Alternating current impedance spectroscopic analysis of biofunctionalized vertically-aligned silica nanospring surface for biosensor applications

In this study, alternating current impedance spectroscopic analysis of the biofunctionalization process of a vertically-aligned (silica) nanosprings (VANS) surface is presented. The VANS surface is functionalized with a biotinylated immunoglobulin G (B-IgG) layer formed by physisorption of B-IgG from the solution phase. Bovine serum albumin passivation of the B-IgG layer reduces additional surface adsorption by blocking the potential sites of weak bond formation via electrostatic and hydrophobic interactions. As avidin acts as a receptor of biotinylated compounds, avidin conjugated glucose oxidase (Av-GOx) binds to the B-IgG layer via biotin. This avidin-biotin bond is a stable bond with high association affinity (Ka = 10¹⁵ M^-1) that withstands wide variations in chemistry and pH. An IgG layer without biotin shows no binding to the Av-GOx, indicating that bonding is through the avidin-biotin interaction. Finally, fluoroscein iso-thiocyanate (FITC) labeled biotinylated bovine serum albumin (B-BSA) added to the Av-GOx surface is used to fluorescently label Av-GOx for fluorescent measurements that allow for the correlation of surface binding with impedance measurements. Modeling of impedance spectra measured after the addition of each biological solution indicates that the bimolecular layers behave as insulating layers. The impedance spectra for the VANS-based sensor are compared to simple parallel capacitor sensors, sans VANS, and serve as controls. VANS-based sensors exhibit a greater magnitude of change between successive bio-layers relative to the controls below 10 kHz. Changes in the magnitudes of the components of the VANS equivalent circuit indicate that the addition of biological layers changes the effective dielectric response of the VANS via the impediment of ionic motion and biomolecule polarization.