Functionalization of GroEL nanocages with hemin for label-free colorimetric assays

The design of functionalized protein nanocages as enzyme mimics is a relatively new and promising field as these biogenic nanocapsules are inherently monodisperse with precise architectures, unattainable by purely synthetic processes. This work explored the catalytic properties of the bacterial GroEL-nanocaged hemin and its performance in label-free colorimetric assays. It is demonstrated that the hemin-GroEL biohybrid has peroxidase-like activity and follows the typical Michaelis-Menten kinetics and ping-pong mechanism in the model sensing processes. The open nature and nanoreactor effect of the GroEL cage and the addition of ATP are shown to significantly influence the catalytic activity. For glucose detection with the hemin-GroEL complex, the linearity between the analyte concentration and UV-vis absorption was determined to range from 0 to 200 μM with a limit of detection (LOD) of ~ 12 μM under the defined conditions. In addition, the colorimetric detection of catechol led to a linear dynamic range of 0-120 μM with a LOD of ~ 17 μM. This indicates that the same assay could be used as a sensing platform for the detection or even oxidative removal of phenolic contaminants in the presence of H₂O₂. Thus, GroEL cage is a valuable tool for the development of nanozymes and practical applications such as clinical analysis and environmental monitoring.