Enzymes sensors

The flow-through sensors described in this Section comply essentially with the definition of biosensor. This word, like every term used to designate devices of scientific and popular note, has been the object of a number of definitions of both generic and specific scope. In a broad sense, a biosensor is any instrument or technique that measures biomolecules. In stricter terms, Rechnitz defines a biosensor as a device that incorporates a biochemical or biological component as a molecular recognition element and yields an analytical signal in response to biomolecules [10]. In between these two 

There is the widespread feeling that biosensors have been overpromoted. In fact, they have suddenly started to be praised as providing solutions to a wide variety of scientific and societal problems. To a great extent, such overpromotion has resulted from overoptimistic market projections by research bodies with little understanding of the tremendous problems still facing biosensors. Such projections are reported as facts in the media, which raises unrealistic expectations. 

On the other hand, the international scope of biosensor research is quite a positive indicator. The relative low equipment costs involved and the wide availability of biological materials has facilitated access of not so wealthy or industrialized countries to biosensor research, which ensures a healthy diversity of approaches and leaves the field open to fresh and, perhaps, radical new ideas. 

These preliminary developments have relied on either metabolic enzyme reactions (viz. those where the substrate is consumed and a product is formed as a result) and bioaffinity reactions (viz. those that are followed via electron density changes). This Section discusses sensors based on immobilized enzymes and both types of reaction metabolic and bioaffinity. 

Given the wide variety of available flow-through sensors using an immobilized enzyme, they are dealt with according to the type of detection involved optical or electroanalytical. Each sensor group is in turn divided according to various criteria. 

An enzyme sensor can be considered as the combination of a transducer and a thin enzymatic layer, which normally measures the concentration of a substrate. The enzymatic reaction transforms the substrate into a reaction product that is detectable by the transducer. An extension of this definition is that the concentration of any substance can be measured provided that its presence affects the rate of an enzymatic reaction this is especially true for enzyme inhibitors. We will first examine the principle of enzyme sensors and the theoretical and practical aspects of their operation. We will then describe the various sensors, classified according to their detection mode. 

transport of the substrate from the bulk of the solution towards the enzymatic layer, 

diffusion of the substrate within this layer, accompanied by the 

conversion of the concentration of the product at this interface into an electrical signal by the transducer. 

The presence of the enzyme ensures the transformation of the substrate into the reaction product according to the following reaction  

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Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Spohn U., Preuschoff F., Blankenstein G., Janasek D., Kula M.R., Hacker A., Chemiluminometric enzyme sensors for flow injection analysis, Anal.Chim. Acta 1995 303 109-120. 

Hikima S., Kakizaki T., Hasebel K., Enzyme sensor for L-lactate using differential pulse amperometric detection, Fresen. J. Anal. Chem. 1995 351(2-3) 237-240. 

Koncki R., Mohr G., Wolfbeis O.S., Enzyme sensor for urea based on novel pH bulk optode membrane, Biosens. Bioelectr. 1995 10 653-659. 

F. Mizutani, S. Yabuki, T. Sawaguchi, Y. Hirata,Y. Sato, and S. Iijima, Use of a siloxane polymer for the preparation of amperometric sensors 0-2 and NO sensors and enzyme sensors. Sens. Actuator B-Chem. 76, 489—193 (2001). 

G. Jeanty and J.L. Marty, Detection of paraoxon by continuous flow system based enzyme sensor. Biosens. Bioelectron. 13, 213-218 (1998). 

A. Seki, F. Ortega, and J.L. Marty, Enzyme sensor for the detection of herbicides inhibiting acetolactate synthase. Anal. Lett. 29,1259—1271 (1996). 

D. Moscone, D. D Ottavi, D. Compagnone, G. Palleschi, and A. Amine, Construction and analytical characterization of Prussian Blue-based carbon paste electrodes and their assembly as oxidase enzyme sensors. Anal. Chem. 73, 2529—2535 (2001). 

J. Pei and X. Li, Amperometric glucose enzyme sensor prepared by immobilizing glucose oxidase on CuPtC16 chemically modified electrode. Electroanalysis 11, 1266-1272 (1999). 

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. 

Figure 5 Five-channel enzyme sensor for the simultaneous determination of glucose, lactate, glutamate, glutamine, and ammonium. MFM, microfiltration module WV, valves P, pumps DC, dialysis cell B, blank reactors MC, reactor D, biosensor flow cell. (Adapted with permission from Ref. 34.)...

Diffusion Currents. Half-wave Potentials. Characteristics of the DME. Quantitative Analysis. Modes of Operation Used in Polarography. The Dissolved Oxygen Electrode and Biochemical Enzyme Sensors. Amperometric Titrations. Applications of Polarography and Amperometric Titrations. 

The Dissolved Oxygen Electrode and Biochemical Enzyme Sensors... 

The type of enzyme sensor described above is highly selective and can be sensitive in operation. There are obvious applications for the determination of small amounts of oxidizable organic compounds. However, it is perhaps too early to give a realistic assessment of the overall importance of enzyme sensors to analytical chemistry. This is especially so because of parallel developments in other biochemical sensors which may be based upon a quite different physical principle. 

Redox enzymes have been assembled in a monolayer on the solid surface by a potential-assisted self-assembling method as well as a thiol-gold selfassembling method. These enzymes are electronically communicated with the solid substrate through a molecular interface of conducting polymer and a covalently bound mediator. Electron transfer type of enzyme sensors have been fabricated by the self-assembling methods. 

Apart from electron promoters a large number of electron mediators have long been investigated to make redox enzymes electrochemically active on the electrode surface. In the line of this research electron mediators such as ferrocene and its derivatives have successfully been incorporated into an enzyme sensor for glucose [3]. The mediator was easily accessible to both glucose oxidase and an electron tunnelling pathway could be formed within the enzyme molecule [4]. The present authors [5,6] and Lowe and Foulds [7] used a conducting polymer as a molecular wire to connect a redox enzyme molecule to the electrode surface. 

Ferrocene-mediated enzyme sensors have usually been obtained by adsorption of the mediator onto the electrode surface because of this insolubility in aqueous solution. Due to the good solubility of ferrecinium cations in aqueous solution complications arise from leakage of the mediator from the electrode surface. 

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. 

Fig.23 Calibration curves for fructose with two different enzyme sensors...

It is difficult to incorporate dehydrogenases that are coupled with NAD(P) into amperometric enzyme sensors owing to the irreversible electrochemical reaction of NAD. We have developed an amperometric dehydrogenase sensor for ethanol in which NAD is electrochemically regenerated within a membrane matrix. 

An electron transfer type of enzyme sensor was thus fabricated by a electrochemical process. Although no appreciable leakage of ADH and MB from the membrane matrix was detected, NAD leaked slightly. To prevent this leakage, the ADH-MB-NAD/polypyrrole electrode was coated with Nation. A calibration curve is presented in Fig.25 for ethanol determination in an aquous solution with the enzyme sensor. Ethanol is selectively and sensitively determined in the concentration range from 0.1 nM to 10 mM. 

In a further development, an ADH-MB-NAD/polypyrrole electrode, a platinum counter electrode and an Ag/AgCl reference electrode were assembled and covered with a gas-permeable polymer membrane to form an gaseous ethanol sensor. This appears to be the first time that a complete enzyme sensor for gaseous ethanol has been fabricated in such a manner with NAD incorporated in immobilized form. 

D. Pfeiffer, F. W. Scheller, C. J. McNeil, and T. Schulmeister, Cascade like exponential substrate amplification in enzyme sensors. Biosensors Bioelectron., 10, 169-180 (1995). 

Among potentiometric enzyme sensors, the urea enzyme electrode is the oldest (and the most important). The original version consisted of an enzyme layer immobilized in a polyacrylamide hydrophilic gel and fixed in a nylon netting attached to a Beckman 39137 glass electrode, sensitive to the alkali metal and NHj ions [19, 2A Because of the poor selectivity of this glass electrode, later versions contained a nonactin electrode [20,22] (cf. p. 187) and especially an ammonia gas probe [25] (cf. p. 72). This type of urea electrode is suitable for the determination of urea in blood and serum, at concentrations from 5 to 0.05 mM. Figure 8.2 shows the dependence of the electrode response... 

Dosivit, Nantes, France MC2 Multisensor Glucose, sucrose, lactose, lactate, ethanol Electrochemical enzyme sensor Agriculture, food... 

Sensors based on integrated reaction and detection are the most varied and numerous among flow-through sensors and those that will predictably experience the greatest development in the near future. Both enzyme sensors and immunosensors are bound to become virtually irreplaceable tools in some areas of social interest including clinical and environmental analysis. While other sensors inspired by those discussed in Sections 3.4 and 3.5 may... 

In principle, there are two possible ways to measure this effect. First, there is the end-point measurement (steady-state mode), where the difference is calculated between the initial current of the endogenous respiration and the resulting current of the altered respiration, which is influenced by the tested substances. Second, by kinetic measurement the decrease or the acceleration, respectively, of the respiration with time is calculated from the first derivative of the currenttime curve. The first procedure has been most frequently used in microbial sensors. These biosensors with a relatively high concentration of biomass have a longer response time than that of enzyme sensors. Response times of comparable magnitude to those of enzyme sensors are reached only with kinetically controlled sensors. 

Many different types of techniques for protein immobilization have been developed using, in most cases, enzyme sensors. Early studies of enzyme biosensors often employed thick polymer membranes (thickness 0.01-1 mm) in which enzymes are physically entrapped or chemically anchored. The electrode surface was covered with the enzyme-immobilized polymer membranes to prepare electrochemical enzyme sensors. Although these biosensors functioned appropriately to... 

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