Fluoro4Graphene: A fluorogenic high-throughput screening platform for property engineering of graphene binding...

Experimental and quantitative methods for studying protein-graphene interactions remain limited, despite their importance for biosensing and biointerface engineering. Here, we report Fluoro4Graphene, a three-step high-throughput fluorescence-based screening assay for the identification of graphene-binding peptides. In this platform, candidate peptides are genetically fused to the phytase of Yersinia mollaretti, which serves as a reporter enzyme by converting 4-methylumbelliferyl phosphate into the fluorescent 4-methylumbelliferone. Robust signal quantification is achieved by transferring the soluble fluorophore to a secondary microtiter plate prior to detection, thereby avoiding graphene-induced fluorescence. The assay is performed directly from crude Escherichia coli lysates in microtiter plates coated with electrochemically exfoliated graphene flakes, eliminating protein purification steps and enabling high sample throughput. The platform exhibits good reproducibility, with a coefficient of variation of approximately 17%, allowing reliable discrimination of small differences in binding. Analytical performance was validated in a directed evolution campaign targeting the material binding peptide Macaque histatin-1, yielding variants with improved graphene surface coverage. The best variant showed a 1.44-fold improvement in the Fluoro4Graphene screening assay. Independent validation using surface plasmon resonance on high-purity graphene confirmed an increase in surface coverage from 106.19 ng/cm² to 154.10 ng/cm². Overall, Fluoro4Graphene provides a robust, reproducible, and scalable analytical tool for the quantitative assessment of protein-graphene interactions.