Why use biosensors to measure free metal ions?
Metal ion sensors: There are quite a variety of sensors of differing kinds designed to detect or quantify various metal ion species. For the most part, we are interested in applications in cell biology, biochemistry, clinical analyses, bio/geochemistry, and environmental analyses, and thus primarily interested in measuring metal ion species dissolved in aqueous media. Sensors capable of such analyses include devices which transduce the presence or level of the metal ion analyte initially as a change in electrical voltage or current, or an optical signal. Particularly for applications in biology, optical sensors are most useful because they are easily introduced into cells and compartments therein, and the analyte levels mapped by imaging in relation to the cell, tissue, or organism. For the most part optical sensors for metal ions are molecules which exhibit changes in color, fluorescence, or chemi- or bioluminescence upon binding the analyte. Please see the discussion of “free” vs bound metal ions in Resources.
Attributes/figures of merit for sensors: For sensors of all types figures of merit include sensitivity, usually defined by the signal to noise ratio and detection limit; selectivity, meaning the ability to respond to the analyte of interest and not potential interferents; dynamic range, meaning the range of analyte levels which can be accurately quantified (and not the signal level difference between free and analyte-bound sensor); and speed of response. Other aspects may be important as well, including cell penetrability, targeting to organelles in cells, toxicity, susceptibility to washout or ejection from cells by multidrug resistance transporters (MDR) or other means, resistance to photobleaching, fluorescent “brightness”, and cost.
Advantages of biosensors for metal ion sensing:the Fierke-Thompson sensors offered by Pokegama are termed biosensors in the oldest sense of the word because they are derived from a biological molecule, a variant of carbonic anhydrase. They thus differ from other, small molecule sensors such as Zn-AF2, FluoZin-3TM, and ZinPyr-1, where typically a metal ion-binding moiety is covalently attached to an absorbing or fluorescing moiety. The biosensors offer several advantages. First, they are very sensitive, having demonstrated quantitation of free Zn2+ or Cu2+ at picomolar levels and below. Secondly, they are very selective, having demonstrated such sensitivity in complex matrices such as cytoplasm and sea water where other divalent metal ions that might potentially interfere are present at billion-fold higher concentrations. They have rapid response: the association rate constants for free Zn2+ and Cu2+ with the E117A variant and wild type human carbonic anhydrase II, respectively, are both within an order of magnitude of diffusion-controlled: e.g., as fast as possible for a reversible binding sensor. The biosensors when configured as fusion proteins can be selectively expressed within certain cell types or organelles therein for localized measurements. Because the metal ion-binding site(s) on the protein may have their affinity, speed, and selectivity modified almost independently of the optical transduction moiety, a sensor can often be configured for a particular application. Finally, the binding of the metal ion may be transduced not only as a change in fluorescence intensity, but as a change in intensity ratios at two different excitation or emission wavelengths, fluorescence anisotropy, fluorescence lifetime, or bioluminescence intensity ratio. Thus biosensors are well suited for many applications of interest.