Scanning Ion Conductance Microscopy of Nafion-Modified Nanopores.

Single nanopores in silicon nitride membranes are asymmetrically modified with Nafion and investigated with scanning ion conductance microscopy, where Nafion alters local ion concentrations at the nanopore. Effects of applied transmembrane potentials on local ion concentrations are examined, with the Nafion film providing a reservoir of cations in close proximity to the nanopore. Fluidic diodes based on ion concentration polarization are observed in the current-voltage response of the nanopore and in approach curves of SICM nanopipette in the vicinity of the nanopore. Experimental results are supported with finite element method simulations that detail ion depletion and enrichment of the nanopore/Nafion/nanopipette environment.

Surface Charge Measurements with Scanning Ion Conductance Microscopy Provide Insights into Nitrous Acid Speciation at the Kaolin Mineral-Air Interface.

Unique surface properties of aluminosilicate clay minerals arise from anisotropic distribution of surface charge across their layered structures. Yet, a molecular-level understanding of clay mineral surfaces has been hampered by the lack of analytical techniques capable of measuring surface charges at the nanoscale. This is important for understanding the reactivity, colloidal stability, and ion-exchange capacity properties of clay minerals, which constitute a major fraction of global soils. In this work, scanning ion conductance microscopy (SICM) is used for the first time to visualize the surface charge and topography of dickite, a well-ordered member of the kaolin subgroup of clay minerals. Dickite displayed a pH-independent negative charge on basal surfaces whereas the positive charge on edges increased from pH 6 to 3. Surface charges responded to malonate addition, which promoted dissolution/precipitation reactions. Results from SICM were used to interpret heterogeneous reactivity studies showing that gas-phase nitrous acid (HONO) is released from the protonation of nitrite at Al-OH2+ groups on dickite edges at pH well above the aqueous pKa of HONO. This study provides nanoscale insights into mineral surface processes that affect environmental processes on the local and global scale.

Imaging with Ion Channels.

We describe the incorporation of gated ion channels into probes for scanning ion conductance microscopy (SICM) as a robust platform for collecting spatial information at interfaces. Specifically, a dual-barrel pipet is used, where one barrel controls the pipet position and the second barrel houses voltage-gated transient receptor potential vanilloid 1 (TRPV1) channels excised in a sniffer-patch configuration. Spatially resolved sensing with TRPV1 channels is demonstrated by imaging a porous membrane where a transmembrane potential across the membrane generates local electric field gradients at pores that activate TRPV1 channels when the probe is in the vicinity of the pore. The scanning routine and automated signal analysis demonstrated provide a generalizable approach to employing gated ion channels as sensors for imaging applications.

Imaging effects of hyperosmolality on individual tricellular junctions.

The use of hyperosmolar agents (osmotherapy) has been a major treatment for intracranial hypertension, which occurs frequently in brain diseases or trauma. However, side-effects of osmotherapy on the brain, especially on the blood-brain barrier (BBB) are still not fully understood. Hyperosmolar conditions, termed hyperosmolality here, are known to transiently disrupt the tight junctions (TJs) at the endothelium of the BBB resulting in loss of BBB function. Present techniques for evaluation of BBB transport typically reveal aggregated responses from the entirety of BBB transport components, with little or no opportunity to evaluate heterogeneity present in the system. In this study, we utilized potentiometric-scanning ion conductance microscopy (P-SICM) to acquire nanometer-scale conductance maps of Madin-Darby Canine Kidney strain II (MDCKII) cells under hyperosmolality, from which two types of TJs, bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs), can be visualized and differentiated. We discovered that hyperosmolality leads to increased conductance at tTJs without significant alteration in conductance at bTJs. To quantify this effect, an automated computer vision algorithm was designed to extract and calculate conductance components at both tTJs and bTJs. Additionally, lowering Ca2+ concentration in the bath facilitates tTJ disruption under hyperosmolality. Strengthening tTJ structure by overexpressing immunoglobulin-like domain-containing receptor 1 (ILDR1) protein abrogates the effect of hyperosmolality. We posit that osmotic stress physically disrupts tTJ structure, as evidenced by super-resolution microscopy. Findings from this study not only provide a high-resolution view of TJ structure and function, but also can inform current osmotherapy and drug delivery strategies for brain diseases.

Nanoelectrochemistry in the study of single-cell signaling.

Label-free biosensing has been the dream of scientists and biotechnologists as reported by Vollmer and Arnold (Nat Methods 5:591-596, 2008). The ability of examining living cells is crucial to cell biology as noted by Fang (Int J Electrochem 2011:460850, 2011). Chemical measurement with electrodes is label-free and has demonstrated capability of studying living cells. In recent years, nanoelectrodes of different functionality have been developed. These nanometer-sized electrodes, coupled with scanning electrochemical microscopy (SECM), have further enabled nanometer spatial resolution study in aqueous environments. Developments in the field of nanoelectrochemistry have allowed measurement of signaling species at single cells, contributing to better understanding of cell biology. Leading studies using nanoelectrochemistry of a variety of cellular signaling molecules, including redox-active neurotransmitter (e.g., dopamine), non-redox-active neurotransmitter (e.g., acetylcholine), reactive oxygen species (ROS), and reactive nitrogen species (RNS), are reviewed here.

A high spatiotemporal study of somatic exocytosis with scanning electrochemical microscopy and nanoITIES electrodes.

Extra-synaptic exocytosis is an essential component of cellular communication. A knowledge gap exists in the exocytosis of the non-redox active transmitter acetylcholine. Using the nano-interface between two immiscible electrolyte solutions and scanning electrochemical microscopy (SECM), a high resolution spatiotemporal study of acetylcholine exocytosis is shown from an individual neuronal soma. The nanoelectrode was positioned ∼140 nm away from the release sites on the soma using an SECM. The quantitative study enables the obtaining of key information related to cellular communication, including extracellular concentration of the neurotransmitter, cellular permeability, Ca2+ dependence on somatic release, vesicle density, number of molecules released and the release dynamics. Measurements were achieved with a high signal to noise ratio of 6-19. The released neurotransmitter with a concentration of 2.7 (±1.0) µM was detected at the nanoelectrodes with radii of 750 nm to 860 nm.