Amperometric biosensors function by the production of a current when a potential is applied between two electrodes. They generally have response times, dynamic ranges and sensitivities similar to the potentiometric biosensors. The simplest amperometric biosensors in common usage involve the Clark oxygen electrode (Figure 1). This consists of a platinum cathode at which oxygen is reduced and a silver/silver chloride reference electrode. When a potential of -0.6 V, relative to the Ag/AgCl electrode is applied to the platinum cathode, a current proportional to the oxygen concentration is produced. Normally both electrodes are bathed in a solution of saturated potassium chloride and separated from the bulk solution by an oxygen-permeable plastic membrane (e.g. Teflon, polytetrafluoroethylene). The following reactions occur:
Ag Anode
4Ag0 + 4Cl- ------------> 4AgCl + 4e- [ 1]
Pt cathode
O2 + 4H+ + 4e- --------------> 2H2O [2]
The efficient reduction of oxygen at the surface of the cathode causes the oxygen concentration there to be effectively zero. The rate of this electrochemical reduction therefore depends on the rate of diffusion of the oxygen from the bulk solution, which is dependent on the concentration gradient and hence the bulk oxygen concentration. It is clear that a small, but significant, proportion of the oxygen present in the bulk is consumed by this process; the oxygen electrode measuring the rate of a process which is far from equilibrium, whereas ion-selective electrodes are used close to equilibrium conditions. This causes the oxygen electrode to be much more sensitive to changes in the temperature than potentiometric sensors. A typical application for this simple type of biosensor is the determination of glucose concentrations by the use of an immobilised glucose oxidase membrane. The reaction results in a reduction of the oxygen concentration as it diffuses through the biocatalytic membrane to the cathode, this being detected by a reduction in the current between the electrodes (fig 4). Other oxidases may be used in a similar manner for the analysis of their substrates (e.g. alcohol oxidase, D- and L-amino acid oxidases, cholesterol oxidase, galactose oxidase, and urate oxidase)
Figure 1. Schematic diagram of a simple amperometric biosensor. A potential is applied between the central platinum cathode and the annular silver anode. This generates a current (I) which is carried between the electrodes by means of a saturated solution of KCl. This electrode compartment is separated from the biocatalyst (here shown glucose oxidase, GOD) by a thin plastic membrane, permeable only to oxygen. The analyte solution is separated from the biocatalyst by another membrane, permeable to the substrate(s) and product(s). This biosensor is normally about 1 cm in diameter but has been scaled down to 0.25 mm diameter using a Pt wire cathode within a silver plated steel needle anode and utilising dip-coated membranes.
Pt anode
H2O2 --------------> O2 + 2H+ + 2e- [3]
Ag cathode
2AgCl + 2e- -----------------> 2Ag0 + 2Cl-[4]
The major problem with these biosensors is their dependence on the dissolved oxygen concentration. This may be overcome by the use of 'mediators' which transfer the electrons directly to the electrode bypassing the reduction of the oxygen co-substrate. In order to be generally applicable these mediators must possess a number of useful properties.
- They must react rapidly with the reduced form of the enzyme.
- They must be sufficiently soluble, in both the oxi dised and r educed forms, to be able to rapidly diffuse between the active site of the enzyme and the electrode surface. This solubility should, however, not be so great as to c au se signifi can t loss of the mediator from the biosensor's microenvironment to the bulk of the solution. However soluble, the mediator should generally be non-t oxic.
- The overpotential for the regeneration of the oxidised m ediator, at the electrode, should be low and independent of pH.
- The reduced form of the mediator s hould not readil y re act with oxygen.
The ferrocenes represent a commonly used family of m
ediators (Figure 3a). Their reactions may be represented as follows,
Electrodes have now been developed which can remove the electrons directly from the reduced enzymes, without the necessity for such mediators. They utilise a coating of electrically conducting organic salts, such as N-methylphenazinium cation (NMP+, Figure 3b) with tetracyanoquinodimethane radical anion (TCNQ.- Figure 3c). Many flavo-enzymes are strongly adsorbed by such organic conductors due to the formation of salt links, utilising the alternate positive and negative charges, within their hydrophobic environment. Such enzyme electrodes can be prepared by simply dipping the electrode into a solution of the enzyme and they may remain stable for several months. These electrodes can also be used for reactions involving NAD(P)+-dependent dehydrogenases as they also allow the electrochemical oxidation of the reduced forms of these coenzymes. The three types of amperometric biosensor utilising product, mediator or organic conductors represent the three generations in biosensor development (Figure 4). The reduction in oxidation potential, found when mediators are used, greatly reduces the problem of interference by extraneous material.
Substrate(2H) + FAD-oxidase ----------> Product + FADH2-oxidasefi [5]
This is followed by the processes:
(a)
biocatalyst
FADH2-oxidase + O2 ---------> FAD-oxidase + H2O2 [6]
electrode
H2O2 ----------> O2 + 2H+ + 2e- [7]
(b)
biocatalyst
FADH2-oxidase + 2 Ferricinium+ -----------> FAD-oxidase + 2 Ferrocene + 2H+ [8]
electrode
2 Ferrocene ----------------> 2 Ferricinium+ + 2e- [9]
(c)
biocatalyst/electrode
The current (i) produced by such amperometric biosensors is related to the rate of reaction (vA) by the expression:
i = nFAvA
where n represents the number of electrons transferred, A is the electrode area, and F is the Faraday. Usually the rate of reaction is made diffusionally controlled by use of external membranes. Under these circumstances the electric current produced is proportional to the analyte concentration and independent both of the enzyme and electrochemical kinetics.
1 comments:
The enzyme urate oxidase (UO) catalyzes the oxidation of uric acid to 5-hydroxyisourate:Uric acid + O2 + H2O → 5-hydroxyisourate + H2O2 → allantoin + CO2. urate oxidase
Post a Comment