Cleavable Peptide Synthesis on Polydimethylsiloxane (PDMS)-based Microarrays and Its Application for PKA Kinase Assay

• Main Authors: Yu-ChiehHsiao, 蕭宇傑
• Other Authors: Shu-Hui Chen
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/45885866078939904334

碩士 === 國立成功大學 === 化學系 === 102 === Solid state synthesis of cleavable peptides on the surface of gold nanoparticle-coated poly(dimethylsiloxane) (PDMS) substrate was demonstrated as an array format using a 5-mer molecule of poly-Trp5 as an example and heterobifunctional crosslinkers as the anchoring layer, t-Boc chemistry/ trifluoroacetic acid (TFA) for protection and de-protection, and reducing agents for peptide cleavage. The method was judiciously designed to use reagents and solvents that are compatible with the PDMS polymer substrate and the formation of the covalent binding during the synthesis steps were monitored by Infrared spectroscopy (IR), Atomic Force Microscopy (AFM) and Fluorescence spectroscopy. The products of each step were characterized by mass spectrometry and the final production yield of the 5-mers was estimated to be near 43%. Due to flexible molding techniques for PDMS fabrication, such peptide synthesis can be implemented inside a traditional 96-well ELISA plate or on a patterned PDMS substrate for assay developments using either the light or MS detection. We applied the method to fabricate kemptide peptide microarrays on PDMS and demonstrated that protein kinase A (PKA) could phosphorylate the serine residue by chemiluminescence detection.