Stretch–Stowage–Growth Strategy to Fabricate Tunable Triply-Amplified Electrochemiluminescence Immunosensor for Ultrasensitive Detection of Pseudorabies Virus Antibody

Triply amplified electrochemical biosensors have attracted particular attention in the detection of low-abundance biomarkers. The universal construction routes for nonenzymatic triply amplified and even multiply amplified biosensors are extremely desirable but remain challenging. Here, we proposed a “stretch–stowage–growth” strategy to tunably fabricate a nonenzymatic triply amplified or multiply amplified electrochemiluminescence (ECL) immunosensor for ultrasensitive determining pseudorabies virus (PrV) antibody. Based on the matrix role of gold nanoparticle-graphene nanosheet (Au–GN) hybrids, carrier role of silicon nanoparticles (SNPs) and bridge role of “biotin–streptavidin–biotin” (B–SA–B) structure, the establishment processes were defined as “stretch”, “stowage”, and “growth”, respectively. Relying on the interaction of antigen–antibody and of B-SA, the “Au–GN/PrV (Ag)/PrV antibody (Ab1)/biotinylated IgG (B-Ab2)/SA/biotinylated Ru(bpy)32+-encapsulated SNPs (B–Ru@SNPs)” triply amplified biosensor could be fabricated and exhibited better analytical performance not only toward monoclonal PrV antibody with a linear detection range from 50 ng mL–1 to 1 pg mL–1 and a detection limit of 0.40 pg mL–1, but also toward actual serum samples when compared with enzyme-linked immunosorbent assay and fluorometry. Furthermore, multiply amplified biosensors could be conveniently fabricated by controllably repeating the combination of B–Ru@SNPs and SA to form the B–SA–B structure. After it was repeated three times, the multiply amplified biosensor stretched to the maximum of signal amplification and achieved a luminescence quantum efficiency about 23.1-fold higher than the triply amplified biosensor. The tunable biosensor exhibits good stability, acceptable reproducibility and accuracy, suggesting its potential applications in clinical diagnostics.