# 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
Further tuning of the sensitivity may be required for potential clinical deployment of the sensing device in the future. Another study employed the sandwich immunoassay technique for dual-detection of HbA1c through electrochemical and optical methods, where the nano-bioprobe was obtained by conjugation of cadmium telluride (CdTe) quantum dots to the secondary antibody. The sandwich immunoassay was performed using an anti-HbA1c antibody as the capture antibody along with the quantum dot-labeled secondary antibody as a dual-mode sensor for the quantification of HbA1c. The sensor employed a small volume of the sample diluted 500 times for the detection and exhibited linearity between 4 and 16% HbA1c [58]. In this line, a bovine serum albumin (BSA)-MWCNT composite crosslinked to a screen-printed carbon electrode by glutaraldehyde was employed as a nano-biosensing interface for the quantification of HbA1c [46]. The anti-HbA1c antibodies were conjugated to the interface along with APBA using EDC-NHS coupling. The resultant immunosensor quantified HbA1c between 2 and 15% in blood samples. The electrode also exhibited good stability over a month when stored in BSA with less than an 8% loss in signal response. Recently, ratiometric studies related to detection of both HbA1c and Hb have been reported [59]. This methodology relied on the sandwich immunoassay, which was assisted by acridine ester-labeling, and the %CV for both repeatability for multiple detections and within-site reproducibility were documented as 1.22 to 2.21% and 2.13 to 3.27%, respectively. In another study, a thiolated aptamer-based sensing label-free detection platform was employed for the detection of HbA1c using an aptamer-conjugated gold electrode. The resulting modified sensor was employed for label-free detection of HbA1c and total HbA in blood using square wave voltammetry. The sensor exhibited a linear range between 100 pg/mL and 100 ng/mL for both HbA and HbA1c. The sensor was also validated using human blood samples, where it detected HbA1c between 6.6 and 10.5% [62]. The same group also conducted a study comparing six different carbon nanointerfaces, namely, carbon (C), graphene (G), graphene oxide (GO), multi-walled carbon nanotube (MWCNT), single-walled carbon nanotube (SWCNT), and carbon nanofibers (CNF), for the aptamer-based determination of HbA1c. The aptamers were non-covalently immobilized on the nano-interfaced electrodes via \(\pi\)-\(\pi\) stacking interactions between the DNA aptamer and the carbon nanomaterial, which restricts the electron transfer. However, upon binding of the target protein, the aptamer dissociates from the surface, resulting in enhanced electron transfer, which was quantified as a measure of HbA1c. The results revealed that the SWCNT-modified sensor showed a superior response when compared to other interfaces in the presence of a ferro/ferricyanide redox couple. The SWCNT displayed an ultra-low LoD of 0.03 pg/mL for HbA1c and a detection range of 0.1 pg/mL to 1000 ng/mL. The sensor also displayed excellent specificity and its electrochemical response remained unaffected by the presence of other proteins encountered in blood, such as STAT3, DOCK8, SMN, and CFTR [63]. In an interesting strategy, a non-enzymatic aptasensor has been fabricated using bacteriorhodopsin-containing purple membrane isolated from bacteria. These were modified with aptamers against Hb and HbA1c. The binding of the protein results in a decrease in the photocurrent measured on the electrode, which was used to quantify the amounts of HbA and HbA1c in the sample. The sensors displayed a dynamic linear range of 0.1-100 ug/mL for both HbA and HbA1c. Additionally, the HbA and HbA1c aptasensors displayed excellent stability of 14 and 28 days, respectively. The sensor performance was validated using 19 human blood samples and the results showed a good agreement with conventional measurements. The detection time of the sensor is 15 min [29].