Session: Current Topics in Biological Chemistry:

Recent quests for biomarker analyses have found a myriad of disease-related biomarkers spanning a wide variety of biomolecules such as nucleic acids (DNA/miRNA) and proteins. Due to the possible overlap of certain biomarkers in more than one disease, simultaneous detection of multiple biomarkers is attractive not only to save time, cost, and resources but also to improve the accuracy of diagnosis. Currently available ensemble-based multiplexed methods such as DNA microarrays and polymerase chain reactions (PCR) often suffer from either complicated design/engineering or a high rate of false positives/negatives. Single molecule techniques can be an asset, in this regard, by providing unique insights into the behavior and interactions of individual molecules. For example, single molecule fluorescence resonance energy transfer (smFRET) is able to quantitatively measure signals from individual molecules and is a sensitive means for detection of biomarkers. However, current methods often require multiple excitation sources and complex data analysis algorithms. Using the toehold-mediated strand displacement approach, we recently developed a FRET-based multiplexed detection method for DNA sequences utilizing only a single donor/acceptor fluorophore pair. This approach allowed for simultaneous detection of 3 different DNA sequences with a detection limit of ~200 pM while still using a single FRET pair. To expand the application of the method to detect microRNAs that are present at low picomolar to femtomolar concentrations in biological samples, we have re-imagined our sensor design that offers a direct binding of target to the sensor and hence does not need go through the competitive toehold displacement process.

Dr. Soma Dhakal

Assistant Professor, Virginia Commonwealth University

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