Enzyme-free colorimetric assay for the detection of uric acid in urine by cobalt tetroxide

A simple and efficient combustion method was employed for the one-step preparation of the porous structural material cobalt tetroxide (Co 3 O 4 ). An enzyme-free colorimetric method for the sensitive detection of uric acid (UA) was established using Co 3 O 4 as a catalyst, 3,3′,5,5′-tetramethylbenzidine (TMB) as a color-developing substrate and UA as a reducing agent. In a weak acid medium, dissolved O 2 underwent catalysis by Co 3 O 4 to generate superoxide radical (·O 2 − ) and singlet oxygen ( 1 O 2 ). The colorless TMB was then oxidized by these radicals to yield a blue oxidation product (TMBox) exhibiting strong absorption at 652 nm. Reduction of TMBox by UA led to a fading of the blue color in the solution and a subsequent decrease in absorbance at 652 nm. Thus, a straightforward and rapid method for UA determination was realized using UV–visible spectroscopy and visual inspection. Optimal color development was achieved at room temperature with 4.0 mg of Co 3 O 4 , pH = 5.0, 20 μL of 8 mmol/L TMB, and a reaction time of 4 min. Under these optimized conditions, the ratio of decrease in absorbance (ΔA) before and after the addition of UA exhibited a good linear relationship with UA concentration, within the linear ranges of 1–10 μmol/L and 10–600 μmol/L, respectively. The limit of detection in the low concentration range was determined to be 0.33 μmol/L. In addition, the method was successfully applied to the determination of UA in human urine, yielding recoveries in the range of 95–105 %, with a relative standard deviation (RSD) of less than 2 %, thus ensuring satisfactory results.