Optical nanosystems for biomolecular reaction studies

Tae-Gon Cha, Purdue University

Abstract

There have been continuous efforts to understand the structures and functions of biomolecules. Biomolecular activities, critical to viability of living organisms, are often accomplished through a series of chemical reactions. A number of methodologies have been developed to probe and study biomolecular reactions, using radioactive, electrochemical, piezoelectric, or magnetic measurements. These techniques, however, are unable to provide non-invasive, comprehensive, and dynamic information about the biomolecular reactions. To overcome these limitations, optical methods have received significant attention because they are capable of acquiring and transferring dynamic information in-situ, in real-time non-intrusively with minimal perturbation to samples during the measurements. This thesis focuses on designing and exploring novel optical nanosystems that directly monitor and analyze various biomolecular reactions including molecular recognition and enzymatic activities. By integrating novel optical nanomaterials such as nucleic acid-functionalized nanocrystals and single-wall carbon nanotubes, we have probed biomolecular reactions at the nanoscale and studied their reaction kinetics. Theoretical kinetic models have been developed to elucidate the mechanisms involved in such molecular reactions and to understand the relevant kinetics. In this study, we have demonstrated two independent platforms - optical biosensors and synthetic DNA motors. In our first model system, efforts have been made to enable optical detection of physiologically important molecules in-situ, in real-time, non-invasively. Here, we used insulin as target analytes and optically monitored their secretion profiles from live pancreatic cells in a temporally and spatially resolved fashion using near-infrared fluorescent carbon nanotubes. The sensors modulate their emission properties upon binding to the analytes. A high selectivity (detection limit of ∼10 nM) is achieved by integrating nanotubes with molecular recognition elements such as insulin-binding aptamers. In theory, the sensitivity may reach the single-molecule level with single nanotube spectroscopy. The sensors are extremely robust such that they can be regenerated via proteolysis indefinitely, and thus, they are suitable for long-term measurements. Our theoretical model reveals the diffusion and binding reaction kinetics of analyte molecules of kr = ∼0.129 sec-1. In our second model system, we have demonstrated a DNA enzyme based synthetic molecular motor that transports inorganic nanoparticles, walking along carbon nanotubes. This system is reminiscent of cargo-carrying, microtubule-based intracellular protein motors such as kinesins and dyneins. In our design, a semiconductor nanocrystal and a carbon nanotube are used as model cargo and track, respectively. The DNA motors extract chemical energy from RNA molecules decorated on the nanotube track and convert that energy to direct processive, autonomous, unidirectional movement through a series of conformation changes. The translocation of the DNA motors has been visualized in real-time using spectrally resolved emission properties of the nanocrystal cargo and carbon nanotube track. We have identified and studied the critical parameters that govern the kinetics of the motor's programmed walking. Such parameters include enzymatic core sequence/structure and recognition arm lengths as well as environmental factors such as buffer temperature, pH, and types and concentration of metal cations. The maximum translocation speed observed in our experiments is approximately 1 nm sec-1. A theoretical kinetic model, based on the probability distributions of single molecule reactions, describes mechanistic details of the motor operation. The numerical calculations indicate that the single turnover time is roughly 1 min, where dissociation of the upper recognition arm and branch migration of the lower recognition arm are the rate-limiting steps for a given enzyme motor. By incorporating photoisomerizable azobenzene moieties into the DNAzyme sequences, we also have shown that the translocation kinetics of DNA motors can be effectively controlled by external light irradiation. The optical nanosystems studied in this thesis form a novel bioanalytical platform to probe and study biomolecular reactions both at the single-molecule and ensemble levels. Our integrated experimental and theoretical study ultimately generates kinetics-based design principles for DNA walkers and optical biosensors. The findings advance our understanding of DNA reactions and should be valuable to the field of DNA nanotechnology and biomolecular diagnostics in general.

Degree

Ph.D.

Advisors

Choi, Purdue University.

Subject Area

Nanotechnology|Optics

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