Antibiyotik Tayinine Yönelik Biyosensör Geliştirilmesi
Özet
Antibiotics are one of the most used drug type in pharmaceutical. But the efficiency of these drugs comes to a standstill point because of the bacterial resistance fact that has become widespread day after day. Inefficient usage of antibacterial drug by human being, widespread usage of antibacterials in animals or existence of antibacterials in environment and food caused increment in antibiotic resistant bacterial strains. Even antibiotics can be given for the purpose of healing and preventing animals from sickness they can also be given for the enhancement of growth and nutritional productivity. Therefore, examining of antibiotic in animals or foodstuffs of animal origin is so important. In this scope, sensitive and fast detection systems are needed for the detection of residual antibiotic level. Simple, fast and sensitive detection of antibiotics is highly important because of the prevention of resistant bacterial strains that may cause health risks and also from the technological point of view prevention of problems at the fermentation step in the food processes. Especially, it is important to perform these analyses fastly at the farm or at the plant, before processing of food. Antibiotic detection methods that are often used in foods such as immunoassays, high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) are very sensitive as well as they are over costing and they need some improved or specialization-required equipment.
In the scope of this thesis, development of a simple, fast, cost-efficient and sensitive as the maximum residual levels (MRL) that have been set by the Europion Union (EU), whole cell based biosensor is aimed. Detection of commonly used antibiotic groups in dairy farming is present in the focus of this study. In accordance with this purpose firstly, it is aimed to form the bacteria, which have a fluorescens feature and will be used in the bioanalysis system. Therefore, expression of green fluorescence protein in the cells of Escherichia coli by the recombination was provided. At this stage, analysis system was improved by the optimization studies for the detection of antibiotic. Antibiotic detection was carried out by following the inhibition of viable bacteria cells at the presence of antibiotic. Changes in fluorescent intensity were pursued in the analysis system. In this context, ampicillin, bacitracin A, benzylpenicillin, furazolidone, gentamicin, lincomycin, neomycin, cefazolin, spectinomycin, spiramycin, streptomycin, sulfadiazine, and tetracycline antibiotics were tested. The calibration curve, limit of detection (LOD) and limit of quantification (LOQ) values were obtained for each tested antibiotic by determining the rate of increment in fluorescent intensity versus varying antibiotic concentrations. Acording to obtained results; developed method can be successfully used for the detection of ampicillin, benzylpenicillin, gentamicin, neomycin and tetracycline within 60 minutes. LOD values for the these antibiotics are 3.33 ppb, 0.29 ppb, 28.00 ppb, 618.36 ppb and 33.17 ppb, respectively and these values are lower than the allowed MRL for each antibiotic.
In the thesis, model system and milk, which poses great risk and is the most audited food from the standpoint of antibiotic level, are investigated and real sample trials were done with milk. Within this scope, milk samples that contain ampicillin, benzylpenicillin, cefazolin, tetracycline, gentamicin and neomycin are used. Obtained recovery results were in the range of 91.00% and 105.85%.
On behalf of the validation studies of the developed biosensor for the detection of antibiotic, accuracy and precision values were investigated. In this context, in a day and inter day repeatability data were examined. Bias values for accuracy, relative standart deviation values for precision were calculated. While bias values were found in the range of -8.00% and 0.64%, RSD values were found in the range of 1.30% and 7.34%.
