A highly selective and sensitive fluorescent probe for mercury ions, its preparation method, and its applications.

Abstract

This invention provides a highly selective and sensitive fluorescent probe for mercury ions, its preparation method, and its applications, belonging to the fields of chemical sensing materials and environmental monitoring technology. This invention connects 4-(1,2,2-triphenylvinyl)phenol, which exhibits aggregation-induced emission, and a guanosine derivative with multiple coordination sites, via a flexible chain to obtain a fluorescent probe with the structure shown in Formula I. This fluorescent probe not only overcomes the problems of large differences in solubility and reactivity between 4-(1,2,2-triphenylvinyl)phenol and guanosine derivatives, making coupling difficult, but also possesses high sensitivity and selectivity; its detection limit reaches 12.24 nM, effectively detecting trace mercury pollution in samples. It can also accurately identify mercury ions in water samples containing multiple high concentrations of metal ions, demonstrating high accuracy in real water samples. It is suitable for the accurate determination of mercury ions in complex environmental water samples and has good practical application value in the field of environmental monitoring. Formula I

Description

Technical Field

[0001] This invention belongs to the field of chemical sensing materials and environmental monitoring technology, specifically relating to a highly selective and highly sensitive mercury ion fluorescent probe, its preparation method, and its application. Background Technology

[0002] Mercury and its compounds, as highly toxic heavy metal pollutants, have long been a focus of attention in environmental science and public health. Mercury exists in nature in various forms, including divalent mercury ions (Hg). 2+ Hg is one of the most common forms of phytosanitary compounds in aquatic environments. Studies have shown that Hg... 2+ Mercury has extremely high lipophilicity, enabling it to penetrate biological membranes and accumulate in critical organs such as the nervous system, kidneys, and brain. Even low-concentration mercury exposure can lead to chronic poisoning, inducing tremors, memory loss, visual impairment, and even irreversible damage to embryonic development. Therefore, establishing a real-time, rapid, and accurate method for detecting mercury ions is of significant practical importance for environmental protection and human health.

[0003] Currently, commonly used methods for mercury ion detection in laboratories mainly include atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS), and electrochemical analysis. Although these traditional methods have low detection limits and wide linear ranges, they still have significant limitations in practical applications. First, these analytical devices are typically expensive and bulky, making real-time on-site monitoring difficult. Second, sample pretreatment is complex and time-consuming, failing to meet the needs of rapid screening. Furthermore, some physicochemical methods can damage the original state of the sample during detection. To overcome these shortcomings, developing a low-cost, easy-to-operate, rapid-response chemical sensing technology with visualization potential has become a research hotspot in recent years.

[0004] Among numerous detection methods, fluorescent probe methods have shown great promise due to their high sensitivity, good selectivity, fast response speed, and ability to achieve non-destructive imaging. However, existing mercury ion fluorescent probes still face many challenges in practical applications. For example, many probes lack selectivity in complex environmental samples (such as industrial wastewater containing multiple competing metal ions) and are easily interfered with by metal ions such as silver and iron; some probes have low sensitivity and cannot capture trace levels of mercury ion changes. In addition, the application of traditional "quenching" probes is greatly limited due to high background signals and a tendency to produce false negative results.

[0005] Therefore, developing a new type of fluorescent probe with high selectivity, high sensitivity, and the ability to accurately identify mercury ions through significant signal changes, in order to overcome the shortcomings of existing detection technologies and improve the accuracy and convenience of mercury ion monitoring, has become a key issue that urgently needs to be addressed in the field of sensing materials. Summary of the Invention

[0006] The purpose of this invention is to provide a mercury ion fluorescent probe with high selectivity and high sensitivity, its preparation method, and its application.

[0007] This invention provides a mercury ion fluorescent probe, the structure of which is as follows: In this context, R1 and R2 are independently C1 to C3 alkyl groups, and n is selected from integers from 1 to 3.

[0008] Furthermore, R1 and R2 are independently methyl groups.

[0009] Furthermore, the structure of the mercury ion fluorescent probe is as follows: .

[0010] This invention also provides a method for preparing a mercury ion fluorescent probe, comprising the following steps: ; (1) Compound 1 was dissolved in an organic solvent, and an aliphatic cyclic dianhydride and a catalyst were added to react and obtain compound 2. (2) Compound 2 and 4-(1,2,2-triphenylvinyl)phenol were dissolved in an organic solvent, and a catalyst and condensing agent were added. After the reaction was completed, the mercury ion fluorescent probe was obtained by washing, drying and purifying.

[0011] Further, the molar ratio of compound 1 to aliphatic cyclocarboxylic anhydride is (4~6):(5~7); the molar ratio of compound 2 to 4-(1,2,2-triphenylvinyl)phenol is 1:(0.8~1.2).

[0012] Further, the organic solvent is selected from at least one of acetone, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, N,N-dimethylacetamide, and acetonitrile; the catalyst is selected from at least one of triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, 4-dimethylaminopyridine, and imidazole; and the condensing agent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and O-benzotriazole-tetramethylurea tetrafluoroborate.

[0013] This invention also provides a kit for qualitative and / or quantitative detection of mercury ions in environmental water bodies, soil extracts, in vitro biological samples, or food samples, comprising the following components: Component 1: The above-mentioned mercury ion fluorescent probe; Component 2: Solvent system; the solvent system is a mixed solution of organic solvent and water; Component 3: Buffer solution.

[0014] Further, the organic solvent is selected from at least one of acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide, and methanol; the buffer solution is selected from at least one of HEPES, PBS, Tris-HCl, MOPS, and citrate-sodium citrate buffer solution; and the pH of the buffer solution is 6.0 to 8.0.

[0015] Furthermore, the pH of the buffer solution is 7.4.

[0016] Furthermore, the organic solvent is acetonitrile, and the volume ratio of acetonitrile to water is 1:1.

[0017] This invention also provides the application of a mercury ion fluorescent probe in the preparation of a mercury ion fluorescent detection kit.

[0018] Furthermore, the mercury ion fluorescence detection kit is used for the qualitative and / or quantitative detection of mercury ions in environmental water bodies, soil extracts, in vitro biological samples, or food samples.

[0019] Furthermore, the mercury ion fluorescence detection kit also includes a solvent system and a buffer solution; the solvent system is a mixed solution of an organic solvent and water; the organic solvent is selected from at least one of acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide, and methanol; the buffer solution is selected from at least one of HEPES, PBS, Tris-HCl, MOPS, and citrate-sodium citrate buffer solution; the pH of the buffer solution is 6.0~8.0.

[0020] Furthermore, the pH of the buffer solution is 7.4.

[0021] Furthermore, the organic solvent is acetonitrile, and the volume ratio of acetonitrile to water is 1:1.

[0022] The fluorescent probe of this invention is formed by the condensation of 4-(1,2,2-triphenylvinyl)phenol (TPE-OH), which exhibits aggregation-induced emission (AIE), and 2',3'-O-isopropylidene guanosine, which has multiple coordination sites, via a succinyl flexible chain. Firstly, the isopropylidene group selectively protects the 2' and 3' hydroxyl groups of the guanosine nucleoside, ensuring that subsequent reactions occur only at the 5' hydroxyl group. Subsequently, a hemisuccinyl group is introduced, providing not only a carboxyl site for the incorporation of the fluorescent group but also introducing a flexible carbon chain, optimizing the probe's coordination space for metal ions; thus providing a structural basis for the accurate detection of mercury ions.

[0023] The probe of this invention not only overcomes the problems of large differences in solubility and reactivity between TPE-OH (hydrophobic structure, large steric hindrance) and guanosine derivatives (strong polarity), making coupling difficult, but also possesses high sensitivity and selectivity. Experiments have shown that the detection limit of the probe reaches the nanomolar level (12.24 nM), far lower than conventional chemical analysis methods, effectively monitoring trace mercury pollution in drinking water sources. Simultaneously, it maintains high identification accuracy even in complex water samples containing multiple high concentrations of coexisting metal ions, significantly reducing the false positive rate in environmental monitoring. Furthermore, it exhibits high accuracy in complex real-world water samples, making it suitable for the accurate determination of mercury ions in real-world environmental water samples, and has significant practical application value in the field of environmental monitoring.

[0024] Furthermore, this invention uses inexpensive and readily available guanine nucleosides as raw materials, and can be synthesized through a simple three-step reaction. The reaction conditions are mild, making it easy to produce on an industrial scale. At the same time, the probe can work stably in common solvent systems such as CH3CN / H2O, without the need for complex instrumentation, which significantly reduces detection costs and operational barriers.

[0025] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0026] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0027] Figure 1 This is the 1H NMR spectrum of compound 2.

[0028] Figure 2 This is the mass spectrum of compound 2.

[0029] Figure 3This is the 1H NMR spectrum of XXG-TPE.

[0030] Figure 4 It is a mass spectrometer of XXG-TPE.

[0031] Figure 5 This is a comparison of the fluorescence response intensity of XXG-TPE to mercury ions in different solvent systems.

[0032] Figure 6 XXG-TPE in the CH3CN / H2O (1:1, v / v) system with Hg 2+ Changes in fluorescence emission spectrum with increasing concentration.

[0033] Figure 7 The fluorescence intensity of XXG-TPE at 468 nm and Hg 2+ Linear relationship fitting curve of concentration.

[0034] Figure 8 This is a test chart showing the selective performance of XXG-TPE against interference from other competing ions. Detailed Implementation

[0035] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0036] Example 1: Probe Synthesis and Characterization 1.1 Probe Synthesis The synthesis route is as follows: ; The specific steps include: (1) Guanosine (2.83 g, 10 mmol) was suspended in 100 mL of acetone, and 2,2-dimethoxypropane (12 mL) was added. Then, under ice bath conditions, perchloric acid (HClO4, 70% by mass, 1.2 mL) was added dropwise. After the addition was completed, the ice bath was removed, and the mixture was stirred vigorously at room temperature for 24 h. The conversion of the raw materials was monitored by thin-layer chromatography (TLC). When the reaction was completed, dilute ammonia was added to neutralize the solution to pH 7-8. At this time, the solution changed from turbid to clear. The acetone was removed by rotary evaporation under reduced pressure. The residue was extracted three times with dichloromethane (100 mL each time). The organic phases were combined, dried with anhydrous sodium sulfate, and purified by silica gel column chromatography after the solvent was evaporated (eluent volume ratio: dichloromethane: methanol = 100:1) to obtain a white foamy solid product compound 1 (2',3'-O-isopropylidene guanosine). (2) Compound 1 (1.61 g, 5 mmol) was dissolved in 30 mL of anhydrous DMF, and succinic anhydride (0.6 g, 6 mmol) was added. After the solid was completely dissolved, triethylamine (TEA, 1.5 mL) was slowly added. The mixture was stirred at room temperature for 12 h. After the reaction was complete as monitored by TLC, the DMF solvent was removed by rotary evaporation under reduced pressure. The remaining oily substance was purified by silica gel column chromatography (eluent volume ratio: dichloromethane: methanol = 100:5) to obtain the white powdery solid product compound 2 (5'-O-hemisuccinyl-2',3'-O-isopropylidene guanosine). (3) Compound 2 (0.42 g, 1 mmol), 4-(1,2,2-triphenylvinyl)phenol (TPE-OH, 0.35 g, 1 mmol) and 4-dimethylaminopyridine (DMAP, 0.02 g) were dissolved in 40 mL of anhydrous dichloromethane. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.23 g, 1.2 mmol) was added at 0 °C. The mixture was stirred for 24 h at room temperature. After the reaction was completed by TLC, the organic phase was washed successively with 0.1 mol / L dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent volume ratio: dichloromethane:methanol = 100:3) to obtain the white powdery solid product XXG-TPE (i.e., the probe).

[0037] 1.2 Characterization of the probe and its intermediate products The proton NMR spectrum of compound 2 is as follows: Figure 1 As shown, the mass spectrometry is as follows Figure 2 As shown; The proton NMR spectrum and mass spectrum of XXG-TPE are as follows: Figure 3 and Figure 4 As shown.

[0038] Experimental Example 1: Solvent responsiveness test of probe (XXG-TPE) to mercury ions 1.1 Experimental Methods Different solvent systems for XXG-TPE were prepared, with XXG-TPE at a concentration of 10 µM in each solvent system; then, 10 µM Hg of XXG-TPE was added to each solvent system. 2+ The changes in fluorescence intensity were tested; different solvent systems were: THF / H2O, Acetone / H2O, DMSO / H2O, DMF / H2O, CH3OH / H2O and CH3CN / H2O solutions with different volume ratios.

[0039] 1.2 Experimental Results like Figure 5As shown, XXG-TPE exhibits a significant solvent dependence. In the CH3CN / H2O (1:1, v / v) system, the addition of Hg... 2+ The fluorescence enhancement was highest afterward. Therefore, this system was chosen as the standard testing environment for all subsequent tests.

[0040] Experimental Example 2: Sensitivity and Detection Limit Test of Probe (XXG-TPE) for Mercury Ions 2.1 Experimental Methods A 10 µM XXG-TPE solution was prepared using CH3CN / H2O (1:1, v / v) as solvent and 10 mM HEPES as buffer solution at pH 7.4. Different concentrations of Hg were then added dropwise to the XXG-TPE solution. 2+ (0~12µM), record the changes in fluorescence spectrum.

[0041] 2.1 Experimental Results like Figure 6 and Figure 7 As shown: with Hg 2+ With increasing concentration, the fluorescence intensity of XXG-TPE at 468 nm showed a significant increasing trend, and the fluorescence intensity was related to Hg. 2+ The concentration showed a good linear relationship in the range of 0–12 μM, with a linear regression equation of F = 74.63x - 10.92 and R0. 2 =0.991, and calculated according to the 3σ / k formula, the limit of detection (LOD) for mercury ions by XXG-TPE is as low as 12.24 nM.

[0042] Experiment Example 3: Selectivity and Anti-interference Test of Probe (XXG-TPE) for Mercury Ions 3.1 Experimental Methods Using CH3CN / H2O (1:1, v / v) as solvent and 10 mM HEPES as buffer solution, pH=7.4, prepare a 10 µM XXG-TPE solution, and then add 25 µM of other common metal ions (such as...) to each solution. Figure 8 (as shown) and 5µM Hg 2+ .

[0043] 3.1 Experimental Results like Figure 8 As shown, only Hg 2+ It can significantly enhance the fluorescence signal of the probe, while other interfering ions do not cause obvious signal fluctuations; even in complex systems where multiple interfering ions coexist, XXG-TPE effectively enhances the fluorescence signal of Hg. 2+ The identification still exhibits extremely high accuracy, which fully demonstrates the effectiveness of the probe of this invention in identifying Hg. 2+ It has high selectivity.

[0044] Experiment Example 4: The probe (XXG-TPE) was used for the detection of mercury ions in real water samples. 4.1 Experimental Methods Using CH3CN / real water sample (1:1, v / v) as solvent and 10 mM HEPES as buffer solution, pH=7.4, a 10 µM XXG-TPE solution was prepared. Using a spiked recovery method, 3 μM or 6 μM Hg was added to water samples taken from laboratory tap water and the Jiang'an River. 2+ Fluorescence detection was performed using XXG-TPE, and the recovery rate was calculated.

[0045] 4.2 Experimental Results As shown in Table 1, in real water samples, the recovery rate of mercury ions by XXG-TPE is 95.8%~108.4%, and the relative standard deviation (RSD) is less than 3%, indicating that the probe of the present invention can effectively overcome the interference of complex water matrices and accurately detect the mercury ion content in complex water samples.

[0046] Table 1. Test results of mercury ion recovery rate of XXG-TPE in real water samples. In summary, this invention links 4-(1,2,2-triphenylvinyl)phenol (TPE-OH) with 2',3'-O-isopropylguanosine via a succinyl flexible chain to obtain the fluorescent probe XXG-TPE. It exhibits a significant solvent dependence; in a CH3CN / H2O (1:1, v / v) system, it shows good resistance to Hg... 2+ It exhibits optimal responsiveness. XXG-TPE is effective against Hg. 2+ With high sensitivity and selectivity, its detection limit reaches the nanomolar level (12.24 nM), far lower than conventional chemical analysis methods, enabling effective monitoring of trace mercury pollution in drinking water sources. Simultaneously, it maintains high identification accuracy even in complex water samples containing multiple high concentrations of coexisting metal ions, significantly reducing false positive rates in environmental monitoring. XXG-TPE also demonstrates high accuracy in complex real-world water samples, making it suitable for the accurate determination of mercury ions in real-world environmental water samples, and possessing significant practical application value in the field of environmental monitoring.

Claims

1. A mercury ion fluorescent probe, characterized in that, The structure of the mercury ion fluorescent probe is as follows: In this context, R1 and R2 are independently C1 to C3 alkyl groups, and n is selected from integers from 1 to 3.

2. The mercury ion fluorescent probe according to claim 1, characterized in that, R1 and R2 are methyl groups independently.

3. The mercury ion fluorescent probe according to claim 2, characterized in that, The structure of the mercury ion fluorescent probe is as follows: 。 4. The method for preparing the mercury ion fluorescent probe according to any one of claims 1 to 3, characterized in that, Includes the following steps: ； (1) Compound 1 was dissolved in an organic solvent, and an aliphatic cyclic dianhydride and a catalyst were added to react and obtain compound 2. (2) Compound 2 and 4-(1,2,2-triphenylvinyl)phenol were dissolved in an organic solvent, and a catalyst and condensing agent were added. After the reaction was completed, the mercury ion fluorescent probe was obtained by washing, drying and purifying.

5. The preparation method according to claim 4, characterized in that, The molar ratio of compound 1 to aliphatic cyclodian anhydride is (4~6):(5~7); the molar ratio of compound 2 to 4-(1,2,2-triphenylvinyl)phenol is 1:(0.8~1.2).

6. The preparation method according to any one of claims 4 to 5, characterized in that, The organic solvent is selected from at least one of acetone, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, N,N-dimethylacetamide, and acetonitrile; the catalyst is selected from at least one of triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, 4-dimethylaminopyridine, and imidazole; the condensing agent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and O-benzotriazole-tetramethylurea tetrafluoroborate.

7. The use of the mercury ion fluorescent probe according to any one of claims 1 to 3 in the preparation of a mercury ion fluorescent detection kit.

8. The application according to claim 7, characterized in that, The mercury ion fluorescence detection kit is used for the qualitative and / or quantitative detection of mercury ions in environmental water bodies, soil extracts, in vitro biological samples, or food samples.

9. The application according to any one of claims 7 to 8, characterized in that, The mercury ion fluorescence detection kit further includes a solvent system and a buffer solution; the solvent system is a mixed solution of an organic solvent and water, wherein the organic solvent is selected from at least one of acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide, and methanol; the buffer solution is selected from at least one of HEPES, PBS, Tris-HCl, MOPS, and citrate-sodium citrate buffer solution; and the pH of the buffer solution is 6.0~8.

0.

10. The application according to claim 9, characterized in that, The organic solvent is acetonitrile, and the volume ratio of acetonitrile to water is 1:1.

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