Review Determination of glycated hemoglobin with special emphasis on biosensing methods Chandra Shekhar Pundir ⇑ , Sheetal Chawla Department of Biochemistry, M. D. University, Rohtak 124 001, Haryana, India article info Article history: Received 20 June 2013 Received in revised form 21 September 2013 Accepted 23 September 2013 Available online 1 October 2013 Keywords: Glycated hemoglobin Biosensors Fructosyl valine Fructosyl amino acid oxidase Amperometric biosensors abstract The glycated hemoglobin (HbA1c) level in blood is a measure of long-term glycemic status in patients with diabetes mellitus. Current clinical methods for determination of the HbA1c level include electropho- resis/electroendosmosis, ion exchange chromatography, high-performance liquid chromatography, boro- nate affinity chromatography, immunoassay, and liquid chromatography–tandem mass spectroscopy in addition to fluorometry and colorimetry. These methods have certain drawbacks such as being complex, time-consuming, and requiring expensive apparatus and trained persons to operate. These drawbacks were overcome by biosensing methods. We review these biosensors, which are based on (i) measurement of electrons, that is, current generated from splitting of hydrogen peroxide released during oxidation of fructosyl valine by immobilized fructosyl amino acid oxidase, which is directly proportional to HbA1c concentration, and (ii) direct measurement of HbA1c by some specific reaction. HbA1c biosensors work optimally within 4 to 1800 s, between pH 7.0 and 9.0 and between 25 and 45 °C, and in the range of 1 to 10,000 lM, with a detection limit between 20 and 500 lM and sensitivity between 4.6 nA and 21.5 lA mM 1 cm 2 and stable over a period of 5 to 90 days. We suggest the ways to modify existing HbA1c biosensors, leading to simple, reliable, and economical sensors ideally suited for point-of-care treatment. Ó 2013 Published by Elsevier Inc. Diabetes mellitus is a chronic metabolic disorder characterized by a rise in blood glucose level called hyperglycemia [1]. Monitor- ing the blood glucose level is required for diabetic patients to maintain it within a normal range, thereby reducing the risk of se- vere complications such as kidney disease, blindness, and nerve damage [2]. Blood glucose monitoring is the main tool to check control of diabetes. The worldwide prevalence of diabetes in 2000 was approximately 2.8%, which is estimated to grow to 4.4% by 2030. This translates to a projected rise of diabetes from 171 million in 2000 to well over 350 million in 2030 [3]. Hence, there is an urgent need for improved treatment of hyperglycemia and other risk factors associated with this metabolic syndrome. If left untreated or improperly managed, diabetes can result in a vari- ety of complications, including heart disease, kidney disease, neu- ropathy, gangrene, gastroparesis, blindness, erectile dysfunction, impotence, and nerve damage. Poor blood glucose control also in- creases the risk of short-term complications of surgery such as poor wound healing. Historically, measurement of glucose level, by fasting plasma glucose and 2-h plasma glucose, has been the universal method for diagnosis of diabetes. However, this approach still suffers from the same problems and difficulties associated with glucose biosensors such as the need for fasting, biological var- iability, and the effects of acute perturbations (e.g., stress or illness related) on glucose levels [4]. Blood glucose also reflects a fluctuat- ing glucose level and is affected by daily diet and requires frequent measurements. Currently, portable-sized glucose measurement devices are being designed for self-use, and these are economical and give quick results. Some of them adopt invasive measurement methods for blood extraction, that is, using a piercing component of a microneedle or a lancet with a disposable strip for sensing and a handheld fixture for readout [5]. The glycated hemoglobin (HbA1c) 1 level, defined as the ratio between HbA1c concentration and total hemoglobin concentration, is considered to be a very useful diagnostic marker for diabetic patients in addition to the measurement of the glucose level [6]. 0003-2697/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ab.2013.09.023 ⇑ Corresponding author. E-mail address: pundircs@rediffmail.com (C.S. Pundir). 1 Abbreviations used: HbA1c, glycated hemoglobin; MBG, mean blood glucose; HPLC, high-performance liquid chromatography; LC–MS/MS, liquid chromatography– tandem mass spectroscopy; CE, capillary electrophoresis; UV/VIS, ultraviolet/visible; CV, coefficient of variation; ICP–MS, inductively coupled plasma–mass spectrometry; CIEF, capillary isoelectric focusing; RSD, relative standard deviation; ESI–MS, electrospray ionization–mass spectrometry; Hb, hemoglobin; 5-HMF, 5-hydroxym- ethylfurfural; ELISA, enzyme-linked immunosorbent assay; FV, fructosyl valine; FAO, fructosyl amino acid oxidase; H 2 O 2 , hydrogen peroxide; Pt, platinum; GCPE, glassy carbon paste electrode; FcBA, ferrocene boronic acid; PPy, polypyrrole; p-APBA, poly- aminophenylboronic acid; FET, field effect transistor; SAM, self-assembled mono- layer; RBC, red blood cell; DEP, dielectrophoretic. Analytical Biochemistry 444 (2014) 47–56 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio