Amperometric Enzymatic Electrodes

The product will diffuse out of the enzyme phase through the exterior membrane E and also diffuse toward the amperometric electrode detector (if one is part of the sensor system). The steady state product concentration within the enzyme phase will be determined by the balance of enzymatic production of product within the enzyme region, volume V, and its disappearance by diffusion or electrode reaction... 

Such amperometric electrodes as the oxygen electrode are also frequently employed in enzymatic analyses involving consumption or release of oxygen... 

When an amperometric electrode is used as the transducer of a biosensor, there is a consumption of reaction products this is the major difference from a potentiometric electrode. The diffusion-reaction equations ((3) and (4)) still apply, assuming that the product concentration at the transducer-active layer interface is zero ([P] = 0). This hypothesis corresponds to the maximal sensitivity of the biosensor. The flow of product towards the transducer can be limited by mass transfer, either in the semi-permeable membrane, which separates the enzyme from the sample medium [139], or in the active enzymatic layer [140]. When the semi-permeable membrane is the limiting factor of a diffusion process, all of the product formed is... 

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],... 

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.)... 

As far as the use of ferrocene molecules as amperometric sensors is concerned, they have found wide use as redox mediators in the so-called enzymatic electrodes, or biosensors. These are systems able to determine, in a simple and rapid way, the concentration of substances of clinical and physiological interest. The methodology exploits the fact that, in the presence of enzyme-catalysed reactions, the electrode currents are considerably amplified.61 Essentially it is an application of the mechanism of catalytic regeneration of the reagent following a reversible charge transfer , examined in detail in Chapter 2, Section 1.4.2.5 ... 

Most of the amperometric flow-through biosensors based on commercially available enzymes are employed to measure consumed or released oxygen by using a Clark electrode or a solid-state electrode to monitor the hydrogen peroxide formed or an enzymatically reduced acceptor. 

In amperometry, the current at the working electrode is proportional to analyte concentration. The amperometric glucose monitor generates H202 by enzymatic oxidation of glucose and the H202 is measured by oxidation at an electrode. A mediator is employed to rapidly shuttle electrons between electrode and analyte. 

The enzyme can be incorporated into an amperometric sensor in a thick gel layer, in which case the depletion region due to the electrochemical reaction is usually confined within this layer. Alternatively, enzyme can be immobilized at the surface of the electrode or even within the electrode material itself, in which case the depletion region extends into the solution in the same way as it would for an unmodified electrode. In the latter case, the enzyme can then be seen as a true electrocatalyst that facilitates the interfacial electron transfer, which would otherwise be too slow. The general principles of the design and operation of these biosensors is illustrated on the example of the most studied enzymatic sensor, the glucose electrode (Fig. 2.14, Section 2.3.1). 

The three types of glucose electrode discussed here illustrate the major facets of design and operation of enzymatic amperometric sensors. Examples of amperometric enzyme electrodes for other substrates are shown in Table 7.3. The actual design details of these sensors depend on the enzyme kinetics involved and on the operating conditions under which they are used. 

The amperometric response, described above, is based on the electrooxidation at the platinum-modified electrode of the enzymatically generated hydrogen peroxide at + 0.60 V vs. Ag/AgCl. 

Fig. 26.2. Amperometric immunosensors set-up using a biotinylated copolymer poly(pyrrole-biotin, pyrrole-lactitob-ionamide) coated platinum or glassy carbon electrodes and three enzymatic markers (GOX-B, PPO-B, HRP-Ab) for the detection of cholera antitoxin. (A) HRP-immunosensor, (B) GOX-B-immunosensor, (C) PPO-B-immunosensor. Mred/Mox = hydroquinone/quinone Gox = biotinylated glucose oxidase PPO — biotinylated polyphenol oxidase HRP-Ab = peroxidase-labeled IgG anti-rabbit antibody.

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