Method for calibrating fluorescent plate readers
Inventors
LIN, Cheng-Tse • Sun, Yi-Ming • HSU, Kuei-Shen • CHANG, Liang-Han • CHUNG, Yao-Kuang • WU, Chin-Yun • Huang, Chin-Shiou
Assignees
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Publication Number
US-12099011-B2
Publication Date
2024-09-24
Expiration Date
Abstract
The present invention relates to a fluorescent polymer cast on the surface of wells of a plate and a plate comprising the fluorescent polymer. The plate is used in a method for calibrating read-out values of plate readers. The method for calibrating the plate readers can effectively reduce the deviation of read-out values among different plate readers.
Core Innovation
The document describes a calibration standard based on a fluorescent polymer that is transparent, dry, solvent-free and porogen-free. The fluorescent polymer is cast on a plurality of wells of a microplate so that a pre-determined amount of a fluorescent dye, Nile Red, is imbedded in the polymer, and the Nile Red is arranged as a concentration gradient that corresponds to a serial dilution scheme.
A fluorescent polymer microplate is prepared and incubated, and then placed in a master plate reader to measure a mean fluorescence intensity of each well. The resulting set of original fluorescence intensity values is used to determine a standard curve of best fit by plotting the original fluorescence intensity values against corresponding amounts of fluorescent polymer through linear regression analysis.
For calibration of read-out values, the document describes measuring mean fluorescence intensity values in a user plate reader using the same type of fluorescent-polymer microplate. A calibration formula of Y=ax+b is obtained based on master and user fluorescence intensity values using a Weighted Least Squares method, and calibrated fluorescence intensity values are obtained from the user read-outs by subtracting b and dividing by a.
Claims Coverage
The partial document includes two independent methods. One method provides a standard curve for quantifying a biomolecule, using linear regression analysis, and the other method calibrates read-out values of a user plate reader, using a Weighted Least Squares method to obtain Y=ax+b and then compute calibrated fluorescence intensity values. Across both independent claims, the microplate is defined by a fluorescent polymer that is transparent, dry, solvent-free and porogen-free and that comprises a pre-determined amount of Nile Red imbedded in a polymer as a concentration gradient.
Fluorescent polymer microplate standard with concentration gradient for a best-fit standard curve
Preparing a microplate comprising a fluorescent polymer cast on a plurality of wells as a concentration gradient corresponding to a serial dilution of a solution, wherein the fluorescent polymer is transparent, dry and solvent-free and porogen-free and comprises a pre-determined amount of Nile Red imbedded in a polymer; incubating the microplate and placing it in a master plate reader to measure mean fluorescence intensity values, and determining a standard curve of best fit by plotting the original fluorescence intensity values against corresponding amounts of fluorescent polymer through linear regression analysis.
Master-to-user plate reader calibration using Y=ax+b and Weighted Least Squares
Calibrating read-out values of a user plate reader by preparing the fluorescent polymer microplate as defined, measuring mean fluorescence intensity values in a master plate reader and in the user plate reader, obtaining a formula of Y=ax+b based on original fluorescence intensity values and current fluorescence intensity read-out values by conducting a Weighted Least Squares method, and obtaining calibrated fluorescence intensity values from the set of current fluorescence intensity read-out values by subtracting b and dividing by a.
Both independent claims rely on a fluorescent-polymer microplate standard and use fluorescence measurements to derive calibration relationships. The standard-curve method uses linear regression to relate mean fluorescence intensity to corresponding amounts of fluorescent polymer for quantifying a biomolecule, while the read-out calibration method uses Weighted Least Squares to obtain Y=ax+b and then compute calibrated fluorescence intensity values from user plate reader read-outs.
Stated Advantages
Reduction of inter-reader deviation, with reported coefficient of variation (CV%) less than 5% and down to about 1% after calibration.
Documented Applications
Calibration workflows for vendor and user plate readers, using the prepared microplate and certified reference materials, including performing calibration across multiple plate readers and comparing standard curves before versus after calibration.
Providing a standard curve for quantifying a biomolecule using the fluorescent polymer microplate and linear regression analysis.
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