Smartphone-Based Paper Microfluidic Particulometry of Norovirus from Environmental Water Samples at the Single Copy Level

Human enteric viruses can be highly infectious and thus capable of causing disease upon ingestion of low doses ranging from 10⁰ to 10² virions. Norovirus is a good example with a minimum infectious dose as low as a few tens of virions, that is, below femtogram scale. Norovirus detection from commonly implicated environmental matrices (water and food) involves complicated concentration of viruses and/or amplification of the norovirus genome, thus rendering detection approaches not feasible for field applications. In this work, norovirus detection was performed on a microfluidic paper analytic device without using any sample concentration or nucleic acid amplification steps by directly imaging and counting on-paper aggregation of antibody-conjugated, fluorescent submicron particles. An in-house developed smartphone-based fluorescence microscope and an image-processing algorithm isolated the particles aggregated by antibody-antigen binding, leading to an extremely low limit of norovirus detection, as low as 1 genome copy/μL in deionized water and 10 genome copies/μL in reclaimed wastewater.