Ultrasensitive and multiplex SERS immunoassay for stroke subtype-specific biomarkers based on graphene oxide-supported nanofilms coated by roughened nanoboxes with extensive high-density hotspots

Herein, we fabricate the graphene oxide-supported nanofilms coated by roughened nanoboxes (GO@AuAgRNB) for the ultrasensitive and simultaneous determination of multiple stroke subtype-specific biomarkers. Initially, Au-Ag roughened nanobox (AuAgRNB) with abundant coupling and tip hotspots is prepared by the partial surface passivation strategy. AuAgRNB is uniformly, densely and firmly assembled onto graphene oxide (GO) by metal-sulfur bonds, generating extensive high-density hotspots. Owing to electromagnetic and chemical enhancement, the surface enhanced Raman scattering (SERS) activity of GO@AuAgRNB is greatly improved with the enhancement factor of 5.78 × 10⁷. Combined with magnetic bead, GO@AuAgRNB was employed to develop a SERS-based immunoassay platform for the simultaneous detection of glial fibrillary acidic protein (GFAP) and retinol binding protein 4 (RBP₄). The platform demonstrates ultra-sensitivity with detection ranges of 0.1 pg/mL-0.1 μg/mL and limits of detection of 0.16 pg/mL for GFAP and 0.10 pg/mL for RBP₄. Furthermore, the platform provides superior anti-interference properties, accuracy, and capability for simultaneous detection and practical application. In the detection of clinical patient samples, the receiver operating characteristic curve analysis shows that cut-off values (RBP₄ = 13.51 μg/mL and GFAP = 2.07 ng/mL) can reliably differentiate patients with ischaemic and haemorrhagic stroke. Overall, the ultrasensitive and multiplex immunoassay platform based on GO@AuAgRNB demonstrates high potential in the clinical diagnosis of stroke subtypes.