Ultrafast tangential micro-mixers for the study of biochemical reactions on the microsecond time scale

Detailed understanding of chemical and enzyme catalysis constitutes a main focus of current biochemical research. Fundamental insight in how (bio)catalysts function, requires knowledge of their three dimensional structure and a wide range of time resolved experiments that monitor the reaction progress. The ultimate aim is the determination of the molecular structure of transition and transient states during the chemical bond-breaking and bond-making step that occurs as part of the overall reaction. Chemists claim to have observed transient or transition states with lifetimes as short as 100-500 femtoseconds. Single steps in enzyme catalysis are usually slower than this, although electron transfer and proton transfer can occur in picoseconds or nanoseconds, respectively. The movements of protein domains which are critical to drive enzyme catalysis because they directly promote the breaking and reforming of chemical bonds, occur at a longer time scale of ~0.1-1 µs. This time range can thus be regarded as the fastest in which formation of enzyme catalytic intermediates occur or protein domains can fold into the native structure of the active enzyme.

To study catalytic mechanisms of enzymes and chemical reactions in detail, the reaction should be initiated so rapidly that the subsequent formation and decay of all reaction intermediates can in fact be detected. Even the fastest present-day continuous-flow mixing equipment is too slow (~45 µs) to monitor the very beginning of enzyme catalysis. In order to design a general kinetic instrument with a much shorter dead-time to mix reactants and observe the reaction progress both the mixer and observation cell need to be miniaturized to micrometer dimensions (~100 µm) while maintaining high mixing efficiency and good optical quality. This thesis deals with the design and development of a new kinetic instrument that can perform, observe and detail, on the μs time scale, the catalytic mechanism of enzymes, in particular those of the oxidoreductases.