Stability of enzyme sensors

To assure the proper handling and optimal functional stability of enzyme sensors the following topics have to be considered. 

A specific problem related to enzyme sensors is their restricted shelf life, since enzymes are not very stable. In dry state, they can be stored for some time. Disposable sensors are activated when they are humidified but lose their activity soon after use. Efforts are being made to improve the stability of enzyme sensors by chemical treatment. It has been proposed that the active substance in polyelectrolytes be immobilized with polysugars (Gibson and Hulbert 1993). The enzyme-polyelectrolyte complex formed seems to be stabilized electrostatically by some kind of Faraday cage. In this way, the enzyme activity remains preserved somewhat longer. 

The stability of enzyme sensors is thus related to the enzyme loading factor a of the active membrane. When the membrane contains more active enzyme, the biosensor is more stable and has a longer lifetime. We will see, however, that it is not always possible to satisfy this... 

Second, sensors are often intended for a single use, or for usage over periods of one week or less, and enzymes are capable of excellent performance over these time scales, provided that they are maintained in a nfild environment at moderate temperature and with minimal physical stress. Stabilization of enzymes on conducting surfaces over longer periods of time presents a considerable challenge, since enzymes may be subject to denaturation or inactivation. In addition, the need to feed reactants to the biofuel cell means that convection and therefore viscous shear are often present in working fuel cells. Application of shear to a soft material such as a protein-based film can lead to accelerated degradation due to shear stress [Binyamin and Heller, 1999]. However, enzymes on surfaces have been demonstrated to be stable for several months (see below). 

The stability of enzyme electrodes is difficult to define because an enzyme can lose some of its activity. Deterioration of immobilized enzyme in the potentiometric electrodes can be seen by three changes in the response characteristics (a) with age the upper limit will decrease (e.g., from 10-2 to 10 3 moll-1), (b) the slope of the analytical (calibration) curve of potential vs. log [analyte] decrease from 59.2 mV per decade (Nernstian response) to lower value, and (c) the response time of the biosensor will become longer as the enzyme ages [59]. The overall lifetime of the biosensor depends on the frequency with which the biosensor is used and the stability depends on the type of entrapment used, the concentration of enzyme in the tissue or crude extract, the optimum conditions of enzyme, the leaching out of loosely bound cofactor from the active site, a cofactor that is needed for the enzymatic activity and the stability of the base sensor. 

One of the most important problems in the development of commercial biosensors is the stabilization of the enzymes used as the biological detection agents in the sensor. Although many different biosensors have been constructed, few have been developed in commercially successful instruments. The molecular parameters which influence the activity and stability of enzymes in die dry state, and when re-hydrated in an electrode surface, have been investigated A new and novel system has been developed, which appears to have broad application to the stabilization of enzymes. The use of this system to stabilize enzymes on electrodes is reported, together with a discussion on the mode of action of stabilization in general. 

AChE was sandwiched with poly(diallyldimethyl-ammonium chloride) layers on the surface of CNTs. The OP biosensor thus developed was used to detect paraoxon (Figure 55.7) as low as 4 x 10 M with a 6 min response time in flow injection analysis. A high stability of the sensors was also a merit of this biosensor no deterioration in the response was observed after 1 week of eontinuous use of the sensor. Only a 15% deerease in the aetivity of the enzyme was observed after 3 weeks, though the authors... 

Enzymes, like most proteins, are unstable molecules. The stability of enzymes is of particular importance to biosensors because the rate of the enzymatically catalysed reaction is especially sensitive to the three-dimensional conformation of the enzyme. Most, if not all, enzymes are fully active only in a hydrated state. However, rarely are enzymes found in a completely aqueous medium in a sensor. Usually, the enzyme is dissolved into a hydrogel, immobilized onto a surface, or entrapped in a polymer matrix, all environments which differ in varying degrees from the natural environment of the enzyme. 

Organelles and tissue of animal or plant origin constitute good sources of biocatalytic materials and the extracted enzymes are often employed in the construction of enzyme sensors. Using these materials in their original state keeps the enzymes in their natural environment and increases the stability of the catalyst... 

The most important step in the development of an enzyme sensor is the firm attachment of the enzyme onto the surface of the working electrode. This process is governed by various interactions between the enzyme and the electrode material, and strongly affects the performance of the biosensor in terms of sensitivity, stability, response time, and reproducibility. This immobilization should be sufficiently strong to provide good mechanical stability of the sensor, but also sufficiently soft to provide optimal conformation freedom of the enzymes that is crucial... 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

The immobilization of enzymes for sensing purposes frequently provides several important advantages an increase of its stability, operational reusability and greater efficiency in consecutive multistep reactions. Sometimes immobilization is accompanied by a certain degree of denaturalization however, the enzyme-matrix interactions may assist in stabilization preventing conformational transitions that favor such process. In some cases excessive bond formation affects the conformation of the active site and the steric hindrances caused by the polymer matrix may render an inactive sensor. 

The first enzyme biosensor was a glucose sensor reported by Clark in 1962 [194], This biosensor measured the product of glucose oxidation by GOD using an electrode which was a remarkable achievement even though the enzyme was not immobilized on the electrode. Updark and Hicks have developed an improved enzyme sensor using enzyme immobilization [194], The sensor combined the membrane-immobilized GOD with an oxygen electrode, and oxygen measurements were carried out before and after the enzyme reaction. Their report showed the importance of biomaterial immobilization to enhance the stability of a biosensor. 

Agents for chemical bleaching rely on different types of peroxides. Potentiometric or amperometric biosensors that detect the highly specific and sensitive reaction of enzymes like katalases with their corresponding substrates can be used for on-line measurement [84]. The sensors can be manufactured with simple technologies at moderate cost, but their stability is not sufficient for integration in household appliances. 

To overcome the poor stability of ferrocene-mediated enzyme sensors, mediator-modified electrodes have been used. In the case of glucose oxidase, the cofactor FAD is deeply buried within the protein matrix. The depth of the active center is estimated to be 0.87 nm. Therefore, one cannot expect that the mediator covalently attached to the electrode surface via a short spacer retain the possibility of closely approaching the cofactor of the enzyme. 

Electron Transfer Type of Dehydrogenase Sensors To fabricate an enzyme sensor for fructose, we found that a molecular interface of polypyrrole was not sufficient to realize high sensitivity and stability. We thus incorporated mediators (ferricyanide and ferrocene) in the enzyme-interface for the effective and the most sensitive detection of fructose in two different ways (l) two step method first, a monolayer FDH was electrochemically adsorbed on the electrode surface by electrostatic interaction, then entrapment of mediator and electro-polymerization of pyrrole in thin membrane was simultaneously performed in a separate solution containing mediator and pyrrole, (2) one-step method co-immobilization of mediator and enzyme and polymerization of pyrrole was simultaneously done in a solution containing enzyme enzyme, mediator and pyrrole as illustrated in Fig.22. 

In terms of enzyme-based sensors for fermentation monitoring, the main problem is the lack of enzyme stability during long-term use. Two recent reviews describe the current state of the art, 62,63) so we will not dwell on their use. 

Stabilization of activated oxidoreductases on time scales of months to years has historically been challenging, and the lack of success in this regard has limited the industrial implementation of redox enzymes to applications that do not require long lifetimes. However, as mentioned in the Introduction, some possibility of improved stability has arisen from immobilization of enzymes in hydrophilic cages formed by silica sol—gels and aerogels, primarily for sensor applications.The tradeoff of this approach is expected to be a lowering of current density because... 

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