Applications

One of the most important applications of Pokegama biosensors is measuring free zinc levels in cells, usually by fluorescence microscopy. Users may simply wish to map the concentrations of free zinc inside the cell(s) of interest, or look at the time rate of change of free zinc inside the cell or a particular organelle, perhaps due to transporter activity.  This Application Note describes how to do that.  

Introducing the Sensor into the Cell: 

For this Application Note we will focus on Pokegama’s excitation ratiometric zinc biosensors, which consist of two components, a fluorescent-labeled apocarbonic anhydrase variant, and a fluorescent sulfonamide (see  Pokegama Web Resources: "Why use biosensors to measure free metal ions?" and "Introduction to Zinc Sensing").  The fluorescent-labeled sensor protein can be introduced into the cell either by expressing it (having inserted the DNA for the fluorescent-protein –carbonic anhydrase fusion protein as a plasmid or other vector into the cell), or by using a TAT peptide to induce the cell to take up the sensor protein (see Products: "Fluorescent Biosensor Proteins").  The TAT peptide approach works with most cells, and is straightforward: the apo-sensor protein is introduced (for instance) as a 1 μM solution in zinc-free buffer to a cell monolayer in a dish; after 30 minutes incubation at 37 C., the protein solution is decanted (the uptake of the fluorescent protein sensor can be verified by fluorescence microscopy at excitation 530 nm, emission 600 nm.  For expression, the sensor DNA ligated into a suitable plasmid or vector such as a lentivirus is mixed with cells for two hours, then expression is induced by addition of IPTG or other suitable reagent; again, the success of the expression is judged by the appearance of the fluorescent protein label in the plate reader or microscope.  We note that with different cell types, conditions may need to be adjusted.  

Introduction of fluorescent sulfonamide into the cell

Calibration of Sensor in the Microscope or Plate Reader

It is essential to calibrate measurements of free zinc in cells (or wherever) 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.   

Calibration inside the Cell 

Calibration within the cell (in situ calibration) is harder than external calibration, for a couple of reasons: the calibration buffers may be sufficiently unphysiological as to perturb the cell, the zinc concentrations may be toxic, and/or the ionophore may have deleterious effects  However, it may be desirable or essential to calibrate in situ if it is known or suspected that there is significant background fluorescence in the cell at the relevant wavelengths, or the pH is much different inside the cell or organelle of interest, or there is some effect of the cellular milieu on the sensor or its fluorescence.   The procedure is the same as measuring the free zinc inside the cell above, except that the cells are immersed in a metal ion buffer of known free zinc concentration (like our MetalloBuffer series), and the cells treated with the ionophore pyrithione (2-thiopyridine) at 5 uM for 30 minutes: the ionophore transfers zinc ion across the cell membrane such that the cytoplasmic free zinc concentration equilibrates with and matches the external free zinc concentration.  Pokegama has under development calibration buffers formulated in widely used basic salts media, and selected common growth media.  

For non-ratiometric sensors it is desirable to use the high affinity cell-penetrant zinc ligand TPEN to drastically lower the free zinc in the interior of the cell to measure the fluorescence of the unbound sensor.  

References

B. J. McCranor, H. Szmacinski, H-H. Zeng, A.K. Stoddard, T.K. Hurst,  C. A. Fierke,  J.R. Lakowicz,  and R. B. Thompson, "Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells with a Cu(II)-Selective Carbonic Anhydrase-Based Biosensor," Metallomics 6, 1034-1042 (2014); DOI: 10.1039/c3mt00305a; NIHMS 585083 PMID: 24671220

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

R. A. Bozym, A. K. Stoddard, C. A. Fierke, and R. B. Thompson, “Measuring picomolar exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor,” ACS Chemical Biology 1(2) 103 – 111 (2006)  PMID: 17163650.

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