Rapid and reliable norovirus assay at pg/mL level using smartphone-based fluorescence microscope and a microfluidic paper analytic device

Detection of norovirus from water samples typically requires extremely low limit of detection (LOD), preferably at single virus particle level, since they can be pathogenic at extremely low concentrations. Complicated equipment and/or lengthy procedures are necessary to concentrate large volume of water sample. In addition, this low LOD requirement have traditionally been associated non-reproducible and less convincing assay results. In this work, rapid and reliable detection of norovirus contamination in water samples was demonstrated using an in-house developed smartphone-based fluorescence microscope and a paper microfluidic analytic device (μPAD). Norovirus was concentrated directly on the μPAD, which was fabricated with polarity filter, to further decrease the LOD. Antibodyconjugated submicron (0.5 μm diameter) fluorescent particles were added to this μPAD, and a smartphone based fluorescence microscope imaged these beads directly from the μPAD. Since the spatial resolution of our smartphonebased fluorescence microscope is > 1 μm, only the beads immunoaggltuinated by norovirus can be identified, providing reliable, reproducible, and visually convincing assay results. Using this novel this method, extremely low LOD was demonstrated, 0.01 pg/mL with a benchtop fluorescence microscope and 10 pg/mL to 100 pg/mL with a smartphone based fluorescence microscope. This novel assay can provide a fully unmanned platform for assaying various waterborne pathogens that require extremely low LOD as well as high reliability, while providing low-cost, ease-ofuse, and user friendliness appropriate for field applications.