Mediated amperometric detection of glucose-6-phosphate dehydrogenase at a poly(vinyl chloride) covered electrode using 1,4-benzoquinone and diaphorase

Abstract

Reduced nicotinamide adenine dinucleotide (NADH), produced from glucose-6-phosphate dehydrogenase (G6PDH) was catalytically oxidized using diaphorase and 1,4-benzoquinone to yield 1,4-hydroquinone. This benzoquinone redox mediator was readily detected at an electrode polarized at +0.5 V (vs. Ag/AgCI) so avoiding the high overpotentials required for a direct electrochemical measurement of NADH. Also, the lipophilic nature of the hydroquinone enabled a novel high selectivity plasticized PVC membrane mounted over the electrode to be exploited. The mediator/PVC combination achieved NADH calibration linear up to at least 1 0 0~~. When used to assay G6PDH activity, a linear calibration from 0.1-1.0 U/mL was produced. The detection system offers the possibility of highly selective measurement of dehydrogenase enzyme activity in biological samples without sample preparation and could provide the basis for simplified homogeneous immunoassays as well as dehydrogenase amperometric enzyme electrodes.

Analytical aspects of biosensors

“…45 Also, existing enzyme multiplied immunoassay technique (EMIT TM )based assays (Syva UK, Maidenhead, UK) have been adapted. 46,47 The NADH generated from the glucose-6-phosphate dehydrogenase reaction can be readily oxidized by mediators such as 2,6dichloroindophenol 48 and 1,4-benzoquinone, 49 the oxidation of which is followed amperometrically at potentials around 150 mV versus Ag/AgCl (see Fig. 6).…”

Specific and Rapid Glucose Detection Using NAD‐dependent Glucose Dehydrogenase, Diaphorase, and Osmium Complex

“…On the contrary, NAD‐GDH shows much better glucose specificity than other GDHs. Although NAD‐GDH allows both oxygen insensitivity and high glucose specificity, direct electrochemical oxidation of NADH (the enzymatic reaction product of NAD‐GDH) requires a high applied potential (∼0.75 V vs Ag/AgCl) . Fast mediated oxidation of NADH using a redox enzyme and an electron mediator is required to lower the applied potential and to perform rapid glucose detection .…”

“…Although NAD‐GDH allows both oxygen insensitivity and high glucose specificity, direct electrochemical oxidation of NADH (the enzymatic reaction product of NAD‐GDH) requires a high applied potential (∼0.75 V vs Ag/AgCl) . Fast mediated oxidation of NADH using a redox enzyme and an electron mediator is required to lower the applied potential and to perform rapid glucose detection . Diaphorase (DI) from Bacillus stearothermophilus (EC 1.6.99.‐) is a promising redox enzyme for this purpose because of (i) its high reaction rate with NADH, (ii) oxygen insensitivity, and (iii) remarkable thermal stability (up to 70 °C) .…”

Development of a Redox Mediated Amperometric Detection System for Immunoassay. Application to Urinary Amphetamine Screening

“…The use of a lower overpotential reduces side reactions, direct NADH oxidation and the adsorption of NADH reaction products at the electrode surface, which inevitably decreases electrode sensitivity [8]. Currently utilized redox mediators such as 1,2-naphthoquinone, benzoquinone, and dichlorophenol indophenol (DCPIP) demonstrate either poor solubility and stability in the aqueous media required for compatibility with the enzyme and clinical sample, or are oxidized at relatively high potentials ( > 450 mV vs. Ag=AgCl) [9]. Some of these problems have been overcome using amperometric dehydrogenase biosensors by adsorption or covalent attachment of the mediator to the electrode surface, or incorporation of the mediator into the electrode material [10].…”

“…Although¯ow-injection techniques have been required for the mediated amperometric detection of NADH [8], we have demonstrated a direct detection method for NADH produced by G6PDH activity, using 1,4-benzoquinone, which is reduced by NADH in the presence of diaphorase [9]. This was carried out at 450 mV (vs. Ag=AgC1).…”