Fluorescent probe for detecting nitrite as well as preparation method and application of fluorescent probe
By using a fluorescent probe prepared from 1,8-naphthalimide and 4-nitrophenylpyruvic acid, the biocompatibility and synthetic complexity issues of existing fluorescent nanoclusters for nitrite detection are resolved, achieving highly sensitive, rapid, and simple nitrite detection suitable for in vivo detection.
Patent Information
- Application Number
- CN202511309138.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-15
- Publication Date
- 2025-11-18
AI Technical Summary
Existing fluorescent nanoclusters and methods for detecting nitrite suffer from problems such as low biocompatibility, complex synthesis, poor solubility, high toxicity, and insufficient environmental adaptability, making it difficult to meet the requirements for high sensitivity and real-time monitoring.
A green, non-toxic, and water-soluble fluorescent probe for the detection of nitrite was prepared via a simple synthetic route using 1,8-naphthalimide as the fluorescent group and 4-nitrophenylpyruvic acid as the leaving group.
A highly sensitive and rapid method for detecting nitrite has been developed, which has good biocompatibility and a simple preparation process, and is suitable for in vivo detection.
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Figure CN120965582A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of fluorescent probe, in particular to a fluorescent probe for detecting nitrite and a preparation method and application thereof. BACKGROUND
[0002] As a kind of chemical pollutants widely existing in natural environment and food processing system, nitrite has become an important monitoring object in the field of global public health safety due to its potential carcinogenic risk and acute toxicity effect. Under certain conditions, this kind of compound can react with amine to generate strong carcinogen nitrosamine, and its toxicity mechanism involves multiple pathological pathways such as inducing DNA oxidative damage and interfering with the oxygen carrying function of hemoglobin. The World Health Organization (WHO) has listed it as one of the four major food pollutants and has formulated strict limit standards (such as the drinking water limit of 3 mg / L). However, traditional detection technology faces multiple technical bottlenecks in practical application, and the development of new detection methods has become an important research direction in the cross field of analytical chemistry and food safety.
[0003] The pollution sources of nitrite show significant diversity characteristics: fertilizer residues in agricultural runoff, abuse of preservatives in food processing, industrial wastewater discharge, etc. all constitute the main input channels. Its toxicological effect has dose-dependent characteristics, and acute poisoning shows high methemoglobinemia, while long-term low-dose exposure is significantly positively correlated with the incidence of digestive tract tumors. It is worth noting that the dynamic transformation characteristics of nitrite in food system (such as the reduction of nitrate during vegetable storage) further increase the complexity of risk control, which puts higher requirements on the real-time monitoring ability of detection technology.
[0004] The current mainstream detection methods have significant differences in sensitivity, operation convenience and environmental adaptability: spectrophotometry (Griess method) is based on the diazotization-coupling color principle of aromatic amine, although it has the advantage of equipment popularity, but its detection limit (~μM level) is difficult to meet the demand of trace analysis. The interference of reducing substances (such as ascorbic acid) and colored components in complex matrix often leads to false positive / negative results, and the pretreatment step is complicated, which restricts the efficiency of on-site detection. Chemiluminescence method uses the catalytic characteristics of nitrite in luminol-H2O2 etc. light emitting system, which effectively avoids the light scattering interference without external excitation light source. However, the reaction system is sensitive to pH value, temperature and other parameters, and the stability problem of luminescent reagent limits the long-term reliability of the method. Electrochemical method modifies the electrode surface to catalyze the oxidation reaction of nitrite, which has the advantage of fast response. However, the repeatability difference of electrode polishing process leads to signal fluctuation (RSD>5%) between batches, and surface passivation phenomenon seriously affects the service life of the sensor, which is difficult to meet the demand of long-term continuous monitoring.
[0005] The new fluorescent detection method based on quantum dots and metal nanoclusters has achieved a breakthrough in detection sensitivity (nM level). The mechanism mainly involves: surface plasmon resonance effect enhances light capture efficiency, ligand-analyte specific interaction causes fluorescence quenching / restoration, and nanometer confinement effect regulates electron transfer path. However, there are still key technical bottlenecks in the existing system: biocompatibility defects: the cytotoxicity of noble metal-based materials (such as Au / Ag NCs) limits their application in vivo detection; complex synthesis process: particle size control requires strict thermodynamic regulation (such as microwave-assisted method), and batch repeatability is poor; insufficient environmental adaptability: hydrophobic nanoparticles are prone to non-specific aggregation in body fluids or food matrices, leading to signal distortion.
[0006] In summary, the existing fluorescent nanoclusters and methods for detecting nitrite have the advantages of fast detection speed, high sensitivity, etc. However, these fluorescent probes or fluorescent detection methods still have certain deficiencies, such as low biocompatibility, complex synthesis, poor solubility, and high toxicity. SUMMARY
[0007] The purpose of the present application is to solve the problems in the prior art and provide a fluorescent probe for detecting nitrite and a preparation method and application thereof.
[0008] A fluorescent probe for detecting nitrite, the fluorescent probe comprising 1,8-naphthalimide as a fluorescent group of the fluorescent probe, 4-nitrophenylpyruvic acid as a leaving group of the fluorescent probe, and the structural formula being:
[0009] Preferably, the synthesis route of the fluorescent probe is:
[0010]
[0011] A preparation method of a fluorescent probe for detecting nitrite, comprising the following steps:
[0012] Step one, 4-amino-1,8-naphthalic anhydride (100 mg, 0.469 mmol) and 4-(2-aminoethyl) morpholine (122.13 mg, 0.938 mmol) are dissolved in 20 mL of anhydrous ethanol, stirred in a round-bottom flask and heated to 90°C reflux for 12 h, vacuumed repeatedly, and nitrogen was introduced three times or more to remove oxygen, then the solvent was removed under vacuum, and the crude product was purified by silica gel column chromatography, using (dichloromethane / methanol = 10:1, v / v) as the eluent to purify the crude product, to obtain a yellow solid compound 1, yield (137 mg, 61.7%); the structural formula of compound 1 is Step two, compound 1 (162.57mg, 0.5mmol), 4-nitrophenylpyruvic acid (195.13mg, 1mmol) and dichlorosulfoxide (380.79mg, 3mmol) were dissolved in 50mL anhydrous ethanol, stirred in a round-bottom flask and heated to 90℃ reflux for 12h, vacuumed repeatedly, and then nitrogen was introduced for more than three times to remove oxygen, then the anhydrous ethanol was evaporated under vacuum, and the crude product was purified by silica gel column with (dichloromethane / methanol = 10:1, v / v) as eluent to obtain a yellow solid probe, yield (70.5mg, 43%).
[0013] The application of the fluorescent probe for detecting nitrite in detecting nitrite, the probe molecule is dissolved in PBS buffer (20.0mM, pH = 7.4), and the test is carried out at room temperature, the fluorescent probe has weak fluorescence in the PBS buffer (20.0mM, pH = 7.4), and the fluorescence emission peak after response to nitrite is at 535nm.
[0014] The beneficial effects of the present application are:
[0015] Compared with the existing fluorescent probe for detecting nitrite, the fluorescent probe prepared by the present application has the advantages of green and non-toxic, good water solubility, simple synthesis, short preparation time, good biocompatibility and the like; the nitrite detection method of the present application has the advantages of low cost, simple preparation, fast detection speed, high sensitivity, low detection limit and the like; through optimization of the preparation conditions and the detection system of the fluorescent probe, the self-assembled fluorescent probe with better performance and stability is obtained. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 The fluorescence spectrum of the fluorescent probe (10.0μM) of the present application in PBS buffer (20.0mM, pH = 7.4) in response to metal ions (200μM K + , Na + , Mg 2+ , Ca 2+ , Fe 2+ , Cu 2+ , Zn 2+ ), biological thiols (Cys, Hcy, GSH), RNS (200μM NO and NO 2- ) and ROS (100μM ClO - , -OH, ONOOCO 2- and 50μM H2O2) with concentrations of 0.1mM. The abscissa is different reagent groups, and the ordinate is the fluorescence intensity.
[0017] Figure 2The fluorescence spectrum of the fluorescent probe (10.0 μM) of the present application in PBS buffer (20.0 mM, pH = 7.4) in response to different concentrations of nitrite (0.0-100.0 μM). Excitation wavelength: 440 nm. The abscissa is the wavelength, and the ordinate is the fluorescence intensity.
[0018] Figure 3 The linear fitting relationship between the fluorescence intensity of the solution at 535 nm and the concentration of nitrite after the fluorescent probe (10.0 μM) of the present application in PBS buffer (20.0 mM, pH = 7.4) was acted on different concentrations of nitrite. The abscissa is the concentration of nitrite, and the ordinate is the fluorescence intensity.
[0019] Figure 4 The relationship between the fluorescence intensity of the solution at 535 nm and the time after the fluorescent probe (10.0 μM) of the present application in PBS buffer (20.0 mM, pH = 7.4) was acted on 100.0 μM of nitrite. The abscissa is the time, and the ordinate is the fluorescence intensity.
[0020] Figure 5 The cell imaging diagram after the fluorescent probe (10.0 μM) of the present application in living cells (A549) was acted on nitrite. DETAILED DESCRIPTION
[0021] Please refer to Figures 1 to 5 As shown in the structure formula, a fluorescent probe for detecting nitrite, the fluorescent probe comprises 1,8-naphthalimide as a fluorescent group of the fluorescent probe, 4-nitrophenyl pyruvic acid as a leaving group of the fluorescent probe, and the structure formula is:
[0022] The synthesis route of the fluorescent probe is as follows:
[0023]
[0024] A preparation method of a fluorescent probe for detecting nitrite, comprising the following steps:
[0025] Step one, 4-amino-1,8-naphthalic anhydride (100 mg, 0.469 mmol) and 4-(2-aminoethyl) morpholine (122.13 mg, 0.938 mmol) are dissolved in 20 mL of anhydrous ethanol, stirred in a round-bottom flask and heated to 90°C reflux for 12 h, repeatedly vacuumed, and then nitrogen is introduced for more than three times to remove oxygen, and then the solvent is removed under vacuum, and the crude product is purified by silica gel column chromatography, and the crude product is purified with (dichloromethane / methanol = 10:1, v / v) as an eluent to obtain a yellow solid compound 1, with a yield (137 mg, 61.7%); the structure formula of the compound 1 is Step two, compound 1 (162.57 mg, 0.5 mmol), 4-nitrophenylpyruvic acid (195.13 mg, 1 mmol) and dichlorosulfoxide (380.79 mg, 3 mmol) were dissolved in 50 mL of absolute ethanol, stirred in a round bottom flask and heated to 90 °C reflux for 12 h, vacuumed and purged with nitrogen three times above, remove oxygen, then evaporate the absolute ethanol under vacuum, and purify the crude product by silica gel column with (dichloromethane / methanol = 10:1, v / v) as eluent to obtain the yellow solid probe, yield (70.5 mg, 43%).
[0026] The application of the fluorescent probe for detecting nitrite in detecting nitrite, the probe molecule is dissolved in PBS buffer (20.0 mM, pH = 7.4), and the test is carried out at room temperature. The fluorescent probe has weak fluorescence in the PBS buffer (20.0 mM, pH = 7.4), and the fluorescence emission peak after response to nitrite is at 535 nm.
[0027] Example 1: Synthesis of compound 1:
[0028] 4-amino-1,8-naphthalene anhydride (100 mg, 0.469 mmol) and 4-(2-aminoethyl) morpholine (122.13 mg, 0.938 mmol) were dissolved in 20 mL of absolute ethanol, stirred in a round bottom flask and heated to 90 °C reflux for 12 h, vacuumed and purged with nitrogen three times above, remove oxygen, then remove the solvent under vacuum, purify the crude product by silica gel column chromatography, and purify the crude product with (dichloromethane / methanol = 10:1, v / v) as eluent to obtain compound 1 as a yellow solid, yield (137 mg, 61.7%), 1H NMR (400 MHz, DMSO-d6) δ 8.61 (dd, J = 8.4, 1.2 Hz, 1H), 8.42 (dd, J = 7.3, 1.0 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.3, 7.3 Hz, 1H), 7.47 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.14 (t, J = 7.0 Hz, 2H), 3.54 (t, J = 4.5 Hz, 4H), 2.54 (s, 2H), 2.46 (d, J = 4.7 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 164.25, 163.33, 153.22, 134.47, 131.51, 130.18, 129.82, 124.47, 122.19, 119.81, 108.63, 107.92, 66.67, 56.28, 53.89, 36.81. HRMS: m / z [C 18 H 20 N3O3]+ calcd 326.14992, found 326.14896.
[0029] Example 2: Synthesis of fluorescent probes:
[0030] Compound 1 (162.57 mg, 0.5 mmol), 4-nitrophenylpyruvic acid (195.13 mg, 1 mmol), and thionyl chloride (380.79 mg, 3 mmol) were dissolved in 50 mL of anhydrous ethanol. The mixture was stirred in a round-bottom flask and refluxed at 90 °C for 12 h. Oxygen was removed by repeatedly evacuating the flask under vacuum and purging with nitrogen three times or more. Then, anhydrous ethanol was evaporated under vacuum, and the crude product was purified by passing it through a silica gel column with dichloromethane / methanol = 10:1, v / v as the eluent to obtain a pale yellow solid probe NP, yield (70.5 mg, 43%). ¹H NMR (400 MHz, Chloroform-d) δ 9.92 (s, 1H), 8.75–8.66 (m, 4H), 8.45–8.37 (m, 2H), 8.33 (dd, J = 8.5, 1.1 Hz, 1H), 7.90 (dd, J = 8.5, 7.3 Hz, 1H), 4.36 (t, J = 6.9 Hz, 2H), 3.68 (t, J = 4.6 Hz, 4H), 2.72 (t, J = 6.9 Hz, 2H), 2.67–2.47 (m, 4H). ¹³C NMR(101MHz,Chloroform-d)δ185.32,163.91,157.92,137.10,136.62,132.87,132.12,131.60,129. 01,127.48,125.74,123.77,123.65,122.95,119.98,118.66,67.07,56.14,53.85,37.35.HRMS:m / z[C 26 H 23 N4O7] + calcd 503.15613,found 503.15546.
[0031] Example 3: Application of bright fluorescent probes:
[0032] The probe was dissolved in 20.0 mM PBSS buffer at pH 7.4 to prepare a probe solution of 1.0 × 10⁻⁵ mol / L. 20 equivalents of nitrite were added to the solution. When the excitation wavelength was 440 nm, the solution showed a clear emission peak at 535 nm. The fluorescent probe of this invention can be used to detect nitrite in intracellular pure water systems.
[0033] It should be noted that the above embodiments are only one of the preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. For those skilled in the art, all forms of equivalent substitutions or changes made to the technical solutions without departing from the essential concept of the present invention should be included within the scope of protection of the present invention.
Claims
1. A fluorescent probe for detecting nitrite, characterized in that: The fluorescent probe includes 1,8-naphthalimide as the fluorophore and 4-nitrophenylpyruvic acid as the leaving group, which has a rapid and sensitive reaction. Its structural formula is:
2. The fluorescent probe for detecting nitrite according to claim 1, characterized in that: The synthetic route of the fluorescent probe is as follows:
3. A method for preparing a fluorescent probe for detecting nitrite, used to prepare the fluorescent probe for detecting nitrite according to any one of claims 1 to 2, characterized in that: Includes the following steps: Step 1: Dissolve 4-amino-1,8-naphthoic anhydride (100 mg, 0.469 mmol) and 4-(2-aminoethyl)morpholine (122.13 mg, 0.938 mmol) in 20 mL of anhydrous ethanol. Stir and reflux at 90 °C for 12 h in a round-bottom flask. Repeat the vacuuming and nitrogen purging process three times or more to remove oxygen. Then, remove the solvent under vacuum and purify the crude product by silica gel column chromatography using (dichloromethane / methanol = 10:1, v / v) as the eluent to obtain compound 1 as a yellow solid, yield (137 mg, 61.7%). The structural formula of compound 1 is as follows: Step 2: Compound 1 (162.57 mg, 0.5 mmol), 4-nitrophenylpyruvic acid (195.13 mg, 1 mmol), and thionyl chloride (380.79 mg, 3 mmol) were dissolved in 50 mL of anhydrous ethanol. The mixture was stirred in a round-bottom flask and heated to 90 °C under reflux for 12 h. The oxygen was removed by repeatedly evacuating the vacuum and purging with nitrogen three times or more. Then, the anhydrous ethanol was evaporated under vacuum, and the crude product was purified by passing it through a silica gel column with dichloromethane / methanol = 10:1, v / v, to obtain a pale yellow solid probe with a yield of 70.5 mg (43%).
4. The application of the fluorescent probe for detecting nitrite according to any one of claims 1 to 2 in the detection of nitrite, characterized in that: The probe molecule was dissolved in PBS buffer (20.0 mM, pH=7.4) and tested at room temperature. The fluorescent probe showed weak fluorescence in PBS buffer (20.0 mM, pH=7.4), and the fluorescence emission peak after responding to nitrite was at 535 nm.