Date of Degree
PhD (Doctor of Philosophy)
Alexei V. Tivanski
The necessity to explore nanoscopic systems is ever increasing in the world of science and technology. This evolving need to study such physically small systems demands new experimental techniques and methodologies. Atomic force microscopy (AFM) is a versatile technique that can overcome many nanoscopic size limitations. AFM has been utilized in the world of nanotechnology to study physiochemical properties of particles, materials, and biomolecules through characterization of morphology, electrical and mechanical properties, binding interactions, and surface tension, among others. The work discussed herein is largely a report of several novel AFM methodologies that were developed to allow new characterization techniques of individual submicrometer particles and single biomolecular interactions.
The effects of atmospheric aerosols on the radiative budget of the earth and climate are largely unknown. For this reason, characterizing the physiochemical properties of aerosols is vital. Since the particles that have relatively long lifetimes in the atmosphere are smaller than one micrometer in size, high resolution microscopy techniques are required to study them. AFM is a suitable technique for single particle studies because it has nanometer spatial resolution, can perform experiments under ambient pressure and variable relative humidity and temperature. These advantages were utilized here and AFM was used to study morphology, organic volume fraction, water uptake, and surface tension of nascent sea spray aerosol (SSA) particles as well as laboratory generated aerosols composed of relevant chemical model systems. The morphology of SSA was found, often times, to be composed of core-shell structure. With complementary microscopy techniques, the composition of the core and the shell was found to be inorganic and organic in nature, respectively. Novel methodology to measure water uptake and surface tension of single substrate deposited particles with AFM was established using chemical model systems. Furthermore, these methodologies were employed on nascent chemically complex SSA particles collected from a biologically active oceanic waveflume experiment. Finally, phase imaging was used to measure organic volume fraction on a single particle basis and was correlated with biological activity. Overall, this suite of single (submicrometer) particle AFM analysis techniques have been established, allowing future systematic studies of increasing complexity aimed at bridging the gap between the simplicity of laboratory generated particles and the complexity of nature.
Another nanotechnology topic of interest is studying single biomolecular interactions. Virtually every biological process involves some amount of minute forces that are required for the biomolecular system to function properly. For example, there are picoNewton forces associated with enzymatic motions that are important for enzyme catalysis. The AFM studies reported here use a model enzyme/drug system to measure the forces associated with single molecule adhesion events. Escherichia Dihydrofolate Reductase (DHFR) is a target of cancer therapeutic studies because it can be inhibited by drugs like methotrexate (MTX) that are structurally similar to the natural folate binder but have much higher binding affinity. One of the obstacles of single molecular recognition force spectroscopy (MRFS) studies is the contribution of non-specific forces that create a source of uncertainty. In this study, DHFR and MTX are bound to the surface and the AFM tip, respectively, using several different linking molecules. These linking molecules included polyethylene glycol (PEG) and double stranded DNA (dsDNA) and the distribution of forces was compared to scenarios were a linker was not employed. We discovered that dsDNA and PEG both allow identification and removal of non-specific interaction forces from specific forces of interest, which increases the accuracy of the measurement compared to directly bound constructs. Traditionally, the linker of choice in the MRFS community is PEG. Here, we introduce dsDNA as a viable linker that offers more rigidity than PEG, which may be desirable in future molecular constructs.
The majority of the work and data presented in this dissertation supports the establishment of new AFM methodologies that can be used to better explore single biomolecular interactions and individual submicrometer particles on the nanoscale.
As the world of science and technology advances, so does the need to study systems that are physically too small to measure with traditional science techniques. For example, particles in the atmosphere (dust and pollution) are smaller than one millionth of a meter in diameter. For comparison, the diameter of a human hair is about one hundred times larger than the average atmospheric particle. Unfortunately, the effects of particles on the climate and the environment remain largely unknown because we need high resolution analysis techniques to be able to study such small scale systems.
Another nanoscopic system of interest is single-biomolecular binding events like a drug binding with an enzyme. These fundamental processes control how enzymes work and how by inhibiting an enzyme with a drug, we can treat diseases like cancer. Studying these processes on a single-molecule basis will gain us insights on their mechanisms and improve our understanding of the catalytic processes of enzymes to ultimately facilitate the development of new and better therapies.
Atomic force microscopy (AFM) is a powerful tool capable of nanoscale imaging and measuring extremely minute forces. The research presented herein describes several novel AFM techniques that were developed to study individual nanoscopic atmospheric particles and biomolecular systems. Ultimately, this work strives to better understand the physicochemical properties of atmospheric particles to determine their effect on climate and environment. Furthermore, we developed new and better methodology to facilitate accurate drug-enzyme binding force detection that will help reveal fundamental insights on single biomolecular systems.
publicabstract, Aerosols, Atomic force microscopy
xxvi, 193 pages
Includes bibliographical references (pages 171-193).
Copyright 2016 Holly VanMetre