# Trends in Quantification of HbA1c Using Electrochemical and Point-of-Care Analyzers
## 2 Sensors for Quantification of HbA1c
### Electrochemical Immunosensors for HbA1c Detection Using Antibodies
The unreacted amine groups of (Ph-NH2) were blocked with OEG, followed by surface modification of AuNP with Ph-NH2, and attachment of GPP (Figure 5). The fabricated sensor upon exposure to anti-HbA1c IgG antibodies resulted in a corresponding change of Rct of the system due to the selective interaction of anti-HbA1c antibodies and GPP. The sensor had a wide detection range between 0 and 23.3% HbA1c and the values were in agreement with the conventional clinical techniques when tested with human samples. In another variant, a label-free amperometric immunosensor, using a modified interface with the redox mediator FDMA between the GPP- and the Ph-NH2-modified AuNPs, was also reported for detection of HbA1c based on the competitive immunoassay technique, where the linear detection range was between 4.6 and 15.1% in human blood samples [52]. Ion-sensitive field-effect transistor (ISFET)-integrated chip-based immunosensors have been investigated for quantification of HbA1c [53]. The detection setup used two gold working electrodes, two gold reference electrodes, and a platinum pseudo-reference electrode. The gold reference electrodes were modified by electropolymerization of polypyrrole (PPy), while the working electrodes were first modified by electropolymerization of Ppy-HAuCl4 composite film, followed by in situ deposition of gold nanoparticles. Subsequently, the working electrodes were incubated in diluted antibody solution to immobilize the antibodies on the working electrode. The sensor detected both HbA1c and Hb and the working range of the sensor was between 5 and 20% HbA1c. When the interface was changed to SAM-modified gold nanospheres that were conjugated to mercaptamine-modified gold electrode, followed by immobilization of antibodies to SAMs using EDC-NHS chemistry,  
Figure 4: Electrochemical immunosensor based on gold as an interface for direct detection of HbA1c [49]. the sensor was able to detect HbA1c between the linear range of 50 and 170.5 ng/mL and total Hb between 166.7 and 570 ng/mL [54]. The same strategy was employed to develop a field-effect transistor chip with electrodes surface-modified with 1,6-hexanedithiol and a nano-gold monolayer linked with anti-HbA1c antibodies. The sensor detected Hb and HbA1c between the ranges of 60 and 180 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 4 and 24 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, respectively [55]. The same group had reported a FET-based device where the gold nanofilm was grown by a seed-mediated technique over the gold electrode containing a mixed SAM. After conjugation of the antibodies to the gold nanoparticles, the FET sensor was employed for detection of Hb and HbA1c. The sensor showed a linear range of 1.67-170.5 ng/mL and 16.7-1705 ng/mL for HbA1c and Hb, respectively [56]. The presence of the nanogold layer was found to confer additional sensitivity towards the detection. These studies suggest that apart from the chemical nature, the mode of formation of the interface could also influence the detection range and sensitivity of the resultant sensor. Figure 5: Immunosensor established by [PERSON] et al. summarizes AuNP-assisted amperometric detection of HbA1c. Reprinted with permission from [52]. A modified sandwich immunoassay protocol with an anti-Hb molecule serving as the capture antibody specific to the total Hb, and an enzyme-labeled detection antibody specific for HbA1c, was studied for quantification of HbA1c. Though four different anti-Hb and two different anti-HbA1c antibodies were investigated in the study, none of the results were in agreement with the actual HbA1c values of the samples. It was hypothesized that the detection site of the anti-HbA1c antibody is hidden inside due to folding, and hence an additional denaturation step using 0.2% dodecyltrimethylammonium bromide was introduced in the protocol to expose the antigen-binding site of the anti-HbA1c antibody. This resulted in improved sensitivity of the immunosensor, that was now able to quantify HbA1c between 5.6 and 10.6% [57].