Fluorescent biosensors are indispensable tools for molecular imaging, detection, and drug screening. Conventionally,fluorescent biosensors were constructed by incorporating fluorophores into ligands. Here, to developligand-independent biosensors, we demonstrated biosensor selection from a fluorophore-modified peptide phagelibrary. In this library, the peptides were designed to form α-helical structures, and one cysteine, the probemodification site, was located at the center of four randomized residues on the same face of the helix. Byconjugation with 4-nitrobenzoxadiazole (NBD), we constructed an NBD-modified phage library. We conductedselection against galectin-3 (Gal-3), a galactose-specific lectin associated with various biological events such astumor metastasis and insulin resistance. After biopanning, we obtained NBD-modified peptides that selectivelybind to Gal-3 from the library. The fluorescence intensity of the hit biosensors increased with the concentrationof Gal-3, and this fluorescent response was visually observed.
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