Enzymatic electrode

Electrode Enzymatic me nib rare Iiveratse enzyme and substrate in solution... 

Acetylcholineesterase Bilayer lipid membranes were prepared by adding a solution of egg phosphatidylcholine and dipalmi-toyl phosphatidic acid dropwise into the surface of aqueous 0.1 M KC1/10 mM HEPES, near the Saran Wrap partition of a two compartment plexiglass cell. A portion of AChE solution in 10 mM Tris hydrochloride buffer solution of pH 7.4 was applied. The electrolyte level was momentarily dropped below the orifice and raised to form a membrane. The membranes were used as transducers for the reaction of AChE with ACh. An external voltage (25 mV) was applied across the membrane between two Ag/AgCl reference electrodes. Enzymatically generated hydronium ion causes transient current due to alteration of the electrostatic field by the ionization of dipalmitoyl phosphatidic acid. The response delay time was directly related to the substrate concentration where acetylcholine can be determined from 1 pM upto mM level. [113]... 

The response characteristics of enzyme electrodes depend on many variables, and an understanding of the theoretical basis of their function would help to improve their performance. Enzymatic reactions involving a single substrate can be formulated in a general way as... 

Improved sensitivity and scope can be achieved by coupling two (or more) enzymatic reactions hi a chain, cycling, or catalytic mechanism (9). For example, a considerable enhancement of the sensitivity of enzyme electrodes can be achieved by enzymatic recycling of the analyte in two-enzyme systems. Such an amplification... 

Ethanol Electrodes The reliable sensing of ethanol is of great significance in various disciplines. The enzymatic reaction of ethanol with the cofactor nicotinamide-adenine dinucleotide (NAD+), in the presence of alcohol dehydrogenase (ADH)... 

Tissue and Bacteria Electrodes The limited stability of isolated enzymes, and the fact that some enzymes are expensive or even unavailable in the pure state, has prompted the use of cellular materials (plant tissues, bacterial cells, etc.) as a source for enzymatic activity (35). For example, banana tissue (which is rich with polyphenol oxidase) can be incorporated by mixing within the carbon paste... 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

The concept of a biocatalytic membrane electrode has been extended to the use of a tissue slice as the catalytic layer. An example of this approach is an electrode for AMP which consists of a slice of rabbit muscle adjacent to an ammonia gas electrode. NHj is produced by enzymatic action of rabbit muscle constituents on AMP The electrode exhibits a linear range of 1.4 x 10 to 1.0 x 10 M with a response time varying from 2.5 to 8.5 min, depending on the concentration. Electrode lifetime is about 28 days when stored between use in buffer with sodium azide to prevent bacterial growth. Excellent selectivity enables AMP to be determined in serum. 

The demand for monitoring common metabolites of diagnostic utility such as glucose, urea and creatinine continue to provide the impetus for a staggering research effort towards more perfect enzyme electrodes. The inherent specificity of an enzyme for a given substrate, coupled with the ability to electrochemically detect many of the products of enzymatic reactions initiated the search for molecule-selective electrodes. 

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-... 

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

There are many examples of enzymatic reactions that yield free ammonium ion and as a consequence an alteration of pH around the glass electrode, e.g.,... 

Tubular electrodes. Blaedel et al.161 were the first to introduce the TBE, a tubular electrode. It was constructed by melting a Pt cylinder in a glass capillary (see Fig. 5.25), the total length of Pt being 25.5 mm and its diameter 0.75 mm. The authors originally used it for an enzymatic determination of glucose by means of differential amperometry based on the following sequential reactions"12 ... 

Electrocatalytic regeneration of NADH has also been performed at polymer-modified electrodes with pendent [(r/s-Cs M cs) R h( bpy )C1]1 55,56 and [Rh(terpy)2]3+ 57 complexes, and coupled to enzymatic reduction with alcohol dehydrogenase.55,57... 

MWNTs favored the detection of insecticide from 1.5 to 80 nM with a detection limit of InM at an inhibition of 10% (Fig. 2.7). Bucur et al. [58] employed two kinds of AChE, wild type Drosophila melanogaster and a mutant E69W, for the pesticide detection using flow injection analysis. Mutant AChE showed lower detection limit (1 X 10-7 M) than the wild type (1 X 10 6 M) for omethoate. An amperometric FIA biosensor was reported by immobilizing OPH on aminopropyl control pore glass beads [27], The amperometric response of the biosensor was linear up to 120 and 140 pM for paraoxon and methyl-parathion, respectively, with a detection limit of 20 nM (for both the pesticides). Neufeld et al. [59] reported a sensitive, rapid, small, and inexpensive amperometric microflow injection electrochemical biosensor for the identification and quantification of dimethyl 2,2 -dichlorovinyl phosphate (DDVP) on the spot. The electrochemical cell was made up of a screen-printed electrode covered with an enzymatic membrane and combined with a flow cell and computer-controlled potentiostat. Potassium hexacyanoferrate (III) was used as mediator to generate very sharp, rapid, and reproducible electric signals. Other reports on pesticide biosensors could be found in review [17],... 

The formal potentials ( ° ) of the three kinds of SODs were found to be dependent on solution pH as displayed in Fig. 6.6. As shown, the formal potential of bovine erythrocyte Cu, Zn-SOD decreases linearly with increasing solution pH with a slope of ca. -60mV/pH from pH 5.8 to pH 9.5 (curve b), indicating one proton and one electron are included in the electrode reaction of Cu, Zn-SOD, which is similar to previously proposed enzymatic catalytic mechanistic scheme of the Cu, Zn-SOD [139— 144], In contrast, the pH dependency of Fe-SOD from E. coli was complicated (curve a) the formal potential changes linearly with solution pH in a range from pH 5.8 to 8.5 with a slope of ca. -60mV/pH, and becomes pH-independent at above pH > 8.5. Previous studies have observed that the Fe (III) form of the protein ionizes with an apparent pKa of 9.0 0.3 and such ionization effect has been interpreted in terms of hydrolysis of a bound water molecule with p/<"a of ca. 8.5 [145], The C -pII profile of... 

FIGURE 6.11 Typical current—time responses of Fe-SOD/MPA-modified Au electrode toward 02 in 25 mM phosphate buffer (02-saturated, pH 7.5) containing 0.002 unit of XOD upon the addition of 50 nM xanthine at +300 (a) and —lOOmV (b). The arrows represent the addition of 10 j,M of Cu, Zn-SOD (a) and 580 units of catalase and 10 pM of Cu, Zn-SOD to the solution (b). The solution was stirred with a magnetic stirrer at 200rpm. Inset mechanism for the amperometric response of SODs/MPA-modified Au electrodes to 02, based on enzymatic catalytic oxidation (a) and reduction (b) of 02 (M metal ions of SODs). (Reprinted from [138], with permission from the American Chemical Society.)... 

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