Controlling Surface Chemistry on the Microscopic and Nanoscopic Scale through Photopatterned Self-Assembled Monolayers

ABSTRACT OF DISSERTATION Controlling Surface Chemistry on the Microscopic and Nanoscopic Scale through Photopatterned Self-Assembled Monolayers by Matthew J. Hynes Doctor of Philosophy in Chemistry Washington University in St. Louis, 2013 Professor Joshua A. Maurer, Chair The development of new patterning strategies for self-assembled monolayers: SAMs) using photolithography described here allows for the production of highly functional substrates for biological applications. Photolithography methods have been developed that utilize either high or low irradiation doses of 325 nm ultraviolet light. Utilizing high power led to the development of photo-induced monolayer desorption in which patterns were generated by thermally ablating glycol-terminated thiol monomers from gold substrates. A direct relationship between laser intensity and surface modulus was observed using scanning probe microscopy: SPM), which was expected since higher laser intensities should remove more glycol monomers from the surface exposing a greater percentage of the bare gold substrate. Conversely, an inverse relationship was determined between laser intensity and surface adhesion. Utilizing direct-write photolithography provided a facile means to generate complex protein patterns containing both gradients and punctate regions. Proteins adsorption to patterned substrates was quantified by surface plasmon resonance imaging: SPRi) and fit to a Boltzmann function, which allowed us to correlate laser intensity with protein adsorption. Thus, the concentration of the protein could be precisely controlled by adjusting the gray scale level in the 8-bit image, since this file is used to modulate the laser intensity during patterning. Moreover, adsorbed neutravidin was detected using a commercial biotin labeled anti-avidin antibody, which allowed for significant signal enhancement over background. The ability to produce complex protein patterns will contribute greatly to creating in vitro models that more accurately mimic an in vivo environment. In order to utilize low irradiation doses, two unique photoprotected thiol monomers were designed and synthesized. A nitroveratryl-protected carboxylic acid thiol monomer was synthesized, which when irradiated at 325 nm, resulted in cleavage of the nitroveratryl groups to produce free carboxylic acids on the surface. Direct-write photolithography provided a means to create complex patterns containing functional group gradients, which were observed directly using SPM. In addition, two different amine molecules were sequentially coupled on to a single substrate with spatial control. Coupling was visualized using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: MALDI TOF) imaging, which demonstrated the utility of this method for generating complex multi-molecule patterns. A new cyclopropenone monomer was also developed, which was used to site-selectively pattern azide terminated molecules. Exposure of the monomer to UV light under an argon atmosphere generated a strained cyclooctyne, which was used for Cu-free [3+2] cycloadditions with azide terminated molecules. Using direct-write photolithography, neutravidin gradients were produced by coupling an azido-biotin monomer to the patterned surfaces and a linear relationship, with an R2 value of 0.993, between laser intensity and protein coupling was found. These patterned surfaces were also visualized using traditional immunohistochemistry by coupling an azido-cRGD peptide to the surface and probing with a primary anti-RGD antibody followed by a fluorescently labeled secondary antibody. Moreover, patterned surfaces with cRGD could be used to control NIH/3T3 cell growth at various concentrations of functional monomer

Biomimetic and bioactive plasma polymer surfaces

Plasma polymer surfaces have been produced and analysed to evaluate their suitability as biomimetic and bioactive surfaces. The conclusions drawn are listed below:• Plasma patterning of surfaces can be achieved by both an "ink and lift-off' or "emboss and lift-off" approaches. Plasma patterning using the "emboss and lift-off' approach improves with increasing force used to emboss the aperture containing device. Plasma polymer patterned surfaces can be used to mimic naturally occurring micro-condensers and a combination of super-hydrophobic and super-hydrophilic surfaces results in the optimal micro-condenser. Super-hydrophilic plasma polymer surfaces are superior in cell adhesion tests to polymers at higher contact angles. Plasma patterning of super-hydrophilic spots onto protein resistant backgrounds leads to patterning of cell growth

Optically Induced Nanostructures

Nanostructuring of materials is a task at the heart of many modern disciplines in mechanical engineering, as well as optics, electronics, and the life sciences. This book includes an introduction to the relevant nonlinear optical processes associated with very short laser pulses for the generation of structures far below the classical optical diffraction limit of about 200 nanometers as well as coverage of state-of-the-art technical and biomedical applications. These applications include silicon and glass wafer processing, production of nanowires, laser transfection and cell reprogramming, optical cleaning, surface treatments of implants, nanowires, 3D nanoprinting, STED lithography, friction modification, and integrated optics. The book highlights also the use of modern femtosecond laser microscopes and nanoscopes as novel nanoprocessing tools

Laser Direct-Write Microfabrication and Patterning.

The ability to generate small structures is central to modern science and technology. In this work, four laser direct-write microfabrication and micropatterning techniques were studied: (a)Laser micromachining of channels in PMMA using a CO2 laser was investigated experimentally and theoretically. Heat transfer models for the channel depth, channel profile, laser power and scanning speed were developed and applied in this work. These models, are in excellent agreement with experimental results, with a maximum deviation of approximately 5% for the range of experimental parameters (laser power, scanning speed) tested. (b)A sub-micrometer resolution laser direct-write polymerization system for creating two-dimensional and three-dimensional structures was developed using a frequency-doubled Nd:YAG laser. Experimental studies and Monte Carlo simulations were conducted to understand the detailed microscale optical scattering, chemical reaction, polymerization, and their influence on critical fabrication parameters. The experimental data are in good agreement with the theoretical model. (c)Direct laser interference was developed for rapid and large area fabrication of two-dimensional and three dimensional periodic structures on photopolymerizable materials with 10ns pulses from a frequency-tripled Nd:YAG laser emitting at 355 nm. Three different photopolymerizable materials were investigated: pentaerythritol triacrylate (PETIA) with photoinitiator N-methyldiethanolamine (N-MDEA); SU-8 with absorber TINUVIN 384-2; and Shipley 1813. (d)A new approach to fabricating nanometer sized cavity arrays on Poly(3,4-ethylene dioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) thin films using laser-assisted near-field pattering was investigated. Periodic nano-cavity arrays were patterned by combining direct laser interference technology and laser induced near-field technology. An analytical model based on Mie theory was developed, the predicted intensity distributions on the substrate indicate a strong near-field enhancement confined to a very small area (nanometer scale).Ph.D.Mechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60759/1/yuandj_1.pd

Microwave assisted processing of metal loaded inks and pastes for electronic interconnect applications

Isotropically conductive adhesives (ICAs) and inks are potential candidates for low cost interconnect materials and widely used in electrical/electronic packaging applications. Silver (Ag)filled ICAs and inks are the most popular due to their high conductivity and good reliability. However, the price of Ag is a significant issue for the wider exploitation of these materials in low cost, high volume applications such as printed electronics. In addition, there is a need to develop systems compatible with temperature sensitive substrates through the use of alternative materials and heating methods. Copper (Cu) is considered as a more cost-effective filler for ICAs and in this work, Cu powders were treated to remove the oxide layer and then protected with a self-assembled monolayer (SAM). The coating was found to be able to limit the re-oxidation of the Cumicron particles. The treated Cu powderswerecombined with one of two different adhesive resins to form ICAs that were stencil printed onto glass substrates before curing. The use of conventional and microwave assisted heating methods under an inert atmosphere for the curing of the Cu loaded ICAs was investigated in detail. The samples were characterised for electrical performance, microstructure and shrinkage as a function of curing temperature (80–150°C) and time. Tracks with electrical conductivity comparable to Ag filled adhesives were obtained for both curing methods and with both resins. It was found that curing could be accelerated and/or carried out at lower temperature with the addition of microwave radiation for one adhesive resin, but the other showed almost no absorption indicating a difference in curing mechanism for the two formulations. [Continues.

Additive manufacturing and surface functionalisation of Ti6Al4V components using self-assembled monolayers for biomedical applications

The ability to provide mass customised and biocompatible implants is increasingly important to improve the quality of life. Additive manufacturing (AM) techniques have obtained increasing popularity and selective laser melting (SLM), a metal-based AM technique with an ability to build complex and well defined porous structures, has been identified as a route to fabricate customised biomedical implants. Surface modification of an implant with a biomolecule is used to improve its biocompatibility and to reduce post-implant complications. In this thesis, the potential of a novel approach to use self-assembled monolayers to modify SLM fabricated surfaces with therapeutic drugs has been evaluated. Although there are numerous studies on the material development, process optimisation and mechanical testing of SLM fabricated parts, the surface chemistry of these parts is poorly understood. Initially, the surface chemistry of SLM as-fabricated (SLM-AF), SLM fabricated and mechanically polished (SLM-MP) and forged and mechanically polished (FGD-MP) parts made of Ti6Al4V was determined using an X-ray photoelectron spectrophotometer (XPS). Later the impact of laser power on the surface chemistry of the parts was also studied. A non-homogeneous surface chemistry was observed due to a change in the distribution of the alloying elements titanium, aluminium and vanadium on the surface oxide layer. Surface modification of the SLM fabricated component would be beneficial to obtain a homogenous surface chemistry, especially for biomedical application. Coating of self-assembled monolayers (SAMs) onto SLM fabricated Ti6Al4V structures was performed to modify their surface chemistry. 16-phosphanohexadecanoic acid monolayers (16-PhDA) were used to modify SLM-AF and SLM-MP surfaces. XPS and static water contact angle measurements confirmed the chemisorption of monolayers on these surfaces. The obtained results confirmed that SAMs were stable on the Ti6Al4V surface for over 28 days before its desorption. It was also witnessed that the stability of monolayers on the rough SLM-AF and smooth SLM-MP surfaces were not significantly different. Later, the 16-PhDA SAM coated Ti6Al4V SLM-MP surface was functionalised with a model drug, Paracetamol. An esterification reaction was performed to functionalise the phosphonic acid monolayers with Paracetamol. Surface characterisation revealed the sucessful attachment of Paracetamol to the SAMs. Bacterial infections from biomedical implants and surgical devices are reported to be a major problem in orthopaedic, dental and vascular surgery. Hence, to further explore the potential of the proposed method, Ciprofloxacin® a broad spectrum antibiotic was immobilised to the SAMs, previously adsorbed on the SLM-MP Ti6Al4V surfaces. Using the proposed approach, approximately 1.12 µg/cm2 of the drug was coated to the surface. Results showed that Ciprofloxacin® is highly stable under the oxidative conditions used in this study. Under in vitro condition, the drug was observed to release in a sustained manner. Antibacterial susceptibility tests revealed that the immobilised Ciprofloxacin® was therapeutically active upon its release. Thus, a novel methodology to fabricate customised and functionalised implants has been demonstrated for an improved biocompatibility and reduced post-implant complications

Additive manufacturing and surface functionalisation of Ti6Al4V components using self-assembled monolayers for biomedical applications

The ability to provide mass customised and biocompatible implants is increasingly important to improve the quality of life. Additive manufacturing (AM) techniques have obtained increasing popularity and selective laser melting (SLM), a metal-based AM technique with an ability to build complex and well defined porous structures, has been identified as a route to fabricate customised biomedical implants. Surface modification of an implant with a biomolecule is used to improve its biocompatibility and to reduce post-implant complications. In this thesis, the potential of a novel approach to use self-assembled monolayers to modify SLM fabricated surfaces with therapeutic drugs has been evaluated. Although there are numerous studies on the material development, process optimisation and mechanical testing of SLM fabricated parts, the surface chemistry of these parts is poorly understood. Initially, the surface chemistry of SLM as-fabricated (SLM-AF), SLM fabricated and mechanically polished (SLM-MP) and forged and mechanically polished (FGD-MP) parts made of Ti6Al4V was determined using an X-ray photoelectron spectrophotometer (XPS). Later the impact of laser power on the surface chemistry of the parts was also studied. A non-homogeneous surface chemistry was observed due to a change in the distribution of the alloying elements titanium, aluminium and vanadium on the surface oxide layer. Surface modification of the SLM fabricated component would be beneficial to obtain a homogenous surface chemistry, especially for biomedical application. Coating of self-assembled monolayers (SAMs) onto SLM fabricated Ti6Al4V structures was performed to modify their surface chemistry. 16-phosphanohexadecanoic acid monolayers (16-PhDA) were used to modify SLM-AF and SLM-MP surfaces. XPS and static water contact angle measurements confirmed the chemisorption of monolayers on these surfaces. The obtained results confirmed that SAMs were stable on the Ti6Al4V surface for over 28 days before its desorption. It was also witnessed that the stability of monolayers on the rough SLM-AF and smooth SLM-MP surfaces were not significantly different. Later, the 16-PhDA SAM coated Ti6Al4V SLM-MP surface was functionalised with a model drug, Paracetamol. An esterification reaction was performed to functionalise the phosphonic acid monolayers with Paracetamol. Surface characterisation revealed the sucessful attachment of Paracetamol to the SAMs. Bacterial infections from biomedical implants and surgical devices are reported to be a major problem in orthopaedic, dental and vascular surgery. Hence, to further explore the potential of the proposed method, Ciprofloxacin® a broad spectrum antibiotic was immobilised to the SAMs, previously adsorbed on the SLM-MP Ti6Al4V surfaces. Using the proposed approach, approximately 1.12 µg/cm2 of the drug was coated to the surface. Results showed that Ciprofloxacin® is highly stable under the oxidative conditions used in this study. Under in vitro condition, the drug was observed to release in a sustained manner. Antibacterial susceptibility tests revealed that the immobilised Ciprofloxacin® was therapeutically active upon its release. Thus, a novel methodology to fabricate customised and functionalised implants has been demonstrated for an improved biocompatibility and reduced post-implant complications

Amphiphilic diblock copolymers for molecular recognition

In this thesis, the synthesis and the characterization of poly(butadiene)-blockpoly( ethylene oxide) copolymers with terminal Me2+-NTA groups (copper or nickel) is described for the first time. A convenient “one-pot” procedure that allows control over the individual block lengths of the copolymer and the end-group functionalization was successfully established. The formation of the metal-polymer complex has been confirmed by EPR and UV/VIS spectroscopy. Mixing of the Ni2+-NTA polymers with the corresponding non functionalized block copolymers at a concentration of 10 mol% does not affect the self-assembly behavior of the mixtures, i.e., in dilute aqueous solutions the polymer mixtures aggregate to vesicular structures (metal-doped vesicles) with identical size distribution as the non functionalized block copolymer vesicles. Vesicles were characterized by dynamic light scattering, static light scattering, small angle X-ray scattering and zeta potential. All measurements led to the conclusion that hollow spheres, i.e. vesicles, with a narrow size distribution and a negative surface potential were generated. Moreover different vesicle shapes as “necklace pearls”, “wormlike micelles” and “spermasomes” can be attributed to different salt solutions or buffers of defined concentrations which suggests a control of morphology. The accessibility of the metal sites at the surface of such vesicles has been tested using fluorescence correlation spectroscopy. The model proteins His10-MBP-FITC and His6-EGFP bind selectively to the Me2+-NTA groups exposed at the surface of the vesicles. While the choice of the buffer significantly influenced the fractions of protein-vesicle conjugates, the interactions of Cu2+- and Ni2+-NTA groups with both His-tagged proteins showed similar values. It should be noted that the experimentally determined dissociation constants of the Me2+-His-Tag complexes were found to be in good agreement with literature data on Ni- NTA functionalized liposomes14, indicating that the polymer brushes at the polymer vesicle surface only slightly interfere with the binding of the proteins. Fluorescence Microscopy was used to visualize the binding of the fluorescent proteins to the functionalized vesicles and images of vesicles with a fluorescent corona were taken. Additionally, atomic force microscopy clearly demonstrated that the polymer adsorbs in an oriented manner on highly oriented pyrolytic graphite surfaces and is able to induce a 2D protein crystallization when Ni-NTA functionalized polymer was used. We believe that these metal-functionalized polymeric membranes have a large potential for the selective immobilization and alignment of proteins at vesicle/planar membrane surfaces. In particular, the high flexibility and compressibility of block copolymer membranes and monolayers could open new possibilities for inducing a 2D protein crystallization. The high cohesion and robustness of block copolymer membranes make them rather insensitive toward mechanical shear or the presence of detergents, increasing their potential utility. In this context, it should also be noted that the pendant double bonds of the poly(butadiene) blocks can be covalently cross-linked, thus freezing the self-assembled structures and providing additional stabilization