# A Review of Electrochemical Sensors for the Detection of Glyated Hemoglobin
## 3 Indirect Type Electrochemical HbA1c Sensors
### FPOX Type  
Compared with other enzymatic HbA1c sensors, FPOX type HbA1c sensors have high specificity because substantial specific measurements of HbA1c can be realized through mutagenesis and modification [104; 105]. As shown in Figure 5C, [PERSON] et al. immobilized a recombinant engineered FPOX enzyme to specifically hydrolyze FVH on an electrode surface modified by CHIT, graphene oxide (GO), and gold nanoparticles (AuNPs). The biosensor showed a linear response within the range of 0.1-2 mM [104]. In addition, to prove that the FPOX-modified electrode can specifically measure FVH, significant changes in electron transfer resistance were observed after incubation of the FPOX-modified electrode with FVH, but there was no response in the control group, indicating the specific measurement of FVH. [PERSON] et al. replaced Arg414 with Lys to form the PnFPOX (FPOX from Phaeosphaeria nodorum) N56A/R414K mutant and modified PnFPOX near FAD with amine-reactive phenazine ethosulfate (arPES), which showed quasi-direct electron transferability [106]. This electrode was combined with an enzyme flow injection analysis (FIA) system. The linear range of the system for both FV and FVH was 20-500 uM, and the sensitivities and detection limits of the system were 0.49 nA\(\cdot\)uM\({}^{-1}\) and 1.3 uM for FV, respectively, and 0.13 nA uM\({}^{-1}\) and 2.0 uM for FVH. The oxidative activity and specificity of PnFPOX for this method are commendable, but the detection range needs to be expanded if this method is to be adopted for clinical measurements of HbA1c. In addition, the PnFPOX electrode was engineered for continuous operation of the FIA system. This case shows that an electrochemical sensor can be combined with an FIA system to develop an integrated measurement system for sample pretreatment and sample electrochemical measurement.  
There are also some sensors that modify both FPOX and FAO, which can selectively determine only glycated N-terminal peptides from the \(\beta\) chain without any interference in the presence of glycated peptides from the \(\alpha\) chain [107]. [PERSON] et al. adopted an FIA system with a flow-type spectrophotometer and electrochemical detector (with FPOX immobilized on amino-alkyl-bonded Uniport C and FAO immobilized on dialdehyde-activated Uniport C) [107]. The total hemoglobin in the sample is determined by spectrophotometry, and  
Figure 5: **(A)** An FAO/AuNP-PTA-TiO\({}_{2}\) nanocomposite was prepared on an ITO electrode (this figure was adapted from [92], with some modifications); **(B)** Preparation method of the FAO/PtNPs/RGO-NWCNT nanocomposite (this figure was adapted from [94], with some modifications); **(C)** CHIT-GO-AuNPs-FPOX nanocomposites were prepared on an FTO glass plate (this figure was adapted from [104], with some modifications).  
then, the FVH released from HbA1c is determined by an electrochemical sensor with FPOX or FAO. As a result, at pH 8.0-8.5, FAO showed high activity against FV but no activity against FVH, and at nearly pH 7.0, FPOX showed the maximum activity of FV and FVH. The FIA system with the FAO reactor showed a linear detection range for FV of 2-200 uM, and the FPOX reactor showed linear detection ranges for FV and FVH of 2-100 uM and 7-110 uM, respectively. However, when blood cell samples were digested by protease, only FVH was released, not FV. Therefore, according to the definition of HbA1c by the International Federation of Clinical Chemistry (IFCC), the combination of protease and FPOX reactor systems can determine only FVH.