Low-concentration ratio type high-concentration enhanced fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, and provides a low-concentration ratio type high-concentration enhanced fluorescent probe as well as a preparation method and application thereof in order to solve the problems that a traditional fluorescent probe depends on a high-concentration use condition and is often faced with fast probe consumption, increased biotoxicity, non-specific target binding and the like in a biological sample. The preparation method of the fluorescent probe comprises the following steps: step 1, preparing phenothiazine dialdehyde; and 2, mixing phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine, and carrying out a reflux reaction to obtain the low-concentration ratio-type high-concentration enhanced fluorescent probe. The probe provided by the invention realizes quantitative detection of HClO through synchronous change of two emission channels, and the detection sensitivity and reliability are remarkably improved; when the concentration is high, an enhanced single-channel fluorescence amplification effect is shown, and strong signal identification and macroscopic visual imaging in specific application are facilitated, so that the dual requirements of trace monitoring and high-throughput screening are met.

Description

Technical Field

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

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

[0003] Therefore, developing analytical tools that can efficiently and sensitively detect HClO is of great significance for early disease diagnosis and pathological mechanism research. Fluorescent probes, due to their advantages such as non-invasiveness, high sensitivity, and real-time monitoring, have become powerful tools for detecting HClO. Currently, a variety of fluorescent probes have been used for the detection of hypochlorous acid, such as probes based on organic dyes, metal nanoparticle probes, etc. 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 being easily interfered by other oxidizing substances. In addition, the detection results of many probes do not have a direct visualization function, which limits their practicality in rapid detection and on-site applications.

[0004] Ratio-type fluorescent probes have received extensive attention in recent years because they can achieve intrinsic self-calibration, reduce background interference, and improve signal accuracy.

[0005] The existing patent publication number is "CN 117603203 A", and the name is "A Ratio Fluorescent Probe and Its Preparation Method and Application". A ratio fluorescent probe based on phenothiazine was synthesized for the specific detection of HClO. It has good selectivity, biocompatibility, and low detection limit. In addition, it also has good mitochondrial targeting ability and can rapidly detect endogenous HClO in living cells and in vivo, which makes it have broad application potential in life sciences.

[0006] However, traditional ratio-type fluorescent probes usually rely on high-concentration usage conditions to obtain sufficient signal intensity and resolution, which often face problems such as fast probe consumption, increased biological toxicity, and non-specific binding to targets in biological samples. Especially in cell or in vivo imaging applications, high-concentration probes may disrupt the cell microenvironment, leading to a shift in the physiological state, thereby affecting the authenticity of the detection results. Therefore, developing fluorescent probes that still have stable ratio response characteristics under low-concentration conditions has become the key to constructing a high-performance biosensing system. Summary of the Invention

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

[0008] To achieve the above object, in the first aspect of the present invention, a low-concentration ratio-type / high-concentration enhanced-type fluorescent probe is provided, and the structural formula is .

[0009] In the second aspect of the present invention, a preparation method of the low-concentration ratio-type / high-concentration enhanced-type fluorescent probe is provided, including Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3 and bromohexylphenothiazine monoaldehyde as raw materials; Step 2: Mix phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine, and carry out reflux reaction to obtain a low-concentration ratio-type / high-concentration enhanced-type fluorescent probe, and the structural formula is .

[0010] Preferably, it includes: Step 1.1: Place DMF in an ice bath, dropwise add POCl3 to DMF, react completely, and then heat 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, carry out reflux reaction, and separate by column chromatography to obtain yellow phenothiazine dialdehyde.

[0011] Preferably, in Step 1.3, reflux reaction is carried out at 90 °C for 24 h.

[0012] Preferably, in Step 1, the molar ratio of DMF, POCl3 and bromohexylphenothiazine monoaldehyde is 10:20:1.

[0013] Preferably, in Step 2, reflux and stir at 80 °C for 3 h.

[0014] In the third aspect of the present invention, an application of the low-concentration ratio-type / high-concentration enhanced-type fluorescent probe prepared according to the above scheme is provided, which is used as a reagent for preparing HClO detection.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. When the probe of the present application detects hypochlorous acid, it emits fluorescence, and visualization detection is achieved by means of techniques such as spectrometers and fluorescence microscopes. Under the condition of low-concentration HClO (0–0.5 equivalents), the probe exhibits an obvious ratio-type fluorescence response. The fluorescence intensity of the probe at about 645 nm decreases significantly, while the fluorescence intensity at about 545 nm increases significantly. At higher concentrations of HClO (0.6–1.5 equivalents), the probe shows an increase in fluorescence intensity at 545 nm, and its fluorescence intensity has a good linear correlation with the HClO concentration.

[0016] And F 545 / F 645 The ratio shows 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 is as low as 21 nM.

[0017] 2. To verify that the probe has high selectivity and specificity in a biological environment, selective and anti-interference experiments were carried out by simulating complex physiological conditions to investigate its performance in the presence of various reactive oxygen species (ROS) and common interfering substances. The probe only shows a significant fluorescence response to HClO, while for other reactive species such as , , , , and (each at a concentration of 100 μM) hardly cause fluorescence changes, fully verifying its excellent selectivity.

[0018] 3. To evaluate the stability of the probe in complex biological matrices, anti-interference experiments were also carried out. The results show that under the same conditions, the presence of these interfering substances does not significantly affect the detection of HClO by the probe, showing good anti-interference ability. In addition, the pH stability and reaction kinetic characteristics of the probe were also evaluated. The results show that the fluorescence intensity of the probe remains stable within the physiological pH range (5.0–8.0), ensuring its feasibility for in vivo applications. The kinetic analysis results show that the probe can respond to HClO within 30 seconds, demonstrating its rapid response ability. And real-time monitoring of endogenous and exogenous HClO was achieved at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention.

[0020] In the drawings: Figure 1 is the of the probe; Figure 2 for the probe ; Figure 3 is the mass spectrum of the probe ( m / z ); Figure 4 In, A: Absorption spectrum of the probe in dimethyl sulfoxide; B: Absorption spectra of the probe in 9 solvents; C: Emission spectrum of the probe in dimethyl sulfoxide; D: Emission spectra of the probe in 9 solvents (where solvents 1 - 9 are benzene, dichloromethane, tetrahydrofuran, ethyl acetate, ethanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and water, conditions: at room temperature; probe (10 μM)); Figure 5 is the HOMO-LUMO electron distribution and energy levels of the probe ( : energy range; f: oscillator strength); Figure 6 In, 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; Figure 7 In, 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); Figure 8 In, A is the bar graph of the selectivity experiment of the probe for HClO ( 、 、 、 、 、 、 ); B is the anti-interference experiment of the probe; C is the stability of the probe at different pH values; D is the response time of the probe to HClO; Figure 9 is the reaction mechanism of the probe with HClO; Figure 10 is the reaction mass spectrum of the probe with HClO (2 μM); Figure 11 is the reaction mass spectrum of the probe with HClO (15 μM); Figure 12Effect of probes with different concentrations on the viability of RAW 264.7 cells (evaluating the cytotoxicity of probes on RAW 264.7 cells by MTT method, the cell survival rate of cells untreated with probes was defined as 100%, and the results were expressed as the mean ± standard deviation of five independent measurements); Figure 13 Fluorescence imaging of low-concentration exogenous HClO in RAW264.7 cells. (A-C respectively represent the relative emission intensities of RAW264.7 cells in the red channel (λem = 650 - 700 nm, λex = 405 nm) and yellow channel (λem = 500 - 600 nm, λex = 405 nm) after staining the cells with 10 μM probe and 0, 2, 5 μM HClO for 0.5 h. Scale bar: 30 µm); Figure 14 Fluorescence imaging of high-concentration exogenous HClO in RAW264.7 cells; Figure 15 Fluorescence imaging of endogenous HClO in RAW264.7 cells. (A represents the relative emission intensities of RAW264.7 cells in the red channel (λem = 650 - 700 nm, λex = 405 nm) and yellow channel (λem = 500 - 600 nm, λex = 405 nm) after staining the cells with 10 μM probe for 0.5 h; B-E respectively represent the relative emission intensities of RAW264.7 cells in the red channel (λem = 650 - 700 nm, λex = 405 nm) and yellow channel (λem = 500 - 600 nm, λex = 405 nm) after pretreating with LPS (2 μg / mL) for different times (2, 4, 6, 8, 10, 12 h) and then incubating with 10 μM probe for 0.5 h. Scale bar: 30 µm). Detailed implementation manners

[0021] The following is combined with Figures 1 - 15 Preferred embodiments of the present invention are described. 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.

[0022] Raw materials and reagents: Phenothiazine, phosphorus oxychloride, malononitrile, and hypochlorous acid were all purchased from Macklin Reagent Company.

[0023] Testing instruments: The nuclear magnetic data was collected using an AVANCE NEO 400M nuclear magnetic resonance spectrometer (Bruker Company, Switzerland). High-resolution mass spectrometry was tested using a Bruker Micro TOFII mass spectrometer. Ultraviolet-visible spectroscopy was tested using a UV-2600 spectrometer (Shimadzu, Japan). Fluorescence spectroscopy was tested using an FLS1000 fluorescence spectrometer (Shimadzu, Japan).

[0024] Example 1: A low-concentration ratio-type high-concentration enhanced fluorescence probe, with the structural formula: .

[0025] Example 2: As follows is the synthetic route of the probe of the present invention: .

[0026] A preparation method of a low-concentration ratio-type high-concentration enhanced fluorescence probe based on the above synthetic route, including: Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3, and bromohexylphenothiazine monoaldehyde as raw materials; including: Step 1.1: Place DMF (1.5 g, 0.02 mol) in an ice bath, and gradually add POCl3 (6.1 g, 0.04 mol, 3.64 mL) dropwise to DMF. After the reaction is complete, heat the solution to room temperature; Step 1.2: Dissolve bromohexylphenothiazine monoaldehyde (0.78 g, 2 mmol) in 10 mL of chloroform; Step 1.3: Add the solution prepared in Step 1.2 to the solution in Step 1.1, reflux and react at 90 °C for 24 h, and perform column chromatography separation with PE / EA = 3:1 to obtain yellow phenothiazine dialdehyde (5.0 g, yield 80%).

[0027] In a 100 mL round-bottom flask, add 80 mg (0.23 mmol) of phenothiazine dialdehyde, 38 mg (0.575 mmol) of malononitrile, 10 mL of ethanol, 5 mL of acetonitrile, 1 mL of DMF, and 2.3 μL of piperidine (0.049 mmol, 0.1 eq), reflux and stir at 80 °C for 3 h to obtain a low-concentration ratio-type high-concentration enhanced fluorescence probe in the form of a red solid (94.3 g, yield 94.3%). As Figure 1 is the of the probe, and it is the nuclear magnetic H spectrum of the structural characterization of the probe. Figure 2 For the probe , is the NMR C spectrum for the structural characterization of the probe. Figure 3 is the mass spectrum of the probe ( m / z ): [M + Na] + calcd for C26H25N5NaS, 458.1415.2300; found, 458.1424. 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. The structural formula of the low-concentration ratio type and high-concentration enhanced type fluorescent probe is: .

[0028] Example 3: Use of the low-concentration ratio type and high-concentration enhanced type fluorescent probe as in Example 1 for the preparation of a HClO detection reagent.

[0029] Detection experiment for hypochlorous acid: Prepare a stock solution of the fluorescent probe with a concentration of 1 mmol / L for standby. During testing, dilute it to 10 μmol / L with 10 mmol / L phosphate buffer (PBS, pH = 7.2), shake well and then conduct the test. The solution should be prepared freshly before use. Prepare a stock solution of 0.1 mol / L hypochlorous acid with ultrapure water ( ) for testing at a concentration of 1 mmol / L. Using as the solvent, prepare a solution with a concentration of 10 mmol / L of , , , , , The mother solution was set aside for use. The fluorescent probe (10 μmol / L) was reacted with HClO and other test substances in PBS solution (pH=7.2) for 40 min, and the changes in absorption and fluorescence spectra were tested respectively.

[0030] Result analysis: 1. Optical properties of low-concentration ratio-type high-concentration enhanced fluorescent probes: Photophysical properties of low-concentration ratio-type high-concentration enhanced fluorescent probes in nine solvents, the corresponding UV-visible spectra and fluorescence spectra are shown in Figure 4 shown.

[0031] 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 shows 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 4 D in the figure 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 solvation phenomenon of the probe is also observed 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 the charge in the molecular structure, making the probe exhibit solvent polarity-dependent absorption and fluorescence characteristics. In order to further clarify its electronic properties, quantum chemical calculations were performed. Figure 5 As shown, 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, explaining the strong solvent dependence observed in the spectrum.

[0032] 2. Spectral detection: 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 1, as HClO (0–8 μM) was gradually added, the absorbance of the probe at approximately 495 nm gradually decreased. Figure 6In Figure B, it shows that the absorbance at 495 nm has a good linear relationship with the concentration of HClO (y = 0.10 - 0.0038x, R² = 0.99).

[0033] In terms of fluorescence properties, as Figure 7 shown in Figure A and Figure 7 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 increased at 545 nm. The fluorescence intensity ratio F 545 / F 645 showed a strong linear correlation with the concentration of HClO (y = 0.25 + 0.33x, R² = 0.95). 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 range of 0–5 μM HClO concentration.

[0034] Under the condition of higher concentration of HClO (6–15 μM), its fluorescence emission spectrum was further studied. With the increase in the concentration of HClO (6–15 μM), the fluorescence emission of the probe at 545 nm gradually increased, and this trend was further confirmed in Figure 7 Figure C. A good linear relationship was shown between the fluorescence intensity and the concentration of HClO, highlighting the high sensitivity of the probe in the detection of HClO ( Figure 7 Figure D). It is worth noting that different from most previously reported fluorescent probes, the probe of this application has two different reaction sites, which can produce different fluorescence responses to low-concentration and high-concentration HClO. This unique property makes the probe a dual-mode fluorescent probe, capable of achieving accurate detection in a wide range of HClO concentrations.

[0035] 3. Selectivity and Specificity: To verify that the probe of the present invention has good selectivity and specificity in the biological environment, by simulating the complex environment in the organism and the influence of other reactive oxygen species and common interfering factors on the probe, selectivity and anti-interference experiments of the probe were carried out for the complex environment in the organism.

[0036] As Figure 8 shown in Figure A, when the probe was in the presence of interfering substances such as HClO, , , , , , etc. (100 μM), only significant signal changes were shown for HClO. To further confirm the stability of the probe in complex biological matrices, anti-interference tests of the probe were carried out ( Figure 8B) In the same environment, the interference factors have no obvious interference on the detection results of HClO. Finally, the pH stability and response kinetics of the probe were studied. The pH stability study ( Figure 8 C) shows that the fluorescence intensity of the probe is stable within the physiological pH range (5.0 - 8.0), meeting the requirements for in vivo detection. The response kinetics analysis ( Figure 8 D) indicates that the probe can complete the response within 30 s. Through the data of various response experiments of the probe to hypochlorous acid, it shows that the probe has good response ability and application prospects for hypochlorous acid.

[0037] 4. Reaction mechanism: Figure 9 The sensing mechanism of the probe for detecting HClO is shown. The probe (10 μM) was mixed with HClO (2 μM) and analyzed by mass spectrometry. As Figure 10 shown, the mass peak corresponding to the reaction of the probe with HClO was observed, and the calculated m / z was 452.1545, with the experimental value being 452.1552. In addition, when the concentrations of the probe and HClO were relatively high (15 μM), the dominant peak at m / z (490.1818) was attributed to the reaction product peak of the probe and HClO ( Figure 11 ). Therefore, the detection mechanism of the probe for HClO is based on the oxidation of the S atom by HClO in the probe. These results indicate that the detection mechanism of the probe for HClO is mainly operated by the sulfoxide product generated under the limited oxidation of HClO, and excessive HClO may lead to the formation of sulfone. After oxidation, the electron-donating ability of the sulfur atom is inhibited, thereby changing the intramolecular charge transfer process and resulting in changes in fluorescence emission.

[0038] 5. Cell imaging: Before the cell imaging experiment, the cytotoxicity of the probe was tested by the MTT method. The results show that even at a concentration of 50 μM, the probe has low toxicity to RAW264.7 cells ( Figure 12 ). Then, the probe was applied to image HClO in living cells under a confocal microscope.

[0039] After culturing RAW264.7 cells with the probe (10 μM) at 37 °C for half an hour, the RAW264.7 cells located by the probe were rinsed 3 times with PBS, and then fluorescence imaging was performed. As can be seen from Figure 13 , as shown in Figure 13 A, after RAW264.7 cells were co-incubated with 10 μM probe for 0.5 h, strong red fluorescence signals were shown in the red channel, while no obvious fluorescence signals were shown in the yellow channel. Figure 13In Figure B, it shows that after RAW264.7 cells were co-incubated with 10 μM probe and 2 μM HClO for 0.5 h, a significant decrease in red fluorescence signal was observed, and an increase in yellow fluorescence signal in the yellow channel. Figure 13 In Figure C, it shows that after RAW264.7 cells were co-incubated with 10 μM probe and 5 μM HClO for 0.5 h, compared with the addition of 2 μM HClO, a significant decrease in red fluorescence signal was observed, and an increase in yellow fluorescence signal in the yellow channel. These cell imaging results indicate that the probe can be used to monitor the changes in the physiological concentration of HClO.

[0040] In addition, RAW264.7 cells in which the probe was located were cultured with high concentrations of HClO (6, 8, 10, 12, 15 μM) for half an hour, and then the changes in fluorescence signals in different channels were observed using a fluorescence microscope. As Figure 14 shown in Figure A - E (where A - E respectively 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 staining with 10 μM probe and 6, 8, 10, 12, and 15 μM HClO for 0.5 h, scale bar: 30 µm), after RAW264.7 cells were co-incubated with 10 μM probe and 6, 8, 10, 12, and 15 μM HClO for 0.5 h, the yellow fluorescence in the yellow channel gradually increased. It indicates that the probe can successfully detect higher concentrations of HClO in cells, which is consistent with the detection in vitro. The above data indicate that the probe can be used to detect HClO in a wide concentration range produced in cells and can be used as an effective tool for the early diagnosis of HClO-related diseases.

[0041] RAW264.7 cells were stimulated with lipopolysaccharide LPS for different times (2, 4, 6, 8, 10, 12 h) and then incubated with 10 μM probe for 0.5 h. The changes in fluorescence signals in the red channel and the yellow channel were observed using a fluorescence microscope. As Figure 15 shown in Figure A, after RAW264.7 cells were incubated with 10 μM probe for 0.5 h, there was a significant signal in the red channel, while there was no signal in the yellow channel. Figure 15 Figure B is the cell imaging diagram of RAW264.7 cells after pretreatment with LPS (2 μg / mL) for 2 h and then incubation with the probe for 0.5 h. It can be seen from the figure that the signal in the red channel decreased and a weak signal appeared in the yellow channel. As Figure 15 shown in C - G, as the LPS stimulation time increased from 2 h to 12 h, the fluorescence signal in the red channel gradually decreased and disappeared after 10 h, while the fluorescence signal in the yellow channel gradually increased. It shows that the probe can preferably detect the changes in the concentration of endogenous hypochlorous acid in real time.

[0042] The above data indicate that the probe can be used to detect HClO produced intracellularly over a wide concentration range and can serve as an effective tool for the early diagnosis of HClO-related diseases.

[0043] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed for the present invention is defined by the appended claims and their equivalents.

Claims

1. A low-concentration ratio-type high-concentration enhanced fluorescence probe, characterized in that, The structural formula is 。 2. The preparation method of the low-concentration ratio type and high-concentration enhanced fluorescence probe according to claim 1, wherein including: Step 1: Prepare phenothiazine dialdehyde using DMF, POCl3 and bromohexylphenothiazine monoaldehyde as raw materials; Step 2: Mix phenothiazine dialdehyde, malononitrile, ethanol, acetonitrile, DMF and piperidine, and carry out reflux reaction to obtain a low-concentration ratio type high-concentration enhanced fluorescence probe, the structural formula of which is: 。 3. The preparation method of the low-concentration ratio type high-concentration enhanced fluorescence probe according to claim 2, wherein Step 1 includes: Step 1.1: Place DMF in an ice bath, add POCl3 dropwise to DMF, react completely, and then heat 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, carry out reflux reaction, and separate by column chromatography to obtain yellow phenothiazine dialdehyde.

4. The preparation method of the low-concentration ratio type high-concentration enhanced fluorescence probe according to claim 3, characterized in that, In Step 1.3, reflux reaction is carried out at 90 °C for 24 h.

5. The preparation method of the low-concentration ratio type high-concentration enhanced fluorescence 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 preparation method of the low-concentration ratio type high-concentration enhanced fluorescence probe according to claim 5, wherein In Step 2, reflux and stir at 80 °C for 3 h.

7. The application of the low-concentration ratio type high-concentration enhanced fluorescence probe according to claim 1, characterized in that, Use as a reagent for detecting HClO.

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