Research Of Aptamer-based Analytical Methods On The Rapid Detection For Pesticides And Veterinary Drug Residues

As a new type of recognition element for biosensor, aptamers are short oligonucleotide sequences that can strongly bind to their targets with high affinity and specificity by undergoing conformational changes. Selection of aptamers is an in vitro process- Systematic Evolution of Ligands by Exponential Enrichment(SELEX) and once selected, they can be synthesized in a controlled fashion with high purity and reproducibility. Theoretically, aptamers have a wide range of targets including toxic substances from small molecule to macromolecular substance like protein, even cell and tissue. Additionally, aptamers are chemically more stable than antibodies, retaining most of their functionality even after multiple regeneration steps. As oligonucleotides can be easily modified with different reactive chemical groups, their immobilization on surfaces can be easily controlled, unlike antibodies. Because of the numerous advantages over antibodies, aptamers have been applied in development of multiple analytical methods based colorimetry, fluorescence, electrochemistry and so on.In this study, a quick and sensitivity aptamer-based gold nanoparticles(Au NPs) colorimetric assay for simultaneous detection of 6 organophosphorous pesticides was firstly developed; a sandwich-format homogeneous detection method for 19-Nortestosterone that utilized the split aptamers for proximitydependent fluorescence resonance energy transfer(FRET) was developed after optimizing the conditions of aptamer; a visual and rapid method for the detection of Cyromazine had been also developed based the study of effect of Au NPs size on the sensitivity of colorimetric detection. The specific works are as follows:1. An Au NP aggregation label-free method was designed using 55-mer single-stranded DNA aptamers binding to organophosphorous pesticides for the detection of organophosphorous pesticides. This is the first time that an aptamer-based Au NP colorimetric assay has been used to quickly determinate 6 kinds of organophosphorous pesticides, and this assay was successfully used in detecting organophosphorous pesticides in river water. Although the sensitivity of this assay is not high enough, it provides a new idea for developing aptamers in the rapid detection of pesticide residues.2. On the basic of our group that the effects of the length of aptamer on the colorimetric methods performance. We found that the split 76-mer-17β-estradiol aptamers, P1 and P2, showed high affinity for 19-Nortestosterone, due to the similarity of their structures. And there was no reports involved aptamer selection for 19-Nortestosterone till now. Hence, a 76-mer 17β-estradiol aptamer was splited into two pieces, P1 and P2, which were respectively labeled with a quencher(BHQ) and afluorophore(6FAM) and then a simple sandwich-format fluorescence resonance energy transfer(FRET)-based homogeneous detection method was developed, which showed high sensitivity for 19-Nortestosterone. The assay showed a wide dynamic range, spanning from 1 μM to 1000 μM, and a detection limit of 5 μM. The practicality of the proposed approach was successfully tested with spiked urine samples. Recovery from 57.94% to 118.76% was reached under 3 spiked concentrations and average relative standard deviation(RSD) was less than 7%, which demonstrated that the method had great reproducibility and reproducibility.3. A visual determination based on Au NPs for Cyromazine was established. A series of Au NPs with different size including 13 nm, 16 nm, 30 nm and 35 nm were compared referring to sensitivity and the finally optimized Au NPs size for visual detection was 30 nm. Due to strong reaction between Cyromazine and Au NPs, the gold nanoparticles could be aggregated in the presence of Cyromazine without any additional specific acceptors. A simple colorimetric visual detection assay for Cyromazine had been developed by using citrate-stabilized gold nanoparticles and applied in river water. The red-to-blue color change of Au NPs in the presence of Cyromazine was easily observed by the naked eyes or measured by a UV-vis spectrometer.