Stability and bandwidth investigation of alternative structures for nanopore sensors

The genetic information carriers, DNA molecules can be thought of as the blueprints of living organisms. This crucial functionality of the DNA mole- cules may explain the drive and momentum for DNA sequencing research. The commonly used parallel sequencing methods require extensive sample preparation, long processing times and expensive chemical reagents. In or- der to realize the goal of $1000 genome sequencing, many alternative meth- ods are proposed. One of the most promising technologies among these is nanopore sequencing. Nanopore sequencing involves the threading of a DNA molecule between two electrolytic reservoirs through a nanometer-sized pore on a synthetic or an organic platform by means of electrophoresis and/or mag- netism. During the threading of DNA molecules, various electrical aspects of the bases are investigated. The minimal label-free sample preparation, possibility of parallelizability and high throughput are the factors that make this method a very promising solution for low-cost, robust and fast DNA se- quencing. In the nanopore sequencing eld, the synthetic platforms have the advantage of durability and mass production value due to the existing sili- con device fabrication technologies. This thesis work focuses on the stability and bandwidth investigation of alternative structures for nanopore sensing. Membranes with various thicknesses of Al2O3, Si3N4 and SiO2 stack con gu- rations were fabricated. The fabricated membranes were analyzed and drilled through by focused e-beam sputtering in TEM. The membranes were tested in 0.1 M and 1 M KCl solutions for IV characteristics, noise level and AC response. The membranes with desirable noise and IV characteristics were further tested for DNA sensing purposes. The membranes featuring Al2O3 insulating layer con gurations yielded low noise, high bandwidth and lim- ited durability in KCl solutions. The low yield in DNA sensing in 1 M KCl solutions using these architectures forms the background and motivation for next generation structures for DNA sensing

Nano to Micro Scale Coulter Counters

As biotechnology advances, personalized medicine has become one of the prominent trends. It can be briefly described as an effort to provide preventative, diagnostic and treatment measures for health problems implemented on an individual basis. Resistive pulse technique is a measurement scheme that has found a wide range of applications in this field. In this dissertation, research on devices that are based on resistive pulse technique from nano to micro scale are presented. Nanopore sensing, one of the major candidate technologies for next-generation DNA sequencing is an example of nano-scale application of this technique. It is a promising technology due to its potential to provide label-free, robust and rapid DNA sequencing. However, there are several challenges in reaching this ultimate goal. We present an architecture for solving the aggregate base detection problem through ubiquitous, cost-effective CMOS fabrication. We describe the challenges and advantages of this approach. Beyond DNA sequencing, there are many exciting potential applications of synthetic nanopores, such as sizing and investigating polymer based constructs. Due to its well understood properties, DNA can be used to build functional nano-mechanical structures. However, DNA nano-structures usually lack a robust validation and quality control method, leading to populations that are poorly characterized in terms of shape and size. In this dissertation, the feasibility of utilizing synthetic nanopores to characterize a high volume of DNA nanotubes is investigated. Next, a micro scale application of resistive pulse technique for cancer diagnosis is explored. Particularly, Circulating Tumor Cells(CTCs) have recently emerged as indicators of cancer metastasis. Thus, efficient detection of CTCs can provide non-invasive biopsy, enable personalized medicine and help understand cancer biology. Currently used immunoassay based CTC detection techniques are inefficient and insufficient to classify extremely heterogeneous CTCs such as Circulating Melanoma Cells(CMCs). Cancer cells have markedly different physical attributes, such as size and stiffness, and can be used to distinguish tumor cells from normal cells. We report a micro-fluidic chip potentially meeting the urgent need to detect individual CTCs in a label-free, fast and inexpensive fashion while maintaining cell viability. We present the design, fabrication and modeling of microfluidic channels that enable the classification of CTCs based on their size and stiffness. We use the device was to classify melanoma (MNT1)and breast cancer (MCF-7) cells both alone and in the presence of blood cells

Tailored Polymeric Membranes for Mycobacterium Smegmatis Porin A (MspA) Based Biosensors.

Nanopores based on protein channels inserted into lipid membranes have paved the way towards a wide-range of inexpensive biosensors, especially for DNA sequencing. A key obstacle in using these biological ion channels as nanodevices is the poor stability of lipid bilayer membranes. Amphiphilic block copolymer membranes have emerged as a robust alternative to lipid membranes. While previous efforts have shown feasibility, we demonstrate for the first time the effect of polymer composition on MspA protein functionality. We show that membrane-protein interaction depends on the hydrophobic-hydrophilic ratio (f-ratio) of the block copolymer. These effects are particularly pronounced in asymmetric protein pores like MspA compared to the cylindrical α-Hemolysin pore. A key effect of membrane-protein interaction is the increased 1/fα noise. After first showing increases in 1/fα behaviour arise from increased substate activity, the noise power spectral density S(f) was used as a qualitative tool for understanding protein-membrane interactions in polymer membranes. Polymer compositions with f-ratios close to lipid membranes caused noise behaviour not observed in lipid membranes. However, by modifying the f-ratio using a modular synthetic approach, we were able to design a block copolymer exhibiting noise properties similar to a lipid membrane, albeit with better stability. Thus, by careful optimization, block copolymer membranes can emerge as a robust alternative for protein-pore based nano-biosensors