Click Coupling on Anodic Niobia and Tantala Nanocolumns for Label-Free Biosensing

Abstract

Efficient covalent immobilization of biomolecules on the surface of nanostructured materials while maintaining their functional properties is a key step in the creation of modern biosensor systems. Niobia and tantala nanocolumns were fabricated by magnetron sputtering of metal targets together with Al on Si wafers, followed by anodizing and dissolution of the porous alumina template. The morphology of the nanocolumns was characterized using scanning electron microscopy, and the surface area enhancement factor was evaluated. The nanostructures were covalently functionalized with clickable azide groups via (3-chloropropyl)trichlorosilane, followed by nucleophilic substitution. Surface functional group load was quantified via a Cu-catalyzed azide–alkyne cycloaddition with a dimethoxytrityl (DMT)-substituted alkyne, followed by photometric detection of the released DMT cation. The use of a high boiling point hydrophobic solvent (o-dichlorobenzene, o-DCB) together with the extended surface area of the nanostructures enabled convenient and reproducible measurements, providing a satisfactory analytical signal (A ≈ 0.2–0.3) from samples of about 1 cm². Both niobia- and tantala-based nanocolumns exhibited comparable functionalization densities of ∼1 nm^–2, calculated with consideration of their geometrical parameters. The feasibility of refunctionalization with azide groups was demonstrated. A biotin derivative bearing a DMT moiety was covalently conjugated to the nanocolumns. The surface load of biotin was quantified, and its biospecific activity was validated through binding of a streptavidin–peroxidase conjugate. The applicability of anodic nanocolumns for the construction of label-free biosensors was demonstrated by biospecific streptavidin adsorption, registered via reflectance spectra.