Low-concentration ratio-type high-concentration enhanced fluorescent probe and its preparation method and application

Abstract

The present invention relates to the technical field of fluorescent probes. In order to solve the problems that traditional fluorescent probes rely on high-concentration usage conditions and often face in biological samples such as rapid probe consumption, increased biological toxicity, and non-specific binding to targets, a low-concentration ratio-type high-concentration enhanced fluorescent probe and its preparation method and application are proposed. The preparation method of the fluorescent probe comprises the following steps: 1: preparing phenothiazine dialdehyde; 2: mixing phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine, and refluxing the mixture to obtain a low-concentration ratio-type high-concentration enhanced fluorescent probe. The probe of the present invention realizes the quantitative detection of HClO through the synchronous change of two emission channels, significantly improving the sensitivity and reliability of the detection; at high concentrations, it exhibits an enhanced single-channel fluorescence amplification effect, which is helpful for strong signal recognition and macroscopic visual imaging in specific applications, thereby taking into account the dual needs of trace monitoring and high-throughput screening.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a low-concentration ratio-type high-concentration enhanced fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hypochlorous acid (HClO), as an endogenous reactive oxygen species (ROS), plays an important role in the human immune defense, inflammatory response, and the occurrence and development of various pathological processes such as atherosclerosis, rheumatoid arthritis, tumors, and neurodegenerative diseases.

[0003] Therefore, the development of analytical tools that can efficiently and sensitively detect HClO is of great significance for early diagnosis of diseases and research on pathological mechanisms. Fluorescent probes have become a powerful tool for detecting HClO due to their advantages such as non-invasiveness, high sensitivity, and real-time monitoring. Currently, a variety of fluorescent probes have been used for the detection of hypochlorous acid, such as probes based on organic dyes and metal nanoparticle probes. These probes have achieved certain results in terms of detection sensitivity and selectivity. However, these probes usually have problems such as long response time, poor selectivity, and susceptibility to interference from other oxidizing substances. In addition, the detection results of many probes do not have direct visualization capabilities, which limits their practicality in rapid detection and field applications.

[0004] Ratiometric fluorescent probes have attracted widespread attention in recent years because they can achieve intrinsic self-calibration, reduce background interference, and improve signal accuracy.

[0005] The patent, CN 117603203 A, entitled "A Ratiometric Fluorescent Probe, Its Preparation Method, and Application," describes the synthesis of a phenothiazine-based ratiometric fluorescent probe for the specific detection of HClO. The probe exhibits excellent selectivity, biocompatibility, and a low detection limit. Furthermore, it exhibits excellent mitochondrial targeting, enabling rapid detection of endogenous HClO in living cells and in vivo, promising broad application potential in life sciences.

[0006] However, traditional ratiometric fluorescent probes typically rely on high concentrations to achieve sufficient signal intensity and resolution, which often leads to problems such as rapid probe consumption, increased biotoxicity, and nonspecific binding to targets in biological samples. Especially in cell or in vivo imaging applications, high probe concentrations can disrupt the cellular microenvironment, causing shifts in physiological states and thus affecting the authenticity of detection results. Therefore, developing fluorescent probes that maintain stable ratiometric response characteristics even at low concentrations has become key to building high-performance biosensing systems. Summary of the Invention

[0007] In order to comprehensively solve the above problems, the present invention aims to design a low-concentration ratio-type to high-concentration enhanced fluorescent probe, which realizes the concentration-dependent detection of the response mode in the design strategy. In actual detection, it demonstrates its excellent selectivity, sensitivity and imaging ability in complex biological environments.

[0008] In order to achieve the above-mentioned object, the first aspect of the present invention provides a low concentration ratio high concentration enhanced fluorescent probe, the structural formula of which is .

[0009] The second aspect of the present invention provides a method for preparing a low concentration ratio high concentration enhanced fluorescent probe, comprising:

[0010] Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3 and bromohexylphenothiazine monoaldehyde as raw materials;

[0011] Step 2: Phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine were mixed and refluxed to obtain a low-concentration ratio-type high-concentration enhanced fluorescent probe with the structural formula: .

[0012] Preferably, including:

[0013] Step 1.1: Place DMF in an ice bath and add POCl3 dropwise to DMF until the reaction is complete. Then, warm the solution to room temperature.

[0014] Step 1.2: Dissolve bromohexylphenothiazine monoaldehyde in chloroform;

[0015] Step 1.3: Add the solution prepared in step 1.2 to the solution in step 1.1, reflux the mixture, and separate by column chromatography to obtain yellow phenothiazine dialdehyde.

[0016] Preferably, in step 1.3, the reaction is refluxed at 90° C. for 24 h.

[0017] Preferably, in step 1, the molar ratio of DMF, POCl3 and bromohexylphenothiazine monoaldehyde is 10:20:1.

[0018] Preferably, in step 2, the mixture is refluxed and stirred at 80° C. for 3 h.

[0019] A third aspect of the present invention provides an application of a low-concentration ratio-type high-concentration enhanced fluorescent probe prepared as described above, as a reagent for preparing HClO detection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The probe described herein emits fluorescence when detecting hypochlorous acid, enabling visual detection using spectrometers, fluorescence microscopy, and other techniques. At low concentrations of HClO (0–0.5 equivalents), the probe exhibits a clear ratiometric fluorescence response, with a significant decrease in fluorescence intensity at approximately 645 nm and a significant increase at approximately 545 nm. At higher concentrations of HClO (0.6–1.5 equivalents), the probe exhibits enhanced fluorescence at 545 nm, with a good linear correlation between fluorescence intensity and HClO concentration.

[0022] And F 545 / F 645 The ratio showed a good linear correlation with the HClO concentration (y = 0.25 + 0.33x, R² = 0.95), and the detection limit of the probe for HClO was as low as 21 nM.

[0023] 2. To verify the probe’s high selectivity and specificity in biological environments, selectivity and anti-interference experiments were conducted by simulating complex physiological conditions to examine its performance in the presence of various reactive oxygen species (ROS) and common interfering substances. The probe only showed a significant fluorescence response to HClO, while for other active species such as 、 、 、 、 and (each concentration was 100 μM) caused almost no fluorescence change, fully verifying its excellent selectivity.

[0024] 3. The probe's stability in complex biological matrices was evaluated, and anti-interference experiments were also conducted. The results showed that, under the same conditions, the presence of these interfering substances did not significantly affect the probe's detection of HClO, demonstrating good anti-interference capabilities. Furthermore, the probe's pH stability and reaction kinetics were evaluated. Results showed that the probe's fluorescence intensity remained stable within the physiological pH range (5.0–8.0), ensuring its feasibility for in vivo application. Kinetic analysis results demonstrated that the probe responded to HClO within 30 seconds, demonstrating its rapid response capability. Real-time monitoring of endogenous and exogenous HClO was achieved at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0026] In the attached figure:

[0027] Figure 1 For probe ;

[0028] Figure 2 For probe ;

[0029] Figure 3 The mass spectrometry of the probe ( m / z );

[0030] Figure 4 A: Absorption spectrum of the probe in dimethyl sulfoxide; B: Absorption spectrum of the probe in nine solvents; C: Emission spectrum of the probe in dimethyl sulfoxide; D: Emission spectrum of the probe in nine solvents (solvents 1-9 are benzene, dichloromethane, tetrahydrofuran, ethyl acetate, ethanol, acetonitrile, NN-dimethylformamide, dimethyl sulfoxide and water, conditions: room temperature; probe (10 μM));

[0031] Figure 5 is the HOMO-LUMO electron distribution and energy level of the probe ( : energy range; f: oscillator strength);

[0032] Figure 6 In the figure, A is the absorption spectrum of the probe (10 μM) after adding HClO (0 ~ 8 μM); B is the absorption of the probe and [HClO] at 485 nm;

[0033] Figure 7 In the figure, A is the fluorescence spectrum of the probe (10 μM) after adding HClO (0 μM - 5 μM); B is the fluorescence spectrum of the probe (10 μM) after adding HClO (6 μM -15 μM); C is the fluorescence spectrum of the probe (10 μM) after adding HClO (0 μM - 15 μM); D is the relationship between the fluorescence intensity of the probe and the concentration of HClO (0 μM - 15 μM);

[0034] Figure 8 In the figure, A is the experimental bar graph of the probe's selectivity to HClO ( 、 、 、 、 、 、 B is the anti-interference experiment of the probe; C is the stability of the probe at different pH; D is the response time of the probe to HClO;

[0035] Figure 9 is the reaction mechanism between the probe and HClO;

[0036] Figure 10 is the mass spectrum of the reaction between the probe and HClO (2 μM);

[0037] Figure 11 is the mass spectrum of the reaction between the probe and HClO (15 μM);

[0038] Figure 12 Effects of different concentrations of probes on the viability of RAW 264.7 cells (MTT assay was used to evaluate the cytotoxicity of probes on RAW 264.7 cells, the viability of cells not treated with probes was defined as 100%, and the results were expressed as the mean ± standard deviation of five separate measurements);

[0039] Figure 13 Fluorescence imaging of RAW264.7 cells in response to low concentrations of exogenous HClO. (A-C represent the relative emission intensities of RAW264.7 cells in the red channel (λem = 650-700 nm, λex = 405 nm) and the yellow channel (λem = 500-600 nm, λex = 405 nm) after cells were stained with 10 μM probe and 0, 2, and 5 μM HClO for 0.5 h, respectively. Scale bar: 30 μm).

[0040] Figure 14 Fluorescence imaging of RAW264.7 cells exposed to high concentrations of exogenous HClO;

[0041] Figure 15 Figure 3 Fluorescence imaging of endogenous HClO in RAW264.7 cells. (A represents the relative emission intensities in the red channel (λem = 650 ~ 700 nm, λex = 405 nm) and the yellow channel (λem = 500 ~ 600 nm, λex = 405 nm) of RAW264.7 cells after staining with 10 μM probe for 0.5 h; B and E represent the relative emission intensities in the red channel (λem = 650 ~ 700 nm, λex = 405 nm) and the yellow channel (λem = 500 ~ 600 nm, λex = 405 nm) of RAW264.7 cells after pretreatment with LPS (2 μg / mL) for different time periods (2, 4, 6, 8, 10, and 12 h) and then incubation with 10 μM probe for 0.5 h. Scale bar: 30 µm. DETAILED DESCRIPTION

[0042] The following combination Figures 1-15 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0043] Raw materials and reagents:

[0044] Phenothiazine, phosphorus oxychloride, malononitrile, and hypochlorous acid were purchased from MacLean Reagent Company.

[0045] Test equipment:

[0046] Nuclear magnetic resonance (NMR) data were acquired using an AVANCE NEO400M NMR spectrometer (Bruker, Switzerland). High-resolution mass spectra were obtained using a Bruker Micro TOFII mass spectrometer. UV-visible spectra were obtained using a UV-2600 spectrometer (Shimadzu, Japan). Fluorescence spectra were obtained using an FLS1000 fluorescence spectrometer (Shimadzu, Japan).

[0047] Example 1:

[0048] Low concentration ratio high concentration enhanced fluorescent probe, the structural formula is: .

[0049] Example 2:

[0050] The following is the synthetic route of the probe of the present invention: .

[0051] The preparation method of the low-concentration ratio-type high-concentration enhanced fluorescent probe based on the above-mentioned synthetic route comprises:

[0052] Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3 and bromohexylphenothiazine monoaldehyde as raw materials; comprising:

[0053] Step 1.1: Place DMF (1.5 g, 0.02 mol) in an ice bath. Add POCl3 (6.1 g, 0.04 mol, 3.64 mL) dropwise to DMF until the reaction is complete. Then, warm the solution to room temperature.

[0054] Step 1.2: Dissolve bromohexylphenothiazine monoaldehyde (0.78 g, 2 mmol) in 10 mL of chloroform.

[0055] Step 1.3: Add the solution prepared in step 1.2 to the solution in step 1.1, reflux at 90°C for 24 h, and separate by column chromatography using PE / EA = 3:1 to obtain yellow phenothiazine dialdehyde (5.0 g, yield 80%).

[0056] Step 2: In a 100 mL round-bottom flask, 80 mg (0.23 mmol) of phenothiazine dialdehyde, 38 mg (0.575 mmol), 10 mL of ethanol, 5 mL of acetonitrile, 1 mL of DMF, and 2.3 μL of piperidine (0.049 mmol, 0.1 eq) were added. The mixture was refluxed at 80 °C for 3 h to obtain a red solid low-concentration ratiometric high-concentration enhanced fluorescent probe (94.3 g, yield 94.3%).

[0057] like Figure 1 For probe , is the H NMR spectrum for structural characterization of the probe. Figure 2 For probe , is the NMR C spectrum of the probe's structural characterization. Figure 3 The mass spectrometry of the probe ( m / z ): [M + Na] + calcd for C26H25N5NaS, 458.1415.2300; found, 458.1424.

[0058] 1 H NMR (400 MHz, DMSO) δ 8.25 (s, 1H), 7.83 (dd, J = 8.8, 2.1 Hz,1H), 7.69 (d, J = 2.1 Hz, 1H), 7.26 – 7.10 (m, 4H), 7.04 (dd, J = 10.9, 4.0Hz, 1H), 3.96 (s, 2H), 3.59 (s, 2H), 1.69 (d, J = 6.7 Hz, 4H), 1.41 (d, J =3.5 Hz, 4H). 13 C NMR (101 MHz, DMSO) δ 159.26, 150.47, 142.58, 132.28, 129.40,128.63, 127.80, 125.90, 124.55, 122.21, 117.20, 116.19, 114.56, 76.51, 47.47,45.71, 32.41, 26.26, 25.66.

[0059] The structural formula of the low concentration ratio high concentration enhanced fluorescent probe is:

[0060] .

[0061] Example 3:

[0062] For example, the low-concentration ratio-type high-concentration enhanced fluorescent probe of Example 1 is used as a reagent for preparing HClO detection.

[0063] Detection experiment of hypochlorous acid:

[0064] Prepare the fluorescent probe into a stock solution with a concentration of 1 mmol / L for later use. When testing, dilute it to 10 μmol / L with 10 mmol / L phosphate buffer (PBS, pH=7.2). Shake well before testing. The solution should be prepared and used immediately. ) was prepared into a stock solution with a concentration of 1 mmol / L for testing. As solvent, prepare the solution with a concentration of 10 mmol / L 、 、 、 、 、 The mother solution was set aside for use. The fluorescent probe (10 μmol / L) was reacted with HClO and other analytes in PBS solution (pH = 7.2) for 40 min, and the changes in absorption and fluorescence spectra were measured.

[0065] Result analysis:

[0066] 1. Optical properties of low-concentration ratio-type high-concentration enhanced fluorescent probes:

[0067] The photophysical properties of the low-concentration ratio-type high-concentration enhanced fluorescent probe in nine solvents, the corresponding UV-visible spectra and fluorescence spectra are as follows Figure 4 shown.

[0068] like Figure 4 In Figure A, the absorption peak of the probe in DMSO is at 498 nm. Figure 4 Figure B shows that the fluorescent probe exhibits multiple absorption bands in different solvents, with the most significant absorption peak located at approximately 490 nm. It is worth noting that when the polarity of the solvent increases, the absorption peak at 490 nm exhibits a significant blue shift. Figure 4 As shown in C, the fluorescence emission peak of the probe in DMSO is located at 649 nm. Figure 4D in the middle shows the fluorescence emission spectra of the probe in different solvents. As the polarity of the solvent increases, the fluorescence emission peak red-shifts, indicating that the probe also observes solvation from the fluorescence spectrum. This is due to the typical D-π-A structure of the probe (the electron-rich phenothiazine group acts as an electron donor (Doner, D), and the cyanide group acts as an electron acceptor (Accepter, A), which promotes the delocalization of charges in the molecular structure, making the probe exhibit solvent polarity-dependent absorption and fluorescence properties. In order to further clarify its electronic properties, quantum chemical calculations were performed. Figure 5 As shown in Figure 3, the HOMO electrons are mainly concentrated on the central phenothiazine unit, while the LUMO electrons are mainly distributed on the electron-withdrawing group cyano, which is attributed to the intramolecular charge transfer (ICT) process and explains the strong solvent dependence observed in the spectrum.

[0069] 2. Spectral detection:

[0070] To verify the feasibility of our probe design, the spectral response of the probe (10 μM) to HClO in PBS (pH = 7.2) was evaluated. Figure 6 As shown in Figure 5A, the absorbance of the probe at approximately 495 nm gradually decreased with the gradual addition of HClO (0–8 μM). Figure 6 Panel B shows that the absorbance at 495 nm showed a good linear relationship with the HClO concentration (y = 0.10 -0.0038x, R² = 0.99).

[0071] In terms of fluorescence properties, such as Figure 7 China A and Figure 7 As shown in Figure B, with the addition of hypochlorous acid (0–5 μM), the fluorescence intensity of the probe at approximately 645 nm decreased significantly, while the fluorescence at 545 nm increased. 545 / F 645 A strong linear correlation was observed with the HClO concentration (y = 0.25 + 0.33x, R² = 0.95), and the detection limit of the probe for HClO was determined to be as low as 21 nM, indicating that the probe has obvious ratiometric fluorescence properties in the 0–5 μM HClO concentration range.

[0072] The fluorescence emission spectrum of the probe was further studied under higher concentrations of HClO (6–15 μM). As the concentration of HClO increased (6–15 μM), the fluorescence emission of the probe at 545 nm gradually increased. This trend was Figure 7 The fluorescence intensity showed a good linear relationship with the HClO concentration, highlighting the high sensitivity of the probe in HClO detection ( Figure 7 (D). Notably, unlike most previously reported fluorescent probes, the probe described in this application possesses two distinct reaction sites, generating distinct fluorescence responses to low and high concentrations of HClO. This unique property makes the probe a dual-mode fluorescent probe, enabling accurate detection across a wide range of HClO concentrations.

[0073] 3. Selectivity and specificity:

[0074] In order to verify that the probe of the present invention has good selectivity and specificity in the in vivo environment, the probe was subjected to selectivity and anti-interference experiments in the complex in vivo environment by simulating the complex in vivo environment and the effects of other reactive oxygen species and common interference factors on the probe.

[0075] like Figure 8 As shown in A, the probe is in HClO, 、 、 、 、 、 In the presence of interfering substances (100 μM), only HClO showed a significant signal change. In order to further confirm the stability of the probe in complex biological matrices, the anti-interference test of the probe was carried out ( Figure 8 In the same environment, the interfering factors have no significant interference on the HClO detection results. Finally, the pH stability and response kinetics of the probe were studied. Figure 8 Middle C) shows that the fluorescence intensity of the probe is stable within the physiological pH range (5.0-8.0), meeting the requirements of in vivo detection. Figure 8 (D) shows that the probe can complete its response within 30 seconds. The data from various experiments on the probe's response to hypochlorous acid demonstrate its excellent response to hypochlorous acid and its potential for application.

[0076] 4. Reaction mechanism:

[0077] Figure 9 The sensing mechanism of the probe for detecting HClO was demonstrated. The probe (10 μM) was mixed with HClO (2 μM) and analyzed by mass spectrometry. Figure 10 As shown in the figure, the mass peak corresponding to the reaction between the probe and HClO was observed, and the calculated m / z was 452.1545, while the experimental value was 452.1552. In addition, when the concentration of the probe and HClO was high (15 μM), the dominant peak at m / z (490.1818) was attributed to the reaction product peak of the probe and HClO ( Figure 11Therefore, the probe's detection mechanism for HClO is based on the oxidation of the sulfur atom with the HClO in the probe. These results suggest that the probe's detection mechanism for HClO is primarily driven by the formation of sulfoxide products under limited HClO oxidation, while excess HClO may lead to the formation of sulfones. After oxidation, the electron-donating ability of the sulfur atom is suppressed, altering the intramolecular charge transfer process and leading to changes in fluorescence emission.

[0078] 5. Cell Imaging:

[0079] Before the cell imaging experiment, the cytotoxicity of the probe was tested by MTT assay. The results showed that the probe had low cytotoxicity to RAW264.7 cells even at a concentration of 50 μM ( Figure 12 The probe was then used to image HClO in living cells using a confocal microscope.

[0080] After culturing RAW264.7 cells with the probe (10 μM) at 37°C for half an hour, the probe-localized RAW264.7 cells were rinsed three times with PBS and then subjected to fluorescence imaging. Figure 13 It can be seen that, as Figure 13 As shown in middle A, after RAW264.7 cells were incubated with 10 μM probe for 0.5 h, they showed a strong red fluorescence signal in the red channel, but no obvious fluorescence signal in the yellow channel. Figure 13 Middle B shows that after RAW264.7 cells were incubated with 10 μM probe and 2 μM HClO for 0.5 h, a significant decrease in the red fluorescence signal was observed, and an increase in the yellow fluorescence signal in the yellow channel was observed. Figure 13 Middle C shows that after RAW264.7 cells were incubated with 10 μM probe and 5 μM HClO for 0.5 h, a significant decrease in red fluorescence signal and an increase in yellow fluorescence signal in the yellow channel were observed compared to the addition of 2 μM HClO. These cell imaging results demonstrate that the probe can be used to monitor changes in physiological HClO concentrations.

[0081] In addition, the probe-localized RAW264.7 cells were cultured with high concentrations of HClO (6, 8, 10, 12, 15 μM) for half an hour, and the changes in fluorescence signals in different channels were observed using a fluorescence microscope. Figure 14As shown in Figures AE (where AE represents the relative emission intensity in the red channel (λem = 650–700 nm, λex = 405 nm) and yellow channel (λem = 500–600 nm, λex = 405 nm) of RAW264.7 cells after staining with 10 μM probe and 6, 8, 10, 12, and 15 μM HClO for 0.5 h, respectively; scale bar: 30 µm), yellow fluorescence in the yellow channel gradually increased after incubation of RAW264.7 cells with 10 μM probe and 6, 8, 10, 12, and 15 μM HClO for 0.5 h. This indicates that the probe can successfully detect high intracellular HClO concentrations, consistent with in vitro detection. These data demonstrate that the probe can detect a wide range of intracellular HClO concentrations and may serve as an effective tool for the early diagnosis of HClO-related diseases.

[0082] RAW264.7 cells were stimulated with lipopolysaccharide (LPS) for different time periods (2, 4, 6, 8, 10, and 12 h) and then incubated with 10 μM probe for 0.5 h. Fluorescence microscopy was used to observe changes in the red and yellow channel fluorescence signals. Figure 15 As shown in A, after RAW264.7 cells were incubated with 10 μM probe for 0.5 h, there was a clear signal in the red channel, but no signal in the yellow channel. Figure 15 Figure B shows the cell development after RAW264.7 cells were pretreated with LPS (2 μg / mL) for 2 h and then incubated with the probe for 0.5 h. As can be seen from the figure, the signal in the red channel is decreasing and a weak signal appears in the yellow channel. Figure 15 As shown in Figure CG, as LPS stimulation time increases from 2 to 12 hours, the fluorescence signal in the red channel gradually decreases and disappears after 10 hours, while the fluorescence signal in the yellow channel gradually increases. This indicates that the probe can effectively detect changes in endogenous hypochlorous acid concentration in real time.

[0083] The above data indicate that the probe can be used to detect a wide range of HClO concentrations produced within cells and can serve as an effective tool for early diagnosis of HClO-related diseases.

[0084] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of a low-concentration ratio-type high-concentration enhanced fluorescent probe in the preparation of an HClO detection reagent, characterized in that: The structural formula of the low concentration ratio high concentration enhanced fluorescent probe is: .

2. The use of the low concentration ratio high concentration enhanced fluorescent probe according to claim 1, characterized in that: The preparation method of the low-concentration ratio high-concentration enhanced fluorescent probe comprises: Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3 and bromohexylphenothiazine monoaldehyde as raw materials; Step 2: Phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine were mixed and refluxed to obtain a low-concentration ratio-type high-concentration enhanced fluorescent probe with the structural formula: 。 3. The use of the low concentration ratio high concentration enhanced fluorescent probe according to claim 2, characterized in that: Step 1 includes: Step 1.1: Place DMF in an ice bath and add POCl3 dropwise to DMF until the reaction is complete. Then, warm the solution to room temperature. Step 1.2: Dissolve bromohexylphenothiazine monoaldehyde in chloroform; Step 1.3: Add the solution prepared in step 1.2 to the solution in step 1.1, reflux the mixture, and separate by column chromatography to obtain yellow phenothiazine dialdehyde.

4. The use of the low concentration ratio high concentration enhanced fluorescent probe according to claim 3, characterized in that: In step 1.3, reflux the reaction at 90 °C for 24 h.

5. The use of the low concentration ratio high concentration enhanced fluorescent probe according to claim 4, characterized in that: In step 1, the molar ratio of DMF, POCl3 and bromohexylphenothiazine monoaldehyde is 10:20:

1.

6. The use of the low concentration ratio high concentration enhanced fluorescent probe according to claim 5, characterized in that: In step 2, the mixture was refluxed at 80 °C with stirring for 3 h.

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