Detectors enzyme electrodes

Fig. 7. Schematic diagram of an enzyme electrode. A, B and C comprise the membrane system, while D is the detector (either amperometric or potentiometric)...

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.

Acetylcholineesterase and choline oxidase 300 pL 0.1 M phosphate buffer (pH 6.5) containing 16 mg BSA and 1 mg each of ChO and AChE were mixed with 30 pL 25% glutaral-dehyde diluted 10 fold with phosphate buffer. The solution was used to coat the surface of a Pt electrode. This enzyme electrode was used for the amperometric measurement of ACh and Ch. Calibration graphs were linear upto 0.09 and 0.08 mM Ch and ACh, respectively. Detection limits were 0.1 pM of both Ch and ACh. Response time was 1 s for both Ch and ACh. The use of the sensor as detector for HPLC analysis for both Ch and ACh was demonstrated. [91]... 

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. ... 

An enzyme electrode is basically a dense package of dialyzer, enzyme reactor, and electrode (detector). Enzymes introduce analytical selectivity due to the specificity of the signal-producing interaction of the enzyme with the analyte. They enhance the equihbrium formation of chemical reactions. For example, splitting of H2O2 is accelerated by a factor of 3 x 10 in the presence of catalase. Turnover numbers can be as fast as 6 X 10 s (carbonic anhydrase) where cat/ m approaches the diffusion limited value of 10 s ... 

Mueller P, Rudin D O, Tien H T and Wescott W 1962 Reconstruction of excitable cell membrane structure in vitro Circulation 26 1167-71 King W H Jr 1964 Piezoelectric sorption detector Ana/, Chem. 36 1735-9 Guilbault G and Montalvo J 1969 A urea specific enzyme electrode J. Am. Chem. Soc. 91 2164-5... 

Enzyme electrode detector for the continuous monitoring of D-glucose... 

Within the last few years there has been an almost explosive surge in the development and applications of electrochemical and optical enzyme sensors. In enzyme electrodes a membrane containing one or several immobilized enzymes is placed in front of the active surface of an electrochemical detector. The analyte is transported by diffusion into the mem-... 

An enzyme electrode (Fig. 1) is a dense package of dialyzer, enzyme reactor, and detector. A typical example would be a... 

Potentiomelric Detectors. Essentially, poten-tiometric detectors consist of ion-selective electrodes (ISEs) to monitor ionic species in chromatographic effluents. Examples of ISEs are glass, solid-state, liquid-membrane, and enzyme electrodes. 

The main limitation to enzymatic procedures is the high cost of enzymes, particularly when used for routine or continuous measurements. This disadvantage has led to the use of immobilized enzyme media in which a small amount of enzyme can be used for the repetitive analysis of hundreds of samples. Two general techniques are used. In one, the sample is passed through a fixed bed of immobilized enzyme and then to the detector. In the second, a porous layer of the immobilized enzyme is attached directly to the surface of the ion-selective electrode, thus forming an enzyme electrode. In such devices, the reaction product reaches the selective membrane surface by diffusion. 

The enzyme is attached to the sensing electrode itself. Solution flow is shown in Figure 2C only to suggest a method of analyte introduction to the biosensor. This system does not require flow past the enzyme and detector but instead relies on diffusion of substrate to the enzyme, and diffusion of the enzymatically generated reduced cofactor fi om the inunobilized enzyme to the working electrode. As will be shown, inuno-biUzation of enzymes on the electrode surface often reduces the detector efficiency because both enzyme conversion efficiency and diffusion of analytes can limit the time response of such a system. This type of system is amenable to the immobilization of the cofactor as well (e.g., wired enzyme electrodes [5-7], which use intrinsic FAD/FADHj as cofactors). 

Thus, FIA systems for lactate sensing have predominantly been developed using electrochemical and optical detectors. Incorporation of other enzyme electrodes and reactors has led to the development of multisensing FIA systems. Finally, FIA systems based on enzyme thermistors have been developed for lactate sensing using LOD. Together, these FIA systems have been used to detect lactate in food samples and human blood samples. 

In 1990, Yao et al. proposed two different alternatives to determine MSG in seasonings using immobilized GIOD a packed-bed reactor and a lab-made flow-through enzyme electrode used as a detector [19]. Both configurations are tested in a FIA system with a simple channel manifold to compare the performance of the enzyme with... 

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. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Due to their response mechanism the polyion-selective electrodes are not sensitive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves the polyionic molecule these sensors can be used for detection of enzyme activity. Polycation protamine is rich in arginine residues that make it a suitable substrate for protease-sensitive electrochemical assays. Real-time detection of trypsine activity was demonstrated with the protamine-selective electrode as a detector [38],... 

It would appear certain that the most important need in LCEC is the development of improved electrode materials. It may be possible in the near future to design an electrode that will give superior performance for certain classes of compounds. Modifying electrode surfaces by covalent attachment of various ligands or electron-transfer catalysts (including enzymes) can provide the key to better amperometric devices for all sorts of analytical purposes. Research in the area of chemically modified electrodes (CMEs) has been reviewed (see Chap. 13) [6,11]. Those interested in improving the performance of electrochemical detectors would do well to study these developments in detail. 

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