Novel Fluorescent Probe and Assay for Quantifying Adenosine and Adenosyl-Containing Compounds in Solution


Project:  D2014-24


Novelty: Use of a novel fluorescent probe and a recombinant protein in development of in vitro assays for determination of adenosine concentration in biochemical reactions.  


Description: Researchers at the University of Toledo have developed a fluorescence polarization (FP) assay that uses nanomolar concentrations of a novel fluorescent probe and nanomolar concentrations of recombinant protein for quantification of adenosine or adenosyl-containing compounds in a biochemical reaction. The recombinant protein developed in the university laboratory can bind adenosine; adenosine derivatives such as 5’ methylthioadenosine, 5’ deoxyadenosine, SAH, and 6-amino-6-deoxyfutalosine; and it is expected that many other adenosyl-based metabolites can also be specifically bound by this protein. These researchers validated the use of this assay to distinguish ATP and ADP/AMP at concentrations relevant to a typical protein kinase assay. The scientists observed no apparent binding of ATP at concentrations as high as 5 mM and saw a significant response to ADP concentrations in the mid nM range. A concentration course using AMP was found similar to the ADP concentration course.



Value Proposition:

·       Can be performed in real time with high precision

·       Option to perform as an endpoint assay

-       Can easily quench the coupled reaction if metals are required

-       Does not require strong acid – malachite green/quinaldine red

·       Broader range of possible adenosyl-containing compounds


Applications: This assay can be applied to any situation where the determination of adenosine concentration is required. Additionally, this system can be coupled to any biochemical reaction that either uses adenosine as a substrate or produces adenosine. Potential applications include:

·       Measurement of kinase activity

·       Measurement of enzymatic activity of deadenylase

·       Measurement of cAMP phosphodiesterase activity


Patent Information:
For Information, Contact:
Katherine Pollard
Licensing Associate
The University of Toledo
Donald Ronning
Vidhi Mishra
Endothelial Dysfunction
Fluorescent Probe