Amplification-Free Dual-Mode Gold Nanoparticle–Nanocluster Platform for Single-Nucleotide Discrimination of SMN1 in Spinal Muscular Atrophy Screening

Abstract

Spinal muscular atrophy (SMA) is a severe genetic disorder primarily caused by homozygous deletion of exon 7 in the SMN1 gene, leading to deficiency of the survival motor neuron (SMN) protein. Accurate detection of SMN1 is challenging because it is highly homologous to SMN2, differing at only a few nucleotides, most notably a single nucleotide substitution in exon 7 that affects splicing. Rapid and reliable detection of SMN1 exon 7 deletion is therefore essential for newborn screening; however, the high sequence homology between SMN1 and SMN2 necessitates highly specific assay designs, which can increase technical complexity in high-throughput workflows, including those based on dried blood spot (DBS) samples. Here, we report an amplification-free dual-mode nanoplasmonic biosensing platform capable of single-nucleotide discrimination based on hybrid interactions between cetyltrimethylammonium bromide-stabilized gold nanoparticles (CTAB-AuNPs) and glutathione-protected gold nanoclusters (GSH-AuNCs). Hybridization-regulated nanoparticle assembly modulates probe adsorption onto AuNPs and promotes interparticle interactions between CTAB-AuNPs and GSH-AuNCs, generating two correlated optical readouts: a ratiometric colorimetric response (A525/A650) arising from plasmonic coupling of AuNPs and fluorescence attenuation (ΔI) originating from the inner filter effect between AuNP absorption and AuNC fluorescence emission. The platform exhibits a linear response over 10–100 nM and achieves limits of detection of 7.29 and 6.12 nM for the colorimetric and fluorescence modes, respectively. Receiver operating characteristic (ROC) analysis demonstrates excellent single-nucleotide discrimination between SMN1 and SMN2, with area under the curve (AUC) values approaching 1.0, indicating high diagnostic accuracy even in complex genomic DNA (gDNA) matrices. The sensor also enables reliable detection in gDNA extracted from DBS samples, highlighting its potential for amplification-free genetic testing and decentralized SMA newborn screening.