## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers
An aptamer sandwich assay on nanoporous gold (NPG) electrode was used to achieve high sensitivity with chronocoulometric detection for detection of thrombin [42]. An electrode of high surface area was created by generating a nanoporous gold layer by applying a square wave potential pulse to a gold disc electrode in a highly basic solution (2 M NaOH). The roughness factor of this electrode as determined using oxide stripping in cyclic voltammetry was \(\sim\)34. The NPG electrode was first modified by thiolated 15-mer aptamer with a TTTTT tail (5\({}^{\prime}\)-SH-(CH\({}_{2}\))\({}_{6}\)-TTTT-GGTTG-GTGTG-GTGG-3\({}^{\prime}\)) and then mercaptohexanol was allowed to assemble to fill in bare spots and provide better orientation of the aptamers. Subsequent or co-addition of mercaptohexanol for self-assembly of oligonucleotides onto Au is an established strategy for providing better orientation and filling in bare spots between aptamers on a gold surface [43]. Optamermodified gold nanoparticles of 3.2 nm diameter were prepared modified by a 3/1 ratio of non-binding aptamer to binding aptamer with the goal of having each modified nanoparticle bind to only one thrombin. Electrochemical impedance spectroscopy was used to characterize the extent of surface modification after each step in electrode modification and sandwich formation through the increase in charge transfer resistance. The detection step used the electrostatic binding of [Ru(NH3)\({}_{6}\)]\({}^{3+}\) to the DNA strands in the outer regions of the sandwich complexes. The binding of greater numbers of aptamer-modified Au nanoparticles to the thrombin captured on the aptamer-modified electrode resulted in the accumulation of a greater amount of [Ru(NH3)\({}_{6}\)]\({}^{3+}\) which was then detected by the amount of charge passed as measured by chronocoulometry upon reduction of [Ru(NH3)\({}_{6}\)]\({}^{3+}\) to [Ru(NH3)\({}_{6}\)]\({}^{2+}\). The difference in charge passed in the presence and absence of thrombin was plotted versus thrombin concentration achieving a detection limit of 30 fM and a linear range from 0.01-22 nM. The selectivity of the biosensor was tested using fibrinogen, prothrombin, and Factor Xa and found to be excellent. The times required for incubation with thrombin and then with the aptamer-modified nanoparticles were reported each as 2 h.  
An approach using magnetic beads was used to perform an aptamer-based electrochemical sandwich assay for thrombin [44]. Streptavidin-coated magnetic beads were first used to bind the biotinylated 15-mer aptamer (primary aptamer) followed by binding of thrombin and then binding of biotinylated 29-mer aptamer (secondary aptamer). Optimization of conditions resulted in the choice of 0.1 uM 29-mer biotinylated aptamer and an incubation time of 15 min. Incubation with thrombin was also for 15 min. Incubation with streptavidin-enzyme conjugate was carried out for 10 min. Each step was performed in an appropriate buffer. The bound sandwich complexes were then attracted to a screen-printed carbon electrode. The amount of thrombin bound was then assayed by measuring the current peak due to reduction of the 1-naphth product obtained by the action of the alkaline phosphatase enzyme on the substrate 1-naphthyl phosphate. A nonlinear dose-response curve was observed. A detection limit of 0.45 nM was obtained, and the assay was useful over the range 0.1-100 nM. Selectivity in the presence of 72 uM human serum albumin was only slightly greater than that obtained using buffer alone as a blank. The response for thrombin spiked into 10\(\times\) diluted serum was similar to that obtained in the buffer. Assays for thrombin using magnetic beads were reported in indirect competitive, direct competitive and product detection formats [45]. In the indirect competitive assay, thrombin conjugated to magnetic beads competed with free thrombin for binding of biotinylated 15-mer aptamer. Subsequently, the biotinylated aptamer captured on the magnetic beads could bind to alkaline phosphatase labeled streptavidin.