Functional Nucleic Acid-Based Colorimetric Biosensors for Pathogen Detection

Abstract

In recent years, the rising incidence of infectious diseases has emerged as a significant and escalating threat to global public health and economic stability. Consequently, the prevention and timely management of infections have become critical priorities. Accurate and rapid pathogen identification at the point of care is essential for guiding appropriate therapeutic interventions and optimizing anti-infective treatment strategies. Traditional diagnostic methods, such as cell culturing, immunoassays, and polymerase chain reaction (PCR), though effective, are often hindered by inherent limitations, including long processing times, operational complexity, cross-reactivity, and the requirement for specialized equipment. These challenges highlight the demand for affordable, sensitive, and rapid detection methods for pathogens. In this context, functional nucleic acid (FNA)-based biosensors have attracted considerable attention, with broad applications in healthcare, food safety, and environmental monitoring. The incorporation of synthetic FNAs, such as DNA aptamers and DNAzymes, as molecular recognition elements (MREs) offer distinct advantages, including cost-efficiency, MRE stability, and exceptional recognition specificity. Among the various detection methods, colorimetric readouts stand out as particularly beneficial for pathogen detection, providing rapid and easily interpretable visual results that are highly suited for point-of-care diagnostics. My research focuses on the development of sensitive and portable FNA-based biosensors for pathogen detection, utilizing three key strategies: the integration of high-affinity FNAs as MREs; the incorporation of urease and palladium-iridium (Pd-Ir) nanocubes as efficient signaling elements; and the design of low-cost, highly sensitive, and easy-to-use biosensing platforms. The first research project compares three aptamer-based and urease-mediated litmus tests (AUTs) for detecting SARS-CoV-2 in saliva samples, using monomeric (dissociation constant, Kd ~10,000 pM), dimeric (Kd ~100 pM), and trimeric (Kd ~10 pM) aptamers. We analyzed their sensitivity and specificity across 48 patient samples and found a linear relationship between sensitivity and the logarithmic binding affinity of the aptamers. Our predictions suggest that achieving 80-100% sensitivity and 100% specificity requires highly affinity aptamers. This underscores the need for developing aptamers with a very high affinity for accurate viral detection in future pandemics. The second research project describes the development of a highly sensitive nanozyme-linked aptamer assay (NLAA) for detecting SARS-CoV-2. This assay leverages the high affinity of a trimeric aptamer and the strong peroxidase-like activity of palladium-iridium (Pd-Ir) nanocubes. The trimeric aptamer-based NLAA achieved a limit of detection (LOD) of 9.3 × 103 cp/mL for pseudoviruses with the SARS-CoV-2 spike protein, which is 172- and 12.9-fold lower than the LODs of the monomeric and dimeric aptamer-based assays, respectively. Testing 60 clinical saliva samples, the trimeric aptamer-based NLAA showed 100% specificity and 86.7% sensitivity. Additionally, trimeric aptamer-conjugated Pd-Ir nanocubes blocked viral entry into host cells with an IC50 (half-maximal inhibitory concentration) of 6.4 pM, 2.7- and 10.1-fold lower than the dimeric and monomeric versions, respectively. This study highlights the potential of aptamer-modified nanomaterials for both diagnostics and therapeutics in future pandemics. The third research project presents a portable and sensitive gold filter tip-based assay (GFTA) for rapid, colorimetric, multiplexed bacterial detection. The assay utilizes specific DNA/RNA substrates, which targets RNase H2 from bacteria of interest. Clostridium difficile (C. difficile) and Salmonella typhimurium (S. typhimurium) were selected as modern organisms to validate our approach due to their bacteria toxicity and the limitations of traditional detection methods. Through in vitro selection, we isolated a substrate, FDRC1-3B, specifically cleaved by RNase H2 from C. difficile (CDH2). The catalytic constant (kcat = 2.53 s−1) of CDH2 for FDRC1-3B is notably higher than that of previously all identified RNA-cleaving fluorogenic DNAzymes. By integrating FDRC1-3B with the GFTA platform, we achieved visible detection of C. difficile in fecal samples at concentrations as low as 1.3 × 103 CFU/mL within 30 minutes, without requiring advanced equipment. Additionally, we developed a S. typhimurium-responsive GFTA using the substrate SSR1-T4, which specifically targets RNase H2 from S. typhimurium. This versatile assay can be adapted for multiplex bacterial detection, offering a valuable tool for identifying various pathogens in resource-limited settings. In summary, my research focuses on developing advanced FNA-based colorimetric biosensing technologies for pathogen detection. This includes creating an aptamer-based urease-mediated litmus test to demonstrate the importance of high-affinity aptamers in clinical diagnostics, developing a sensitive nanozyme-linked aptamer assay for SARS-CoV-2 using trimeric aptamers and Pd-Ir nanocubes, and designing a portable gold filter tip-based colorimetric assay for multiplex bacterial detection with a specific DNA/RNA substrate for bacterial RNase H2. These innovations provide practical, rapid, and sensitive solutions for pathogen detection, especially in resource-limited settings.