Developing a FRET-based Cd2+ biosensor for toxicological investigations on Cd2+ within living cells

• Main Authors: Po-Hsun Chen, 陳柏勳
• Other Authors: De-Ming Yang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/59632893956945063041

碩士 === 國立陽明大學 === 生醫光電研究所 === 100 === Cadmium is one of the common heavy metal pollutants in nature. Cadmium usually enters the body through ingestion and inhalation. Long-term accumulation of low dose cadmium causes damages of human organs in body, especially kidney, and even increases the risk of cancers. Methods like chemical dyes or the utilization of isotope always are restricted by relatively poor ion selectivity and the dangerous radiation. To study the cadmium entry in cells, we can develop a protein-based cadmium biosensor that can monitor the cadmium in cytoplasm. In this study, we applied the strategy of detecting fluorescent resonance energy transfer (FRET) and produced the live-cell cadmium biosensor, YCcadRD57. Cd2+-binding protein cadR (from Pseudomonas putida) which belongs to MerR family (associated with metal resistance in bacteria) was inserted between the fluorescent protein pair ECFP(△C11) and cp173Venus as the key sensing center. The in vitro experiments show the relatively high ion selectivity and broad dynamic range (2.8~4.7) of YCcadRD57, with the dissociation constent Kd= 250 nM. The in vivo results also indicated that YCcadRD57 could sense and monitor the cadmium in the living cells. On the cytosolic Cd2+ entry issue, we found that nifedipine, verapamil and nickel could inhibit the Cd2+ entry with the help of YCcadRD57. In summary, we successfully developed a Cd2+ indicator which can monitor dynamics of Cd2+ inside living cells.