title: Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.
abstract: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Jesus
authors.initials: J
authors.lastname: Rodriguez-Manzano
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Karymov
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Stefano
authors.initials: S
authors.lastname: Begolo
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Selck
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Dmitriy V
authors.initials: DV
authors.lastname: Zhukov
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Erik
authors.initials: E
authors.lastname: Jue
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
authors.forename: Rustem F
authors.initials: RF
authors.lastname: Ismagilov
mesh_terms: D010582:Bacteriophage lambda / Q000302:isolation & purification; D040421:Cell Phone; D003124:Colorimetry / Q000295:instrumentation; D004396:Coloring Agents / Q000032:analysis; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004867:Equipment Design; D016174:Hepacivirus / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D012313:RNA / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D018401:Sample Size
chemical_list: D004396:Coloring Agents; D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA; D004247:DNA
keywords: cell phone camera; digital; isothermal; microfluidics; nanoliter reactions; ratiometric visual readout; single-molecule counting