Amperometric 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. 

In Fig. 2.10, the boundary between the enzyme-containing layer and the transducer has been considered as having either a zero or a finite flux of chemical species. In this respect, amperometric enzyme sensors, which have a finite flux boundary, stand apart from other types of chemical enzymatic sensors. Although the enzyme kinetics are described by the same Michaelis-Menten scheme and by the same set of partial differential equations, the boundary and the initial conditions are different if one or more of the participating species can cross the enzyme layer/transducer boundary. Otherwise, the general diffusion-reaction equations apply to every species in the same manner as discussed in Section 2.3.1. Many amperometric enzyme sensors in the past have been built by adding an enzyme layer to a macroelectrode. However, the microelectrode geometry is preferable because such biosensors reach steady-state operation. 

Amperometric enzyme sensors for the detection of cyanobacterial toxins in environmental samples... 

In analogous fashion enzymes with their highly sophisticated natural selectivity have been covalently immobilized (particularly on nylon mesh) to provide long-life amperometric enzyme sensors. 

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. 

Examples of substrates that can be detected by amperometric enzyme sensors... 

Relevant issues still to be addressed in constructing amperometric enzyme sensors either using the electrical wiring of enzymes with redox polymers or with flexible polymeric electron mediators are sensor efficiency, accuracy, reproducibility, selectivity, insensitivity to partial pressure of oxygen, detectivity (signal-to-noise ratio) as well as sensor hfetime and biocompatibility [47]. Then we can address manufacturability and the cost of use of either in vitro or in vivo sensors. 

Borgmann, S., Hartwich, G., Schulte, A., and Schuhmann, W. (2006) Amperometric enzyme sensors based on direct and mediated electron transfer, in Electrochemistry of Nucleic Acids and Proteins. Towards Electrochemical Sensors for Genomics and Proteomics (eds E. Palecek, F. Scheller, and J. Wang), Elsevier, Amsterdam, pp. 599-655. 

Diffusion barriers are typically used where the chemical reaction responsible for sensing is slow relative to the diffusion rate of the analyte to the active portion of the sensor. As shown above, a gas-permeable membrane can act as a diffusion barrier in some cases for gas sensors. Diffusion barriers are also often used in amperometric enzyme sensors which exploit the native selectivity of an enzyme-substrate reaction to measure the concentration of the substrate. If the substrate is at a high concentration, the enzyme may become saturated, especially if the enzyme turnover rate is low. In this condition, the signal will plateau and no longer be dependent upon the substrate concentration. A diffusion barrier between the enzyme layer and the sample reduces the flux of the substrate to the enzyme and, thus, prevents saturation of the enzyme and increases the linear range of the sensor. 

Fig. 10. Differential amperometric enzyme sensor. (1) glass plate, (2) Au-layer (150nm) or Cr-layer (20 nm), (3) deactivated GOD-layer, (4) common cathode, (5) active GOD-layer. Abbreviations Up, polarization voltage I, measured current 1, compensation current Rf, feedback resistors of current-to-voltage converters R, subtractor-circuit resistors.

Figure 77. Operating principle of a mediator-modified am-perometric biosensor, involving an oxidase or a dehydrogenase (in the case of a reductase, electron transfer would proceed from the electrode to the analyte substrate) a) Amperometric enzyme sensor b) Mediator-modified electrode c) Redox electrode...

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