Copper ion detection fluorescent probe LCY as well as preparation method and application thereof

Abstract

The invention discloses a copper ion detection fluorescent probe LCY as well as a preparation method and application thereof, the copper ion detection fluorescent probe LCY takes benzothiazole-2-acetonitrile as a fluorophore, and the structure of the copper ion detection fluorescent probe LCY is shown in the specification. The fluorescent probe LCY with the m-aminophenol derivative as a parent is prepared through condensation of the m-aminophenol derivative and benzothiazole-2-acetonitrile, and the probe is novel in structure, has specific selection on copper ions, is short in response time and high in sensitivity, has selectivity on glyphosate, can effectively detect glyphosate residues and has good application prospects. More importantly, the preparation method of the probe is simple, and the obtained product is solid powder, easy to store and good in stability. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe LCY for copper ion detection, a preparation method thereof, and an application thereof. Background Art

[0002] Glyphosate is a broad-spectrum organophosphorus herbicide that is widely used in modern agriculture due to its high efficiency, low cost and low toxicity. However, the growing popularity of glyphosate-resistant crops has significantly increased its use, resulting in worrying levels of environmental residues. Long-term exposure to glyphosate through the food chain has been associated with serious health problems, including breast cancer cell proliferation, impaired brain function, respiratory dysfunction, liver damage, and even death. To address these risks, the maximum contamination level of glyphosate in drinking water has been set at 5.325 μM (900 μg / L). Glyphosate binds to Cu through its phosphonates and amino groups. 2+ ions show strong coordination and form stable complexes. 2+ The developed fluorescent probes or organic sensors may have potential benefits in determining glyphosate residues.

[0003] Copper (Cu 2+ ) is an essential heavy metal that plays a vital role in human health by supporting red blood cell production, nerve cell function, and the immune system. 2+ Abnormal ion levels can lead to the onset of serious diseases such as Parkinson's disease, anemia, Wilson's disease and acute liver damage. In addition, industrial and agricultural activities lead to the uncontrolled release of copper ions into the environment, posing a significant risk to aquatic ecosystems. 2+ Ions are also important analytes that require the development of sensitive chemical sensors.

[0004] Atomic absorption spectrometry (AAS), inductively coupled plasma emission spectrometry, inductively coupled plasma mass probe and electrochemical methods are currently the most effective methods for copper ion detection. However, the cost of each test of these methods is high, and the sample requirements of the instrument test are also high, which leads to a long measurement time. Relatively speaking, the probes synthesized by synthesizing fluorescent organic small molecules have the advantages of low cost, easy operation, specific selection, high sensitivity, short response time and visualization. Therefore, a Cu ion probe with high sensitivity, specific selection and low detection limit was designed and synthesized. 2+ Fluorescent probes have very important application value. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a fluorescent probe LCY for copper ion detection. The fluorescent probe LCY has specific selection for copper ions, a low detection limit, and a short response time, and can quickly detect trace amounts of copper ions in environmental systems.

[0006] The present invention also provides a preparation method and application of the copper ion detection fluorescent probe LCY.

[0007] Technical solution: To achieve the above-mentioned purpose of the invention, the present invention provides a copper ion detection fluorescent probe LCY, wherein the copper ion detection fluorescent probe LCY uses benzothiazole-2-acetonitrile as a fluorescent group, and its structure is shown in the following formula I:

[0008]

[0009] The method for preparing the copper ion detection fluorescent probe LCY of the present invention comprises the following steps:

[0010] m-Aminophenol and 1-bromo-3-chloropropane are dissolved in an organic solvent and subjected to an electrophilic substitution reaction under catalysis to obtain the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol; then, 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol is used as a raw material and subjected to a Vilsmeier reaction in a phosphorus oxychloride solution to obtain the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde; then, the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde is reacted with the 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-8-ol as a raw material. 6,7-Tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and tert-butyldimethylsilyl chloride undergo nucleophilic substitution to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde; finally, the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde is condensed with benzothiazole-2-acetonitrile to obtain the fluorescent probe LCY.

[0011] The reaction route for preparing the copper ion detection fluorescent probe LCY is as follows:

[0012]

[0013] The m-aminophenol and 1-bromo-3-chloropropane are added and dissolved in an organic solvent DMF, and then sodium carbonate is added as a catalyst. The mixture is refluxed and stirred at not less than 80° C. for 12-15 hours. The DMF is removed by extraction, and the organic phase is recovered, dried, and then subjected to column chromatography to obtain the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol.

[0014] The intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-8-ol is dissolved in an organic solvent DMF, and then added to a DMF solution of phosphorus oxychloride, stirred at room temperature for 0.5-1h, heated to no less than 100°C and refluxed for 1-2h. After the reaction is complete, the reaction solution is cooled to room temperature, saturated brine is added and stirred, and the obtained solid is filtered and dried to obtain the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde.

[0015] The intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and tert-butyldimethylsilyl chloride are dissolved in an organic solvent DCM, and then organic bases TEA and DMAP are added. The mixture is stirred at room temperature for 4-5 hours. After the reaction is complete, the mixture is washed with saturated brine, and the organic phase is recovered, dried, and distilled under reduced pressure to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde.

[0016] The intermediate 8-((tert-butyldimethylsilyl)oxy)2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and benzothiazole-2-acetonitrile are dissolved in an organic solvent, anhydrous ethanol, with piperidine as a catalyst, and stirred at room temperature for 4-5 hours. After the reaction is completed, the obtained reaction product is filtered, washed, and dried to obtain the fluorescent probe LCY.

[0017] Preferably, the preparation process of LCY comprises:

[0018] m-Aminophenol, 1-chloro-3-bromopropane, and Na₂CO₃ were added to a round-bottom flask, followed by DMF. The mixture was refluxed at 80°C for 12 hours, resulting in the system changing from colorless to red. After the reaction, the mixture was cooled to room temperature, added with saturated brine, and extracted with ethyl acetate. The organic phase was recovered, dried, and distilled under reduced pressure. Column chromatography with a 5:1 ratio of PE to EA was performed to obtain the crude product. The crude product was dissolved in EA, concentrated, and then PE was added, shaken, and refrigerated overnight. The mother liquor was filtered off the next day and dried to yield the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol as a white crystalline solid.

[0019] Slowly add POCl3 dropwise to DMF in an ice bath. Stir for 5 minutes after addition. Then add the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-8-ol solution prepared in DMF. Stir at room temperature for 30 minutes, then reflux at 100°C for 30 minutes. After cooling to room temperature, add saturated brine. After stirring, a green solid appears. Filter and dry to obtain the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde.

[0020] The intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde, tert-butyldimethylsilyl chloride, and DMAP were dissolved in DCM. TEA was then slowly added and stirred at room temperature for 4 hours. After the reaction, the mixture was washed once with saturated brine. The organic phase was recovered, dried, and evaporated under reduced pressure to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde as a brown solid.

[0021] Dissolve 8-((tert-Butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde and benzothiazole-2-acetonitrile in EtOH, then add piperidine. Stir at room temperature to produce a red precipitate. Filter the reaction mixture and wash with ethanol once to obtain a bright red solid, the target fluorescent probe molecule LCY.

[0022] Preferably, the reaction path of the preparation is as follows:

[0023]

[0024] The invention discloses an application of the copper ion detection fluorescent probe LCY in the detection of copper ions and glyphosate.

[0025] Furthermore, the copper ion detection fluorescent probe LCY is used to detect copper ions and glyphosate in a solution.

[0026] Furthermore, the copper ion detection fluorescent probe LCY is used in the preparation of reagents or tools for detecting copper ions and glyphosate in a solution.

[0027] The copper ion detection fluorescent probe LCY of the present invention is prepared into fluorescent test paper and used in detecting trace copper ions in a solution.

[0028] The invention relates to an application of the copper ion detection fluorescent probe LCY in the preparation of a reagent for detecting trace copper ions in cells.

[0029] The present invention provides a multifunctional fluorescent sensor, LCY, for the simultaneous and sequential detection of glyphosate and copper ions, addressing key issues in environmental monitoring and public health. LCY, chemically identified as (E)-2-(benzothiazol-2-yl)-3-(8-((tert-butyldimethylsilyl)oxy)-julodin-9-yl)acrylonitrile, was synthesized via a direct aldol condensation reaction, ensuring synthetic simplicity and structural versatility. It exhibited remarkable dual analyte sensitivity in near-aqueous media, with a limit of detection (LOD) of 53.8 nM for glyphosate and 53.8 nM for Cu. 2+ The detection limit of LCY is 148nM, which exceeds many existing chemical sensors. 2+ Afterwards, it undergoes a rapid and distinct color change from yellow to purple-red. LCY and copper ions are in a 1:1 stoichiometric ratio, accompanied by a fluorescence "off" effect driven by a chelation-enhanced quenching (CHEQ) mechanism. The present invention uses benzothiazole-2-acetonitrile as the fluorescent group and prepares the benzothiazole-based fluorescent probe LCY via an aldol condensation reaction.

[0030] In addition, LCY has a great influence on Cu 2+ and HPO4 2- The reversibility of Cu 2+ These outstanding features make LCY suitable for practical applications, such as rapid, on-site detection of Cu 2+ Portable test strips and real-time analysis of Cu in complex environments 2+ The invention provides a smartphone-assisted sensing platform for oxaline and glyphosate, and the preparation method of the fluorescent probe is simple, the raw materials are easily obtained, and the obtained product is a solid powder that is easy to store.

[0031] The present invention designs a fluorescent probe LCY with a brand-new structure and a benzothiazole-2-acetonitrile fluorescent group based on an m-aminophenol derivative. In the parent structure of the copper ion detection fluorescent probe, the m-aminophenol derivative has a rigid planar structure and a large π bond conjugated system, and benzothiazole-2-acetonitrile is a very good fluorescent group. Organosilane reagents are usually stable to common functional groups and reaction conditions, and can be selectively chemically transformed in multifunctional molecules. On the other hand, their selectivity can also be used to selectively bind to or destroy specific ions or functional groups to achieve the purpose of detecting specific ions or groups. At the same time, compared with the probes in the prior art that use a m-aminophenol derivative structure as a parent, the present probe has a shorter synthesis route, high yield and high purity, and the raw materials are easily available and low cost, and it is very sensitive to Cu. 2+ With unique selectivity and low detection limit, fluorescent test paper has been successfully prepared and used to detect trace amounts of Cu in aqueous solution. 2+ .

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] The present invention prepares a fluorescent probe LCY with a meta-aminophenol derivative as a parent by condensing a meta-aminophenol derivative with benzothiazole-2-acetonitrile. The probe has a novel structure, is specific for copper ions, has a short response time, and is highly sensitive to Cu in the solution. 2+ It exhibits high sensitivity, high selectivity and a low detection limit (148nM). It is also selective for glyphosate and can effectively detect glyphosate residues with a detection limit of 53.8nM. More importantly, the probe preparation method is simple, and the resulting product is a solid powder that is easy to store and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The fluorescence spectra of the copper ion fluorescent probe prepared in Example 1 at different water contents;

[0035] Figure 2 The copper ion fluorescent probe prepared in Example 1 was added to a solution of (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) to obtain a concentration of copper ion (Cu 2+ )'s UV absorption spectrum and color change diagram ( Figure 2 Yellow on the left and purple on the right in the illustration);

[0036] Figure 3 Graph showing the selective fluorescence spectra of the copper ion fluorescent probe prepared in Example 1 to different metal ions in a (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) solution;

[0037] Figure 4 The fluorescent probe prepared in Example 1 was tested for different concentrations of copper ions (Cu 2+ )'s fluorescence spectrum response diagram and color change diagram;

[0038] Figure 5 Graph showing the fluorescence response of the fluorescent probe prepared in Example 1 to selective interference detection of different metal ions in a (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) solution;

[0039] Figure 6 The fluorescent probe prepared in Example 1 was mixed with (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) and copper ions (Cu 2+ ) Job-plot curve of complexation ratio;

[0040] Figure 7This is a response time diagram of the fluorescent probe prepared in Example 1 when detecting copper ions;

[0041] Figure 8 The fluorescent probe prepared in Example 1 was mixed with (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) and copper ions (Cu 2+ ) Fluorescence response graph at different pH values (2 to 12);

[0042] Figure 9 The fluorescent probe prepared in Example 1 and the optimal configuration diagram after the fluorescent probe is complexed with copper ions;

[0043] Figure 10 The color change diagram of the fluorescent test paper prepared with the fluorescent probe prepared in Example 1 when testing different concentrations of copper ions;

[0044] Figure 11 The fluorescent probe complex [LCY-Cu 2+ ]Selective fluorescence spectra of different pesticides in (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) solution;

[0045] Figure 12 The fluorescent probe complex [LCY-Cu 2+ ] Fluorescence spectrum response diagram of glyphosate at different concentrations in (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) solution;

[0046] Figure 13 This is a graph showing the cytotoxicity of the fluorescent probe prepared in Example 1 to copper ions;

[0047] Figure 14 This is a cell experiment diagram of the fluorescent probe prepared in Example 1 on copper ions;

[0048] Figure 15 This is a mobile phone detection image of copper ions by the fluorescent probe prepared in Example 1;

[0049] Figure 16 The fluorescent probe prepared in Example 1 1 H NMR spectrum;

[0050] Figure 17 The fluorescent probe prepared in Example 1 13 C NMR spectrum;

[0051] Figure 18 is the mass spectrum MS spectrum of the fluorescent probe prepared in Example 1;

[0052] Figure 19This is the infrared IR spectrum of the fluorescent probe prepared in Example 1. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to the accompanying drawings and examples.

[0054] Unless otherwise specified, the experimental methods used in this invention are conventional methods. The materials and reagents used in the experiments, unless otherwise specified, can be obtained from commercial sources. All the following reagents used in the examples were commercially available of analytical or chemical purity.

[0055] The various metal ion solutions in the embodiments are prepared by adding deionized water to a hydrochloride chemical reagent with a purity of more than 99%, such as cupric chloride dihydrate, cuprous chloride, etc.

[0056] The specific synthetic route of the fluorescent probe LCY based on m-aminophenol derivatives:

[0057] (1) Preparation of intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol

[0058] m-Aminophenol (2.2 g, 20 mmol), 1-chloro-3-bromopropane (10 mL, 50 mmol), and Na₂CO₃ (4.24 g, 40 mmol) were added to a round-bottom flask. 15 mL of DMF was then added and the mixture was refluxed at 80°C for 12 h, resulting in the color of the reaction changing from colorless to red. After the reaction was completed, the mixture was cooled to room temperature, added with saturated brine, and extracted with ethyl acetate. The organic phase was recovered, dried, and evaporated under reduced pressure. The crude product was separated by column chromatography using a 5:1 ratio of PE to EA to obtain the crude product. The crude product was dissolved in EA, concentrated, and then shaken with 10 mL of PE. The mixture was refrigerated overnight. The mother liquor was filtered off the next day and dried to obtain the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol (1.50 g, 40%) as a white crystalline solid.

[0059] The structural formula of the obtained intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol is:

[0060]

[0061] (2) Preparation of the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde

[0062] 0.5 mL of POCl3 was slowly added dropwise to 5 mL of DMF in an ice bath. After the addition was complete, the mixture was stirred for 5 minutes. A solution of the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol (0.946 g, 5 mmol) prepared in 5 mL of DMF was then added. The mixture was stirred at room temperature for 30 minutes, and then refluxed at 100°C for 30 minutes. After cooling to room temperature, 20 mL of saturated brine was added. After stirring for 1.5 hours, a green solid appeared. After filtration and drying, the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde (0.93 g, 85%) was obtained.

[0063] The structural formula of the obtained intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde is:

[0064]

[0065] (3) Preparation of the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde

[0066] The intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde (434.54 mg, 2 mmol), tert-butyldimethylsilyl chloride (452.16 mg, 3 mmol), and DMAP (12 mg, 0.1 mmol) were dissolved in 15 mL of DCM. TEA (303.54 mg, 3 mmol) was slowly added and stirred at room temperature for 4 hours. After the reaction, the mixture was washed four times with saturated brine. The organic phase was recovered, dried, and evaporated under reduced pressure to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde (0.6017 g, 90%) as a brown solid.

[0067] The structural formula of the obtained intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde is:

[0068]

[0069] (4) Preparation of copper ion fluorescent probe molecule LCY

[0070] Dissolve 8-((tert-Butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde (331.53 mg, 1 mmol) and benzothiazole-2-acetonitrile (191.64 mg, 1.1 mmol) in 10 mL of EtOH, then add 0.25 mL of piperidine. Stir at room temperature for 4 hours to produce a red precipitate. The reaction mixture was filtered and washed twice with ethanol to obtain a bright red solid fluorescent probe molecule LCY (0.2955 g, 61%).

[0071] The obtained fluorescent probe molecule LCY structural formula is:

[0072]

[0073] Characterization data of this fluorescent probe 1 HNMR(400MHz, CDCl3)δ8.20(s,1H),7.98(s,1H),7.92–7.88(m,1H),7.77–7.75(m,1H),7.39–7.35(m,1H),7.28–7.22(m,1H),3.25–3.2 2(m,2H),3.22–3.18(m,2H),2.71–2.66(m,2H),2.57(t,J=6.2Hz,2H),1.93–1.86(m,2H),1.86–1.79(m,2H),1.05(s,9H),0.09(s,6H). 13 C NMR (101MHz, CDCl3) δ164.83,153.00,152.50,147.11,142.08,133.32,125.41,125.23,123.58,121.69,120.25,11 7.81,115.14,111.14,109.83,93.39,49.21,48.77,26.48,25.04,21.38,20.49,20.00,17.57,-4.42.MS:Calcdfor C 28 H 33 N3OSSi:[M+H] + 488.21; Found, 488.22

[0074] The hydrogen spectrum of the fluorescent probe prepared in Example 1 ( 1 H NMR), carbon spectrum ( 13 C NMR), mass spectrometry (MS) and infrared spectra are shown in Figure 16 , Figure 17 , Figure 18 , Figure 19 As shown, it shows that the fluorescent probe of the present invention was successfully prepared.

[0075] Example 2

[0076] The copper ion detection fluorescent probe LCY prepared in Example 1 was configured with DMSO to a 1 mM probe stock solution, and each metal ion was configured with deionized water to a 3 mM metal ion stock solution. 30 μL of the probe stock solution and 50 μL of the metal ion stock solution were added to 3 mL of a blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) and detected using a fluorescence spectrometer and an ultraviolet spectrophotometer. The test showed that the maximum excitation wavelength of the fluorescent probe was 552 nm, and the maximum emission wavelength was 479 nm. The specific test results are as follows:

[0077] like Figure 1 As shown in Figure 2, the fluorescence spectra of the copper ion detection fluorescent probe for different water content analysis. 30 μL of probe stock solution (1 mM) and 50 μL of Cu 2+ were added to the cuvette. 2+ The stock solution (3mM) was prepared by changing the ratio of deionized water to dimethyl sulfoxide (DMSO) in the cuvette (the volume ratio of deionized water to dimethyl sulfoxide was 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0), with a total volume of 3mL. The fluorescence intensity at 552nm was obtained and plotted against the water component ratio. Figure 1 It can be seen that LCY-Cu 2+ It exhibits weak fluorescence in pure DMSO. When the percentage of water in DMSO is continuously added from 0 to 40%, LCY-Cu 2+ The fluorescence intensity of LCY-Cu increases gradually. When the water content in the DMSO aqueous medium increases from 50% to 100%, 2+ This observation suggests that LCY detects Cu by a fluorescence “off” strategy. 2+ The most suitable medium for the ion is a DMSO / H2O mixture with a volume fraction of 1:9, that is, in a near-aqueous medium. Therefore, it can be determined that the optimal buffer ratio for the fluorescent probe and copper ions is DMSO:H2O:Hepes = v / v / v = 1:9:0.1 (pH = 8.0).

[0078] Take two cuvettes, add 3 mL of a mixed solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1) containing the probe stock solution (30 μL, 1 mM) to each cuvette, and then add 50 μL (3 mM) of copper ion stock solution to one of the cuvettes for UV spectrum testing. Figure 2As shown in the figure, the fluorescent probe itself has ultraviolet absorption at wavelength λ = 479nm. When copper ions are added, the ultraviolet absorption at 479nm gradually weakens. Moreover, the addition of copper ions changes the color of the probe solution from yellow to purple-red. The results show that the probe has a strong affinity for Cu 2+ It has high sensitivity and selectivity.

[0079] like Figure 3 As shown, the fluorescent probe for copper ion detection has selective fluorescence spectra for various common metal ions. To 3 mL of a blank solution (DMSO:H2O:Hepes) (v / v / v = 1:9:0.1), 30 μL (1 mM) of the probe stock solution and 50 μL (3 mM) of the metal ion stock solution were added. The experimental results show that only with the addition of copper ions does the fluorescence intensity at 552 nm of the fluorescence spectrum significantly decrease. Moreover, the fluorescence intensity is significantly weaker than when other metal ions are added, indicating that the fluorescent probe of the present invention has good selectivity for copper ions.

[0080] like Figure 4 As shown in the figure, the copper ion detection fluorescent probe is sensitive to different concentrations of copper ions (Cu 2+ ) is a fluorescence spectrum response diagram. 30 μL (1 mM) of the probe stock solution and 0 to 100 μL (0, 1, 2, 3...10, 15...50, 60...100 μL) of copper ion solution (3 mM copper ion stock solution) were added to 3 mL of blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1). The fluorescent probe itself has strong fluorescence in the solution, but as the copper ion concentration increases, the fluorescence at 552 nm decreases continuously with the increase of copper ion concentration, that is, the fluorescence intensity decreases with the increase of copper ion concentration, and is accompanied by obvious color changes. This shows that the probe LCY prepared in Example 1 of the present invention has a strong fluorescence to Cu 2+ Has a strong binding preference, LCY-Cu 2+ The stability of the complex leads to the chelation-enhanced quenching (CHEQ) effect, which is a very effective quenching probe.

[0081] like Figure 5 As shown in the figure, the fluorescence intensity bar graph of the copper ion detection fluorescent probe after reacting with copper ions in the presence of different interfering metal ions. To 3 mL of blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1), 30 μL (1 mM) of the probe stock solution and 50 μL (3 mM) of any other metal ion (Al 3+ ,Co 2+ , Pd 2+ , Ni 2+ , Zn 2+ , Cr 3+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Pd 2+ , Sn 2+ , Fe 2+ , K + , Ca 2+ , Sr 2+ , Na + , Ag + and Cd 2+ ) stock solution, and finally add 50 μL of Cu 2+ The results showed that, except for cuprous ions, other metal ions had no significant interference in the recognition of copper ions by the copper ion fluorescent probe of the present invention, indicating that the probe prepared by the present invention has good specificity.

[0082] like Figure 6 As shown in the figure, the interaction between the probe and Cu was studied by Job's plot method. 2+ To determine the binding rate, a certain volume of probe stock solution (1 mM) and Cu were added to 3 mL of blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1). 2+ The stock solution (3mM) was prepared so that the total concentration of the copper ion detection fluorescent probe and copper ions was 50μM. By changing the concentration ratio of the two (the molar ratio of the copper ion detection fluorescent probe and copper ions was 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1), the difference between the fluorescence intensity at 552nm and the combined fluorescence intensity of the copper ion fluorescent probe at this concentration was obtained, and the ratio of ions to the total concentration was plotted. Figure 5 It can be seen that when the proportion of copper ions is 0.5, the vertical axis reaches the highest value, which can be determined that the fluorescent probe and copper ions form a stable complex at a ratio of 1:1 [LCY-Cu 2+ ].

[0083] like Figure 7 As shown, 30 μL (1 mM) of probe stock solution and 50 μL (3 mM) of Cu were added to 3 mL of blank buffer (DMSO:H2O:Hepes) (v / v / v=1:9:0.1). 2+ The fluorescence intensity of the probe rapidly increased to the maximum value and reached a stable value within 1 minute. In addition, the fluorescence intensity of the probe remained basically unchanged in the following 10 minutes, which shows that the probe has a strong affinity for Cu 2+ The detection is stable enough.

[0084] like Figure 8As shown, 1M HCl and 1M NaOH were used to adjust the DMSO / H2O solutions (1 mL) containing the probe LCY stock solution (10 μL) and the 2+ [LCY-Cu 2+ stock solution (10 μL), respectively. The [LCY-Cu 2+ stock solution was prepared by dissolving the probe and copper chloride dihydrate in secondary distilled water to concentrations of 10 μM and 50 μM, respectively, to give different pH change values. Then, the fluorescence intensities of these two systems were measured over different pH ranges. The probe LCY itself had almost no fluorescence intensity in the pH range from 2 to 12, but the 2+ [LCY-Cu 2+ system showed a significant decrease in fluorescence (552 nm) intensity in the pH range of 8.0 - 11.0, and the fluorescence intensity reached a minimum at pH = 10. [LCY-Cu 2+ showed less obvious decrease in fluorescence signal under acidic conditions (pH < 6.0), probably because the complexation sites of the fluorescent probe LCY were protonated and not easily complexed with copper ions. [LCY-Cu

[0085] In the weakly alkaline condition (8.0 < pH < 10.0), the fluorescence signal was very weak, but as the alkalinity increased (pH > 10.0), the fluorescence intensity slowly recovered but was not as good as the state before adding copper ions, probably because Cu(OH)2 precipitation formed, slightly reducing the Figure 9 [LCY-Cu 2+ concentration. Therefore, the most suitable pH range for LCY is 8 - 10.

[0086] As Figure 10 shown, the optimal configuration diagrams and their corresponding energy level diagrams of the fluorescent probe and the

[0087] As Figure 11 [LCY-Cu 2+] complex to some common pesticides (glyphosate, glufosinate, thiophosphate, phoxim). To 3mL of blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1), 30μL (1mM) of probe stock solution and 50μL (3mM) of copper ion stock solution were added, shaken evenly, and then 50μL of pesticide stock solution (3mM) was added thereto. The experimental results showed that after the addition of glyphosate, the fluorescence intensity at 552nm of the fluorescence spectrum was significantly restored. Moreover, the fluorescence intensity was significantly stronger than that when other pesticides were added, indicating that the fluorescent probe complex of the present invention [LCY-Cu 2+ ] has good selectivity for glyphosate.

[0088] like Figure 12 As shown, the fluorescence spectrum response diagram of the copper ion detection fluorescent probe to different concentrations of glyphosate. To 3 mL of blank solution (DMSO:H2O:Hepes) (v / v / v=1:9:0.1), 30 μL (1 mM) of probe stock solution and 50 μL (3 mM) of copper ion stock solution were added, and the mixture was shaken evenly. Then, 0 to 100 μL (0, 1, 2, 3...10, 15...50, 60...100 μL) of glyphosate stock solution (3 mM glyphosate stock solution) was added thereto. The fluorescent probe complex [LCY-Cu 2 + The fluorescence intensity in the solution itself is very low, but as glyphosate increases, the fluorescence at 552 nm continuously increases with the increase in glyphosate concentration. That is, the fluorescence intensity increases with increasing glyphosate concentration, accompanied by a significant color change. This indicates that the probe complex prepared in Example 1 of the present invention has a strong detection effect for glyphosate and can be used for glyphosate detection.

[0089] like Figure 13 As shown, PK-15 living cells were seeded into 96-well culture plates at a density of approximately 7,000 cells per well and allowed to adhere for 12 hours at 37°C in a humidified atmosphere of 5% CO2. Then, 100 μL of fresh culture medium with different LCY concentrations (0, 2, 4, 6, 8, and 10 μM) was added to each well and cultured for 24 hours. Finally, 10 μL of MTT reagent was added to each well and cultured for another 3 hours. Figure 13 LCY was initially tested at a concentration range of 0, 2, 4, 6, 8, 10, 20, 50, and 100 M by CCK-8 assay to determine its cytotoxicity against live PK-15 cells. These cell viability results showed that LCY had low cytotoxicity at concentrations of 0-50 M, and further studies of LCY in biological systems were conducted.

[0090] like Figure 14As shown, PK-15 living cells were incubated with LCY (10 μM) in growth medium at 37°C for 30 minutes. Excess LCY on the cell surface was then removed after washing three times with sterile PBS buffer. Then, the LCY-prepared PK-15 living cells were further exposed to 10 μM Cu 2+ The solution was left for another 30 min and then washed with PBS to remove excess Cu. 2+ Finally, all prepared cell samples were detected using confocal fluorescence microscopy. Cells treated with LCY alone showed no color change under 488nM blue light. However, cells treated with both LCY and Cu showed no color change under 488nM blue light. 2+ The treated cells showed blue under 488 nM blue light irradiation, proving that the probe of the present invention can effectively detect copper ions in cells.

[0091] like Figure 15 As shown, Cu based on RGB mode 2+ Linear calibration curve of concentration and color intensity. 30 μL of probe stock solution and 0, 20, 40, 60, 80, and 100 μL of copper ion solution (3 mM) were added to 3 mL of blank solution DMSO:H2O:Hepes (v / v / v=1:9:0.1). 2+ A high-resolution photo of the solution under 365nm UV light was taken and converted into corresponding RGB data using the "Color Assist" application on the mobile phone. The color intensity was calculated using the formula I = 0.3R + 0.59G + 0.11B. The results showed that in the range of 0-100μM, the color intensity was consistent with that of Cu 2+ There is a good linear relationship between the concentrations.

[0092] The above experiments show that the present invention uses benzothiazole-2-acetonitrile as a fluorescent group to prepare a fluorescent probe LCY with a meta-aminophenol derivative through a condensation reaction. The probe is sensitive to Cu in the solution. 2+ It exhibits high sensitivity and selectivity, and can also detect the residues of the pesticide glyphosate in the environment.

Claims

1. A fluorescent probe LCY for copper ion detection, characterized in that: The copper ion detection fluorescent probe LCY uses benzothiazole-2-acetonitrile as a fluorescent group, and its structure is shown below:

2. A method for preparing the copper ion detection fluorescent probe LCY according to claim 1, characterized in that: The steps include: m-Aminophenol and 1-bromo-3-chloropropane are dissolved in an organic solvent and subjected to an electrophilic substitution reaction under catalysis to obtain the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol; then, 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol is used as a raw material and subjected to a Vilsmeier reaction in a phosphorus oxychloride solution to obtain the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde; then, the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde is reacted with the 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-8-ol as a raw material. 6,7-Tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and tert-butyldimethylsilyl chloride undergo nucleophilic substitution to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde; finally, the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde is condensed with benzothiazole-2-acetonitrile to obtain the fluorescent probe LCY.

3. The preparation method according to claim 2, characterized in that The reaction route for preparing the copper ion detection fluorescent probe LCY is preferably as follows:

4. The preparation method according to claim 2, characterized in that The m-aminophenol and 1-bromo-3-chloropropane are added and dissolved in an organic solvent, DMF, and then sodium carbonate is added as a catalyst. The mixture is refluxed and stirred at not less than 80° C. for 12-15 hours. The DMF is removed by extraction, and the organic phase is recovered, dried, and then subjected to column chromatography to obtain the intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol.

5. The preparation method according to claim 2, characterized in that The intermediate 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-8-ol is dissolved in an organic solvent DMF, and then added to a DMF solution of phosphorus oxychloride. The mixture is stirred at room temperature for 0.5-1 hour, heated to no less than 100° C. and refluxed for 1-2 hours. After the reaction is complete, the reaction solution is cooled to room temperature, saturated brine is added and stirred, and the obtained solid is filtered and dried to obtain the intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde.

6. The preparation method according to claim 2, characterized in that The intermediate 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and tert-butyldimethylsilyl chloride are dissolved in an organic solvent DCM, and then organic bases TEA and DMAP are added. The mixture is stirred at room temperature for 4-5 hours. After the reaction is complete, the mixture is washed with saturated brine, and the organic phase is recovered, dried, and distilled under reduced pressure to obtain the intermediate 8-((tert-butyldimethylsilyl)oxy)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde.

7. The preparation method according to claim 2, characterized in that The intermediate 8-((tert-butyldimethylsilyl)oxy)2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carboxaldehyde and benzothiazole-2-acetonitrile are dissolved in an organic solvent, anhydrous ethanol, and piperidine is used as a catalyst. The mixture is stirred at room temperature for 4-5 hours. After the reaction is completed, the obtained reaction product is filtered, washed, and dried to obtain the fluorescent probe LCY.

8. Use of the copper ion detection fluorescent probe LCY according to claim 1 in detecting copper ions and glyphosate.

9. Use of the copper ion detection fluorescent probe LCY according to claim 1 in preparing fluorescent test paper for detecting trace copper ions in a solution.

10. Use of the copper ion detection fluorescent probe LCY according to claim 1 in preparing a reagent for detecting trace copper ions in cells.

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