Author ORCID Identifier
https://orcid.org/0000-0003-2849-1959
Defense Date
2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Soma Dhakal
Abstract
Foodborne diseases have become a major problem for public health. Staphylococcus aureus is among the top five most common foodborne pathogens causing thousands of deaths each year. The most critical challenge more than ever is that S. aureus is rapidly developing antibiotic resistance, making it harder to control its fatal infections. Therefore, efficient and reliable detection of S. aureus is important not only to minimize the clinical burden and hospitalizations, but also to save lives. Several detection strategies for S. aureus have been developed from conventional culture-based methods to novel portable biosensors. However, even the most efficient detection methods are susceptible to false results, cross reactivity, and limited specificity. To address this gap, we have developed DNA-based sensors to detect pathogenic biomarkers, particularly the iron regulated surface determinant protein A (IsdA) and 16S ribosomal gene using single-molecule fluorescence microscopy and fluorescence measurements.
Among several markers, surface proteins are considered to be the most important markers due to their specific roles in the survival and colonization of the bacterium on hosts. We have developed a detection platform for a key surface protein, IsdA, using a combination of computationally developed aptamer and single-molecule fluorescence resonance energy transfer (smFRET). IsdA is a surface protein in S. aureus that facilitates heme uptake by binding to human hemoproteins and removing heme molecules. We demonstrated that detection can be achieved even in the presence of interfering proteins with sensitivity as low as 11 pM. We further optimized the sensor design and achieved an ∼18-fold higher sensitivity while enabling single-step detection. Molecular dynamics (MD) simulations show distinct conformational flexibility of the unbound aptamer and a reduced flexibility for the aptamer-IsdA complex, corresponding to the experimentally observed higher FRET efficiencies. The FRET-based single-molecule aptasensor that we developed has great potential for rapid monitoring of S. aureus. Further, the developed approach has the potential to be broadly applicable across diverse fields of biotechnology including environmental monitoring, forensic analysis, and clinical diagnostics.
The detection was also tested using the DNA logic gate platform after optimizing a series of logic gates based on simple to complex logical operations. The use of DNA structures in creating multimodal logic gates bears high potential for building molecular devices and computation systems. However, due to the complex designs or complicated working principles, the implementation of DNA logic gates within molecular devices and circuits is still quite limited. We successfully designed simple four-way DNA logic gates that can serve as multimodal platforms for simple to complex operations. Using the proximity quenching of the fluorophore–quencher pair in combination with the toehold-mediated strand displacement (TMSD) strategy, we have successfully demonstrated that the fluorescence output, which is a result of gate opening, solely relies on the oligonucleotide(s) input. We further demonstrated that this strategy can be used to create multimodal (tunable displacement initiation sites on the four-way platform) logic gates including YES, AND, OR, and the combinations thereof. The four-way DNA logic gates developed here bear high promise for building biological computers and next-generation smart molecular circuits with biosensing capabilities. Furthermore, the previously designed four-way DNA logic gate system was leveraged as a biosensing platform by incorporating an IsdA-specific aptamer as the toehold strand within the four-way DNA junction. A YES gate configuration was employed for the detection of the S. aureus IsdA protein. Upon binding of IsdA to the aptamer, the aptamer strand was released from the DNA junction, triggering a fluorescence increase over time. This target-induced fluorescence response confirmed the successful operation of the logic gate and demonstrated the potential of four-way DNA logic gates as programmable biosensing platforms for protein detection.
In parallel, we also developed a gene-based sensor using the 16S ribosomal RNA (rRNA) gene containing hypervariable regions that allows species-specific detection. We introduced a novel detection of S. aureus via a specific gene segment - 16SSAIII, a 16S ribosomal RNA domain containing a hypervariable region and species-specific sequence, making it the most suitable region for identification of pathogens. The detection is achieved via a change in the FRET signal upon binding of the target to a custom-designed DNA platform containing multiple binding sites. The developed detection platform integrates three identical donor-acceptor FRET pairs to enable enhanced binding and improved sensitivity. This strategy enabled detection down to 1 pM without target amplification and labeling, and 1 fM with increased incubation time and ionic strength. The FRET-based single-molecule detection of S. aureus that we reported here can be adapted for reliable and sensitive detection of a wide range of pathogens ensuring food safety, and may find applications in clinical diagnostics, detection of viruses, environmental monitoring, and beyond.
Overall, we have developed non-enzymatic pathogen detection platforms for S. aureus using either protein-aptamer interaction or a simple complementary DNA hybridization approach. These sensing platforms are adaptable for detecting a wide range of pathogens and thus have promising applications in the food industry and beyond.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-3-2026