# Current Status of HbA1c Biosensors
## 2 HbA1c Biosensors
### Antibody-Modified Biosensors
#### 2.2.3 Immobilization of HbA1c Antibody  
The immobilization of the anti-HbA1c antibody on the electrode surface is the most direct and specific way to bind HbA1c (Figure 2C). By covalently immobilizing HbA1c-specific antibody on the surface of a gold working electrode treated with the SAM of 3-Mercaptopropionic acid (MPA), the binding of HbA1c was monitored by differential pulse voltammetry (DPV) using ferricyanide/ferrocyanide as the redox mediator [46]. The single-use disposable three-electrode immunosensor shows a linear range of 7.5-20 \(\upmu\)g/mL of HbA1c in 0.1 M phosphate buffer solution and 0.1-0.25 mg/mL of HbA1c in undiluted human serum. Hence, this biosensor has a great potential for application to in vitro measurement of HbA1c for diabetic management.  
[PERSON] and co-workers constructed a series of ion-sensitive field effect transistor (ISFET)-based HbA1c immunosensors composed of a FET-based sensor chip and a disposable extended-gate electrode chip by using complementary metal-oxide-semiconductor transistor (CMOS) and micro-electronic mechanical system (MEMS) techniques. Much effort has put on the method development of HbA1c/Hb antibody immobilization on the gold working electrode, including polypyrrole (PPy)-Au NPs composite film [47], mixed self-assembled monolayers (SAMs), seed mediated growth of nanogold film [48], and mixed SAMs wrapped with a nanogold array [49; 50; 51; 52]. The PPy-Au NPs composite gold electrode fabricated by electro-polymerization technique exhibits a linear response over 4\(\sim\)18 \(\upmu\)g/mL of HbA1c [47]. Although the response time is fast (less than 1 min), the stability and signal reproducibility of the sensor need further improvement.  
The mixed SAMs wrapped with nano-gold array interface for antibody immobilization are prepared as follows: (i) form two-layer thiol-containing SAMs (16- and 3-mercaptoethanolacanoic acid) on the gold nanospheres; (ii) modify the gold electrode with SAM of mercaptoethylamine; (iii) bioconjugate the mixed SAMs/Au nano-spheres with modified-Au electrode in the presence of NHS and EDC; (iv) and covalently bind the HbA1c or Hb antibodies on the mixed SAMs/Au electrode via the amide linkage in the presence of NHS and EDC.  
The mixed SAMs/Au nanospheres array-modified electrode provides several valuable features. For example, the two-layer structure of SAMs on Au nano-spheres reduces the steric hindrance for biomolecule binding. Given the uniformly distribution of nano-spheres on the electrode surface with an increased large surface area-to-volume ratio, the sensitivity and signal reproducibility of this micro immunosensor are improved. In addition, by immobilizing HbA1c and HbA antibody separately, the FET-based immunosensors can simultaneously measure HbA1c and HbA concentrations without a separation procedure which has great advantages for the development of HbA1c POC device.  
A sandwich immunoassay configuration is constructed by using specific monoclonal HbA1c antibody to capture the antigen followed with the addition of high fluorescent CdTe quantum dots (QDs)-labeled polyclonal secondary antibodies to measure HbA1c by both optical and electrochemical means [53]. The QD-based electrochemical assay showed a linear increase in current with increasing levels of HbA1c (4-16%) and a high correlation coefficient of 96% compared to that of the standardized HPLC method. Furthermore, this method requires a very small sample volume and is applicable for rapid, reproducible, and cost-effective analysis of HbA1c in clinical samples.