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)), were coloaded onto graphene oxide (GO) through simple incubation, forming a 2D nanoscale recognition probe (Ru(bpy)/Apt@GO). Owing to the size exclusion effect of VMSF towards the 2D nanoscale probe, the electrochemical signal of Ru(bpy)/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) from GO, resulting in their presence in the solution. The efficient electrostatic enrichment towards Ru(bpy) by negatively charged VMSF allows for high electrochemical signals of free Ru(bpy) 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.