Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Soma Dhakal


In recent years, biomarkers have drawn tremendous interest in the fields of clinical diagnostics and biotechnology. In fact, recent quests for biomarker analyses have discovered a myriad of disease-related biomarkers spanning a wide variety of biomolecules such as proteins, nucleic acids, and small molecule ligands. While the detection of only one target at a time is the most common practice in biosensing today, it has been shown that the accuracy of diagnosis increases significantly by measuring the level of multiple biomarkers instead of just one due to the possible overlap of certain biomarkers in more than one disease. However, currently available ensemble multiplexed methods such as DNA microarrays and polymerase chain reactions (PCR) often suffer from complicated design/engineering, semi-quantitative analysis, or a high false positive rate. 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 by relying on the emission from one or more donor/acceptor FRET pairs. However, current methods require multiple excitation sources and complex data analysis algorithms. To address this knowledge gap, using prism-based total internal reflection fluorescence (pTIRF) microscopy, we developed a FRET-based multiplexed detection method for DNA sequences utilizing only a single donor/acceptor fluorophores pair. By tuning the minimum distance separation between a FRET pair through a careful design, we showed that multiple FRET values can be achieved depending on the distance set. We utilized interconvertible hairpin-based sensors (iHabSs) where detection was dependent on a toehold-mediated displacement of a probe strand by the target. 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 microRNA biomarkers 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.

Further, we implemented and refined a program called MASH-FRET (a MATLAB-based multifunctional analysis software for handling smFRET data) and evaluated its capability of identifying seemingly overlapped FRET populations. The motivation was to expand the multiplexing capability of predominately used single-molecule FRET techniques, which utilize only one FRET pair. Through MASH-FRET-enabled bootstrap-based analysis (also called BOBA-FRET) of experimental and simulated data, we first demonstrated that the statistical confidence of poorly resolved FRET populations can be readily determined. Using simulated data sets, we then demonstrated that the program can easily identify FRET populations that are separated only by ~0.1 FRET level, indicating the possibility of up to ~9-fold multiplexing. Overall, we showed that our streamlined MASH-FRET platform has great promise to increase the multiplexing capability of smFRET techniques without the need for complicated experimental set ups such as multiple FRET pairs and/or multiple excitation sources.


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Available for download on Monday, May 11, 2026