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

Development of optical microchip sensor for biomolecule detection

Optical sensors play vital roles in many applications in today’s world. Photonic technologies used to design and engineer optical sensing platforms can provide distinctive advantages over conventional detection techniques. For instance, when compared to electronic and magnetic sensing systems, optical sensors require physically smaller equipment and have the capability for delivering more analytical information (e.g. spectroscopic signatures). In addition, demand for low-cost and portable bio-analyte detections is a growing area for applications in healthcare and environmental fields. Among other factors to achieve reliable results in terms of selectivity and sensitivity is key for the detection of bio-analytes with analytical relevance. Commonly used bio-analytical techniques (e. g. high performance liquid chromatography) have been appropriately designed based on qualitative and quantitative analysis. However, the requirement of expensive equipment, and complexity of procedures (e.g. biomolecule labelling, calibrations, etc.) restrict the board applicability and growth of these techniques in the field of biosensing. Optical sensors tackle these problems because they enable selective and sensitive detection of analytes of interest with label-free, real-time, and cost-effective processes. Among them, optical interferometry is increasingly popular due label-free detection, simple optical platforms and low-cost design. An ideal substrate with high surface area as well as biological/chemical stability against degradation can enable the development of advanced analytical tools with broad applicability. Nanoporous anodic alumina has been recently envisaged as a powerful platform to develop label-free optical sensors in combination with different optical techniques. This thesis presents a high sensitive label-free biosensor design combining nanoporous anodic alumina (NAA) photonic structures and reflectometric interference spectroscopy (RIfS) for biomedical, food and agricultural applications. NAA is a suitable optical sensing platform due to its optical properties; a high surface area; its straightforward, scalable, and cost-competitive fabrication process, and its chemical and mechanical stability towards biological environments. Our biosensor enables real-time screening of any absorption and desorption event occurring inside the NAA pores. A proper selection of bio-analytes were able to be detected using this platform which offers unique feature in terms of simplicity and accuracy. The most relevant components of this thesis are categorised as below: 1. Self-ordered NAA fabrication and detection of an enzymatic analyte as a biomarker for cancer diagnosis: Fabrication of NAA photonic films using two step electrochemical anodization and chemical functionalisation. Detection of trace levels of analyte enzyme and its quantification by selective digestion. The NAA photonic film with the enzyme acts as a promising combination for a real-time point-of-care monitoring system for early stages of disease. 2. NAA rugate filters used to establish the binding affinity between blood proteins and drugs: Design, fabrication, and optimisation of NAA anodization parameters using sinusoidal pulse anodization approach (i.e. anodization offset and anodization period) to produce rugate filter photonic crystals that provide two comparative sensing parameters. Establishment of highly sensitive and selective device capable for drug binding assessments linked to treating a wide range of medical conditions. 3. NAA bilayers and food bioactive compound detection: Design, fabrication, and optimisation of NAA anodization parameters (i.e. anodization time and number of anodization steps) to obtain NAA bilayered photonic structures that display the effective response of NAA geometry with different types of nano-pore engineering. The photonic properties of the NAA bilayer were studied at each layer of nano-structure under specific binding of human serum albumin and quercetin as target agent. 4. Single nucleotide polymorphism (SNP) detection: The design and implementation of a Ligation-Rolling Circle Amplification assay to detect a single nucleotide polymorphism associated with insecticide resistance in a pest beetle species, Tribolium castaneum. This proof-of-concept SNP detection assay has the potential to provide a method compatible with a biosensor platform such as NAA. This demonstrates the first step towards the potential development of a genotyping biosensor, and a real-world application of insect insecticide resistance monitoring. The results presented in this thesis are expected to enable innovative developments on NAA sensing technology that could result in highly sensitive and selective detection systems for a broad range of bio-analytes detections.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Chemical Engineering, 201

Real-time Profiling of Solid-State Nanopores During Solution-Phase Nanofabrication

We describe a method for simply characterizing the size and shape of a nanopore during solution-based fabrication and surface modification, using only low-overhead approaches native to conventional nanopore measurements. Solution-based nanopore fabrication methods are democratizing nanopore science by supplanting the traditional use of charged-particle microscopes for fabrication, but nanopore profiling has customarily depended on microscopic examination. Our approach exploits the dependence of nanopore conductance in solution on nanopore size, shape, and surface chemistry in order to characterize nanopores. Measurements of the changing nanopore conductance during formation by etching or deposition can be analyzed using our method to characterize the nascent nanopore size and shape—beyond the typical cylindrical approximation—in real-time. Our approach thus accords with ongoing efforts to broaden the accessibility of 3 nanopore science from fabrication through use: it is compatible with conventional instrumentation and offers straightforward nanoscale characterization of the core tool of the field

NANOHARVESTING AND DELIVERY OF BIOACTIVE MATERIALS USING ENGINEERED SILICA NANOPARTICLES

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism

DESIGN, MICROFABRICATION, AND TESTING OF ALL-PMMA, NANOPORE-BASED ELECTROPHORETIC FLOW DETECTORS FOR BIOMEDICAL APPLICATIONS

ABSTRACT Detection of and discrimination between different nanoparticles and biomolecules are vital steps in analytical, biochemical, and diagnostic biomedical procedures used in life sciences. Synthetic micro/nanopores in solid-state membranes form an emerging class of single-molecule detectors capable of detecting and probing the properties of particles and biomolecules with high throughput and resolution: The particles or biomolecules to be analyzed are added to an electrolyte solution in one of the two reservoirs of the detector system separated by a thin membrane containing a single micro/nanopore. An outer electric field induces an open-pore ionic current (Iopen) through the pore, dragging the particles with itself. Transient changes occur when a particle slightly smaller than the pore translocates through the pore. This electrical signal can be analyzed to derive information regarding to the particle or biomolecule size and even its morphology, concentration in the solution, and the affinity for the pore. Many detectors are based on self-assembled, naturally occurring protein pores in lipid bilayer membranes. Most solid-state pore-based detectors reported in literature use artificial pores in silicon nitride or silicon oxide membranes. Applying polymers as a membrane potentially offers advantages over the aforementioned types, including good electrical insulation, improved wettability thanks to higher hydrophilicity, and long-term stable yet low-cost and disposable devices. The present study aims at exploiting such advantages by developing the proof-of-concept for a single-material, all-polymer, nanopore detector allowing the continuous variation of target pore size in the range from micrometers to a few nanometers for best pore size adaption to the biomolecules to be investigated. The research comprises materials selection, system design, development of a fabrication and assembly sequence, device fabrication, and functional device testing. Poly (methyl methacrylate) (PMMA) was selected as it combines advantageous microfluidic properties know from competing materials, such as polyimide, polystyrene, polycarbonate, or polyethylene terephthalate, with outstanding micropatterning capabilities. The membrane thickness is set to be 1 µm, based on a compromise between robustness during fabrication and operation on one side, and electrochemical performance on the other. After spincoating the membrane onto a sacrificial wafer, pores with diameters of typically several hundred nanometers are patterned by electron beam lithography. In combination with thermal post processing leading to polymer reflow, diameters one order of magnitude smaller can be achieved. The present study focuses on 450 nm and 22 nm pores, respectively. Besides these pores fabricated in a top-down approach, self-assembled -hemolysin protein pores of 1.5 nm diameter are integrated in a combined top-down and bottom-up approach so that single digit, double digit, and triple digit nanometer pores are available. Systems integration is achieved by capillary-forced based release from the sacrificial substrate and the application of UV-initiated glue. Test sequences proved and qualified the device functionality: Electrical characterization was performed in aqueous KCl electrolyte solution. The devices exhibit a stable, time-independent ionic current. The current-voltage curves are linear and scale with the electrolyte concentration. System verification was performed using silica nanospheres of 100 nm and 150 nm diameter as known test particles. Translocation through a 450 nm pore induced current blockades for about 1 ms with an amplitude of 30 pA to 55 pA for 100 nm particles and in excess of 70 pA for 150 nm particles. This is in close agreement with results obtained by a mathematical model used in this study. Biomolecules relevant to many life science applications, double-stranded DNA (dsDNA) and bovine serum albumin (BSA) were subsequently analyzed to prove the device concept. Post-processed pores of 22 nm diameter were used at 600 mV driving voltage and 0.1 molar electrolyte in a slightly acidic regime of pH = 6. Typical current blockade amplitudes for complete translocations of dsDNA are Iblock = 22 pA for a translocation time of tD = 0.2 ms, and an almost threefold current blockade (Iblock = 60 pA) for the larger BSA molecules, respectively. The results demonstrate that the PMMA-based nanopores are sensitive enough to not only detect translocating biomolecules, but to also sense them by distinguishing between different biomolecules. The molecule-specific and distinct translocation signals through the pores using both, standardized silica nanoparticles and biomolecules of different dimensions, prove the concept of an all-PMMA electrophoretic flow detector with adjustable pore diameters. Devices with pore diameters covering three orders of magnitude in the nanometer range were successfully built, tested, and characterized. The results suggest such detectors are promising candidates for biomolecule detecting applications

Environmental topology and water availability modulates the catalytic activity of beta-Galactosidase entrapped in a nanosporous silicate matrix

In the present work we studied the catalytic activity of E. coli β-Gal confined in a nanoporous silicate matrix (Eβ-Gal) at different times after the beginning of the sol-gel polymerization process. Enzyme kinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps of the reaction mechanism for their β-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through 1H-NMR, and scanning electron microscopy analysis of the gel nanostructure, were performed. In conjunction, results provided evidence that water availability is crucial for the modulation observed in the catalytic activity of β-Gal as long as water participate in the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrate concentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (kcB). More structured water populations (lower T2) correlates with higher catalytic rate constants (kcB). The T2-kcB negative correlation observed along the aging of gels within the 15-days period assayed reinforces the coupling between water structure and the hydrolysis catalysis inside gels.Fil: Burgos, Martha Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Velasco, Manuel Isaac. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Física Enrique Gaviola. Universidad Nacional de Córdoba. Instituto de Física Enrique Gaviola; ArgentinaFil: Acosta, Rodolfo Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Física Enrique Gaviola. Universidad Nacional de Córdoba. Instituto de Física Enrique Gaviola; ArgentinaFil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentin

Label-free single-molecule nanopore analysis of protein post-translational modifications

Despite significant advancements and refinements in single-molecule nanopore technology for DNA and RNA sequencing, its applicability to protein analysis remains limited. Proteins, consisting of 20 diverse building blocks, undergo post-translational modifications, fold into complex three-dimensional structures, and lack uniform charge distribution. These factors complicate signal interpretation and hinder their capture and movement through nanopore sensors. In the endeavor to enhance protein sequencing methodologies, ongoing research predominantly classifies into two primary strategies. Firstly, there are initiatives aimed at emulating the ordered translocation events observed in DNA/RNA nanopore sequencing, a process guided by enzymatic mechanisms. These approaches frequently employ molecular motors, necessitating preliminary sample processing steps to introduce recognition tags and labels. Alternatively, more straightforward strategies center on the examination of short peptides. Nonetheless, a substantial challenge persists in decoding the signal and reconstructing the original protein sequence from these peptide fragments, presenting a notable bottleneck in this methodology. Electro-osmotic flow (EOF), which occurs when an applied potential induces liquid motion in narrow tube-like conduits, is a phenomenon reported in nanopores for decades. EOF generates forces capable of increasing the capture rate and intra-pore residence time of neutral molecules and folded protein substrates. Despite promising results, limited work has explored the utilization of EOF for driving proteins across nanopores in a manner compatible with sequence-level readouts. In this study, we capitalize on the robust and well-characterized heptameric protein nanopore, αHL. We engineer this nanopore by introducing charged residues near its constriction, creating a series of ion-selective pores with varying EOF strengths. We then use a model protein, Trx, to construct concatemeric molecules featuring 2, 4, 6, 8, and 9 repetitions of a Trx-linker unit, resulting in chain lengths ranging from approximately 300 to over 1200 amino acids. These constructs are transported across the engineered pores in a label-free, enzyme-less manner under mild denaturing conditions. This process generates distinct current signatures corresponding to the number of units within each construct, indicating a mechanism of co-translocational unfolding. In an effort to assess the applicability of this approach, we introduce three types of post-translational modifications (PTMs)—phosphorylations, glutathionylation, and a glycosylation mimic—at five different positions within the middle Trx-linker unit of a nonameric concatemer. We engineer a universal modification site at these locations. The detection and discrimination of the three different modifications are successfully demonstrated at two of these positions. While this approach may not provide de novo sequence information, with further optimizations, it holds the potential to become a powerful method for fingerprinting PTMs. It could enable the characterization of underivatized full-length proteoforms from cells and tissues, serving as a valuable tool for proteomic analysis

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

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