## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers
Thrombin samples were drop-cast onto the electrode and incubated at 4 \({}^{\circ}\)C for 1 h. The electrodes were then incubated with the enzyme and antibody-modified gold nanoparticles for 40 min. Detection of the bound nanoparticles was done using chronoamperometry to measure the current from the reduction of 3% H\({}_{2}\)O\({}_{2}\). The detection of thrombin using this scheme was found to give a linear range from 10 pM to 100 nM with a detection limit of 1.5 pM. Thrombin spiked into human serum was able to be detected over a linear range from 100 pM-100 nM. An ultrasensitive and highly specific electrochemical aptamer-based assay for thrombin was designed based on the capture of Fe\({}_{3}\)O\({}_{4}\)/Au core-shell magnetic nanoparticles (AuMNPs) onto an Au electrode using a magnet [52]. AuMNPs were modified with the thiolated capture aptamers. The thiolated detection aptamer was immobilized on Au nanoparticles together with horseradish peroxidase (HRP). The formation of the sandwich structure including thrombin and the two aptamer labeled nanoparticles took place in solution and was followed by the capture of these sandwich complexes from 30 \(\upmu\)L of the solution drop-cast onto the Au working electrode and collected onto the electrode surface using a magnet. After the addition of 1 mL of solution containing hydroquinone and H\({}_{2}\)O\({}_{2}\), detection of benzoquinone by DPV was done after a 5 min incubation. The assay was able to detect thrombin over a linear range of 0.1-60 pM and with a detection limit of 30 fM as shown in Figure 2.  
The use of a sandwich between the primary aptamer and antibody-modified magnetic nanoferrite nanoparticles was introduced as a means of achieving much faster analysis times [53]. Screen-printed electrodes (SPCE) were modified with a conducting polymer (poly-(2,2'.5',5',5'-terthiophene-3'-p-benzoic acid)) and the amine-terminated aptamer conjugated to the polymer using EDC/NHS chemistry. The nanoferrite particles were modified by starch with attached streptavidin proteins for binding of biotinylated antibodies. The hydroxyl groups of the nanoparticles were activated by carbonyl-diimidazole for binding to toluidine blue O (TBO) which would serve as a redox marker. The analysis parameters were optimized and found to be 3 min for thrombin binding followed by 4 min of binding of the magnetic nanoparticles with the magnetic field followed by 10 s of reversed magnetic field to remove magnetic nanoparticles not bound by antibody-thrombin interaction. The current due to reduction of TBO was used as the signal and a linear range of 1-500 nM was found with a detection limit of 0.49 nM. An approach to signal amplification in an aptamer sandwich assay for thrombin was based on having the reporter aptamer bound to gold nanoparticles decorating the surface of microparticles of hollow CeO\({}_{2}\) capable of catalyzing the reduction of H\({}_{2}\)O\({}_{2}\)[54]. Indium tin oxide (ITO) electrodes were dip-coated with Ag nanowires (d = 80 nm, l = 20 \(\upmu\)m) for three cycles and then modified with the amine-terminated capture aptamer. The highly conductive Ag nanowires helped to enhance the electrochemical signal. The hollow CeO\({}_{2}\) microparticles (d-1 \(\upmu\)m) were prepared by template synthesis and then served as templates for decoration with Au nanoparticles onto which the thiolated reporter aptamer was bound. The detection was achieved using DPV in 10 mM H\({}_{2}\)O\({}_{2}\) after a 40 min thrombin binding time. The arrangement of the components for this assay is depicted in Figure 3. A linear relation was found between the current due to H\({}_{2}\)O\({}_{2}\) reduction and log [thrombin] over the range 0.5 pM-30 nM with a detection limit of 0.25 pM. A summary of the results obtained for the detection of thrombin using aptamers and electrochemical sandwich assay approaches is presented in Table 1. In certain studies, both a very low detection limit and a wide linear range have been reported, conditions needed for a successful assay.