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

Area of Science:

• Biochemistry
• Biotechnology
• Analytical Chemistry

Background:

• Metal ion detection is vital for food safety, environmental monitoring, and cellular studies.
• Fluorescent proteins (FPs) are promising biosensors due to their intrinsic fluorescence and sensitivity to metal binding.
• The tKeima FP offers advantages like a large Stokes shift and spectral stability for detecting analytes in complex matrices.

Purpose of the Study:

• To investigate the metal-quenching mechanism of the tKeima fluorescent protein.
• To quantify the binding affinities and detection limits of tKeima for biologically relevant metal ions (Fe2+, Fe3+, Cu2+).
• To evaluate tKeima's potential as a biosensor for metal ion detection in complex samples.

Main Methods:

• Metal titration experiments were performed to analyze tKeima quenching by Fe2+, Fe3+, and Cu2+.
• Langmuir isotherm modeling was used to determine dissociation constants (Kd) and maximum quenching capacities (Bmax).
• Stern-Volmer analysis and sphere-of-action models were applied to elucidate the quenching mechanisms.

Main Results:

• tKeima exhibited distinct quenching responses to Fe2+, Fe3+, and Cu2+ with varying affinities and detection limits.
• Dynamic quenching was predominant for Fe2+ and Cu2+, while Fe3+ showed a significant static quenching component.
• tKeima demonstrated partial fluorescence recovery with EDTA and moderate selectivity against common interfering ions.

Conclusions:

• The study clarifies the metal-quenching mechanisms of tKeima, highlighting its utility for detecting specific metal ions.
• tKeima's properties make it a suitable platform for developing robust biosensors for environmental and biological applications.
• Further optimization could enhance tKeima's selectivity and sensitivity for broader metal ion sensing applications.