# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Conventional Electrode Substrates
#### 3.1.1 Enzyme-Based Immunosensors
Transduction was designed using competition equilibrium for the binding sites of the immobilized antibody, between the target ACTH and a biotinylated ACTH (Figure 2A,B). The electroanalytical response was generated by using an AlkP-labelled streptavidin and 1-naphthyl phosphate as enzyme substrate. Differential pulse voltammetry (DPV) was used to monitor the enzyme activity (instead of classical CV, to suppress the capacitive component). A very low LoD of 18 fs mL\({}^{-1}\) was obtained. A competitive immunostaining approach was also followed by [PERSON] et al. [17] in 2013, for determination of sulfonamide (SA or SPY) and tetracycline (TC), two antibiotics which could be present in milk. The originality was to use Protein G coupled to 4-aminobenzcoic acid electrografted on the electrode, as anchoring point for oriented immobilization of anti-SA and anti-TC (Figure 2C,D). Using HRP-labelled TC, they obtained a LoD of ca. 1 nM (ca. 200-500 pg mL\({}^{-1}\)) for both SA and TC. As for the previous ACTH competitive detection, no linear range was given, probably because it is relatively difficult to obtain such linear response with competitive transduction. For detection of cancer antigen 125 (CA-125), [PERSON] et al. [18] described a Ru(NH3)\({}_{6}\)\({}^{3+}\)-mediated glucose oxidase (GOx) labelling instead of routinely used enzymes such as HRP or AlkP. The LoD, for an incubation period of 5 min, was slightly lower 0.1 U mL\({}^{-1}\) for CA-125, comparable to the other reported electrochemical immunosensors. However, the authors claimed a shorter incubation time compared to HRP or AlkP amplifications. Figure 1: (**A**) (**a**) Au microelectrodes obtained by electroreduction of HAuCl\({}_{4}\) in 5 \(\upmu\)m apertures; (**b**) Immunorecognition. HIV-1 antigens are covalently immobilized on a SU-8 layer, then target antibodies are bound, followed by secondary AlkP-labelled anti-IgG binding. p-Aminophenyl phosphate (pAPP) is added as substrate; (**c**) Transduction: (**c**) AlkP converts pAPP to p-aminophenol which is electrooxidized at the electrode and produces a current proportional to the amount of target antibody bound to the sensor; (**d**) Resulting DPV. Reprinted with permission from [12]. Copyright 2013 American Chemical Society; (**B**) _Above_. Detection principle of the screen-printed bi-analyte array, including a BSA (reference) electrode. _Below_. Change in current for different concentrations of AFP, for blank (lowest current) to 50 ng mL\({}^{-1}\) (highest current); calibration curve in inset. Adapted from [15] with permission from The Royal Society of Chemistry. More generally, for enzyme-based amplification routes, the turnover of the enzyme (more precisely the K\({}_{\rm cat}\)/K\({}_{\rm M}\) ratio, which should be as high as possible) is a crucial parameter for a good amplification. [PERSON] et al. [19] described an electrochemical immunosensor for detection of tumor necrosis factor \(\alpha\) (TNF-\(\alpha\)) with a strategy to avoid non-specific adsorption. For this purpose, they used an original layer of phenylphosphorly choline (PPC) and phenylbutyric acid (PBA). The capture antibody was grafted on the working ITO electrode along with this anti-adsorption layer and, in a sandwich configuration, the signaling (labelled) antibody was coupled to HRP. H\({}_{2}\)O\({}_{2}\) was added in solution as well as ferrocene (Fc) to recycle HRP. The immunosensor was shown to detect TNF-\(\alpha\) with a LoD of 10 pg mL\({}^{-1}\) with a wide linear range between 0.01 ng mL\({}^{-1}\) to 500 ng mL\({}^{-1}\). More recently, [PERSON] et al. [20] reported in 2017 a tyrosine kinase immunosensor involving a sandwich architecture with a capture antibody covalently immobilized on poly(pyrrolepropionic acid)-modified electrodes and a HRP-labeled secondary antibody. The LoD was 337 pg mL\({}^{-1}\). Due to the relative instability, limited robustness and severe limitation of the operating conditions required for most enzymes, a strategy to get rid of enzymes is to design non-amplified sensors.