Method for fluorometric determination of the concentrations of substances in a sample and arrangement for implementing this method
First Claim
1. A method for the fluorometric determination of the concentration of a number n of individual constituent substances contained in a sample by using fluorescent indicator substances, said method comprising(1) providing at least one fluorescent indicator substance which is non-specific relative to at least one of said individual constituent substances and which has different quenching constants with respect to said individual constituent substances, the unquenched fluorescence intensity and the different quenching constants of each fluorescent indicator substance with respect to each of said individual constituent substances being known,(2) associating said fluorescent indicator substances with a sample containing a number n of individual constituent substances,(3) taking a number n of quenched fluorescence intensity measurements of said sample, and(4) determining the concentrations of said individual constituent substances and the ratio of individual constituent substance concentrations in said sample using the known unquenched fluorescence intensities of each fluorescent indicator substance, the different quenching constants of each fluorescent indicator substance, and the measured quenched fluorescence intensities obtained in step (3).
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Abstract
For the simultaneous measurement of the concentrations of several substances a number of fluorescence measurements corresponding to the number of substances to be tested are performed using at least one fluorescent indicator which is non-specific relative to at least one of the substances to be tested, each fluorescent indicator having different quenching constants with regard to the individual substances quenching its intensities.
From the known, unquenched fluorescence intensities of the fluorescent indicators employed, the quenched fluroescence intensities obtained by measuring, and from the quenching constants that are known, or rather, have been determined beforehand by graphical methods or calculation, the concentrations and/or concentration ratios of the individual substances are obtained.
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Citations
18 Claims
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1. A method for the fluorometric determination of the concentration of a number n of individual constituent substances contained in a sample by using fluorescent indicator substances, said method comprising
(1) providing at least one fluorescent indicator substance which is non-specific relative to at least one of said individual constituent substances and which has different quenching constants with respect to said individual constituent substances, the unquenched fluorescence intensity and the different quenching constants of each fluorescent indicator substance with respect to each of said individual constituent substances being known, (2) associating said fluorescent indicator substances with a sample containing a number n of individual constituent substances, (3) taking a number n of quenched fluorescence intensity measurements of said sample, and (4) determining the concentrations of said individual constituent substances and the ratio of individual constituent substance concentrations in said sample using the known unquenched fluorescence intensities of each fluorescent indicator substance, the different quenching constants of each fluorescent indicator substance, and the measured quenched fluorescence intensities obtained in step (3).
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9. An apparatus for the fluometric determination of the concentration of a number n of individual constituent substances contained in a sample, said apparatus comprising
a sample chamber for containing a sample having a number n of individual constituent substances therein, means containing a number n of fluorescent indicator substances which are quenchable by said individual constituent substances, at least one of said fluorescent indicator substances responding non-specifically to at least one of said individual constituent substances, each fluorescent indicator substance having quenching constants which are different for each of said individual constituent substances, a light means for directing excitation light towards said means containing said fluorescent indicator substances and causing said fluorescent indicator substances to emit fluorescent radiation, a detector means for detecting the fluorescent radiation emitted by said fluorescent indicator substances, and an evaluation unit means connected to said detector means for determining the concentrations and the concentration ratios of said individual constituent substances.
Specification