Reading Out Single-Molecule Digital RNA and DNA IsothermalAmplification in Nanoliter Volumes with Unmodified Camera Phones

摘要

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 lowas 5 nL. We validate this methodology using SlipChip-based digitalsingle-molecule isothermal amplification with λDNA as a modeland hepatitis C viral RNA as a clinically relevant target. The innovativecombination of isothermal amplification chemistry in the presenceof a judiciously chosen indicator dye and ratiometric image processingwith SlipChip technology allowed the sequence-specific visual readoutof single nucleic acid molecules in nanoliter volumes with an unmodifiedcell phone camera. When paired with devices that integrate samplepreparation and nucleic acid amplification, this hardware-agnosticapproach will increase the affordability and the distribution of quantitativediagnostic and environmental tests.