# Current Status of HbA1c Biosensors
## 1 Introduction  
With the worldwide improvement in the living conditions of humans in general, accompanied by problematic changes in diet and life-style habits such as reduced exercise, diabetes mellitus (DM) has become a major epidemic disease. According to the latest report by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), there were 415 million people with diabetes around the world, of which 47% of the patients were unaware of their state of illness [1]. Glycated hemoglobin (HbA1c) is a typical glycosylated protein in the body, and its abundance reflects the average blood glucose level over two to three months, corresponding to the 100- to 120-day lifespan of erythrocytes. The HbA1c level can not only be used by diabetes patients to monitor their long-term glucose management in a way that is not affected by fluctuations of the blood-glucose level, but also can be used by doctors to assess potential risks of diabetes complications of patients. In 2010 and 2011 the American Diabetes Association (ADA) and World Health Organization (WHO) have recommended a diagnostic cut-off point of \(\geq\)6.5% HbA1c as one of three diagnostic criteria for diabetes, respectively [2; 3]. The glycosylation of hemoglobin occurs via a sequential two-step non-enzymatic reaction. Firstly, the neutral amino groups from N-terminal residues or from the side chain of Lys residues in hemoglobin (Hb) interact with the aldehyde or ketone groups in sugar molecules to form reversible Schiff base intermediates. Then the intermediate undergoes an irreversible intermolecular Amadori rearrangement to generate a more stable ketoamine structure [4]. The probabilities of glycosylation are dependent on the local pKa of the amino groups as well as the charge and steric effects by neighboring residues [5]. In general, the pKa values of \(\alpha\)-amino groups of N-terminal residues, especially the one of \(\beta\)Val1 (close to 7), are lower than those of \(\varepsilon\)-amino groups of Lys residues in Hb. In addition, a positively charged cavity around the \(\beta\)Val1 moiety has a strong affinity to attract sugar molecules. Due to the high concentration of glucose in the blood stream, the most abundant form of the glycosylated hemoglobins (GHbs) is the glucose adduct named HbA1c. The HbA1c level is defined as the ratio of HbA1c to the total Hb concentration, and the physiological range of HbA1c in the whole-blood samples is 3-13 mg/mL in terms of concentration or 5% to 20% in terms of percentage of total Hb. The ADA-recommended diagnostic criteria of HbA1 cfor diabetes and prediabetes are shown in Table 1[6]. For pregnant diabetic individuals, it is strongly recommended that they have a stringent control on their HbA1c to minimize risks such as congenital malformations, overweight infants, and complications of pregnancy [7]. Accurate and precise methods to detect HbA1c are thus required for better diagnosis and management control of DM. In the past several decades, a variety of HbA1c detection methods has been developed, such as immunoassay [8], ion-exchange chromatography [9], boronate affinity chromatography [10], electrophoresis [11; 12], and a colorimetric method [13]. Those methods can be generalized into three basic chemical principles based on charge differences, structural differences and chemical reactivity. Most of these methods are subject to at least one or more types of interferences from Hb variants including various forms of Hb (HbC, HbS, HbE, HbD, and HbF) and/or the modification of Hb (cabamylated Hb, acetylated Hb, labile HbA1c) [14]. The global standardization of HbA1c was conducted by the IFCC and evaluated by different designated comparison methods developed by the National Glycohemoglobin Standardization Program (NGSP) and others, resulting in a laboratory-based gold reference method of HbA1c [15].