FLOW-THROUGH AND FLOW INJECTION ANALYSIS USING ELECTROPOLYMERIZED ENZYME ELECTRODES FOR FOOD AND ENVIRONMENTAL QUALITY CONTROL
Contract no: HPMF – CT – 2000 -000725
New nonconducting polymers based on dihydroxynpahthalenes (DHN), 2-(4-aminophenyle)-ethylenamine (AP-EA) have been synthesised on the surface of Pt electrodes to assemble fast-response and interference free amperometric sensors for hydrogen peroxide.
For nitrite determination studies for obtaining of selective sensors based on 1,5-DAN and 1,8-DAN were carried out. The electropolymerization was performed by cyclic voltammetry. The electropolymerization conditions were optimised according to the monomer. Studies on reproducibility, interferences, response time, buffers, storage and operational time of the sensors have been carried out as well in a batch system, as in a flow injection system. The permselectivity of the polymers towards the target compound (hydrogen peroxide or nitrite) and life time were the main characteristics used for the selection of the best films.
For enzyme electrodes assembling a polymeric film based on the copolymerisation of 2,6-DHN with AP-EA was used because of its high permselectivity for hydrogen peroxide and high stability during time.
Different strategies were studied and optimised for oxidases immobilization based on covalent attachment with different reagents, cross-linking, entrapment during electropolymerisation, etc. Glucose oxidase (GOD) was used as a model enzyme. A new technique for enzyme immobilisation was developed based on the simultaneous enzyme entrapment and covalent attachment via glutaraldehyde during the film synthesis.
The versatility of the new copolymer and of the immobilization method was demonstrated by assembling sensors based on L-lactate oxidase (L-LOD), alcohol oxidase (AOD), L-aminoacid oxidase (L-AAOD), tyrosinase and xanthine oxidase (XOD). The assembled enzyme electrodes were characterised by a very good reproducibility, high stability during storage and in operational conditions.
Structure and morphology of the polymeric films were established using XPS and SEM techniques.
Each type of sensors prepared was integrated in a flow injection system designed in function of the conditions required for the determination of the target compound. A flow system based on solenoidal valves and controlled by a special software was developed.
The analytical characteristics (concentration range, detection limit, sensitivity, reproducibility, throughput, life time) for the developed flow methods were evaluated.
A fast, simple, robust and inter-changeable flow system based on electropolymerized sensors was developed for determination of nitrite, glucose, lactate, ethanol and aminoacids.
The nitrite sensor based on poly(1,8-DAN) was applied for the analysis of drinking and mineral waters. Also, nitrite determination in cured meat was performed. Glucose and ethanol were analyzed in fruit juices, wines and other commercially available products using the GOD/Copolymer/Pt and AOD/copolymer/Pt sensors integrated in the flow injection system. A good agreement between the new methods and AOAC methods was obtained.
The most important achievement of the studies performed is the developing of some new sensors based on non-conducting films characterized by a high selectivity, good sensitivity and an exceptional stability during storage and in operational conditions.