Oriented Antibody Immobilization Vs Random Covalent Binding: Improving Reproducibility for Label-Free Electrochemical Immunosensing of Multi-Allergens on Silicon Chips

The development of compact biosensing devices capable of sensitive and selective concomitant detection of two or more analytes in a single sample has enormous potential for point-of-care and -use applications. Silicon microfabrication technologies outperform other fabrication processes in terms of reproducibility, miniaturization capability, and design adjustability. In this work, we present a novel miniaturized fully integrated multiplexed silicon-based sensing platform for rapid and label-free detection of milk and egg allergens in water. After modifying the microelectrodes with gold nanostructures and chitosan-gold nanocomposite hydrogel layer, we compared random covalent binding with boronic acid-assisted oriented strategy for antibody immobilization to the surface. The Boronic affinity-based approach exhibited a high reproducible and reliable immuno-response towards casein as a major milk allergen. The stepwise surface modifications characterized using various analytical techniques, confirming the successful attachment of antibodies to the surface. We then successfully adapted the biosensor surface for ovalbumin detection, an egg allergen, demonstrating multiplexed allergen detection and universal immobilization strategy for any IgG type antibodies, opening a wide range of potential applications in portable immunosensing for various target analytes beyond food allergens.