## 3 Other Electrochemical Sandwich Assays Using Two Aptamers
The surface of a glassy carbon electrode (GCE) was drop-cast with a 0.1% chitosan solution containing carbon aerogel (CA) mesoporous carbon material incorporating MoS\({}_{2}\) nanoflowers that provided excellent conductivity and increased the surface area. The chitosan was used to immobilize gold nanoparticles modified with the thiolated capture aptamer. The reporter and capture aptamer were the same 45-mer with hexanethiol at the 5'-end. The PDGF protein form detected was a dimer with two identical disulfide-linked subunits and hence using the same aptamer for capture and reporting was suitable. The reporter aptamers were attached to gold nanoparticles together with 6-ferrocenyl hexanethiol. The assay was performed using a 2-h incubation time in the PDGF solution with the capture aptamer-modified electrode followed by an additional 2 h incubation with the reported aptamer-modified gold nanoparticles, providing the results shown in Figure 4. Cyclic voltammetry or DPV was done in the presence of dissolved [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) serving as a mediator. A detection limit of 0.3 pM was achieved along with a linear range from 0.001-10 nM. The determination of PDGF using the biosensor in three human serum samples diluted into PBS from healthy donors and two from patients was found to agree well, better than 5%, with the amount determined by a standard ELISA assay. Figure 4: **(A)** CV of different electrodes at a scan rate of 50 mV s\({}^{-1}\): (a) GCE, (b) MoS\({}_{2}\)/CA/GCE, (c) AuNPs/MoS\({}_{2}\)/CA/GCE, (d) Atp1/AuNPs/MoS\({}_{2}\)/CA/GCE, (e) BSA/Apt1/AuNPs/MoS\({}_{2}\)/CA/GCE; (**B**) Nyquist plots of bare GCE (a), MoS\({}_{2}\)/CA/GCE (b), AuNPs/MoS\({}_{2}\)/CA/GCE; (**C**) CV of BSA/Apt1/AuNPs/MoS\({}_{2}\)/CA/GCE (a), PGDF-BB/BSA/Apt1/AuNPs/MoS\({}_{2}\)/CA/GCE (b), Fc-AuNPs-Apt2/PGDF/BB/BSA/Apt1/AuNPs/MoS\({}_{2}\)/CA/GCE; (**D**) DPV response to the different volume proportions of AuNP-modified Fc (5 mM) and Apt 2 (1 mM); (E) Plot of Q-t curves of the (a) GCE and (b) AuNPs/MoS\({}_{2}\)/CA/GCE. Inset plot of Q-t\({}^{1/2}\) curves on (a) GCE and (b) AuNPs/MoS\({}_{2}\)/CA/GCE. Reprinted with permission from reference [55]. Copyright Elsevier 2015. Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2) protein is a potential biomarker for pancreatic adenocarcinoma (PADC), and hepatocellular carcinoma in combination with other biomarkers. Two different NGAL aptamers were used in direct sandwich and competitive assay formats on Au electrodes and magnetic beads. A sandwich assay using capture aptamer on magnetic beads and a reporter aptamer tagged with biotin or with 6-carboxyl fluorescein that would bind to either a peroxidase-streptavidin conjugate or to a peroxidase-anti-FC-peroxidase conjugate was found to show large currents in the absence of NGAL, attributed to possible aptamer hybridization. Using truncated aptamers, similar currents were seen in the presence or presence of NGAL [56]. Detection of lipocalin-2, also known as a biomarker for renal failure, was achieved using an electrochemical aptamer sandwich assay [57]. A graphite screen-printed electrode was modified by a layer of conducting polymer formed by electropolymerization onto which a layer of gold nanoparticles was formed by electrodeposition. The gold nanoparticles were modified by a first aptamer that would bind lipocalin-2. After the binding of lipocalin-2, a second aptamer conjugated to biotin was bound to form a sandwich. Streptavidin conjugated to alkaline phosphatase was applied and using 1-naphthylphosphate as an enzyme substrate yielded 1-naphth that could then be detected by oxidation in DPV yielding a linear range from 1.0-1000 ng mL\({}^{-1}\) and a detection limit of 0.3 ng mL\({}^{-1}\). An electrochemical aptasensor for immunoglobulin E (IgE) was fabricated using enzyme-linked aptamer in a sandwich assay format [58].