Development of biosensor using pyrrolidinyl PNA for screening of hepatitis C virus DNA

Abstract

In this work, a new paper-based analytical device (PAD) that relied on fluorescence measurement was developed for label-free detection of hepatitis C virus (HCV) DNA. Pyrrolidinyl peptide nucleic acid (acpcPNA) was covalently modified onto the patterned cellulose paper to act as a specific probe for capturing the HCV DNA target according to Watson−Crick base-paring rules. The single-stranded (ssDNA)-specific dye which can be electrostatically attached to the surface-bound DNA was employed as the signaling element for the fluorescence-based detection via a smartphone gadget and an iOS application. To acquire the optimal sensitivity, several experimental parameters, namely acpcPNA probe concentration, hybridization time, incubation time, and the amount of fluorescent dye were examined. Under the optimized conditions, a linear relationship (R2 = 0.9956) between the change of green fluorescent color and the amount of HCV DNA was observed in the range of 5 to 100 pmol. The detection limit of the target sequence (55 nucleotides) was found to be 5 pmol. Moreover, this proposed sensor exhibited excellent discrimination between complementary and mismatch oligonucleotide targets. To assess the efficiency of the proposed sensor for real DNA sample analysis, it was utilized for the detection of HCV DNA in PCR-amplified samples with satisfactory results. Hence, this developed device could be applied as an alternative method which indicates the possibility to develop a point-of-care (POC) platform for screening of HCV DNA for biomedical purposes.​