CN102590192A - Chemical luminescence enhanced type method for detecting pesticide residues - Google Patents
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Abstract
一种化学发光增强型检测农药残留的方法，包括类酶活性粒子的制备和鲁米诺化学发光体系的建立，所述的化学发光增强是将含至少一个磷硫单键的系列浓度有机磷农药样品和自由基清除剂一道加入类酶活性粒子溶液中，然后加入化学发光剂鲁米诺溶液，此时与只加入等量自由基清除剂的空白相比，化学发光强度明显增强，据此确立样品浓度与化学发光强度之间的线性关系。本发明首次利用表面配位抑制自由基清除的原理，设计出化学发光增强型传感器，可用于特定农药成分的敏感检测。以有机磷农药灭线磷为例，其线性范围为0.1～1000nmol/L和1～100μmol/L，检出限为0.1nmol/L。A method for detecting pesticide residues with enhanced chemiluminescence, comprising the preparation of enzyme-like active particles and the establishment of a luminol chemiluminescent system, wherein the enhanced chemiluminescence is a series of concentrations of organophosphorus pesticides containing at least one phosphorus-sulfur single bond The sample and the free radical scavenger were added to the enzyme-like active particle solution together, and then the chemiluminescent agent luminol solution was added. At this time, compared with the blank with only the same amount of free radical scavenger, the chemiluminescent intensity was significantly enhanced, thus establishing Linear relationship between sample concentration and chemiluminescence intensity. The invention utilizes the principle of surface coordination to inhibit free radical scavenging for the first time to design a chemiluminescent enhanced sensor, which can be used for sensitive detection of specific pesticide components. Taking the organophosphorus pesticide methimphos as an example, its linear range is 0.1-1000nmol/L and 1-100μmol/L, and the detection limit is 0.1nmol/L.
Description
一、技术领域 1. Technical field
本发明涉及一种农用化学品的分析方法，特别涉及一种痕迹量农用化学品的化学发光分析检测方法，具体地说是一种利用表面配位抑制自由基清除原理发展的化学发光增强型检测农药残留的方法。The present invention relates to an analysis method of agrochemicals, in particular to a chemiluminescence analysis and detection method of traces of agrochemicals, in particular to a chemiluminescence enhanced detection method developed using the principle of surface coordination to inhibit free radical scavenging methods of pesticide residues.
二、背景技术 2. Background technology
农药作为一种重要的农业生产资料，在防治作物病、虫、草害，保证农业丰收，促进高产、优质、高效现代化农业发展等方面发挥着不可替代的作用。然而，无节制的农药使用已经对生态坏境、食品安全和人体健康构成了严重威胁，同时也成为国际贸易争端的焦点之一。由于实际样品中农药残留组分含量往往很小，共存组分复杂，因此建立快速、高灵敏、高选择性的分析方法在农残检测中显得尤为迫切，这对于保护生态环境、保障人类健康、减少农业损失、促进经济发展具有十分重要意义。我国的农药残留分析技术始于20世纪50年代，近年来出现了不少行之有效的鉴别和分析检测方法。目前，农产品中农药残留的实验室分析与检测主要依赖于大型仪器来完成，例如，气相色谱法、液相色谱法、气-质联用分析法等。这些方法灵敏度高，但是所需要仪器昂贵，样品前处理复杂、耗时长并且无法做到现场检测。未来的农残检测要求向快速现场、廉价、减少溶剂、减少对环境污染、微型化和自动化方向发展，这是国家和社会亟待解决的挑战性难题。As an important means of agricultural production, pesticides play an irreplaceable role in preventing and controlling crop diseases, insects, and weed damage, ensuring agricultural harvests, and promoting the development of high-yield, high-quality, and efficient modern agriculture. However, the uncontrolled use of pesticides has posed a serious threat to the ecological environment, food safety and human health, and has also become one of the focuses of international trade disputes. Since the content of pesticide residue components in actual samples is often very small and the coexisting components are complex, it is particularly urgent to establish a fast, highly sensitive, and highly selective analysis method in the detection of pesticide residues, which is very important for protecting the ecological environment, ensuring human health, It is of great significance to reduce agricultural losses and promote economic development. my country's pesticide residue analysis technology began in the 1950s, and many effective identification and analysis methods have emerged in recent years. At present, the laboratory analysis and detection of pesticide residues in agricultural products mainly rely on large-scale instruments, such as gas chromatography, liquid chromatography, gas-mass spectrometry analysis, etc. These methods have high sensitivity, but the required instruments are expensive, the sample pretreatment is complex, time-consuming and cannot be detected on-site. The future detection of pesticide residues requires rapid on-site, low cost, less solvent, less environmental pollution, miniaturization and automation. This is a challenging problem that the country and society need to solve urgently.
化学发光分析以其灵敏度高、仪器设备简单、分析快速和容易实现自动化等优点，在生化分析、环境监测及食品安全等领域发挥着重要作用。近年来，利用化学发光方法检测农药残留也受到人们的关注。Roda等人利用农药抑制乙酰胆碱酶(AchE)活性的原理，采用流动注射化学发光法检测了对氧磷、涕灭威等有机磷农药，对氧磷和涕灭威的检测限分别是0.75μg·L-1和4μg·L-1。与其它检测系统相比，该方法简单、快速，而且有更高的灵敏度和重现性。Ayyagari等报道了基于碱性磷酸酯酶催化化学发光检测了乐果。碱性磷酸酯酶可以催化含磷酸酯发生去磷酸化作用，乐果抑制磷酸酯酶的行为，产生微弱的发光信号，检测限为500ppb。Gao等将酶固定在纤维尖端催化反应，产生的发光信号通过同一根纤维收集、传递到CCD检测器检测。此外，Pande等利用生物共聚物固定碱性磷酸酯酶在一个玻璃毛细管内壁，对甲基对硫磷等进行了检测，检测限达500-700ppb。这些检测农药残留的化学发光体系都利用酶来改善检测敏感性和选择性，因此使用寿命短、价格昂贵，不利于推广使用。此外，有人报道利用有机磷农药形成氧化能力更强的过氧化磷酸酯发展出化学发光增强型农残检测传感器，然而这种增强检测方法敏感性和选择性均较差。目前，利用表面配位抑制自由基清除原理构成的化学发光增强型传感器尚无文献报道，其对农药残留的检测更是无人涉及。Chemiluminescence analysis plays an important role in the fields of biochemical analysis, environmental monitoring and food safety due to its advantages of high sensitivity, simple equipment, fast analysis and easy automation. In recent years, the use of chemiluminescence to detect pesticide residues has also attracted people's attention. Roda et al. used the principle that pesticides inhibit the activity of acetylcholinease (AchE), and used flow injection chemiluminescence to detect organophosphorus pesticides such as paraoxon and aldicarb. The detection limits of paraoxon and aldicarb were 0.75 μg. L -1 and 4 μg·L -1 . Compared with other detection systems, the method is simple, fast, and has higher sensitivity and reproducibility. Ayyagari et al. reported the detection of dimethoate based on alkaline phosphatase-catalyzed chemiluminescence. Alkaline phosphatase can catalyze the dephosphorylation of phosphate-containing esters. Dimethoate inhibits the action of phosphatase and produces a weak luminescent signal with a detection limit of 500ppb. Gao et al. immobilized the enzyme on the tip of the fiber to catalyze the reaction, and the luminescent signal generated was collected by the same fiber and transmitted to the CCD detector for detection. In addition, Pande et al. used biological copolymers to immobilize alkaline phosphatase on the inner wall of a glass capillary to detect methyl parathion, etc., with a detection limit of 500-700ppb. These chemiluminescent systems for detecting pesticide residues all use enzymes to improve detection sensitivity and selectivity, so the service life is short and the price is expensive, which is not conducive to popularization. In addition, it was reported that organophosphorus pesticides were used to form phosphoperoxides with stronger oxidation ability to develop chemiluminescence-enhanced pesticide residue detection sensors. However, this enhanced detection method has poor sensitivity and selectivity. At present, there is no literature report on the chemiluminescence-enhanced sensor based on the principle of surface coordination to inhibit free radical scavenging, and no one has been involved in the detection of pesticide residues.
三、发明内容 3. Contents of the invention
本发明针对现有技术的上述不足，旨在提供一种快速检测灭线磷、丙溴磷等含至少一个硫磷单键的有机磷农药(硫赶式硫代磷酸酯或二硫代磷酸酯)残留量的方法，所要解决的技术问题是利用农药在类酶活性粒子表面的配位去抑制自由基清除以实现化学发光增强检测。The present invention aims at the above-mentioned deficiencies of the prior art, and aims to provide a rapid detection of organophosphorus pesticides (phosphorothioate or phosphorodithioate) containing at least one single bond of phosphion, such as methimphos, profenofos, etc. ) residue method, the technical problem to be solved is to use the coordination of pesticides on the surface of enzyme-like active particles to inhibit free radical scavenging to achieve chemiluminescence enhanced detection.
所述的化学发光信号是由于类酶活性粒子催化水中的溶解氧产生过氧负离子，继而氧化化学发光剂鲁米诺产生化学发光；这时若加入自由基清除剂，由于产生的自由基被消耗(亦称自由基清除)，因此化学发光信号减弱直至消失(猝灭)。发光信号减弱直至猝灭与自由基清除剂含量之间存在一定的线性关系。The chemiluminescent signal is due to the enzyme-like active particles catalyzing the dissolved oxygen in water to produce peroxide negative ions, and then oxidizing the chemiluminescence agent luminol to produce chemiluminescence; if a free radical scavenger is added at this time, the free radicals produced will be consumed (also known as free radical scavenging), so the chemiluminescence signal is weakened until it disappears (quenching). There is a certain linear relationship between the luminescent signal weakening until quenching and the content of free radical scavenger.
含至少一个磷硫单键的有机磷农药在类酶活性粒子表面的配位作用有抑制自由基清除剂清除自由基的作用，因此与只加入自由基清除剂的空白相比，化学发光强度明显增强，据此可确立样品浓度与化学发光强度之间的线性关系。The coordination of organophosphorus pesticides containing at least one phosphorus-sulfur single bond on the surface of enzyme-like active particles has the effect of inhibiting free radical scavengers from scavenging free radicals, so compared with the blank only adding free radical scavengers, the chemiluminescence intensity is obvious Enhancement, whereby a linear relationship between sample concentration and chemiluminescent intensity can be established.
本发明正是利用类酶活性粒子催化溶解氧形成过氧负离子以及表面配位抑制自由基清除等性质，将其联合应用到化学发光检测中，开发出一种化学发光增强型检测方法。The present invention utilizes the properties of enzyme-like active particles to catalyze dissolved oxygen to form peroxide negative ions and surface coordination to inhibit free radical scavenging, and applies them to chemiluminescence detection to develop a chemiluminescence-enhanced detection method.
所述的化学发光增强是指以只加入自由基清除剂的鲁米诺化学发光体系(空白)为参考，将农药和自由基清除剂一起加入到类酶活性粒子溶液后，再加入发光剂鲁米诺溶液，这时体系化学发光强度与空白相比明显增强，当自由基清除剂的含量和鲁米诺的含量一定时，随着农药浓度的增加，化学发光强度逐渐由弱到强。待测样品浓度变化范围对应化学发光强度变化的过程。这就是说通过化学发光增强模式确立起待测样品浓度与化学发光强度之间的线性关系，从而实现待测样品的定量检测。The chemiluminescence enhancement refers to taking the luminol chemiluminescence system (blank) with only free radical scavengers as a reference, adding the pesticide and the free radical scavenger to the enzyme-like active particle solution together, and then adding the luminescent agent Luminol. Minol solution, at this time, the chemiluminescence intensity of the system was significantly enhanced compared with the blank. When the content of free radical scavenger and luminol were constant, the chemiluminescence intensity gradually changed from weak to strong with the increase of pesticide concentration. The concentration range of the sample to be tested corresponds to the process of changing the intensity of chemiluminescence. That is to say, the linear relationship between the concentration of the sample to be tested and the intensity of chemiluminescence is established through the enhanced mode of chemiluminescence, thereby realizing the quantitative detection of the sample to be tested.
本发明技术方案的关键是确立待测样品浓度的增加与化学发光强度由弱到强的变化之间的线性关系，发光强度的变化是以空白体系为参考比较得到的。The key to the technical solution of the present invention is to establish the linear relationship between the increase of the concentration of the sample to be tested and the change of chemiluminescence intensity from weak to strong, and the change of luminescence intensity is obtained by comparing with the blank system as a reference.
本发明的技术方案包括类酶活性粒子的制备和鲁米诺化学发光体系的建立，与现有技术的区别是所述的化学发光增强是将含至少一个磷硫单键的系列浓度有机磷农药样品和自由基清除剂一道加入类酶活性粒子溶液中，然后加入化学发光剂鲁米诺溶液，此时与只加入等量自由基清除剂的空白相比，化学发光强度明显增强，据此确立样品浓度与化学发光强度之间的线性关系。The technical scheme of the present invention includes the preparation of enzyme-like active particles and the establishment of a luminol chemiluminescence system. The difference from the prior art is that the enhancement of chemiluminescence is a series of concentrations of organophosphorus pesticides containing at least one phosphorus-sulfur single bond. The sample and the free radical scavenger were added to the enzyme-like active particle solution together, and then the chemiluminescent agent luminol solution was added. At this time, compared with the blank with only the same amount of free radical scavenger, the chemiluminescent intensity was significantly enhanced, thus establishing Linear relationship between sample concentration and chemiluminescence intensity.
所述的鲁米诺化学发光体系是由鲁米诺水溶液(水中有溶解氧)和类酶活性粒子所构成。若在该体系中加入自由基清除剂则构成空白体系。The luminol chemiluminescent system is composed of luminol aqueous solution (dissolved oxygen in water) and enzyme-like active particles. If a free radical scavenger is added to the system, a blank system is formed.
所述的类酶活性粒子是具有类酶催化活性的纳米粒子，选自磁性Fe3O4、CoFe2O4粒子或ZnO、ZnS等半导体粒子或Au、Pb等贵金属粒子以及各种复合结构。The enzyme-like active particles are nanoparticles with enzyme-like catalytic activity, selected from magnetic Fe 3 O 4 , CoFe 2 O 4 particles, semiconductor particles such as ZnO, ZnS, noble metal particles such as Au, Pb, and various composite structures.
所述的自由基清除剂选自乙醇或甲醇。The free radical scavenger is selected from ethanol or methanol.
在本发明中由于化学发光源自两种溶液的混合，为获得最大的化学发光强度，每种溶液存有一最佳使用浓度，鲁米诺溶液最佳使用浓度为1×10-4M，对磁性Fe3O4纳米粒子最佳使用浓度为25μg/mL。In the present invention, since the chemiluminescence originates from the mixing of two solutions, in order to obtain the maximum chemiluminescence intensity, each solution has an optimum concentration, and the optimum concentration of the luminol solution is 1×10 -4 M, for The optimum concentration of magnetic Fe 3 O 4 nanoparticles is 25μg/mL.
当选择磁性Fe3O4纳米粒子和质量百分浓度99％乙醇为自由基清除剂时，可以将含至少一个磷硫单键有机磷农药样品溶于99％乙醇后加入浓度为25μg/mL、pH＝2的磁性Fe3O4纳米粒子溶液中，然后加入浓度1×10-4M的鲁米诺溶液，与只加入等量99％乙醇的空白相比，发光强度明显增强，据此确立样品浓度和发光强度之间的线性关系。When selecting magnetic Fe 3 O 4 nanoparticles and mass percent concentration of 99% ethanol as free radical scavengers, the organic phosphorus pesticide sample containing at least one phosphorus-sulfur single bond can be dissolved in 99% ethanol and then added at a concentration of 25 μg/mL, pH = 2 magnetic Fe 3 O 4 nanoparticle solution, and then add luminol solution with a concentration of 1 × 10 -4 M, compared with the blank that only adds the same amount of 99% ethanol, the luminescence intensity is significantly enhanced, and it is established accordingly Linear relationship between sample concentration and luminescence intensity.
当线性关系确立后就可按常规操作对实样进行农药残留的检测。When the linear relationship is established, the real sample can be tested for pesticide residues according to routine operations.
本发明的优点和积极效果：Advantage and positive effect of the present invention:
本发明首次利用表面配位抑制自由基清除的原理，设计出化学发光增强型传感器，可用于特定农药成分的敏感检测。以有机磷农药灭线磷为例，其线性范围为0.1～1000nmol/L和1～100μmol/L，检出限为0.1nmol/L。The invention utilizes the principle of surface coordination to inhibit free radical scavenging for the first time to design a chemiluminescent enhanced sensor, which can be used for sensitive detection of specific pesticide components. Taking the organophosphorus pesticide methimphos as an example, its linear range is 0.1-1000nmol/L and 1-100μmol/L, and the detection limit is 0.1nmol/L.
本发明方法具有化学发光传感器的一切优点，如无需大型仪器，方便快速，灵敏度高，效果显著。The method of the invention has all the advantages of the chemiluminescent sensor, such as no need for large-scale instruments, convenient and fast, high sensitivity and remarkable effect.
化学发光抗干扰能力差，由于实样中成分复杂因此会严重干扰化学发光信号。在该方法中，可通过磁性分离的方法克服实样成分的干扰。因此不需要富集等复杂的前处理过程，操作更简单，成本更低。The anti-interference ability of chemiluminescence is poor. Due to the complex components in the real sample, it will seriously interfere with the chemiluminescence signal. In this method, the interference of real sample components can be overcome by magnetic separation. Therefore, complex pre-treatment processes such as enrichment are not required, and the operation is simpler and the cost is lower.
四、附图说明 4. Description of drawings
图1是利用表面配位抑制自由基清除原理构筑的化学发光增强型传感器示意图。Figure 1 is a schematic diagram of a chemiluminescent enhanced sensor constructed using the principle of surface coordination to inhibit free radical scavenging.
图2是对灭线磷的化学发光增强检测以及对应的线性关系图。Fig. 2 is the chemiluminescent enhanced detection of phenimin and the corresponding linear relationship diagram.
图3是化学发光增强型传感器对果汁、绿茶中添加农药的检测原理和结果图。Figure 3 is a diagram showing the principle and results of the detection of pesticides added in fruit juice and green tea by the enhanced chemiluminescence sensor.
图4是化学发光增强型传感器的选择性测试图。Fig. 4 is a selectivity test diagram of a chemiluminescent enhanced sensor.
五、具体实施方式 5. Specific implementation
现以质量百分浓度99％乙醇为自由基清除剂和磁性Fe3O4纳米粒子为例非限定实施例叙述如下：Now take 99% ethanol as a free radical scavenger and magnetic Fe3O4 nanoparticles as an example , and the non-limiting examples are described as follows:
1、磁性Fe3O4纳米粒子的制备1. Preparation of Magnetic Fe3O4 Nanoparticles
参考文献L.Z.Gao et al，Nat.Nanotech.，2007，2，577报道的共沉淀方法。Co-precipitation method reported by reference L.Z.Gao et al, Nat.Nanotech., 2007, 2, 577.
将10μL的HCl加入20mL去离子水中，通氮除氧30分钟后，加入2.4g的FeCl2·4H2O和4.6g的FeCl3·6H2O，超声溶解后经过滤转移到80mL通氮除氧过的去离子水中，并保持氮气气氛。随后，将10mL的氨水逐滴加到上述溶液中，溶液迅速变黄，且颜色逐渐加深，进而变黑。滴加完毕，将体系升温至40℃，反应0.5小时，再升温到85℃反应2小时。经离心洗涤后重新分散到去离子水中，备用。从XRD花样可以看出得到的粒子是立方相Fe3O4，晶粒尺寸10nm。使用前将磁性粒子稀释到pH＝2的去离子水中，浓度为25μg/mL。Add 10 μL of HCl to 20 mL of deionized water, pass nitrogen to remove oxygen for 30 minutes, add 2.4 g of FeCl 2 4H 2 O and 4.6 g of FeCl 3 6H 2 O, dissolve by ultrasonic and transfer to 80 mL of nitrogen for removal by filtration. Oxygenated deionized water, and maintain a nitrogen atmosphere. Subsequently, 10 mL of ammonia water was added dropwise to the above solution, and the solution quickly turned yellow, and the color gradually deepened, and then turned black. After the dropwise addition was completed, the temperature of the system was raised to 40° C. for 0.5 hours of reaction, and then the temperature was raised to 85° C. for 2 hours of reaction. After centrifugal washing, redisperse into deionized water and set aside. It can be seen from the XRD pattern that the obtained particles are cubic phase Fe 3 O 4 with a grain size of 10nm. The magnetic particles were diluted into deionized water with pH=2 at a concentration of 25 μg/mL before use.
2、灭线磷浓度与发光强度之间线性关系的确定2. Determination of the linear relationship between the concentration of anphenphos and the luminous intensity
将灭线磷溶解在乙醇溶液中，配成系列浓度(1-60μM)的溶液。将30μL的磁性粒子稀释液加入化学发光仪96孔板的孔中，再加入30μL的灭线磷乙醇溶液，振荡10s后，加入90μL鲁米诺溶液测试该孔的化学发光，以最强峰为标准。为确定化学发光检测信号，需以单纯加入30μL乙醇的体系发光为参考，通过对比可以看出，灭线磷增强了化学发光，并且随着灭线磷浓度的增加化学发光强度逐渐增加，如图2A所示。因此可通过计算化学发光的增强量来定量分析灭线磷的浓度，图2B所示，其线性范围有两部分构成，分别为0.1～1000nmol/L和1～100μmol/L，检出限为0.1nmol/L。Dissolve methamidophos in ethanol solution to make solutions with series concentration (1-60μM). Add 30 μL of magnetic particle diluent into the wells of a chemiluminescence instrument 96-well plate, then add 30 μL of phenimin ethanol solution, shake for 10 seconds, add 90 μL of luminol solution to test the chemiluminescence of the well, and take the strongest peak as standard. In order to determine the chemiluminescence detection signal, the luminescence of the system simply added with 30 μL of ethanol should be used as a reference. It can be seen from the comparison that the chemiphos enhances the chemiluminescence, and the chemiluminescence intensity gradually increases with the increase of the phenimin concentration, as shown in the figure 2A. Therefore, the concentration of animaphos can be quantitatively analyzed by calculating the enhanced amount of chemiluminescence. As shown in Figure 2B, the linear range consists of two parts, which are 0.1-1000nmol/L and 1-100μmol/L respectively, and the detection limit is 0.1 nmol/L.
3、对葡萄汁和绿茶中添加农药的检测3. Detection of pesticides added in grape juice and green tea
如果直接检测葡萄汁中的农药分子，由于内部成分的干扰将不能获得化学发光信号。而在本方法中，通过磁性粒子的吸附-分离-再分散的过程(图3)，可有效克服实样成分的干扰，从而实现对实际样品的检测。典型的，将含一定浓度农药灭线磷的乙醇溶液添加进葡萄汁中，搅拌5分钟后加入磁性粒子，继续搅拌10分钟使农药配位结合到粒子表面，然后，通过外加磁场将磁性粒子分离，弃去果汁溶液再添加一定量的去离子水(pH＝2)，控制粒子浓度为25μg/mL。按照同样的步骤，在葡萄汁中加入不含农药的乙醇溶液得到参考粒子溶液。按照步骤2进行化学发光测试与检测，由于农药分子已经配位结合到磁性粒子上，因此只需加入30μL的乙醇即可。同样的过程也被用于绿茶中添加农药的检测，但是由于更加复杂的成分，需要进行两次分离-再分散的过程。If the pesticide molecules in grape juice are detected directly, the chemiluminescent signal cannot be obtained due to the interference of internal components. However, in this method, through the adsorption-separation-redispersion process of magnetic particles (Figure 3), the interference of real sample components can be effectively overcome, thereby realizing the detection of actual samples. Typically, an ethanol solution containing a certain concentration of the pesticide cyprophos is added to the grape juice, stirred for 5 minutes, then the magnetic particles are added, and the stirring is continued for 10 minutes to coordinate the pesticide to the surface of the particles, and then the magnetic particles are separated by an external magnetic field. , discard the juice solution and add a certain amount of deionized water (pH=2) to control the particle concentration to 25 μg/mL. Following the same procedure, add a pesticide-free ethanol solution to the grape juice to obtain a reference particle solution. Carry out chemiluminescence test and detection according to
4、磁性Fe3O4纳米粒子的选择性测试4. Selectivity test of magnetic Fe 3 O 4 nanoparticles
化学发光敏感性高，但选择性差。本发明对磁性Fe3O4纳米粒子选择灭线磷EP、丙溴磷PF、敌百虫DL、毒死蜱CP、甲基对硫磷PM、2，4-二氯苯氧乙酸2，4-D进行测试。如图4所示，在浓度均为1×10-4M的条件下，灭线磷引起化学发光强度18倍的增强，丙溴磷的增强倍数为10，而其它农药对化学发光基本没影响。对比上述几种农药的分子结构，可以看出该体系可以选择性检测含有硫磷单键的农药成分如灭线磷、丙溴磷。Chemiluminescence has high sensitivity but poor selectivity. The present invention selects methimphos EP, profenofos PF, trichlorfon DL, chlorpyrifos CP , methyl parathion PM, 2,4-
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103389301A (en) * | 2013-08-01 | 2013-11-13 | 辛焕发 | Method for fast detecting pesticide residue |
| CN110186912A (en) * | 2019-05-20 | 2019-08-30 | 济南大学 | A method of based on thiocholine coreaction promotor electrogenerated chemiluminescence aptamer sensor and measurement chlopyrifos |
| CN113791064A (en) * | 2021-09-08 | 2021-12-14 | 武汉谱信环保科技有限公司 | Rapid detection method of quinalphos pesticide residue |
| CN115308180A (en) * | 2022-08-08 | 2022-11-08 | 青岛农业大学 | Fluorescent zinc oxide quantum dot, and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993023756A1 (en) * | 1992-05-08 | 1993-11-25 | Monitor Diagnostics, Inc. | Use of nonmetallic tetrapyrrole molecules and novel signal solutions in chemiluminescent reactions and assays |
| WO2002022861A1 (en) * | 2000-09-13 | 2002-03-21 | California Institute Of Technology | Oxygenase enzymes and screening method |
| CN1664567A (en) * | 2005-03-14 | 2005-09-07 | 陕西师范大学 | Chemiluminescent organophosphorus pesticide residue analyzer and its detection method |
| CN101148584A (en) * | 2007-11-15 | 2008-03-26 | 上海交通大学 | Methods for catalyzing and enhancing chemiluminescence with nanoparticles |
| CN101706431A (en) * | 2009-06-09 | 2010-05-12 | 华中农业大学 | Chemiluminescence method for detecting organophosphorus pesticide |
| CN102053085A (en) * | 2009-10-28 | 2011-05-11 | 中国科学院高能物理研究所 | Method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence |
| CN102191034A (en) * | 2010-03-03 | 2011-09-21 | 中国科学技术大学 | N-(4-aminobutyl)-N-ethylisoluminol luminescence functionalized nanogold, and preparation method and application thereof |
| CN102221529A (en) * | 2011-03-31 | 2011-10-19 | 吉林大学 | Method for rapidly detecting residuals of organophosphorus pesticides in vegetables by utilizing Au nano-particle colorimetric method |
-
2012
- 2012-02-17 CN CN201210037336.6A patent/CN102590192B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993023756A1 (en) * | 1992-05-08 | 1993-11-25 | Monitor Diagnostics, Inc. | Use of nonmetallic tetrapyrrole molecules and novel signal solutions in chemiluminescent reactions and assays |
| WO2002022861A1 (en) * | 2000-09-13 | 2002-03-21 | California Institute Of Technology | Oxygenase enzymes and screening method |
| CN1664567A (en) * | 2005-03-14 | 2005-09-07 | 陕西师范大学 | Chemiluminescent organophosphorus pesticide residue analyzer and its detection method |
| CN101148584A (en) * | 2007-11-15 | 2008-03-26 | 上海交通大学 | Methods for catalyzing and enhancing chemiluminescence with nanoparticles |
| CN101706431A (en) * | 2009-06-09 | 2010-05-12 | 华中农业大学 | Chemiluminescence method for detecting organophosphorus pesticide |
| CN102053085A (en) * | 2009-10-28 | 2011-05-11 | 中国科学院高能物理研究所 | Method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence |
| CN102191034A (en) * | 2010-03-03 | 2011-09-21 | 中国科学技术大学 | N-(4-aminobutyl)-N-ethylisoluminol luminescence functionalized nanogold, and preparation method and application thereof |
| CN102221529A (en) * | 2011-03-31 | 2011-10-19 | 吉林大学 | Method for rapidly detecting residuals of organophosphorus pesticides in vegetables by utilizing Au nano-particle colorimetric method |
Non-Patent Citations (6)
| Title |
|---|
| ANDREW L. ROSE AND T. DAVID WAITE: "Chemiluminescence of Luminol in the Presence of Iron(II) and Oxygen:Oxidation Mechanism and Implications for Its Analytical Use", 《ANAL.CHEM.》 * |
| HUI WEI AND ERKANG WANG: "Fe3O4 Magnetic nanoparticles as peroxidase mimetics and their applications in H2O and Glucose detection", 《ANAL.CHEM.》 * |
| 刘晓宇等: "蔬菜中有机磷农药辛硫磷的化学发光分析研究", 《分析化学》 * |
| 林洁华等: "基于固定化纳米金增强化学发光双酶传感器测定葡萄糖", 《中国科学(B辑:化学)》 * |
| 熊志刚等: "磁性纳米粒子固定葡萄糖氧化酶修饰电极电致化学发光葡萄糖传感器", 《分析化学》 * |
| 饶志明等: "流动注射化学发光测定甲基对硫磷", 《分析化学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103389301A (en) * | 2013-08-01 | 2013-11-13 | 辛焕发 | Method for fast detecting pesticide residue |
| CN110186912A (en) * | 2019-05-20 | 2019-08-30 | 济南大学 | A method of based on thiocholine coreaction promotor electrogenerated chemiluminescence aptamer sensor and measurement chlopyrifos |
| CN110186912B (en) * | 2019-05-20 | 2021-07-06 | 济南大学 | A kind of electrochemiluminescence aptamer sensor based on thiocholine co-reaction promoter and method for determination of chlorpyrifos |
| CN113791064A (en) * | 2021-09-08 | 2021-12-14 | 武汉谱信环保科技有限公司 | Rapid detection method of quinalphos pesticide residue |
| CN113791064B (en) * | 2021-09-08 | 2024-03-12 | 武汉谱信环保科技有限公司 | Rapid detection method for quinfos pesticide residue |
| CN115308180A (en) * | 2022-08-08 | 2022-11-08 | 青岛农业大学 | Fluorescent zinc oxide quantum dot, and preparation method and application thereof |
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