Efficient glucose detection in anaerobic solutions using an enzyme-modified electrode designed to detect H2O2: implications for biomedical applications

Abstract

The finding that a 'first generation' glucose oxidase modified poly( o-phenylenediamine) coated Pt electrode, designed t o detect H202' responded t o glucose in N2-saturated solutions with a sensitivity similar to that of air-saturated media is of considerable significance for the application of biosensors in biological systems where O2 availability is severely restricted.Ortho-Phenylenediamine (PD , 1,2-diaminobenzene) forms a self-sealing, highly insulating thin (ca. 10 nm) membrane containing trapped enzyme molecules when electropolymerised onto a Pt anode in enzyme-electrolyte solution.*,* When glucose oxidase (GOx) is used, the GOx-modified polyphenylenediamine (PPD) coated Pt (Pt/PPD/GOx) electrodes are considered 'first generation' sensors as they detect glucose by oxidising H202 formed in the presence of the natural co-substrate for GOx, dioxygen [reactions.(1)-(3)].G O X F A D H~ + 0 2 -+ GOx/FAD + HzO?A number of different laboratories have demonstrated that Pt/PPD/GOx electrodes possess a variety of properties indicating potential suitability for monitoring glucose levels in biomedical applications. 1-10 These properties include fast response time, linearity over the relevant range of concentration, effective elimination of interference by reducing agents such as ascorbic acid, freedom from protein and lipid fouling, stability in vivo, and ease of miniaturisation. However, since the mechanism of electrochemical signal generation involves oxidation of H202 [reaction (3)] formed from the reaction of O2 with reduced enzyme [reaction (2)], changes in ambient oxygen tension may mimic changes in glucose concentration, undermining the reliability of the sensor to monitor glucose unambiguously. We therefore investigated the sensitivity of Pt/PPD/GOx electrodes to glucose for different concentrations of O2 in solution.GOx was immobilised in poly(o-phenylenediamine) films by potentiostatic electropolymerisation of the monomer on the bare disk end of a freshly cut Teflon-insulated Pt wire (125-250 pm diameter) as described in detail recently.8Briefly, a deoxygenated solution of the monomer (300 mmol dm-3) and GOx ( 5 mg cm-3) was prepared in phosphate buffered saline (PBS, pH 7.4). The working electrode potential was maintained at +0.65 V vs. SCE during the electropolymerisation for 15 min using a large Pt wire as auxiliary electrode. All experiments using these Pt/PPD/GOx electrodes were performed in a standard three-electrode glass electrochemical cell containing 20 ml PBS thermostated at 25 5 1 "C. To attain effective anaerobic conditions, all solutions were vigorously purged with 02-free N2 (average O2 content 2 ppm, maximum O2 content 5 ppm) for at least 30 min before recording began and a N2 atmosphere maintained over the cell thereafter. In experiments involving solution O,, either atmospheric air or pure O2 from a gas cylinder was bubbled through the PBS. The mean k standard error is reported with n = number of electrodes or number of electrodes times determinations. Background current recorded in PBS wi...

An amperometric glucose-oxidase/poly(o-phenylenediamine) biosensor for monitoring brain extracellular glucose: in vivo characterisation in the striatum of freely-moving rats

“…These results support our previous in vitro O 2 interference studies (Lowry et al, 1994b) and confirm that Pt/PPD/GOx sensors are not affected by changes in O 2 over physiologically relevant concentration ranges for the brain. A similar lack of O 2 interference has also been observed by other groups; Asai et al (1996) have used glutamate oxidase and a dialysis electrode incorporating a PPD coated Pt wire to monitor brain extracellular glutamate during acute ischemia and found that the sensor operates reliably despite the lack of O 2 around the dialysis electrode and Garguilo and Michael (1994) have found that the availability of O 2 in brain ECF is sufficient for from the reaction of O 2 , the natural cosubstrate for GOx, with the reduced form of the enzyme (GOx/ FADH 2 ) which is generated by reaction with glucose (see reactions 1 and 2 above).…”

“…An increase in brain O 2 levels accompanying an insulin induced fall in brain glucose has also been reported by Silver and Ereciñ ska (1994). The different time courses of changes in glucose and oxygen observed with insulin suggest that changing O 2 levels in vivo do not influence the Pt/PPD/GOx signal supporting our previously published in vitro work on O 2 interference (Lowry et al, 1994b). A more detailed investigation of this type of interference is presented below (see Section 3.4).…”

“…A similar lack of O 2 interference has also been observed by other groups; Asai et al (1996) have used glutamate oxidase and a dialysis electrode incorporating a PPD coated Pt wire to monitor brain extracellular glutamate during acute ischemia and found that the sensor operates reliably despite the lack of O 2 around the dialysis electrode and Garguilo and Michael (1994) have found that the availability of O 2 in brain ECF is sufficient for from the reaction of O 2 , the natural cosubstrate for GOx, with the reduced form of the enzyme (GOx/ FADH 2 ) which is generated by reaction with glucose (see reactions 1 and 2 above). We have previously demonstrated in vitro that these biosensors respond to glucose in O 2 -depleted (N 2 -saturated) solutions with a sensitivity similar to that of air-saturated media (200 vM O 2 ) (Bourdillon et al, 1982;Zhang and Wilson, 1993), suggesting that in brain tissue where the range of O 2 tension is 5-50 vM (Feng et al, 1988;Zimmerman and Wightman, 1991), glucose monitoring should be free of O 2 interference (Lowry et al, 1994b).…”

Characterization in vitro and in vivo of the oxygen dependence of an enzyme/polymer biosensor for monitoring brain glucose

“…These attributes include fast response time, linearity over the relevant concentration range, effective elimination of interference by endogenous reducing agents such as ascorbic acid, freedom from protein and lipid fouling, stability in vivo, and ease of miniaturisation (O'Neill and Lowry, 2000). In a preliminary publication, we demonstrated that Pt/ PPD/GOx sensors might also be free of O 2 interference in applications both in vitro and in vivo by showing that they responded to glucose with similar sensitivity over a wide range of pO 2 values in vitro (Lowry et al, 1994b). As this phenomenon has important implications both for the development of biosensors and for their operation in biological systems, where O 2 availability is restricted, we examine it now in more detail.…”

Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor

“…An important stage in the development of this biosensor was the in vitro and in vivo characterisation of the sensor, which demonstrated that it has a fast response time, is linear over the relevant concentration range, is free of protein and lipid fouling, and has minimal interference from endogenous species (Lowry and O'Neill, 1994;Lowry et a!., 1994b;.…”