Taylor Dispersion SPR Analysis for Aggregate Quantification
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Solution Overview
Problem
Current methods for estimating aggregate formation and percentage in heterogeneous solutions require multiple steps and struggle with biomolecules like proteins, DNA, and peptides due to the lack of easily prepared known aggregate standards, necessitating calibration-free methods for determining aggregate presence and percentage.
Innovation Solution
A method using Surface Plasmon Resonance (SPR) analysis under Taylor Dispersion conditions, involving pulse or sigmoidal injections, to determine the presence, percentage, and number of monomers in aggregates by fitting specific equations to the SPR bulk refractive index response curves, eliminating the need for initial calibration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If calibration methods using known aggregate standards are used, then measurement precision of aggregate percentage is improved, but ease of operation deteriorates due to the inability to easily prepare known aggregate standards for many biomolecules
Solution Approach 1:
The method uses the biomolecule itself to generate the calibration curve by analyzing samples with known total concentrations but unknown aggregate percentages. The system self-calibrates by comparing Taylor dispersion profiles across multiple concentrations, eliminating the need for external known aggregate standards that are difficult to prepare for many biomolecules.
Solution Approach 2:
The method changes the concentration parameter of the biomolecule samples while keeping other conditions constant. By analyzing Taylor dispersion profiles at multiple known total concentrations and observing how the dispersion parameters change with concentration, the method can calculate aggregate percentage without requiring known aggregate standards, thus resolving the contradiction between measurement precision and ease of operation.
2Measurement precision
If multiple step methods are used for aggregate analysis, then measurement precision is improved, but productivity deteriorates due to the time-consuming multi-step process
Solution Approach 1:
The method combines multiple analysis steps into a single Taylor dispersion measurement. Instead of performing separate experiments for diffusion coefficient determination, concentration measurement, and aggregate calculation, the method simultaneously obtains all necessary information from one Taylor dispersion profile by fitting the experimental data to theoretical equations, thus improving productivity while maintaining measurement precision.
Solution Approach 2:
The Taylor dispersion measurement serves multiple functions simultaneously: it determines total analyte concentration, calculates aggregate percentage, and characterizes diffusion properties. This multi-functional approach eliminates the need for multiple separate analytical steps, resolving the contradiction between measurement precision and productivity by achieving both in a single experiment.
3Ease of operation
If calibration-free methods are used, then ease of operation is improved, but measurement precision deteriorates due to the lack of known standards for comparison
Solution Approach 1:
The method replaces the mechanical calibration system (physical standards) with a theoretical model-based system. Instead of comparing samples to physical known aggregate standards, the method uses mathematical models of Taylor dispersion to calculate aggregate percentage from the dispersion profile, achieving both ease of operation and measurement precision through theoretical rather than empirical calibration.
Solution Approach 2:
The method uses changes in dispersion parameters with concentration as a built-in reference system. By monitoring how Taylor dispersion characteristics change with known total concentration changes, the method creates an internal calibration mechanism that maintains measurement precision without requiring external standards, thus resolving the contradiction between ease of operation and measurement precision.
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 qualitative and quantitative determination of aggregate presence and percentage in heterogeneous solutions without prior calibration, effectively addressing the challenges with biomolecules by distinguishing between monomer and aggregate forms through dispersion coefficients analysis.
Implementation Method 1
performing SPR analysis on a sample of said solution containing said monomer and aggregate by injecting said sample under Taylor Dispersion conditions
Implementation Method 2
injecting said sample under Taylor Dispersion conditions using a pulse injection technique; determining the SPR bulk refractive index response curve under Taylor Dispersion conditions
Implementation Method 3
determining the Dm for said monomer; Dm=analyte diffusion coefficient of monomer species (m2/s); Da=analyte diffusion coefficient of aggregate species (m2/s)
Data Source
AI summary
Disclosed is a method for preparing dispersion gradients and an SPR injection method for determining full kinetics and affinity analysis in the presence of a competitor molecule. The SPR injection provides a dispersion gradient of two or more samples to a SPR flow cell and detector.