Investigations into Protein-Surface Interactions via Atomic Force Microscopy and Surface Plasmon Resonance

Protein surface interactions are important in many diverse applications. In this dissertation nonspecific and specific interactions of two proteins (fibrinogen and F1-ATP synthase) with a variety of surfaces have been investigated via atomic force microscopy and surface plasmon resonance. Chapter one provides background information on protein surfaces interactions. Chapter 2 summarizes the techniques and surfaces utilized in the investigations in the following chapters. Chapter 3 provides background and investigations on nonspecific fibrinogen to surfaces. Fibrinogen is an important plasma protein involved in the blood clotting cascade. To improve design of materials for biodevices and implants, more knowledge about the interactions controlling fibrinogen adsorption is essential. Nonspecific adsorption of fibrinogen was investigated on model surfaces of graphite and mica as well as on self-assembled monolayer (SAM) via atomic force microscopy (AFM) to determine conformation. Complementary studies were performed via surface plasmon resonance (SPR) to investigate the dynamics of this adsorption process on gold, and an amine-, carboxyl-, methyl- and hydroxyl-terminated SAM films. Chapter 4 provides background and investigation into F1-Adenosine triphosphate synthase (ATPase) adsorption to surfaces. ATPase is a tiny molecular motor which synthesizes ATP. This motor is of interest in the fabrication of hybrid nanobiodevices. Incorporation of this protein into devices requires precise control over immobilization properties such as location, concentration, orientation, and function. Orientation of ATPase adsorbed nonspecifically on a mica surface was observed via AFM. Control over placement within the device was investigated via nanopatterning of a 1-dodecene SAM surface. Control over orientation was performed via engineering a landing pad within a resist matrix with AFM. This involved patterning a dithiol into a methyl resist matrix and addition of maleimide-NTA with coordination to nickel ions and histidine tags in the protein. The chemistry of this process was validated with SPR and fluorescence microscopy. Information on the kinetic of ATPase-his binding to the NTA surface was obtained. Hopefully information learned from these investigations enables the development of enhanced biocompatible materials design and control over the fabrication of functional hybrid nanobiodevices

Investigations of Structure / Property Interrelationships of Organic Thin Films Using Scanning Probe Microscopy and Nanolithography

Studies of the surface assembly and molecular organization of organic thin films were studied using scanning probe microscopy (SPM) and scanning probe lithography (SPL). Systems of organic thin films such as n-alkanethiols and pyridyl functionalized porphyrins were characterized at the molecular level, and measurements of the conductive properties of polythiophenes containing in-chain cobaltabisdicarbollides were accomplished. Understanding the self-organization and mechanisms of self-assembly of organic molecules provides fundamental insight for structure/property interrelationships. Investigations of the surface assembly of 5,10-diphenyl-15,20-di-pyridin-4-yl-porphyrin (DPP) on Au(111) were done using SPL methods of nanoshaving and nanografting. Automated computer designs were developed for nanofabrication to provide local characterizations of the thickness of DPP films and nanostructures. Nanolithography was accomplished using DPP films as either matrix self-assembled monolayers (SAMs) or as molecules for nanofabrication. Results presented in this dissertation demonstrate that DPP forms compact layers on Au(111), which can be used for inscribing nanopatterns of n-alkanethiols. Arrays of DPP nanopatterns with precise geometries and alignment were fabricated within n-alkanethiols by nanografting, demonstrating nanoscale lithography with pyridyl porphyrins can be accomplished to produce an upright surface orientation on Au(111) mediated by nitrogen-gold chemisorption. Beyond research investigations, the applicability of atomic force microscopy (AFM) and advancements with automated SPL were applied for teaching undergraduate chemistry laboratories to introduce the fundamentals of surface chemistry and molecular manipulation. New classroom activities were developed for the Chemistry 3493 Physical Chemistry laboratory to give students “hands-on” training with AFM. Undergraduates were trained to prepare nanopatterns of n-alkanethiols using software to control the position, force and speed of the AFM tip for nanolithography experiments. The sensitivity and nanoscale resolution of current sensing AFM was applied for studies of the conductive properties of electropolymerized thin films of polythiophenes with cobaltabisdicarbollide moieties. Images acquired with AFM furnished views of the morphology of different polymers prepared on gold surfaces. Surface maps of the conductivity of electropolymerized films were acquired with AFM current images. These studies provide new insight of the effects of the bound cobaltabisdicarbollide moiety and coordinated metal centers for the electronic properties of the resulting conducting materials

Nanoscale Studies of Proteins and Thin Films Using Scanning Probe Microscopy

Nanostructures of organosilanes, thin metal films, and protein nanopatterns were prepared and analyzed with atomic force microscopy (AFM). Organosilanes with designed functional groups were used to selectively pattern green fluorescent protein at the nanoscale using protocols developed with particle lithography. Mesospheres are deposited onto a substrate to produce a surface mask. Organosilanes are deposited to form a matrix film surrounding nanopores for depositing proteins. The nanopatterns were characterized using AFM, after steps of particle lithography for directly visualizing surface changes. Studies with AFM also provide a compelling tool for teaching undergraduates to introduce concepts of nanoscience. An undergraduate laboratory was developed with particle lithography to introduce the concepts of nanoscience and surface chemistry. Nanopatterns of organosilane films are prepared using protocols of particle lithography. An organic thin film is applied to the substrate using steps of either heated vapor deposition or immersion in solution. At the molecular level, two types of sample morphology can be made depending on the step for depositing organosilanes. Experience with advanced AFM instrumentation is obtained for data acquisition, digital image processing and analysis. Skills with chemical analysis are gained with bench methods of sample preparation. Concepts such as the organization of molecules on surfaces and molecular self-assembly are demonstrated with the visualization of nanopatterns prepared by students. Experiments with particle lithography can be used as a laboratory module or for undergraduate research projects, and are suitable for students with a multidisciplinary science background. The kinetics and properties of thin gold films during dewetting were studied using AFM. Thin films of gold with varying initial thickness were first deposited onto fire polished glass slides and imaged with AFM. Next, the films were annealed for two hours, and then imaged after annealing. Gold islands with varying degrees of separation were formed. Surface plasmon spectroscopy was also used to analyze the gold films. To further this study, a kinetic study was done. Two gold thin films of 10 nm each were imaged after being annealed for 15, 30, 45, 60 and 120 minutes. It was found that after the first 15 minutes of annealing, gold islands were observed

Designing surface chemistries for in situ AFM investigations of biomolecular reactions with proteins at the nanoscale

In situ atomic force microscopy (AFM) characterizations and lithography can be applied to investigate the orientation, reactivity and stability of protein molecules adsorbed on nanostructures of self-assembled monolayers at near-physiological conditions. Automated nanografting was used to fabricate regular arrays of nanopatterns of ù-functionalized n-alkanethiols with designated terminal chemistries. After writing nanopatterns, protein binding occurs selectively on carboxylate-terminated nanopatterns via covalent bonds that are formed using N-ethyl-N\u27(dimethylaminoporpyl)-carbodiimide and N-hydroxysuccinimide activation. The amine groups of lysine residues of proteins bond covalently to nanopatterns of carboxylate-terminated alkanethiol self-assembled monolayers, to form a robust surface attachment for sustained contact-mode AFM imaging during biochemical reactions. Staphylococcal protein A (SpA) furnishes a generic foundation for binding immunoglobulins for nanometer scale sandwich assays. The self-assembly of á,ù-alkanedithiols onto Au(111) was investigated using AFM. When SAMs of 1,8-octanedithiol or 1,9-nonanedithiol are grown naturally from solution, different surface orientations are observed in comparison to methyl-terminated n-alkanethiols. Local views from AFM images reveal a layer of mixed orientations in which the majority of á,ù-alkanedithiol molecules adopt an orientation parallel to the surface with both thiol endgroups bound to Au(111). Results from AFM studies reveal that the chemisorption of thiol endgroups of dithiols inhibits the phase transition from a lying-down to a standing orientation during natural self-assembly. Another method for producing protein nanostructures is particle lithography. Monodisperse mesospheres can be applied to rapidly prepare millions of exquisitely uniform nanometer-sized structures of proteins on flat surfaces using conventional benchtop chemistry steps of mixing, centrifuging, evaporation and drying. The natural self-assembly of monodisperse spheres provides a high throughput and efficient route to prepare circular geometries over millimeter scale areas. The spontaneous assembly of silica or latex mesospheres into organized crystalline layers on flat substrates supplies a structural frame to direct the placement of proteins. Nanopatterns of ferritin, apoferritin, immunoglobulin G and bovine serum albumin were produced with particle lithography. The applicability of particle lithography to generate arrays of protein nanostructures on surfaces such as mica(0001), glass and Au(111) was demonstrated. The morphology and diameter of the protein nanostructures can be tailored by selecting the ratios of protein-to-particles and the diameters of spheres

USING CROSS-SECTIONED MULTILAYER POLYMER FILM AND SURFACE MODIFICATION TO FORM CHEMICALLY PATTERNED SUBSTRATES

Highly layered structures are important to micro- and nanofabrication technologies for understanding and controlling surface structures through manipulation of chemical and physical interactions. The objective of this work was to develop a new approach to create micro- and nanopatterned surfaces using multilayer polymer films of commercially available and inexpensive polymers instead of inorganic substrates. As an example, linear low density polyethylene (LLDPE) and ethylene-co-acrylic acid copolymer (EAA) were used as alternating inert and reactive polymers, respectively. Thin cross-sections of the multilayer molded sheets were prepared by ultra-microtoming. As a precursor to the multilayer work, surface modification of EAA was conducted to carefully control the chemical functionality on the surface by a variety of methods. Dansyl cadaverine and polyethylene glycol (PEG) derivatives were grafted on the surface of EAA film and in its subsurface region through formation of amides and esters, respectively. First, EAA film was activated with PCl5 and then the acid chloride was reacted with dansyl cadaverine or a PEG derivative. Moreover, two other reaction schemes were developed to covalently graft PEG chains on EAA surfaces. The schemes involved surface grafting of linker molecules l-lysine or polypropyleneamine dendrimer (AM64), with subsequent covalent bonding of PEG chains to the linker molecules. Combining the data from ATR-FTIR, XPS, and contact angle goniometry, it was found that the PEG chains were grafted on the surface of the EAA film and larger surface coverage was achieved when the dendrimer was used as intermediate layer. Research was then conducted on the EAA-LLDPE multilayer cross-sectioned templates. Regionally confined chemical functionality was confirmed by grafting an amine-terminated biotin to the alternating layers of EAA. Subsequently, fluorescently labeled streptavidin selectively adsorbed on the biotin-modified EAA layers. As a further development, polyelectrolyte multilayers (PEM) were adsorbed on the nanopatterned surfaces to significant increase the areal density of reactive groups. Using PAH and PAA as the polyelectrolytes, the EAA nano-stripes were successfully modified by PEM films, forming a nanopatterned template with alternating hydrophilic and hydrophobic regions. This kind of nano-striped surface could serve as a template for many applications, including biomedical, separation, and electronics

Surface studies of organic thin films using scanning probe microscopy and nanofabrication

Porphyrins and metalloporphyrins have unique chemical and electronic properties and thus provide useful model structures for nanoscale studies of the role of chemical structure for electronic properties. Porphyrins have been proposed as viable materials for molecular-based information-storage devices, gas sensors, photovoltaic cells, organic light-emitting diodes and molecular wires. The function and efficiency of porphyrins in devices is largely attributable to molecular architecture and how the molecules are self-organized. Modifications of the porphyrin macrocycle, peripheral groups or bound metal ions can generate a range of electrical, photoelectrical or magnetic properties. The conductive properties are greatly influenced at the molecular level by the organization of porphyrins into supramolecular arrays, aggregates, and nanocrystals on surfaces. Conductive-probe atomic force microscopy (CP-AFM) has been used extensively for studies of alkanes, phenylalkanes and arenethiols; however, the conductive properties of porphyrins have not been studied as rigorously. Characterizations with CP-AFM are becoming prevalent for molecular electronics studies because of the dual capabilities for obtaining physical measurements and structural information with unprecedented sensitivity. For CP-AFM, the tip is placed directly on the sample surface, at a designated force. To acquire current-voltage (I-V) spectra, a conductive tip is grounded, and a bias is applied to the substrate. For this dissertation, cobaltcarborane porphyrins were synthesized using a ring-opening zwitterionic reaction to produce isomers with different numbers of carborane clusters per macrocycle. Particle lithography was used to prepare regular arrangements of well-defined nanopatterns of porphyrin nanocrystals on conductive substrates. Nanopatterned SAMs of alkanethiols and organosilanes were used successfully to direct the nanocrystals of porphyrins on the surface and characterized with contact and tapping mode imaging of AFM. Our goals were to elucidate the role of molecular structure, packing and orientation for the conductive properties of porphyrins. Understanding how the self-organization and surface assembly influence electrical properties and reliable measurements of conductive properties when these molecules are coordinated to different metals and surfaces will provide information for developing predictive models

Quantitative electroanalysis of host-guest binding at organized supramolecular interfaces

As a young and important class of supramolecular host-guest chemistry, the macrocyclic cucurbit[n]uril (CB[n]) hosts consisting of one hydrophobic inner cavity and multiple carbonyl portals have shown dramatically increased research interests since 1980s, with tens of thousand publications focusing on their synthesis, distinct structural features, exceptional physical and chemical properties. More importantly, their excellent host-guest recognition behavior leads to their great application potentials in many fields, such as nanofabrication, biomedical/pharmaceutical science, analytical chemistry, catalytic chemistry, and adaptive chemistry, which have been explored extensively in the past two decades. Particularly, CB[7], an attractive member of CB[n] family, shows ultra-strong host guest binding ability towards small aromatic or ring-structured organic compounds, which is mainly attributed to its proper-sized inner cavity. As a representative, the host-guest complexes formed between CB[7] and various redox-active ferrocene (Fc) derivatives have extremely high binding affinities (109 to 1012 M-1), which have been employed as an alternative of natural binding pairs (e.g., antigen-antibody, biotin-avidin) for fabricating versatile functional molecular and biomolecular interfaces. In order to gain further understanding of this particular host-guest binding pair formed at molecular interfaces, in this thesis, based on both conventional cyclic voltammetry and advanced structural characterizations, the binding thermodynamics and kinetics were investigated on mixed ferrocenylundecanethiolate/octanethiolate self-assembled monolayers on gold as a highly-organized model system. The results show that the inclusion binding behavior of this host-guest pair, while significantly affected by the surface, still has satisfactory stability for practical application. In addition, the broad potential of this new interfacial Fc@CB[7] host-guest binding system is manifested as nanoscale probes for the distribution of Fc terminal groups on SAMs (as an indicative of their structural heterogeneity), as an environmental regulator of long-range electron transfer process, and as an electrochemical sensor for pharmaceutical drugs via competitive host-guest assay strategy. It is expected that this new interfacial host-guest binding system can be further explored for fabricating well-controlled, ratiometric electrochemical biosensors

Surface and Electrical Characterization of Conjugated Molecular Wires

University of Minnesota Ph.D. dissertation. SEptember 2016. Major: Material Science and Engineering. Advisor: C.Daniel Frisbie. 1 computer file (PDF); xvii, 252 pages.This thesis describes the surface and electrical characterization of ultrathin organic films and interfaces. These films were synthesized on the surface of gold by utilizing layer by layer synthesis via imine condensation. Film growth by imine click (condensation) chemistry is particularly useful for molecular electronics experiments because it provides a convenient means to obtain and extend π-conjugation in the growth direction. However, in the context of film growth from a solid substrate, the reaction yield per step has not been characterized previously, though it is critically important. To address these issues, my research focused on a comprehensive characterization of oligophenyleneimine (OPI) wires via Rutherford backscattering spectrometry (RBS), X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry (SE), reflection-absorption infrared spectroscopy (RAIRS), and cyclic voltammetry (CV). In addition, we had the unique opportunity of developing the first of its kind implementation of nuclear reaction analysis (NRA) to probe the intensity of carbon atoms after each addition step. Overall the combination of various techniques indicated that film growth proceeds in a quantitative manner. Furthermore, the NRA experiment was optimized to measure the carbon content in self-assembled monolayers of alkyl thiols. The results indicated well-resolved coverage values for ultrathin films with consecutive steps of 2 carbon atoms per molecule. Another fundamental problem in molecular electronics is the vast discrepancy in the values of measured resistance per molecule between small and large area molecular junctions. In collaboration with researchers at the National University of Singapore, we addressed these issues by comparing the electrical properties of OPI wires with the eutectic gallium indium alloy (EGaIn) junction (1000 µm2), and conducting probe atomic force microscopy (CP-AFM) junction (50 nm2). Our results showed that intensive (i.e., area independent) observables such as crossover length, activation energy, and decay constants agreed very well across the two junction platforms. On the other hand, the extensive (area dependent) resistance per molecule values was 100 times higher for EGaIn junction verses CP-AFM after normalizing to contact area. This was most likely due to differences in metal-molecule contact resistances. My contribution to this collaborative work is in synthesis and timely delivery of OPI wires. The structure-property relationships of OPI wires with 5 terminal F atoms were studied extensively by XPS. The results show similar crossover behavior obtained by molecular junction experiments. Saturated spacers (conjugation disruption units) were introduced into the molecular backbone, and their effects on the intensity of F 1s counts were measured. Overall, there was good correlation between the position and number of saturated units verses F 1s peak area. Even though core hole spectroscopy and time dependent density functional theory (TDDFT) calculations are required to fully understand the charge transport dynamics, the preliminary results point to a new ultrahigh vacuum method of measuring charge transfer rates. Overall, these experiments open significant opportunities to synthesize ultra-thin films and characterize a variety of donor-block-acceptor and metal complex systems in molecular electronics

Gold Surface Nanostructuring for Separation and Sensing of Biomolecules

Detecting biomolecules in physiological environments is critical to health care and environmental monitoring. In this work, we study and use gold surfaces for biomolecule detection while incorporating nanoscale components—specifically, self-assembled monolayers (SAMs) of alkanethiols and gold nanostructured shells—with the goal of improving biomolecule detection methods. Using SAMs to functionalize gold surfaces can offer control over biomolecule binding density and orientation while still keeping the biomolecules near the sensing surface. Using surface IR spectroscopy, x-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) modeling, we found that SAMs of short-chain and long-chain amine-terminated alkanethiols on gold had different sulphur binding environments. We also found that protein binding and recognition on the two different SAMs varied with SAM chain length and was also influenced by the presence of a cross-linker. In the second part of this work, we synthesized gold nanostructured shells on magnetic particles for combined separation and detection of biomolecules. We demonstrated their use as substrates for surface-enhanced Raman spectroscopy (SERS) As a proof-of-concept, we demonstrated the use of these particles to detect oligonucleotide binding and hybridization with SERS using a Raman-tagged oligonucleotide hairpin probe

Ultra-low voltage electrowetting

Electrowetting, the manipulation of surface wettability with an electric field, is an emerging technology used in next generation displays and cameras. This has been made possible by the development of ‘electrowetting -on- die lectric’ by Berge in 1993. Howev er, such a system operates on large voltages poorly suited to portable devices. In recent years, theoretical and experimental results have suggested that electrowetting using the interface between two immiscible electrolyte solutions (ITIES) may provide a solution to this problem. By applying less than 1 V to such a system, it is possible to induce substantial changes in the wettability — and hence the shape — of liquid droplets. However, there is a large degree of hysteresis in such a system meaning that there is a poor correlation between droplet shape and applied potential. Furthermore, the stability of the ITIES over long periods is of concern. This thesis attempts to address the current problems with ITIES electrowetting highlighted above. By moving to smoother and more lubricated surfaces, a substantial reduction in hysteresis was seen. These surfaces were produced by template stripping. In addition, several other surfaces were prepared as potential electrowetting substrates. These involved surface functionalisation by plasma treatment or the reduction of diazonium compounds; preparation of ultra smooth glassy carbon and preparation of a hydrophobic conducting polymer. The potential range over which an ITIES is stable was also improved with the use of a novel mixed organic solvent phase. By optimising the electrode and electrolyte compositions, an electrowetting system operating on less than 1 V with a contact angle range of 53 o and a gap of only 100 mV between forward and reverse scans was possible. Other electrowetting systems with no hysteresis were also developed, although these did not operate within the potential limits defined by the onset of Faradaic processes.Open Acces