Applications of Capillary Electrophoresis with Laser Induced Fluorescence Detection System in Aptamer Based Assays

• Main Authors: Wen-Shang Chou, 周文商
• Other Authors: 莊曜宇
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/51079841314599934444

碩士 === 臺灣大學 === 電機工程學研究所 === 96 === This study establishes a new protein detection method, which is for fast, high sensitive, and accurate. The method uses a new class of biomolecules for molecular recognition to replace antibody which is expensive and bulky. Combining aptamer with nanoparticle constitutes a special probe. In this study, gold nanoparticles are conjugated with aptamers and fluorescently labeled oligonucleotides to detect the presence of target protein and to amplify the signals. This method can be extended to simultaneously detect multiple protein targets by incorporating barcode oligonucleotides of different lengths on the gold nanoparticles. A capillary electrophoresis (CE) instrument with laser-induced fluorescence (LIF) was developed. LIF is a very sensitive method enabling femto-molr detection limit. I also establish a scanning system for detection of multiple capillaries. Comparing to other separation methods, CE is efficient and has high resolution to separate 50 mer DNA. This new model system works like enzyme-link immunosorbent assay (ELISA) in liquid phase. By using two different aptamers recognizing different eitpopes of a target protein, this method identifies the protein target with high specificity. Parametric studies revealed that the established capillary electrophoresis system had high sensitivity and resolution. The studies concluded that smaller diameter of capillary yielded high zeta potential and the number of theoretical plates can be increased by longer alkaline treatment. The signal-to-noise ratio of the detection system can be optimized by using objective lens of higher numerical aperture and spatial filter as well as bandpass filter. With optimization, the system could reach detection limit in femto-molar concentration. To detect the target protein, aptamer probes recognizing the first epitope of the target protein were immobilized on gold nanoparticles, Apt-GNP. The Apt-GNP nanoparticles were also treated to have fluorescently labeled beacon oligonucleotides in the GNP. The aptamer molecules recognizing the second epitope of the target protein were immobilized on magnetic nanoparticles, Apt-MNP. In the presence of target protein, the Apt-GNP an Apt-MNP form a sandwich complex with the target protein. After they were captured by a magnet, the beacon oligonucleotides on the GNP were cleaved by dithiothreitol (DTT) and detected by the CE system with LIF detector to reveal the presence of target proteins in the sample.