Journal Articles

Open-source microscope add-on for structured illumination microscopy

M. T. M. Hannebelle; E. Raeth; S. M. Leitao; T. Lukes; J. Pospisil et al. 

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.|Researchers developed an open-hardware structured illumination microscopy add-on. This affordable upgrade provides super-resolution capabilities for normal optical microscopes. Detailed instructions enable easy reproduction to help democratize advanced microscopy.

Nature Communications. 2024-02-20. Vol. 15, num. 1, p. 1550. DOI : 10.1038/s41467-024-45567-7.

Nanofluidic logic with mechano-ionic memristive switches

T. Emmerich; Y. Teng; N. Ronceray; E. Lopriore; R. Chiesa et al. 

Neuromorphic systems are typically based on nanoscale electronic devices, but nature relies on ions for energy-efficient information processing. Nanofluidic memristive devices could thus potentially be used to construct electrolytic computers that mimic the brain down to its basic principles of operation. Here we report a nanofluidic device that is designed for circuit-scale in-memory processing. The device, which is fabricated using a scalable process, combines single-digit nanometric confinement and large entrance asymmetry and operates on the second timescale with a conductance ratio in the range of 9 to 60. In operando optical microscopy shows that the memory capabilities are due to the reversible formation of liquid blisters that modulate the conductance of the device. We use these mechano-ionic memristive switches to assemble logic circuits composed of two interactive devices and an ohmic resistor.

Nature Electronics. 2024-03-19. DOI : 10.1038/s41928-024-01137-9.

AI-driven detection and analysis of label-free protein aggregates

K. A. Ibrahim 

In this Tools of the Trade article, Khalid Ibrahim (Radenovic and Lashuel labs) describes a tool for the artificial intelligence (AI)-driven detection of cellular aggregates that bypasses the need for fluorescent labelling.

Nature Reviews Molecular Cell Biology. 2024-02-08. DOI : 10.1038/s41580-024-00708-0.


Label-Free Techniques for Probing Biomolecular Condensates

K. A. Ibrahim; A. S. Naidu; H. Miljkovic; A. Radenovic; W. Yang 

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years’ exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools’ key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

Acs Nano. 2024-04-12. DOI : 10.1021/acsnano.4c01534.


Journal Articles

Synthesis of Fluorescent Cyclic Peptides via Gold(I)-Catalyzed Macrocyclization

X-Y. Liu; W. Cai; N. Ronceray; A. Radenovic; B. Fierz et al. 

Rapid and efficient cyclization methods that form structurally novel peptidic macrocycles are of high importance for medicinal chemistry. Herein, we report the first gold(I)-catalyzed macrocyclization of peptide-EBXs (ethynylbenziodoxolones) via C2-Trp C–H activation. This reaction was carried out in the presence of protecting group free peptide sequences and is enabled by a simple commercial gold catalyst (AuCl·Me2S). The method displayed a rapid reaction rate (within 10 min), wide functional group tolerance (27 unprotected peptides were cyclized), and up to 86% isolated yield. The obtained highly conjugated cyclic peptide linker, formed through C–H alkynylation, can be directly applied to live-cell imaging as a fluorescent probe without further attachment of fluorophores.

Journal of the American Chemical Society. 2023. Vol. 145, num. 49, p. 26525-26531. DOI : 10.1021/jacs.3c09261.

Selective Growth of van der Waals Heterostructures Enabled by Electron-Beam Irradiation

J. Sitek; K. Czerniak-Losiewicz; A. P. Gertych; M. Giza; P. Dabrowski et al. 

Van der Waals heterostructures (vdWHSs) enable the fabricationof complex electronic devices based on two-dimensional (2D) materials.Ideally, these vdWHSs should be fabricated in a scalable and repeatableway and only in the specific areas of the substrate to lower the numberof technological operations inducing defects and impurities. Here,we present a method of selective fabrication of vdWHSs via chemicalvapor deposition by electron-beam (EB) irradiation. We distinguishtwo growth modes: positive (2D materials nucleate on the irradiatedregions) on graphene and tungsten disulfide (WS2) substrates,and negative (2D materials do not nucleate on the irradiated regions)on the graphene substrate. The growth mode is controlled by limitingthe air exposure of the irradiated substrate and the time betweenirradiation and growth. We conducted Raman mapping, Kelvin-probe forcemicroscopy, X-ray photoelectron spectroscopy, and density-functionaltheory modeling studies to investigate the selective growth mechanism.We conclude that the selective growth is explained by the competitionof three effects: EB-induced defects, adsorption of carbon species,and electrostatic interaction. The method here is a critical steptoward the industry-scale fabrication of 2D-materials-based devices.

Acs Applied Materials & Interfaces. 2023-07-07. Vol. 15, num. 28, p. 33838-33847. DOI : 10.1021/acsami.3c02892.

Confinement-Controlled Water Engenders Unusually High Electrochemical Capacitance

S. Melnik; A. Ryzhov; A. Kiselev; A. Radenovic; T. Weil et al. 

The electrodynamicsof nanoconfined water have been shownto changedramatically compared to bulk water, opening room for safe electrochemicalsystems. We demonstrate a nanofluidic “water-only” batterythat exploits anomalously high electrolytic properties of pure waterat firm confinement. The device consists of a membrane electrode assemblyof carbon-based nanomaterials, forming continuously interconnectedwater-filled nanochannels between the separator and electrodes. Theefficiency of the cell in the 1-100 nm pore size range showsa maximum energy density at 3 nm, challenging the region of the currentmetal-ion batteries. Our results establish the electrodynamic fundamentalsof nanoconfined water and pave the way for low-cost and inherentlysafe energy storage solutions that are much needed in the renewableenergy sector.

Journal Of Physical Chemistry Letters. 2023-07-17. DOI : 10.1021/acs.jpclett.3c01498.

Nature-Inspired Stalactite Nanopores for Biosensing and Energy Harvesting

A. Chernev; Y. Teng; M. Thakur; V. Boureau; L. Navratilova et al. 

Nature provides a wide range of self-assembled structures from the nanoscale to the macroscale. Under the right thermodynamic conditions and with the appropriate material supply, structures like stalactites, icicles, and corals can grow. However, the natural growth process is time-consuming. This work demonstrates a fast, nature-inspired method for growing stalactite nanopores using heterogeneous atomic deposition of hafnium dioxide at the orifice of templated silicon nitride apertures. The stalactite nanostructures combine the benefits of reduced sensing region typically for 2-dimensional material nanopores with the asymmetric geometry of capillaries, resulting in ionic selectivity, stability, and scalability. The proposed growing method provides an adaptable nanopore platform for basic and applied nanofluidic research, including biosensing, energy science, and filtration technologies.

Advanced Materials. 2023-07-06. DOI : 10.1002/adma.202302827.

The Three-Phase Contact Potential Difference Modulates the Water Surface Charge

V. Artemov; L. Frank; R. Doronin; P. Staerk; A. Schlaich et al. 

The surface charge of an open water surface is crucialfor solvationphenomena and interfacial processes in aqueous systems. However, themagnitude of the charge is controversial, and the physical mechanismof charging remains incompletely understood. Here we identify a previouslyoverlooked physical mechanism determining the surface charge of water.Using accurate charge measurements of water microdrops, we demonstratethat the water surface charge originates from the electrostatic effectsin the contact line vicinity of three phases, one of which is water.Our experiments, theory, and simulations provide evidence that a junctionof two aqueous interfaces (e.g., liquid-solid and liquid-air)develops a pH-dependent contact potential difference Delta phi due to the longitudinal charge redistribution between two contactinginterfaces. This universal static charging mechanism may have implicationsfor the origin of electrical potentials in biological, nanofluidic,and electrochemical systems and helps to predict and control the surfacecharge of water in various experimental environments.

Journal Of Physical Chemistry Letters. 2023-05-16. Vol. 14, num. 20, p. 4796-4802. DOI : 10.1021/acs.jpclett.3c00479.

The Three-Phase Contact Potential Difference Modulates the Water Surface Charge

V. Artemov; L. Frank; R. Doronin; P. Staerk; A. Schlaich et al. 

The surface charge of an open water surface is crucialfor solvationphenomena and interfacial processes in aqueous systems. However, themagnitude of the charge is controversial, and the physical mechanismof charging remains incompletely understood. Here we identify a previouslyoverlooked physical mechanism determining the surface charge of water.Using accurate charge measurements of water microdrops, we demonstratethat the water surface charge originates from the electrostatic effectsin the contact line vicinity of three phases, one of which is water.Our experiments, theory, and simulations provide evidence that a junctionof two aqueous interfaces (e.g., liquid-solid and liquid-air)develops a pH-dependent contact potential difference Delta phi due to the longitudinal charge redistribution between two contactinginterfaces. This universal static charging mechanism may have implicationsfor the origin of electrical potentials in biological, nanofluidic,and electrochemical systems and helps to predict and control the surfacecharge of water in various experimental environments.

Journal Of Physical Chemistry Letters. 2023-05-16. Vol. 14, num. 20, p. 4796-4802. DOI : 10.1021/acs.jpclett.3c00479.

Nanoscale thermal control of a single living cell enabled by diamond heater-thermometer

A. M. Romshin; V. Zeeb; E. Glushkov; A. Radenovic; A. G. Sinogeikin et al. 

We report a new approach to controllable thermal stimulation of a single living cell and its compartments. The technique is based on the use of a single polycrystalline diamond particle containing silicon-vacancy (SiV) color centers. Due to the presence of amorphous carbon at its intercrystalline boundaries, such a particle is an efficient light absorber and becomes a local heat source when illuminated by a laser. Furthermore, the temperature of such a local heater is tracked by the spectral shift of the zero-phonon line of SiV centers. Thus, the diamond particle acts simultaneously as a heater and a thermometer. In the current work, we demonstrate the ability of such a Diamond Heater-Thermometer (DHT) to locally alter the temperature, one of the numerous parameters that play a decisive role for the living organisms at the nanoscale. In particular, we show that the local heating of 11-12 degrees C relative to the ambient temperature (22 degrees C) next to individual HeLa cells and neurons, isolated from the mouse hippocampus, leads to a change in the intracellular distribution of the concentration of free calcium ions. For individual HeLa cells, a long-term (about 30 s) increase in the integral intensity of Fluo-4 NW fluorescence by about three times is observed, which characterizes an increase in the [Ca2+](cyt) concentration of free calcium in the cytoplasm. Heating near mouse hippocampal neurons also caused a calcium surge-an increase in the intensity of Fluo-4 NW fluorescence by 30% and a duration of similar to 0.4 ms.

Scientific Reports. 2023-05-26. Vol. 13, num. 1, p. 8546. DOI : 10.1038/s41598-023-35141-4.

Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds

S. M. Leitao; V. Navikas; H. Miljkovic; B. Drake; S. Marion et al. 

In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio by two orders of magnitude compared with free translocations. We demonstrate the method’s versatility by assessing DNA-protein complexes, DNA rulers and DNA gaps, achieving down to single-nucleotide gap detection. In single-molecule characterization, the near-infinite re-read capability on the same region of interest has the potential to unlock greater sensing capacity. A nanopore-based method, named scanning ion conductance spectroscopy, provides complete control over the translocation speed and nanopore position along a selected region and can detect a single 3 angstrom gap in a long strand of DNA.

Nature Nanotechnology. 2023-06-19. DOI : 10.1038/s41565-023-01412-4.

Optical imaging of the small intestine immune compartment across scales

A. L. Planchette; C. Schmidt; O. Burri; M. G. de Agueero; A. Radenovic et al. 

A workflow for 3D characterization of the mouse small intestine with optical projection tomography allows the identification of sparsely-distributed regions of interest in large volumes while retaining compatibility with high-resolution microscopy modalities.

Communications Biology. 2023-03-31. Vol. 6, num. 1, p. 352. DOI : 10.1038/s42003-023-04642-3.

High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing

M. Thakur; N. Cai; M. Zhang; Y. Teng; A. Chernev et al. 

Nanopores in two-dimensional (2D) membranes hold immense potential in single-molecule sensing, osmotic power generation, and information storage. Recent advances in 2D nanopores, especially on single-layer MoS2, focus on the scalable growth and manufacturing of nanopore devices. However, there still remains a bottleneck in controlling the nanopore stability in atomically thin membranes. Here, we evaluate the major factors responsible for the instability of the monolayer MoS2 nanopores. We identify chemical oxidation and delamination of monolayers from their underlying substrates as the major reasons for the instability of MoS2 nanopores. Surface modification of the substrate and reducing the oxygen from the measurement solution improves nanopore stability and dramatically increases their shelf-life. Understanding nanopore growth and stability can provide insights into controlling the pore size, shape and can enable long-term measurements with a high signal-to-noise ratio and engineering durable nanopore devices.

Npj 2D Materials And Applications. 2023-02-23. Vol. 7, num. 1, p. 11. DOI : 10.1038/s41699-023-00373-5.

Substitutional p‐type Doping in NbS2‐MoS2 Lateral Heterostructures Grown by MOCVD

Z. Wang; M. Tripathi; Z. Golsanamlou; P. Kumari; G. Lovarelli et al. 

Advanced Materials. 2023-01-16.  p. 2209371. DOI : 10.1002/adma.202209371.

Conference Papers

Defect engineering of 2D material for biosensing applications

W. Yang; J. Sulzle; Y. Shimoda; E. Mayner; M. Macha et al. 

2023-02-10.  p. 278A-278A. DOI : 10.1016/j.bpj.2022.11.1584.

Imaging of interactions of biomolecules with nanomaterials with interferometric scattering microscopy

J. Sulzle; W. Yang; Y. Shimoda; E. S. Mayner; M. Macha et al. 

2023-02-10.  p. 153A-153A.


Nanopore-based scanning system and method

A. Radenovic; G. Fantner; S. Mendes Leitão; V. Navikas 

Nanopore-based scanning system including a probe structure comprising a nanopore; suction means configured to draw an end of a (bio)molecule inside the nanopore and inside the probe structure, single or multiple times; and displacement means configured to mechanically displace the probe structure and the nanopore relative to the one (bio)molecule along a direction following a direction of extension of the (bio)molecule while the (bio)molecule is located inside the nanopore and inside the probe structure, or configured to mechanically displace at least one support holding the (bio)molecule relative to the nanopore along a direction following a direction of extension of the (bio)molecule while holding the at least one support and while the (bio)molecule is located inside the nanopore.




Minimum dataset for “Liquid-activated quantum emission from pristine hexagonal boron nitride for nanofluidic sensing”

N. Ronceray; A. Radenovic; Y. You; E. Glushkov; M. Lihter et al. 

Frames and (linked) localization table used to produce Fig. 2 of the manuscript Details are given in ‘README.txt’. The rest of the data is provided with the paper at the publisher website.


Dataset for the paper High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing

M. Thakur; N. Cai; M. Zhang; Y. Teng; A. Chernev et al. 

Information regarding the Dataset, corresponding to the paper: “Thakur, M., Cai, N., Zhang, M. et al. High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing. npj 2D Mater Appl 7, 11 (2023).” This folder contains the raw data and complete package of codes used to analyze, view, save, and plot data for the publication titled “High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing”. The code folder, “OpenNanopore-nanopore-tools”, can be used to plot raw data which corresponds to the figures in the paper and supplementary information.



Journal Articles

High-Throughput Nanopore Fabrication and Classification Using Xe-Ion Irradiation and Automated Pore-Edge Analysis

M. Macha; S. Marion; M. Tripathi; M. Thakur; M. Lihter et al. 

Large-area nanopore drilling is a major bottleneck in state-of-the-art nanoporous 2D membrane fabrication protocols. In addition, high-quality structural and statistical descriptions of as-fabricated porous membranes are key to predicting the corresponding membrane-wide permeation properties. In this work, we investigate Xe-ion focused ion beam as a tool for scalable, large-area nanopore fabrication on atomically thin, free-standing molybdenum disulfide. The presented irradiation protocol enables designing ultrathin membranes with tunable porosity and pore dimensions, along with spatial uniformity across large-area substrates. Fabricated nanoporous membranes are then characterized using scanning transmission electron microscopy imaging, and the observed nanopore geometries are analyzed through a pore-edge detection and analysis script. We further demonstrate that the obtained structural and statistical data can be readily passed on to computational and analytical tools to predict the permeation properties at both individual pore and membrane-wide scales. As an example, membranes featuring angstrom-scale pores are investigated in terms of their emerging water and ion flow properties through extensive all-atom molecular dynamics simulations. We believe that the combination of experimental and analytical approaches presented here will yield accurate physics-based property estimates and thus potentially enable a true function-by-design approach to fabrication for applications such as osmotic power generation and desalination/filtration.

Acs Nano. 2022-09-26. Vol. 16, num. 10, p. 16249–16259. DOI : 10.1021/acsnano.2c05201.

Wafer-scale MoS2 with water-vapor assisted showerhead MOCVD

M. Macha; H. G. Ji; M. Tripathi; Y. Zhao; M. Thakur et al. 

Among numerous thin film synthesis methods, metalorganic chemical vapor deposition performed in a showerhead reactor is the most promising one for broad use in scalable and commercially adaptable two-dimensional material synthesis processes. Adapting the most efficient monolayer growth methodologies from tube-furnace systems to vertical-showerhead geometries allows us to overcome the intrinsic process limitations and improve the overall monolayer yield quality. Here, we demonstrate large-area, monolayer molybdenum disulphide growth by combining gas-phase precursor supply with unique tube-furnace approaches of utilizing sodium molybdate pre-seeding solution spincoated on a substrate along with water vapor added during the growth step. The engineered process yields a high-quality, 4-inch scale monolayer film on sapphire wafers. The monolayer growth coverage, average crystal size and defect density were evaluated using Raman and photoluminescence spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and scanning transmission electron microscopy imaging. Our findings provide a direct step forward toward developing a reproducible and large-scale MoS2 synthesis with commercial showerhead reactors.

Nanoscale Advances. 2022-09-02. DOI : 10.1039/d2na00409g.

Stress induced delamination of suspended MoS2 in aqueous environments

M. Macha; M. Thakur; A. Radenovic; S. Marion 

Applying hydrostatic pressure with suspended 2D material thin membranes allows probing the effects of lateral strain on the ion and fluid transport through nanopores. We demonstrate how both permanent and temporary delamination of 2D materials can be induced by pressure and potential differences between the membrane, causing a strong mechanosensitive modulation of ion transport. Our methodology is based on in situ measurements of ionic current and streaming modulation as the supporting membrane is indented or bulged with pressure. We demonstrate how indirect measurements of fluid transport through delaminated MoS2 membranes is achieved through monitoring streaming current and potential. This is combined with TEM images of 2D material membranes before and after aqueous measurements, showing temporary delamination during mechanical or electrical stress. The obtained results allow a better understanding of measurements with supported 2D materials, i.e. avoiding misinterpreting measured data, and could be used to probe how the electrical field and fluid flow at the nanoscale influence the adhesion of supported 2D materials.

Physical Chemistry Chemical Physics. 2022-07-29. Vol. 24, num. 33, p. 19948-19955. DOI : 10.1039/d2cp02094g.

High Performance Semiconducting Nanosheets via a Scalable Powder-Based Electrochemical Exfoliation Technique

R. A. Wells; M. Zhang; T-H. Chen; V. Boureau; M. Caretti et al. 

The liquid-phase exfoliation of semiconducting transition metal dichalcogenide (TMD) powders into 2D nanosheets represents a promising route toward the scalable production of ultrathin high-performance optoelectronic devices. However, the harsh conditions required negatively affect the semiconducting properties, leading to poor device performance. Herein we demonstrate a gentle exfoliation method employing standard bulk MoS2 powder (pressed into pellets) together with the electrochemical intercalation of a quaternary alkyl ammonium. The resulting nanosheets are produced in high yield (32%) and consist primarily of mono-, bi-, triatomic layers with large lateral dimensions (>1 mu m), while retaining the semiconducting polymorph. Exceptional optoelectronic performance of nanosheet thin-films is observed, such as enhanced photoluminescence, charge carrier mobility (up to 0.2 cm(2) V-1 s(-1) in a multisheet device), and photon-to-current efficiency while maintaining high transparency (>80%). Specifically, as a photoanode for iodide oxidation, an internal quantum efficiency up to 90% (at +0.3 V vs Pt) is achieved (compared to only 12% for MoS2 nanosheets produced via ultrasonication). Further using a combination of fluorescence microscopy and high-resolution scanning transmission electron microscopy (STEM), we show that our gently exfoliated nanosheets possess a defect density (2.33 x 10(13) cm(-2)) comparable to monolayer MoS2 prepared by vacuum-based techniques and at least three times less than ultrasonicated MoS2 nanoflakes. Finally, we expand this method toward other TMDs (WS2, WSe2) to demonstrate its versatility toward high-performance and fully scalable van der Waals heterojunction devices.

Acs Nano. 2022-03-15. Vol. 16, num. 4, p. 5719-5730. DOI : 10.1021/acsnano.1c10739.

Engineering Optically Active Defects in Hexagonal Boron Nitride Using Focused Ion Beam and Water

E. Glushkov; M. Macha; E. Raeth; V. Navikas; N. Ronceray et al. 

Hexagonal boron nitride (hBN) has emerged as a promising material platform for nanophotonics and quantum sensing, hosting optically active defects with exceptional properties such as high brightness and large spectral tuning. However, precise control over deterministic spatial positioning of emitters in hBN remained elusive for a long time, limiting their proper correlative characterization and applications in hybrid devices. Recently, focused ion beam (FIB) systems proved to be useful to engineer several types of spatially defined emitters with various structural and photophysical properties. Here we systematically explore the physical processes leading to the creation of optically active defects in hBN using FIB and find that beam-substrate interaction plays a key role in the formation of defects. These findings are confirmed using transmission electron microscopy, which reveals local mechanical deterioration of the hBN layers and local amorphization of ion beam irradiated hBN. Additionally, we show that, upon exposure to water, amorphized hBN undergoes a structural and optical transition between two defect types with distinctive emission properties. Moreover, using super-resolution optical microscopy combined with atomic force microscopy, we pinpoint the exact location of emitters within the defect sites, confirming the role of defected edges as primary sources of fluorescent emission. This lays the foundation for FIB-assisted engineering of optically active defects in hBN with high spatial and spectral control for applications ranging from integrated photonics, to nanoscale sensing, and to nanofluidics.

Acs Nano. 2022-03-22. Vol. 16, num. 3, p. 3695-3703. DOI : 10.1021/acsnano.1c07086.

Stable Al2O3 Encapsulation of MoS2 ‐FETs Enabled by CVD Grown h‐BN

A. Piacentini; D. Marian; D. S. Schneider; E. González Marín; Z. Wang et al. 

Molybdenum disulfide (MoS2) has great potential as a two-dimensional semiconductor for electronic and optoelectronic application, but its high sensitivity to environmental adsorbents and charge transfer from neighboring dielectrics can lead to device variability and instability. Aluminum oxide (Al2O3) is widely used as an encapsulation layer in (opto)-electronics, but it leads to detrimental charge transfer n-doping to MoS2. Here, this work reports a scalable encapsulation approach for MoS2 field-effect transistors (FETs) where hexagonal boron nitride (h-BN) monolayers are employed as a barrier layer in-between each of the Al2O3 and MoS2 interfaces. These devices exhibit a significant reduction of charge transfer, when compared to structures without h-BN. This benefit of h-BN in the gate stack is confirmed by ab initio density functional theory calculations. In addition, the devices with h-BN layers show very low hysteresis even under ambient operating conditions.

Advanced Electronic Materials. 2022-04-29.  p. 2200123. DOI : 10.1002/aelm.202200123.

Three-step, transfer-free growth of MoS2/WS2/graphene vertical van der Waals heterostructure

J. Sitek; I. Pasternak; K. Czerniak-Losiewicz; M. Swiniarski; P. P. Michalowski et al. 

Van der Waals heterostructures (vdWHSs) provide a unique playground to study fundamental physics and practical applications of two-dimensional (2D) materials. However, most 2D heterostructures are prepared by transfer, hindering their technological implementation. Here, we report the first chemical vapour deposition of monolayered MoS2/WS2/graphene vertical vdWHS without transfer step. By atomic force microscopy, photoluminescence, Raman spectroscopy, and secondary ion mass spectroscopy, we confirmed the vertical stacking of three different 2D materials. The use of WS2, graphene, and sapphire as growth substrates allowed us to describe the 2D materials growth process better. We determined that for the synthesis of 2D materials, only the chemical potential of the crystal formation and the substrate-layer adhesion energy are relevant factors. In addition, we used MoS2/WS2/graphene vdWHS to fabricate a photoresponsive memory device, showing the application potential of such heterostacks. Our results clarify the growth mechanisms of 2D materials and pave the way for the growth of more complex vdWHSs.

2D Materials. 2022-04-01. Vol. 9, num. 2, p. 025030. DOI : 10.1088/2053-1583/ac5f6d.

Statistical distortion of supervised learning predictions in optical microscopy induced by image compression

E. Pomarico; C. Schmidt; F. Chays; D. Nguyen; A. Planchette et al. 

The growth of data throughput in optical microscopy has triggered the extensive use of supervised learning (SL) models on compressed datasets for automated analysis. Investigating the effects of image compression on SL predictions is therefore pivotal to assess their reliability, especially for clinical use. We quantify the statistical distortions induced by compression through the comparison of predictions on compressed data to the raw predictive uncertainty, numerically estimated from the raw noise statistics measured via sensor calibration. Predictions on cell segmentation parameters are altered by up to 15% and more than 10 standard deviations after 16-to-8 bits pixel depth reduction and 10:1 JPEG compression. JPEG formats with higher compression ratios show significantly larger distortions. Interestingly, a recent metrologically accurate algorithm, offering up to 10:1 compression ratio, provides a prediction spread equivalent to that stemming from raw noise. The method described here allows to set a lower bound to the predictive uncertainty of a SL task and can be generalized to determine the statistical distortions originated from a variety of processing pipelines in AI-assisted fields.

Scientific Reports. 2022-03-02. Vol. 12, num. 1, p. 3464. DOI : 10.1038/s41598-022-07445-4.

Zero-Bias Power-Detector Circuits based on MoS2 Field-Effect Transistors on Wafer-Scale Flexible Substrates

E. Reato; P. Palacios; B. Uzlu; M. Saeed; A. Grundmann et al. 

The design, fabrication, and characterization of wafer-scale, zero-bias power detectors based on 2D MoS2 field-effect transistors (FETs) are demonstrated. The MoS2 FETs are fabricated using a wafer-scale process on 8 mu m-thick polyimide film, which, in principle, serves as a flexible substrate. The performances of two chemical vapor deposition MoS2 sheets, grown with different processes and showing different thicknesses, are analyzed and compared from the single device fabrication and characterization steps to the circuit level. The power-detector prototypes exploit the nonlinearity of the transistors above the cut-off frequency of the devices. The proposed detectors are designed employing a transistor model based on measurement results. The fabricated circuits operate in the Ku-band between 12 and 18 GHz, with a demonstrated voltage responsivity of 45 V W-1 at 18 GHz in the case of monolayer MoS2 and 104 V W-1 at 16 GHz in the case of multilayer MoS2, both achieved without applied DC bias. They are the best-performing power detectors fabricated on flexible substrate reported to date. The measured dynamic range exceeds 30 dB, outperforming other semiconductor technologies like silicon complementary metal-oxide-semiconductor circuits and GaAs Schottky diodes.

Advanced Materials. 2022-02-17.  p. 2108469. DOI : 10.1002/adma.202108469.

Conference Papers

Bacterial nanopores open the future of data storage

C. Cao; L. F. Krapp; A. Agerova; A. Al Ouahabi; A. Radenovic et al. 

In the era of “big data” finding solutions for data storage alternative to those based on silicon or magnetic tapes is an urgent need for our society. The development of polymers that can store information at the molecular level has opened up new opportunities for ultrahigh density data storage, long-term archival, anti-counterfeiting systems and molecular cryptography. Biological pores of bacterial origin hold the promise to accurately decode the digital information encoded in tailored-made polymers opening up promising possibilities to develop writing-reading technologies to process digital data using a biological-inspired platform.

2022-01-01. International Electron Devices Meeting (IEDM), San Francisco, CA, Dec 03-07, 2022. DOI : 10.1109/IEDM45625.2022.10019421.


2D MoS2 Nanopores: Wafer-scale Fabrication and Monolayer Stability for Long-term Single-Molecule Sensing

M. Thakur / A. Radenovic (Dir.)  

Biologically inspired solid-state nanopores are artificial openings or apertures in thin membranes similar to natural protein ion channels in a lipid bilayer of cell membranes. In solid-state nanopores, a thin insulating membrane with single or multiple pores separates two conductive salt solutions. When an electric field is applied across this membrane, electrically charged species such as ions pass through these nanopore(s), generating a nanopore ion current. In essence, nanopores are single-molecule sensors and valuable tools for studying biophysics. For instance, an intrinsically charged biomolecule such as DNA can be electrophoretically threaded through the nanopore, transiently blocking the ionic current characteristic of the molecule passing through the pore. With progress in two-dimensional (2D) materials, the marriage of nanopores with 2D materials – “2D nanopores” have emerged as a new class of ultra-thin membrane solid-state nanopores. Molybdenum disulfide (MoS2) is a 2D material with an atomic thickness (0.7 nm) that approaches the inter-base distance of two DNA bases and is a lucrative 2D material for the DNA sequencing application. However, there are inherent challenges and bottlenecks with using MoS2 due to sensitive fabrication and inherent challenges of the 2D materials leading to low device yield. In this thesis, I will demonstrate ways to improve high-throughput production and the development of more reliable and durable nanopore devices. In the second chapter, I will introduce MoS2 material as a 2D nanopore system and elaborate fabrication of nanopore substrates. I will discuss various problems and issues related to substrate fabrication, transfer, nanopore-creation, and nanopore measurements. Finally, I list a step-by-step protocol and troubleshooting guide for early-stage 2D nanopore researchers. In the third chapter, I specifically focus on “chip-scale” transfer strategies for MoS2 grown using chemical vapor deposition (CVD). I introduce two transfer approaches – direct-transfer and stamp-assisted- with just water as a medium. I will demonstrate these transfer approaches and discuss their advantages and limitations. Furthermore, I discuss hydrocarbon contamination with 2D materials and their implications in nanofluidics. In the fourth chapter, I demonstrate a scalable transfer from chip-scale to a larger “wafer-scale” for batch fabrication of nanopore substrates for single-molecule DNA sensing. With PDMS-based polymer, I will demonstrate 3-inch monolayer MoS2 transfer on nanopore substrates with 128 nanopore devices with high transfer efficiency (>70%). Moreover, the technique is etchant-free, and growth substrates are recyclable after transfer. In the fifth chapter, I study nanopore instability and address those issues. I address the delamination issue by chemically modifying Si-substrates with an organosilicon that increases the adherence of monolayer MoS2 layers. This surface pre-treatment helped reinforce 2D layer attachment to the substrate, increasing the nanofluidic devices’ durability. Further, we show that the nanopore enlargement due to dissolved oxygen in an aqueous solution can be considerably reduced in a low dissolved oxygen concentration in solution. These strategies improved MoS2 nanopore stability and enabled long-term DNA sensing. This will pave way toward more durable 2D nanopore sensors.

Lausanne, EPFL, 2022. 

Molybdenum disulphide nanoporous membranes as nanofluidic platforms – large-area engineering and study

M. D. Macha / A. Radenovic (Dir.)  

The work demonstrated in this thesis represent the path towards developing the large-scale fabrication of two-dimensional nanoporous membrane devices and use thereof as platforms to study nanoscale physics of water and ion flow through confined channels. Methods developed here are used to fabricate molybdenum disulfide (MoS2) membrane devices and investigate the fundamental physics of nanopore systems and thin films in aqueous solutions. In particular, the second chapter presents the accomplishments in the large-area MoS2 synthesis via chemical vapor deposition. Process modifications such as the use of spin-coated precursors/growth promoters and addition of active gases such as H2O vapor or moderate amount of O2 enable to scale up the growth reaction into the synthesis of a continuous monolayer films on 2-, 3- and 4-inch substrates. Third chapter shows the application of focused ion beam irradiation for engineering, imaging and studying optically active defects in MoS2 and hexagonal boron nitride films. On top of that, the ion irradiation protocol was used for establishing the tunable and large-throughout nanopore drilling process on suspended MoS2 membranes. With the high-resolution, scanning electron transmission microscopy, nanopores were investigated with an analysis script and classified based on the pore geometries and edge composition. Coupled with molecular dynamics simulations the membranes populated with ~1nm nanopores were assessed in the context of emerging ion- and water- permeation properties. The last, fourth chapter presents the application of nanoporous membranes discussed in previous chapters in the nanofluidics study application. A particular emphasis is put on the application of hydrostatic pressure in nanopore experiments as an additional measurement probe. Experimental results shown in this chapter uncover the non-linear signals originating from nanobubbles pinning, improper wetting and membrane adhesion issues. With the assistance of hydrostatic pressure, the nanoporous MoS2 suspended membranes are then further investigated to explore the ion transport properties and nanopores behaviour under applied strain in the context of artificial mechanosensing as well as osmotic power generation in salinity gradients. The ion irradiated, atomically thin films proved to be a highly attractive membrane materials and demonstrated a great promise in broad nanofluidic applications. Insights acquired during the thesis study were used to advance the state-of-art knowledge in field of the synthesis and application of 2D membranes in the nanofluidics research.

Lausanne, EPFL, 2022. 

Multidisciplinary Investigation of the Gut-Brain Ecosystem in a Model of Alzheimer’s Disease

A. L. Planchette / A. Radenovic; A. J. Macpherson (Dir.)  

The search for an understanding of the causal elements that lead to neurodegenerative diseases has motivated researchers for decades. Today, Alzheimer’s disease is the most prevalent form of dementia and affects approximately fifty million people worldwide, with only 5% of patients carrying causal genetic mutations. In recent years, studies of animal models as well as observations in humans have brought the gut microbiome to the forefront of AD research, as a significant contributor to the development of Alzheimer’s disease. In fact, the gut microbiome is now understood to be a modulator of health and disease in numerous areas of physiology, including energy metabolism, immunity, endocrinology, and most relevant to this thesis, brain health and cognition. The gut microbiome is a complex environment composed of symbiotic bacteria, fungi and viruses (the microbiota) that produce a wide range of signalling molecules that are interpreted both by members of the microbiota and by the host (the microbiome). The host-gut interface is an ecosystem that consists of bi-directional signals and pressures applied by both constituents in order to maintain symbiotic homeostasis. Significant deleterious effects on health occur when homeostasis is lost, which may take place due to a variety of factors. These include host genetic factors and environmental factors, such as diet. Modulation of the gut microbiome has become a potentially viable therapeutic avenue for Alzheimer’s disease, though it currently remains at the early stage of discovery. In this thesis, I characterize the evolution of Alzheimer’s disease in the 5xFAD mouse model, with a focus on the gut microbiome and on cognitive decline and brain biochemistry. Gut microbiota modulaiton was implemented using germ-free and low-complexity bacterial cocktails, in which I outline the effects microbiota modulation on hallmarks of AD and provide insights into the metabolite profile of the brain altered by the microbiota and AD. The work in this thesis was equally motivated by the development of diagnostic tools using microscopy techniques. Biochemical assessments of disease status and progression provide information pertaining to “what” is interacting and “how much” this affects disease. With microscopy, we may gain the additional knowledge of “where” physiological constituents are and how they interact with the surrounding environment. I describe a novel multi-modal microscopy pipeline that enables the observation of signals of interest in large volumes and at high resolution with a capacity to cross-reference regions of interest in both modalities. The pipeline, known as gutOPT, consists of a sample preparation workflow compatible with mesoscale optical projection tomography (OPT) of fluorescently-labelled targets and subsequent imaging of pre-selected regions of interest in high-resolution modalities, such as confocal microscopy.

Lausanne, EPFL, 2022. 


Journal Articles

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics

S. M. Leitao; B. Drake; K. Pinjusic; X. Pierrat; V. Navikas et al. 

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

Acs Nano. 2021-11-23. Vol. 15, num. 11, p. 17613-17622. DOI : 10.1021/acsnano.1c05202.

Rhesus Blood Typing within a Few Seconds by Packing-Enhanced Nanoscattering on Individual Erythrocytes

-S. Chen; S. J. Davis; M-L. Chang; C-H. Hung; A. Radenovic et al. 

A method for the ABO and Rhesus (Rh) blood group typing from individual erythrocytes is proposed in this study. Blood-group-specific antibodies immobilized to gold nanoparticles (BG-AuNP) were utilized for the identification of blood groups from individual erythrocytes by objective-type dark-field microscopy (OTDFM). The scattering of free BG-AuNP and their Brownian motion as well as BG-AuNP attached on erythrocytes is easily observed by OTDFM. The strong scattering intensity caused by BG-AuNP packing-enhanced nanoscattering (PENS) on erythrocytes is first demonstrated. PENS combined with OTDFM allows us to identify blood groups within 5 s for all blood group antigens including A, B, D, C, c, E, and e. This was immediately identified by mixing with BG-AuNP without any washing step or waiting for hemoagglutination. Therefore, PENS combined with OTDFM demonstrates feasibility and advantages for use in emergency transfusions where the blood group of patients is unknown. Moreover, matching RhD+ in the case of emergency transfusions may also be beneficial in reducing the shortage of RhD- red blood cell concentrate in the case of a population with a high frequency in RhD-.

Analytical Chemistry. 2021-11-16. Vol. 93, num. 45, p. 15142-15149. DOI : 10.1021/acs.analchem.1c03590.

Superconducting 2D NbS2 Grown Epitaxially by Chemical Vapor Deposition

Z. Wang; C-Y. Cheon; M. Tripathi; G. M. Marega; Y. Zhao et al. 

Metallic two-dimensional (2D) transition metal dichalcogenides (TMDCs) are attracting great attention because of their interesting low-temperature properties such as superconductivity, magnetism, and charge density waves (CDW). However, further studies and practical applications are being slowed down by difficulties in synthesizing high-quality materials with a large grain size and well-determined thickness. In this work, we demonstrate epitaxial chemical vapor deposition (CVD) growth of 2D NbS2 crystals on a sapphire substrate, with a thickness-dependent structural phase transition. NbS2 crystals are epitaxially aligned by the underlying c-plane sapphire resulting in high-quality growth. The thickness of NbS2 is well controlled by growth parameters to be between 1.5 and 10 nm with a large grain size of up to 500 μm. As the thickness increases, we observe in our NbS2 a transition from a metallic 3R-polytype to a superconducting 2H-polytype, confirmed by Raman spectroscopy, aberration-corrected scanning transmission electron microscopy (STEM) and electrical transport measurements. A Berezinskii–Kosterlitz–Thouless (BKT) superconducting transition occurs in the CVD-grown 2H-phase NbS2 below the transition temperature (Tc) of 3 K. Our work demonstrates thickness and phase-controllable synthesis of high-quality superconducting 2D NbS2, which is imperative for its practical applications in next-generation TMDC-based electrical devices.

ACS Nano. 2021-11-10. Vol. 15, num. 11, p. 18403–18410. DOI : 10.1021/acsnano.1c07956.

Anomalous interfacial dynamics of single proton charges in binary aqueous solutions

J. Comtet; A. Rayabharam; E. Glushkov; M. Zhang; A. Avsar et al. 

Our understanding of the dynamics of charge transfer between solid surfaces and liquid electrolytes has been hampered by the difficulties in obtaining interface, charge, and solvent-specific information at both high spatial and temporal resolution. Here, we measure at the single charge scale the dynamics of protons at the interface between an hBN crystal and binary mixtures of water and organic amphiphilic solvents (alcohols and acetone), evidencing a marked influence of solvation on interfacial dynamics. Applying single-molecule localization microscopy to emissive crystal defects, we observe correlated activation between adjacent ionizable surface defects, mediated by the transport of single excess protons along the solid/liquid interface. Solvent content has a nontrivial effect on interfacial dynamics, leading at intermediate water fraction to an increased surface diffusivity, as well as an increased affinity of the proton charges to the solid surface. Our measurements evidence the notable role of solvation on interfacial proton charge transport.

Science Advances. 2021-10-01. Vol. 7, num. 40, p. eabg8568. DOI : 10.1126/sciadv.abg8568.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy

P. Werther; K. Yserentant; F. Braun; K. Grussmayer; V. Navikas et al. 

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Acs Central Science. 2021-09-22. Vol. 7, num. 9, p. 1561-1571. DOI : 10.1021/acscentsci.1c00703.

Experimental Combination of Super-Resolution Optical Fluctuation Imaging with Structured Illumination Microscopy for Large Fields-of-View

A. C. Descloux; K. S. Grussmayer; V. Navikas; D. Mahecic; S. Manley et al. 

All fluorescence super-resolution microscopy techniques present trade-offs between, for example, resolution, acquisition speed, and live-cell compatibility. Structured illumination microscopy (SIM) improves the resolution through successive imaging of the sample under patterned illumination. SIM can be fast and typically uses low light levels well suited for live cell imaging. However, in its linear form, the resolution gain of SIM is limited by the pattern frequency to a 2-fold improvement over the diffraction limit. Super-resolution optical fluctuation imaging (SOFI) is another low-light level method that achieves higher resolution through the computation of spatiotemporal cross-cumulants of a time series of stochastically blinking fluorescent emitters. The resolution is theoretically enhanced by a factor n, where n is the cumulant order. In practice, it is restricted to smaller orders due to limited signal-to-noise and the need for many frames for good statistics. Here, we demonstrate the experimental combination of SOFT with SIM, where we use SOFI as a source of nonlinearity to further enhance the SIM resolution. We present two implementations of SIM combined with self-blinking dyes for SOFT. We first introduce a new Michelson SIM setup for achromatic high-efficiency (40%) illumination and fast structured pattern projection. We use the setup to acquire single- and two-color SIM data of blinking emitters with up to 2.4-fold image resolution increase and discuss the SOFI-SIM reconstruction challenges. We applied the same concept to realize SOFI-SIM on a flat-fielded, high-throughput instant SIM (iSIM) setup, achieving similar resolution enhancement and demonstrating the versatility of our approach. We established an experimental proof-of-principle of a wide-field combination of SOFT with SIM and iSIM for large fields-of-view, improving SIM resolution without increased complexity of the setup.

Acs Photonics. 2021-08-18. Vol. 8, num. 8, p. 2440-2449. DOI : 10.1021/acsphotonics.1c00668.

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

V. Navikas; S. Mendes Leitão; K. S. Grussmayer; A. C. Descloux; B. F. Drake et al. 

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution.

Nature Communications. 2021-07-27. Vol. 12, num. 1, p. 4565. DOI : 10.1038/s41467-021-24901-3.

Direct Growth of Hexagonal Boron Nitride on Photonic Chips for High-Throughput Characterization

E. Glushkov; N. Mendelson; A. Chernev; R. Ritika; M. Lihter et al. 

Adapting optical microscopy methods for nanoscale characterization of defects in two-dimensional (2D) materials is a vital step for photonic on-chip devices. To increase the analysis throughput, waveguide-based on-chip imaging platforms have been recently developed. Their inherent disadvantage, however, is the necessity to transfer the 2D material from the growth substrate to the imaging chip, which introduces nonuniform material coverage and contamination, potentially altering the characterization results. Here we present a unique approach to circumvent these shortfalls by directly growing a widely used 2D material (hexagonal boron nitride, hBN) on silicon nitride chips and optically characterizing the defects in the intact as-grown material. We compare the direct growth approach to the standard PMMA-assisted wet transfer method and confirm the clear advantages of the direct growth. While demonstrated with hBN in the current work, the method can be extended to other 2D materials.

Acs Photonics. 2021-07-21. Vol. 8, num. 7, p. 2033-2040. DOI : 10.1021/acsphotonics.1c00165.

High resolution optical projection tomography platform for multispectral imaging of the mouse gut

C. Schmidt; A. L. Planchette; D. Nguyen; G. Giardina; Y. Neuenschwander et al. 

Optical projection tomography (OPT) is a powerful tool for three-dimensional imaging of mesoscopic biological samples with great use for biomedical phenotyping studies. We present a fluorescent OPT platform that enables direct visualization of biological specimens and processes at a centimeter scale with high spatial resolution, as well as fast data throughput and reconstruction. We demonstrate nearly isotropic sub-28 mu m resolution over more than 60 mm 3 after reconstruction of a single acquisition. Our setup is optimized for imaging the mouse gut at multiple wavelengths. Thanks to a new sample preparation protocol specifically developed for gut specimens, we can observe the spatial arrangement of the intestinal villi and the vasculature network of a 3-cm long healthy mouse gut. Besides the blood vessel network surrounding the gastrointestinal tract, we observe traces of vasculature at the villi ends close to the lumen. The combination of rapid acquisition and a large field of view with high spatial resolution in 3D mesoscopic imaging holds an invaluable potential for gastrointestinal pathology research. (C) 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Biomedical Optics Express. 2021-06-01. Vol. 12, num. 6, p. 3619-3629. DOI : 10.1364/BOE.423284.

Adaptive optics enables multimode 3D super-resolution microscopy via remote focusing

V. Navikas; A. C. Descloux; K. S. Grussmayer; S. Marion; A. Radenovic 

A variety of modern super-resolution micro-scopy methods provide researchers with previouslyinconceivable biological sample imaging opportunities ata molecular resolution. All of these techniques excel atimaging samples that are close to the coverslip, howeverimaging at large depths remains a challenge due to aber-rations caused by the sample, diminishing the resolution ofthe microscope. Originating in astro-imaging, the adaptiveoptics (AO) approach for wavefront shaping using adeformable mirror is gaining momentum in modern mi-croscopy as a convenient approach for wavefront control.AO has the ability not only to correct aberrations but alsoenables engineering of the PSF shape, allowing localiza-tion of the emitter axial position over several microns. Inthis study, we demonstrate remote focusing as another AO benefit for super-resolution microscopy. We show theability to record volumetric data (45×45×10 μm), whilekeeping the sample axially stabilized using a standard widefield setup with an adaptive optics addon. We processed the data with single-molecule localization routines and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution.

Nanophotonics. 2021-06-10. Vol. 10, num. 9, p. 2451-2458. DOI : 10.1515/nanoph-2021-0108.

Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

A. C. Descloux; K. S. Grussmayer; A. Radenovic 

Localization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

Communications Biology. 2021-05-11. Vol. 4, num. 1, p. 550. DOI : 10.1038/s42003-021-02086-1.

Super-resolved Optical Mapping of Reactive Sulfur-Vacancies in Two-Dimensional Transition Metal Dichalcogenides

M. Zhang; M. Lihter; T-H. Chen; M. Macha; A. Rayabharam et al. 

Transition metal dichalcogenides (TMDs) represent a class of semiconducting two-dimensional (2D) materials with exciting properties. In particular, defects in 2D-TMDs and their molecular interactions with the environment can crucially affect their physical and chemical properties. However, mapping the spatial distribution and chemical reactivity of defects in liquid remains a challenge. Here, we demonstrate large area mapping of reactive sulfur-deficient defects in 2D-TMDs in aqueous solutions by coupling single-molecule localization microscopy with fluorescence labeling using thiol chemistry. Our method, reminiscent of PAINT strategies, relies on the specific binding of fluorescent probes hosting a thiol group to sulfur vacancies, allowing localization of the defects with an uncertainty down to 15 nm. Tuning the distance between the fluorophore and the docking thiol site allows us to control Foster resonance energy transfer (FRET) process and reveal grain boundaries and line defects due to the local irregular lattice structure. We further characterize the binding kinetics over a large range of pH conditions, evidencing the reversible adsorption of the thiol probes to the defects with a subsequent transitioning to irreversible binding in basic conditions. Our methodology provides a simple and fast alternative for large-scale mapping of nonradiative defects in 2D materials and can be used for in situ and spatially resolved monitoring of the interaction between chemical agents and defects in 2D materials that has general implications for defect engineering in aqueous condition.

Acs Nano. 2021-04-27. Vol. 15, num. 4, p. 7168-7178. DOI : 10.1021/acsnano.1c00373.

From Water Solutions to Ionic Liquids with Solid State Nanopores as a Perspective to Study Transport and Translocation Phenomena

S. Marion; N. Vucemilovic-Alagic; M. Spadina; A. Radenovic; A-S. Smith 

Solid state nanopores are single-molecular devices governed by nanoscale physics with a broad potential for technological applications. However, the control of translocation speed in these systems is still limited. Ionic liquids are molten salts which are commonly used as alternate solvents enabling the regulation of the chemical and physical interactions on solid-liquid interfaces. While their combination can be challenging to the understanding of nanoscopic processes, there has been limited attempts on bringing these two together. While summarizing the state of the art and open questions in these fields, several major advances are presented with a perspective on the next steps in the investigations of ionic-liquid filled nanopores, both from a theoretical and experimental standpoint. By analogy to aqueous solutions, it is argued that ionic liquids and nanopores can be combined to provide new nanofluidic functionalities, as well as to help resolve some of the pertinent problems in understanding transport phenomena in confined ionic liquids and providing better control of the speed of translocating analytes.

Small. 2021-05-06.  p. 2100777. DOI : 10.1002/smll.202100777.

Electrochemical Functionalization of Selectively Addressed MoS2 Nanoribbons for Sensor Device Fabrication

M. Lihter; M. Graf; D. Ivekovic; M. Zhang; T-H. Shen et al. 

Tailoring the surface properties of 2D materials, such as transition metal dichalcogenides (TMDCs), at the nanoscale is becoming essential in the fabrication of various 2D material-based nanoelectronic devices. Due to the chemical inertness of their basal plane, the surface modification of 2D TMDCs is limited to their defective sites, often requiring special treatments, such as the conversion of the TMDC from its semiconducting into its metallic phase. In this work, we show that the basal plane of a semiconducting 2D TMDC, molybdenum disulfide (MoS2) can be modified electrochemically by electrografting of aryl-diazonium salt. To demonstrate the advantages of this method at the nanoscale, we perform electrografting of 3,5-bis(trifluoromethyl)benzenediazonium tetrafluoroborate on predefined MoS2 nanoribbons by addressing them individually via a different electrode. The ability to selectively address individually contacted 2D layers opens the possibility for specific surface modification of neighboring 2D nanostructures by different functional groups. This method could be extended to other aryl-diazonium compounds, and other 2D semiconducting materials.

Acs Applied Nano Materials. 2021-02-26. Vol. 4, num. 2, p. 1076-1084. DOI : 10.1021/acsanm.0c02628.

Wetting of nanopores probed with pressure

S. Marion; M. Macha; S. J. Davis; A. Chernev; A. Radenovic 

Nanopores are both a tool to study single-molecule biophysics and nanoscale ion transport, but also a promising material for desalination or osmotic power generation. Understanding the physics underlying ion transport through nano-sized pores allows better design of porous membrane materials. Material surfaces can present hydrophobicity, a property which can make them prone to formation of surface nanobubbles. Nanobubbles can influence the electrical transport properties of such devices. We demonstrate an approach which uses hydraulic pressure to probe the electrical transport properties of solid state nanopores. We show how pressure can be used to wet pores, and how it allows control over bubbles or other contaminants in the nanometer scale range normally unachievable using only an electrical driving force. Molybdenum disulfide is then used as a typical example of a 2D material on which we demonstrate wetting and bubble induced nonlinear and linear conductance in the regimes typically used with these experiments. We show that by using pressure one can identify and evade wetting artifacts.

Physical Chemistry Chemical Physics. 2021-02-28. Vol. 23, num. 8, p. 4975-4987. DOI : 10.1039/d1cp00253h.

Conference Papers

Decoding Digital Information Stored in Polymer by Nanopore

C. Cao; L. Krapp; A. Ouahabi; A. Radenovic; J-F. Lutz et al. 

2021-02-12.  p. 98A-98A. DOI : 10.1016/j.bpj.2020.11.802.


Exploring optically active defects in wide-bandgap materials using fluorescence microscopy

E. Glushkov / A. Radenovic (Dir.)  

Defects in solid-state systems can be both detrimental, deteriorating the quality of materials, or desired, thanks to the novel functionality they bring. Optically active point defects, producing fluorescent light, are a great example of the latter. Naturally existing in various materials, of which the so-called wide-bandgap materials constitute a major part, they can be used as sensors, single-photon emitters or even quantum bits. As the defects preferentially absorb only specific wavelengths of light, the whole material can acquire a visible macroscopic color, becoming the more intense the more there are defects in its lattice. Due to this fact such defects are commonly referred to as “color centers”. The most famous example of color centers is the nitrogen-vacancy (NV) center in diamond, consisting of a nitrogen atom that substitutes carbon next to a vacancy (a missing carbon atom) in the diamond lattice. From the 1990s NV centers have been at the forefront of the second quantum revolution, enabling countless experimental demonstrations of quantum phenomena, even at room temperature. Being currently widely used everywhere from secured telecommunication networks to living cells, diamond NV centers have sparked a persistent interest into novel fluorescent defects, both in diamond (e.g. silicon-, germanium- and tin-vacancy centers) and in other wide-bandgap materials. Very recently a novel class of material platforms hosting fluorescent defects has emerged — namely layered van der Waals (vdW) materials, which can be thinned down to an ultimate single-atom thickness, opening the door into the realm of two dimensional (2D) materials. This area of research has virtually exploded after the discovery of graphene in 2004, followed by continuous reports of the superb mechanical, electrical and optical properties of graphene-based devices. The whole family of graphene-like vdW materials was rapidly and continuously expanding with new members (graphene oxide, fluorographene, borophene, transition-metal dichalcogenides (TMDCs), layered perovskites, etc.), each of which was enabling various functionalities. This thesis explores the properties of newly discovered color centers in a layered vdW wide-bandgap semiconductor – hexagonal boron nitride (hBN). These optically-active defects have shown themselves as exceptionally bright single-photon emitters (SPEs) and optically-addressable spin defects that hold a great promise for quantum sensing and quantum information processing. In this work I have shown how optical super-resolution techniques (specifically, the single-molecule localization microscopy, SMLM) can be used to study the properties of emitters in hBN, including their spectra and temporal dynamics. By engineering a specialized waveguide-based imaging platform I managed to overcome certain limitations of SMLM-based imaging and further showed how the very same imaging platform can be used for the nanophotonic on-chip integration of hBN via direct growth. In addition, I have explored the behaviour of hBN defects in aqueous solutions and how there they can be used as nanoscale charge sensors, tracking the diffusion of single protons. Finally, I developed a novel method for the deterministic engineering of optically-active defects in hBN via focused ion beam (FIB) irradiation. All together these findings pave the way for the use of optically-active defects in hBN for applications in nanophotonics, nanofluidics and nanoscale sensing

Lausanne, EPFL, 2021. 

Biophysical applications of correlative scanning probe and super-resolution microscopy

V. Navikas / A. Radenovic (Dir.)  

Imaging live cells in their native environment is crucial for the understanding of complex biological phenomena. Modern optical microscopy methods such as fluorescence super-resolution microscopy are increasingly combined with complementary, label-free techniques to put the high-resolution fluorescence information into cellular context. Common high-resolution imaging approaches used in combination with fluorescence imaging such as electron microscopy and atomic force microscopy (AFM) were originally developed for solid-state material characterization and thus are not straightforwardly applicable to image live cells for sustained periods of time. In this thesis, I am focusing on advancing state-of-the-art correlative scanning ion-conductance microscopy (SICM) combined with novel super-resolution imaging techniques. The work presented in this thesis demonstrates various technological advancements for both imaging modalities as well as experimental proof-of-principle studies. The first part of the work focuses on a novel experimental combination of SICM and super-resolution optical fluctuation imaging for single-cell imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. We employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction resolution. In order to increase the imaging depth for thick samples, a novel remote focusing modality based on an adaptive optics device was developed. Here, we use a deformable mirror to acquire multiple image planes of the labelled cells and we process the data using a single-molecule localization routine and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution within a whole imaging volume. Second, we demonstrate the advantages of a new SICM probe manufacturing method allowing the batch production of glass nanopipettes used for SICM imaging and sensing. Finally, we show a new application of SICM tailored for single-molecule characterization based on electrical signatures while performing controlled translocations of surface-immobilized DNA molecules. The method is developed to be compatible with single-molecule fluorescence imaging and was applied for the detection of various analytes in a correlative manner. We believe that a synergy between fundamentally different imaging modalities is going to become necessary in an age of computational microscopy to ensure an unbiased data interpretation in the rapidly progressing field of biological research.

Lausanne, EPFL, 2021. 


Journal Articles

Microscopic Detection Analysis of Single Molecules in MoS2 Membrane Nanopores

M. Xiong; M. Graf; N. Athreya; A. Radenovic; J-P. Leburton 

A systematic microscopic analysis of the various resistive effects involved in the electronic detection of single biomolecules in a nanopore of a MoS2 nanoribbon is presented. The variations of the transverse electronic current along the two-dimensional (2D) membrane due to the translocation of DNA and protein molecules through the pore are obtained by model calculations based on molecular dynamics (MD) and Boltzmann transport formalism, which achieved good agreement with the experimental data. Our analysis points to a self-consistent interaction among ions, charge carriers around the pore rim, and biomolecules. It provides a comprehensive understanding of the effects of the electrolyte concentration, pore size, nanoribbon geometry, and also the doping polarity of the nanoribbon on the electrical sensitivity of the nanopore in detecting biomolecules. These results can be utilized for fine-tuning the design parameters in the fabrication of highly sensitive 2D nanopore biosensors.

ACS Nano. 2020-11-06. Vol. 14, num. 11, p. 16131-16139. DOI : 10.1021/acsnano.0c08382.

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

V. Navikas; S. M. Leitão; 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. 2020-07-02. Vol. 3, num. 8, p. 7829-7834. DOI : 10.1021/acsanm.0c01345.

Prospects of Observing Ionic Coulomb Blockade in Artificial Ion Confinements

A. Chernev; S. Marion; A. Radenovic 

Nanofluidics encompasses a wide range of advanced approaches to study charge and mass transport at the nanoscale. Modern technologies allow us to develop and improve artificial nanofluidic platforms that confine ions in a way similar to single-ion channels in living cells. Therefore, nanofluidic platforms show great potential to act as a test field for theoretical models. This review aims to highlight ionic Coulomb blockade (ICB)-an effect that is proposed to be the key player of ion channel selectivity, which is based upon electrostatic exclusion limiting ion transport. Thus, in this perspective, we focus on the most promising approaches that have been reported on the subject. We consider ion confinements of various dimensionalities and highlight the most recent advancements in the field. Furthermore, we concentrate on the most critical obstacles associated with these studies and suggest possible solutions to advance the field further.

Entropy. 2020-12-01. Vol. 22, num. 12, p. 1430. DOI : 10.3390/e22121430.

Aerolysin nanopores decode digital information stored in tailored macromolecular analytes

C. Cao; L. F. Krapp; A. Al Ouahabi; N. F. König; N. Cirauqui et al. 

Digital data storage is a growing need for our society and finding alternative solutions than those based on silicon or magnetic tapes is a challenge in the era of “big data.” The recent development of polymers that can store information at the molecular level has opened up new opportunities for ultrahigh density data storage, long-term archival, anticounterfeiting systems, and molecular cryptography. However, synthetic informational polymers are so far only deciphered by tandem mass spectrometry. In comparison, nanopore technology can be faster, cheaper, nondestructive and provide detection at the single-molecule level; moreover, it can be massively parallelized and miniaturized in portable devices. Here, we demonstrate the ability of engineered aerolysin nanopores to accurately read, with single-bit resolution, the digital information encoded in tailored informational polymers alone and in mixed samples, without compromising information density. These findings open promising possibilities to develop writing-reading technologies to process digital data using a biological-inspired platform.

Science Advances. 2020-12-09. Vol. 6, num. 50, p. eabc2661. DOI : 10.1126/sciadv.abc2661.

Pressure-Induced Enlargement and Ionic Current Rectification in Symmetric Nanopores

S. J. Davis; M. Macha; A. Chernev; D. M. Huang; A. Radenovic et al. 

Nanopores in solid state membranes are a tool able to probe nanofluidic phenomena or can act as a single molecular sensor. They also have diverse applications in filtration, desalination, or osmotic power generation. Many of these applications involve chemical, or hydrostatic pressure differences which act on both the supporting membrane, and the ion transport through the pore. By using pressure differences between the sides of the membrane and an alternating current approach to probe ion transport, we investigate two distinct physical phenomena: the elastic deformation of the membrane through the measurement of strain at the nanopore, and the growth of ionic current rectification with pressure due to pore entrance effects. These measurements are a significant step toward the understanding of the role of elastic membrane deformation or fluid flow on linear and nonlinear transport properties of nanopores.

Nano Letters. 2020-11-11. Vol. 20, num. 11, p. 8089-8095. DOI : 10.1021/acs.nanolett.0c03083.

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.

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.

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, p. 3023. DOI : 10.1038/s41467-020-16841-1.

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.

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.

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

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

Super-resolution microscopy of defects in a two-dimensional material unveils the transport of single proton charges at solid/water interfaces. 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. Vol. 15, p. 598–604. 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.

High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

A. Descloux; M. Mueller; V. Navikas; A. Markwirth; R. Van den Eynde et al. 

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with highspeed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.

Nanophotonics. 2020-01-01. Vol. 9, num. 1, p. 143-148. DOI : 10.1515/nanoph-2019-0346.


Recent Advances and Prospects in the Research of Nascent Adhesions

B. H. Stumpf; A. Ambriovic-Ristov; A. Radenovic; A-S. Smith 

Nascent adhesions are submicron transient structures promoting the early adhesion of cells to the extracellular matrix. Nascent adhesions typically consist of several tens of integrins, and serve as platforms for the recruitment and activation of proteins to build mature focal adhesions. They are also associated with early stage signaling and the mechanoresponse. Despite their crucial role in sampling the local extracellular matrix, very little is known about the mechanism of their formation. Consequently, there is a strong scientific activity focused on elucidating the physical and biochemical foundation of their development and function. Precisely the results of this effort will be summarized in this article.

Frontiers In Physiology. 2020-12-04. Vol. 11, p. 574371. DOI : 10.3389/fphys.2020.574371.


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. 

Development of novel experimental and computational methods for three-dimensional coherent and super-resolution microscopy

A. C-F. R. Descloux / A. Radenovic (Dir.)  

Optical microscopy is one widely used tool to study cell functions and the interaction of molecules at a sub-cellular level. Optical microscopy techniques can be broadly divided into two categories: partially coherent and incoherent. Coherent microscopy techniques are usually label-free and provide diffraction limited structural information about the sample refractive index distribution though the measurement of phase delay induced by the sample. Since they can be very conservative in the illumination power directed toward the sample, they exhibit very low photo-toxicity and are suitable for both high speed and time lapse imaging. Fluorescence microscopy is an incoherent microscopy method which uses biochemistry techniques to label cellular structures with fluorescent molecules which emit light when excited by a laser. Fluorescence microscopy provides diffraction limited high specificity imaging but is limited in time due to photo-bleaching and photo-toxicity. Super-resolution microscopy is a sub-category of fluorescence microscopy which manipulates and exploits some properties of the fluorescent molecules to achieve high specificity sub-diffraction imaging. Super-resolution comes however at the price of an increased total acquisition time, which limits the applications of super-resolution microscopy to relatively slow cellular processes. The ideal microscope however does not exist; due to the limited spatio-temporal bandwidth of far-field microscopy, there will always be unavoidable trade-offs. There is therefore a need to find new ways to ensure that the methods are reaching their optimal performances and a need to study how different methods can complement each others. I start by presenting a new method for three-dimensional quantitative phase retrieval. I derive a model for the image formation of three-dimensional bright field images and extrapolate a novel expression allowing to retrieve the phase distribution from a bright field image stack. Using a unique image-splitting multi-plane prism that allows to acquire 8 distinct focal planes in a single exposure, I demonstrate three-dimensional quantitative phase imaging at 200 Hz . Finally, I show the association of three-dimensional super-resolution SOFI with phase imaging. To improve the imaging speed and lower the illumination intensity, I combine the same prism platform with a high-speed structured illumination. Since the structured illumination presented is using a digital micro-mirror device, about 90 \% of the laser light is diffracted outside of the optical path. To improve the illumination efficiency but keep its speed and flexibility, I present a new approach for achromatic high power high speed SIM, based on a Michelson interferometer. With the access to high illumination power density, I also show the first experimental combination of SIM with SOFI using a self-blinking dye. Motivated by the absence of tools to objectively judge the performance of the microscopy methods I develop, I present a novel algorithm for image resolution estimation. The method estimate the resolution by correlating the image with several filtered version of itself without any external parameters. Finally, in the context of the AD-gut consortium, I show a practical application of deep neural networks used to assists the segmentation and mapping of the microscopy image of enzymatically labeled DNA molecules.

Lausanne, EPFL, 2020. 


Benchmarking of single imaging datasets

A. Descloux; A. Radenovic 

A method of estimating a value (311-313) indicative of a signal component of an input imaging dataset (190) includes determining a normalized Fourier-domain representation of the input imaging dataset (190); and for each one of multiple low-pass filters (320) having varying cut-off frequencies (301, 301-1, 301-2): determining the respective low-pass filtered normalized Fourier-domain representation of the input imaging dataset (190) based on the normalized Fourier-domain representation of the input imaging dataset and performing a respective comparison of the respective low-pass filtered normalized Fourier-domain representation of the input imaging dataset with the input imaging dataset; and based on results (302, 303) of the comparisons associated with the low-pass filters: estimating the value (311-313) indicative of the signal component of the input imaging dataset (190).




Journal Articles

Waveguide-Based Platform for Large-FOV Imaging of Optically Active Defects in 2D Materials

E. Glushkov; A. Archetti; A. Stroganov; J. Comtet; M. Thakur et al. 

Single-molecule localization microscopy (SMLM) is a powerful tool that is routinely used for nanoscale optical imaging of biological samples. Recently, this approach has been applied to study optically active defects in two-dimensional (2D) materials. Such defects can not only alter the mechanical and optoelectronic properties of 2D materials but also bring new functionalities, which make them a promising platform for integrated nanophotonics and quantum sensing. Most SMLM approaches, however, provide a field of view limited to similar to 50 x 50 mu m(2), which is not sufficient for high-throughput characterization of 2D materials. Moreover, the 2D materials themselves pose an additional challenge as their nanometer-scale thickness prevents efficient far-field excitation of optically active defects. To overcome these limitations, we present here a waveguide-based platform for large field-of-view imaging of 2D materials via total internal reflection excitation. We use this platform to perform large-scale characterization of point defects in chemical vapor deposition-grown hexagonal boron nitride on an area of up to 100 x 1000 mu m(2) and demonstrate its potential for correlative imaging and high throughput characterization of defects in 2D materials.

Acs Photonics. 2019-12-01. Vol. 6, num. 12, p. 3100-3107. DOI : 10.1021/acsphotonics.9b01103.

Transverse Detection of DNA Using a MoS2 Nanopore

M. Graf; M. Lihter; D. Altus; S. Marion; A. Radenovic 

Classical nanopore sensing relies on the measurement of the ion current passing through a nanopore. Whenever a molecule electrophoretically translocates through the narrow constriction, it modulates the ion current. Although this approach allows one to measure single molecules, the access resistance limits the spatial resolution. This physical limitation could potentially be overcome by an alternative sensing scheme taking advantage of the current across the membrane material itself. Such an electronic readout would also allow better temporal resolution than the ionic current. In this work, we present the fabrication of an electrically contacted molybdenum disulfide (MoS2) nanoribbon integrated with a nanopore. DNA molecules are sensed by correlated signals from the ionic current through the nanopore and the transverse current through the nanoribbon. The resulting signal suggests a field-effect sensing scheme where the charge of the molecule is directly sensed by the nanoribbon. We discuss different sensing schemes such as local potential sensing and direct charge sensing. Furthermore, we show that the fabrication of freestanding MoS2 ribbons with metal contacts is reliable and discuss the challenges that arise in the fabrication and usage of these devices.

Nano Letters. 2019-12-01. Vol. 19, num. 12, p. 9075-9083. DOI : 10.1021/acs.nanolett.9b04180.

Nanoscale Selective Passivation of Electrodes Contacting a 2D Semiconductor

M. Lihter; M. Graf; D. Ivekovic; A. Radenovic 

2D semiconducting materials have become the central component of various nanoelectronic devices and sensors. For sensors operating in liquid, it is crucial to efficiently block the electron transfer that occurs between the electrodes contacting the 2D material and the interfering redox species. This reduces current leakages and preserves a good signal-to-noise ratio. Here, a simple electrochemical method is presented for passivating the electrodes contacting a monolayer of MoS2, a representative of transition metal dichalcogenide semiconductors. The method is based on blocking the electrode surface by a thin and compact layer of electronically nonconductive poly(phenylene oxide), PPO, formed by electrochemical polymerization of phenol. Since the phenol polymerization occurs in the potential window where MoS2 is electrochemically inactive, the PPO deposition is area-selective, limited to the electrode surface. The deposited PPO film is characterized by electrochemical, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy techniques. The applicability of this method is demonstrated by coating the electrodes of a MoS2-based field-effect transistor coupled with a nanopore. The highly selective deposition, the simple approach, and the compatibility with MoS2 makes this method a good strategy for efficient insulation of micro- and nanoelectrodes contacting 2D semiconductor-based devices.

Advanced Functional Materials. 2019-12-11.  p. 1907860. DOI : 10.1002/adfm.201907860.

Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores

C. Cao; N. Cirauqui; M. J. Marcaida; E. Buglakova; A. Duperrex et al. 

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double beta-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.

Nature Communications. 2019-10-29. Vol. 10, p. 4918. DOI : 10.1038/s41467-019-12690-9.

Wafer-scale MOCVD growth of monolayer MoS2 on sapphire and SiO2

H. Cun; M. Macha; H. Kim; K. Liu; Y. Zhao et al. 

High-quality and large-scale growth of monolayer molybdenum disulfide (MoS2) has caught intensive attention because of its potential in many applications due to unique electronic properties. Here, we report the wafer-scale growth of high-quality monolayer MoS2 on singlecrystalline sapphire and also on SiO2 substrates by a facile metal-organic chemical vapor deposition (MOCVD) method. Prior to growth, an aqueous solution of sodium molybdate (Na2MoO4) is spun onto the substrates as the molybdenum precursor and diethyl sulfide ((C2H5)(2)S) is used as the sulfur precursor during the growth. The grown MoS2 films exhibit crystallinity, good electrical performance (electron mobility of 22 cm(2)center dot V-1 center dot s(-1)) and structural continuity maintained over the entire wafer. The sapphire substrates are reusable for subsequent growth. The same method is applied for the synthesis of tungsten disulfide (WS2). Our work provides a facile, reproducible and cost-efficient method for the scalable fabrication of high-quality monolayer MoS2 for versatile applications, such as electronic and optoelectronic devices as well as the membranes for desalination and power generation.

Nano Research. 2019-10-01. Vol. 12, num. 10, p. 2646-2652. DOI : 10.1007/s12274-019-2502-9.

Identifying microbial species by single-molecule DNA optical mapping and resampling statistics

A. Bouwens; J. Deen; R. Vitale; L. D’Huys; V. Goyvaerts et al. 

Single-molecule DNA mapping has the potential to serve as a powerful complement to high-throughput sequencing in metagenomic analysis. Offering longer read lengths and forgoing the need for complex library preparation and amplification, mapping stands to provide an unbiased view into the composition of complex viromes and/or microbiomes. To fully enable mapping-based metagenomics, sensitivity and specificity of DNA map analysis and identification need to be improved. Using detailed simulations and experimental data, we first demonstrate how fluorescence imaging of surface stretched, sequence specifically labeled DNA fragments can yield highly sensitive identification of targets. Second, a new analysis technique is introduced to increase specificity of the analysis, allowing even closely related species to be resolved. Third, we show how an increase in resolution improves sensitivity. Finally, we demonstrate that these methods are capable of identifying species with long genomes such as bacteria with high sensitivity.

NAR Genomics and Bioinformatics. 2019-10-05. Vol. 2, num. 1, p. lqz007. DOI : 10.1093/nargab/lqz007.

Facile Production of Hexagonal Boron Nitride Nanoparticles by Cryogenic Exfoliation

Ngoc My Hanh Duong; E. Glushkov; A. Chernev; V. Navikas; J. Comtet et al. 

Fluorescent nanoparticles with optically robust luminescence are imperative to applications in imaging and labeling. Here we demonstrate that hexagonal boron nitride (hBN) nanoparticles can be reliably produced using a scalable cryogenic exfoliation technique with sizes below 10 nm. The particles exhibit bright fluorescence generated by color centers that act as atomic-size quantum emitters. We analyze their optical properties, including emission wavelength, photon-statistics, and photodynamics, and show that they are suitable for far-field super-resolution fluorescence nanoscopy. Our results provide a foundation for exploration of hBN nanoparticles as candidates for bioimaging, labeling, as well as biomarkers that are suitable for quantum sensing.

Nano Letters. 2019-08-01. Vol. 19, num. 8, p. 5417-5422. DOI : 10.1021/acs.nanolett.9b01913.

Parameter-free image resolution estimation based on decorrelation analysis

A. C. Descloux; K. S. Grussmayer; A. Radenovic 

Super-resolution microscopy opened diverse new avenues of research by overcoming the resolution limit imposed by diffraction. Exploitation of the fluorescent emission of individual fluorophores made it possible to reveal structures beyond the diffraction limit. To accurately determine the resolution achieved during imaging is challenging with existing metrics. Here, we propose a method for assessing the resolution of individual super-resolved images based on image partial phase autocorrelation. The algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in super-resolution microscopy.

Nature Methods. 2019-08-26. Vol. 16, p. 918–924. DOI : 10.1038/s41592-019-0515-7.

2D MoS2 nanopores: ionic current blockade height for clustering DNA events

A. D. Carral; C. S. Sarap; K. Liu; A. Radenovic; M. Fyta 

2D nanopores can be used to electrophoretically drive DNA molecules, which can in turn be identified through measurable electronic current blockades. In this work, we use experimental data from molybdenum disulfide nanopores threading DNA nucleotides and propose a methodological approach to interpret DNA events. Specifically, the experimental ionic traces are used to train an unsupervised machine learning model for identifying distinct molecular events through the 2D nanopore. For the first time, we propose a clustering of experimental 2D nanopore data based on the ionic current blockade height and unrelated to the traditional dwell time for each DNA event. Within this approach, the blockade level information is implicitly included in the feature space analysis and does not need to be treated explicitly. We could show the higher efficiency of the blockade height over the traditional dwell time also in coping with sparse nanopore data sets. Our approach allows for a deep insight into characteristic molecular features in 2D nanopores and provides a feedback mechanism to tune these materials and interpret the measured signals. It has, thus, a high impact on the efficiency of 2D nanopore-based DNA sequencers.

2D Materials. 2019-10-01. Vol. 6, num. 4, p. 045011. DOI : 10.1088/2053-1583/ab2c38.

Light-Enhanced Blue Energy Generation Using MoS2 Nanopores

M. Graf; M. Lihter; D. Unuchek; A. Sarathy; J-P. Leburton et al. 

Blue energy relies on the chemical potential difference between solutions of high and low ionic concentrations, potentially providing an independent energy source at estuaries around the world. The energy conversion relies on reverse electrodialysis via ion-selective membranes. A novel generation of these membranes is based on nanopores in atomically thin material. Single nanopores in molybdenum disulfide (MoS2)-based membranes have shown record-high power outputs in alkaline conditions. By increasing the surface charge of MoS2 membranes by light, we can double the osmotic power generated by a single nanopore at a neutral pH. The increased surface charge at the pore rim enhances the ion selectivity and leads to a larger osmotic voltage (dominating in small pores), while the increased surface charge of the membrane enhances the surface conductance, leading to a larger osmotic current (dominating in larger pores). The combination of these effects could efficiently boost the energy generation using membranes containing arrays of nanopores of varying sizes.

Joule. 2019-06-19. Vol. 3, num. 6, p. 1549-1564. DOI : 10.1016/j.joule.2019.04.011.

Supervised learning to quantify amyloidosis in whole brains of an Alzheimer’s disease mouse model acquired with optical projection tomography

D. Nguyen; V. Uhlmann; A. L. Planchette; P. J. Marchand; D. Van de Ville et al. 

Alzheimer’s disease (AD) is characterized by amyloidosis of brain tissues. This phenomenon is studied with genetically-modified mouse models. We propose a method to quantify amyloidosis in whole 5xFAD mouse brains, a model of AD. We use optical projection tomography (OPT) and a random forest voxel classifier to segment and measure amyloid plaques. We validate our method in a preliminary cross-sectional study, where we measure 6136 +/- 1637, 8477 +/- 3438, and 17267 +/- 4241 plaques (AVG +/- SD) at 11, 17, and 31 weeks. Overall, this method can be used in the evaluation of new treatments against AD. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Biomedical Optics Express. 2019-06-01. Vol. 10, num. 6, p. 3041-3060. DOI : 10.1364/BOE.10.003041.

Fabrication and practical applications of molybdenum disulfide nanopores

M. Graf; M. Lihter; M. Thakur; V. Georgiou; J. Topolancik et al. 

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores’ efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2–3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2–3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.

Nature Protocols. 2019-03-22. Vol. 14, num. 4, p. 1130-1168. DOI : 10.1038/s41596-019-0131-0.

Wide-Field Spectral Super-Resolution Mapping of Optically Active Defects in Hexagonal Boron Nitride

J. Comtet; E. Glushkov; V. Navikas; J. Feng; V. Babenko et al. 

Point defects can have significant impact on the mechanical, electronic, and optical properties of materials. The development of robust, multidimensional, high-throughput, and large-scale characterization techniques of defects is thus crucial for the establishment of integrated nanophotonic technologies and material growth optimization. Here, we demonstrate the potential of wide-field spectral single-molecule localization microscopy (SMLM) for the determination of ensemble spectral properties as well as the characterization of spatial, spectral, and temporal dynamics of single defects in chemical vapor deposition (CVD)-grown and irradiated exfoliated hexagonal boron-nitride materials. We characterize the heterogeneous spectral response of our samples and identify at least two types of defects in CVD-grown materials, while irradiated exfoliated flakes show predominantly only one type of defects. We analyze the blinking kinetics and spectral emission for each type of defects and discuss their implications with respect to the observed spectral heterogeneity of our samples. Our study shows the potential of wide-field spectral SMLM techniques in material science and paves the way toward the quantitative multidimensional mapping of defect properties.

Nano Letters. 2019-03-13. Vol. 19, num. 4, p. 2516-2523. DOI : 10.1021/acs.nanolett.9b00178.

Waveguide-PAINT offers an open platform for large field-of-view super-resolution imaging

A. Archetti; E. Glushkov; C. Siebenl; A. Stroganov; A. Radenovic et al. 

Super-resolution microscopies based on the localization of single molecules have been widely adopted due to their demonstrated performance and their accessibility resulting from open software and simple hardware. The PAINT method for localization microscopy offers improved resolution over photoswitching methods, since it is less prone to sparse sampling of structures and provides higher localization precision. Here, we show that waveguides enable increased throughput and data quality for PAINT, by generating a highly uniform similar to 100 x 2000 mu m(2) area evanescent field for TIRF illumination. To achieve this, we designed and fabricated waveguides optimized for efficient light coupling and propagation, incorporating a carefully engineered input facet and taper. We also developed a stable, low-cost microscope and 3D-printable waveguide chip holder for easy alignment and imaging. We demonstrate the capabilities of our open platform by using DNA-PAINT to image multiple whole cells or hundreds of origami structures in a single field of view.

Nature Communications. 2019-03-20. Vol. 10, p. 1267. DOI : 10.1038/s41467-019-09247-1.

Detecting topological variations of DNA at single-molecule level

K. Liu; C. Pan; A. Kühn; A. P. Nievergelt; G. Fantner et al. 

In addition to their use in DNA sequencing, ultrathin nanopore membranes have potential applications in detecting topological variations in deoxyribonucleic acid (DNA). This is due to the fact that when topologically edited DNA molecules, driven by electrophoretic forces, translocate through a narrow orifice, transient residings of edited segments inside the orifice modulate the ionic flow. Here we utilize two programmable barcoding methods based on base-pairing, namely forming a gap in dsDNA and creating protrusion sites in ssDNA for generating a hybrid DNA complex. We integrate a discriminative noise analysis for ds and ss DNA topologies into the threshold detection, resulting in improved multi-level signal detection and consequent extraction of reliable information about topological variations. Moreover, the positional information of the barcode along the template sequence can be determined unambiguously. All methods may be further modified to detect nicks in DNA, and thereby detect DNA damage and repair sites.

Nature Communications. 2019. Vol. 10, p. 3. DOI : 10.1038/s41467-018-07924-1.


2D materials as an emerging platform for nanopore-based power generation

M. Macha; S. Marion; V. V. R. Nandigana; A. Radenovic 

Osmotic power generation, the extraction of power from mixing salt solutions of different concentrations, can provide an efficient power source for both nanoscale and industrial-level applications. Power is generated using ion-selective channels or pores of nanometric dimensions in synthetic membrane materials. 2D materials such as graphene and MoS2 provide energy extraction efficiencies that are several orders of magnitude higher than those of more established bulky membranes. In this Review, we survey the current state of the art in power generation with both 2D materials and solid-state devices. We discuss the current understanding of the processes underlying power generation in boron nitride nanotubes and 2D materials, as well as the available fabrication methods and their impact on power generation. Finally, we overview future directions of research, which include increasing efficiency, upscaling single pores to porous membranes and solving other issues related to the potential practical application of 2D materials for osmotic power generation.

Nature Reviews Materials. 2019-09-01. Vol. 4, num. 9, p. 588-605. DOI : 10.1038/s41578-019-0126-z.

Fluorescent Nanodiamonds as Versatile Intracellular Temperature Sensors

E. Glushkov; V. Navikas; A. Radenovic 

Temperature is a widely known phenomenon, which plays an extremely important role in biological systems. Its behavior on the macro-scale has been quite well investigated and understood, thanks to the availability of reliable and precise thermometers such as thermocouples and infrared cameras. However, temperature measurements on the subcellular scale present an ongoing challenge due to the absence of universal nanoscale temperature sensors. Recent work on fluorescent nanodiamonds has revealed their unique ability to measure temperature with high spatial and temporal resolution, of particular importance in the intracellular environment. This review summarizes recent progress in the field and highlights the future directions for intracellular temperature sensing using fluorescent nanodiamonds

CHIMIA International Journal for Chemistry. 2019-02-01. Vol. 73, num. 1, p. 73-77. DOI : 10.2533/chimia.2019.73.


2D nanopores: fabrication, energy harvesting and field-effect sensing

M. Graf / A. Radenovic (Dir.)  

Solid-state nanopores are man-made, nano-sized openings in membranes separating two chambers containing an electrolyte solution. When applying an electric field across the membrane, the nanopore provides the only path for mobile ions to pass from one side of the membrane to the other. This current of ions is highly dependent on the pore size, membrane thickness, and surface charge. Small modulations in the system can lead to a large current modulation. We take advantage of this by translocating biomolecules through the nanopore. Typically, molecules such as DNA have an intrinsic charge in solution and will be attracted by the electric field generated around the nanopore. If the size of the pore allows it, they will thread into the pore and translocate to the opposite chamber. Generally, the thinner the membrane, the more ion current is generated and therefore the larger is the recorded signal. The isolation of mono-atomic crystals of carbon, also known as graphene, at the beginning of the 21st century, sparked much interest in using 2D materials for nanopore sensors. The thickness of these materials is approaching the distance between two bases in a DNA molecule, which raised the hopes of sequencing DNA when it passes through the orifice. First, I will introduce the reader to 2D nanopores made in MoS2. In the last few years, I gained a lot of insights into 2D-nanopore fabrication. Therefore, I will first detail how MoS2-nanopore devices can be fabricated reliably and I will discuss potential pitfalls that researchers might encounter when manufacturing these devices. We realized that nanopores in atomically thin MoS2 not only provide a system with low resistance to ionic current but also exhibit excellent ion-selectivity. Therefore, we developed an energy-harvesting system based on the osmotic pressure generated when concentration gradients are applied across the membrane. Converting this osmotic energy through reverse-electrodialysis relies on ion-selective membranes. By exploiting the photo-excitability of MoS2 membranes, I will show that we can raise the ion selectivity of the membrane by a factor of 5 while staying at a neutral pH and conclude that the observed effect is due to a change in the surface charge caused by light-induced charge generation. Although ionic sensing with nanopores allows the precise measurements of single molecules, the spatial resolution in ionic sensing is limited by the access resistance. This limitation can potentially be overcome by an alternative sensing scheme independent from the ionic current. I will present a supplementary sensing scheme taking advantage of the semiconducting properties of MoS2. I fabricated freestanding nanoribbons of monolayer MoS2 in which a nanopore is drilled. The nanoribbons are then contacted through metal leads, which allow measuring the current through the material itself. The ionic current and the transverse current are recorded simultaneously and show correlated current modulations when DNA molecules translocate through the nanopore. The precise sensing mechanism of these devices is currently not well understood but is believed to originate from the charged molecules themselves or from local potential changes near the nanopore. I will discuss the challenges in fabricating such devices and propose possible explanations for the observed current traces.

Lausanne, EPFL, 2019. 


Journal Articles

Transverse Detection of DNA in a MoS2 Nanopore

M. Graf; K. Liu; A. Sarathy; J-P. Leburton; A. Radenovic 

Biophysical Journal. 2018. Vol. 114, num. 3, p. 180a. DOI : 10.1016/j.bpj.2017.11.1005.

Single step synthesis of Schottky-like hybrid graphene – titania interfaces for efficient photocatalysis

Z. Yi; A. Merenda; L. Kong; A. Radenovic; M. Majumder et al. 

The development of 2D nanomaterial coatings across metal surfaces is a challenge due to the mismatch between the metal microstructure and the nanoscale materials. The naturally occurring thin oxidative layer present across all metal surfaces, may lead to low adherence and connectivity. In this paper, graphene/titania/Titanium hybrid films were for the first time fabricated by a single step chemical vapour deposition process across Titanium foils. The presence of graphene as a dopant was found to enhance the photocatalytic performance of the final products, applied to the degradation of organic molecules and to lead to Schottky-like junction formation at the metal/oxide interface. These Schottky junctions, where vacancies are present across the titania material due to the graphene doping and where Ti3+ ions are predominantly located, yield enhanced catalytic performance. The highest degradation rate was found to be 9.66 × 10−6 min−1, achieved by the sample grown at 700 °C for 5 min, which was 62% higher than the sample just treated at that temperature without graphene growth. This work provides evidence that graphene may be grown across pure Titanium metal and opens new avenues in biomedical devices design, tribological or separation applications.

Scientific Reports. 2018-05-25. Vol. 8, num. 1, p. 8154. DOI : 10.1038/s41598-018-26447-9.

Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations

P-L. Chang; M. Graf; C-H. Hung; A. Radenovic 

Here we present the realization of a novel fluorescence detection method based on the electromigration of fluorescent molecules within a nanocapillary combined with the laser excitation through a platinum (Pt)-coated nanocapillary. By using the Pt nanocapillary assisted focusing of a laser beam, we completely remove the background scattering on the tip of the electrophoretic nanocapillary. In this excitation geometry, we demonstrate a 1000-fold sensitivity enhancement (1.0 nM to 1.0 pM) compared to the detection in microcapillaries with epifluorescence illumination and fluorescence spectrophotometry. Due to a significant electroosmotic flow, we observe a decelerating migration of DNA molecules close to the tip of the electrophoretic nanocapillary. The reduced DNA translocation velocity causes a two-step stacking process of molecules in the tip of the nanocapillary and can be used as a way to locally concentrate molecules. The sensitivity of our method is further improved by a continuous electrokinetic injection of DNA molecules followed by sample zone stacking on the tip of the nanocapillary. Concentrations ranging from 0.1 pM to 1.0 fM can be directly observed on the orifice of the electrophoretic nanocapillary. This is a 1000-fold improvement compared to traditional capillary electrophoresis with laser-induced fluorescence.

Nano Letters. 2018-04-04. Vol. 18, num. 5, p. 3165-3171. DOI : 10.1021/acs.nanolett.8b00831.

Imaging of Optically Active Defects with Nanometer Resolution

J. Feng; H. Deschout; S. Caneva; S. Hofmann; I. Lončarić et al. 

Nano Letters. 2018-02-02. Vol. 18, num. 3, p. 1739–1744. DOI : 10.1021/acs.nanolett.7b04819.

Centimeter-Sized Single-Orientation Monolayer Hexagonal Boron Nitride With or Without Nanovoids

H. Cun; A. Hemmi; E. Miniussi; C. Bernard; B. Probst et al. 

Nano Letters. 2018-01-09. Vol. 18, num. 2, p. 1205-1212. DOI : 10.1021/acs.nanolett.7b04752.


Osmotic power generator

J. Feng; A. Radenovic 

An osmotic power generator comprising an active membrane supported in a housing, at least a first chamber portion disposed on a first side of the active membrane for receiving a first electrolyte liquid and a second chamber portion disposed on a second side of the active membrane for receiving a second electrolyte liquid, a generator circuit comprising at least a first electrode electrically coupled to said first chamber, and at least a second electrode electrically coupled to said second chamber, the first and second electrodes configured to be connected together through a generator load receiving electrical power generated by a difference in potential and an ionic current between the first and second electrodes. The active membrane includes at least one pore allowing ions to pass between the first and second sides of the membrane under osmosis due to an osmotic gradient between the first and second electrolyte liquids to generate said difference in potential and ionic current between the first and second electrodes.

US10801478; EP3475567; US2019226463; EP3475567; WO2018002099; EP3263896.



Journal Articles

Investigating Focal Adhesion Substructures by Localization Microscopy

H. Deschout; I. Platzman; D. Sage; L. Feletti; J. P. Spatz et al. 

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin β3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e., fibronectin-coated substrates and specifically biofunctionalized nanopatterned substrates). To quantify the substructure of FAs, we developed a clustering method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 μm2 , contain 10–100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density.

Biophysical Journal. 2017. Vol. 113, num. 11, p. 2508-2518. DOI : 10.1016/j.bpj.2017.09.032.

Geometrical Effect in 2D Nanopores

K. Liu; M. Lihter; A. Sarathy; S. Caneva; H. Qiu et al. 

A long-standing problem in the application of solid-state nanopores is the lack of the precise control over the geometry of artificially formed pores compared to the well-defined geometry in their biological counterpart, that is, protein nanopores. To date, experimentally investigated solid-state nanopores have been shown to adopt an approximately circular shape. In this Letter, we investigate the geometrical effect of the nanopore shape on ionic blockage induced by DNA translocation using triangular h-BN nanopores and approximately circular molybdenum disulfide (MoS2) nanopores. We Observe a striking geometry dependent ion scattering effect, which is further corroborated by a modified ionic blockage model. The well-acknowledged ionic blockage Model is derived from uniform ion permeability through the 2D nanopore plane and hemisphere like access region in the nanopore vicinity. On the basis of our experimental results, we propose a modified ionic blockage model, which is highly related to the ionic profile caused by geometrical variations. Our findings shed light on the rational design of 2D nanopores and should be applicable to arbitrary nanopore shapes.

Nano Letters. 2017. Vol. 7, num. 17, p. 4223-4230. DOI : 10.1021/acs.nanolett.7b01091.

Conference Papers

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

H. Deschout; T. Lukes; A. Sharipov; L. Feletti; T. Lasser et al. 

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

2017. SPIE Photonics West, San Francisco, 27 January-2 February 2017. DOI : 10.1117/12.2252865.


Nanocapillaries combined with optical tweezers as a single molecule technique for studying DNA-protein complexes

R. Bulushev / A. Radenovic (Dir.)  

Interactions of proteins with DNA are essential for carrying out DNA’s biological functions and performing a cellular cycle. Such processes as DNA replication, expression and repair are performed by an organised action of various proteins. To better understand the function of protein machinery many methods have been developed over the years. They can be divided into two categories: single molecule and bulk techniques. In comparison to bulk experiments, where the effect of an ensemble of proteins is measured, single molecule techniques analyse each molecule one by one. This fact allows to detect rare events and avoid averaging over the population. Moreover, some single molecule techniques can be used for mechanical manipulation of biomolecules, i.e. twisting, stretching, etc. The objective of this thesis was to make a single molecule technique combining nanocapillaries and optical tweezers for the characterisation of DNA-protein complexes in physiological conditions. There were three main steps in this thesis: 1) building and characterisation of the setup 2) using it for detection and characterisation of DNA-protein complexes and 3) localisation and discrimination of DNA-protein complexes. On the first step of the project we combined two single molecule techniques: optical tweezers and glass nanocapillaries. We characterised the electrophoretic force acting on DNA in this setup by using nanocapillaries with openings of different sizes, at different applied voltages and with DNA molecules of different lengths. We observed that the position-dependent electrophoretic force acting on the DNA depends on all above-mentioned parameters. We modelled the system and found out that this effect is due to a non-uniform distribution of the potential inside the nanocapillary, which originates from its elongated shape. After having built and characterised the setup, we detected proteins bound to DNA during their controlled translocation through the opening. The proteins were visualised by a sudden decrease in the force acting on the bare DNA followed up by its slow restoration when the capillary was moved further away. We made a stochastic model to explain this force profile. From the fits of the model to experimental results we extracted the effective charges of DNA-protein complexes inside the nanocapillary. In the case of all three proteins (RecA, EcoRI and RNAP) the effective charge was of opposite sign than the one in solution. We attributed this fact to the dominant impact of the drag force in comparison to the electrostatic force inside the nanocapillary. On the last step of the project we showed the ability to localise and discriminate DNA-protein complexes in our setup using dCas9 and RNAP proteins. During controlled translocation of the DNA-protein complexes we measured multiple parameters, including protein’s location on the DNA, work required to translocate the complex, and conductance change. We demonstrated that the measured location of the proteins is shifted from the designed binding site. We made a model that explained this phenomenon and that can account for the shift in our experiments. In addition, protein-specific work and conductance parameters allowed us to discriminate between RNAP and dCas9 proteins.

Lausanne, EPFL, 2017. 


Molecular sensing device

J. Feng; K. Liu; A. Radenovic 

Molecular sensing system including: a sensing device (5) comprising at least one support layer (10), and an active layer (6) mounted on said support layer and having at least one nano-pore (12) configured for translocation of a molecular analyte (18) therethrough; an electrically conducting liquid (4) in contact with the active layer in a region around said nano-pore; and a signal processing circuit (7) comprising an ionic current circuit (8) configured to generate and measure an ionic current (Ii) in the electrically conducting liquid influenced by the translocation of the molecular analyte through the nano-pore. The molecular sensing device of the invention allows for single-nucleotide discrimination and detection of the specific sequence within ssDNA.

EP3105584; US10648965; US2017059547; EP3105584; WO2015121394; EP2908128.




Manufacturing of orifices in glass like materials, e.g. Nanocapillaries, and objects obtained according to this process

L. J. Steinbock; A. Radenovic 

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique14,17. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules12,31. However, this has not yet been shown for glass having a tubular shape, for instance nanocapillaries. In contrast to their solid-state nanopore counterparts25, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. Nanocapillaries made out of glass-like materials such as quartz or borosilicate glass can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrink rates. This allows to fine control the shrink rate and to stop the shrinking process at any desired diameter. A shrunken nanocapillary may detect DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. The invention opens a new path to detect small and short molecules such as proteins or RNA with nanocapillaries and also increase the sensitivity of other techniques such as SNOM or SCIM, which also rely on conical glass capillaries.

EP2969995; US2016016840; EP2969995; WO2014141168.


Nanopore forming method and uses thereof

J. Feng; K. Liu; A. Radenovic; Y. Astier 

Method for making nanopores in thin layers or monolayers of transition metal dichalcogenides comprising the steps of provinding a suspended transistion metal dichalcogenide layer (1) in an electrolyte (2), applying a transmembrane voltage (V) high enough to lead to the breakdown of the layer (1), monitoring the ionic current (Ii) and reducing the transmembrane voltage (V) when the ionic current (Ii) has reached a predefined cut-off value (Ip).

US2023374693; US11753738; US2022380930; US11401625; EP3268736; US2021189585; US10947637; US2019323143; US10364507; US2018073161; EP3268736; WO2016142925; EP3067693.




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