Rapid detection of aflatoxin B1 by a bifunctional protein crosslinker-based surface plasmon resonance biosensor

•A novel bifunctional protein crosslinker was developed for gold substrate-based immunosensors.•The protein crosslinker enabled a rapid self-oriented immobilization of antibody on gold substrates.•The protein crosslinker-based SPR immunosensor could detect aflatoxin B1 sensitively and selectively.

The aim of this study was the development of a bifunctional protein crosslinker-based surface plasmon resonance (SPR) biosensor for rapid detection of aflatoxin B1 (AFB1), a potent carcinogen. A fusion protein was obtained by genetically fusing gold binding protein (GBP) that binds strongly to gold surfaces to protein G (ProG) that interacts with the Fc portion of antibodies. It was used as a bifunctional crosslinker for rapid self-oriented immobilization of antibodies on gold substrates without any chemical treatment. SPR analyses demonstrated the binding of the GBP-ProG crosslinker to the gold surface was superior to that of an only ProG via currently used self-assembled monolayers of alkanethiol due to the GBP property. As a result, anti-AFB1 antibodies were 36% more immobilized on the GBP-ProG layer than the ProG layer. When the GBP-ProG crosslinker-based SPR chips were fabricated with the best density (100 μg/mL) of anti-AFB1 antibodies, they could detect AFB1 as low as 1 μg/mL in both buffer and corn extracts and selectively detect it with negligible SPR responses in control toxins (zearalenone and ochratoxin A). These results mean the GBP-ProG is more useful than the thiolated chemical linkers for development of gold substrate-based immunosensors, and this GBP-ProG crosslinker-based immunosensor could detect small molecules effectively.