Development of quantitative enzymatic method and its validation for the assay of glucose in human serum

• Published in: Clinical biochemistry Vol. 45; no. 1-2; pp. 139 - 143
• Main Authors: Nagaraja, Padmarajaiah, Honnur Krishna, Shivakumar, Anantharaman, Shrestha, Ashwinee K.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-01-2012; Elsevier
• Subjects: Adsorption; Aniline Compounds - analysis; Biological and medical sciences; Biological Assay - methods; Blood Glucose - analysis; Calibration; Clinical Laboratory Techniques; Dose-Response Relationship, Drug; Glucose; Glucose - analysis; Glucose oxidase; Glucose Oxidase - chemistry; Horseradish peroxidase; Humans; Hydrogen peroxide; Hydrogen Peroxide - chemistry; Investigative techniques, diagnostic techniques (general aspects); Kinetics; Medical sciences; Peroxidase - chemistry; Polymers - analysis; Reproducibility of Results; Serum - metabolism; Serum glucose; Temperature; Glucose oxidase; Hydrogen peroxide; HRP; Horseradish peroxidase; Serum glucose; Glucose; EDTA; A740; Kpow; EU; DMA; Keff; V0; GOD; Human; Validation; Enzyme; Enzymatic method; Peroxidases; Clinical biology; Development; Serum; Peroxidase; Oxidoreductases; Quantitative analysis
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2011.11.007

To develop a simple, rapid, sensitive and affordable assay method for the determination of glucose in blood samples using a novel approach.
A spectrophotometric method for glucose quantification in human serum samples based on self-coupling of activated 2,5-dimethoxyaniline (DMA) in the presence of peroxidase (POD)/glucose oxidase (GOD) and H2O2 is described. H2O2 generated in situ by catalytic reaction between GOD and glucose, activates DMA in the presence of POD to form a green-colored product, which has a strong absorption at λmax=740nm at room temperature (30°C) in a 100mmol/L acetate/acetic acid buffer of pH 4.2.
The linearity ranges for the quantification of glucose by rate and one-time detection method are 0.017–0.740 and 0.017–0.478mmol/L, respectively. Within-day and day-to-day precision were 0.98–1.4% (n=10) and 1.33–2.89% (n=15), respectively. Glucose recoveries ranged from 96.6 to 102%, indicating minimal interference by commonly present interferants in serum samples. Accuracy results were between 90 and 102%. The detection and quantification limits of glucose were 2.376 and 7.923μmol/L, respectively. The proposed method has good correlation coefficient of 0.999 with the enzymatic kit method.
This is a rapid and convenient method to determine serum glucose using simple spectrophotometer with excellent recovery and minimal interference by interferants in serum samples with low detection limit. Therefore, this method can be considered for adoption by the clinical diagnostic laboratories.
► Enzymatic pathway is proposed for quantification of clinically important biomarker. ► 2,5-dimethoxyaniline is used as a chromogenic probe. ► The efficacy of the method has been proved by evaluating kinetic parameters. ► Bland-Altman plot has been used for the validation of the proposed method.