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

## TL;DR

tKeima is a fluorescent protein platform that detects metal ions like Fe2+, Fe3+, and Cu2+ with high sensitivity and specificity, making it useful for biosensing applications.

## Contribution

tKeima introduces a novel fluorescent protein platform with a large Stokes shift for detecting metal ions under complex conditions.

## Key findings

- tKeima shows dissociation constants of ~2700 μM for Fe2+, ~3100 μM for Fe3+, and ~880 μM for Cu2+.
- Dynamic quenching dominates for Fe2+ and Cu2+, while Fe3+ exhibits a stronger static quenching component.
- tKeima has moderate selectivity and partial fluorescence restoration with ethylenediaminetetraacetic acid.

## Abstract

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.