Development of Microfluidic Mixing Techniques to study Enzymatic Reactions Público
Kise, Drew Phillip (2016)
Abstract
Enzymes are biological catalysts with far-reaching implications to the sustaining of biological processes and life as we know it. However, even after many years of extensive research on enzymes, scientists still have questions as to how enzymes are able to do what they do. It is now known that dynamics, in fact, play a role ranging from molecular vibrations to large conformational changes. Therefore, technologies are needed that can be used to study the dynamic processes in enzymes. Microfluidic mixing has been continually developed over the past couple of decades to become one such technique used for enzymatic studies. In this dissertation, developments in both microfluidic mixers, the instruments utilizing the mixers, and applications to enzymatic reactions are discussed. New fabrication methods for microfluidic mixers have been created that can produce mixers in a cost-efficient manner. Also, an infrared imaging system has been developed to apply microfluidic mixing with infrared spectroscopy. Finally, the developments are applied to biological systems. The newly developed infrared imaging was used a sandwich-format microfluidic mixer to study a pH jump experiment with adenosine monophosphate, which both proved the utility of the instrument and established the fast mixing time, defining the timescale of reaction kinetics that can be viably studied. Next, fast mixing was employed to study reaction kinetics in the catalytic cycle of the enzyme dihydrofolate reductase. A common model among enzymatic studies, the hydride step of DHFR catalysis was resolved in mixing experiments by following the intrinsic emission of the cofactor, NADPH. Along with resolving the kinetics of the pH dependent hydride transfer step, a second process was also resolved that was attributed to a pH independent conformational change in DHFR to ready itself for the transfer of a hydride from NADPH to substrate, dihydrofolate. Finally, the implications and future directions of the developed technologies are then discussed.
Table of Contents
Chapter 1: Introduction
Chapter 2: Microfluidic mixer design and optimization. 10
Section 2.1 Introduction to microfluidic mixer design. 10
Section 2.2 The 2D fast microfluidic mixer design and calibration. 15
Section 2.3 Optimization of 2D mixer design. 21
Section 2.4 Other production methods for 2D microfluidic mixers. 26
Section 2.5 Advances in 3D capillary mixer. 47
Chapter 3: Development of an infrared imaging system to follow submillisecond mixing reactions. 52
Section 3.1 Introduction. 52
Section 3.2 The sandwich-style mixer. 53
Section 3.3 Development of Homemade Infrared Imaging System. 58
Section 3.4 Application of infrared imaging system. 68
Section 3.5 Modifications to infrared imaging system. 81
Chapter 4: Applications of microfluidic mixing to biological systems. 85
Section 4.1: Introduction to biological kinetics methodology from micro- to milliseconds. 85
Section 4.2: Introduction to DHFR catalysis. 88
Section 4.3: DHFR microfluidic mixing experiments. 92
Chapter 5: Conclusions. 103
References. 107
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