Comparative Colorimetric Oligonucleotide Sensing using Gold Nanoparticles and Toluidine Blue O: Insights from Representative Viral Sequences.

Colorimetric nucleic acid sensing platforms offer attractive advantages for low-instrumentation detection; however, their performance is strongly governed by the nature of macromolecular interactions involved in signal transduction. In this work, we present a comparative evaluation of two distinct colorimetric sensing strategies, gold nanoparticle (Au NP) agglomeration and toluidine blue O (TBO)-based metachromasia for oligonucleotide detection using synthetic, amplification-relevant targets. Specific oligonucleotide sequences derived from hepatitis C virus (HCV) genotypes 1 and 3 have been employed as representative model targets to enable controlled assessment of sequence discrimination and sensing behavior. Target oligonucleotides are selectively captured on probe-functionalized magnetite nanoparticles, followed by strand separation and release of single-stranded complementary DNA (cDNA) for downstream colorimetric detection. In the Au NP-based system, target-induced electrostatic screening and bridging effects have led to nanoparticle agglomeration, resulting in characteristic localized surface plasmon resonance shifts observable by UV-visible spectroscopy. In contrast, the TBO-based system has exhibited concentration-dependent metachromatic responses arising from electrostatically driven association and agglomeration of the cationic dye along the negatively charged phosphate backbone of the released cDNA. Direct comparison under identical experimental conditions revealed distinct differences in sensitivity, signal evolution, and sequence discrimination between the two platforms, highlighting how fundamentally different macromolecular interaction pathways govern colorimetric response generation. Rather than constituting a disease-specific diagnostic assay, this study provides mechanistic and design-level insights into post-amplification-compatible colorimetric oligonucleotide sensing, offering a generalizable framework for the rational development of colorimetric nucleic acid sensors.