# Trends in Quantification of HbA1c Using Electrochemical and Point-of-Care Analyzers
## 5 Biosensors Based on FV Detection
Other redox mediators, such as ferrocene with peroxidase and Prussian blue, were also investigated for quantification of HbA1c on the PVA-SbQ/Pt electrode. The linear range obtained in both cases was only between 0.1 and  
Figure 6: General representation of fructosyl amino acid oxidase (FAO) enzyme-based electrochemical sensors for HbA1c. 0.3 mM [72]. These results clearly indicate that the nature of the redox mediator also plays a critical role in the detection characteristics of the working electrode. Use of nanointerfaces has resulted in an improved response and lesser loss of the electrons generated during the redox reactions at the electrode-electrolyte interface. Some of these nanoparticles with high surface area to volume ratios also provide immobilization sites for the enzymes, resulting in a higher active surface area. Several types of nanointerfaces have been explored both independently and as composites in electrochemical sensors for quantification of HbA1c. In one such study, the FAO enzyme was immobilized in an interface comprising zinc nanoparticles dispersed in polypyrrole film over a gold electrode (FAO/ZnONNPs/PPy/Au) and employed for the quantification of HbA1c [73]. The sensor exhibited a LoD of 50 \(\upmu\)M and a wide linear range between 0.1 and 3 mM. Other interfaces containing core-shell magnetic bio-nanoparticles (MNP) coated with the amine-containing polymer chitosan have also been studied as immobilization matrices for the FAO enzyme over gold electrodes (FAO/MNPs/Au) for the quantification of HbA1c [74]. The sensor exhibited a linear range up to 2 mM, with a rapid response time below 4 s and a LoD of 100 \(\upmu\)M. The biosensor exhibited good reusability up to 250 measurements over a period spanning 3 months when maintained at a temperature of 4 \({}^{\circ}\)C to prevent enzyme denaturation. However, the clinically defined high levels of HbA1c are above 40 mM and hence the linear ranges of these sensors need further improvement for clinical relevance. Multiple nanostructures comprising gold nanoparticles (AuNPs) entrapped in phosphotungstic acid-coordinated TiO\({}_{2}\) nanotubes have been investigated as a composite interface to facilitate electron transfer at the electrode-electrolyte interface (FAO/SNPs-PTA-TiO\({}_{2}\)/ITO) for an improved sensing response for the quantification of FV (Figure 7) [75]. The sensor exhibited a linear range of 0.5 \(\upmu\)M to 2 mM with a response time of 3 s and a LoD of 0.5 \(\upmu\)M. The sensor performance was found to be unaffected in the presence of interferents such as bilirubin, urea, cysteine, triglycerides, and ascorbic acid, which resulted in less than a 10% response from the working electrode. However, both uric acid and glucose elicited an electrochemical response between 15 and 18% from the working electrode. Though a rapid response was achieved, the linear range requires further improvement and a lower response to glucose for clinical use. Along similar lines, FAO bio-conjugated to gold and platinum nanoparticles was electrodeposited over a poly(indole-5-carboxylic acid)-modified gold electrode. The authors evaluated the fabricated biosensor for its stability, and it was found that the sensor retains 50% of its initial activity until 12 weeks after fabrication [76]. The sensor did not  
Figure 7: The final working electrode, FAO/AuNPs-PTA-TiO\({}_{2}\)/ITO, employed for quantification of FV. Reprinted with permission from [75]. respond to interferents such as bilirubin, urea, cysteine, glucose, ascorbic acid, and triglycerides, and displayed a linear range between 0.1 uM and 1 mM of HbA1c. The biosensor was also successfully tested in human blood samples for HbA1c measurement using a ferro/ferricyanide redox couple. The sensor, however, showed a >10% response to uric acid. The same group had also reported a nanointerface with Au nanoparticles (AuNPs) embedded in N-doped graphene nanosheets (GN) with the FAO enzyme coated on a fluorine-doped tin oxide (FTO) glass electrode (FAO/AuNPs/GNs/FTO).