Citric acid-based fluorescent dye with water-induced long-life characteristic as well as preparation method and application of citric acid-based fluorescent dye

Abstract

The invention relates to the technical field of fluorescence imaging, and provides a citric acid-based fluorescent dye with a water-induced long service life characteristic and a preparation method and application thereof, and the structural formula of the dye is shown as TPA-ER. The citric acid-based fluorescent dye provided by the invention has a remarkable water-induced long service life characteristic in a physiological water environment, and the service life is gt; the method has the advantages that the fluorescence intensity is 15ns, the FLIM analysis can be carried out in a long-life window, the interference of short-life endogenous background fluorescence is effectively reduced, and the contrast ratio and the quantitative accuracy of imaging are obviously improved.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging technology, specifically to a citrate-based fluorescent dye with water-induced long lifetime properties, its preparation method, and its application. Background Technology

[0002] Fluorescence lifetime imaging (FLIM) enables quantitative analysis by detecting the average residence time of fluorescent molecules in the excited state. Because its signal is less affected by probe concentration, excitation light intensity, or light scattering from biological tissue, it exhibits higher accuracy and reliability than intensity imaging in bioanalysis applications. However, the performance of FLIM technology largely depends on the development of high-performance fluorescent probes.

[0003] Currently, commonly used long-lived probes (such as rare-earth complexes) often face problems such as poor biocompatibility and difficulty in controlling optical properties. In contrast, small organic molecule dyes have excellent biocompatibility and are easy to modify, but their fluorescence lifetimes are generally short (usually 0-5 ns), which overlaps significantly with the lifetime of cellular autofluorescence (0-7 ns), resulting in low signal-to-background ratio and limited detection sensitivity. On the other hand, probes constructed based on such dyes typically have a small lifetime response amplitude to changes in the intracellular microenvironment (often less than 1 ns), which to some extent limits the contrast and quantitative accuracy of FLIM imaging.

[0004] Crucially, water, as a major component of living systems, further shortens the fluorescence lifetime of most organic dyes through non-radiative transitions such as enhanced molecular vibration and collisional quenching, severely limiting the imaging contrast and quantitative capabilities of FLIM in physiological environments. Therefore, developing high-performance fluorescent probes that can maintain long lifetimes in aqueous environments and exhibit significant responses to changes in the cellular microenvironment has become a key challenge in advancing FLIM technology for precise bioimaging. Summary of the Invention

[0005] The present invention aims to overcome the above-mentioned shortcomings of the prior art and provide a citrate-based fluorescent dye with long fluorescence lifetime, its preparation method and application.

[0006] The citrate-based fluorescent dye provided by this invention is characterized by its unique "water-induced long lifetime" property. Specifically, the fluorescence lifetime of this dye in an aqueous environment is significantly extended compared to its fluorescence lifetime in organic solvents, reaching 19.5 ns, thereby enabling high signal-to-noise ratio fluorescence lifetime imaging within a long lifetime range (e.g., greater than 10 nanoseconds). The fluorescence lifetime of this dye is highly sensitive to changes in the surrounding water content, and can effectively reflect changes in the hydration state of the microenvironment through quantitative changes in lifetime. In addition, the dye also possesses endoplasmic reticulum targeting capability, specifically accumulating in the endoplasmic reticulum of living cells. Based on the above characteristics, the fluorescent dye of this invention is particularly suitable for fluorescence lifetime imaging within living cells, and can be used to monitor, for example, the dynamic changes in the hydrophobic microenvironment accompanying early endoplasmic reticulum stress, providing a novel and effective probe tool for real-time research and quantitative analysis of related pathophysiological processes.

[0007] This invention also provides a method for preparing the fluorescent dye, which uses readily available raw materials, has mild reaction conditions, simple steps, and is easy to implement.

[0008] The technical solution adopted in this invention is as follows: This invention provides a citrate-based fluorescent dye with a long fluorescence lifetime in a physiological aqueous environment, the structure of which is as follows:

[0009] TPA-ER.

[0010] This invention provides a method for preparing the compound TPA-ER, comprising the following steps: Potassium carbonate was added to compound TPA-N-COOH and the mixture was stirred and reacted. Then N-(2-bromoethyl)-4-methylbenzenesulfonamide was added and the mixture was heated and reacted to obtain compound TPA-ER. The synthesis route is as follows: .

[0011] In some embodiments, the reaction conditions for compound TPA-N-COOH with potassium carbonate are: stirring at 30-40°C for 20-30 min; the heating temperature can be any one of 30, 35, and 40°C or any range between two of these values. The heating time can be any one of 20, 25, and 30 min or any range between two of these values.

[0012] In some embodiments, the heating reaction is carried out under the condition of stirring at 30-40°C for 18-24 hours; the heating temperature can be any one of 30, 35, and 40°C or a range between any two values. The heating time can be any one of 18, 20, 22, and 24 hours or a range between any two values.

[0013] In some embodiments, the molar ratio of TPA-N-COOH, potassium carbonate, and N-(2-bromoethyl)-4-methylbenzenesulfonamide is 1:2:1.5; in some embodiments, the reaction solvent is an organic solvent, such as N,N-dimethylformamide.

[0014] In some embodiments, after the heating reaction is completed, the process further includes washing, extraction, drying, and / or purification steps; in some embodiments, after the heating reaction is completed, the process includes water washing, dichloromethane extraction, drying with anhydrous sodium sulfate, and / or column chromatography separation and purification. In some embodiments, in the column chromatography separation and purification step, the chromatographic solvent is petroleum ether (PE):ethyl acetate (EA) = 1:1, V / V.

[0015] In some embodiments, the reaction involves heating citric acid with 1,2-diphenylethylenediamine to obtain the compound TPA-N-COOH.

[0016] In some embodiments, the heating reaction is carried out under the condition of stirring at 120-140°C for 4-8 hours; the heating temperature can be any one of 120, 125, 130, 135, and 140°C, or a range between any two values. The heating time can be any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 hours, or a range between any two values.

[0017] In some embodiments, the molar ratio of citric acid to 1,2-diphenylethylenediamine is 1:1.

[0018] In some embodiments, after the heating reaction is completed, the process further includes washing, extraction, drying, and / or purification steps. In some embodiments, after the heating reaction is completed, the temperature is lowered to room temperature, and then the mixture is dissolved in anhydrous methanol, washed with water, extracted with dichloromethane, dried with anhydrous sodium sulfate, and / or purified by column chromatography. In some embodiments, in the column chromatography purification step, the chromatographic solvent is dichloromethane (DCM):methanol (MeOH) = 8:1, V / V.

[0019] The citrate-based fluorescent dye provided in this invention is suitable for fluorescence lifetime imaging. The compound TPA-ER, prepared by the method described above, can achieve fluorescence lifetime imaging in living cells over a long lifetime (greater than 10 nanoseconds), responds to changes in water content and can be used for microenvironment analysis, while also possessing endoplasmic reticulum targeting capabilities.

[0020] The technical solution of this invention has the following advantages: 1. This invention provides a citrate-based fluorescent dye with water-induced long lifetime characteristics, the structural formula of which is shown in TPA-ER. The provided citrate-based fluorescent dye exhibits significant "water-induced long lifetime" characteristics (lifetime > 15 ns) in physiological aquatic environments, enabling FLIM analysis within a long lifetime window, effectively reducing interference from short-lived endogenous background fluorescence, and significantly improving imaging contrast and quantitative accuracy.

[0021] Furthermore, this dye combines endoplasmic reticulum targeting capability with high sensitivity to water content, enabling it to specifically locate in the endoplasmic reticulum of living cells. By analyzing changes in fluorescence lifetime, it can monitor in real time the dynamic changes in the hydrophobic microenvironment accompanying processes such as early endoplasmic reticulum stress, providing a novel and effective molecular probe for pathophysiological research.

[0022] Furthermore, the dye uses citric acid as its backbone, is safe with no significant cytotoxicity, exhibits excellent biocompatibility, and is suitable for complex biological systems.

[0023] Furthermore, the preparation method provided by this invention has a simple synthetic route, is easy to operate, uses inexpensive and readily available raw materials, and has low production costs, which is conducive to large-scale preparation and application promotion. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 The 1H NMR spectrum of the dye TPA-N-COOH in Example 1; Figure 2 The 1H NMR spectrum of the dye TPA-ER in Example 1; Figure 3 The carbon NMR spectrum of the dye TPA-ER in Example 1; Figure 4 This is the high-resolution mass spectrum of the dye TPA-ER in Example 1; Figure 5The fluorescence emission spectrum of the dye TPA-ER in DMSO in Example 2 is shown. Figure 6 The fluorescence lifetime test results of dye TPA-ER in different solvents in Example 3 are shown in (A) spectral results and (B) bar graph. Figure 7 The fluorescence lifetime of dye TPA-ER in Example 4 in a 1,4-dioxane / water mixed system (water volume fraction 0%-100%) varies with water content (A) spectral results and (B) dot plot. Figure 8 The images show (A) fluorescence lifetime imaging (scale bar of each image is 20 μm) and the corresponding (B) lifetime spectrum results of the dye TPA-ER in Example 5 in a 1,4-dioxane / water mixed system (water volume fraction 0%-100%). Figure 9 This is a cytotoxicity diagram of the dye TPA-ER in Example 6; Figure 10 This is a comparison image of intracellular fluorescence lifetime of the dye TPA-ER under normal and hyperosmolar culture conditions in HeLa cells, as shown in Example 7. Figure 11 The results of (A) confocal imaging (scale bar of each image is 20 μm) and (B) colocalization analysis of dye TPA-ER and commercial endoplasmic reticulum dye ER-Tracker Red in HeLa cells in Example 8 are shown. Figure 12 The following are examples of fluorescence lifetime imaging and corresponding lifetime spectra and normal distribution of the dye TPA-ER in a starvation-induced endoplasmic reticulum stress model in HeLa cells, as shown in Example 9. Figure 13 The following are examples of fluorescence lifetime imaging and corresponding lifetime spectra and normal distribution of the dye TPA-ER in the endoplasmic reticulum stress model of HeLa cells induced by tunicamycin in Example 10: (A) fluorescence lifetime imaging and (B) lifetime spectrum and (C) normal distribution. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0027] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1: Synthesis of TPA-ER, a water-induced long-lifetime fluorescent dye with citrate-based properties This embodiment provides a method for synthesizing a citrate-based fluorescent dye with water-induced long lifetime properties, the chemical structural formula of which is as follows:

[0029] TPA-ER.

[0030] The synthetic route of TPA-ER is as follows: .

[0031] The synthesis of TPA-ER includes the following steps: (1) Citric acid (10 mmol) and 1,2-diphenylethylenediamine (10 mmol) were mixed and stirred at 140 °C for 4 h. After the reaction was completed, the mixture was cooled to room temperature and dissolved in anhydrous methanol. The reaction solution was washed with water, extracted with dichloromethane, dried with anhydrous sodium sulfate, and purified by column chromatography (the chromatography solvent was DCM:MeOH=8:1, V / V) to obtain the compound TPA-N-COOH.

[0032] Its 1H NMR spectrum is as follows Figure 1 As shown, 1 H NMR (600 MHz, DMSO- d 6) δ 8.27 (s, 1H), 7.44-7.32 (m, 8H), 7.23-7.22 (d, 2H), 5.98 (s, 1H), 5.93 (s, 1H), 5.29 (d, 1H), 4.81 (d, 1H).

[0033] (2) Synthesis of compound TPA-ER: The purified intermediate TPA-N-COOH (0.6 mmol) and potassium carbonate (1.2 mmol) were dissolved in N,N-dimethylformamide (25 mL) and stirred at 40 °C for 30 minutes. Then, compound N-(2-bromoethyl)-4-methylbenzenesulfonamide (0.9 mmol) was added and the reaction was continued for 20 hours. After the reaction was completed, the mixture was washed with water, extracted with dichloromethane, dried with anhydrous sodium sulfate, and purified by column chromatography (the chromatographic solution was PE:EA=1:1, V / V) to finally obtain the fluorescent dye TPA-ER (the compound name is 2-((4-methylphenyl)sulfonamide)ethyl 5-oxo-2,3-diphenyl-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylate).

[0034] Its hydrogen NMR spectrum is as follows Figure 2 As shown, 1 H NMR (600 MHz, DMSO-d 6) δ 8.49 (s, 1H), 7.71-7.69 (d, 2H), 7.45-7.32 (m, 10H), 7.24-7.23 (d, 2H), 6.10 (s, 1H), 5.91 (s,1H), 5.33 (s, 1H), 4.86 (s, 1H), 4.19 (t, 2H), 3.11 (t, 2H), 2.36 (s, 3H).

[0035] Its carbon NMR spectrum is as follows Figure 3 As shown, 13 C NMR (151 MHz, DMSO- d 6) δ 170.82, 159.93,154.24, 146.18, 143.65, 143.17, 142.08, 138.14, 130.15, 129.53, 129.31,128.80, 126.91, 126.19, 126.07, 106.12, 80.53, 68.05, 66.54, 60.23, 41.78,21.42.

[0036] Its high-resolution mass spectrometry, such as Figure 4 As shown, HRMS (ESI, m / z): Calculates C 21 H 18 N₂O₃[M+H] + : 530.1750, found: 530.1747.

[0037] Example 2: Fluorescence Spectroscopy Measurement This embodiment investigated the fluorescence emission performance of the citrate-based fluorescent dye TPA-ER.

[0038] Take 1 μL of 10 mM fluorescent dye TPA-ER stock solution (dissolved to the corresponding concentration using DMSO) and add it to 1 mL of different solvents (Dioxane, DCM, EA, DMF, MeOH, DMSO, H2O) to obtain a 10 μM working solution; use a fluorescence spectrometer to excite with a 360 nm laser and scan the emission spectrum from 370 to 700 nm to obtain the fluorescence emission spectrum of the dye.

[0039] like Figure 5 As shown in the fluorescence emission spectrum, the maximum emission wavelength of the dye TPA-ER in aqueous solution is 465±5 nm.

[0040] Example 3 Solvent Effect Test This embodiment investigates the fluorescence lifetime of the citrate-based fluorescent dye TPA-ER in different solvents.

[0041] Take 1 μL of 10 mM TPA-ER fluorescent dye stock solution and add it to 1 mL of different solvents (Dioxane (1,4-dioxane), DCM (dichloromethane), EA (ethyl acetate), DMF (N,N-dimethylformamide), MeOH (methanol), DMSO (dimethyl sulfoxide), H2O) to obtain a 10 μM working solution; use a fluorescence spectrometer to determine the fluorescence lifetime of the dye.

[0042] like Figure 6 As shown in Figures A and B, TPA-ER exhibits a long fluorescence lifetime (>10 ns) in different solvents, with the maximum value of approximately 19.5 ns in aqueous solution.

[0043] Example 4: Verification of Water-Induced Long Lifetime Characteristics This example investigated the fluorescence lifetime of the citrate-based fluorescent dye TPA-ER in systems composed of 1,4-dioxane and water at different volume ratios (0% to 100%).

[0044] Take 1 μL of 10 mM TPA-ER fluorescent dye stock solution (dissolved to the corresponding concentration using DMSO) and add it to 1 mL of a mixture of 1,4-dioxane and water to obtain a 10 μM working solution; use a fluorescence spectrometer to determine the fluorescence lifetime of the dye.

[0045] like Figure 7 As shown in Figures A and B, the fluorescence lifetime of the dye TPA-ER continuously increases with the increase of water content in the test system, clearly confirming its "water-induced long lifetime" phenomenon.

[0046] Example 5: Feasibility Verification of Fluorescence Lifetime Imaging This embodiment examines the fluorescence lifetime imaging of the citrate-based fluorescent dye TPA-ER in systems composed of 1,4-dioxane and water at different volume ratios (0% to 100%).

[0047] Take 1 μL of 10 mM TPA-ER fluorescent dye stock solution (dissolved to the corresponding concentration using DMSO) and add it to 1 mL of a mixture of 1,4-dioxane and water to obtain a 10 μM working solution; then place it in a confocal dish and acquire and analyze the signal using a fluorescence lifetime imaging microscope.

[0048] like Figure 8As shown in Figures A and B, the TPA-ER can output clear fluorescence lifetime images, and its lifetime values ​​are highly consistent with the solution test results, proving that it is suitable for fluorescence lifetime imaging analysis applications.

[0049] Example 6 Cytotoxicity Test This example investigated the cytotoxicity of the citrate-based fluorescent dye TPA-ER.

[0050] Cell culture: HeLa cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin and 100 μg / mL streptomycin. The cell culture incubator conditions were 5% CO2 and 37℃.

[0051] Cytotoxicity assay: Cytotoxicity was determined using the CCK-8 cell counting method. HeLa cells at appropriate densities were seeded into 96-well plates and cultured overnight. After complete cell adhesion, the cells were incubated for 24 h with a series of TPA-ER solutions at final concentrations (1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM) dissolved in complete culture medium. The control group consisted of cell samples without dye, and the blank group consisted of cells in complete culture medium. After incubation, the original culture medium was replaced with complete culture medium containing 10% CCK-8 solution, and incubation continued for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated based on the absorbance results (cell viability % = (experimental group - blank group) / (control group - blank group) × 100%).

[0052] like Figure 9 As shown, even at a concentration of 60 μM, the survival rate of HeLa cells remained above 80%. These results indicate that the TPA-ER dye has good biocompatibility and can be used for fluorescence imaging in biological systems such as cells.

[0053] Example 7 Intracellular fluorescence lifetime imaging This embodiment investigates the fluorescence lifetime imaging of the citrate-based fluorescent dye TPA-ER under normal and hyperosmolar culture conditions.

[0054] The ability of the dye TPA-ER to reflect changes in intracellular water content was investigated using HeLa cells, and the steps are as follows: Take 1 μL of 10 mM TPA-ER fluorescent dye stock solution (dissolved to the appropriate concentration using DMSO) and add it to 1 mL of culture medium (DMEM high-glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a 10 μM working solution. Add 1 mL of the working solution to each well containing 4 × 10⁴ μM of TPA-ER fluorescent dye stock solution.5 Cells were placed in 12-well plates containing either untreated cells or hypertonic cells (hypertonic cells were cultured in complete medium containing 1.8 wt% sodium chloride for 24 h) and cultured for another 60 min in an incubator. Signals were acquired and analyzed using a fluorescence lifetime imaging microscope at an excitation wavelength of 375 nm.

[0055] like Figure 10 As shown, in cells cultured under normal conditions, TPA-ER exhibits a long red fluorescence lifetime signal; while in cells cultured under hypertonic conditions, due to water outflow, TPA-ER exhibits a short yellow-green fluorescence lifetime signal. This indicates that TPA-ER can enter living cells to perform fluorescence lifetime imaging and can accurately reflect changes in intracellular water content through fluorescence lifetime signals.

[0056] Example 8 Subcellular localization verification This example examines the results of confocal imaging using the citrate-based fluorescent dye TPA-ER and the commercially available endoplasmic reticulum dye ER-Tracker Red, co-stained with these dyes.

[0057] The following steps were taken to investigate the intracellular localization of the dye TPA-ER using HeLa cells: Add 1 μL of 10 mM TPA-ER stock solution to 1 mL of DMEM high-glucose medium (containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin), and then add 1 μL of ER-Tracker Red dye (10 mM) and mix thoroughly to obtain a working solution containing both TPA-ER and ER-Tracker Red. Add 1 mL of the working solution to each well containing 4 × 10⁴ TPA-ER and ER-Tracker Red. 5 The cells were mixed evenly in a 12-well plate containing 100 live cells and incubated for 30 min. Fluorescence intensity imaging was performed using a laser scanning confocal microscope at excitation wavelengths of 405 nm and 615 nm.

[0058] like Figure 11 As shown in Figures A and B, the fluorescence signal of TPA-ER highly overlaps with the signal of ER-Tracker Red. Colocalization analysis showed a Pearson correlation coefficient of 0.89, confirming that TPA-ER is specifically located in the endoplasmic reticulum of cells.

[0059] Example 9: Application in a starvation-induced endoplasmic reticulum stress model This embodiment examines the application of the citrate-based fluorescent dye TPA-ER in fluorescence lifetime imaging analysis in a starvation-induced endoplasmic reticulum stress model.

[0060] The experiment used HeLa cells to investigate the ability of the dye TPA-ER to monitor changes in the hydrophobicity of the endoplasmic reticulum microenvironment under stress. The steps are as follows: Add 1 μL of 10 mM TPA-ER fluorescent dye stock solution to 1 mL of culture medium (DMEM high-glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a 10 μM working solution. Add 1 mL of the working solution to each well containing 4 × 10⁴ μM TPA-ER solution. 5 Cells were placed in 12-well plates containing either untreated or stress-treated cells (stress-treated cells were cultured in EBSS starvation medium (commercially available) for 24 h) and cultured for another 60 min in an incubator. Signals were acquired and analyzed using a fluorescence lifetime imaging microscope at an excitation wavelength of 375 nm.

[0061] like Figure 12 As shown in Figures A, B, and C, the endoplasmic reticulum (ER) of normal cells exhibits a uniform, long-lived signal (approximately 16 ns, red). After starvation stress, the ER structure becomes fragmented, and large areas of short-lived signal regions appear (green), suggesting that the hydrophobicity of the ER microenvironment may increase under stress.

[0062] Example 10: Application in a tunicamycin-induced endoplasmic reticulum stress model This embodiment examines the application of the citrate-based fluorescent dye TPA-ER in fluorescence lifetime imaging analysis in a tunicamycin-induced endoplasmic reticulum stress model.

[0063] The experiment used HeLa cells to investigate the ability of the dye TPA-ER to reflect changes in the hydrophobicity of the endoplasmic reticulum microenvironment during early endoplasmic reticulum stress. The steps are as follows: Add 1 μL of 10 mM TPA-ER fluorescent dye stock solution to 1 mL of culture medium (DMEM high-glucose medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin) to obtain a 10 μM working solution. Add 1 mL of the working solution to each well containing 4 × 10⁴ μM TPA-ER solution. 5 Cells were placed in 12-well plates containing either untreated or stress-treated cells (stress-treated cells were cultured in complete medium containing 5 μM / 10 μM tunicamycin for 24 h) and cultured for another 60 min in an incubator. Signals were acquired and analyzed using a fluorescence lifetime imaging microscope at an excitation wavelength of 375 nm.

[0064] like Figure 13As shown in Figures A, B, and C, with the increasing intensity of tunicamycin-induced stress, the long-lived fluorescent signal regions within the cells continuously decreased, while the short-lived signal regions correspondingly expanded. This change in lifetime is synchronized with the fragmentation process of the endoplasmic reticulum morphology, further indicating that the fluorescence lifetime of TPA-ER can dynamically and sensitively reflect changes in the hydrophobicity of the endoplasmic reticulum microenvironment in the early stages of stress.

[0065] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A citrate-based fluorescent dye with water-induced long lifetime properties, characterized in that, The structural formula of the dye is shown below: TPA-ER.

2. A method for preparing a citrate-based fluorescent dye with water-induced long lifetime properties as described in claim 1, characterized in that, Includes the following steps: Compound TPA-N-COOH was reacted with potassium carbonate by stirring, and then N-(2-bromoethyl)-4-methylbenzenesulfonamide was added and heated to give compound TPA-ER. The synthesis route is as follows: 。 3. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 2, characterized in that, The reaction conditions for compound TPA-N-COOH with potassium carbonate are 30-40℃ with stirring for 20-30 min.

4. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 2 or 3, characterized in that, The heating reaction is carried out at 30-40℃ with stirring for 18-24 hours.

5. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 2 or 3, characterized in that, The molar ratio of TPA-N-COOH, potassium carbonate, and N-(2-bromoethyl)-4-methylbenzenesulfonamide is 1:2:1.

5.

6. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 2 or 3, characterized in that, After the heating reaction is completed, the process also includes washing, extraction, drying and / or purification steps; in the purification step, column chromatography is used for separation and purification, and the chromatographic solution is petroleum ether: ethyl acetate = 1:1, V / V.

7. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 2 or 3, characterized in that, The process also includes the step of heating citric acid with 1,2-diphenylethylenediamine to obtain the compound TPA-N-COOH. 。 8. The method for preparing a citrate-based fluorescent dye with water-induced long lifetime characteristics according to claim 7, characterized in that, The heating reaction conditions are 120~140℃ with stirring for 4-8 hours; And / or, the molar ratio of citric acid to 1,2-diphenylethylenediamine is 1:1; And / or, after the heating reaction is completed, the process further includes washing, extraction, drying and / or purification steps; in the purification step, column chromatography is used for separation and purification, and the chromatographic solution is dichloromethane:methanol = 8:1, V / V.

9. The use of the compound prepared by the method of preparing the water-induced long-lifetime citrate-based fluorescent dye as described in claim 1 or any one of claims 2-8 in the field of fluorescence imaging.

10. The use according to claim 9, characterized in that, The aforementioned uses include: (1) Applications in the field of fluorescence lifetime imaging; (2) Monitor the hydration status of the intracellular microenvironment; (3) Monitor the dynamic changes of the hydrophobic microenvironment accompanying early endoplasmic reticulum stress.

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