| Background and objective:China is a big country of production and consumption of pesticides. Pesticides play an important role in pest control, removing weeds, controlling human and livestock diseases, increasing production of agricultural products, et al. However, using pesticides widely and unreasonably, the negative impact of pesticides residues on human health, ecological environment and foreign trade exposed gradually. It has been widely concerned that pesticides residues endanger food security, human and livestock safety, ecological environment by a variety of ways. When pesticides widely used in agriculture entered environment by sedimentation, runoff and other ways, they caused many environmental problems, such as water resource pollution, damage to aquatic ecosystem, food safety, et al. In order to protect human’s health and prevent environmental pollution of pesticides, it is of particular significance and necessity to develop a rapid, sensitive and reliable quantitative analysis method for pesticides. Organophosphate pesticides and carbamate pesticides are two types of pesticides which are widely used in agriculture in China. They can inhibit acetylcholinesterase activity, cause poisonings, and even endanger life of human being. Classical instrumental analytical techniques for pesticides analysis involve gas chromatography, high-performance liquid chromatography, gas chromatography coupled with mass spectrometry, or liquid chromatography with mass spectrometry. Although chromatography based methods are sensitive and reliable, they require sophisticated equipment, skilled analysts and time-consuming sample preparation steps. Moreover, organic solvents used in the detection process may lead to environmental pollution. Rapid determination technology is an effective way to monitor pesticide residues. Technologies based on immunoassay for determination of pesticides residues have many advantages, such as good-selectivity, fast-speed and high-sensitivity, which can realize fast detection of pesticides residues. Recently, most of immunoassays focus on detection of single pesticide. There might exist many kinds of pesticides in the samples at the same time. Suspension arrays have increasingly gained attention in multiplex analysis of biomarkers, drug screening, food detection and environmental monitoring. Compared with the common single analyte assays, suspension arrays using encoded microbeads as solid supports have the advantages of enhanced detection throughput, shortened analytical time, decreased sampling volume, improved test efficiency, reduced cost and multiplexing capability. In our study, the techniques for multiplex detection of organophosphorus pesticides and carbamate pesticides have been developed using suspension arrays based on Silica Colloidal Crystal Beads and Silica-Hydrogel Hybrid Microbeads.Research contents and results:(1) Fabrication of Silica Colloidal Crystal Beads (SCCBs)SCCBs with controllable sizes were produced by the microfluidic device using monodispersed silica nanoparticles. The reflection peaks of SCCBs can be varied by changing the diameters of silica nanoparticles. These SCCBs can be used as coding microcarriers for detection of pesticides with a suspension array.(2) Simultaneous detection of fenitrothion and Chlorpyrifos-methyl with a photonic suspension array based on SCCBsThe photonic suspension array for simultaneous detection of the selected pesticides was a competitive immunoassay based on the increase of the fluorescein signals from the conjugates of pesticides labeled by fluorescein isothiocyanate (FITC) when they were bound by the specific monoclonal antibodies of pesticides immobilized on different types of SCCBs. If the sample contained the target pesticides, the FITC-labeled conjugates would compete with the target pesticides for binding with the monoclonal antibodies and the fluorescein signals would decrease when more target pesticides were available. After experimental conditions optimized, standard curves for multiplex detection of pesticides were plotted and LODs were 0.25 ng/mL and 0.40 ng/mL, respectively. The proposed suspension array allowed simultaneous detection of chlorpyrifos-methyl and fenitrothion in the ranges of 0.40 ng/mL to 735.37 ng/mL and 0.25 ng/mL to 1024 ng/mL. The chemicals chosen to estimate the specificity of the suspension array were chloryrifos, bromophos,3,5, 6-trichloro-2-pyridinol, triazophos, methidathion, fenthion, paraoxon, chlorthion, parathion and parathion-methyl. The cross-reactivity values of the various potential cross reactants were very small and fell below 5%. Thus, it could be considered that the suspension array was very specific. The recovery rates for fenitrothion and chlorpyrifos-methyl were 81.6%～92.2% and 82.4%～97.3%, respectively. The developed method showed high sensitivity, selectivity, reproducibility, wide linear range and low detection of limit. Grape, lettuce and cabbage were chosen for recovery studies. This method has potential application for practical, effective and high throughput analysis of pesticides residues.(3) Multiplex detection of five kinds of organophosphorus pesticides and carbamate pesticides with a suspension array based on Silica-Hydrogel Hybrid Microbeads (SHHMs)Based on SCCBs, five types of SHHMs with different characteristic reflection peaks were produced as microcarriers for suspension array. Five kinds of organophosphorus pesticides and carbamate pesticides, which have been widely used, were chosen as target analytes. The suspension array is based on an indirect competition immunoassay and a biotin-streptavidin signal amplification system for pesticides detecting. The selected pesticides are detected through the competition of the specific monoclonal antibodies between the multiple pesticides and the antigens (the pesticides conjugated with BSA), which are immobilized on different types of SHHMs in solution. If the sample contained the target pesticides, the antigens on the surface of SHHMs would compete with the target pesticides for binding with monoclonal antibody and secAb-biotin, which are subsequently recognized by streptavidin-R-phycoerythrin. Then, the fluorescein signals would decrease when more target pesticides were available. The sensitivity and stability of suspension array technology was improved by optimizing and screening the experimental conditions (the amount of antigens, antibodies, incubation time and so on). LODs for fenitrothion, chlorpyrifos-methyl, fenthion, carbaryl and metolcarb were measured to be 0.02 ng/mL,0.012 ng/mL,0.04 ng/mL,0.05 ng/mL and 0.1 ng/mL, respectively. The ranges of detection were 0.02-1562.5 ng/mL,0.012-937.5 ng/mL,0.04-1250 ng/mL,0.05-819.2 ng/mL and 0.1-218.7 ng/mL for fenitrothion, chlorpyrifos-methyl, fenthion, metolcarb and carbaryl, respectively. All the determination coefficients for these five pesticides were greater than 0.99, demonstrating excellent correlations. The chemicalschosen to estimate the specificity of the suspension array were chloryrifos, triazophos, parathion and parathion-methyl, methidathion, bromophos, methomyl, carbofuran, propoxur, fenobucarb, isoprocarb. The cross-reactivity values of all the different pesticides were very small, typically below 5%. There was no cross-reaction between the different antibodies and any of the other heterologous pesticides. Hence, these organophosphorus pesticides and carbamate pesticides did not interfere with the performance of the multiplex immunoassay. Clearly, the suspension array was very specific. More importantly, we demonstrate the real application of this assay by detecting spiked pesticides in agricultural products and water samples. The recovery rates for apple, lettuce, cabbage, cucumber, tomato and water samples were in the range of 88.9～106.2%、82.6～106.3%,85.4～105.5%,88.7～104.3%,85.6-103.5%, 83.3%～102.2%, respectively. These results obtained with our suspension array were in good agreement with those of LC-MS/MS, which were in the range 85.9～104.3%, 88.4～105.6%,86.7～103.6%,84.6～102.7%and 85.3～105.6%,85.6～95.3%, respectively. Therefore, this new type of suspension array has proven to be useful in monitoring pesticides in fruits, vegetables and water samples. Compared with traditional liquid chromatography-tandem mass spectrometry, the suspension array based on SHHMs is simple, rapid and reliable. |
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