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There is a need for the development of new technologies for the early detection of disease. Diverse initiatives are underway in academia and the pharmaceutical and biotechnology industries to develop highly-sensitive, high-throughput methods to detect disease-relevant biomarkers at the single-cell level. Biomarkers can define the progress of a disease or efficacy of disease treatment, and can include nucleic acids (RNA, DNA), proteins, small molecules, or even specific cells. While discovery research in this area is accelerating, there are a number of current experimental limitations. Most existing methodologies require a relatively large sample size. Also, amplification-based detection technologies are destructive to sample, and errors in amplification can occur, leading to an incorrect diagnosis. Nanomaterial-based devices (nanodevices) offer the promise of label-free, amplification-free detection strategies. Such nanodevices could allow analysis of minute biological samples without the requirement for amplification or incorporation of reporter groups. Loss of sample, due to handling and processing would be minimized and the sample could be recovered for further analysis. Atomic force microscopy (AFM) allows topographic imaging and compressibility/elasticity measurement of biomolecules on solid supports. AFM can enable assays of ligand binding with single molecule detection capability. Certain nucleic acid types, in particular double-stranded (ds) RNA, can act as a biomarker for specific cancers (e.g. leukemia) and viral infection. dsRNA also is of interest since it is a conserved structural feature of precursors to gene-regulatory RNAs, including micro (mi) RNAs and short interfering (si) RNAs. This project demonstrates a single-step, label-free, amplification-free approach for detecting the interaction of biomolecules that bind and/or process dsRNA, using a nanomanipulated, self-assembled monolayer (SAM) of a ds[RNA-DNA] chimera as imprinting matrix, a reference nuclease as imprinting agent, and AFM for imprint-readout. The action of the dsRNA-specific enzyme, ribonuclease III (RNase III), as well as the binding of an inactive, dsRNA-binding RNase III mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA] chimera, as measured by atomic force microscopy, provides a distinct digital output for each type of input. These findings provide the basis for developing imprinting-based nano-biosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale. RNA-DNA heteroduplexes are biomarkers for specific inflammatory conditions of genetic origin, and also are the product of capture of an RNA (e.g., miRNA) by a complementary DNA sequence. The approach used here to detect RNA-DNA hybrids is based on the ability of alkylthiol-modified ssDNA molecules to form monolayers and nanomatrices on gold surfaces (as described above) with density-dependent thickness, which increases upon formation of RNA-DNA hybrids following addition of a complementary oligoribonucleotide. Changes in hybrid matrix thickness can be measured by AFM, using a reference monolayer. RNA-DNA hybrid formation as well as subsequent processing by RNase H can be observed as a height increase or decrease, respectively, of the monolayer. When Mg2+ is omitted to prevent RNA cleavage, but not protein binding, a significant height increase is observed. The height increase is not observed with the corresponding ssDNA or ssRNA nanomatrices, and only occurs with nanomatrices having a hybrid density above a defined threshold. The data indicate formation of a stable multimeric RNase H assembly on the hybrid nanomatrix which provides a robust signal that is nondestructive to the RNA. The implications of these findings are discussed with respect to development of novel detection methodologies for RNA, dsRNA, and RNA-DNA hybrids. / Chemistry

Detailed studies are presented of thin films containing a polycation, a nonlinear optically (NLO) active chromophore, and a synthetic hectorite that self-assemble into the noncentrosymmetric structure required for second order nonlinear optical responses. UV/Vis spectroscopy and ellipsometry were used as probes to monitor film growth for upto 25 deposition cycles. Exceptionally homogeneous films were obtained with regular film growth for up to the 25 cycles deposited. ISAM films self-assemble from polyelectrolyte solutions due to coulombic interactions between a charged substrate and the charged polymer in solution. Charges accumulating at the surface restrict further growth due to charge overcompensation at the surface. The entire process occurs relatively quickly as compared to other competing assembly techniques such as Langmuir-Blodgett assembly and covalent self-assembly. Previous studies indicated that second harmonic signal diminishes after the deposition of the first few bilayers. This is potentially due to adjacent layer interpenetration, which becomes prominent moving further away from the hard substrate interface. Laponite RD, a synthetic hectorite was introduced in the films in an effort to minimize interpenetration of adjacent bilayers and hence maintain chromophore orientation in every bilayer of the ISAM film. The film was deposited in quadlayers that have the following sequence: Polycation–Laponite–Polycation–Chromophore. This study is unique in its approach as it investigates the possible implications of film interpenetration on the NLO-activity of ISAM films that can be easily fabricated. It also shows the effects of different interfaces on the NLO-activities of the films. We have investigated the effect of changing the polycation from poly(allylamine hydrochloride) (PAH) and poly(diallyldimethylammonium chloride) (PDDA) and the solution pH to see how these variables affect the growth and NLO properties of ISAM films. At lower pH values (specify relevant range of values), for both polycations, intrachain and interchain repulsion is strong due to little electrostatic screening. This results in polycation deposition in relatively thin, train-like layers. At higher pH levels (specify relevant range of values here) the electrostatic screening is greater due to a higher effective ionic strength. At these conditions, intrachain and interchain repulsion is reduced and so the polymers adsorb to form thicker layers with more loops and tails than for the case at lower pH. This also results in a higher density of the chromophore in the film. Extremely smooth surfaces of Laponite RD in film were obtained as confirmed by AFM imaging on glass. Regular quadlayer growth monitored by UV/Vis spectroscopy and ellipsometry was observed for up to 25 quadlayers. Second harmonic generation (SHG) signal was not conclusively affected by the presence of laponite as the decrease of signal was seen after the first few layers in the laponite containing films. This decrease was however was not as sharp in the films containing laponite as in the films that did not contain laponite. It was also noted that the SHG was not so much affected by the number of layers deposited but more so by the distance of the chromophore layer from the hard glass interface. This study thus brings to light the very important effect of the glass interface on the NLO-activity of these films. / Master of Science

Ionically self-assembled monolayer (ISAM) films are typically an assemblage of oppositely charged polymers built layer by layer through coulombic attraction utilizing an environmentally friendly process to form ordered structures that are uniform, molecularly smooth, and physically robust. ISAM films have been shown to be capable of the noncentrosymmetric order requisite for a second-order nonlinear optical response. However, films fabricated with a nonlinear optical (NLO) polymer result in significant cancellation of the chromophore orientations. This cancellation occurs by two mechanisms: competitive orientation due to the ionic bonding of the polymer chromophore with the subsequent polycation layer, and random orientation of the chromophores within the bulk of each polyanion layer. A reduction in film thickness accompanied by an increase in net polar ordering is one possible avenue to obtain the second-order nonlinear optical susceptibility chi(2) necessary for electro-optic devices. In this thesis, we will discuss the structural characteristics of ISAM films and explore three novel approaches to obtain the desired characteristics for nonlinear optical response. One approach involves the variation of solution parameters of several different cationic polymers separately from the polyanion solution in order to reduce the competitive chromophore orientation at the layer interfaces and to reduce the thickness of the inactive polycation layer. We have found that the complexity of ISAM films does not allow large chi(2) values in polyion-based films, and that the selection of the polymer cation is vital to achieve second harmonic generation (SHG) at all. The second approach involves the incorporation of dianionic molecules into ISAM films in order to eliminate both competitive chromophore orientation and random chromophore orientation inherent with polymer chromophores. We have also studied the effects of complexing dianionic chromophores with beta-cyclodextrin in order to increase solubility and improve chromophore orientation. This approach fails because the outermost monolayer of dianionic chromophore is only tethered to the preceding polycation layer by a single ionic bond for each molecule, so each chromophore can by dissociated during the following immersion into the cation solution. Finally, we have introduced a novel approach of hybrid covalent / ionic self-assembly which overcomes these disadvantages and yields a substantial increase in chi(2) due to the chromophore being locked in place to the preceding polycation layer by a covalent bond. The films fabricated in this manner yield a chi(2) that rival any polymer-polymer films despite the very low first-order molecular hyperpolarizability beta of the incorporated monomer. This suggests that incorporation of high beta molecules may result in significant improvement of chi(2), holding high promise for the hybrid covalent / ionic self-assembly technique. / Master of Science

The reactivity of chemical warfare agents (CWAs) and CWA simulants on organic and oxide surfaces is not currently well understood, but is of substantial importance to the development of effective sensors, filters and sorbent materials. Polyurethane coatings are used by the armed forces as chemical agent resistive paints to limit the uptake of CWAs on surfaces, while the use of metal oxides has been explored for decontamination and protection purposes. To better understand the chemical nature of the interactions of organophosphonate simulants with these surfaces, an ultra-high vacuum environment was used to isolate the target interactions from environmental gaseous interferences. The use of highly-characterized surfaces, coupled with molecular beam and dosing capabilities, allows for the elucidation of adsorption, desorption, and reaction mechanisms of CWA simulants on a variety of materials. Model urethane-containing organic coatings were designed and applied toward the creation of well-ordered thin films containing carbamate linkages. In addition, novel trisilanolphenyl-polyhedral oligomeric silsesquioxane (POSS) molecules were used to create Langmuir-Blodgett films containing reactive silanol groups that have potential use as sensors and coatings. The uptake and reactivity of organophosphonates and chlorophosphates on these surfaces is the focus of this study. Surfaces were characterized before and after exposure to the phosphates using a number of surface sensitive techniques including: contact angle goniometry, reflection-absorption infrared spectroscopy (RAIRS), X-ray photoelectron spectroscopy (XPS) and temperature-programmed desorption (TPD) measurements. In conjunction with surface probes, uptake coefficients were monitored according to the King and Wells direct reflection technique. The integration of these analytical techniques provides insight and direction towards the design of more effective chemical agent resistant coatings and aids in the development of more functional strategies for chemical warfare agent decontamination and sensing. / Ph. D.

Constructing a detailed understanding of the heterogeneous oxidation of atmospheric organic aerosols, both from a mechanistic and kinetic perspective, will enable researchers to predict the fate and lifetime of atmospheric gases and the particles with which they interact. In an effort to develop a more complete understanding of the interfacial reactions of ozone with vinyl-containing organic thin films, self-assembled monolayers that contain vinyl groups positioned precisely at the gas/surface interface were synthesized as model systems for atmospheric organic aerosols. To isolate the reactions of background gases with ozone or surface products, an ultrahigh vacuum surface analysis instrument was designed and constructed to explore the reactions of ozone with the atmospheric model systems. The surface reactions can be monitored in real-time with reflection absorption infrared spectroscopy (RAIRS) and mass spectrometry. The chemical identity of adsorbates on a surface can also be determined before or after a reaction with X-ray photoelectron spectroscopy (XPS). Disordering of the monolayers concurrent with the disappearance of the vinyl group was observed with RAIRS. New bands within the RAIR spectra were observed and assigned to carbonyl or carboxylic acids bound to the surface. Little oxidation of the sulfur head groups and no significant loss of carbon during the reaction was observed with XPS. A mechanism is proposed that includes the cross linking of the hydrocarbon chains within the monolayer, which impedes further oxidation of the sulfur head group and limits desorption of the chains. By RAIRS, the kinetics of the oxidation of the vinyl groups were tracked and an observed rate constant was determined by monitoring the changes in IR absorbance of the C=C bond. With the aid of the rate constant, an initial reaction probability for the collisions of ozone with vinyl groups positioned precisely at an interface was determined. The reaction probability is approximately three orders of magnitude greater than the reaction probability for an analogous gas-phase reaction, which demonstrates that the gas/surface interface plays an important role in this reaction. The results presented in this thesis should help develop a more detailed understanding of the interfacial reactions of pure ozone at surfaces. / Ph. D.

This dissertation describes the impact of defects on monolayer properties for self-assembled monolayers (SAMs) created by interfacial patterning methods. When forming a two-dimensional interfacial pattern with n-alkanethiols on gold, the desired electrochemical properties are those of a homogeneous, solution adsorbed monolayer. However, even well-ordered SAMs contain a small degree of defects, especially at domain boundaries where two nucleating domains converge. Patterning a surface creates user-defined domain boundaries within the monolayer, potentially having a significant impact on the properties of the interface. This dissertation investigates the effect that user-created domain boundaries have on the properties of a monolayer, as studied by electrochemical impedance spectroscopy. Two patterning methods are investigated for creating user-defined domain boundaries: the soft lithography method of contact printing and site-selective reductive desorption. The electrochemical properties of homogeneous contact printed monolayers are measured and compared to those of monolayers prepared by solution adsorption. The contact printed monolayers are found to have dramatically different impedance behavior from the solution prepared monolayers, consistent with the contact printed monolayers having greater defect density. In addition, these studies show that the overall defect density depends on the concentration of the solutions used for contact printing. In this work, simple patterns are created by contact printing a pattern onto the substrate and then backfilling the remaining gold substrate by solution adsorption. Backfilling with the same alkanethiol used to create the pattern generates a homogeneous monolayer; however, it is found that the contact printed/backfilled monolayer has an impedance intermediate between the homogeneous contact printed and the homogeneous solution adsorbed monolayer. This result suggests that the backfilling process also saturates the pinhole defects associated with the contact printed areas. In addition to exploring defects that arise from contact printing, simple patterns with user-defined defects, created by site-selective reductive desorption (SSRD), were also investigated. Following the backfill step, the impedance behavior of the SSRD produced patterns was similar to that of the impedance of the initial pattern before backfilling. This important result implies that the domain boundaries play the most important role in defining the overall impedance of the patterned interface. / Ph. D.

Ultrahigh vacuum (UHV) surface science experiments were designed to study reaction kinetics and mechanisms of gas-phase NO₃ radicals with well-organized, highly characterized, organic thin films. The surface reactions were monitored in situ with reflection-absorption infrared spectroscopy (RAIRS). The oxidation states of surface-bound molecules were identified with X-ray photoelectron spectroscopy (XPS). Consumption of vinyl groups was observed concurrently with formation of organic nitrates in RAIRS. XPS spectra showed little oxidation of sulfur head groups. The observed rate constant was determined based on the consumption of carbon-carbon double bonds and the formation of organic nitrates. Using this rate constant, the initial reaction probability was determined to be (3 ± 1) X 10⁻³. This reaction probability is approximately two orders of magnitude higher than that for the reactions between the same surface and pure O₃, which is due to the higher electron affinity of NO₃ relative to O₃. These results led to the development of a proposed mechanism that involves electrophilic addition of NO₃ to the double bonds. Reactions between NO₃ and a methyl-terminated SAM were also monitored in situ with RAIRS. In the CH3-SAM studies, hydrogen abstraction was observed during NO3 exposure. The results presented in this thesis should help develop an understanding of the fundamental interfacial reaction dynamics of NO₃ radicals with organic surfaces. / Master of Science

Reactions of strongly oxidizing pollutants with unsaturated hydrocarbon surfaces are important to many areas of scientific interest. For example, reactions of unsaturated hydrocarbons on the surface of tropospheric aerosols could have a great effect on the oxidizing capacity of the troposphere while the reaction products could be involved in the formation of clouds and smog. These reactions are also important in understanding the toxic effect inhalation of these pollutants have on the pulmonary surfactant of the lung, the only amicable air-water interface of the body. The fatty acids of this surfactant are as much as 30% unsaturated, and exposure to oxidizing pollutant is known to alter both the composition and function of the surfactant. Understanding the reaction mechanism will further the knowledge of how this toxicity occurs. While the reactions of strongly oxidizing pollutants, such as ozone and nitrogen dioxide, with alkenes in the gas and solution phases are well known, the interfacial reaction mechanisms of these species is not fully understood. The goal of this study is to determine the reaction mechanism when an unsaturated hydrocarbon monolayer at the gas-surface interface is exposed to gas phase nitrogen dioxide. An olefin-terminated thiol was synthesized and a self-assembled monolayer on Au(111) made and characterized using Reflection-Absorption Infrared Spectroscopy (RAIRS). This unsaturated surface was then exposed to NO2 at a pressure of 1x10-4 mbar in a UHV (Ultrahigh Vacuum) chamber. Time-resolved RAIRS was preformed in situ to monitor the reaction during exposure. X-ray Photoelectron Spectroscopy and RAIRS determined the surface reaction product as an aldehyde. While the mechanism can not be precisely determined, two mechanisms involving either the hydrogen abstraction or radical addition of the NO2 to yield an aldehyde are proposed. / Master of Science

Scanning Electrochemical Microscopy (SECM) is most commonly used to spatially resolve reaction rates, image surface topography and surface reactivity. In this research, SECM is applied to various chemical systems in order to resolve local reaction chemistry and to produce patterns with dimensions of tens of microns in n-alkanethiol passivated gold substrates. Upon completing construction of the instrumentation, SECM was applied to capillary electrophoresis to accurately and reproducibly place the electrode directly above a very small capillary opening. Feedback SECM was then used to image and pattern surfaces, effectively distinguishing between insulating and conductive domains. Finally, the size of desorbed features patterned on a passivated gold substrate were studied as a function of both applied potential and ionic strength. Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End-capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal-to-noise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using SECM, the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal-to-noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal-to-noise does not dramatically decrease until the electrode is more than 30 μm above the 10 μm inner-diameter capillary. Limits of detection for 2,3-dihydroxybenzoic acid were found to be 8.2 fmol when aligned manually, and 3.8 fmol when the SECM is used to automatically align the microelectrode. SECM was then used to image a series of multi-disk electrode arrays in order to demonstrate the ability of the instrument to discriminate between conductive and insulating domains. Upon demonstrating the capacity of the SECM to image very small domains of conductor on an insulating substrate, n-alkanethiol passivated gold surfaces were patterned using site-selective desorption. A number patterns, potentially useful for enzyme deposition, were subsequently produced in the passivated gold substrate. The feature size of the desorbed domains was monitored as a function of applied potential and the ionic strength of the solution used for desorption. Results showed that applying a more negative potential or increasing the ionic strength of the solution increased the magnitude of the electric field at the surface of the passivated gold substrate and resulted in a more complete, larger desorption. Both ionic strength and applied desorption potential prove to be parameters useful for controlling the size of patterned features in site selective desorption. / Master of Science