Sample Clarification for Low-Background Fluorescence Analysis
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Solution Overview
Problem
Fluorescence-based assays, such as fluorescence polarization assay (FPA), are unreliable for samples with high riboflavin content like milk and egg yolk due to significant background autofluorescence and opacity, which skew results and hinder accurate analyte detection.
Innovation Solution
Treatment of samples with riboflavin binding protein (RBP) to quench autofluorescence, combined with irradiation and clarification methods including coagulation of casein and centrifugation/filtration, using non-aqueous polar solvents to maintain analytes like progesterone in solution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence-based assays are used to detect analytes in samples with high riboflavin content, then analyte detection is performed, but background autofluorescence increases and skews results
Solution Approach 1:
The patent extracts and removes riboflavin from the sample using riboflavin-binding protein, which specifically binds to riboflavin and allows its separation from the analyte. This extraction approach eliminates the harmful autofluorescence while preserving the target analyte for accurate detection.
Solution Approach 2:
The patent introduces riboflavin-binding protein as an intermediary substance that mediates between the harmful riboflavin and the detection system. The binding protein selectively binds riboflavin, preventing it from contributing to background fluorescence while allowing the analyte to be detected without interference.
2Measurement precision
If samples with high protein content are used for fluorescence analysis, then analyte detection is attempted, but sample opacity increases and resists fluorescence analysis
Solution Approach 1:
The patent extracts and removes proteins from the sample through precipitation and centrifugation steps. This extraction eliminates the opacity-causing proteins while maintaining the analyte in solution, allowing light to pass through the sample for accurate fluorescence measurement.
Solution Approach 2:
The patent changes the physical-chemical parameters of the sample by adjusting pH and adding precipitating agents to alter protein solubility. These parameter changes cause proteins to precipitate out of solution, reducing sample opacity while keeping the analyte accessible for detection.
3Object-generated harmful factors
If clarification methods are applied to remove interfering components, then background fluorescence is reduced, but analytes may be lost during processing
Solution Approach 1:
The patent uses riboflavin-binding protein as a selective intermediary that binds only to riboflavin, not to the analyte. This selective binding allows removal of interfering components through the intermediary without causing loss of the target analyte during the clarification process.
Solution Approach 2:
The patent applies different treatment conditions to different components in the sample. Riboflavin is bound by specific protein at certain concentrations and pH levels, while the analyte remains in solution under these same conditions. This local quality approach allows selective removal of interferents while preserving the analyte.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Reduces background fluorescence, clarifies samples, and maintains analytes for accurate fluorescence detection, enabling reliable detection of hydrophobic analytes like progesterone in milk products.
Implementation Method 1
treating the sample to reduce riboflavin-dependent autofluorescence in the sample, adding a tracer to the sample, and detecting fluorescence from the tracer in the sample. Treating the sample to reduce riboflavin-dependent fluorescence can comprise adding riboflavin binding protein to the sample
Implementation Method 2
the clarifying comprises coagulating casein in the sample and removing the coagulated casein from the sample. The coagulating can comprises adding a rennet to the sample in an amount sufficient to coagulate the casein
Implementation Method 3
The clarifying can comprise centrifuging the sample, filtering the sample, or a combination thereof
Implementation Method 4
the clarifying comprises adding a non-aqueous polar solvent to the sample. The non-aqueous polar solvent can comprise an alcohol. The non-aqueous polar solvent is preferably added to the sample in an amount sufficient to maintain the analyte in the sample
Data Source
AI summary
Methods and reagents for processing samples for fluorescence analysis. Processing methods include treating samples containing riboflavin to reduce riboflavin-dependent autofluorescence by adding riboflavin binding protein to the sample, irradiating the sample, or a combination thereof. Processing methods also include clarifying samples by coagulating, precipitating, and/or otherwise removing proteins and other components that interfere with fluorescence analysis without removing the analyte. Fluorescence analysis methods include fluorescence polarization analysis (FPA) and others. Reagents suitable for performing the disclosed methods are provided.