# Trends in Quantification of HbA1c Using Electrochemical and Point-of-Care Analyzers
## 5 Biosensors Based on FV Detection
This modification showed promising results in the electrochemical detection of FV as an indirect measure of HbA1c between the concentration range of 0.3 uM and 2 mM [77]. In another variant, reduced graphene oxide (rGO) mixed with multiwalled carbon nanotubes (MWCNT) was electrodeposited over a gold (Au) working electrode, followed by electrodeposition of platinum nanoparticles and drop-casting of FAO to form the working electrode. The FAO/PtNPs/rGO-MWCNT/Au was successfully employed for quantification of HbA1c between 0.05 and 1000 uM, with a LoD of 0.1 uM [78]. Screen-printed electrodes have emerged as a portable, flexible, and disposable option for clinical diagnosis. In a proof-of-concept study, recombinant FAO from _E. coli_ was immobilized over an iridium-carbon ink screen-printed electrode that was employed for measurement of FV with a sensitivity of 0.0215 mAmM\({}^{-1}\) cm\({}^{-2}\)[79]. Along similar lines, a single-use disposable screen-printed electrode setup for FV and HbA1c was developed by drop-casting FAO on graphite ink screen-printed electrodes [80]. The fabricated disposable sensor was employed for the quantification of FV and was also evaluated for its sensing performance using human blood samples. The biosensor showed effectiveness for quantification of fructosyl valine (FV) as well as human blood samples, with a wide linear range of 1 to 8 mM and a sensing time of 60 s.  
Recently, fructosyl peptide oxidase (FPO or FPOX)-based HbA1c sensors have also gained traction as a substitute for the more extensively studied FAO enzyme. A composite interface comprising chitosan (CHIT), graphene oxide (GO), and gold nanoparticles (AuNPs) was employed to immobilize FPOX by drop-casting to obtain the final working electrode, FPOX/CHIT-GO-AuNPs/FTO [81] (Figure 8). The sensor displayed a LoD of 0.3 uM and quantified FV between 0.1 and 2 mM. The performance of the sensor was studied using human blood samples and the values were found to be in agreement with those obtained using a commercially available FPOX-based biochemical assay kit. Figure 8: Modified working electrode FPOX/CHIT-GO-AuNPs/FTO for amperometric detection of FV. Reprinted with permission from [81]. A dual-enzyme-based microfluidic chip was fabricated for the detection of glucose and HbA1c. Gold electrodes patterned by photolithography were treated with cysteamine followed by enzyme conjugation through glutaraldehyde crosslinking. Glucose oxidase and FAO were employed for the specific detection of glucose and HbA1c, respectively. The chip employed di-electrophoresis for separation of blood plasma and had separate detection areas for measurement of glucose and HbA1c that regulated sample movement by micropumps and microvalves. The sensor detected glucose between 40 and 200 mg/dL and HbA1c in the range 0-7% [82]. This automated chip with slight modifications to extend the detection range of HbA1c may find use for clinical diagnosis in the future. An electro-chemiluminescent biosensor fabricated using a composite nanointerface comprising Au nanoparticles and titania nanotubes (TiNT) with immobilized FAO coated over an ITO electrode was employed for detection of FV. The H\({}_{2}\)O\({}_{2}\) released by the enzyme-catalyzed redox reaction was used to convert luminol, resulting in emission of electromagnetic radiation that was further used to enhance the electrochemical response. The sensor exhibited a linear range of detection between 4.0 nM and 0.72 \(\upmu\)M of FV and an ultrahigh sensitivity with a detection limit of 3.8 nM. The sensor retained its sensing performance over 20 days when stored at 4 \({}^{\circ}\)C. The sensor showed good correlation with the results obtained using a commercial HbA1c enzymatic kit when tested with human blood samples [84]. Recently, a label-free affinity sensor based on a field-effect transistor (FET) was fabricated for the detection of FV.