P 2.69: NEW IMMUNOSENSOR FOR PENICILLIN DETERMINATION IN PHARMACEUTICAL FORMULATIONS AND RIVER WATERS

Several kinds of immunosensors for clinical and environmental purposes were developed in recent years in view of the possibility of generating a large number of antibodies for the analysis of numerous chemical species. Originally, if a low molecular weight chemical substance produced by microorganisms showed growth-inhibitory effects on other microorganism in high dilution it was defined as an antibiotic. However in the present context any chemical substance either of microbial, synthetic, or semi–synthetic origin, used in the chemical treatment of infections, is called an antibiotic. The discovery of penicillin by Alexander Fleming in 1928 and the subsequent isolation of the compound by other researchers opened up vast horizons in the field of chemotherapy. Penicillin was used on a large scale in the years following its discovery up until recent times. However, within four years after its introduction, its widespread use on humans and animals induced bacterial resistance infections. It thus became increasing necessary to monitor on a large scale the presence of this and other antibiotics in wastewater, river water and even in surface water tables. Of course, penicillin determination is an important stage also in the control of biotechnological production processes as well as in the quality control of penicillin pharmaceutical preparations. A number of analytical methods for penicillin control were developed especially involving chromatographic techniques. Nevertheless the need for rapid and inexpensive tests has also encouraged the use of biosensor methods. In previous years, for instance, also several of the authors of the present communication developed some useful ISEs for analysing β-lactamic antibiotics [1], although their selectivity was not particularly high. In more recent years therefore more specific enzymatic biosensors have been developed for the analysis of penicillin and other β-lactamic antibiotics using penicillinase as enzyme and as transducer a sensor for pH measurement of different types: potentiometric (glass [2-4], metal-oxide [5], membrane electrodes [6], ISFET [7]) or optical (fluorescence[8] and absorbance [9]) optodes. The difficulty encountered in developing this type of enzymatic sensor is that found in all those biosensors in which a pH variation produced by an enzymatic reaction is to be measured even when having to operate practically in a pH buffered solution. The availability of antibodies for β-lactamic antibiotics on the market is thus now encouraging the development of immunosensors that do not have this drawback and which also have good selectivity and offer the advantage of being able to analyse real samples without any need for pre-treatment. The present communication describes the development of a new immunosensor for penicillin. In actual fact two different competitive procedures were used for penicillin determination, in which the antigen (Penicillin) or the antibody (anti-Penicillin), respectively, were conjugated with horseradish peroxidase enzyme, using a biotinylation process. After optimizing the ‘competitive’ measurement procedure, the penicillin immunosensor was used to determine penicillin in several types of pharmaceutical formulations sold in chemist’s shops but also found in river water. Lastly, selectivity vis-à-vis other antibiotics and the affinity constant values were determined and discussed.