Most information about enzyme kinetics has been garnered by traditional bulk reactions that average the behavior of large enzyme populations. Only in recent years single molecule techniques were developed that shed light on the individual behavior of enzyme molecules in a population. Performing single molecule experiments one by one is cumbersome and statistically not representative of the whole population. To obtain better statistics hundreds of single enzyme molecules can be separated and enclosed in a large array of confined femtoliter containers. Furthermore, no surface immobilzation of the enzyme is required, such that steric hindrance or partial enzyme inactivation are avoided. According to the Poisson distribution, one enzyme molecule in 20 containers yields a maximum of only a single enzyme molecule per container while the rest are empty. The catalytic activity of these individual enzyme molecule results in the turnover of a non-fluorescent substrate to a fluorescent product that can be measured simultaneously by fluorescence microscopy.
The topics listed below provide an overview of my previous work on single enzyme molecule kinetics in the Walt group at Tufts University. Single molecule experiments with various enzyme molecules in new femtoliter arrays will follow up. The substrate turnover of these enzyme molecules will be monitored individually by fluorescence microscopy to elucidate their static heterogeneity and single molecule inhibition mechanisms. My long-term goal is to move the field of single enzyme molecule kinetics forward toward evolutionary questions and enzymes of biomedical importance.
Bioanalysis based on organic fluorophores or quantum dots is limited by autofluorescence and light scattering from biological materials that increase with shorter wavelengths. These limitations can be elegantly avoided by using nanoparticles made of upconverting photoluminescent materials (UCLNPs). UCLNPs can be excited with near-infrared light from a simple continuous light source and emit higher energy wavelengths with large anti-Stokes shifts. Thus, luminescence is measured without background. One of the most efficient types of UCLNPs consists of a NaYF4 host lattice doped with the lanthanide ions Yb3+ and Er3+. Due to overlapping lanthanide energy levels, Yb3+ absorbs excitation light at 980 nm and then sensitizes Er3+ in two sequential non-radiative energy transfer steps. The activator Er3+ displays dual and well separated emission bands of green (543 nm) and red (657 nm) light.