# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Diffusing Redox Probes
Upon recognition of the carbohydrate antigen and using DPV to probe the electroactivity of Fe(CN\({}_{6}\))\({}^{3-/4-}\) added in solution, a LoD of 12 mU mL\({}^{-1}\) was achieved, and a linear range between 0.1 and 20 U mL\({}^{-1}\). In 2014, [PERSON] et al. [59] reported an immunosensor for human chorionic gonadotropin (hCG), a key hormone for pregnancy diagnostic. Multi-layer epitaxial graphene (MEG) was grown on SiC substrates, then patterned using electron beam lithography to make channels. These short channels were then functionalized with 3-aminopropyltriethoxysilane (APTES) to covalently bind the anti-hCG antibody (Figure 10A,B). Detection was made by monitoring the redox current of diffusing Fe(CN\({}_{6}\))\({}^{3-/4-}\) on the graphene sheet, or by monitoring changes in the channel resistance upon exposure to hCG. The LoD was 0.62 ng mL\({}^{-1}\) and the linear response was in the range 0.6-6 ng mL\({}^{-1}\). In 2015, [PERSON] et al. [60] used AuNPs-modified graphene to bind PSA antibodies. CV was used with Fe(CN\({}_{6}\))\({}^{3-/4-}\) as diffusing redox probe to quantify PSA binding on immobilized anti-PSA. The LoD was 0.6 ng mL\({}^{-1}\) and the linear range was obtained for up to 10 ng mL\({}^{-1}\). Also in 2015, [PERSON] et al. [61] described an original paper substrate modified by reduced graphene oxide (RGO), ZnO nanorods and capture antibodies. They used a sandwich assay where the signaling antibodies were coupled to graphene sheets and AgNPs. With H\({}_{2}\)O\({}_{2}\) as redox probe and CV for measuring H\({}_{2}\)O\({}_{2}\) electroreduction, the authors demonstrated a linear response between 1 pg mL\({}^{-1}\) and 110 ng mL\({}^{-1}\). This sensor was also employed for human chorionic gonadotropin (linearity between 2 uU mL\({}^{-1}\) and 120 mU mL\({}^{-1}\)) and to carcinoembryonic antigen (CEA) (linearity between 1 pg mL\({}^{-1}\) and 100 ng mL\({}^{-1}\)). In 2016, [PERSON] et al. [62] also reported PSA detection but for a sandwich-type device. AuNPs-decorated APTES-functionalized graphene sheets (Au/APTES/GR) were employed as substrate, and Cu\({}_{2}\)O NPs decorated with ferrocene were employed as label. Cu\({}_{2}\)ONPs presented a good electrocatalytic towards Fc and hydrogen peroxide (added in solution) electrooxidation and electroreduction, respectively (Figure 10C,D). The linear range was between 0.05 and 100 pg mL\({}^{-1}\) and the LoD of 0.05 pg mL\({}^{-1}\). Last example of PSA detection, [PERSON] et al. [63] reported in 2017 AgNPs-functionalized RGO for immobilization of anti-PSA antibodies. Using CV with FeCN\({}_{6}\)\({}^{3-/4-}\) as diffusing redox probe (Figure 11A), the authors demonstrated peak current changes which varied linearly with PSA concentration between 1 and 1000 ng mL\({}^{-1}\), with a LoD of 0.01 ng mL\({}^{-1}\). Also using Fe(CN\({}_{6}\))\({}^{3-/4-}\) as redox probe, [PERSON] et al. [64] reported RGO sheets decorated by cysteine and CuONPs deposited on Au electrode, for detection of _Escherichia coli_ O157: H7. With EIS to measure changes in electron transfer resistance, they found a detection range from 10 CFU mL\({}^{-1}\) to 10\({}^{8}\) CFU mL\({}^{-1}\) and a LoD of 3.8 CFU mL\({}^{-1}\). [PERSON] et al. [65] described a sandwich-type electrochemical immunosensor for detection of squamous cell carcinoma antigen (SCCA). Graphene nanosheets were immobilized on the electrode surface and functionalized with \(\beta\)-cyclodextrintrin, used to immobilize capture antibodies. Signaling antibodies were modified with Pt/PdCu nanocubes anchored on graphene sheets. Pt/PdCu exhibited high electrocatalytic activity toward the reduction of H\({}_{2}\)O\({}_{2}\) added in solution (Figure 11B). The results showed linearity between 0.1 pg mL\({}^{-1}\) and 1 ng mL\({}^{-1}\), with a LoD of 25 fg mL\({}^{-1}\). Pd/V\({}_{2}\)O\({}_{5}\) also presents a catalytic activity towards H\({}_{2}\)O\({}_{2}\) reduction, demonstrated by [PERSON] et al. [66], in 2016, for a non-enzymatic sandwich-type immunosensor for carcinoembryonic antigen (CEA) detection.