Aptamer-based approaches for multiplexed sensing applications

Abstract

Biosensors offer high specificity, sensitivity, real-time recognition, and rapid response across various applications, including Medical Diagnostics, Environmental Monitoring, Food Safety, agriculture, Point-of-Care diagnostics, and Research and Development. Ensuring the robustness of biosensors is critical for their commercial viability, stability, reproducibility, specificity, and sensitivity. Among the biosensors, DNA-based approaches, particularly aptamers, stand out due to their high thermal stability, chemical modifiability, and programmable structural assemblies, offering advantages in molecular detection applications. The robustness of aptamers and advancements in DNA nanotechnology enhance the potential of DNA-based aptasensors for diverse molecular detection purposes.

In this dissertation, we begin by reviewing different aptasensing strategies that reveal a fascinating interplay of operating conditions influencing key performance metrics. Optimization led to nearly three orders of magnitude improvement in Limits of detection (LOD) and better repeatability. The positive relationship observed between optimization and performance metrics, along with the stability and lower RSD values seen in optimized assays, highlights the potential of parameter optimization in the development of robust aptasensors. DNA nucleotides' key properties—secondary structure and complex formation—are crucial and determined by free energy. Hence, in our next phase, the Nearest-neighbour (NN) model, estimating free energy, is implemented in MATLAB for DNA folding and binding free energy calculations, validated against literature values.

Further, we introduce a detection method that combines aptamers' target-specific capabilities with graphene oxide's fluorophore quenching efficiency. This method, effective for lead detection using fluorescence imaging, is adaptable to T30695-aptamer of different lengths. We explored a bead-based strand displacement aptamer sensing technique utilizing T30695, known for its high selectivity. This technique efficiently detects Pb2+ ions under optimal conditions, showing a robust correlation between fluorescence reduction and Pb2+ ion concentration, with a calculated dissociation constant of 7.83nM. While demonstrating exceptional selectivity for Pb2+ ions, we identified conditions that may lead to false positives and emphasize the technique's specificity when duplex-forming divalent cation concentrations are low compared to Pb2+ ions.

We then explored the detection of Thrombin molecules using the same bead-based strand displacement aptamer sensing strategy with Thrombin aptamer and were successful in detecting thrombin with a dissociation constant value of 158.75nM. Subsequently, we investigated the challenges encountered in extending the strategy for multiplexed detection of Pb2+ and Thrombin molecules. Our findings underscore the complexity of optimizing aptamer-based multiplexing strategies, emphasizing the need for meticulous experimental design and interpretation for reliable results.