Contact pin-printing of albumin-fungicide conjugate for silicon nitride-based sensors biofunctionalization: Multi-technique surface analysis for optimum immunoassay performance

Highlights: • Contact pin-printing of overlapping probe spots and spotting by hand are compared. • Contact pin-printing favors probe immobilization with two-fold higher surface density. • Incomplete monolayer develops to bilayer as printing solution concentration increases. • Blocking molecules complete probe monolayer but reduce probe bilayer. • Surface immunoreaction increases with probe concentration in printing solution. - Abstract: Mass fabrication of integrated biosensors on silicon chips is facilitated by contact pin-printing, applied for biofunctionalization of individual Si₃N₄-based transducers at wafer-scale. To optimize the biofunctionalization for immunochemical (competitive) detection of fungicide thiabendazole (TBZ), Si₃N₄ surfaces are modified with (3-aminopropyl)triethoxysilane and examined after: immobilization of BSA-TBZ conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin (BSA), and immunoreaction with a mouse monoclonal antibody against TBZ. Nanostructure, surface density, probe composition and coverage uniformity of protein layers are evaluated with Atomic Force Microscopy, Spectroscopic Ellipsometry, Time-of-Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy. Contact pin-printing of overlapping probe spots is compared with hand spotted areas. Contact pin-printing resulted in two-fold increase of immobilized probe surface density as compared to hand spotting. Regarding BSA-TBZ immobilization, an incomplete monolayer develops into a bilayer as the concentration of BSA-TBZ molecules in the printing solution increases from 25 to 100 μg/mL. Upon blocking, however, a complete protein monolayer is formed for all the BSA-TBZ concentrations used. Free surface sites are filled with BSA for low surface coverage with BSA-TBZ, whereas loosely bound BSA-TBZ molecules are removed from the BSA-TBZ bilayer. As a consequence immunoreaction efficiency increases with the printing probe concentration.