Label-Free Homogeneous Electrochemical Aptasensor Based on Size Exclusion/Charge-Selective Permeability of Nanochannel Arrays and 2D Nanorecognitive Probe for Sensitive Detection of Alpha-Fetoprotein

The labeling-free and immobilization-free homogeneous aptamer sensor offers advantages including simple operation, low cost, and high sensitivity, demonstrating great potential in rapid detection of tumor biomarkers in biological samples. In this work, a labeling-free and immobilization-free homogeneous aptamer sensor was conveniently fabricated by combining size exclusion and charge-selective penetration of a nanochannel-modified electrode and two-dimensional (2D) nanorecognition probe which can realize selective and highly sensitive detection of alpha-fetoprotein (AFP) in serum. Vertically ordered mesoporous silica film (VMSF) with ultra-small, uniform, and vertically aligned nanochannels was easily grown on the simple, low-cost, and disposable indium tin oxide (ITO) electrode. Through π-π interaction and electrostatic force, the AFP aptamer (Apt) and electrochemical probe, tris(bipyridine)ruthenium(II) (Ru(bpy)₃²⁺), were coloaded onto graphene oxide (GO) through simple incubation, forming a 2D nanoscale recognition probe (Ru(bpy)₃²⁺/Apt@GO). Owing to the size exclusion effect of VMSF towards the 2D nanoscale probe, the electrochemical signal of Ru(bpy)₃²⁺/Apt@GO could not be detected. In the presence of AFP, the specific binding of AFP to the aptamer causes the dissociation of the aptamer and Ru(bpy)₃²⁺ from GO, resulting in their presence in the solution. The efficient electrostatic enrichment towards Ru(bpy)₃²⁺ by negatively charged VMSF allows for high electrochemical signals of free Ru(bpy)₃²⁺ in the solution. Linear determination of AFP ranged from 1 pg/mL to 1000 ng/mL and could be obtained with a low limit of detection (LOD, 0.8 pg/mL). The high specificity of the adapter endowed the constructed sensor with high selectivity. The fabricated probe can be applied in direct determination of AFP in serum.