Metallic nanoparticles bioassay for Enterobacter cloacae P99 β-lactamase activity and inhibitor screening

Abstract

This article presents a simple colorimetric assay for Enterobacter cloacae P99 β-lactamase activity detection and its inhibitors screening on the basis of silver and gold nanoparticles aggregation. In the presence of E. cloacae P99 β-lactamase, the β-lactam ring in the cephalosporin substrate was opened and resulted in releasing the active sites-modified linker which induced significant aggregation of silver or gold nanoparticles based on the electrostatic interactions and metal-thiols conjugation between the flexible linker and citrates on the surfaces of silver and gold nanoparticles. This aggregation process was associated with a concomitant color change of the nanoparticles solution and a red-shift of the particle surface plasmon resonance band, which was monitored by the naked eye or UV-Vis spectrophotometry. With this simple and convenient colorimetric assay, the activity of E. cloacae P99 β-lactamase with a concentration as low as 16 pM could be easily visualized on the basis of gold nanoparticles aggregation. Silver nanoparticles provide a more sensitive assay toward the E. cloacae P99 β-lactamase by which the lowest enzyme concentration down to 5.0 pM could be determined. Moreover, this effective colorimetric assay was also found useful for quantitative screening of E. cloacae P99 β-lactamase inhibitors. Both the silver and gold nanoparticles exhibited identical trends for the E. cloacae P99 β-lactamase inhibition screening, which were consistent with the results as determined by the standard assay. The results clearly indicated that the silver and gold nanoparticle based colorimetric assay may offer a new way to accurately evaluate the effect on the inhibition of bacterial drug resistance. Furthermore, the quantitative measurements presented in this work may also open the way for other relevant applications in prodrug development for cancer treatment.