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Capillary stamping for bioanalytics and spatial manipulation of protein-protein interactions in live cellsPhilippi, Michael 27 September 2021 (has links)
Capillary stamping is a versatile patterning platform to create micron/sub-micron features on surfaces. When used in combination with mesoporous silica stamps, dot arrays with length scale characteristics matching those of various biomolecular organizations on living cells can be printed. Therefore, different types of ink with functional molecules were printed onto a glass surface and assessed toward their capability to enable an analysis of cellular interactions. Among the evaluated patterned surfaces were dot arrays generated with heterocyclic silanes, which react in a ring-opening reaction upon contact with hydroxyl-terminated surfaces and allow post-modifications of the stamped dot array. Similarly, functionalized proteins were stamped from an aqueous solution, analyzed in regards to specific geometric descriptors and overall contrast between dot and background. After the establishment of a robust patterning system, the stamped substrates were used to spatially manipulate protein-protein interactions in live cells. With the introduction of optogenetics, namely the photoactivatable iLID-system into HeLa cells, protein recruitment from the cytosol to the membrane-bound domains upon irradiation with light was investigated. The technique was also utilized to explore the determinants of Wnt signalosome formation. Wnt co-receptor Lrp6 expressed at the surface of living cells was successfully assembled into nanodot arrays. Strikingly, the co-receptor Fzd8 and the cytosolic scaffold proteins Axin1 and Disheveled2 were spontaneously recruited into the nanodot array to form spatially defined signalosomes in the absence of ligand pointing toward Liquid-Liquid Phase Separation driven signalosome assembly. Immunofluorescence staining confirmed ligand-independent Wnt/β-catenin signaling activated the nanodot arrays.
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Capillary nanostamping with spongy mesoporous silica stampsSchmidt, Mercedes 03 June 2019 (has links)
Many lithographic methods to pattern surfaces both by a mechanical manipulation of the surface or by printing functionalities in the form of particles or molecules have been developed and used in research. Examples for contact-lithographic methods are soft lithography and polymer-pen lithography. One of the main drawbacks of the these methods is the lateral dimension of the obtained pattern. Due to limitations of stamps, materials and the methods themselves, feature sizes of arrays consisting of discrete spots in the sub-micrometer range remain challenging. Another factor in the state-of-the-art contact-lithographic methods is the ex situ adsorption of ink prior to the stamping procedure and thus, an uninterrupted flow of ink cannot be guaranteed. As the variety of imaginable inks is wide and the appropriate solvent often appears to be of organic nature, state-of-the-art contact-lithographic methods are unable to print these inks. The elastomeric polymer stamps used within contact-lithographic methods swell or dissolve in contact with organic solvents. Often, contact-lithographic methods require expensive equipment or defined conditions, e.g. high vacuum or a solvent-enriched humidity, and cannot be carried out in a simple and efficient way under ambient conditions.
In this work, a new approach to generate patterned structures with feature sizes in the sub-micrometer range and spot-to-spot distances in the one-micron range is presented. Stamps with an integrated, continuous pore system generate the patterns while the ink is supplied through the capillaries of the stamp. The method of capillary nanostamping provides a simple and low-cost stamping procedure by the synthesis of spongy mesoporous silica stamps. Due to a continuous pore system within the stamp, the ink can be supplied continuously and even without a refilling system, the stamp itself serves as ink reservoir. This provides a continuous or intermittently ink supply for a stamping process with several stamping cycles without the need to refill the stamp. A new stamp or re-inking after one stamping cycle is not necessary. The stamping process is carried out manually by hand under ambient conditions. Due to the silica network, the stamps can be infiltrated with organic solvents.
The development of spongy mesoporous silica stamps for capillary nanostamping is presented in this work by demonstrating the progress from pure silica stamps in a typical well-known sol-gel synthesis to spongy and flexible silica stamps with a reduced network bonding and hydrophobic internal residues. For the proof of concept of capillary nanostamping with spongy mesoporous silica stamps, several different inks are stamped. All inks are chosen with respect to a potential application and consist of a volatile organic solvent to proof the stability of the stamps against these solvents, and a non-volatile component, which remains on the substrate surface after precipitation and drying of the solvent. As ink, a dispersion of C60 fullerenes in toluene is stamped onto perfluorinated glass slides. A solution of 1-dodecanethiol in ethanol is stamped onto a gold-coated glass with the outcome of a heterogeneous surface. As a model for nanoparticles, nanodiamonds dispersed in isopropanol are stamped and subsequently functionalized with a fluorescent dye in a click-reaction. A polymer and two different block copolymers dissolved in toluene/chloroform are stamped onto differently functionalized substrate surfaces to analyze the dependency of the nature of the substrate on the stamping results. In a final experiment, a solution of 17α-ethinylestradiol in acetonitrile is stamped as a model for an active pharmaceutical ingredient and subsequently detached from the substrate surface to obtain a defined nanodispersion.
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Planar Lensing Lithography: Enhancing the Optical Near Field.Melville, David O. S. January 2006 (has links)
In 2000, a controversial paper by John Pendry surmised that a slab of negative index material could act as a perfect lens, projecting images with resolution detail beyond the limits of conventional lensing systems. A thin silver slab was his realistic suggestion for a practical near-field superlens - a 'poor-mans perfect lens'. The superlens relied on plasmonic resonances rather than negative refraction to provide imaging. This silver superlens concept was experimentally verified by the author using a novel near-field lithographic technique called Planar Lensing Lithography (PLL), an extension of a previously developed Evanescent Near-Field Optical Lithography (ENFOL) technique. This thesis covers the computational and experimental efforts to test the performance of a silver superlens using PLL, and to compare it with the results produced by ENFOL. The PLL process was developed by creating metal patterned conformable photomasks on glass coverslips and adapting them for use with an available optical exposure system. After sub-diffraction-limited ENFOL results were achieved with this system additional spacer and silver layers were deposited onto the masks to produce a near-field test platform for the silver superlens. Imaging through a silver superlens was achieved in a near-field lithography environment for sub-micron, sub-wavelength, and sub-diffraction-limited features. The performance of PLL masks with 120-, 85-, 60-, and 50-nm thick silver layers was investigated. Features on periods down to 145-nm have been imaged through a 50-nm thick silver layer into a thin photoresist using a broadband mercury arc lamp. The quality of the imaging has been improved by using 365 nm narrowband exposures, however, resolution enhancement was not achieved. Multiple layer silver superlensing has also been experimentally investigated for the first time; it was proposed that a multi-layered superlens could achieve better resolution than a single layer lens for the same total silver thickness. Using a PLL mask with two 30-nm thick silver layers gave 170-nm pitch sub-diffraction-limited resolution, while for a single layer mask with the same total thickness (60 nm) resolution was limited to a 350-nm pitch. The proposed resolution enhancement was verified, however pattern fidelity was reduced, the result of additional surface roughness. Simulation and analytical techniques have been used to investigate and understand vi ABSTRACT the enhancements and limitations of the PLL technique. A Finite-Difference Time- Domain (FDTD) tool was written to produce full-vector numerical simulations and this provided both broad- and narrowband results, allowing image quality as a function of grating period to be investigated. An analytical T-matrix method was also derived to facilitate computationally efficient performance analysis for grating transmission through PLL stacks. Both methods showed that there is a performance advantage for PLL over conventional near-field optical lithography, however, the performance of the system varies greatly with grating period. The advantages of PLL are most prominent for multi-layer lenses. The work of this thesis indicates that the utilisation of plasmonic resonances in PLL and related techniques can enhance the performance of near-field lithography.
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