# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Nanostructured Carbon Substrates
#### 3.2.1 Carbon Nanotubes
Immunosensing was measured based on the catalytic activity of the HRP with H\({}_{2}\)O\({}_{2}\) added in solution and BP acting as mediator immobilized on the electrode surface. Using DPV to recycle Prussian Blue, the authors found that peak current was linearly related to \(\alpha\)-fetoprotein in the range 0.05-10.0 ng mL\({}^{-1}\), with a LoD of 10 pg mL\({}^{-1}\). In the same spirit, [PERSON] et al. [38] described cobalt phthalocyanine (CoPc)-functionalized MWCNTs as label for signaling antibodies (Ab\({}_{2}\)) in a sandwich-type immunosensor, for detection of procalcitonin (a peptide biomarker of severe sepsis). The originality and pertinence of this approach was that the electrochemical signal directly originates from the CoPc without the addition or any redox mediator in solution or any label, which makes such kind of tranduction scheme more efficient for a real application. However, to enhance sensitivity, choline oxidase (ChOx) was added on the electrode. H\({}_{2}\)O\({}_{2}\) produced by this enzyme was catalytically oxidized by CoPc, resulting in a signal amplification (Figure 5C,D). Using DPV, they reported linearity from 0.01 to 100 ng mL\({}^{-1}\) and a LoD of 1 pg mL\({}^{-1}\), but choline had to be added in solution, which did not make this system reactant-free. * Enzyme-Free Immunosensors  
For enzyme-free detection of aflatoxin-B\({}_{1}\), [PERSON] et al. [39] reported a method of functionalization of an ITO electrode by electrophoretic deposition of MWCNTs (lying flat on the electrode surface) onto which aflatoxin antibodies were grafted. Using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) as redox probe diffusing in  
Figure 5: **(A)** Preparation of the immunoelectrode, and reactions occurring at the electrode surface for determination of E. sakazakii and E. coli O157:H7. Thi(ox) and Thi(red) are the oxidized and reduced forms of thionine, respectively; **(B)** CVs after incubation with _E. sakazakii_ (10\({}^{10}\) cfu mL\({}^{-1}\)) (left) and _E. coli_ O157:H7 (right). Curves a1 and b1 were obtained before incubation; curves a2 and b2 were obtained after incubation. Reprinted from [32], Copyright 2013, with permission from Elsevier; **(C)** **(a)** Preparation procedure of the ChOx/Ab\({}_{2}\)/CoPc-MWCNTs bioconjugates; **(b)** Transduction and amplification mechanisms; **(D)** DPV responses after incubation with PCT (from 0.01 to 100 ng mL\({}^{-1}\)), and corresponding calibration curve of the anodic peak current. Reprinted from [38], Copyright 2016, with permission from Elsevier. solution and CV as electrochemical technique, their immunosensor was sensitive to the association of the aflatoxin-B1 on the immobilized Ab, showing a LoD of 0.08 ng mL\({}^{-1}\) and a linear detection range between 0.25 and 1.4 ng mL\({}^{-1}\). The most interesting example, however, was given by [PERSON] et al. [40] in 2012. They reported a sensor for detection of endosulfan (an organochlorine insecticide) by electrografting of a mixed layer of 4-aminophenyl and phenyl with the aryldiazonium route, for covalent grafting of SWCNTs on conventional GC electrodes. They also used the diazonium electrografting route for functionalization of these SWCNTs with anti-adsorption chains such as poly(ethylene glycol) (PEG). Ferrocendimethylamine was attached to the upper end of SWNTs through amide bonding followed by the attachment of the endosulfan hapten to which an antibody bind (Figure 6A,B). Association/dissociation of the antibody on the sensing interface causes a modulation of the ferrocene electroactivity. SWV was used to sense this electroactivity change, with a linear detection range of ca. 0.01-20 pg mL\({}^{-1}\). It must be emphasized that, through this competitive assay, without the use of any diffusing redox probe in solution nor additional reactant, they achieved a excellent LoD of 0.01 pg mL\({}^{-1}\); this approach certainly paved the way for enzyme-free, reactant-free, diffusion-free immunosensors.