## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers
An early report made use of electrochemical impedance spectroscopy (EIS) as the detection method for an assay and used the increase in charge transfer resistance (R\({}_{\text{ct}}\)) as the response versus thrombin concentration [38]. In the assay, a gold electrode was modified by thiolated 15-mer aptamer (5\({}^{\prime}\)-HS-(CH\({}_{2}\))\({}_{6}\)-GGTTGGTGTGG-3\({}^{\prime}\)) and mercaptohexanol or mercaptoeethanol were used to fill in bare spots on the electrode surface. The use of mercaptoeethanol was found to be preferred as it gave the best response in EIS. Incubation with thrombin solutions of varying concentrations took place for 60 min. A sandwich structure was then formed by incubation with aptamer-modified gold nanoparticles that had their surface filled in by rhodamine-6G which presumably interacts with the nanoparticle surfaces through its two N-H groups. The gold nanoparticles modified by DNA are negatively charged and thus repel the [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\) probes used in the buffer for conducting impedance spectroscopy. In this work, the same aptamer was used on the surface and on the nanoparticles. The ratio of R\({}_{\text{ct}}\) for thrombin solution to R\({}_{\text{ct}}\) of a blank solution was plotted versus thrombin concentration and a detection limit of 0.02 nM was achieved with a linear range from 0.05-18 nM. BSA used as a control showed a minimal response. The presence of the adsorbed rhodamine 6G was found to increase the response and was also noted as of potential use as a Raman probe for detection of thrombin binding using surface-enhanced Raman spectroscopy (SERS). Two different anti-thrombin aptamers that recognized different binding sites of the targeted thrombin were assembled on the surface of the gold electrode for the fabrication of the electrochemical sensor system [39]. Glucose dehydrogenase (GDH) from _Burkholdelia cepacian_ was used as the label on the detecting aptamer. The two different aptamers were the 15-mer aptamer, 5\({}^{\prime}\)-GGTTGGTGTGG-3\({}^{\prime}\) and the other was a 29-mer aptamer, 5\({}^{\prime}\)-AGTCCGTGGTAGGGCAGGTTGGGGGTGACT-3\({}^{\prime}\) with K\({}_{\text{d}}\) values of 26 nM and 0.5 nM, respectively. The thiolated aptamer (29-mer) was immobilized on the surface of the gold electrode followed by the target thrombin and the GDH labeled (15-mer) second aptamer. BSA was used as the control protein. The lower limit of the detection of this system was 1 \(\upmu\)M. In a thrombin detection study using potentiometric detection, gold electrodes were first modified by a thiolated primary aptamer and filled in with 6-mercaptoethanol(40). After binding with thrombin for 1 h, the bound thrombin was exposed to CdS quantum dots modified by the secondary aptamer. The CdS quantum dots were oxidized for 15 min using 0.01 M H\({}_{2}\)O\({}_{2}\) and then the Cd\({}^{2+}\) ions were detected using a solid-contact ion-selective microelectrode. The EMF vs. log(thrombin, ppm) was nearly linear and useful for detection over a range of 10-250 ppb with a detection limit of 5 ppb (0.14 nM). Another impedetric study made use of Au nanoparticle-modified sheets of reduced graphene oxide (rGO) to provide amplification of the charge transfer resistance response to formation of the capture aptamer-thrombin-reporter aptamer sandwich [41]. Gold electrodes were modified with thiolated capture aptamer and filled with dithiothreitol. In a one-pot reaction, rGO was generated from graphene oxide and decorated with 7.5 nm Au nanoparticles. The thiolated reporter aptamer was bound to these gold nanoparticles. Incubation with thrombin was done for 90 min and the incubation with the rGO-Au nanoparticle conjugates required 5 h to reach saturation. The increase in charge transfer resistance to thrombin concentration was linear from 0.3-50 nM with a detection limit of 0.01 nM. In addition to flat gold surfaces, electrodes with nanostructured surfaces or assemblies of gold nanoparticles have been used to enhance surface area as an additional means to achieve signal amplification.