Head-to-tail: hybridization and single-mismatch discrimination in metallic nanoparticle-DNA assembly

Gold nanoparticles functionalized with DNA (or AuNP-DNA) have been widely used in the detection of target single-stranded DNA (ssDNA). However, the bulk size of the AuNPs brings significant constraints to the hybridization compared to the free DNA system. In this context, a head-to-head alignment of the AuNPs is thermodynamically unfavourable, and the least restricted tail-to-tail structure has been applied extensively. It is envisaged that a head-to-tail alignment would allow the tail AuNP-DNA probe to hybridize to the target DNA at a higher efficiency than that of the head-probe. Therefore, in this work, we first investigate the ability of the head-to-tail AuNP-DNA system to discriminate single nucleotide mismatch DNA based on the coupling of the localized surface plasmon resonance of AuNPs via simple dark field microscopy. Our findings prove that the tail-probe exhibits considerably greater mismatch-discrimination than the head-probe. Next, the analysis of single-mismatch loci on the tail-probe shows that the GC-mismatch located in the middle of recognition site gives the greatest distinction against perfectly matched targets. Finally, the head-to-tail assay is extended to perform hybridization on targets with non-hybridizing elongated sequences. The results show that while the non-hybridizing elongated sequence reduces the nanoassembly formation efficiency, it enhances the single-mismatch discrimination.