An Enzyme Electrode for Extended Linearity Citrate Measurements Based on Modi®ed Polymeric Membranes Andrew Maines, + Mamas I. Prodromidis, ++ Stella M. Tzouwara-Karayanni, ++ Miltiades I. Karayannis,* ++ David Ashworth, + and Pankaj Vadgama + + Universityof Manchester, Department of Clinical Biochemistry, Hope Hospital, Eccles Old Road, Salford, M6 8HD, UK ++ Department of Chemistry, University of Ioannina, G-45110 Ioannina, Greece; e-mail: mkaragia@cc.uoi.gr Received: November 10, 1999 Final version: February 28, 2000 Abstract Pyruvate oxidase (POD), oxaloacetate decarboxylase (AOCD), and citrate lyase (CL) were coimmobilized (or immobilized separately) in a series of polymeric membranes for the construction of amperometric bisensors of pyruvic, oxaloacetic, or citric acid in concentrated samples. For oxaloacetic acid, POD, and OACD were coimmobilized on dialysis membranes and were used in a multimembrane con®g- uration with an inner spin-coated cellulose acetate (CA) membrane modi®ed with isopropyl myristate (IPM) and an outer diffusion restricted membrane of cellulose acetate modi®ed with a cationic surfactant. These membranes were also tested in a POD laminate con- tainingthecofactorsFADandTPPformonitoringtheresponsestabilitywithdurationofexposuretoanexternalelectrolyteandfoundtobe effective in reagentless mode, with good pyruvate response stability over 3h without compromise of signal size. Coimmobilization of POD=OACDandCLondifferenttypesofhighproteinbindingmembranesuchasmixedcelluloseester(HA)wasinvestigated.Therelative optimum concentrations of several activators (divalent cations) and cofactors such as FAD and thiamine pyrophosphate (TPP) were investigated with the probe assembled as a pyruvate biosensor. An extended linearity up to 100mM citric acid was achieved. Keywords: Amperometric biosensors, Citrate, Modi®ed polymeric membranes, Extended linearity 1. Introduction Citric acid occurs as a three-fold negatively charged anion (citrate)atphysiologicalpH-valuesandisfoundinallanimaland vegetable cells in low amounts. High amounts of citric acid are found in several fruits such as citrus fruit, berries, and kiwi fruit [1]. Citric acid directly takes part in the production of energy through the tricarboxylic acid cycle, as well as in several processes associated with the metabolism of fatty acids, carbo- hydrates and certain amino acids. Furthermore, owing to its innocuousnatureitisoftenusedasanantibacterialsubstanceand as an additive for the control of pH [2]. Several methods have been proposed for the determination of citric acid, based on gas chromatography [3], liquid chromato- graphy [4], and ion chromatography [5]. These are time consuming procedures, and sample clean up is required in order to separate citric acid from other coexisting tricarboxylic acids. Other approaches based on conductimetric [6] or spectro- photometricmethods[7]oronchelatingpropertiesofcitratewith copper ions using an ISFET urease sensor [8], suffer from selectivity as they are based on nonspeci®c reactions with carboxylic acids. Theneedforsimplerseparationcouldbepotentiallysolved,by using a high speci®city enzyme such as citrate lyase [9]. Several approaches have been proposed [10±14], however, the majority of them using soluble citrate lyase as the enzyme is ready inac- tivated by complex formation with divalent cations and also the enol-form of oxaloacetate, itself the product of the action on citrate [15]. In cases where CL has been immobilized the proposed sensors needs recalibration every six runs and the enzyme reactivated after incubation wth adenosine 5-tripho- sphate (ATP) and CH 3 COOH [12] or a 50% loss of the initial activity after 12±15 sample injections has been reported [10]. Another problem associated with the above mentioned approa- chesistheinterferenceeffectofascorbicacid.Useofanascorbic oxidase reactor [10] or measurements using plain membranes are some of the proposed routes to the elimination of interferent species [12]. The proposed method is based on a sequence of the enzymes CL, OACD and POD, according to the following scheme: Citrate  ? CL Oxaloacetate  CH 3 COOH 1 Oxaloacetate  ? OACD Pyruvate  CO 2 2 Pyruvate  HPO 2 4  O 2  ? POD TPP; Mg 2 acetyl-phosphate  CO 2  H 2 O 2 3 The main development in this work is our use of cofactor retaining membranes that allow reagentless, interference free assay with a complex multicomposite of enzymes. The citrate electrodeusedisachallengingmodelsystem,thathasprovedthe value of a cofactor retention strategy suitable for a wide range of enzymes. The proposed method having these features provides a fast,easytouseandreliableanalyticaldevicesuitableforroutine analysis providing the analytical simpli®cation of biosensors. 2. Experimental 2.1. Apparatus All measurements were performed using an amperometric cell suppliedbyRankBros.(Bottisham,Cambs,UK),consistingofa platinum anode and an annular silver pseudo-reference, with an overlying Perspex sample well incorporated, enabling direct magneticstirringtobeemployedovertheelectrodes.Polarization of the working electrode was performed using an ``in house'' potentiostat (Dept. of Chemistry Workshop, University of 1118 Electroanalysis 2000, 12, No. 14 # WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2000 1040-0397/00/1410±1118 $17.50.50=0