Glass Nanopipette Research Articles

PH-modulated ion-current rectification in a cysteine-functionalized glass nanopipette

The unique properties of nanopores depend largely on the functionalization of the pore surface, which has always been a great challenge. In this study, a facile method for functionalizing the surface of a glass nanopipette to establish thiol-based linkage without involving Au film coating was successfully developed by utilizing the thiol–maleimide click reaction which works rapidly and with high specificity. Cysteine was employed as a model molecule to functionalize the nanopipette, resulting in pH-modulated ion-current rectification behavior which showed a good reproducible response to pH. Due to the widespread existence of thiol groups in nature, this nanopipette functionalization method has the potential and versatility to be used in a wide range of applications, including mimicking biological ion channels and fabricating sensing devices.

Combined Ion Conductance and Atomic Force Microscope for Fast Simultaneous Topographical and Surface Charge Imaging.

We report here an advanced approach for simultaneous and independent submicroscale imaging of local surface charge and topography using microchanneled cantilevers, also known as FluidFM nanopipette probes. These hollow cantilevers with a 300 nm opening are employed for ion current measurements that provide access to the local properties of the electrical double layer using the phenomenon of ion current rectification, while also taking advantage of the force sensing capabilities for accurate probe vertical positioning and topography imaging. The independent nature of this atomic force microscope (AFM) feedback opens up a possibility to significantly increase the sensitivity for probing local surface charges in a wider range of salt concentrations, especially in electrolytes of low ionic strength (below 10 mM), where classical local ion conductance measurements with glass nanopipettes would suffer from inaccuracies and instabilities, but where the electrical double layer extends further into the liquid medium and has stronger effect on the measured ion currents for charge imaging. We demonstrate that the measurements with FluidFM do not compromise the positioning accuracy and enable accurate and simultaneous topographical and charge imaging in contact mode (similar to AFM) at high scanning rates, approaching thousands of pixels per second, therefore overtaking state-of-the-art techniques for charge mapping by at least 2 orders of magnitude (the probes reach translation rates of 120 μm s-1 equating to 2 ms per image pixel). We also reveal experimentally the physical limit of this high speed scanning, constrained by the rate of ion redistribution in surface-induced rectification required for double layer sensing and charge mapping.

Analysis of 2D DNA Origami with Nanopipettes

AbstractNanopipettes are a useful tool for the detection and analysis of biological molecules at the single‐molecule level. Here, we employ nanopipettes fabricated from glass capillaries to identify differently structured 2D DNA origami through the distinctive amplitude and dwell time of the ion current peak resulting from the translocation of the origami through the nanopipette pore. We demonstrate that the ion current peak originating from frame‐like DNA origami comprises two individual peaks in contrast to solid tiles of similar size that only lead to a single peak in the ion current. Interestingly, the fine details of the shape of the double‐peak characteristic of the translocation of DNA origami frames are correlated with the structural features of the DNA origami. In particular, the size of the central cavity governs the lag time between the two constituent peaks of the double‐peak. This work demonstrates the ability of glass nanopipettes to identify and characterize differently structured DNA origami of similar size, advancing the potential of nanopipettes for high‐sensitivity single‐molecule studies.

(Europe Section Allessandro Volta Medal) From Nanobiosensors to Scanning Bipolar Electrochemistry

Measurement of physiological relevant molecules in and at individual cells with minimal disturbance of the cellular function is a key challenge in single-cell analysis. Ad­vantages of nanoscale electrochemical probes include their high spatial and tempo­ral resolution as well as a low substrate consumption. Carbon nanoelectrodes (CNEs) with a tunable radius between a few and several hundreds of nanometres can be fabricated by pyrolytic decomposition of alkane gas in glass nanopipettes. Their needle-type shape and small size enable electro­chemical measurements inside of single living cells. Two potential applications of CNEs namely for the design of nanometer-sized amperometric biosensors, and as the basis for gate-modulated reduced graphene oxide based transistors are presented. However, the probability that a nanosensor is positioned close to a release site is tiny. Hence, we propose to combine scanning electrochemical microscopy (SECM) with bipolar electrochemistry (BPE) as a new approach to image heterogeneous electrochemical surface processes. The core of the system is a closed bipolar electrochemistry configuration comprising as the bipolar electrode (BPE) a microelectrode as SECM tip (cathodic pole of the BPE) connected with a microelectrode tip (anodic pole of the BPE) in the second half cell. Reduction processes enabled by a suitably applied feeder electrode voltage difference at the SECM tip in the sample generation/tip collection mode are transduced into an optical read-out e.g. electrochemiluminescence at the other pole which is visualized with a high sensitive CCD camera. Evidently, this configuration can be extended to multi-barrel SECM tips thus being able to simultaneously visualize the release of compounds at multiple locations while positioning the multi-barrel microelectrode above a release site of interest. Acknowledgement : All coworkers who have contributed to this research are acknowledged namely Jan Clausmeyer, Vera Eßmann, Carla Santana Santos, Tsvetan Tarnec, Thomas Erichsen, Miriam Marquitan, Patrick Wilde, Thomas Quast, Corina Andronescu, Federica Mariani. The author is grateful to the project "Graphtivity" funded in the framework of the FLAG-ERA Joint Transnational Call (JTC) 2015 (Graphene Flagship) and the Deutsche Forschung­gemeinschaft in the framework of the cluster of excellence RESOLV (EXc1069).

Ion-current-rectification-based customizable pH response in glass nanopipettes via silanization

This study shows that it is possible to customize the ion-current-rectification (ICR)-based pH response in glass nanopipettes by adjusting the ratio of 3-aminopropyldimethylethoxysilane (APTES) and (3-triethoxysilylpropyl) succinic acid anhydride (TESP-SA) used in silanization. The silanization time and concentration were optimized by investigating the rectification ratio of the nanopipettes. For each APTES/TESP-SA silane ratio, analysis of variance (ANOVA) tests indicated that the rectification ratio of the nanopipette had good linearity in the pH range 3–8. Moreover, nanopipettes silanized with equal concentrations of amino and carboxylic groups gave the highest pH response of 1.06 × 10−4 pH −1. This method for customizing the ICR-based pH response in nanopipettes has the potential and versatility to be used in applications such as mimicking biological ion channels and developing nanosized pH sensing devices.

A low voltage nanopipette dielectrophoretic device for rapid entrapment of nanoparticles and exosomes extracted from plasma of healthy donors

An insulator-based dielectrophoresis (iDEP) is a label-free method that has been extensively utilized for manipulation of nanoparticles, cells, and biomolecules. Here, we present a new iDEP approach that can rapidly trap nanoparticles at the close proximity of a glass nanopipette’s tip by applying 10 V/cm direct current (DC) across the pipette’s length. The trapping mechanism was systemically studied using both numerical modeling and experimental observations. The results showed that the particle trapping was determined to be controlled by three dominant electrokinetic forces including dielectrophoretic, electrophoretic and electroosmotic force. Furthermore, the effect of the ionic strength, the pipette’s geometry, and the applied electric field on the entrapment efficiency was investigated. To show the application of our device in biomedical sciences, we demonstrated the successful entrapment of fluorescently tagged liposomes and unlabeled plasma-driven exosomes from the PBS solution. Also, to illustrate the selective entrapment capability of our device, 100 nm liposomes were extracted from the PBS solution containing 500 nm polystyrene particles at the tip of the pipette as the voltage polarity was reversed.

A Multiparameter pH‐Sensitive Nanodevice Based on Plasmonic Nanopores

AbstractWith controllable mass transfer and special optical properties, plasmonic nanopores may be applied as a nanodevice and possibly create a new generation of single molecule detection technique based on plasmon‐enhanced spectra. In the present study, gold nanoparticles self‐assemble into a gold porous sphere (GPS) on the tip of a glass nanopipette with the help of i‐motif DNA thiolated by both ends as linker molecules. The gaps among neighboring gold nanoparticles are considered as plasmonic nanopores. The size of the formed nanopores can be tuned by the folded–unfolded conformational change of i‐motif DNA upon pH adjustment from 4.5 to 7.0. Based on its tunable structural property, the GPS shows reversible changes in ionic current, potential, and surface‐enhanced Raman scattering signal. The GPS is further used to probe regional pH in single cells. The successful application of GPS in multiparameter pH probing and single cell analysis suggests that the new physical properties of the self‐assembled plasmonic nanopores can be used for fabricating multiple types of nanodevices and nanosensors.

Redox Reaction on the Surface/Edge Strturtures of Graphene/Graphite

Graphene, a single atomic layer of graphite, has been widely studied because of its unique electrical, optical, electrochemical, and elastic properties. With respect to the electrochemical property, it is also expected for energy harvesting and storage applications such as hydrogen generation for fuel cells and ion transport for secondary batteries. Indeed, the graphene’s exotic phenomena come from its ideal structure such as edge states, two-dimensional surface, defects. To understand their intrinsic mechanism due to structure effects, it is required to analyze and visualize their electrochemical reaction by a spatially resolved electrochemical analysis technique at microscopic scale. In general, electrochemical analytical tools are conducted at mesoscopic scale. Scanning probe microscopies have been also utilized for analyzing their properties to satisfy the issue in resolution. Scanning tunneling microscopy and conductive atomic force microscopy can analyze at atomic scale precisely, but their information is mainly limited to electronic properties which do not directly reflect to their electrochemical properties. In this study, to overcome the issue, scanning electrochemical cell microscopy with sub-micrometer resolution, i.e. nanoSECCM, was utilized as a direct technique for electrochemical reactions [1]. A glass nano-pipette was applied as a probe. The pipettes were filled electrolyte and a reference electrode (5 mM hexaammineruthenium chloride and 25 mM KCl in phosphate buffer or deionized water for electrolyte, Ag/AgCl for a reference electrode). The nanoSECCM was applied to cleaved graphite or graphene on silicon substrates. When the pipette was approached to the sample surface, a meniscus was created as an electrochemical cell. Through the cell, redox reaction of ruthenium ion inside electrolyte was measured. By scanning the pipette with the same process at next measurement location, redox reaction on the graphene/graphite surface were visualized as shown in Fig 1. [1] Y. Takahashi, A. Kumatani et al. Nat. Comm. 5:6450 (2014). Figure 1

Levitated Plasmonic Nanoantennas in an Aqueous Environment.

We report on the manipulation of a plasmonic nanoantenna in an aqueous solution using an electrostatic trap created between a glass nanopipette and a substrate. By scanning a trapped gold nanosphere in the near field of a single colloidal quantum dot embedded under the substrate surface, we demonstrate about 8-fold fluorescence enhancement over a lateral full width at half-maximum of about 45 nm. We analyze our results with the predictions of numerical electromagnetic simulations under consideration of the electrostatic free energy in the trap. Our approach could find applications in a number of experiments, where plasmonic effects are employed at liquid-solid interfaces.

Insight into Ion Transfer through the Sub-Nanometer Channels in Zeolitic Imidazolate Frameworks.

A crack-free sub-nanometer composite structure for the study of ion transfer was constructed by in situ growth of ZIF-90 [Zn(ICA)2 , ICA=Imidazole-2-carboxaldehyde] on the tip of a glass nanopipette. The potential-driven ion transfer through the sub-nanometer channels in ZIF-90 is strongly influenced by the pH of the solution. A rectification ratio over 500 is observed in 1 m KCl solution under alkaline conditions (pH 11.58), which is the highest value reported under such a high salt concentration. Fluorescence experiments show the super-high rectification ratio under alkaline conditions results from the strong electrostatic interaction between ions and the sub-nanometer channels of ZIF-90. In addition to providing a general pathway for further study of mass-transfer process through sub-nanometer channels, the approach enable all kinds of metal-organic frameworks (MOFs) to be used as ionic permselectivity materials in nanopore-based analysis.

Insight into Ion Transfer through the Sub‐Nanometer Channels in Zeolitic Imidazolate Frameworks

AbstractA crack‐free sub‐nanometer composite structure for the study of ion transfer was constructed by in situ growth of ZIF‐90 [Zn(ICA)2, ICA=Imidazole‐2‐carboxaldehyde] on the tip of a glass nanopipette. The potential‐driven ion transfer through the sub‐nanometer channels in ZIF‐90 is strongly influenced by the pH of the solution. A rectification ratio over 500 is observed in 1 m KCl solution under alkaline conditions (pH 11.58), which is the highest value reported under such a high salt concentration. Fluorescence experiments show the super‐high rectification ratio under alkaline conditions results from the strong electrostatic interaction between ions and the sub‐nanometer channels of ZIF‐90. In addition to providing a general pathway for further study of mass‐transfer process through sub‐nanometer channels, the approach enable all kinds of metal–organic frameworks (MOFs) to be used as ionic permselectivity materials in nanopore‐based analysis.

Resistive-pulse and rectification sensing with glass and carbon nanopipettes.

Along with more prevalent solid-state nanopores, glass or quartz nanopipettes have found applications in resistive-pulse and rectification sensing. Their advantages include the ease of fabrication, small physical size and needle-like geometry, rendering them useful for local measurements in small spaces and delivery of nanoparticles/biomolecules. Carbon nanopipettes fabricated by depositing a thin carbon layer on the inner wall of a quartz pipette provide additional means for detecting electroactive species and fine-tuning the current rectification properties. In this paper, we discuss the fundamentals of resistive-pulse sensing with nanopipettes and our recent studies of current rectification in carbon pipettes.

Argon gas flow through glass nanopipette

We have observed the flow of argon gas through a glass nanopipette in vacuum. A glass nanopipette with an inner diameter of 100 nm and a shank length of 3 mm was set between vacuum chambers, and argon gas was introduced from the top of the nanopipette to the bottom. The exit pressure was monitored with an increase in entrance pressure in the range of 50–170 kPa. Knudsen flow was observed at an entrance pressure lower than 100 kPa, and Poiseuille flow was observed at an entrance pressure higher than 120 kPa. The proposed pressure-dependent gas flow method provides a means of evaluating the glass nanopipette before using it for various applications including nanodeposition to surfaces and femtoinjection to living cells.

Angular Approach Scanning Ion Conductance Microscopy

Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.

Nonlinear dependence of the ion current rectification factor on bias voltage in conical nanopipettes

Ion current rectification (ICR), which is the departure of the experimentally measured current-voltage curves from ohmic behavior, has recently drawn intensive interests from both a fundamental point of view and microfluidic-based applications. In this case, the rectification factor (RF), which is defined as the absolute value of the quotient between the currents recorded for one voltage polarity and the currents recorded for the same absolute value of voltage at the opposite polarity, is one of the most important parameters to describe the rectification behavior. Herein, we interestingly observed that RF is strongly dependent on the bias voltage from both the experimental and theoretically simulated results, and this dependence exhibits nonlinear deviation with increasing the rectification degree. At the same surface charge density, the linear deviation increases with decreasing the electrolyte concentration. When the electrolyte concentration remains the same, the linear deviation increases with increasing the surface charge density. The simulated results demonstrate that the electro-osmotic flow is not the dominating factor to this nonlinear phenomenon. This nonlinear deviation is considered to possibly originate from the inhomogeneous conductivity and the nonlinear change of conductance with the bias potential. This study is not only useful to understanding the ICR behavior at a conical glass nanopipette, but also potentially offers a new parameter to describe the rectification degree with this nonlinear relationship.

Dipole Effects on Ion Transport Demonstrated in Aprotic Solvents

Dipole interactions play a significant role in biological systems, influencing key functions such as protein folding and selective transport of ions through channels, among many others. Possible importance of the presence of dipoles in systems of synthetic nanopores has not yet been explored. Here, we report experiments and modeling of ionic current through nanopores in polymer films as well as glass nanopipettes. In order to probe importance of dipoles for ionic transport, experiments were performed with aprotic solvents characterized with dipole moments between 5D and 1.7D. LiClO4 was used as the electrolyte due to its high solubility in a wide range of solvents. The conical nanopores used in the experiments rectify the curent with the direction and degree being very sensitive to the surface properties of the pore walls. Current-voltage curves in aprotic solvents with high dipole moments provided evidence that the solvent and possible cation adsorption caused formation of an effectively positive surface potential, which was further confirmed by scanning ion conductance microscopy. Continuum modeling of ion current using models developed for biological systems confirmed that presence of dipoles on pore walls can indeed modify local ionic concentrations and the recorded current.

High-speed detection of DNA translocation in nanopipettes.

We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.

ナノ電気化学セル顕微鏡を用いた電極表面の局所電気化学測定

Scanning electrochemical cell microscopy with a single barrel nanopipette (nanoSECCM), which is one of scanning electrochemical microscope (SECM) families, is a powerful tool for analyzing electrochemical activities (e.g. redox reaction and lithium-ion (de) intercalation processes) at localized area. A glass nano-pipette is used as a probe of nanoSECCM filled with electrolyte and a reference electrode. As the pipette is in proximity of sample surface, a meniscus is created. Through the meniscus, electrochemical activities can be obtained at localized area on the sample. Further, as the pipette scans the sample surface, electrochemical activities can be also visualized. Our experiments demonstrate that nanoSECCM is applicable to investigation of current response related to lithium-ion transport, redox cycling on various types of electrodes.

High-bandwidth detection of short DNA in nanopipettes.

Glass or quartz nanopipettes have found increasing use as tools for studying the biophysical properties of DNA and proteins, and as sensor devices. The ease of fabrication, favourable wetting properties and low capacitance are some of the inherent advantages, for example compared to more conventional, silicon-based nanopore chips. Recently, we have demonstrated high-bandwidth detection of double-stranded (ds) DNA with microsecond time resolution in nanopipettes, using custom-designed electronics. The electronics design has now been refined to include more sophisticated control features, such as integrated bias reversal and other features. Here, we exploit these capabilities and probe the translocation of short dsDNA in the 100 bp range, in different electrolytes. Single-stranded (ss) DNA of similar length are in use as capture probes, so label-free detection of their ds counterparts could therefore be of relevance in disease diagnostics.

Real-time Monitoring of Discrete Synaptic Release Events and Excitatory Potentials within Self-reconstructed Neuromuscular Junctions.

Chemical synaptic transmission is central to the brain functions. In this regard, real-time monitoring of chemical synaptic transmission during neuronal communication remains a great challenge. In this work, in vivo-like oriented neural networks between superior cervical ganglion (SCG) neurons and their effector smooth muscle cells (SMC) were assembled in a microfluidic device. This allowed amperometric detection of individual neurotransmitter release events inside functional SCG-SMC synapse with carbon fiber nanoelectrodes as well as recording of postsynaptic potential using glass nanopipette electrodes. The high vesicular release activities essentially involved complex events arising from flickering fusion pores as quantitatively established based on simulations. This work allowed for the first time monitoring in situ chemical synaptic transmission under conditions close to those found in vivo, which may yield important and new insights into the nature of neuronal communications.