Electrodes three enzyme

Horseradish peroxidase (HPP), choline oxidase and acetylcholineesterase A three enzyme layered assembly on Au electrodes or Au quartz crystal, consisting of HRP, ChO and AChE is used to sense ACh by the HRP-mediated oxidation of 3,3, 5,5 -tetramethyl benzidine (1) by H202 and the formation of the insoluble product (2) on the respective transducers. Acetylcholine is hydrolyzed by AChE to choline that is oxidized by ChO and 02 to yield the respective betaine and H202. The amount of generated H202 and the resulting insoluble product on the transducer correlates with the concentration of acetylcholine in the samples. 

A more complex biosensor for acetylcholine has been developed by Larsson et al. [154]. Three enzymes, AChE, ChOX, and HPR, have been coimmobilized in an Os-based redox polymer on solid graphite electrodes. After a careful optimization of the immobilization procedure, the biosensor, inserted into a flow cell of very small volume, was integrated into a flow injection system, and some samples of microdialysate, taken from rat brains before and after stimulation with KCl, were analysed. Even if a clear increase in signal could be noted, it was not possible to distinguish whether it was due to an increase in choline or in acetylcholine, since the biosensor responded to both metabolites. 

Miyahara et al. (1985) developed an integrated enzyme FET based on a silicon-on-sapphire (SOS) sensor for simultaneous determination of glucose and urea. Three ISFETs and two metal insulator semiconductor FETs (MISFETs) were integrated on a surface area of 2.5 mm x 2.5 mm (Fig. 54). One of the ISFETs served as reference sensor in order to compensate the signals caused by pH changes of the solution the two others were covered by GOD and urease, respectively. The MISFETs can be used as pH electrodes. For enzyme immobilization the chip was covered with a laminated photosensitive layer of 75 pm thickness and,... 

The use of a second downstream electrode to monitor chemical fluxes at the working electrode is proving to be an important technique for the investigation of electrode mechanisms. This is particularly true for electrodes which have a more complicated structure than a simple metallic surface. Examples are modified electrodes, oxide electrodes, or enzyme electrodes. For these more complex systems, the separate measurement of the fluxes at the electrolyte-electrode interface provides unique and valuable information. Double electrodes can be constructed for all three hydrodynamic systems. A crucial parameter for such a double electrode is the collection efficiency, N, which, in the steady state, relates the flux of material detected as a limiting current on the downstream electrode to the flux of material generated on the upstream electrode. The collection efficiency is a function of the geometry of the electrode and is given for all three systems by [4, 9]... 

Yamato, H., M. Ohawa, and W. Wernet. 1995. A polypyrrole/three enzyme electrode for creatinine detection. Anal Chem 67 2776. 

When the three-enzyme sequence based on creatinine amidohydrolase is used, any creatine present can interfere with the determination of creatinine, so two sensors are used one to determine the total creatine plus creatinine and one to determine just creatine (by only using creatine amidinohydrolase and sarcosine oxidase). Creatinine is determined by difference. Amperometric sensors are generally based on this sequence and do not suffer from interferences. They are usually designed to respond to peroxide, though some have used oxygen electrodes. Typically, Pt electrodes are used. A sensor for just creatine only requires the creatine amidinohydrolase and sarcosine oxidase sequence. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

The three dimensional redox epoxy offers some of the advantages of both the freely diffusing s stems and the immobilized systems. As in freely diffusing mediator based systems, not only the electrode adsorbed enzyme molecules, but also those remote from the electrode, yet connected by the redox polymer are electroactive. At the same time, there is no need to add mediator to the sample, and the mediator can not leach out or contaminate the sample. 

Fig. 9 (a) Structural models of the three enzymes. A is an overview of the tunnel network B is a close-up of the tuimel near the active site in the WT. C, D and E are close-ups of the MM and FI mutants, as indicated. In C, an arrow points to the second conformation of M122. A conserved hydrophilic cavity is shown in blue in E. (b) Comparison of the kinetics of CO inhibition of H2 oxidation in PFV experiments. The current (i) has been normalised by its value I o, measured before CO was added. Left shows the short-term change in current, whereas the end of the relaxation is shown on Right. The dimensionless volumic fractions of solutions saturated under 1 atm of CO at 25°C and injected at time 0. Electrode rotation rate 2 krpm, pH 7. Adapted with permission from [77]. Copyright (2008) PNAS... 

In 1964, Yahiro et al. demonstrated enzyme mediation to platinum electrodes [21], They used three enzymes GOx, D-amino acid oxidase, and alcohol dehydrogenase (ADH). If iron was introduced into the system with either GOx or o-amino acid oxidase, an electrical potential was observed. However, this was not the case with ADH, which is dependent on a nicotinamide adenine dinucleotide (NAD) cofactor (see below). 

Gutierrez-Sanchez, C. Jia, W. Beyl, Y. Pita, M. Schuhmann, W. De Lacey, A. L. Stoica, L. Enhanced direct electron transfer between laccase and hierarchical carbon microfibers/carbon nanotubes composite electrodes. Comparison of three enzyme immobilization methods. Electrochim. Acta 2012,82, 218-223. 

This is normal for microorganisms that contain a number of enzymatic systems. A pNHa electrode with immobilized Streptococcus faecium detects arginine through the presence of three enzymes [60], which catalyse successively the following reactions ... 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

---