The synergetIc effect of redox mediators and peroxidase in a bienzymatic biosensor for glucose assays in FIA

Abstract

Amperometric biosensors based on enzymes are interesting due to their high sensitivity, excellent selectivity, simplicity, low cost and rapid response. The most frequently used enzymatic methods for glucose determination employ glucose oxidase (GOD), due to its high selectivity towards ß-D-glucose. In the presence of GOD, the oxidation of glucose occurs, producing H 2 O 2 . Most commonly, the electrochemical detection is performed through the anodic oxidation of H 2 O 2 [1]. A drawback with this detection technique is that high overvoltages are required, and an applied potential in the range of 0.6-1.1 V vs. Ag/AgCl is necessary. Working at such high potentials increases the risk of interference from easily oxidisable compounds such as ascorbate, ureate, acetaminophen, paracetamol, phenols, etc. which are common compounds in biological samples.A possible way of improving the selectivity of such a biosensor is to couple a second enzyme (peroxidase) to the oxidase, creating a bienzymatic sensor. Peroxidase catalyses the electrochemical reduction of H 2 O 2 produced by GOD through a direct electron transfer reaction at a much lower applied potential compared with electrochemical oxidation of H 2 O 2 and maximum catalytic current can be obtained at carbon electrodes in a potential range of -0.1 and 0 V [2-14]. However, the kinetics of the direct electron transfer from an electrode to the peroxidase is a sluggish process [15][16][17] hence the advantage of using a mediator.The introduction of a mediator in the bienzymatic system (GOD/HRP) leads to an acceleration of the electron transfer, a decrease in the applied working potential (the reduction of the mediator is detected at the electrode) and an increase in sensitivity. The use of mediators is a well-known technique although the way of adding the mediator to the bienzymatic system (oxidase/peroxidase) may vary. A number of different mediators have been used as additions into sample solutions such as, potassium hexacyanoferrate (II) [18][19][20][21][22][23][24] [44,45], and Nafion-Nmethyl phenazinium [46]. The advantages of using the redox polymers are several, but mainly they result in more stable biosensors since leaking of the mediator from the electrode is minimised and higher and faster responses are observed due to the proximity between the enzyme and the mediator. Although, choosing an appropriate bulk matrix and mediator concentration these properties can be achieved with nonpolymeric mediators and recently a ferrocene mediated bienzymatic graphite-Teflon electrode has been developed for batch and flow-injection measurements [47]. On the other hand several biosensors based on monoenzymatic electrodes 649The synergetIc effect of redox mediators and peroxidase in a bienzymatic biosensor for glucose assays in FIA Abstract. A bienzymatic biosensor incorporating a mediator has been developed in order to achieve a fast and selective detection of glucose in a flow injection system. The working electrode is based on a carbon paste matrix bulk modified with glucose ...

Carbon Paste Electrodes in Facts, Numbers, and Notes: A Review on the Occasion of the 50‐Years Jubilee of Carbon Paste in Electrochemistry and Electroanalysis

“…Similar procedures are being utilized in stripping analysis, where the beneficial effect of the preconcentration step at a CMCPE can be combined yet with highly sensitive detection in the presence of electrocatalysts [102]. Finally, even a CPbiosensor with two enzymes [180], can be quoted here as another example of multiple modification, attaining the desired sensitivity under actual conditions.…”

Electrocatalytic oxidation of ascorbate by heme-FeIII/heme-FeII redox couple of the HRP and its effect on the electrochemical behaviour of an l-lactate biosensor

“…It is obvious that the critical step in improving both sensitivity and selectivity of LOD/HRP-modified carbon paste electrode is the effective regeneration of the active form of HRP. The effect of a mediator on the analytical performances of the bienzymatic GOD/HRP electrode has already been studied, and it has been demonstrated that the introduction of ferrocene significantly increased the amperometric response [14]. In this study, the use of ferrocene in the LOD/HRP-modified CPE was also found beneficial in terms of sensitivity because the highest response with mediated electron transfer of HRP was obtained.…”

Characterization of an hrp-aox-polyaniline-graphite composite biosensor

“…Rondeau, et al [38] identified the optimal proportion of two enzymes in the biosensor composite by monitoring electrical response signals to establish idealized conditions for glucose oxidase : horseradish peroxidase immobilized with a modified carbon paste for in order to increase the selectivity, sensitivity, accuracy and stability [38]. The intensity of the electrochemical signal response was analyzed by Alpat and Telefoncu [39] who measured the amount of alcohol dehydrogenase immobilized on the electrode surface (47.1 to 200 U cm -2 ) and found that the linear response was between 0.01 mM and 0.04 mM for 117.6 U cm -2 .…”