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應用微奈米流體元件於單分子DNA複合體之分析

為了解決W221的問題，作者史利南 這樣論述：

Single molecule DNA studies have developed into a useful tool over the past two decades. Unlike conventional experiments that provide collective information of an ensemble, it gives us the opportunity to manipulate individual DNA molecules, observe and analyze their behavior upon interaction with o

ther biomolecules like proteins. DNA is a semi-flexible polymer, which exists in its physiological random-coil state in a bulk environment, making direct visualization of DNA and its interactions to proteins or drugs challenging. Stretching and immobilization of DNA molecules thus becomes a critical

requirement for single molecule studies. Techniques like optical trapping, magnetic trapping, flow stretching, molecular combing, and confinement induced stretching using micro- and nanofluidic devices have been widely used to stretch and immobilize DNA molecules.In this thesis, we developed two in

dependent micro/nanofluidic devices that share a similar fabrication scheme, but with different approaches for effective DNA immobilization and stretching, other than the previously demonstrated techniques. The first one is a proof-of-concept method adopting nanoslit, bioconjugation, and single mole

cule imaging for direct mapping of transcriptional factor (TF) binding sites on field-stretched single DNA molecules. TFs are proteins that bind to specific loci of DNA using DNA binding domains to carry out the process of transcription. Hence, the identification of TF binding sites is essential for

understanding the regulatory circuits that control cellular processes such as cell division and differentiation as well as metabolic and physiological balance. The nanoslit devices used in this work are fabricated in fused silica substrate, with tens of nanometers in depth, and are conformably seal

ed by a polymer-coated coverslip. This device is used for the mapping of TF (E. coli RNA polymerase holoenzyme, or RNAP) binding sites along lambda phage DNA, where two promoters and three pseudo-promoters sites of E. coli RNAP have been identified with ~ 100 nm resolution. The advantages of our nan

oslit device include multiplexing, quick mapping of TF binding sites, and the ability to distinguish real complexes from non-specific ones.The second work is the demonstration of a new DNA combing scheme using oxygen (O2) plasma-modified polysilsesquioxane (PSQ) polymer coated surface to effectively

immobilize and stretch DNA molecules. The increase in surface roughness and surface potential due to low-pressure O2 plasma treatment are proposed to be the major factors in effective DNA combing. The advantage of this method is extremely simple, and the property of the surface can be fine-tuned w

ith O2 plasma treatment, to anchor DNA molecule at the end or multiple points. Plasma treated PSQ surface can be directly used for room-temperature bonding of micro/nanofluidic devices, which in turn can be used directly for DNA combing experiments without further surface modifications. In this work

, we used our DNA combing platform for fluorescence single molecule imaging of anti-cancer drug cisplatin complexed DNA molecules. Our observations show intra-strand crosslinking of DNA molecules in the presence of cisplatin, leading to DNA condensation.Both platforms enable single molecule DNA stud

ies and open up the possibility of multitude of DNA interaction studies involving other biomolecules, like proteins, or drugs. In addition, we have also demonstrated the possibility of carrying out transverse current measurements using a device with a tandem array of electrode nanogaps embedded co-

planar into a nanofluidic channel, to map DNA binding sites of proteins, which, with further development, could well serve as a tool for single molecule DNA analysis and a wide variety of other applications.