2 research outputs found
Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader
Nucleic acids, DNA
and RNA, provide important fingerprint information
for various pathogens and have significant diagnostic value; however,
improved approaches are urgently needed to enable rapid detection
of nucleic acids in simple point-of-care formats with high sensitivity
and specificity. Here, we present a system that utilizes a series
of toehold-triggered hybridization/displacement reactions that are
designed to convert a given amount of RNA molecules (i.e., the analyte)
into an amplified amount of signaling molecules without any washing
steps or thermocycling. Fluorescent probes for signal generation were
designed to consume products of the catalytic reaction in order to
push the equilibrium and enhance the assay fold amplification for
improved sensitivity and reaction speed. The system of toehold-assisted
reactions is also modeled to better understand its performance and
capabilities, and we empirically demonstrate the success of this approach
with two analytes of diagnostic importance, i.e., influenza viral
RNA and a micro RNA (miR-31). We also show that the amplified signal
permits using a compact and cost-effective smartphone-based fluorescence
reader, an important requirement toward a nucleic-acid-based point-of-care
diagnostic system
Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout
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