Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Nickel (Ni²⁺) is essential in trace amounts but toxic at excess levels.
• Current analytical methods for Ni²⁺ are often invasive or toxic, limiting mechanistic studies.
• Understanding Ni²⁺ imbalance and toxicity requires advanced detection tools.

Purpose of the Study:

• To develop a genetically encoded probe for real-time detection of Ni²⁺ in live cells.
• To overcome limitations of existing invasive and toxic analytical methods for nickel.
• To enable investigation of Ni²⁺ dynamics and toxicity mechanisms in cellular environments.

Main Methods:

• Construction of a genetically encoded Förster Resonance Energy Transfer (FRET)-based probe (FProNiM) using the bacterial periplasmic nickel-binding protein NikA.
• Incorporation of fluorescent protein variants ECFP and Venus flanking the NikA protein.
• Expression and testing of the FProNiM sensor in various cell types (E. coli, yeast, mammalian cells) and validation using confocal microscopy.

Main Results:

• FProNiM demonstrated specificity for Ni²⁺ and insensitivity to pH fluctuations (5.0–8.0).
• The probe exhibited a dissociation constant (K_d) of 21.6 μM, enabling detection of Ni²⁺ from 0.1 to 5000 μM.
• Real-time detection of Ni²⁺ concentration changes with subcellular resolution was achieved in E. coli, yeast, and mammalian cells.

Conclusions:

• FProNiM is a robust and specific sensor for real-time intracellular Ni²⁺ detection.
• The probe facilitates non-invasive monitoring of nickel ion dynamics in live cells.
• This technology provides a valuable tool for studying nickel homeostasis and toxicity mechanisms.