# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Nanostructured Carbon Substrates
#### 3.2.1 Carbon Nanotubes  
\(\bullet\) Enzyme-Based Immunosensors  
In 2013, [PERSON] et al. [32] described an electrochemical immunosensor for enterobacterial detection using a carbon SPE modified by multi-walled carbon nanotubes (MWCNTs)/alginate/chitosan composite onto which HRP-labeled capture antibodies were immobilized. Detection of the Ag/Ab binding was transduced using CV with thionine (Thi) and H\({}_{2}\)O\({}_{2}\) diffusing in solution, without the use of any secondary antibody (Figure 5A,B). They found a linear range from \(10^{4}\) to \(10^{10}\) CFU mL\({}^{-1}\) and a LoD of \(5\times 10^{3}\) CFU mL\({}^{-1}\). [PERSON] et al. [33] reported the use of conventional oxidized CNTs immobilized on polyethyleneimine to bind anti-cTnT (cardiac troponin T, a diagnostic biomarker for myocardial infarction or heart muscle cell death) capture antibodies. After a conventional sandwich assay with a HRP-labelled anti-cTnT and upon addition of H\({}_{2}\)O\({}_{2}\) in solution, they measured the cathodic peak current of Fe(CN)\({}_{6}\)\({}^{3-/4-}\) also added in solution and achieved a LoD of 0.033 ng mL\({}^{-1}\) and a linear range between 0.1 and 10 ng mL\({}^{-1}\). In 2016, still using a diffusing redox probe and not an immobilized one, [PERSON] et al. [34] described an electrochemical immunosensor for detection of a mycotoxin (aflatoxin B\({}_{1}\)) based on SWCNT/chitosan electrodes, using a conventional indirect competitive binding with an AlkP-labelled secondary IgG antibody. AlkP was used to catalyze the hydrolysis of \(\alpha\)-naphthyl phosphate (added in solution), which in turn produced an electrochemical signal at the electrode. Using DPV, they found a linear response between 0.01 and 100 ng mL\({}^{-1}\), with a LoD of 3.5 pg mL\({}^{-1}\) for aflatoxin B\({}_{1}\). At last for immunoassay using diffusing redox probes, [PERSON] et al. [35] reported in 2016 an electrochemical immunosensor for the determination of Transforming Growth Factor \(\beta\)1 cytokine using MWCNT-modified SPE. MWCNTs were functionalized by azide-alkyne click chemistry for covalent coupling of alkyne-functionalized anti-TGF. The target was detected through sandwich immunoassay with HRP-labeled anti-TGF. The affinity reaction was monitored amperometrically at \(-0.20\) V using the HQ/H\({}_{2}\)O\({}_{2}\) system. Linearity was obtained between 5 and 200 pg mL\({}^{-1}\) and the LoD was 1.3 pg mL\({}^{-1}\). It could be more pertinent not to add diffusing redox probes in solution, for the sake of simplicity, reproducibility, or simply to make the sensors compatible with in-situ measurements for which redox mediators could not be added to the analyzed medium. This is what [PERSON] et al. [36] reported using thionine as redox probe integrated on the electrode substrate. They developed a sensor for detection of prostate specific antigen (PSA) based on immobilization of a PSA antibody using a composite of MWCNTs and an ionic liquid (1-butyl-methylpyrrolidinium bis(trifluoromethyl-sulfonyl)imide, [C\({}_{4}\)mpyr][NTf\({}_{2}\)]). Anti-PSA was immobilized in this composite as well as thionine (Thi), and HRP-labeled anti-PSA was used in a sandwich type immunoassay with H\({}_{2}\)O\({}_{2}\) as substrate. Using DPV to oxidize Thi at the electrode, the linear range was between 1 and 40 ng mL\({}^{-1}\), with a  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Target** & **Probe** & **LoD** & **Method** & **Reference** \\ \hline HE4 & Ag & 0.06 ng L\({}^{-1}\) & DNA/DNA  
LoD of 20 pg mL\({}^{-1}\). More recently, [PERSON] et al. [37] reported a poly-L-lysine/SWCNT-modified electrode coated with Prussian blue (BP) for \(\alpha\)-fetoprotein detection. Poly-L-lysine is a positively charged synthetic polymer of L-lysine containing amino groups able to bind bioactive materials onto an electrode surface, or to link some active groups such as epoxy groups, hydroxyl groups, or carboxyl groups present on the SWCNTs surface. All these components were crosslinked with glutaraldehyde to stabilize the interface.