# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Metal or Metal Oxide Nanoparticles
#### 3.4.1 Enzyme-Based Immunosensors  
There is only one example of antibody sensor, published in 2013. [PERSON] et al. [104] described a signal-amplified sensor for IgG using TiO\({}_{2}\) nanotube array, for their large surface area, high pore volume and good electrochemical conductivity. AuNPs were functionalized with HRP-tagged antibodies and immobilized on the nanotubes. This architecture allowed for a higher surface concentration of HRP-labelled Ab compared to flat electrodes, which led to higher amplified electrochemical signals from the catalytic reaction of HRP relative to hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) and its electroreduction of the AuNPs. It exhibited a detection range from 0.1 to 10\({}^{5}\) ng mL\({}^{-1}\) with a LoD of 0.01 ng mL\({}^{-1}\). All other works described immunosensors for antigens, mostly proteins. In 2013, [PERSON] et al. [105] described bimetallic AuPt nanochains (NC) onto which HRP-conjugated anti-CEA signaling antibodies were attached, giving \"hairy\" enzyme-labeled nanoparticles (HRP-anti-CEA-NCAuPt). Detection of CEA was performed in a sandwich-type immunoassay format, with H\({}_{2}\)O\({}_{2}\) as enzyme substrate and Prussian Blue (PB) as mediator, both added in solution. PB was immobilized under a AuNPs layer on the electrode (Figure 19A). The linear detection range was 0.01-200 ng mL\({}^{-1}\) and the LoD 0.11 pg mL\({}^{-1}\). Figure 19: **(A)** (**a**) Preparation of the HRP-anti-CEA-AuPt nanochains as label; (**b**) sandwich-type recognition; (**c**) transduction. (**B**) Corresponding LSV (I) and calibration (II) curves for CEA at different concentrations in PBS + 0.8 mM H\({}_{2}\)O\({}_{2}\). Reprinted from [105], Copyright 2013, with permission from Elsevier. (**C**) Detection of CEA with AuNPs-Ab\({}_{2}\)-GOx as label. (**D**) EIS (III) for CEA at different concentrations: (a) 5, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50, (g) 60, (h) 70 and (i) 80 ng mL\({}^{-1}\) in 0.1 M KCl + 5.0 mM Fe(CN)\({}_{6}\)\({}^{3-/4-}\); (IV) corresponding calibration curve. Reprinted from [106], Copyright 2016, with permission from Elsevier. [PERSON] et al. [106] described more recently a CEA immunosensor, based on an original transduction scheme based on a sandwich architecture. Signaling antibodies were modified with AuNPs, GOx and concanavalin A and macroporous carbon modified with capture antibodies as signal collector (substrate) (Figure 19C). Concanavalin A was used because its surface charge varies significantly with pH. Therefore, when Fe(CN)\({}_{6}\)\({}^{3-/4-}\) was used as redox mediator in solution, the GOx activity, generating local acidification, changed the neat charge of the NPs and repulsed the redox probe from the NPs surface, therefore lowered the current. Using EIS, the charge transfer resistance changed proportionally toward CEA concentration from 4 pg mL\({}^{-1}\) to 50 ng mL\({}^{-1}\) with a LoD of 1.3 pg mL\({}^{-1}\). [PERSON] et al. [107] presented also in 2016 an electrochemical immunosensor for detection of three tumor markers: CA125, CEA, and PSA. They used as substrate a temperature-responsive polymer (poly(N-isopropylacrylamide--PNIPAAm) for temperature-induced regeneration of the electrode/electrolyte interface (Figure 20A). HRP-labeled antibodies were coupled to polypyrrole NPs for signaling. A linear range from the pg mL\({}^{-1}\) to 100 ng mL\({}^{-1}\) was reported, with LoDs in the pg mL\({}^{-1}\) range depending on the target. [PERSON] et al. [108] also reported a sensor for PSA. The paper-based device was fabricated by sequentially growing AuNPs on cellulose fibers, then MnO\({}_{2}\) nanowires, to form a 3D network with a large surface area. GOx was used as enzyme label and 3,3\({}^{\prime}\),5,5\({}^{\prime}\)-tetramethylbenzidine (TMB) as a redox mediator. The linear detection range, obtained by amperometry at \(-\)0.2 V, was between 5 pg mL\({}^{-1}\) and 100 ng mL\({}^{-1}\) with a LoD of 1 pg mL\({}^{-1}\). Detection of AFP was also reported. For example, [PERSON] et al.