Development of liposomal aptamers for the use in high throughput biosensing

• Main Authors: Tzu‐Cheng Sung, 宋子承
• Other Authors: Chien‐Sheng Chen
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/2ujbdw

博士 === 國立中央大學 === 系統生物與生物資訊研究所 === 104 === In this research, we developed liposomal aptamers for the use in high throughput biosensing. In the first part, the crucial vitamin biotin was used for model analytes for developing a liposomal aptamer as a competitor for replacement assay, which is an effective assay format for high throughput biosensing. We demonstrated the assay format in the 96-well plate, which provides high sample throughput for biosensing. In the second part, we developed a liposomal aptamer for binding to rabbit antibodies, which is common biorecognition elements in biosensing. Therefore, this anti-rabbit antibody liposomal aptamer can be used for multiplexed biosensing for simultaneous detections of many different analytes. We demonstrate that both liposomal aptamers have the potential to be used to minimize the assay time and simplified the assay protocol to be used in high throughput biosensing In the first part, we developed a liposomal aptamer as the competitor of biotin for the detection of biotin in 96-wells plate format replacement assay. The limit of detection by using ST-21 and ST-21M liposomal aptamer were 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This demonstrates the successful use of aptamer as a competitor of analyte in a replacement assay. It also shows the replacements assay without any washing procedure is fast enough and with a potential to use in the high throughput biosensing. In the second part, we developed a liposomal aptamer as a secondary reagent for immunoassays. The R18 liposomal aptamer can bind with rabbit antibody, which is commonly used biorecognition elements in biosensing. In this research, the R18 liposomal aptamer can be used both on the 96-well plate and chips. LOD for R18 liposomal aptamer was 80 ng/ml, and the dynamic range was three orders of magnitude (80 ng/ml to 50 µg/ml) on the 96-well plate. The LOD for R18 liposomal aptamer on the chip was 18.2 ng/ml. The dynamic range was three orders of magnitude (18.2 ng/ml to 10 µg/ml) on the chip. We further used the R18 liposomal aptamer on high density proteome microarrays. We observed that the R18 liposomal aptamer showed high resolution and clear signals with dose response on the chip and also can R18 showed specific binding to the rabbit antibodies. There results demonstrate the R18 liposomal aptamer can be as a seconadry reagent for the use in high throughput immunoassays. In summary, liposomal aptamers can be ideal competitors for the replacement biosensing and effective secondary reagents for immunoassays. They are both applicable for high-throughput biosensing.