Characterization of the RNAP Binding Sites on a λ DNA using a Solid State Nanopore Combined with a Tuning Fork Force Sensing Probe Tip

In this dissertation, the binding positions of RNAP holoenzyme on a λ DNA are characterized using an apparatus that integrates a Solid State Nanopore with a Tuning Fork based Force sensing probe (SSN-TFFSP). The SSN-TFFSP system combines the measurement of ionic current through a solid-state nanopore with a DNA tethered probe tip. The position of the tip is sensed by a tuning fork force sensor and is controlled with a nanopositioning system. With this apparatus, translocation speed of DNA through solid state nanopores has been brought down to 100 μs/base. Such a controlled movement of DNA through a solid state nanopore can provide enough temporal resolution to determine the individual binding site of a RNAP on a λ DNA. Three signals measured simultaneously from this apparatus were: ionic current through a nanopore, tip position, and tip vibrational amplitude. These signals were measured when the probe tip was approaching towards the nanopore and was being lifted away from the pore. The λ DNA+ RNAP complex tethered to the probe tip can be captured by the electric field near a nanopore. The nanopore current signal measured during the capture of RNAP bound λ DNA provides new insights to the dynamics of λ DNA+RNAPcomplex molecules inside the nanopore. The binding positions of RNAP on a λ DNA are measured directly from the tip position signal corresponding to the distinct current drop within λ DNA current blockage level. The resolution limit of this apparatus is estimated to be 100 nm or 300 bp for RNAP binding sites. The resolution limit was further compared with the free translocation data set of λ DNA+RNAPcomplex through the solid state nanopore

Doctor of Philosophy

dissertationThis dissertation presents experimental and computational investigations of nanoparticle transport and ion current rectification in conical-shaped glass nanopore membranes (GNMs). Chapter 1 provides an overview of the Coulter counter or "resistive pulse" method, ion current rectification, and finite-element simulations used in solving mass transfer problems in conical-shaped nanopores. Chapter 2 describes a fundamental study of the electrophoretic translocation of charged polystyrene nanoparticles in conical-shaped pores contained within glass membranes using the Coulter counter principle, in which the time-dependent current is recorded as the nanoparticle is driven across the membrane. Particle translocation through the conical-shaped nanopore results in a direction-dependent and asymmetric triangularshaped resistive pulse. The simulation and xperimental results indicate that nanoparticle size can be differentiated based on pulse height. Chapter 3 presents experimental, theoretical, and finite-element simulation investigations of the pressure-driven translocation of nanoparticles across a conicalshaped GNM. Analytical theory and finite-element simulation for pressure-driven flow through a conical-shaped pore were developed to compute the volumetric flow rate, the position-dependent particle velocity, and the particle translocation frequency. The translocation frequencies computed from theory and simulation were found to be in agreement with experimental observations. Chapter 4 reports the pressure-dependent ion current rectification that occurs in conical-shaped glass nanopores in low ionic strength solutions. Because the pressureinduced flow rate is proportional to the third power of the nanopore orifice radius, the pressure-driven flow can eliminate rectification in nanopores with radii of ?200 nm but has a negligible influence on rectification in a nanopore with a radius of ?30 nm. The dependence of the i-V response on pressure is due to the dependence of cation and anion distributions on convective flow within the nanopore. Chapter 5 describes pressure-reversal methods to capture and release individual nanoparticles. One (or more) particle is driven through the orifice of a conical-shaped nanopore by pressure-induced flow. A reverse of flow, following the initial translocation, drives the particle back through the nanopore orifice in the opposite direction. The sequence of particle translocations in the capture step is preserved and can be read out in the release step. The observed instantaneous transfer rate and return probability are in good agreement with finite-element simulations of particle convection and diffusion in the confined geometry of the nanopore

Engineered nanofluidic platforms for single molecule detection, analysis and manipulation

Since the pioneering studies on single ion-channel recordings in 1976, single molecule methods have evolved into powerful tools capable of probing biological systems with unprecedented detail. In this work, we build on the versatility of a type of nanofluidic devices, called nanopipettes, to explore novel modes of single molecule detection and manipulation with the aim of improving spatial and temporal control of biomolecules. In particular, a novel nanopore configuration is presented, where biomolecules were individually confined into a zeptoliter volume bridging two adjacent nanopores at the tip of a nanopipette. As a result of this confinement, the transport of biomolecules such as DNA and proteins was slow down by nearly three orders of magnitude, leading to an improved sensitivity and superior signal-to-noise performances compared to conventional nanopore sensing. Active ways of controlling the transport of biomolecule by combining the advantages of nanopore single-molecule sensing and Field-Effect Transistors are also presented. These hybrid platforms were fabricated in a simple two step process which integrates a gold electrode at the apex of a nanopipette. We show that these devices were effective in modulating the charge density of the nanopore and in actively switching "on" and "off" the transport of DNA through the nanopore. Finally, a nanoscale dielectrophoretic nanotweezer device has been developed for high resolution manipulation and interrogation of individual entities. Two closely spaced carbon nanoelectrodes were embedded at the apex of a nanopipette. Voltage and frequency applied to the electrodes generated a highly localized force capable of trapping and manipulating a broad range of biomolecules. These dielectrophoretic nanotweezers were suitable for probing complex biological environments and a new technique for minimally invasive single-cell nanobiopsy was established. Such study provides encouraging results on how nanopipettebased platforms can be integrated as a future tool for routinely interrogating molecules at the nanoscale.Open Acces

Selective single molecule sensing in nanopore

Nanopores have emerged as one of the powerful tool for single molecule detection as it offers advantages such as label free and minimal sample preparation; this stimulates many exciting applications in biophysics and molecular sensing. The sensing principle can be achieved by electrophoretically driving biomolecules in solution through a nanometer pore. The ability to detect and measure abundance of many proteins precisely and simultaneously is an important and predictive biological research. To date, quantification of multiple proteins in buffer or purified samples have been successfully demonstrated in ELISA or tagged with fluorescence probes to detect specific molecules however, they rely on sophisticated and expensive diagnostics platform, as well as long assay time which hinder the process of simple and parallel screening multiple proteins at the single molecule level. Perhaps the biggest challenge for nanopore protein detection is the lack of selectivity, making it challenging to differentiate between multiple analytes; let alone trying to obtain meaningful data from complex biological samples such as human serum. Therefore, there is a need to develop strategies whereby such detection modalities can be used with unprocessed biological samples where thousands of different background proteins exists, often at much higher concentrations than the target analyte making detection exceptionally challenging. We present in this thesis some novel strategies that can improve the selectivity of the nanopore, allowing efficient detection and analysis of biomolecules in solutions accurately. Specifically, detection of multiple proteins via the use of aptamers attached onto a DNA carrier and in particular utility in detecting in biological sample in a low costs, scalable and sensitive manner. We were able to detect multiplex proteins, differentiate different protein size as well as accurately locate the proteins bound to the carrier without the need for extensive sample preparation and amplification, allowing direction sensing of proteins in unmodified samples. This thesis also introduces a process where having a control over the pore dimensions to ensure the dimensions or probe molecules match the pore size allowing good signal to noise ratio and enhancement in sensing ability. We show that Al2O3 atomic layer deposition (ALD) modified nanopipettes is capable to reduce the pore diameter down to 7.5 nm while allowing batch production of reproducible pipettes. Importantly, the sensing abilities were not affected by the ALD deposition. The other strategies demonstrate precise opening of the nanopore by electroetching the graphene nanoflakes (GNFs) that coated the nanopipette. The pore opening process enable in situ nanopore size control while performing DNA translocation, broadly functioning the nanopore devices. Overall the combined findings from the above strategies provided an incredible insight on the sensing and molecular biophysics of proteins and DNA at the single molecule level. The use of aptamer modified carrier increases the sensitivity and selectivity of the nanopore platform and enable potential applications for sensing of multiple proteins biomarker with single molecule sensitivity.Open Acces

Nanopores with Fluid Walls for Characterizing Proteins and Peptides.

Nanopore-based, resistive-pulse sensing is a simple single-molecule technique, is label free, and employs basic electronic recording equipment. This technique shows promise for rapid, multi-parameter characterization of single proteins; however, it is limited by transit times of proteins through a nanopore that are too fast to be resolved, non-specific interactions of proteins with the nanopore walls, and poor specificity of nanopores for particular proteins. This dissertation introduces the concept of nanopores with fluid walls and their applications in sensing and characterization of proteins, disease-relevant aggregates of amyloid-beta peptides, and activity of membrane-active enzymes. Inspired by lipid-coated nanostructures found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to a nanopore such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision, well-defined control of protein transit times, simultaneous multi-parameter characterization of proteins, and an ability to monitor phospholipase D. Using these bilayer-coated nanopores with lipids presenting a ligand, proteins binding to the ligand were captured, concentrated on the surface, and selectively transported to the nanopore, thereby, conferring specificity to a nanopore. These assays enabled the first combined determination of a protein’s volume, shape, charge, and affinity for the ligand using a single molecule technique. For non-spherical proteins, the dipole moment and rotational diffusion coefficient could be determined from a single protein. Additionally, the fluid, biomimetic surface of a bilayer-coated nanopore was non-fouling and enabled characterization of Alzheimer’s disease-related amyloid-beta aggregates. The presented method and analysis fulfills a previously unmet need in the amyloid research field: a method capable of determining the size distributions and concentrations of amyloid-beta aggregates in solution. The experiments presented here demonstrate that the concept of a nanopore with fluid walls enables new nanopore-based assays. In particular, it demonstrates the benefits of this concept for simultaneous, multi-parameter characterization of proteins with a single-molecule method; this technique may, therefore, be well-suited for identification of proteins directly in complex biological fluids. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single-molecule assays and protein characterization.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96049/1/ecyus_1.pd

Metallic Nanopores for Single Molecule Biosensing

This thesis describes a novel approach to the fabrication and characterisation of metallic nanopores and their application for the detection of single DNA molecules. Metallic nanopores with apparent diameters below 20 nm are produced using electrochemical deposition and real-time ionic current feedback. Beginning with large nanopores (diameter 100-200 nm) milled into gold silicon nitride membranes using a focused ion beam, platinum metal is electrodeposited onto the gold surface, thus reducing the effective pore diameter. By simultaneously observing the ion current feedback, the shrinking of the nanopore can be monitored and terminated at any pre-defined value of the pore conductance in a precisely controlled and reproducible way. The ion transport properties of the metallic nanopore system are investigated by characterising the pore conductance at varying potentials across the nanopore and concentrations of electrolyte. The results are compared to conventional bare silicon nitride nanopore systems. Chemical modification at the nanopore surface is also studied using thiolisation to reduce the capacitive charging effects observed with metallic nanopores. Further to this, impedance measurements are carried out to study the resistive behaviour exhibited in these systems. An equivalent circuit model is proposed to validate the results obtained from the experimental studies. To evaluate the suitability of these nanopores for applications in single-molecule biosensing, translocation experiments using λ-DNA are performed. DNA molecules are electrokinetically driven through the nanopore under an applied electric field, hence as the DNA translocates through the pore, current blockade events are detected. Each event is the result of a single molecular interaction of DNA with the nanopore and is characterised by its dwell time and amplitude. Characterisation studies and noise analysis towards the applicability of metallic nanopores as single molecule detectors are also studied and compared to current bare silicon nitride pore systems

Fabrication and characterization of plasmonic nanopores for Raman detection of biomolecules

In the last two decades solid-state nanopores have been widely investigated for the development of efficient and functional sensing platforms that enable real-time identification of biomolecules and even sequencing. Besides the typical electrical readout, single solid-state nanopores have been integrated with plasmonic nanostructures to add additional sensing modalities by using optical techniques such as Surface-enhanced Raman Spectroscopy (SERS). However, these nanosensors often exhibit low throughput which limits their use for real biological applications. Here, a large area and low-cost approach to produce transferrable arrays of plasmonic nanopores is presented. The devices are characterized by optical measurements and tested for the detection of biomolecules by using SERS

Nanopore Analysis of Peptides and Proteins

Nanopore sensing is a single-molecule technique capable of detecting peptide and protein molecules by monitoring the change in current generated by their interaction with protein or solid-state nanopores in an applied electric field. The interaction of a small globular protein HPr and two of its mutants, with the aerolysin nanopore were analyzed and compared with earlier results obtained with the α-hemolysin nanopore. The HPr molecules interact differently with the two nanopores while the anatomy and net charge of the pores affect their translocation parameters. Cleavage of insulin’s disulfide bonds with the reducing agent TCEP and the release of the component polypeptides could also be detected by nanopore analysis. An alternating current field superimposed on the direct current field inhibited the translocation of a peptide with a permanent dipole moment, while another peptide with no dipole moment was less affected. The detection of conformational changes in peptides and small proteins caused by metal ion binding also proved possible. A Zn-finger protein was able to translocate the α-hemolysin pore in the absence of Zn(II), while mostly bumping events were observed when Zn(II) was added. By comparison, the FSD-1 protein, which folds into a Zn-finger motif by hydrophobic interactions alone, was not able to translocate. The metal binding ability of three prion peptides was studied with an α-hemolysin pore. The results clearly indicated that Cu(II) and Zn(II) bound to all three peptides and caused conformational changes reflected in their interaction parameters with the α-hemolysin pore. The interaction of HPr, calmodulin and maltose binding protein with 7 nm and 5 nm diameter silicon nitride (SixNy) pores indicated that protein molecules with dimensions comparable to, or larger than the pore diameter do not translocate. However, smaller proteins are able to translocate in a folded conformation. Finally, the formation of prion/antibody complexes was successfully detected with an 11 nm SixNy pore but not with a 19 nm pore. The results underline the importance of choosing a pore with a suitable diameter in relation to the size of the analytes

Nanofluidic Pathways for Single Molecule Translocation and Sequencing -- Nanotubes and Nanopores

abstract: Driven by the curiosity for the secret of life, the effort on sequencing of DNAs and other large biopolymers has never been respited. Advanced from recent sequencing techniques, nanotube and nanopore based sequencing has been attracting much attention. This thesis focuses on the study of first and crucial compartment of the third generation sequencing technique, the capture and translocation of biopolymers, and discuss the advantages and obstacles of two different nanofluidic pathways, nanotubes and nanopores for single molecule capturing and translocation. Carbon nanotubes with its constrained structure, the frictionless inner wall and strong electroosmotic flow, are promising materials for linearly threading DNA and other biopolymers for sequencing. Solid state nanopore on the other hand, is a robust chemical, thermal and mechanical stable nanofluidic device, which has a high capturing rate and, to some extent, good controllable threading ability for DNA and other biomolecules. These two different but similar nanofluidic pathways both provide a good preparation of analyte molecules for the sequencing purpose. In addition, more and more research interests have move onto peptide chains and protein sensing. For proteome is better and more direct indicators for human health, peptide chains and protein sensing have a much wider range of applications on bio-medicine, disease early diagnoses, and etc. A universal peptide chain nanopore sensing technique with universal chemical modification of peptides is discussed in this thesis as well, which unifies the nanopore capturing process for vast varieties of peptides. Obstacles of these nanofluidic pathways are also discussed. In the end of this thesis, a proposal of integration of solid state nanopore and fixed-gap recognition tunneling sequencing technique for a more accurate DNA and peptide readout is discussed, together with some early study work, which gives a new direction for nanopore based sequencing.Dissertation/ThesisDoctoral Dissertation Physics 201

Probing chemical structures and physical processes with nanopores

This thesis develops and applies the nanopore tool to probe chemical structures and physical processes at the single-molecule level: from single ions to DNA molecules. Nanopore experiments electrically measure the ionic transport through the pore and its modulation from the local environment which can be caused by translocations of an analyte such as objects like DNA molecules or change of the physical conditions such as surface charge. Its precision relies on the physical dimension of the nanopore probe. In this thesis, the atom by atom engineering of single-layer molybdenum disulfide (MoS2) nanopores was achieved using transmission electron microscopy (TEM) or controlled electrochemical reaction (ECR), which further enabled the following investigations. On the translational side, the key driver of the application of nanopores is single molecule DNA sequencing. The sequence of DNA can be extracted based on the modulation of ionic current through the pore by individual nucleotides. To this end, we realized for the first time with solid-state nanopores, identification of all four types of nucleotides by introducing an ionic liquid based viscosity gradient system to control the translocation dynamics. This method provides a potential route for sequencing with solid-state nanopores. On the fundamental side, nanopore experiments could probe physics of single ion transport and with subnanometer pores, we discovered Coulomb blockade for the first time in ionic transport, as the counterpart of quantum dots, and proposed a new mesoscopic understanding for biological ion channel transport. From an engineering perspective, measurement with a single nanopore can avoid averaging over many pores and allow accurately identifying individual parameters for membrane-based processes. With single-layer MoS2 nanopores, we realized the first exploration of a two-dimensional (2D) membrane for osmotic power generation. This thesis demonstrates that nanoscopic, atomically thin pores allow for the exploration of applications in DNA sequencing and investigations of fundamental ion transport for biological ion channels and membrane-based processes