Tunable Elastomeric Nano-Channels from Guided Fracture for Biopolymer Super Resolution Imaging

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Chiu, Han-Ching
Takayama, Shuichi
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This thesis describes the development of a polydimethylsiloxane (PDMS) based micro-, nano-fluidics system compatible with super-resolution imaging to capture and linearize extracted chromatin, for epigenetics studies. Native chromatin are DNA folded and tightly coiled around proteins, to access the proteins of interest, chromatin must be unwrapped via linearization. An array of cracks ranging from micron-scale to nano-scale is fabricated by subjecting a two-layer elastomeric materials system to an applied tensile strain. The morphology of the generated surface cracks is determined by the pre-patterned photoresist features in a PDMS substrate and the spin coated thin film on its surface. The depths of the cracks are governed by the thickness and properties of the spin-coated surface layer, while the locations of crack initiation are determined by pre-patterned features in the bulk substrate. The surface cracks are sealed with a thin PDMS film by plasma bonding. This creates channels from the surface cracks, where the channel widths can be tuned via an automated stretcher that applies a uniaxial strain. This tunable device can be used to capture biopolymers such as DNA and chromatin. The linearization of captured biopolymers is achieved by a combination of nano-confinement in small conduits and the squeezing hydrodynamic flow which results from the strain being released from the stretcher. Various fluorescent dyes are investigated to overcome the oxygen rich PDMS environment that is not conducive to super-resolution microscopy. Tetrahymena thermophila, a protozoan, is chosen as the model system for chromatin extraction and the protein of interest is labeled with super-resolution compatible dye. Thus, direct super resolution imaging of proteins on biopolymers in the tunable nano-channels is achieved. Visualization of the linearized chromatin pointed towards dispersive segregation for histone deposition. We believe that this linearization platform can be adapted for visualization of other protein modifications and eventually provide valuable epigenetic information.
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