An enzymatic paper-based biosensor for ultrasensitive detection of DNA.

Point-of-care Nucleic acid testing (POCNAT) has become an attractive technique for DNA identification in resource-limited settings, offering a rapid system for urgent clinical applications. In this study, a chemiluminescence-based lateral flow biosensor (CL-LFB) was developed for the quantitative analysis of DNA, without labeling and amplification. The developed biosensor employs a two-step hybridization, a primary hybridization of 5'-biotinylated detector probe to the target DNA and a secondary hybridization of the resulting complex with the immobilized capture probe. Quantitative analysis of DNA was provided via HRP-catalyzed reaction with the chemiluminescense substrate followed by imaging with a complementary metal-oxide-semiconductor (CMOS) digital camera. The assay performance was investigated using a synthetic target, 16S rRNA gene (775 bp) and the whole genome derived from Escherichia coli (E.coli). A detection limit of 1.5 pM for the synthetic target and 0.4 ng/ml for 16S rRNA gene was obtained. In spite of LFBs limitations for the detection of large DNA fragments, the proposed assay provided a low-cost, fast, and sensitive tool for PCR-free diagnosis of small and larger fragments of nucleic acids.