The aim of this diploma work is to improve selectivity and sensitivity in DNA-chips by utilizing fluorescence resonance energy transfer (FRET) between conjugated polyelectrolytes (CPEs) and fluorophores. Leclerc and co-workers have presented successful results from studies of super FRET between fluorophore tagged DNA and a CPE during hybridisation of the double strand. Orwar and co-workers have constructed a DNA-chip using standard photo lithography creating a pattern of the hydrophobic photoresist SU-8 and cholesterol tagged DNA (chol-DNA). This diploma work will combine and modify these two ideas to fabricate a improved DNA-chip. Immobilizing of DNA onto surface has been done by using soft lithography. Hydrophobic pattern arises from the poly(dimethylsiloxane) (PDMS) stamp. The hydrophobic pattern will attract chol-DNA that is adsorbed to the chip. Different sets of fluorophores are covalently bound to the DNA and adding CPEs to the complex will make FRET occur between CPE and bound fluorophore. We will here show that the specificity in DNA hybridization by using PDMS patterning was high. FRET clearly occurred, especially with the CPEs as donor to the fluorophore Cy5. The intensity of FRET was higher when the fluorophore and the CPE were conjugated to the same DNA strand. The largest difference in FRET intensity between double stranded and single stranded complexes was observed with the CPE tPOMT. Super FRET has been observed but not yet fully proved. The FRET efficiency was lower with the fluorophore Alexa350 as donor compared to the Cy5/CPE complex. Most of the energy transferred from Alexa350 was extinguished by quenching.

Micro-Electro-Mechanical Systems (MEMS) micromirrors have been developed for more than two decades along with the development of MEMS technology. They have been used into many application fields: optical switches, digital light projector (DLP), adoptive optics (AO), high definition (HD) display, barcode reader, endoscopic optical coherence tomography (OCT) and confocal microscope, and so on. Especially, MEMS mirrors applied into endoscopic OCT and confocal microscope are the intensive research field. Various actuation mechanisms, such as electrostatic, electromagnetic, electro bimorph thermal, electrowetting, piezoelectric (PZT) and hybrid actuators, are adopted by different types of micromirrors. Among these actuators, the electrostatic is easily understood and simple to realize, therefore, it is broadly adopted by a large number of micromirrors. This thesis reports the design, fabrication, and characterization of a 2-D Silicon-on-insulation (SOI) MEMS micromirror with sidewall (SW) electrodes for endoscopic OCT or confocal microscope imaging. The biaxial MEMS mirror with SW electrodes is actuated by electrostatic actuators. The dimension of mirror plate is 1000micron×1000micron, with a thickness of a 35micron. The analytical modeling of SW electrodes, fabrication process, and performance characteristics are described. In comparison to traditional electrostatic actuators, parallel-plate and comb-drive, SW electrodes combined with bottom electrodes achieve a large tilt angle under a low drive voltage that the comb-drive does and possess fairly simple fabrication process same as that of the parallel-plate. A new fabrication process based on SOI wafer, hybrid bulk/surface micromachined technology, and a high-aspect-ratio shadow mask is presented. Moreover, the fabrication process is successfully extended to fabricate 2×2 and 4×4 micromirror arrays. Finally, a biaxial MEMS mirror with SW electrodes was used into Confocal MACROscope for imaging. Studied optical requirements in terms of two optical configurations and frequency optimization of the micromirror, the biaxial MEMS mirror replaces the galvo-scanner and improves the MACROscope. Meanwhile, a new Micromirror-based Laser Scanning Microscope system is presented and allows 2D images to be acquired and displayed.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein α (C/EBPα) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBPα. Additionally, the original clone found to interact with C/EBPα consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBPα mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBPα, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBPα-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBPα-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBPα. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBPα was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBPα, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBPα.

This research delineates the design of a nanolithographic process for nanometer scale surface patterning. The process involves the combination of serial atomic force microscope (AFM) based nanolithography with the parallel patterning capabilities of soft lithography. The union of these two techniques provides for a unique approach to nanoscale patterning that establishes a research knowledge base and tools for future research and prototyping.To successfully design this process a number of separate research investigations were undertaken. A custom 3-axis AFM with feedback control on three positioning axes of nanometer precision was designed in order to execute nanolithographic research. This AFM system integrates a computer aided design/computer aided manufacturing (CAD/CAM) environment to allow for the direct synthesis of nanostructures and patterns using a virtual design interface. This AFM instrument was leveraged primarily to study anodization nanolithography (ANL), a nanoscale patterning technique used to generate local surface oxide layers on metals and semiconductors. Defining research focused on the automated generation of complex oxide nanoscale patterns as directed by CAD/CAM design as well as the implementation of tip-sample current feedback control during ANL to increase oxide uniformity. Concurrently, research was conducted concerning soft lithography, primarily in microcontact printing (µCP), and pertinent experimental and analytic techniques and procedures were investigated.Due to the masking abilities of the resulting oxide patterns from ANL, the results of AFM based patterning experiments are coupled with micromachining techniques to create higher aspect ratio structures at the nanoscale. These relief structures are used as master pattern molds for polymeric stamp formation to reproduce the original in a parallel fashion using µCP stamp formation and patterning. This new method of master fabrication provides for a useful alternative to conventional techniques for soft lithographic stamp formation and patterning. / Dissertation

<p>The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.</p><p>In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.</p><p>In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.</p><p>In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.</p> / Dissertation

Atomic force microscopy (AFM) can be used to acquire high-resolution topographical images of surfaces, but has the additional capability of detecting the local nanometer scale mechanical properties. For these reasons, it becomes a standard research tool in the surface science recently. In this paper, we used AFM to measure the several properties of two different human hepatocellular carcinoma cell lines, Hep G2 ( known as well differentiated and more highly carcinomatous hepatoma cell lines ) and SK-HEP-1 ( known as poorly differentiated and more lightly carcinomatous hepatoma cell lines ) cells fixed on the glass substrate, which including the surface morphology and the relationship between the cantilever deflections and loading forces ( force curve ). Considered the heterogeneous characteristics of the cell surface, the preferred experimental method is to make pixel-by-pixel force curves in a designated area ( force map ) , both adhesion forces and elasticity associated with different locations on the cell surfaces will be obtained. Finally, we use Hertzian model to calculate Young's modulus of Hep G2 and SK-HEP-1 respectively. Based on these results, we can understand the surface properties of two human hepatocarcinoma cell lines with different differentiated stage. The results showed the difference of the morphology, height, cell migration, degree of cell aggregation, roughness, elasticity, adhesive force of two cells. SK-HEP-1 cell has the wide distance of the folds, better cell migration, homogeneous properties of elasticity. It can be assumed that the SK-HEP-1 cells have a dense network structure of actin filaments under the cell membrane like branches (branched networks); Hep G2 cell has the narrow distance of the folds, poor cell migration, heterogeneous properties of elasticity. It can be assumed that the Hep G2 cells have the individual actin filaments and cross-linked network structure of actin filaments under the cell membrane. The above results can be speculated that the elastic properties of the membrane surface will be influenced of actin filaments.

Supported lipid bilayers (SLBs) are unique model systems for biological membranes. SLBs can be formed by fusing liposomes on solid substrates, which can be characterized by a variety of surface analytical techniques, such as Atomic Force Microscopy (AFM), X-ray diffraction, Quartz Crystal Microbalance (QCM), etc. In this study we used AFM to investigate the dynamic process of the formation of SLBs from liposomes in solutions containing metal ions and phase separation between different lipids as a function of temperature. Divalent cations, Ni2+ in particular, was found to be critical to the deposition of bilayers. Lipid rafts are plasma membrane microdomains rich in sphingolipid and cholesterol forming a liquid ordered phase surrounded by a liquid disordered phase. Lipid rafts are insoluble in cold non-ionic detergents, also called Detergent Resistant Membranes (DRMs). The interaction behaviors between detergent (Triton X-100) and mixed bilayers (DOPC/DPPC and DOPC/SpM) were studied by AFM. The way lipid bilayers were solubilized by Triton X-100 was quite different below and above its critical micelle concentration (CMC), and the SpM domains were found to be resistant to detergent extraction in the cold.

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The adhesion and removal of particles from surfaces is a contemporary problem in many industrial applications like Semiconductor manufacturing, Bioaerosol removal, Pharmaceuticals, Adhesives and Petroleum industry. The complexity of the problem is due to the variety of factors like roughness, temperature, humidity, fluid medium etc. that affect pull-off of particles from surfaces. In particle removal from surfaces using fluid motion, the dynamic effects of particle separation will play an important role. Thus it is essential to study the dynamic effects of particle removal. Velocity of pull-off and force duration effects are two important dynamic factors that might affect pull-off. Particle adhesion studies can be made using the Atomic Force Microscope (AFM). The velocity of pull-off and force duration can be varied while making the AFM measurements. The objective of the current work is to obtain the dependence of pull-off force on pull-off velocity. Experiments were conducted using AFM and the data obtained from the experiments is processed to obtain plots for pull-off force vs. particle size and pull-off force vs. pull-off velocity. The pull-off force is compared with the predictions of previous contact adhesion theories. A velocity effect on pull-off force is observed from the experiments conducted.

This research presents a detection scheme for analyzing the temperature distribution produced by the Joule heating effect nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system¡Xtotal internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the inner channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective-type TIRFM, microscope cover glass are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM makes the intensity information obtained is not sensitive to the channel depth variation which ususally biases the measured results while using conventional epi-fluorescence microscope (Epi-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within hundreds of nanometer of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. In order to investigate the temperature distribution produced by the Joule heating effect for electrokinetically driven microchips, this study not only measures the temperature on the microchannel wall by the proposed TIRFM but also measures the temperature inside the microchannel by an Epi-FM. In addition, this research presents a method to reduce the Joule heating effect and enhance the separation efficiency of DNA biosamples in a chip-based capillary electrophoresis (CE) system utilizing pulse DC electric fields. Since the average power consumption is reduced by the pulse electric fields, the Joule heating effect can be significantly reduced. Results indicate the proposed TIRFM method provides higher measurement sensitivity over the Epi-FM method. Significant temperature difference along the channel depth measured by TIRFM and Epi-FM is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the Joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a simulation model for precisely predicting the Joule heating effect in microfluidic chips. Moreover, in the research of reducing the Joule heating effect and enhancing the separation efficiency in a chip-based CE system utilizing pulse electric fields, the experimental and numerical investigations commence by separating a mixed sample comprising two fluoresceins with virtually identical physical properties. The separation level is approximately 2.1 times higher than that achieved using a conventional DC electric field. The performance of the proposed method is further evaluated by separating a DNA sample of Hae III digested £XX¡V174 ladder. Results indicate the separation level of the two neighboring peaks of 5a (271 bp) and 5b (281 bp) in the DNA ladder is as high as 120% which is difficult to be achieved using a conventional CE scheme. The improved separation performance is attributed to a lower Joule heating effect as a result of a lower average power input and the opportunity for heat dissipation during the zero-voltage stage of the pulse cycle. Overall, the results demonstrate a simple and low-cost technique for achieving a high separation performance in CE microchips.