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.

Formation of uniform reaction volumes using concentric amphiphilic microparticles

“…These benefits are particular attractive for clinical trials, drug screening, and diagnostics, where sample volumes can be small or precious. Digital nucleic acid detection using compartmentalized amplification of single nucleic acids with PCR or Loopmediated isothermal amplification (LAMP) [54] should also be compatible with dropicle systems given the similar volumes to current systems and biocompatibility of the PEG layer and surrounding oil. In addition, by modifying the PEGDA layer with cell adhesive-moieties such as biotinylated collagen, [38] our amphiphilic particles could serve as cell carriers, enabling single cell culture and analysis, including the accumulation of secretions [55] or RNA in the small volume of a dropicle for secretion or gene expression analysis.…”

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

“…Isothermal methods, such as loop-mediated isothermal amplification (LAMP), are attractive for nucleic acid amplification tests (NAATs) in point-of-care and limited-resource settings (1,2). LAMP in particular shows promise as a NAAT with fewer hardware requirements compared with polymerase chain reaction (PCR) (3).…”

Portable Multiplex Optical Assays

“…Reprinted from refs. (a), (b), (c), (d), (e), (f), (g), (h), and (i) with permission: Copyright 2012, Elsevier Ltd. (a); Copyright 2012 and 2011, The Royal Society of Chemistry (b and g); CC‐BY 4.0 open access publication (c and f); CC‐BY‐NC‐ND 4.0 open access publication (d); ACS AuthorChoice License open access publication (e); Copyright 2006, Springer Science Business Media (h); Copyright 2014, American Chemical Society (i).…”

“…A higher sensitivity with field‐portable design was demonstrated using isothermal (mostly room temperature) amplification strategies. A loop‐mediated and toehold‐assisted nucleic acid amplification methods have boosted the fluorescent readout . While the operation principle of this chemical assay was relatively complex, target samples were conjugated to a nucleic acid capture probe, which was then amplified by a series of reactions without the need for thermal cycling to overcome a drawback of portable designs.…”