Surface-Initiated Activators Generated by Electron Transfer for Atom Transfer Radical Polymerization in Detection of DNA Point Mutation

摘要

Amplification-by-Polymerization reportedly offers a sensitive and detector-free approach for DNA detection. However, the requirement for an oxygen-free environment when classic radical polymerization reactions are used in signal amplification significantly limits the mobility of this approach for point-of-need applications. We report here the employment of a purge-free controlled/"living" polymerization reaction, activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP), to achieve signal amplification upon DNA hybridization. Its aptitude in simplifying assay procedure and shortening assay turn-around has been demonstrated in this report, which substantiates the feasibility of using Amplification-by-Polymerization for high throughput or portable screening of genetic mutations. In addition, employment of water-soluble ascorbic acid as the reducing agent has overcome the hurdles encountered by heterogeneous AGET ATRP reactions. Optimization of AGET ATRP in the presence of oligonucleotides has been conducted where tris[(2-pyridyl)methyl]amine (TPMA) was selected as the catalyst ligand for its mild reaction rate. Effective polymer growth has been achieved when the concentration of the Cu(II) catalyst was controlled at 20 mM and ascorbic acid at 18 mM. The propagation and termination reaction constants have been derived, purporting the speculated controlled growth kinetics during polymer grafting. A linear relationship between the grafted polymer film thickness and the amount of captured DNA target sequences has been established, which provides the quantification basis during DNA detection. Detection of DNA sequences with single-point mutations has been successful regardless of the mutation site.