Polypeptide Size Determination via Reference Calibration
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Solution Overview
Problem
Conventional methods for acquiring information on the size of target polypeptides using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) suffer from significant measurement errors, leading to inaccuracies in size determination.
Innovation Solution
A method involving the acquisition of diffusion times for both the target polypeptide and a set of fluorescently labeled reference polypeptides with known sizes, using FCS or FCCS, to accurately determine the size of the target polypeptide by referencing the diffusion times of the reference polypeptides, along with a reagent kit containing these reference polypeptides for calibration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional FCS or FCCS methods are used to acquire diffusion time of target polypeptide, then measurement can be performed, but measurement error is large and size determination accuracy is poor
Solution Approach 1:
The patent applies preliminary action by preparing reference polypeptides with known sizes and predetermined diffusion times before the actual measurement. These reference polypeptides are used to create a calibration relationship in advance, which then enables accurate size determination of the target polypeptide. The reference polypeptides serve as pre-established standards that correct the measurement system before analyzing the unknown sample.
Solution Approach 2:
The patent uses reference polypeptides as intermediaries between the measurement system and the target polypeptide. These reference polypeptides with known diffusion times act as mediators that bridge the gap between raw diffusion time measurements and accurate size determination. By comparing the target polypeptide's diffusion time against the reference polypeptides, the system indirectly determines the target's size with high accuracy.
2Measurement precision
If proportional calculation using diffusion time of dye and specimen protein is used, then size estimation can be performed, but measurement error remains large
Solution Approach 1:
The patent applies parameter changes by transitioning from using diffusion time of dyes as the reference parameter to using diffusion times of reference polypeptides with known sizes. This change in the reference parameter from small dye molecules to larger polypeptide molecules provides a more appropriate calibration standard, thereby improving the accuracy of size estimation for the target polypeptide.
3Measurement precision
If calibration curve is prepared using polystyrene beads of different sizes, then diffusion time determination can be performed, but accuracy in size calculation is insufficient
Solution Approach 1:
The patent applies parameter changes by replacing polystyrene beads with reference polypeptides as the calibration standard. This change from inorganic beads to biological polypeptides provides a more appropriate reference system for measuring target polypeptides, as the reference polypeptides share similar physical and chemical properties with the target molecules, thereby improving measurement accuracy.
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
This approach significantly enhances the accuracy of size information acquisition for target polypeptides compared to existing methods, as demonstrated by reduced differences between predicted and theoretical values, particularly in various solvent environments.
Implementation Method 1
acquiring a diffusion time of a fluorescently labeled target polypeptide and a diffusion time of each of a plurality of fluorescently labeled reference polypeptides by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy
Implementation Method 2
a diffusion time of a fluorescently labeled target polypeptide is acquired by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy
Data Source
AI summary
Disclosed is a method for acquiring information of a target polypeptide, comprising: acquiring a diffusion time of a fluorescently labeled target polypeptide and a diffusion time of each of a plurality of fluorescently labeled reference polypeptides by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, and acquiring information on size of the fluorescently labeled target polypeptide from the diffusion time of the fluorescently labeled target polypeptide with reference to the diffusion times of the plurality of fluorescently labeled reference polypeptides, wherein information on size of each of the plurality of fluorescently labeled reference polypeptides is known, and the sizes of the plurality of reference polypeptides are different from each other.