Publications

2020

Journal Articles

Towards artificial mechanosensing

S. Marion; A. Radenovic 

Carbon nanotubes with single-digit diameter embedded in a solid artificial membrane show pressure-sensitive ionic conductance that is similar to the mechanically activated currents of biological ion channels.

Nature Materials. 2020-10-01. Vol. 19, num. 10, p. 1043-1044. DOI : 10.1038/s41563-020-00811-5.

High-Throughput Nanocapillary Filling Enabled by Microwave Radiation for Scanning Ion Conductance Microscopy Imaging

V. Navikas; S. M. Leitao; S. Marion; S. J. Davis; B. Drake et al. 

Solid state nanopores provide a highly sensitive tool for single-molecule sensing and probing nanofluidic effects in solutions. Glass nanopipettes are a cheap and robust type of solid-state nanopore produced from pulling glass capillaries with opening orifice diameters down to below tens of nanometers. Sub-50 nm nanocapillaries allow an unprecedented resolution for translocating single molecules or for scanning ion conductance microscopy imaging. Due to the small opening orifice diameters, such nanocapillaries are difficult to fill with solutions, compromising their advantages of low cost, availability, and experimental simplicity. We present a simple and cheap method to reliably fill nanocapillaries down to sub-10 nm diameters by microwave radiation heating. Using a large statistic of filled nanocapillaries, we determine the filling efficiency and physical principle of the filling process using sub-50 nm quartz nanocapillaries. Finally, we have used multiple nanocapillaries filled by our method for high-resolution scanning ion conductance microscopy imaging.

ACS Applied Nano Materials. 2020-08-28. Vol. 3, num. 8, p. 7829-7834. DOI : 10.1021/acsanm.0c01345.

Self-Blinking Dyes Unlock High-Order and Multiplane Super-Resolution Optical Fluctuation Imaging

K. Grussmayer; T. Lukes; T. Lasser; A. Radenovic 

Most diffraction-unlimited super-resolution imaging critically depends on the switching of fluorophores between at least two states, often induced using intense laser light and specialized buffers or UV radiation. Recently, so-called self-blinking dyes that switch spontaneously between an open, fluorescent “on” state and a closed, colorless “off” state were introduced. Here, we exploit the synergy between super-resolution optical fluctuation imaging (SOFI) and spontaneously switching fluorophores for 2D and 3D imaging. SOFI analyzes higher order statistics of fluctuations in the fluorophore emission instead of localizing individual molecules. It thereby tolerates a broad range of labeling densities, switching behavior, and probe brightness. Thus, even dyes that exhibit spontaneous blinking characteristics that are not suitable or suboptimal for single molecule localization microscopy can be used successfully for SOFI-based super-resolution imaging. We demonstrate 2D imaging of fixed cells with almost uniform resolution up to 50-60 nm in 6th order SOFI and characterize changing experimental conditions. Next, we investigate volumetric imaging using biplane and eight-plane data acquisition. We extend 3D cross-cumulant analysis to 4th order, achieving super-resolution in 3D with up to 29 depth planes. Finally, the low laser excitation intensities needed for single and biplane self-blinking SOFI are well suited for live-cell imaging. We show the perspective for time-resolved imaging by observing slow membrane movements in cells. Self-blinking SOFI thus provides a more robust alternative route for easy-to-use 2D and 3D high-resolution imaging.

Acs Nano. 2020-07-28. Vol. 14, num. 7, p. 9156-9165. DOI : 10.1021/acsnano.0c04602.

Polymer Coatings to Minimize Protein Adsorption in Solid-State Nanopores

S. Awasthi; P. Sriboonpeng; C. Ying; J. Houghtaling; I. Shorubalko et al. 

Nanopore-based resistive-pulse recordings represent a promising approach for single-molecule biophysics with applications ranging from rapid DNA and RNA sequencing to “fingerprinting” proteins. Based on advances in fabrication methods, solid-state nanopores are increasingly providing an alternative to proteinaceous nanopores from living organisms; their widespread adoption is, however, slowed by nonspecific interactions between biomolecules and pore walls, which can cause artifacts and pore clogging. Although efforts to minimize these interactions by tailoring surface chemistry using various physisorbed or chemisorbed coatings have made progress, a straightforward, robust, and effective coating method is needed to improve the robustness of nanopore recordings. Here, covalently attached nanopore surface coatings are prepared from three different polymers using a straightforward “dip and rinse” approach and compared to each other regarding their ability to minimize nonspecific interactions with proteins is compared. It is demonstrated that polymer coatings approach the performance of fluid lipid coatings with respect to minimizing these interactions. Moreover, these polymer coatings enable accurate estimates of the volumes and spheroidal shapes of freely translocating proteins; uncoated or inadequately coated solid-state pores do not have this capability. In addition, these polymer coatings impart physical and chemical stability and enable efficient and label-free characterization of single proteins without requiring harsh cleaning protocols between experiments.

Small Methods. 2020-07-16.  p. 2000177. DOI : 10.1002/smtd.202000177.

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

K. S. Grussmayer; S. Geissbuehler; A. Descloux; T. Lukes; M. Leutenegger et al. 

Super-resolution opticalfluctuation imaging provides a resolution beyond the diffraction limitby analysing stochasticfluorescencefluctuations with higher-order statistics. Usingnthorderspatio-temporal cross-cumulants the spatial resolution and the sampling can be increased upton-fold in all spatial dimensions. In this study, we extend the cumulant analysis into thespectral domain and propose a multicolor super-resolution scheme. The simultaneousacquisition of two spectral channels followed by spectral cross-cumulant analysis andunmixing increases the spectral sampling. The number of discriminablefluorophore species isthus not limited to the number of physical detection channels. Using two color channels, wedemonstrate spectral unmixing of threefluorophore species in simulations and experimentsinfixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for anoptimized spectralfilter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conservingthe spatial super-resolution property.

Nature Communications. 2020-06-15. Vol. 11, num. 1. DOI : 10.1038/s41467-020-16841-1.

Direct observation of water-mediated single-proton transport between hBN surface defects

J. Comtet; B. Grosjean; E. Glushkov; A. Avsar; K. Watanabe et al. 

Aqueous proton transport at interfaces is ubiquitous and crucial for a number of fields, ranging from cellular transport and signalling, to catalysis and membrane science. However, due to their light mass, small size and high chemical reactivity, uncovering the surface transport of single protons at room temperature and in an aqueous environment has so far remained out-of-reach of conventional atomic-scale surface science techniques, such as scanning tunnelling microscopy. Here, we use single-molecule localization microscopy to resolve optically the transport of individual excess protons at the interface of hexagonal boron nitride crystals and aqueous solutions at room temperature. Single excess proton trajectories are revealed by the successive protonation and activation of optically active defects at the surface of the crystal. Our observations demonstrate, at the single-molecule scale, that the solid/water interface provides a preferential pathway for lateral proton transport, with broad implications for molecular charge transport at liquid interfaces.

Nature Nanotechnology. 2020-05-25. DOI : 10.1038/s41565-020-0695-4.

Wafer-Scale Fabrication of Nanopore Devices for Single-Molecule DNA Biosensing using MoS2

M. Thakur; M. Macha; A. Chernev; M. Graf; M. Lihter et al. 

Atomically thin (2D) nanoporous membranes are an excellent platform for a broad scope of academic research. Their thickness and intrinsic ion selectivity (demonstrated for example in molybdenum disulfide-MoS2) make them particularly attractive for single-molecule biosensing experiments and osmotic energy harvesting membranes. Currently, one of the major challenges associated with the research progress and industrial development of 2D nanopore membrane devices is small-scale thin-film growth and small-area transfer methods. To address these issues, a large-area protocol including a wafer-scale monolayer MoS2 synthesis, Si/SiNx substrate fabrication and wafer-scale material transfer are demonstrated. First, the 7.62 cm wafer-scale MOCVD growth yielding homogenous monolayer MoS2 films are introduced. Second, a large number of devices are fabricated in one batch by employing the wafer-scale thin-film transfer method with high transfer efficiency (>70% device yield). The growth, the transfer quality and cleanliness are investigated using transmission electron microscopy, atomic force microscopy and Raman spectroscopy. Finally, the applicability and robustness of the large-area protocol is demonstrated by performing a set of double-stranded DNA translocation experiments through as-fabricated MoS2 nanopore devices. It is believed that the shown approach will pave the way toward wafer-scale, high-throughput use of 2D nanopores in various applications.

Small Methods. 2020-05-11.  p. 2000072. DOI : 10.1002/smtd.202000072.

Nanocapillary confinement of imidazolium based ionic liquids

S. Marion; S. J. Davis; Z-Q. Wu; A. Radenovic 

Room temperature ionic liquids are salts which are molten at or around room temperature without any added solvent or solution. In bulk they exhibit glass like dependence of conductivity with temperature as well as coupling of structural and transport properties. Interfaces of ionic liquids have been found to induce structural changes with evidence of long range structural ordering on solid-liquid interfaces spanning length scales of 10-100 nm. Our aim is to characterize the influence of confinement on the structural properties of ionic liquids. We present the first conductivity measurements on ionic liquids of the imidazolium type in single conical glass nanopores with confinements as low as tens of nanometers. We probe glassy dynamics of ionic liquids in a large range of temperatures (-20 to 70 degrees C) and nanopore opening sizes (20-600 nm) in silica glass nanocapillaries. Our results indicate no long range freezing effects due to confinement in nanopores with diameters as low as 20 nm. The studied ionic liquids are found to behave as glass like liquids across the whole accessible confinement size and temperature range.

Nanoscale. 2020-04-28. Vol. 12, num. 16, p. 8867-8874. DOI : 10.1039/d0nr01164a.

Theses

Fundamental Applications of Nanopores: Controlled DNA Translocations to Nanofluidics

S. J. Davis / A. Radenovic; S. Marion (Dir.)  

Nanopores are nanometer sized openings that are the connection between two electrolyte filled reservoirs. The measurement of the ion transport flowing through such a pore allows to probe physically or biologically interesting phenomena. These range from the passage of biological molecules, to the modulation of current due to multiple physical effects when the nanopore undergoes mechanical strain or pressure induced flow. Many types of nanopores exist: biological protein pores engineered to be able to sequence DNA, glass nanocapillaries easily interfaced with optical tools, or silicon nitride membrane pores which are a standard tool of recent nanotechnology. This thesis is split into two parts. The first focuses on the use of glass nanocapillaries which allow the facile combination of nanopore experiments with optical tweezers. Optical tweezers are a well established single molecule tool that allow precise force measurement on biologically relevant scales. They are used here for the control of DNA passing through the nanopore. Their ability to measure small scale forces allows the detailed investigation of DNA binding proteins, and attempts to measure the force of DNA passage through biological pores. Extensions of these experiments to the elastic behaviour of DNA during its passage through a nanopore reveal the effects of flow generated by the charged surface of nanopores themselves. This motivates attempts to control such flows as well as the second part of the thesis. The second part of the thesis focuses on nanofluidics, the role of fluid flow and ion transport at the nanoscale. Using a setup combining pressure with nanopores it is possible to probe, via precise measurements of the conduction of the nanopore, the wetting state of the pore. Contamination phenomena are shown to be abundant with such small systems and a description of their effects on standard measurements such as direct current current-voltage curves is given. Following this, pressure is applied to perfectly filled pores and, thanks to a new alternating current detection method, is shown to be able to discern the effect of pressure induced strain at the pore as well as the coupling of hydraulic flow with electrical properties of the pore. Finally, extensions beyond aqueous solvents are explored in both nanocapillaries and silicon nitride pores. For this, room-temperature ionic liquids are used. These liquids are known from previous studies to behave differently at surfaces and in nano-confinement. The nanopore system both with and without added pressure is shown to be a good tool for investigating such phenomena.

Lausanne, EPFL, 2020. 

Electrochemical and morphological engineering of 2D materials for nanopore sensing

M. Lihter / A. Radenovic (Dir.)  

Nanopores are nanometer-sized holes that were initially proposed for DNA sequencing. Several years ago sequencing was made possible with biological nanopores. However, solid-state nanopores have plenty of advantages to offer compared to their biological counterparts. This thesis focuses on nanopores made in 2D materials, and their sensing capabilities. They provide an outstanding spatial resolution and a good signal-to-noise ratio (SNR) for sensing. As a 2D semiconductor, molybdenum disulfide (MoS2) exhibits unique (opto)electronic and electrochemical properties. The electronic band structure of a semiconducting MoS2 results in highly sensitivity to its chemical environment and external electric field. Therefore, monitoring the transverse current through the MoS2 during analyte translocation can provide a powerful sensing mechanism. As an n-type semiconductor, MoS2 exhibits a characteristic electrochemical behavior: it shows electrochemical activity when polarized cathodically, while in the anodic region becomes electrochemically inactive. This thesis covers five topics in which I demonstrate how the properties of 2D materials can be used for designing advanced sensing platforms for biosensing. Fabrication of MoS2 Nanopores. Here, we give an overview of a reliable fabrication process optimized for the production of high-quality devices. We discuss the main issues, how to solve and avoid them. In the end, we demonstrate applications of MoS2 nanopores for sensing and for osmotic power generation. Geometrical Effect in 2D Nanopores. We compare two different pore geometries: triangular pores in hexagonal boron-nitride (h-BN) vs. approximately circular pores in MoS2. In h-BN nanopores, we observe a lower conductance drop caused by DNA translocation, than expected from a conventional conductance model. As an explanation, we propose a reduced ion-concentration and ion-mobility inside the pore, supported by molecular dynamics simulations. Transverse Detection of DNA in MoS2 Nanopore. This chapter presents the realization of a hybrid nanopore-FET device, consisting of an MoS2 ribbon and a nanopore drilled in it. Such a device acts as a field-effect transistor (FET), where the transverse current through the MoS2 is modulated by the translocating molecule and the ionic voltage applied across the pore. We show that the transverse current is more sensitive to DNA translocation than the ionic current. Passivation of Electrodes Contacting MoS2. The method is based on the electrochemical deposition of a polymer, which blocks the electron transfer from the surface of the electrodes to ions in solution. To avoid the deposition on MoS2, we used poly(phenylene oxide) (PPO) that polymerizes in the potential window where MoS2 is inactive. Deposition is compatible with MoS2 and highly area-selective, even at the nanoscale, as we demonstrate on the nanoelectrodes of a nanopore-FET device. Electrochemical Modification of MoS2. By polarizing MoS2 cathodically, it is possible to electrochemically deposit a thin layer of aryl-diazonium compounds on MoS2 surface. This approach allows to introduce specific chemical groups on MoS2 and nanopore rim, which could be used for specific recognition of analytes as they translocate through the pore. Furthermore, since the deposition occurs only on the MoS2 to which the voltage was applied, this could enable specific functionalization of adjacent nanoribbons in nanopore-FET devices.

Lausanne, EPFL, 2020. 

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