An enzymatic microfluidic sensor for β-lactam antibiotics based on cross-linked ancestral β-lactamase crystals

A microfluidic enzymatic sensor based on cross-linked crystals of an ancestral β-lactamase (βLa-CLECs) was developed for the detection of β-lactam antibiotics under continuous flow conditions. The sensor is presented in a modular configuration, consisting of a separated microprobe for enzymatic catalysis, allowing for precise enzyme loading, and a photonic lab-on-a-chip (PhLoC) platform for detection by in-line spectrophotometric measurements. The exceptional thermal stability of the reconstructed Precambrian β-lactamase made it suitable for the steps required in device fabrication and carrier-free immobilization treatment, while its inherent promiscuity, i.e., its capability to degrade a variety of lactam antibiotics, including man-made third-generation antibiotics, broadens its potential application. Full catalytic activity of the ancestral enzyme was retained after immobilization, and inhibition by antibiotics such as ampicillin and sulbactam was detected at concentrations as low as parts per billion. These results support the use of ancient enzymes as stable and responsive biorecognition elements in cost-effective, high-throughput analytical systems targeting environmental pollutants and pharmaceutical compounds.