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Stable and efficient conjugates between antibodies and gold nanoparticles (GNP-Ab) are sought to develop highly sensitive and robust biosensors with applications in medicine, toxicology, food safety controls, and targeted drug delivery. Several strategies have been proposed for directing the antibody attachment to GNPs thus preserving antibody activity, including covalently coupling the antibody to a polymer grafted on GNP surface and exploiting the high affinity of bioreceptors as mediators for the binding. Both approaches also allow for shielding GNPs with a protective layer that guarantees the robustness of the conjugate. Notwithstanding, antibodies freely adsorb to GNP with high binding efficiency. The nonspecific adsorption is far more simple, fast, and inexpensive than any mediated coupling. Therefore, it is preferred for most applications, although it is considered to produce GNP-Ab with a limited activity. In this work, we compared three strategies for producing GNP-Ab, such as (i) covalent coupling mediated by a chemical layer, (ii) affinity-based binding mediated by a biomolecular layer composed of Staphylococcal protein A, and (iii) direct attachment via adsorption. The so-prepared GNP-Ab were employed as probes in a colorimetric lateral flow immunoassay (LFIA) for measuring salivary cortisol as a model biosensor that relies on the use of active GNP-Ab conjugates. Unexpectedly, the biosensors fabricated using the three probes were completely comparable in terms of their ability to measure salivary cortisol. Furthermore, we observed that the sensitivity of the LFIA primarily depended on the amount of the antibody bound to GNPs rather than on the method by which it was bound. The probes prepared using both the direct adsorption approach and mediated coupling via the biochemical mediator enabled development of point-of-care devices for the fast, sensitive, and reliable measurement of human salivary cortisol.

Direct vs Mediated Coupling of Antibodies to Gold Nanoparticles: The Case of Salivary Cortisol Detection by Lateral Flow Immunoassay