Enzyme Molecules as Nanomotors and Micropumps

Open Access
Author:
Sengupta, Samudra
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 06, 2013
Committee Members:
  • Ayusman Sen, Dissertation Advisor
  • Thomas E Mallouk, Committee Member
  • John V Badding, Committee Member
  • Peter J Butler, Special Member
Keywords:
  • enzyme
  • single-molecule
  • diffusion
  • nanomotor
  • chemotaxis
  • micropump
Abstract:
The predominant goal that motivated this research was to design artificial enzyme-powered motors that can move autonomously in solution utilizing energy from catalytic reactions. Enzyme-based biological motors perform specific cellular functions, such as DNA synthesis and vesicular transport, with great precision and efficiency. In all cases, the movement arises from the harnessing of chemical free energy released through enzymatic turnover of substrates. A fundamental question that arises is whether a single enzyme molecule can generate sufficient mechanical force through substrate turnover to cause its own movement and, more significantly, whether the movement can become directional through the imposition of a gradient in substrate concentration, a situation that parallels the chemotaxis of whole cells. Positive answers to these questions have important implications in areas ranging from biological transport to the design of “intelligent,” enzyme-powered, autonomous nano- and micromotors, which are expected to find applications in bottom-up assembly of structures, pattern formation, cargo delivery at specific locations, roving sensors, and related functions. The dissertation starts by discussing the evolution of catalytic nano/micromotors and micropumps. The physics involved in powering objects at the nanoscale has been briefly reviewed. A detailed survey on the existing artificial micromotors and micropumps has been given, starting from the preliminary nanomotor design to the present day design modifications and their applications in the field of microscale delivery vehicles and assembly. The limitations of these artificial systems have been discussed with emphasis on how they can be improved using biocatalysts to power miniature devices. Using fluorescence correlation spectroscopy, it was shown that the diffusive movement of single enzyme molecules of urease and catalase increases in the presence of their respective substrates, urea and hydrogen peroxide, in a concentration-dependent manner. The increase in diffusion of both the enzymes is highly attenuated in the presence of the respective enzyme inhibitors, thereby, suggesting the role of catalysis in enhanced diffusive behavior of enzyme molecules. The mechanisms involved in the anomalously high diffusion were explored. The impulse force generated during single enzymatic turnover was also estimated. This dissertation highlights the chemotactic behavior of enzyme molecules, a phenomenon observed in biological systems like bacteria and cells. By employing a microfluidic device to generate substrate concentration gradient, it was demonstrated that ensembles of catalase and urease enzyme molecules spread towards areas of higher substrate concentration, a form of chemotaxis at the single molecule scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, the migration of catalase towards glucose oxidase was induced. The potential of DNA polymerase to function as a nanomotor and micropump has been explored. The DNA polymerase is responsible for synthesizing DNA, a key component for running the biological machinery. The diffusive movement of a molecular complex of DNA template and DNA polymerase was enhanced during nucleotide incorporation into the growing DNA template. The molecular complex also exhibits collective migration towards areas of higher nucleotide and cofactor concentrations. Further, by immobilizing the molecular complex on a patterned surface, it was demonstrated that fluid and tracer particles could be pumped in a directional manner with the pumping speed increasing in the presence of cofactor in the surrounding fluid. The role of DNA polymerase as a micropump opens up an entirely new avenue of designing miniature fluid pumps using enzymes as engines. An enzyme that moves by generating a continuous surface force in a fluid should, when fixed in place, function as a micropump moving fluid and colloids in a directed manner. It was established that by immobilizing enzymes on a patterned gold surface, ATP-independent, non-mechanical enzyme-powered micropumps can be fabricated that function in the presence of their substrates. The pumping velocity increases in a substrate-concentration-dependent manner. These pumps demonstrate both spatial and temporal regulation of fluid and colloid transport. Further, these pumps can be triggered by the presence of specific analytes in solution, opening up the possibility of designing enzyme based smart devices that act both as a sensor and a pump. Finally, a proof-of-concept device, powered by enzymes, was designed that acts as a scaffold for stimuli responsive autonomous delivery of small molecules. This dissertation concludes with an outlook on the field of artificial nano/micromotors and micropumps. It also highlights how the research findings emphasized in this dissertation aid in making a big leap towards improvising this field both from a fundamental and an application standpoint.