## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers
Incubation with thrombin in phosphate buffer was carried out for one hour at 37 \({}^{\circ}\)C and then after washing exposure to the dispersion of aptamer-modified gold nanoparticles on doped graphene oxide took place for 80 min. The biotinylated aptamer was then bound to horseradish peroxidase-labeled avidin. Electrochemical detection of the oxidation of hydroquinone by H\({}_{2}\)O\({}_{2}\) by DPV was used as the response to increasing thrombin concentration. The detection limit achieved in phosphate buffer was 25 fM and a linear range of response was found from 100 fM to 10 nM. Selectivity was excellent when tested against human serum albumin, \(\alpha\)-fetoprotein, and carcinoembryonic antigen. Agreement between thrombin spiked into human serum samples from 50 fM to 50 pM was very good. In addition to alkaline phosphatase and horseradish peroxidase, other enzymes could be used to develop aptamer-based electrochemical assays. In one study, an enzyme for which the substrate is simply glucose was used to develop an assay [49]. An electrochemical sandwich assay for thrombin was based on pyroquinolineglucose dehydrogenase, (PQQ)GDH. (PQQ)GDH was conjugated to biotinylated 29-mer aptamer using glutaraldehyde and used as the reporter. The 15-mer aptamer was used in a thiolated form and self-assembled onto a gold electrode surface. The enzyme-labeled detection aptamer was incubated together with thrombin in buffer and after washing the enzyme activity to added glucose was measured. Using 62 mM glucose in 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and chronoamperometric detection, a linear range from 40-100 nM was found with a detection limit of 10 nM. Some electrochemical assays for thrombin have combined the use of an aptamer and an antibody. Both possibilities, aptamer as capture probe and antibody as capture probe have been reported. In one study [50], a nanostructured film of chitosan and gold nanoparticles was formed by electrodeposition from a solution of chitosan and HAuCl\({}_{4}\) at \(-1\) to \(-3\) V (vs. SCE, saturated calomel electrode) for 2-6 min giving nanoparticles of diameter around 50 nm. A polyclonal sheep anti-thrombin antibody was then drop-cast onto the electrode, allowed to react, and then the remaining reactive sites blocked using BSA. The assay involved binding of thrombin to the antibody-modified electrode followed by binding of a 22-mer aptamer (170 nM) and then intercalation of methylene blue (MB) between the base pairs of the aptamer. The parameters for film formation and the concentrations of KCl and MgCl\({}_{2}\) in the buffer solution were optimized, as the presence of Mg\({}^{2+}\) promotes aptamer folding into stable G-quadruplexes that results in improved binding to thrombin. Using DPV to measure the peak current from the reduction of MB versus thrombin concentration gave a linear range from 1-60 nM and a detection limit of 0.5 nM. In contrast, an electrochemical sandwich assay combining an anti-thrombin antibody and 15-mer aptamer in which the aptamer served as the capture agent was reported [51]. The 15-mer aptamer had a long T tail of 20 base pairs terminated by  
Figure 1: Scheme of an electrochemical method for the determination of thrombin based on aptamers forming a sandwich structure on an Au electrode covered by electrodeposited Au nanoparticles. The dissolution of CdS nanoparticles linked around gold nanoparticle labels releases Cd\({}^{2+}\) ions that are detected by differential pulse voltammetry. Reprinted with permission from reference [46]. Copyright 2010 Elsevier. biotin on the 5'-end. Au nanoparticles of 13 nm diameter were prepared, and then antithrombin antibody and horseradish peroxidase were physisorbed onto the nanoparticles. Screen-printed electrodes (SPCE) were treated with NH\({}_{4}\)OH to generate amine groups and then Au nanoparticles were adsorbed onto the surface and ferrocenedicarboxylic acid was added as a mediator. Streptavidin was then adsorbed onto the electrode surface followed by adsorption of the biotinylated aptamer.