# Trends in Quantification of HbA1c Using Electrochemical and Point-of-Care Analyzers
## 2 Sensors for Quantification of HbA1c
### Electrochemical Sensors for Direct HbA1c Detection Using Boronate Chemistry  
For HbA1c sensing, the electrochemical sensors work on the principle of boronic acid interactions with the diol groups in HbA1c, which are absent in normal Hb, thereby conferring specificity for the detection (Figure 2). The frequently used boronic acid derivatives are ferrocene boronic acid (FcBA), aminophenyl boronic acid (APBA), formylphenyl boronic acid (FPBA), thiophene-3-boronic acid (T3 BA), etc. These boronic acid derivatives have been sometimes used in tandem with metallic nanoparticles for increased conductivity and sensing performance (Table 1). In an earlier work, an amperometric sensor employing FcBA as the capture element was used for HbA1c detection. The pyrolytic graphite working electrode (PGE) modified with zirconium dioxide (ZrO2) nanoparticles was used to immobilize Hb or HbA1c from the samples. The working electrode was then incubated in FcBA solution and the amount of HbA1c was measured through the electrochemical response of FcBA using square wave voltammetry (SWV). The electrochemical response of heme was used to quantify total hemoglobin (HB) in the PGE without the addition of FcBA. The sensor exhibited a linear response between 6.8 and 14% of HbA1c. The sensor performance was validated using whole blood samples from human volunteers and compared with the conventional HPLCmethod. A variation ranging from \(-10.7\) to 31% was observed [33]. The limitations of this system are the tedious and long sample analysis time, the narrow detection range, as well as greater deviations from the values reported by the conventional methods. Screen-printed electrodes have garnered much attention in the clinical diagnosis domain owing to their cost-effectiveness and disposability. [PERSON] et al. [34] designed a novel HbA1c sensor using a screen-printed reticulated vitreous carbon (RVC) electrode. For fabrication of the sensor, RVC foam was compressed to form a working electrode of diameter 3 mm and length 5 mm, which was further incubated in HNO\({}_{3}\) and H\({}_{2}\)SO\({}_{4}\) solution for 1 h at 85 \({}^{\circ}\)C. Then, APBA was immobilized on the working electrode using an ethyl (dimethyl amino propyl) carbodiimide-N-hydroxysuccinimide (EDC-NHS) coupling reaction. Finally, the electrode was immersed in glycine solution to block the unreacted carboxylic ends of RVC. This modified RVC was set into a cylindrical polyether ether ketone (PEEK) to form the flow cell. The electrochemical properties of the electrode were studied in the flow injection assay system. The results showed that the sensor exhibited a detection range of 0.2-12.0 mg/mL HbA1c with a limit of detection (LoD) of 89 \(\upmu\)g/mL at a low potential of +0.2 V. The electrode was also stable and lost only 4.4% of the signal intensity after 100 cycles of use. Another disposable APBA-based sensor fabricated on screen-printed carbon electrodes (SPCE) comprised of a layer containing electrodeposited gold nanoparticles (AuNPs) that was dipped in a 1 mM TTBA (2,2':5',5\"-terthiophene-3'-p-benzoic acid) solution. The TTBA was then electro-polymerized by potential cycling twice from 0.0 to +1.4 V (Ag/AgCl) in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) at a scan rate of 0.1 V/s, followed by immobilization of APBA using EDC-NHS chemistry. The final working electrode (APBA-pTTBA/AuNPs/SPCE) when subjected to electrochemical studies demonstrated the specificity and stability of the HbA1c owing to the highly stable conducting polymer layer. The sensor had a LoD of the sensor of 0.052% and showed good correlation with conventional HPLC in the HbA1c values measured for diabetic and normal patients [35]. In a similar strategy, 4-mercaptoethanol-phosphoric acid (4-MPBA) was immobilized by drop-casting on a SPCE working electrode modified with an electrodeposited layer of gold  
Figure 2: Pictorial representation of broadly used boronic acid-assisted electrochemical sensors for HbA1c detection. nanofluowers (AuNF).