# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Diffusing Redox Probes
#### 3.3.2 Enzyme-Based Immunosensors
Upon addition of HRP-labeled anti-cortisol  
Figure 16: (**A**) Sandwich immunoassay for IgG detection using HRP-AuNP for oxidation of aniline into polyaniline; PANi is detected by DPV. Adapted with permission from [91]. Copyright 2014 American Chemical Society; (**B**) DPV responses of the immunosensor toward different concentrations of IgG (I) and (II) corresponding calibration curve. Curves a–g correspond to IgG concentrations from 0.01 to 500 ng mL\({}^{-1}\) (**C**) Fabrication process of HRP-Ab\({}_{2}\)/Thi/AuNPs and electrochemical transduction; (**D**) (III) Calibration curves for CEA in pH 7.0 PBS + 2 mmol L\({}^{-1}\) H\({}_{2}\)O\({}_{2}\) for different electrode materials: (a) 3D-AuNPs/GR (inset: DPV curves), (b) GR, (c) AuNPs. (IV) Interfererents and associated currents. Reprinted from [93], Copyright 2013, with permission from Elsevier. antibodies, a competition occurred, which was quantified by measuring the HRP activity by DPV (o-phenylenediamine and H\({}_{2}\)O\({}_{2}\) as substrate and co-substrate, respectively, diffusing in solution). This immunosensor displayed a detection range from 0.1 to 1000 ng mL\({}^{-1}\) and a LoD of 0.05 ng mL\({}^{-1}\). Detection of protein biomarkers was also described. [PERSON] et al. [99] reported detection of matrix metalloproteinase-2 (MMP-2) by immobilizing AuNPs on N-doped graphene sheets, onto which capture antibodies were immobilized. Signaling antibodies were coupled to GO sheets decorated by HRP-labelled signaling antibodies (Figure 17A). Upon addition of Thi and H\({}_{2}\)O\({}_{2}\) in solution, DPV was used to measure the HRP activity through Thi reduction. They found a linear range from 0.5 pg mL\({}^{-1}\) to 50 ng mL\({}^{-1}\), with a LoD of 0.1 pg mL\({}^{-1}\). These works show that two approaches could be followed to improve the sensors signal. The first approach consists in using a surface area which is increased by using a nanostructured substrate where probes are immobilized at a high surface density, then a classical labelling of secondary antibodies brings amplification. A second approach, different, tries to increase the surface concentration of the labels by immobilizing them on NPs or other nanostructures such as nanosheet; it is not the surface density of probes which is increased, but that of the redox label. In 2015, [PERSON] et al. [100] reported the detection of HIV-p24 protein (the protein envelope of HIV) using MWCNTs-silica as a matrix for HRP and capture antibodies, and GO as carrier of Thi and HRP-modified signaling antibodies. In a sandwich immunoreaction, HRP/MWCNTs/SiO\({}_{2}\) and HRP/Thi/GO captured onto the electrode surface produced an amplified electrocatalytic response by electroreduction of the Thi\({}_{\text{Ox}}\) produced by HRP. The increase of current was proportional to the HIV-p24 concentration between 0.5 pg mL\({}^{-1}\) and 8.5 ng mL\({}^{-1}\), with a LoD of 0.15 pg mL\({}^{-1}\). At last, [PERSON], [PERSON] et al. [101] presented the detection of adiponectin (APN), an hormone involved in glucose and lipid metabolism with a central role in the regulation of insulinoresistance, with a sandwich-type assay involving a metal complexes-based polymer (Mix & Go\({}^{\text{TM}}\)) for immobilization of the anti-APN capture antibody, and HRP-anti-APN as signaling antibody (Figure 17C). Using amperometry at \(-\)200 mV and the H\({}_{2}\)O\({}_{2}\)/HQ redox system in solution, they obtained a linear calibration within 0.5-10.0 ug mL\({}^{-1}\); the LoD was 60 ng mL\({}^{-1}\). There is one example of carbohydrate immunosensor. [PERSON] et al. [102] reported a sandwich-type sensor for carbohydrate antigen (CA19-9), using AuNPs-functionalized GO as substrate where capture antibodies were immobilized, and Au@PdNPs-functionalized GR as signal enhancer, bearing thionine (Thi) and HRP-labelled signaling antibodies.