Biosensors and Bioelectronics 26 (2011) 3438–3443 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios An electrochemical biosensor for fructosyl valine for glycosylated hemoglobin detection based on core–shell magnetic bionanoparticles modified gold electrode Sheetal Chawla, Chandra Shekhar Pundir ∗ Department of Biochemistry, M.D. University, Rohtak, Haryana 124 001, India article info Article history: Received 22 December 2010 Accepted 14 January 2011 Available online 22 January 2011 Keywords: Fructosyl valine Fructosyl amino-acid oxidase Immobilization Amperometric biosensor Core–shell magnetic bionanoparticles abstract A high-performance amperometric fructosyl valine (FV) biosensor was developed, based on immobi- lization of fructosyl amino-acid oxidase (FAO) on core–shell magnetic bionanoparticles modified gold electrode. Chitosan was used to introduce amino groups onto the surface of core–shell magnetic bio- nanoparticles (MNPs). With FAO as an enzyme model, a new fructosyl valine biosensor was fabricated. The biosensor showed optimum response, when operated at 50 mV s -1 in 0.1 M potassium phosphate buffer, pH 7.5 and 35 ◦ C. The biosensor exhibited excellent sensitivity [the detection limit is down to 0.1 mM for FV], fast response time (less than 4 s), wide linear range (from 0 to 2 mM). Analytical recovery of added FV was 95.00–98.50%. Within batch and between batch coefficients of variation were <2.58% and <5.63%, respectively. The enzyme electrode was used 250 times over 3 months, when stored at 4 ◦ C. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Over the past several decades, diabetes mellitus has become a major health problem worldwide, reaching epidemic proportions in many developing countries as well as in minority groups in the developed world (Bennett et al., 1971; King and Rewers, 1993). Dia- betes can be associated with serious complications and premature death. Glycosylated hemoglobin (HbA 1c ) is the major glycohe- moglobin species in human blood. The HbA 1c level, defined as the ratio between HbA 1c concentration and total hemoglobin concen- tration, is considered to be a very useful diagnostic marker for diabetic patient in addition to the measurement of the glucose level (John, 2003). Glycation of hemoglobin has been associated with cardiovascular disease, nephropathy and retinopathy in diabetes mellitus. Since the lifetime of hemoglobin in blood is approxi- mately 2–3 months, the HbA 1c level provides a good indication of glucose level over this period of time. The determination of HbA 1c in clinical practice is now accepted as the gold standard of glycemic assessment, providing an objective and retrospec- tive view of diabetes control within 2 months preceding blood collection (Krishnamurti and Steffes, 2001). Therefore, a cost- effective measurement of HbA 1c level is essential for management of diabetic patient (Goldstein et al., 2004). Many methods have been proposed for the routine measurement of the HbA 1c . These methods are based on different analytical principle such as ion- ∗ Corresponding author. Tel.: +91 9416492413. E-mail address: pundircs@rediffmail.com (C.S. Pundir). exchange chromatography, electrophoresis, isoelectric focusing, high-performance liquid chromatography (HPLC) and immunoas- say (Hageman and Kuehn, 1977; Goldstein et al., 1986; John, 1997; Turner et al., 1999). However, these suffer from certain draw- backs such as time-consuming, labor-intensive, expert handling, pretreatment of sample etc. Electrochemical methods for clinical diagnosis have advantages such as good selectivity, relatively low cost and the potential for miniaturization and automation (Marko- varga et al., 1995; Wang, 1999). Also, electrochemical measurement of HbA 1c level is a convenient method for the point-of care appli- cation and can be integrated with the current glucose meter for the diabetic patient management. Hence, there is great interest to develop an electrochemical enzyme sensor for HbA 1c . The enzy- matic method for HbA 1c assay consists three steps: (1) proteolysis of the HbA 1c -subunits to release fructosyl amino acid [viz. fructo- syl valine (FV)], (2) the oxidative deglycation of the produced FV and (3) the detection of the enzymatically produced hydrogen perox- ide. A fructosyl amino-acid oxidase (FAO) catalyzes the oxidative deglycation of fructosyl amino acids to produce the correspond- ing amino acids, glucosone and hydrogen peroxide (Horiuchi et al., 1989) as follows: Fru-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-ser ... Protease -→ Fructosyl valine + (amino acid) n-1 (N-terminal residue of -chain in HbA 1c ) (FV) (His, Leu, Thr) (1) FV + O 2 + H 2 O FAO -→Valine + D-glucosone + H 2 O 2 (2) 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.01.021