Multiplexed Aptamer Assay Background Signal Reduction
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Solution Overview
Problem
Multiplexed aptamer assays face challenges in reducing background signal due to unanticipated aptamer-aptamer interactions, which can limit sensitivity and specificity.
Innovation Solution
The use of aptamers with releasable tags, such as photocleavable biotin, immobilized on a solid support, followed by washes to remove aptamer aggregates, and subsequent partitioning and release of aptamer-target affinity complexes, helps reduce background signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiplexed aptamer assays are performed with multiple aptamers in solution, then the ability to detect multiple target molecules is improved, but background signal increases due to unanticipated aptamer-aptamer interactions
Solution Approach 1:
The assay is divided into separate solution-based target interaction steps and solid-phase detection steps. Aptamers interact with targets in solution where they remain mobile and accessible, then complexes are transferred to solid support for detection. This segmentation prevents aptamer-aptamer interactions while maintaining multiplexed detection capability.
Solution Approach 2:
A solid support acts as an intermediary between the solution-phase aptamer-target interaction and the detection system. The solid support captures the complexes without requiring aptamers to be immobilized, thereby preventing unwanted aptamer-aptamer interactions while enabling signal detection.
2Measurement precision
If aptamers are immobilized on solid support to enable detection, then detection capability is improved, but specific target/aptamer interactions are reduced due to restricted aptamer conformational freedom
Solution Approach 1:
The assay separates the interaction phase (in solution with mobile aptamers) from the detection phase (on solid support). This allows aptamers to maintain their conformational freedom and binding specificity during interaction, while still enabling detection when complexes are captured on the solid support.
Solution Approach 2:
The aptamer-target complexes themselves serve as the means for capture on the solid support. The complexes are formed in solution and then naturally captured when the solution is contacted with the solid support, eliminating the need for pre-immobilized aptamers and preserving interaction specificity.
3Reliability
If solution-based target interaction is used to maintain aptamer flexibility, then binding specificity is improved, but separation of components becomes more difficult
Solution Approach 1:
A solid support serves as an intermediary that simplifies separation. The solution-phase interaction maintains specificity, then the solid support selectively captures the aptamer-target complexes, automatically separating them from unbound components without complex separation procedures.
Solution Approach 2:
The solid support extracts or captures the aptamer-target complexes from the solution phase. This extraction step simplifies the separation process by selectively removing complexes from the solution, leaving unbound components behind without requiring complex separation machinery.
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 effectively reduces background signal, maintaining specific target/aptamer interactions, thereby enhancing the sensitivity and specificity of aptamer-based assays.
Implementation Method 1
The attachment of the aptamer to the solid support is accomplished by contacting a first solid support with the aptamer and allowing the releasable first tag included on the aptamer to associate, either directly or indirectly, with an appropriate first capture agent that is attached to or part of the first solid support
Implementation Method 2
Washes with a solution buffered to pH 11 remove aptamer/aptamer aggregates, thereby reducing assay background
Implementation Method 3
The aptamer-target affinity complex and uncomplexed aptamer that has associated with the probe on the solid support is then partitioned from the remainder of the mixture
Implementation Method 4
eluting aptamers from the solid support with one or more buffered solutions comprising a chaotropic salt that disrupts aptamer/aptamer interactions but supports aptamer/aptamer aggregates and DNA hybridization
Implementation Method 5
wash buffers comprising an organic solvent that disrupts aptamer-aptamer interactions
Data Source
AI summary
The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer) where the aptamer-aptamer interactions are significantly reduced or eliminated while maintaining the aptamer-target interaction.