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Zinc ion is of growing interest in medicine and biology generally, and especially in the ischemic brain and other tissues. We have developed ratiometric fluorescence-based biosensors for the study of zinc in these systems; the biosensors use apocarbonic anhydrase variants as recognition elements that offer high sensitivity and selectivity. We report continuous in situ, in vivo measurement of nanomolar extracellular zinc in the brain of an animal model of ischemia using a ratiometric fiber optic biosensor. We also report the development of an expressible excitation ratiometric indicator of zinc ion suitable for use in cells that exhibits picomolar sensitivity. Finally, we also report the discovery that the Zn complex of the chelator TPEN seems to be comparably apoptogenic to the free chelator itself.

We describe new methods for the study of zinc in biological specimens. Intracellular free zinc was determined at levels down to picomolar using an excitation ratiometric fluorescence-based biosensing approach using a carbonic anhydrase variant as transducer. A new fiber optic sensor suitable for in vivo use is also described using laser excitation and an emission ratiometric approach; the zinc concentration range of sensor response can be selected to fit the application.

A continuing issue in chemical oceanography and environmental monitoring is the need for frequent and continuous monitoring of analytes in complex matrices such as sea water and ground waters. Particularly for analytes at trace levels such as Cu(II) in sea water, sampling and analysis of discrete specimens is costly, slow, labor intensive, employs ship time inefficiently, and risks error by contamination. We have developed a fluorescence lifetime- based fiber optic biosensor which demonstrates real time determination of free Cu(II) in coastal waters, in situ, with a subpicomolar detection limit.

Recently, there has been substantial interest in reducing the levels of toxic heavy metals in wastewater effluents from activities such as shipyards. Of particular interest is copper, which comprises tens of percent by weight of the hundreds of pounds of antifouling paint coating the bottom of a large vessel, but which is toxic to commercially important shellfish at sub-part per billion levels. As a result wastewater effluents must be monitored closely with sensor(s) capable of rapidly and accurately detecting excess copper in time to prevent release. We have pursued a fluorescence-based biosensing approach to obtain sub-ppb sensitivity for Cu(II) and immunity from interference from other cations abundant in sea water, such as Ca, Mg, and Sr. Our approach uses a protein, apocarbonic anhydrase II, as a very sensitive and selective ligand for Cu(II) which transduces the (reversible) binding of the metal as a change in fluorescence intensity, lifetime, or anisotropy, the first two of which may be conveniently measured through optical fiber. Thus we have been able to measure sub-ppb levels of Cu added to sea water, and to characterize the speciation of the Cu(II) to some degree, due to the presence of other ligands.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In developing fluorescence-based biosensors for rapidly determining metal ions such as zinc or copper in complex media such as sea water, serum, cerebrospinal fluid, or the interior of a cell, several issues must be considered. Among these are the selectivity, sensitivity, ease of calibration, speed of response, reversibility, stability, and the ease of immobilization onto a solid substrate. While the first three have been dealt with in the design of our transducer molecule, apocarbonic anhydrase II, the others remain to be considered. In this paper we examine the stability of the apoprotein to storage at various temperatures and pHs with a view to establishing storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a fluorescent-labeled apo-CA variant on quartz to determine its sped, sensitivity, and kinetics of response.

The response time of biosensors which reversibly bind an analyte such as a metal ion is necessarily limited by the kinetics with which the biosensor transducer binds the analyte. In the case of the carbonic anhydrase-based biosensor we have developed the binding kinetics are rather slow, with the wild type human enzyme exhibiting an association rate constant ten thousand-fold slower than diffusion-controlled. By designed and combinatorial means the transducer may be mutagenized to achieve nearly diffusion-controlled association rate constants, with commensurate improvement in response. In addition, a variant of apocarbonic anhydrase has been immobilized on quartz, and is shown to response rapidly to changes in free copper ion in the picomolar range.

b1c University Medical Center, Durham, NC 27710ABSTRACTRecently, we have shown that metal ions free in solution may be determined at low levels by fluorescenceanisotropy (polarization) measurements. Anisotropy measurements enjoy the advantages of wavelength ratiometrictechniques for determining metal ions such as calcium, because anisotropy measurements are ratiometric as welLFurthermore, fluorescence anisotropy may be imaged in the microscope. An advantage ofanisotropy notdemonstrated for wavelength ratiometric approaches using indicators such as Fura-2 and lndo-1 is that underfavorable circumstances anisotropy-based determinations exhibit a much broader dynamic range in metal ionconcentration. Determinations offree Zn(ll) in the picomolar range are demonstrated.

Recent work has suggested that free Cu(II) may play a role in syndromes such as Crohn's and Wilson's diseases, as well as being a pollutant toxic at low levels to shellfish and sheep. Similarly, Zn(II) has been implicated in some neural damage in the brain resulting from epilepsy and ischemia. Several high sensitivity methods exist for determining these ions in solution, including GFAAS, ICP-MS, ICP-ES, and electrochemical techniques. However, these techniques are generally slow and costly, require pretreatment of the sample, require complex instruments and skilled personnel, and are incapable of imaging at the cellular and subcellular level. To address these shortcomings we developed fluorescence polarization (anisotropy) biosensing methods for these ions which are very sensitivity, highly selective, require simple instrumentation and little pretreatment, and are inexpensive. Thus free Cu(II) or Zn(II) can be determined at picomolar levels by changes in fluorescence polarization, lifetime, or wavelength ratio using these methods; these techniques may be adapted to microscopy.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In comparison to metal ions in aqueous solutions, there are few methods for analysis of small anions such as cyanide, cyanate, carbonate, sulfide, and nitrate. Yet such analytes are important as environmental pollutants and as reagents and byproducts of industrial processes, paper manufacture, and mining. For some time we have been developing fluorescence-based fiber optic biosensors for metal ions such as zinc, cobalt, copper, mercury, nickel and cadmium, using the unparalleled selectivity and avidity of a metalloenzyme, human carbonic anhydrase. In the cases of Cu2+, CO2+, and Ni2+, we made use of the characteristic weak d-d absorbance bands of these metals when bound in the active site of the enzyme to serve as a fluorescence energy transfer acceptor for a suitably positioned fluorescent label attached to the enzyme. For this approach the intensity and lifetime of the fluorophore reflect the degree of energy transfer, and therefore the concentration of the metal. To measure certain anions such as cyanide and cyanate, we made use of the well-known perturbation of the d-d absorbance of Co2+ when an anion inhibitor becomes bound and inhibits the enzyme. These changes in absorbance modify the overlap integral with a suitable fluorescent label, and thereby the degree of energy transfer, resulting in a perturbation of the intensity and lifetime.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In order to gain wavelength and analyte flexibility, we have recently altered the transduction approach of our fluorescence-based biosensor. Briefly, binding of metal ions such as zinc to the active site of carbonic anhydrase is transduced by metal-dependent binding of a colored inhibitor to a fluorescent derivative of the enzyme; in the absense of metal the inhibitor does not bind and the label fluorescence is unquenched, but at higher metal concentrations the inhibitor binds, energy transfer occurs with moderate efficiency and the fluorescent label exhibits reduced intensity and lifetime. Inasmush as Forster energy transfer is distance dependent the position of the fluorescent label on the surface of the enzyme has some impact on the performance of the sensor. We designed, produced, and expressed site-selective mutants of carbonic anhydrase which could be unambiguously derivatized with suitable fluorescent labels, and which gave much improved responses to zinc ion compared with randomly derivatized wild type enzyme.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.