CN115356318B - Fluorescence detection method of tobacco pesticide residue bud inhibition pellet - Google Patents
Fluorescence detection method of tobacco pesticide residue bud inhibition pellet Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 55
- 241000208125 Nicotiana Species 0.000 title claims abstract description 49
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 49
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 40
- 239000000447 pesticide residue Substances 0.000 title claims abstract description 35
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims abstract description 158
- 238000001514 detection method Methods 0.000 claims abstract description 69
- 239000003446 ligand Substances 0.000 claims abstract description 32
- 239000003068 molecular probe Substances 0.000 claims abstract description 23
- 239000000523 sample Substances 0.000 claims description 99
- 239000007864 aqueous solution Substances 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 29
- XJHCXCQVJFPJIK-UHFFFAOYSA-M caesium fluoride Chemical compound [F-].[Cs+] XJHCXCQVJFPJIK-UHFFFAOYSA-M 0.000 claims description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 claims description 20
- 238000002189 fluorescence spectrum Methods 0.000 claims description 19
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- MGAAUJYLQGQIOK-UHFFFAOYSA-N 2,7-dibromoacridine Chemical compound C1=CC(Br)=CC2=CC3=CC(Br)=CC=C3N=C21 MGAAUJYLQGQIOK-UHFFFAOYSA-N 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 16
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- CHQVQXZFZHACQQ-UHFFFAOYSA-M benzyl(triethyl)azanium;bromide Chemical compound [Br-].CC[N+](CC)(CC)CC1=CC=CC=C1 CHQVQXZFZHACQQ-UHFFFAOYSA-M 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
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- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 2
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- ONUFESLQCSAYKA-UHFFFAOYSA-N iprodione Chemical compound O=C1N(C(=O)NC(C)C)CC(=O)N1C1=CC(Cl)=CC(Cl)=C1 ONUFESLQCSAYKA-UHFFFAOYSA-N 0.000 description 2
- ZQEIXNIJLIKNTD-UHFFFAOYSA-N methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alaninate Chemical compound COCC(=O)N(C(C)C(=O)OC)C1=C(C)C=CC=C1C ZQEIXNIJLIKNTD-UHFFFAOYSA-N 0.000 description 2
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- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical compound COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 2
- BAZVSMNPJJMILC-UHFFFAOYSA-N triadimenol Chemical compound C1=NC=NN1C(C(O)C(C)(C)C)OC1=CC=C(Cl)C=C1 BAZVSMNPJJMILC-UHFFFAOYSA-N 0.000 description 2
- FFSJPOPLSWBGQY-UHFFFAOYSA-N triazol-4-one Chemical compound O=C1C=NN=N1 FFSJPOPLSWBGQY-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 239000005983 Maleic hydrazide Substances 0.000 description 1
- BGRDGMRNKXEXQD-UHFFFAOYSA-N Maleic hydrazide Chemical compound OC1=CC=C(O)N=N1 BGRDGMRNKXEXQD-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a fluorescence detection method of tobacco pesticide residue bud inhibition pellet, which adopts acridine ligand as a fluorescence detection molecular probe; the acridine ligand is a2, 7-di (pyridine-4) acridine molecule. The beneficial effects of the invention are as follows: according to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and the non-radiative transition of the acridine ligand molecule is inhibited by the intermolecular acting force between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, the precedent of measuring the sprouting inhibition pill pesticide residue by using a small molecular fluorescent probe is opened, and the defect that the sprouting inhibition pill pesticide residue is detected by other detection modes in the prior art is overcome.
Description
Technical Field
The invention belongs to the technical field of detection of tobacco pesticide residues, and particularly relates to a fluorescence detection method of tobacco pesticide residue bud inhibition pellets based on 2, 7-di (pyridine-4) acridine molecules.
Background
The bud-inhibiting pellet, also called maleic hydrazide, is a low-toxicity plant growth regulator and is widely applied to tobacco to inhibit the growth of axillary buds. Although no exact results of research have demonstrated that the use of budding inhibition can induce cancer, it has been demonstrated that the use of budding inhibition in large doses results in genetic mutations. Therefore, the residual amount of the buddhist is increasingly concerned. The Chinese regulations that the Maximum Residual Limit (MRL) of the bud-suppressing pellet in garlic, onion and shallot is 15mg/kg, and the MRL of the bud-suppressing pellet in potato is 50mg/kg; the international food code prescribes that the MRL of the bud-suppressing pill in garlic, onion and shallot is 15mg/kg, and the MRL of the bud-suppressing pill in potato is 50mg/kg; the European Union prescribes that the MRL of the bud-suppressing pill in the garlic is 15mg/kg; the U.S. prescribes that the MRL of the bud inhibition pellet in the onion is 15mg/kg, and the MRL of the bud inhibition pellet in the potato is 50mg/kg; the International center for the research of tobacco science (CORESTA) prescribes a Guided Residual Limit (GRL) of 80mg/kg for the sprouting inhibitor in tobacco.
There are various methods for analyzing the residue of the xidan: distillation spectrophotometry (AOAC), high performance liquid chromatography-ultraviolet detection (HPLC-UV), high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography, capillary electrophoresis, polarography, and the like, and also methods such as flow injection and pulse voltammetry, among which the first 3 analysis methods are most commonly used. However, the AOAC method has high requirements on a distillation device, complex sample pretreatment, complex operation, large alkali consumption and serious instrument corrosion; the HPLC-UV and HPLC-MS method not only uses expensive equipment, high detection cost and long detection period, but also needs professional operators to operate, so that the method is difficult to popularize. How to realize the rapid, simple and convenient and low-cost detection of the pesticide residues of the prosaposin becomes a problem to be solved in the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a fluorescence detection method of tobacco pesticide residue bud inhibition pellets.
The aim of the invention is achieved by the following technical scheme: a fluorescence detection method of tobacco pesticide residue bud inhibition pellet adopts acridine ligand as fluorescence detection molecular probe;
further, the acridine ligand is a2, 7-bis (pyridine-4) acridine molecule;
further, the synthesis of the 2, 7-bis (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring and reacting for 2-4 days at 80-100 ℃ under the atmosphere of N 2, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine;
Further, the synthesis step of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: adding acridine into benzyl triethyl ammonium bromide-methanol solution, and stirring the reaction for 10 to 20 hours at the temperature of between 50 and 70 ℃ by using a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and dichloromethane, and evaporating in vacuum at 40-60 ℃ to obtain light yellow solid 2, 7-dibromoacridine;
Further, the method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample;
further, the step of preparing the probe aqueous solution comprises dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution;
Further, the pretreatment of tobacco samples comprises the following steps: weighing 1.5-2.5 g of tobacco powder sample, adding hydrochloric acid solution with the volume of 15-25 mL and the concentration of 3-5 mol/L, magnetically stirring and extracting for 0.5-1.5 h at the temperature of 100-140 ℃ in an oil bath, cooling to room temperature, centrifuging, and taking supernatant to obtain a sample to be detected;
Further, the step of drawing a standard curve is to add the stock solution of the buddhist into the aqueous solution of the probe to obtain a series of aqueous solutions of the buddhist and the probe with concentration, carry out ultrasonic treatment to obtain a stable suspension, set 320nm as the maximum excitation wavelength and 480nm as the maximum emission wavelength by adopting a fluorescence spectrometer, and record and collect fluorescence spectra within the range of 320-690 nm;
Further, the sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690nm by adopting a fluorescence spectrometer;
The further technical proposal is that,
The residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
The invention has the following advantages:
1. According to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and non-radiative transition of the acridine ligand molecule is inhibited through intermolecular acting force between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, a precedent for measuring the sprouting inhibition pill pesticide residue by using a small molecular fluorescent probe is created, and the defect that the sprouting inhibition pill pesticide residue is detected by other existing detection modes is overcome;
2. The invention preferably adopts self-designed and synthesized organic micromolecule 2, 7-di (pyridine-4) acridine (DPA for short) as a fluorescent probe, and the bud-suppressing pill molecule (MH for short) can inhibit non-radiative transition of the DPA molecule through hydrogen bond interaction with the DPA molecule, so that a fluorescence enhancement mechanism of the DPA is effectively started, and the DPA is used as a fluorescence detection probe of the bud-suppressing pill by utilizing the characteristic, so that the rapid detection of the tobacco pesticide residue bud-suppressing pill can be rapidly, conveniently and cost-effectively completed;
3. The detection method has the advantages of rapid detection, simple and convenient operation and low cost; the response time of the detection method is 1min, and the peak time of the liquid-phase method of the anti-sprouting pellet is 8min; the high performance liquid chromatograph is very simple and convenient to operate, and the fluorescence spectrum can be collected and recorded after a sample to be measured is placed; the fluorescence spectrometer in the range of 320-690nm can be collected only by 6 ten thousand, instrument consumables are not needed in the detection process, the maintenance cost is extremely low, and the method has good application prospect.
4. The detection method can resist detection interference of various pesticide coexistence substances, has good method selectivity, can detect the extracted sample filtrate on the machine without purification, and greatly simplifies the pretreatment flow of the bud-suppressing pill compared with the existing pretreatment method.
Drawings
FIG. 1 is a flow chart of the preparation of DPA molecules.
FIG. 2 is a nuclear magnetic resonance spectrum of DPA molecule.
FIG. 3 is a nuclear magnetic hydrogen spectrum of DPA molecule.
FIG. 4 is a mass spectrum of DPA molecules.
FIG. 5 is a schematic structural diagram of DPA molecule.
FIG. 6 is a schematic diagram of the crystal structure of a DPA molecule;
Wherein a is a three-dimensional diagram of a crystal structure; b is a crystal structure from the front direction; c is a crystal structure in a lateral direction, and d is a crystal structure in a longitudinal direction.
FIG. 7 is a graph of fluorescence spectra of MH, DPA and DPA containing MH.
FIG. 8 shows the results of the optimization of the DPA probe concentration.
Fig. 9 is a response time optimization result.
Fig. 10 is a graph of the detection linear range and the detection limit.
Fig. 11 is a selective histogram of DPA.
FIG. 12 shows nuclear magnetic patterns of DPA, MH and a mixture of the two in different proportions;
Wherein: DPA/MH is 1:2; DPA/MH is 1:1.5; DPA/MH is 1:1, a step of; DPA/MH is 1:0.5; mh; dpa.
FIG. 13 is a graph showing fluorescence lifetime decay.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
In addition, the embodiments of the present invention and the features of the embodiments may be combined with each other without collision.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or are directions or positional relationships conventionally put in use of the inventive product, or are directions or positional relationships conventionally understood by those skilled in the art, are merely for convenience of describing the present invention and for simplifying the description, and are not to indicate or imply that the apparatus or element to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
Example 1: a fluorescence detection method of tobacco pesticide residue bud inhibition pellet adopts acridine ligand as fluorescence detection molecular probe; according to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and non-radiative transition of the acridine ligand molecule is inhibited through intermolecular acting force between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, and a precedent for measuring the pesticide residue of the sprouting inhibition pill based on a small molecular fluorescent probe is created; is favorable for solving the defect that other detection methods detect the pesticide residue of the prosaposin.
The acridine ligand is a2, 7-di (pyridine-4) acridine molecule; the invention preferably adopts self-designed and synthesized organic micromolecule 2, 7-di (pyridine-4) acridine (DPA for short) as a fluorescent probe, and the anti-sprouting pellet molecule (MH for short) can inhibit non-radiative transition of the DPA molecule through hydrogen bond interaction with the DPA molecule, so that a fluorescence enhancement mechanism of the DPA is effectively started, and by utilizing the characteristic, the DPA is used as a fluorescence detection probe of the anti-sprouting pellet, so that the rapid detection of the tobacco pesticide residue anti-sprouting pellet can be completed quickly, conveniently and at low cost.
The synthesis of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring for 2-4 days at 80-100 ℃ under the atmosphere of N 2, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine; wherein, the 2, 7-dibromoacridine is 0.67mg,2.0mmol; pyridine-4-boronic acid is 0.59g,2.4mol; cesium fluoride (CsF) of 2.8g,18mmol; tetrakis (triphenylphosphine) palladium (Pd (PPh 3)4) 0.130g,0.12mmol;
The synthesis steps of the 2, 7-di (pyridine-4) acridine molecule comprise: adding acridine into benzyl triethyl ammonium bromide-methanol solution, and stirring the reaction for 10 to 20 hours at the temperature of between 50 and 70 ℃ by using a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and dichloromethane, and evaporating in vacuum at 40-60 ℃ to obtain light yellow solid 2, 7-dibromoacridine; wherein, the benzyl triethyl ammonium bromide (TEBA) was 12g,44mmol; methanol was 100mL; the acridine content was 2.16g,12mmol.
The method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample; the detection method has the advantages of rapid detection, simple and convenient operation and low cost; the response time of the detection method is 1min, and the peak time of the liquid-phase method of the anti-sprouting pellet is 8min; the high performance liquid chromatograph is very simple and convenient to operate, and the fluorescence spectrum can be collected and recorded after a sample to be measured is placed; the fluorescence spectrometer in the range of 320-690nm can be collected only by 6 ten thousand, instrument consumables are not needed in the detection process, the maintenance cost is extremely low, and the method has good application prospect.
The preparation of the probe aqueous solution comprises the step of dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution.
The pretreatment of tobacco samples comprises the following steps: 1.5-2.5 g of tobacco powder sample is weighed, hydrochloric acid solution with the volume of 15-25 mL and the concentration of 3-5 mol/L is added, the solution is magnetically stirred and extracted for 0.5-1.5 h under the condition of oil bath of 100-140 ℃, cooled to room temperature and centrifuged, and supernatant fluid is taken to obtain the sample to be measured. According to the invention, through a proper sample pretreatment method, the tobacco powder sample is extracted by the hydrochloric acid solution, and the extracted sample filtrate can be detected on machine without purification. In addition, the detection method can avoid detection interference of coexisting matters by utilizing the hydrogen bond action of the molecular probe and the anti-sprouting pellet, and has good method selectivity.
The step of drawing the standard curve is that the stock solution of the buddhist is added into the probe aqueous solution to obtain a series of concentration buddhist-probe aqueous solution, the stable suspension is obtained by ultrasonic treatment, a fluorescence spectrometer is adopted, 320nm is set as the maximum excitation wavelength, 480nm is set as the maximum emission wavelength, and the fluorescence spectrum is recorded and collected within the range of 320-690 nm.
The sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690 nm.
The residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
Example 2: a fluorescence detection method of tobacco pesticide residue bud inhibition pellet adopts acridine ligand as fluorescence detection molecular probe; according to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and the non-radiative transition of the acridine ligand molecule is inhibited through the hydrogen bond action between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, a precedent for measuring the sprouting inhibition pill pesticide residue by using a small molecular fluorescent probe is opened, and the defect that the sprouting inhibition pill pesticide residue is detected by other detection modes in the prior art is overcome.
The acridine ligand is a 2, 7-di (pyridine-4) acridine molecule; the invention preferably adopts self-designed and synthesized organic micromolecule 2, 7-di (pyridine-4) acridine (DPA for short) as a fluorescent probe, and the anti-sprouting pellet molecule (MH for short) can inhibit non-radiative transition of the DPA molecule through hydrogen bond interaction with the DPA molecule, so that a fluorescence enhancement mechanism of the DPA is effectively started, and the DPA is used as a fluorescence detection probe of the anti-sprouting pellet by utilizing the characteristic, so that the rapid detection of the tobacco pesticide residue anti-sprouting pellet can be rapidly, conveniently and cost-effectively completed.
The synthesis of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring at 80 ℃ for 2 days under the atmosphere of N 2, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine; wherein, the 2, 7-dibromoacridine is 0.67mg,2.0mmol; pyridine-4-boronic acid 0.59g,2.4 mol; cesium fluoride (CsF) of 2.8g,18mmol; tetrakis (triphenylphosphine) palladium (Pd (PPh 3) 4) 0.130g,0.12mmol;
The synthesis steps of the 2, 7-di (pyridine-4) acridine molecule comprise: acridine is added into benzyl triethyl ammonium bromide-methanol solution, and the reaction is stirred for 10 hours at 50 ℃ by a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and dichloromethane, and evaporating in vacuum at 40 ℃ to obtain light yellow solid 2, 7-dibromoacridine; wherein, the benzyl triethyl ammonium bromide (TEBA) was 12g,44mmol; methanol was 100mL; the acridine content was 2.16g,12mmol.
The method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample; the detection method has the advantages of rapid detection, simple and convenient operation and low cost; the response time of the detection method is 1min, and the peak time of the liquid-phase method of the anti-sprouting pellet is 8min; the high performance liquid chromatograph is very simple and convenient to operate, and the fluorescence spectrum can be collected and recorded after a sample to be measured is placed; the fluorescence spectrometer in the range of 320-690nm can be collected only by 6 ten thousand, instrument consumables are not needed in the detection process, the maintenance cost is extremely low, and the method has good application prospect.
The preparation of the probe aqueous solution comprises the step of dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution.
The pretreatment of tobacco samples comprises the following steps: 1.5g of tobacco powder sample is weighed, hydrochloric acid solution with the volume of 15mL and the concentration of 3mol/L is added, the mixture is magnetically stirred and extracted for 0.5h at the temperature of 100 ℃ in an oil bath, and the mixture is cooled to room temperature and centrifuged, and supernatant fluid is taken to obtain the sample to be detected. According to the invention, through a proper sample pretreatment method, the tobacco powder sample is extracted by the hydrochloric acid solution, and the extracted sample filtrate can be detected on machine without purification. In addition, the detection method can avoid detection interference of coexisting matters by utilizing the hydrogen bond action of the molecular probe and the anti-sprouting pellet, and has good method selectivity.
The step of drawing the standard curve is that the stock solution of the buddhist is added into the probe aqueous solution to obtain a series of concentration buddhist-probe aqueous solution, the stable suspension is obtained by ultrasonic treatment, a fluorescence spectrometer is adopted, 320nm is set as the maximum excitation wavelength, 480nm is set as the maximum emission wavelength, and the fluorescence spectrum is recorded and collected within the range of 320-690 nm.
The sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690 nm.
The residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
Example 3: a fluorescence detection method of tobacco pesticide residue bud inhibition pellet adopts acridine ligand as fluorescence detection molecular probe; according to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and the non-radiative transition of the acridine ligand molecule is inhibited through the hydrogen bond action between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, a precedent for measuring the sprouting inhibition pill pesticide residue by using a small molecular fluorescent probe is opened, and the defect that the sprouting inhibition pill pesticide residue is detected by other detection modes in the prior art is overcome; .
The acridine ligand is a 2, 7-di (pyridine-4) acridine molecule; the invention preferably adopts self-designed and synthesized organic micromolecule 2, 7-di (pyridine-4) acridine (DPA for short) as a fluorescent probe, and the anti-sprouting pellet molecule (MH for short) can inhibit non-radiative transition of the DPA molecule through hydrogen bond interaction with the DPA molecule, so that a fluorescence enhancement mechanism of the DPA is effectively started, and the DPA is used as a fluorescence detection probe of the anti-sprouting pellet by utilizing the characteristic, so that the rapid detection of the tobacco pesticide residue anti-sprouting pellet can be rapidly, conveniently and cost-effectively completed.
The synthesis of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring at 100 ℃ for 4 days under N 2 atmosphere, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine; wherein, the 2, 7-dibromoacridine is 0.67mg,2.0mmol; pyridine-4-boronic acid is 0.59g,2.4mol; cesium fluoride (CsF) of 2.8g,18mmol; tetrakis (triphenylphosphine) palladium (Pd (PPh 3) 4) 0.130g,0.12mmol;
The synthesis steps of the 2, 7-di (pyridine-4) acridine molecule comprise: acridine is added into benzyl triethyl ammonium bromide-methanol solution, and the reaction is stirred for 20 hours at 70 ℃ by a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and dichloromethane, and evaporating in vacuum at 60 ℃ to obtain light yellow solid 2, 7-dibromoacridine; wherein, the benzyl triethyl ammonium bromide (TEBA) was 12g,44mmol; methanol was 100mL; the acridine content was 2.16g,12mmol.
The method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample; the detection method has the advantages of rapid detection, simple and convenient operation and low cost; the response time of the detection method is 1min, and the peak time of the liquid-phase method of the anti-sprouting pellet is 8min; the high performance liquid chromatograph is very simple and convenient to operate, and the fluorescence spectrum can be collected and recorded after a sample to be measured is placed; the fluorescence spectrometer in the range of 320-690nm can be collected only by 6 ten thousand, instrument consumables are not needed in the detection process, the maintenance cost is extremely low, and the method has good application prospect.
The preparation of the probe aqueous solution comprises the step of dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution.
The pretreatment of tobacco samples comprises the following steps: weighing 2.5g of tobacco powder sample, adding hydrochloric acid solution with the volume of 25mL and the concentration of 5mol/L, magnetically stirring and extracting for 1.5h at the temperature of 140 ℃ in an oil bath, cooling to room temperature, centrifuging, and taking supernatant to obtain a sample to be detected. According to the invention, through a proper sample pretreatment method, the tobacco powder sample is extracted by the hydrochloric acid solution, and the extracted sample filtrate can be detected on machine without purification. In addition, the detection method can avoid detection interference of coexisting matters by utilizing the hydrogen bond action of the molecular probe and the anti-sprouting pellet, and has good method selectivity.
The step of drawing the standard curve is that the stock solution of the buddhist is added into the probe aqueous solution to obtain a series of concentration buddhist-probe aqueous solution, the stable suspension is obtained by ultrasonic treatment, a fluorescence spectrometer is adopted, 320nm is set as the maximum excitation wavelength, 480nm is set as the maximum emission wavelength, and the fluorescence spectrum is recorded and collected within the range of 320-690 nm.
The sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690 nm.
The residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
Example 4: a fluorescence detection method of tobacco pesticide residue bud inhibition pellet adopts acridine ligand as fluorescence detection molecular probe; according to the invention, the acridine ligand is used as a molecular probe for fluorescence detection, and the non-radiative transition of the acridine ligand molecule is inhibited through the hydrogen bond action between the sprouting inhibition pill molecule and the molecular probe, so that the acridine ligand can be used for fluorescence detection, a precedent for measuring the sprouting inhibition pill pesticide residue by using a small molecular fluorescent probe is opened, and the defect that the sprouting inhibition pill pesticide residue is detected by other detection modes in the prior art is overcome; .
The acridine ligand is a 2, 7-di (pyridine-4) acridine molecule; the invention preferably adopts self-designed and synthesized organic micromolecule 2, 7-di (pyridine-4) acridine (DPA for short) as a fluorescent probe, and the anti-sprouting pellet molecule (MH for short) can inhibit non-radiative transition of the DPA molecule through hydrogen bond interaction with the DPA molecule, so that a fluorescence enhancement mechanism of the DPA is effectively started, and the DPA is used as a fluorescence detection probe of the anti-sprouting pellet by utilizing the characteristic, so that the rapid detection of the tobacco pesticide residue anti-sprouting pellet can be rapidly, conveniently and cost-effectively completed.
The synthesis of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring for 2-4 days at 80-100 ℃ under the atmosphere of N 2, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine; wherein, the 2, 7-dibromoacridine is 0.67mg,2.0mmol; pyridine-4-boronic acid is 0.59g,2.4mol; cesium fluoride (CsF) of 2.8g,18mmol; tetrakis (triphenylphosphine) palladium (Pd (PPh 3) 4) 0.130g,0.12mmol;
The synthesis steps of the 2, 7-di (pyridine-4) acridine molecule comprise: adding acridine into benzyl triethyl ammonium bromide-methanol solution, and stirring the reaction for 10 to 20 hours at the temperature of between 50 and 70 ℃ by using a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and dichloromethane, and evaporating in vacuum at 40-60 ℃ to obtain light yellow solid 2, 7-dibromoacridine; wherein, the benzyl triethyl ammonium bromide (TEBA) was 12g,44mmol; methanol was 100mL; the acridine content was 2.16g,12mmol.
The method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample; the detection method has the advantages of rapid detection, simple and convenient operation and low cost; the response time of the detection method is 1min, and the peak time of the liquid-phase method of the anti-sprouting pellet is 8min; the high performance liquid chromatograph is very simple and convenient to operate, and the fluorescence spectrum can be collected and recorded after a sample to be measured is placed; the fluorescence spectrometer in the range of 320-690nm can be collected only by 6 ten thousand, instrument consumables are not needed in the detection process, the maintenance cost is extremely low, and the method has good application prospect.
The preparation of the probe aqueous solution comprises the step of dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution.
The pretreatment of tobacco samples comprises the following steps: weighing 2g of tobacco powder sample, adding hydrochloric acid solution with the volume of 20mL and the concentration of 4mol/L, magnetically stirring and extracting for 1h at the temperature of 120 ℃ in an oil bath, cooling to room temperature, centrifuging, and taking supernatant to obtain a sample to be detected. According to the invention, through a proper sample pretreatment method, the tobacco powder sample is extracted by the hydrochloric acid solution, and the extracted sample filtrate can be detected on machine without purification. In addition, the detection method can avoid detection interference of coexisting matters by utilizing the hydrogen bond action of the molecular probe and the anti-sprouting pellet, and has good method selectivity.
The step of drawing the standard curve is that the stock solution of the buddhist is added into the probe aqueous solution to obtain a series of concentration buddhist-probe aqueous solution, the stable suspension is obtained by ultrasonic treatment, a fluorescence spectrometer is adopted, 320nm is set as the maximum excitation wavelength, 480nm is set as the maximum emission wavelength, and the fluorescence spectrum is recorded and collected within the range of 320-690 nm.
The sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690 nm.
The residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
Example 5: DPA structural characterization
The synthesized compound is structurally determined by utilizing nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum, and the specific data are as follows:
Hydrogen spectrum data ：1H NMR(400MHz,DMSO-d6)δ:9.29(s,1H),8.75(d,6H,J＝5.6Hz),8.31(t,4H,J＝10.8,9.5Hz),7.98(d,4H,J＝5.2Hz);
Carbon spectrum data: 13 C NMR (400 MHz, DMSO-d 6) delta 150.9,149.2,146.4,138.6,134.8,130.5,130.1,127.5,126.8,122.0;
mass spectrometry data: HRMS (ESI) m/z calculated for C 23H15N3[M+H]+, 334.1339; found,334.1334.
The molecular structure of DPA was analyzed from the above carbon, hydrogen and mass spectra data as shown in FIG. 5.
DPA crystal is prepared by toluene/ethyl acetate solvent gas phase diffusion method, and from crystal structure diagram, as can be seen from FIG. 6, dimer exists in DPA molecule, molecular layers are piled up by pi-pi interaction, and the distance is。
Example 6: feasibility of fluorescence detection method
To verify the feasibility of this fluorescence sensing method, fluorescence spectra of MH aqueous solution, DPA aqueous solution, and DPA aqueous solution containing MH were examined, and the results are shown in fig. 7. The maximum emission wavelength of the DPA solution is 480nm, the maximum excitation wavelength is 320nm, the bud inhibition pellet solution has almost no fluorescence, and after the target analyte bud inhibition pellet (100 mol/L) is added into the 10mol/L DPA probe solution, the fluorescence of the DPA aqueous solution can be obviously enhanced, so that the method can realize the detection of the bud inhibition pellet.
Example 7: optimization of detection conditions
(1) Optimization of DPA Probe concentration
After the bud inhibition pellet is added into the DPA probe solution, the fluorescence of the DPA aqueous solution can be obviously enhanced, and DPA aqueous solutions with the concentration of 1, 5, 10, 50 and 100mol/L are prepared, and the fluorescence enhancement condition before and after the bud inhibition pellet solution with the concentration of 0.1mol/L is compared, and the result is shown in figure 8. The results in FIG. 8 show that the enhancement of the addition of the budding inhibitor molecule is most pronounced when the DPA probe concentration is 10mol/L, so that the DPA probe concentration in the reaction base solution is optimally 10mol/L.
(2) Optimization of response time
The response time is an important factor for measuring the detection method, and the response time of the detection method is examined. 0.05mM of the budding inhibitor was added to 10mol/L of the DPA probe aqueous solution, and the fluorescence intensities of the solutions 0, 1, 2, 3, 4,5, 7, 9, 11, 13, 15 and 17min after the addition of the budding inhibitor were recorded, and the results are shown in FIG. 9. FIG. 9 shows that the fluorescence intensity is increased with the increase of the equilibrium time, the fluorescence is obviously enhanced at 1min, and the subsequent change of the fluorescence intensity is smaller, which indicates that the optimal detection time of the buddlest fluorescence is 1min, and the method has faster response time.
Example 8: analytical performance of the detection method
In order to determine the linear range of DPA detection bud inhibition pellet molecules, the influence of different bud inhibition pellet concentrations on the fluorescence intensity of DPA aqueous solution is explored, and the DPA aqueous solution with the bud inhibition pellet concentration of 0.1 mu M-1mM is prepared and measured. As a result, as shown in FIG. 10, the fluorescence intensity of the DPA aqueous solution gradually increased with increasing concentration of the budding inhibitor, and the fluorescence intensity was linear with the budding inhibitor concentration in 0.5. Mu.M-0.1 mM, and the linear equation was I=7.36C+750, and the linear correlation coefficient R 2 =0.996.
The detection was continued 10 times with the lowest concentration of the budding inhibition standard solution (0.5. Mu.M), and the Standard Deviation (SD) of the measurement result was 0.033, so that the detection limit of the method was 3SD=0.1. Mu.M (0.11 mg/Kg), and the quantitative limit was 10SD=0.33. Mu.M (0.37 mg/Kg).
Example 9: process selectivity
To investigate the detection performance of DPA on the budding inhibition under the condition that other pesticides coexist, a series of interference pesticides such as acetamiprid, triazolone, iprodione, thiophanate-methyl, metalaxyl and triadimenol and budding inhibition coexist (DPA and budding inhibition concentration is 1X 10 -5 mol/L, other pesticide concentration is 1X 10 -4 mol/L) are prepared, and the fluorescence intensity is measured, and the result is shown in figure 11. In the figure, the composition comprises the following components of the anti-sprouting agent, the acetamiprid, the anti-sprouting agent, the triazolone, the anti-sprouting agent, the iprodione, the anti-sprouting agent, the thiophanate-methyl, the anti-sprouting agent, the metalaxyl, and the triadimenol in turn from left to right. According to the results of FIG. 11, under the coexistence condition of other pesticides with the concentration of 10 times, the effect on the fluorescence intensity of the liquid to be detected is small, and DPA detection of the anti-bud pellets has better anti-interference capability.
Example 10: principle of response
In order to explore the detection mechanism of DPA on the sprouting inhibition pill, DPA, sprouting inhibition pill and the mixture of the two in different molar ratios are dissolved in deuterated DMSO, and the mechanism is explored through nuclear magnetism. As shown in FIG. 12, the two hydroxyl hydrogens of the budding inhibition pellet have larger coupling effect, and after the budding inhibition pellet is mixed with DPA molecules, the hydroxyl hydrogen coupling effect of the budding inhibition pellet is obviously weakened. Indicating that hydrogen bonds are formed between the DPA molecules and the budding inhibition molecules, so that the coupling effect between the hydroxyl hydrogen of the budding inhibition molecules is weakened. The fluorescence lifetime of the aqueous DPA solutions before and after addition of the budding inhibition was tested, as shown in fig. 13, and the fluorescence lifetime of the solution was shortened after addition of the budding inhibition, indicating that non-radiative transitions were inhibited between the DPA molecules and the budding inhibition molecules by hydrogen bonding, thereby enhancing the fluorescence of DPA.
Example 11: actual sample detection
The tobacco leaf sample to be tested is subjected to MH detection by the method of the invention after the original content of the sample is measured by HPLC (high performance liquid chromatography), and is subjected to standard adding recovery, and the result is shown in Table 1. As can be seen from Table 1, when the tobacco leaf samples are detected, the standard adding recovery rate of MH with high, medium and low concentration is 98.3-106.7%, and the Relative Standard Deviation (RSD) is 1.4-4.1%, which shows that the method can be used for detecting actual samples.
Table 1 detection of MH in actual samples (n=5)
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (6)
1. A fluorescence detection method of tobacco pesticide residue bud inhibition pellet is characterized in that: the method specifically comprises the following steps: preparing a probe aqueous solution; pretreatment of tobacco leaf samples; drawing a standard curve; detecting a sample, wherein an acridine ligand is used as a fluorescent detection molecular probe; the acridine ligand is a2, 7-di (pyridine-4) acridine molecule; the synthesis of the 2, 7-di (pyridine-4) acridine molecule comprises the following steps: dissolving 2, 7-dibromoacridine, pyridine-4-boric acid, cesium fluoride and tetrakis (triphenylphosphine) palladium in 1, 2-dimethoxyethane, stirring and reacting for 2-4 days at 80-100 ℃ under the atmosphere of N 2, and purifying the crude product by chromatography to obtain yellow powder, namely 2, 7-bis (pyridine-4) acridine; the synthesis steps of the 2, 7-di (pyridine-4) acridine molecule comprise: adding acridine into benzyl triethyl ammonium bromide-methanol solution, and stirring the reaction for 10 to 20 hours at the temperature of between 50 and 70 ℃ by using a reflux condenser; cooling to room temperature, filtering the crude product, washing with pyridine and methylene dichloride, and evaporating in vacuum at 40-60 ℃ to obtain light yellow solid 2, 7-dibromoacridine.
2. The fluorescence detection method of the tobacco pesticide residue bud suppression pellet according to claim 1, which is characterized in that: the preparation of the probe aqueous solution comprises the step of dispersing 2, 7-di (pyridine-4) acridine in deionized water to obtain the probe aqueous solution.
3. The fluorescence detection method of the tobacco pesticide residue bud suppression pellet according to claim 1, which is characterized in that: the pretreatment of tobacco samples comprises the following steps: 1.5-2.5 g of tobacco powder sample is weighed, hydrochloric acid solution with the volume of 15-25 mL and the concentration of 3-5 mol/L is added, the solution is magnetically stirred and extracted for 0.5-1.5 h under the condition of oil bath of 100-140 ℃, cooled to room temperature and centrifuged, and supernatant fluid is taken to obtain the sample to be measured.
4. The fluorescence detection method of the tobacco pesticide residue bud suppression pellet according to claim 1, which is characterized in that: the step of drawing the standard curve is that the stock solution of the buddhist is added into the probe aqueous solution to obtain a series of concentration buddhist-probe aqueous solution, the stable suspension is obtained by ultrasonic treatment, a fluorescence spectrometer is adopted, 320nm is set as the maximum excitation wavelength, 480nm is set as the maximum emission wavelength, and the fluorescence spectrum is recorded and collected within the range of 320-690 nm.
5. The fluorescence detection method of the tobacco pesticide residue bud suppression pellet according to claim 1, which is characterized in that: the sample detection comprises the steps of adding a sample solution to be detected into a probe aqueous solution, performing vortex oscillation, setting 320nm as a maximum excitation wavelength, setting 480nm as a maximum emission wavelength, and recording and collecting fluorescence spectra within a range of 320-690 nm.
6. The fluorescence detection method of the tobacco pesticide residue bud suppression pellet according to claim 1, which is characterized in that: the residual amount of the bud inhibition pellet is calculated by the following formula:
wherein:
r represents the residue of the bud inhibition pellet in terms of dry basis, mg/Kg;
C, calculating a standard working curve to obtain a result, namely mu mol/L;
V-volume of extract, mL;
m is the molar mass, g/mol of the sprout inhibition pellet;
m-mass of sample to be measured g.
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Non-Patent Citations (1)
| Title |
|---|
| 王春琼等.基于2,7-二（4-吡啶基）吖啶的荧光探针灵敏检测烟草农药残留抑芽丹.《烟草科技》.2023,全文. * |
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