**6. Carbonic anhydrase-based biosensing of metal ions**

In the last years the affinity of carbonic anhydrase for metal ions has been applied for the development of fluorescence based biosensors for determination of free metal ions in solution using variants of human carbonic anhydrase (apoCA). In particular, Cu2+, Co2+, Zn2+, Cd2+, and Ni2+ have been determined at concentration down the picomolar range (Fierke and Thompson, 2001; Thompson and Jones, 1993; Mey et al., 2011) by changes in fluorescence emission (Thompson et al., 2000) and excitation wavelength ratios (Thompson et al., 2002), lifetimes (Thompson and Patchan, 1995), and anisotropy (polarization) (Elbaum et al., 1996; Thompson et al., 2000). The sensitivity, selectivity, analyte binding, kinetics and stability of the biosensors have been improved by subtle modification of the protein structure by directed mutagenesis (Kiefer et al., 1995; Hunt et al., 1999; DiTusa et al., 2001; McCall et al., 2004; Burton et al, 2000). These studies have hallowed the development of highly selective and sensitive fluorescence-based biosensors for Zn2+ e Cu2+, which have been shown to be viable approach in some important applications. In fact, the CA-based Cu2+ biosensor has been used to obtain real-time measurement of free Cu(II) at picomolar concentrations in seawater (Zeng et al., 2003), while the CA-base Zn2+ biosensor has been used for measurement of free Zn ion at picomolar levels in cultured cells (Bozym et al, 2004).
