Fe/Pt-doped carbon nanoparticles with peroxidase-like activity for point-of-care determination of uric acid

A pasting-3D microfluidic paper-based analytical device (P-3D μPAD) was developed. It enabled an efficient cascade reaction between urate oxidase (UOX) and Fe/Pt-doped carbon nanoparticles (Fe/Pt-CNPs) for visual colorimetric detection of uric acid (UA). The novel Fe/Pt-CNP nanozyme performed high peroxidase-like activity toward 3,3',5,5'-tetramethylbenzidine (TMB) and H₂O₂ with Michaelis - Menten constants (K_m) of 0.97 and 2.30 mM, respectively. The UOX-Fe/Pt-CNP system was incorporated into P-3D μPAD: the 1st layer was UOX hydrolysis reaction under alkaline pH, and the 2nd layer was Fe/Pt-CNPs catalyzing H₂O₂ to oxidize TMB at acidic pH. The separated two layers allowed cascade reaction under different working pH without sacrificing the activity of UOX or Fe/Pt-CNPs. The images were captured and analyzed by the camera and "Color Recognition" application using a  smartphone. The linear range of P-3D μPAD for UA was 25-1000 μM with a limit of detection of 10 μM which met the requirements for clinical applications. The high accuracy of P-3D μPAD for UA detection in invasive (blood) and noninvasive (saliva) samples has been confirmed by the biochemical analyzer. This work offers a sensitive, flexible, affordable, and disposable tool for on-site UA level monitoring and provides new insight into natural enzyme and nanozyme tandem systems for biosensing.