Chemistry: analytical and immunological testing – Metal or metal containing
Reexamination Certificate
1998-04-30
2001-09-04
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Metal or metal containing
C436S074000, C436S079000, C436S080000, C436S081000, C436S083000, C436S084000, C436S166000, C436S172000
Reexamination Certificate
active
06284544
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to homogeneous methods for the detection, determination, and quantitative analysis of Cu (II), Zn (II) and other transition metal ions in solution and is based on the measurement of changes in fluorescence polarization (anisotropy) in response to metal ion concentration.
2. Background Art
Although many analytical methods exist for the quantitative analysis of metals, none utilize fluorescence polarization (anisotropy). Present analytical methods include graphite furnace atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, mass spectroscopy, and numerous electrochemical and luminescence techniques, including phosphorescence, chemiluminescence and fluorescence. Common to all these methods are requirements for complex, expensive, sizable and fragile instrumentation; skilled operators; and elaborate experimental and separation procedures to achieve acceptable precision and accuracy.
At present, one skilled in the art would not find it obvious to utilize fluorescence polarization (anisotropy) for the quantitative analysis of metal ions in solution. It is well known in the art that most metal ions are not photoluminescent in solution. Even if we consider transition metal ions which have weak absorption bands in the uv/visible
ear IR; the excitation of these metal ions with plane polarized light generally effects only depolarized fluorescence due to the radial symmetry of the absorbing species. Alternatively, if an indirect approach is used wherein the binding of a metal ion to a metallo-fluorescent indicator is monitored, one also finds little or no change in fluorescence polarization (anisotropy), as there is no change in the rotational motion of the fluorescent indicator in response to the binding of a metal ion. Even if the binding of the metal ion perturbs the fluorescence lifetime of the metal-free fluorescent indicator, the rotational motion of the indicator will probably be sufficiently rapid in comparison to its rate of emission that its emission will be completely depolarized whether the metal ion is present or not, resulting in an imperceptible change in fluorescence polarization. These representative and other difficulties have prevented the application of fluorescence polarization (anisotropy) to metal ion analysis in aqueous solutions.
The present invention extends for the first time the use of fluorescence polarization (anisotropy) for the detection and quantitative analysis of metal ions in aqueous solutions. This invention comes more than twenty-five years after the first application of fluorescence polarization (anisotropy) to the detection and quantitation of fluorescent labeled happens and antigens with suitable antibodies. The present invention is not constrained by immunoassay reaction kinetics between labeled and unlabeled antigen or hapten but is simply predicated on the use of a photoluminescent indicator that will emit polarized light in a measurably different manner on the formation of a macromolecule-metal ion complex. A complex which in the preferred embodiments relies on either the metal dependent binding of a photoluminescent indicator to a macromolecule, or the binding of a metal ion to a macromolecule that has previously been labeled with a photoluminescent label. Additionally, the present invention is not constrained by the difficulties in raising antibodies to metal ions in solution.
3. Fluorescence Based Methods of Analysis
Assays for chemical entities based on changes in fluorescence intensity are well known. Ullman et al. (1981) (U.S. Pat. No. 4,261,968) discloses a proximity-dependent quenching homogeneous immunoassay wherein a fluorescent-labeled antibody is brought in contact with a sample containing a ligand (antigen or hapten) and a quencher-labeled ligand; the free ligand (of unknown concentration) and the quencher-labeled ligand (of known concentration) compete for the same antibody binding site. Quenching of the label's fluorescence emission is inversely proportional to the unlabeled antigen concentration, and serves as the measurement variable for determining the concentration of free ligand. Key to this method is the use of a ligand comprising two chromophores, termed a fluorescer-quencher pair with non-polymeric ligands being in the molecular weight range of 125 to 2,000, well above the atomic weights of most metal ions of interest. In relation to the present invention, Ullman does not teach the analysis of free metal ions in solution, or the use of polarization (anisotropy) as the measurement variable.
Numerous fluorometric methods for the analysis of metal ions in solution are known and vary primarily in terms of signal transduction. Metal ion concentrations may be transduced as changes in fluorescence intensity or as changes in the ratios of fluorescence intensities at two different wavelengths, or as changes in fluorescence lifetimes. Although, lifetime and ratiometric methods are qualitatively similar with respect to their freedom from spurious variations in fluorescence intensity, the physical measurement of fluorescence lifetime is costlier and technically more difficult than a simple intensity measurement, especially in the context of imaging applications. Regardless of these drawbacks, lifetime analysis has been preferred for reasons that include its broad dynamic test range in sample concentrations which may exceed five orders of magnitude in some cases (Szmacinski, H. and J. R. Lakowicz (1993) Anal. Chem. 65, 1668-1674); Thompson, R. B. and M. W. Patchan (1993) in
Proc. of the SPIE Conference on Chemical, Biochemical and Environmental Fiber Optic Sensors V.
R. A. Lieberman (Ed.) Bellingham, Wash., pp 296-306). Lifetime-based analysis also minimizes errors associated with optics, fluorophore concentration and detector sensitivity.
Methods based on the measurement of changes in fluorescence intensity include:
U.S. Pat. No. 4,762,799 (1988) to Seitz et al. which discloses an ionophore-based cation detection system wherein the concentration of selected alkali metal ions in an aqueous sample may be determined by detecting the fluorescence emission of a fluorescent anionic material.
U.S. Pat. No. 5,154,890 (1992) to Mauze and Rupp which discloses a homogeneous method for detecting the concentration of potassium ions in solution where the ion concentration is made proportional to an increase in fluorescence intensity on the formation of a complex comprising a fluorophore labeled molecule which selectively binds potassium ions.
U.S. Pat. No. 5,516,864 (1996) to Kuhn and Haugland which in part discloses the use of xanthylium-based dyes with metal-binding N,N′-diaryldiaza crown ethers, the best known class of ionophores, for the detection of alkali-metal ions in aqueous solution by measuring spectral changes in the fluorescence emission of an indicating dye.
These intensity based methods, however, are all sensitive to artifacts, as any change in fluorescence intensity, regardless of its origin may be misinterpreted as a change in concentration. Changes in light scattering, or variations in excitation light intensity and or photobleaching may easily be misinterpreted as a change in the concentration of the metal ion. Although the accuracy and precision of these methods may be improved through the use of internal fluorescent standards, or with the monitoring of excitation intensity or with the use of kinetic methods of analysis, careful and even repeated calibration of reagents and instrumentation are required to minimize spurious variations in signal intensity.
Recognizing these limitations, Tsien and other researchers developed indicators whose fluorescent properties such as excitation or emission maxima, shifted in conjunction with the binding of analyte, permitting concentration to be described not in terms of absolute fluorescence intensity but rather in terms of a ratio of fluorescence intensities at different wavelengths (Tsien, R. Y. (1989) Annu. Rev. Neurosci. 12, 227-253); Thompson, R. B. and E
Christianson David
Elbaum Daniel
Feliccia Vincent L.
Ge Zhengfang
Maliwal Badri P.
Birch & Stewart Kolasch & Birch, LLP
Soderquist Arlen
University of Pennsylvania
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