Ratiometric fluorometer

Details Ratiometric fluorometer Ratiometric fluorometer

1999-11-30

2003-05-13

Pham, Hoa Q. (Department: 2877)

Optics: measuring and testing

For light transmission or absorption

Of fluent material

C356S432000, C356S039000

Reexamination Certificate

active

06563585

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to an analytic apparatus and particularly to a ratiometric fluorometer for measuring the concentration of various analytes in a sample.
BACKGROUND
Currently, the predominant technique for measuring dissolved oxygen in a sample is a polarographic method involving the monitoring of the modified Clark electrode. This approach involves a relatively expensive, inefficient, and inaccurate method of measuring oxygen concentrations. In particular, the Clark electrode method is adversely affected by signal drift which in turn affects the long term stability of the electrode. Furthermore, this method is adversely affected by flow rates as a result of the electrode consuming amounts of the oxygen to be analyzed and thus causing the measurements of the oxygen concentration to be unreliable at best. Finally, while the Clark electrode method is a long-standing polarographic approach for measuring dissolved oxygen, practitioners find this method is susceptible to electrical interference that adversely affects the accuracy of the oxygen concentration measurements.
Recently, optical methods in measuring the oxygen concentration of a sample have been employed in an attempt to overcome the limitations of the Clark electrode. These optical techniques attempt to measure the oxygen induced changes in the emission intensity of a sample to determine the oxygen concentration in that sample. Optical measurement techniques are based on the premise that long-lived states of many emissive transition metal complexes (emissive dyes) are quenched at oxygen concentrations of environmental, industrial, and biomedical interest. Emissive dyes, once polymer encapsulated or dissolved in the media being analyzed, can be used to measure oxygen in the gas phase as well as in an aqueous or biomedia form. These emissive dyes include several tris(diimine)ruthenium (II) complexes and metalloporphyrins that, once polymer encapsulated or dissolved in the media under investigation, may be used for oxygen measurement. However, systems emphasizing oxygen induced changes in emission intensity have incurred various problems that have resulted in inaccurate measurements of oxygen concentration of the sample and therefore unreliable results. Measurements obtained by such systems are adversely affected by changes in optical clarity, fluctuations in the source detector, and photobleaching of the emitter. These non-analyte induced variations in emission intensity of the sample require a relatively expensive and extremely complex system that continuously restandardizes intensity based sensors in an attempt to obtain accurate measurements.
A second technique used to measure oxygen concentration in a sample utilizes a frequency modulated excitation to irradiate the sample. Use of a system embodying a frequency modulated excitation apparatus enables a lifetime dependent phase-shift to be used to measure quenching of a long-lived emissive state and thereby obtain the oxygen concentration. Although a frequency modulated phase-based method may be used to eliminate many of the problems associated with emission intensity techniques, the implementation of such methods based on frequency modulation requires an extremely expensive and complex system.
A third technique used to measure the oxygen concentration of a sample utilizes a two-dye method. An optical device is used to measure the emission intensity of both dyes and obtain an intensity ratio used to measure the oxygen concentration of the sample. Unfortunately, systems utilizing the two-dye method are adversely affected by photobleaching of one or both dyes. This non-analyte induced variation in intensity leads to gross miscalculations of the intensity ratio and results in inaccurate oxygen concentration measurements.
In addition to oxygen concentration measurements, optical methods have been employed to measure pH, Ca
2+
, Mg
2+
, Zn
2+
, heavy metals and transmembrane potentials of samples which are very important in the biomedical field. Currently, conventional steady-state methods are used to determine these types of measurements. However, steady-state systems embodying optical measurement techniques are prone to errors due to losses in the optical path, photobleaching, scattering, and background light. Furthermore, many conventional measurement systems employ techniques that require electrical contact with the object under investigation in order for a measurement to be obtained. Frequent recalibration is needed due to such non-analyte induced variations in emission intensity of the sample. As a result of these problems and limitations, very strict experimental conditions need to be upheld during the measurement process resulting in complex measurement systems and an expensive and inefficient process that yields error-prone results.
The publication entitled “A Unique Analyzer Combining a Dual Emission Probe and a Low-Cost Solid State Ratiometric Fluorometer”, with a publication date of September 1999, by Yordan Kostov, Kelly A. Van Houten, Peter Harms, Robert S. Pilato, and Govind Rao, is hereby incorporated by reference. In addition, the publication entitled “Low-cost Device for Ratiometric Fluorescence Measurements”, with a publication date of December 1999, by Yordan Kostov and Govind Rao, is hereby incorporated by reference.
For example, U.S. Pat. No. 3,804,535, issued Apr. 16, 1974 to Rodriguez, the disclosure of which is hereby incorporated by reference, discloses a dual wavelength photometer apparatus for measuring an analyte in a sample. The measurement of an oxygenation characteristic of a blood sample is described as a typical use of the apparatus. Light sources are sequentially directed through a sample of blood at a predetermined recurring rate and the difference in intensity of the emerging resultant beams (called the reference light beam and measure light beam) is measured. The only light beam affected by the oxygenation of the blood sample is the measure light beam allowing the oxygen content of the blood sample to be measured. However, measuring an analyte of a sample by measuring the difference in intensity has proven to be a problematic and unreliable method. As discussed above, such intensity based measurements are adversely affected by changes in optical clarity due to losses in the optical path between the reference and measurement light sources and the photometer, photobleaching, scattering and background light, and fluctuations in the source and detector. Moreover, these non-analyte induced variations in intensity make continual restandardization of the intensity based circuit shown in FIG. 1 of the '535 Patent a requirement.
Such an instrument is described in U.S. Pat. No. 4,803,049 issued to Hirschfeld et al., the disclosure of which is hereby incorporated herein by reference. Patent No. '049 discloses a pH-sensitive optrode (optrode) for monitoring the pH of a sample of physiological fluids, such as blood. An organic dye that fluoresces when excited by a light having a particular wavelength and whose fluorescence emission intensity varies with the levels of pH in physiological fluids, such as blood, is utilized to generate a fluorescent signal used to measure the pH of a blood sample. The organic dye molecules are covalently attached to a support material that in turn is in contact with the blood sample. When illuminated, the organic dye disposed on the support is caused to fluoresce. The intensity of the organic dye fluorescence varies with the levels of pH of the blood sample. However, as discussed above, this intensity based method of pH measurement is adversely affected by changes in optical clarity due to losses in the optical path between the support material and the photomultiplier tube, scattering and background light, and fluctuations in the source and detector. Moreover, photobleaching of the dye leads to gross changes in the fluorescence intensity of the dye resulting in unreliable and inaccurate pH measurements.
For the foregoing reasons, there is a need for a

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