Enzyme-mediated individual nanoparticle release assay.

Analytical Biochemistry
James R GlassD A Schultz

Abstract

Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzy...Continue Reading

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Citations

Aug 25, 2007·Nanomedicine·Hongwei LiaoJason H Hafner
Sep 30, 2010·Small·Caterina MinelliMolly M Stevens
Jan 26, 2021·Analytical and Bioanalytical Chemistry·Pavel KhramtsovMikhail Rayev

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