# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Metal or Metal Oxide Nanoparticles
#### 3.4.1 Enzyme-Based Immunosensors
[109] reported a microfluidic bead-based immunosensor that used multienzyme-nanoparticle amplification and quantum dots labels. Microbeads were functionalized with capture antibodies and AuNPs were functionalized with HRP-labelled signaling antibodies. Upon a sandwich immunoreaction, the activity of HRP was monitored and used as quantification signal. This immunosensor presented a LoD of 0.2 pg mL\({}^{-1}\) AFP and showed a 500-fold increase in detection limit compared to the off-chip test, which demonstrated the pertinence to use flow cells instead of conventional ones. More recently (2016), [PERSON] et al. [110] also reported a device for AFP detection, using TiO\({}_{2}\) nanotubes functionalized by HRP, PANi and AuNPs. Protein G was crosslinked on the AuNPs for oriented immobilization of the capture anti-PSA antibody. Upon a sandwich-type immunoreaction, the HRP-labelled signaling antibody was captured on the surface and using DPV with HQ and H\({}_{2}\)O\({}_{2}\) as a redox probe in solution, the authors obtained a linear range between 0.01 and 350 ng mL\({}^{-1}\) and a LoD of 1.5 pg mL\({}^{-1}\). This work, along with other works dealing with concanavalin A, underlines the great importance to take care of the orientation of the probe Ag for reaching the lowest limits of detection. Applied to another kind of protein, [PERSON] et al. [111] described detection of TNF-\(\alpha\) using peptide-based self-assembled nanomaterials and GOx-modified gold nanorods (GNR). Anti-TNF-\(\alpha\) antibodies were immobilized on Fc-peptide nanowires on the electrode surface, the Fc moiety acting as redox mediator for GOx. Secondary anti-TNF-\(\alpha\) antibodies were labelled with GNR and GOx in a sandwich-type assay (Figure 20B). The SWV response towards glucose oxidation was used as signal to quantify TNF-\(\alpha\), with a wide linear range from 0.005 to 10 ng mL\({}^{-1}\). Still using metal or metal-oxide NPs for their catalytic properties, [PERSON] et al. [112] reported an electrochemical immunosensor for interferon-\(\gamma\) detection based on PDDA and AuNPs to provide a high-area interface, and HRP-labeled antibody-conjugated AuNPs (HRP-Ab\({}_{2}\)-AuNP) as signal tag. The oxidation of HQ by H\({}_{2}\)O\({}_{2}\) was monitored by re-reduction of BQ into HQ at the electrode. The linear range was observed from 0.1 to 10,000 pg mL\({}^{-1}\), with a LoD of 0.05 pg mL\({}^{-1}\). Metal NPs can also simply be used for their high surface-to-volume ratio. For example, [PERSON] et al. [113] described ZnO nanorods grown on PDMS for sensing H1N1, H5N1, and H7N9 influenza retrovirus simultaneously, using an original array architecture (Figure 21). They have shown that ZnO nanorods, in addition to their high specific area, have an isoelectric point of ca. 9.5 which allowed to interact electrostatically with capture antibodies which have a lower isoelectric point. The originality is that the substrate used for the immunoreaction is not directly the electrode but the PDMS wall of the microfluidic cell, in front of a working non-modified gold electrode. Using HRP-labelled signaling antibodies and TMB as substrate, they achieved a LoD of 1 pg mL\({}^{-1}\) for each virus, with a linear detection range (measured through current-time curves) of 1-10 ng mL\({}^{-1}\). Very recently, [PERSON] et al. [114] reported polyvinyl alcohol-functionalized AgNPs ( AgNPs-PVA) used in a microfluidic immunosensor for quantification of epithelial cell adhesion molecule (EpCAM),  
Figure 21: (**A**) Schematic representation of the structure of the ZnO nanorods microfluidic immunosensor for influenza virus detection; (**B**) Chronoamperometric curves measured from each electrode of the arrayed sensor, for an antigen mixture of (**a**) 1 pg mL\({}^{-1}\) H1N1 and 100 pg mL\({}^{-1}\) H5N1; (**b**) 10 pg mL\({}^{-1}\) H5N1 and 100 pg mL\({}^{-1}\) H7N9; and (**c**) 10 pg mL\({}^{-1}\) H1N1 and 1 pg mL\({}^{-1}\) H7N9, respectively. Reprinted from [113], Copyright 2016, with permission from Elsevier.