Enzyme biosensors substrate concentration

Electrodes based on enzyme activity. These are selective and sensitive devices that may be used to measure substrate concentrations. A biosensor based on glucose oxidase is used to measure the concentration of glucose by detecting the production of H202. 

Amperometric biosensors based on flavin-containing enzymes have been studied for nearly 30 years. These sensors typically undergo several chemical or electrochemical steps which produce a measurable current that is related to the substrate concentration. In the initial step, the substrate converts the oxidized flavin adenine dinucleotide (FAD) center of the enzyme into its reduced form (FADH2). Because these redox centers are essentially electrically insulated within the enzyme molecule, direct electron transfer to the surface of a conventional electrode does not occur to a substantial degree. The classical" methods (1-4) of indirectly measuring the amount of reduced enzyme, and hence the amount of substrate present, rely on the natural enzymatic reaction ... 

The electrochemical activation of enzyme electrodes results in the electrobiocata-lyzed oxidation or reduction of a substrate specific to the biocatalyst. The rate of the biotransformation is dependent on the substrate concentration, hence these assemblies provide a basis for the construction of analytical biosensors [160]. The... 

The kinetic behaviour of electrochemical biosensors is most commonly characterized using the dependence of the steady-state amperometric current on the substrate concentration. This type of analysis has some limitations because it does not allow for a decoupling of the enzyme-mediator and enzyme-substrate reaction rates. The additional information required to complete the kinetic analysis can be extracted either from the potential dependence of the steady-state catalytic current or from the shift of the halfwave potential with substrate concentration [154]. Saveant and co-workers [155] have presented the theoretical analysis of an electrocatalytic system... 

Figure 16. Cyclic voltammograms of the affinity biosensors as a function of reacted avidin concentration (A) 0, (B) 1 ng/mL, (C) 10 ng/mL, (D) 100 ng/mL, (E) 1 pg/mL, and (F) 10 pg/mL. Cyclic voltammograms were obtained in the presence of 30 pg/mL of GOx as a signal generator and 10 mM glucose as a substrate (G) background voltammogram in the absence of enzyme and substrate. All curves were registered in deoxygenated 0.1 M phosphate buffer (pH 7.2) solution under argon atmosphere. Potential scan rate was 5 mV/s. (Adapted from Ref. [172]).

The above approach for measurement of urea using an enzyme-based potentiometric biosensor assumes that the turnover of urea to ammonium at steady state provides a constant ratio of ammonium ions to urea, independent of concentration. This is rarely the case, especially at higher substrate concentrations, resuitmg in a nonlinear sensor response. The hnearity of the sensor is also limited by the fact tiiat hydrolysis of urea produces a local alkaline pH in the vicinity of the ammonium-sensing membrane, partially converting NH to NH3 (pKa = 9.3). Ammonia (NH3) is not sensed by the ISE. The degree of nonlinearity may be reduced by placement of a semipermeable membrane between enzyme and sample to restrict diffusion of urea to the immobilized enzyme layer. 

The kinetics of enzyme-catalyzed reactions can be very complex, and the mathematical representations for the effect of the concentrations of substrate, product, cofactors, and inhibitors are presented in a variety of textbooks in this field [1]. The exact form of this dependence of enzyme activity on these factors might have a profound effect on the behavior of an enzyme biosensor. However, one can delineate general rules of thumb concerning the properties of enzymes for the preliminary design of enzyme-based sensors. 

In these circumstances of membrane permeability limitations, shown as curves 1 and 2 in figure 7.11 at external substrate concentrations below 0.01 M, the response of the sensor will be completely independent of the enzyme prop>erties. A sensor operated in this regime would be independent of factors affecting enzyme behavior, such as denaturation, temperature, and pH. This is the preferred regime for operating an enzyme biosensor. 

Biosensors have recently become an area of great Interest, especially in clinical chemistry. These sensors can be used to determine analyte concentrations in clinical samples, such as blood serum and urine. Electrochemical biosensors are constructed by incorporating a biochemical system, such as an enzyme, with an electrode. Amperometrlc biosensors often use oxidase enzymes, such as glucose oxidase, as the sensing enzyme. A major product of these types of enzymatic reactions is hydrogen peroxide, which can be oxidized at eun electrode surface. The current produced by the oxidation of hydrogen peroxide is directly proportional to the enzymatic substrate concentration. 

Biosensors require highly active enzymes/biomolecules therefore, the immobilisation methods must be chosen in such a way that they can achieve a high sensitivity and functional stability. This is important for economic reasons also. The measurable activity gives an idea about the biocatalytic efficiency of an immobilised enzyme. The rate of substrate conversion should rise linearly with enzyme concentration. The measured reaction rates depend not only on the substrate concentration and the kinetic constants (Michaelis Menten constant) and (maximum velocity of the reaction) but also on the immobilisation effects. The following effects have been observed [157] due to the immobilisation process ... 

From the analysis of the coupling of enzyme reaction and mass transfer, the following conclusions may be drawn for the design of biosensors. The substrate concentration at which deviations from the analytically usable linear measuring range occur depends on the extent of diffusion limitation. Under kinetic control, a linear dependence may only be expected for very low substrate concentrations. Under diffusion control, the decrease of substrate concentration in the enzyme layer caused by slow substrate diffusion results in an extended linear range. It has to be considered, however, that for two-substrate reactions deviations from linearity may also be produced by cosubstrate limitation. 

The external diffusion limitation (EDL) indicates that substrate transport through diffusion (stagnant) layer is a rate-limiting process [10]. At internal diffusion limitation (IDL), the substrate diffusion through the external diffusion layer is fast, and process is limited by the diffusion inside an enzyme membrane. The disadvantage of these approximate solutions is an error at the boundaries between the different approximate treatments. It is helpful to illustrate this approach by reference to a trivial problem of the substrate conversion in the biocatalytical membrane of the biosensor and at the concentration of the substrate less than the Michaelis-Menten constant Km)- The calculated profile of substrate concentration at steady-state or stationary conditions is shown in Fig. 1. 

Biosensors based on membrane electrodes are attractive from several perspectives. First, complex organic molecules can be determined with the convenience, speed, and ease that characterize ion-selective measurements of inorganic species. Second, enzyme-catalyzed reactions occur under mild conditions of temperature and pH and at relatively low substrate concentrations. Third, combining the selectivity of the enzymatic reaction and the electrode response provides results that are free from most interferences. 

The response of an enzyme sensor in the steady state depends largely on the ratio of the substrate concentration [5] to the enzyme Michaelis constant K. When [S K is large, the reaction rate reaches a maximal value V,, which is proportional to the number of active sites of the immobilized enzyme. The reaction rate is independent of the substrate concentration, and the product concentration at the contact with the electrode is the same for all high substrate concentration. The quantify of enzyme in the layer determines the linear zone in the response to the substrate concentration. This zone corresponds to first-order kinetics with respect to substrate concentration, whereas the region with a plateau has zeroth-order kinetic. When the substrate concentration is very high([5] K ), the biosensor is no longer capable of determining the substrate but may determine inhibitors which affect the minimal rate of the enzymatic reaction... 

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