Rodriguez-Manzano, Jesus and Karymov, Mikhail A. and Begolo, Stefano and Selck, David A. and Zhukov, Dmitriy V. and Jue, Erik and Ismagilov, Rustem F. (2016) Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones. ACS Nano, 10 (3). pp. 3102-3113. ISSN 1936-0851 . PMCID PMC4819493. http://resolver.caltech.edu/CaltechAUTHORS:20160223-132834891
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Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.
|Additional Information:||© 2016 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: November 20, 2015. Accepted: February 3, 2016. Publication Date (Web): February 22, 2016. This research was supported by DARPA Cooperative Agreement HR0011-11-2-0006. The content of this article does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. Multivolume SlipChip design and analysis were based on work supported by NIH Grant R01EB012946 administered by the National Institute of Biomedical Imaging and Bioengineering. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. R.F.I. holds an Innovation in Regulatory Science Award from BWF. This material is also based upon work supported by National Science Foundation Graduate Research Fellowships DGE-1144469 (to D.V.Z. and E.J). We thank T. Schlappi for multivolume device design calculations and N. Shelby for contributions to writing and editing this manuscript. R.F.I. has a financial interest in SlipChip Corp. J.R.-M. and M.A.K. contributed equally to this work. The authors declare the following competing financial interest(s): Rustem F. Ismagilov has a financial interest in SlipChip Corp.|
|Subject Keywords:||nanoliter reactions, single-molecule counting, cell phone camera, digital, isothermal, microfluidics, ratiometric visual readout|
|PubMed Central ID:||PMC4819493|
|Official Citation:||Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones Jesus Rodriguez-Manzano, Mikhail A. Karymov, Stefano Begolo, David A. Selck, Dmitriy V. Zhukov, Erik Jue, and Rustem F. Ismagilov ACS Nano 2016 10 (3), 3102-3113 DOI: 10.1021/acsnano.5b07338|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||George Porter|
|Deposited On:||23 Feb 2016 21:48|
|Last Modified:||02 Mar 2017 21:54|
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