BCN Clickable Fluorophore Linkers for Mild Rapid Fluorescence Quenching
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Solution Overview
Problem
Cellular samples from fine needle aspirates are scant and delicate, limiting the number of stains that can be applied, and existing fluorescent cycling methods are not compatible with these samples, requiring harsh conditions that degrade them.
Innovation Solution
Utilization of bicyclononyne (BCN)-based clickable fluorophore linkers that enable ultrafast quenching of fluorescence through click reactions with tetrazine or azide-functionalized quenchers, enhancing stability and reaction kinetics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If harsh destaining/quenching conditions are used for fluorescent cycling, then fluorescence quenching performance is improved, but cellular sample stability deteriorates
Solution Approach 1:
The patent changes the chemical parameters of the quenching system by introducing bicyclononyne (BCN)-based fluorophore linkers that enable photoinduced electron transfer (PET) quenching. This allows fluorescence quenching to occur under mild conditions (room temperature, neutral pH) rather than harsh conditions, thus maintaining cellular sample stability while achieving reliable quenching performance.
Solution Approach 2:
The BCN-based fluorophore linker acts as an intermediary between the fluorophore and the quenching mechanism. It facilitates the photoinduced electron transfer process that enables fluorescence quenching without requiring harsh chemical conditions, thereby protecting the cellular sample from degradation while achieving effective quenching.
2Reliability
If existing fluorescent cycling methods are applied to cellular samples, then fluorescence imaging capability is improved, but sample processing time increases
Solution Approach 1:
The patent modifies the reaction kinetics parameters by using BCN-based linkers that enable rapid photoinduced electron transfer. This accelerates the quenching process from hours to minutes, significantly reducing sample processing time while maintaining fluorescence imaging capability.
Solution Approach 2:
The patent employs a direct and rapid quenching mechanism through PET-mediated electron transfer that skips intermediate steps required by conventional methods. This rushing through the quenching process achieves effective fluorescence imaging in a fraction of the time required by traditional approaches.
3Reliability
If trans-cyclooctene-based probes are used, then quenching performance is achieved, but oxidation and photo-stability deteriorates
Solution Approach 1:
The patent changes the chemical structure parameter from trans-cyclooctene to bicyclononyne (BCN) moiety. This structural modification enhances the probe's resistance to oxidation and photo-degradation while maintaining effective quenching performance, as the BCN-based probes show superior stability after environmental exposure.
Solution Approach 2:
The patent creates a composite probe system combining the BCN-based fluorophore linker with appropriate fluorophores and quenchers. This composite structure leverages the enhanced stability of the BCN moiety to protect the entire probe system from oxidation and photo-degradation while preserving quenching functionality.
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
Achieves superior oxidation- and photo-stability, allowing for rapid quenching of fluorescence in cellular samples, enabling multiplexed imaging of up to 20-30 markers within one hour with enhanced reaction rates.
Implementation Method 1
a tetrazine- or an azide-functionalized quencher can be clicked to the BCN moiety, thereby quenching fluorescence and turning off the fluorophore
Implementation Method 2
the BCN-based probes within the present claims show superior oxidation- and photo-stability after environmental exposure (e.g., ambient light, microscopy illumination, and air) and higher quenching performance
Data Source
AI summary
The present disclosure provides bicyclononyne based compounds and methods to prepare an antibody conjugate with a fluorophore, as well as the methods of using these conjugates for cellular imaging. In one example, the conjugate may be coupled with a quencher to absorb fluorescence from the fluorophore.