Enzyme films

Enhanced thermal stability enlarges the areas of application of protein films. In particular it might be possible to improve the yield of reactors in biotechnological processes based on enzymatic catalysis, by increasing the temperature of the reaction and using enzymes deposited by the LB technique. Nevertheless, a major technical difficulty is that enzyme films must be deposited on suitable supports, such as small spheres, in order to increase the number of enzyme molecules involved in the process, thus providing a better performance of the reactor. An increased surface-to-volume ratio in the case of spheres will increase the number of enzyme molecules in a fixed reactor volume. Moreover, since the major part of known enzymatic reactions is carried out in liquid phase, protein molecules must be attached chemically to the sphere surface in order to prevent their detachment during operation. 

The entrapment method is based on confining the enzyme within the lattice of a polymeric matrix. Polyacrylamide gels have successfully yielded stable enzyme films with a high retention of activity... 

Besides catalyzing styrene and benzaldehyde, CYP enzymes play an important role in the metabolism of endogenous compounds as well as in pharmacokinetics and toxicokinetics. Joseph [228] developed a biosensor with human CYP3A4 as a novel drugscreening tool. It was constructed by assembling enzyme films on Au electrodes by alternate adsorption of a layer of CYP3A4 on top of a layer of PDDA. The biosensor was applied to detect verapamil, midazolam, quinidine, and progesterone. 

TABLE 6.9. Fluxes Equations in an Enzyme Film Containing n 1 Inactive and N — n Active Layers... 

A consequence of the fact that the diffusion layer is much thicker than the enzyme film is that the fluxes in the solution are negligible compared to the fluxes in the film. The two time-dependent integral equations relating the fluxes and the concentrations at the film-solution interface may be thus be... 

The concentrations of Q and P are normalized to the values they would have if the film were exposed to a concentration of Q or P equal to the bulk concentration of cosubstrate, Cp, taking into account the two partition coefficients, Kp and kq. The kinetic parameter A measures the competition within the enzyme film between diffusion represented by the term 8qD/1 and the rate term The current is normalized toward the parameters of the diffusional transport of the cosubstrate in the solution in the solution. The set of equations listed in Table 6.10 ensues. 

One of the promising potentials of SECM for biosensor research is the possibility to investigate immobilized enzymes independent of the communication to the electrode onto which they are immobilized. In fact, not too seldom, the immobilization of proteins onto electrode surfaces inhibits fast electron transfer reactions. SECM can be used to probe the enzymatic activity from the solution side of an immobilized enzyme film with a UME that is free of any cover layer. When designing an SECM experiment for the investigation of immobilized enzymes, one should consider the following guidelines. 

In this paper, we have evaluated three different protocols for the preparation of AQ-enzyme film using choline and glucose oxidases and mixture of AQ 29D and AQ 55D (1 1). This AQ mixture is recommended by Eastman Kodak to increase the adherency of the film to a surface such as a platinum electrode (17). The values 29 and 55 represent the glass transition temperature of each polymer. Also, the main structural difference between the two polymers is that, in the case of AQ 55D, an aliphatic glycol moiety replaces the cycloaliphatic glycol moiety found in the AQ29 (17,18). 

Film Fabrication. The platinum electrode (0.28 cm area) was fabricated and cleaned as previously described (19). Thin films of AQ-enzyme were prepared by dissolving an amount of the enzyme, as indicated below, in 10 il of 1.5% AQ polymers solution at room temperature. Two aliquots of 5 il were deposited atop the platinum electrode and the first aliquot was allowed to dry before the second addition. This procedure corresponds to the first protocol. In addition, for the second protocol, 10 il of the 0.5% Nafion solution was casted atop the dried AQ-enzyme film and the methanol was allowed to evaporate at room temperature. The third protocol consisted in the deposition of 10 il of a 1% of AQ solution containing the enzyme, atop the platinum electrode followed by heating in an oven at 50°C during 30 min. In each case, 2 U of glucose oxidase were used. 

The activity of immobilized alcohol dehydrogenase was probed and modified by changing the local pH [133], The tip was positioned close to the enzyme film and used to increase the local pH by reducing water and producing hydroxide ions. A significant increase in enzyme activity was observed at higher pH. The opposite effect, that is, local inactivation of the immobilized enzyme (diaphorase) by chlorine or bromine species electrogenerated at the tip, also has been reported [134]. 

Fig. 1 Conceptual representation of DNA/enzyme films used for toxicity sensing. (View this art in color at www. dekker.com.)...

Schenkman, J.B. Rusling, J.F. Toxicity screening 28. by electrochemical detection of DNA damage by metabolites generated in-situ in ultrathin DNA-enzyme films. J. Am. Chem. Soc. 2003,125, 1431 —... 

Lowe and co-workers investigated covalent immobilization of enzyme in polypyrrole film [123,124]. First, pyrrole-modified enzyme was prepared by reacting glucose oxidase with either W-(3-aminopropyl)pyrrole or N- 2-carboxyethyl)pyrrole, then electropolymerized enzyme films, which covalently immobilize enzyme were deposited at platinum disk electrodes from solutions of free pyrrole and native or pyrrole modified enzyme of equivalent activity. They observed that sensors constructed of covalently electropolymerized GOx films demonstrated higher enzyme activity than those using entrapped native... 

Two of the electrochemical techniques used in protein film voltammetry are shown in Fig. 4-3. In cyclic voltammetry the electrode potential is swept in a linear manner back and forth between two limits. The rate at which the potential is scanned defines the time scale of the experiment and this can be varied from < 1 mV s to > 1000 V s . This is a very large dynamic range, and it is possible to carry out both steady-state and transient experiments on the same sample of enzyme. " Cyclic voltammetry is important because it provides the big picture and produces a signal that links the reaction or active site of interest to a particular potential. In chronoamperometry, the current is monitored at a constant potential following a perturbation such as a step to this potential or addition of a substrate. This experiment is important because it separates the potential and time dependencies of a response. In both types of experiment, it is usually important to be able to rotate the electrode in order to control transport of the substrate and product to and from the enzyme film. 

Figure 4-3. Electrochemical techniques and the redox-linked chemistries of an enzyme film on an electrode. Cyclic voltammetry provides an intuitive map of enzyme activities. A. The non-turnover signal at low scan rates (solid lines) provides thermodynamic information, while raising the scan rate leads to a peak separation (broken lines) the analysis of which gives the rate of interfacial electron exchange and additional information on how this is coupled to chemical reactions. B. Catalysis leads to a continual flow of electrons that amphfles the response and correlates activity with driving force under steady-state conditions here the catalytic current reports on the reduction of an enzyme substrate (sohd hne). Chronoamperometry ahows deconvolution of the potenhal and hme domains here an oxidoreductase is reversibly inactivated by apphcation of the most positive potential, an example is NiFe]-hydrogenase, and inhibition by agent X is shown to be essentially instantaneous.

Eremenko A., Kurochkin I., Chernov S., Barmin A., Yaroslavov A., Mosk-vitina T. Monomolecular enzyme films stabilized by amphiphilic polyelectrolytes for biosensor devices. Thin Solid Films 1995 260 212-216. 

The LbL technique is undoubtedly one of the best methods to incorporate biological components into man-made devices. Therefore, sensor applications must be one of the most promising subjects for LbL assemblies of biomaterials. For example, Leblanc and coworkers used several bilayers of chitosan and poly(thiophene-3-acetic acid) as cushion layers for stable enzyme films [187]. The first five bilayers of the cushion layer allowed for better adsorption of organophosphorus hydrolase than the corresponding adsorption on a quartz slide. The immobilized enzyme becomes more stable and can be used under harsher conditions. The assembled LbL films can be used for spectroscopic detection of paraoxon, an organophosphorus compound. This cushion layer strategy provides a well-defined substrate-independent interface for enzyme immobilization, in which the bioactivity of the enzyme is not compromised. This leads to fast detection of paraoxon and quick recovery times. 

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