Talanta 66 (2005) 1281–1286 Automated determination of glucose in soluble coffee using Prussian Blue–glucose oxidase–Nafion ® modified electrode Ivanildo Luiz de Mattos ∗ , Madalena Carneiro da Cunha Areias Departamento de Qu´ ımica Fundamental - DQF, Universidade Federal de Pernambuco, Cidade Universit´ aria, 50740-540 Recife, PE, Brazil Received 8 July 2004; received in revised form 20 January 2005; accepted 24 January 2005 Available online 24 February 2005 Abstract A highly selective, fast and stable biosensor for determination of glucose in soluble coffee has been developed. The biosensor electrode consist of a thin film of ferric hexacyanoferrate (Prussian Blue or PB) electrodeposited on the glassy carbon electrode (GCE) (to provide a catalytic surface for the detection of hydrogen peroxide) glucose oxidase immobilized on top of the electrode and a Nafion ® polymer layer. The stability of the PB film and the biosensor was evaluated by injecting standard-solution (50 MH 2 O 2 and 0.5 mM glucose) during 4 h in a flow-injection system with the electrodes polarized at -50 mV versus Ag/AgCl. The system is able to handle about 60 samples per hour and is very stable and suitable for industrial control. Determination of glucose in the range 2.5 and 15% (w/v) in phosphate buffer with precision (r.s.d. < 1.5%) has been achieved and is in agreement with the conventional procedures. Linear calibration in the range of 0.15 and 2.50 mM with detection limits of ca. 0.03 mM has been obtained. The morphology of the enzyme glucose oxidase on the modified electrode has been analyzed by scanning electron microscopy (SEM) measurements. © 2005 Elsevier B.V. All rights reserved. Keywords: Modified electrode; Prussian Blue; Biosensor; Soluble coffee; Glucose 1. Introduction The glucose content is a very important parameter in food industry for evaluating the quality of commercial instant cof- fee [1]. It indicates the degree of maturity [2] and can assess the commercial value of the product [2,3]. Among glucose determinations, are widely used several chromatographic procedures (anion-exchange chromatography [4], capillary zone electrophoresis [5], paper chromatography [6] and liquid chromatography [3,6]) normally with electrochemical detection. Alternatively, the use of Fehling’s reagent [6] and enzyme electrode [7] can be used but when a fast deter- mination is needed, procedures involving flow systems are preferable [8]. For industrial control, the use of automated methods is desirable and the flow injection analysis (FIA) is worthwhile [9–11]. For that purpose, the combination of the FIA system with biosensors became attractive due to its ∗ Corresponding author. Tel.: +55 8121268440; fax: +55 8121268442. E-mail address: ilmattos@ufpe.br (I.L. de Mattos). versatility, simplicity and suitability for large-scale analyses [12–15]. The uses of biosensors are increasing in several appli- cations [12–21], and the reason for that is the fact that its presents high easy fabrication procedure, low cost and porta- bility [12–21]. One of the main factors contributed for biosen- sor development was the use of the electrochemical cata- lysts (mediators) such as ferrocene [14,22], ferrocene deriva- tives [14,22], meldola blue [14,22], methylene green [23], bienzyme systems [24,25] or transition metal hexacyano- ferrates [26–29]. This last one attracted widespread atten- tion due to its characteristics to form well-defined electroac- tive films on electrodes [28,29]. One example is the PB, Fe 4 [Fe(CN) 6 ]·nH 2 O – a classical mixed-valence compound which present the reduced form (Prussian White) with cat- alytic activity for the reduction of hydrogen peroxide even in the presence of molecular oxygen [27–31]. This mixed valence compound seems to be suitable mediator for the de- velopment of biosensors based on oxidases for analytical ap- plications [32–38]. 0039-9140/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2005.01.044