# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Magnetic Nanoparticles
#### 3.5.2 Enzyme-Free Immunosensors  
In 2012, [PERSON] et al. reported a simple way to monitor cancer circulating cells (CTCs) using nanoparticles [153]. They combine capturing antibody-functionalized magnetic beads (MBs) and labeling through antibody-modified AuNPs (antibody: anti-EpCAM, Epithelial Cell Adhesion Molecule) able to electrocatalytic the hydrogen evolution reaction (HER). A LoD of 2.2 \(\times\) 10\({}^{2}\) cells was achieved for human colon adenocarcinoma cells (CaCo2) (Figure 28), without the need for a reactant to be added in solution. [PERSON] et al. [154] reported in 2013 a non-enzymatic sandwich-type magnetic electrochemical immunosensor for determination of aflatoxin B1. They used gold-coated Fe\({}_{3}\)O\({}_{4}\) magnetic MBs functionalized with both 3-((2-mercaptoethanol)methyl)benzene-1,2-diol and the specific aflatoxin antibody. The GC working electrode was also modified with the capture anti-aflatoxin B\({}_{1}\). Binding of the antigen brought the modified MBs on the electrode surface. Oxidation of the immobilized benzene-1,2-diol (catechol moiety) was used as redox indicator using DPV (Figure 29). Aflatoxin B\({}_{1}\) was detected within the range 0.6-110 ng mL\({}^{-1}\), with a LoD of 0.2 ng mL\({}^{-1}\). Also in 2013, [PERSON] et al. [155] described a non-competitive _E. coli_ immunosensor using MB-modified capture antibodies for concentration of _E. coli_ from the solution bulk onto a nanoporous alumina membrane. Quantification of _E. coli_ was made by monitoring the membrane impedance after collection. This method gave a LoD of 10 CFU mL\({}^{-1}\). Still for _E. coli_ detection, [PERSON] et al. [156] described an immunosuppressive assay based on the electrocatalytic properties of gold nanoparticles (AuNPs) towards hydrogen evolution, combined with superparamagnetic microbeads (MBs) as capture pre-concentration and purification platform. After capture and sandwiching with secondary antibody-modified AuNPs, transduction was made using chronoamperometry on SPCEs. LoDs of 148, 457 and 309 CFU mL\({}^{-1}\) were obtained in buffer solution, minced beef and tap water samples, respectively (Figure 30). Figure 28: Electrochemical detection of Caco2 cells using MBs/antiEpCAM for selective capture. **(a,b)** Effect of the number of cells on the analytical signal for different target (Caco2) and control cells (THP-1) proportions for **(a)** simultaneous labeling with AuNPs/antiEpCAM and **(b)** simultaneous labeling with AuNPs/anti-CEA. 100% corresponds to a cell amount of 5 \(\times\) 104; **(c)** Calibration plot for an increasing number of Caco2 cells (in the absence of THP-1) with simultaneous labeling with AuNPs/anti-CEA. Reprinted with permission from [153]. Copyright 2012 American Chemical Society. More classically, [PERSON] et al. [157] described Pt-Fe\({}_{3}\)O\({}_{4}\) MBs for detection of squamous cell carcinoma antigen (SCC-Ag). Curiously, Pt/Fe\({}_{3}\)O\({}_{4}\) MBs were not used for their magnetic properties but to enhance the catalytic electroreduction of H\({}_{2}\)O\({}_{2}\) added in solution. Nitrogen-doped graphene sheets were used to immobilize anti-SCC capture antibodies whereas signaling antibodies were adsorbed onto the Pt/Fe\({}_{3}\)O\({}_{4}\) NPs. Using a sandwich type immunoassay, the sensor gave a linear range between 0.05 and 18 ng mL\({}^{-1}\), with a LoD of 15 pg mL\({}^{-1}\). In 2014, [PERSON] et al. [158] also reported a non-enzymatic  
Figure 30: Schematic experimental design. **(A)**_E. coli_ was captured by MBs modified with primary antibodies, and subsequently labelled with AuNPs carrying secondary antibodies; **(B)** Left: detection of labeled _E. coli_ through the hydrogen evolution reaction electrocatalyzed by the AuNP labels. Right: Chronoamperograms registered in 1 M HCl, during the HER applying a constant voltage of 1.0 V for increasing _E.