# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Magnetic Nanoparticles
#### 3.5.1 Enzyme-Based Immunosensors  
In 2012, [PERSON] et al. [146] reported electrochemical immunodetection of PSA using gold-coated magnetic Fe\({}_{3}\)O\({}_{4}\) nanoparticles (Au/Fe\({}_{3}\)O\({}_{4}\)) dispersed in the analytical medium. These nanoparticles were decorated with an alkylthiol-modified PSA antibody and incubated with a medium containing PSA. After completion of this step, an HRP-labeled secondary antibody was added, then the Ab\({}_{2}\)-PSA-Ab\({}_{1}\)-NPs were magnetically collected on a gold electrode. After addition of H\({}_{2}\)O\({}_{2}\) as substrate and ferrocenemethanol as redox mediator for HRP, an amperometric current was measured at +0.15 V vs. Ag/AgCl (Figure 26A). The LoD was found to be 100 fg mL\({}^{-1}\), much lower than the LoD obtained by the same authors for planar electrodes (ca. 1 pg mL\({}^{-1}\)). Also in 2012, [PERSON] et al. [147] reported a method for detection of CEA using a combination of magnetic NPs covered by an electropolymerized poly(\(o\)-phenylenediamine) film and silver nanoparticles onto which capture antibodies were immobilized. On the other side, HRP-labelled signaling antibodies were immobilized on AuNP-modified GO sheets, onto which magnetic NPs were also immobilized. The sandwich complexation was initiated by magnetic recollection of the GO sheets (Figure 26C). Upon addition of H\({}_{2}\)O\({}_{2}\), the authors found a LoD of 1.0 pg mL\({}^{-1}\) and linearity between 10 pg mL\({}^{-1}\) and 10 pg mL\({}^{-1}\) by probing the electroactivity of the poly(\(o\)-phenylenediamine) film with DPV at pH 5. [PERSON] et al. [148] reported a direct competitive immunosensor for ochratoxin A (OCA) using magnetic beads (MBs) of ca. 1 \(\upmu\)m in diameter functionalized with OCA antibodies. Using a competitive assay with incubation in a solution containing HRP-conjugated ochratoxin A antibodies and free OCA, MBs were magnetic collected on the electrode, HQ and H\({}_{2}\)O\({}_{2}\) were added and the HRP activity was measured by DPV. The LoD was ca. 0.10 ng L\({}^{-1}\). In 2013, [PERSON] et al. [149] reported a sandwich-type electrochemical immunosensor for detection of avian leukosis virus (ALVs-I) using \(\beta\)-cyclodextrin-reinforce (\(\beta\)-CD-Fc) host-guest complex on Fe\({}_{3}\)O\({}_{4}\) MBs. Capture antibodies were coupled to graphene sheets immobilized on the working electrode whereas signaling antibodies and GOx were immobilized on the MBs along with (\(\beta\)-CD-Fc). Fc was used as redox mediator for GOx recycling (Figure 27A). Upon addition of glucose in the medium, the detection range was from 200-3000 TCID\({}_{50}\) mL\({}^{-1}\) (TCID\({}_{50}\): 50% tissue culture infective dose), with a low LoD of 150 TCID\({}_{50}\) mL\({}^{-1}\). More recently in 2016, [PERSON] et al. [150] described an impedimetric immunosensor based on the use of MBs for separation and a screen-printed interdigitated microelectrode for detection of Escherichia  
Figure 26: (**A**) Sandwich immunodetection of PSA using Ab1-Au@MNPs, magnetic collection and enzymatic transduction; (**B**) Calibration plots of reduction current versus PSA concentration using planar Ab\({}_{1}\)-modified gold electrode (black dots) and Ab\({}_{1}\)-Au@MNPs (black dots), anti-biotin immobilised on thiosctic acid modified Au@MNPs (red squares) and thiosctic acid modified Au@MNPs exposed to Ab\({}_{1}\) without EDC/NHS activation (blue triangles). Reproduced from [146] with permission from The Royal Society of Chemistry. (**C**) Steps required for (**a**) the magnetic capture of PSA using Ab\({}_{1}\)-Au@MNPs; (**b**) construction of the GO-based label; and (**c**) sandwich immunodetection of CEA; (**D**) Calibration plots of the magnetic immunoassay toward CEA standards in pH 5.3 acetic acid salt-buffered saline buffer + 3.0 mM H\({}_{2}\)O\({}_{2}\). Inset: corresponding linear and DPV curves. Reprinted from [147], Copyright 2012, with permission from Elsevier. coli and _Salmonella_ Typhimurium.