Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f )azepine as chromogenic probe Padmarajaiah Nagaraja * , Anantharaman Shivakumar, Ashwinee Kumar Shrestha Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India article info Article history: Received 2 June 2009 Available online 15 August 2009 Keywords: MBTH–DBZ Horseradish peroxidase Glucose oxidase Glucose assay Catalytic parameters Human serum sample abstract A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H 2 O 2 ) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H- benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H 2 O 2 by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H 2 O 2 is found between 5 Â 10 À6 and 45 Â 10 À6 mol L À1 at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 Â 10 6 M À1 min À1 and 9.81 Â 10 À4 min À1 , respectively. The catalytic constant (k cat ) and specificity constant (k cat /K m ) at saturated concentration of the cosubstrates were 163.2 min À1 and 4.156 Â 10 6 L mol À1 min À1 , respectively. This method can be incorporated into biochemical analysis where H 2 O 2 undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum. Ó 2009 Elsevier Inc. All rights reserved. Development of sensitive and selective analytical methods for the assay of hydrogen peroxide (H 2 O 2 ) 1 is important in food, phar- maceutical, clinical, industrial, environmental, and biochemical fields [1–3]. In clinical analyses, biomarkers such as glucose, triglyc- eride, oxalate, creatinine, maltose, cholesterol, and uric acid are rou- tinely measured using enzymatic methods [4], where the amount of H 2 O 2 produced by the oxidase corresponding to the analyte (e.g., glucose oxidase and uricase for glucose and uric acid, respectively) is measured indirectly based on the color reaction with the aid of peroxidase. Hence, H 2 O 2 is an important target in the determination of vital compounds in clinical analyses. Consequently, it is necessary to develop more sensitive methods for measuring H 2 O 2 . Several methods are available for the quantification of trace amount of H 2 O 2 , including electroanalytical techniques [5,6], fluo- rimetry, luminescence [7], nanotechnology [8], high-performance liquid chromatography (HPLC) [9], and mimetic peroxidase behav- ior of metal complexes [10]. However, they have a few constraints; for example, instruments used in fluorimetry and luminescence are very expensive and less versatile. The selectivity of luminescence is poor. The electroanalytical technique needs several steps to immo- bilize enzyme on the solid support, and this may reduce enzyme activity. During the incorporation of enzyme within electropoly- merized polymers or carbon paste, a large enzyme will not be used, resulting in the waste of an expensive biocatalyst [11]. Also, the application and development of electrochemical biosensors for H 2 O 2 are limited by denaturation of horseradish peroxidase (HRP) and lack of an ideal approach for immobilization of the enzyme on an electrode surface [12]. Enzymatic assay by spectro- photometer is economical and easy to handle, and reagents used in the assay are inexpensive. Some of the enzymatic methods used in the quantification of H 2 O 2 include 4-aminoantipyrine (AP)/phe- nol [13], o-phenylenediamine and catechol [14], pyrogallol [15], 4-amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one (DAP) with N-ethyl-N-sulfopropylaniline sodium salt [16], o-dianisidine, benzidine, p-phenylenediamine [17], 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) [18], and tetramethyl-p- phenylenediamine (TMPD) [19], but these reagents have some lim- itations such as carcinogenicity and mutagenicity of o-dianisidine and benzidine and broader linearity range of pyrogallol and guaia- col due to their autoxidation. The poor sensitivity of AP is a com- mon disadvantage, and phenol tends to polymerize due to air oxidation; the product formed is an enzyme denaturing agent. TMPD registers serious interference from the antioxidants due to the radical cation nature of the oxidized form during its oxidation; 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.07.053 * Corresponding author. Fax: +91 821 2421263. E-mail address: drpn58@yahoo.co.in (P. Nagaraja). 1 Abbreviations used: H 2 O 2 , hydrogen peroxide; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; AP, 4-aminoantipyrine; DAP, 4- amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one; TMPD, tetramethyl-p- phenylenediamine; MBTH, 3-methyl-2-benzothiazolinonehydrazone hydrochloride; DBZ, 10,11-dihydro-5H-benz(b,f)azepine; GOD, glucose oxidase; UV–Vis, ultraviolet– visible. Analytical Biochemistry 395 (2009) 231–236 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio