Capillary Electrophoresis Using Dual-Wavelength Internal Standards
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Solution Overview
Problem
Capillary electrophoresis methods for analyzing biological molecules, such as proteins, face challenges in achieving high reproducibility of absolute detection times between experiments, limiting the versatility of information obtained.
Innovation Solution
A method using standard substances (markers) for electrophoresis, where analytes and standards are subjected to electrophoresis simultaneously, with optical signals detected at different wavelengths, allowing for the derivation of equations correlating isoelectric points or molecular weights with detection times, and using tryptophan-free standards labeled with fluorophores for distinct detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If capillary electrophoresis is used to analyze proteins, then robust analysis capability is achieved, but reproducibility of absolute detection times between experiments is not high
Solution Approach 1:
The patent introduces standard substances (markers) as intermediary reference materials that migrate alongside analytes during electrophoresis. These markers serve as mediators to establish a correlation between migration distance and isoelectric point, enabling accurate determination of analyte properties independent of absolute detection time variations between experiments
Solution Approach 2:
The patent changes the measurement parameter from absolute detection time to relative migration distance correlated with marker positions. By deriving equations that relate analyte migration distance to the positions of standard substances with known isoelectric points, the method transforms the measurement approach to achieve reproducibility without relying on consistent absolute detection times across different experiments
2Measurement precision
If standard substances are used simultaneously with analytes in the same separation space, then determination accuracy of isoelectric points is improved, but detection complexity increases due to need for multiple wavelength detection
Solution Approach 1:
The patent applies local quality by assigning different optical properties to different components: standard substances are selected or labeled to exhibit optical signals at specific wavelengths distinct from the analyte. This allows the detection system to differentiate between markers and analytes by detecting at different wavelengths, with each wavelength optimized for detecting specific substance types
Solution Approach 2:
The patent employs a multi-wavelength detection system that serves multiple functions simultaneously: it detects both analytes and standard substances in the same run, distinguishes between different substance types through wavelength-specific detection, and enables accurate determination of isoelectric points through comparative analysis of migration distances at different wavelengths
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
Enables precise determination of isoelectric points and molecular weights of proteins with improved reproducibility and versatility, facilitating accurate analysis of biological molecules.
Implementation Method 1
subjecting a sample containing the analyte and standards to electrophoresis simultaneously in the same separation space
Implementation Method 2
detecting the analyte with an optical signal derived from tryptophan
Implementation Method 3
detecting the standard with an optical signal of a different wavelength from the analyte
Data Source
AI summary
The present disclosure provides a method for analyzing proteins and polypeptides by electrophoresis using standards as internal standards. In one aspect, the present disclosure provides a method for measuring an analyte by electrophoresis, the method including the steps of preparing standards that do not contain tryptophan, subjecting a sample containing the analyte and the standards to electrophoresis simultaneously in the same separation field, detecting the analyte with an optical signal derived from tryptophan and detecting the standards with an optical signal of a wavelength different from that of the analyte, and measuring the analyte based on the optical signal. In one embodiment, the electrophoresis is capillary electrophoresis.