Specific Nucleic Acid Detection Using a Nanoparticle Hybridization Assay

Abstract

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has become a global health crisis, leading to significant public health challenges. Since symptoms overlap with other respiratory infections, prompt and accurate diagnosis is essential to manage the disease. Two issues arise from using PCR-based diagnostic tests for SARS-CoV-2: false positives at high cycle numbers from early nonspecific amplification and the amplification of virus nucleic acids present in trace amounts in the collected specimen that are not necessarily associated with active infection. These issues, standard across all tests that use nucleic acid amplification, necessitate the development of alternative diagnostic approaches. This thesis presents a novel approach to developing a molecular biosensor for detecting SARS-CoV-2 RNA, leveraging gold nanoparticles (AuNPs) combined with two oligonucleotides. The biosensor relies on forming aggregates of oligo-AuNPs in the presence of targeted viral nucleic acid sequences. Initial testing involved conjugating AuNPs with two designed oligonucleotides, reverse complements of specific SARS-CoV-2 target sequences. Notably, nanoparticle aggregate detection was performed by darkfield videomicroscopy, detecting nanoparticle tracks and estimating the aggregates’ diffusion coefficients. Ancillary identification of nanoparticle aggregation occurred by detecting a ~6 nm shift in the surface plasmon resonance peak measured with nano spectrophotometry and a mobility shift detected by agarose gel electrophoresis. To maximize target hybridization and reduce aggregation of oligo-coated nanoparticles in the presence of off-target nucleic acid sequences, two oligomers chosen for nanoparticle conjugation were optimized for hybridization efficiency with the chosen target and for lack of self- and cross-hybridization through a parameter-limited Primer-BLAST search and thermodynamic and structure analysis using NUPACK. Experimental results demonstrated that oligomers hybridized with a specific DNA sequence that complements the RdRp RNA gene sequence from the published virus genome. After successfully conjugating the oligomers to 10 nm diameter gold nanoparticles, the nanoparticles aggregated in the presence of target DNA, confirmed by spectrophotometry, nanoparticle, tracking, and gel electrophoresis. Computational models of nanoparticle diffusion and nanoparticle aggregation as systems of first-order reactions confirmed findings from experiments: that larger and aggregated nanoparticles produced lower diffusion coefficients and that more targets in the reaction mixture produced larger aggregates. Integrating primers with gold nanoparticle-based biosensors highlights the potential for developing rapid and portable diagnostic tools for SARS-CoV-2 detection. In conclusion, this thesis proposes and provides some data supporting a novel approach for detecting nucleic acids in solution without amplification: darkfield videomicroscopy with nanoparticle tracking and diffusion coefficient estimation. The steps needed to make this approach viable and the generalization of the assay beyond the detection of SARS-CoV-2 nucleic acids was discussed. The insights gained have implications for developing sensitive, rapid, and portable diagnostic platforms for various infectious diseases and detecting other types of nucleic acids.