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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.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/µL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

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