## 2 Electrochemical Sandwich Assays for Thrombin Using Aptamers  
Thrombin, which is a serine protease present in the blood during ongoing coagulation, serves as a biomarker for diseases including Alzheimer's [24] and cancer [25]. Thrombin is by far the most studied analyte in electrochemical sandwich assays using aptamers because two complimentary thrombin binding aptamers have been developed. A 15-mer aptamer was first discovered [26] and later a 29-mer [27], each binding to different sites of the protein. The 15-mer forms a Gquadruplex that interacts with the fibrinogen recognition exosite by mainly electrostatic interactions [28] and has a reported K\({}_{\text{d}}\) value of \(\sim\)100 nM. The 29-mer aptamer binds to the heparin-binding exosite of thrombin with hydrophobic interactions and duplex formation being significant and resulting in K\({}_{\text{d}}\) = 0.5 nM [29]. These two sites on the protein are well separated. Numerous approaches have been reported for the detection of thrombin using aptamers, including optical, electrochemical and chromatographic affinity-based assays [30]. Thrombin can be an important biomarker for diseases that involve changes or abnormalities in the coagulation process [31]. Thrombin is an important component of the coagulation cascade [32]. Thrombin is a serine protease homologous to other serine proteases such as chymotrypsin [33]. The precursor to thrombin, prothrombin, circulates in the blood at a concentration of 1.4 uM. The relevant physiological range of thrombin concentration is low nanomolar to low micromolar [34]. The appeal of thrombin as a target, as noted above, is that there are two aptamers available that target two different, spatially separated sites on the protein making formation of a sandwich structure readily achieved. Some electrochemical assays have made use of only one aptamer, such as an assay based on aptamer conformational change upon thrombin binding [35]. A 32-mer aptamer for thrombin would undergo a conformational change from unfolded to G-quartet conformation that binds to thrombin. The distance of a redox label, methylene blue conjugated to the 3'-end, from a gold electrode surface increased upon thrombin binding. The peak current in a differential pulse voltammetry sweep would decrease with increasing thrombin concentration. Analysis using the Laviron equation [35] revealed that binding of thrombin reduced the kinetic rate constant from 88 s\({}^{-1}\) to 58 s\({}^{-1}\), attributed to a change in the electron transfer pathway due to a binding-induced conformational change. A linear response for thrombin spiked in fetal calf serum was seen from 6.4-768 nM. Other methods aside from Electrochemistry used to detect thrombin using aptamers have included a guided-mode resonance (GMR) sensor [36] which is an optical method based on the change in refractive index showing up as a shift in transmittance of a grating structure. The SiO\({}_{2}\) on the GMR sensor surface was modified with 3-aminopropyltriethoxysilane and then conjugated to the 29-mer for thrombin, which is the one of higher binding affinity. This sensor achieved a detection limit of 0.19 \(\upmu\)M and a linear range of 0.25-1.0 \(\upmu\)M. Using a Biacore SPR chip, a multiple sandwich assay for thrombin was reported that achieved a detection limit of 0.1 aM (attomolar), and a range from 0.1-4.0 aM [37]. In this approach, a gold sensor chip was first modified with a mixture of mercaptoundecanoic acid and a PEGylated alkanethiol followed by conjugation of an anti-thrombin antibody using EDC/NHS chemistry. The thrombin bound to the antibody was then exposed to the 29-mer binding sequence thrombin aptamer but with a polyadenine tail of 30 units in length. The SPR response was enhanced using a dual nanoparticle strategy, where gold nanorods (length = 50 \(\pm\) 10 nm, width = 10 \(\pm\) 2 nm) modified by poly(thymine) chains of 20 units would then bind to roughly spherical gold nanoparticles (50 \(\pm\) 5 nm) modified by poly(adenine) chains of 30 units.