Micro- and Nanotechnology-Based Platforms to Study Biology at Small Scale: From DNAs to Single Cells

摘要

This thesis describes technology platforms for various biological applications at nano- and microscale. The first platform is the silicon nanowire (SiNW) field-effect-transistor (FET)-based biosensor. SiNW FETs have unique features such as label-free, real-time, and electrical measurement, which will be demonstrated with DNA and protein sensing. We further demonstrate that using different surface chemistry can modulate the sensitivity and dynamic range of the sensor. Debye screening, one of the major bottlenecks of the technology, is shown to be circumvented by using electrostatically immobilized capture DNA for DNA sensing and a small synthetic capture agent, peptide, for protein sensing. A model for the detection of analyte by SiNW sensors is also developed and utilized to extract DNA binding kinetic parameters, which shows the potential of the platform as a more sensitive version of surface plasmon resonance (SPR).ududThe second part of this thesis focuses on a more practical and easily expandable technology, the microfluidics-based platform, to perform a single-cell-based protein analysis. We develop a flow patterning technology to generate highly parallel DNA barcodes that can be further utilized as a handle to immobilize protein capture agents, such as antibodies. As a first step, a protocol to make high-quality DNA micro-barcodes with an excellent uniformity is introduced. The uniform DNA barcode patterns enable us to perform protein detection from single cells in a microfluidic device that spans the whole glass microscope slide. A data set from about thousand experiments can be collected from a single test with the developed microfluidic device, owing to the good quality of DNA barcodes and DNA Encoded Antibody Libraries (DEAL) technology. This platform further demonstrates that multi-parameter protein detection at the single-cell level presents cellular heterogeneity which leads to new findings in biology. A quantitative version of the Le Chatelier’s principle, as derived using information theory, is applied to analyze a large amount of data from this platform. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein–protein interaction network. ududLastly, another application of microfluidics is demonstrated for studying interfacial chemistry on lung surfactant systems under oxidative stress, along with mass spectrometry (MS) and molecular dynamic (MD) simulation results. The findings from the MS and MD simulations provide mechanistic details for the reaction of ozone with unsaturated phospholipids, leading to possible damage of the pulmonary system by ROS or direct ozone exposure. These investigations focus on molecular transformations that occur as a result of oxidative stress. Such molecular transformations can have a strong influence on the physical properties of the pulmonary surfactant (PS) system (i.e., the surface tension and elasticity of the interface), and therefore understanding how chemical transformations influence such physical properties can provide key insights into how the PS system responds to environmental challenges. Thus, we also propose utilizing microbubbles as a model system for investigating the physical transformations of the PS system when exposed to environmental challenges. The chemical composition change, along with physical property change, is analyzed by altered bubble size and oscillatory behavior which can provide an improved understanding of the physics of a PS system when it is subjected to oxidative stress.