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.