A population of identical proteins has the same amino acid sequence, but there may be subtle differences in local folding that lead to variations in activity. Single molecule studies allow us to understand these subtle differences. Single molecule experiments are usually time consuming and difficult because only a few molecules are observed in one experiment. To address this problem, we have developed an assay where we can simultaneously measure the activity of multiple individual molecules of a protease, α-chymotrypsin. The assay utilizes a synthetic chymotrypsin substrate that is non-fluorescent before cleavage by chymotrypsin, but is intensely fluorescent after. To study the activity of individual enzymes, the enzyme and substrate are encapsulated in micron-sized droplets of water surrounded by silicone oil. On average, each micro-droplet contains less than one enzyme. The fluorescence of these droplets is recorded over time using a microscope and a CCD camera system. Software tracks individual droplets over time and records fluorescence. The kinetics of individual chymotrypsin molecules is calculated through the increase of fluorescence intensity of the same individual droplet over time. The activity profiles of the individual enzymes and the bulk sample of the enzyme are very similar. This validates the assay and demonstrates that the average of a few individual molecules can be representative of the behavior of the bulk population.
The particular form of electrochemiluminescence (ECL) used for analytical assays relies upon the discovery that tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)<sub>3</sub><sup>2+</sup>] emits a 620 nm photon when adjacent to an electrode held at about one volt relative to Ag/AgCl. This reaction occurs within nanometers of the electrode. The enormous economic investment in nanoscale lithography tools is leading to tools capable of routinely producing 32 nm features by 2009. We propose that these two technologies could be combined to produce a nanoscale microscopy system. We constructed a macroscopic test-bed and performed tests on it to explore the feasibility of such a system. We tested an ECL solution containing 1 mM Ru(bpy)<sub>3</sub><sup>2+</sup> 0.2 mM ammonium oxalate monohydrate in a 0.1 M ammonium acetate buffer at pH 5.0. Using this solution, we found that the ECL light was most intense at an applied voltage of 1.6 Volts, that the effect had excellent reproducibility and that the time to reach maximum intensity was several seconds after applying a voltage.