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

Protein sequencing strategy in nanotechnology by classical and quantum atomistic models

Il mio lavoro di ricerca ha avuto l’obiettivo di studiare le proprietà principali dell’interazione tra materiale biologico e superfici inorganiche. Per tale scopo è stato utilizzato uno approccio basato sulla teoria del funzionale densità (DFT), lo studio è stato svolto nell’ambito del calcolo ad alte prestazioni utilizzando un approccio quanto-meccanico da principi primi, in modo da poter descrivere al meglio le interazioni chimico-fisiche a livello molecolare e sub-molecolare. Il tutto è stato applicato in dispositivi di ultima generazione per sequenziamento di catene biologiche, basate sulla tecnologia a nano-poro; in questi sensori nano-strutturati vi è un analisi detta a singola-molecola, il tipo e i modi d’interazioni tra dispositivo e target d’analizzare sono fondamentali e determinano la variazione del nostro segnale in uscita. Il funzionamento è relativamente semplice: si applica agli estremi della superfice con il poro una differenza di potenziale, e si misura la variazione della corrente quando il foro è occupato; le dimensioni del poro fanno si che si possa analizzare una molecola alla volta. Il materiale scelto per la realizzazione di questi dispositivi è stato il grafene per le sue proprietà elettroniche e la sua geometria; le catene biologiche scelte sono sequenze di amminoacidi; questa scelta si basa sulla possibile evoluzione di questi dispositivi, finora utilizzati per il sequenziamento di DNA (commercializzato dalla Oxford Nanopores Technologies), e sull’importanza dell’identificazione della sequenza e della struttura delle proteine, visto la connessione a patologie neurodegenerative come Parkinson e Alzheimer. Più in dettaglio il mio lavoro di ricerca è partito da studi precedenti dove venivano analizzate filamenti di DNA con la traslazione di basi nucleiche in nano-pori biologici per il sequenziamento; si sono studiati i cambiamenti caratteristici di corrente quando il target si avvicina alla superficie o attraversava il poro in modo da ottenere un'analisi rapida a singola-molecola. Si è provato, così, ad applicare lo stesso principio su sequenze di peptidi, per la loro importanza a livello medico-scientifico, con nanostrutture allo stato solido, visti i vantaggi di quest'ultimi rispetto a quelli biologici (miglior rapporto segnale rumore e una vita media più lunga). Sono state effettuate simulazioni Ab-Initio per caratterizzare sia le proprietà elettroniche superficiali, osservando la densità degli stati (DOS), e sia l'effetto quantistico del tunneling degli elettroni al variare della molecola interagente con la superficie. Per fare ciò si è studiata la corrente elettronica trasversale al piano del poro, su un ribbon di grafene, correlando le variazioni della nube elettronica con la molecola target. Per raggiungere questi obiettivi abbiamo: • definito modelli atomistici di interazione tra amminoacidi e bordi di un nano-poro di grafene; • utilizzato simulazioni atomistiche/molecolari per ottimizzare la morfologia (grandezza) e struttura (forma) più adeguata del poro; • studiato il funzionamento elettronico del nano-poro in fase di traslocazione degli amminoacidi attraverso esso. In particolare ci si è concentrati sul calcolo della conduttività trasversale attraverso la metodologia della Non-Equilibrium Green Function (NEGF) e l'approccio Landauer-Buttiker La ricerca è stata articolata in due fasi: I Fase: Design del nano-poro di grafene Nonostante diversi nano-pori siano già studiati con tecniche sperimentali, l’approccio teorico-modellistico basato su simulazioni molecolari atomistiche della struttura del nano-poro ha reso possibile avere una rigorosa caratterizzazione fisica e chimica del sistema; questa caratterizzazione è diventata la base per il successivo processo di ottimizzazione del dispositivo (passando da un nano-poro a un nano-gap). Dal punto di vista teorico-computazionale, si è confrontato il comportamento, strutturale del passaggio all’interno del sensore di diversi amminoacidi e si è progettato un nano-gap adatto alla valutazione degli effetti di traslocazione. II Fase: Caratterizzazione del segnale Si è studiata la variazione del “segnale” ottenuto, per caratterizzarlo al meglio e abbassare il rapporto segnale rumore. Attraverso varie analisi di post-processing si è andata a vedere la corrente elettronica elastica ed anelastica e si è aggiunta l’analisi della corrente ionica con simulazioni di dinamica molecolare classica

Real-time detection of dopamine -- aptamer interactions in a nanopore: a label-free toolkit for study of nucleic-acid-based molecular sensors.

Understanding how small molecules regulate nucleic acid structures is important in both biomechanism elucidation and biotechnological applications. Through the conformational variation, native nucleic acid motifs can be used as the targets to screen therapeutic compounds; In vitro selected aptamers can be used to detect small molecule biomarkers such as neurotransmitters and hormones, and ligand-triggered riboswitches can be designed to control gene expressions. All these applications need a rapid universal platform to detect nucleic acid conformational change in response to small molecule binding. Here we propose a label-free, non-invasive, and modular aptamer-inlaid nanopore capable of revealing time-resolved single nucleic acid molecule conformational transitions at the millisecond resolution. When a dopamine aptamer is docked in the MspA protein pore, the ion current through the pore can characteristically vary as the aptamer transitions between different conformations, recording a sequence of current fingerprints for binding and release of single neurotransmitter molecules from the aptamer. Without the need to mix the aptamer and the ligand, the sensor can quantify the target neurotransmitter, discriminate between different neurotransmitters, assay nucleic acid-ligand interactions, elucidate the ligand selectivity mechanism and pinpoint the ligand docking motifs in the aptamer, offering a potential nanopore toolbox for multiple small molecule biomarkers detection and screening nucleic acid-targeted small molecule regulators. Finally, we optimize the sensitivity of the nanopore sensor by employing divalent ions.Includes bibliographical references

Ion conduction characteristics in small diameter carbon nanotubes and their similarities to biological nanochannels

In this study, we designed a series of experiments to determine the factors governing ion permeation through individual carbon nanotubes (CNTs) less than 1.5 nm in diameter and 20 µm in length. We then rationalize the experimental results by using a model, which is drawn from previous literature on protein ion channels and is centered around a simplified version of the Gouy-Chapman theory of electrical double layer. Lastly, we experimentally demonstrate and discuss the general similarities in ion permeation characteristics between CNTs and biological ion-selective pores. The role of many potential factors influencing the ion transport is assessed by taking two experimental approaches: (1) studying the effect of electrolyte concentration and composition on channel conductance and reversal potential, and (2) examining a second type of nanochannel as a parallel ion conduction pathway within the same device architecture and measurement set-up, which we refer to as leakage devices. This helps to differentiate the effect of CNT on ionic transport from any other possible source. Taken together, these two experimental methods provide strong evidence that the electrostatic potential arising from ionized carboxyl groups at the nanopore entrance has a significant effect on ionic permeation in a manner consistent with a simple electrostatic mechanism

Physics of Ionic Conduction in Narrow Biological and Artificial Channels

The book reprints a set of important scientific papers applying physics and mathematics to address the problem of selective ionic conduction in narrow water-filled channels and pores. It is a long-standing problem, and an extremely important one. Life in all its forms depends on ion channels and, furthermore, the technological applications of artificial ion channels are already widespread and growing rapidly. They include desalination, DNA sequencing, energy harvesting, molecular sensors, fuel cells, batteries, personalised medicine, and drug design. Further applications are to be anticipated.The book will be helpful to researchers and technologists already working in the area, or planning to enter it. It gives detailed descriptions of a diversity of modern approaches, and shows how they can be particularly effective and mutually reinforcing when used together. It not only provides a snapshot of current cutting-edge scientific activity in the area, but also offers indications of how the subject is likely to evolve in the future

Electrokinetic Transport, Trapping, and Sensing in Integrated Micro- and Nanofluidic Devices

Thesis (Ph.D.) - Indiana University, Chemistry, 2009Microfluidics is rapidly becoming a mature field, and improved fabrication methods now routinely produce sub-micrometer features. As device dimensions shrink, physical phenomena that are negligible at larger length scales become more important, and by integrating nanofluidic elements with microchannels, new analytical techniques can be developed based on the unique behavior of matter at the nanoscale. This work addresses the fabrication, operation, and application of in-plane nanochannels and out-of-plane nanopores in lab-on-a-chip devices. In planar nanofluidic devices, we demonstrate a method to produce micro- and nanoscale features simultaneously with a single UV exposure step and evaluate flow control and sample dispensing with nanofluidic cross structures. Modification of the pinched injection method makes it applicable to variable-volume, attoliter-scale injections, including the smallest volume electrokinetically-controlled injections to date. As an alternative approach, track-etch nanopore membranes are explored as out-of-plane nanofluidic components. The random distribution of pores in these membranes is overcome by lithographic and microchannel-based methods to isolate and address specific pores. Microfluidic isolation improves mass transport to the pore(s), provides easy coupling of electrical potentials, and facilitates additional sample processing steps up- and downstream. These integrated microchannel-nanopore devices are used for diffusion-based dispensing, electrokinetic trapping, and resistive pulse sensing. In a high pore density device, diffusion-based dispensing establishes a stable chemical gradient for bacterial chemotaxis assays. For lower pore density devices, the nanopores are the most resistive components in the fluidic circuit, and application of an electric potential produces localized regions of high electric field strength and field gradient. These high field regions are applied to electrokinetic trapping of particles and cells in multiple-pore devices and to single particle detection by resistive pulse sensing in devices with a single isolated pore. To better understand factors influencing ion current in single nanoscale conduits, we systematically examine ion current rectification as a function of pore diameter, ionic strength, and pH to improve understanding of ion current through nanopores and to characterize preferred operating parameters for sensing applications. These results are applied to detection of virus capsids, and future work is proposed to investigate capsid assembly

Single-molecule DNA detection in nanopipettes using high-speed measurements and surface modifications

Inspired by transmembrane pores found in cell membranes and the operating principle of the Coulter counter used for cell counting, nanopore biosensors have emerged as a tool for single-molecule detection. This thesis describes single-molecule DNA detection through resistive pulse sensing using nanopipettes, a novel subclass of solid-state nanopores. In the first part of this thesis, double-stranded (ds) DNA-nanopipette surface interactions were probed in 1 M KCl electrolyte using DNA molecules with lengths ranging from 48.5 to 4 kilobase pair (kbp). A custom-built current amplifier was employed for low-noise and high-bandwidth measurements. Results from these experiments were used to theoretically rationalise DNA-surface interactions and suggest that dsDNA adsorption to the nanopipette surface prior to translocation through the pore is likely to be an important factor in the process. Subsequently, initial investigations to probe DNA-surface interactions were carried out by modifying the surface charge of nanopipettes using silanes. Additionally, experiments were performed to detect shorter dsDNA lengths. In 1 M KCl electrolyte, 200 base pair (bp) long dsDNA was successfully detected using the low-noise and high-bandwidth current amplifier. However detection of 100 bp long dsDNA required the use of 2 or 4 M LiCl electrolyte. Attention was finally shifted to the detection of 100 bp dsDNA in 1 M KCl electrolyte using functionalised lipid bilayer coated nanopipettes. Additional techniques were employed to prepare and characterise the lipid bilayers, including atomic force microscopy (AFM) and dynamic light scattering (DLS). The promising preliminary results provide a framework for further experiments using functionalised lipid bilayers to coat nanopipettes. Overall, results of the aforementioned research presented in this thesis demonstrate high-speed single-molecule detection of DNA and provide novel insights into the translocation dynamics of DNA molecules in nanopipettes and the sensing capabilities of nanopipettes.Open Acces

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

Computational modeling of biological nanopores

Throughout our history, we, humans, have sought to better control and understand our environment. To this end, we have extended our natural senses with a host of sensors-tools that enable us to detect both the very large, such as the merging of two black holes at a distance of 1.3 billion light-years from Earth, and the very small, such as the identification of individual viral particles from a complex mixture. This dissertation is devoted to studying the physical mechanisms that govern a tiny, yet highly versatile sensor: the biological nanopore. Biological nanopores are protein molecules that form nanometer-sized apertures in lipid membranes. When an individual molecule passes through this aperture (i.e., "translocates"), the temporary disturbance of the ionic current caused by its passage reveals valuable information on its identity and properties. Despite this seemingly straightforward sensing principle, the complexity of the interactions between the nanopore and the translocating molecule implies that it is often very challenging to unambiguously link the changes in the ionic current with the precise physical phenomena that cause them. It is here that the computational methods employed in this dissertation have the potential to shine, as they are capable of modeling nearly all aspects of the sensing process with near atomistic precision. Beyond familiarizing the reader with the concepts and state-of-the-art of the nanopore field, the primary goals of this dissertation are fourfold: (1) Develop methodologies for accurate modeling of biological nanopores; (2) Investigate the equilibrium electrostatics of biological nanopores; (3) Elucidate the trapping behavior of a protein inside a biological nanopore; and (4) Mapping the transport properties of a biological nanopore. In the first results chapter of this thesis (Chapter 3), we used 3D equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at https://github.com/willemsk/phdthesis-tex