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

Area of Science:

• Biomaterials Science
• Analytical Chemistry
• Biotechnology

Background:

• Studying protein-graphene interactions is crucial for biosensing and biointerface engineering.
• Existing experimental and quantitative methods for these interactions are limited.
• Developing high-throughput screening assays is essential for advancing this field.

Purpose of the Study:

• To develop a novel, high-throughput fluorescence-based screening assay for identifying graphene-binding peptides.
• To enable robust and quantitative assessment of protein-graphene interactions.
• To facilitate advancements in biosensing and biointerface engineering.

Main Methods:

• Developed Fluoro4Graphene, a three-step fluorescence assay using a phytase reporter enzyme.
• Performed assay directly from crude Escherichia coli lysates in microtiter plates coated with graphene flakes.
• Quantified fluorescence by transferring the soluble fluorophore to a secondary plate to avoid graphene interference.

Main Results:

• The Fluoro4Graphene assay demonstrated good reproducibility (CV ~17%) for discriminating binding differences.
• Directed evolution using the assay yielded peptide variants with improved graphene surface coverage.
• The best variant showed a 1.44-fold improvement in the assay and a 45% increase in surface coverage validated by SPR.

Conclusions:

• Fluoro4Graphene is a robust, reproducible, and scalable analytical tool for quantitative assessment of protein-graphene interactions.
• The assay eliminates protein purification steps and enables high sample throughput.
• This platform significantly advances the study of protein-graphene interactions for various applications.