# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Conventional Electrode Substrates
#### 3.1.2 Enzyme-Free Immunosensors  
[PERSON] et al. [21] developed gold SPE electrodes modified with specific thiolated antibodies for direct detection of infection biomarkers, using electrochemical impedance spectroscopy (EIS), more particularly measuring the charge transfer resistance of FeCN\({}_{6}\)\({}^{3-7/4-}\) on the gold electrode depending on the presence or not of the targeted antigen (TREM-1, MMP-9 and HSL). Limits of detection were between the pM and the nM range, depending on the target. Also with an enzyme-free transduction procedure, [PERSON] et al. [22] described a label-free electrochemical competitive  
Figure 2: **(A)** Reactions involved in the ACTH immunosensor using SPEs modified with phenylboronic acid; **(B)** DPV obtained with the Strept-AlkP-Biotin-ACTH/anti-ACTH/SPCE electrode for 0 (1), 0.05 (2), 0.50 (3), and 1.00 (4) pg mL\({}^{-1}\) ACTH. Reprinted from [16], Copyright 2012, with permission from Elsevier; **(C)** Above, scheme of the immunosensor principle for SA and TC antibiotics. Below, surface chemistry involved for covalent binding of Protein G; **(D)** Calibration curves obtained with the SPY/TC immunosensor for SPY and TC in a 1:1 PBS:milk mixture. Reprinted from [17], Copyright 2013, with permission from Elsevier. immunosensor based on an electroactive conducting polymer coupled with a molecule close to atrazine (a common pesticide) (Figure 3A,B). This quinone-based polymer presented a current decrease following anti-atrazine antibody complexation, and a current increase after atrazine addition in solution, with a very low detection limit of 1 pM, i.e., 0.2 pg mL\({}^{-1}\) estimated by square wave voltammetry (SWV). One originality relies on the fact that the redox probe is not diffusing in solution but immobilized on the electrode surface, and another originality is the competitive equilibrium between an immobilized mimic of the target (so-called hapten) and the diffusing target to detect. Compared to many other examples in the literature, it should be noted that this non-amplified method gave an electroactivity increase upon recognition of the target. The same year (2012), a similar idea was developed by [PERSON] et al. [23] They reported an electrochemical immunosensor for detecting glycosylated hemoglobin (HbA1c) based on glassy carbon (GC) electrodes modified with a mixed layer of oligo(phenylethylene) and oligo(ethylenediylcol), obtained by electrografting of the corresponding aryldiazonium salts. 1,1\({}^{\prime}\)-Di(aminomethyl)ferrocene and an epitope a pentapeptide, glycosylated-VHLTP (GPP) were covalently attached to oligo(phenylethylene) (GPP is a peptide mimetic to HbA1c, to which an anti-HbA1c antibody could bind). As for [PERSON] et al. HbA1c was detected by a competitive assay based on the competition for binding to anti-HbA1c between the analyte in solution, HbA1c, and the surface bound GPP peptide. However, exposure of the GPP-modified interface to the mixture of anti-HbA1c IgG antibody and HbA1c resulted in the attenuation of Fc electroactivity due to steric hindrance generated by the antibody bound to the surface (Figure 3D,E), and not to an increase in electroactivity as reported by [PERSON] et al. The authors found that HbA1c could be detected from 4.5% to 15.1% of total hemoglobin in serum. The same authors, the same year, adapted this method to AuNPs-modified surfaces (reference cited later in the text). Still in order to avoid addition of a diffusing redox probe in solution, [PERSON] et al. [24] reported later a similar approach, based on an electroactive polymer onto which an antibody was coupled, to detect bisphenol A (BPA) by competitive binding assay with a detection limit of 2 pg mL\({}^{-1}\) using SWV. A current decrease was obtained upon anti-BPA binding and an opposite current increase upon BPA addition in solution. The same authors described a similar approach for detection of acetaminophen. [25] The detection limit was ca. 10 pM (1.5 pg mL\({}^{-1}\)).