Electrode multienzyme

Multienzyme Electrodes. Coupling the reactions of two or more immobilized enzymes increases the number of analytes that can be measured. An electro-inactive component can be converted by an enzyme to a substrate that is subsequentiy converted by a second enzyme to form a detectable end product (57). For example, a maltose [69-79-4] sensor uses the enzymes glucoamylase and glucose oxidase, which convert... 

Multienzyme electrodes can increase sensitivity from micromolar to nanomolar detection levels (53,57). In this case the substrate is converted to a detectable product by one enzyme, then that product is recycled into the initial substrate by another enzyme resulting in an amplification of the response signal. For example, using lactate oxidase and lactate dehydrogenase immobilized in poly(vinyl chloride), an amplification of 250 was obtained for the detection oflactate (61). 

Multienzyme tylon Electrodes. Di- and polysaccharides require more than one enzyme to realise the amperanetrically detectable hydrogen peroxide and even glucose really needs the back up of mutarotase with glucose oxidase. It is fortunate that all the necessary enzymes can be immobilized simultaneously on just one nylon net. Thus a viable starch electrode has been fabricated (6) from a nylon net immersed in a cocktail of glucose oxidase, mutarotase and amylogluoosidase (Figure 1). Its response to a continuous flow of 0.1% m/v starch remained steady for over a period of 60 h. 

Multienzyme systems have been used in carbon paste electrodes, providing bio-catalytic cascades that result in an analytical amperometric signal. For example, acetylcholine esterase (AChE) and choline oxidase (ChOx) have been co-immobilized in carbon pastes, either with monomeric TTF [145] or flexible ferrocene-containing polymers [146] as electron mediators. The primary reaction includes the hydrolysis of acetylcholine biocatalyzed by AChE, then the choline produced is oxidized by the electrically contacted ChOx giving an analytical amperometric signal corresponding to the acetylcholine concentration. 

The last type of redox recycling is substrate recycling rather than true redox recycling. Two enzymes are used, with the product of the first enzyme reaction serving as substrate for the second enzyme. In turn, the product of the second reaction acts as the substrate for the first enzyme. Both the degradation of a co-substrate and the production of a product like hydrogen peroxide can be quantified electrochemically. This concept can be applied to other electrochemical transducers, such as ion selective electrodes, because label conversion is not accomplished with the electrode. Amplification factors of 3-48,000 were reported by Scheller and colleagues [55-58] for ampero-metric multienzyme electrodes with the appropriate substrates. 

The inactivation of certain enzymes such as cholesterol oxidase (CO) on interaction with Au nanoparticles is overcome by immobilizing CO and cholesterol esterase (CE) in a carragenean hydrogel and coupling with Au particles loaded with HRP. This multienzyme device has been shown to be very useful in the determination of cholesterol in serum and whole blood [172]. The sensor operates at low potentials avoiding major interferences. The presence of the hydrogel enables the analysis of whole blood with no fouling of the electrode surface. 

The linear approach described here is expandable to multienzyme electrodes as well as multilayer electrodes. At least for the stationary case, multilayer models of bienzyme electrodes may be easily treated, too. The whole system is readily adaptable to potentiometric electrodes (Carr and Bowers, 1980). It must be noted, however, that the superiority over purely numerical solution procedures decreases with increasing number of enzyme species and in the multilayer model. The advantage in calculation speed using the sum formulas described (e.g., in Section 2.5.2) amounts to about two orders of magnitude. With multilayer electrodes and formulas containing double and triple sums it is reduced to one order of magnitude. 

The lactate oxidation catalyzed by LMO forms the basis of several other multienzyme electrodes (Fig. 86). The LDH-LMO sensor has also been used to assay the activity of alanine aminotransferase (ALAT, EC 2.6.1.2) and pyruvate kinase (PK, EC 2.7.1.40) (Weigelt 1987 Weigelt et al., 1988). The sample was added to the NADH-containing measuring solution and when the steady state signal for endogenous lactate and pyruvate was attained the substrates of the enzyme to be determined... 

Multienzyme Electrodes for Nucleic Acid Compounds, Phosphate and Fatty Acids... 

Since not all enzyme-catalyzed reactions involve changes in the level of compounds detectable at an electrode, such as oxygen, or hydrogen peroxide, only a limited number of substances can be determined with one-enzyme sensors. One way of overcoming this problem is to couple the catalytic activities of different enzymes either in sequence, in competing pathways, or in cycles. In general, enzymes to be used in multienzyme electrodes should fulfil the following requirements ... 

Enzyme-inhibition analysis based on mediatorless multienzyme electrodes was proposed making use of the cholinester-ase/choline oxidase/peroxidase system. 

In general, enzymes to be used in multienzyme electrodes should fulfill the following requirements ... 

Other researchers have followed related strategies as described above for detection of IL-6. For example, Wang et. al. [75] reported an amperometric immunosensor to detect interleukin-6 (lL-6) using a AuNP-Poly-dopamine sensor platform and multienzyme-antibody functionalized AuNPs on carbon nanotubes. They obtained a DL of 1 pg mL for lL-6 in buffer. Du et. al. [76] used AuNP-modified screen printed carbon electrode to detect p53 phosphorylated at Ser392 (phospho-p53 ) along with multi-enzyme labeled graphene oxide (GO). 

Mani, V., Chikkaveeraiah, B.V., Patel, V., Gutkind, J.S., Rusling, J.F. Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification. ACSNano 3, 585-594 (2009)... 

In some cases, multienzyme cascades are used where an artificial metabolic pathway has been created on an electrode to take a specific substrate and electrocataly tic ally convert it to produce energy [5,12-14]. With these systems, coulometry is useful for determining coulombic efficiency, as well as combining coulometry with nuclear magnetic resonance (NMR) analysis to determine reaction intermediates and products, and elucidate bottlenecks in the artificial metabolic pathway, or to determine the energy density of a fuel cell. Please refer to Chapter 5 for further information regarding multienzyme cascades. 

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