Office of Technology Transfer – University of Michigan

Fluorescent Dye Conjugate

Technology #5608

This is a dendrimer conjugated fluorescent dye that can be used as a bioorthogonal chemical reporter for probing cellular processes. Following a ‘click’ reaction between an azide group on this dye and an alkyne moiety that has been incorporated into a macromolecule or metabolite in a cell, this fluorophore undergoes a 21-fold increase in fluorescence intensity. Given this excellent signal-to-noise ratio, purification steps normally required to remove background fluorescence generated by unreacted reporter are eliminated, and assay robustness and throughput are significantly improved. The market for fluorescent reagents in life sciences research is over $1 billion and growing. High quality fluorescent dyes and probes greatly facilitate cellular analysis and imaging, and reagent performance has a tremendous impact on the results and timelines of scientific endeavors.

Fluorescent Dye Conjugate with Improved Physical and Chemical Properties

Researchers at the Michigan Nanotechnology Institute developed a polymer-conjugated azidocoumarin dye that has superior physical characteristics and improved in vitro assay performance compared to the small molecule reagent. Specifically, this fluorescent dye conjugate demonstrates better aqueous solubility and higher signal-to-noise ratios by showing more complete fluorescence quenching in the unreacted state. This dendrimer-conjugated azidocoumarin was designed as a ‘click’ reagent to react with alkyne-containing target biomolecules such that they could be analyzed by a fluorescence detector. Following reaction with an alkyne-derivatized molecule, the conjugated coumarin showed a 21-fold fluorescence intensity increase compared to an 8.5-fold increase observed for the unconjugated fluorophore. This difference was primarily attributable to a decrease in unreacted fluorophore fluorescence of the dendrimer-conjugated coumarin. Furthermore, this polymer conjugate showed superior attributes when tested in a cell proliferation reporter assay. Briefly, 5-ethynyl-2’-deoxyuridine was incorporated into newly synthesized DNA in a cancer cell line, and subsequent labeling of the DNA with dendrimer-conjugated azidocoumarin required no washing procedure to accurately reflect 5-ethynyl-2’-deoxyuridine incorporation. In contrast, cells labeled with azidocoumarin small molecule had to be washed extensively to remove unreacted fluorophore before signal intensity could be directly correlated with alkynyl nucleoside incorporation. Elimination of purification steps enhances the efficiency of this assay such that it can now be used for high-throughput profiling of biological systems.

Applications

  • Molecular profiling of biological systems
  • Fluorescence-based biomolecule detection
  • ‘Click’ chemical reporter
  • High-throughput cellular assay

Advantages

  • Improved assay signal-to-noise ratio
  • Improved water solubility of fluorescent dye
  • Elimination of difficult and time consuming purification procedures