2 research outputs found
Adaptive PCR Based on Hybridization Sensing of Mirror-Image l‑DNA
Polymerase chain
reaction (PCR) is dependent on two key hybridization
events during each cycle of amplification, primer annealing and product
melting. To ensure that these hybridization events occur, current
PCR approaches rely on temperature set points and reaction contents
that are optimized and maintained using rigid thermal cycling programs
and stringent sample preparation procedures. This report describes
a fundamentally simpler and more robust PCR design that dynamically
controls thermal cycling by more directly monitoring the two key hybridization
events during the reaction. This is achieved by optically sensing
the annealing and melting of mirror-image l-DNA analogs of
the reaction’s primers and targets. Because the properties
of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions
required for effective primer annealing and product melting during
each cycle without interfering with the reaction. This hybridization-sensing
approach adapts in real time to variations in reaction contents and
conditions that impact primer annealing and product melting and eliminates
the requirement for thermal calibrations and cycling programs. Adaptive
PCR is demonstrated to amplify DNA targets with high efficiency and
specificity under both controlled conditions and conditions that are
known to cause traditional PCR to fail. The advantages of this approach
promise to make PCR-based nucleic acid analysis simpler, more robust,
and more accessible outside of well-controlled laboratory settings
Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests
Multi-antigen rapid diagnostic tests
(RDTs) are highly informative,
simple, mobile, and inexpensive, making them valuable point-of-care
(POC) diagnostic tools. However, these RDTs suffer from several technical
limitationsî—¸the most significant being the failure to detect
low levels of infection. To overcome this, we have developed a magnetic
bead-based multiplex biomarker enrichment strategy that combines metal
affinity and immunospecific capture to purify and enrich multiple
target biomarkers. Modifying antibodies to contain histidine-rich
peptides enables reversible loading onto immobilized metal affinity
magnetic beads, generating a novel class of antibodies coined “Capture
and Release” (CaR) antibody reagents. This approach extends
the specificity of immunocapture to metal affinity magnetic beads
while also maintaining a common trigger for releasing multiple biomarkers.
Multiplex biomarker enrichment is accomplished by adding magnetic
beads equipped with CaR antibody reagents to a large sample volume
to capture biomarkers of interest. Once captured, these biomarkers
are magnetically purified, concentrated, and released into a RDT-compatible
volume. This system was tailored to enhance a popular dual-antigen
lateral flow malaria RDT that targets <i>Plasmodium falciparum</i> histidine-rich protein-II (HRPII) and <i>Plasmodium</i> lactate dehydrogenase (<i>p</i>LDH). A suite of <i>p</i>LDH CaR antibody reagents were synthesized, characterized,
and the optimal CaR antibody reagent was loaded onto magnetic beads
to make a multiplex magnetic capture bead that simultaneously enriches <i>p</i>LDH and HRPII from <i>Plasmodium falciparum</i> parasitized blood samples. This system achieves a 17.5-fold improvement
in the dual positive HRPII/pan-<i>p</i>LDH detection limits
enabling visual detection of both antigens at levels correlating to
5 p/ÎĽL. This front-end sample processing system serves as an
efficient strategy to improve the sensitivity of RDTs without the
need for modifications or remanufacturing