Flame etching enhances the sensitivity of carbon-fiber microelectrodes

Small sensors are useful for in vivo measurements and probing small spaces. In this paper, we compare two methods of fabrication of small, cylindrical carbon-fiber microelectrodes: flame-etching and electrochemical etching. With both methods, microelectrodes can be fabricated with tip diameters of 1 to 3 microm. Electrodes were tested with fast-scan cyclic voltammetry. Flame etching resulted in electrodes that have larger S/N ratios and higher currents per unit area for 1 microM dopamine than normal carbon-fiber microelectrodes or electrochemically etched electrodes. Therefore, the increased sensitivity is not just a property of size. The flame-etched surfaces had nanometer-scale surface features that were not observed on the other electrodes and exhibited increased sensitivity for other electroactive compounds found in the brain, including ascorbic acid, DOPAC, and serotonin. Faster kinetics and a faster response to a step change in dopamine were also observed, when the applied waveform was -0.4 to 1.0 V and back at 400 V/s. The sensitivity of the flame-etched electrodes was enhanced by overoxidizing the surface. The flame-etched electrodes were used to detect dopamine release in anesthetized rats after a single stimulation pulse. The small flame-etched electrodes will facilitate measurements of low concentrations in discrete brain regions or small organisms.     

Fabrication and characterization of a nanometer-sized optical fiber electrode based on selective chemical etching for scanning electrochemical/optical microscopy

We have already reported a method for fabricating ultramicroelectrodes (Suzuki, K. JP Patent, 2004-45394, 2004). This method is based on the selective chemical etching of optical fibers. In this work, we undertake a detailed investigation involving a combination of etched optical fibers with various types of tapered tip (protruding-shape, double- (or pencil-) shape and triple-tapered electrode) and insulation with electrophoretic paint. Our goal is to establish a method for fabricating nanometer-sized optical fiber electrodes with high reproducibility. As a result, we realized pencil-shaped and triple-tapered electrodes that had radii in the nanometer range with high reproducibility. These nanometer-sized electrodes showed well-defined sigmoidal curves and stable diffusion-limited responses with cyclic voltammetry. The pencil-shaped optical fiber, which has a conical tip with a cone angle of 20 degrees , was effective for controlling the electrode radius. The pencil-shaped electrodes had higher reproducibility and smaller electrode radii (r(app) < 1.0 nm) than those of other etched optical fiber electrodes. By using a pencil-shaped electrode with a 105-nm radius as a probe, we obtained simultaneous electrochemical and optical images of an implantable interdigitated array electrode. We achieved nanometer-scale resolution with a combination of scanning electrochemical microscopy SECM and optical microscopy. The resolution of the electrochemical and optical images indicated sizes of 300 and 930 nm, respectively. The neurites of living PC12 cells were also successfully imaged on a 1.6-microm scale by using the negative feedback mode of an SECM.     

Fast-scan voltammetry of cyclic nitroxide free radicals

Fast-scan cyclic voltammetry at carbon fiber microelectrodes is used to detect the cyclic nitroxide 2,2,6,6-tetramethylpiperidinyl-1-oxy free radical (TEMPO) and three analogs. The electrochemical behavior of the TEMPO analogs at unmodified carbon fiber electrodes is found to differ greatly from their behavior at glassy carbon electrodes. After the electrode is coated with the polymer Nafion, the electrodes exhibit increased sensitivity to TEMPO and 4-amino-TEMPO. Voltammograms of the nitroxides at Nafion-coated electrodes indicate that the oxidized form (oxoammonium ion) and the free radical form have greatly different mobilities through the polymeric coating. Response times to changes in nitroxide concentration vary from subsecond at bare electrodes (all four analogs) and 4-hydroxy-TEMPO at modified electrodes to 1-3 s for TEMPO and 4-amino-TEMPO at modified electrodes. The detection limit for 4-amino-TEMPO is 50 μM at an unmodified electrode and 5 μM at a Nafion-coated carbon fiber electrode. The sensitivity of the Nafion-modified electrode to TEMPO, 4-hydroxy-TEMPO, and 4-amino-TEMPO can be improved by choosing a resting potential at which the oxoammonium ion form of the nitroxide is preconcentrated into the Nafion film. Using fast-scan cyclic voltammetry and the modified carbon fiber electrodes, the reaction of two nitroxide free radicals with ascorbate can be monitored. This work shows that fast-scan voltammetry at microelectrodes is a sensitive method that can be used to follow reactions of cyclic nitroxide free radicals in solution.     

基于多功能加工平台的微细电解加工

电解加工在加工过程中因难以控制加工形状而很少应用在微细加工领域,为了对微细电解加工可行性进行探索,设计了多功能微细加工平台,利用多功能微细加工平台可为微细电解加工在线制作电极,采用低加工电压、低浓度的钝化电解液、高频窄脉冲电源和高速旋转的微细电极,进行了微细电解加工实验,取得了很好的工艺效果,加工间隙是影响加工精度的关键因素,设计了一个加工间隙控制伺服系统,以保证微小的加工间隙,在厚为100μm的不锈钢薄片上用微细电解打孔加工出直径为65μm的微小孔,利用微细电解加工时电极无损耗,提出采用简单圆柱微细电极进行微细电解铣削,加工出较高精度的微结构,取得了较好的工艺效果,从而验证了该微细电解加工装置的微细加工能力和方法的可行性。     

Multiwalled carbon-nanotube-functionalized microelectrode arrays fabricated by microcontact printing: platform for studying chemical and electrical neuronal signaling

A facile method is proposed for the deposition of multiwalled carbon nanotube (MWCNT) layers onto microelectrode arrays by means of a microcontact printing technique, leading to the fabrication of MEAs characterized by well defined electrical and morphological properties. Using polydimethyl siloxane stamps, produced from different mold designs, a flexibility of printing is achieved that provides access to microscale, nanostructured electrodes. The thickness of MWCNT layers can be exactly predetermined by evaluating the concentration of the MWCNT solution employed in the process. The electrode morphology is further characterized using laser scanning and scanning electron microscopy. Next, by means of impedance spectroscopy analysis, the MWCNT-electrode contact resistance and MWCNT film resistance is measured, while electrochemical impedance spectroscopy is used to estimate the obtained electrode-electrolyte interface. Structural and electrochemical properties make these electrodes suitable for electrical stimulation and recording of neurons and electrochemical detection of dopamine. MWCNT-functionalized electrodes show the ability to detect micromolar amounts of dopamine with a sensitivity of 19 nA μm(-1) . In combination with their biosensing properties, preliminary electrophysiological measurements show that MWCNT microelectrodes have recording properties superior to those of commercial TiN microelectrodes when detecting neuronal electrical activity under long-term cell-culture conditions. MWCNT-functionalized microelectrode arrays fabricated by microcontact printing represent a versatile and multipurpose platform for cell-culture monitoring.     

Microscopic imaging with electrogenerated chemiluminescence

The use of electrogenerated chemiluminescence (ECL) at microelectrodes as a light source for scanning optical microscopy is demonstrated. Cone-shaped microelectrodes were constructed by flame etching carbon fibers to a fine point. ECL generated in solution at such electrodes was forced to the apex of the conducting surface by using high-frequency (20-kHz) potential pulses and high concentrations of ECL reagents in the solution. ECL arose from the reaction of 9,10-diphenylanthracene radical cation with the radical anion of benzonitrile, the solvent. The electrode/light source was raster-scanned a finite distance above the sample surface, and images were generated with standard scanning probe software by collecting the transmitted light with a microscope objective. These images compared favorably to optical images of the same sample. A resolution of approximately 600 nm was achieved with this arrangement even though a feedback loop was not employed to control the tip distance from the sample. The source was sufficiently bright (1.82 pW) that well-defined transmittance spectra could be obtained at individual locations on the sample.     

Carbon composite microelectrodes fabricated by electrophoretic deposition

Single-walled carbon nanotubes were deposited on one end of the etched carbon fiber electrodes by an electrophoretic method. The carbon nanotube bundles formed a dense network on the carbon fiber surface. The electrochemical properties of the composite carbon electrodes were studied in the buffered neutral solutions. The results in cyclic voltammetry's characteristic indicate that the electrons on the electrodes transfer very fast. Furthermore, the redox reactions of dopamine (DA) on the composite electrodes show good sensitivity. When the DA concentration was 0.02 mM, the peak current in differential pulse technique reached 1.33 microA after performing the background subtraction. In addition, the simultaneous detection of DA and ascorbic acid (AA) showed that the interference effect was not observed. It was suggested that the carbon composite microelectrodes have potential applications as electrochemical sensors inside a single cell.     

Fabrication of nanopore array electrodes by focused ion beam milling

Single nanopore electrodes and nanopore electrode arrays have been fabricated using a focused ion beam (FIB) method. High aspect ratio pores (approximately 150-400-nm diameter and 500-nm depth) were fabricated using direct-write local ion milling of a silicon nitride layer over a buried platinum electrode. This local milling results in formation of a recessed platinum electrode at the base of each nanopore. The electrochemical properties of these nanopore metal electrodes have been characterized by voltammetry. Steady-state voltammograms were obtained for a range of array sizes as well as for single nanopore electrodes. High-resolution scanning electron microscopy imaging of the arrays showed that the pores had truncated cone, rather than cylindrical, conformations. A mathematical model describing diffusion to an electrode located at the base of a truncated conical pore was developed and applied to the analysis of the electrode geometries. The results imply that diffusion to the pore mouth is the dominant mass transport process rather than diffusion to the electrode surface at the base of the truncated cone. FIB milling thus represents a simple and convenient method for fabrication of prototype nanopore electrode arrays, with scope for applications in sensing and fundamental electrochemical studies.     

Etched carbon-fiber electrodes as amperometric detectors of catecholamine secretion from isolated biological cells

Voltammetric electrodes with micrometer dimensions have been fabricated from carbon fibers etched to a conical shape and insulated with poly(oxyphenylene) following literature procedures. The resultant electrode has a tip radius in the micron range. The response of this electrode is compared to a carbon-fiber electrode prepared by sealing a carbon-fiber electrode in a glass pipet (electrode radius greater than 3.5 microns). While the etched electrodes did not follow electrochemical theory as well as the glass-encased electrode, the etched electrode was found to be suitable for the amperometric measurement of the secretion of catecholamines from isolated bovine adrenal cells. Comparable results are only obtained when the two different electrodes are placed 1 micron from the cell surface. When the etched electrode is placed further away, less secretion is observed, because of diffusion and accompanying dilution.     

Steady-state limiting currents at finite conical microelectrodes

The investigation of steady-state diffusion-limiting currents at finite conical microelectrodes is reported. Such electrodes are of particular interest as probes in studies of kinetic reactions, measurements in microenvironments, and high-resolution electrochemical imaging. The diffusion-limiting currents were calculated using numerical (finite element) analysis and compared to those obtained at inlaid disk and hemispheroidal microelectrodes. Time-dependent simulations demonstrating the approach of the diffusion current to a steady-state value are also reported. The steady-state diffusion-limiting currents obtained were found to be a strong function of the electrode geometry, including the aspect ratio of the cone and the thickness of the insulating sheath. Thus, simple, approximate analytical expressions which account for these geometrical dependencies in the simulated steady-state diffusion-limited currents are also reported. As a limiting case, an analytical approximation was obtained for the steady-state current to a microdisk as a function of the insulator thickness. The use of these approximate equations in calculating electrode radii from steady-state diffusion-limiting currents is demonstrated, and good agreement was found with previously reported experimental studies. Use of the frequently implemented hemispherical theory to analyze steady-state diffusion-limiting currents obtained with finite conical microelectrodes is shown to result in an experimentally acceptable underestimation of the electrode radius.     

化学镀制备高温质子导体镍电极及其电化学性能

采用化学镀在高温质子导体CaZr_0.9In_0.1O_3-δ (CZI)的电解质陶瓷表面沉积金属镍电极, 通过SEM显微结构分析比较了酸刻蚀和还原工艺对电极形貌以及电极-电解质界面的影响。结果表明, 使用HNO₃-HCl混合刻蚀液, 并以水合肼为还原剂的二次化学镀可获得颗粒均匀细小且界面结合良好的镍电极。通过电化学阻抗谱并结合浓差电池等方法研究比较了以化学镀镍电极和涂覆焙烧铂电极为电极, CZI为电解质的对称电池的电导率和质子迁移率。工作温度为800℃时, 镍电极高温质子导体的总电导率为4.131×10^-4 S/cm, 并且工作温度在400℃以上时, 镍电极对称电池的质子迁移率均接近100%。这些结果表明, 二次化学镀制备的镍电极具有与铂电极相近的电化学性能, 而成本则更低, 可以取代Pt电极用于高温质子导体的电化学器件中。     

Measurement of enzyme activity in single cells by voltammetry using a microcell with a positionable dual electrode

The electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual microelectrode. The microcell array with the nanoliter-scale microcells was constructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radius Au disk working electrode and a approximately 80-microm-radius Ag/AgCl reference electrode. Peroxidase was chosen as the model enzyme. Factors that concern electrochemical single-cell analysis in microcells such as solution evaporation, interference of soluble oxygen, electrode size, solution volume, and electrode fouling were investigated and discussed. The 20 or 100 nL of detection volume was found to be suitable for peroxidase determination in single neutrophils or single acute promyelocytic leukemia cells without interference from intracellular macromolecules and electrode fouling, when the dual electrode with a 10-microm-radius Au disk working electrode was used. Cells were perforated with digitonin before transferring them into the microcells, to lyse cells easily. The perforated cells were transferred into the microcells by pushing a microscope slide on a drop of the cell suspension on the microcell array. After a single cell in the microcell was lysed using a freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 x 10(-3) mol/L hydroquinone and 2.0 x 10(-3) mol/L H2O2 as the substrates of the enzyme-catalyzed reaction was added. The microcell array was positioned in a constant-humidity chamber to prevent evaporation. Then the dual electrode was inserted into the microcell by means of a scanning electrochemical microscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-state current on the voltammogram after subtracting the blank and using the calibration curve.     

Carbon-ring microelectrode arrays for electrochemical imaging of single cell exocytosis: fabrication and characterization

Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 μm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.     

Scanning electrochemical microscopy with slightly recessed nanotips

Slightly recessed nanoelectrodes were prepared by controlled etching of nanometer-sized, flat Pt electrodes. By using high-frequency (e.g., 2 MHz) ac voltage, the layer of Pt as thin as greater, approximately >3 nm was removed to produce a cylindrical cavity inside the insulating glass sheath. The etched electrodes were characterized by combination of voltammetry and scanning electrochemical microscopy (SECM) to determine the radius and the effective depth of the recess. The theory was developed for current versus distance curves obtained with a recessed tip approaching either a conductive or an insulating substrate. Good agreement between the theoretical and experimental approach curves indicated that recessed nanotips are suitable for quantitative feedback mode SECM experiments.     

基于碳膜的柔性神经微电极阵列加工

为了实现长期安全有效的神经电刺激,避免电极腐蚀情况,提出了一种基于热解光刻胶形成碳膜作为导电材料的柔性视网膜神经微电极阵列．首先,通过高温热解图形化的光刻胶形成导电碳膜,然后利用光敏性聚酰亚胺（Durimide 7510）作为基底材料,通过光刻结合转膜、键合和牺牲层腐蚀技术,实现了具有柔性特征的碳膜神经微电极阵列．实验结果显示这种碳膜电极的电荷存储能力达到6．625mC／cm2．是金电极的12．4倍,显示了良好的电化学稳定性和电荷注入能力,有利于其长期植入和安全有效地工作,可应用于新一代人工视网膜系统的构建．     

Miniaturized electrochemical flow cells

Several novel types of miniaturized electrochemical flow cells are described. The flow cells are fabricated in fluorinated ethylene propylene using a novel technique where channels with inner diameters down to 13 microm are integrated with electrodes. The channel is formed by shrinking and simultaneous melting of a heat shrink/melt tubing around a channel template (a tungsten wire) and electrodes followed by removal of the channel template. The technique allows incorporation of different electrode materials of different sizes. The electrode configuration consists of one or two working electrodes inside the channel and a counter electrode located in the channel outlet reservoir. Electrode configurations with different channel and working electrode sizes, different electrode materials including carbon fibers, glassy carbon rods, poly(tetrafluoroethylene)/carbon composite material, and platinum wires, and different arrangements have been assembled. Hydrodynamic voltammograms in dual-electrode (generator-collector) experiments indicate good potential control for cells with 25-microm channels, while there is some iR drop in cells with 13-microm channels. Cells prepared with a cylindrical working electrode tangent and perpendicular to a flow channel show a flow rate dependence consistent with thin-layer cell behavior. Electrode areas can be made in the range of 10(-10)-10(-8) m2.     

SiC-C fiber electrode for biological sensing

Microelectrodes implanted in the brain can act as transducers for the neuronal impulses. In this work a composite fiber (SiC-C) electrode was studied for neuronal activity sensing and for biochemical detection of electroactive neurotransmitters. An electrolytic etching technique was also developed for fabricating electrode tips from SiC-C composite fiber. SEM was used to study different tip geometries and shapes. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the electrochemical characterization and modeling. Almost square voltammograms showed that the current generated was due to the charging and discharging of the capacitive double layer without any significant redox reaction. The electrodes showed capacitive interaction with the surrounding electrolyte solution which is highly desirable for safe charge transfer. Biochemical sensing of neurotransmitters including dopamine hydrochloride and vitamin C was done with the SiC-C composite electrodes and oxidation currents were found to vary linearly with concentration. In vivo action potential recordings from anesthetized rat's brain with very high signal to noise ratio were obtained.     

Electrochemical multianalyte immunoassays using an array-based sensor

A novel amperometric biosensor for performing simultaneous electrochemical multianalyte immunoassays is described. The sensor consisted of eight iridium oxide sensing electrodes (0.78 mm(2) each), an iridium counter electrode, and a Ag/AgCl reference electrode patterned on a glass substrate. Four different capture antibodies were immobilized on the sensing electrodes via adsorption. Quantification of proteins was achieved using an ELISA in which the electrochemical oxidation of enzyme-generated hydroquinone was measured. The spatial separation of the electrodes enabled simultaneous electrochemical immunoassays for multiple proteins to be conducted in a single assay without amperometric cross-talk between the electrodes. The simultaneous detection of goat IgG, mouse IgG, human IgG, and chicken IgY was demonstrated. The detection limit was 3 ng/mL for all analytes. The sensor had excellent precision (1.9-8.2% interassay CV) and was comparable in performance to commercial single-analyte ELISAs. We anticipate that chip-based sensors, as described herein, will be suitable for the mass production of economical, miniaturized, multianalyte assay devices.     

Plasma-deposited fluorocarbon films: insulation material for microelectrodes and combined atomic force microscopy-scanning electrochemical microscopy probes

Pinhole-free insulation of micro- and nanoelectrodes is the key to successful microelectrochemical experiments performed in vivo or in combination with scanning probe experiments. A novel insulation technique based on fluorocarbon insulation layers deposited from pentafluoroethane (PFE, CF3CHF2) plasmas is presented as a promising electrical insulation approach for microelectrodes and combined atomic force microscopy-scanning electrochemical microscopy (AFM-SECM) probes. The deposition allows reproducible and uniform coating, which is essential for many analytical applications of micro- and nanoelectrodes such as, e.g., in vivo experiments and SECM experiments. Disk-shaped microelectrodes and frame-shaped AFM tip-integrated electrodes have been fabricated by postinsulation focused ion beam (FIB) milling. The thin insulation layer for combined AFM-SECM probes renders this fabrication technique particularly useful for submicro insulation providing radius ratios of the outer insulation versus the disk electrode (RG values) suitable for SECM experiments. Characterization of PFE-insulated AFM-SECM probes will be presented along with combined AFM-SECM approach curves and imaging.     

碳基电化学双层电容器及复合电源系统的研制

碳基电化学超电容器作为一种新一代储能系统具有广泛的应用.多种测试及研究手段表明本文制备的炭材料具有适合电化学超电容器用途的特殊结构和型貌.直流充放电、循环伏安以及交流阻抗等实验显示了使用该材料组装的电容器具有良好的电化学性能,活性物质的比容量为168.5F/g,在大功率充放电条件下的活性物质的能量密度>5.0Wh/kg,同时具有10⁵以上的循环寿命,脉冲放电实验证明超电容器能有效改善镍氢电池的大电流脉冲放电性能.