Fluorescent Dyes Multimerized on Branched Polyether Scaffolds
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Solution Overview
Problem
Conventional small molecule fluorescent dyes have limited brightness due to self-quenching and are unable to achieve phycobiliprotein-like fluorescence intensity without the drawbacks of phycobiliproteins, such as stability and availability issues, and existing multichromophore constructs face challenges with non-covalent binding and quenching.
Innovation Solution
Fluorescent dyes multimerized on branched polyether scaffolds, such as multi-arm polyethylene glycols, which provide high fluorescence intensity without noticeable quenching, stability against environmental conditions, and allow for a high degree of fluorophore labeling on biomolecules like antibodies without loss of activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If multiple fluorescent dye molecules are conjugated to biomolecules to increase brightness, then fluorescence intensity should increase proportionally, but self-quenching and dimer formation cause fluorescence intensity to decrease at higher degrees of labeling
Solution Approach 1:
The patent segments the fluorescent label into two distinct components: a multimeric scaffold containing multiple fluorophore units and a separate biomolecule (e.g., antibody). The scaffold is designed with a specific structure (e.g., polymeric, dendritic, or supramolecular assembly) that spatially separates the fluorophores to prevent self-quenching while maintaining high brightness. This segmentation allows the fluorophores to remain isolated from each other on the scaffold, eliminating the self-quenching effect that occurs when multiple dyes are randomly conjugated to biomolecules.
Solution Approach 2:
The patent introduces a multimeric scaffold as an intermediary structure between the fluorophores and the biomolecule. This scaffold acts as a mediator that holds multiple fluorophores in a controlled arrangement, preventing direct interaction between dye molecules that would cause self-quenching. The scaffold's structure (e.g., polyethylene glycol chains, dendritic arms, or DNA origami frameworks) ensures optimal spacing and orientation of fluorophores, allowing them to maintain individual fluorescence while collectively providing high brightness.
2Stability of the object's composition
If substituents are added to flat aromatic dye molecules to reduce dimerization, then water solubility increases, but deviation from linear proportionality between fluorescence intensity and degree of labeling persists
Solution Approach 1:
The patent segments the fluorescent system into a multimeric scaffold with controlled architecture and separate fluorophore units. Instead of relying on substituents on individual flat aromatic dyes, the scaffold provides the necessary spacing and solubility through its overall structure (e.g., polyethylene glycol chains, dendritic branches). This segmentation allows fluorophores to be positioned at optimal distances from each other, ensuring linear proportionality between degree of labeling and fluorescence intensity while maintaining water solubility through the hydrophilic scaffold structure.
Solution Approach 2:
The patent changes the structural parameters of the fluorescent system by transitioning from monomeric dyes with chemical substituents to multimeric scaffolds with controlled physical architecture. By adjusting parameters such as scaffold generation, arm length, fluorophore density, and spatial arrangement, the patent achieves both water solubility and linear fluorescence intensity proportionality. The multimeric scaffold's parameters can be tuned to optimize both solubility and fluorescence linearity independently of each other.
3Illumination intensity
If the number of functionalization sites on biomolecules is increased to achieve higher brightness, then conjugate brightness improves, but biomolecule activity is lost
Solution Approach 1:
The patent segments the high-brightness function into a separate multimeric scaffold component rather than requiring multiple functionalization sites on the biomolecule. The scaffold contains multiple fluorophore units (e.g., 10-100+ fluorophores per scaffold) that provide high brightness while the biomolecule requires only a single or few attachment points to the scaffold. This segmentation preserves biomolecule activity by minimizing the number of modifications needed on the biomolecule while achieving the desired brightness through the scaffold's inherent multimeric structure.
Solution Approach 2:
The patent creates a composite fluorescent conjugate consisting of a multimeric scaffold material combined with a biomolecule. The scaffold material provides the high-brightness function through its multiple fluorophore units, while the biomolecule provides the specific binding function. This composite structure allows the two components to fulfill their respective functions independently, with the scaffold bearing the burden of providing high brightness without requiring extensive functionalization of the biomolecule, thus preserving biomolecule activity.
4Illumination intensity
If phycobiliproteins are used to achieve high fluorescence intensity, then brightness increases, but stability against environmental conditions and photostability are limited
Solution Approach 1:
The patent replaces the protein-based phycobiliprotein structure with synthetic or semi-synthetic multimeric scaffolds that are chemically more stable. These scaffolds (e.g., polyethylene glycol-based, dendritic, or DNA/RNA frameworks) provide the same high-brightness function through multiple fluorophore units but with enhanced stability against environmental conditions such as temperature, pH, and proteolytic degradation. The synthetic scaffold materials are designed to be robust and long-lasting, eliminating the stability limitations of protein-based systems while maintaining the desired fluorescence intensity.
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 phycobiliprotein-like fluorescence intensity with low unspecific background staining and stability, enabling high degrees of fluorophore labeling on biomolecules like antibodies, surpassing conventional dyes and maintaining activity.
Implementation Method 1
Fluorescent dyes multimerized on branched polyether scaffolds, such as multi-arm polyethylene glycols, which provide high fluorescence intensity without noticeable quenching
Data Source
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AI summary
The invention is directed to a fluorescent dye according to the general formula I: with C is a core moiety comprising 20 to 200 atoms; S same or different ether residues comprising 1 to 10 carbon atoms; n is an integer ranging from 2 to 500; m is an integer ranging from 0 to 500; x is an integer ranging from 2 to 50; y is an integer ranging from 1 to 50; R R same or different residue comprising a reactive group capable of forming a covalent bond with a biomolecule; F same or different fluorophores covalently bound to (S)n. The fluorescent fluorescent dyes can be conjugated to a biomolecule and used for flow cytometry and/or by fluorescence microscopy.