Communication A redox hydrogel integrated PQQ–glucose dehydrogenase based glucose electrode M. Alkasrawi, a I. C. Popescu,† a V. Laurinavicius, b B. Mattiasson a and E. Cs¨ oregi* a a Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. E-mail: Elisabeth.Csoregi@biotek.lu.se; Fax: +46 46 2224713 b Institute of Biochemistry, Mokslininku str. 12, 2600-Vilnius, Lithuania Received 6th September 1999, Accepted 11th November 1999 The present work describes the design and characteristics of a glucose electrode based on a newly isolated and purified, oxygen-independent, pyrrole quinoline quinone glucose dehydrogenase (PQQ–GDH) integrated in a redox hydrogel (poly(1-vinylimidazole) complexed with Os(4-4A-dimethylbi- pyridine) 2 Cl) using poly(ethylene glycol)diglycidyl ether (PEGDGE) as a crosslinker. The influence of the buffer’s nature and the presence of PQQ and Ca 2+ ions on the enzyme stability were considered. The characteristics of the optimal electrode design were compared to those obtained for similarly prepared, glucose oxidase based electrodes. The optimal electrode configuration inserted in a single manifold flow injection system, was characterised by a linear range up to 100 mM, a sensitivity of 5.9 mA M 21 cm 22 , and a detection limit of 5 mM (calculated as three times the signal-to-noise ratio). Introduction Selective, sensitive and reliable glucose biosensors are required not only for biomedical applications 1–4 but also for biotechno- logical process control. 5–7 Hundreds of biosensor designs based on the commercially available enzymes glucose oxidase (GOx) and/or NAD-dependent glucose dehydrogenase (NAD–GDH) are reported yearly. 8–12 These biosensors display some basic drawbacks: the GOx-based ones are dependent on the oxygen content 13 and often use diffusional mediators such as ferrocene derivatives, 14 while the NAD–GDH based ones require the addition of the soluble cofactor, and often a mediator as well, 15 which complicate the biosensor construction and decrease their long-term stability. The above mentioned drawbacks have prompted research targeting the isolation and purification of new enzymes catalysing glucose oxidation, especially those having the bound, oxygen-independent PQQ cofactor (PQQ–GDH). There are only a few published works describing such biosensors. 16–18 These were either based on a PQQ–GDH, 16 which is not available any more, or showed low sensitivity also requiring the presence of soluble mediators such as ferrocene and its derivatives, 17 or phenazine derivatives. 18 Biosensor designs based on redox hydrogels were found to display high sensitivity and improved characteristic such as better stability, since the enzyme is effectively entrapped in a highly permeable crosslinked redox network containing a fast mediating redox couple. 16,19–24 Therefore, in this work, a newly isolated and purified PQQ–GDH was integrated into a redox hydrophilic polymer, [1-poly(vinylimidazole) complexed with Os(4-4A-dimethylbipyridine) 2 Cl (PVI 10 –dme-Os)], and the flow injection bioelectrochemical characteristics of the obtained glucose bioelectrode were evaluated and compared with those obtained for similarly constructed GOx-based ones. Experimental Reagents The PQQ-dependent glucose dehydrogenase (PQQ–GDH) from Erwinia sp. 34-1 (EC 1.1.1.1.47; 22 U mg 21 solid;‡ MW 88.4 kDa) was purified as previously described. 25 Glucose oxidase (GOx; EC 1.1.3.4; 240 U mg 21 solid) was purchased from Feinbiochemica (Heidelberg, Germany; Cat. no. 22738), and glucose from BDH Chemicals Ltd. (Poole, England; Cat. no. 10117I). Methoxatin (pyrrolo-quinoline quinone) (PQQ) was purchased from Fluka (Tyres ¨ o, Sweden; Cat. no. 64682), while phenazine methosulfate (Cat. no. 100955) (PMS) and N- 2-hydroxyethylpiperazine-n-2-ethanesulfonic acid (Cat. no. 101926) (HEPES) were from ICN Biomedicals Inc. (Aurora, OH, USA). Tris[hydroxymethyl]aminomethane enzyme grade (TRIS) was obtained from Amersham (Cleveland, OH, USA; Cat. no. 22674), poly(ethylene glycol)diglycidyl ether (PEGDGE) from Polysciences Inc. (Warrington, PA, USA; Cat. no. 08210), and polyethylenimine (PEI) from Sigma (St. Louis, MO, USA; Cat. no. 3143). Poly(vinylimidazole) complexed with Os(4,4A dimethylbipyridine) 2 Cl (PVI 10 –dme-Os) was syn- thesised according to a previously published procedure, 15 where the subscript 10 indicates the number of vinylimidazole monomer units complexed to an osmium centre. Acetate buffer, pH 5.56, (AB) and phosphate buffer, pH 7.0 (PB) solutions were prepared using CH 3 COONa, acetic acid, Na 2 HPO 4 ·2H 2 O and NaH 2 PO 4 ·H 2 O, all purchased from Merck (Darmstadt, Ger- many). All solutions were prepared using HPLC-grade water produced in a Milli-Q system (Millipore, Bedford, MA, USA), if not otherwise stated. Electrode preparation Rods of spectroscopic graphite (type RW001; 3.03 mm diameter; Ringsdorff Werke GmbH., Bonn, Germany) were polished on wet fine emery paper (Tufback, Durite P1200, Allar, Sterling Heights, MI, USA) rinsed with distilled water and dried at 25 ± 2 °C for 5 min. The different types of modified graphite electrodes were obtained placing a defined amount of a premixed solution onto the graphite surface. The coating solutions had the following composition: type I electrodes: 4.8 mL of PQQ–GDH (5 mg mL 21 in PB) with and without 1.2 mL of PEI (5 mg mL 21 ); type II electrodes: 4.8 mL of PQQ–GDH (5 mg mL 21 in AB), 1.45 mL of PVI 10 –dme-Os (5 mg mL 21 ) and 1.45 mL of PEGDGE (1 mg mL 21 ) freshly prepared and used within 15 min. The resulting hydrogel contained PQQ– GDH, PVI 10 –dme-Os and PEGDGE in 70/25/5% (w/w) ratio; type III electrodes: had the same composition as type II ones, excepting that PQQ–GDH was replaced by the same amount of GOx. All electrodes were coated with the same amount of enzyme. The modified electrodes were allowed to dry for 1 h at 25 ± 2 °C † On leave from University ‘Babes-Bolyai’, Department of Physical Chemistry, RO-3400 Cluj-Napoca, Romania. ‡ 1 U = 16.67 nkat. This journal is © The Royal Society of Chemistry 1999 Anal. Commun., 1999, 36, 395–398 395