Dual-Marker EV Isolation Using Oligonucleotide Stapling

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Solution Overview

Problem

Existing methods for isolating extracellular vesicles (EVs) face challenges due to the variability of surface protein expression across cell types, leading to inconsistent results and the need for protocol standardization, particularly in flow cytometry and immunoaffinity capture techniques.

Innovation Solution

A method involving the use of a library of oligonucleotide-conjugated binding reagents that target multiple surface markers to capture EVs, utilizing a 'stapling' technique to ensure high specificity and purity by forming a stable complex through rolling circle amplification, allowing for the release of non-targeted EVs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of operation

If single marker methods are used for EV isolation, then the process is simpler, but specificity and reliability of EV identification deteriorate

Engineering Contradiction:

Improvesimplicity of isolation processVSAvoidspecificity of EV identification

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The patent divides the EV isolation process into multiple sequential steps: initial capture using one marker, release of non-specifically bound EVs, and secondary capture using a different marker. This segmentation allows the system to achieve high specificity through multiple filters while maintaining operational simplicity through standardized protocol steps.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces an intermediary release step between two capture steps. This intermediary action allows non-specifically bound EVs to be released while specifically bound EVs remain attached, enabling the system to differentiate between specific and non-specific binding without requiring complex real-time detection systems.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If multiple markers are used for EV isolation, then specificity of EV identification improves, but device complexity and protocol difficulty increase

Engineering Contradiction:

Improvespecificity of EV identificationVSAvoidcomplexity of isolation protocol

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent employs markers with multiple functions: they serve as both capture targets and release triggers. The same marker can be used for initial capture and later for selective release, reducing the need for additional reagents and simplifying the overall protocol while maintaining high specificity through dual-marker verification.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent utilizes changes in binding parameters (affinity, specificity) between different markers to achieve selective isolation. By choosing markers with distinct binding characteristics, the system can capture EVs with one marker and selectively release non-specifically bound EVs with another, achieving high specificity without requiring complex device modifications.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If single marker capture is used, then productivity is higher, but measurement precision and EV characterization accuracy deteriorate

Engineering Contradiction:

Improvethroughput of EV isolationVSAvoidaccuracy of EV characterization

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent performs preliminary capture of EVs using one marker, followed by selective release and secondary capture. This preliminary action allows the system to process large numbers of EVs efficiently while ensuring high measurement precision through the secondary verification step, maintaining both productivity and accuracy.

Inventive Principle:

Principle #10Preliminary action

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 achieves highly specific and efficient isolation of EVs, reducing variability and improving the accuracy of EV detection and analysis by ensuring that only targeted EVs remain bound to the surface.

Implementation Method 1

A method involving a library of oligonucleotides and binding reagents conjugated to oligonucleotides is used to capture EVs by targeting at least two surface markers

Methodology Applied

Scientific EffectOligonucleotide hybridization:

Implementation Method 2

employing rolling circle amplification and oligonucleotide-based 'stapling' to enhance specificity and stability of EV isolation

Methodology Applied

Scientific EffectRolling circle amplification:

Implementation Method 3

employing rolling circle amplification and oligonucleotide-based 'stapling' to enhance specificity and stability of EV isolation

Methodology Applied

Scientific EffectOligonucleotide hybridization and stapling:

Data Source

PatentUS12631624B2Methods for isolating surface marker displaying agents

Publication Date: 2026.05.19 MESO SCALE TECH LLC

AI summary

The invention relates to method and kits for highly specific isolation of surface marker displaying agents by targeting at least two surface markers. The invention further relates to methods and kits for analyzing surface marker displaying agents and their contents.