# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Metal or Metal Oxide Nanoparticles
#### 3.4.2 Enzyme-Free Immunosensors
0.4 ng L\({}^{-1}\)). Detection of more classical antigens were also reported with metal or metal oxide-based immunosensors, such as CEA, AFP or PSA. For example, in 2012, [PERSON] et al. [127] described a triple signal amplification strategy for immunosensing of CEA using AuNPs-decorated poly(styrene-co-acrylic acid) microbeads, onto which signaling antibodies were immobilized. On the other side, the working electrode, a chitosan/RGO-modified GC electrode, was functionalized by the capture antibody. After sandwich-type immunoreaction, the anchored AuNPs further induced the chemical deposition of silver for electrochemical stripping analysis of target antigen (Figure 23B). This method allowed a linear range from 0.5 pg mL\({}^{-1}\) to 0.5 ng mL\({}^{-1}\) and a LoD of 0.1 pg mL\({}^{-1}\). [PERSON] et al. [128] also described two CEA immunosensors using silver enhancement on AuNPs. The first proposed approach was a label-free impedimetric immunosensor based on polyanthranilic acid-modified graphite SPE where anti-CA 125 were immobilized. Using DPV, without amplification, a LoD of 8 U mL\({}^{-1}\) was reported. The second approach was based on a sandwich format where a secondary anti-CA 125 antibody, labeled with AuNPs, was added to induced the Ag deposition from a silver solution. The quantity of AgNPs was measured by ASV, which gave a LoD of 2 U mL\({}^{-1}\). [PERSON] et al. [129] reported a more classical approach with the use of a poly(2-aminothiophen)-modified GC electrode to immobilize AuNPs onto which anti-CEA was immobilized. With Fe(CN)\({}_{6}\)\({}^{3-/4-}\) as redox probe diffusing in solution, DPV was used to characterize the recognition of CEA on its antibody. The immunosensor displayed a linear response with CEA from 1 fg mL\({}^{-1}\) to 10 ng mL\({}^{-1}\) and a LoD of 0.015 fg mL\({}^{-1}\). Still using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) as diffusing redox probe, [PERSON] et al. [130] described an immunosensor for CEA where AuNPs were grafted on a GC electrode by the \"double\" diazonium route already proposed by [PERSON] et al. the same year, as illustrated on Figure 24A. Anti-CEA was directly adsorbed on these AuNPs. Using CV, the linear detection range was from 10 fg to 100 ng mL\({}^{-1}\) with a LoD of 3 fg mL\({}^{-1}\). These two latter works demonstrated very low detection limits, significantly lower than that reported for more evolved detection schemes. In particular, a LoD of 0.014 fg mL\({}^{-1}\) for CEA (of around 200 kD) is equivalent to 7 \(\times\) 10\({}^{-23}\) mol mL\({}^{-1}\). At this level of concentration, one has 40 molecules per mL, which impose a particular attention to the experimental conditions. Still for CEA detection, [PERSON] et al. [131] reported porous PtNPs able to absorb metal ions used for their electrochemical signals. PtNPs were capped with 2-aminoethanethiol then complexed with Cd\({}^{2+}\) and Cu\({}^{2+}\) to form PtNPs-Cd\({}^{2+}\) and PtNPs-Cu\({}^{2+}\) hybrids. Anti-CEA and anti-AFP were labeled with these hybrids and used in a sandwich immunoassay. Using DPV for reduction of the metal ions present on the electrode, the authors could identify and quantify antibodies. A linear response was measured from 0.05 ng mL\({}^{-1}\) to 200 ng mL\({}^{-1}\) for both CEA and AFP, with LoDs of 2 pg mL\({}^{-1}\) for CEA and 50 pg mL\({}^{-1}\) for AFP. Also for detection of AFP, [PERSON] et al. [132] described Pd nanoplates onto which AFP antibodies were immobilized, this assembly itself immobilized on a GC electrode. Using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) in solution as redox probe, AFP was detected from 0.01 to 75.0 ng mL\({}^{-1}\) with a LoD of 4 pg mL\({}^{-1}\). [PERSON] et al. [133] reported an immunosensor based on Chi-poly(acrylic acid) nanospheres doped with various metal ions such as copper, cadmium, lead and zinc ions. In the same spirit as reported above with porous PtNPs, those nanospheres were used to graft antibodies and to produce an electrochemical signal by reduction of ions into the corresponding metals at the working electrode, using SWV.