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

[Image: see text] Human enteric viruses can be highly infectious and thus capable of causing disease upon ingestion of low doses ranging from 10(0) to 10(2) 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.