Fluorescent lifetime assays for non-invasive quantification of analytes such as glucose
First Claim
1. A method of using a population of fluorescent sensor molecules to measure the concentration of a polyhydroxylate analyte (A) in a solution, wherein the population of fluorescent sensor molecules are present in species that ate not bound to the polyhydroxylate analyte (FS) and species that are bound to the polyhydtoxylate analyte (AFS), the method comprising:
- (a) determining the total fluorescence of the solution;
(b) determining the relative fluorescence contribution that the FS species and the AFS species make to the total fluorescence of the solution;
(c) using the relative fluorescence contribution values of FS and AFS as determined in step (b) to calculate the relative abundances of FS and AFS in the solution; and
(d) correlating the relative abundances of FS and AFS in the solution as calculated in step (c) with the concentration of the polyhydroxylate analyte so that the concentration of the polyhydroxylate analyte in the solution is determined.
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Abstract
The invention disclosed herein provides fluorescence based methods for the determination of polyhydroxylated analyte concentrations as well as optical polyhydroxylate analyte sensors and sensor systems. In particular, the invention provides methods of quantifying the abundances or concentrations of polyhydroxylate analyte by measuring changes in the fluorescence lifetimes. The methods of the invention are based on the observation that fluorescent sensor molecules capable of binding a polyhydroxylated analyte such as glucose have distinct fluorescent lifetimes depending upon whether they are in a form that is either bound to analyte or a form that is not bound to the analyte. The distinct and measurable differences in the fluorescence lifetimes of the different fluorescent sensor species can be used to determine the relative abundance of the bound and unbound fluorescent sensor species, a parameter which can then be correlated to the concentration of the analyte.
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Citations
35 Claims
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1. A method of using a population of fluorescent sensor molecules to measure the concentration of a polyhydroxylate analyte (A) in a solution, wherein the population of fluorescent sensor molecules are present in species that ate not bound to the polyhydroxylate analyte (FS) and species that are bound to the polyhydtoxylate analyte (AFS), the method comprising:
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(a) determining the total fluorescence of the solution;
(b) determining the relative fluorescence contribution that the FS species and the AFS species make to the total fluorescence of the solution;
(c) using the relative fluorescence contribution values of FS and AFS as determined in step (b) to calculate the relative abundances of FS and AFS in the solution; and
(d) correlating the relative abundances of FS and AFS in the solution as calculated in step (c) with the concentration of the polyhydroxylate analyte so that the concentration of the polyhydroxylate analyte in the solution is determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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16. A method of optically sensing the presence of a polyhydroxylate analyte in a sample, the method comprising:
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(a) placing a fluorescent sensor molecule (FS) in contact with the sample, wherein the fluorescent sensor molecule reversibly binds to the polyhydroxylate analyte, the fluorescent sensor molecule comprising a first fluorescence lifetime corresponding to the fluorescent sensor molecule bound to the polyhydroxylate analyte (FSA) and a second fluorescence lifetime corresponding to the fluorescent sensor molecule not bound to the polyhydroxylate analyte, and wherein the fluorescence lifetimes of FSA and FS contribute relatively to a detectable fluorescence lifetime for the sample;
(b) exposing a population of the fluorescent sensor molecules to the sample;
(b) exciting the fluorescent sensor molecules in the sample with radiation;
(c) detecting a resulting emission beam emanating from the fluorescent sensor molecules in the sample, wherein the emission beam varies with the concentration of the polyhydroxylate analyte; and
(e) correlating the resulting emission beam to the presence of the polyhydroxylate analyte in the sample, wherein the concentration of the polyhydroxylate in the sample is determined.
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Specification