Optical enzyme sensors

In this section, only the important differences resulting from the optical detection are highlighted. The optimum conditions for the operation of optical enzyme sensors, particularly the thickness of the enzyme layer, could be found again by solving the set of diffusion-reaction equations for a given geometry. There is, however, an... 

An example of an optical enzyme sensor (Arnold, 1985) in a bifurcated optical fiber is shown in Fig. 9.32. The bifurcated fiber delivers and collects light to and from the site of the enzymatic reaction. The enzyme, alkaline phosphatase (AP), catalyzes hydrolysis of p-nitrophenyl phosphate to p-nitrophenoxide ion which is being detected (A = 404 nm). 

Aizawa presented an overview on the principles and applications of the electrochemical and optical biosensors [61]. The current development in the biocatalytic and bioaffinity bensensor and the applications of these sensors were given. The optical enzyme sensor for acetylcholine was based on use of an optical pH fiber with thin polyaniline film. 

Arnold et al. (1987) described an optoelectronic ethanol sensor based on fluorimetric detection of NADH formed in the reaction catalyzed by ADH. The enzyme was fixed to the inner surface of a membrane permeable to volatile substances, which separated the sample from the internal sensor solution. This solution contained NADH and semicarbazide, so that no reagent had to be added to the sample. The arrangement was named an internal optical enzyme sensor . 

Within the last few years there has been an almost explosive surge in the development and applications of electrochemical and optical enzyme sensors. In enzyme electrodes a membrane containing one or several immobilized enzymes is placed in front of the active surface of an electrochemical detector. The analyte is transported by diffusion into the mem-... 

NH groups left available for further interaction with PSS. The transfer was very poor in both cases and resulted in inhomogeneously coloured films. However, the simple alternation of TB and PAA yields much better quality films although still patchy. Further work in this direction is underway. We hope to produce composite PESA films containing different pairs enzyme/indicator for the development of optical enzyme sensor-array in near future. 

Optical enzyme sensors are designed preferably as extrinsic sensors, i.e. as op-todes. There are, however, some examples of intrinsic sensors. As an example, an optical fibre has been described which was manufactured on the basis of a polystyrene fibre coated with adsorbed enzyme and indicator molecules. The colour changes brought about by the enzymatic reaction was detected using the evanescent field. 

Optical sensors that are sensitive to pH and ammonia can save as a basis for the production of optical enzyme sensors [8]. Certain enzymes catalyse reactions with a variation in pH (penicillinase, cholinesterase, glucose oxidase or urease, etc.), or with liberation of ammonia (urease, glutaminase, amino acid oxidase) and are simply deposited onto the optical sensors. 

Optical enzyme sensors do not necessarily require a colored immobilized reagent. Sometimes the product of the reaction is tically absorbent. One example is the simple optical sensor that uses immobilized alkaline phosphatase to catalyse the following reaction [9] ... 

Bioluminescence and chemiluminescence are very powerful analytical tools, since in addition to the direct measurement of ATP, NAD(P)H or hydrogen peroxide, any compound or enzyme involved in a reaction that generates or consumes these metabolites can be theoretically assayed by one of the appropriate light-emitting reactions. Some of these possibilities have been exploited for the development of optical fibre sensors, mainly with bacterial bioluminescence and with luminol chemiluminescence. 

Another approach, developed in our laboratory, consists of the compartmentalization of the sensing layer25"27. This concept, only applicable for multi-enzyme based sensors, consist in immobilizing the luminescence enzymes and the auxiliary enzymes on different membranes and then in stacking these membranes at the sensing tip of the optical fibre sensor. This configuration results in an enhancement of the sensor response, compared with the case where all the enzymes are co-immobilized on the same membrane. This was due to an hyperconcentration of the common intermediate, i.e. the final product of the auxiliary enzymatic system, which is also the substrate of the luminescence reaction, in the microcompartment existing between the two stacked membranes. 

The detection modes commonly applied in enzyme-based optical fiber sensors are based on one of the following principles ... 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

Optical fiber sensors that use enzymes can operate in the direct or indirect detection mode. In the first case, the optical properties of the reactives, intermediates or products of the biocatalyzed reaction can be monitored using the optical fibers. In the second type, an optochemical transducer generates the optical changes. 

As it is shown in Figure 8, the enzyme can be immobilized in the vicinity (membrane, beads, etc) or on the surface of the fiber for optical fiber sensor development. Alternatively, it can be placed in a reactor and use the optode... 

Enzymatic reactions coupled to optical detection of the product of the enzymatic reaction have been developed and successfully used as reversible optical biosensors. By definition, these are again steady-state sensors in which the information about the concentration of the analyte is derived from the measurement of the steady-state value of a product or a substrate involved in highly selective enzymatic reaction. Unlike the amperometric counterpart, the sensor itself does not consume or produce any of the species involved in the enzymatic reaction it is a zero-flux boundary sensor. In other words, it operates as, and suffers from, the same problems as the potentiometric enzyme sensor (Section 6.2.1) or the enzyme thermistor (Section 3.1). It is governed by the same diffusion-reaction mechanism (Chapter 2) and suffers from similar limitations. 

The enzyme is immobilized on a nylon mesh, which also acts as a diffuse reflector for the light. The dynamic range of this sensor is between 1(T5 and 10 3M. Although the primary process that determines the steady-state concentration of the p-nitrophenoxide ion is the diffusion-reaction mechanism (which is governed by concentrations of all participating species), the detection of its concentration is again subject to the limitations of optical sensing of ionic species (Section 9.4.1). There are many similar optical enzyme biosensor schemes that utilize detection of... 

Furthermore, samples are not usually optically clear.On the other hand, electrochemical monitoring of these compounds can be done by biosensors. Many reports on enzyme sensors have been published for clinical and food analyses(1,2). The enzyme sensors consisted of the immobilized enzyme and an electrochemical device. However, enzymes are unstable and expensive. Therefore, the enzyme sensors are not suitable for industrial process and environmental control. 

Moreno-Bondi MC, Benito-Pena E (2005) Fundamentals of enzyme-based sensors. In Martellucci S, Baldini F (eds) Optical chemical sensors. Springer-Kluwer, New York (in press)... 

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

---