Chem. Educator 1999, 4, 137–140 137 © 1999 Springer-Verlag New York, Inc.,S1430-4171(99)04317-5, 10.1007/s00897990317a, 440137sa.pdf A Practical Approach to Potentiometric Biosensors Based on Consolidated Composites: Construction and Evaluation of a D- Amygdalin Biosensor Arben Merkoçi, Esteve Fàbregas and Salvador Alegret * Grup de Sensors & Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain, guest1@gsb.uab.es Abstract: A simple procedure to be used in an analytical chemistry laboratory by undergraduate students to prepare a potentiometric biosensor for D-amygdalin is given. The membrane material is prepared by simple compression of a solid sensing mixture (β-glucosidase mixed with Ag 2 S and AgI). This new technology has some advantages. It presents a simple way to prepare a biosensor membrane and this methodology adapts well to mass production technology. Simple polishing before each new measurement can renew the membrane. This type of biosensor produced by consolidated biocomposites can serve as a base material for different biosensing schemes. Using this technique, students can easily envision the functioning of a potentiometric biosensor where the classic detection mechanism as of an I-ISE is combined with the biological recognition of an enzyme. Different kinds of materials are used to prepare biosensor membranes. Enzymes may be immobilised within an insoluble gel matrix (physical entrapment), to a support matrix via covalent bonds, or by a multifunctional reagent (chemical entrapment) or they can be adsorbed directly onto the surface [1]. Electrochemical composite materials, however, represent an attractive alternative for fabrication of biosensors [2–7]. They are formed by the combination of two or more different phases. As presented in Figure 1, composite electrodes can be classified according to how the phases are distributed within the composite material. For example, a conducting composite electrode surface can be prepared as an ordered array or as a random arrangement (ensemble) of conducting regions separated by an insulator [5]. The random composite mixtures are classified according to the distribution of the conductor within the composite matrix. If the conductor particles are distributed randomly within the composite matrix the composite is said to be of the dispersed type. If the conductor extends throughout the composite in a random fashion with regions of pure insulator and pure conductor that do not intermix, the composite is said to be of the consolidated type. According to this classification we will introduce a practical approach to the design of a D-amygdalin biosensor based on the consolidation of a silver salt mixture (Ag 2 S, AgI) and the enzyme β-glucosidase. The first step consists of the preparation of the electroactive salt mixture of Ag 2 S and AgI [8]. The second step is the mixing of this salt mixture with the β-glucosidase enzyme and the thorough homogenisation of these dry components using an agate mortar. The third step is the consolidation of all the components at high pressure using a pellet die and a hydraulic press. The fourth step is the biosensor construction. This type of biosensor offers several advantages. Because they have a single active surface the reaction occurs directly in the liquid phase at the interface in contact with the biocomposite membrane; therefore, it is faster than other types of biosensor using active membranes attached directly to the transducer. Furthermore, this kind of biocomposite membrane can be polished, renewing the enzyme active surface leading to improved reproducibility. To evaluate the biosensor, D-amygdalin substrate is used as the analyte. Amygdalin is a potentially toxic cyanogenic glycoside. The name amygdalin (d-mandelonitrile-β-d- glucosido-6-β-d-glucoside) is currently used interchangeably with laetrile. It occurs in seeds; mainly in bitter almonds, but also in peaches and apricots [9, 10]. These seeds have a high protein content and can be used as a food or feed ingredient; however, they contain approximately 50 nmol of the potentially toxic cyanogenic glycosides amygdalin and prunasin per mg [11]. Cyanoglycosides yield glucose, benzaldehyde, and hydrocyanic acid when hydrolysed in vitro by mineral acids or in vivo by enzymes (see reaction 1). An application of this biosensor in diluted and undiluted samples is possible considering the concentration of amygdalin in these seeds. The most important application would be in food technology wastewater control. The process effluents in food technology that involve amygdalin materials are regarded as highly toxic and cyanide must be destroyed prior to its disposal to aquatic environments. An amygdalin biosensor can substitute for the gas chromatographic detection of amygdalin [12], which is a more costly technique. Consider a biosensor using the previously mentioned membrane in contact with an aqueous solution of D- amygdalin. The hydrolysis of the substrate is catalysed by the enzyme leading to the liberation of cyanide at the membrane– solution interface as presented in the following equation: -glucosidase 2 D-amygdalin + H O benzaldehyde + 2 glucose + HCN β → (1) The presence of cyanide generates the following concurrent reaction at the membrane-solution interface: