Excitation ratiometric fluorescent biosensor for zinc ion at picomolar levels

Zinc is a metal ion of increasing significance in several biomedical fields, including neuroscience, immunology, reproductive biology, and cancer. Fluorescent indicators have added greatly to our understanding of the biology of several metal ions, most notably calcium. Despite substantial efforts, only recently have zinc indicators been developed which are sufficiently selective for use in the complex intra- and extracellular milieus, and which are capable of quantifying the free zinc levels with some degree of reliability. However, these indicators (such as FuraZin-1 and Newport Green DCF) have only modest sensitivity, and there is growing evidence that significantly lower levels of free zinc may be biologically relevant in some instances. We have adapted the peerless selectivity and sensitivity of a carbonic anhydrase-based indicator system to an excitation ratiometric format based on resonance energy transfer: i.e., where the zinc ion level is transduced as the ratio of fluorescence intensities excited at two different excitation wavelengths, which is preferred for fluorescence microscopy. The system exhibits more than a 60% increase in the ratio of intensity excited at 365 nm to that excited at 546 nm (emission observed at 617 nm). The detection limit is about 10 pM in zinc buffered systems, a 10-1000-fold improvement on the Fura indicators (which respond to Ca and Mg as well), and a 10 000-fold improvement on the recently described FuraZin-1.