Aptamer-Based Reagents for Multiplex Immunoassays

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Solution Overview

Problem

Multiplex immunoassays face challenges such as the need for highly specific capture ligands, cross-reactivity, matrix interference, and the complexity and cost of developing assays for multiple analytes, particularly in clinical diagnostics where only a few biomarkers are required for disease diagnosis.

Innovation Solution

The use of aptamer-based reagents selected through a magnetic-assisted differential affinity method, specifically window-MARAS, which employs biofunctionalized magnetic particles and oscillating magnetic fields to differentiate and quantify multiple analytes in a single assay, reducing the need for multiple capture ligands and minimizing interference.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If multiple highly specific capture ligands are used for multiplex detection, then the ability to detect multiple analytes is improved, but the complexity and cost of assay development increases

Engineering Contradiction:

Improveability to detect multiple analytesVSAvoidcomplexity of assay development

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

A single aptamer is designed to bind multiple different analytes with different affinities, replacing the need for multiple separate capture ligands. This universal aptamer can detect various analytes including CRP, HBsAg, and HCV NS3 in a single multiplex assay, significantly reducing assay development complexity while maintaining the ability to detect multiple targets

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention exploits differences in binding affinity (a physical parameter) between the aptamer and different analytes to achieve multiplex detection. By controlling washing stringency and using oscillating magnetic fields with specific frequencies, analytes with different affinities can be selectively detected, allowing one aptamer to replace multiple capture ligands

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If multiple capture ligands are used for multiplex immunoassay, then the number of detectable analytes increases, but cross-reactivity between capture ligands and analytes increases

Engineering Contradiction:

Improvenumber of detectable analytesVSAvoidcross-reactivity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The aptamer is designed with specific local binding characteristics for different analytes, creating distinct binding pockets with different affinities. This local differentiation in binding properties allows the single aptamer to specifically recognize multiple analytes without cross-reactivity, as each analyte binds to a specific region of the aptamer with unique affinity characteristics

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention uses oscillating magnetic fields with varying frequencies to dynamically control the detection process. Different frequencies selectively disrupt or maintain binding between the aptamer and analytes based on their affinity differences, allowing sequential detection of multiple analytes while preventing cross-reactivity through dynamic control of binding conditions

Inventive Principle:

Principle #15Dynamics

3Adaptability or versatility

If traditional multiplex immunoassay methods are used, then multiple analytes can be detected, but interference from matrix effect increases

Engineering Contradiction:

Improvedetection of multiple analytesVSAvoidinterference from matrix effect

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and isolates the aptamer-analyte binding interaction from the complex biological matrix by using magnetic particles to capture the complexes. This physical separation allows selective detection of target analytes while excluding interfering substances in the serum or plasma matrix, reducing matrix effect interference in multiplex detection

Inventive Principle:

Principle #2Taking out (Extraction)

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 efficient, cost-effective multiplex detection and quantitation of multiple analytes with high specificity, reducing assay complexity and interference, making it suitable for clinical applications by utilizing aptamers with tailored affinities for various target analytes.

Implementation Method 1

window-MARAS, which employs biofunctionalized magnetic particles and oscillating magnetic fields to differentiate and quantify multiple analytes

Methodology Applied

Scientific EffectMagnetic field: Magnetic Field

Data Source

PatentUS10976313B2Method of multiplex immunoassays utilizing differential affinity and methods for synthesizing aptamer-based reagents for multiplex immunoassays

Publication Date: 2021.04.13 HONG KS

AI summary

Multiplex immunoassays utilize the differential affinities among the conjugation pairs between the capture ligands and target analytes are proposed. Window magnetic-assisted rapid aptamer selection (window-MARAS) methods for selecting aptamers with desirable affinity toward the target analytes and methods for generating reagents for multiplex immunoassays or multiplex detection in one assay by utilizing the selected aptamers as capture ligands in reagents are described and used to demonstrate the feasibility of multiplex immunoassays based on the differential affinity of conjugation pairs between the capture ligands and target analytes.