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Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope

Abstract

Norovirus is a widespread public health threat and has a very low infectious dose. This protocol presents the extremely sensitive mobile detection of norovirus from water samples using a custom-built smartphone-based fluorescence microscope and a paper microfluidic chip. Antibody-conjugated fluorescent particles are immunoagglutinated and spread over the paper microfluidic chip by capillary action for individual counting using a smartphone-based fluorescence microscope. Smartphone images are analyzed using intensity- and size-based thresholding for the elimination of background noise and autofluorescence as well as for the isolation of immunoagglutinated particles. The resulting pixel counts of particles are correlated with the norovirus concentration of the tested sample. This protocol provides detailed guidelines for the construction and optimization of the smartphone- and paper-based assay. In addition, a 3D-printed enclosure is presented to incorporate all components in a dark environment. On-chip concentration and the assay of higher concentrations are presented to further broaden the assay range. This method is the first to be presented as a highly sensitive mobile platform for norovirus detection using low-cost materials. With all materials and reagents prepared, a single standard assay takes under 20 min. Although the method described is used for detection of norovirus, the same protocol could be adapted for detection of other pathogens by using different antibodies.

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Fig. 1: Hardware and assay procedure.
Fig. 2: Outline of the protocol.
Fig. 3: Rendered SolidWorks images.
Fig. 4: On-chip concentration procedure.
Fig. 5: Adaptive thresholding.
Fig. 6: Processing smartphone images.
Fig. 7: Expected results with different types of environmental water samples.
Fig. 8: Expected results without and with on-chip concentration.
Fig. 9: Expected results with adaptive thresholding.

Data availability

The data that support the findings of this study either were previously published in ref. 3 (Figs. 6 and 7, with source data provided in Supplementary Data 3) or are available upon reasonable request from the corresponding author (Figs. 8 and 9, with source data is provided in Supplementary Data 4 and 5). The video clip demonstrating the enclosure assembly is available in Supplementary Video 1. The video clip demonstrating the assay procedure is available in Supplementary Video 2.

Code availability

The SolidWorks designs of paper microfluidic chip and microscope enclosure are available in Supplementary Data 1. MATLAB Mobile, MATLAB, and ImageJ macro codes are available in Supplementary Data 2, along with raw sample images. These codes are also deposited and publicly available at https://github.com/jeongyeolyoon/yoon-lab. The code in this protocol has been peer-reviewed.

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Acknowledgements

The authors thank S. Perea for his contribution to an earlier version of this work. This work was funded by the University of Arizona National Science Foundation Water and Environmental Technology (WET) Center (award number IIP-1361815) and Tucson Water.

Author information

Affiliations

Authors

Contributions

J.-Y.Y. conceived the overall concept with input from S.C., L.E.B., W.Q.B., and K.A.R. S.C., L.E.B. and C.M.J. designed, fabricated, and tested the paper microfluidic chips, smartphone-based fluorescence microscope and its 3D-printed enclosure, with input from J.-Y.Y. Protocols for optimizing antibody conjugation to particles, paper types, and on-chip concentration were conceived and developed by S.C., L.E.B. and J.-Y.Y., with input from W.Q.B. and K.A.R. Image-processing algorithms and codes were conceived and developed by S.C., L.E.B., and A.G. with input from J.-Y.Y. Water and norovirus sample preparations and procedures were developed and tested by C.M.M. and W.Q.B. L.E.B. and J.-Y.Y. wrote the manuscript with input from S.C., W.Q.B. and K.A.R. All authors have given approval of the final version of the manuscript.

Corresponding author

Correspondence to Jeong-Yeol Yoon.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Suresh Neethirajan, Daniel Olson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Chung, S. et al. ACS Omega 6, 11180−11188 (2019): https://doi.org/10.1021/acsomega.9b00772

Ulep, T.-H. et al. Biosens. Bioelectron. 153, 112042 (2020): https://doi.org/10.1016/j.bios.2020.112042

Key data used in this protocol

Chung, S. et al. ACS Omega 6, 11180−11188 (2019): https://doi.org/10.1021/acsomega.9b00772

Supplementary information

Supplementary Information

Supplementary Figs. 1–5.

Reporting Summary

Supplementary Data 1

The SolidWorks designs of paper microfluidic chip and microscope enclosure

Supplementary Data 2

MATLAB Mobile code, MATLAB code, and ImageJ macro code, along with raw image examples

Supplementary Video 1

Video clip demonstrating the enclosure assembly

Supplementary Video 2

Video clip demonstrating the assay procedure

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Chung, S., Breshears, L.E., Gonzales, A. et al. Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope. Nat Protoc 16, 1452–1475 (2021). https://doi.org/10.1038/s41596-020-00460-7

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