Fluorescence-based simultaneous dual oligo sensing of HCV genotypes 1 and 3 using magnetite nanoparticles

Nucleic acid tests (NATs) have gained an important position in biosensing in the context of the increasing need to meet the stringent requirements for accurate diagnosis of infectious diseases with high sensitivity and selectivity. Recently, the development of new strategies towards multiplex detection of analytes in a single assay is gaining impetus since such an approach would lead to high throughput analysis, leading to substantial benefits in terms of time, infrastructure, labor, and cost. In this work, we demonstrate a facile fluorescence-based simultaneous dual oligo sensing of genotypes 1 and 3 by employing two target sequences (36-mers each) derived from the NS4B and NS5A regions of HCV genome, respectively. A set of 18-mer amine-tagged probes and another set of 18-mer fluorescently-labeled probes that were complementary to each half of the 36-mer target sequences were designed. The amine-tagged probes were immobilized over aldehyde-derivatized magnetite nanoparticles (NPs) via imine bond formation, which was characterized using X-ray photoelectron spectroscopy (XPS) and energy dispersive spectroscopy (EDS) mapping techniques. The successful hybridization between the two probes with their target followed by magnetic removal of the NPs from the solution enabled quantitative analysis of the target by measuring the fluorescence intensity of the residual concentration of the fluorescently-tagged probe. In this manner, the targets corresponding to genotypes 1 and 3 were simultaneously detected with the detection limit in the range of 10-15 nM. The current strategy can potentially be amalgamated with existing nanotechnology-based techniques towards multiplex oligo sensing of several pathogens.