Introduction

As has been pointed out by many others, the total concentration of zinc or other metal ions in aqueous media of many types (aqueous buffers, groundwater, seawater, cytoplasm, culture media, etc.) is often less important in understanding biological or geochemical effects of the metal ion than the "free" (also known as "exchangeable", "mobile", or "labile") concentration which is weakly bound to more labile ligands.  This is because when the dissolved metal ion is tightly bound to (often chelating) ligands, it is generally less able to exchange ligands with others and thus exert effects, especially biological effects.  Thus it is frequently more useful to measure the free form; please also note that the distinction between free and tightly bound is not hard and fast, but more commonly empirical and operational. 

Measurement of Free Zinc Ion in Aqueous Media with Biosensors

Measurement of free zinc using Pokegama carbonic anhydrase-based fluorescence biosensors in many aqueous samples that themselves have the capacity to buffer the free metal ions is straightforward: the fluorescent apobiosensor is dissolved in the medium at one micromolar concentration or less, together with a few micromolar of  a suitable sulfonamide (e.g., Pokegama D-0005 4-(5-dimethylamino-phenyl-2-oxazolyl)benzenesulfonamide in DMF), and the fluorescence ratio (exc 380 and 530 nm, emission 600+ nm) measured.  Samples that have capacity to buffer the zinc concentration include most biological fluids and growth media, as well as cellular cytoplasm; some groundwater and seawater may also be capable of buffering metal ion concentrations.  By comparison, many simple buffers have little or no capacity to buffer zinc; important exceptions are citrate, Tris, phosphate, and Bicine buffers.  Thus the procedure described above fails with a reasonably pure natural water sample or simple buffer like MES or HEPES, since adding the sensor at near micromolar concentrations binds nearly all the available metal without becoming significantly saturated, producing an erroneous result: in these circumstances it is necessary to use a fiber optic or other sensor that employs only a small absolute amount of sensor to interrogate the sample.  

Calibration of Sensor in Aqueous Media

It is essential to calibrate measurements of free zinc using buffers of known free zinc concentration such as Pokegama MetalloBuffers.  The reason is that optical properties of filters, monochromators, detectors, and other components of the instruments differ, such that the actual measured fluorescence intensity ratio values corresponding to particular free zinc concentrations also differ among microscopes, plate readers, and spectrofluorometers.  

Other important caveats  for such measurements include using calibration buffers that reflect both the pH and ionic strength of the medium under test: for instance, the higher pH and ionic strength of sea water usually require special buffers.  Oftentimes cell growth media contain serum albumin and other fairly strong metal ion ligands that buffer metal ions; moreover, media that contain, for instance, fetal calf serum not only add ligands but zinc ion (and other metals) as well; Pokegama has calibration products for these applications under development.  

References

R.B. Thompson, H.-H. Zeng, D. Ohnemus, B. McCranor, M.L. Cramer, J. Moffett, “Instrumentation for fluorescence-based fiber optic biosensors,” in Methods in Enzymology: Fluorescence Spectroscopy Vol. 450 (L. Brand and M.L. Johnson, editors) New York: Elsevier, pp 303 – 329 (2008). PMID 19152867

D. Wang, T. K.  Hurst, R. B. Thompson, and C. A. Fierke “Genetically Encoded Ratiometric Biosensors to Measure Intracellular Exchangeable Zinc in Escherichia coli” J. Biomed. Opt. 16(8) 087011/1-11 (2011) [DOI: 10.1117/1.3613926. PMID: 21895338 PMCID: PMC3166341