# 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  
Antibodies against HbA1c have been employed as specific capture agents to selectively retain HbA1c on the electrode surface. The captured HbA1c could result in increased impedance or could be quantified using an enzyme-linked secondary antibody for generating the electrochemical response. [PERSON] et al. [49] developed a disposable electrochemical immunosensor for the quantification of HbA1c using a three-electrode system (Figure 4). A polyethylene terephthalate (PET) sheet served as the flexible substrate for the electrode system, over which the Ag/AgCl reference electrode was printed as a thick film, while gold films deposited by sputtering formed the working and counter electrodes. The gold electrode surface pretreated sequentially in KOH, H\({}_{2}\)SO\({}_{4}\), and HNO\({}_{3}\) solutions was modified with a SAM of 3-mercaptoethanol(MPA) that served to immobilize anti-HbAlc antibodies via EDC-NHS coupling. The detection of HbA1c using differential pulse voltammetry (DPV) resulted in a linear range of 7.5-20 \(\mu\)g/mL. The sensor was validated in undiluted serum samples, where the linear range changed to 100-250 \(\mu\)g/mL. However, the reported range for diabetic individuals is greater than 9 mg/mL. Therefore, it is evident that the fabricated sensor needs additional modifications to enable detection of HbA1c in the clinically relevant range. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Electrochemical Technique** & **Interface** & **Detection Range (HbAlc)** & **LoD** & **Ref.** \\ \hline \multirow{8}{*}{**Amperometric**} & APBA-pTTBA/AuNP/SPCE & 0.1–1.5\% & 0.052\% & [35] \\ \cline{2-5}  & APBA/RVC & 0.2–12 mg/mL & 89 \(\mu\)g/mL & [34] \\ \cline{2-5}  & MPBA/AuNF/SPCE & 2–20\% & \multirow{2}{*}{0.65\%} & [36] \\  & & 5–1000 \(\mu\)g/mL & & \\ \cline{2-5}  & FPBA/PAMAM/DTSP/Au (GOx Backfilling) & 2.5–15\% & NA & [37] \\ \cline{2-5}  & TiO\({}_{2}\)(G) NW@PAPBA-Au/ITO & 2–10\% & 0.17\% & [47] \\ \cline{2-5}  & AQBA/SPCE & 31.2–500 nM & 4.2 nM & [40] \\ \cline{2-5}  & ZrO\({}_{2}\)-DDAB/PG & 6.8–14\% & NA & [33] \\ \cline{2-5}  & PAPBA/SPCE & 0.975–156 \(\mu\)M & NA & [39] \\ \hline \multirow{8}{*}{**Impedometric**} & APBA/ESM & 2.3–14\% & 0.19\% & [41] \\ \cline{2-5}  & APBA/ESM & 2.3–14\% & 0.21\% & [48] \\ \cline{2-5}  & APBA/GA/Cys/Au & 0.1–8.36\% & 0.024\% & [42] \\ \cline{2-5}  & T3 BA/Au & 4.5–11.5\% & NA & [43] \\ \cline{2-5}  & GO-APBA/GCE & 8\% & NA & [44] \\ \cline{2-5}  & T3 BA & 10–100 ng/\(\mu\)L & 1 ng/\(\mu\)L & [45] \\ \cline{2-5}  & APBA/BSA/MWCNTs/GA/SPCE & 4–15\% & 1.2\% & [46] \\ \hline \hline \end{tabular}  
* NA: Not applicable.  
\end{table} 
Table 1: Boronic acid-based electrochemical sensors for direct detection of HbA1c. In another interesting approach for the quantification of HbA1c using anti-HbA1c IgG antibodies, a mixed layer of oligo (phenyl ethynylene) molecular wires (MW) and an oligo (ethylene glycol) (OEG) were employed as the interface. The distal end of the MW was modified by non-covalent addition of the HbA1c analogue glycosylated pentapeptide (GPP) and the redox mediator 1,1'-di(aminomethyl)ferrocene (FDMA). The sensor used the competitive inhibition assay for the quantification of HbA1c, where the surface-bound GPP and free HbA1c compete for the antibodies in the solution. The binding of the antibodies to the interface resulted in an attenuation in the ferrocene electrochemical response. Higher concentrations of HbA1c resulted in an increase in Faradaic currents due to lesser anti-HbA1c on the electrode interface, and vice versa. The measurement of the change in charge transfer resistance (Rct) was used for the quantification of the HbA1c between 4.5 and 15.1% [50]. The same group also reported another method to quantify HbA1c based on EIS measurements [51]. In this method, the glassy carbon electrode was modified with 4-aminophenyl (Ph-NH2) and AuNP.