Substrate recycling

The concentrations of a number of analytically relevant compounds, such as creatinine, pyruvate, hormones, and drugs, are often so low that their assay with enzyme electrodes requires large sample volumes or is totally impossible. The detection limit of usual enzyme electrodes is 

Analyte Enzymes Form of application Transducer Amplification factor References 

ADP/ATP pyruvate kinase + hexokinase membrane with LDH+LMO O2 electrode 220 Wollenberger (1987b) 

Ethanol alcohol oxidase + alcohol dehydrogenase membrane O2 electrode Hopkins (1985) 

Benzoquinone/ hydroquinone cytochrome b2 + laccase membrane O2 electrode 500 Scheller et al. (1987b) 

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Amplification of the sensitivity of substrate or co-en me recycling is especially efficient in thermometric analysis since all the reactions involved frequently contribute to increasing the overall temperature change. One case in point is the determination of lactate or pyruvate by substrate recycling using co-immobilized lactate oxidase and lactate dehydrogenase [160]. 

Fig. 16.8. Schematic effect of substrate on microelectrode tip response in SECM (a) nonconducting substrate hindered diffusion and reduction in current (b) conducting substrate recycling (positive feedback) and current enhancement (c) conducting substrate recycling with current enhancement attenuated by slow electrode kinetics at substrate electrode.

Hu, Y. Y, He, S. S., Wang, X., Duan, Q. H Grundke-Iqbal, I., Iqbal, K., Wang,). (2002). Levels of nonphosphorylated and phosphorylated tau in cerebrospinal fluid of Alzheimer s disease patients an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay. Am J. Pathol. 160,1269-1278. 

Coche-Guerente, L., Labbe, P., Mengeaud, V. (2001). Amplification of amperometric biosensor responses by electrochemical substrate recycling. 3. Theoretical and experimental study of the phenol-polyphenol oxidase system immobilized in Laponite hydrogels and layer-by-layer self-assembled structures. Anal. Chem. 73 3206-18. 

Eppley et at. (1977) measured in situ rates of assimilation of NH4 , urea and N03 in the upper photic zone on several expeditions to the central North Pacific gyre near the Climax site using N tracer techniques. Their experiments were based on 24-h incubations and did not account for isotope dilution (substrate recycling during the incubation period). Assimilation of NOa in surface waters was negligible due to low ambient NOs concentrations, but potential uptake rates under conditions of NOs saturation were in the range of 1—8 nM d h... 

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. 

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

Figure 17.5 A schematic illustration of a substrate recycling biosensor S1/2 substrates C /2 cosubstrates E1/2 enzymes P product (reproduced with the permission of Elsevier Science Publishers BV).

The use of alkaline phosphatase as an enzyme label allows enhancement of the sensitivity by using phosphoenolpyruvate as substrate and the utilization of a separate detection column in the ET unit for the determination of the product (pyruvate) by substrate recycling. This is accomplished by using the substrate recycling system described above [18] comprising the coimmobilized enzymes lactate dehydrogenase (reduces pyruvate to lactate under the consumption of... 

Figure 20.4 A principle scheme for a TELISA with enzymatic substrate recycling detection. Column 1 contains anti-insulin antibody (Ab) bound to Sepharose. Column 2 contains the three enzymes lactate dehydrogenase (LDH), lactate oxidase (LOD), and catalase (CAT) coimmobilized on CPG. PEP is phosphoenolpyruvate.

Kiba N, Tomiyasu T and Furusawa M 1984 Flow enthalpimetric determination of glucose based on oxidation of 1,4-benzoquinone with use of immobilized glucose oxidase column Talanta 31 131-2 Scheller F, Siegbahn N, Danielsson B and Mosbach K 1985 High-sensitivity enzyme thermistor assay of L-lactate by substrate recycling Anal. Chem. 57 1740-3... 

Figure 22.5 An example of reactions involved in an enzyme-catalyzed recycling processes for amplification of the sensitivity. In the left part (A) of the figure the enzyme-pair hexokinase and pyruvate kinase is used for recycling of the coenzyme ATP/ADP. In the right part (B), substrate recycling of pymvate or lactate is accomplished using the enzyme-pair lactate oxidase/lactate dehydrogenase. A multiplication effect is obtained by combination of A and B resulting in a very high sensitivity [27], The calorimetric sensitivity is further inreased by including catalase (cat).

F. Scheller, N. Siegbahn, B. Danielsson, and K. Mosbach, High-Sensitivity Enzyme Thermistor Determination of L-Lactate by Substrate Recycling. Anal. Ghent., 57 (1985) 1740. 

Amplification and Filtering of Chemical Signals 777 Table 14-9. Substrate recycling in biosensors. 

Yield increase due to substrate recycling. Biotechnol Lett 7(12) 877-882 Kasche V (1986) Mechanism and yields in enzyme catalysed equihbrium and kineticaUy controUed synthesis of 3-lactam antibiotics, peptides and other condensation products. Enzyme Microb Technol 8 4-16... 

In qualitative kinetic studies, Hodge was also able to show that the linear soluble phosphinated polystyrene reagent was only slightly less reactive than a similar DVB cross-linked polystyrene reagent with the same alcohol substrate. Recycling of the soluble polystyrene reagent was not explicitly described. 

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