The fabrication of a potentiometric penicillin biosensor for the detection of ß-lactam antibiotics in pharmaceutical preparations and milk
2017-01-16T01:38:31Z (GMT) by
A sensitive penicillin potentiometric biosensor was developed for the detection of ß-lactam antibiotics in pharmaceutical formulations and in milk. This study outlines the approaches undertaken for the immobilisation of penicillinase (P'nase) in a number of polymer matrices. The first approach involved the immobilisation of P'nase in polypyrrole (PPy) by galvanostatic polymerisation of pyrrole (Py). The optimum conditions established for the formation of these films were 0.03 M Py, 50 U/mL P'nase, an applied current density of 0.9 mA/cm2, polymerisation time of 40 s and 0.01 M penicillin (Pen) in the monomer solution. The incorporation of Pen in the monomer solution is important for the attainment of a steady state response. The biosensor was applied to raw milk samples spiked with penicillin G, as well as 3 different antibiotics, namely; abocillin 125 mg, flucoxacillin 500 mg and amoxycillin 875 mg. The use of a PPy bilayer configuration was also investigated as a means of improving the sensitivity of the single layer biosensor. This was achieved through the immobilisation of P'nase in both the inner and outer layers which enhanced the response of the biosensor. The optimum conditions for the formation of the bilayer were: (a) outer layer: 0.1 M Py, 19 U/mL P'nase, 0.01 M Pen, current density of 0.9 mA/cm2 and polymerisation time of 40 s; (b) inner layer: 0.1 M Py, 0.1 M KNO" current density of 0.9 mA/cm2, polymerisation time of 40 sand 19 lJImL P'nase. The presence of the enzyme in the film was verified with the use of scanning electron microscopy (SEM) and x-ray photo electron spectroscopy (XPS). The minimum detectable penicillin concentration with the bilayer potentiometric biosensor was 0.3 /lM and the linear concentration range was 7.5 -146 /lM. The VII bilayer biosensor was applied to the determination of penicillin in amoxycillin 500 mg and the average percentage recovery was 113 ± 24% indicating that the reproducibility was not very good, but similar observations were made for other bilayer configurations. Also the application of the biosensor to the determination of penicillin in milk was fraught with problems of non-specific binding of penicillin to the milk proteins resulting in poor reproducibility and low percentage recoveries. The self-limiting growth of the non-conducting polymer, polytyramine (PTy), was exploited for the fabrication of a sensitive biosensor. The optimum conditions for the formation of the; PTy-P'nase film were 0.03 M tyramine (Ty), 37 U/mL P'nase, 0.01 M KN03, 3 mM Pen, current density of 0.8 mA/cm2 and an electropolymerisation time of 40 s. The linear concentration range obtained with this biosensor was 3 -283 /lM and the minimum detectable concentration was 0.3 /lM. The average percentage recovery for amoxycillin of 102 ± 6%, was in close agreement with the average percentage recovery of 105 ± 5% obtained with the standard titrimetric method. Satisfactory percentage recoveries were also achieved for the detection of penicillin G in milk, particularly for concentrations :s 5 ppm penicillin G. Other immobilisation methodologies, such as the cross-linking of penicillinase with glutaraldehyde (GLA) and bovine serum albumin (BSA), were also examined in this study. The optimum conditions established for the formation of the BSA-GLA-P'nase were 0.006% w/v BSA, 0.012% v/v GLA and 8 U/mL of P'nase. The linear concentration range obtained with the BSA-GLA-P'nase biosensor was 3 -283 /lM which is the same as that obtained with the PTy-P'nase electrode. The minimum detectable concentration obtained with the 13SA-GLA-P'nase biosensor was 0.3 /lM. VIII Relatively good percentage recoveries were obtained for the detection of penicillin in milk. The average percentage recovery of penicillin in amoxycillin of 103 ± 5% was also in close agreement with the results obtained with the standard titrimetric method. P'nase was also cross-linked with poly (vinyl alcohol) (PVA) and BSA. The optimum conditions for the of BSA-PVA-P'nase film were: 2.5% w/v PVA, 0.006% w/v BSA, 2.4 mM Pen and 16 U/mL P'nase. The minimum detectable concentration was 1.7 )lM. The linear concentration range obtained for the BSA-PV A film was 7.5 -283 )lM. The BSA-PV A-P'nase biosensor successfully detected penicillin in amoxycillin with an average percentage recovery of 97 ± 12 %. Higher penicillin concentrations (10 ¬20 ppm) were detected more successfully than lower penicillin concentrations (≤ 5 ppm). A comparison hetween the use of PTy/BSA-GLA and the PPy/BSA-PV A electrodes was undertaken. The minimum detectable concentration achieved with both electrodes was 3.3 )lM. The linear concentration range obtained with the PPyIBSA-PV A and the PTy/BSA-GLA electrodes were 7.5 -89 )lM and 7.5 -283 )lM, respectively. The percentage recoveries ohtained with these two electrodes was similar for the detennination of penicillin in amoxycillin. The average percentage recovery obtained with the PPy/BSA-PV A electrode was 102 ± 15%, while that achieved with PTy/BSA-GLA was 100 ± 19%. These results indicated that the use of bilayer films with both conducting and non-conducting polymers, can be adequately used for the fabrication of penicillin potentiometric biosensors, but further improvement in the reproducibility is required when applied to real samples.