Paper BI+NC-WeM12
Optical Fiber Microarrays for Single Molecule Detection

Wednesday, October 22, 2008, 11:40 am, Room 202

Optical fiber microarrays have been employed for the detection of single enzyme molecules. Single enzyme molecules were enclosed with fluorogenic substrate in an array of 50,000 individually addressable microchambers etched into a glass optical fiber bundle. The large array size provided excellent statistics. The substrate turnover in the microchambers was monitored with epifluorescence microscopy. We have observed a broad distribution of discrete turnover rates of single ß-galactosidase molecules that can be attributed to different enzyme conformations. When a slow-binding inhibitor was added to single ß-galactosidase molecules inhibited and active states of ß-galactosidase could be clearly distinguished. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme’s catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics. Furthermore, we have applied the optical fiber microarray to the detection of single horseradish peroxidase molecules. These monomeric enzyme molecules exhibit a narrower distribution of turnover rates than the tetrameric ß-galactosidase, which could be explained by the number of catalytic sites involved in substrate turnover.