## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers
In the direct competitive assay, 15-mer aptamer-modified magnetic beads competed to bind thrombin from solution in the presence of biotinylated thrombin and then alkaline phosphatase labeled streptavidin was bound onto the captured thrombin. The magnetic beads were then captured on the electrode surface by a magnetic field, washed, and the activity of alkaline phosphatase for the substrate 1-naphthyl phosphate was measured by differential pulse voltammetry detection of the 1-napthol product. The direct competitive assay achieved a detection limit of 430 nM. The indirect competitive assay was subject to too much non-specific adsorption of the alkaline phosphatase labeled streptavidin to be useful as an assay. The enzymatic activity of thrombin captured onto aptamer-modified magnetic beads was assayed using the substrate \(\beta\)-Ala-Gly-Arg-p-nitroaniline by detection of the released p-nitroaniline by its reduction peak using DPV. A detection limit of 175 nM was achieved using this approach. This study confirmed that the detection limit of 0.45 nM achieved using the sandwich assay approach [45] was superior, and additionally, the sandwich assay displayed outstanding selectivity in the presence of 72 uM human serum albumin. A novel electrochemical biosensor based on the aptamer and the signal amplification by gold nanoparticles was fabricated for the determination of thrombin [46], as shown in Figure 1. The aptamer was immobilized on the Au NPs coated gold electrode for about 16 h followed by 6-mercapto-1-hexanol for about 1 h to block the uncovered parts of the surface. The aptamer-modified electrode then interacted with different concentrations of the thrombin followed by incubation in the solution contain AuNP probes. AuNP probes were prepared by incubating 25 nm Au particles in the solution containing the aptamer and CdS-ssDNA for 16 h with gentle shaking. The resulting sandwich complex was treated with 1.0 M HNO\({}_{3}\) to dissolve the CdS nanoparticles. DPV measurements of the dissolved Cd\({}^{2+}\) were performed to measure anodic stripping peak currents. Thrombin was detected in this assay in the linear range of 1.0 fM to 10 pM with the detection limit of 0.55 fM of the target protein. Detection of the thrombin was done by an aptasensor with platinum nanoparticles (PtNPs) decorated carbon nanocages (CNCs) as signal labels [47]. PtNPs of 3-5 nm in diameter were dispersed on the surface of CNCs. The thiolated aptamer was immobilized on the surface of the gold electrode to capture the thrombin, and then aptamer functionalized PtNPs/CNCs nanocomposites were used to fabricate a sandwich aptasensor. The aptasensor was tested by recording the electrocatalytic reduction current toward H\({}_{2}\)O\({}_{2}\) by CV, EIS and DPV. The thrombin probe used was 5'-SH-(CH\({}_{2}\))\({}_{6}\)GGTTGGGTGTGGTTGG-3'. The linear response of the aptasensor ranged from 0.05 pM to 20 nM with a low limit of detection of 10 fM. The aptasensor provided excellent specificity toward thrombin in the mixture of other proteins including BSA, Hb, L-vys, lysozyme, and spiking of thrombin with different concentrations into the 10-fold diluted serum. Graphene doped with sulfur and nitrogen has improved electrical conductivity and was combined with gold nanoparticles and two aptamers to produce a highly sensitive electrochemical sandwich assay for thrombin [48]. One of the thrombin aptamers was thiolated on the 5'-end with a poly(T) tail (5'-SH-(CH\({}_{2}\))\({}_{6}\)-TTTTTTTTTGGTGTGGTTGG-3'). A glassy carbon electrode was modified by casting the doped graphene oxide dispersed with Au nanoparticles and then modifying it with the thiolated aptamer. A second dispersion was prepared of doped graphene oxide-modified with gold nanoparticles and then modified with an aptamer both thiolated on the 3'-end and biotinylated on the 5'-end (5'-biotim-AGTAGGCGGGCGTATGATTTTTTTTTTTTTTTT-SH-3').