# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Conventional Electrode Substrates
#### 3.1.1 Enzyme-Based Immunosensors  
Enzymes are generally used not for detection itself but for transduction and amplification, taking profit of the enzyme turn-over (for one event of biorecognition, one antibody or antigen captured, several molecules of enzyme product are produced at the vicinity of the electrode surface and electrochemically detected). HRP, horse radish peroxidase, is probably the most used enzyme label in immunosensors because it is commercially available and catalyzes the oxidation of numerous chromogenic substrates using H\({}_{2}\)O\({}_{2}\) as co-substrate. However, some other enzymes are also encountered such as alkaline phosphatase (AlkP) and glucose oxidase (GOx). * Detection of antibodies  
The term \"immunosensor\" could mean that the target could be either an antigen (Ag) or an antibody (Ab). Even if the most popular approach remains targeting Ag using antibodies, some works report Ab detection, as for example in the framework of serological diagnosis, i.e., the research and the determination of specific antibodies linked to a pathogenic infection. In 2013, [PERSON] et al. [12] described an interesting route to detect human immunodeficiency virus (HIV) antibodies by immobilization of antigenic peptides derived from a complex transmembrane protein, HIV-1 gp41 or HIV-2 gp36, covalently attached to a SU-8 substrate (a negative epoxy photoresist) close to microelectrodes. The detection of HIV antibodies was achieved using an alkaline phosphatase (AlkP)-conjugated secondary antibody (Figure 1A). The linear detection range was reported between 1 ng mL\({}^{-1}\) and 1 ug mL\({}^{-1}\), with a limit of detection (LoD) of 1 ng mL\({}^{-1}\) (6.7 pM). More recently in 2016, [PERSON] et al. [13] used a composite graphite-epoxy substrate into which an HRP-labelled antibody was incorporated, for detection of IgG. Transduction was classical, with a competitive assay using H\({}_{2}\)O\({}_{2}\) and hydroquinone (HQ) in solution. Amperometric measurements (reduction of benzoquinone BQ into HQ at the electrode) led to a high LoD of 1.4 ug mL\({}^{-1}\) and a relatively reduced linear range up to 2.8 ug mL\({}^{-1}\). There is no other recent cited work reporting enzyme-based immunosensor for detection of antibodies, the literature being now more focused on nanostructured electrodes rather than on classical substrates. * Detection of antigens  
Detection of antigens on such substrates is still popular. For example, in 2012, [PERSON] et al. [14] described an electrochemical immunosensor for estradiol sensing, based on carbon screen-printed electrodes (SPE) sequentially modified with \(p\)-aminobenzoic acid, streptavidin and biotinylated anti-estradiol. Transduction was performed by applying a competitive immunoassay between peroxidase-labeled estradiol (HRP-estradiol) and estradiol for the binding sites of the immobilized antibodies. The reaction between estradiol and biotinylated anti-estradiol was amperometrically detected by addition of H\({}_{2}\)O\({}_{2}\) in the presence of HQ. The linear range was between 1 and 250 pg mL\({}^{-1}\) and the LoD was 0.77 pg mL\({}^{-1}\). Also in 2012, [PERSON] et al. [15] reported an array of carbon SPE for simultaneous detection of several tumor biomarkers such as carcinoembryonic antigen (CEA) and \(\alpha\)-fetoprotein (AFP, a tumor markers used in the early diagnosis of cancer). Electrodes were modified by grafting \(p\)-phenylenediamine via the diazonium route, followed by crosslinking the primary capture antibody using a Schiff base reaction. Transduction was made by using a sandwich assay with HRP-labelled secondary antibodies. (Figure 1B). The detection range was from 0.10 to 50 ng mL\({}^{-1}\) and the LoD of ca. 40 pg mL\({}^{-1}\). Still in 2012, [PERSON] et al. [16] described a competitive electrochemical immunosensor for adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified carbon SPE used to efficiently immobilize ACTH antibodies.