Electrochemiluminescence-Based Detection of β-Lactam Antibiotics and β-Lactamases

Bacterial resistance to clinically administered β-lactam antibiotics is usually caused by β-lactamases, enzymes that hydrolytically inactivate the antibiotics. This paper describes the use of electrogenerated chemiluminescence (ECL) to detect β-lactam antibiotics and their hydrolysis by β-lactamases. All 10 tested antibiotics were detected on the basis of their ability to participate in an ECL reaction with ruthenium(II) tris(bipyridine). In every case, antibiotic-promoted ECL changed when the antibiotic was hydrolyzed by β-lactamases or NaOH. Standard curves of antibiotic concentration versus ECL intensity showed that antibiotics could be quantitated to low micromolar concentrations. Substrate profiles were generated for four β-lactamases using six structurally diverse β-lactam antibiotics. ECL-based antibiotic detection was accomplished in untreated whole milk, and β-lactamases were detected in crude bacterial broth culture. Because several structurally diverse antibiotics were detectable by ECL, this method may become valuable for the detection of many or all β-lactam antibiotics and their inactivation by β-lactamases.