Enzyme electrode in flow systems

Enzyme electrode in flow system Closed-loop Packed bed... 

From the fact that the dehydrogenases depend on a soluble cofactor follows that the enzyme need not be immobilized in close proximity to the detection device. There are several examples in flow systems (FLA and LC) where the enzymatic reaction and NADH production occur in an immobilized enzyme reactor (18,66, 104-106,118,119). The NADH is then transported downstream to the electrode situated in an amperometric flow through cell. As we have reported, the enzyme... 

The utilization of a DNA matrix to aid enzyme immobilization is a promising alternative for the development of new biosensors and its application in flow systems. A DNA-tyrosinase carbon paste electrode [95] showed excellent performance for detection of cathecol in a FIA system and suggests that other enzymatic biosensors can benefit from the presence of DNA. 

An appropriate approach for comfortable on-line process monitoring is the combination of enzyme electrodes with flow injection analysis. For the determination of glucose and lactate this is incorporated in the OLGA system (Biometra Gottingen, Germany). 

The application of enzyme electrodes in a flow system enables two biosensors to be placed in a tubular flow-cell facing each other to measure two analytes simultaneously, e.g., glucose and urea [318]. 

A modified carbon paste electrode (CPE) using asolectin, cytochrome c, and cytochrome oxidase were applied for amperometric determination of cyanide [56]. The modified CP matrix mimics a biological membrane environment. The sensor, polarized at —0.15 V versus Ag/AgCl, generates the reduced form of cytochrome c, which in turn is oxidized by the enzyme cytochrome oxidase. The resulting current is related to the enzyme activity and is depressed by inhibitors of cytochrome oxidase such as cyanide. Concentrations of cyanide as low as 0.5 pM can be measured with half-maximal response at about 12 pM. The inhibition is reversible and reproducible (RSD = 4%), allowing cyanide determination for more than 2 months using the same probe. Possible use of this biosensor in flow systems was illustrated. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

The sensing microzone of the flow-through sensor depicted in Fig. 5.9.B1 integrates gas-diffusion and detection with two analytical reactions [28], viz. (a) the urease-catalysed formation of ammonium ion by hydrolysis of urea (the analyte), which takes places on a hydrophilic enzyme membrane in contact with the sample-donor stream, which contains a gel where the enzyme is covalently bound and (b) an acid-b reaction that takes place at the microzone on the other side of the diffusion membrane and involves Bromothymol Blue as indicator. This is a sandwich-type sensor including a hydrophilic and a hydrophobic membrane across which the sample stream is circulated —whence it is formally similar to some enzyme electrodes. Since the enzymatic conversion of the analyte must be as efficient as possible, deteetion (based on fibre optics) is performed after the donor and acceptor streams have passed through the sensor. Unlike the previous sensor (Fig. 5.9.A), this does not rely on the wall-jet approach in addition, each stream has its own outlet and the system includes two sensing microzones... 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Amperometric sensors monitor current flow, at a selected, fixed potential, between the working electrode and the reference electrode. In amperometric biosensors, the two-electrode configuration is often employed. However, when operating in media of poor conductivity (hydroalcoholic solutions, organic solvents), a three-electrode system is best (29). The amperometric sensor exhibits a linear response versus the concentration of the substrate. In these enzyme electrodes, either the reactant or the product of the enzymatic reaction must be electroactive (oxidizable or reducible) at the electrode surface. Optimization of amperometric sensors, with regard to stability, low background currents, and fast electron-transfer kinetics, constitutes a complete task. 

Enzyme-linked electrochemical techniques can be carried out in two basic ways. The first approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. In the second approach, the enzyme is immobilized at the electrode. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of the substrate to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system that is amenable to in vivo analysis. Alternatively, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near the electrode. Enzyme electrodes are an extremely important area of bioelectrochemical analysis, and many reviews are available in the literature. ... 

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