Preparation method and detection method of near-infrared fluorescent probe for detecting cysteine
By synthesizing naphthalimide-based fluorescent materials and optimizing the preparation process, a near-infrared fluorescent probe NA-BH was designed, which solved the problem of insufficient overall performance of existing fluorescent probes and achieved high selectivity, rapid response and low detection limit for cysteine detection, which is suitable for live cell imaging.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-04-10
AI Technical Summary
Existing fluorescent probes struggle to simultaneously achieve large Stokes shift, rapid response, high selectivity, and low detection limit, especially in the near-infrared region where their overall performance is insufficient, limiting their application in the detection of cysteine in vivo or living cells.
Naphthalimide-based fluorescent materials were synthesized using compounds such as 4-bromo-1,8-naphthalic anhydride. Fluorescent probes were designed through specific nucleophilic substitution reactions. By utilizing the photoinduced electron transfer (PeT) mechanism, specific recognition groups were introduced, and the preparation process was optimized to form the near-infrared fluorescent probe NA-BH.
This invention enables the development of a fluorescent probe with high selectivity, rapid response, and low detection limit. It possesses good biocompatibility and anti-interference properties, is suitable for live-cell imaging, and provides a real-time, dynamic monitoring tool for cysteine.
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Figure CN121824422A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical imaging technology, specifically relating to a method for preparing a near-infrared fluorescent probe for detecting cysteine and a method for detecting it. Background Technology
[0002] Cysteine is a sulfur-containing non-essential amino acid that plays a vital role in the body. The total concentration of Cys in the blood plasma of normal individuals is 80-200 µM. Abnormal Cys concentrations in the human body can lead to various diseases. Low concentrations primarily affect antioxidant capacity, protein synthesis, and immune function; high concentrations are associated with metabolic abnormalities, stone formation, organ damage, and cardiovascular risks. Therefore, accurate, sensitive, and rapid detection of Cys is crucial for a deeper understanding of physiological and pathological processes within the body and for the early diagnosis of diseases.
[0003] Among the many methods for detecting Cys, traditional methods such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), and electrochemical analysis generally suffer from limitations such as complex operation, high cost, long detection time, and cumbersome sample pretreatment. These limitations make it difficult to meet the needs of dynamic monitoring of Cys in living organisms or cells, thus restricting their widespread application. In contrast, fluorescent probe detection technology, especially near-infrared (NIR) fluorescent probes, has become an ideal choice for Cys detection due to its advantages such as high sensitivity, rapid response, ease of operation, strong tissue penetration, and low background interference. However, although some progress has been made in reported Cys fluorescent probes, it is generally difficult to simultaneously achieve multiple ideal properties such as large Stokes shift, rapid response, high selectivity, and low detection limit, especially in the near-infrared region where reports of probes simultaneously possessing these advantages are still relatively few. Summary of the Invention
[0004] In view of this, the purpose of this invention is to provide a method for preparing a near-infrared fluorescent probe for detecting cysteine and a detection method thereof, so as to solve the problem that current fluorescent probes are difficult to achieve simultaneously large Stokes shift, fast response, high selectivity and low detection limit.
[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a near-infrared fluorescent probe for detecting cysteine, comprising the following steps: S1. Weigh 4-bromo-1,8-naphthalenedic anhydride and dissolve it in anhydrous ethanol. Then add n-butylamine and stir to dissolve. Heat under reflux at 78 °C for 2.5 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with ethanol to obtain the yellow product 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (NA-1). S2. Dissolve NA-1 and potassium carbonate in methanol and stir thoroughly to dissolve. Heat under reflux at 65 °C for 12 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with ice-distilled water to obtain a yellow solid product 2-butyl-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (NA-2). S3. Weigh NA-2 and dissolve it in hydroiodic acid. Heat the mixture under reflux at 127 °C for 12 h. After the reaction is complete, return the mixture to room temperature, filter under reduced pressure, wash the filter cake with ice water, and then wash it with petroleum ether to obtain the pale yellow product 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (NA-3). S4. Weigh NA-3 and hexamethylenetetramine and mix them. Dissolve them in trifluoroacetic acid. Stir at 80 °C for 10 h under nitrogen protection. After cooling to room temperature and adding ice water, a solid precipitates out. Filter under reduced pressure and wash with ice water to obtain a yellow solid product 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carboxaldehyde (NA-4). S5. NA-4, 2-(3,5,5-trimethylcyclohexane-2-en-1-yl)malonitrile and the catalyst were mixed and dissolved in anhydrous ethanol. The catalyst was pyridine. The mixture was refluxed at 78 °C for 12 h under nitrogen protection. The solvent was removed under reduced pressure. The product was purified by silica gel column chromatography to obtain the red product (E)-2-(3-(2-(2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-yl)vinyl)-5,5-dimethylcyclohexane-2-en-1-yl)malonitrile (NA-OH). S6. Weigh NA-OH and dissolve it in anhydrous dichloromethane. Add triethylamine dropwise at 0 °C under a nitrogen atmosphere and stir for 90 min. Then add 2,4-dinitrobenzenesulfonyl chloride and stir at room temperature for 4 h. Rotary evaporation yields a yellow solid crude product. The product is purified by silica gel column chromatography and dried to obtain a yellowish-brown solid (E)-2-butyl-5-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl 2,4-dinitrobenzenesulfonate (NA-BH), which is the near-infrared fluorescent probe for detecting cysteine.
[0006] Furthermore, the chemical structural formula of the fluorescent probe is shown below: Further, in step S1, the 4-bromo-1,8-naphthalenedic anhydride is dissolved in a molar ratio of 1:1.25 with n-butylamine.
[0007] Further, in step S2, the molar ratio of NA-1 to potassium carbonate is 1:4.
[0008] Further, in step S3, the molar ratio of NA-2 to hydroiodic acid is 1:52.
[0009] Further, in step S4, the molar ratio of NA-3 to hexamethylenetetramine is 1:3.
[0010] Further, in step S5, the eluent phase for column chromatography purification consists of dichloromethane and methanol in a volume ratio of 100:1, the molar ratio of NA-47 and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile is 1:1, and the silica gel is 200-300 mesh. In step S6, the eluent for column chromatography purification consists of petroleum ether and ethyl acetate in a volume ratio of 8:1, and the molar ratio of NA-OH, 2,4-dinitrobenzenesulfonyl chloride and triethylamine is 1:1.5:1. The silica gel is 200-300 mesh.
[0011] Furthermore, a method for detecting cysteine in biological samples includes the following steps: a) Introduce a near-infrared fluorescent probe into the biological sample to be tested; b) Induces fluorescence signal generation; c) Detect the resulting fluorescence signal to determine the presence or concentration of cysteine.
[0012] Furthermore, the biological sample is selected from living cells, tissues, body fluids, or organisms.
[0013] Furthermore, the detection method is used for imaging and visual tracking of endogenous and exogenous cysteine in living cells.
[0014] The beneficial effects of this invention are as follows: 1. The fluorescent probe of the present invention is designed and synthesized based on the photoinduced electron transfer (PeT) mechanism. Through a specific nucleophilic substitution reaction between 2,4-dinitrobenzenesulfonyl chloride (DNBS) and cysteine, the probe achieves a fluorescence "off-on" response.
[0015] 2. The fluorescent probe provided by this invention has strong selectivity and high detection efficiency; moreover, the fluorescent probe prepared by this invention has pH stability; it has a fast response time to recognize cysteine and has the ability to monitor dynamic changes in cysteine.
[0016] 3. The fluorescent probe provided by this invention has strong anti-interference properties and can accurately identify cysteine from a variety of interfering ions in acetonitrile; it is suitable for live cell imaging and has good biocompatibility; moreover, the detection method of this invention is simple to operate, uses inexpensive solvents, and is convenient for post-processing.
[0017] 4. The fluorescent probe provided by this invention has the characteristics of long emission wavelength (near infrared), large Stokes shift, fast response and high selectivity, and can be used to realize real-time, dynamic and efficient monitoring of endogenous and exogenous cysteine in living cells, providing a more powerful tool for cysteine-related life science research and clinical diagnosis.
[0018] 5. The naphthalimide compounds used in this invention are a class of high-performance fluorescent materials, possessing characteristics such as a large Stokes shift, high fluorescence quantum yield, and good photostability, making them ideal fluorophores for constructing fluorescent probes. By rationally modifying the parent structure of naphthalimide and introducing specific recognition groups, selective recognition and fluorescence response of target analytes can be achieved. Based on this, this invention successfully prepared a near-infrared fluorescent probe for the efficient and rapid detection of cysteine through optimized screening.
[0019] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description
[0020] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration: Figure 1 This is a synthetic route diagram of the fluorescent probe (NA-BH) of the present invention; Figure 2 (a) is the proton NMR spectrum of the fluorescent probe NA-BH of this invention; Figure 2 (b) is the carbon NMR spectrum of the fluorescent probe NA-BH of this invention; Figure 3 (a) Fluorescence spectra of 15 common ions identified by the fluorescent probe of the present invention; Figure 3 (b) Comparison of fluorescence enhancement at 732 nm for 15 common ions identified by the fluorescent probe of the present invention; Figure 4 The fluorescence spectra of the fluorescent probe NA-BH of the present invention recognizing cysteine at different times are shown. Figure 5 The UV and fluorescence titration diagrams of the fluorescent probe NA-BH recognizing cysteine in this invention are shown. Figure 6 This is a diagram showing the limit of detection for cysteine recognition by the fluorescent probe NA-BH of this invention. Figure 7 This is a pH fluorescence response diagram of the fluorescent probe NA-BH of the present invention recognizing cysteine; Figure 8 (a) Cytotoxicity assessment diagram of the fluorescent probe of the present invention in HeLa cells; Figure 8 (b) Real-time intracellular fluorescence imaging of exogenous cysteine by the fluorescent probe of the present invention; Figure 8 (c) Fluorescence imaging of endogenous cysteine in HeLa cells at different concentrations of the fluorescent probe of the present invention. Detailed Implementation
[0021] like Figure 1-8 As shown, the present invention provides a method for preparing a near-infrared fluorescent probe for detecting cysteine and a method for detecting it.
[0022] Example 1: A method for preparing a near-infrared fluorescent probe for detecting cysteine.
[0023] S1. In a 100 mL round-bottom flask, 4-bromo-1,8-naphthalenedic anhydride (5.60 g, 0.02 mol) was dissolved in 60 mL of anhydrous ethanol. Then, n-butylamine (2.47 mL, 0.025 mol) was added and stirred thoroughly. The mixture was then heated under reflux at 120 °C for 2.5 h. The solution turned into a deep yellow turbid liquid. After the reaction was completed as monitored by TLC, the mixture was cooled to room temperature, filtered under reduced pressure, and washed with ethanol to obtain the yellow product NA-1. NA-1 is 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione. S2. Weigh intermediate NA-1 (5.00 g, 15.05 mmol) and potassium carbonate (8.509 g, 61.57 mmol) and dissolve them in 150 mL of methanol. Heat under reflux at 120 °C for 12 h. After the reaction is completed by monitoring by TLC plate, cool to room temperature, filter under reduced pressure, and wash with ice water to obtain yellow product NA-2. NA-2 is 2-butyl-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione. S3. Weigh intermediate NA-2 (0.8 g, 2.8 mmol) and dissolve it in hydroiodic acid (20 mL, 146 mmol). Heat under reflux at 127 °C for 12 h. After the reaction is complete, return to room temperature, filter under reduced pressure, wash the filter cake with ice water and then wash with petroleum ether to obtain the pale yellow product NA-3. The NA-3 is 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione. S4. Weigh NA-3 (1.5 g, 5.57 mmol) and hexamethylenetetramine (2.4 g, 17 mmol), mix them, dissolve them in 50 mL of trifluoroacetic acid, stir at 80 °C for 10 h under nitrogen protection, cool to room temperature and add ice water, a solid precipitates out, filter under reduced pressure and wash with ice water to obtain yellow solid product NA-4, wherein NA-4 is 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carboxaldehyde; S5. NA-4 (148 mg, 0.49 mmol), 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile (94.0 mg, 0.50 mmol), and 5 drops of piperidine were dissolved in 10 mL of ethanol. The mixture was refluxed at 78 °C for 12 h under nitrogen protection. After the reaction was completed as monitored by TLC, the solvent was removed under reduced pressure. The crude product was purified by column chromatography to obtain the red product NA-OH. The NA-OH is (E)-2-(3-(2-(2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-yl)vinyl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile; S6. Weigh NA-OH (0.3 g, 0.65 mmol) and dissolve it in anhydrous dichloromethane. Add triethylamine (100 µL, 0.71 mmol) dropwise at 0 °C under a nitrogen atmosphere and stir for 90 min. Then add 2,4-dinitrobenzenesulfonyl chloride (189 mg, 0.71 mmol) and stir at room temperature for 4 h. After the reaction is completed as monitored by TLC, rotary evaporation is used to obtain a yellow solid crude product. The product is purified by silica gel column chromatography and dried into a yellowish-brown solid NA-BH. The NA-BH is (E)-2-butyl-5-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl 2,4-dinitrobenzenesulfonate, which is the near-infrared fluorescent probe for detecting cysteine.
[0024] The proton NMR spectrum of the near-infrared fluorescent probe of this invention: 1H NMR (400 MHz, CDCl3) δ 8.83 (s,1H), 8.74 (s,1H),8.56 (t,J = 6.9 Hz,2H),8.28 (d,J = 8.6 Hz,1H), 8.04 (d,J = 8.5 Hz,1H),7.69 (t,J = 7.9 Hz,1H),7.44 (d,J = 16.1 Hz,1H),7.21 (d,J = 6.4 Hz,1H),6.88 (s,1H),4.13 (t,J = 7.4Hz,2H), 2.57 (s,2H), 2.39 (s, 2H), 1.66 (dd,J = 14.8, 7.2 Hz,2H), 1.37 (dd,J= 13.9,5.8 Hz,2H), 1.04 (s,6H), 0.92 (t,J = 7.3 Hz,3H). Carbon NMR spectrum of the near-infrared fluorescent probe of this invention: 13C NMR (101 MHz, CDCl3) δ167.56,162.28, 161.68,151.10,145.06,133.48,132.91,132.33,131.08,128.12,127.98, 127.79,127.67,126.92,126.86,126.12,125.44,124.87,122.23,121.83, 119.85, 111.97, 111.12, 80.13, 76.31, 76.20, 76.00, 75.68, 59.40, 41.86, 38.12, 30.97, 29.11, 28.68, 26.93, 19.30, 12.81. Example 2: A method for detecting cysteine using a near-infrared fluorescent probe.
[0025] P1. Accurately weigh the fluorescent probe NA-BH prepared in Example 1 and dissolve it in acetonitrile (ACN) solution to prepare a 1 mM solution for later use. P2. Prepare stock solutions of cysteine and other amino acids using deionized water. Add 20 µL of the 1 mM probe NA-BH stock solution and acetonitrile solution to centrifuge tubes to dilute the system to a total volume of 1.5 mL. Finally, add 0.5 mL of cysteine / common biomolecule or ion solutions of different concentrations. P3. After shaking to mix evenly, immediately collect fluorescence spectrum test data. The fluorescence test parameters are set as follows: excitation slit 5 nm, emission slit 5 nm, excitation wavelength 520 nm.
[0026] The results are as follows Figure 3 As shown, when the fluorescent probe is reacted with common biomolecules or ions including Al³⁺, Ca²⁺, Na⁺, Mg²⁺, Ba²⁺, K⁺, Co²⁺, Cu²⁺, Fe²⁺, Ni²⁺, HCO3⁻, NH4⁺, Thr, Lys, and Glu, as well as cysteine (Cys), only cysteine shows significant fluorescence enhancement at 732 nm. This indicates that the probe has strong selectivity for cysteine and is suitable for the analysis of cysteine in complex environments.
[0027] Example 3: Kinetic test of the reaction process between near-infrared fluorescent probe and cysteine.
[0028] like Figure 4 As shown, when the excitation wavelength is 520 nm, a graph is plotted with the fluorescence intensity at 732 nm as the ordinate and time as the abscissa.
[0029] After the addition of cysteine, the near-infrared fluorescent probe showed a gradual increase in fluorescence at 732 nm, and the fluorescence intensity tended to stabilize after 6 minutes. This indicates that the near-infrared fluorescent probe of the present invention has a fast response speed and can quickly detect the content of cysteine.
[0030] Example 4: Near-infrared fluorescent probe detection of cysteine fluorescence spectrometry titration test.
[0031] like Figure 5 As shown, when excited by 520 nm light, the addition of cysteine (0-400 µM) significantly enhanced the fluorescence intensity at 732 nm.
[0032] like Figure 6 As shown, when the cysteine concentration increased from 0 µM to 20 µM, the two showed a good linear fit (R2=0.985), and the detection limit was 10.7 nM. Example 5: Study on pH stability of cysteine detected by near-infrared fluorescent probe.
[0033] Near-infrared fluorescent probes were mixed with cysteine in phosphate buffer solutions of different pH values, and their fluorescence intensity was measured.
[0034] like Figure 7 As shown, when the system pH ≥ 7.0, the fluorescence intensity of the NA-BH probe tends to stabilize and remains at a relatively stable level. This indicates that the probe of the present invention has good stability under physiological conditions and is suitable for detection in biological systems.
[0035] Example 6: Application of near-infrared fluorescent probes in cell imaging.
[0036] Near-infrared fluorescent probes were used in HeLa cells for the following experiments: Cytotoxicity assessment: The effect of near-infrared fluorescent probes at different concentrations on the survival rate of HeLa cells was evaluated using the MTT assay. After incubating HeLa cells with a 0-20 µM near-infrared fluorescent probe for 24 h, the cell viability remained above 85%, indicating that the near-infrared fluorescent probe had no significant toxic effect on HeLa cells.
[0037] like Figure 8 As shown in (a), it exhibits good biocompatibility and can be applied to live cell imaging.
[0038] Real-time imaging of exogenous cysteine: like Figure 8 As shown in (b), after NEM treatment, HeLa cells were co-incubated with cysteine and monitored by time-lapse imaging using a live-cell fluorescence microscope.
[0039] The results showed that the intracellular fluorescence signal intensity gradually increased over time, and remained essentially unchanged after 35 min. Furthermore, the increase in fluorescence signal showed a significant positive correlation with time. This indicates that the near-infrared fluorescent probe of this invention can rapidly respond to and effectively monitor exogenous cysteine.
[0040] Imaging of endogenous cysteine: like Figure 8 As shown in (c), HeLa cells were incubated and imaged under different near-infrared fluorescent probe concentration gradients.
[0041] The results showed that endogenous cysteine illuminated the cells and emitted red fluorescence, and the fluorescence signal in HeLa cells was enhanced with the increase of near-infrared fluorescent probe concentration.
[0042] This demonstrates that the near-infrared fluorescent probe of the present invention can effectively identify and monitor endogenous cysteine in cells and can be used to assess its relative concentration level in cells.
[0043] In summary, this invention provides a simple detection method that uses inexpensive solvents and facilitates convenient post-processing.
[0044] Finally, it should be noted that the above preferred 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 through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for preparing a near-infrared fluorescent probe for detecting cysteine, characterized in that: Includes the following steps: S1. Weigh 4-bromo-1,8-naphthalic anhydride and dissolve it in anhydrous ethanol. Then add n-butylamine and stir to dissolve. Heat under reflux at 78 °C for 2.5 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with ethanol to obtain the yellow product 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione. S2. Dissolve 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione and potassium carbonate in methanol and stir thoroughly to dissolve. Heat under reflux at 65 °C for 12 h. After the reaction is complete, return to room temperature, filter under reduced pressure, and wash with ice-distilled water to obtain the yellow solid product 2-butyl-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione. S3. Weigh 2-butyl-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione and dissolve it in hydroiodic acid. Heat the mixture under reflux at 127°C for 12 h. After the reaction is complete, return the mixture to room temperature, filter under reduced pressure, wash the filter cake with ice water, and then wash it with petroleum ether to obtain the pale yellow product 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione. S4. Weigh 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione and hexamethylenetetramine, mix them, dissolve them in trifluoroacetic acid, stir at 80 °C for 10 h under nitrogen protection, cool to room temperature and add ice water, a solid precipitates out, filter under reduced pressure and wash with ice water to obtain the yellow solid product 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carboxaldehyde; S5. 2-Butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carboxaldehyde, 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malonitrile and the catalyst were mixed and dissolved in anhydrous ethanol. The catalyst was pyridine. The mixture was refluxed at 78 °C for 12 h under nitrogen protection. The solvent was removed under reduced pressure. The product was purified by silica gel column chromatography to give the red product (E)-2-(3-(2-(2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile; S6. Weigh (E)-2-(3-(2-(2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-yl)vinyl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile and dissolve it in anhydrous dichloromethane. Add triethylamine dropwise at 0 °C under a nitrogen atmosphere and stir for 90 min. Then add 2,4-dinitrobenzenesulfonyl chloride and stir at room temperature for 4 minutes. Rotary evaporation yielded a yellow solid crude product, which was purified by silica gel column chromatography and dried to obtain a yellowish-brown solid (E)-2-butyl-5-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl 2,4-dinitrobenzenesulfonate (NA-BH), which is the near-infrared fluorescent probe for detecting cysteine.
2. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: The chemical structural formula of the fluorescent probe is shown below: 。 3. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: In step S1, the 4-bromo-1,8-naphthalenedic anhydride is dissolved in a molar ratio of 1:1.25 with n-butylamine.
4. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: In step S2, the molar ratio of NA-1 to potassium carbonate is 1:
4.
5. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: In step S3, the molar ratio of NA-2 to hydroiodic acid is 1:
52.
6. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: In step S4, the molar ratio of NA-3 to hexamethylenetetramine is 1:
3.
7. The method for preparing a near-infrared fluorescent probe for detecting cysteine according to claim 1, characterized in that: In step S5, the eluent for column chromatography purification consists of dichloromethane and methanol in a volume ratio of 100:1, the molar ratio of NA-4 and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile is 1:1, and the silica gel is 200-300 mesh. In step S6, the eluent for column chromatography purification consists of petroleum ether and ethyl acetate in a volume ratio of 8:1, and the molar ratio of NA-OH, 2,4-dinitrobenzenesulfonyl chloride and triethylamine is 1:1.5:
1. The silica gel is 200-300 mesh.
8. A method for detecting cysteine in biological samples, employing a near-infrared fluorescent probe as described in any one of claims 1-7, characterized in that: Includes the following steps, a) Introduce a near-infrared fluorescent probe into the biological sample to be tested; b) Induces fluorescence signal generation; c) Detect the resulting fluorescence signal to determine the presence or concentration of cysteine.
9. A method for detecting cysteine in biological samples according to claim 8, characterized in that: The biological samples are selected from living cells, tissues, body fluids, or organisms.
10. The method for detecting cysteine in biological samples according to claim 9, characterized in that: The detection method is used for imaging and visual tracking of endogenous and exogenous cysteine in living cells.