Enzyme electrodes ethanol

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

An electron transfer type of enzyme sensor was thus fabricated by a electrochemical process. Although no appreciable leakage of ADH and MB from the membrane matrix was detected, NAD leaked slightly. To prevent this leakage, the ADH-MB-NAD/polypyrrole electrode was coated with Nation. A calibration curve is presented in Fig.25 for ethanol determination in an aquous solution with the enzyme sensor. Ethanol is selectively and sensitively determined in the concentration range from 0.1 nM to 10 mM. 

M.J. Lobo Castanon, A.J. Miranda Ordieres and P. Tunon Blanco, Amperometric detection of ethanol with poly-(o-phenylenediamine)-modified enzyme electrodes, Biosens. Bioelectron., 12(6) (1997) 511-520. 

Figure 3.23 Typical amperometric (readout during automated flow injection assays of ethanol solutions of increasing concentrations in 2 x 1CL5M steps at a carbon paste enzyme electrode detector. Curves a-h 2 x 10 SM - 1.6 x lCUM ethanol.

Piovesta s alcohol oxidase, from the yeast Pichia pastor is. Described as "equally active on methanol and ethanol" (D. Ranasiak arid T. Hopkins, Provest a, personal communication) it is "one-third as active no ethanol than on methanol.." on an enzyme electrode (Hopkins and Muller (in )). The commercial product is virtually devoid of catalase and has a pH stability profile (5. -8. 3) that see/n.s to rule out its use in beer or wines. Alcohol oxidase is still of interest... 

Several other sensors are available that are based on the amperometric measurement of hydrogen peroxide produced by enzymatic reactions. The analytes measured include sucrose, lactose, ethanol, and L-lactate. A different enzyme is, of course, required for each species. In some cases, enzyme electrodes can be based on measuring oxygen or on measuring pH. 

In an externally buffered enzyme electrode (Fig. 45), substrate-free buffer is continuously pumped between the dialysis membrane and the enzyme layer (Cleland and Enfors, 1984), i.e., the sample is diluted before it reaches the enzyme. The intensity of the buffer flow may be used to adjust the measuring range and sensitivity. The configuration of the sensor permits it to be sterilized. While the membrane is protected by continuously flowing buffer, the rest of the sensor can be sterilized for 1 h in a solution of 95% ethanol and 5% H2SO4. 

Between 15 and 20 analyzers based on enzyme electrodes are on the market worldwide. They are one-parameter instruments for the measurement of glucose, galactose, uric add, choline, ethanol, lysine, lactate, pesti-ddes, sucrose, lactose, and the activity of a-amylase (Table 23). They provide for a negligible enzyme consumption of less than 1 pg per sample. 

Other examples of amperometric enzyme electrodes based on the measurement of oxygen or hydrogen peroxide include electrodes for the measurement of galactose in blood (galactose oxidase,enzyme), oxalate in urine (oxalate oxidase), and cholesterol in blood serum (cholesterol oxidase). Ethanol is determined by reacting with a cofactor, nicotinamide adenine dinucleotide (NAD" ) in the presence of the enzyme alcohol dehydrogenase to produce the reduced form of NAD", NADH, which is electrochemically oxidized. Lactate in blood is similarly determined (lactate dehydrogenase enzyme). 

Varela H, Ferrari MD, Belobradjic L et al. (1997) Skin unhairing proteases of Bacillus subtilis production and partial characterization. Biotechnol Lett 19 755-758 Verduyn C, Zomerdijk TPL, van Etijken JHP et al. (1984) Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode. J Appl Microbiol Biotechnol 19(3) 181-185... 

This equation represents a useful model for interpreting data from flow experiments on immobilized enzyme electrodes (in this case, aleohol dehydrogenase) in which an electron mediator (i.e., NAD" ) is limiting and the substrate (i.e., ethanol) is present in excess. In this scenario, bulk NAD is pumped to the electrode and its reduced counterpart (NADH) is measured spectrophotometrically in the outflow. Specifically, half of the equation is measured experimentally and the remainder is plotted in the form of a Lineweaver-Burk plot, wherein a value for the combined mass transfer parameter Mnad+is assumed. As the equation has three unknowns. 

Blaedel W.J. and Engstrom R.C. (1980) Reagentless enzyme electrodes for ethanol, lactate and malate. Anal. Chem., 52, 1691-1697. 

FIGURE 3-21 Typical amperometric readout during automated flow injection assays of ethanol at an enzyme carbon-paste electrode. Peaks a through h 2 x 10 5 M to 1.6 x 10 4 M ethanol. 

Many dehydrogenase enzymes catalyze oxidation/reduction reactions with the aid of nicotinamide cofactors. The electrochemical oxidation of nicotinamide adeniiw dinucleotide, NADH, has been studied in depthThe direct oxidation of NADH has been used to determine concentration of ethanol i s-isv, i62) lactate 157,160,162,163) pyTuvate 1 ), glucose-6-phosphate lactate dehydrogenase 159,161) alanine The direct oxidation often entails such complications as electrode surface pretreatment, interferences due to electrode operation at very positive potentials, and electrode fouling due to adsorption. Subsequent reaction of the NADH with peroxidase allows quantitation via the well established Clark electrode. 

The alcohol tolerance of O2 reduction by bilirubin oxidase means that membraneless designs should be possible provided that the enzymes and mediators (if required) are immoblized at the electrodes. Minteer and co-workers have made use of NAD -dependent alcohol dehydrogenase enzymes trapped within a tetraaUcylammonium ion-exchanged Nafion film incorporating NAD+/NADH for oxidation of methanol or ethanol [Akers et al., 2005 Topcagic and Minteer, 2006]. The polymer is coated onto an electrode modified with polymethylene green, which acts as an electrocatalyst... 

Minteer and co-workers have also exploited the broad substrate specificity of PQQ-dependent alcohol dehydrogenase and aldehyde dehydrogenase from Gluconobacter species trapped within Nahon to oxidize either ethanol or glycerol at a fuel cell anode [Arechederra et al., 2007]. Although the alcohol dehydrogenase incorporates a series of heme electron transfer centers, it is unlikely that many enzyme molecules trapped within the mediator-free Nahon polymer are electronically engaged at the electrode. 

In a further development, an ADH-MB-NAD/polypyrrole electrode, a platinum counter electrode and an Ag/AgCl reference electrode were assembled and covered with a gas-permeable polymer membrane to form an gaseous ethanol sensor. This appears to be the first time that a complete enzyme sensor for gaseous ethanol has been fabricated in such a manner with NAD incorporated in immobilized form. 

Biosensors constructed for ethanol and D-glucose measurements in beverages were built using ferrocene compounds as electron transfer mediators between the coenzyme PQQ of immobilized enzymes glucose (GDH) and alcohol (ADH) dehydrogenases and a carbon electrode surface <2003JOM(668)83>. 

Ethanol (2-200) x 10 5M Ethanol 5.3 x 10 6M RSD = 8.3% in = 10, after polishing the electrode surface) RSD = 6.5% in = 9, different biosensors) The useful lifetime of a single AOD-HRP-ferrocene composite electrode was of approximately 15 days with no need of enzyme stabilisers in the electrode matrix. Both batch and FIA modes. 

---