CN112113943A - Method for detecting paraquat - Google Patents
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- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 37
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000010931 gold Substances 0.000 claims abstract description 42
- 229910052737 gold Inorganic materials 0.000 claims abstract description 42
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 15
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 8
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 6
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 6
- 229960004853 betadex Drugs 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 24
- 239000000523 sample Substances 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000337 buffer salt Substances 0.000 claims description 10
- 230000000873 masking effect Effects 0.000 claims description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 7
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- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 229910052979 sodium sulfide Inorganic materials 0.000 description 3
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
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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 1
- MARUHZGHZWCEQU-UHFFFAOYSA-N 5-phenyl-2h-tetrazole Chemical compound C1=CC=CC=C1C1=NNN=N1 MARUHZGHZWCEQU-UHFFFAOYSA-N 0.000 description 1
- 239000005875 Acetamiprid Substances 0.000 description 1
- 229910004613 CdTe Inorganic materials 0.000 description 1
- 239000005944 Chlorpyrifos Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000005958 Fenamiphos (aka phenamiphos) Substances 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
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- 239000005916 Methomyl Substances 0.000 description 1
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- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
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- VDQVEACBQKUUSU-UHFFFAOYSA-M disodium;sulfanide Chemical compound [Na+].[Na+].[SH-] VDQVEACBQKUUSU-UHFFFAOYSA-M 0.000 description 1
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- 229940097068 glyphosate Drugs 0.000 description 1
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- YWTYJOPNNQFBPC-UHFFFAOYSA-N imidacloprid Chemical compound [O-][N+](=O)\N=C1/NCCN1CC1=CC=C(Cl)N=C1 YWTYJOPNNQFBPC-UHFFFAOYSA-N 0.000 description 1
- 229940056881 imidacloprid Drugs 0.000 description 1
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- 210000004072 lung Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- UHXUZOCRWCRNSJ-QPJJXVBHSA-N methomyl Chemical compound CNC(=O)O\N=C(/C)SC UHXUZOCRWCRNSJ-QPJJXVBHSA-N 0.000 description 1
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- RLBIQVVOMOPOHC-UHFFFAOYSA-N parathion-methyl Chemical compound COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C=C1 RLBIQVVOMOPOHC-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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"
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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"
- G01N2021/6432—Quenching
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Abstract
Description
技术领域technical field
本发明涉及一种检测百草枯的方法,属于分析化学技术领域。The invention relates to a method for detecting paraquat, belonging to the technical field of analytical chemistry.
背景技术Background technique
农药在农业领域中的应用可以在保证农作物产量同时,也会带来农药残留问题。百草枯是一种能够快速杀灭一年生杂草的除草剂除草,但因其对人体可产生不可逆的肺组织性损伤等病理症状,对人体器官伤害大,无特效的解毒药。目前已将百草枯的使用类别变更为禁用农药。为应对农业生产中可能存在违法使用百草枯的风险,建立一种能够快速实现定量-半定量检测的检测方法就显得较为重要。The application of pesticides in the agricultural field can not only ensure the yield of crops, but also bring about the problem of pesticide residues. Paraquat is a herbicide that can quickly kill annual weeds, but because it can cause irreversible lung tissue damage and other pathological symptoms to the human body, it is harmful to human organs and has no specific antidote. The use category of paraquat has now been changed to a banned pesticide. In order to deal with the risk of illegal use of paraquat in agricultural production, it is important to establish a detection method that can quickly realize quantitative and semi-quantitative detection.
目前,所开发的农药快速检测方法以酶抑制作用为基础较多。酶抑制法一般为广谱性方法,且存在生物酶稳定性受环境因素影响较大,保存要求高等缺点。直接利用纳米材料实现农药特异性检测的方法具有体系简单、特异性高等优点。其中,针对百草枯农药有基于纳米材料的比色、化学发光、表面拉曼增强、以及荧光检测方法的报道。其中,典型的例子是采用半导体量子点(如碲化镉CdTe)实现荧光检测百草枯的方法,但是材料纳米材料制备相对复杂、且需要应用重金属,然而重金属非常难以被生物降解,相反却能在食物链的生物放大作用下,成千百倍地富集,最后进入人体,造成慢性中毒,对人体有危害。At present, the rapid detection methods of pesticides developed are mostly based on enzyme inhibition. The enzyme inhibition method is generally a broad-spectrum method, and there are disadvantages that the stability of biological enzymes is greatly affected by environmental factors, and the preservation requirements are high. The method of directly using nanomaterials to realize the specific detection of pesticides has the advantages of simple system and high specificity. Among them, there are reports of colorimetric, chemiluminescence, surface Raman enhancement, and fluorescence detection methods based on nanomaterials for paraquat pesticides. Among them, a typical example is the use of semiconductor quantum dots (such as cadmium telluride CdTe) to realize the method of fluorescence detection of paraquat, but the preparation of nanomaterials is relatively complex and requires the application of heavy metals. However, heavy metals are very difficult to be biodegraded. Under the bio-amplification of the food chain, it is enriched thousands of times, and finally enters the human body, causing chronic poisoning, which is harmful to the human body.
发明内容SUMMARY OF THE INVENTION
为了解决上述问题,本发明的主要目的在于建立一种基于金纳米簇的荧光检测百草枯的方法。该发明的检测原理为:通过从金纳米簇到百草枯的静电转移而使得金纳米簇的荧光发生淬灭,同时,由于金纳米簇的双配基修饰具有荧光增强作用和增强抗干扰能力,于实现可靠的定量检测百草枯。In order to solve the above problems, the main purpose of the present invention is to establish a method for detecting paraquat by fluorescence based on gold nanoclusters. The detection principle of the invention is as follows: the fluorescence of gold nanoclusters is quenched by electrostatic transfer from gold nanoclusters to paraquat. for reliable quantitative detection of paraquat.
本发明的第一个目的是提供一种检测百草枯的方法,所述检测方法是采用金纳米簇作为百草枯的检测试剂,对待测样品进行荧光检测;所述金纳米簇是采用巯基-β-环糊精SH-β-CDs对谷胱甘肽还原金制备进行修饰制备得到的。The first object of the present invention is to provide a method for detecting paraquat. The detection method is to use gold nanoclusters as a detection reagent for paraquat, and perform fluorescence detection on the sample to be tested; the gold nanoclusters use sulfhydryl-β - Cyclodextrin SH-β-CDs are prepared by modifying the preparation of glutathione-reduced gold.
在本发明的一种实施方式中,所述金纳米簇的制备方法为:将2mL的HAuCl4(10mM)、0.3mL的GSH(100mM)溶液和7.7mL超纯水混合。在温和搅拌下将混合物加热至70℃,反应24小时。向所得到的淡黄色溶液中,以1:1比例加入乙醇;溶液由澄清变为浑浊,将溶液8000rpm下离心15min,分离沉淀并超声溶解,经0.22μM滤膜过滤,得到谷胱甘肽还原金溶液。向所得到的谷胱甘肽还原金溶液中加入SH-β-环糊精(终浓度5mM),50℃下孵育3h;所得到的溶液经超滤管(10kDa)离心浓缩,制备得到金纳米簇。In one embodiment of the present invention, the preparation method of the gold nanoclusters is: mixing 2 mL of HAuCl 4 (10 mM), 0.3 mL of GSH (100 mM) solution and 7.7 mL of ultrapure water. The mixture was heated to 70°C with gentle stirring and reacted for 24 hours. To the obtained pale yellow solution, ethanol was added at a ratio of 1:1; the solution changed from clarification to turbidity, the solution was centrifuged at 8000 rpm for 15 min, the precipitate was separated and dissolved by ultrasonic, and filtered through a 0.22 μM filter to obtain glutathione reduction. Gold solution. SH-β-cyclodextrin (final concentration 5mM) was added to the obtained glutathione-reduced gold solution, and incubated at 50°C for 3h; the obtained solution was centrifuged and concentrated by an ultrafiltration tube (10kDa) to prepare gold nanoparticles cluster.
在本发明的一种实施方式中,所述检测方法是:将待测样品溶液中加入金纳米簇溶液,混合反应后再采用荧光分光光度计进行检测,通过外标法计算得出待测样品中百草枯的含量。In one embodiment of the present invention, the detection method is: adding gold nanocluster solution to the solution of the sample to be tested, mixing and reacting, and then using a fluorescence spectrophotometer for detection, and calculating the sample to be tested by the external standard method paraquat content.
在本发明的一种实施方式中,所述金纳米溶液需采用缓冲液调节pH为8.5-9.0。In an embodiment of the present invention, the pH of the gold nano solution needs to be adjusted to 8.5-9.0 with a buffer.
在本发明的一种实施方式中,所述缓冲溶液包括甘氨酸缓冲盐、Tris-HCl或磷酸盐缓冲溶液。In one embodiment of the present invention, the buffer solution includes glycine buffered saline, Tris-HCl or phosphate buffered solution.
在本发明的一种实施方式中,所述荧光检测条件为激发宽带为20nm,狭缝宽度为20nm,激发波长为392nm,检测610nm发射峰值处荧光值。In an embodiment of the present invention, the fluorescence detection conditions are that the excitation broadband is 20 nm, the slit width is 20 nm, the excitation wavelength is 392 nm, and the fluorescence value at the emission peak of 610 nm is detected.
在本发明的一种实施方式中,若待测样品中含有金属离子,须先向待测样品中加入金属离子掩蔽剂,再加入金纳米簇进行检测。In an embodiment of the present invention, if the sample to be tested contains metal ions, a metal ion masking agent must be added to the sample to be tested, and then gold nanoclusters are added for detection.
本发明的第二个目的是提供一种上述检测方法在检测食品、河水、湖水或污水中农药含量方面的应用。The second object of the present invention is to provide an application of the above-mentioned detection method in the detection of pesticide content in food, river water, lake water or sewage.
本发明的第三个目的是提供一种检测水中百草枯含量的方法,所述方法是先向待测样品中加入金属离子掩蔽剂,再采用上述方法检测待测样品中百草枯含量。The third object of the present invention is to provide a method for detecting the content of paraquat in water, the method is to first add a metal ion masking agent to the sample to be tested, and then use the above method to detect the content of paraquat in the sample to be tested.
在本发明的一种实施方式中,所选择金纳米簇的浓度为0.02-0.2μM。In one embodiment of the present invention, the concentration of gold nanoclusters is selected to be 0.02-0.2 [mu]M.
在本发明的一种实施方式中,所选择的缓冲盐为Gly-NaOH(5-20mM)。In one embodiment of the present invention, the buffer salt of choice is Gly-NaOH (5-20 mM).
在本发明的一种实施方式中,所述的掩蔽剂为Na2S和cDCTA的混合液,Na2S浓度为0.1-1.0mM,cDCTA浓度为1-10mM。In an embodiment of the present invention, the masking agent is a mixture of Na2S and cDCTA , the concentration of Na2S is 0.1-1.0 mM, and the concentration of cDCTA is 1-10 mM.
在本发明的一种实施方式中,所述方法步骤为:In one embodiment of the present invention, the method steps are:
(1)金纳米簇溶液预备:将制备的金纳米簇由甘氨酸缓冲盐(5-20mM)调整pH为9.0,稀释至适当浓度:(1) Preparation of gold nanocluster solution: The prepared gold nanoclusters were adjusted to pH 9.0 by glycine buffer (5-20 mM), and diluted to an appropriate concentration:
(2)标准曲线的准备:将百草枯标准溶液与纳米簇溶液混合孵育1min,建立金纳米簇的荧光淬灭率信号与百草枯浓度的之间对应关系的标准曲线0.02-0.2μM;(2) Preparation of standard curve: The paraquat standard solution and the nanocluster solution were mixed and incubated for 1 min to establish a standard curve of 0.02-0.2 μM of the corresponding relationship between the fluorescence quenching rate signal of gold nanoclusters and the concentration of paraquat;
(3)样品的检测:将样品溶液中加入由硫化钠(Na2S)和反式环己二胺四乙酸(cDCTA)配置的掩蔽剂,和金纳米簇溶液混合反应,将计算后的荧光淬灭率信号带入标准曲线,计算得出检测样品中百草枯的浓度。(3) Sample detection: Add a masking agent configured by sodium sulfide (Na 2 S) and trans-cyclohexanediaminetetraacetic acid (cDCTA) to the sample solution, and mix and react with the gold nanocluster solution, and the calculated fluorescence The quenching rate signal was brought into the standard curve, and the concentration of paraquat in the test sample was calculated.
本发明的有益效果:Beneficial effects of the present invention:
本发明所构建的金纳米簇荧光检测百草枯的方法,其检测限为5.0ng/mL,线性范围为5.0-360ng/mL;其它常见有机磷农药在相同检测浓度500ng/mL浓度下,百草枯对于纳米簇的检测淬灭率在80%,其他农药的淬灭率均低于5%,纳米簇加入金属离子屏蔽剂后离子的干扰降低,对5uM已检测的金属离子的淬灭率响应均小于5%。该方法具有高灵敏、高选择性、检测快速等特点。The gold nanocluster fluorescence detection method for paraquat constructed in the present invention has a detection limit of 5.0 ng/mL and a linear range of 5.0-360 ng/mL; other common organophosphorus pesticides have the same detection concentration of 500 ng/mL. The detection quenching rate of nanoclusters is 80%, and the quenching rates of other pesticides are all lower than 5%. After adding metal ion shielding agent to nanoclusters, the interference of ions is reduced, and the quenching rate responses to 5uM detected metal ions are all less than 5%. The method has the characteristics of high sensitivity, high selectivity, and rapid detection.
附图说明Description of drawings
图1是金纳米簇的制备以及百草枯的检测原理图。Figure 1 is a schematic diagram of the preparation of gold nanoclusters and the detection of paraquat.
图2是修饰HS-β-CDs后纳米簇的荧光变化。Figure 2 is the fluorescence change of nanoclusters after modification of HS-β-CDs.
图3是百草枯检测荧光曲线。Figure 3 is the paraquat detection fluorescence curve.
图4是百草枯检测标准曲线。Figure 4 is the standard curve for the detection of paraquat.
图5是β-CD/GSH-AuNCs对各种农药的选择性响应。Figure 5 shows the selective response of β-CD/GSH-AuNCs to various pesticides.
图6是β-CD/GSH-AuNCs对各种金属离子的响应。Figure 6 is the response of β-CD/GSH-AuNCs to various metal ions.
图7是GSH-AuNCs对各种金属离子的响应。Figure 7 shows the responses of GSH-AuNCs to various metal ions.
图8是A,B,C,D,E,F分别是百草枯在不同浓度Na2HPO4-NaH2PO4,Tris-HCl,柠檬酸-Na2HPO4缓冲盐中对纳米簇淬灭率响应和在不同pH下Na2HPO4-NaH2PO4,Tris-HCl,Gly-NaOH的淬灭率响应。Figure 8 is A, B, C, D, E, F, respectively, the quenching of nanoclusters by paraquat in different concentrations of Na 2 HPO 4 -NaH 2 PO 4 , Tris-HCl, citric acid-Na 2 HPO 4 buffer salt Rate response and quench rate response of Na 2 HPO 4 -NaH 2 PO 4 , Tris-HCl, Gly-NaOH at different pH.
图9是EDTA对各种离子的屏蔽作用。Figure 9 shows the shielding effect of EDTA on various ions.
图10是cDCTA对各种金属离子的屏蔽作用。Figure 10 shows the shielding effect of cDCTA on various metal ions.
图11是cDCTA和Na2S对各种金属离子的屏蔽作用。Figure 11 shows the shielding effect of cDCTA and Na 2 S on various metal ions.
具体实施方式Detailed ways
以下对本发明的优选实施例进行说明,应当理解实施例是为了更好地解释本发明,不用于限制本发明。The preferred embodiments of the present invention will be described below, and it should be understood that the embodiments are used to better explain the present invention and are not intended to limit the present invention.
实施例1:金纳米簇的制备方法Example 1: Preparation method of gold nanoclusters
将2mL的HAuCl4(10mM)、0.3mL的GSH(100mM)溶液和7.7mL超纯水混合。在温和搅拌下将混合物加热至70℃,反应24小时。向所得到的淡黄色溶液中,以1:1比例加入乙醇;溶液由澄清变为浑浊,将溶液8000rpm下离心15min,分离沉淀并超声溶解,经0.22μM滤膜过滤,得到谷胱甘肽还原金溶液GSH-AuNCs。向所得到的谷胱甘肽还原金溶液中加入SH-β-环糊精(终浓度5mM),50℃下孵育3h;所得到的溶液经超滤管(10kDa)离心浓缩,制备得到金纳米簇β-CD/GSH-AuNCs,收集浓缩液(0.03mM)于4℃下保存,金纳米簇反应原理如图1所示。得到的金纳米簇的粒径为1.0~3.0nm,激发波长在350~400nm之间,发射波长为620nm。修饰前后的金纳米簇的荧光强度见图2,这说明采用巯基-β-环糊精修饰可提高百草枯的结合作用。2 mL of HAuCl 4 (10 mM), 0.3 mL of GSH (100 mM) solution and 7.7 mL of ultrapure water were mixed. The mixture was heated to 70°C with gentle stirring and reacted for 24 hours. To the obtained pale yellow solution, ethanol was added at a ratio of 1:1; the solution changed from clarification to turbidity, the solution was centrifuged at 8000 rpm for 15 min, the precipitate was separated and dissolved by ultrasonic, and filtered through a 0.22 μM filter to obtain glutathione reduction. Gold solution GSH-AuNCs. SH-β-cyclodextrin (final concentration 5mM) was added to the obtained glutathione-reduced gold solution, and incubated at 50°C for 3h; the obtained solution was centrifuged and concentrated by an ultrafiltration tube (10kDa) to prepare gold nanoparticles For cluster β-CD/GSH-AuNCs, the concentrated solution (0.03mM) was collected and stored at 4°C. The reaction principle of gold nanoclusters is shown in Figure 1. The particle size of the obtained gold nanoclusters is 1.0-3.0 nm, the excitation wavelength is between 350-400 nm, and the emission wavelength is 620 nm. The fluorescence intensities of the gold nanoclusters before and after modification are shown in Figure 2, which indicates that the modification with thiol-β-cyclodextrin can improve the binding effect of paraquat.
实施例2:一种应用金纳米簇检测河水/湖水中百草枯含量的方法Example 2: A method for detecting paraquat content in river/lake water using gold nanoclusters
应用实施例1制备得到的金纳米簇检测河水/湖水中百草枯含量,金纳米簇的制备以及百草枯的检测原理图见图1。The gold nanoclusters prepared in Example 1 were used to detect paraquat content in river/lake water. The preparation of gold nanoclusters and the detection principle of paraquat are shown in Figure 1 .
1、标准曲线的建立1. Establishment of standard curve
(1)标准溶液的制备:(1) Preparation of standard solution:
900uL Gly-NaOH缓冲液+50uL百草枯农药的标准液或样品液+50uL纳米簇(0.05μM),混匀后900uL Gly-NaOH buffer + 50uL paraquat pesticide standard solution or sample solution + 50uL nanoclusters (0.05μM), after mixing
(2)荧光光谱检测:(2) Fluorescence spectrum detection:
采用荧光光度计进行检测,检测条件为:激发波长为390nm,收集620nm处发射信号。Fluorescence photometer was used for detection, and the detection conditions were as follows: the excitation wavelength was 390 nm, and the emission signal was collected at 620 nm.
(3)线性关系、检测限的确定:(3) Determination of linear relationship and detection limit:
检测纳米簇荧光强度计为F1,纳米簇不添加农药的荧光强度计为F0,绘制标准曲线如图3和4,该标准曲线为y=0.0254x+0.0037(5-150ng/mL),R2=0.9956;y=0.1367x+0.014,(150-360ng/mL)R2=0.9837,线性范围为5-360ng/mL,定量限为5ng/mL。The fluorescence intensity of the detected nanoclusters was measured as F 1 , and the fluorescence intensity of the nanoclusters without pesticides was measured as F 0 , and the standard curve was drawn as shown in Figures 3 and 4. The standard curve was y=0.0254x+0.0037 (5-150ng/mL), R 2 =0.9956; y=0.1367x+0.014, (150-360 ng/mL) R 2 =0.9837, the linear range was 5-360 ng/mL, and the limit of quantification was 5 ng/mL.
2、检测河水/湖水中百草枯含量的方法2. Method for detecting paraquat content in river/lake water
(1)样品预处理:将河水/湖水经过微孔滤膜过滤,同时,以添加掩蔽剂,掩蔽剂含有200uM Na2S和2mM cDCTA,样品与掩蔽剂体积比为1:1。(1) Sample pretreatment: filter the river water/lake water through a microporous membrane, and at the same time, add a masking agent, the masking agent contains 200uM Na 2 S and 2mM cDCTA, and the volume ratio of sample to masking agent is 1:1.
(2)荧光光谱检测:(2) Fluorescence spectrum detection:
采用荧光光度计进行检测,检测条件为:激发波长为390nm,收集620nm处发射信号。Fluorescence photometer was used for detection, and the detection conditions were as follows: the excitation wavelength was 390 nm, and the emission signal was collected at 620 nm.
实施例3:方法的准确性和特异性Example 3: Accuracy and specificity of the method
1、方法的准确性1. The accuracy of the method
向湖水样品中加入标准添加百草枯的浓度为160ng/mL,3次平行测定实际测得百草枯平均浓度为155.6ng/mL,测得的回收率为97.2%,相对标准偏差(RSD)=2.0%。The concentration of standard paraquat added to the lake water sample was 160ng/mL. The actual average concentration of paraquat measured in three parallel determinations was 155.6ng/mL, and the measured recovery was 97.2%. Relative standard deviation (RSD)=2.0 %.
2、方法的特异性2. The specificity of the method
(1)考察了常见有机磷农药以及金属离子对检测的影响。有机磷农药选取了苯线磷、啶虫脒、毒死蜱、草甘膦、甲基对硫磷、水胺硫磷、灭多威和吡虫啉9种农药在相同浓度下(500ng/mL),结果显示它们不会影响对于百草枯的检测(如图5所示)。(1) The influence of common organophosphorus pesticides and metal ions on the detection was investigated. The organophosphorus pesticides selected 9 pesticides, fenamiphos, acetamiprid, chlorpyrifos, glyphosate, methyl parathion, amphos, methomyl and imidacloprid at the same concentration (500ng/mL). They did not affect the detection of paraquat (as shown in Figure 5).
(2)考察了常见金属离子对检测的影响,如图6和图7所示,结果表明经HS-β-CD修饰后抗金属和S2-能力提高。(2) The influence of common metal ions on the detection was investigated, as shown in Figures 6 and 7. The results showed that the anti-metal and S 2- ability was improved after modification by HS-β-CD.
实施例4:不同盐浓度对于百草枯淬灭金纳米簇荧光的影响Example 4: The effect of different salt concentrations on the quenching of gold nanocluster fluorescence by paraquat
参照实施例2的方法进行检测,区别仅在于,调整体系缓冲盐和缓冲盐浓度,采用Na2HPO4-NaH2PO4,Tris-HCl,柠檬酸-Na2HPO4将缓冲盐浓度分别调整为5mM,10mM,15mM,20mM,25mM,30mM其它条件相同,结果见图8。Detect with reference to the method of Example 2, the difference is only in that the buffer salt and buffer salt concentration of the system are adjusted, and the buffer salt concentration is adjusted respectively by using Na 2 HPO 4 -NaH 2 PO 4 , Tris-HCl, citric acid-Na 2 HPO 4 For 5mM, 10mM, 15mM, 20mM, 25mM, 30mM and other conditions are the same, the results are shown in Figure 8.
由图8A,8B,8C可知,高浓度的缓冲盐会影响百草枯对于纳米簇的荧光淬灭,因此选取5mM缓冲盐浓度作为最优。It can be seen from Figures 8A, 8B, and 8C that a high concentration of buffer salt will affect the fluorescence quenching of paraquat on nanoclusters, so 5mM buffer salt concentration is selected as the optimum.
实施例5:pH对于百草枯淬灭金纳米簇荧光的影响Example 5: The effect of pH on the quenching of gold nanocluster fluorescence by paraquat
参照实施例2的方法进行检测,区别仅在于,调整体系pH,采用Na2HPO4-NaH2PO4,Tris-HCl,Gly-NaOH将缓冲盐调整体系为不同的pH,其它条件相同,结果见图8。Detected with reference to the method of Example 2, the difference is only that the pH of the system was adjusted, and the buffer salt was adjusted to different pHs by using Na 2 HPO 4 -NaH 2 PO 4 , Tris-HCl, Gly-NaOH, and other conditions were the same, the results See Figure 8.
由图8D,8E,8F可知,当体系pH=9.0时百草枯对于纳米簇的荧光淬灭达到最大,因此选取pH=9.0的Tris-HCl,Gly-NaOH缓冲盐作为最优。8D, 8E, 8F, when the pH of the system is 9.0, the fluorescence quenching of the nanoclusters by paraquat reaches the maximum, so Tris-HCl and Gly-NaOH buffer salts with pH=9.0 are selected as the optimal.
实施例6:金属离子屏蔽剂的选择Example 6: Selection of metal ion shielding agent
参照实施例2的方法进行检测,区别仅在于,添加浓度为10uM的金属离子,将掩蔽剂分别更换成EDTA,cDCTA,Na2S和cDCTA体系,其它条件相同,结果分别如图9、10和11。通过筛选,选择了同时加入反式环己乙二胺四乙酸和硫化钠作为金属离子屏蔽剂,加入屏蔽剂后,对等13种常见金属离子屏蔽效果很好。Detected with reference to the method of Example 2, the only difference was that the metal ions with a concentration of 10uM were added, and the masking agent was replaced with EDTA, cDCTA, Na 2 S and cDCTA systems respectively, other conditions were the same, and the results were shown in Figure 9, 10 and 11. Through screening, the simultaneous addition of trans-cyclohexaneethylenediaminetetraacetic acid and sodium sulfide was selected as the metal ion shielding agent. After adding the shielding agent, the shielding effect of 13 common metal ions was very good.
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.
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