One-pot, solid-phase immunosensing platform consisting of a nanometer-thick Au/TiO2 photocatalytic film and Cy5/capture antibody/gold nanorod conjugates

Abstract

There is a demand for one-pot, portable (solid-phase), sensitive, and user-friendly immunosensors for future point-of-care (POC) self-testing. However, current immunoassays such as the enzyme-linked immunosorbent assay (ELISA) typically involve several complicated steps, and they are not readily adaptable by nonexpert users. Herein, we present a rapid (∼30 min) one-pot, solid-phase immunosensor, based on nanomaterials, by combining a nanometer-thick Au/TiO2 photocatalytic film and Cy5/capture antibody/gold nanorod (GNR) conjugates immobilized on a membrane (the fluorescence of Cy5 was enhanced by the GNR). The one-pot immunoassay is started by adding a drop of a mixture containing 4-chloro-1-naphthol (CN), a horseradish peroxidase (HRP)-labeled detection antibody, and an antigen onto the one-pot immunosensor and illuminating UV light. The UV illumination on the Au/TiO2 film results in the production of H2O2, which promotes a CN precipitation reaction. 4-Chloro-1-naphthol precipitates produced by the HRP, which was bound to the conjugates via the antibodies and antigens, could preliminarily quench Cy5 fluorescence via Forster resonance energy transfer, because of their proximity to Cy5. The sensitivity of the developed one-pot immunosensor was similar to that of a commercially available ELISA kit. Given the increasing interest in the early diagnosis of various diseases, including cancers, dementia, and coronavirus disease 2019, the application of nanomaterials such as a porous thin-film photocatalyst and GNR-based fluorescent probes could pave way for the development of next-generation POC biosensors.