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Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays. | LitMetric

Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays.

J Am Chem Soc

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States.

Published: September 2020

AI Article Synopsis

Article Abstract

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide -acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472518PMC
http://dx.doi.org/10.1021/jacs.0c06599DOI Listing

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