RESUMO
Stabilisation of electrochemically deposited Prussian blue (PB) films on glassy carbon (GC) electrodes has been investigated and an enhancement in the stability of the PB films is reported if the electrodes are treated with tetrabutylammonium toluene-4-sulfonate (TTS) in the electrochemical activation step following the electrodeposition. A multi-enzyme PB based biosensor for sucrose detection was made in order to demonstrate that PB films can be coupled with an oxidase system. A tri-enzyme system, comprising glucose oxidase, mutarotase and invertase, was crosslinked with glutaraldehyde and bovine albumin serum on the PB modified glassy carbon electrode. The deposited PB operated as an electrocatalyst for electrochemical reduction of hydrogen peroxide, the final product of the enzyme reaction sequence. The electrochemical response was studied using flow injection analysis for the determination of sucrose, glucose and H(2)O(2). The optimal concentrations of the immobilisation mixture was standardised as 8U of glucose oxidase, 8U of mutarotase, 16U of invertase, 0.5% glutaraldehyde (0.025mul) and 0.5% BSA (0.025mg) in a final volume of 5mul applied at the electrode surface (0.066cm(2)). The biosensor exhibited a linear response for sucrose (4-800muM), glucose (2-800muM) and H(2)O(2) (1-800muM) and the detection limit was 4.5, 1.5 and 0.5muM for sucrose, glucose and H(2)O(2), respectively. The sample throughput was ca. 60 samples h(-1). An increase in the operational and storage stability of the sucrose biosensor was also noted when the PB modified electrodes were conditioned in phosphate buffer containing 0.05M TTS during the preparation of the PB films.
