Double-recognition fluorescent molecular probe, preparation method thereof and application of double-recognition fluorescent molecular probe in detection of lead ions and / or glyphosate

Abstract

The invention discloses a double-recognition fluorescent molecular probe, a preparation method thereof and application of the double-recognition fluorescent molecular probe in detection of lead ions and / or glyphosate, and relates to the technical field of detection of lead ions and pesticide glyphosate. The double-recognition fluorescent molecular probe disclosed by the invention is a Schiff base type probe which takes dicyanoisophorone and benzoyl hydrazine as fluorophores. The invention also relates to application of the double-recognition fluorescent molecular probe in lead ion sensing detection. The complex of the double-recognition fluorescent molecular probe and lead ions can be used for sensing detection of glyphosate. The double-recognition fluorescent molecular probe provided by the invention has the advantages of easily available raw materials and simple operation. According to the present invention, the quantitative detection of the lead ions / or the glyphosate by the probe through the different functions displayed by the ultraviolet and fluorescence spectra is achieved, the reversibility is strong, the multi-time cyclic utilization can be achieved, the selectivity is good, the influence by other metal ions is not easily generated, the detection limit is low, and the excellent application imaging is provided in animal bodies such as cells, zebra fish and the like.

Description

Technical Field

[0001] The invention relates to the technical field of lead ion and pesticide glyphosate detection, and in particular to a dual-recognition fluorescent molecular probe and a preparation method thereof, and 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 source of lead pollution is lead used in industries such as gasoline and battery manufacturing. Lead plays a vital role in 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 disease and the nervous system, leading to carcinogenic, mutagenic and teratogenic effects. The maximum intake of lead ions in drinking water allowed by the World Health Organization is 10μM. Therefore, it is very important to track and detect lead ions in a timely manner.

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

[0004] In recent years, various technologies for detecting lead have been developed, 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 range of these instruments usually depend on the sample preparation method, operating conditions, and instruments, and they also have disadvantages such as time-consuming, bulky, and expensive. Therefore, compared with other reported detection technologies, people are increasingly interested in the detection of organic fluorescent molecular probes in the field of biological environment. 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 purpose of the present invention is to overcome the problems of complex synthesis steps, poor selectivity and low sensitivity of the lead ion and glyphosate detection probes in the prior art, and to provide a dual-recognition fluorescent molecular probe and a preparation method thereof and an application in the detection of lead ions and / or glyphosate.

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

[0007] In order to achieve the above object, 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 shown in the following formula 1:

[0008]

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

[0010] The second aspect of the present invention provides a method for preparing the dual recognition fluorescent molecular probe described in the first aspect, which is obtained by reacting compound III with benzoyl hydrazide. The synthesis reaction equation is shown in Formula 2 below:

[0011]

[0012] Furthermore, the above technical solution, the method for preparing the dual recognition fluorescent molecular probe, specifically comprises the following steps:

[0013] Compound III is added to ultra-dry ethanol, followed by benzoyl hydrazide, and the mixture is heated to reflux for reaction; after the reaction is stopped, the mixture is cooled to room temperature, the obtained product is filtered, and the obtained filter cake is slurried and purified with anhydrous ethanol to obtain the target product.

[0014] Specifically, in the above technical solution, the amount of the super dry ethanol is not specifically limited, as long as the uniform dissolution of compound III can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of compound III to super dry 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 heating reflux reaction time is 4-8h.

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

[0018] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0019] Step 2, reacting compound I with p-hydroxybenzaldehyde to prepare compound II;

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

[0021] Specifically, the synthesis reaction equation of the compound III is shown in Formula 3 below:

[0022]

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

[0024] Dissolve isophorone in anhydrous ethanol, then add malononitrile and piperidine in sequence, and heat, stir and reflux under nitrogen atmosphere to react; after the reaction is completed, cool to room temperature, pour the obtained product into ice water to generate a large amount of precipitate, filter, and separate and purify the obtained solid by column chromatography to obtain the 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 scheme, the specific synthesis steps of compound II in step S2 are as follows:

[0029] Add p-hydroxybenzaldehyde to anhydrous ethanol, then add compound I and piperidine in sequence, heat and reflux until the raw material point disappears, and then stop the reaction; spin dry the obtained product, and then separate and purify it by column chromatography to obtain the compound II.

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

[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 scheme, the specific synthesis steps of compound III in step S3 are as follows:

[0034] Compound II is dissolved in trifluoroacetic acid, and then hexamethylenetetramine is added, and the mixture is heated under reflux for reaction. After the reaction is completed, the obtained product is cooled to room temperature, and then slowly poured into ice water, and the obtained product is filtered and purified by column chromatography to obtain the compound III.

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

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

[0037] The third aspect of the present invention provides the use 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 lead ion sensing detection.

[0038] In some embodiments of the present invention, the dual recognition fluorescent molecular probe is used for sensing and detecting lead ions in water environments, 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 and 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 the lead ions.

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

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

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

[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 spectroscopy or fluorescence spectroscopy.

[0046] Existing fluorescent probes only use fluorescence spectra to quantitatively detect metal ions. The dual-recognition fluorescent molecular probe of the present invention can provide a variety of different data by combining ultraviolet ratio quantitative detection with fluorescence spectrum quantitative detection, increasing the accuracy and reliability of lead ions and glyphosate detection. 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 the fluorescence color under 365nm ultraviolet light. This dual observation mode makes it a chromogenic sensing fluorescent probe suitable for different lighting conditions.

[0047] Through the above technical solution, the beneficial technical effects achieved 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 undergo color changes, making it a highly specific indicator suitable for visual colorimetric detection. At the same time, the probe has a 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 perform repeated detections without compromising 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, the raw materials are easily available, and the operation is simple.

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

[0051] Lead ion: UV detection limit is 1.29×10 -7 M, the fluorescence detection limit is 3.23×10 -8 M;

[0052] Glyphosate: UV detection limit is 3.94×10 -7 M, the fluorescence detection limit is 3.65×10 -8 M. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

[0057] Figure 4 In the application example 1 of the present invention: (A) is H-2+Pb 2+ or other metal ions; (B) is the fluorescence spectrum of [H-2-Pb 2+ ]+Fluorescence spectrum of pesticides or anions.

[0058] Figure 5 H-2 and H-2+Pb in Application Example 1 of the present invention 2+ and [H-2-Pb 2+ ]+UV absorption spectrum of glyphosate.

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

[0060] Figure 7 In the application example 3 of the present invention: (A) is H-2+Pb 2+ Fluorescence titration curve of [H-2-Pb 2+ ]+fluorescence titration curve of glyphosate.

[0061] Figure 8 In the application example 3 of the present invention: (A) H-2+Pb 2+UV titration curve of [H-2-Pb 2+ ]+UV titration curve of glyphosate.

[0062] Fig. 9 The H-2 and Pb in the application example 4 of the present invention are 2+ and reversibility detection diagram of glyphosate.

[0063] Fig.10 The probes H-2 and H-2+Pb in Application Example 5 of the present invention are 2+ and [H-2-Pb 2+ ]+The effect of glyphosate in sunlight.

[0064] Fig.11 The probes H-2 and H-2+Pb in Application Example 5 of the present invention are 2+ and [H-2-Pb 2+ ]+The effect of glyphosate under 365nm ultraviolet light.

[0065] Fig.12 The present invention uses the probe H-2 in Example 6 to detect Pb in living cells. 2+ and imaging of glyphosate applications.

[0066] Fig.13 The present invention uses the probe H-2 in Example 7 to detect Pb in zebrafish 2+ and imaging of glyphosate applications. DETAILED DESCRIPTION

[0067] The present invention relates to the technical field of lead ion and pesticide glyphosate detection, and discloses a dual-recognition fluorescent molecular probe, a preparation method thereof, and an application thereof in detecting lead ions and / or glyphosate. The dual-recognition fluorescent molecular probe is a Schiff base-type probe with dicyanoisophorone and benzoylhydrazide 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 sensing detection of glyphosate. 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 probe through the different functions displayed by ultraviolet and fluorescence spectra, has strong reversibility, can be recycled multiple times, has good selectivity, is not easily affected by other metal ions, has a low detection limit, and also presents excellent application imaging in cells and animals such as zebrafish.

[0068] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which 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 method for preparing the dual recognition fluorescent molecular probe described in the first aspect, which is obtained by reacting compound III with benzoyl hydrazide. The synthesis reaction equation is shown below:

[0072]

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

[0074] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0075] Step 2, reacting 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 use 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 lead ion sensing detection.

[0079] In some embodiments of the present invention, the dual recognition fluorescent molecular probe is used for sensing and detecting lead ions in water environments, 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 the dual recognition fluorescent molecular probe and 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 the lead ions.

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

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

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

[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 spectroscopy and fluorescence spectroscopy.

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

[0088] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0089] Example 1

[0090] This embodiment provides a dual-recognition fluorescent molecular probe, which is a Schiff base probe with dicyanoisophorone and benzoylhydrazide as fluorescent groups, and its structural formula is shown below:

[0091]

[0092] Example 2

[0093] This example provides a method for preparing the dual recognition fluorescent molecular probe described in Example 1, and the reaction scheme is as follows:

[0094]

[0095] The method for preparing the dual recognition fluorescent molecular probe comprises the following steps:

[0096] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I; specifically, 6.91 g of isophorone (50.1 mmol) was dissolved in 100 mL of anhydrous ethanol, followed by sequentially adding 3.84 g of malononitrile (60.1 mmol) and 0.3 mL of piperidine (3.0 mmol), and heating and stirring the mixture under reflux for 6 h under nitrogen protection, and the reaction was stopped after the isophorone was completely reacted by monitoring by TLC; the reaction was allowed to stand and cool to room temperature, and then the reaction solution was poured into 200 mL of ice water to generate a large amount of off-white precipitate, and the solid obtained by suction filtration was separated and purified by column chromatography to obtain 7.23 g of a white crystalline solid, namely compound I;

[0097] Step 2, compound I is reacted with p-hydroxybenzaldehyde to prepare compound II; specifically, p-hydroxybenzaldehyde (10.2 mmol) is added to a round-bottom flask containing 40 mL of anhydrous ethanol, and then 10.5 mmol of compound I and 3.0 mmol of piperidine are added in sequence, and the mixture is heated under reflux to react until the raw material spot disappears as monitored by the TLC spot plate. After reacting for 5 hours, the reaction is stopped; the product obtained after the reaction solution is spin-dried is separated and purified by column chromatography to obtain compound II;

[0098] Step 3, reacting compound II with hexamethylenetetramine to prepare compound III; specifically, dissolving compound II (1.5 mmol) in 10 mL of trifluoroacetic acid, then adding 1.5 mmol of hexamethylenetetramine, heating to reflux, monitoring the reaction completion by TLC, and stopping the reaction; cooling the reaction solution to room temperature and then slowly pouring it into 50 mL of ice water, filtering the obtained product, and separating and purifying it by column chromatography to obtain compound III;

[0099] Step 4, reacting compound III with benzoyl hydrazide to obtain the target product; the specific method is to add 100 mg of compound III into a round-bottom flask containing 20 mL of ultra-dry ethanol, and then add 1.1 times the equivalent of compound III of benzoyl hydrazide, and heat to reflux for 5 hours until the reaction is complete as monitored by TLC; stop the reaction, cool the reaction liquid to room temperature, and a large amount of precipitate is generated, which is filtered, and the filter cake is slurried and purified with ethanol to obtain the target product - a colorimetric-fluorescent dual-channel molecular probe for recognizing 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, high-resolution mass spectrometry (HRMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR) to characterize it. The high-resolution mass spectrum of probe H-2 is shown in Figure 1 As shown, the high-resolution mass spectrum of probe H-2 is Figure 1 As shown, [M+H + ] + The molecular formula is C 27 H24 N4O2, the theoretical value is 437.1978, and the actual value found in the mass spectrum is 437.1981. 1 H NMR Figure 2 As shown, the NMR of probe H-2 13 CNMR Figure 3 shown. 1 H NMR (500MHz, DMSO-d6) δ (ppm) 12.22 (s, 1H), 11.75 (s, 1H), 8.65 (s, 1H), 7.93 (dd, J = 23.3, 4.5Hz, 3H), 7.72 (dd, J = 8.6, 1.8Hz, 1H), 7.63 (t, J = 7. 3Hz,1H),7.56(t,J=7.5Hz,2H),7.31(s,2H),7.00(d,J=8.6Hz,1H),6.8 5(s,1H),3.33(s,2H),2.62(s,2H),2.56(s,2H),2.53–2.46(m,4H)ppm. 13 C NMR(101MHz,DMSO-d6)δ170.75,163.36,159.35,156.81,147.84,137.86,133.19,132.53,1 31.18,130.06,129.04,128.17,127.85,122.36,119.66,117.70,114.49,113.70,75.90ppm.

[0101] Example 3

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

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

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

[0105] Metal cations, anions, pesticides, etc. are all configured into 0.01 mol / L aqueous solutions.

[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+ ) solution, so that the final cation concentration is 5×10 -5 mol / L, fluorescence spectra such as Figure 4 (A) is shown. Figure 4 As can be seen in (A), the addition of Pb 2+ After addition, the fluorescence intensity increased significantly. The addition of other metal ions did not cause significant changes in fluorescence intensity. 2+ It can enhance the fluorescence of probe H-2, which is unique compared with other ions, so probe H-2 can specifically recognize Pb 2+ .

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

[0110] Figure 5 Solution A and Pb 2+ The UV spectra of solution B and solution B after glyphosate was added. 2+ After adding glyphosate to solution B, the absorption peak at 428 nm increased and red-shifted to 475 nm. After adding glyphosate to solution B, the absorption peak at 475 nm decreased and blue-shifted to 427 nm.

[0111] The above results show that Pb 2+ It can enhance the fluorescence of probe H-2, which is unique compared with other ions. Probe H-2 can specifically recognize Pb 2+ Glyphosate can make the probe [H-2-Pb 2+ ] fluorescence quenching, which is unique compared with other ions. The probe [H-2-Pb 2+ ] can specifically recognize glyphosate.

[0112] Application Example 2

[0113] Detection of Pb using Solution A 2+ (5×10 -5 mol / L) for 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+ Competitiveness: The fluorescence intensity at 538 nm after adding 25 μL of different metal cations to 5 mL of solution A, and the fluorescence intensity after adding other metal cations to solution A and then adding an equal amount of Pb 2+The fluorescence intensity of the system at 538 nm is as follows: Figure 6 (A) As shown. It can be seen that probe H-2 has a strong affinity for Pb 2+ The recognition has good anti-interference ability, and the presence of other metal ions in the system will not affect its recognition effect.

[0114] Solution B was used to detect glyphosate (1×10 -4 mol / L) for different pesticides, amino acids or anions (1×10 -4 mol / L)(glyphosate, parathion, DDVP, dimethoate, parathion methyl, malathion, BF4 - ,NO3 - ,S2O3 2- ,HPO4 2- ,HSO3 - ,I - ,ClO4 - ,CrO4 2- ,F - ,HCO3 - ,Cl - ,Br - ,CN - ) competitiveness, such as Figure 6 As shown in (B), it can be seen that the probe [H-2-Pb 2+ ] The complex has a 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] The different concentrations of lead ions (0-2.4×10 -5 mol / L) / glyphosate (0-3.0×10 -5 mol / L) was added to solution A / solution B, and the changes in its fluorescence spectrum were detected, such as Figure 7 shown.

[0117] Figure 7 (A) shows the fluorescence spectrum change of the system and Pb 2+ The relationship between the concentration of lead ions and the fluorescence intensity gradually weakened with the increase of lead ion concentration. The change of fluorescence intensity was obvious, which proved that probe H-2 can be used for Pb 2+ The quantitative detection is clear and easy to identify.

[0118] like Figure 7 As shown in (B), with the increase of glyphosate concentration, the fluorescence intensity gradually weakened, and the change of fluorescence intensity was obvious, which proved that [H-2-Pb 2+ ] The complex can be used for quantitative detection of glyphosate, and the phenomenon is obvious and easy to identify.

[0119] Different concentrations of lead ions (0-1.5×10 -5 mol / L) / glyphosate (0-1.6×10 -5 mol / L) was added to solution A / solution B, and the changes in its UV spectrum were detected, such as Figure 8 shown.

[0120] Figure 8 (A) shows the change of the UV spectrum of the system and Pb 2+ With the increase of lead ion concentration, the 428nm ultraviolet absorbance gradually increased and red-shifted to 470nm. The changes in the ultraviolet spectrum were obvious, proving that probe H-2 can be used for Pb 2+ The quantitative detection is clear and easy to identify.

[0121] like Figure 8 As shown in (B), with the increase of glyphosate concentration, the 470nm UV absorbance gradually weakened and blue-shifted to 430nm. The UV spectrum changed significantly, proving that [H-2-Pb 2+ ] The complex can be used for quantitative detection of glyphosate, and the phenomenon is obvious and easy to identify.

[0122] Application Example 4

[0123] like Fig. 9 As shown, Pb was added alternately to solution A. 2+ (Concentration is 1×10 -5 mol / L) and glyphosate (concentration of 2×10 -5 mol / L) and then detect the reversible change of its fluorescence intensity at 650nm.

[0124] The results showed that probe H-2 had a strong affinity for Pb 2+ The recognition of [H-2-Pb 2+ ]The recognition of the complex glyphosate has good reversibility and can be used repeatedly.

[0125] Application Example 5

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

[0127] Fig.11 To add 1×10 -5 mol / L lead ions and then add 2×10-5 The color of the solution of 100 mol / L glyphosate under 365 nm ultraviolet light changes. It can be seen that the color changes 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 light.

[0128] Application Example 6

[0129] HeLa cells were treated in DMEM medium containing penicillin (100 units / mL), streptomycin (100 μg / mL) and 10% fetal bovine serum at 37°C for 20 minutes at room temperature. The probe was prepared in a 10 mM H-2 stock solution containing DMSO and diluted to a concentration of 10 μM in the medium. Pb was diluted with ultrapure water. 2+ The probes H-2 (10 μM) and H-2-Pb were prepared as 10 mM and diluted to 10 μM with culture medium. Subsequently, HeLa cells were directly incubated with 0.01 M Hepes buffer for 20 min as a control group. First, for the experimental group, HeLa cells were incubated with probes H-2 (10 μM) and H-2-Pb 2+ The cells were cultured in different solutions (10μM, 10μM) and one group was selected for comparison. Then, different concentrations of Pb were added to the two solutions. 2+ (5 and 10 μM) and Glyp (10 and 20 μM), and the cells were further incubated for 20 minutes. After the specified incubation period, the cells used for analysis were washed three times with HEPES solution and then imaged under laser confocal. The fluorescence excitation wavelength was 405 nm and 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 28°C for 24 hours. Larval zebrafish were divided into three main groups, one of which was in Hepes buffer solution as a control. The first group of zebrafish were exposed to different solutions of H-2 (10 μM), H-2+Pb 2+ (10μM, 5μM), H-2+Pb 2+ (10μM, 10μM), 4 minutes each time. Similarly, the second group of zebrafish were incubated in H-2-Pb 2+ (10μM,10μM)H-2-Pb 2+ +Glyp (10μM, 10μM, 10μM), H-2-Pb 2+ +Glyp (10μM, 10μM, 20μM), 4 minutes each time. Before imaging, larval zebrafish were rinsed three times with HEPES buffer solution, then fixed in a culture dish, and then imaged under laser confocal fluorescence imaging parameters were the same as those for cell imaging. The fluorescence excitation wavelength was 405nm and collected in the range of 620-670nm.

[0131] Fluorescence images of lead ions and glyphosate detected by probe H-2 in Hela cells, such as Fig.12 shown.

[0132] like Fig.12 As shown in (A), probe H-2 detects Pb in Hela cells 2+ Fluorescence images of Hela cells (purchased from Cytochrome Plc) were incubated with Hepes buffer solution, H-2 (10 μM), H-2 (10 μM) + Pb 2+ (5μM), H-2(10μM)+Pb 2+ (10μM) after incubation. 2+ With the increase of concentration, the red fluorescence signal gradually increased.

[0133] like Fig.12 As shown in (B), the probe [H-2-Pb 2+ ] Detection of glyphosate fluorescence images in Hela cells: Hela cells were incubated with Hepes buffer solution, H-2-Pb 2+ (10μM), H-2-Pb 2+ (10μM)+glyphosate (10μM), H-2-Pb 2+ The changes in fluorescence imaging after incubation with (10μM) + glyphosate (20μM). As the concentration of glyphosate increases, the red fluorescence signal gradually weakens.

[0134] The results showed that the probe H-2 can be used to monitor and study the dynamic changes of metal lead ion levels in the cell environment in real time. 2+ ] The complex can realize the actual tracking detection of glyphosate in cells.

[0135] Probe [H-2-Pb 2+ ] Fluorescence images of lead ions and glyphosate detected in zebrafish, such as Fig.13 shown.

[0136] like Fig.13 As shown in (A), the probe [H-2-Pb 2+ ] Detection of Pb in zebrafish 2+ Fluorescence images of Hela cells incubated with Hepes buffer solution, H-2 (10 μM), H-2 (10 μM)-Pb 2+ (10μM)+、H-2(10μM)+Pb 2+ (10μM) after incubation. 2+ With the increase of concentration, the fluorescence signal in the red channel gradually increased.

[0137] like Fig.13As shown in (B), the probe [H-2-Pb 2+ ] Detection of glyphosate in zebrafish: Zebrafish fry were incubated with Hepes buffer solution, H-2-Pb 2+ (10μM), H-2-Pb 2+ (10μM)+glyphosate (10μM), H-2-Pb 2+ The changes in fluorescence imaging after incubation with (10μM) + glyphosate (20μM). As the concentration of glyphosate increases, the fluorescence of the red channel becomes weaker and weaker.

[0138] The results showed that the probe H-2 achieved the imaging capability of detecting lead ions in zebrafish, and [H-2-Pb 2+ ] complex has high efficiency in detecting glyphosate and other residual pollutants in zebrafish.

[0139] The present invention designs and synthesizes a dual-specific recognition fluorescent molecular probe for lead ions and glyphosate. The fluorescence intensity of probe H-2 is weak in the absence of lead ions, but it is significantly enhanced after adding lead ions. 2+ The fluorescence of the complex was restored after the addition of glyphosate, which showed high selectivity and anti-interference. In addition, the detection limit of probe H-2 for lead ions was low, with a UV detection limit of 1.29×10 -7 M, the fluorescence detection limit is 3.23×10 -8 M. [H-2-Pb 2+ The detection limit of the complex for glyphosate is low, and the UV detection limit is 3.94×10 -7 M, 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 are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A dual recognition fluorescent molecular probe, characterized in that: The structural formula of the dual recognition fluorescent molecular probe is shown in Formula 1:

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

3. The method for preparing the dual recognition fluorescent molecular probe according to claim 1, characterized in that: It is obtained by reacting compound III with benzoyl hydrazide, and its synthesis reaction equation is shown in the following formula 2:

4. The method for preparing a dual recognition fluorescent molecular probe according to claim 3, characterized in that: The specific steps include: Compound III is added to ultra-dry ethanol, followed by benzoyl hydrazide, and the mixture is heated to reflux for reaction; after the reaction is stopped, the mixture is cooled to room temperature, the obtained product is filtered, and the obtained filter cake is slurried and purified with anhydrous ethanol to obtain the target product.

5. The method for preparing a dual recognition fluorescent molecular probe according to claim 3 or 4, characterized in that: The molar ratio of the compound III to benzoyl hydrazide is 1:1.

1.

6. The method for preparing a dual recognition fluorescent molecular probe according to claim 3 or 4, characterized in that: The preparation method of compound III comprises the following steps: Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I; Step 2, reacting compound I with p-hydroxybenzaldehyde to prepare compound II; Step 3: react compound II with hexamethylenetetramine to prepare the compound III.

7. Use of the dual-recognition fluorescent molecular probe according to claim 1 or 2 or the dual-recognition fluorescent molecular probe prepared by the preparation method according to any one of claims 3 to 6 in lead ion sensing detection.

8. A complex of a dual-recognition fluorescent molecular probe and a lead ion, characterized in that: The dual recognition fluorescent molecular probe is the dual recognition fluorescent molecular probe described in claim 1 or 2 or the dual recognition fluorescent molecular probe prepared by the preparation method described in any one of claims 3-6.

9. Use of the complex of the dual recognition fluorescent molecular probe and lead ions as claimed in claim 8 in glyphosate sensing detection.

10. The use according to claim 9, characterized in that: The sensing assay is a selective sensing assay or a competitive sensing assay.

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