Urea

Tin tetrachloride hydrate (SnCl₄·5H₂O) and thioacetamide were obtained from Macklin Biochemical Co., Ltd (Shanghai, China). Dicyandiamide, urea and nitric acid were all purchased from Damao Chemical Reagent Factory (Tianjin, China). NHS and EDC were both purchased from Aladdin Reagent Database Inc. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (Beijing, China). PSA and anti-PSA were both purchased from Shanghai Linc-Bio Science Co., Ltd., China. All other chemicals were analytical grade and used without further purification. Phosphate buffer solution (PBS) is a mixed solution of disodium hydrogen phosphate (Na₂HPO₄·12H₂O, 0.1 mol·L⁻¹) and potassium phosphate monobasic (KH₂PO₄, 0.1 mol·L⁻¹), which was used for the preparation of the antibody, antigen and washing buffer solution. All aqueous solutions were prepared by using ultrapure water (Milli-Q, Millipore).

Sporosarcina pasteurii in a freeze-dried form from China General Microbiological Culture Collection Center (CGMCC) was used as ureolytic bacteria for catalyzing urea hydrolysis. The strain was transferred to a liquid medium which consists of yeast extract (Oxoid Ltd., United Kingdom) of 20 g/L, urea (Damao Chemical Reagent Factory, China) of 20 g/L, NH₄Cl (Chengdu Chron Chemicals Co. Ltd., China) of 10 g/L, MnSO₄·H₂O (Shanghai Aladdin Biochemical Technology Co. Ltd., China) of 10 mg/L, and NiCl·6H₂O (Tianli Chemical Reagent Co. Ltd., China) of 24 mg/L and was cultured at 180 rpm and 30°C for 24 h. pH for the liquid medium was measured being 8.8. The activated ureolytic bacteria were mixed with glycerol using a ratio of 7:3 and stored at −20°C (Achal et al., 2012 (link)). They were used as the inoculum in subsequent test tube experiments.