Thickness of the Enzyme Layer

The time required to reach steady-state potential reading is dependent on the enzyme layer thickness because of the diffusion parameter for the substrate to reach the active sites of the enzyme and of the electroactive species to diffuse through the membrane to the sensor. A mathematical model relating the thickness of the membrane, d, the diffusion coefficient, D, the Michaelis constant, K, and the maximum velocity of the enzyme reaction, has been developed  

Membranes as thin as possible are recommended for the best results, and this is helped by the use of highly active enzyme. 

There is a twofold effect on increasing the enzyme activity in the layer that is in close proximity to the electrode surface. First, a complete conversion of the substrate into products will be ensured secondly, the response time of the electrode will be affected. The increase in the amount of enzyme affects the thickness of the membrane. This results in an increase in the time required for the substrate to diffuse through the membrane. Hence, for the best results, it is recommended that an enzyme of as high an activity as possible is used to ensure rapid kinetics by achieving the thinnest possible membrane. 

---

Once the theoretical curves have been fitted (Figs. 2.10 and 2.11), it is possible to plot the concentration profiles of all the species included in the model and to determine the optimum thickness of the enzyme layer (Fig. 2.12). Because the Thiele modulus is the controlling parameter in the diffusion-reaction equation, it is obvious from (2.22) that the optimum thickness will depend on the other constants and functions included in the Thiele modulus. For this reason, the optimum thickness will vary from one enzyme and one kinetic scheme to another. 

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... 

The electrodeposition process allowed large numbers of sensors to be coated simultaneously due to the electrical nature. Yet 3ie enzyme was precisely deposited only on the surface of the conductor exposed to the solution and file flow of current. Finally, the thickness of the enzyme layer was controlled by varying the electrical deposition parameters. 

Almost all the FDH molecules on the electrode surface seemed to retain the enzyme activity because of the mild immobilization at less extreme potential. The enzyme activity of immobilized FDH was dependent on the thickness of polypyrrole membrane because a thicker membrane could prevent the enzyme substrate from diffusing into the membrane matrix. Therefore, it was very important to make the polypyrrole membrane as thin as possible to minimize the effect on substrate diffusion and to ensure the complete coverage of the enzyme layer. 

A pulsed system, called Time-Clock System, has been developed. It comprises a solid dosage form coated with a hydrophobie surfactant layer to which a water-soluble polymer is attached to improve adhesion to the core [66]. The thickness of the outer layer determines the time required to disperse in an aqueous environment. Following the dispersion of the outer layer, the eore becomes available for dispersion. An advantage is that eommon pharmaceutical excipients can be used to manufacture this system. Studies performed on human volunteers showed that the lag time was not affeeted by gastrie residence time. Furthermore, the dispersion of the hydrophobic film was not influenced by the presence of intestinal digestive enzymes or by the mechanieal aetion of the stomach. 

If external diffusion dominates the overall rate, the process obviously reduces the observed enzyme activity. The flux N through the stagnating film at the surface can be expressed as in Eq. (5.54), where 8 signifies the thickness of the stagnating layer and ks is the mass transfer coefficient of the respective solute ks can be estimated by the simple relationship of Eq. (5.55). 

The thickness of the mucus layer changes in function of age. In rats a subsequent increase of microclimate pH from 6 to 6.5 is observed with increasing age [91 The influence of gastrointestinal mucin on enzyme absorption is not well documented, but it is an intestinal diffusion barrier for many nutrients [10],... 

As noted earlier in this chapter, the apparent Km values of immobilized enzymes vary with the thickness of the diffusion layer surrounding the particles. In packed-bed enzyme reactors, the thickness of this layer varies with the mobile phase flow rate. Faster flow rates produce smaller diffusion layers and therefore K m values that more closely approximate the true Km of the enzyme. This effect has also been observed with the ficin-CM-cellulose reactor, and plots of K m against flow rate Q obtained at different mobile phase flow rates are shown in Figure 4.14. 

Changes in enzyme bulk concentration may induce a large reduction in reaction rate within the membrane reactor. Figures 7.7 and 7.8 give evidence of the existence of enzyme deactivation phenomena which can be misleading in the evaluation of reactor performances, and/or of the thickness of the boundary layer close to the membrane surface in accordance with Equation 12. 

In the kinetic scheme of a biocatalyst electrode given in Fig. 12, the concentration polarization of the mediator occurs only within a finite thickness of the immobilized layer. Since the solution is usually stirred, the concentration polarization of the substrate will be neglected at the outside of the membrane and then the substrate polarization is restricted within the immobilized layer and the membrane. As a result, the catalytic current reaches a steady state after a certain period of time in any case. The enzymic reaction in the immobilized layer with excess amounts of Mox is expressed by... 

Conventional enzyme electrodes employ disorete-maorosoopio membranes to overcome problems associated with interferences, enzyme immobilization, and electrode fouling. While these types of enzyme electrodes have been commercially developed, there are some limitations with this approach. Some sensors use three relatively thick membranes, resulting in a slow smd complex diffusion path for reactants reaching the enzyme and hydrogen peroxide reaching the electrode. Slow diffusion in this type of system adversely affects the response and recovery time, decreasing sampling rate. Each sensor must be individually constructed, and this construction technique is limited to two-dimensional surfaces. In addition, for sensors that have complex and slow diffusion paths, rates of diffusion must remain constant, otherwise calibration of the biosensor, and more important the maintenance of calibration, are difficult. A variety of factors can influence rates of diffusion, and consequently the performance of the enzyme layer and the performance of the sensor. These complicated, and most often uncharacterizable, properties have made the development of roost biosensors difficult. 

The effect of the wetting characteristics of the membrane in the hydrolytic activity of immobilized lipase was evaluated by Bouwer and co-workers [113]. These authors suggested that the larger thickness of the reaction layer in hydrophilic membranes, as compared to hydrophobic membranes, allowed the retention of the full enzyme activity. A lipase adsorbed onto polypropylene membranes in a hollow-fiber system was used for the selective hydrolysis of menhaden oil [112]. The experimental set-up led to the release of about 88% of the fatty acid residues and retention of more than 90% of the aimed eicosapen-taenoic and docosahexaenoic acids in a space-time of 3.5 h, while the half-life of the enzyme, under ideal operational conditions was 170 h. The enzymatic synthesis of an aspartame precursor in an organic-aqueous membrane-assisted two-liquid phase system has been continuously studied by Isono and co-workers [121, 149,156,201]. Productivity was increased from 6.6 to 8.4kgm d [121]. 

---