Determination of DNA sequences following dot blot hybridization format employing filter paper immobilized with quaternized polymer brushes and peptide nucleic acid

Abstract

In this research, a new paper-based platform for colorimetric detection of specific DNA sequences employing a new conformationally constrained pyrrolidinyl peptide nucleic acid (acpcPNA) as a probe has been developed. The filter paper was modified to be positively charged with grafted polymer brushes of QPDMAEMA to be used for selective capturing of the negatively charged DNA, but not the neutral acpcPNA, by electrostatic interacton. Only whe the sequences of the DNA and modifier labeled acpePNA probe (m-PNA) are complementray, the probe can be immobilized through hybridization with the surface-bound DNA. The presence of DNA-PNA hybrids can be detected by colorimetric assay via either enzymatic or polymerization amplificaton. In enzymatic amplification mode, it is obvious that this assay was capable of discriminating a single base mismatch at a detection limit of at least 10 fmole. Moreover, the QPDMAEMA grafted filter paper exhibited a superior performance to the commercial membranes, nylon 66 and nitrocellulose. In polymerization amplification mode, it was demonstrated that the desired red copolymer of HEMA-Rh B and PEGMA grew from the initiator adsorbed on paper with minimal background from non-specific adsorption of unbound copolymer. However, the signal amplification of PNA-DNA hybridization in picomol level was not observed by naked eyes because concurrent ARGET ATRP/RAFT for this assay required initiator with a mount greater than 1 nmol.