Fluorescent molecular probe, preparation method thereof and application of complex of fluorescent molecular probe and zinc ions in glyphosate sensing detection

Abstract

The invention relates to a fluorescent molecular probe, a preparation method thereof and application of a complex of the fluorescent molecular probe and zinc ions in glyphosate sensing detection. The invention belongs to the field of fluorescent molecular probes. The method overcomes the defects that glyphosate detection in the prior art is time-consuming, cumbersome, expensive, only qualitative and the like. The fluorescent molecular probe HMB prepared by the invention is a Schiff base type probe taking dicyanoisophorone and benzoyl hydrazine as fluorophores, and the fluorescent molecular probe has the advantages of easily available raw materials and simple operation. The invention further provides a complex HMB-Zn < 2 + > of the fluorescent molecular probe and zinc ions, HMB and HMB-Zn < 2 + > both have a chromogenic sensing function and can show color changes under sunlight and ultraviolet light, and HMB-Zn < 2 + > has a specific recognition function on glyphosate and can be used as a specific indicator for existence of glyphosate in a real water sample and rice roots. And the sensor can also be used for detecting glyphosate in a water body environment and plant tissues.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent molecular probes, and particularly relates to a fluorescent molecular probe, a preparation method thereof, and an application of a complex of the fluorescent molecular probe and zinc ions in the sensing detection of glyphosate. Background Art

[0002] Glyphosate is an organophosphorus compound, specifically a phosphonate ester, which is mainly used to kill weeds, especially annual broad-leaved weeds that compete with crops. Glyphosate is widely used in agriculture and forestry due to its high activity, low cost, and high efficiency. However, glyphosate has strong water solubility and a long half-life, and will remain in large amounts in the environment and food. Glyphosate is also considered to have certain toxicity to the human body and is considered to cause respiratory, myocardial, central nervous system, and muscle dysfunction, and even death in severe cases, which seriously threatens the ecosystem and human health. Therefore, the World Health Organization has restricted the maximum residue level of glyphosate in drinking water to 900 μg / L. Therefore, the research and development of glyphosate safeners is of great significance for production and life. And there is no rapid, efficient, and convenient glyphosate detection technology in the prior art. Therefore, a convenient technology to assist the research and development of glyphosate safeners is of great significance for the ecological environment and human health.

[0003] In recent years, various technologies for detecting glyphosate have been developed to a certain extent, such as high performance liquid chromatography, gas chromatography-mass spectrometry, atomic absorption spectrometry, atomic emission spectrometry, capillary electrophoresis, fluorescence spectroscopy, X-ray fluorescence spectroscopy, and inductively coupled plasma mass spectrometry. However, the detection of these instruments usually has problems such as complex sample processing, long detection time, and high cost. The fluorescent molecular probe technology has significant advantages such as simple operation, high sensitivity, low cost, and convenience. People are becoming more and more interested in the organic fluorescent molecular probe detection technology in the biological environment field. In the prior art, fluorescent probes only quantitatively detect metal ions by fluorescence spectroscopy. Therefore, the development of fluorescent probes that can be used for specific detection of glyphosate is of great significance in the biological environment and chemical analysis. Summary of the Invention

[0004] The purpose of the present invention is to solve the disadvantages of time-consuming, cumbersome, expensive, and only qualitative detection of glyphosate in the prior art. The present invention provides a fluorescent molecular probe, a preparation method thereof, and an application of a complex of the fluorescent molecular probe and zinc ions in the sensing detection of glyphosate.

[0005] The technical solution of the present invention is as follows:

[0006] One of the purposes of the present invention is to provide a fluorescent molecular probe (HMB), and the structural formula of the HMB is as follows:

[0007]

[0008] The second object of the present invention is to provide a preparation method of HMB, and the method is as follows:

[0009] S1: Dissolve isophorone in absolute ethanol, add malononitrile and piperidine, and heat and stir for reflux reaction under nitrogen protection to obtain compound I;

[0010] S2: Add 6-hydroxy-2-naphthaldehyde to absolute ethanol, then add compound I and piperidine, and heat to reflux to obtain compound II;

[0011] S3: Dissolve the compound II obtained in S2 in trifluoroacetic acid, add hexamethylenetetramine, and heat to reflux to obtain compound III;

[0012] S4: Add the compound III obtained in S3 to ethanol, add benzoyl hydrazide, heat to reflux, filter, and purify to obtain HMB.

[0013] Preferably, in S1, the molar ratio of isophorone to malononitrile is 5:6, and the heating and stirring time is 5-8 h.

[0014] Preferably, in S2, the molar ratio of compound I to 6-hydroxy-2-naphthaldehyde is 1:1.

[0015] Preferably, in S3, the molar ratio of compound II to hexamethylenetetramine is 1:1.

[0016] Preferably, in S4, the molar ratio of compound III to benzoyl hydrazide is 1:1.1, and the heating and stirring time is 5-8 h.

[0017] Preferably, the reaction route is as follows:

[0018]

[0019] The third object of the present invention is to provide a complex (HMB-Zn 2+ ) of the above HMB and zinc ions, and the structural formula of the HMB-Zn 2+ is as follows:

[0020]

[0021] The fourth object of the present invention is to provide an application of the above HMB-Zn 2+ in the sensing detection of glyphosate;

[0022] Preferably, the sensing detection is selective sensing detection and competitive sensing detection.

[0023] Preferably, the sensing detection is visual qualitative detection, ultraviolet light detection and fluorescence detection.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] (1) The fluorescent molecular probe HMB prepared in the present invention is a novel compound that has not been reported before. The raw materials of HMB provided by the present invention are easily accessible, and the synthesis steps are simple. The HMB of the present invention can combine ultraviolet ratio quantitative detection and fluorescence spectrum quantitative detection to provide a variety of different data, increasing the accuracy and reliability of the detection of glyphosate by the complex of the fluorescent molecular probe and zinc. As a cheap and easy-to-operate highly selective detection technology, the HMB prepared in the present invention can greatly assist the research of glyphosate safeners.

[0026] (2) The HMB prepared in the present invention has a chromogenic sensing function. It can not only cause a change in the color of the solution under sunlight, but also have a change in fluorescence color under the irradiation of a 365 nm ultraviolet lamp. The ultraviolet spectrum can provide higher contrast and sensitivity, and this characteristic makes it a chromogenic sensing fluorescent probe suitable for different lighting conditions.

[0027] (3) The HMB-Zn prepared in the present invention 2+ has a specific recognition function for glyphosate, and the detection is very convenient and simple. It can be qualitatively determined only by the naked eye, and can also be quantitatively determined by ultraviolet light skin and fluorescence spectrum. It can be used as a specific indicator for the presence of glyphosate in real water samples and rice roots, and can also be used as a sensor for detecting glyphosate in water environment and plant tissues. The HMB-Zn of the present invention 2+ can provide real-time qualitative and quantitative information through the changed color and fluorescence detection, making it a highly specific indicator suitable for visual colorimetric detection.

[0028] (4) The HMB-Zn prepared in the present invention 2+ realizes the quantitative detection of different functional glyphosates by the complex of the probe and zinc through ultraviolet and fluorescence spectra. The HMB-Zn prepared in the present invention 2+ has good selectivity for glyphosate, is not easily affected by other ions, and has a low detection limit, and can be widely used in the detection of glyphosate in complex environments.

[0029] Glyphosate: The fluorescence detection limit is 1.02×10 -8 M. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the high-resolution mass spectrum of the fluorescent molecular probe HMB synthesized in Example 1 of the present invention;

[0031] Figure 2 is the nuclear magnetic resonance 1 HNMR spectrum of the fluorescent molecular probe HMB synthesized in Example 1 of the present invention;

[0032] Figure 31H NMR of the fluorescent molecular probe HMB synthesized in Example 1 of the present invention 13 13C NMR spectrum;

[0033] Figure 4 For HMB + Zn prepared in Example 2 2 High-resolution mass spectrometry detection chart;

[0034] Figure 5 For HMB-Zn in Example 4 2+ + Fluorescence spectra of pesticides, amino acids and anions;

[0035] Figure 6 For HMB, HMB + Zn 2+ and [HMB-Zn 2+ + Ultraviolet absorption spectra of glyphosate;

[0036] Figure 7 For the competitive detection chart of glyphosate against different pesticides, amino acids or anions detected by solution B in Example 5;

[0037] Figure 8 For the fluorescence titration curve of HMB-Zn 2+ + glyphosate in Example 6;

[0038] Figure 9 For HMB, HMB + Zn in Example 7 2+ and HMB-Zn 2+ + Effect diagram of glyphosate under sunlight in Example 7;

[0039] Figure 10 For HMB, HMB + Zn in Example 7 2+ and HMB-Zn 2+ + Effect diagram of glyphosate under 365 nm ultraviolet lamp irradiation in Example 7;

[0040] Figure 11 For the high-efficiency detection ability diagram of glyphosate residues in rice roots in Example 8. Detailed implementation manners

[0041] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used, unless otherwise specified, are all conventional materials, reagents, methods and instruments in the art, and those skilled in the art can obtain them through commercial channels.

[0043] Example 1: Preparation of fluorescent molecular probe HMB:

[0044]

[0045] Preparation of Compound I: Dissolve isophorone (50.0 mmol) in 100 mL of absolute ethanol, add malononitrile (60.0 mmol) and piperidine (3.0 mmol), heat and stir under reflux in a nitrogen atmosphere for 6 h. Monitor the complete reaction of isophorone by TLC. Let the reaction stand and cool to room temperature, then pour the reaction solution into 200 mL of ice water. A large amount of off-white precipitate will form. Filter the obtained solid and separate and purify it by column chromatography to obtain Compound I;

[0046] Preparation of Compound II: Add 6-hydroxy-2-naphthaldehyde (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 under reflux until the disappearance of the starting material spots is monitored by TLC plate, stop the reaction, rotary evaporate the reaction solution, and separate and purify it by column chromatography to obtain Compound II;

[0047] Preparation of Compound III: Dissolve Compound II (1.5 mmol) in 10 mL of trifluoroacetic acid, then add 1.5 mmol of hexamethylenetetramine, heat under reflux, monitor the complete reaction by TLC, and stop the reaction; cool the reaction solution to room temperature and then pour it into 50 mL of ice water, filter the product, and separate and purify it by column chromatography to obtain Compound III;

[0048] Preparation of fluorescent molecular probe HMB: Add 100 mg of Compound III to a round-bottom flask containing 20 mL of ultradry ethanol, then add benzohydrazide in an amount 1.1 times the molar amount of Compound III, heat under reflux for 6 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 will form, filter, and purify the filter cake by slurrying with ethanol to obtain fluorescent molecular probe HMB.

[0049] The HMB obtained in this example is a new compound not reported before. Perform high-resolution mass spectrometry (HRMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR) detection on it, and the results are as shown in Figure 1 、 Figure 2 and Figure 3 . The molecular formula of HMB is C 31 H 26 N 4 O 2 , [M + H + + The theoretical value is 487.2134, from Figure 1 ​As can be seen, the actual value found in the mass spectrometry is 487.2135, and the error is within the allowable range, proving that HMB was successfully prepared.

[0050] HMB Nuclear Magnetic Resonance 1 1H NMR is as Figure 2 shown below: 1 1H NMR (600 MHz, DMSO-d 6 ) δ 12.86 (s, 1H), 12.25 (s, 1H), 9.48 (s, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.09 (s, 1H), 8.00 (d, J = 7.6 Hz, 3H), 7.91 (d, J = 8.9 Hz, 1H), 7.65 (t, J = 7.3 Hz, 1H), 7.59 (t, J = 7.5 Hz, 2H), 7.48 (d, J = 16.1 Hz, 1H), 7.38 (d, J = 16.1 Hz, 1H), 7.27 (d, J = 8.9 Hz, 1H), 6.88 (s, 1H), 2.55 (s, 4H), 1.03 (s, 6H) ppm.

[0051] Nuclear Magnetic Resonance of HMB 13 13C NMR is as Figure 3 shown below: 13 13C NMR (151 MHz, DMSO-d 6 ) δ 170.68, 162.91, 159.32, 156.35, 147.07, 137.75, 133.71, 133.01, 132.63, 132.56, 131.75, 130.42, 129.62, 129.14, 128.29, 128.08, 126.21, 123.07, 121.96, 120.03, 114.38, 113.60, 109.63, 76.53, 42.72, 40.52, 40.40, 40.26, 40.12, 39.98, 39.84, 39.70, 39.56, 38.59, 32.14, 27.93 ppm.

[0052] Example 2: Preparation of the Complex of HMB with Zinc Ion HMB+Zn 2+ Preparation:

[0053] HMB and zinc ions in equimolar amounts were mixed with a DMSO / water (5:5, v / v, 0.01 M Hepes, pH = 7.0) buffer solution system to prepare a complex HMB-Zn 2+ with a concentration of 1×10 -5 mol / L.

[0054] For the HMB+Zn prepared in this example2+ High-resolution mass spectrometry (HRMS) detection was carried out, and the results are as follows Figure 4 shown, [HMB + Zn 2+ + 2H 2 O - 2H + The theoretical value is 584.1402, and the actual value in the mass spectrum is 584.1588, with the error within the allowable range.

[0055] Example 3:

[0056] The HMB prepared in Example 1 was dissolved in DMSO, and a DMSO / water (5:5, v / v, 0.01 M Hepes, pH = 7.0) system with a concentration of 1×10 -5 mol / L was prepared, denoted as Solution A.

[0057] Example 4:

[0058] HMB + Zn 2+ Selectivity detection of glyphosate:

[0059] The HMB-Zn prepared in Example 2 2+ was formulated into a DMSO / water (5:5, v / v, 0.01 M Hepes, pH = 7.0) system with a concentration of 1×10 -5 mol / L, denoted as Solution B.

[0060] Pesticides, amino acids, and anions (glyphosate, pretilachlor, thiamethoxam, phenylalanine, glutamine, glutathione, metronidazole, chloramphenicol, BF 4 - , NO 3 - , S 2 O 3 2- , HPO 4 2- , HSO 3 - , I - , ClO 4 - , F - , HCO 3 - , Cl - , Br - , CN - etc.) were added to Solution B to make the concentration of these ions in the final solution 1×10 -4 mol / L), and the results are as shown in Figure 5 the fluorescence spectrum. It can be seen from Figure 5 that the addition of glyphosate makes HMB-Zn 2+The fluorescence of the system was significantly enhanced, while the addition of other pesticides, amino acids or anions had little effect on HMB-Zn 2+ system fluorescence. The above results indicate that glyphosate can enhance the fluorescence of HMB-Zn 2+ , which is unique compared with other pesticides and anions. Therefore, the probe HMB-Zn 2+ can specifically recognize glyphosate.

[0061] Figure 6 are the UV spectra of solution A (HMB), solution B (HMB-Zn 2+ ), and solution B with the addition of 1×10 -4 mol / L glyphosate (HMB-Zn 2+ + glyphosate). As can be seen from Figure 6 , after adding Zn 2+ to solution A, the UV absorption decreased significantly, and the absorption peak red-shifted from 440 nm to 455 nm. The absorbance of the absorption peak increased after adding glyphosate to solution B.

[0062] The above results indicate that Zn 2+ can quench the fluorescence of the probe HMB, which is unique compared with other ions. After the probe HMB is complexed with Zn 2+ , pesticides, amino acids or anions are detected. Among them, glyphosate can restore the fluorescence of the probe HMB-Zn 2+ , which is unique compared with other ions. The probe HMB-Zn 2+ can specifically recognize glyphosate.

[0063] Example 5: Competitive detection of glyphosate by solution B against different pesticides, amino acids or anions:

[0064] Solution B was used to detect the competition of glyphosate (1×10 -4 mol / L) against different pesticides, amino acids or anions (1×10 -4 mol / L) (glyphosate, pretilachlor, thiamethoxam, oxyfluorfen, phenylalanine, glutamine, cysteine, glutathione, metronidazole, erythromycin, streptomycin, sodium amoxicillin, chloramphenicol, Cl - , Br - , SCN - , S 2 O 3 2- , NO 3 - , HSO 3 - , SO4 2-The competitiveness of (etc.). The fluorescence intensities at 620 nm of solution B and solution B after adding different pesticides, amino acids or anions were detected respectively, as well as the fluorescence intensity at 620 nm of the system after adding glyphosate to solution B first and then adding the same amount of other pesticides, amino acids or anions. The results are as Figure 7 shown. It can be seen from Figure 7 that the [HMB-Zn 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.

[0065] Example 6:

[0066] Different concentrations of glyphosate (0 - 10 eq) were added to solution B, and the changes in its fluorescence spectrum were detected, as Figure 8 shown.

[0067] Figure 8 It shows the relationship between the change in the fluorescence spectrum of the system and the concentration of glyphosate. As the concentration of glyphosate increases, the fluorescence intensity gradually increases, and the change in fluorescence intensity is obvious, proving that HMB-Zn 2+ can be used for the quantitative detection of glyphosate, and the phenomenon is obvious and easy to distinguish.

[0068] The detection limit is an important direct index representing the sensitivity of the probe, and can be calculated according to the following equation.

[0069]

[0070] Where SD refers to the standard deviation of the blank sample, K is the signal-to-noise ratio, generally taken as 3, and S is the slope of the linear fitting curve of the analyte concentration and the probe fluorescence intensity obtained from the probe concentration titration experiment.

[0071] After calculation, the fluorescence detection limit of glyphosate in the present invention is 1.02×10 -8 M.

[0072] Example 7:

[0073] Visual colorimetry was used to test the detection of glyphosate by HMB-Zn 2+ :

[0074] After adding 1×10 -5 mol / L zinc ions to solution A and then adding 1×10 -4 mol / L glyphosate, the color of the solution under sunlight was observed. The results are as Figure 9 shown. It can be seen from Figure 9 that on the left, HMB is light yellow, in the middle, HMB-Zn 2+ is dark red, and on the right, HMB-Zn 2++Glyphosate is light orange, indicating HMB, HMB-Zn 2+ and HMB-Zn 2+ +The solution color of glyphosate is significantly different under sunlight, proving that the probe of the present invention can detect glyphosate by visual colorimetry after complexing with zinc.

[0075] Add 1×10 -5 mol / L zinc ions to solution A and then add 2×10 -4 mol / L glyphosate. The solution color under the irradiation of a 365 nm ultraviolet lamp is as shown in Figure 10 . As can be seen from Figure 10 , HMB on the left is bright red, HMB-Zn 2+ in the middle is dark purple, and HMB-Zn 2+ +glyphosate on the right is red, indicating that the solution colors of HMB, HMB-Zn 2+ and HMB-Zn 2+ +glyphosate are significantly different under ultraviolet lamp irradiation, proving that the probe of the present invention can detect glyphosate by visual colorimetry after complexing with zinc.

[0076] Example 8:

[0077] High detection ability of glyphosate residue pollutants in rice roots:

[0078] Immerse the rice grains in pure water at 26°C for 36 h to promote germination. Subsequently, transfer the germinated grains to a nylon net immersed in Hoagland nutrient solution. The rice roots are divided into two main groups, with one group in Hepes buffer solution as a control. The second group of rice roots is exposed to [HMB-Zn 2+ (10 μM), [HMB-Zn 2+ (10 μM)+glyphosate (50 μM) for 4 min each time. Before imaging, the rice roots are rinsed three times with HEPES buffer solution, then fixed in a petri dish, and then imaged by laser confocal fluorescence imaging. The fluorescence excitation wavelength is 405 nm and is collected in the range of 600 - 650 nm. The results are as shown in Figure 11 . As can be seen from Figure 11 , HMB-Zn 2+ achieves the imaging ability to detect glyphosate in rice roots, proving the high detection ability of HMB-Zn 2+ to detect glyphosate residue pollutants in rice roots, indicating that the prepared HMB-Zn 2+ of the present invention can be applied to the high detection of glyphosate residue pollutants in rice roots.

[0079] Example 9:

[0080] Detection of glyphosate in various aqueous solutions and aqueous solutions of soil extracts:

[0081] In the experiment, Songhua River water, tap water, aqueous solution of honeysuckle extract, aqueous solution of liquorice tablet extract, and aqueous solution of soil extract were selected to study the probe HMB-Zn 2+ The detection ability in real environmental water samples. The real water samples were filtered to remove insoluble impurities, and then the glyphosate solution prepared from the filtered extract was mixed with the probe HMB-Zn 2+ to prepare a test solution for fluorescence measurement. The fluorescence hardly changed, indicating that the glyphosate content in the extract was very low and did not reach the detection limit of HMB-Zn 2+ Subsequently, glyphosate solutions with concentrations of 1, 2, and 5×10 -5 mol / L were prepared with real water samples for the standard addition recovery experiment, and the results are shown in Table 1.

[0082] Table 1 Detection of glyphosate in various aqueous solutions and aqueous solutions of soil extracts

[0083]

[0084] As shown in Table 1, the standard addition recovery rates in real water samples were 95.0 - 113.1%, and the relative standard deviations were all less than 2%. Therefore, the HMB-Zn prepared in the present invention 2+ can be used for quantitative detection of the glyphosate content in environmental water samples.

[0085] The above are only the preferred specific embodiments of the present invention. These specific embodiments are all different implementation manners based on the overall concept of the present invention, and the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A fluorescent molecular probe, characterized in that: The fluorescent molecular probe structural formula is as follows:

2. The method for preparing the fluorescent molecular probe according to claim 1, characterized in that: The method comprises the following steps: S1: Dissolve isophorone in anhydrous ethanol, add malononitrile and piperidine, and heat and stir under nitrogen protection to reflux to obtain compound I; S2: Add 6-hydroxy-2-naphthaldehyde to anhydrous ethanol, then add compound I and piperidine, and heat under reflux to obtain compound II; S3: dissolving compound II obtained in S2 in trifluoroacetic acid, adding hexamethylenetetramine, and heating under reflux to obtain compound III; S4: Add compound III obtained in S3 to ethanol, add benzoyl hydrazide, heat to reflux, filter, and purify to obtain a fluorescent molecular probe.

3. The method according to claim 2, characterized in that The molar ratio of isophorone to malononitrile in S1 is 5:6, and the heating and stirring time is 5 to 8 hours.

4. The method according to claim 2, characterized in that: The molar ratio of compound I to 6-hydroxy-2-naphthaldehyde in S2 is 1:

1.

5. The method according to claim 2, characterized in that: The molar ratio of compound II to hexamethylenetetramine in S3 is 1:

1.

6. The method according to claim 2, characterized in that The molar ratio of compound III to benzoyl hydrazide in S4 is 1:1.1, and the heating and stirring time is 5 to 8 hours.

7. A complex of the fluorescent molecular probe and zinc ion according to claim 1, characterized in that: The complex structure of the fluorescent molecular probe and zinc ion is as follows:

8. The complex of the fluorescent molecular probe and zinc ion HMB-Zn according to claim 7 2+ Application in glyphosate sensing detection.

9. The use according to claim 8, characterized in that: The sensing assays are selective sensing assays and competitive sensing assays.

10. The use according to claim 8 or 9, characterized in that: The sensing detection includes visual qualitative detection, ultraviolet light detection and fluorescence detection.

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