# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Diffusing Redox Probes  
[PERSON] et al. [52] described in 2012 an immunosensor for \(\beta\)-lactoglobulin (a milk antigen) using graphene-modified SPE. They demonstrated electrografting of an aryl diazonium salt on graphene as substrate, for covalent grafting of the \(\beta\)-lactoglobulin capture antibodies through a Schiff base reaction (Figure 9). CV and DPV were carried out using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) added in solution to characterize Ab/Ag association. Currents decreased linearly with increasing the concentration of \(\beta\)-lactoglobulin due to the steric hindrance generated by the antibody-antigen complex on the modified electrode surface. With this classical transduction scheme, the LoD was 0.85 pg mL\({}^{-1}\) and the dynamic range was from 1 pg mL\({}^{-1}\) to 100 ng mL\({}^{-1}\). In 2013, [PERSON] et al. [53] reported an enzyme-free immunosensor for detection of \(\alpha\)-fetoprotein (AFP), using a sandwich-like format with catalytic AuPdNPs-labeled antibodies and \(N\)-doped graphene for grafting of the capture antibody on the electrode. AuPdNPs were used for their catalytic properties towards H\({}_{2}\)O\({}_{2}\) added in solution. The LoD was 5 pg mL\({}^{-1}\) and the linear range between 0.05 and 30 ng mL\({}^{-1}\). Still in 2013, [PERSON] et al. [54] also described a label-free amperometric  
Figure 9: (**A**) Electroreduction of a diazonium salt on a SPE, for covalent immobilization of \(\beta\)-lactoglobulin antibodies; (**B**) DPVs of the immunosensor incubated with different concentrations of \(\beta\)-lactoglobulin (inset: calibration curve); (**C**) Comparison of the DPV response of the graphene-modified electrodes to 1000 ng mL\({}^{-1}\) ovalbumin, BSA, casein, lysozyme, and 100 ng mL\({}^{-1}\)\(\beta\)-lactoglobulin. Reprinted from [52], Copyright 2012, with permission from Elsevier. immunosensor for AFP detection, based on a TiO\({}_{2}\)/graphene/chitosan/AuNPs film deposited on a GC electrode. Antibodies were immobilized by adsorption on the AuNPs. The Ab/Ag interaction at the electrode interface generated a steric hindrance, resulting in the decrease of DPV signals when Fe(CN)\({}_{6}\)\({}^{3-/4-}\) was added in solution. The detection range was between 0.1 and 300 ng mL\({}^{-1}\) and the LoD of 0.03 ng mL\({}^{-1}\). Again, for AFP detection, [PERSON] et al. [55] inserted anti-AFP and SWCNTs inside the channels of mesoporous silica (MPS) and immobilized this MPS/SWCNTs/anti-AFP assembly on graphene using a layer-by-layer approach. Capture of AFP was probed by DPV using ferroceencarboxylic acid diffusing in solution. The authors reported detection of AFP within a linear range from 0.1 to 100 ng mL\({}^{-1}\) and a LoD of 0.06 ng mL\({}^{-1}\). In 2015, [PERSON] et al. [56] described a GR/SnO\({}_{2}\)/Au nanocomposite used to immobilize anti-AFP. Using Ru(NH\({}_{3}\))\({}_{6}\)\({}^{3+}\) as redox probe added in solution, the authors demonstrated that the DPV peak currents decreased due to the interaction between Ab and Ag on the electrode. AFP was quantified between 0.02 and 50 ng mL\({}^{-1}\), with a LoD of 0.01 ng mL\({}^{-1}\). In 2016, last example for AFP detection, [PERSON] et al. [57] presented a sandwich-type electrochemical immunosensor where AuNPs were electrodeposited on GCE to bind capture anti-AFP. Graphene oxide functionalized with CeO\({}_{2}\)NPs and PdNPs was utilized as labels for labelling secondary anti-AFP. PdNPs played the role of catalyst towards H\({}_{2}\)O\({}_{2}\) reduction. The immunosensor exhibited a linear range from 0.1 pg mL\({}^{-1}\) to 50 ng mL\({}^{-1}\) with a LoD of 0.033 pg mL\({}^{-1}\). Back in 2013, [PERSON] et al. [58] reported label-free detection of carbohydrate antigen 15-3 (CA 15-3), a biomarker of breast cancer. Capture antibodies were immobilized on N-doped graphene-modified electrodes.