The directed transport of microparticles in microfluidic devices is vital for efficient bioassays and fabrication of complex microstructures. There remains, however, a need for methods to propel and ...steer microscopic cargo that do not require modifying these particles. Using theory and experiments, we show that catalytic surface reactions can be used to deliver microparticle cargo to specified regions in microchambers. Here reagents diffuse from a gel reservoir and react with the catalyst-coated surface. Fluid density gradients due to the spatially varying reagent concentration induce a convective flow, which carries the suspended particles until the reagents are consumed. Consequently, the cargo is deposited around a specific position on the surface. The velocity and final peak location of the cargo can be tuned independently. By increasing the local particle concentration, highly sensitive assays can be performed efficiently and rapidly. Moreover, the process can be repeated by introducing fresh reagent into the microchamber.
Recent experimental results have shown that enzymes can diffuse faster when they are in the presence of their reactants (substrate). This faster diffusion has been termed enhanced diffusion. ...Fluorescence correlation spectroscopy (FCS), which has been employed as the only method to make these measurements, relies on analyzing the fluctuations in fluorescence intensity to measure the diffusion coefficient of particles. Recently, artifacts in FCS measurements due to its sensitivity to environmental conditions have been evaluated, calling prior enhanced diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes. Herein, we use a technique of direct single molecule imaging to observe the diffusion of individual enzymes in solution. This technique is less sensitive to intensity fluctuations and deduces the diffusion coefficient directly based on the trajectory of the enzyme. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if the enzyme oligomerization state changes during its catalytic turnover. We find that the diffusion increase is independent of the total concentration of enzymes and the presence of substrate does not change the oligomerization state of enzymes.
Surface-immobilized enzyme systems can be used as micropumps in the presence of enzyme-specific substrates. In other words, the enzymes transduce chemical energy from the reaction into fluid motion. ...This discovery enables the design of non-mechanical, self-powered nano/microscale pumps that precisely control flow rate and turn on and off in response to specific analytes. In order to obtain spatio-temporal control over enzyme micropumps with different architectures, it is essential to understand in detail how solutal and thermal buoyancy affect the speed and pumping directionality. Presented are the results of separately probing the effects of solutal and thermal buoyancy on the behavior of phosphatase-based micropumps through experiments and modeling. One of the key outcomes of this study is that even though the reactions catalyzed by phosphatases are fairly exothermic, the primary driver behind pumping is the difference between the densities of the products versus the reactants. To further investigate the mechanisms driving enzyme micropumps, both glutamate dehydrogenase (GLDH) and alcohol dehydrogenase (ADH), enzymes that catalyze reversible reactions with the assistance of cofactors, were studied. These types of reactions are important to consider as a part of the fundamental understanding of pumps because many of the enzymes found in nature catalyze reversible reactions, including enzymes that are involved in metabolism, by which cells can harvest energy to perform important functions. GLDH and ADH pumps have been studied with the substrates only (in the absence of the respective cofactors) to understand the pumping behavior when these enzymes are not catalyzing any reaction. The resulting dehydrogenase pumping behavior in the presence of only substrates implies that the interaction between substrates and enzyme is sufficient to power the enzyme microfluidic pumping. Supplementary experiments were done to confirm that the interaction of enzyme and substrate can produce sufficient mechanical work. Fluorescence correlation spectroscopy (FCS) was done to show that the diffusion of free enzymes increases in the presence of only substrates. In addition, isothermal titration calorimetry (ITC) was done to determine the thermodynamic parameters of GLDH and its substrates. Another goal of this work was the development of an alternative enzyme pump architecture, particle-based enzyme pumps, which can potentially be used in directed fluid transport. Different enzyme particle pumps were studied in the presence of their substrates and proved more versatile in comparison to the 2D surface-patterned pumps previously studied. In one study, enzymes were anchored onto microparticles resulting in enhanced and directed fluid pumping. In a final study, enzymes were encapsulated inside virus-like particles and demonstrated their ability to move and power the motion of other particles, similar to a mobile pump. These two systems could aid in the development of enzyme pump networks, which can be explored for its application in fluidic delivery.
Recent experimental results have shown that active enzymes can diffuse faster when they are in the presence of their substrates. Fluorescence correlation spectroscopy (FCS), which relies on analyzing ...the fluctuations in fluorescence intensity signal to measure the diffusion coefficient of particles, has typically been employed in most of the prior studies. However, flaws in the FCS method, due to its high sensitivity to the environment, have recently been evaluated, calling the prior diffusion results into question. It behooves us to adopt complimentary and direct methods to measure the mobility of enzymes in solution. Herein, we use a novel technique of direct single-molecule imaging to observe the diffusion of single enzymes. This technique is less sensitive to intensity fluctuations and gives the diffusion coefficient directly based on the trajectory of the enzymes. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if enzyme oligomerization state changes during catalytic turnover. We find that the diffusion increase is independent of the total background concentration of enzyme and the catalysis of substrate does not change the oligomerization state of enzymes.
PDF
