Metal ion sensors and methods of detecting metal ions

A method of detecting metal ions in a biological sample, comprising:

• (i) providing a polypeptide metal ion sensor comprising an engineered green fluorescent polypeptide (GFP) having a heterologous metal ion binding site, wherein said engineered GFP is a variant of amino acid sequence SEQ ID NO:
• 37 and having the amino acid substitutions corresponding to L22V and I218M and, when having a metal ion species bound thereto, has an elevated fluorescence output compared to the polypeptide SEQ ID NO:
• 37 binding to the same metal ion species at 37° C., wherein the amino acid sequence of said engineered GFP comprises any one of the amino acid sequences selected from the group consisting of sequences having 95% similarity to any one of SEQ ID NOs:
• 71, 72, 73, 74, 79, 81, and 82, or wherein said engineered GFP has 100% similarity to SEQ ID NO:
• 80 or said engineered GFP consists of SEQ ID NO:
• 75;
• (ii) delivering the polypeptide metal ion sensor or an expression vector having an nucleic acid sequence encoding said metal sensor to a biological sample;
• (iii) detecting a first fluorescent signal emitted by said sensor;
• (iii) generating a physiological or cellular change in the biological sample;
• (iv) detecting a second fluorescent signal emitted by said sensor after step (iii);
• and (v) comparing the first and second fluorescent signals, wherein a ratiometric change in at least one of a wavelength, an intensity, and lifetime between the first and second fluorescent signals indicates a change in the rate of release or intracellular concentration of a metal ion in the sample.