Rational design of mini protein mimicking uricase: Encapsulation in ZIF-8 for uric acid detection

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction.