Improving the Signal-to-Noise of Nanopore Sensors

Over the last five years, solid state nanopore technology advanced to rival biological pores as a platform for next generation DNA sequencing. Fabrication improvements led to a reduction in nanopore diameter and membrane thickness, offering high precision sensing. Custom electronics were developed concomitant with low capacitance membranes for low-noise, high-bandwidth measurements. These advances improved our ability to detect small differences between translocating molecules and to measure short molecules translocating at high speeds. This work focuses specifically on the challenge of maximizing the signal magnitude generated by the solid state nanopore. One way that this can be achieved is by thinning the membrane. We prove that it is possible to differentiate between DNA homopolymers by using nanopores with \u3c 6 nm thickness and \u3c 2 nm diameter. The results imply that solid state nanopores offer higher signal-to-noise than what is currently achieved with biological pores. Attempts to reduce membrane thickness further by making nanopores in 2D materials proved to be limited by wetting and noise considerations. Instead, we developed an electron-irradiation-based thinning technique to thin Si-based films to the limit of their stability in order to determine the intrinsic limit of their detection capabilities. At these small thicknesses, we discovered unexpected blocked current structure in the translocation events, which we hypothesize to be related to the DNA molecule blocking current flow before entering the nanopore. Then we outline an alternative technique for high signal-to-noise single-molecule measurement by using a nanopore to localize the molecule near a charge sensor. The design of such a device required the development of a technique to make nanopores without damaging the sensor. Results from measurements of these devices in solution are reported, along with discussion of methods for improving the sensitivity. In the last section we report on somewhat unrelated experiments that involve imaging charge flow through structured quantum dot films. We use a combination of AFM, EFM, and TEM to map the topography, charge flow, and structural features in high resolution. We show that charge flow patterns can be clearly correlated with structural details in the film

Development of Polymer-Based In-Plane Nanopore for DNA Sequencing

Mechanically robust solid-state nanopores have the potential to be the next generation DNA sensing platforms. However, mass production and limited base-calling accuracy are the hurdles for solid-state nanopore based DNA sensing. In order to solve these problems, a polymer dual-nanopore device fabricated via high throughput nanoimprint lithography (NIL) was proposed to sequence DNA by time-of-flight (ToF) measurement. As a proof of concept, this study presents mononucleotides discrimination via ToF measurement using polymer in-plane dual-nanopore device. First, fabrication of polymer in-plane nanopore with controllable dimensions was studied in consideration of experimental conditions and materials selection. Then, surface charge density effect on DNA translocation through in-plane nanopore was studied numerically and experimentally using fabricated nanopore devices on PEGDA, PMMA and COC. λ-DNA sensing was only observed in PEGDA device with a surface charge density lower than the threshold surface charge density predicted by COMSOL simulation. With demonstrated single molecule sensing ability, mononucleotides were introduced to PEGDA dual-nanopore with 500 nm flight tube and discriminated under various conditions. At pH 8.0, mononucleotides were driven by eletrophoretic motion and their ToF was in a decreasing order of dGMP \u3e dAMP \u3e dCMP \u3e dTMP. At pH 10.0, mononucleotides were driven by electroosmotic flow (EOF) due to a higher surface charge density on nanochannel walls and ToF was in the same order as pH 8.0 with an average identification accuracy of 55%. Dual-nanopore device with 1 μm flight tube was then used to improve the average identification accuracy to 75%. Finally, dGMP and dTMP in a mix solution were dicriminated by their ToF difference

Enhancing the temporal and spatial resolution of solid-state nanopore single-molecule sensors

Thesis (Ph.D.)--Boston UniversitySince the first report of single-molecule detection using the biological nanopore alpha-hemolysin in 1996, nanopores have grown substantially more versatile. The genetic and chemical modification of biological nanopores and the fabrication of synthetic nanopores in solid-state membranes have enabled detection of analytes ranging in size from single nucleotides to large protein complexes. Among the most promising applications of nanopores is single-molecule sequencing, which has the potential to become a routine part of medical care, is compatible with long read lengths, and can detect epigenetically modified bases. Yet in order to further develop nanopores as useful tools for basic research as well as commercial applications, their temporal and spatial limitations must be addressed. Free electrophoretic threading of nucleic acids through a nanopore allows for discrimination based on large features (e.g., molecular length), but is too fast to resolve smaller features (e.g., single nucleotide identity). The first aim of this research is to enhance the temporal resolution of nanopores by tuning their electrostatic interaction with translocating molecules via chemical modification of the nanopore surface. To this end, we designed and fabricated pH-sensitive chemically coated nanopores to slow the translocation of DNA molecules. A practical nanopore sensing device relies on taking measurements from many pores in parallel to provide sufficient robustness (through redundancy) and throughput. Optical detection facilitates parallel throughput, but requires coupling between an analyte feature and a fluorescence source. The second aim is to enhance nanopore spatial resolution via optical detection of chemically activated fluorescence signals associated with single nanopores under total internal reflection (TIR) illumination. We performed numerical simulations of the concentration field of donor molecules near a nanopore and showed that nanopores are theoretically capable of discriminating between features separated by ~ 1 nm or less, a distance that far exceeds the resolution offered by TIR illumination. Finally, we use fluorescence signals to detect unlabeled DNA translocation through spatially addressed nanopores. With this aim we experimentally validate our theoretical predictions and demonstrate a novel highly parallel near-field chemo-optical detection scheme

Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA

The remarkable molecule that encodes genetic information for all life on earth—DNA—is a polymer with unusual physical properties. The mechanical and electrostatic properties of DNA are utilized extensively by cells in the replication, regular maintenance, and expression of their genetic material. This can be illustrated by considering the journey of a typical gene regulating protein, the lac repressor, which recognizes a particular gene and prevents its expression. First, the large electrostatic charge density of DNA provides an energetic track that guides the repressor’s search for its target binding site. Next, as the protein moves along the DNA, it attempts to deform the DNA. The repressor is only able to form an active complex with DNA that has the right sequence-dependent flexibility. Finally, the repressor is believed to form a very small DNA loop that prevents the gene from being expressed. The stability of the loop can be expected to depend sensitively on the global flexibility of DNA. Thus, the key to understanding the some of the most important cellular processes lies in understanding the physical properties of DNA. Single-molecule experiments allow direct observation of the behavior of individual DNA molecules, but act on length and timescales that are often too large and fast to observe underlying DNA and DNA–protein dynamics. Acting on length and timescales that complement single-molecule experiments, molecular dynamics simulations provide a high-resolution glimpse into the mechanics of a biomolecular world. Here, several simulation studies are presented, each of which quantified one or more properties of DNA. Specifically, the repulsive forces between parallel duplex DNA molecules were measured; the short-ranged, attractive end-to-end stacking energy was obtained; a single-stranded DNA model was developed that reproduced experimental measurements of its extension upon applied force; and finally the nature of single-stranded DNA binding to a single-stranded DNA binding protein was investigated. These works represent important steps towards larger simulations of more biologically complete DNA–protein systems

Lysenin Channels as Single Molecule Nano-Sensors and Nano-Switches for Controlled Membrane Permeability

Pore-forming toxins secreted by various evolutionarily distant organisms are important components of their innate defense mechanisms. These toxins may kill the target cells by inserting un-regulated channels into the plasma membrane. Tampering with the otherwise well-controlled membrane permeability alters cell homeostasis by contributing to un-controlled dissipation of both chemical and electrical gradients, which is often an essential component of virulence mechanisms leading to cell death. However, the same ability to create nanoscopic conducting pathways, i.e. nanopores, has been exploited for creating powerful tools in nano-biotechnology. Single nano-channels reconstituted in artificial planar lipid membranes are extremely versatile sensors that are capable of detection, identification, and characterization of single molecules. In addition, the changes in permeability induced by pore-forming toxins reconstituted in artificial and natural lipid membrane systems are exploited for numerous biomedical, scientific, and bio-technological applications. Many of these applications, underlying principles, and limitations are briefly described in the introduction section of this dissertation. To overcome many of the restrictions presented by currently used pore-forming toxins, we propose to use lysenin channels for both stochastic sensing and for the achievement of controlled membrane permeability. Lysenin is a pore-forming toxin extracted from the earthworm E. foetida that inserts large nanopores in natural and artificial lipid membranes containing sphingomyelin. Chapter 2 of the presented work is focused on exploring the use of single lysenin nanopores for stochastic sensing of human angiotensin II, a short hormone peptide,which is highly relevant for the pathophysiology of cardiovascular diseases. Besides a traditional analysis of the interactions between lysenin channels and peptides, we succeeded to employ high sensitivity liquid chromatography – mass spectroscopy analyses to demonstrate the passage of un-altered peptide molecules through open lysenin channels. In Chapter 3 we exploit the unique regulatory mechanisms presented by lysenin channels to achieve controlled permeability over artificial and natural lipid membranes. We demonstrate that ATP molecules may reversibly regulate the macroscopic ionic conductance of lysenin channels inserted into planar lipid membranes. Lysenin reconstitution into spherical membranes (liposomes) enables a two-way control over membrane permeability by using multivalent metal cations capable of inducing reversible ligand-induced gating of the channels. Live cell analyses demonstrate that lysenin channels allow transport of non-permeant molecules in Jurkat leukemia and ATDC5 chondrogenic cells. In addition, extended control over membrane permeability is achieved by using chitosan molecules as irreversible blockers of the macroscopic conductance. Survival rate estimations indicate that the permeabilized cells maintain a satisfactory viability rate for further use. Therefore lysenin may be used for the controlled transport of ions and molecules in living systems

Modification and regulation of biomolecules in vitro and in silico

The focus in functional and dynamics studies of biomolecules, such as protein or DNA, has been very much on their own structures and energy landscapes. However, in real biological systems, biomolecules are usually modified or regulated by several external factors, including surface coating, hydration condition, and local environment. For example, by covalently coupling a Poly(ethylene glycol) (PEG) on a protein surface, the stability of the host protein could be largely enhanced. This method, called PEGylation, has been widely used in pharmaceutical industry to protect protein drugs and increase their circulation life-time since the 1990s. However, the mechanism of protein-PEG interaction is yet to be understood. On the other hand, in developing one of the advanced DNA sequencing techniques --- nanopore sequencing, different mechanisms have been introduced to the nanopore system to regulate the conformation and motion of DNA molecules, attempting to achieve a better signal-to-noise ratio in reading DNA sequence. This dissertation aims to study the above two topics from both experiments and molecular dynamics simulations. The first part focuses on developing nanopore sequencing techniques. We have developed a method combining continuum modeling results of a nano-scale system and a coarse-grained DNA model to study the DNA translocation through a nanopore in a device scale. We use this method to develop advanced DNA sequencing techniques, including plasmonic nanopore and double nanopores. Both of them show great potential as novel approaches for DNA sequencing. The protein-PEG interaction is addressed in the second part. We show that the conjugated PEG affects the thermodynamic stability and local structure of the host protein WW domain, but not that of Lambda repressor. A reoccurring and cooperative folding of a PEG molecule onto the protein surface is revealed by molecular dynamics simulations. Specific PEG-binding motifs on the protein surface are identified

Kinetics and dynamics of electrophoretic translocation of polyelectrolytes through nanopores

The idea of sequencing a DNA based on single-file translocation of the DNA through nanopores under the action of an electric field has received much attention over the past two decades due to the societal need for low cost and high-throughput sequencing. However, due to the high speed of translocation, interrogating individual bases with an acceptable signal to noise ratio as they traverse the pore has been a major problem. Experimental facts on this phenomenon are rich and the associated phenomenology is yet to be fully understood. This thesis focuses on understanding the underlying principles of polymer translocation, with an emphasis on pore-polymer interactions, polymer architecture, and polymer chain fluctuations. Langevin dynamics simulations are used to study a variety of polymer and pore designs. For a uniformly charged linear polymer, a nanopore with charge patterns along its length is proposed. Variation in the charge pattern length reveals the existence of a critical length at which the polymer is trapped, causing a significant delay during the pore emptying stage. This trapping is modeled using an appropriate free energy landscape and the Fokker-Planck formalism. The predictions of this theory are in qualitative agreement with the simulation results across different pore and polymer lengths. Moreover, a linear polymer with charge patterns along its backbone passing through such a charge-patterned pore shows rich kinetic behavior; a significant delay is introduced even in the pore entrance and threading stages due to pattern matching, suggesting the use of pore-polymer interactions to slow down translocation. In a related study, the translocation of charged star polymers through an uncharged pore is simulated. Star polymers with different functionalities show rich translocation kinetics while passing through such a pore. The mean translocation time varies non-monotonically with the polymer functionality, suggesting the use of nanopores as a filtering and analytical technique for star polymers. Recent experiments have suggested the use of phi29 polymerase in conjunction with a protein pore (α-Hemolysin) in the presence of an electric field to slow down the polymer translocation speed, enabling reasonably successful base-calling. The role of polymer chain fluctuations inside the nanopore is evaluated using Langevin dynamics simulations on models of this construct. By monitoring the contributions of the conformational fluctuations of the polymer, the diffusional behavior of monomers of the chain under the speed resulting from the polymerase activity and externally imposed voltage gradients is computed. The simulations show that even if the translocation speed is slowed down considerably by using the polymerase-nanopore construct, the conformational fluctuations of ssDNA inside the pore are always present at high levels, resulting in high levels of noise in the detection signal