Although soluble enzymes can be used as excellent reagents for the analysis of inorganic and organic compounds, they face a serious challenge when attempts are made to utilize them in complex matrices, like blood or crude water. Problems center about the effect of activators, inhibitors, other substrates, pH, and temperature on the soluble enzyme. However, upon immobilization most of these effects can be eliminated or minimized. For example, an enzyme with a narrow pH range of 4–6 can be transformed upon insolubilization to a more viable reagent with a broad pH range of 4–10. Also, following immobilization the enzymes are much more stable; they can be heated to 37, 40 or 50°C, with little loss of activity; and the activity persists after several thousand analysis are performed. However, the biggest advantage, analytically speaking, of immobilization, is that the insolubilized reagent becomes a much more selective reagent. No longer do many activators and inhibitors have an effect; only the most powerful can actually attack the enzyme.

Analytical Uses of Immobilized Enzymes

Dans cette electrode, les enzymes sont immobilisees a l'interieur d'une membrane attachee a un detecteur electrochimique de l'oxygene, cosubstrat de la reaction enzymatique. Le modele mathematique presente concerne les phenomenes de reaction et de diffusion ayant lieu a l'interieur de la membrane

Model of a two-substrate enzyme electrode for glucose.

An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide promoted by the immobilized peroxidase starts already at about +600 mV vs SCE at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off at about -200 mV vs SCE. Amperometric biosensors for hydrogen peroxide can be constructed with these kind of peroxidase modified electrodes. By co-immobilizing a hydrogen peroxide producing oxidase with the peroxidase, amperometric biosensors can be made responding for the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Sensors for L- and D-amino acids are shown with preliminary results obtained for L- and D-phenylalanine, respectively.

Amperometric biosensors for detection of L‐ and D‐amino acids based on coimmobilized peroxidase and L‐ and D‐amino acid oxidases in carbon paste electrodes

The rapid growth in the number of enzymes and the reactions associated with them, ensures that a wide variety of enzyme catalyzed analysis procedures are now available. The development of immobilized enzymes probes (I.E.P.) is related to advances in both immobilization technology and in the improvement of numerous sensing devices. Potentiometric, amperometric, enthalpimetric, and chemiluminescent sensors have been employed as transducers for enzyme electrodes. Most of them have been designed for organic and biological substrates for which simple analyses were not available. Nevertheless, few electrodes have been constructed for inhibitor determination. Other immobilized enzyme systems developed for analysis of enzyme inhibitors will be treated under reactor systems since they are generally used in a flow-through reactor configuration. On the other hand, if some type of electrode is used to measure the rate of an enzyme catalyzed reaction which takes place in solution, this is not a true enzyme electrode. The concept of an enzyme electrode was introduced by Hicks and Updike. The basic features of an I.E.P. are a thin enzyme layer held in close proximity to the active surface...

Immobilized enzyme probes for determining inhibitors

Preconcentration and determination of ferrocenecarboxaldehyde at a chemically modified platinum electrode

The construction of electrodes, the behavior of which could be accurately analyzed and optimized is described. Two methods were used to construct such enzyme electrodes. A direct binding method, where a cation electrode was dipped into a solution of enzyme, albumin, and glutaraldehyde: a thin layer of cross-linked protein coated the electrode bulb. A two-step method, where an active membrane was first made by cross-linking these proteins by glutaraldehyde on a glass plate, then fitting with a silicone membrane onto a gas electrode (O2 or CO2). Both methods were used to construct glucose and urease electrodes measuring either NH4+ ions or CO2. Glucose oxidase and L-amino acid oxidase-pO2 electrodes were also developed. The theoretical and experimental behavior of these enzymatic membrane electrodes were compared using the analysis of product concentrations resulting from the association of diffusion with enzyme reaction in the active layer, and the optimum operating conditions were determined.

Tran-Minh C

Papers

Construction and study of electrodes using crosslinked enzymes