tKeima: A Large-Stokes-Shift Platform for Metal Ion Detection.

Detection of metal ions under complex and heterogeneous conditions is crucial for food safety, environmental monitoring, and cellular studies. Fluorescent proteins (FPs) are attractive biosensors due to their ease of expression, strong emission without external cofactors, and fluorescence quenching upon metal binding. tKeima features a large Stokes shift, pH sensitivity, and spectral stability, reducing background interference and enabling metal detection in complex samples. Here, we examined tKeima quenching toward biologically relevant metal ions (Fe2+, Fe3+, and Cu2+). Metal titration fitted to the Langmuir isotherm yielded dissociation constants (Kd) of 2710.7 ± 178.6 μM (Fe2+), 3112.0 ± 176.7 μM (Fe3+), and 881.9 ± 76.2 μM (Cu2+), with maximum quenching capacities (Bmax) of 133.8 ± 2.4%, 128.3 ± 2.5%, and 109.2 ± 1.2%, respectively. Limits of detection were 396.0 μM (Fe2+), 428.6 μM (Fe3+), and 457.7 μM (Cu2+), and linear quenching responses were observed up to ~1000, 1500, and 1000 μM, respectively. Sphere-of-action combined with Stern-Volmer analysis indicated primarily dynamic quenching for Fe2+ and Cu2+, whereas Fe3+ showed a stronger static component. tKeima showed partial fluorescence restoration with ethylenediaminetetraacetic acid and moderate selectivity against interfering ions. These findings clarify tKeima's metal-quenching mechanism and support its use as a platform for metal-responsive biosensors.