Bio-microdevices are miniaturised devices based on biologically derived components (e.g., DNA, proteins, and cells) combined or integrated with microfabricated substrates. These devices are of interest for numerous applications, ranging from drug discovery, to environmental monitoring, to tissue engineering. Before a bio-microdevice can be fully developed, specific fabrication issues need to be addressed. One of the most important is the spatial immobilization of selected biomolecules in specific micro-areas of the device. Among the biomolecules of interest, the controlled immobilization of proteins to surfaces is particularly challenging due to the complexity of these macromolecules and their tendency to lose bioactivity during the immobilization step. The present Thesis reports on three novel micropatterning techniques for the spatial immobilization of proteins with bioactivity retention and improved read-out of the resulting micropatterns. The technologies developed are based on three different micropatterning approaches, namely 1) direct-writing UV laser microablation (proLAB), 2) a novel microcontact printing method (�CPTA) and 3) a replica molding method combined with bead selfassembly (BeadMicroArray). The first two technologies, proLAB and �CPTA, are an implementation of existing techniques (laser ablation and �CP, respectively), whereas the third, i.e., the BeadMicroArray, is a totally new technique and type of patterning platform. 'ProLAB' is a technology that uses a micro-dissection tool equipped with a UV laser (the LaserScissors�) for ablating a substrate made of a layer of ablatable material, gold, deposited over a thin polymer layer. The latter layer is transparent to the laser but favours protein adsorption. In the present work microchannels were chosen as the structure of interest with the aim of arranging them in 'bar-codes', so to create an 'information-addressable' microarray. This platform was fabricated and its application to specific antigen binding demonstrated. The second technique that was developed is a microstamping method which exploits the instability of a high-aspect ratio rubber stamp fabricated via soft-lithography. The technique is denominated microcontact printing trapping air (�CPTA) since the collapsing of a rubber stamp made of an array of micro-pillars over a plane glass surface resulted in the formation of a large air gap around the entire array. The method can be successfully employed for printing micro-arrays of proteins, maintaining biological activity. The technique was compared with robotic spotting and found that microarrays obtained with the �CPTA method were more homogeneous and had a higher signal-tonoise ratio. The third technique developed, the BeadMicroArray, introduces a totally new platform for the spatial addressable immobilization of proteins. It combines replica molding with microbead self-assembling, resulting in a platform where diagnostic beads are entrapped at the tip of micropillars arranged in a microarray format. The fabrication of the BeadMicroArray involves depositing functional microbeads in an array of V-shaped wells using spin coating. The deposition is totally random, and conditions were optimised to fill about half the array during spin coating. After replica molding, the resulting polymer mold contains pyramid-shaped posts with beads entrapped at the very tip of the post. Thanks to the fabrication mode involved, every BeadMicroArray fabricated contains a unique geometric code, therefore assigning a specific code to each microarray. In the present work it was demonstrated that the functionality of the beads after replica molding remains intact, and that proteins can be selectively immobilized on the beads, for instance via biorecognition. The platform showed a remarkable level of selectively which, together with an efficient blocking towards protein non-specific adsorption, lead to a read-out characterized by a very good signal-to-noise. Also, after recognition, a code was clearly visible, therefore showing the encoding capacity of this unique microarray.

Optical waveguides, quantum dot emitters, and flat top beam shapers were designed and fabricated by two soft lithographic techniques; micro transfer molding (microTM) and vacuum assisted microfluidics (VAM). Optical waveguides were fabricated through a microTM technique that utilizes a poly dimethylsiloxane (PDMS) stamp. Generation of the flexible stamp required development of a channel waveguide pattern mask, defined by maskless lithography, and followed by construction of a three dimensional channel waveguide master, acquired through contact lithography on a glass substrate coated with SU-8 photoresist. Creation of a positive imprint replicating mold was accomplished through prepolymer PDMS solution settling and curing around the master. Waveguide fabrication was achieved through PDMS conformal contact on, and subsequent curing of, ultraviolet (UV) polymer resins on a silicon substrate. A slight modification of the microTM PDMS stamp, whereby inlet and outlet tunnels were incorporated, resulted in a novel VAM structure and correspondingly waveguides. Waveguide propagation losses were determined to be 1.14 dB/cm and 0.68 dB/cm for the microTM and VAM fabricated waveguides, respectively. A lithographic approach employing quantum dots doped in SU-8 photoresist has led to the realization of a new quantum dot emitter. Uniform coating of a doped material on a silver coated substrate was followed by contact mask lithography. Evaporation of a thin silver layer, upon development of the resultant quantum dot doped channel waveguide structure, facilitates confined emission. Successful edge emitting was demonstrated with blue laser pumping. The lithographic fabrication of such quantum dot emitter is successfully replaced by soft lithographic VAM technique. A flat top beam shaper, whose profile was developed on cured UV polymer resins, was fabricated by microTM technique. The master used for the development of the PDMS stamp was produced through an iterative wet etching process capable of achieving etching depths as small as a few nanometers. Comparisons between the reference wet etched beam shaper and the microTM based beam shaper produced near identical output flat top beams from incident Gaussian beams. Through this research work, successful soft lithographic fabrication of optical waveguides, quantum dot emitters, and flat top beam shapers were demonstrated. The vast potential exhibited by these and other related technologies show great promise for cost-effective mass production of various micro optics and integrated photonic components.

Microfluidics is gaining in importance due to its wide ranging benefits and applicability in chemical and biological analysis. Although traditional microfluidic devices are created with glass or silicon based fabrication technologies, polymer based devices are gaining in popularity. Soft lithography and replica molding are techniques for the rapid prototyping of such devices, utilizing Polydimethylsiloxane (PDMS) as the dominant material. Other benefits include its low costs and ease of fabrication. Even though soft lithography is a well researched and developed fabrication process, new applications have been discovered in which the technology can be applied. Often, changes in the fabrication process are necessary for their application in other areas of research. This thesis will address several microfluidic applications using soft lithography. These areas of research include microfluidic thermometry, isoelectric focusing (IEF), and micromixers. In microfluidic thermometry, a novel thin film PDMS/Rhodamine B has been developed allowing whole-chip temperature measurements. In addition, compatibility problems between Rhodamine B and PDMS microfluidic devices were resolved. The thin film fabrication process, experimental results, and issues with its use are discussed. Future work and attempts at improving the thin film performance are also provided. IEF involves applications in which samples are separated according to its electrostatic charge. Two types of IEF applications are shown in which soft lithography has been shown to be beneficial to its development and performance. In isoelectric focusing with the use of thermally generated pH gradients, soft lithography allows for the rapid design, production and testing of different channel layouts. In general, due to PDMS insulation and overall low heat transfer rates, the temperatures detected are more gradual than those previously reported in literature. IEF using carrier ampholytes are also discussed, with preliminary results in which devices fabricated using soft lithography are compared to commercially available IEF cartridges. Its fabrication issues are discussed in detail. In micromixers, soft lithography fabrication issues and overall integration with flow mechanisms is discussed. In general it is difficult to perform mixing in the microscale due to the predominantly laminar flow and flow rate restrictions. Channel geometry is insignificant, as can be seen through numerical simulations.

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.

Microfluidics is gaining in importance due to its wide ranging benefits and applicability in chemical and biological analysis. Although traditional microfluidic devices are created with glass or silicon based fabrication technologies, polymer based devices are gaining in popularity. Soft lithography and replica molding are techniques for the rapid prototyping of such devices, utilizing Polydimethylsiloxane (PDMS) as the dominant material. Other benefits include its low costs and ease of fabrication. Even though soft lithography is a well researched and developed fabrication process, new applications have been discovered in which the technology can be applied. Often, changes in the fabrication process are necessary for their application in other areas of research. This thesis will address several microfluidic applications using soft lithography. These areas of research include microfluidic thermometry, isoelectric focusing (IEF), and micromixers. In microfluidic thermometry, a novel thin film PDMS/Rhodamine B has been developed allowing whole-chip temperature measurements. In addition, compatibility problems between Rhodamine B and PDMS microfluidic devices were resolved. The thin film fabrication process, experimental results, and issues with its use are discussed. Future work and attempts at improving the thin film performance are also provided. IEF involves applications in which samples are separated according to its electrostatic charge. Two types of IEF applications are shown in which soft lithography has been shown to be beneficial to its development and performance. In isoelectric focusing with the use of thermally generated pH gradients, soft lithography allows for the rapid design, production and testing of different channel layouts. In general, due to PDMS insulation and overall low heat transfer rates, the temperatures detected are more gradual than those previously reported in literature. IEF using carrier ampholytes are also discussed, with preliminary results in which devices fabricated using soft lithography are compared to commercially available IEF cartridges. Its fabrication issues are discussed in detail. In micromixers, soft lithography fabrication issues and overall integration with flow mechanisms is discussed. In general it is difficult to perform mixing in the microscale due to the predominantly laminar flow and flow rate restrictions. Channel geometry is insignificant, as can be seen through numerical simulations.

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 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.</p><p>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.</p><p>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.</p><p>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.</p>

Cell encapsulation is a promising concept in regenerative medicine and stem cell treatment of diseases. Cells encapsulated in hydrogels have shown to yield better therapeutic outcome over cells in suspension. Microfluidic platforms have facilitated the process of cell encapsulation through the controlled mixing of aqueous cell solution and hydrogel with an immiscible liquid to yield a monodispersed population of microcapsules at a high throughput. However, given that the microfluidic process of placing cells in microcapsules is completely random, yielded samples are often riddled with empty microcapsules, raising the need for a post-encapsulation purification step to sort empty microcapsules from cell-laden ones. Sorting of microcapsules can be achieved through several techniques, most desirable of which are electrokinetic such as dielectrophoresis (DEP) and electrophoresis (EP). The advantages of DEP and EP techniques are that they support label-free sorting and yield a high throughput. However to achieve true effective DEP or EP sorting, there is a need to understand how empty microcapsules react to these electrokinetic forces versus occupied microcapsules. This study developed microfluidic devices for characterising the electrokinetic effects on microcapsules using DEP and EP. Results of both characterization techniques showed notable differences in the response of empty microcapsules versus cell-laden ones, reinforcing their potentials for sorting. Furthermore, this study proposed designs for microcapsules sorting devices that leverage EP and DEP.

The diploma thesis deals with cellular membrane model preparation on microfluidic devices. It summarizes means of microfluidic device fabrication, phospholipid bilayer formation mechanisms, optimization techniques and characterization methods of those systems. It focuses on free-standing planar lipid bilayers which are easily accessible by a number of different characterization methods and at the same time exhibit good stability and variability. The aim of this work is to design and prepare a microfluidic chip on which a planar lipid bilayer can be prepared. It therefore presents microfluidic device prepared by soft lithography of PDMS adapted for model membrane formation by self-assembly of phospholipids at the interface of aqueous and organic phases created by the architecture of the microfluidic device. Formation of the model membrane was visualized by optical microscopy and fluorescence-lifetime imaging microscopy.

The investigation of the physical properties of deoxyribonucleic acid under confinement is an essential step for the all-embracing understanding of the replication and transcription in living cells as well as for the development of the biomimetic nanotechnology. The following report addresses the measure-ment and interpretation of the intramolecular diffusion along stretched λ-DNA-molecules. This work comprises the fabrication of submicroscopic channels via softlithography, the integration of the chan-nels in an experimental setup with a fluorescence microscope and a source-measurement unit, and the experiments with the DNA-stretching in electrical field. The important results are the development of a measuring assembly with stable, softlithographic structures in Ormostamp, the direct imaging and measurement of the intermolecular diffusion along stretched DNA-molecules in channels with cross sections down to 100 x 300 nm2, and the qualitative and quantitative analysis on the basis of models of polymer physics.