# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Conventional Electrode Substrates
#### 3.1.2 Enzyme-Free Immunosensors
These approaches present the great advantage to use a simple design, with few reactants, all immobilized on the sensing electrode. However, other more complicated design could also perform well. One of them, using DNA, was reported by [PERSON] et al. [26]. They described detection of human epididymis-specific protein 4 (HE4) with a chitosan-titanium carbide-modified ITO electrode (Chi-TiC/ITO) onto which AuNPs were deposited. The capture antibody was adsorbed onto the Au and TiC NPs. For transduction and amplification, secondary antibodies were labelled with DNA strands, followed by rolling circle amplification (RCA). Using doxorubicin as DNA intercalator and DPV for detection, the redox current responded to HE4 linearly in the concentration range of 3-300 pM, with a LoD of 0.06 pM (respectively 3-300 ng L\({}^{-1}\) and 0.06 pg mL\({}^{-1}\)) (Figure 3C). This is a good example of enzyme-free amplification where the authors tried to increase the surface density of the redox probe by multiple intercalation within the DNA strands. The high surface density of doxorubicin achieved by this strategy provided high currents, so high sensitivity. These approaches could appear very complicated. For this reason, Electrochemical Impedance Spectroscopy (EIS) combined with a diffusing redox probe stayed popular. [PERSON] et al. [27] described the immobilization of anti-okadaic acid antibody on 4-carboxyphenyl film. The Ab/Ag binding was transduced simply using electrochemical impedance spectroscopy with FeCN\({}_{6}\)\({}^{3-/4-}\) as diffusing redox probe. The increase in electron transfer resistance was linearly proportional to the okadaic acid concentration in the range 0.195-12.5 ug L\({}^{-1}\), with a LoD of 0.3 ng mL\({}^{-1}\). In 2013, [PERSON] et al. [28] described a similar procedure for epidermal growth factor receptor (EGFR) detection, by immobilizing anti-EGFR antibody on dithiobissucccinimidyl propionate (DTSP) SAM on Au electrodes. EIS measures with Fe(CN)\({}_{6}\)\({}^{3-/4-}\) exhibited a linear range from 1 pg mL\({}^{-1}\) to 100 ng mL\({}^{-1}\) and a LoD of 1 pg mL\({}^{-1}\)[PERSON] et al. [29] also presented the same procedure but replaced conventional Au electrode by microfabricated interdigitated ones. As a proof-of-concept, cortisol antibodies were immobilized using the same SAM as previously described. Cortisol (MW 362 g mol\({}^{-1}\)) was detected using CV over a linear range of 10 pM to 100 nM (3.6 pg mL\({}^{-1}\)-36 ng mL\({}^{-1}\)). This example is probably the occasion to recall here that the relative size of the target molecule compared to that of an antibody should determine the choice of the transduction architecture of an electrochemical immunosensor. Indeed, the most popular Ab reported in biosensors are immunoglobulins G (IgGG), with a typical molecular weight of 150 kDa, i.e., a volume of between 300 and 700 nm\({}^{3}\)) or a projected area of ca. 60 nm\({}^{2}\). This should be compared to the molecular weight of the target antigen. If this one is a for example a small protein of 30 kDa, it corresponds to a projected area of 22 nm\({}^{2}\), i.e., ca 30% of the antibody's. But for a molecule (such as pesticide, industrial or pharmaceutical pollutant) of 200 g mol\({}^{-1}\), this ratio falls to 1% of the antibody's projected surface, which is negligible  
Figure 3: (**A**) Strategy for the electrochemical detection of atrazine based on the change in electroactivity of a polymer film poly(juglone-ATZ); (1) polymer/hapten-modified electrode; (2) after complexation with anti-ATZ; (3) after addition of ATZ in solution; (**B**) Calibration curve measured from SWV at \(-\)450 mV/SCE, after addition of ATZ from 1 pM up to 10 \(\upmu\)M. Adapted from [22], Copyright 2012, with permission from Elsevier; (**C**) RCA-based immunosensor for HE4 detection.