Chemistry: analytical and immunological testing – Metal or metal containing – Li – na – k – rb – cs – fr – be – mg – ca – sr – ba – ra
Reexamination Certificate
2000-09-20
2002-10-29
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Metal or metal containing
Li, na, k, rb, cs, fr, be, mg, ca, sr, ba, ra
C436S177000
Reexamination Certificate
active
06472221
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of optically measuring sodium or potassium concentrations in solutions.
BACKGROUND OF THE INVENTION
Measurement of electrolytes in the blood is an important aspect of clinical chemistry. The most common techniques used for measuring electrolytes in aqueous environments are flame photometry or ion selective electrodes (ISE). Both flame photometry and ISE are highly evolved technologies which provide good precision and accuracy over a wide range of concentrations. These methods require good operator skills and meticulous maintenance of equipment for optimal performance. Additionally, these methods require the handling of blood, which is expensive and associated with significant health risks to the operator.
In recent years there has been an increased emphasis on the use of optical probes for clinical chemistry. Optical methods have been developed to monitor blood gases (pH, pCO
2
and pO
2
) in whole blood. Optical techniques can be relatively inexpensive, have excellent signal to noise ratio and because they are virtually instantaneous in their response time they can provide immediate answers for point-of-care testing.
Fluorescence assays have been shown to be approximately three orders of magnitude more sensitive than absorption methods and the fluorescence assays also permit analysis using smaller amounts of probe in the assay solution. Moreover, in contrast to absorption methods, fluorescence probes do not require additional chemical reagents and complex sample manipulation.
At present, most fluorescence assays are based on the change in fluorescence intensity which occurs in response to an analyte. While fluorescence intensity measurements are simple and accurate in the laboratory, these are often inadequate in real world situations. A significant disadvantage of intensity-based sensing is the problem of referencing. The intensity depends on a number of instrumental factors and on the probe concentration. For instance, the intensity for a given sensor can depend on the details of the optical correction efficiency or on the concentration of the fluorophore in the sensor itself. Hence, frequent recalibration is needed for most intensity-based measurements.
A method in which a luminescent ligand is added to a sample to be analyzed in the form of a photoluminescent probe having intrinsic analyte-induced lifetime changes is known in the art. Lifetime measurements are advantageous over intensity measurements because they can be performed in optically dense samples or turbid media and are independent of and/or insensitive to photo bleaching, probe wash out or optical loss. The lifetime changes are measured using known time-resolved or phase-modulation fluorometry methods. A description of the phase modulation fluorometry methods are found in U.S. Pat. No. 5,624,847 ('847 patent) which is incorporated by reference herein in its entirety. The step of adding a luminescent ligand (i.e., probe) to the sample to be analyzed requires matching a particular probe to a particular analyte, so that at least a portion of the sample will be non-covalently bound to the probe resulting in both bound and unbound species of the probe.
While the use of lifetime-based sensing and phase-modulation fluorometry disclosed in the prior art is useful for determining analytes in certain solutes, the problems of quantification associated with other solutions, particularly extracellular ones, were not previously recognized. There has been a clinical need to extend the use of lifetime-based sensing and phase-modulation fluorometry to such solutions.
SUMMARY OF THE INVENTION
One of the problems not previously recognized is the difficulty of measuring alkali metal ions that have similar properties and are difficult to distinguish one in the presence of the other. In blood, the mean concentrations of sodium and potassium are 140 mM and 4.5 mM, respectively. It is difficult to achieve selective detection of K
+
in the presence of a 30 fold excess of chemically similar Na
+
.
Development of a K
+
sensor using as ionophore, like valinomycin, based on the inner filter effect (H. He, H. Li, G. Mohr, B. Kovác, T. Werner, and O. S. Wolfbeis, Novel Type of Ion-Selective Fluorosensor Based on the Inner Filter Effect:
An Optrode for Potassium.
Anal. Chem.
65, 123-127, 1993) or energy transfer (J. N. Roe, F. C. Szoka, and A. S. Verkman, Optical measurement of aqueous potassium concentration by a hydrophobic indicator in lipid vesicles.
Biophys. Chem.
33, 295-302, 1989; J. N. Roe, F. C. Szoka, and A. S. Verkman, Fibre optic sensor for detection of potassium using fluorescence energy transfer.
Analyst
115, 353-368, 1990) has been attempted. One difficulty with energy transfer sensing is that the extent of energy transfer strongly depends on acceptor concentration, so that the sensor will require frequent calibration. This problem can potentially be circumvented by using covalently linked donors and acceptors. However, few such sensors have appeared due to the difficulties with chemical synthesis.
The present invention provides a method of optically measuring Na
+
or K
+
in a sample such as blood, containing concentrations of up to 6.5 mM of K
+
and of up to 160 mM of Na
+
. A photoluminescent ligand probe having intrinsic sodium- or potassium-induced lifetime changes is added to the sample to be analyzed. The probe is non-covalently bound to the ionic solute of either sodium or potassium to form a Na
+
-bound or K
+
-bound probe species wherein bound and unbound species of the probe exist in the sample and the probe has intrinsic Na
+
-induced or K
+
-induced lifetime changes. The sample is excited with radiation and the resulting emission beams from the bound and unbound species are detected. The apparent luminescence lifetime of the emission is calculated to determine concentration of either Na
+
or K
+
in the sample. Because of the similar chemical properties of Na
+
and K
+
, probes are utilized such that the presence of high levels of Na
+
allows the measurement of K
+
and the presence of K
+
allows the measurements of Na
+
.
REFERENCES:
patent: 5134232 (1992-07-01), Tsien et al.
patent: 5624847 (1997-04-01), Lakowicz et al.
patent: 5759767 (1998-06-01), Lakowicz et al.
patent: 98/16656 (1998-04-01), None
A. Minta et al, “Flourescent Indicators forCystolic Sodium” The Journal of BiologicalChemistry, 1989, vol. 264, pp. 19449-19457.*
A. T. Harootunian et al, “Fluorescence RatioImaging of Cystolic Free Na+ in IndividualFibroblasts and Lymphocytes” The Journal ofBiological Chemistry, 1989, vol. 264, pp. 19458-19467.*
H. Satoh et al, “Quantification odIntracellular Free Sodium Ions by Using aNew Fluorescent Indicator, Sodium-BindingBenzofuran Isophthalate in Guinea PigMyocytes” Biochemical and BiophysicalResearch Communications, 1991, vol. 175,pp. 611-616.*
J. R. Lakiwicz et al, “FluorescenceLifetime-Based Sensing of Blood Gases andCations” SPIE, 1992, vol. 1648, pp. 150-163.*
R. Crossley et al, “Synthesis and Propertiesof a Potential Extracellular FluorescentProbe for Potassium” Journal of the ChemicalSociety Perkin Transactions 2, 1994, pp. 1615-1623.*
G. P. Amorino et al, “Intracellular Na+Measurements Using Sodium Green Tetraacetatewith Flow Cytometry” Cytometry, 1995, vol. 21, pp. 248-256.*
H. Szmacinski et al. “Sodium Green as a Potential Probe forIntracellular Sodium Imaging Based on Fluorescence Lifetime” Analytical Biochemistry, 1997, vol. 250, pp. 131-138.*
Enrico Gratton, Resolution of Mixtures of Fluorophores Using Variable-Frequency Phase and Modulation Data, Biophys. J., 1984, pp. 479-486, vol. 46.
R.P. Haugland, Alternative Fluorescence Techniques for Measuring Na+ and K+, Handbook of Fluorescent and Research Chemicals, 1996, p. 575, Molecular Probes, Inc. Eugene, OR.
Haurui He et al., Novel Type of Ion-Selective Fluorosensor Based on the Inner Filter Effect: An Optrode for Potassium, Analytical Chemistry, 1
Lakowicz Joseph R.
Szmacinski Henryk
Rothwell Figg Ernst & Manbeck
Soderquist Arlen
University of Maryland Baltimore
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