One way to stop malignant cells in their tracks is to interfere with the activity of enzymes required to produce uncontrolled cell division, angiogenesis, or metastasis. A new assay that uses nanoparticle release to detect and quantify changes in enzyme activity could greatly speed the search for molecules that can alter the activity of enzymes that play a role in cancer.
Reporting its work in the journal Analytical Biochemistry, a team of investigators led by David Schultz, Ph.D., at the University of California, San Diego, describes its use of plasmon resonance spectroscopy to detect the release of individual gold nanoparticles that are cleaved from a surface through the activity of a target enzyme. The nanoparticles are tethered to a solid surface using a linker that is the substrate for the target enzyme. Enzyme activity is monitored by observing a change in plasmon resonance resulting from the release of single nanoparticles, affording exquisite sensitivity for even low levels of enzyme activity.
In a demonstration of the technique, the researchers used DNA as a tether and measured the activity of an enzyme that cuts this DNA at a specific site. Individual nanoparticle release was counted by measuring a decrease in plasmon resonance using dark-field microscopy. Nanoparticles released through the action of the DNA cleavage enzyme float out of the microscope’s field of view, and the resulting drop in optical signal intensity is measured easily. Particle release was apparent within 2 minutes of adding the enzyme to the immobilized linker-nanoparticle assay system.
In a second proof-of-concept experiment, the investigators used as a tether a particular piece of DNA that is normally a substrate for an enzyme that adds a methyl group to DNA; DNA methylation is involved in regulating gene expression. In this case, DNA methylation blocks DNA cleavage by a second enzyme whose activity is not being monitored. However, any changes in the activity of the DNA methylating enzyme impacts the activity of the second enzyme, which is reflected in the number of gold nanoparticles released. This same system was then used successfully to assay inhibitors of DNA methylation.
The investigators note that this assay system should be amenable to miniaturization and is appropriate for use in high-throughput screening systems. They also believe that this system could be adapted for use in microfluidic systems, suggesting that the nanoparticle-release assays could eventually find their way into bedside monitoring systems.
This work is detailed in a paper titled, “Enzyme-mediated individual nanoparticle release assay.” Investigators from Seashell Technology, in La Jolla, CA, also participated in this study. An abstract of this paper is available through PubMed.
"Source"_[ http://nanotechwire.com/news.asp?nid=3395]
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