Extracellular Vesicle Isolation Using Dual Surface Marker Binding
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for isolating extracellular vesicles (EVs) face challenges due to the variability in expression of surface proteins across different cell types, leading to inconsistent isolation and characterization of EVs, particularly in complex biofluids like blood, which complicates the identification of specific cell populations and their cargo.
Innovation Solution
A method involving the use of multiple surface markers to selectively bind EVs to a surface through capture and anchoring reagents, followed by rolling circle amplification and hybridization with oligonucleotides to form stable complexes, allowing for the release of unwanted components and isolation of EVs of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single surface marker is used to isolate EVs, then the isolation process is simple, but the specificity and consistency of EV isolation deteriorates due to variability in surface protein expression across different cell types
Solution Approach 1:
The patent divides the EV isolation process into two distinct binding steps: first binding EVs to the surface via capture reagents targeting one surface marker, then stabilizing the bound EVs through anchoring reagents targeting a second surface marker. This segmentation allows each step to contribute to overall specificity while keeping individual steps relatively simple.
Solution Approach 2:
The patent performs preliminary binding of EVs to the surface using capture reagents before applying anchoring reagents. This preliminary action allows for initial enrichment of EVs while maintaining the option to further stabilize specific subsets through the second binding step, improving overall isolation consistency.
2Measurement precision
If multiple surface markers are used to selectively bind EVs, then the specificity of EV isolation improves, but the device complexity and procedure difficulty increases
Solution Approach 1:
The patent merges the detection and isolation functions into a single integrated process. By using capture reagents and anchoring reagents that simultaneously bind to surface markers and attach EVs to the surface, the method combines what would traditionally be separate detection and isolation steps into one unified procedure, reducing overall complexity.
Solution Approach 2:
The patent employs reagents with multiple functions: capture reagents both detect and initially bind EVs to the surface, while anchoring reagents both stabilize the binding and provide specificity through secondary marker recognition. This multi-functionality reduces the need for additional specialized reagents or steps.
3Measurement precision
If multiple binding steps are performed to ensure specific EV isolation, then the accuracy of EV characterization improves, but the time required for the isolation process increases
Solution Approach 1:
The patent implements continuous binding actions where capture reagents and anchoring reagents are applied in sequence without interrupting the overall isolation process. The surface-bound EVs from the first step are directly stabilized by the second step, maintaining continuous productive action and minimizing idle time between steps.
Solution Approach 2:
The patent uses periodic addition of reagents in a structured sequence: first capture reagents are added and allowed to bind, then anchoring reagents are added to stabilize. This periodic, rhythmically structured approach optimizes binding efficiency at each stage while maintaining overall process efficiency.
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 enables highly specific and efficient isolation of EVs, reducing variability and improving the accuracy of EV characterization by ensuring that only EVs with both markers remain bound to the surface, while others are eluted, thereby enhancing the reliability of EV analysis.
Implementation Method 1
selectively binding the EV of interest to: (i) a capture reagent releasably bound to the surface
Implementation Method 2
binding the anchoring reagent to the binding reagent, thereby forming a complex on the surface
Implementation Method 3
binding a circular oligonucleotide template to the primer oligonucleotide to form an amplicon by rolling circle amplification
Implementation Method 4
hybridizing the anchoring oligonucleotide to the amplicon to form a second complex on the surface
Data Source
AI summary
The invention relates to method and kits for highly specific isolation of extracellular vesicles (EVs) by targeting at least two EV surface markers. The invention further relates to methods and kits for analyzing EVs and their contents.