AIE fluorescent probe based on tetraphenyl ethylene as well as synthesis method and application of AIE fluorescent probe

Abstract

The invention discloses a tetraphenyl ethylene-based AIE fluorescent probe as well as a synthesis method and application thereof, and belongs to the technical field of fluorescent probe detection. The preparation method of the AIE fluorescent probe based on tetraphenylethylene comprises the following steps: mixing 1-bromo-1, 2, 2-triphenylethylene, 4-formylphenylboronic acid, a catalyst and an organic solvent, and carrying out a heating reaction to obtain 4-(1, 2, 2-triphenylethylene) benzaldehyde; the preparation method comprises the following steps: dissolving 4-(1, 2, 2-triphenylethylene) benzaldehyde and 2, 9-dimethyl-1, 10-phenanthroline in an acetic acid solution, and carrying out a heating reaction so as to obtain the AIE fluorescent probe based on tetraphenyl ethylene. The synthesized novel AIE fluorescent probe can be specifically combined with Cu < 2 + >, so that efficient and sensitive detection of glyphosate and imaging of glyphosate in living cells are realized, and a raw material and a theoretical basis are provided for detection of glyphosate residues in foods and environments.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence probe detection, and particularly relates to an AIE fluorescence probe based on tetraphenylethylene, its synthesis method and application. Background Art

[0002] Glyphosate (N-(phosphonomethyl)glycine, Glyp) is one of the most widely used organophosphorus herbicides, which is used to inhibit the growth of various unwanted plants. Its excessive residues in food, water and soil pose a serious threat to human health and the ecological environment.

[0003] So far, the methods for studying glyphosate mainly include physicochemical techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE) and enzyme-linked immunosorbent assay (ELISA). These methods play a very important role in studying the structural information of glyphosate. However, these methods usually require expensive instruments, time-consuming sample pretreatment and high detection costs, which will seriously restrict their application in on-site monitoring and real-time analysis of actual samples. Therefore, it is very necessary to develop low-cost, convenient and user-friendly methods to accurately and sensitively detect Glyp in real samples. Small molecule fluorescence probes have become an effective method for detecting glyphosate due to their high cell permeability, washing-free and adjustable photophysical properties. However, the existing small molecule fluorescence probes have the problem of low sensitivity. Therefore, it is very necessary to develop a highly sensitive glyphosate fluorescence probe. Summary of the Invention

[0004] The purpose of the present invention is to provide an AIE (aggregation-induced emission) fluorescence probe based on tetraphenylethylene, its synthesis method and application to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: an AIE fluorescence probe based on tetraphenylethylene (abbreviated as TPEOP), and its structural formula is shown as follows:

[0007]

[0008] The TPEOP of the present invention has unique aggregation-induced emission (AIE) characteristics. This AIE characteristic enables it to have the potential of high photoluminescence (PL) in solvents, so it can show high fluorescence intensity in solvents itself. Moreover, the phenanthroline unit in TPEOP can specifically bind to Cu 2+ , forming a TPEOP-Cu 2+ complex, resulting in the fluorescence quenching of TPEOP, thereby realizing the detection of Cu 2+Sensitive detection. In addition, glyphosate molecules have multiple phosphoric acid and amino groups that can complex with Cu in the TPEOP-Cu 2+ complex 2+ . Therefore, when glyphosate is contained in the solution system containing TPEOP-Cu 2+ , the fluorescence of TPEOP can be restored, thus realizing the sensitive detection of glyphosate.

[0009] The second technical solution of the present invention: The synthesis method of the above-mentioned AIE fluorescent probe based on tetraphenylethylene includes the following steps:

[0010] Dissolve 4-(1,2,2-triphenylethynyl)benzaldehyde and 2,9-dimethyl-1,10-phenanthroline in acetic acid solution, and heat for reaction to obtain the AIE fluorescent probe based on tetraphenylethylene.

[0011] Furthermore, the dosage ratio of 4-(1,2,2-triphenylethynyl)benzaldehyde, 2,9-dimethyl-1,10-phenanthroline and acetic acid solution is 1 mmol: 1-2 mmol: 1-2 mL.

[0012] Furthermore, the temperature of the heating reaction is 100-120 °C and the time is 6-8 h.

[0013] Furthermore, the concentration of the acetic acid solution is 99.5 wt%.

[0014] Furthermore, the preparation steps of 4-(1,2,2-triphenylethynyl)benzaldehyde include: mixing 1-bromo-1,2,2-triphenylethylene, 4-formylphenylboronic acid, a catalyst and an organic solvent, and heating for reaction to obtain 4-(1,2,2-triphenylethynyl)benzaldehyde.

[0015] Furthermore, the catalyst includes tetrakis(triphenylphosphine)palladium and an inorganic base.

[0016] Optionally, the inorganic base is K2CO3.

[0017] Furthermore, the organic solvent includes tetrahydrofuran.

[0018] Furthermore, the molar ratio of 1-bromo-1,2,2-triphenylethylene to 4-formylphenylboronic acid is 1: 1-2.

[0019] Furthermore, the temperature of the heating reaction is 55-60 °C and the time is 6-8 h.

[0020] Furthermore, the dosage ratio of 1-bromo-1,2,2-triphenylethylene to the organic solvent is 1 mmol: 15-25 mL.

[0021] Further, the dosage ratio of 1-bromo-1,2,2-triphenylethylene to tetrakis(triphenylphosphine)palladium is 1 mmol:10 mg, and the molar ratio of 1-bromo-1,2,2-triphenylethylene to the inorganic base is 1:1.

[0022] Technical solution three of the present invention: The above-mentioned tetraphenylethylene-based AIE fluorescent probe is used for detecting Cu 2+ in it.

[0023] Technical solution four of the present invention: The above-mentioned tetraphenylethylene-based AIE fluorescent probe is used for preparing a fluorescent probe for detecting glyphosate.

[0024] Technical solution five of the present invention: A fluorescent probe for detecting glyphosate, the raw materials include the above-mentioned tetraphenylethylene-based AIE fluorescent probe and Cu 2+ , that is, the fluorescent probe for detecting glyphosate is a complex of TPEOP and Cu 2+ , simply referred to as TPEOP-Cu 2+ .

[0025] Technical solution six of the present invention: The above-mentioned tetraphenylethylene-based AIE fluorescent probe or the above-mentioned fluorescent probe for detecting glyphosate is used for detecting glyphosate.

[0026] Technical solution seven of the present invention: The above-mentioned tetraphenylethylene-based AIE fluorescent probe or the above-mentioned fluorescent probe for detecting glyphosate is used for preparing a fluorescent detection product for fluorescent imaging of glyphosate in living cells.

[0027] The present invention discloses the following technical effects:

[0028] The present invention discloses a tetraphenylethylene-based AIE fluorescent probe, which can specifically bind to Cu 2+ , and realize the sensitive detection of Cu 2+ . The TPEOP-Cu 2+ formed after the AIE fluorescent probe binds to Cu 2+ can also be used as a fluorescent probe for detecting glyphosate, and realize the efficient and sensitive detection of glyphosate. It is found through experiments that the probe TPEOP-Cu 2+ has a high affinity for glyphosate, and is a novel glyphosate fluorescent probe with excellent performance, high stability and strong selectivity. The probe TPEOP-Cu 2+ can be used as an indicator for glyphosate to quantitatively detect glyphosate and image glyphosate in living cells, providing raw materials and theoretical basis for detecting the residue of glyphosate in food and environment. Description of the drawings

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 It is the fluorescence spectrum of the probe TPEOP in solution systems with different compositions.

[0031] Figure 2 It is for the ultraviolet absorption spectra of the probe TPEOP alone and when the probe TPEOP coexists with Cu 2+ (i.e., TPEOP + Cu 2+ ).

[0032] Figure 3 It is for the fluorescence detection results of the probe TPEOP at different Cu 2+ concentrations. Among them, A is the fluorescence spectrum of TPEOP at different Cu 2+ concentrations, B is the strongest emission intensity of TPEOP at different Cu 2+ concentrations (at 500 nm), and C is the linear relationship diagram of the detection of Cu 2+ by TPEOP.

[0033] Figure 4 It is for the response performance test results of glyphosate on the probe TPEOP-Cu 2+ . Among them, A is the influence of different concentrations of glyphosate on the fluorescence intensity of the probe TPEOP-Cu 2+ , and B is the linear relationship diagram of the detection of glyphosate by the probe TPEOP-Cu 2+ .

[0034] Figure 5 It is for the reversibility test results of the probe TPEOP-Cu 2+ to glyphosate.

[0035] Figure 6 It is for the influence results of pH value on the continuous recognition of Cu 2+ and glyphosate by TPEOP.

[0036] Figure 7 It is for the selectivity test results of the probe TPEOP-Cu 2+ to different pesticides.

[0037] Figure 8 It is for the cytotoxicity evaluation results of the probe TPEOP.

[0038] Figure 9 It is for the probe TPEOP and the probe TPEOP-Cu2+ Fluorescence imaging ability test results of glyphosate in living cells. Among them, A shows the cell imaging situation after culturing cells only with the probe TPEOP, and B shows the use of the probe TPEOP and Cu 2+ (i.e., the probe TPEOP-Cu 2+ ) for culturing cells and then imaging the cells, and C shows the cell imaging situation after culturing cells with the probe TPEOP-Cu 2+ and glyphosate and then imaging the cells. Detailed implementation manners

[0039] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0043] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0044] It should be noted that the aspects not described in detail in the present invention are all conventional operation means in the art and are not the focus of the present invention.

[0045] As the first aspect of the present invention, the present invention provides a tetraphenylethylene-based AIE fluorescent probe, and its structural formula is shown as follows:

[0046]

[0047] As the second aspect of the present invention, the present invention provides a synthesis method of the above-mentioned tetraphenylethylene-based AIE fluorescent probe, including the following steps:

[0048] (1) Synthesis of 4-(1,2,2-triphenylethynyl)benzaldehyde

[0049] The synthesis route is as follows:

[0050]

[0051] The synthesis steps are as follows: Mix 1-bromo-1,2,2-triphenylethylene, 4-formylphenylboronic acid, a catalyst and an organic solvent, and heat and react at 55 - 60 °C for 6 - 8 h to obtain 4-(1,2,2-triphenylethynyl)benzaldehyde;

[0052] (2) Synthesis of the tetraphenylethylene-based AIE glyphosate fluorescent probe

[0053] The synthesis route is as follows:

[0054]

[0055] The synthesis steps are as follows: Dissolve 4-(1,2,2-triphenylethynyl)benzaldehyde and 2,9-dimethyl-1,10-phenanthroline in an acetic acid solution, and heat and react at 100 - 120 °C for 6 - 8 h to obtain the tetraphenylethylene-based AIE fluorescent probe (abbreviated as TPEOP).

[0056] As a preferred embodiment of the present invention, the dosage ratio of 4-(1,2,2-triphenylethynyl)benzaldehyde, 2,9-dimethyl-1,10-phenanthroline and the acetic acid solution is 1 mmol: 1 - 2 mmol: 1 - 2 mL.

[0057] As a preferred embodiment of the present invention, the catalyst includes tetrakis(triphenylphosphine)palladium and an inorganic base.

[0058] As a preferred embodiment of the present invention, the inorganic base is K2CO3.

[0059] As a preferred embodiment of the present invention, the organic solvent includes tetrahydrofuran.

[0060] As a preferred embodiment of the present invention, the molar ratio of 1-bromo-1,2,2-triphenylethylene and 4-formylacetophenone is 1: 1 - 2.

[0061] As a preferred embodiment of the present invention, the dosage ratio of 1-bromo-1,2,2-triphenylethylene to the organic solvent is 1 mmol: 15 - 25 mL.

[0062] As a preferred embodiment of the present invention, the dosage ratio of 1-bromo-1,2,2-triphenylethylene to tetrakis(triphenylphosphine)palladium is 1 mmol: 10 mg, and the molar ratio of 1-bromo-1,2,2-triphenylethylene to the inorganic base is 1:1.

[0063] As a third aspect of the present invention, the present invention provides the above-mentioned tetraphenylethylene-based AIE fluorescent probe for use in detecting Cu 2+ in it.

[0064] As a preferred embodiment of the present invention, the steps of the application include:

[0065] (1) Mix TPEOP with Cu 2+ standard solutions with different concentrations. After reacting for 30 min, measure the fluorescence intensity at a wavelength of 500 nm, establish the quantitative relationship between the fluorescence intensity and the Cu 2+ concentration, draw a standard curve, and obtain a regression equation;

[0066] (2) Measure the fluorescence intensity of the sample determination solution according to the operation in step (1), substitute it into the regression equation, and calculate the concentration of Cu 2+ in the sample determination solution.

[0067] As a fourth aspect of the present invention, the present invention provides the above-mentioned tetraphenylethylene-based AIE fluorescent probe for use in preparing a fluorescent probe for detecting glyphosate.

[0068] As a fifth aspect of the present invention, the present invention provides a fluorescent probe for detecting glyphosate, the raw materials of which include the above-mentioned tetraphenylethylene-based AIE fluorescent probe and Cu 2+ , that is, the fluorescent probe for detecting glyphosate is a complex of TPEOP and Cu 2 + , abbreviated as TPEOP-Cu 2+ .

[0069] As a sixth aspect of the present invention, the present invention provides the above-mentioned tetraphenylethylene-based AIE fluorescent probe or the above-mentioned fluorescent probe for detecting glyphosate for use in detecting glyphosate.

[0070] As a preferred embodiment of the present invention, the steps of the application include:

[0071] (1) Mix TPEOP and Cu 2+Mix with glyphosate standard solutions of different concentrations. After reacting for 30 min, measure the fluorescence intensity at a wavelength of 500 nm, establish the quantitative relationship between the fluorescence intensity and the glyphosate concentration, plot the standard curve, and obtain the regression equation.

[0072] (2) Digest Cu in the test sample 2+ Extract glyphosate from the test sample to obtain the sample determination solution. Measure the fluorescence intensity according to the operation in step (1), substitute it into the regression equation, and calculate the concentration of glyphosate in the test sample solution to be measured.

[0073] As the seventh aspect of the present invention, the present invention provides the application of the above-mentioned AIE fluorescent probe based on tetraphenylethylene or the above-mentioned fluorescent probe for detecting glyphosate in the preparation of a fluorescent detection product for fluorescent imaging of glyphosate in living cells.

[0074] Cu involved in the specific implementation manner of the present invention 2+ Is added in the form of any soluble copper salt in the art.

[0075] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0076] In the following examples, if room temperature or normal temperature is involved, it specifically refers to 20 - 30 °C.

[0077] All raw materials used in the following examples of the present invention are ordinary commercially available products.

[0078] Example 1

[0079] A synthesis method of an AIE fluorescent probe based on tetraphenylethylene, the steps are as follows:

[0080] (1) Synthesis of 4-(1,2,2-triphenylethynyl)benzaldehyde

[0081] Add 335 mg of 1-bromo-1,2,2-triphenylethylene and 230 mg of 4-formylphenylboronic acid to 20 mL of tetrahydrofuran, stir evenly, then add 10 mg of tetrakis(triphenylphosphine)palladium and 138 mg of K2CO3 as catalysts, and heat and stir the reaction at 60 °C for 7 h under nitrogen protection. After the reaction is completed, extract with dichloromethane 3 times, collect the organic phase, and further evaporate; then use ethyl acetate (EA) and petroleum ether (PE) (volume ratio 1:20) as the eluent, and separate by column chromatography to obtain a yellow solid (yield about 62.5%), which is 4-(1,2,2-triphenylethynyl)benzaldehyde.

[0082] Perform nuclear magnetic resonance detection on the prepared 4-(1,2,2-triphenylethynyl)benzaldehyde, and the results are as follows: 11H NMR (400 MHz, CDCl3) δ (ppm): 7.05 - 7.07 (6H, m), 7.15 (9H, s), 7.23 (2H, d, J = 4.0 Hz), 9.93 (1H, s).

[0083] 13 13C NMR (100 MHz, CDCl3) δ (ppm): 126.86, 126.89, 127.05, 127.75, 127.93, 129.16, 131.23, 131.29, 131.94, 134.29, 139.77, 142.90, 143.00, 143.05, 150.56.

[0084] MS (ESI) m / z: calcd for C 27 H 20 O [M + H]+ m / z 361.15, found 361.17.

[0085] (2) Synthesis of AIE Fluorescent Probe Based on Tetraphenylethylene

[0086] Dissolve 360 mg of 4-(1,2,2-triphenylethenyl)benzaldehyde and 410 mg of 2,9-dimethyl-1,10-phenanthroline in 2 mL of acetic acid solution (concentration: 99.5 wt%). Heat the reaction mixture at 110 °C for 7 h under nitrogen protection. After the reaction, remove acetic acid, extract with ethyl acetate three times, collect the organic phase, and further evaporate it. Then, use EA and PE (volume ratio 1:10) as the eluent and separate by column chromatography to obtain a yellow solid (yield: about 45%), which is the AIE fluorescent probe based on tetraphenylethylene (abbreviated as probe TPEOP).

[0087] Perform nuclear magnetic resonance detection on the prepared probe TPEOP, and the results are as follows: 1 1H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.64 (s, 2H), 7.55 (d, J = 4.2 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.19 (s, 1H), 7.10 - 6.95 (m, 18H), 2.89 (s, 3H).

[0088] Example 2

[0089] The probe TPEOP prepared in Example 1 was added to a solution system composed of different proportions of dimethyl sulfoxide (DMSO) and PBS (pH = 7.4) to study the AIE properties of the probe TPEOP. Specifically, the probe TPEOP was added to each group of solutions, and fluorescence spectroscopy analysis was performed (excitation wavelength: 400 nm, maximum emission wavelength: 500 nm, the same below). The addition concentration of the probe TPEOP in each group of solutions was 10.0 μM.

[0090] The results are as Figure 1 shown. Among them, the lines marked with 5% - 100% represent that the volume concentration of DMSO in the mixed solution gradually increases from 5 vol% to 100 vol% along the arrow direction. It can be seen that in the range of 0% - 95% PBS content, when the PBS content is 0%, the probe TPEOP shows a weak fluorescence intensity at 500 nm. When the PBS content increases, the fluorescence intensity gradually increases, and reaches the maximum value when the PBS content is 95%. The results show that the probe TPEOP forms aggregates in a poor solvent system and exhibits good AIE effects. Moreover, it was found during the experiment that when the proportion of DMSO in the mixed solution is 10 vol%, TPEOP is just completely dissolved. Therefore, in order to better measure copper ions and glyphosate in aqueous solutions subsequently, the subsequent detection experiments were carried out in a mixed solution with 10 vol% DMSO.

[0091] To further evaluate the ability of the probe TPEOP to quantitatively detect Cu 2+ , the probe TPEOP (10.0 μM) was interacted with PBS (containing 10 vol% DMSO, pH = 7.4) and different concentrations of Cu 2+ for 30 min (that is, the probe TPEOP was added to the mixed solution of DMSO and PBS containing 10 vol% DMSO at a concentration of 10.0 μM, and different final concentrations of Cu 2+ were added, and then reacted at 37 °C for 30 min), and then ultraviolet absorption spectroscopy and fluorescence spectroscopy analysis were performed.

[0092] Figure 2 is the ultraviolet absorption spectrum. Among them, TPEOP represents that the concentration of Cu 2+ is 0, and TPEOP + Cu 2+ represents the absorption spectrum curve after adding Cu 2+ with a final concentration of 100 μM. It can be seen that when TPEOP is incubated with Cu 2+ , the absorption peak of the absorption spectrum of TPEOP at 380 nm does not change significantly, further indicating that the color of TPEOP does not change under daylight after adding Cu 2+ .

[0093] Figure 3 Fluorescence detection results of probe TPEOP at different Cu 2+ concentrations. Among them, A is the fluorescence spectrum of TPEOP at different Cu 2+ concentrations, B is the strongest emission intensity of TPEOP at different Cu 2+ concentrations (at 500 nm), and C is a linear relationship diagram of TPEOP's detection of Cu 2+ with different concentrations as the abscissa and the strongest emission intensity of TPEOP at 500 nm as the ordinate (showing the standard curve and regression equation). As can be seen from 2+ , as the concentration of Cu Figure 3 added to the probe TPEOP solution increases, the fluorescence intensity at 500 nm gradually decreases. When the Cu 2+ concentration increases to 600 nM, the fluorescence intensity weakens by nearly 86 times compared with that without adding Cu 2+ , indicating that TPEOP has a dynamic response to the Cu 2+ concentration (A in 2+ ). Moreover, the probe TPEOP has a very good linear relationship of concentration response to Cu Figure 3 in the range of 0 - 80 nM. The calibration equation is y = 6.13E6 - 54145x (nM), and R 2+ = 0.992. The LOD (limit of detection) of Cu 2 is calculated by the formula LOD = 3σ / k (the ratio of the noise level (3σ) to the method sensitivity (k)) to be 17 nM. It shows that the probe TPEOP has a high affinity for Cu 2+ (C in 2+ ). Figure 3

[0094] Example 3

[0095] Detect the response performance of the probe TPEOP-Cu 2+ to different concentrations of glyphosate. The specific steps are as follows:

[0096] First, add glyphosate PBS solutions with the same concentration but different volumes to a 1 mL PE tube, then add the probe TPEOP and Cu 2+ respectively and mix evenly. Finally, add PBS solvent (10 mM, pH = 7.4) to make up the total volume to 1 mL to obtain multiple groups of mixed solutions with the same TPEOP-Cu 2+ concentration but different glyphosate concentrations (the final mixed concentration of TPEOP-Cu 2+ is 10.0 μM, and the final glyphosate concentration is 0 - 5.5 μM). Incubate at 37 °C for 30 min, and then perform fluorescence emission spectrum testing on it.

[0097] ​The fluorescence emission spectrum is as shown in Figure 4 A in it. When the glyphosate concentration is 0 μM, the fluorescence intensity of the probe TPEOP-Cu 2+ is very weak. However, as the glyphosate concentration continuously increases, the fluorescence intensity of the probe TPEOP-Cu 2+ at 500 nm also increases with the increase of the glyphosate concentration. When the glyphosate concentration increases to about 5.0 μM, the fluorescence intensity of the probe TPEOP-Cu 2+ at 500 nm basically no longer increases with the increase of the glyphosate concentration and reaches the maximum value. It shows that the saturation binding concentration of the probe TPEOP-Cu 2+ to glyphosate is about 5.0 μM.

[0098] Taking the different glyphosate concentrations as the abscissa and the strongest emission intensity of the probe TPEOP-Cu 2+ at 500 nm as the ordinate, a linear relationship graph (showing the standard curve and regression equation) of the detection of glyphosate by the probe TPEOP-Cu 2+ is plotted, as shown in Figure 4 B in it. It can be found that the linear relationship between the probe TPEOP-Cu 2+ and glyphosate with a concentration of 0 - 5 μM is very good, and its R 2 = 0.996. The detection limit of glyphosate is calculated to be 9.8 nM. It shows that the probe TPEOP-Cu 2+ has a high affinity for glyphosate.

[0099] From the above experimental results, it can be seen that the probe TPEOP-Cu 2+ is a novel AIE fluorescent probe for glyphosate with excellent performance.

[0100] Example 4

[0101] To detect the reversibility of the probe TPEOP-Cu 2+ to glyphosate, the specific steps are as follows:

[0102] At 37 °C, prepare a TPEOP solution (the solvent is a mixed solution of DMSO and PBS containing 10 vol% DMSO, pH = 7.4; the TPEOP concentration is 10.0 μM), detect its fluorescence signal at 500 nm, add Cu 2+ (the added concentration is 600 nM) to the TPEOP solution, detect the change of the fluorescence signal at 500 nm after acting for 30 min, and then add glyphosate (the added concentration is 5 μM), and detect the change of the fluorescence signal at 500 nm after acting for 30 min. Repeat the process of alternately adding Cu 2+ and glyphosate to form a cycle (the added amount each time is the same). The test results are as shown in Figure 5 It can be seen that when Cu2+ When added alternately with glyphosate to the TPEOP solution, TPEOP can stably cycle 3 times with no significant change in fluorescence intensity. The results of the reversibility experiment not only prove that TPEOP has high cycle stability, but also further confirm that Cu 2+ in the Cu 2+ composition is complexed by glyphosate and the fluorescence of TPEOP is restored.

[0103] Example 5

[0104] Detect the influence of pH value on the probe TPEOP-Cu 2+

[0105] To verify the effectiveness of the probe TPEOP-Cu 2+ this example uses fluorescence spectroscopy to explore the influence of different pH values on the sequential recognition of Cu 2+ and glyphosate by TPEOP. Specifically: A 10 μM probe TPEOP solution (with the solvent being a mixed solution of DMSO and PBS containing 10 vol% DMSO) was used to measure the fluorescence intensity in the pH range of 2.0 - 8.0 as a control; Cu 2+ (added concentration 600 nM) was added to the probe TPEOP solution at different pH values, and the fluorescence intensity was detected. Glyphosate (added concentration 5 μM) was added to the probe TPEOP-Cu 2+ solution at different pH values, and the fluorescence intensity was detected.

[0106] The results are as Figure 6 shown. When pH < 6, TPEOP has no obvious fluorescence, while when pH > 7, the fluorescence intensity of TPEOP significantly increases, which may be caused by the destruction of the TPEOP structure. The fluorescence of the TPEOP-Cu 2+ system shows an obvious quenching state in a wide range of 2 - 8. The detection of glyphosate by the probe TPEOP-Cu 2+ has the best performance under neutral (7.2 - 7.4) conditions.

[0107] Example 6

[0108] Selectivity test of the probe TPEOP-Cu 2+

[0109] In this example, the selectivity of the probe TPEOP-Cu 2+ for different pesticides was studied to confirm the selectivity of the probe TPEOP-Cu 2+ for glyphosate. Specifically: A 10.0 μM probe TPEOP-Cu 2+ ​​The solution (the solvent is a mixed solution of DMSO and PBS containing 10 vol% DMSO, pH = 7.4) was incubated with different pesticides at 37 °C for 30 min and then fluorescence tests were performed.

[0110] To verify the possibility that the TPEOP-Cu 2+ complex can specifically recognize glyphosate, in this example, the TPEOP-Cu 2+ complex was used to conduct spectral studies on 11 pesticides, and the results are as Figure 7 shown. The experimental results show that the fluorescence intensity increased by 41 times after the addition of glyphosate, probably because the phosphonate and carboxylate groups of glyphosate endow glyphosate with an efficient ability to chelate with Cu 2+ . The probe TPEOP-Cu 2+ showed basically no response to the other 10 pesticides.

[0111] Example 7

[0112] Cytotoxicity evaluation

[0113] The WST-8 reagent was used to evaluate the cytotoxicity of the probe TPEOP to HeLa cells. The specific operation steps are as follows:

[0114] TPEOP solutions with different concentrations (0 μM - 30 μM) (the solvent of the three solutions is a mixed solution of DMSO and PBS containing 10 vol% DMSO, pH = 7.4) were added to different wells of cells with a density of about 30%, and incubated for 24 h. After washing 3 times with PBS, the WST-8 reagent was added, and the absorbance at 490 nm was recorded with an enzyme-linked immunosorbent assay (ELISA) reader after incubation for about 3 h to explore the cell viability.

[0115] The results are as Figure 8 shown, indicating that after incubation with the probe TPEOP at different concentrations for 24 h, the survival rate of HeLa cells exceeded 95%, which shows that the probe TPEOP has good biocompatibility.

[0116] Example 8

[0117] Based on the excellent performance of the probe TPEOP in vitro, in this example, the ability of the probe TPEOP to perform fluorescence imaging of glyphosate in living cells was studied. The specific operation steps are as follows:

[0118] A 10.0 μM probe TPEOP solution (the solvent of the three solutions is a mixed solution of DMSO and PBS containing 10 vol% DMSO, pH = 7.4), or a 10.0 μM TPEOP-Cu 2+ mixed solution, or a mixed solution of the probe TPEOP-Cu 2+ and glyphosate (TPEOP-Cu 2+Cultivate HeLa cells with a density of approximately 40% at a concentration of 10.0 μM and a glyphosate concentration of 10 μM, then perform confocal fluorescence imaging and obtain confocal fluorescence images.

[0119] The results are as Figure 9 shown, where A shows the case of cell imaging after cultivating cells only with the probe TPEOP, B shows the case of cell imaging after cultivating cells with the probe TPEOP and Cu 2+ (i.e., the probe TPEOP-Cu 2+ ), and C shows the case of cell imaging after cultivating cells with the probe TPEOP-Cu 2+ and glyphosate. It can be seen that there is strong fluorescence after adding glyphosate into the cells, and this result indicates that the probe TPEOP can effectively perform fluorescence imaging of glyphosate in vivo.

[0120] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A tetraphenylethylene-based AIE fluorescent probe, characterized in that, The structural formula of the tetraphenylethylene-based AIE fluorescent probe is as follows:

2. The synthesis method of the AIE fluorescent probe based on tetraphenylethylene according to claim 1, wherein It includes the following steps: Dissolve 4-(1,2,2-triphenylethynyl)benzaldehyde and 2,9-dimethyl-1,10-phenanthroline in acetic acid solution, and heat for reaction to obtain the tetraphenylethylene-based AIE fluorescent probe.

3. The synthesis method according to claim 2, wherein, The dosage ratio of the 4-(1,2,2-triphenylethynyl)benzaldehyde, 2,9-dimethyl-1,10-phenanthroline and acetic acid solution is 1 mmol: 1-2 mmol: 1-2 mL; And / or, the temperature of the heating reaction is 100-120 °C, and the time is 6-8 h.

4. The synthesis method according to claim 2, characterized in that, The preparation steps of the 4-(1,2,2-triphenylethynyl)benzaldehyde include: mix 1-bromo-1,2,2-triphenylethylene, 4-formylphenylboronic acid, a catalyst and an organic solvent, and heat for reaction to obtain the 4-(1,2,2-triphenylethynyl)benzaldehyde.

5. The synthesis method according to claim 4, characterized in that, The catalyst includes tetrakis(triphenylphosphine)palladium and an inorganic base; And / or, the organic solvent includes tetrahydrofuran; And / or, the molar ratio of the 1-bromo-1,2,2-triphenylethylene and 4-formylphenylboronic acid is 1: 1-2; And / or, the temperature of the heating reaction is 55-60 °C, and the time is 6-8 h.

6. Use of a tetraphenylethylene-based AIE fluorescent probe as described in claim 1 for detecting Cu 2+ in it.

7. Application of a tetraphenylethylene-based AIE fluorescent probe as described in claim 1 in the preparation of a fluorescent probe for detecting glyphosate.

8. A fluorescence probe for detecting glyphosate, characterized in that, The raw materials include the tetraphenylethylene-based AIE fluorescent probe as described in claim 1 and Cu 2+ .

9. Application of a tetraphenylethylene-based AIE fluorescent probe as described in claim 1 or a fluorescent probe for detecting glyphosate as described in claim 8 in the detection of glyphosate.

10. Application of a tetraphenylethylene-based AIE fluorescent probe as described in claim 1 or a fluorescent probe for detecting glyphosate as described in claim 8 in the preparation of a fluorescent detection product for fluorescence imaging of glyphosate in living cells.

Images