Multiplex Hybridization Colorimetric Sensor for Wild Type and Variant RNA Biomarkers

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Laboratory-based assays such as reverse transcriptase-polymerase chain reaction (RT-PCR) of respiratory secretions are being used routinely to detect viral nucleic acids. The RT-PCR assay, however, has many limitations such as the need for high purity samples, gene isolation and amplification, highly trained personnel, sophisticated clean facilities for sample processing, and access to expensive laboratory instruments. To combat the ongoing pandemic there is an urgent need for accessible, affordable, and visual molecular diagnostics that serve resource-limited regions across the globe. Such advanced technology would also facilitate early disease diagnosis, prognosis, and post-occurrence conditions through precise measurements of specific biomarker targets. In contrast to most DNA sensors, visual colorimetric sensors do not require costly instruments or a laboratory setting to diagnose an infection or a disorder. Despite these advantages, existing limitations on the effective use of colorimetric sensors include poor detection limit thresholds and the reliance on one target molecule, thus resulting in false-negative detection of variants. Hence, we propose in this application a multiplex sensor approach that will inherently amplify detection signals by targeting together a panel of RNA markers in saliva and serum and thus increase the probability of positive identification and mitigate false negative detection. Our approach involves the design of multi-oligonucleotide probes carrying magnetically-separable, antifouling iron oxide core/gold shell nanoparticles to selectively capture the target SARS-CoV-2 wild type RNA and the two most common N501Y and E484K variants in saliva and serum (Step 1, 10 min) as the model system for broader applications to any other biomarkers targets. A second complementary oligonucleotide (biotinylated) hybridization and magnetic separation (Step 2, 5 min), and subsequent selective binding of streptavidin-peroxidase molecules to the target RNAs hybridized biotin oligonucleotides and magnetic separation (Step 3, 5 min) will allow visual and spectrometer color readouts in the presence of added hydrogen peroxide and tetramethylbenzidine substrate as supported with the presented preliminary results from the proposed new approach. A calibrated color reference scale featuring color intensity profiles like a pH indicator paper will be devised for a yes/no identification of RNA biomarkers-based infections in real samples. Validation of the colorimetric sensor with the standard nucleic acid-based PCR techniques is proposed. Our research teams will be composed primarily of undergraduate students. In addition to establishing an R15 undergraduate club at our school, to offer virus sensor research exposure for several hundred undergraduates, we will conduct in-class demonstrations in general chemistry classes, Biomedical Engineering, and Process and Product Design classes (taught by the PIs). All undergraduate students will thus gain significant biomedical research experience through active involvement in the proposed project and thus enhance the research environment of Oklahoma State University.