## 3 Other Electrochemical Sandwich Assays Using Two Aptamers  
Strategies using two aptamers have been applied to develop assays for a number of other analytes ranging from small molecules to biomolecules to cells but with a particular focus on protein biomarkers. The surfaces on which the primary (capture) aptamer is immobilized have included self-assembled monolayers on Au electrode surfaces, electrodeposited Au nanoparticles, and on a variety of nanostructured surfaces and nanocomposite materials of high surface area or improved conductivity supported on electrodes such as glassy carbon. The secondary aptamer (detection, or reporter aptamer) has been applied in many forms, including conjugated to enzymes through the biotin-streptavidin interaction and also bound around nanoparticles to provide amplification or for the nanoparticles themselves to be used as electrochemical tags. The aptamers themselves are often modified, being thiolated, biotinylated, or bearing a redox-active tag. In the simplest approach for a sandwich assay using two aptamers, the most common electrochemical measurement technique used is differential pulse voltammetry to detect an enzyme product produced by an enzyme associated with the detection aptamer in the presence of added substrate. Another approach uses chronocoulometry to detect charge associated with the binding of a redox marker by electrostatic interactions. These two basic assay architectures are shown in Scheme 2. In more elaborate architectures in which a double-stranded DNA is formed in a hybridization step, a redox marker that intercalates into the dsDNA can be used. \begin{table}
\begin{tabular}{l l l l l} \hline \hline
**Components of Sandwich Assay for Thrombin** & & & & & \\ (**In** = Thrombin, SAM = Self-Assembled & & & & \\ Monolayer) & & & & & \\ \hline Au/thiolated aptamer/Th/Pt nanoparticle & Differential pulse & & & & \\ decorated carbon nanocages & & & & & \\ \hline Glassy carbon electrode/graphene oxide sheets & & & & & \\ doped with nitrogen and sulfur decorated with & & & & & \\ gold nanoparticles/thiolated & & & & & \\ aptamer/Th/graphene oxide sheets doped with & & & & & \\ nitrogen and sulfur decorated with & & & & & \\ nanoparticles modified by thiolated & & & & \\ aptamer and libuited biotinylated & & & & & \\ DNA/avidin-nseradish peroxidase conjugate & & & & & \\ \hline Au/thiolated aptamer/Th/pyrroquinoline & & & & & \\ quinone glucose dehydrogenase labeled secondary aptamer & & & & & \\ aptamer & & & & & \\ \hline Glassy carbon electrode/chitosan-nanogold & & & & & \\ film/cross-linked antibody/Th/secondary & & & & & \\ aptamer and methylene blue & & & & & \\ \hline Screen-printed carbon electrode/Au nanoparticles & & & & & \\ and reference dicarboxylic acid & & & & \\ mediator/streptavidin/bio/biotinylated & & & & & \\ aptamer/Th/Au nanoparticles modified with & & & & & \\ horseradish peroxidase-antibody conjugates & & & & & \\ \hline Au/cone-shell Fe\({}_{3}\)O\({}_{4}\)/Au magnetic nanoparticles & & & & & \\ modified with primary aptamer/Th/Au nanoparticles labeled with secondary aptamer and & & & & & \\ nanoparticles labeled with secondary aptamer and & & & & \\ horseradish peroxidase & & & & & \\ \hline Screen-printed carbon electrodes/conducting & & & & & \\ polymer layer conjugated to primary & & & & & \\ aptamer/Th/stepetravidin-starch coated magnetic nanoparticles and biotinylated secondary aptamer, & & & & \\ toluidine blue O & & & & & \\ \hline Indium in oxide electrodes/Ag & & & & & \\ nanowires/amino-functionalized primary & & & & & \\ aptamer/Th/Au nanoparticles on hollow CeO\({}_{2}\) & & & & & \\ structures with secondary aptamer & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._A two aptamer sandwich assay was developed for a form of platelet-derived growth factor (PDGF), useful as a biomarker for cancer and renal function deterioration [55].