Sequence-Dependent PNA–AgNP Bioconjugation Enables Electrochemical Discrimination of a Single-Base Mutation in Zika Virus RNA

Abstract Single-nucleotide polymorphisms (SNPs) in RNA viruses such as Zika virus (ZIKV) can alter viral pathogenicity and are therefore critical molecular markers for surveillance. Here, we report an electrochemical hydrogel biosensor that discriminates a clinically relevant ZIKV SNP through a sequence-dependent bioconjugation mechanism between peptide nucleic acids (PNAs) and silver nanoparticles (AgNPs). A single guanine-to-thymine substitution in the PNA probe produces distinct AgNP coordination modes, resulting in markedly different nanoparticle aggregation states, colorimetric signatures, and silver oxidation profiles. Transmission electron microscopy reveals that guanine-containing PNAs induce AgNP clustering through strong nucleobase–silver coordination, whereas thymine-containing PNAs preserve dispersed nanoparticles. These coordination patterns persist upon hybridization with complementary or mismatched ZIKV RNA, establishing PNA–AgNP bioconjugation as the primary determinant of the sensing response. Integration of this mechanism into an AgNP-hydrogel matrix enables the sensitive and selective electrochemical detection of short synthetic targets (15 bp) with an LOD of 0.004 nM, LOQ of 0.015 nM, and 7.4% RSD, alongside full-length 10.8 kbp viral RNA. This work demonstrates how nucleobase-specific PNA–nanoparticle interactions can be harnessed to achieve robust SNP discrimination in RNA viruses.