Chemistry: analytical and immunological testing – Metal or metal containing – Organometallic compound determined
Reexamination Certificate
1999-03-15
2001-05-01
Wessendorf, T. (Department: 1627)
Chemistry: analytical and immunological testing
Metal or metal containing
Organometallic compound determined
C436S077000, C436S074000, C436S073000, C436S501000, C530S413000, C530S412000, C530S350000
Reexamination Certificate
active
06225127
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention generally relates to the detection, determination, and quantitation of certain ions and small molecules. The approach is based upon quenching of a fluorescent label attached to a macromolecule, often due to fluorescence energy transfer to a colored inhibitor or certain metal ions bound to the macromolecule.
BACKGROUND OF THE INVENTION
For scientific, regulatory, or other applications, many persons, institutions, and agencies often require analyses of samples to determine whether such small molecule and ionic analytes are present. Examples of such analyses include the determination of metal ions in sea water to understand the processes of chemical oceanography; determination of toxic materials such as Hg(II), Ni(II), CN
−
, or HS
−
in groundwater or wastewater; detection of the corrosion of metal alloys by the presence of Co(II), Zn(II), or Cu(II) in condensates; or the presence of metals in lubricating oil as an indicator of machinery wear and incipient failure.
Many methods are known in the art for such analyses. For metal ions, such methods include graphite furnace atomic absorption spectrophotometry, inductively coupled plasma atomic emission spectroscopy and mass spectroscopy, various electrochemical means, and fluorescence spectroscopy using metallofluorescent indicators. For common anionic analytes there are fewer techniques available; they include ion chromatography, mass spectrometry, and electrochemical means.
Most of these techniques involve analysis of single or multiple discrete samples in a specialized instrument which may not be close to the sample. This is a particular drawback for analytical tasks that require a continuous or quasicontinuous determination of the analyte with real time readout of the result; require samples to be collected from remote, inaccessible, or hazardous environments; or require such extensive sampling that it is prohibitively costly. For many of these methods a sensor capable of remotely, continuously, and selectively monitoring the analyte of interest in situ is required, and subsequent reporting of the results of the analysis back to the operator in real time.
Improvements have been made in the development of fluorescence-based sensors for a variety of applications (Thompson, R. B. (1991) in
Topics in Fluorescence Spectroscopy
, Vol. 2:
Principles
, Lakowicz, J. R. (Ed.) Plenum Press, NY; Wolfbeis, O. S. (Ed.) (1992)
Fiber Optic Chemical Sensors and Biosensors
Vols. I and II, CRC Press, Boca Raton, Fla.; Lakowicz, J. R., and Thompson, R. B. (Eds.) (1993)
Proc. of the SPIE Conference on Advances in Fluorescence Sensing Technology
Vol. 1885, Society of Photooptical Instrumentation Engineers, Bellingham, Wash.) A central issue in the development of such sensors has been the means of transduction, whereby the presence or relative amount of the chemical analyte is transduced as a change in the fluorescence which may be quantitated. Thus, workers in the field have mainly transduced analyte levels as changes in fluorescence intensity (Thompson (1991); Saari, L. A., and Seitz, W. R. (1982)
Anal. Chem.
54, 821; Thompson, R. B. and Ligler, F. S. (1991) in
Biosensors with Fiber Optics.
, Wise, D., and Wingard, L. (Eds.) pp. 111-138, Humana Press, Clifton, N.J.), or ratios of fluorescence intensity at two different wavelengths (Tsien, R. Y. (1989)
Ann. Rev. Neurosci.
12, 227; Opitz, N., and Lubbers, D. W. (1984)
Adv. Exp. Med. Biol.
180, 757; Thompson, R. B. and Jones, E. R. (1993)
Anal. Chem.
65, 730-4; and U.S. Pat. No. 5,545,517).
The ratio approach has proven particularly popular because it is robust in avoiding many of the artifacts associated with simple intensity measurements. The major limitation of the ratio approach has been the limited number of ratiometric fluorescent indicators (Haugland, R. P. (1992)
Handbook of Fluorescent Probes and Research Chemicals
, Molecular Probes, Eugene, Oreg.).
Recently, many groups have shown that transducing the level of analyte as a change in fluorescence lifetime is a robust and flexible approach to optical sensing (Demas, J. N., (1994) in
Topics in Fluorescence Spectroscopy
, Vol. 4:
Probe Design and Chemical Sensing
, Lakowicz, J. R. (Ed.) Plenum Press, NY; Lippitsch, M. E., Pusterhofer, J., Leiner, M. J. P. and Wolfbeis, O. S. (1988)
Anal. Chim. Acta
205, 1-6; Keating, S. M., and Wensel, T. G. (1991)
Biophys. J.
59, 186-202; Lakowicz, J. R., Szmacinski, H., and Karakelle, M. (1993)
Anal. Chim. Acta
272, 179-186; Szmacinski, H., and Lakowicz, J. R. (1993)
Anal. Chem.
65, 1668-74; Lakowicz, J. R. (1992)
Laser Focus World
28(5) 60-80; Thompson, R. B. and Patchan, M. W. (1993) in
Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V.
Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.; Ozinskas, A. J., Malak, H., Joshi, J., Szmacinski, H., Britz, J., Thompson, R. B., Koen, P. A., and Lakowicz, J. R. (1993)
Anal. Biochem.
213, 264-270). Lifetime-based sensing can exhibit a dynamic range of greater than five orders of magnitude in analyte concentration (Szmacinski, H., and Lakowicz, J. R. (1993)
Anal. Chem.
65, 1668-74; Thompson, R. B. and Patchan, M. W. (1993) in
Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V.
Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.). Lifetime-based sensing has been adapted to and has particular advantages for fiber optic sensors.
The development of fluorescence-based fiber optic biosensors is well documented. In particular, optical designs which optimize fluorescence measurements through optical fiber are described (U.S. Pat. No. 5,141,132), a sensor suited for the determination of anesthetics and other lipid-soluble analytes (U.S. Pat. No. 5,094,819), and an optical design which optimizes the sensitivity of so-called evanescent-wave sensors (U.S. Pat. No. 5,061,897) have been described. The advantages of fiber optic sensors for remote, continuous monitoring of analytes in environments that are hazardous or inaccessible are well known (Thompson, R. B. (1991) in
Topics in Fluorescence Spectroscopy
, Vol. 2:
Principles
, Lakowicz, J. R. (Ed.) Plenum Press, NY; Wolfbeis, O. S. (Ed.) (1992)
Fiber Optic Chemical Sensors and Biosensors
Vols. I and II, CRC Press, Boca Raton, Fla.; Thompson, R. B. and Ligler, F. S. (1991) in
Biosensors with Fiber Optics.
Wise, D., and Wingard, L. (Eds.) pp. 111-138, Humana Press, Clifton, N.J.).
Recently, a fiber optic biosensor for metal ions in aqueous solution that takes advantage of the very selective binding of particular metals by the enzyme carbonic anhydrase II from mammalian erythrocytes has been described. (Thompson, R. B. and Jones, E. R. (1993)
Anal. Chem.
65, 730-4; Thompson, R. B. and Patchan, M. N. (1993) in
Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V.
Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.; and U.S. Pat. No. 5,545,517). It was shown that metal-dependent binding of a fluorescent aryl sulfonamide inhibitor, dansylamide, could be transduced as a change in the ratio of fluorescence emission intensities at two different wavelengths (450 and 550 nanometers (R. B. Thompson and E. R. Jones,
Anal. Chem.
65: 730-4 (1993)), or changes in the fractional contributions of two fluorescence lifetimes arising from the bound and free forms of the dansylamide (see
FIG. 1
) (R. B. Thompson and M. W. Patchan,
J. Fluorescence
5:123-30 (1995)). These fluorescence observables are related to the fraction of enzyme with dansylamide bound to it, which is equal to the fraction of enzyme with Zn(II) in its active site and simply related to the concentration of the analyte Zn(II) by the law of mass action. These methods demonstrate rapid determination of Zn(II) at nanomolar levels in aqueous solutions.
However, dansylamide has some properties
Ge Zhenfang
Patchan Marcia W.
Thompson Richard B.
University of Maryland Baltimore
Wessendorf T.
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