In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

Nanopore-based single-molecule analysis of biomolecules such as DNA and proteins is a subject of strong scientific and technological interest. In recent years, solid state nanopores have been demonstrated to possess a number of advantages over biological (e.g., ion channel protein) pores due to the relative ease of tuning the pore dimensions, pore geometry, and surface chemistry. However, solid state fabrication methods have been limited in their scalability, automation, and reproducibility. In this work, a wafer-scale fabrication method is first demonstrated for reproducibly fabricating large arrays of solid-state nanopores. The method couples the high-resolution processes of electron beam lithography (EBL) and atomic layer deposition (ALD). Arrays of nanopores (825 per wafer) are successfully fabricated across a series of 4' wafers, with tunable pore sizes from 50 nm to sub-20 nm. The nanopores are fabricated in silicon nitride films with thicknesses varying from 10 nm to 50 nm. ALD of aluminum oxide is used to tune the nanopore size in the above range. By careful optimization of all the processing steps, a device survival rate of 96% is achieved on a wafer with 50 nm silicon nitride films on 60- 80 micron windows. Furthermore, a significant device survival rate of 88% was obtained for 20 nm silicon nitride films on order 100 micron windows. In order to develop a deeper understanding of nanopore fabrication-structure relationships, a modeling study was conducted to examine the physics of EBL, in particular: to investigate the effects of beam blur, dose, shot pattern, and secondary electrons on internal pore structure. Under the operating conditions used in pore production, the pores were expected to taper to a substantially smaller size than their apparent size in SEM. This finding was supported by preliminary conductance readings from nanopores.

Alpha-synuclein (AS) is a natively unfolded protein whose structure is extremely sensitive to its environment. The hallmark of Parkinson’s disease (PD) is aggregation and deposition of AS in inclusion bodies. Formation of misfolded AS monomers which are partially folded is the first and critical stage in fibrillation of AS and is a good target for designing therapeutic strategies. Characterization the biochemical properties of partially folded intermediates induced by fibrillization and anti- fibrillization agents will help to design drugs as new inhibitors of AS misfolding and aggregation. Nanopore analysis is an emerging technique for studying the molecular mechanism of protein misfolding. This technique was used to characterize the conformational change of AS in the presence of two groups of chemicals; anti-parkinsonian small molecules (dopamine and nicotine) and Parkinson’s developing toxin (Cu(II) and methamphetamine). Other biophysical techniques such as NMR spectroscopy and isothermal titration calorimentry (ITC) were able to confirm the nanopore analysis results and also to study other biophysical properties of the partially folded intermediates such as the binding constant of the interaction and the secondary structure content. The results from nanopore analysis showed that both groups of ligands shifted the blockade current peak of AS (centered at -86 pA) to lower blockade currents but in a different manner. Anti-parkinsonian drugs shifted the blockade current of AS to intermediate peaks between -40 to -80 pA but Parkinson developing toxins shifted the peak to a lower blockade current centered at -25 pA which suggests a more compact conformation. Thus nanopore analysis distinguished the different conformation induced by different ligands. Furthermore nanopore analysis with AS fragments showed that these ligands bind to different regions of AS. NMR spectroscopy of AS in the presence of dopamine and nicotine isomers was in agreement with the nanopore analysis and showed conformational changes of AS in a concentration dependent manner. CD spectroscopy results showed that the secondary structure of AS alone and in the presence of ligands was mostly random coil and suggests a loop formation model for the interaction of ligands with AS. The results of this thesis showed the application of nanopore analysis as a real-time and label-free technique to screen a library of ligands for designing misfolding inhibitors for PD treatment. The result of a synergic experiment with nicotine and caffeine showed that combination of these anti-parkinsonian small molecules would be a promising new drug for treatment of PD.

Nanopores (1 – 10 nm diameter) constructed in solid-state membranes, have shown promise as next-generation biopolymer analysis devices offering both high resolution and high throughput. One promising application of nanopores is in the analysis of nucleic acids, such as DNA. This involves translocation experiments in which DNA is placed in an ionic solution and is forced through a nanopore with the aid of an applied electric field. The modulation of ionic current through the pore during DNA translocation can then be correlated to various properties of the biopolymer such as the length. To optimally design and operate nanopore devices, it would be advantageous to develop an accurate computer simulation methodology to predict the physics of the translocation process. Hence, I have developed a physically accurate, computationally efficient simulation methodology to predict and analyze the physics of biopolymer translocation through solid-state (silicon nitride) nanopores. The overall theme of this thesis is to use this simulation methodology to thoroughly investigate important issues in the physics underlying translocation experiments and thereby determine the effects of key structural and operation parameters, such as nanopore dimensions, applied voltage, hydrodynamic interactions, solvent viscosity, and the polymer chain length. The results from these simulation studies can assist in not only proper nanopore design, but also help determine the proper experimental environments and parameters for nanopore operation.

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

abstract: Nanofluidic devices in which one single-walled carbon nanotube (SWCNT) spans a barrier between two fluid reservoirs were constructed, enabling direct electrical measurement of the transport of ions and molecules. Ion current through these devices is about 2 orders of magnitude larger than that predicted from the bulk resistivity of the electrolyte. Electroosmosis drives excess current, carried by cations, and is found to be the origin of giant ionic current through SWCNT as shown by building an ionic field-effect transistor with a gate electrode embedded in the fluid barrier. Wetting of inside of the semi-conducting SWCNT by water showed the change of its electronic property, turning the electronic SWCNT field-effect transistor to "on" state. These findings provide a new method to investigate and control the ion and molecule behavior at nanoscale. / Dissertation/Thesis / Ph.D. Physics 2011

Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible. Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.

The 21st century marks the defining point of human history in terms of technological advancement. In 2014, we were at the edge of acquiring a complete understanding of the fundamental construct to all life forms. The capability to manipulate and recreate lives as desired will soon be at our hands and will eventually lead to the redefinition of life and humanity. This brave new world, for better or worse, will be stitched together by scientific breakthroughs in many disciplines. Nanopore fluidic system – and microfluidic in general – might be one of the key puzzles towards the future. It is seen as a likely candidate for the next generation of rapid and low-cost genetic sequencing technology, which will allow us to gain thorough insight into the genetic code of every living organism on earth. It can also have the capability to individually detect and manipulate virtually any biological molecules, possibly allowing it to be a universal diagnostic tool or a bio-molecule synthesiser. The future of nanopore fluidic system is prosperous, but the difficulties are equally challenging. Currently, both biological and solid-state nanopores are non-trivial to create. For instance, a small solid-state nanopore can only be fabricated with expansive machinery in a low-yield, low-throughput manner. To overcome this challenge, a new set of methods involving high electric field to fabricate and enlarge a solid-state nanopore has been developed. It was found that a nanopore, when subjected to a high electric field, can be enlarged in angstrom increments and cleared of unidentified obstructions that cause low-frequency ionic current fluctuations. It was also found that an intact solid-state membrane, when subjected to a high electric field for a period of time, can leave a single nanopore imprinted onto it. The process of creation is best describe as a dielectric breakdown event and can be modeled by the percolation theory for dielectric breakdown. The resulting nanopores are cylindrical in shape and are shown to be equally capable of single molecule sensing compare to pores created by other methods. To accommodate future nanopore designs and applications and to examine the scope of applicability of the new fabrication approach, more advanced nanopore devices were created on some dual-layer solid-state membranes comprising of a metallic and a dielectric layer. Experiments indicated that the method could indeed create nanopore on such advanced membranes. It was further shown that the metallic layer receded further than the dielectric layer, forming a hollow conical shape at the opening of the dielectric nanopore. Such metalized bi-layer nanopore system was found to interact strongly with short single stranded DNA molecules, resulting in prolonged DNA translocation time. A simple picture of the mechanism was proposed to explain the observation. Lastly, to extend the limit of the new fabrication approach, I attempted to fabricate nanopore on complex multi-layer membranes involving a graphene film sandwiched in several dielectric materials. It was found that the quality of the graphene film and the transfer method were vital to the success of this project. Nevertheless, preliminary results indicated that the new method could create a nanopore through this complex multi-layer membrane. The new method to fabricate and tune both simple and complex nanopores is amongst the simplest, the least costly and the most efficient one that one can imagine. The research work has already sparked a dramatic increase in scientific throughput in our laboratory and other laboratories we had collaboration with. It fueled more than a dozen projects and involved close to a thousand nanopores in total. Such projects are far from possible if they were to rely on conventional fabrication methods. However, these are insignificant if we consider the new method is simple enough that, for the very first time, general public can easily access nanofabrication and single-molecule manipulation technology. The liberation of nanotechnology to the general public symbolically marks the beginning of a brave new world.

Monolayer molybdenum disulfide (MoS2) is a three-atom-thick direct band gap semiconductor, which has received considerable attention for use as a channel material in atomically thin transistors, photodetectors, excitonic LED’s, and many other potential applications. It is also a mechanically exceptional material with a large stiffness and flexibility, and can withstand very large strains (11%) before rupture. In this dissertation we investigated the mechanics of the stiffness and adhesion forces in atomically thin MoS2 membranes, and how biaxial strains can be used to induce large modulations in the band structure of the material. First, we used chemical vapor deposition (CVD) to grow MoS2 crystals that are highly impermeable to gas, and used a pressure difference across suspended membranes to induce large biaxial strains. We demonstrated the continuous and reversible tuning of the optical band gap of suspended monolayer membranes by as much as 500 meV, and induced strains of as much as 5.6% before rupture. We observed the effect of strain on the energy and intensity of the peaks in the photoluminescence (PL) and Raman spectra and found their linear strain tuning rates, then report evidence for the strain tuning of higher level optical transitions. Second, we determined the Young’s modulus and works of separation and adhesion of MoS2 membranes, and found that adhesion hysteresis is an important effect in determining the behavior of our systems. Finally, we investigated the use of atomically thin materials as nanofiltration membranes, by perforating the material with nanopores which selectively permit the transport of smaller molecules while rejecting larger ones. We studied ion transport through nanopores in graphene membranes and demonstrate that in-situ atomic force microscope measurements in liquid are a powerful way to reveal occlusions and contaminants around the pores - work which will aid future researchers in further unveiling the properties of these fascinating systems.

Solid-state nanopore sensors are an emerging platform for performing single-molecule characterization of biomolecules such as DNA. With the advent of Controlled Breakdown (CBD), creating a simple, tunable, ultra-low concentration sensing device in situ has enabled their direct integration with a host of platforms. The simplicity and sensitivity in performing measurements allows nanopore-based technologies to find uses which enhance existing methods. One such promising avenue for nanopore-based sensing is in the detection of infectious diseases, where early and accurate identification of the causative pathogen is essential for successful patient outcomes. Conventional assays, while effective, often have limitations in speed, cost, or target sensitivity, and may benefit from nanopore sensing approaches. However, solid-state nanopores currently lack the ability to discriminate between biomarkers sharing identical size and charge densities, such as sequentially-heterogeneous strands of DNA. Addressing the weakness of both conventional assays and nanopores could come from combining nanopore sensing with the polymerase chain reaction (PCR), a well-established and highly-selective nucleic acid amplification scheme. PCR is designed to produce large quantities of identical fragments of DNA, known as amplicons, if a user-defined parent copy is present. After the PCR process has finished, the signals produced by this population of amplicons on a nanopore sensor should therefore be indicative to the presence of a DNA biomarker in the starting sample. As PCR reactions can use a mix of different proteins, detergents, and other molecules, the challenge lies in ensuring the compatibility of these reagents with a nanopore, and determining whether the background signal they produce can be discriminated from an amplicon signal. To this end, this thesis investigates PCR-nanopore compatibility, and experimentally demonstrates a nanopore signal-classification technique to successfully identify the presence of chromosomal DNA from Group A Streptococcus (GAS), an infectious bacterium responsible for strep throat, in samples derived from clinical extracts.