A Method For The Detection Of Thrombin And Mercury Ions Based On Signal Amplification Of Biofunctionalized Nanomaterials

For the past few years, the nanotechnology is growing very fast, in environmental protection and clinical diagnostic and other areas, more sensitive, accurate, simple and economic detection methods for biological macromolecules and heavy metal ions have been the focus of Life Analytical Chemistry and Environmental Analytical Chemistry.Signal amplifying detection technology which based on the biocompatibility and surface affect of nanoparticles have realized highly sensitive and fast detection of biological molecules and heavy metal ions.In this thesis, several signal amplifying detection technology platforms have developed. Nano-gold, magnetic particles were used as solid phase carrier, aptamers were used as recognition means, HRP and rare earths chalate were used as the signal groups.The developed methods have high sensitivity for analysis of thrombin and heavy metal ions. This present thesis consists of three parts as following:1. Time-resolved fluorescence detection of thrombin based on biofunctionalized gold nanoparticles Herein, we prepared the compound of BSA-Eu3+ according to the reaction between BSA and the chelate of Eu3+.The compound and thiol-modified aptamer were conjugated onto the surface of Au NPs to form a dual-codified probe. Then the other aptamer of thrombin is bound to the surface of magnetic nanoparticles. At the presence of thrombin,the two aptamers recognize the thrombin to form a structure of aptamer-thrombin-aptamer.According to that a high sensitively method to detect thrombin was formed which based on Time-resolved Fluorescence. Under the optimal conditions, the limit detection for thrombin of this method is up to 0.78 p M, the linear range is from 1 p M to 100 p M. This method has been applied to the analysis of practical samples.2. A sandwich assay for detection thrombin based on biofunctionalized gold nanoparticles?Au NPs? and HRP Herein, HRP and thiol-modified aptamer were conjugated with Au NPs to form a dual-codified probe, which is both with signal amplification and molecular recognition function. Then the other aptamer of thrombin is covalently bound to the surface of magnetic nanoparticles. At the presence of thrombin, the two aptamers recognized the thrombin to form a structure of aptamer-thrombin-aptamer. Then a high sensitively method to detect thrombin was formed which based on enzyme catalysis to TMB. In this thesis,magnetic particles which are good for separation and concentration are used as the carrier of aptamer and avoid the complex processes of washing and centrifugation. A series of exploration for the experimental conditions were carried out, the linear range is from 0.1n M to 10 n M and the detection limit for thrombin is reach to 0.07 n M under the optimal conditions. This method has been applied to the analysis of practical samples.3. Dual signal amplification system based on Au NPs by Time-resolved fluorescence to detect Hg²⁺Herein, the compound of streptavidin and the chelate of Eu3+?SA-Eu3+? was synthesized first. The thiol-modified special oligo sequence for detection Hg²⁺ and SA-Eu3+ were conjugated onto the surface of Au NPs. One SA can conjugate with four biotin-modified oligo sequences. When the Hg²⁺existing, the sequences were hybridized with the sequences which combine on to the magnetic nanoparticles. The magnetic particles were used as carrier and the time-resolved fluorescence was used as detection method. Under the optimal conditions, the limit detection for Hg²⁺is 0.65 p M, the linear range is from1 p M to 1000 p M. We have demonstrated Hg²⁺ quantification with this sensitive method in real samples.