# Trends in Quantification of HbA1c Using Electrochemical and Point-of-Care Analyzers
## 5 Biosensors Based on FV Detection  
Antibody-based immunosensors for HbA1c are expensive, with a poor shelf-life and greater susceptibility to loss of function due to denaturation of the antibodies. The use of enzyme-based sensing elements offers the advantage of specificity and a robust electrochemical response that can be directly correlated with the analyte concentrations (Table 3). Fructosyl amino acid oxidase (FAO) has been isolated from both bacterial sources, such as _Corynebacterium_ and _Agrobacteriumium_, as well as fungal species, such as _Aspergillus_, _Penicillium_, _Fusarium_, and _Pichia_. These differ in their substrate specificity, with the bacterial enzymes showing high specificity towards alpha glycated amino acids [69]. The fructosyl peptide oxidase (FPOX) enzyme isolated from fungal species such as _Eupenicillin terrenum_ and _Coniochaeta_ sp. has shown significant sequence similarity with FAO. It also catalyzes the oxidation of the fructosyl valine-histidine peptide (FV) obtained from the protease digestion of HbA1c to form glucose and valyl histidine, with the concomitant reduction of the FAD coenzyme to FADH\({}_{2}\). The reoxidation of FADH\({}_{2}\) occurs with the formation of H\({}_{2}\)O\({}_{2}\) that is detected electrochemically, either directly or indirectly, in the presence of redox mediators (Figure 6). Biosensors based on immobilization of the enzyme on suitable matrices offer benefits such as portability, reusability, and rapid detection of HbA1c, as well as being less expensive than immunosensors. Immobilization of enzymes on the electrode surface offers a solution to many issues, such as leaching of the enzyme, maintenance of enzyme stability, and the shelf-life of the sensor. The choice of immobilization strategy will therefore be a major influence on the sensing performance. Another facet that has emerged in recent decades towards improved performance of enzyme-based sensors is the fabrication of electrodes with nanointerfaces. This has resulted in additional advantages in terms of increased stability, mechanical strength, improved conductivity, promotion of catalytic transformation of redox species, and inhibition of oxidation of interferants, thereby improving the signal-to-noise ratios. Nanoparticles also aid in increasing the electro-active surface area, promoting increased loading of the enzyme, and leading to an enhanced electron transfer at the electrode-electrolyte interface. Various electrochemical sensors with different electron conducting interfaces and enzyme immobilization matrices have been designed for HbA1c detection using the FV method. The sensing performance of these enzyme-based HbA1c biosensors is dependent upon the efficiency of enzyme immobilization as well as the source of the enzyme employed, which in turn influences the specificity of detection. Most of these amperometric biosensors exhibit a response time within 120 s between the pH window of 7.0 and 7.5, which is in the physiological pH range. Most electrochemical amperometric sensors reported in the literature could be operated in the temperature range of 25 \({}^{\circ}\)C to 45 \({}^{\circ}\)C, with a linear range of detection between 0.1 mM and 700 mM [70]. In an extensive study, a FAO enzyme extracted from marine yeast _Pichia_ sp. N1-1 strain was immobilized in a special water-soluble polymer, namely, poly(vinyl alcohol)-stylbazole (PVA-SbQ), and the resultant thin film was deposited over a platinum working electrode and photo-crosslinked. When used for quantification of HbA1c in the presence of the redox mediator methoxy phenazine methosulfate, this sensor exhibited a detection range of 0.05-1.8 mM and a sensitivity of 0.42 \(\upmu\)AmM\({}^{-1}\). When FAO was immobilized in a carbon paste electrode and crosslinked using 1% glutaraldehyde, the sensing range obtained for HbA1c in the presence of the redox mediator was between 0.2 and 2.7 mM, with a sensitivity of 1.1 \(\upmu\)AmM\({}^{-1}\)[71].