title: Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.
abstract: Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Janay E
authors.identifier: 0000-0001-5911-8116
authors.initials: JE
authors.lastname: Kong
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Derek
authors.initials: D
authors.lastname: Tseng
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Jingzi
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Pan
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewinski
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Omai B
authors.initials: OB
authors.lastname: Garner
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Aydogan
authors.identifier: 0000-0002-0717-683X
authors.initials: A
authors.lastname: Ozcan
authors.affiliation: Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and ¶Jonsson Comprehensive Cancer Center, University of California Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Dino
authors.initials: D
authors.lastname: Di Carlo
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry; D040421:Cell Phone / Q000191:economics; D004247:DNA / Q000032:analysis*; D005453:Fluorescence; D007364:Intercalating Agents / Q000737:chemistry*; D015394:Molecular Structure; D009282:Naphthalenesulfonates / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000295:instrumentation; D019095:Point-of-Care Systems / Q000191:economics; D013050:Spectrometry, Fluorescence / Q000191:economics / Q000295:instrumentation
chemical_list: D007364:Intercalating Agents; D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate; D004247:DNA
keywords: intercalating dyes; loop-mediated isothermal amplification; mobile-phone fluorescence microscopy; nucleic acid detection; point-of-care assay