Single Molecule Enzyme Detection And Application To Immunoassay: Implications For Personalized Medicine
2010 ISE INTERNATIONAL CONFERENCE ON ENZYMES, CRETE, GREECE
Okrongly, D. Quanterix Corporation, Cambridge, MA
Quanterix was founded in 2007 based on single molecule detection of enzyme labels and fits well with the theme of the 2010 ISE Meeting: New Roles for Old Molecules: Enzymes in Personalized Medicine. The ability to observe the behavior of single enzyme molecules was first described by Rotman in 1961, using ß–D–galactosidase to cleave a fluorogenic substrate dispersed as microdroplets in silicone oil. This provocative finding was followed up over the years by others using various detection systems, and has led to a better understanding of the heterogeneity of enzymatic activity displayed at the single molecule level. In 2005, Rissin and Walt demonstrated the analytical potential of single enzyme molecule systems. They demonstrated that single molecules of streptavidin covalently coupled to ß–D–galactosidase (SBG) could be dispersed into an optical array composed of 24,000 individual femtoliter reaction chambers coated with biotin. After sealing the chambers with a fluorogenic substrate solution trapped inside, a digital readout of the array was obtained by counting the number of fluorescent reaction vessels, indicating that the well had captured an SBG molecule. The number of fluorescent wells was demonstrated to be proportional to the concentration of SBG present in the original solution and was consistent with a Poisson distribution at low concentrations where single molecules were in each well and Gaussian distribution at higher concentrations where more than one enzyme molecule was in each vessel. The sensitivity was an astounding 2.6 aM (2.6 x 10–18 M). This observation paved the way for more complex systems to be developed, where a capture antibody–ligand–detector antibody complex is sequentially formed in the vessel, and the detector antibody (coupled with biotin), is labeled by the SBG. Quanterix is utilizing this technology to develop high sensitivity assays for the clinical diagnostics market. Data for several representative assays with over 1000x more sensitivity than standard immunoassays will be presented, along with the implications for personalized medicine.