# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Nanostructured Carbon Substrates
#### Other Carbon Materials  
CNTs are not the only carbon-based nanoparticles. For example, [PERSON] et al. [44] reported an immunosensor using carbon nanospheres and AuNPs, which were used for labeling secondary antibodies in a sandwich-type immunoassay format. For transduction, these AuNPs were electro-oxidized to produce AuCl\({}_{4}\)\({}^{-}\) at the electrode surface, detected by DPV. The high-loading capability of AuNPs on carbon nanospheres led to obvious signal amplification. Using human immunoglobulin G (IgG) as model target, they obtained a linear dependence on the logarithm of target concentration ranging from 10 pg mL\({}^{-1}\) to 10 pg mL\({}^{-1}\), with a LoD of ca. 10 pg mL\({}^{-1}\). No external reactants were necessary. Using carbon nanohorns instead of nanospheres, [PERSON] et al. [45] reported a sensor for determination of 8-isoprostane, a biomarkers of lipid peroxidation in the human body, derived of essential fatty acids (Figure 7A,B). A competitive immunoassay involving HRP-labeled 8-isoprostane was designed and detection was based on the steric hindrance generated by bound 8-isoprostane on the electrode surface, expected to impede diffusion of the redox species (i.e., H\({}_{2}\)O and BQ). A linear response was obtained up to 700 pg mL\({}^{-1}\) with a LoD of 12 pg mL\({}^{-1}\). Figure 6: (**A**) SWNT-modified GC electrodes for detection of endosulfan; (**B**) SWVs for GC-Ph-NH\({}_{2}\)/SWNT/PEG/FDMA/endosulfan/anti-endosulfan electrode after incubation in with 0, 0.02, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, and 4 ppb endosulfan, respectively. Adapted with permission from [40]. Copyright 2012 American Chemical Society; (**C**) Immunosensing architecture used for tissue transglutaminase. 3-IP: 3-indoxyl phosphate disodium salt; (**D**) CVs obtained for anti-tTG at various concentrations. _Inset_: relationship between the peak current and the anti-tTG concentration. Reprinted from [42], Copyright 2012, with permission from Elsevier. Figure 7: _Cont._  
Another procedure, which guaranties diffusion hindering (then efficient transduction) independently of the size of the targets and probes, was interestingly reported by [PERSON] et al. [46] who described detection of \(\alpha\)-fetoprotein (AFP) with the use of single-walled carbon nanohorms. Based on a sandwich-type immunoreaction, a bienzymatic (HRP and GOx) cascade was used for transduction and amplification (Figure 7C,D). 4-Chloro-1-naphthol, used a redox cosubstrate for HRP, was catalytically oxidized by H\({}_{2}\)O\({}_{2}\) to yield to an insoluble product on the electrode surface, which was probed with Fe(CN)\({}_{6}\)\({}^{4-7/3-}\) using CV and EIS. The sensor showed a wide linear range from 1 pg mL\({}^{-1}\) to 60 ng mL\({}^{-1}\) and a LoD of 0.33 pg mL\({}^{-1}\). Recently (2016), [PERSON] et al. [47] showed that such nanocarbon structured could be featured into integrated devices. Indeed, they described a multiplexed electrochemical immunosensor for label-free detection of three cardiac markers (C-reactive protein, cardiac troponin-I and myoglobin) using carbon nanofibers as electrodes. Carbon nanofibers were grown vertically using plasma enhanced chemical vapor deposition (PE-CVD), onto which capture antibodies were coupled by conventional carbodiimide chemistry. The complexation of the cardiac markers on the corresponding antibodies were characterized using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) as redox probe (Figure 8A). No detection limits were given but this device finds its originality in the nanofibers displayed in a relatively dense array (however not electrochemically addressable individually), providing a very high surface area for Ab immobilization. Figure 8: **(A)** Biosensor chip for CRP, cTnT and myoglobin; **(a)** SEM image of an array of 9 electrodes, and **(b)** of an individual electrode; **(c)** AFM image of an array carbon nanofibers (bright dots); **(d)** AFM profile of the carbon nanofibers height; **(e)** 3D AFM micrograph after antibody immobilization.