Enzyme Linked Electrochemical Techniques

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

A wide variety of enzymes have been used in conjunction with electrochemical techniques. The only requirement is that an electroactive product is formed during the reaction, either from the substrate or as a cofactor (i.e. NADH). In most cases, the electroactive products detected have been oxygen, hydrogen peroxide, NADH, or ferri/ferrocyanide. Some workers have used the dye intermediates used in classical colorimetric methods because these dyes are typically also electroactive. Although an electroactive product must be formed, it does not necessarily have to arise directly from the enzyme reaction of interest. Several cases of coupling enzyme reactions to produce an electroactive product have been described. The ability to use several coupled enzyme reactions extends the possible use of electrochemical techniques to essentially any enzyme system. 

---

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique... 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

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.

Since the early 1970 s there has been much interest shown in the development of non-isotopic immunoassays. The main reasons for this stem from the perceived dangers of using radio-labelled substances (as required in radioimmunoassay), and the search for more selective, sensitive and precise methods of analysis. Much work has therefore been devoted to the development of fluorescent and enzyme-linked immunoassays, but it is only in recent years that there has been strong interest in the application of electrochemical techniques in this regard. ... 

Figure 14.2. Some detection principies used in the double-surface DNA hybridization techniques. (A) Label-free detection of target DNA (tDNA). [B] Labeling of tDNA. Redox labels are covalently attached to the tDNA strand outside the segment or on a secondary DNA stiund recognized by the capture probe. After hybridization and separation, the electroactive tags are determined electrochemically [e.g., hy ex situ adsorptive stripping voltammetry [a]. Alternatively, electrochemical enzyme-linked immunoassay can be used for detection of labeled tDNA at the MB surface (b).

Multiple enzyme systems, where one enzyme produces an electrochemically inactive product that is consumed as a substrate by another enzyme to form an active product, have been successfully used to extend enzyme selectivity. The selectivity of immunochemical systems has been employed by implementation of enzyme-linked assays. Direct coupling of redox relay centers of enzymes to conductive electrodes has been achieved by a technique known as molecular wiring and avoids the indirect analysis of products of enzyme-substrate reactions. This fast and sensitive technique measures current flow and is commercially available. 

Electroactive labels introduced into DNA also possess electrochemical signals at less extreme potentials than intrinsic DNA responses. An example is electroactive osmium tetraoxide with 2,2 -bipyridine bound to free 3 -ends of the ss regions created by a DNA repair enzyme exonuclease III, which responds to the extent of DNA damage [25]. The technique is capable of detection of one lesion per 10 nucleotides in supercoiled plasmid DNA. DNA-hybridization biosensors were proposed for studies of DNA damage by common toxicants and pollutants where voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated target DNA [26]. 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

---