Tsutomu Horiuchi

Interdigitated array microelectrodes as electrochemical sensors

We investigated electrochemical measurements with interdigitated array (IDA) electrodes in both stationary solutions and flow systems. In a stationary solution, we achieved a very low detection limit of 10 pM of reversible redox species by using substitutional stripping voltammetry, which is a new type of stripping voltammetry using an IDA microelectrode. In flow systems, current enhancement by redox cycling is less effective than that in a stationary solution. The flow rate dependence of redox cycling is constant in the amperometric region, varies with coulometric yield in the quasi-amperometric region, and is inversely proportional to the 2/3 power of the volume flow rate in the coulometric region. A low detection limit of 5 fg (32 amol) is obtained for dopamine due to the high current density and low background noise level (0.1 pA) at the carbon-based IDA microelectrode used as a detector for liquid chromatography. A new separation approach is demonstrated which combines electrochemical detection and a molecular template. The electrode is first partly covered with print molecules and then modified with silane coupling reagent. The catechol-imprinted electrode shows the usual diffusion-limited cyclic voltammogram of catechol and has a diminished response against all catecholamines. The selectivity between catechol and epinephrine is about 100 when the electrode is used as an electrochemical detector in liquid chromatography.

Fabrication and electrochemical features of new carbon based interdigitated array microelectrodes

Abstract A new type of interdigitated array (IDA) microelectrode based on carbon film was developed. A conductive carbon film was prepared on a platinum coated Si wafer by the pyrolysis of 3,4,9,10-perylenetetracarboxylic dianhydride. The IDA microelectrode was fabricated by photolithographic techniques from the carbon/platinum composite film. The IDA electrode had 50 microband pairs. The width of each microband was 3 μm with a length of 2 mm, and the gap between two electrode bands was 2 μm. The electrode was characterized by cyclic voltammetry with water soluble ferrocene and ruthenium hexa-amine. The potential window without redox species was found to be similar to that of a conventional glassy carbon electrode. The IR drop was negligible in a measurement with 10 μM water soluble ferrocene at a moderate scan rate. Generation—collection voltammograms of water soluble ferrocene and ruthenium hexa-amine show clear limiting currents and their response in the negative potential range was superior to that of conventional metal film IDA electrodes.

Continuous monitoring of L-glutamate released from cultured nerve cells by an online sensor coupled with micro-capillary sampling.

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7-15 nM and a linear range of 50 nM to 10 microM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by the KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.

Determination of acetylcholine and choline with platinum-black ultramicroarray electrodes using liquid chromatography with a post-column enzyme reactor

Abstract A method for the highly sensitive determination of acetylcholine (ACh) and choline (Ch) was developed using platinum (Pt) black microarray electrodes as detectors in a microbore liquid chromatography (LC) with a post column enzyme reactor. The electrodes were prepared by plating a gold (Au) film electrode with sub-μm Pt-black particles. Since hydrogen peroxide generated by the enzymatic reaction of ACh and Ch was oxidized only at the Pt-black microarray electrode, each Pt-black particle (typically 0.1–0.2 μm in size) operated as an ultramicroelectrode. A high signal-to-noise ratio was achieved because of the high current density at the Pt-black microarray electrodes and because the Au film has a much lower baseline noise than the Pt. Detection limits of 5.7 (ACh) and 6.0 (Ch) fmol were obtained, with a wide linear range. The ACh and Ch signals with an Au film electrode modified with Pt-black particles retained more than 70% of their initial value after 5 days with continuous potential application. This is better stability than for a bare platinum electrode which retained only 40% of its initial response under comparable conditions.

NADH and glutamate on-line sensors using Os-gel-HRP/GC electrodes modified with NADH oxidase and glutamate dehydrogenase

We have developed a highly sensitive and selective on-line biosensor for detecting the reduced form of nicotinamide adenine dinucleotide (NADH) produced by the enzymatic reactions of dehydrogenases with various substrates such as glutamate. The sensor consists of a glassy carbon electrode modified with an osmium-polyvinylpyridine-based bottom layer containing horseradish peroxidase, and a bovine serum albumin (BSA)–gluteraldehyde (Glut) top layer containing NADH oxidase (NOX) or glutamate dehydrogenase (GluDH) and NOX. We assembled the modified electrode in a thin-layer radial flow cell and sample solution was continuously introduced into the cell with a syringe pump. We optimized the sensitivity of the NADH sensor by adjusting the glutaraldehyde amount in the immobilized layer, the applied potential and the pH of buffer solution. We examined the flow-rate effect on the current response and the conversion efficiency of NADH at the modified electrode. As a result, we achieved a sensitivity of 48.8 nA cm−2 μM−1, a detectable concentration range of 25 nM∼10 μM and a detection limit of 20 nM (S/N=3) for the NADH sensor. The interference from ascorbic acid and other electroactive interferents can be greatly reduced since the sensor can be operated below 0 mV versus Ag/AgCl. The NADH sensor is relatively stable since it retains 70% of its original response after 1 month if stored at 2–8°C in a dry state after use. Furthermore, we fabricated a glutamate sensor by coimmobilizing GluDH and NOX in the BSA–Glut top layer. The detectable glutamate concentration range is from 0.1 to 10 μM and the detection limit is 0.1 μM (S/N=3). Our glutamate dehydrogenase-based sensor offers good selectivity as regards other amino acids.

Analysis of electrochemical processes using surface plasmon resonance

Abstract We simultaneously employed voltammetry and surface plasmon resonance (SPR) measurements and monitored electrochemical processes on gold electrode. SPR is sensitive not only to the adsorbed layer on the gold electrode, but also to the dielectric properties of the solution phase. Therefore, an electrochemically modulated diffusion layer can be detected by SPR. We tested the relationship between the time differential SPR data and the electrode current for two electrochemical processes and confirmed the possibility of using electrochemical SPR measurement.

Selective detection of L-glutamate using a microfluidic device integrated with an enzyme-modified pre-reactor and an electrochemical detector

A microfluidic device integrated with a nanoliter volume enzyme pre-reactor and an enzyme-modified electrode was developed for the highly selective continuous measurement of glutamate (Glu). The device consists mainly of two glass plates. One plate incorporates an electrochemical cell that consists of working electrode (WE), reference electrode (RE) and counter electrode (CE). The WE is modified with a bilayer film of Os-polyvinylpyrridine-based mediator containing horseradish peroxidase (Os-gel-HRP). The WE was operated at -50 mV versus Ag. The other plate has a thin layer flow channel integrated with a pre-reactor. The reactor has a number of micropillars (20 microm in diameter, 20 microm high and separated from each other by a 20 microm gap) modified with ascorbate oxidase (AAOx) to eliminate L-ascorbic acid (AA). The enzymatic oxidation of AA is superior to that obtained with our previously reported pre-electrolysis type micro-reactor since electrochemically reversible transmitters such as catecholamines do not provide a cathodic current at the WE. In addition, the high operation potential of the pre-reactor causes unknown electroactive species, which also cause interference at the detection electrode. As a result, we were able to detect 1 microM Glu continuously at a low flow rate even when AA concentration was 100 microM.

Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance.

Three types of imaging, namely layer structure, electrochemical reaction, and enzyme sensor response, were achieved by applying surface plasmon resonance (SPR) measurement to an electrochemical biosensor. We constructed glucose oxidase based mediator type sensors on a gold electrode by spotting the mediator that contained horseradish peroxidase and spin coating the glucose oxidase film. The layer structure of the sensor was imaged by means of angle scanning SPR measurement. The single sensor spot (about 1 mm in diameter) consisted of about 100 x 100 pixels and its spatial structure was imaged. The multilayer structure of the enzyme sensor had a complex reflectance-incident angle curve and this required us to choose a suitable incident angle for mapping the redox state. We chose an incident angle that provided the most significant reflection intensity difference by using data obtained from two angle scanning SPR measurements at different electrode potentials. At this incident angle, we controlled the electrochemical states of the spotted mediator in cyclic voltammetry and imaged the degree to which the charged site density changed. Finally, we mapped the enzymatic activity around the mediator spot by the enzymatic reoxidation of pre-reduced mediator in the presence of glucose.

Evidence for laser action driven by electrochemiluminescence

Emission of light from excited-state dye molecules can be driven by the electron transfer between electrochemically generated anion and cation radicals — a process known as electrochemiluminescence (ECL). ECL has been investigated for both display and laser applications. The latter is of particular interest as, in contrast to conventional dye lasers, a laser operating by this principle would not require an additional laser source optically to pump the dye into the required excited state, and may offer additional advantages in terms of power, tunability and range of available wavelengths. But the pumping rate hitherto achieved by ECL is two orders of magnitude lower than the optical pumping threshold. Here we describe a device structure designed to enhance the efficiency of the ECL process, and present evidence that laser action has been realized in such a structure: the ECL spectrum is strongly modulated by the device structure, the output intensity shows a clear threshold as the drive current increases, and spectral narrowing is observed as the intensity increases.

Real-time electrochemical imaging using an individually addressable multi-channel electrode

We developed a real-time electrochemical imaging method that uses a multiple enzyme-modified microelectrode. The method will enable the investigation of the functions of biological materials and cells. To test its effectiveness, we imaged the two-dimensional concentration distribution for hydrogen peroxide and L-glutamate in a standard solution. The multiple electrode consists of an 8 x 8 array of 30 x 30 microm2 carbon micro electrode. Each electrode was connected to a 64-channel potentiostat that could apply a potential to all electrodes at the same time. The multiple electrode was coated with an Os-polyvinylpyridine based polymer (Os-gel) containing horse radish peroxidase (HRP) to detect hydrogen peroxide, which is a very common product of oxidase enzyme. When measuring glutamate, which is a well-known neurotransmitter in the mammalian central nerve system, we modified the electrode with a bilayer of Os-gel-HRP and GluOx. The detection limit of our method was 1 microM and images of the glutamate concentration-distribution changes induced by local injection of glutamate through microcapillary were obtained in real time.