Journal of Electroanalytical Chemistry 418 (I 996) 147- I5 1 ELSEVIER A new amperometric biosensor for fructose using a carbon paste electrode modified with silica gel coated with Meldola’s Blue and fructose Sdehydrogenase Carlos A.B. Garcia, Graciliano de Oliveira Neto, Lauro T. Kubota * , Luciane A. Grandin Insrituto de Quimica, Unicwnp. CP 6154, 13083 Campinrrs, SP, Brazil Received 15 November 1995; revised 14 March 1996 Abstract A carbon paste modified with silica gel coated with Meldola’s Blue and fructose 5dehydrogenase was used to construct a new biosensor for fructose. An efficient electron transfer mediation from the reduced enzyme to the carbon paste electrode was verified, at a low applied potential of 20mV vs. SCE, where interfering reactions are minimized. A linear response range for 0.1 to 0.8mmoll-’ fructose was adjusted by the equation I = O.OlSO( + 0.006) + 0.618( + 0.011) [fructose], with a correlation coefficient of 0.999 and an rsd of 0.68% for n = 7. The biosensor was used to determine fructose in sweets and fruit jellies and the results present good recovery, higher than 96% in both cases. A stable response is observed over two months, when stored in a refrigerator, or for 300 measurements in continuous use. Krywordst Biosensors; Fructose; Meldola’s Blue mediator, Modified silica gel; Fructose 5-dchydrogenase 1. Introduction Nowadays, the necessity for determination of sugar in food and biological fluids for quality control and disease diagnostics, such as in diabetes, has received much atten- tion in clinical and industrial analysis [ 1,2]. Several meth- ods for glucose and fructose determination have been developed in recent years [3,4]. Analytical techniques such astitration [5], polarimetry [6], gas-liquid chromatography [7,8], spectrophotometry [9,10], enzymatic analysis [l 11, NMR spectrometry [12], fluorimetry [13] and electrochem- istry [14- 183 have been described in the literature for determination of sugars in food and blood. However, various studies are being carried out to obtain faster and more selective methods for monitoring sugar species in situ [19]. A great number of biosensors have been developed for clinical and food analysis [20,21]. However, few papers concerning a fructose sensor have been published, al- though this sugar is frequently usedas a dietetic sweetener. Presumably, this is related to the need to usethree kinds of enzyme (hexokynase, EC 2.7.1.1, phosphoglucose iso- * Corresponding author. merase, EC 5.1.3.9 and glucose-6-phosphate dehydroge- nase, EC 1.1.1.49) plus two co-enzymes(ATP and NAJIP), which increase the cost and complexity of the analysis [=I. Yamada et al. [23] discovered the enzyme o-fructose 5-dehydrogenase (FDH, EC 1 .I .99.11) some years ago, and confirmed that the enzyme catalyzes the oxidation of D-fIUCtOSe to 5-ceto-o-fructose. Recently, Prado and Sampietro [24] described the purification and properties of o-fructose 5-dehydrogenase from Gluconobacter lndus- n-ius. They verified the possibility of its use in the quanti- tative determination of D-frUCtOSe. The reaction catalyzed by the enzyme can be represented as D-fructose + acceptor(ox) FDH + 5-ceto-o-fructose + acceptor( red) Several acceptors can be used in this reaction in the same way as for glucose oxidase. Thus, acceptors such as fer- rocene [25], quinone [26], phenoxazine [27] can be usedto construct a biosensor. However, in the majority of the works, the mediators are not stable and are leached from the support. The surfaces of these electrodes need to be renewed after some measurements. So, the immobilization procedure is very important to obtain an efficient and 0022-0728/96/$15.00 Copyright 0 1996 Elsevier Science S.A. All tights reserved. PII SOO22-0728(96)04775-4