Quinone Methide Precursors for Low-Background Multiplex Signal Amplification
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Solution Overview
Problem
Current signal amplification methods in immunohistochemistry (IHC) and in situ hybridization (ISH) suffer from increased background signals, limiting the detection of low-abundance cellular markers, and lack alternatives to horseradish peroxidase-based tyramide signal amplification (TSA) that can concurrently use chromogenic and fluorescence-based detection.
Innovation Solution
The use of quinone methide analog precursors (QMPs) that separate detectable label function from quinone methide generation and nucleophile stabilization, allowing for enzyme-mediated signal amplification without significant background noise, enabling chromogenic or fluorescence-based detection of multiple targets simultaneously or sequentially.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If signal amplification is performed using conventional methods (e.g., TSA), then detection sensitivity is improved, but background signal increases obscuring faint signals
Solution Approach 1:
The invention divides the amplification system into separate functional components: a first reagent that generates a reactive intermediate and a second reagent that provides the detectable label. This segmentation allows the signal generation and labeling functions to be performed separately, reducing non-specific background signal while maintaining amplification sensitivity.
Solution Approach 2:
The invention introduces a reactive intermediate species as a mediator between the enzyme-substrate interaction and the final detectable signal. The first reagent generates this intermediate, which then reacts with the second reagent to produce the amplified signal. This intermediary step provides temporal and spatial control over signal generation, reducing background noise.
2Reliability
If HRP-based TSA is used for signal amplification, then robust detection is achieved, but alternative ADRE systems are limited
Solution Approach 1:
The invention creates a universal amplification system that can work with multiple different analyte-dependent reporter enzymes (ADREs), not just HRP. The reagent pair (first reagent generating reactive intermediate, second reagent providing detectable label) can be adapted to various enzymes including phosphatases, glycosidases, and proteases, making the system universally applicable across different detection platforms.
3Adaptability or versatility
If amplification reagents are applied to multiple targets, then comprehensive diagnostic capability is improved, but reagent compatibility and signal differentiation become complex
Solution Approach 1:
The invention enables different reagent pairs to be used for different targets, with each pair having specific local characteristics (different reactive intermediates and detectable labels). This allows simultaneous or sequential detection of multiple targets with distinct signal properties, simplifying multiplexed assays while maintaining comprehensive diagnostic capability.
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
QMPs provide improved signal quality with reduced off-target staining, allowing for robust detection of multiple targets in tissue samples, enhancing detection sensitivity and correlation with morphological features.
Implementation Method 1
contacting the biological sample with a first labeling conjugate comprising a first enzyme. The first enzyme converts the first QMP into a first reactive quinone methide analog (QM)
Implementation Method 2
which covalently binds to the biological sample proximally to or directly on the first target
Implementation Method 3
tyramide is converted to a highly-reactive and short-lived radical intermediate that reacts preferentially with electron-rich amino acid residues on proteins
Data Source
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AI summary
Disclosed herein are novel quinone methide analog precursors and embodiments of a method and a kit of using the same for detecting one or more targets in a biological sample. The method of detection comprises contacting the sample with a detection probe, then contacting the sample with a labeling conjugate that comprises an enzyme. The enzyme interacts with a quinone methide analog precursor comprising a detectable label, forming a reactive quinone methide analog, which binds to the biological sample proximally to or directly on the target. The detectable label is then detected. In some embodiments, multiple targets can be detected by multiple quinone methide analog precursors interacting with different enzymes without the need for an enzyme deactivation step.