Research On Selection, Modification And Detection Methods Of Aptamers To Chlorpyrifos And Mercury Ion

Pesticide residue and heavy metal pollution are the important factors of destroying nature and ecological environment and harming to human health. The effective detection methods are the premise requirement. At present, the common detection methods for pesticide such as chlorpyrifos are high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and for heavy metal such as mercury ions are atomic absorption spectroscopy, atomic fluorescence spectroscopy and so on, all of which have the advantage of high sensitivity and strong selectivity but the disadvantage of expensive equipment and complex sample processing. It’s necessary to develop a new rapid, convent and sensitive detection method.The aptamer can form into the specific and complex three-dimensional structures by the effect of hydrogen bondings, electrostatic and Van der Waals interactions, through which aptamers can well-fittingly bind to a wide variety of targets. It’s become a new trend to build methods based on aptamers, because aptamers have the biochemical characteristics of a wide range of targets, strong affinity, high specificity, convenience and quick for preparation and modification.The paper is to study the effect between aptamers with targets including chlorpyrifos and mercury ion separately and establish the corresponding detection methods. The main contents of this paper are as follows:1. Selection, identification and structures analysis of aptamers binding to chlorpyrifosSELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to select the specific aptamer of chlorpyrifos with carrier of streptavidin agarose resins. Several single-stranded DNA aptamers had been selected for their ability to bind chlorpyrifos with high specificity from the single DNA library with91nt. Through the use of fluorescent label technique, the selection efficiency, affinity and specificity of selected aptamer had been investigated. The result showed that after15rounds in vitro selection, the efficiency of selection can reach to44.00%and9aptamers were successfully selected for their ability to bind chlorpyrifos with high affinity, in which aptamer N23had the best affinity to chlorpyrifos, and also the binding ability of aptamer N23to Isocarbophos, Profenofos and Oethoate were evaluated. With secondary structures of4aptamers with higher affinity being analyzed by software of DNAMAN, it was predicated that the loopTTCTT, loopATAT and loopGCGC might be the binding sites.2. Study on the modification of the selected aptamer and corresponding detection methodTo improve the affinity of the selected aptamers binding to chlorpyrifos, three oligonucleotides, named M1, M2and M3separately, were obtained by linking and combinating the loopTTCTT, loopATAT and loopGCGC. Through the use of fluorescent label technique, the affinity of modificated aptamers had been investigated. The result showed that compared with N23, Ml had much higher affinity to chlorpyrifos. Based on aptamer Ml, a fluorescence assay method of chlorpyrifos was developed, which displayed the linear range for the chlorpyrifos concentration detection was0.0625-0.5mmol/L with a detection limit of0.0625mmol/L and the correlation coefficient of0.9942.3. Aptamer binding to mercury ion based on structure recognition from the modification of nickel ion RNA aptamer.Most of metal ions aptamers are RNA which is unstable and easy to degradate, and there have been no detection methods based on RNA aptamers for metal ions by now. To expand the scope of application of RNA aptamers of metal ions, it is a novel idea to replace and shear the bases to get stable and active DNA aptamers originating from RNA aptamers. Two DNA aptamers, respectively named DNA1-B and Nl, were obtained from the process of modificating the RNA aptamer of nickel ions. Through the use of fluorescent label technique, the affinity of modificated aptamers to different metal ions had been investigated. The result showed that N1could bind specifically to mercury ion with typical second structure, which was not usually presented in the reported specific nucleic acid aptamers of mercury ion. For aptamer N1, the fluorescence assay results showed that linear response toward mercury ion concentration ranged from1.25ppm to20ppm, with the limit of detection0.625ppm (mg/kg).4. Modification of DNA aptamer based on Nl and the establishment of detection method for mecury ionDNA oligonucleotides were obtained from the modification to N1based on the double principle that mercury ion could bind to the special secondary structures of the N1and the T base, naming N4, N5, N6and N7respectively. The fluorescence assay method was used to identify the activity of the four DNA oligonucleotides to mercury ion. The results showed that all of the four had much higher affinity compared with N1, and N5had the best affinity and specificity to mercury ion which displayed the linear range for the mercury ion concentration detection was0.156ppm to2.5ppm with a detection limit of78ppb. The result of detecting the mercury ion added into the water sample was good with spike recovery rate of85%-90.5%.