Assay Marker Composition for Broad Dynamic Range Detection
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Solution Overview
Problem
Current assays face limitations in dynamic range, particularly when the concentration range of an analyte in a sample is unknown or broad, leading to challenges in detection and quantification, as existing methods often result in saturated signals and require multiple assays or extensive dilutions, which are inefficient and costly.
Innovation Solution
A set of markers with predefined ratios of differently labelled reporter molecules is used, allowing a single reporter molecule composition to detect a broad concentration range of analytes without the need for sample dilutions or altering assay parameters, thereby extending the dynamic range of the assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a single marker is used for analyte detection, then the assay is simple to perform, but the dynamic range is limited and signal saturation occurs
Solution Approach 1:
The single marker is segmented into multiple markers with different binding affinities (e.g., high, medium, low affinity markers). Each marker segment targets the same analyte but operates in a different concentration range, preventing signal saturation and extending the overall dynamic range while maintaining assay simplicity through a unified detection system.
Solution Approach 2:
Multiple markers are designed to perform the same detection function (binding to the analyte) but with different operational characteristics (binding affinities). This multi-functionality allows the marker set to collectively cover a broad dynamic range, from low to high analyte concentrations, while each individual marker maintains its specialized role.
2Measurement precision
If multiple assays are performed to cover broad concentration ranges, then the dynamic range coverage is improved, but the time and cost increase
Solution Approach 1:
Multiple markers with different binding affinities are merged into a single assay system. All markers are applied simultaneously to the sample, and their combined signals provide comprehensive coverage of the concentration range. This eliminates the need for sequential assays or serial dilutions, reducing time and operational complexity while maintaining broad dynamic range coverage.
Solution Approach 2:
The marker composition is pre-designed with specific ratios of high, medium, and low affinity markers optimized for broad dynamic range detection. This preliminary optimization allows the assay to automatically adapt to unknown analyte concentrations without requiring preliminary sample dilution series or concentration range estimation, saving time and simplifying the workflow.
3Measurement precision
If sample dilutions are performed to extend detection range, then the dynamic range is extended, but the complexity and number of steps increase
Solution Approach 1:
The marker composition performs self-adjustment based on the analyte concentration in the sample. The high affinity markers automatically dominate at low concentrations, while low affinity markers dominate at high concentrations, without requiring external intervention such as dilution. This self-service mechanism extends the detection range while maintaining a simple, single-step assay procedure.
Solution Approach 2:
The assay exploits changes in binding affinity parameters across different marker types to achieve dynamic range extension. By varying the binding affinity parameter (Kd) while keeping the target the same, the system naturally adapts to different analyte concentrations, eliminating the need for physical sample manipulation like dilution and reducing procedural complexity.
Data Source
AI summary
Methods for detecting and quantifying an analyte employ a pair of proximity probes, each comprising a proteinaceous target-binding domain coupled to a nucleic acid domain (NAD), which NADs interact when the proximity probes have bound in proximity to their respective target; and a set of markers, wherein each marker is a nucleic acid molecule comprising a binding domain and a reporter domain giving a detectable signal, can interact with said NADs to form a nucleic acid molecule from which a detectable signal is generated, or with a nucleic acid molecule generated by interaction of said NADs, cannot interact with said NADs simultaneously with another marker in the set, generates a signal that is distinguishable from another marker signal, and is present in an amount capable of detecting analyte at a range of concentrations differing from the range of concentrations detectable by other markers.