Desing and Development of a Receptor Assay Based on the Inhibition of Carboxypeptidase Activity Coupled with and Electrochemical D-Alanine sensor for the Detection of β-Lactamic Antibiotics.

ALARCÓN, S. H.; RIVAS, G.; FEO MANGA, J.; COMPAGNONE, D.; PEPE, A.

Roma

Simposio; XIII AISEM: Associazione Italiana Sensori e Microsistemi; 2008

AISEM

The B-lactam group is one of the most important family of antibiotic used in veterinary medicine in the treatment of septicaemia, urinary and pulmonary infections. The presence of penicillin residues in food of animal origin (milk, meat) can have several drawbacks: unfavourable microbiological effects in the dairy industry, possible hypersensitivity reaction to the consumer and antibiotic resistance. Maximum residue limits (MRL) were set at 4 ?g.kg-1 for penicillin G, penethamate (penicillins G), amoxicillin and ampicillin (penicillins A) and at 30 ?g.kg-1 for cloxacillin, oxacillin, dicloxacillin and nafcillin in milk (penicillins M).The MRLs are defined for the intact ?-lactam and not for their metabolites or degradation products. According to that, the antibody used for immunochemical methods (e.g. enzyme immunoassay) should be specific for the intact ?-lactam ring. Until recently it was difficult to obtain antibodies against intact ?-lactam antibiotic. In the other hand the use of penicillin binding proteins (PBPs) makes this detection possible because they interact with intact ?-lactam  exclusively and have high affinities for both, penicillins and cephalosporins. The use of a receptor protein, instead of antibodies, allows to obtain a generic assay, specific for the active form of the ?-lactam structure.Enzyme immunoassay methods are currently available for the detection of specific compounds in food of animal origin. These methods are generally more specific than microbial inhibition tests and give results more rapidly. The use of liquid chromatography is better for confirmation because of its specificity and possible quantification, but it is time-consuming and more expensive. Biosensors could be an alternative to commonly used immunoassays because the immunisation procedure led to the open–ring forms of the ?-lactam and consequently to antibodies against hydrolysed penicillins.The purpose of the present study was to investigate the applicability of a biosensor in combination with a ?-lactam receptor protein for detection of ?-lactam antibiotics.Two assays based on the same receptor protein, a DD-carboxypeptidase from Streptomyces R39 (PBP R39) were developed using a glassy carbon paste and screen printed electrodes with electrochemical revelation. The assay was based on the enzymatic activity of the receptor protein. 1- The DD-carboxypeptidase activity of PBP R39 results in the hydrolysis of a 3-peptide (Ac-L-Lys-D-Ala-D-Ala) into a 2-peptide (Ac-L-Lys-D-Ala) and D-Ala, reaction that is inhibited in the presence of b-lactam. 2- The liberated D-Ala is oxidised by the action of a D-Amino acid oxidase (immobilized on the sensor), which in turn produces hydrogen peroxide measured by amperometric detection at – 100 mV    1-      PBP R39 + L-Lys-D-Ala-D-Ala ? L-Lys-D-Ala + D-Ala    2-            D-Ala + D-AAOx ? Piruvic Acid + H2O2In the first time we optimized two electrochemical sensors devices for the determination of D-Alanine constructed by inmobilization of D-amino acid oxidase on composite prepared by dispersion of glassy carbon and copper microparticles (GCC) and on the screen-printed electrode with Prussian blue and nafion layers (SPEs). The performance analytical obtained were: for GCC (pH = 8.4, T = 45 C), Linear range, 50 – 3000 ?M, LOD 20 ?M and ?i = 1000 nA and for SPEs (pH = 8.4, T = 25 C), Linear range, 10 – 600 ?M, LOD 5 ?M and ?i = 200 nA.Therefore, we describes the development of an alternative route for the rapid quantification of B-lactam residue using the  glassy carbon composite modified whit cooper microparticles.The analytical characteristic were: Linear range is the 2.5 – 80 ng.mL-1, I50 = 23 ng.ml-1 and ?i = 200 nA. The LOD of the assay was determined to be 1 ng.mL-1 based on the standard deviation of the blanc inhibition assay: (% ILOD =  3 SD).