FILTER PAPER GRAFTED WITH PNA-CONTAINING COPOLYMER BRUSHES FOR COLORIMETRIC DNA SEQUENCE DETERMINATION

Abstract

This work aims to develop a filter paper-based platform for colorimetric DNA detection employing peptide nucleic acid (PNA) probe. Filter paper functionalized with poly(glycidyl methacrylate-co-poly(ethylene glycol) methacrylate) (P(GMA-co-PEGMA)) was first prepared via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. PNA probes (Ac-GGAACCTGCGCG-LysNH2) were then immobilized on P(GMA-co-PEGMA) through ring-opening of epoxide groups in the GMA repeat units by amino groups in the PNA’s structure. The success of P(GMA-co-PEGMA) grafting on filter paper and subsequent PNA immobilization was confirmed by Fourier transform-infrared spectroscopy. As monitored by fluorescence microscopy, the PNA conjugation can be promoted under basic condition. Signal amplification relies on sandwich-hybridization assay employing biotinylated PNA probe (b-PNA) having a sequence of b-o-o-AACACACAGACT-SerOH as reporter probe together with horseradish peroxidase-labeled streptavidin (SA-HRP). The sensing platform showed the best performance in preventing non-specific adsorption from the non-complementary DNA and discriminating between complementary and single base mismatch targets at a detection limit of at least 100 fmol. It was later found that increasing ionic strength of DNA hybridization step by NaCl addition not only can increase the signal intensity which can be visualized by naked eye, but also suppressed non-specific adsorption.