addition of hydrazine, the fluorescence intensity of probe 1 at 615 nm decreases sharply. The fluorescence intensity of probe 1 at 615 nm decreases sharply 2 minutes after the addition of hydrazine. When the reaction time is extended to 3 minutes, no significant change in fluorescence intensity is observed. These results indicate that probe 1 can be used for the rapid detection of hydrazine.

[0061] Figure 1 Absorption spectrum (A) and fluorescence spectrum (B) of probe 1 (10 μM) before and after the addition of hydrazine (10 equivalents); (C) Fluorescence spectrum of probe 1 (10 μM) under different equivalents of hydrazine; (D) Fluorescence quenching response curve of probe 1 (10 μM) at 615 nm wavelength (λex = 413 nm) over time, corresponding to the detection results after the addition of 1.5 equivalents of hydrazine.

[0062] Select an experiment;

[0063] Fluorescence spectroscopy was used to evaluate the selectivity of probe 1 for hydrazine, which coexists with other substances. Adding 1.5 equivalents of hydrazine to probe 1, along with different substances (10 equivalents) including acetic anhydride−, chlorine−, bromine−, iodine−, phosphate−, sodium+, barium+, calcium+, potassium+, zinc+, manganese+, copper+, iron+, lead+, sulfuric acid−, phenylalanine, lysine, proline, leucine, cysteine, glycine, isoleucine, and tyrosine, did not result in any detectable change in emission intensity at 615 nm. Figure 2 The results showed that probe 1 exhibited significant fluorescence quenching of hydrazine and was almost unaffected by other substances.

[0064] Figure 2 The fluorescence intensity of probe 1 (10 μM) at 615 nm was compared with that of different analytes (10 equivalents) or analytes (10 equivalents) and (1.5 equivalents) hydrazine. ax: none, AcO−, Cl−, Br−, I−, HPO4−, Na+, Ba2+, Ca2+, K+, Zn2+, Mn2+, Cu2+, Fe2+, Pb2+, HSO3−, Phe, Lys, Pro, Leu, Cys, Gly, Ile and Tyr.

[0065] Reaction mechanism study

[0066] To obtain information about this "shutdown" mechanism, high-resolution mass spectrometry (HRMS) analysis was performed on the reaction solution of probe 1 and hydrazine. HRMS showed that the molecular ion peak ([M+H]+) of probe 1 was 385.1816 at m / z (Figure). Figure 4 A new ion peak ([M+H]+) was observed at m / z 351.1619 in the mixture of hydrazine and probe 1. Figure 5 This peak is attributed to the hydrazone product (compound 7, Scheme 2). Furthermore, compound 7 is also detected by 1H NMR (…). Figure 6 and 13C NMR spectrum ( Figure 7 Successfully characterized. ¹H NMR (400 MHz, DMSO-d6) δ 9.16 (s, ¹H), 8.68 (d, J = 1.8 Hz, ¹H), 8.14 (d, J = 7.7 Hz, ¹H), 8.12 (d, J = 8.5 Hz, ¹H), 7.96 (dd, J = 8.5 Hz, 1.6 Hz, ¹H), 7.81–7.74 (m, 4H), 7.72 (d, J = 8.5 Hz, 1H), 7.63–7.61 (m, 2H), 7.53–7.50 (m, 1H), 6.83 (s, 2H), 3.96 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 152.9, 146.7, 142.4, 139.9, 138.5, 135.4, 132.4, 129.5, 129.3, 128.7, 127.6, 127.3, 127.1, 126.3, 124.3, 123.5, 120.8, 120.2, 118.2, 110.2, 28.2. Based on the above information and in conjunction with previous reports, we propose a “shutdown” mechanism for probe 1 in Scheme 2. First, electron-rich hydrazine attacks the malononitrile group of probe 1 to generate intermediate A. Subsequently, the residual malononitrile anion leaves to form intermediate B. Finally, the deprotonation of intermediate B generates a hydrazone group, referencing... Figure 8 .

[0067] In vitro biocompatibility

[0068] Before applying probe 1 to biomedical imaging, we first investigated its biocompatibility in MCF-10 and MCF-7 cells. Cell viability was assessed using the MTT assay, revealing that the probe showed biocompatibility with MCF-10 cells after 72 hours of culture at different concentrations. Figure 9 A) and MCF-7 ( Figure 9 B) No cells showed significant toxicity, and cell viability remained above 77%. This indicates that our probe is safe and non-toxic at the cellular level.

[0069] In vivo fluorescence imaging

[0070] Based on the excellent in vitro sensing performance of probe 1 for hydrazine, we further explored its imaging application in live animals. In the experiment, after intratumoral injection of hydrazine into tumor-bearing MCF-7 nude mice, probe 1 was intravenously injected 10 minutes later, and then fluorescence images of each nude mouse were recorded in real time using an IVIS imaging system. Figure 11 As shown, probe 1 responded rapidly to hydrazine stimulation, significantly inhibiting the fluorescence signal in tumor tissue 0.5 hours after injection, and remaining fluorescence-free for 24 hours. The calculated NTTR value of probe 1 reached 1.73 ± 0.45 at 0.5 hours after injection and remained at a high level for the following 48 hours (1.92 ± 0.52 at 12 hours and 1.89 ± 0.42 at 24 hours, see...). Figure 10 This indicates that the probe has the potential to detect hydrazine under physiological conditions.

[0071] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of an indoloquinoline-based fluorescent probe in the preparation of a hydrazine detection reagent, characterized in that, The fluorescent probe is 2-(4-(6-methyl-6H-indolo[2,3-b]quinolin-9-yl)benzylidene)malononitrile, and the specific structural formula is as follows: 。 2. Use according to claim 1, characterized in that: The fluorescent probe also includes an application in hydrazine imaging in vivo and in vitro.

3. The method for preparing the indolequinoline-based fluorescent probe according to any one of claims 1-2, characterized in that: The method comprises the following steps: S1, synthesizing 2-((5-bromo-1-methyl-1H-indol-3-yl)methyl) aniline; S2, synthesizing 9-bromo-6-methyl-6H-indolo[2,3-b]quinoline; S3, synthesizing 4-(6-methyl-6H-indolo[2,3-b]quinolin-9-yl)benzaldehyde; S4, synthesizing the target probe compound; The chemical formula is as follows: 。 4. The method for preparing the fluorescent probe according to claim 3, characterized in that: In the S1, the synthesis of 2-((5-bromo-1-methyl-1H-indol-3-yl)methyl) aniline is performed by adding trifluoroacetic acid dropwise into a 1,2-dichloroethane solution containing 2-aminophenylmethanol and 5-bromo-1-methyl-1H-indole under nitrogen protection, then the mixture is heated and stirred in an oil bath at 50°C for 12 hours, when the reaction is completed by monitoring thin layer chromatography, saturated aqueous sodium carbonate solution is added, and the mixture is extracted with dichloromethane three times, the combined organic layers are dried with anhydrous sodium sulfate, filtered and concentrated, and the obtained mixture is purified by silica gel column chromatography to obtain the product.

5. The method for preparing the fluorescent probe according to claim 3, characterized in that: In the S2, the step is to slowly add iodobenzenediacetic acid into a hexafluoroisopropanol solution containing the compound prepared in S1 at room temperature, then the mixture is stirred for 2 hours, when the reaction is completed by monitoring thin layer chromatography, 20 mL of water is added, and the mixture is extracted with ethyl acetate, the combined organic layers are dried with anhydrous sodium sulfate, filtered and concentrated, and the obtained mixture is purified by silica gel column chromatography to obtain 9-bromo-6-methyl-6H-indolo[2,3-b]quinoline.

6. The method for preparing the fluorescent probe according to claim 3, characterized in that: In the S3, the step is to dissolve the compound 9-bromo-6-methyl-6H-indolo[2,3-b]quinoline, 4-formylphenylboronic acid, potassium carbonate and tetrakis(triphenylphosphine)palladium in tetrahydrofuran under nitrogen protection, heat the mixture to 55°C and stir for 12 hours, when the reaction is completed by monitoring thin layer chromatography, the solvent is evaporated under reduced pressure, and the product is purified by silica gel column chromatography to obtain 4-(6-methyl-6H-indolo[2,3-b]quinolin-9-yl)benzaldehyde.

7. The method for preparing the fluorescent probe according to claim 3, characterized in that: In the S4, the step is to add 4-(6-methyl-6H-indolo[2,3-b]quinolin-9-yl)benzaldehyde and malononitrile into a 50 mL three-necked round-bottom flask, the flask is vacuumized and purged with nitrogen three times, then piperidine, glacial acetic acid and ethanol are injected into the flask, the mixture is refluxed at 85°C overnight under nitrogen protection, and the probe 2-(4-(6-methyl-6H-indolo[2,3-b]quinolin-9-yl)benzylidene)malononitrile is collected after cooling to room temperature and washed with petroleum ether three times.

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