The proposed biosensor was implemented in a FIA system that requires only minimum operator intervention. The FIA system was used for the determination of aspartame in commercial pharmaceutical formulations and food without any pretreatment other than sample solubilisation/dilution with a buffer solution.2.?Experimental Section2.1. Chemicals and MaterialsAlcohol oxidase AOX (from Hansenula sp. 7.7 UI/mg solid), carboxyl esterase CaE (from porcine liver, 17 UI/mg solid), aspartyl phenylalanine methyl ester (aspartame), bovine serum albumin BSA, glutaraldehyde GA (25% solution in water), potassium phosphate monobasic, sodium phosphate dibasic and potassium chloride were purchased from Sigma-Aldrich (St.Louis, MO, USA). Methanol was obtained from Merck (Darmstadt, Germany).

Standard solutions of methanol and aspartame were prepared daily in water.The amperometric measurements were made with a 0.1 M phosphate buffer solution (PBS) pH 7.3 supplemented with 0.05 M potassium chloride. All aqueous solutions were prepared with purified water (18 M��?cm?1; Millipore, Billerica, MA, USA). The soft drinks samples were purchased from a local supermarket. The pharmaceutical formulations were obtained from a local pharmacy.2.2. InstrumentsAll amperometric measurements were performed using a PGSTAT302N potentiostat/galvanostat (Metrohm-Autolab, Utrecht, The Netherlands) controlled by a PC with the software Entinostat Nova 1.8. The electromagnetic noise produced by magnetic stirring during batch measurements was reduced using the filter from the ECD module set to 1 s.

Cobalt-phthalocyanine (CoPC) screen-printed electrodes were kindly provided by BIOMEM-University of Perpignan, France [24]. An Ag/AgCl pseudoelectrode and a carbon auxiliary electrode were printed alongside with the working electrode. The monocanal FIA manifold was constructed with: a Gilson Minipuls 3 peristaltic pump (Gilson, Middleton, WI, USA), a methacrylate wall-jet flow cell (DropSens, Oviedo, Spain) for electrodes, a sample injection valve (Omnifit, Cambridge, UK) with a 50 ��L sample loop, connectors and PTFE tubing with 1 mm i.d.2.3. Biosensor PreparationThe methanol biosensors were prepared as follows: 7.7 IU of AOX was dissolved in 20 ��L PBS and mixed with 5 ��L of 0.6% BSA and 5 ��L of 1.5% glutaraldehyde. Four ��L of the resulting solution was carefully spread on the surface of the working electrode. The aspartame biosensors were prepared using a similar procedure based on a bienzymatic solution containing 7.7 IU AOX and 18.7 IU CaE in the 20 ��L PBS. The electrodes were dried at room temperature for 1 h and then were stored in sealed plastic bags at ?20 ��C.2.4. Amperometric MeasurementsAspartame is first cleaved by carboxyl esterase in methanol and a dipeptide: L-Asp-L-Phe.