Immobilised Enzyme Electrodes

Ref. Analyte Matrix Enzyme/immobilisation Electrode configuration/ Mediator... 

In the first approach, enzymes are used. As for GOD, oxygen consumption is measured. An enzyme-immobilised electrode is not essential in this system but an oxygen electrode is required for measuring changes in the dissolved oxygen concentration. 

The second approach uses an enzyme-immobilised electrode and the commonly used immobilising matrices are polymers. The enzyme is immobilised close to the electrode, and so only a small amount of reaction product is needed. Thus response time and sensitivity are improved. This system is polarised at a suitable redox potential. 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

Membranes can be used as well as a supporting material for immobilisation (e.g. polycarbonate membranes with created amino groups on the surface that allow covalent binding with glutaraldehyde). Entrapment of enzymes on electrode surfaces can be carried out with polymeric membranes such as polyacrylamide and gelatine, or by electropolymerisation of small monomers (o-phenylenediamine). Enzyme encapsulation within a sol-gel matrix has also been reported. 

Finally, self-assembled monolayers (SAMs) on gold electrodes constitute electrochemical interfaces of supramolecular structures that efficiently connect catalytic reactions, substrate and product diffusion and heterogeneous electron transfer step when enzymes are immobilised on them. Resulting enzyme-SAM electrodes have demonstrated to exhibit good performance and long-term enzyme stability. 

Ref. Matrix Enzyme/immobilisation method Electrode configuration/ applied potential Mediator... 

Following immobilisation, the beads were dispersed in an aqueous solution of HEC and cast onto Pt electrodes. Activity tests showed that leaching of immobilised enzyme was 2.5 times slower than that of free enzyme dispersed in HEC. Comparisons of activity to acetylthiocholine after 72 h constant operation showed a large stability enhancement for enzymes immobilised on both silica and carbon when compared to dispersion in HEC [36]. 

G.S. Nunes, G. Jeanty and J.-L. Marty, Enzyme immobilisation procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides. Compartive study, Anal. Chim. Acta, 523 (2004) 107-115. 

Regarding the drying time, the enzyme-modified electrodes dried for 30 min provided higher absorbance values than the ones dried for 22 h. Hence, short drying times were enough to immobilise the enzyme and longer times only accelerated the enzyme inactivation. At this stage it is... 

The enzyme immobilisation was carried out on 7,7,8,8-tetra-cyanoquinodimethane (TCNQ)-modified screen-printed electrodes. TCNQ allows the electrochemical oxidation of thiocholine, the product of the reaction between acetylthiocholine and the enzyme, at +100 mV (vs. Ag/AgCl) (Fig. 16.6). The enzymatic activity of the acetylcholinesterase can thus be monitored by electrochemical methods. 

Enzyme immobilisation was performed within a custom-made, low-volume (3 ml) PTFE cell, which housed working, reference and counter electrodes. All water used was purified with a ELGA Purelab UHQ purifier. 

Perhaps the original hope for these polymers was that they would act simultaneously as immobilisation matrix and mediator, facilitating electron transfer between the enzyme and electrode and eliminating the need for either O2 or an additional redox mediator. This did not appear to be the case for polypyrrole, and in fact while a copolymer of pyrrole and a ferrocene modified pyrrole did achieve the mediation (43), the response suggested that far from enhancing the charge transport, the polypyrrole acted as an inert diffusion barrier. Since these early reports, other mediator doped polypyrroles have been reported (44t45) and curiosity about the actual role of polypyrrole or any other electrochemically deposited polymer, has lead to many studies more concerned with the kinetics of the enzyme linked reactions and the film transport properties, than with the achievement of a real biosensor. 

Tlie electrochemically-derived kinetic constants can then be compared to those obtained by conventional methods, to judge how the activity of the enzyme immobilised on the electrode compares with that observed in solution. Even when the population of electroactive enzyme is too small to measure reliably (generally below 2 pmol cm for one-electron signals on a graphite electrode) a catalytic wave can be observed whose current... 

Enzyme-based electrodes ISEs can be used in conjunction with immobilised enzymes to make them selective for enzyme substrates. These electrodes are not true ion-selective electrodes but are based upon them. Such an electrode has a two-step mechanism an... 

A number of biomolecules have been physically immobilised on conducting polymers [66,112, 116-119]. This is the simplest method of enzyme immobilisation. Since the binding forces involved are hydrogen bonds, van der Waals forces, etc., porous conducting polymer surfaces are most commonly used. The pre-adsorption of an enzyme monolayer prior to the electrodeposition of the polymer, [120] and two-step enzyme adsorption on the bare electrode surface and then on PPy film [121] have also been investigated. 

Polymerisation based on the electrochemical oxidation of a given monomer from a solution containing the enzyme is the simplest method of enzyme immobilisation in a polymer at the working electrode surface and results in the formation of conducting or non conducting... 

Thus, immobilisation should not lead to enzyme deactivation, make access to the enzyme active site difficult or let enzyme leach into the surrounding solution. The more common methodology for immobilisation of enzymes on electrode surfaces involves cross-linking with glutaraldehyde, aided by the protein bovine serum albumin (BSA) in order to increase the interaction with the enzyme molecules. In this section two other strategies will be described. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

In nature there are many redox enzymes which interact with biologically important molecules and in doing so eject or accept electrons. Consequently, it has long been realised that if such redox enzymes could be immobilised at an electrode surface, and rapid electron transfer between the two facilitated. 

In some systems, for example, the immobilised enzyme is sandwiched between an external cellulose acetate membrane that blocks the passage of heavy molecules and an internal polycarbonate membrane that allows passage of the transformed products to the electrode. 

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