Direct electrochemistry behavior of cytochrome c/L-cysteine modified electrode and its electrocatalytic oxidation to nitric oxide

Cytochrome c (Cyt c) was successfully immobilized on L-cysteine modified gold electrode by multicyclic voltammetry method. The electrochemical behavior of Cyt c on the L-cysteine modified electrode was explored. In 0.10 M, pH 7.0 phosphate buffer solution (PBS), Cyt c showed a quasi-reversible electrochemical redox behavior with E(pc)=0.180 V, E(pa)=0.208 V (versus Ag/AgCl). The Cyt c/L-cysteine modified electrode gave an excellent electrocatalytic activity towards the oxidation of nitric oxide, and the catalysis currents were proportional to the nitric oxide concentration in the range of 7.0 x 10(-7) to 1.0 x 10(-5) M, the linear regression equation is I (microA)=-0.124-0.003 C(NO) (microM), with a correlation coefficient 0.996, The detection limit was 3.0 x 10(-7) M (times the ratio of signal to noise, S/N=3).     

A Novel Nitric Oxide Cellular Biosensor Based on Red Blood Cells Immobilized on Gold Nanoparticles

Abstract

We have developed a novel nitric oxide (NO) cellular biosensor based upon the immobilization of red blood cells (RBCs) onto nanometer‐size colloidal gold that is attached to an electrochemically pretreated glassy carbon electrode via the bridging of an ethylenediamine monolayer. The biosensor has been characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), and electrochemistry. The immobilized RBCs display an excellent electrocatalytic response to nitric oxide. The electrocatalytic currents are proportional to the NO concentration in the range from 1.0×10^–8 to 1.0×10^–6 M and the detection limit is as low as 5.0×10^–9 M (S/N=3). Furthermore, the biosensor is very stable and relatively free of potential interference.     

Ion chromatography on-chip

On-chip separation of inorganic anions by ion-exchange chromatography was realized. Micro separation channels were fabricated on a silicon wafer and sealed with a Pyrex cover plate using standard photolithography, wet and dry chemical etching, and anodic bonding techniques. Quaternary ammonium latex particles were employed for the first time to coat the separation channels on-chip. Owing to the narrow depths of the channels on the chip, 0.5-10 microm, there were more interactions of the analytes with the stationary phase on the chip than in a 50-microm I.D. capillary. With off-chip injection (20 nl) and UV detection, NO2-, NO3-, I-, and thiourea were separated using 1 mM KCl as the eluent. The linear ranges for NO2- and NO3- are from 5 to 1000 microM with the detection limits of 0.5 microM.     

A microchip-based endothelium mimic utilizing open reservoirs for cell immobilization and integrated carbon ink microelectrodes for detection

This paper describes the fabrication and characterization of a microfluidic device that utilizes a reservoir-based approach for endothelial cell immobilization and integrated embedded carbon ink microelectrodes for the amperometric detection of extracellular nitric oxide (NO) release. The design utilizes a buffer channel to continuously introduce buffer or a plug of stimulant to the reservoir as well as a separate sampling channel that constantly withdraws buffer from the reservoir and over the microelectrode. A steel pin is used for both the fluidic connection to the sampling channel and to provide a quasi-reference electrode for the carbon ink microelectrode. Characterization of the device was performed using NO standards produced from a NONOate salt. Finally, NO release from a layer of immobilized endothelial cells was monitored and quantified using the system. This system holds promise as a means to electrochemically detect extracellular NO release from endothelial cells in either an array of reservoirs or concurrently with fluorescence-based intracellular NO measurements.     

Myoglobin‐Clay Electrode for Nitric Oxide (NO) Detection in Solution

Sodium montmorillonite was prepared via a colloidal chemical approach and deposited onto glassy carbon electrodes (GCE). Myoglobin was immobilized on the clay membrane modified electrode by spontaneous adsorption. Characterization of the myoglobin/clay/glassy carbon electrode (Mb/clay/GCE) showed a quasi‐reversible, electrochemical redox behavior of the adsorbed protein with a formal potential of ?0.380±0.010 V (vs. Ag/AgCl). The heterogeneous electron transfer rate constant was found to be strongly influenced by the buffer concentration. The Mb/clay/GCE was stable for several days in solution. The interaction of the immobilized Mb with nitric oxide (NO) is characterized by coordination chemistry. The reaction was found to be reversible and could be applied for NO detection in the nanomolar concentration range by a voltammetric analysis. In addition a mixed protein electrode with co‐immmobilized myoglobin (Mb) and cytochrome c (Cyt.c) was developed. By choice of the electrode potential both proteins can be addressed independently.     

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.     

Amperometric Nitric Oxide Sensor Based on Poly(Thionine)/Nafion‐Modified Electrode and Its Application in Monitoring Nitric Oxide Release from Rat Kidney

Abstract

A novel amperometric nitric oxide (NO) sensor based on a glassy carbon electrode modified with thionine and Nafion films has been developed. The oxidation peak current of NO increased significantly at the poly(thionine)/Nafion‐modified glassy carbon electrode (GCE), which can be used for the detection of NO. The oxidation peak current was linear with the concentration of nitric oxide over the range from 3.6×10^?7 to 6.8×10^?5 mol · L^?1, and the detection limit was 7.2×10^?8 mol · L^?1. This nitric oxide sensor showed high selectivity to nitric oxide determination, and some potential interference could be eliminated effectively. The nitric oxide sensor has been applied to monitor NO release from rat kidney stimulated by L‐arginine. The results indicated the applicability of the NO sensor to biomedical samples.     

Planar nitric oxide (NO)-selective ultramicroelectrode sensor for measuring localized NO surface concentrations at xerogel microarrays

A planar ultramicroelectrode nitric oxide (NO) sensor was fabricated to measure the local NO surface concentrations from NO-releasing microarrays of varying geometries. The sensor consisted of platinized Pt (25 microm) working electrode and a silver paint reference electrode coated with a thin silicone rubber gas permeable membrane. An internal hydrogel layer separated the Pt working electrode and gas permeable membrane. The total diameter of the sensor was 相似文献   

Construction and Characterization of a New Flexible and Nonbreakable Nitric Oxide Microsensor

Nitric oxide (NO) is an important molecule in many different physiological phenomena. Investigation of nitric oxide production in vivo requires a specialized sensor capable of real‐time concentration measurement, with a high spatial resolution of NO gas production. In this study, a flexible microsensor is developed specifically for measurement of production of nitric oxide. The new sensor consists of a Pt/Ir working electrode coupled with an integrated Ag/AgCl reference electrode. The sensor is coated with a series of NO‐selective membrane polymers to prevent potential amperometric response due to interfering species. Presented experimental data demonstrates the ability for NO detection between 100 and 400 nM concentrations with a linear response (R²=0.9997). The detection limit of the sensor is 2.14 nM (S/N=3). Various selectivity experiments are indicative of a resistance to interfering species such as dopamine, norepinephrine, L ‐arginine.     

Cytochrome c′ from Chromatium vinosum on gold electrodes

Cytochrome c′ from Chromatium vinosum (CVCP) was immobilized at a surface-modified gold electrode. Characterization of the CVCP electrode showed a quasi-reversible, diffusionless electrochemical redox behavior of the surface adsorbed protein with a formal potential of −128±5 mV vs. Ag/AgCl. The heterogeneous electron transfer rate constant of adsorbed CVCP was determined to be about 50 s⁻¹. Different immobilization strategies were compared. The interaction of the immobilized CVCP with nitric oxide (NO) was investigated and applied for a primary amperometric detection of NO in solution.     

Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes

An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 microm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO(+) and prevents interferences from negatively charged compounds such as NO(2)(-). For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 microm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca(2+)-channel inhibitors for testing Ca(2+)-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines.     

Fabrication and evaluation of a 3-dimensional microchip device where carbon microelectrodes individually address channels in the separate fluidic layers

A fabrication method that results in a 3-dimensional fluidic device containing poly(dimethylsiloxane) (PDMS) -embedded microelectrodes that individually address each layer is described. The two electrode-containing layers and the polycarbonate membrane are reversibly sealed together, eliminating the need for plasma oxidation during device assembly, while enabling simultaneous amperometric detection in membrane-separated fluidic channels. The electrodes were characterized using microchip-based flow analysis. It was found that PDMS-embedded electrodes have a limit of detection (400 nM for catechol) that is 5-fold lower than that reported for microchip-based flow analysis with similar electrodes in a hybrid PDMS-glass device. The selectivity of the carbon ink microelectrodes can be tuned by a simplified modification procedure; this was demonstrated by the selective detection of nitric oxide over possible interferents. Finally, the ability to monitor processes occurring in separate layers of a 3-dimensional device was shown by the simultaneous detection of catechol on either side of the polycarbonate membrane. The electrode response in each fluidic channel was found to be linear as a function of concentration and the transport between layers could be controlled by varying the linear velocities of each fluidic channel. The ability to fabricate and operate this type of 3-dimensional device will be useful for the development of cell-based in vivo mimics that involve the transport of molecular messengers and/or pharmaceuticals across layers of immobilized cells.     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

Electrochemical and electrocatalytic behaviors of Safranin O/Nafion film deposited on the glassy carbon electrode

The glassy carbon (GC) electrode modified with Nafion and Safranin O (SFO) was prepared and its electrochemical properties were investigated. The SFO molecules were strongly and irreversibly adsorbed on the Nafion — modified GC surface. The electrochemical behavior and mechanism for interactions of the SFO molecules with the Nafion film were investigated through cyclic voltammetric method. The electrocatalytic reduction of nitric oxide was performed at this modified electrode by cyclic voltammetric and hydrodynamic amperometric techniques. The Nafion membrane played a duel role as a matrix for the SFO immobilization and also helped to partition the nitric oxide from the solution phase. The diffusion coefficient of NO at the SFO/Nafion/GC modified electrode was calculated using chronoamperometry. The dependence of response currents on the concentration of NO was examined and was linear in the range of 0.05–1.9 mM of NO.     

A novel amperometric sensor for the determination of nitric oxide, and its application in rat liver cells

A novel amperometric nitric oxide sensor with a wide linear range, low detection limit and fast response time was developed. The sensor was fabricated using a poly-brilliant cresyl blue (PBCB)/Nafion film modified glassy carbon electrode (GCE). The PBCB on the working GCE surface dramatically improves the oxidation response of nitric oxide and lowers the required potential for the two-step oxidation of NO. Otherwise, Nafion coated onto the film electrode surface does not only improve the selectivity of the sensor, but also further lowers the active energy of the direct three-electron oxidation of NO to nitrate. The effect of the preanodic time and the film thickness of the electropolymerization on the nitric oxide response as well as the volume of Nafion coated onto the surface and the potential for amperometric detection were optimized. This novel sensor has been applied to the determination of NO released from rat liver cells, and the result is satisfactory. Correspondence: Sheng Shui Hu, Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China     

A New Nitric Oxide Gas Sensor Based on Reticulated Vitreous Carbon/Nafion and Its Applications

Reticulated vitreous carbon (RVC), and Nafion membrane are used to fabricate a composition electrode to measure nitric oxide (NO) concentration amperometrically in the gas phase. Limit of detection was found to be 6 ppb at an applied voltage of 0.66 V (vs. mercury sulfate reference electrode) with average response time of less than 30 seconds. The response of the sensor was linearly dependent on the concentration over the whole tested range from 19 ppb‐50 ppm of NO. Simplicity in electrode fabrication and consistent performance between individual sensors make RVC and Nafion attractive materials for detecting very low levels of nitric oxide gas in routine analysis.     

A Nitric Oxide Biosensor Based on Myoglobin Adsorbed on Multi‐Walled Carbon Nanotubes

Myoglobin (Myb) of horse heart is incorporated on multi‐walled carbon nanotubes (MWNTs) and immobilized at a glassy carbon (GC) electrode surface. Its electrochemical behavior and enzyme activity are characterized by employing electrochemical methods. The results indicate that MWNTs can obviously promote the direct electron transfer between Myb and electrode, and that the Myb on MWNTs behaves as an enzyme‐like activity towards the electrochemical reduction of nitric oxide (NO). Accordingly, an unmediated NO biosensor is constructed. Experimental results reveal that the peak current related to NO is linearly proportional to its concentration in the range of 2.0×10^?7–4.0×10^?5 mol/L. The detection limit is estimated to be 8.0×10^?8 mol/L. Considering a relative standard deviation of 2.1% in seven independent determinations of 1.0×10^?5 mol/L NO, this biosensor shows a good reproducibility. The biosensor based on Myb/MWNTs modified electrode can be used for the rapid determination of trace NO in aqueous solution with a good stability, nice selectivity and easy construction.     

基于表面活性剂单分子层修饰碳糊电极的一氧化氮电化学传感器及其应用

报道了一种表面活性剂单分子层修饰碳糊电极,并用于NO的高灵敏电化学检测。研究表明,表面活性剂通过烷基链在电极表面形成的疏水性单分子层微环境对NO的电化学响应具有较好的促进作用。其中,阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)对NO的电化学氧化表现出最强的催化活性和增敏作用。在Nafion膜覆盖的CTAB修饰碳糊电极上,NO的安培响应与其浓度在3.6×10-8～1.8×10-5mol/L范围内呈良好的线性关系,检出限为1.8×10-8mol/L。该电极作为低成本、高灵敏的NO电化学传感器,被成功应用于大鼠肺组织细胞中NO释放的实时监测。     

Electrooxidation of Nitric Oxide at a Glass Carbon Electrode Modified with Functionalized Single-walled Carbon Nanotube

The electrocatalytic oxidation of nitric oxide(NO) at a glass carbon electrode(GC) modified with functionalized single-walled carbon nanotubes(SWCNTs) was investigated by cyclic voltammetry(CV) and electrochemical impedance spectroscopy(EIS).It was found that the SWCNT modified electrode could speed greatly up the electron transfer rate compared with the bare GC electrode.After the SWCNT was treated with alkali or mixed acids,the reaction rate and activation energy of NO electrooxidation were changed to different extent.Chemical modification of the SWCNT surface is one of the most powerful methods to change the sensitivity of NO electrooxidation reaction.The modified electrode with SWCNT obtained by the firstly alkali treatment and then the mixed acids treatment was the best one for NO electrooxidation,the result of CV was also confirmed by that of EIS.The anodic processes of NO were recognized more clearly by exploring the reaction mechanism of NO electrooxidation at the SWCNT modified electrode.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.