Analysis of Polyethylene Glycol in the α-Hemolysin Nanopore

Nanopores have been shown to be a useful analytical tool for single molecule detection. They have been used to study the composition of DNA and other molecules of interest. These pores are usually α-hemolysin which is a toxin from Staphylococcus aureus or more recently nanoscale synthetic solid state pores. Now we are beginning to look at other molecules or proteins by sending them into the nanopores and measuring a characteristic partial current blockade. In this thesis we look at polyethylene glycol (PEG) as it enters and blocks current through a single alpha hemolysin pore. We report the effects of ionic strength, PEG size, and applied voltage on the depth and duration of the current blockades. We also apply autocorrelation analysis on the arrival times of PEG molecules to the pore see if we can identify if the PEG is translocating through the pore or escaping from the same side it enters. This suggests a new approach to current blockade analysis

Force-induced acoustic phonon transport across single-digit nanometre vacuum gaps

Heat transfer between bodies separated by nanoscale vacuum gap distances has been extensively studied for potential applications in thermal management, energy conversion and data storage. For vacuum gap distances down to 20 nm, state-of-the-art experiments demonstrated that heat transport is mediated by near-field thermal radiation, which can exceed Planck's blackbody limit due to the tunneling of evanescent electromagnetic waves. However, at sub-10-nm vacuum gap distances, current measurements are in disagreement on the mechanisms driving thermal transport. While it has been hypothesized that acoustic phonon transport across single-digit nanometre vacuum gaps (or acoustic phonon tunneling) can dominate heat transfer, the underlying physics of this phenomenon and its experimental demonstration are still unexplored. Here, we use a custom-built high-vacuum shear force microscope (HV-SFM) to measure heat transfer between a silicon (Si) tip and a feedback-controlled platinum (Pt) nanoheater in the near-contact, asperity-contact, and bulk-contact regimes. We demonstrate that in the near-contact regime (i.e., single-digit nanometre or smaller vacuum gaps before making asperity contact), heat transfer between Si and Pt surfaces is dominated by force-induced acoustic phonon transport that exceeds near-field thermal radiation predictions by up to three orders of magnitude. The measured thermal conductance shows a gap dependence of $d^{-5.7\pm1.1}$ in the near-contact regime, which is consistent with acoustic phonon transport modelling based on the atomistic Green's function (AGF) framework. Our work suggests the possibility of engineering heat transfer across single-digit nanometre vacuum gaps with external force stimuli, which can make transformative impacts to the development of emerging thermal management technologies.Comment: 9 pages with 4 figures (Main text), 13 pages with 7 figures (Methods), and 13 pages with 6 figures and 1 table (Supplementary Information

Scanning electrochemical cell microscopy : a versatile technique for nanoscale electrochemistry and functional imaging

Scanning electrochemical cell microscopy (SECCM) is a new pipette-based imaging technique purposely designed to allow simultaneous electrochemical, conductance, and topographical visualization of surfaces and interfaces. SECCM uses a tiny meniscus or droplet, confined between the probe and the surface, for high-resolution functional imaging and nanoscale electrochemical measurements. Here we introduce this technique and provide an overview of its principles, instrumentation, and theory. We discuss the power of SECCM in resolving complex structure-activity problems and provide considerable new information on electrode processes by referring to key example systems, including graphene, graphite, carbon nanotubes, nanoparticles, and conducting diamond. The many longstanding questions that SECCM has been able to answer during its short existence demonstrate its potential to become a major technique in electrochemistry and interfacial science

Electro-spinning/netting: A strategy for the fabrication of three-dimensional polymer nano-fiber/nets.

Since 2006, a rapid development has been achieved in a subject area, so called electro-spinning/netting (ESN), which comprises the conventional electrospinning process and a unique electro-netting process. Electro-netting overcomes the bottleneck problem of electrospinning technique and provides a versatile method for generating spider-web-like nano-nets with ultrafine fiber diameter less than 20 nm. Nano-nets, supported by the conventional electrospun nanofibers in the nano-fiber/nets (NFN) membranes, exhibit numerious attractive characteristics such as extremely small diameter, high porosity, and Steiner tree network geometry, which make NFN membranes optimal candidates for many significant applications. The progress made during the last few years in the field of ESN is highlighted in this review, with particular emphasis on results obtained in the author's research units. After a brief description of the development of the electrospinning and ESN techniques, several fundamental properties of NFN nanomaterials are addressed. Subsequently, the used polymers and the state-of-the-art strategies for the controllable fabrication of NFN membranes are highlighted in terms of the ESN process. Additionally, we highlight some potential applications associated with the remarkable features of NFN nanostructure. Our discussion is concluded with some personal perspectives on the future development in which this wonderful technique could be pursued

Voltage gated inter-cation selective ion channels from graphene nanopores

With the ability to selectively control ionic flux, biological protein ion channels perform a fundamental role in many physiological processes. For practical applications that require the functionality of a biological ion channel, graphene provides a promising solid-state alternative, due to its atomic thinness and mechanical strength. Here, we demonstrate that nanopores introduced into graphene membranes, as large as 50 nm in diameter, exhibit inter-cation selectivity with a ~20x preference for K+ over divalent cations and can be modulated by an applied gate voltage. Liquid atomic force microscopy of the graphene devices reveals surface nanobubbles near the pore to be responsible for the observed selective behavior. Molecular dynamics simulations indicate that translocation of ions across the pore likely occurs via a thin water layer at the edge of the pore and the nanobubble. Our results demonstrate a significant improvement in the inter-cation selectivity displayed by a solid-state nanopore device and by utilizing the pores in a de-wetted state, offers an approach to fabricating selective graphene membranes that does not rely on the fabrication of sub-nm pores

Nanofluidic platforms for sensing applications in biomedical and environmental fields

Nowadays, nanofluidic platforms are powerful tools for carrying out fundamental studies on molecular-scale phenomena. Typically, the use of these systems results in being crucial both in biomedical and environmental fields. In fact, they are widely exploited for many applications such as detection, concentration, sorting, counting and sizing of several nano-objects such as nanoplastics, viruses, antibodies and DNA. This is the context in which my Ph.D. research project is inserted. I have worked on the development of elastomeric nanofluidic platforms, equipped with different nanostructures, that are the functional areas of the entire fluidic system, useful for different applications among which single nanoparticles detection, high-sensitivity immunoassay analysis and DNA sensing. Typically, nanofluidic platforms are composed of two U-shaped microchannels connected by nanostructures with suitable geometries. All devices were fabricated starting from a pre-patterned silicon mold on which nanostructures were etched using the Focus Ion Beam (FIB) milling technique. Then, the molds were replicated through a, Poly(DiMethylSiloxane)(PDMS) based, double REplica Molding (REM) technique. Although FIB is a high-resolution but expensive technology with REM technique, that is a low-cost and simple approach, I was able to fabricate many polymeric replicas with high precision re-using the mold for several times. This combination allowed obtaining high-resolution nanofluidic platforms reducing fabrication costs, a method that is potentially applicable to processes with high production rate. However, when the dimension shrinks from micro to nanoscale, PDMS presents significant limits. In particular, polymeric nanostructures suffer from the \u201croof collapse\u201d phenomenon that occurs when the replica is sealed with a glass substrate, a necessary procedure to obtain watertight devices. It is possible to overcome this problem both by exploiting the Junction Gap Breakdown (JGB) technique and by using hard-PDMS (h-PDMS) during the fabrication process. During my Ph.D. research activity, I have initially worked on an asymmetric structure that was a funnel-shaped nanochannel in which the tip, after experiencing \u201croof-collapse\u201d, was re-opened, thanks to the Junction Gap Breakdown procedure. From an electrical investigation of the devices fabricated with this strategy, we observed an ion current rectification characteristic and analyzing the electro-kinetic transport properties we observed that, in few minutes, intra-funnel accumulation occurs, and this phenomenon results in being stronger for low ionic strength solutions. Combining intra-funnel accumulation of biomolecules, governed by electro-hydrokinetic phenomena, that occurs applying high voltage across the device, and an appropriate functionalization of nanochannel polymeric surface with antibodies, it was possible to decrease sensing limit for the detection of one or several targeted antigens for clinical diagnostics. It was possible to identify through fluorescence optical microscopy and electrical measurements, the uptake of a specific antigen, diluted in solution (down to 1 pg/ml), to the nanochannel surface when functionalized with antibodies. So, in this condition, we successfully detected antigen-antibody binding on the nanostructure surface, a promising step for realizing a high-sensitivity nanofluidic immuno-assay sensor. Successively, I have developed other nanofluidic devices equipped with symmetric nanostructures for single-particle sensing. These devices were made using h-PDMS (hard- PDMS) in order to confer higher rigidity to the nanostructures, i.e. the functional part of the device, avoiding collapse problems. H-PDMS was used in exploiting a \u201cfocused drop-casting\u201d approach in order to make only the nanostructure region stiffer, while leaving the other regions of the device flexible enough to avoid the formation of cracks along the device. Combining the nanoscale dimension of the sensing gate with the Resistive Pulse Sensing (RPS) technique, it was possible to analyze single nanoparticles (NPs) and the motion of single \u3bb-DNA molecules through the nanochannel as transient variations in ionic current during the translocation events, allowing a real-time, label-free and high-sensitivity detection. In particular, it was possible to demonstrate the possibility of counting nano-objects depending on selected characteristics (i.e. charge and size ranging from 40 nm to 100 nm) that is a crucial step, useful in many fields such as medicine (drug delivery, imaging, cell-secreted carriers), environment (groundwater remediation, nanoplastics detection) and food production (nano-agrochemicals, nano-encapsulated additives, anti-microbials)

How to detect when cells in space perceive gravity

It is useful to be able to measure when and whether cells detect gravity during spaceflights. For studying gravitational physiology, gravity perception is the response the experimentalist needs to measure. Also, for growing plants in space, plant cells may have a non-directional requirement for gravity as a development cue. The main goals of spaceflight experiments in which gravity perception would be measured are to determine the properties of the gravity receptor and how it is activated, and to determine fundamental characteristics of the signal generated. The main practical difficulty with measuring gravity sensing in space is that gravity sensing cannot be measured with certainty on earth. Almost all experiments measure gravitropic curvature. Reciprocity and intermittent stimulation are measurements which were made to some degree on earth using clinostatting, but which would provide clearer results if done with microgravity rather than clinostatting. These would be important uses of the space laboratory for determining the nature of gravity sensing in plants. Those techniques which do not use gravitropic curvature to measure gravity sensing are electrophysiological. The vibrating probe would be somewhat easier to adapt to space conditions than the intracellular microelectrode because it can be positioned with less precision. Ideally, a non-invasive technique would be best suited if an appropriate measure could be developed. Thus, the effect of microgravity on cultured cells is more likely to be by large-scale physical events than gravity sensing in the culture cells. It is not expected that it will be necessary to determine whether individual cultured cells perceive gravity unless cells grow abnormally even after the obvious microgravity effects on the culture as a whole can be ruled out as the cause

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology