Azide-Based Cleavable Linker for Iterative Biological Detection
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Solution Overview
Problem
Conventional fluorescence imaging techniques face limitations such as partial photobleaching, specimen degradation from harsh chemicals, and increased background due to mis-hybridization and non-specific binding, which hinder consistent protein quantification and comprehensive DNA, RNA, and protein analysis.
Innovation Solution
A system and method utilizing azide-based cleavable linkers to tether fluorophores to probes, allowing for efficient chemical cleavage with tris(2-carboxyethyl)phosphine (TCEP), enabling iterative detection of biological molecules with high on/off ratios and minimal sample alteration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If photobleaching is used to remove fluorescence signals, then fluorescence signal removal is achieved, but partial photobleaching during imaging and incomplete signal removal occur
Solution Approach 1:
The fluorophore is extracted from the probe through chemical cleavage of the azide-based linker by TCEP, separating the detectable moiety from the probe. This allows complete and controlled removal of the fluorescence signal without affecting the probe's ability to bind targets, enabling reliable iterative detection cycles with consistent quantification.
2Reliability
If harsh chemical reagents are used to remove fluorophores, then fluorescence signal removal is achieved, but specimen degradation occurs
Solution Approach 1:
The chemical conditions for fluorophore removal are optimized by using TCEP at controlled concentrations and temperatures. This moderate chemical approach achieves complete fluorophore cleavage while minimizing damage to the specimen, allowing multiple iterative cycles without significant sample degradation.
3Reliability
If DNA displacement reactions are used for iterative detection, then fluorescence signal removal is achieved, but mis-hybridization and non-specific binding increase background
Solution Approach 1:
An azide-based chemical linker serves as an intermediary between the fluorophore and the probe, enabling controlled chemical cleavage by TCEP. This chemical mediation approach avoids DNA hybridization issues entirely, providing clean signal removal without mis-hybridization or non-specific binding, thus maintaining low background in iterative detection cycles.
4Productivity
If multiple cycles of staining and imaging are performed, then comprehensive target profiling is achieved, but sample degradation accumulates
Solution Approach 1:
The fluorophore is completely extracted from the probe through chemical cleavage after each imaging cycle, removing the source of potential damage while preserving the intact probe for subsequent staining cycles. This enables multiple iterative detection cycles (profiling of 100+ targets) without cumulative sample degradation, as the gentle TCEP cleavage conditions do not affect the probe's binding 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
Enables comprehensive profiling of over 100 different targets in single cells with efficient fluorophore removal and retention of antigenicity, overcoming the limitations of conventional techniques by allowing multiple cycles of staining, imaging, and cleavage without degrading the sample.
Implementation Method 1
The linker is configured to be cleaved in the presence of tris(2-carboxyethyl)phosphine (TCEP)
Implementation Method 2
exciting the sample at a first wavelength, detecting the emission of a second wavelength from the sample
Data Source
AI summary
A system for the iterative detection of biological molecules includes a probe, and a fluorophore tethered to the probe with an azide-based linker. The linker is configured to be cleaved in the presence of tris(2-carboxyethyl)phosphine (TCEP), and the on/off ratio between a signal measured before treatment with TCEP and a signal measured after treatment with TCEP is at least about 20:1.