Bis-MPA dendritic scaffolds with orthogonal features provide enhance fluorescent and chemoselective labeling of biomolecules such as the Human IgG Fc antibodies
Cost-efficient fluorescent dyes with versatile multicolor labeling properties have become a powerful tool for bioimaging and diagnostic. While widely employed, its inherently low sensitivity is deemed as a major drawback. To overcome this, there is a need to identify dyes with strong fluorescence signals and offers accurate conjugation to biomolecules without jeopardizing the bioactivity of the conjugated product. Independent of conjugation chemistry, cyanine-based dyes are found to be one of the most promising near-infrared (NIR) dyes, ideal for in vivo and in vitro optical imaging. A natural strategy would be to use orthogonal linkers that are able to carry multiple strong fluorescent dyes while having only one single selective reactive group for site-specific conjugation to the selected biomolecule. In this context, dendrons, as highly branched and monodisperse scaffold with multiple peripheral end groups and a single reactive focal point seem to be an ideal option for an orthogonal reaction. PAMAM dendrons and Bis-MPA dendrons are both possible options but due to the biodegradable and biocompatible nature of Bis-MPA dendron, it will be a more desirable scaffold for biomedical application.
In this research paper, bis-MPA based dendrons as versatile fluorescent amplifiers for enhanced detection of biomolecules were proposed and tested with Human IgG Fc antibody. Three fluorescent dendrons (G0-Cy5, G1-Cy5, and G3-Cy5) with single hydrazine functionality focal point, a TEG extender between the hydrazine group and the fluorescent Cy5 moieties at the periphery were synthesized. The fluorescence signal of G0-Cy5, G1-Cy5, and G3-Cy5 was evaluated by Ultraviolet-Visible and Fluorescence Spectroscopic Technique. The G1-Cy5 and G3-Cy5 with 2 and 8 units of sulfo-Cy5 dyes respectively were found to fulfill the prerequisites for enhancing fluorescence signals but not the monovalent G0-Cy5. The most favorable signal amplification was G1-Cy5 with enhanced fluorescence by one order of magnitude when compared to G0-Cy5, which makes it ideal for macromolecules labeling applications such as immunoassays signal amplification. The hydrazine groups on the dendrons were able to selectively react with the aldehyde carboxyl on the Human IgG Fc to generate hydrazine linkages without compromising antibody activity or cause further oxidative damage. However, G3-Cy5 was found to induce strong fluorescence quenching when conjugated with Human IgG Fc due to a restricted biomolecular environment. Hence to overcome this, G3-Cy5 could be conjugated with a biomolecule with less restricted environment and be exploited as a strong fluorescent enhancer for bioimaging and diagnostic applications.
Most importantly, the presented synthetic strategy is not limited to Cy5, other fluorescence dyes with higher fluorescent emission can be used as alternatives.
For in-depth information please refer to the research paper below: >>>Design of Multivalent Fluorescent Dendritic Probes for Site-Specific Labeling of Biomolecules
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