Nanoscale Intelligent Imaging Based on Real-Time Analysis of Approach Curve by Scanning Electrochemical Microscopy

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.9b02361.Scanning electrochemical microscopy (SECM) enables high-resolution imaging by examining the amperometric response of an ultramicroelectrode tip near a substrate. Spatial resolution, however, is compromised for non-flat substrates, where distances from a tip far exceed the tip size to avoid artifacts caused by the tip–substrate contact. Herein, we propose a new imaging mode of SECM based on real-time analysis of approach curve to actively control nanoscale tip–substrate distances without contact. The power of this software-based method is demonstrated by imaging an insulating substrate with step edges using standard instrumentation without combination of another method for distance measurement, e.g., atomic force microscopy. An ~500 nm-diameter Pt tip approaches down to ~50 nm from upper and lower terraces of a 500 nm-height step edge, which are located by real-time theoretical fitting of experimental approach curve to ensure the lack of electrochemical reactivity. The tip approach to step edge can be terminated at <20 nm prior to the tip–substrate contact as soon as the theory deviates from the tip current, which is analyzed numerically afterward to locate the inert edge. The advantageous local adjustment of tip height and tip current at the final point of tip approach distinguishes the proposed imaging mode from other modes based on standard instrumentation. In addition, the glass sheath of Pt tip is thinned to ~150 nm to rarely contact the step edge, which is unavoidable and instantaneously detected as an abrupt change in the slope of approach curve to prevent the damage of fragile nanotip

Evolution of Otx paralogue usages in early patterning of the vertebrate head

AbstractTo assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates

Probing High Permeability of Nuclear Pore Complexes by Scanning Electrochemical Microscopy: Ca2+ Effects on Transport Barriers

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http:doi.org/10.1021/acs.analchem.9b00796.The nuclear pore complex (NPC) solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell to play important biological and biomedical roles. However, it is not well-understood chemically how this biological nanopore selectively and efficiently transports various substances, including small molecules, proteins, and RNAs by using transport barriers that are rich in highly disordered repeats of hydrophobic phenylalanine and glycine intermingled with charged amino acids. Herein, we employ scanning electrochemical microscopy to image and measure the high permeability of NPCs to small redox molecules. The effective medium theory demonstrates that the measured permeability is controlled by diffusional translocation of probe molecules through water-filled nanopores without steric or electrostatic hindrance from hydrophobic or charged regions of transport barriers, respectively. However, the permeability of NPCs is reduced by a low millimolar concentration of Ca2+, which can interact with anionic regions of transport barriers to alter their spatial distributions within the nanopore. We employ atomic force microscopy to confirm that transport barriers of NPCs are dominantly recessed (∼80%) or entangled (∼20%) at the high Ca2+ level in contrast to authentic populations of entangled (∼50%), recessed (∼25%), and “plugged” (∼25%) conformations at a physiological Ca2+ level of submicromolar. We propose a model for synchronized Ca2+ effects on the conformation and permeability of NPCs, where transport barriers are viscosified to lower permeability. Significantly, this result supports a hypothesis that the functional structure of transport barriers is maintained not only by their hydrophobic regions, but also by charged regions

Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time

The morphology of a live cell reflects the organization of the cytoskeleton and the healthy status of the cell. We established a label-free platform for monitoring the changing morphology of live cells in real time based on scanning electrochemical microscopy (SECM). The dynamic morphology of a live human bladder cancer cell (T24) was revealed by time-lapse SECM with dissolved oxygen in the medium solution as the redox mediator. Detailed local movements of cell membrane were presented by time-lapse cross section lines extracted from time-lapse SECM. Vivid dynamic morphology is presented by a movie made of time-lapse SECM images. The morphological change of the T24 cell by non-physiological temperature is in consistence with the morphological feature of early apoptosis. To obtain dynamic cellular morphology with other methods is difficult. The non-invasive nature of SECM combined with high resolution realized filming the movements of live cells

Voltammetric Ion Selectivity of Thin Ionophore-Based Polymeric Membranes: Kinetic Effect of Ion Hydrophilicity

The high ion selectivity of potentiometric and optical sensors based on ionophore-based polymeric membranes is thermodynamically limited. Here, we report that the voltammetric selectivity of thin ionophore-based polymeric membranes can be kinetically improved by several orders of magnitude in comparison with their thermodynamic selectivity. The kinetic improvement of voltammetric selectivity is evaluated quantitatively by newly introducing a voltammetric selectivity coefficient in addition to a thermodynamic selectivity coefficient. Experimentally, both voltammetric and thermodynamic selectivity coefficients are determined from cyclic voltammograms of excess amounts of analyte and interfering ions with respect to the amount of a Na⁺- or Li⁺-selective ionophore in thin polymeric membranes. We reveal the slower ionophore-facilitated transfer of a smaller alkaline earth metal cation with higher hydrophilicity across the membrane/water interface, thereby kinetically improving voltammetric Na⁺ selectivity against calcium, strontium, and barium ions by 3, 2, and 1 order of magnitude, respectively, in separate solutions. Remarkably, voltammetric Na⁺ and Li⁺ selectivity against calcium and magnesium ions in mixed solutions is improved by 4 and >7 orders of magnitude, respectively, owing to both thermodynamic and kinetic effects in comparison with thermodynamic selectivity in separate solutions. Advantageously, the simultaneous detection of sodium and calcium ions is enabled voltammetrically in contrast to the potentiometric and optical counterparts. Mechanistically, we propose a new hypothetical model that the slower transfer of a more hydrophilic ion is controlled by its partial dehydration during the formation of the adduct with a “water finger” prior to complexation with an ionophore at the membrane/water interface

Nanoelectrochemistry

This book provides an overview of nanoscale electrochemistry in the context of well-established concepts and principles of electrochemistry. It presents fundamental theory with the emphasis on differences between charge-transfer and mass-transfer processes on the nanoscale and analogous reactions occurring at macroscopic electrochemical interfaces. The rigorous theoretical treatment enables readers to gain an in-depth understanding of nanoelectrochemical systems discussed. The book also covers modern electrochemical tools and techniques that enable the fabrication and characterization of elec