Colorimetric\nHybridization Sensor for DNA Mimic of\na SARS-CoV‑2 RNA Marker: Direct and Inverse Bioanalysis

This article presents a colorimetric visual biosensor\ndesigned\nfor direct application in undiluted biofluids, which holds significant\npromise for point-of-need applications. Unlike traditional biosensors\nthat struggle with heavily diluted sample matrices, the presented\nbiosensor does not require any instrumentation or trained personnel,\nmaking it highly practical. The sensor features an oligonucleotide\nprobe covalently attached to magnetically separable magnetite (Fe<sub>3</sub>O<sub>4</sub>) particles. This probe selectively captures\na DNA mimic of the SARS-CoV-2 RNA sequence via a base-pair hybridization.\nThe DNA mimic oligomer sequence was tested in a buffer solution, undiluted\nserum, and undiluted salivary biofluids. A second complementary hybridization\nsequence with a biotin tag was used to bind the target oligomer already\nhybridized to the magnetic particle-conjugated capture probe. Subsequent\ndetection of the target oligomer was accomplished through high-affinity\nselective binding of streptavidin-peroxidase labels with the detection\nprobe biotin units for visual colorimetric detection in the presence\nof 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide.\nInverse assaying of the unbound-free streptavidin-peroxidase labels\nleft in the detection reagent solution offered a reverse trend to\nthe target oligomer concentration, as anticipated. We obtained detection\nlimits of 1 fM (buffer assay), 1 pM (undiluted serum assay), and 1\npM (undiluted saliva assay) and with the linear ranges of 1 fM–10\nnM (buffer assay), 1 pM–1 nM (undiluted serum assay), and 1\npM–1 nM (undiluted saliva assay), respectively. The assays\nin different biofluids allowed for the estimation of the analytical\nperformance and the effect of sample matrices on the detection limits\nand calibration sensitivity.