Colorimetric Hybridization Sensor for DNA Mimic of a SARS-CoV-2 RNA Marker: Direct and Inverse Bioanalysis

This article presents a colorimetric visual biosensor designed for direct application in undiluted biofluids, which holds significant promise for point-of-need applications. Unlike traditional biosensors that struggle with heavily diluted sample matrices, the presented biosensor does not require any instrumentation or trained personnel, making it highly practical. The sensor features an oligonucleotide probe covalently attached to magnetically separable magnetite (Fe₃O₄) particles. This probe selectively captures a DNA mimic of the SARS-CoV-2 RNA sequence via a base-pair hybridization. The DNA mimic oligomer sequence was tested in a buffer solution, undiluted serum, and undiluted salivary biofluids. A second complementary hybridization sequence with a biotin tag was used to bind the target oligomer already hybridized to the magnetic particle-conjugated capture probe. Subsequent detection of the target oligomer was accomplished through high-affinity selective binding of streptavidin-peroxidase labels with the detection probe biotin units for visual colorimetric detection in the presence of 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Inverse assaying of the unbound-free streptavidin-peroxidase labels left in the detection reagent solution offered a reverse trend to the target oligomer concentration, as anticipated. We obtained detection limits of 1 fM (buffer assay), 1 pM (undiluted serum assay), and 1 pM (undiluted saliva assay) and with the linear ranges of 1 fM–10 nM (buffer assay), 1 pM–1 nM (undiluted serum assay), and 1 pM–1 nM (undiluted saliva assay), respectively. The assays in different biofluids allowed for the estimation of the analytical performance and the effect of sample matrices on the detection limits and calibration sensitivity.