Optimization of DNA detection using purple membrane-based photoelectric chips and application in Micro-DNA analysis

• Main Authors: Xiang-He Wang, 王翔禾
• Other Authors: Hsiu-Mei Chen
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/6xy3nr

碩士 === 國立臺灣科技大學 === 化學工程系 === 107 === With the advance of science and technology as well as the completion of the human genome project, many diseases are understood at the DNA sequence level. Therefore, the demand for genetic testing is increasing. This thesis continued the previous gene detection chip project of this laboratory to optimize the preparation and applications of the chip. The chip used bacteriorhodopsin (BR) residing in Halobacterium salinarum purple membrane (PM) as the signal transducer, a light-driven proton pump transporting protons from the intracellular to the extracellular side of PM and subsequently generating a proton gradient to power electronic circuits. There are three parts in this thesis. First, the optimization of the chip preparation and detection was investigated. Tuning the ssDNA1' probe immobilization concentration and adding an annealing step not only reduced the detection error but also achieved a lowest detection concentration of the single-stranded target gene (ssDNA2') at 0.5 fM DNA concentration. Further optimization of the conjugation ratio of the target gene on gold nanoparticles (AuNPs) at different sizes led to a detection limit of < 0.5 fM particle concentration of ssDNA2'-AuNPs, the complex of the target gene and 80 nm AuNPs. Secondly, the optimally prepared gene chip was applied to real-time monitoring DNA synthesis. The detection limits of ssDNA2' and ssDNA2'-AuNPs in the flow-injection real-time detection system were 50 μM DNA concentration and 50 fM particle concentration, respectively. Photocurrent declination was clearly observed due to the protons released during the chain elongation synthesis at the 3’-end of the target ssDNA2'. The real-time monitoring of DNA synthesis in anther larger-volume static detection cell effectively reduced the lag phase from 15 min to 7.5 min. In addition, the photocurrent reduction profiles of the flow-injection and static detection systems were almost identical. Finally, the feasibility of applying the current gene chip on micro-RNA detection was preliminarily investigated. A brain-tumor miR17-DNA detection chip was prepared, yielding a lowest detection limit of 5 fM DNA concentration. The lowest detection limit was further decreased to 0.5 fM, the average normal miR17-RNA concentration in human blood, when another competitive assay was conducted with the addition of a miR17-DNA-AuNPs complex, imply the future application of the prepared miR17-DNA detection chip in clinical assays.