Temporal resolution in electrochemical imaging on single PC12 cells using amperometry and voltammetry at microelectrode arrays

Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.     

Mechanical mapping of single membrane proteins at submolecular resolution

The capacity of proteins to carry out different functions is related to their ability to undergo conformation changes, which depends on the flexibility of protein structures. In this work, we applied a novel imaging mode based on indentation force spectroscopy to map quantitatively the flexibility of individual membrane proteins in their native, folded state at unprecedented submolecular resolution. Our results enabled us to correlate protein flexibility with crystal structure and showed that α-helices are stiff structures that may contribute importantly to the mechanical stability of membrane proteins, while interhelical loops appeared more flexible, allowing conformational changes related to function.     

Imaging of nonuniform current density at microelectrodes by electrogenerated chemiluminescence

The chemiluminescence arising from reaction of electrogenerated radical cations of 9,10-diphenylanthracene (DPA) and benzonitrile (solvent) radical anions has been used to image microelectrodes with dimensions in the micrometer range. Experimental conditions including supporting electrolyte, DPA concentration, and excitation frequency were optimized to affect high luminescent intensity. In solutions of high resistance, the light was found to be temporally delayed with respect to the applied potential due to the increased time required to charge the double layer. Spatially nonuniform light at disk- and band-shaped microelectrodes was observed under certain conditions, with the highest intensity occurring at the region of the electrode with highest curvature. The optimum condition for observation of the nonuniform light was with very high electrode currents. Under this condition, the current density approaches that of the primary current distribution, a circumstance where spatially nonuniform potentials occur. This phenomenon was also examined at a conical electrode as a method of reducing the emission area. A submicrometer-size light source was obtained at high frequencies with an electrode that had a significantly larger uninsulated area.     

Infrared spectroscopic mapping of single nanoparticles and viruses at nanoscale resolution

We demonstrate that scattering near-field microscopy (s-SNOM) can determine infrared "fingerprint" spectra of individual poly(methyl methacrylate) nanobeads and viruses as small as 18 nm. Amplitude and phase spectra are found surprisingly strong, even at a probed volume of only 10(-20) l, and robust in regard to particle size and substrate. This makes infrared spectroscopic s-SNOM a versatile tool for chemical and-in the case of protein-secondary-structure identification.     

Detection of hydrogen peroxide at mesoporous platinum microelectrodes

Mesoporous (H(I)-ePt) platinum microelectrodes electrodeposited from the hexagonal (H(I)) lyotropic liquid crystalline phase are shown to be excellent amperometric sensors for the detection of hydrogen peroxide over a wide range of concentrations. Good reproducibility, high precision, and accuracy of measurements are demonstrated. Mesoporous microelectrodes retain the high rates of mass transport typical of conventional microelectrodes, and their high real surface area greatly enhances their catalytic activity. This unique combination of properties overcomes the limitations of previous amperometric hydrogen peroxide sensors and yields outstanding qualitative and quantitative results.     

Reusable platinum nanoparticle modified boron doped diamond microelectrodes for oxidative determination of arsenite

Boron doped diamond (BDD) macro- and microelectrodes were modified by electrodeposition of platinum nanoparticles using a multipotential step electrodeposition technique and used for the oxidative determination of arsenite, As(III). The formation of Pt nanoparticles was evident from cyclic voltammetry measurement, whereas AFM and SEM revealed the size and size distribution of deposited Pt nanoparticles. Raman spectroscopy illustrated a correlation between the typical BDD signature and the number of platinum deposition cycles. Linear sweep voltammetry performed with the modified BDD microelectrode outperformed its macrocounterpart and resulted in very low detecting currents with enhanced signal-to-noise ratios. With linearity up to 100 ppb and a detection limit of 0.5 ppb, the electrochemical system was applicable for processing tap and river water samples. Over 150 repetitive runs could be performed, and electrochemical etching of platinum allowed the reuse of the BDD microelectrode. The presence of copper and chloride ions, the two most severe interferents at levels commonly found in groundwater, did not interfere with the assay.     

Subsecond adsorption and desorption of dopamine at carbon-fiber microelectrodes

High-repetition fast-scan cyclic voltammetry and chronoamperometry were used to quantify and characterize the kinetics of dopamine and dopamine-o-quinone adsorption and desorption at carbon-fiber microelectrodes. A flow injection analysis system was used for the precise introduction and removal of a bolus of electroactive substance on a sub-second time scale to the disk-shaped surface of a microelectrode that was fabricated from a single carbon fiber (Thornel type T650 or P55). Pretreatment of the electrode surfaces consisted of soaking them in purified isopropyl alcohol for a minimum of 10 min, which resulted in S/N increasing by 200-400% for dopamine above that for those that were soaked in reagent grade solvent. Because of adsorption, high scan rates (2,000 V/s) are shown to exhibit equivalent S/N ratios as compared to slower, more traditional scan rates. In addition, the steady-state response to a concentration bolus is shown to occur more rapidly when cyclic voltammetric scans are repeated at short intervals (4 ms). The new methodologies allow for more accurate determinations of the kinetics of neurotransmitter release events (10-500 ms) in biological systems. Brain slice and in vivo experiments using T650 cylinder microelectrodes show that voltammetrically measured uptake kinetics in the caudate are faster using 2,000 V/s and 240 Hz measurements, as compared to 300 V/s and 10 Hz.     

Capillary electrophoretic separation of nuclei released from single cells

We report here the first capillary electrophoresis analysis of intact nuclei released on-column from single cells. Expression of the nuclear-targeted protein nuDsRed2 and the plasma membrane-bound farnesylated enhanced green fluorescent protein in cultured human DeltaH2-1 cells allowed fluorescent monitoring of the fate of these subcellular compartments upon injection of a single cell into the separation capillary. On-column treatment with digitonin allowed for the separation of the plasma membrane from the nucleus as indicated by their selective laser-induced fluorescence detection in two separate spectral regions. The data suggest that less than 0.1% of the plasma membrane remains bound to individually detected nuclei. In digitonin-treated cells, the electropherograms consisted of a prominent fluorescent peak attributed to nuDsRed2 localized to the nucleus and a collection of weakly fluorescent events (barely distinguishable from scattering) that seem to indicate additional localization of this protein to other subcellular regions. Taken together, this report points to the feasibility of studying intact organelles released from a single mammalian cell by capillary electrophoresis, which is a prerequisite to understanding the relevance of subcellular heterogeneity in biological systems.     

Triangulation of simple arbitrarily shaped polyhedra by cutting off one vertex at a time

We describe a heuristic method of triangulating arbitrarily shaped polyhedra without the addition of Steiner points. The polyhedra are simple, with each vertex connected to at least 3 other vertices (ie, coplanarity and colinearity are not considered). They may, however, be convex or concave and consist of dozens or even hundreds of facets. This makes the treatment universal enough to well meet the requirements of models used to simulate fractured rock masses. Certain concepts are defined in the work, eg, adjacent vertices, polygon of adjacent vertices, and closed cone of a vertex. A polygon of adjacent vertices of an apex can be subdivided into a set of nonoverlapping triangles without adding any vertices. These triangles, together with the apex, form tetrahedra whose union is the closed cone of the apex. The polyhedron is thus the union of the closed cones. Subsequently, we triangulate the polyhedron by gradually removing the closed cones of its vertices. The number of vertices of the polyhedron decreases by one each time a closed cone is removed. A block with n vertices can produce no more than n?3 tetrahedra. We present the analysis procedure and discuss the core issues of the method proposed.     

Printed carbon microelectrodes for electrochemical detection of single vesicle release from PC12 cells

We present a disposable system for recording neurotransmitter release from individual cells in vitro. A simple yet reliable microelectrode fabrication process is introduced using screen-printed carbon paste. It allows rapid fabrication of devices at low costs without standard clean-room technology. We demonstrate functionality of the system by real-time observation of vesicle release from single PC12 (rat pheochromocytoma) cells. The cells are cultured directly on the chip and can be used for immediate or long-term in vitro experiments. Thus, our approach may serve as a platform for pharmacological cell culture studies.     

Simulation of two-electron homogeneous electrocatalysis for steady-state voltammetry at hemispherical microelectrodes

Expanded space grid digital simulation of second-order, two-electron homogeneous electrocatalysis was extended to slow scan voltammetry at hemispherical microelectrodes. Predictions of the simulations are examined for reversible and quasireversible heterogeneous charge transfer of catalyst for a range of homogeneous catalytic rate constants (k1) and electrode radii. Working curves of catalytic efficiency vs long k1 were generated assuming reacting species with equal diffusion coefficients. As electrode radii in the less than 10-microns range decrease, progressively larger homogeneous catalytic rates are needed to yield analytically significant amplification of limiting currents. Simulations using hemispherical radii of (2/pi)rd can be used to predict catalytic efficiencies for microdisk electrodes with radii rd. Simulated working curves were used to estimate a log k1 of 3.88 +/- 0.55 (M-1 s-1) for electron transfer from the anion radical of 9,10-diphenylanthracene to 4,4'-dibromobiphenyl from steady-state catalytic efficiencies obtained at carbon microdisk electrodes. This value was in good agreement with 3.90 +/- 0.16 M-1 s-1 found previously by cyclic voltammetry.     

Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.     

Copper determination in urine by flow injection analysis with electrochemical detection at platinum disk microelectrodes of various radii

The incorporation of platinum disk microelectrodes of various radii (2.5-50 microns) in a well-jet flow cell offers reduced limits of detection for the determination of copper in urine by flow injection analysis compared with standard methods based on a conventional sized glassy carbon disk macroelectrode (radius 1.5 mm), in a thin-layer cell. The radius of the platinum disk microelectrode was found to be critical with respect to both the limit of detection and flow rate dependence. An optimal radius value of 28 microns was found with detection limits increasing with both larger and smaller electrode radii. In contrast, as theoretically expected, a diminished flow rate dependence was observed the smaller the radii of the platinum disk microelectrodes. Sample cleanup and preparation is conveniently achieved by the use of Sep-Pak cartridges and formation of a copper dithiocarbamate complex. The metal complex is easily oxidized at platinum disk microelectrodes in acetonitrile, which was the solvent used in the flow injection method of analysis.     

激光三角扫描法检测微刻槽

本文介绍一种激光三角扫描法微刻槽检测系统。该系统以激光三角法为基础 ,采用激光光斑能量细分方法 ,有效地解决了激光三角法由于光斑直径过大而引起的横向分辨力不足的问题 ,并容易做到微型化 ,适用对任意非镜面材料表面划痕的在线测量     

Collection and expansion of single cells and colonies released from a micropallet array

The ability to selectively grow out individual cells possessing unique characteristics from within a mixed population is of widespread importance for biomedical investigations. Generation of genetically engineered cell lines, transformation studies, cell-based assays, and stem cell studies are examples where single-cell cloning is of immense value. The vast majority of mammalian cells grow adherent to a surface; therefore, positive selection followed by cloning of cells while the cells remain adherent to their growth surface is an important goal. We recently demonstrated a microfabricated cell array combined with laser-based release of individual array elements for positive selection of single cells. In the current work, a strategy to collect single cells for clonal expansion is described. The system enabled cloning of individual cells with 80-90% efficiency. Single cells were selected and cloned from small populations of fewer than 10,000 cells. Strategies used by cells to migrate from the pallets to form colonies on the surface of the collection device were examined. Implementation of encoded array elements made it possible to follow specific cells throughout the selection, collection, and cloning procedure. Thus, a particular cell can be identified by any number of imaging techniques, isolated, and clonally expanded to generate a homogeneous cell line or a pure sample for genetic or biochemical analysis.     

People's well-being at the core of newly released national standards

正A number of national standards concerning people's wellbeing were officially released on October 20 in Yiwu of Zhejiang Province,covering standards for many daily necessities such as shoes,spectacle frame,knitted sportwear,watch,and household clothes dryer.Several standards boast the first of its kind in such areas as     

Coulometric detection of irreversible electrochemical reactions occurring at Pt microelectrodes used for neural stimulation

The electrochemistry of 50 μm diameter Pt electrodes used for neural stimulation was studied in vitro by reciprocal derivative chronopotentiometry. This differential method provides well-defined electrochemical signatures of the various polarization phenomena that occur at Pt microelectrodes and are generally obscured in voltage transients. In combination with a novel in situ coulometric approach, irreversible H(2) and O(2) evolution, Pt dissolution and reduction of dissolved O(2) were detected. Measurements were performed with biphasic, charge-balanced, cathodic-first and anodic-first current pulses at charge densities ranging from 0.07 to 1.41 mC/cm(2) (real surface area) in phosphate buffered saline (PBS) with and without bovine serum albumin (BSA). The extent to which O(2) reduction occurs under the different stimulation conditions was compared in O(2)-saturated and deoxygenated PBS. Adsorption of BSA inhibited Pt dissolution as well as Pt oxidation and oxide reduction by blocking reactive sites on the electrode surface. This inhibitory effect promoted the onset of irreversible H(2) and O(2) evolution, which occurred at lower charge densities than those in PBS. Reduction of dissolved O(2) on Pt electrodes accounted for 19-34% of the total injected charge in O(2)-saturated PBS, while a contribution of 0.4-12% was estimated for in vivo stimulation. These result may prove important for the interpretation of histological damage induced by neural stimulation and therefore help define safer operational limits.     

Direct determination of diffusion coefficients of substrate and product by chronoamperometric techniques at microelectrodes for any level of ionic support.

A new method for the determination of the diffusion coefficients of both the substrate, DS, and the product, DP, of an electrode process has been developed. The method proposed is based on the analysis of the transient currents and can be applied to some reactions of the type SzS = PzP + ne and, in contrast to the concept based on the steady-state current, to any ratio of the concentrations of supporting electrolyte and substrate. The diffusion coefficients can be evaluated sequentially from the two parts of the double-potential step chronoamperogram, since the magnitude of the normalized chronoamperometric current of the first step depends on the DS value, while that of the second step is controlled by both DS and DP values. The corresponding, easy-to-use equations and procedures are given in the paper. The equations were derived on the basis of the numerical simulation data. The proposed methods of determination of diffusion coefficients for the substrates and products have been examined experimentally with the charged and uncharged ferrocene derivatives under diffusional and mixed diffusion-migration conditions. Only the chronoamperometric DS values obtained for the substrates could be compared to those determined from the steady-state diffusional current. It was found that for the systems investigated the agreement between these two methods was good. In this limited comparison, the standard deviations for the transient techniques were slightly larger than those obtained for the excess supporting electrolyte steady-state voltammetry.     

Studies on stress-induced changes at the subcellular level by Raman microspectroscopic mapping

Raman microspectroscopic mapping enables one to study the chemical composition and molecular structure of subcellular components in individual cells without the need for labeling. Lung fibroblast cells were prepared under normal conditions and under stress, which was induced by 24 h of exposure to glyoxal. Raman microspectroscopic maps were recorded from fixed cells with 785-nm excitation and with 1-microm step width. Cluster analysis was applied to generate pseudocolor images of the cell morphology. Raman maps revealed that the cell nucleus shrinks in stressed cells, called pyknosis, which refers to an early stage of apoptosis. The intensity of nucleic acid bands decreased in cluster-averaged Raman spectra of the nucleus and cytoplasm, which is consistent with degradation and conformational changes of DNA and RNA. During a later stage of apoptosis, Raman maps indicate a rounding of cells, a further intensity decrease of nucleic acids bands, fragmentation of the nucleus, disappearance of lipid bodies, and formation of blisters at the cell surface. Whereas the peripheral membrane of the undisturbed cell is composed of lipids and cholesterol, the blisters have a higher protein content with nucleic acids incorporated. The results demonstrate that Raman spectroscopic mapping might become a powerful tool in cell biology for single cell analysis.