Biological Sample Fractionation Using Stepwise Centrifugation and SEC
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Solution Overview
Problem
Existing methods for molecular-level analysis of biological samples face challenges in achieving high resolution separation and fractionation of substances with varying sizes and densities within a limited time frame, particularly due to limitations in centrifugation and chromatography techniques, which either require excessive time or compromise separation resolution.
Innovation Solution
A method combining stepwise centrifugation, density gradient centrifugal separation, and size-exclusion chromatography to fractionate biological samples into multiple fractions with high resolution, utilizing incremental centrifugal forces and controlled density gradients to separate and recover samples based on size and density.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If stepwise centrifugation method is used to separate biological samples, then separation can be performed into multiple fractions, but the separation resolution is insufficient for structures with similar densities and sizes
Solution Approach 1:
The patent segments the centrifugation process into multiple distinct stages: initial low-speed centrifugation to remove large debris, followed by density gradient centrifugation to separate components by density, and finally size-exclusion chromatography to separate by size. Each stage targets a specific size/density range, progressively resolving structures that would be indistinguishable in a single centrifugation step.
Solution Approach 2:
The patent transitions from one-dimensional separation (single centrifugation parameter) to multi-dimensional separation by combining density gradient centrifugation (separating by density) with size-exclusion chromatography (separating by size). This adds a second separation dimension, enabling resolution of structures with similar density but different sizes, or similar size but different densities.
2Manufacturing precision
If the number of centrifugation steps is increased to enhance separation fineness, then separation resolution improves, but the time required for separation operations exceeds the limited time available to maintain sample freshness
Solution Approach 1:
The patent performs preliminary low-speed centrifugation to remove large cellular debris and organelles before proceeding to density gradient centrifugation. This preliminary separation eliminates the need for numerous subsequent centrifugation steps to remove large particles, significantly reducing total processing time while maintaining high resolution for the target molecular structures.
Solution Approach 2:
The patent introduces density gradient media as an intermediary substance that facilitates separation. The density gradient automatically stratifies components by density during centrifugation, providing high-resolution separation in a single step that would otherwise require multiple centrifugation cycles, thereby reducing processing time.
3Manufacturing precision
If density gradient centrifugation is used to purify specific components, then separation resolution for components with different densities improves, but it is difficult to accurately recover all particles distributed along the continuous density gradient
Solution Approach 1:
The patent segments the density gradient into multiple discrete fractions by collecting eluate at different heights during size-exclusion chromatography. This fractionation ensures that particles distributed along the continuous density gradient are systematically recovered from different zones, achieving both high resolution and complete recovery of all particle populations.
Solution Approach 2:
The patent employs size-exclusion chromatography as a universal recovery method that can retrieve all particle sizes and densities from the density gradient centrifugation step. The chromatography column universally separates components by size regardless of their density, ensuring complete recovery of all particles that were stratified by the density gradient.
4Manufacturing precision
If size-exclusion chromatography is used to separate soluble components, then separation of molecules by size is achieved, but the separation resolution is insufficient to distinguish monomers, dimers, and higher-order oligomers
Solution Approach 1:
The patent performs preliminary density gradient centrifugation to concentrate and pre-separate molecular complexes by density before applying them to size-exclusion chromatography. This preliminary concentration and partial separation enriches the target molecules in specific density fractions, allowing the chromatography column to achieve high resolution separation of oligomeric states with a standard-length column rather than requiring an excessively long column.
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 high-resolution fractionation of biological samples with wide-ranging sizes and densities in a short time, providing reproducible and quantitative analysis of substance types and sizes in each fraction.
Implementation Method 1
a centrifugal separation unit that separates a cell homogenate into a supernatant component and a pellet component
Implementation Method 2
a density gradient centrifugal separation unit that separates a predetermined amount of a density gradient sample into a plurality of fractions by density gradient centrifugal separation
Implementation Method 3
separates a predetermined amount of a density gradient sample into a plurality of fractions by density gradient centrifugal separation
Implementation Method 4
a size-exclusion chromatography unit that separates a supernatant component into a plurality of fractions by size-exclusion chromatography
Data Source
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AI summary
This method for separating and fractionating a biological sample includes: a homogenization step for preparing a cell homogenate; a first centrifugation step for separating the cell homogenate into a first supernatant component and a first pellet component; a stepwise centrifugation step for separating an (m-1)-th supernatant component into an m-th supernatant and an m-th pellet component, wherein m is an integer from 2 to n; a density gradient centrifugation step for subjecting a density gradient sample, which is prepared by adding the m-th pellet component to a density gradient reagent, to density gradient centrifugation; a density gradient centrifugal recovery step for fractionating the density gradient sample in a density gradient centrifuge tube into am fractions in order from the top or bottom side; and a chromatography step for fractionating an n-th supernatant component into b fractions by size exclusion chromatography.