A dual-recognition fluorescent molecular probe, its preparation method and application in detecting lead ions and / or glyphosate

Abstract

The present invention discloses a dual-recognition fluorescent molecular probe and its preparation method and application in the detection of lead ions and / or glyphosate, relating to the technical field of the detection of lead ions and the pesticide glyphosate. The dual-recognition fluorescent molecular probe of the present invention is a Schiff base type probe with dicyanoisophorone and benzoylhydrazine as fluorescent groups. The present invention also relates to the application of the dual-recognition fluorescent molecular probe in the sensing detection of lead ions. The complex of the dual-recognition fluorescent molecular probe and lead ions can be used for the sensing detection of glyphosate. The raw materials of the dual-recognition fluorescent molecular probe provided by the present invention are easily obtained and the operation is simple. The present invention realizes the quantitative detection of lead ions / or glyphosate by the different functions demonstrated by the probe through ultraviolet and fluorescence spectra, with strong reversibility, can be recycled multiple times, and has good selectivity, is not easily affected by other metal ions, has a low detection limit. In addition, it also shows excellent application imaging in animals such as cells and zebrafish.

Description

Technical Field

[0001] The present invention relates to the technical field of lead ion and glyphosate detection, and particularly relates to a dual-recognition fluorescent molecular probe, a preparation method thereof, and an application thereof in detecting lead ions and / or glyphosate in Chinese herbal medicines. Background Art

[0002] Lead is one of the non-degradable heavy metal pollutants in natural environments such as water and soil. The main sources of lead pollution are lead used in industries such as gasoline and battery manufacturing. Lead plays a crucial role in fields such as the environment, chemistry, and biology, but at the same time, it poses a threat to human health. It is reported that the long-term accumulation of lead ions in different organs of the human body can cause serious harm to kidney diseases and the nervous system, resulting in carcinogenic, mutagenic, and teratogenic effects. The maximum intake of lead ions in drinking water allowed by the World Health Organization is 10 μM. Therefore, it is very important to timely track and detect lead ions.

[0003] Glyphosate is an organophosphorus herbicide that mainly inhibits the conversion of shikimic acid to phenylalanine, tyrosine, and tryptophan by inhibiting enolpyruvylshikimate-3-phosphate synthase in plants, thereby interfering with protein synthesis and causing plant death. Due to its high activity, low cost, and high efficiency, it is widely used in agriculture and forestry. However, due to the characteristics of glyphosate having a long half-life and strong water solubility, a large amount of residues have been found in the environment and food, which cause serious harm to the ecosystem and human health. At the same time, glyphosate is considered a potential carcinogen that can cause respiratory, myocardial, central nervous system, and muscle dysfunction, and even lead to death. In addition, the World Health Organization limits the maximum residue level of glyphosate in drinking water to 5.325 μM (900 μg / L). Therefore, the research and development of glyphosate safeners are of great significance for production and life. At present, there is no rapid, efficient, and convenient glyphosate detection technology. Therefore, there is an urgent need for a convenient technology to assist in the research and development of glyphosate safeners.

[0004] In recent years, various technologies for detecting lead have been developed to a certain extent, such as atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES), capillary electrophoresis (CE), fluorescence spectroscopy, X-ray fluorescence spectroscopy (XRF), and inductively coupled plasma mass spectrometry (ICP-MS). However, the detection range and detection limit of these instruments usually depend on the sample preparation method, operating conditions, and the instrument, and at the same time, they also have disadvantages such as time-consuming, bulky, and expensive. Therefore, compared with other reported detection technologies, people are becoming more and more interested in the detection of organic fluorescent molecular probes in the biological environment field. Fluorescent molecular probes show significant advantages such as simple operation, high sensitivity, low cost, and convenience. Therefore, the development of fluorescent probes that can be used for the specific detection of lead and glyphosate is of great significance in biological environment and chemical analysis. Summary of the Invention

[0005] The object of the present invention is to overcome the problems existing in the prior art, such as complex synthesis steps, poor selectivity and low sensitivity of the probes for detecting lead ions and glyphosate, and to provide a dual-recognition fluorescent molecular probe, a preparation method thereof, and an application thereof in detecting lead ions and / or glyphosate.

[0006] The present invention provides a dual-recognition fluorescent detection agent for lead ions and / or glyphosate, which is simple, rapid, highly specific and highly sensitive, and has broad application prospects.

[0007] To achieve the above object, in the first aspect of the present invention, a dual-recognition fluorescent molecular probe is provided, wherein the structural formula of the dual-recognition fluorescent molecular probe is shown as Formula I below:

[0008]

[0009] Specifically, in the above technical solution, the dual-recognition fluorescent molecular probe is a Schiff base type probe with dicyanoisophorone and benzoyl hydrazide as fluorescent groups.

[0010] In the second aspect of the present invention, a preparation method of the dual-recognition fluorescent molecular probe described in the first aspect is provided, which is obtained by reacting compound III with benzoyl hydrazide, and the synthesis reaction equation is shown as Formula II below:

[0011]

[0012] Further, in the above technical solution, the preparation method of the dual-recognition fluorescent molecular probe specifically includes the following steps:

[0013] Add compound III into ultradry ethanol, then add benzoyl hydrazide, and carry out a heating reflux reaction; after stopping the reaction, cool to room temperature, filter the obtained product, and slurry and purify the obtained filter cake with absolute ethanol to obtain the target product.

[0014] Specifically, in the above technical solution, the dosage of the ultradry ethanol may not be specifically limited as long as it can achieve the uniform dissolution of compound III. For example, in the preferred embodiment of the present invention, the dosage ratio of compound III to ultradry ethanol is 100 mg: 20 mL.

[0015] Preferably, in the above technical solution, the molar ratio of compound III to benzoyl hydrazide is 1:1.1.

[0016] Preferably, in the above technical solution, the time of the heating reflux reaction is 4 - 8 h.

[0017] Further, in the above technical solution, in the preferred embodiment of the present invention, the preparation method of compound III is as follows, including the following steps:

[0018] Step 1: Prepare compound I using isophorone and malononitrile as reaction raw materials;

[0019] Step 2: React compound I with p-hydroxybenzaldehyde to prepare compound II;

[0020] Step 3: React compound II with hexamethylenetetramine to prepare the said compound III.

[0021] Specifically, the synthesis reaction equation of the said compound III is as shown in Formula III below:

[0022]

[0023] Furthermore, in the above technical solution, the specific synthesis steps of compound I in step S1 are as follows:

[0024] Dissolve isophorone in absolute ethanol, then sequentially add malononitrile and piperidine, and heat and stir under reflux in a nitrogen atmosphere; after the reaction is completed, cool to room temperature, pour the obtained product into ice water, a large amount of precipitate is formed, filter by suction, and purify the obtained solid by column chromatography to obtain the said compound I.

[0025] Preferably, in the above technical solution, the molar ratio of isophorone to malononitrile is 5:6.

[0026] Preferably, the molar ratio of isophorone to piperidine is 50:3.

[0027] Preferably, the reflux reaction time is 6 h.

[0028] Furthermore, in the above technical solution, the specific synthesis steps of compound II in step S2 are as follows:

[0029] Add p-hydroxybenzaldehyde to absolute ethanol, then sequentially add compound I and piperidine, heat under reflux until the raw material spots disappear, and stop the reaction; spin-dry the obtained product, and then separate and purify it by column chromatography to obtain the said compound II.

[0030] Specifically, in the above technical solution, the dosage of absolute ethanol may not be specifically limited as long as it can achieve uniform dissolution of p-hydroxybenzaldehyde. For example, in the preferred embodiment of the present invention, the dosage ratio of p-hydroxybenzaldehyde to absolute ethanol is 10 mmol:40 mL.

[0031] Preferably, in the above technical solution, the molar ratio of compound I to p-hydroxybenzaldehyde is 1:1.

[0032] Preferably, in the above technical solution, the molar ratio of compound I to piperidine is 10:3.

[0033] Furthermore, in the above technical solution, the specific synthesis steps of the compound Ⅲ in step S3 are as follows:

[0034] Dissolve the compound Ⅱ in trifluoroacetic acid, then add hexamethylenetetramine, and heat under reflux; after the reaction is completed, cool the obtained product to room temperature, then slowly pour the obtained product into ice water, and successively carry out filtration, column chromatography separation and purification to obtain the compound Ⅲ.

[0035] Specifically, in the above technical solution, the dosage of the trifluoroacetic acid may not be specifically limited as long as it can achieve the uniform dissolution of the compound Ⅱ. For example, in the preferred embodiment of the present invention, the dosage ratio of the compound Ⅱ to the trifluoroacetic acid is 1.5 mmol: 10 mL.

[0036] Preferably, in the above technical solution, the molar ratio of the compound Ⅱ to hexamethylenetetramine is 1:1.

[0037] The third aspect of the present invention provides the application of the dual-recognition fluorescent molecular probe described in the first aspect or the dual-recognition fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of lead ions.

[0038] In some embodiments of the present invention, the dual-recognition fluorescent molecular probe is used for the sensing detection of lead ions in water environment, living cells and living zebrafish.

[0039] The fourth aspect of the present invention provides a complex of the dual-recognition fluorescent molecular probe described in the first aspect or the dual-recognition fluorescent molecular probe obtained by the preparation method described in the second aspect and lead ions.

[0040] In the present invention, the preparation method of the complex may include: mixing the dual-recognition fluorescent molecular probe with an equimolar amount of lead ions in a mixed solution of DMF and water to obtain a complex of the dual-recognition fluorescent molecular probe and lead ions.

[0041] The fifth aspect of the present invention provides the application of the complex described in the fourth aspect in the sensing detection of glyphosate.

[0042] In some embodiments of the present invention, the complex is used for the sensing detection of glyphosate in water environment, living cells and living zebrafish.

[0043] In some embodiments of the present invention, the sensing detection is selective sensing detection or competitive sensing detection.

[0044] In some embodiments of the present invention, the sensing detection is visual qualitative detection, ultraviolet light detection, fluorescence detection or reversible detection.

[0045] In some embodiments of the present invention, the sensing detection is quantitative detection under ultraviolet spectrum or fluorescence spectrum.

[0046] Existing fluorescent probes only use fluorescence spectroscopy to quantitatively detect metal ions. The dual-recognition fluorescent molecular probe of the present invention can combine ultraviolet ratio quantitative detection with fluorescence spectroscopy quantitative detection to provide a variety of different data, increasing the accuracy and reliability of the detection of lead ions and glyphosate. The ultraviolet spectrum can provide higher contrast and sensitivity. The most intuitive phenomenon is that the dual-recognition fluorescent molecular probe of the present invention has a chromogenic sensing function, which not only causes a change in the color of the solution under sunlight, but also produces a change in fluorescence color under the irradiation of a 365 nm ultraviolet lamp. This dual observation mode makes it a chromogenic sensing fluorescent probe suitable for different lighting conditions.

[0047] Through the above technical solutions, the beneficial technical effects obtained by the present invention are as follows:

[0048] (1) Based on specific recognition, the dual-recognition fluorescent molecular probe of the present invention can be used as a specific indicator for the presence of lead ions in different water environments, living cells, and zebrafish. It can provide real-time qualitative and quantitative information through fluorescence detection, and the color change makes it a highly specific indicator suitable for visual colorimetry detection. At the same time, the probe has high reversibility and repeatability for the detection of lead ions, and can also be used as a sensor for detecting lead ions in the environment, living cells, and living zebrafish, and can be detected repeatedly without damaging its performance.

[0049] (2) The synthesis of the dual-recognition fluorescent molecular probe provided by the present invention only requires two steps or even one step to synthesize, with easily available raw materials and simple operations.

[0050] (3) The present invention realizes the quantitative detection of lead ions and glyphosate by the different functions of the probe shown by ultraviolet and fluorescence spectra, with strong reversibility, multiple recycling, good selectivity, not easily affected by other ions, and low detection limit, and can be widely used to detect lead ions and / or glyphosate existing in complex environments.

[0051] Lead ions: The ultraviolet detection limit is 1.29×10 -7 M, and the fluorescence detection limit is 3.23×10 -8 M;

[0052] Glyphosate: The ultraviolet detection limit is 3.94×10 -7 M, and the fluorescence detection limit is 3.65×10 -8 M. Description of the Drawings

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. 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.

[0054] Figure 1 It is the high-resolution mass spectrum of the dual-recognition fluorescent molecular probe H-2 synthesized in Example 1 of the present invention.

[0055] Figure 2 It is the nuclear magnetic resonance 1 H NMR spectrum of the dual-recognition fluorescent molecular probe H-2 synthesized in Example 1 of the present invention.

[0056] Figure 3 It is the nuclear magnetic resonance 13 CNMR spectrum of the dual-recognition fluorescent molecular probe H-2 synthesized in Example 1 of the present invention.

[0057] Figure 4 For Application Example 1 of the present invention: (A) is the fluorescence spectrum of H-2 + Pb 2+ or other metal ions; (B) is the fluorescence spectrum of [H-2 - Pb 2+ + pesticide or anion.

[0058] Figure 5 For Application Example 1 of the present invention, the ultraviolet absorption spectra of H-2, H-2 + Pb 2+ and [H-2 - Pb 2+ + glyphosate.

[0059] Figure 6 For Application Example 2 of the present invention: (A) is the competitive detection graph of the probe H-2 for different metal cations in the DMF / water (4:6, v / v, 0.01M Hepes, pH = 7.4) system, and (B) is the competitive detection graph of [H-2 - Pb 2+ for different anions and pesticides in the DMF / water (4:6, v / v, 0.01M Hepes, pH = 7.4) system.

[0060] Figure 7 For Application Example 3 of the present invention: (A) is the fluorescence titration curve of H-2 + Pb 2+ ; (B) is the fluorescence titration curve of [H-2 - Pb 2+ + glyphosate.

[0061] Figure 8 For Application Example 3 of the present invention: (A) H-2 + Pb 2+UV titration curve; (B) is the UV titration curve of [H-2-Pb 2+ + glyphosate.

[0062] Figure 9 This is the reversibility detection graph of H-2 with Pb 2+ and glyphosate in Application Example 4 of the present invention.

[0063] Figure 10 This is the effect diagram of probe H-2, H-2+Pb 2+ and [H-2-Pb 2+ + glyphosate under sunlight in Application Example 5 of the present invention.

[0064] Figure 11 This is the effect diagram of probe H-2, H-2+Pb 2+ and [H-2-Pb 2+ + glyphosate under irradiation of 365nm UV lamp in Application Example 5 of the present invention.

[0065] Figure 12 This is the application imaging diagram of probe H-2 for detecting Pb 2+ and glyphosate in living cells in Application Example 6 of the present invention.

[0066] Figure 13 This is the application imaging diagram of probe H-2 for detecting Pb 2+ and glyphosate in zebrafish in Application Example 7 of the present invention. Detailed implementation manners

[0067] The present invention relates to the technical field of lead ion and glyphosate pesticide detection, and discloses a dual-recognition fluorescent molecular probe and its preparation method and application in detecting lead ions and / or glyphosate. The dual-recognition fluorescent molecular probe is a Schiff base type probe with dicyanoisophorone and benzoylhydrazine as fluorescent groups. The present invention also relates to the application of the dual-recognition fluorescent molecular probe in lead ion sensing detection. The complex of the dual-recognition fluorescent molecular probe and lead ions can be used for glyphosate sensing detection. The raw materials of the dual-recognition fluorescent molecular probe provided by the present invention are easy to obtain and the operation is simple. The present invention realizes the quantitative detection of lead ions / or glyphosate by the different functions of the probe shown through ultraviolet and fluorescence spectra, has strong reversibility, can be recycled multiple times, and has good selectivity, is not easily affected by other metal ions, has a low detection limit, and in addition, also presents excellent application imaging in animals such as cells and zebrafish.

[0068] In the ranges disclosed in the present invention, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0069] The first aspect of the present invention provides a dual-recognition fluorescent molecular probe, wherein the structural formula of the dual-recognition fluorescent molecular probe is as follows:

[0070]

[0071] The second aspect of the present invention provides a preparation method of the dual-recognition fluorescent molecular probe described in the first aspect, which is obtained by reacting compound Ⅲ with benzohydrazide, and the synthetic reaction equation is shown as follows:

[0072]

[0073] Compound Ⅲ used in the present invention is disclosed in the prior art. For example, compound Ⅲ is synthesized by the synthesis method disclosed in the application publication number CN117945994A, and the method includes the following steps:

[0074] Step 1: Prepare compound Ⅰ using isophorone and malononitrile as reaction raw materials;

[0075] Step 2: React compound Ⅰ with p-hydroxybenzaldehyde to prepare compound Ⅱ;

[0076] Step 3: React compound Ⅱ with hexamethylenetetramine to prepare compound Ⅲ.

[0077] In some embodiments of the present invention, the progress of the reaction is monitored by TLC.

[0078] The third aspect of the present invention provides the application of the dual-recognition fluorescent molecular probe described in the first aspect or the dual-recognition fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of lead ions.

[0079] In some embodiments of the present invention, the dual-recognition fluorescent molecular probe is used for the sensing detection of lead ions in water environment, living cells and living zebrafish.

[0080] The fourth aspect of the present invention provides a complex of the dual-recognition fluorescent molecular probe described in the first aspect or the dual-recognition fluorescent molecular probe obtained by the preparation method described in the second aspect and lead ions.

[0081] In the present invention, the preparation method of the complex may include: mixing a dual-recognition fluorescent molecular probe with an equimolar amount of lead ions in a mixed solution of DMF and water to obtain a complex of the dual-recognition fluorescent molecular probe and lead ions.

[0082] The fifth aspect of the present invention provides an application of the complex described in the fourth aspect in the sensing detection of glyphosate.

[0083] In some embodiments of the present invention, the complex is used for sensing detection of glyphosate in an aqueous environment, living cells, and living zebrafish.

[0084] In some embodiments of the present invention, the sensing detection is selective sensing detection and competitive sensing detection.

[0085] In some embodiments of the present invention, the sensing detection is visual qualitative detection, ultraviolet light detection, fluorescence detection, and reversible detection.

[0086] In some embodiments of the present invention, the sensing detection is quantitative detection under ultraviolet spectra and fluorescence spectra.

[0087] The present invention will be described in detail below through examples.

[0088] For those not specified in the following examples and comparative examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial channels.

[0089] Example 1

[0090] A dual-recognition fluorescent molecular probe provided in this example, the molecular probe is a Schiff base type probe with dicyanoisophorone and benzoylhydrazine as fluorescent groups, and its structural formula is shown as follows:

[0091]

[0092] Example 2

[0093] This example provides a preparation method of the dual-recognition fluorescent molecular probe described in Example 1, and the reaction route is as follows:

[0094]

[0095] The preparation method of the above-mentioned dual-recognition fluorescent molecular probe includes the following steps:

[0096] Step 1: Prepare compound I using isophorone and malononitrile as reaction raw materials; specifically, dissolve 6.91 g of isophorone (50.1 mmol) in 100 mL of absolute ethanol, then sequentially add 3.84 g of malononitrile (60.1 mmol) and 0.3 mL of piperidine (3.0 mmol), heat and stir under nitrogen protection for reflux reaction for 6 h. Monitor the complete reaction of isophorone by TLC and stop the reaction; let the reaction stand and cool to room temperature, then pour the reaction solution into 200 mL of ice water to form a large amount of grayish-white precipitate. The solid obtained by suction filtration is separated and purified by column chromatography to obtain 7.23 g of white crystalline solid, which is compound I;

[0097] Step 2: React compound I with p-hydroxybenzaldehyde to prepare compound II; specifically, add p-hydroxybenzaldehyde (10.2 mmol) to a round-bottom flask containing 40 mL of absolute ethanol, then sequentially add 10.5 mmol of compound I and 3.0 mmol of piperidine, heat and reflux the reaction until the disappearance of the raw material spots is monitored by TLC plate. After reacting for 5 h, stop the reaction; the product obtained after rotary evaporation of the reaction solution is separated and purified by column chromatography to obtain compound II;

[0098] Step 3: React compound II with hexamethylenetetramine to prepare compound III; specifically, dissolve compound II (1.5 mmol) in 10 mL of trifluoroacetic acid, then add 1.5 mmol of hexamethylenetetramine, heat and reflux, monitor the complete reaction by TLC and stop the reaction; cool the reaction solution to room temperature and then slowly pour it into 50 mL of ice water. The product obtained by filtration is separated and purified by column chromatography to obtain compound III;

[0099] Step 4: React compound III with benzoylhydrazine to obtain the target product; specifically, add 100 mg of compound III to a round-bottom flask containing 20 mL of ultradry ethanol, then add 1.1-fold equivalent of benzoylhydrazine of compound III, heat and reflux for 5 h until the reaction is monitored to be complete by TLC; stop the reaction, cool the reaction liquid to room temperature, a large amount of precipitate is generated, filter, and the filter cake is purified by slurrying with ethanol to obtain the target product - a colorimetric-fluorescent dual-channel molecular probe for identifying lead ions and glyphosate.

[0100] The target compound H-2 obtained in this example is a new compound that has not been reported before. After purification, it is characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR). The high-resolution mass spectrum of probe H-2 is as shown in Figure 1 shown, the high-resolution mass spectrum of probe H-2 is as shown in Figure 1 shown, [M+H + + has a molecular formula of C 27 H​24 N4O2, with a calculated theoretical value of 437.1978 and an actual value of 437.1981 found in the mass spectrum. The nuclear magnetic resonance of probe H-2 1 H NMR is as Figure 2 shown. The nuclear magnetic resonance of probe H-2 13 13C NMR is as Figure 3 shown. 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.22 (s, 1H), 11.75 (s, 1H), 8.65 (s, 1H), 7.93 (dd, J = 23.3, 4.5 Hz, 3H), 7.72 (dd, J = 8.6, 1.8 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.5 Hz, 2H), 7.31 (s, 2H), 7.00 (d, J = 8.6 Hz, 1H), 6.85 (s, 1H), 3.33 (s, 2H), 2.62 (s, 2H), 2.56 (s, 2H), 2.53–2.46 (m, 4H) ppm. 13 13C NMR (101 MHz, DMSO-d6) δ 170.75, 163.36, 159.35, 156.81, 147.84, 137.86, 133.19, 132.53, 131.18, 130.06, 129.04, 128.17, 127.85, 122.36, 119.66, 117.70, 114.49, 113.70, 75.90 ppm.

[0101] Example 3

[0102] This example is used to illustrate the preparation of the complex of the target compound H-2 with lead ions [H-2 + Pb 2+ .

[0103] The target compound H-2 synthesized in Example 2 was dissolved in DMF and diluted to a DMF / H2O (4:6, v / v, 0.01 M Hepes, pH = 7.4) solution with a final concentration of 1×10 -5 mol / L, denoted as solution A.

[0104] 5 mL of the solution A was taken and mixed with 5 μL of lead ions (1×10 -5 mol / L) to prepare the complex [H-2 - Pb 2+ , and a DMF / H2O (4:6, v / v, 0.01 M Hepes, pH = 7.4) system with a concentration of 1×10 -5 mol / L was prepared, denoted as solution B.

[0105] Metal cations, anions, pesticides, etc. are all prepared into aqueous solutions with a concentration of 0.01 mol / L.

[0106] Unless otherwise specified, the solution A, solution B, metal cations, anions, pesticides, etc. used in the following application examples are all prepared in this example.

[0107] Application Example 1

[0108] Add different metal cations (Fe 3+ , Fe 2+ , Ba 2+ , Ni 2+ , Mg 2+ , Hg 2+ , K + , Ag + , Ca 2+ , Na + , Co 2+ , Mn 2+ , Cd 2+ , Zn 2+ , Cr 3+ , Cu 2+ and Pb 2+ ) solutions to make the final cation concentration 5×10 -5 mol / L. The fluorescence spectrum is as shown in (A) of Figure 4 . It can be seen from (A) of Figure 4 that after adding Pb 2+ , the fluorescence intensity increases significantly. The addition of other metal ions does not cause obvious changes in fluorescence intensity. Pb 2+ can enhance the fluorescence of the probe H-2 and is unique compared with other ions. Therefore, the probe H-2 can specifically recognize Pb 2+ .

[0109] Add pesticides or anions (glyphosate, parathion, DDVP, dimethoate, methyl parathion, malathion, BF4 - , NO3 - , S2O3 2- , HPO4 2- , HSO3 - , I - , ClO4 - , CrO4 2- , F - , HCO3 - , Cl - , Br - , CN - ) to solution B to make the final concentration of these ions 1×10 -4 mol / L. The fluorescence spectrum is as shown in Figure 4as shown in (B) of. It can be seen that the addition of glyphosate causes the fluorescence of the [H-2-Pb 2 + system to be significantly quenched, while the addition of other pesticides, amino acids or anions has little effect on the fluorescence of the system. The above results indicate that glyphosate can quench the fluorescence of the [H-2-Pb 2+ complex, which is unique compared to other pesticides and anions. Therefore, the probe [H-2-Pb 2+ can specifically recognize glyphosate.

[0110] Figure 5 are the ultraviolet spectra of solution A, after adding Pb 2+ to it, and solution B after adding glyphosate to it. It can be seen that after adding Pb 2+ , the ultraviolet absorption peak at 428 nm increases and redshifts to 475 nm. After adding glyphosate to solution B, the absorbance of the absorption peak at 475 nm decreases and blueshifts to 427 nm.

[0111] The above results indicate that Pb 2+ can enhance the fluorescence of the probe H-2, which is unique compared to other ions. The probe H-2 can specifically recognize Pb 2+ . Glyphosate can quench the fluorescence of the probe [H-2-Pb 2+ , which is unique compared to other ions. The probe [H-2-Pb 2+ can specifically recognize glyphosate.

[0112] Application Example 2

[0113] Using solution A to detect the competitiveness of Pb 2+ (5×10 -5 mol / L) against different metal cations (5×10 -5 mol / L) Fe 3+ , Fe 2+ , Ba 2+ , Ni 2+ , Mg 2+ , Hg 2+ , K + , Ag + , Ca 2+ , Na + , Co 2+ , Mn 2+ , Cd 2+ , Zn 2+ , Cr 3+ , Cu 2+ : The fluorescence intensity of 5 mL of solution A at 538 nm after adding 25 μL of different metal cations to it, and the fluorescence intensity after adding an equal amount of Pb to solution A after first adding other metal cations2+ The fluorescence intensity of the post-system at 538 nm, as Figure 6 shown in (A) of 2+ . It can be seen that the probe H-2 has good anti-interference ability for the recognition of Pb

[0114] . Using solution B to detect the competitiveness of glyphosate (1×10 -4 mol / L) against different pesticides, amino acids or anions (1×10 -4 mol / L) (glyphosate, parathion, DDVP, dimethoate, methyl parathion, malathion, BF4 - , NO3 - , S2O3 2- , HPO4 2- , HSO3 - , I - , ClO4 - , CrO4 2- , F - , HCO3 - , Cl - , Br - , CN - ), as shown in (B) of Figure 6 . It can be seen that the [H-2-Pb 2 + complex has good anti-interference ability for the recognition of glyphosate, and the presence of other pesticides or anions in the system will not affect its recognition effect.

[0115] Application Example 3

[0116] Adding lead ions (0 - 2.4×10 -5 mol / L) / glyphosate (0 - 3.0×10 -5 mol / L) of different concentrations to solution A / solution B, and detecting the changes in their fluorescence spectra, as shown in Figure 7 .

[0117] Figure 7 (A) in shows the relationship between the change in the fluorescence spectrum of the system and the concentration of Pb 2+ . As the concentration of lead ions increases, the fluorescence intensity gradually weakens, and the change in fluorescence intensity is obvious, proving that the probe H-2 can be used for the quantitative detection of Pb 2+ , and the phenomenon is obvious and easy to distinguish.

[0118] As shown in (B) of Figure 7 , as the concentration of glyphosate increases, the fluorescence intensity gradually weakens, and the change in fluorescence intensity is obvious, proving that [H-2-Pb 2+The complex can be used for the quantitative detection of glyphosate, and the phenomenon is obvious and easy to distinguish.

[0119] Lead ions at different concentrations (0 - 1.5×10 -5 mol / L) / glyphosate (0 - 1.6×10 -5 mol / L) were added to Solution A / Solution B, and the changes in their ultraviolet spectra were detected, as Figure 8 shown.

[0120] Figure 8 (A) in shows the relationship between the change in the ultraviolet spectrum of the system and the Pb 2+ concentration. As the concentration of lead ions increases, the ultraviolet absorbance at 428 nm gradually increases and redshifts to 470 nm. The changes in the ultraviolet spectrum are obvious, proving that the probe H-2 can be used for the quantitative detection of Pb 2+ and the phenomenon is obvious and easy to distinguish.

[0121] As Figure 8 (B) in shows, as the concentration of glyphosate increases, the ultraviolet absorbance at 470 nm gradually decreases and blueshifts to 430 nm. The changes in the ultraviolet spectrum are obvious, proving that the [H-2-Pb 2+ complex can be used for the quantitative detection of glyphosate, and the phenomenon is obvious and easy to distinguish.

[0122] Application Example 4

[0123] As Figure 9 shown, Pb was alternately added to Solution A 2+ (concentration: 1×10 -5 mol / L) and glyphosate (concentration: 2×10 -5 mol / L), and then the reversible change in the fluorescence intensity at 650 nm was detected.

[0124] The results show that the probe H-2 has good reversibility in the recognition of Pb 2+ , and the [H-2-Pb 2+ complex has good reversibility in the recognition of glyphosate and can be used repeatedly.

[0125] Application Example 5

[0126] Figure 10 It was to add 1×10 -5 mol / L lead ions to Solution A and then add 1×10 -5 mol / L glyphosate, and observe the color change of the solution under sunlight. It can be seen that the color changes from light yellow on the left to deeper orange in the middle and then back to light yellow on the right. The probe of the present invention can detect lead ions and glyphosate by visual colorimetry.

[0127] Figure 11After adding 1×10 -5 mol / L lead ions to solution A and then adding 2×10 -5 mol / L glyphosate, the color change of the solution was observed under a 365 nm ultraviolet lamp. It can be seen that the color changed from orange on the left to red in the middle and then to orange on the right. The probe of the present invention can also achieve visual qualitative detection of lead ions and glyphosate under ultraviolet irradiation.

[0128] Application Example 6

[0129] At 37 °C, Hela cells were cultured in DMEM medium containing penicillin (100 units / mL), streptomycin (100 μg / mL) and 10% fetal bovine serum and treated at room temperature for 20 minutes. The probe was prepared in 10 mM H-2 mother liquor containing DMSO and diluted to a medium with a concentration of 10 μM. Pb 2+ was prepared to 10 mM and diluted to 10 μM with the medium. Subsequently, HeLa cells were directly incubated with 0.01 M Hepes buffer for 20 minutes as a control group. First, for the experimental group, HeLa cells were cultured in different solutions of probe H-2 (10 μM) and H-2-Pb 2+ (10 μM, 10 μM), and a group was taken out for comparison. Then, different concentrations of Pb 2+ (5 and 10 μM) and Glyp (10 and 20 μM) were added to the two solutions respectively, and the cells were further cultured for 20 minutes. After the specified incubation period, the cells for analysis were washed three times with HEPES solution and then imaged under a laser confocal microscope. The fluorescence excitation wavelength was 405 nm and was collected in the range of 620 - 670 nm.

[0130] Four-day-old zebrafish were incubated in embryo medium containing 1-phenyl-2-thiourea (PTU) at a temperature of 28 °C for 24 hours. The juvenile zebrafish were divided into three main groups, and one group was used as a control in Hepes buffer solution. The first group of zebrafish was exposed to H-2 (10 μM), H-2 + Pb 2+ (10 μM, 5 μM), H-2 + Pb 2+ (10 μM, 10 μM) in different solutions for 4 minutes each time. Similarly, the second group of zebrafish was incubated in H-2-Pb 2+ (10 μM, 10 μM) H-2-Pb 2+ + Glyp (10 μM, 10 μM, 10 μM), H-2-Pb 2+In +Glyp (10 μM, 10 μM, 20 μM), each time for 4 minutes. Before imaging, the larval zebrafish were rinsed three times with HEPES buffer solution, then fixed in a petri dish, and then imaged under a laser confocal microscope. The fluorescence imaging parameters were the same as those for cell imaging. The fluorescence excitation wavelength was 405 nm, and the collection was in the range of 620 - 670 nm.

[0131] The fluorescence images of lead ions and glyphosate in Hela cells detected by probe H-2 are shown as Figure 12 follows.

[0132] As Figure 12 shown in (A) of 2+ , the fluorescence images of Pb in Hela cells detected by probe H-2: Hela cells (purchased from Saibakang) were cultured with HEPES buffer solution, H-2 (10 μM), H-2 (10 μM) + Pb 2+ (5 μM), H-2 (10 μM) + Pb 2+ (10 μM) respectively, and the changes in their fluorescence imaging were observed. As the concentration of Pb 2+ increased, the red fluorescence signal gradually enhanced.

[0133] As Figure 12 shown in (B) of 2+ , the fluorescence images of glyphosate in Hela cells detected by probe [H-2-Pb 2+ (10 μM): Hela cells were cultured with HEPES buffer solution, H-2-Pb 2+ (10 μM), H-2-Pb 2+ (10 μM) + glyphosate (10 μM), H-2-Pb

[0134] The results showed that probe H-2 could be used to monitor and study the dynamic changes in the level of metal lead ions in the cell environment in real time, and the [H-2-Pb 2+ complex could achieve the actual tracking and detection of glyphosate in cells.

[0135] The fluorescence images of lead ions and glyphosate in zebrafish detected by probe [H-2-Pb 2+ are shown as Figure 13 follows.

[0136] As Figure 13 shown in (A) of 2+ , the fluorescence images of Pb in zebrafish detected by probe [H-2-Pb 2+ : Hela cells were cultured with HEPES buffer solution, H-2 (10 μM), H-2 (10 μM) - Pb2+ (10 μM)+, H-2(10 μM)+Pb 2+ (10 μM) after cultivation, the changes in its fluorescence imaging. As Pb 2+ concentration increases, the fluorescence signal in the red channel gradually increases.

[0137] As Figure 13 shown in (B) of 2+ , the fluorescence images of glyphosate detection in zebrafish: Zebrafish larvae were cultured with Hepes buffer solution, H-2-Pb 2+ (10 μM), H-2-Pb 2+ (10 μM)+glyphosate(10 μM), H-2-Pb 2+ (10 μM)+glyphosate(20 μM) respectively, and the changes in their fluorescence imaging were observed. As the glyphosate concentration increases, the fluorescence in the red channel becomes weaker and weaker.

[0138] The results show that the probe H-2 realizes the imaging ability for detecting lead ions in zebrafish, and the [H-2-Pb 2+ complex has the ability to efficiently detect residual pollutants such as glyphosate in zebrafish.

[0139] The present invention designs and synthesizes a dual-recognition specific recognition fluorescent molecular probe for lead ions and glyphosate. The fluorescence intensity of the probe H-2 is weak in the absence of lead ions, but significantly increases after the addition of lead ions. The [H-2-Pb 2+ complex restores fluorescence after the addition of glyphosate, with high selectivity and anti-interference ability. In addition, the detection limit of the probe H-2 for lead ions is low, the ultraviolet detection limit is 1.29×10 -7 M, and the fluorescence detection limit is 3.23×10 -8 M. The [H-2-Pb 2+ complex has a low detection limit for glyphosate, the ultraviolet detection limit is 3.94×10 -7 M, and the fluorescence detection limit is 3.65×10 -8 M. It can effectively detect lead ions and glyphosate in water environment, living cells and zebrafish with high sensitivity, which has certain guiding significance for production activities.

[0140] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Application of a complex of a dual-recognition fluorescent molecular probe and lead ions in glyphosate sensing detection, characterized in that: The sensing detection is selective sensing detection or competitive sensing detection, and the structural formula of the dual-recognition fluorescent molecular probe is shown as Formula I below:

2. The application according to claim 1, characterized in that: The dual-recognition fluorescent molecular probe is a Schiff base type probe with dicyanoisophorone and benzoylhydrazine as fluorescent groups.

3. The application according to claim 1, characterized in that: The preparation method of the dual-recognition fluorescent molecular probe is obtained by reacting compound III with benzoylhydrazine, and its synthesis reaction equation is shown as Formula II below:

4. The application according to claim 3, wherein: The specific preparation method includes the following steps: Add compound III into ultradry ethanol, then add benzoylhydrazine, and carry out a heating reflux reaction; after stopping the reaction, cool it to room temperature, filter the obtained product, and pulp and purify the obtained filter cake with absolute ethanol to obtain the target product.

5. The application according to claim 3 or 4, characterized in that: The molar ratio of compound III to benzoylhydrazine is 1:1.

1.

6. The application according to claim 3 or 4, characterized in that: The preparation method of compound III includes the following steps: Step 1: Prepare compound I using isophorone and malononitrile as reaction raw materials; Step 2: React compound I with p-hydroxybenzaldehyde to prepare compound II; Step 3: React compound II with hexamethylenetetramine to prepare the said compound III.

Images