Study On The Cocaine Detection Based On Aptamer Recognition And Biologic Enzyme Signal Amplification

Aptamer has caught more and more attention in the target molecule (small molecule or protein) identification and testing because of the high affinity and specificity, low molecular weight, non-immunogenicity characteristics. And the signal amplification technology is particularly important to detect the molecules that can not be tested by direct amplification in a low concentration. In vitro,the nucleic acid amplification is one of the most important means of biotechnology research. The biologic enzyme-based signal amplification technologies such as fluorescence quantitative PCR (FQ-PCR) and rolling circle amplification (RCA) are the most commonly used signal amplification technology. In this study, the novel and sensitive detection method had been designed combining aptamer recognition with the biologic enzyme-based signal amplification. The major contents of the thesis were as follows:1 An aptamer-based and FQ-PCR amplification method for the detection of cocaineIn this chapter, a novel method based on aptamer identification and fluorescence quantitative PCR signal amplification technology for detection of cocaine was presented. In order to achieve low background and high signal/noise ratio, we used magnetic bead separation technology to achieve a complete separation and enrichment of PCR template chain. The sensitivity had been greatly improved. The template chain was amplified by 2n times by the fluorescence quantitative PCR technology, further improving the detection sensitivity. As a result of these two combined effects, 9 fmol level of cocaine could be detected using this method, and showed good specificity. In the optimum conditions, Ct exhibited a linear dependence on cocaine concentration in a wide range from 10-10 to 10-6 mol/L with a detection limit of 3×10-11 mol/L. 2 An aptamer-based and RCA amplification method for the detection of cocaineIn the present study, the method for highly sensitive detection of cocaine was developed based on aptamer identification and RCA signal amplification. When cocaine was in present, aptamer was specially combined with cocaine so that the supplementary B chain was replaced from aptamer. The B chain was directly used to cyclize probe C, and realized the quantitative detection of cocaine through the RCA technology. In this study, the detection limit of 20 pmol was achieved. When the DNA chain could also be adapted to surfaces of the chips, the quantitative RCA was proved difficult, further emphasizing the importance of real-time amplification methods for sensitive quantization in homogeneous phase solution. In addition, a single Au nanoparticle could be loaded with hundreds of DNA, which had greatly improved the sensitivity. In conclusion, this method exhibited excellent sensitivity, selectivity, good specificity, and provided a new idea and pattern for the molecular detection under complex biological environment.