A simple, fast, sensitive, convenient and economical accurate enzymatic chromogenic sensor is developed for the quantification of clinically important biomarker uric acid (UA) in human serum samples. The method is based on quantification of UA using 3-hydroxytyramine (3-HT) as a novel chromogenic substrate by using Peroxidase (POD) and, Uricase (UOx) in the presence of dissolved oxygen. UOx catalyzes the oxidation of UA where H2O2 is generated in-situ to produce allointon, and CO2. The POD in oxidative coupling of peroxide in presence of 3-HT, forms an intense orange colored product with λmax at 500 nm. The reaction was carried out in citric acid-tripotassium-citrate buffer (12.5 mM) of pH 6.8 at room temperature. The standard curve for UA was found linear in the range of 12.3–297.3 μM and 3.07–396.5 μM by rate and fixed time method, respectively. The apparent molar absorptivity for UA was 0.1003×104 L/mol/cm and its determination has a CV of 2.87 (n=6). The LOD and LOQ for UA were 1.5 μM and 2.9 μM, respectively. Within-day precision was 1.50–3.07 % (n = 7) and day-to-day precision was 3.10–4.16 % (n = 7). The accuracy ranges for UA having concentrations of 24.7 μM, 198 μM and 297.3 μM were 87-101.50 %, 90-105 % and 89-107 % respectively. The UA recovery range by the proposed method was 87-107 % with a mean recovery of 96.87 %. The proposed method has good correlation coefficient of 0.981 with the standard method. This is a rapid and convenient method to determine serum UA using simple spectrophotometer with excellent recovery and minimal interference by interferants with low detection limits. Therefore, this method can be automated for flow injection or high-throughput or ELISA based assays in clinical diagnostic laboratories.
Keywords: Uric acid, Enzymatic, Clinical biomarker, Oxidative coupling, Serum analysis
Abbreviations used: UA: uric acid; 3-HT: 3-hydroxytyramine; POD: Peroxidase; UOx: Uricase; CV: Co-efficient of variations; LOD: limits of detection; LOQ: limits of quantification
Dr. Vijaylakshmi Edalli received her PhD degree in Biodegradation and bio-remediations using enzymes, from the Karnatak University, Dharwad, under the guidance of Prof. C. M. Kamanavalli, Professor of Bio-Chemistry, Karnatak University, Dharwad. She is currently works as an Assistant Professor of Chemistry, in SDVS Sangh’s, S. S. Arts College and T. P. Science Institute, at Sankeshwar affiliated to Rani Channamma University, Belagavi district, Karnataka state, from India. Her research focuses on environmental bio-remediation studies and method development based on enzyme assays. She is also interested in nano technological and its sensing applications.