On Column Binding a Real-Time Biosensor for β-lactam Antibiotics Quantification

This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of beta -lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled beta -lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It((R))2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01-0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 mu M). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples.