Integrating Functional Nucleic Acids and Anti-Fouling Polymers for Advanced Electrochemical Detection in Complex Media

Rapid, sensitive, and reliable detection of disease biomarkers in complex biological samples remains a major challenge for the practical use of electrochemical biosensors. While such sensors offer key advantages including high sensitivity, low cost, and portability, their performance is often significantly hindered in real-world applications due to biofouling and matrix interference. In complex samples, non-specific adsorption of proteins, lipids, and other biomolecules onto the electrode surface can degrade signals, reduce sensitivity, and compromise sensor stability. To address these challenges, we developed zwitterionic anti-fouling polymer coatings for electrochemical biosensor surfaces that prevent non-specific interactions with both the electrode and the biorecognition elements. In parallel, anti-fouling poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-coated magnetic beads were used to protect biorecognition elements from biofoulants while enabling selective target capture and efficient separation of released reporter DNA for sensing. These anti-fouling polymeric systems were then integrated with functional nucleic acids, including DNAzymes and aptamers, to achieve high specificity and strong target recognition. Using this combined strategy, we developed an anti-fouling polymer-coated biosensor functionalized with an aptamer that enabled direct detection of SARS-CoV-2 in unprocessed saliva. A similar approach employing a sandwich assay format using magnetic beads and anti-fouling polymer-coated electrodes was also applied to detect a low-abundance cancer biomarker, VEGF165, in unprocessed plasma. Signal normalization between a target-specific aptamer and a mutant control aptamer was demonstrated to enable improved accuracy across diverse human plasma samples. Subsequently, anti-fouling POEGMA-coated magnetic beads were applied for the detection of Fusobacterium nucleatum in unprocessed saliva. Key challenges associated with bacterial biosensing in unprocessed biological fluids were identified and addressed, including the adaptation of fluorogenic DNAzymes (originally designed with quenchers and fluorophores) to enable compatibility with electrochemical readouts. Building on these advances, an integrated platform combining anti-fouling magnetic beads, DNAzymes, and a flow-based electrochemical device incorporating wrinkled gold electrodes was developed to detect Clostridioides difficile in human stool samples. The sensor design specifically enhanced mass transport while minimizing surface fouling, enabling sensitive detection in processed stool samples using an optimized sample preparation protocol. Overall, this thesis presents a suite of anti-fouling-enabled biosensing technologies for the direct detection of target analytes in complex biological media. These approaches have strong potential for translation into clinical laboratory settings, supporting rapid and early disease detection to improve patient care and treatment outcomes.