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

Area of Science:

• Analytical Chemistry
• Biotechnology
• Pharmacology

Background:

• Accurate detection of penicillin G residues is crucial for food safety and clinical diagnostics.
• Existing methods for antibiotic residue detection can be time-consuming and lack sensitivity.
• Penicillin G detection relies on its interaction with penicillin-binding proteins (PBPs).

Purpose of the Study:

• To develop rapid, accurate, and practical tools for detecting penicillin G antibiotic residues.
• To establish novel assays based on the binding mechanism of beta-lactam antibiotics to PBPs.
• To compare the performance of a real-time PCR-based method with an SDS-PAGE method for penicillin G detection.

Main Methods:

• Developed assays utilizing in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) from Streptococcus pneumoniae.
• Employed fluorescent-labeled beta-lactam analogue Bocillin FL as a competitive substrate.
• Utilized a real-time PCR-based fluorescence detection method and SDS-PAGE with fluorescence quantification.

Main Results:

• Both developed methods demonstrated a broad range of linearity and high sensitivity.
• The real-time PCR-based method detected penicillin G in the range of 0.01-0.2 nM with high accuracy.
• The SDS-PAGE method showed sensitivity ranging from 0.015 to 2 µM.

Conclusions:

• The on-column real-time PCR assay is a fast, highly sensitive, and accurate method for detecting penicillin G.
• This novel assay, based on binding inhibition using a fluorescent competitor, can be adapted for screening penicillin G in biological and environmental samples.
• The developed real-time PCR method offers a significant improvement in speed and sensitivity compared to the SDS-PAGE method.