Effects of Confinement and Crowding on Enzyme Kinetics Utilizing Lab-on-a-chip Devices

Analysis of chemical systems requires speed, reliability, accuracy and precision. With advances in technology, portability is also becoming a useful requirement for analytical instrumentation. Smaller, more portable instrumentation can be advantageous for the speed of analysis, especially for environmental and point-of-care monitoring. In this context, lab-on-a-chip devices are useful because they can incorporate sample preparation, injection, separation and detection into portable instrumentation. These chips also aid in the reduction of sample and reagent waste, because they use low volumes of reagent. At the nanoscale, small volumes mean that reagents experience increased wall and molecule-to-molecule interactions which can enhance kinetic measurements that mimic in vivo conditions, specifically those meant to probe the effects of molecular confinement and crowding.

This dissertation focuses on the design of complex fluidic networks with sub-100 nm geometries, providing confined environments for in vitro enzyme kinetics studies. These devices are fabricated using conventional metal deposition and photolithography in both hybrid polymer as well as in borosilicate glass devices and are designed to provide real-time optical analysis of reaction kinetics. A fluidic gradient mixer is designed and fabricated, so that it can be employed as a means to examine multiple concentration dependent enzyme reactions simultaneously. A series of bifurcated and trifurcated channels is employed to mix different starting analyte concentrations and produce a concentration gradient spanning the two. The fluidic gradient mixer is used to study effects of confinement of enzymes in free solution and immobilized on channel surfaces by limiting the channel size and maximizing aspect ratios. The effect of molecular crowding on enzyme kinetics studies are measured by increasing solution viscosity using varying concentrations of a viscogen, sucrose. These fluidic chips fabricated to implement this strategy may also be used to examine two or more enzyme reactions in a single experiment on a single chip. The information provided in this dissertation provides the groundwork to developing more complex biomacromolecule studies in nanometer scale geometries for both optical and mass spectrometric detection schemes.