A novel fluorescent paper-based DNA sensor employing a highly specific pyrrolidinyl peptide nucleic acid (acpcPNA) probe was developed for the sensitive and selective detection of hepatitis C virus (HCV). The acpcPNA was covalently immobilized onto partially oxidized cellulose paper via reductive alkylation between the amine and the aldehyde groups. The fluorescence-based detection was performed by monitoring the fluorescence signal response of a fluorescent dye that selectively binds to the single-strand region of the DNA target over the PNA probe employing a custom-made portable fluorescent camera gadget in combination with a smartphone camera. Under the optimal conditions, a linear relationship between the fluorescence change in the green channel and the amount of HCV DNA from 5 to 100 pmol with a correlation coefficient of 0.9956, and the limit of detection of 5 pmol were obtained for short synthetic oligonucleotides. The acpcPNA probe exhibited very high selectivity for the complementary oligonucleotides over the single-base-mismatched, two-base-mismatched, and non-complementary DNA targets. Benefitting from the signal amplification achieved through the numerous binding sites for the dye provided by the overhanging tail of long ssDNA target sequences, this system was successfully applied to detect the HCV complementary DNA (cDNA) obtained from clinical samples with satisfactory results. The proposed fluorescent paper-based sensor demonstrated a great potential to be used as a low-cost, simple, label-free, sensitive, and selective DNA sensor for point-of-care applications.
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