# Recent Advances in Electrochemical Immunosensors
## 3 Discussion
### Conventional Electrode Substrates
#### 3.1.2 Enzyme-Free Immunosensors
Reprinted from [26], Copyright 2012, with permission from Elsevier; (**D**) Scheme of the competitive inhibition assay for detecting HbA1c (anti-HbA1c: blue Y; HbA1c: red triangle; GPP: pink triangle, surface bound); (**E**) _Above_, a representative SWV for a FDMA-modified electrodes after (a) grafting of GPP and (b) incubation in 2 \(\upmu\)g mL\({}^{-1}\) anti-HbA1c and 13.5% HbA1c. _Below_, corresponding calibration curve. Reprinted from [23] with permission from The Royal Society of Chemistry. and cannot play a significant role in changing the steric hindrance at the solution/electrode interface; for such situations, other transduction schemes should be considered. It is possible to go beyond this steric hindrance limitation and play on electrostatic repulsions rather than on the size of the target. For detection of a bulky protein (porcine serum albumin), [PERSON] et al. [30] reported a carbon nanofiber-modified SPE electrofunctionalized with a 4-carboxyphenyl diazonium salt onto which antibodies were covalently bound. Taking profit of the strong affinity of serum albumins towards anions, an anionic redox probe was used in solution. An increase in cathodic peak current was measured after immunocomplex formation between antibodies and proteins. The linear range was from 0.5 to 500 pg mL\({}^{-1}\) and the LoD of 0.5 pg mL\({}^{-1}\). Reducing the size of the capture antibody by using only Ab fragments or an analog of protein (a peptidomimetic) is also a solution. Using Fe(CN)\({}_{6}\)\({}^{3-/4-}\) as an electroactive diffusing probe, [PERSON] et al. [31] reported detection of hemagglutinin from avian influenza virus H5N1. Gold electrodes were modified with a SAM of 4,4\({}^{\prime}\)-thiobisbenzenet thiol (TBBT), itself modified by gold nanoparticles (AuNPs) and single chain variable fragments of antibodies (scFv) against hemagglutinin H5 (Figure 4A). Interactions between the fragment of antibodies and hemagglutinin were sensed by EIS, giving a LoD of 0.6 pg mL\({}^{-1}\) and a linear range from 4.0 to 20.0 pg mL\({}^{-1}\). This fragment makes 25 kDa and corresponds to the variable domains; it is the smallest fragment that holds a complete binding site of an antibody and therefore keeps its specificity (Figure 4B). Figures of merit discussed in this section are summarized in Table 2. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Target** & **Probe** & **LoD** & **Method** & **Reference** \\ \hline AbHIV & Peptides & 1 ng mL\({}^{-1}\) & AlkP/DPV & [12] \\ IgG & Ag & 1.4 \(\mu\)g mL\({}^{-1}\) & HRP/CV—Sandwich & [13] \\ Estradiol & Ab & 0.77 pg mL\({}^{-1}\) & HRP/CV—Competitive & [14] \\ CEA & Ab & 40 pg mL\({}^{-1}\) & HRP—Sandwich & [15] \\ ACTH & Ab & 18 kg mL\({}^{-1}\) & AlkP/DPV—Competitive & [16] \\ Sulfonamide & Ab & 200–500 pg mL\({}^{-1}\) & HRP—Competitive & [17] \\ CA-125 & Ab & 0.1 U mL\({}^{-1}\) & GOx—Sandwich & [18] \\ TNF-\(\alpha\) & Ab & 10 pg mL\({}^{-1}\) & HRP/CV—Sandwich & [19] \\ Tyrosine kinase & Ab & 337 pg mL\({}^{-1}\) & HRP—Sandwich & [20] \\ TREM-1 & Ab & 30 pg mL\({}^{-1}\) & FeCN\({}_{6}\)\({}^{3-/4-}\)/EIS & [21] \\ Atrazine & Hapten & 0.2 ng L\({}^{-1}\) & ECP/SW—Competitive & [22] \\ HbA1c & Peptide & - & Fc/CV—Competitive & [23] \\ Bisphenol A & Hapten & 2 pg mL\({}^{-1}\) & ECP/SW—Competitive & [24] \\ Acetaminophen & Hapten & 1.5 pg mL\({}^{-1}\) & ECP/SW—Competitive & [25] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Figures of merit of enzyme-based and enzyme-free immunosensors using conventional electrode substrates. Figure 4: (**A**) Hemagglutinin H5 immunosensor based on a SAM of 4,4\({}^{\prime}\)-thiobisbenzenet thiol carrying single chain variable fragments (scFv) of antibodies as probes. Reprinted from [31], Copyright 2016, with permission from Elsevier; (**B**) Schematic representation of a scFv fragment. A scFv fragment makes 25 kDa and corresponds to the VH + VL domains; it is the smallest fragment that holds a complete binding site of an antibody and therefore provides its specificity.