Difunctional fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a difunctional fluorescent probe as well as a preparation method and application thereof. The difunctional fluorescent probe NDBC is successfully designed and synthesized by taking naphthalimide as a fluorophore, taking acylhydrazine as a connecting agent and taking benzothiophene as a fluorophore through acylhydrazine condensation reaction; the NDBC can be used for continuously detecting Hg < 2 + > and nitenpyram based on'off-on 'fluorescence signal change. When Hg < 2 + > is added into an NDBC solution, yellow fluorescence is rapidly quenched, and the detection limit of Hg < 2 + > is 0.12 mu M. After nitenpyram is introduced into the NDBC-Hg < 2 + > complex, the fluorescence recovers to the original level, and the detection limit of nitenpyram is 0.31 [mu] M. In an actual water sample, the recovery rate of the nitenpyram is 95.82%-101.15%, and the recovery rate of the nitenpyram is 0.83%-4.59%. In addition, fluorescence imaging of Hg < 2 + > and nitenpyram in living cells and zebra fish bodies is successfully realized.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and particularly relates to a bifunctional fluorescent probe, its preparation method, and its application. Background Technology

[0002] With the rapid development of industry and agriculture, large amounts of heavy metals and pesticide residues are released into soil and water bodies. Mercury ions (Hg) 2+ Hg is a well-known toxic heavy metal that accumulates through water transfer and the food chain, posing significant environmental and health risks. It negatively impacts not only the growth and reproduction of aquatic and terrestrial plants but also damages the human nervous system and kidneys, and affects the digestive and immune systems. Long-term exposure to Hg... 2+ It can cause irreversible damage and may endanger life.

[0003] Pesticide residues are considered a major global problem. Acetaminophen, with its advantages of high efficiency, low toxicity, systemic absorption, and no cross-resistance, is widely used in the control of pests in crops such as rice and vegetables. Hg in industrial wastewater... 2+ The presence of Hg and the accumulation of acetamiprid residues in soil pose a significant environmental challenge. Improper handling can lead to water and soil pollution, ultimately entering the human body through the food chain. Therefore, developing methods for monitoring Hg is crucial. 2+ New technologies for acetamiprid are crucial.

[0004] Traditional analytical techniques for detecting heavy metal ions and pesticide residues remain fundamental in environmental monitoring, food safety, and analytical chemistry. Specifically, techniques targeting mercury ions (Hg) are crucial. 2+ For the detection of Hg, atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) are widely used. In the detection of the neonicotinoid pesticide acetamiprid, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) are considered the gold standard methods. In addition, high-performance liquid chromatography (HPLC) with multiple detectors and enzyme-linked immunosorbent assay (ELISA) are also commonly used for routine screening and quantitative analysis. These traditional analytical techniques suffer from low selectivity, complex operation, high cost, and the inability to detect Hg at the cellular or subcellular level. 2+ And the limitations of acetamiprid. Fluorescence methods, with their rapid reaction, intuitive visual detection, and non-toxic fluorescent probes, show great promise in the development of portable detection tools. Although detection of Hg has been reported... 2+ Alternatively, acetamiprid may be used as a fluorescent probe, but few probes can simultaneously detect Hg. 2+ And acetamiprid. Furthermore, it can be detected in organisms such as zebrafish for Hg. 2+ The use of acetamiprid is also very limited. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bifunctional fluorescent probe, its preparation method, and its application. This invention designs and synthesizes a naphthalimide-based bifunctional fluorescent probe for detecting Hg. 2+ And acetamiprid. The probe NDBC (i.e., a bifunctional probe) specifically interacts with Hg. 2+ This combination induces a light quenching effect. Subsequently, acetamiprid can capture Hg from the complex through strong chelation. 2+ This leads to fluorescence recovery and triggers an "on" reaction. This "off-on" mode allows for continuous detection of Hg. 2+ And acetamiprid; in addition, NDBC and NDBC-Hg 2+ Complex in the detection of Hg in living cells 2+ The method exhibits excellent imaging capabilities and low cytotoxicity in the detection of acetamiprid, expanding its application in bioanalysis. This method can detect Hg in real water samples. 2+ It can be detected in conjunction with acetamiprid. Furthermore, NDBC can monitor Hg levels in zebrafish in real time. 2+ And the residual amount of acetamiprid.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a bifunctional fluorescent probe, the chemical structure of which is shown below: .

[0008] Secondly, the present invention also provides a method for preparing the aforementioned bifunctional fluorescent probe, comprising the following steps:

[0009] Intermediate compound 2 was coupled with benzothiophene-2-carboxylic acid to obtain a bifunctional fluorescent probe;

[0010] Thirdly, the present invention also provides a bifunctional fluorescent probe for recognizing Hg in cells, soil, wastewater, or solution. 2+ Applications.

[0011] Fourthly, the present invention also provides an NDBC-Hg 2+ Application of the complex in recognizing acetamiprid in cells, soil, wastewater or solution.

[0012] Fifthly, the present invention also provides an application of the aforementioned bifunctional fluorescent probe in cell imaging.

[0013] Sixthly, the present invention also provides an NDBC-Hg 2+ Application of the complex in cell imaging after treatment with acetamiprid.

[0014] The bifunctional fluorescent probe, its preparation method, and its application of the present invention have the following advantages compared with the prior art:

[0015] This invention utilizes naphthalimide as the fluorescent group, acylhydrazine as the linker, and benzothiophene as the fluorescent group to successfully design and synthesize a bifunctional fluorescent probe, N'-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)benzo[b]thiophene-2-formylhydrazine (NDBC), via an acylhydrazine condensation reaction. NDBC can continuously detect Hg based on "off-on" fluorescence signal changes. 2+ And acetamiprid. When Hg 2+ When added to NDBC solution, the yellow fluorescence was quenched abruptly, and the effect on Hg... 2+ The detection limit was 0.12 μM. (In NDBC-Hg) 2+ After introducing acetamiprid into the complex, the fluorescence returned to its original level, and the detection limit for acetamiprid was 0.31 μM. In actual water samples, the recoveries of acetamiprid were 95.82%–101.15% and 0.83%–4.59%, respectively. Furthermore, the detection of Hg in live cells and zebrafish was successfully achieved. 2+ Fluorescent imaging of acetamiprid. Therefore, the fluorescent probe of this invention can be used to detect Hg in the environment. 2+ And acetamiprid. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The infrared spectrum of the bifunctional fluorescent probe NDBC prepared in Example 1;

[0018] Figure 2 The proton spectrum of the bifunctional fluorescent probe NDBC prepared in Example 1 ( 1 H NMR spectrum;

[0019] Figure 3 The carbon spectrum of the bifunctional fluorescent probe NDBC prepared in Example 1 ( 13 C NMR spectrum;

[0020] Figure 4 The image shows the high-resolution mass spectra (HRMS) of the bifunctional fluorescent probe NDBC prepared in Example 1.

[0021] Figure 5The effect of solvent on the fluorescence spectrum and UV-Vis absorption spectrum of NDBC;

[0022] Figure 6 The effect of ethanol-water mixed solvent ratio on the fluorescence properties of NDBC;

[0023] Figure 7 The effects of different cations and anions on the fluorescence and UV-Vis absorption spectra of NDBC;

[0024] Figure 8 For other metal ions to Hg 2+ The effects of fluorescence interference, different pH values ​​on NDBC, and the addition of Hg were observed. 2+ The effect of subsequent fluorescence interference;

[0025] Figure 9 The fluorescence emission characteristics of NDBC vary with Hg 2+ Fluorescence spectra of concentration gradient changes and NDBC fluorescence intensity versus Hg 2+ Quantitative correlation of concentration;

[0026] Figure 10 Determination of NDBC and Hg using Job's plot method 2+ Binding rate, NDBC to Hg 2+ of 1 1H NMR titration, NDBC and Hg 2 + Interaction mechanism;

[0027] Figure 11 To add Hg 2+ ESI-MS spectrum of NDBC after NDBC;

[0028] Figure 12 For NDBC and NDBC-Hg 2+ fluorescence lifetime, NDBC and NDBC-Hg 2+ The ultraviolet-visible absorption spectrum;

[0029] Figure 13 For different pesticides to target NDBC-Hg 2+ The influence of fluorescence response and UV-Vis absorption characteristics of the composite system;

[0030] Figure 14 For other pesticides against NDBC-Hg 2+ Detection of fluorescence interference effects of acetamiprid, NDBC-Hg 2+ Fluorescence spectra of solutions after adding different concentrations of acetamiprid and NDBC-Hg 2+ The linear relationship between fluorescence intensity and acetamiprid concentration;

[0031] Figure 15 Laser confocal fluorescence microscopy analysis of HepG2 cells under different culture conditions;

[0032] Figure 16 Fluorescence imaging images of zebrafish under different conditions. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more comprehensive description of the invention will be provided below in conjunction with specific embodiments. Preferred embodiments of the invention are given in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0034] The order in which the embodiments are described below is not intended to limit the preferred order of the embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0035] This invention provides a bifunctional fluorescent probe, the chemical structure of which is shown below: .

[0036] The preparation method of the above-mentioned bifunctional fluorescent probe includes the following steps:

[0037] Intermediate compound 2 was coupled with benzothiophene-2-carboxylic acid to obtain a bifunctional fluorescent probe;

[0038] The structural formula of intermediate compound 2 is shown below:

[0039] .

[0040] In some embodiments, the method for preparing intermediate compound 2 includes:

[0041] 4-Bromo-1,8-naphthoic anhydride was reacted with n-butylamine to give intermediate compound 1;

[0042] Intermediate compound 1 was reacted with hydrazine hydrate to obtain intermediate compound 2;

[0043] The structural formula of intermediate compound 1 is shown below:

[0044] .

[0045] In some embodiments, 4-bromo-1,8-naphthoic anhydride is dissolved in ethanol, n-butylamine is added, and the mixture is refluxed under an inert atmosphere to give intermediate compound 1;

[0046] Intermediate compound 1 and hydrazine hydrate were dissolved in ethylene glycol monomethyl ether and refluxed under an inert atmosphere (such as nitrogen, helium, neon, argon, etc.) to obtain intermediate compound 2;

[0047] Intermediate compound 2 and benzothiophene-2-carboxylic acid were dissolved in acetonitrile, and coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, and N,N-diisopropylethylamine were added. The mixture was refluxed to obtain a bifunctional fluorescent probe.

[0048] In some embodiments, in the step of preparing intermediate compound 1, the molar volume ratio of 4-bromo-1,8-naphthoic anhydride, n-butylamine, and ethanol is (0.01~0.02) mol:(0.06~0.07) mol:(25~35) mL, the reflux reaction temperature is 120~125℃, and the time is 6~8 h;

[0049] In the step of preparing intermediate compound 2, the molar volume ratio of intermediate compound 1, hydrazine hydrate, and ethylene glycol monomethyl ether is (1.6~2.6) mmol:(2~3) mL:(25~35) mL, the reflux reaction temperature is 130~135℃, and the time is 0.5~1 h;

[0050] The molar volume ratio of intermediate compound 2, benzothiophene-2-carboxylic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, N,N-diisopropylethylamine, and acetonitrile was (5~10) mmol:(5~10) mmol:(0.01~0.02) mol:(0.01~0.02) mol:(0.02~0.03) mol:(20~25) mL. In the step of obtaining the bifunctional fluorescent probe by reflux reaction, the reflux reaction temperature was 85~90℃ and the time was 10~12 h.

[0051] Specifically, the synthetic route of the bifunctional fluorescent probe of the present invention is as follows:

[0052]

[0053] Among them, 4-bromo-1,8-naphthoic anhydride (CAS No. 81-86-7, molecular formula C) 12H5BrO3) was dissolved in ethanol, and n-butylamine (CAS Registry No. 109-73-9, chemical formula C4H) was added. 11 N), reacted under an inert atmosphere by reflux to give intermediate compound 1 (compound number 1 in the above synthetic route); intermediate compound 1 was dissolved with hydrazine hydrate (N2H4·H2O) in ethylene glycol monomethyl ether (CAS No. 109-86-4, molecular formula C3H8O2), and reacted under an inert atmosphere by reflux to give intermediate compound 2 (compound number 2 in the above synthetic route); intermediate compound 2 and benzothiophene-2-carboxylic acid (CAS No. 6314-28-9, molecular formula: C9H6O2S) were dissolved in acetonitrile solvent, and coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, CAS No. 1892-57-5, chemical formula C8H) was added respectively. 17 N3), hydroxybenzotriazole (HOBt, chemical formula C6H5N3O, CAS number 2592-95-2), N,N-diisopropylethylamine (DIEA, chemical formula C8H5N3O) 19 N, CAS No. 7087-68-5).

[0054] Based on the same inventive concept, the present invention also provides the above-mentioned bifunctional fluorescent probe for recognizing Hg in cells, soil, wastewater or solution. 2+ Applications.

[0055] In some embodiments, when the bifunctional fluorescent probe identifies Hg in the solution 2+ When used, the solvent in the solution includes ethanol.

[0056] Based on the same inventive concept, this invention also provides an NDBC-Hg 2+ Application of the complex in recognizing acetamiprid in cells, soil, wastewater, or solution; among which, NDBC-Hg 2+ The coordination compounds were obtained by the following methods:

[0057] The above-mentioned bifunctional fluorescent probe was added to a solution containing Hg. 2+ In the solution, NDBC-Hg was obtained. 2+ Coordination compounds.

[0058] Based on the same inventive concept, the present invention also provides an application of the above-mentioned bifunctional fluorescent probe in cell imaging.

[0059] Based on the same inventive concept, the present invention also provides the above-mentioned NDBC-Hg 2+ Application of the complex in cell imaging after treatment with acetamiprid.

[0060] This invention utilizes naphthalimide as the fluorescent group, acylhydrazine as the linker, and benzothiophene as the fluorescent group to successfully design and synthesize a bifunctional fluorescent probe, N'-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)benzo[b]thiophene-2-formylhydrazine (NDBC), via an acylhydrazine condensation reaction. NDBC can continuously detect Hg based on "off-on" fluorescence signal changes. 2+ And acetamiprid. When Hg 2+ When added to NDBC solution, the yellow fluorescence was quenched abruptly, and the effect on Hg... 2+ The detection limit was 0.12 μM. (In NDBC-Hg) 2+ After introducing acetamiprid into the complex, the fluorescence returned to its original level, and the detection limit for acetamiprid was 0.31 μM. In actual water samples, the recoveries of acetamiprid were 95.82%–101.15% and 0.83%–4.59%, respectively. Furthermore, the detection of Hg in live cells and zebrafish was successfully achieved. 2+ Fluorescent imaging of acetamiprid. Therefore, the fluorescent probe of this invention can be used to detect Hg in the environment. 2+ And acetamiprid.

[0061] The following specific embodiments further illustrate the bifunctional fluorescent probe of the present invention, its preparation method, and its application. This section further explains the content of the present invention in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0062] All reagents used in the following examples were analytical grade (AR) and commercially available without further purification; measurements from 400 to 4000 cm⁻¹ were recorded using the KBr method on a Bruker ALPHA-T infrared spectrophotometer (Bruker Corp., USA). -1 Fourier transform infrared (FT-IR) spectra within the specified range. Using DMSO-d6 as solvent and tetramethylsilane (TMS) as an internal standard, the spectra were obtained on a Bruker AV600 NMR spectrometer (Bruker, Bremen, Germany). 1 H NMR and 13C10 NMR spectroscopy. High-resolution mass spectrometry (HRMS) was performed using an I-Class / Xevo G2-XS QTOF high-resolution time-of-flight liquid chromatography-mass spectrometer (Waters, USA). Fluorescence spectra were obtained using an F4700 fluorescence spectrophotometer (Hitachi, Ltd., Tokyo, Japan). Absorption spectra were obtained using a Shimadzu UV-2700 UV-Vis spectrophotometer (Bruker Corp., Billerica, MA, USA). Fluorescence lifetimes were obtained using an FLS1000 steady-state and lifetime fluorescence spectrometer (Edinburgh Instruments Ltd., Livingston, UK). pH values ​​were measured using a pH - 3c pH meter (Inesa, Beijing, China). Cell images were recorded using a LEICA SP2 confocal laser scanning microscope (LEICA, Germany). A Spark10M multimode microplate reader (Tecan, Switzerland) was used. All experiments were performed at room temperature using 10 mm quartz cells.

[0063] In the following examples, the HEPES buffer solution was prepared as follows: 119.2 g of HEPES was dissolved in 400 mL of ultrapure water, the pH was adjusted to 7.0 with 1.0 M NaOH solution, and then diluted with 500 mL of ultrapure water to prepare 2-[4-(2-hydroxyethyl)piperate-1-yl]ethanesulfonic acid (HEPES) buffer solution.

[0064] Example 1

[0065] This embodiment provides a method for preparing a bifunctional fluorescent probe, including the following steps:

[0066] S1. Dissolve 4.0 g (0.01 mol) of 4-bromo-1,8-naphthoic anhydride in 25 mL of ethanol, then add 6 mL (0.06 mol) of n-butylamine dropwise, and reflux at 120 °C for 6 h under nitrogen protection. After the reaction is complete, cool to room temperature and recrystallize from ethanol to obtain a yellow solid, which is intermediate compound 1.

[0067] S2. Intermediate compound 1 (2.0 g, 1.6 mmol) and hydrazine hydrate (2.0 mL, 5.3 mmol) were co-dissolved in 25 mL of ethylene glycol monomethyl ether solvent. The mixture was refluxed in an oil bath at 130 °C under N2 protection for 30 min. After naturally cooling to room temperature, the precipitated solid product was collected by filtration. The crude product was purified by silica gel column chromatography with ethyl acetate / petroleum ether (2:3, V / V) to finally obtain intermediate compound 2.

[0068] S3. Intermediate compound 2 (5 mmol) and benzothiophene-2-carboxylic acid (5 mmol) were dissolved in 20 mL of acetonitrile. Coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.01 mol), hydroxybenzotriazole (0.012 mol), and N,N-diisopropylethylamine (0.02 mol) were added, respectively. The mixture was refluxed at 85 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The obtained solid was purified by silica gel column chromatography using CH2Cl2 / CH3OH (15:1, V / V) as the eluent to obtain yellow-green solid NDBC with a yield of 44.3% and a melting point of 252.1~252.5 °C, which is the bifunctional fluorescent probe (NDBC).

[0069] Bifunctional fluorescent probe structure characterization

[0070] Figure 1 The infrared spectrum of the bifunctional fluorescent probe prepared in Example 1;

[0071] Figure 2 The proton spectrum of the bifunctional fluorescent probe prepared in Example 1 ( 1 H NMR spectrum;

[0072] Figure 3 The carbon spectrum of the bifunctional fluorescent probe prepared in Example 1 ( 13 C NMR spectrum;

[0073] Figure 4 The image shows the HRMS mass spectrum of the bifunctional fluorescent probe prepared in Example 1.

[0074] Through IR, 1 H NMR, 13 The structural features of the bifunctional fluorescent probe were analyzed by C NMR and HRMS.

[0075] The infrared spectral data are as follows:

[0076] IR (KBr, v, cm -1 ): 3277.06-3238.48 (NH), 3057.17-2931.80 (CH), 1697.36 (C=O);

[0077] Hydrogen spectrum (H NMR) 1 The H NMR (H NMR) data are as follows:

[0078] 1H NMR (600 MHz, DMSO-d6) δ 11.17 (s, 1H, NH), 9.89 (s, 1H, NH), 8.76 (m, 1H, Ar-H), 8.52 (m, 1H, Ar-H), 8.37-8.33 (m, 2H, Ar-H), 8.10 (m, 1H,Ar-H), 8.05 (m, 1H, Ar-H), 7.82 (m, 1H, Ar-H), 7.56-7.48 (m, 2H, Ar-H), 7.05(m, 1H, Ar-H), 4.03 (t, J = 7.5 Hz, 2H, CH), 2.53 – 2.49 (m, 3H, CH), 1.60(m, 2H, CH), 1.35 (m, 2H, CH), 0.93 (t, J = 7.4 Hz, 3H, CH);

[0079] Carbon spectrum (CMR) 13 The C NMR (chromatogram) data is as follows:

[0080] 13C NMR (151 MHz, DMSO-d6) δ 166.25, 166.22, 166.20, 160.50, 145.55,145.50, 142.37, 142.32, 137.26, 137.25, 135.90, 128.61, 128.56, 128.51,127.70, 127.69, 127.66, 127.65, 127.63, 127.30, 127.28, 127.01, 126.96,126.94, 126.93, 126.22, 126.19, 126.16, 126.09, 126.04, 125.97, 125.95, 125.91, 125.89, 125.86, 125.85, 125.80, 125.48, 125.46, 125.41, 125.40, 123.57, 123.55, 123.52, 123.35, 123.34, 123.33, 123.28, 123.27, 119.10, 119.08, 119.04, 111.66, 111.64, 111.61, 111.59, 40.97, 40.95, 40.94, 40.92, 29.07, 29.05, 29.03, 29.01, 28.99, 19.65, 19.63, 19.60, 19.57, 13.65, 13.63, 13.62, 13.59;

[0081] The HRMS mass spectrometry data are as follows:

[0082] HRMS (ESI) [M+H] + The theoretical value is 444.1383, and the actual measured value is [M+H]. + The value is 444.1378, with an error of -1.12 ppm.

[0083] Based on the above data, the target compound prepared in Example 1 is N'-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)benzo[b]thiophene-2-formylhydrazine (NDBC).

[0084] Effects of different solvents on the photophysical properties of NDBC

[0085] Using CH3CN, DMSO, DMF, EtOH, CH3OH, CH3COCH3, and H2O as solvents, NDBC was added to each solvent to achieve a concentration of 10. -5M was used to obtain the probe solution; the fluorescence spectrum and UV-Vis absorption spectrum of the probe solution were tested, and the results are as follows. Figure 5 As shown.

[0086] Figure 5 In the image (a), the effect of the solvent on the fluorescence spectrum of NDBC is shown, and in the image (b), the effect of the solvent on the UV-Vis absorption spectrum of NDBC is shown.

[0087] from Figure 5 As can be seen, the luminescence properties of NDBC are significantly related to the solvent. When ethanol is used as the solvent, the fluorescence intensity and UV-Vis absorbance of NDBC reach the optimal level.

[0088] The effects of different ethanol / water solvent ratios on the fluorescence spectra of NDBC were further investigated; specifically, ethanol and water were used as a mixed solvent, and NDBC was added to the mixed solvent to achieve a concentration of 10. -5 M, the probe solution was obtained, and the fluorescence spectrum of the probe solution was tested. The results are as follows: Figure 6 As shown; wherein, the volume ratio of ethanol and water in the mixed solvent is adjusted to (1~9):(9~1).

[0089] from Figure 6 As can be seen, when the volume ratio of ethanol to water is 5:5 (i.e., 1:1), the fluorescence intensity of the mixed system reaches its peak and exhibits a characteristic red shift. Based on photophysical performance and biocompatibility evaluation, CH3CH2OH:HEPES (using HEPES buffer instead of water, with a volume ratio of ethanol to HEPES buffer of 1:1, V / V, pH = 7.0) was finally selected as the optimal detection system.

[0090] NDBC vs Hg 2+ Specific identification

[0091] Using KCl, NaCl, AgCl, CuCl2, CaCl2, MgCl2, ZnCl2, SnCl2, FeCl2, PbCl2, HgCl2, CoCl2, MnCl2, CuCl2, BaCl2, NiCl2, FeCl3, AlCl3, and CrCl3 as raw materials, 10... -2 M metal ion solution; concentrations of 10 were prepared in ultrapure water. -2 M contains F - Cl - ,Br - Cl - I - CN - SCN - NO2 - NO3 - ClO4- SO4 2- HSO4 - H2PO4 - S2O3 2- CrO4 2- SO3 2- HPO4 2- CO3 2- HCO3 - P2O7 4- S2O5 2- Tetrabutylammonium salt solution.

[0092] Mix equal volumes of EtOH (ethanol) and HEPES buffer, then add NDBC to bring the concentration to 10. -5 M, yielding the probe solution (pH 7) (corresponding to...) Figure 7 (CK); The probe solution was mixed with metal ion solutions containing different metal ions (the concentration of metal ions after mixing was 50 μM) to prepare multiple 10 mL test samples, and UV-Vis and fluorescence spectroscopy were performed respectively; The probe solution was also mixed with tetrabutylammonium salt solutions containing different anions (the concentration of anions after mixing was 50 μM) to prepare multiple 10 mL test samples, and UV-Vis and fluorescence spectroscopy were performed respectively; The results are as follows. Figure 7 As shown.

[0093] Figure 7 (a) and (c) show the fluorescence spectra of NDBC for different cations and anions, and a comparison of photographs of cations and anions under UV light; (b) and (d) show the UV-Vis absorption spectra of NDBC, and everyday photographs of cations and anions under natural light.

[0094] from Figure 7 As can be seen from the data, the addition of 50 μM Hg... 2+ Subsequently, the fluorescence of the NDBC probe was quenched, and its color darkened. In contrast, the fluorescence intensity remained almost unchanged after the addition of other metal ions. Figure 7 (a) Add Hg 2+ The absorbance increases after the addition of other metal ions, but the absorbance remains almost unchanged. Figure 7 (b). After the addition of anion exchange solution, the fluorescence spectrum of probe NDBC ( Figure 7 c) and ultraviolet absorption spectrum ( Figure 7 (d) showed almost no change. The above results indicate that, compared to other ions, Hg... 2+ The fluorescence of the NDBC probe can be selectively quenched. Therefore, the NDBC probe can specifically recognize Hg in the environment. 2+ .

[0095] Metal ions and different pH values ​​affect the detection of Hg by NDBC. 2+ Impact of interference performance

[0096] Following the method described above, mix equal volumes of EtOH (ethanol) and HEPES buffer, then add NDBC to achieve a concentration of 10. -5 M, yielding the probe solution (pH 7) (corresponding to...) Figure 8 (CK), then add Hg 2+ (The concentration was 50 μM, added in the form of HgCl2), then other metal ion solutions were added (to bring the concentration of other metal ions to 50 μM), and the effects of these other metal ions on the detection of Hg by NDBC were tested. 2+ Fluorescence interference.

[0097] like Figure 8 As shown in (a), CK+Hg 2+ +other metal ions indicates that Hg has been added to the probe solution. 2+ The fluorescence spectrum after adding other metal ions, CK+other metal ions represents the fluorescence spectrum of the probe solution with only other metal ions added; furthermore, Figure 8 The x-coordinate corresponding to (a) is Hg. 2+ At that time, CK+other metal ions means CK+Hg 2+ The fluorescence was quenched, CK+Hg 2+ +other metal ions, also means CK+Hg 2+ The fluorescence was quenched.

[0098] from Figure 8 As can be seen in (a), NDBC-Hg 2+ Multiple competing metal ions are introduced sequentially into the system. The coexistence of metal ions in the solution affects the Hg of NDBC. 2+ The fluorescence response signal was not significantly affected, confirming that the probe NDBC has good anti-interference performance.

[0099] Following the method described above, mix equal volumes of EtOH (ethanol) and HEPES buffer, then add NDBC to achieve a concentration of 10. -5 M, to obtain the probe solution, then add Hg 2+ (Concentration 50 μM), then adjust the pH of the probe solution to 2-12 (using HCl or NaOH), and test the fluorescence spectra at different pH values. The results are as follows. Figure 8 As shown in (b).

[0100] from Figure 8 As can be seen in (b), NDBC-Hg 2+The system exhibited good fluorescence stability within the pH range of 2.0–7.0. However, the fluorescence intensity decreased significantly when pH > 7.0, which may be due to changes in the molecular structure of the probe under alkaline conditions. Comprehensive analysis indicated that the detection system performed best under weakly acidic to neutral conditions (pH = 2.0–7.0). Considering the future imaging applications of NDBC in biological models, pH = 7.0 was chosen as the pH condition for subsequent experiments. Simultaneously, the NDBC probe was used to detect Hg. 2+ Reversibility tests and time response tests.

[0101] Furthermore, Hg 2+ Reversibility detection of EDTA, such as Figure 8 As shown in (c), EtOH (ethanol) and HEPES buffer are mixed in equal volumes, and NDBC is added to make the concentration 10. -5 M, to obtain the probe solution, then add Hg 2+ (At a concentration of 50 μM), the fluorescence intensity of the system dropped sharply. After adding an equal amount of EDTA, the fluorescence intensity recovered. The addition of EDTA enabled the reversible cycle of the cyclic experiment to be repeated at least five times, indicating that the probe NDBC can be reused for analysis.

[0102] Figure 8 (d) represents NDBC versus Hg 2+ Time response; by Figure 8 As shown in (d), the fluorescence intensity of NDBC remained stable over a long period, demonstrating that the probe itself possesses a certain degree of photostability. When Hg is added to the system... 2+ Subsequently, the fluorescence of the system quenched to a plateau within 70 seconds and remained stable thereafter. In summary, the NDBC probe can rapidly detect Hg. 2+ .

[0103] Quantitative measurement of Hg using probe NDBC 2+ ability

[0104] To determine the quantitative measurement of Hg using the probe NDBC 2+ The ability of NDBC to react with different concentrations (0–300 μM) of Hg was measured using fluorescence titration spectroscopy. 2+ The solution response. Specifically, equal volumes of EtOH (ethanol) and HEPES buffer were mixed, and NDBC was added to bring the concentration to 10. -5 M, to obtain the probe solution, then add Hg 2+ (Setting its concentration to 0~300 μM), its fluorescence spectrum was measured, and the results are as follows. Figure 9 As shown.

[0105] from Figure 9 As can be seen in (a), with Hg2+ As the concentration increases, the fluorescence intensity of the solution gradually decreases, especially when Hg... 2 + When the concentration reached 23 μM, the fluorescence intensity tended to stabilize and no longer decreased. Therefore, a concentration titration based on fluorescence spectroscopy was used to establish the relationship between NDBC fluorescence intensity and Hg. 2+ Quantitative linear relationship of concentration (e.g.) Figure 9 As shown in (b), Y = -71.24x + 2576.73 (R 2 =0.99). The LOD value for Hg was calculated using the formula LOD = 3σ / k. 2+ The fluorescence detection limit was 0.12 μM. These results indicate that, under environmental conditions, the NDBC bioprobe can reliably quantify Hg in a low concentration range. 2+ .

[0106] probe NDBC and Hg 2+ Integration method and sensing mechanism

[0107] To investigate the relationship between NDBC and Hg 2+ The interaction mechanism was investigated, and the Job's plot method was used to determine the interaction between NDBC and Hg. 2+ Binding rate, probe NDBC for Hg 2+ of 1 1H NMR titration and ESI-MS experiments. Results are as follows: Figures 10-11 As shown.

[0108] from Figure 10 As can be seen from this, the probe NDBC and Hg 2+ The convergence at a concentration ratio of 0.5 indicates that NDBC and Hg... 2+ The complexation pattern is 1:1, such as Figure 10 As shown in (a). ¹H NMR titration experiments indicate that, Figure 10 Throughout the measurement process shown in (b), the proton signal and peak height within the spectrum remained stable without change. Based on these experimental observations, the chemical coordination theory was used to hypothesize that Hg 2+ It may form coordination bonds with the S and carbonyl groups in the NDBC molecule. To further support the probe's interaction with Hg... 2+ The specific binding ability of Hg was detected and analyzed using ESI-MS technology, and the addition of Hg was tested. 2+ ESI-MS spectrum of NDBC after ( Figure 11 (As shown). The results indicate that NDBC and Hg 2+ The highest mass-to-charge ratio was observed, at 679.4110; this experiment confirmed that NDBC specifically binds to Hg. 2+ Based on the above experimental results, it is inferred that NDBC and Hg...2+ The interaction mechanism, such as Figure 10 As shown in (c).

[0109] Figure 12 In (a), NDBC and NDBC-Hg are represented. 2+ (a) fluorescence lifetime of NDBC and NDBC-Hg; (b) fluorescence lifetime of NDBC and NDBC-Hg. 2+ The UV-Vis absorption spectrum; where CK represents: EtOH (ethanol) and HEPES buffer are mixed in equal volumes, and NDBC is added to make the concentration 10. -5 M, to obtain the probe solution; NDBC-Hg 2+ This indicates the addition of Hg to the probe solution. 2+ (Concentration is 50 μM).

[0110] from Figure 12 As can be seen in (a), the addition of Hg 2+ Subsequently, the fluorescence lifetime of the probe NDBC decreased from 3.7491 ns to 1.4268 ns (a decrease of 61.9%), and its UV absorption band also showed enhancement and a red shift. Figure 1 (b)). The significant shortened lifetime indicates dynamic quenching via impact inactivation, while the altered ground-state absorption indicates static quenching via complex formation. The shortened fluorescence lifetime is functionally advantageous, enabling rapid imaging and real-time monitoring applications. This result demonstrates the effectiveness of adding Hg to NDBC. 2+ The detection process combines dynamic quenching and static quenching mechanisms.

[0111] probe NDBC-Hg 2+ Spectroscopic study of the complex of acetamiprid

[0112] To prove NDBC-Hg 2+ The specific recognition of the complex by acetamiprid was determined by NDBC-Hg. 2+ Fluorescence selectivity experiments of the complexes for different pesticides were conducted, and the results are as follows: Figure 13As shown. Specifically, weigh out the following pesticides: Diflufenican, Pyroclostrobin, Flunicamid, Fomesafen, Hexazinone, Mesotrione, Emamectin, Fipronil, Cyhalothrin, Metribuzin, Clothiadin, Thiamethoxam, Glyphosate, Nitenpyram, Clethodim, Atrazine, and Oxyfluorfen. Use DMSO as a solvent to prepare each pesticide to a concentration of 10. -2 M standard solution. Mix EtOH (ethanol) and HEPES buffer in equal volumes, then add NDBC to make the concentration 10. -5 M, yielding a probe solution (pH 7), was then added with Hg. 2+ (Concentration 50 μM) Standard solutions containing different pesticides were then added to the probe solution (to bring the pesticide concentration to 50 μM). The NDBC-Hg levels were then tested when different pesticides were introduced. 2+ Fluorescence response behavior of the composite system Figure 13 (a) and UV-Vis absorption characteristics ( Figure 13 (b)

[0113] from Figure 13 It can be seen from this that NDBC-Hg 2 + The complex significantly enhanced the fluorescence of acetamiprid, essentially restoring the original fluorescence intensity of the NDBC probe. In contrast, other insecticides did not cause significant changes. These results confirm the NDBC-Hg... 2+ The complex exhibits high specificity for the recognition of acetamiprid.

[0114] Figure 14 (a) represents the effects of other pesticides on NDBC-Hg. 2+ The experiment to detect interference from acetamiprid; specifically, following the method described above, equal volumes of EtOH (ethanol) and HEPES buffer were mixed, and NDBC was added to achieve a concentration of 10. -5 M was used to obtain the probe solution (pH 7), and then Hg was added. 2+(Added at a concentration of 50 μM in the form of HgCl2), then Nitenpyram (acetamiprid, to a concentration of 50 μM) was added, followed by other pesticide solutions (to a concentration of 50 μM). The effects of these other pesticides on NDBC-Hg were then tested. 2+ Detection of fluorescence interference effects of acetamiprid. Figure 14 (a), CK+Hg 2+ +Nitenpyram+various otherpesticides indicates that Hg has been added to the probe solution. 2+ Fluorescence spectra of CK+Hg after the addition of other pesticides following acetamiprid. 2 + "+ various other pesticides" indicates that Hg was added to the probe solution. 2+ The fluorescence spectrum after adding other pesticides was then observed. Figure 14 When the x-coordinate corresponding to (a) is Nitenpyram, CK+Hg 2+ + various other pesticides means CK+Hg 2+ +Nitenpyram, fluorescence recovered; CK+Hg 2+ +Nitenpyram+various other pesticides also indicates CK+Hg 2+ +Nitenpyram, fluorescence recovered.

[0115] from Figure 14 The results show that the probe NDBC-Hg 2+ Even when the surrounding environment is disturbed by other pesticides, the complex can still specifically identify and detect the target pesticide, which fully demonstrates the effectiveness of the probe NDBC-Hg in complex pesticide environments. 2+ The complex remains stable and efficient in detecting acetamiprid. The addition of acetamiprid triggers fluorescence recovery. This is due to the nitro oxygen and heterocyclic nitrogen atom pairing Hg in acetamiprid. 2+ The binding affinity of nitrile is higher than that of NDBC. Therefore, nitrile can competitively bind NDBC from NDBC-Hg. 2+ Displaced from the complex, forming a more stable acetamiprid-Hg 2+ Complex. The displaced NDBC probe detaches from Hg. 2+ The quenching effect of Hg restores its fluorescence, thereby recovering the observed signal. 2+ A mediated substitution strategy constructs a highly specific cascaded sensing system: Hg 2+Firstly, it acts as a quencher, and then as a bridge for recognizing secondary targets. Compared to a single quenching or enhancement mode, this "off-on" signal switching has a higher signal-to-noise ratio.

[0116] Furthermore, NDBC-Hg 2+ Fluorescence spectra of solutions after adding different concentrations of acetamiprid and NDBC-Hg 2+ The linear relationship between fluorescence intensity and acetamiprid concentration is as follows: Figure 14 As shown in (b) to (c), the specific method is as follows: Following the method described above, mix equal volumes of EtOH (ethanol) and HEPES buffer, and add NDBC to make the concentration 10. -5 M was used to obtain the probe solution (pH 7), and then Hg was added. 2+ (Concentration of 50 μM, added in the form of HgCl2), Nitenpyram (acetamiprid) was added to make its concentration 0~300 μM, and the fluorescence spectrum was tested.

[0117] from Figure 14 As can be seen from (b) to (c), NDBC-Hg 2+ The fluorescence intensity of NDBC-Hg showed an increasing trend with increasing concentration of acetamiprid; further analysis revealed that NDBC-Hg 2+ The fluorescence intensity showed a good linear relationship with the concentration of acetamiprid in the range of 0–75 µM, and the linear regression equation was Y = 18.47x + 940.47 (correlation coefficient R). 2 = 0.99). Based on this linear relationship, NDBC-Hg was calculated. 2+ The detection limit for acetamiprid was 0.31 µM.

[0118] NDBC-Hg 2+ Detection of acetamiprid in environmental samples using complexes

[0119] Based on NDBC-Hg 2+ The complex (prepared by mixing equal volumes of EtOH (ethanol) and HEPES buffer, then adding NDBC to a concentration of 10) -5 M was used to obtain the probe solution (pH 7), and then Hg was added. 2+The specific fluorescent response of nitenpyram (at a concentration of 50 μM, added in the form of HgCl2) to nitenpyram was used to detect nitenpyram in environmental samples, and a standard addition method was employed for spiked recovery experiments. Nitenpyram standard solutions at concentrations of 20 µM, 40 µM, and 60 µM were added to target samples (laboratory tap water, Songhua River water, and Nenjiang River water; all water samples were filtered through a 0.22 µm membrane to remove major impurities and large solids), with each concentration determined in triplicate. The experimental results are shown in Table 1. Table 1 shows that the spiked recoveries of nitenpyram ranged from 95.82% to 101.15%, with relative standard deviations (RSDs) ranging from 0.83% to 4.59%. These results indicate that the nitenpyram detection method based on the NDBC fluorescent probe has high reliability and accuracy, and has significant application potential in practical sample analysis.

[0120] Table 1 - NDBC-Hg 2+ System detection of acetamiprid in actual samples

[0121]

[0122] Cytotoxicity and Imaging Analysis

[0123] NDBC was detected using a cell counting kit-8 (CCK-8, a commercially available cell viability dye). The solution was prepared by mixing equal volumes of EtOH (ethanol) and HEPES buffer, then adding NDBC to achieve a concentration of 10. -5 M, yielding the probe solution (pH 7, i.e., NDBC solution) and NDBC-Hg. 2+ Cytotoxicity of the complex (prepared as above) to HepG2 cells. HepG2 cells were seeded in 96-well microplates at a density of 100 μL of culture medium per well. After cell attachment, the original culture medium was removed, and the test drug solution (i.e., NDBC or NDBC-Hg) was added. 2+ (Compound). After 24 h, add 10 μL of CCK-8 to each well and incubate for 40 min at 37℃ in a 5% CO2 incubator. Measure the absorbance of each well at 450 nm using a microplate reader. Cell viability = A s A represents the absorbance of the experimental group. b Absorbance of the blank group (containing no cells but CCK-8), A c The absorbance is for the control group.

[0124] This invention determined the concentrations of NDBC solution and NDBC-Hg. 2+ Cytotoxicity of the complex against HepG2 cells. NDBC treatment group and NDBC-Hg 2 +The cell viability rates in the treatment groups were 90.41% and 90.28%, respectively. The experimental data show that NDBC and NDBC-Hg... 2 + Cell viability remained high in all treated cell groups. This indicates that both probes exhibited low cytotoxicity after 12 hours of incubation at 10 µM concentration. The results confirm the effectiveness of NDBC and NDBC-Hg. 2+ Their good biocompatibility in live-cell imaging suggests their potential application in more complex biological experiments and related research.

[0125] Further research was conducted on the probe NDBC solution (prepared using the same method as above) and NDBC-Hg. 2+ The complex (prepared by the same method as above) was used to detect Hg in HepG2 cells. 2+ The interaction with acetamiprid. Specifically, HepG2 cells were gently washed with phosphate-buffered saline (PBS) (3 times, 1 minute each time). The control group received no treatment; the NDBC group: 10 µM of NDBC solution prepared with ethanol and HEPES buffer (1:1) was added to the cells, ensuring the solution completely covered the cells. The cells were incubated at 37°C for 30 minutes in the dark. After incubation, the cells were washed 3 times with ethanol and HEPES buffer (1:1), 1 minute each time, to remove unbound probes; NDBC + Hg 2+ Group: NDBC solution (10 µM) prepared with ethanol and HEPES buffer (1:1) and Hg were added to the cells. 2+ (10 µM) mixed solution. Incubate at 37°C for 30 minutes, protected from light. After incubation, wash three times with ethanol and HEPES buffer (1:1), 1 minute each time, to remove unbound probe; NDBC+Hg 2+ +Nitenpyram group: NDBC solution (10 µM) prepared with ethanol and HEPES buffer (1:1) was added to cells, along with Hg. 2+ A mixture of 10 µM ethanol and nitenpyram (10 µM) was prepared. The mixture was incubated at 37°C for 30 minutes, protected from light. After incubation, the cells were washed three times with ethanol and HEPES buffer (1:1), 1 minute each time, to remove unbound probes. The final wash solution was discarded, and an appropriate amount of anti-fluorescence quenching mounting medium was added to the cells for observation using a laser confocal microscope.

[0126] Figure 15 The results show ( Figure 15 In the middle, Fluorescence represents fluorescence images, Bright field represents bright field images, and Merged represents merged images; the blank group showed no fluorescence (i.e., ...). Figure 15 (balank), processed separately using NDBC (i.e. Figure 15Cells of the NDBC (Neuro-Nutrient Breast Cells) emit green fluorescence, while the addition of Hg... 2+ (Right now Figure 15 NDBC+Hg 2+ After adding acetamiprid (i.e., nitenpyram) to the above system, the fluorescence in the cells was quenched. Figure 15 NDBC+Hg 2+ After Nitenpyram addition, the fluorescence intensity of the cells returned to its original level. This trend is consistent with the results of previous concentration titration experiments. This invention confirms the efficacy of NDBC and NDBC-Hg. 2+ Hg in living cells 2+ The detection of nitenpyram and acetamiprid was effective, demonstrating their potential applications in biosensing. The NDBC probe not only maintains cell viability but also effectively detects Hg in living cells. 2+ The presence of acetamiprid and other similar products indicates good overall performance and biocompatibility. Zebrafish imaging experiment.

[0127] One day after hatching, the zebrafish were divided into three groups. The third group was incubated with 10 μM acetamiprid for 30 min, while the other two groups were incubated with HEPES buffer. Specifically, the first group of zebrafish was given only 10 μM NDBC (NDBC solution (10 µM) prepared using ethanol and HEPES buffer (1:1)). Figure 16 The second group received both 10 μM NDBC and 10 μMHg. 2+ (NDBC solution (10 µM) prepared using ethanol and HEPES buffer (1:1) and Hg) 2+ (10 µM) mixed solution, corresponding Figure 16 NDBC+ Hg 2+ The third group was given 10 μM NDBC and Hg. 2+ Complex (NDBC solution (10 µM) prepared using ethanol and HEPES buffer (1:1), Hg) 2+ A mixed solution of (10 µM) and nitenpyram (10 µM), corresponding to Figure 16 NDBC+Hg 2+ +Nitenpyram). Samples were incubated for 45 minutes. Subsequently, zebrafish were anesthetized with tricaine and washed three times with HEPES buffer to remove excess analytes. Fluorescence imaging was performed using laser confocal scanning microscopy.

[0128] This invention utilizes a zebrafish in vivo fluorescence imaging system to systematically evaluate the detection of Hg by the NDBC probe. 2+ And the potential of acetamiprid. Zebrafish, as a representative aquatic model organism, has been widely used in fluorescence imaging research in agriculture and biomedicine due to its many unique advantages. From Figure 16 The fluorescence imaging results clearly show that zebrafish treated with the NDBC probe exhibited extremely significant green fluorescence signals. Due to Hg... 2+ The quenching effect of NDBC-Hg 2+ The fluorescence of the complex completely disappeared. However, in the above system, the fluorescence of cells treated with acetamiprid was restored. The experimental results indicate that the NDBC probe has good penetration properties in biological tissues and can effectively monitor Hg in vivo. 2+ The achievement of determining the residual levels of acetamiprid provides essential experimental support for the development of novel biosensors.

[0129] In summary, NDBC was designed and synthesized using 1,8-naphthalimide as the basic fluorescent group, based on the probe-analyte recognition principle and splicing strategy, by introducing a hydrazine segment and a benzothiophene unit. Hg was achieved through an "off-on" mode. 2+ The method enables continuous detection of acetamiprid (detection limit 0.12 μM) and nitenpyram (detection limit 0.31 μM), with a reversible detection process. In actual water samples, the recovery rate of nitenpyram was 95.82%–101.15%, with an RSD ≤ 4.59%. These results indicate that the method is suitable for high-precision analysis. In vivo cell and zebrafish imaging experiments demonstrated that NDBC can monitor Hg in zebrafish in real time. 2+ The method is highly sensitive, selective, and biocompatible, and can be used to monitor Hg and its residues. 2+ And acetamiprid has effective detection performance, and is Hg 2+ This provides a new method for the detection of acetamiprid and holds promise for developing into a rapid method for detecting Hg. 2+ Visualization tools for acetamiprid.

[0130] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A bifunctional fluorescent probe, characterized in that, The chemical structure of the bifunctional fluorescent probe is shown below: .

2. A method for preparing a bifunctional fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Intermediate compound 2 was coupled with benzothiophene-2-carboxylic acid to obtain a bifunctional fluorescent probe; The structural formula of intermediate compound 2 is shown below: 。 3. The method for preparing the bifunctional fluorescent probe as described in claim 2, characterized in that, The method for preparing the intermediate compound 2 includes: 4-Bromo-1,8-naphthoic anhydride was reacted with n-butylamine to give intermediate compound 1; Intermediate compound 1 was reacted with hydrazine hydrate to obtain intermediate compound 2; The structural formula of intermediate compound 1 is shown below: 。 4. The method for preparing the bifunctional fluorescent probe as described in claim 3, characterized in that, 4-Bromo-1,8-naphthoic anhydride was dissolved in ethanol, and n-butylamine was added. The mixture was refluxed under an inert atmosphere to give intermediate compound 1. Intermediate compound 1 was dissolved in hydrazine hydrate in ethylene glycol monomethyl ether and refluxed under an inert atmosphere to give intermediate compound 2. Intermediate compound 2 and benzothiophene-2-carboxylic acid were dissolved in acetonitrile, and coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, and N,N-diisopropylethylamine were added. The mixture was refluxed to obtain a bifunctional fluorescent probe.

5. The method for preparing the bifunctional fluorescent probe as described in claim 4, characterized in that, In the step of preparing intermediate compound 1, the molar volume ratio of 4-bromo-1,8-naphthoic anhydride, n-butylamine, and ethanol is (0.01~0.02) mol:(0.06~0.07) mol:(25~35) mL, the reflux reaction temperature is 120~125℃, and the time is 6~8 h; In the step of preparing intermediate compound 2, the molar volume ratio of intermediate compound 1, hydrazine hydrate, and ethylene glycol monomethyl ether is (1.6~2.6) mmol:(2~3) mL:(25~35) mL, the reflux reaction temperature is 130~135℃, and the time is 0.5~1 h; The molar volume ratio of intermediate compound 2, benzothiophene-2-carboxylic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxybenzotriazole, N,N-diisopropylethylamine, and acetonitrile was (5~10) mmol:(5~10) mmol:(0.01~0.02) mol:(0.01~0.02) mol:(0.02~0.03) mol:(20~25) mL. In the step of obtaining the bifunctional fluorescent probe by reflux reaction, the reflux reaction temperature was 85~90℃ and the time was 10~12 h.

6. A bifunctional fluorescent probe as described in claim 1 for recognizing Hg in cells, soil, wastewater, or solution. 2+ Applications.

7. The application as described in claim 6, when the bifunctional fluorescent probe identifies Hg in the solution. 2+ When used, the solvent in the solution includes ethanol.

8. An NDBC-Hg 2+ The application of complexes in the identification of acetamiprid in cells, soil, wastewater, or solution; among which, The NDBC-Hg 2+ The coordination compounds were obtained by the following methods: The bifunctional fluorescent probe as described in claim 1 is added to a solution containing Hg. 2+ In the solution, NDBC-Hg was obtained. 2+ Coordination compounds.

9. The application of a bifunctional fluorescent probe as described in claim 1 in cell imaging.

10. An NDBC-Hg 2+ Application of the complex in cell imaging after acetamiprid treatment, the NDBC-Hg 2+ The coordination compounds were obtained by the following methods: The bifunctional fluorescent probe as described in claim 1 is added to a solution containing Hg. 2+ In the solution, NDBC-Hg was obtained. 2+ Coordination compounds.

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