Enzyme Biosensor Stability

Enzymes are proteins. They are sensitive to denaturation by pH, temperature, or aging. Enzymes have an optimal pH range in which their activity is maximal this pH range should be compatible with the transducer. Moreover, most of the biological systems have a very narrow range of temperature (15-40 C). The most important problem and main drawback for industrial exploitation is the short lifetime associated with the biological elements. 

The gradual loss in activity generates a decrease in. Stability of enzyme biosensor is related to enzyme loading, and in general, if the membrane contains more active enzyme, the biosensor is more stable and has a longer lifetime. However it is not always possible to satisfy this condition and a compromise must be adopted [20,21]. 

The removal of interferences has also been the other important aspect for the wide use of biosensors for industrial processes. This problem could be solved by using multilayer membranes. The main role of the membrane is to prevent interferences from passing into the bioactive layer. Cellulose acetate membrane allows only molecule of the size of hydrogen peroxide to cross and contact the platinum anode, thus preventing interference from ascorbic acid or uric acid, for example, at the fixed potential in the case of a glucose biosensor. 

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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. 

A.L. Crumbliss, J.Z. Stonehuerner, R.W. Henkens, J. Zhao, and J.P. O Daly, A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood. Biosens. Bioelectron. 8, 331-337 (1993). 

Utilization of whole cells and tissues in biosensor has increasingly been used. Enzyme stability, availability of different enzymes and reaction systems, and characteristics of cell surface are the advantages of using cells and tissues in biosensor designs. Multi-step enzyme reactions in cells also provide mechanisms to amplify the reactions that result in an increase in the detectability of the analytes. The presence of cofactors such as NAD, NADP, and metals in the cells allows the cofactor-dependent reactions to occur in the absence of reagents. (34, 50, 69). However, the diffusion of analytes through cell wall or membrane imposes constraint to this type of biosensors and results in a longer response time compared to the enzyme biosensors. 

Enzyme biosensors containing pol3mieric electron transfer systems have been studied for more than a decade. One of the earlier systems was first reported by Degani and Heller [1,2] using electron transfer relays to improve electrochemical assay of substrates. Soon after Okamoto, Skotheim, Hale and co-workers reported various flexible polymeric electron transfer systems appUed to amperometric enz5une biosensors [3-16], Heller and co-workers further developed a concept of wired amperometric enzyme electrodes [17—27] to increase sensor accuracy and stability. 

When constructing biosensors, which are to be used continuously in vivo or in situ, maintaining sensor efficiency while increasing sensor lifetime are major issues to be addressed. Researchers have attempted various methods to prevent enzyme inactivation and maintain a high density of redox mediators at the sensor surface. Use of hydrogels, sol-gel systems, PEI and carbon paste matrices to stabilize enzymes and redox polymers was mentioned in previous sections. Another alternative is to use conductive polymers such as polypyrrole [123-127], polythiophene [78,79] or polyaniline [128] to immobilize enzymes and mediators through either covalent bonding or entrapment in the polymer matrix. Application to various enzyme biosensors has been tested. 

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Eremenko A., Kurochkin I., Chernov S., Barmin A., Yaroslavov A., Mosk-vitina T. Monomolecular enzyme films stabilized by amphiphilic polyelectrolytes for biosensor devices. Thin Solid Films 1995 260 212-216. 

Since hydrogen peroxide is the product of reactions catalysed by a huge number of oxidase enzymes and is essential in food, pharmaceutical, and envitonmental analysis, its detection was and remains a necessity. Many attempts have been made in order to develop a biosensor that would be sensitive, stable, inexpensive and easy to handle. The most popular and efficient of them are amperometric enzyme biosensors, which utihsed different types of mediators and enzymes, mosdy peroxidase and catalase. Unfortunately many of the sensors developed do not mea the requirements for a practical device, which has a balance of technological charaaeristics (sensitivity, reliability, stability) and commercial adaptability (easy of mass production and low price). Thus a window of opportunity still remains open for future development. We hope that the present work will inspire other researches for further advances in the area of biosensors, in particular sensors for detection of such an important analyte as hydrogen peroxide. 

A reference electrode, such as the standard calomel electrode (SCE), is placed next to the enzyme electrode. The reference electrode may be combined with the working electrode, as is the case in the pH-sensitive glass electrode. The electrodes are connected to a millivoltmeter for potentiometric measurements, or to a potendostat for amperometric measurements. The system is connected to a recorder which monitors the biosensor stability and the progression of its response curve towards a steady state. This recorder may be replaced with a data acquisition unit which also gives the slopes of the response curves and relates them directly to the analyte concentration thereby increasing the rate of measuremenL... 

Enzyme electrodes belong to the family of biosensors. These also include systems with tissue sections or immobilized microorganism suspensions playing an analogous role as immobilized enzyme layers in enzyme electrodes. While the stability of enzyme electrode systems is the most difficult problem connected with their practical application, this is still more true with the bacteria and tissue electrodes. 

The work in the biosensor industry permitted the testing and proved of stability and reproducibility of enzymes, within the conditions employed in that area. Enzymes with demonstrated stability include lactate oxidase, malate dehydrogenase, alcohol oxidase, and glutamate oxidase. 

Enzyme-based optical sensor applications will be further described in this book. They are still the most widespread optical biosensors but work is needed to overcome limitations such as shelf life, long term stability, in situ measurements, miniaturization, and the marketing of competitive devices. 

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