Heterogeneous electrochemical enzyme

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. 

Samples from patients receiving digoxin therapy were analyzed by the heterogeneous immunoassay LCEC method. Samples were diluted 5/1 with pooled human plasma in order to eliminate an observed antibody matrix effect. Digoxin standards in pooled human plasma were treated similarly and a standard curve constructed. Digoxin levels in patient samples were determined by reference to the standard curve. The values obtained for the samples by the electrochemical enzyme immunoassay method were compared to those obtained by radioimmunoassay. The results for the 54 samples analyzed are presented in Fig. 8. A good correlation was obtained between the two methods (r = 0.93). 

M. Stoytcheva, Electrochemical evaluation of the kinetic parameters of a heterogeneous enzyme reaction in presence of metal ions. Electroanalysis 14, 923-927 (2002). 

K.R. Wenmeyer, H.B. Halsall, W.R. Heineman, C.P. Voile, and I.W. Chen, Competitive heterogeneous enzyme immunoassay for digoxin with electrochemical detection. Anal. Chem. 58, 135-139 (1986). 

Finally, self-assembled monolayers (SAMs) on gold electrodes constitute electrochemical interfaces of supramolecular structures that efficiently connect catalytic reactions, substrate and product diffusion and heterogeneous electron transfer step when enzymes are immobilised on them. Resulting enzyme-SAM electrodes have demonstrated to exhibit good performance and long-term enzyme stability. 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

The numerical values for ki. .. k4 vary with RG. For instance, for RG = 10, the following values provide the analytical function Jfei = 0.40472, k2 = 1.60185, k3 = 0.58819, and k4 = -2.37294 [12]. The analytical approximations for hindered diffusion provide a way to determine d from experimental approach curves. For this purpose, one can use an irreversible reaction at the UME (often 02 reduction). In such a case, Fig. 37.2, curve 1 is obtained irrespective of the nature of the sample. Besides the mediator flux from the solution bulk, there might be a heterogeneous reaction at the sample surface during which the UME-generated species O is recycled to the mediator R. The regeneration process of the mediator might be (i) an electrochemical reaction (if the sample is an electrode itself) [9], (ii) an oxidation of the sample surface (if the sample is an insulator or semiconductor) [14], or (iii) the consumption of O as an electron acceptor in a reaction catalyzed by enzymes or other catalysts immobilized at the sample surface [15]. All these processes will increase (t above the values in curve 1 of Fig. 37.2. How much iT increases, depends on the kinetics of the reaction at the sample. If the reaction of the sample occurs with a rate that is controlled by the diffusion of O towards the sample, Fig. 37.2, curve 2 is recorded. If the sample is an electrode itself, such a curve is experimentally obtained if the sample potential... 

In the present section, analogies and similarities will be noted between enzymes and heterogeneous catalysts in the concept of the active site and metal-protein/metal-support analogies the possession of size and shape selectivity the similarity or identity in kinetics between the two processes the use of electrochemical organization on a molecular or supramolecular level the possibility of... 

Clark and Lyons [1] have primarily suggested a model of electrochemical sensor, based on the concept of an enzyme heterogenized on the electrode surface. 

One form of heterogeneous immunoassay is called enzyme-linked immunosorbent immunoassay (ELISA). In one instance, electrochemical immunoassay was performed for anti-ferritin (antibody) in a PDMS/PMMA chip. First, DTSSP was self-assembled on the gold electrode deposited on the PMMA plate. Then horse spleen ferritin (antigen) was attached to the DTSSP layer. A 100-p.g/mL solution of anti-horse ferritin (rabbit serum) was added. Then a secondary anti-rabbit antibody (HRP-linked) was introduced. A substrate (4-CN) was finally added which was converted to a precipitate product. The precipitate caused a reduction... 

Heterogeneous immunoassay has also been conducted without the use of an enzyme label. For instance, electrochemical immunoassay of mouse IgG (antigen) was carried out in glass chip. The chip contained magnetic beads coated with the sheep anti-mouse antibody. After flowing in the secondary antibody (rat antimouse conjugated with PAPP), electrochemical oxidative detection of PAP was achieved (i.e., PAP was oxidized to p-quinoneimine) [1016]. 

The mechanism and theory of bioelectrocatalysis is still under development. Electron transfer and variation of potential in the electrodeenzyme-electrolyte system has therefore to be investigated. Whether the enzyme is soluble and the electron transfer process occurs through a mediator, or whether there is direct enzyme immobilization on the electrode surface, the homogeneous process in the enzyme active centre has to be described by the laws of enzyme catalysis, and the heterogeneous processes on the electrode surface by the laws of electrochemical kinetics. Besides this there are other aspects outside electrochemistry or... 

All electrodes react with their environment via the surfaces in ways which will determine their electrochemical performance. Properly selected surface modification can effectively enhance the electrode heterogeneous catalysis property, especially selectivity and activity. The bulk materials can be chosen to provide mechanical, chemical, electrical, and structural integrity. In this part, several surface modification methods will be introduced in terms of metal film deposition, metal ion implantation, electrochemical activation, organic surface coating, nanoparticle deposition, glucose oxidase (GOx) enzyme-modified electrode, and DNA-modified electrode. 

In summary, it can be stated that methods of electrochemical detection are very applicable to enzyme immunoassays. Broadly speaking, the above examples demonstrate that homogeneous EIA are faster and simpler but often less sensitive and more subject to interference than heterogeneous EIA. The latter are less sensitive to interference and electrode fouling because the measuring chamber in front of the electrode is rinsed before determination of the marker activity. However, none of the methods described is suitable for continuous measurement. 

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