## 3 Other Electrochemical Sandwich Assays Using Two Aptamers
The selectivity against four other common blood proteins (C-reactive protein, human serum albumin, myoglobin, immunoglobulin G) was tested and the signal associated with each of these four proteins was found to be less than 20% of the signal due to cTnI. Another electrochemical biosensor for cTnI was designed based on the reduction of H\({}_{2}\)O\({}_{2}\) by hydrazine [66]. SPCE were modified with 5 nm Au nanoparticles by drop-casting followed by potential cycling to achieve electrostatic deposition. A layer of conducting polymer was then formed by potential cycling in the presence of the 5,2'.5',2\"-terthiophene-3\"-carboxylic acid monomer. The Tro4 aptamer was conjugated to the polymer layer through the 5'-amine end using EDC/NHS chemistry and served as the capture aptamer. The Tro6 aptamer was 5'-phosphate modified and then linked to adipic acid dihydro through a phosphoramidate bond. The reaction between the hydrazine labeled Tro6 detection aptamer and H\({}_{2}\)O\({}_{2}\) was detected using chronoamperometry. A detection limit of 1.0 pM was achieved and a linear range of 1-100 pM. The limit of detection was below that of commercial immunoassay kits for cTnI. A highly sensitive electrochemical sandwich assay for human epidermal growth factor 2 (HER2), a biomarker for breast cancer, was developed using two aptamers and a combination of nanoflowers functioning as nanozymes [67]. Thiolated DNA nanotetrahedrons were assembled onto a gold electrode presenting the capture aptamer. Nanoflowers of Mn\({}_{3}\)O\({}_{4}\) coated by a layer of poly(diallydimethylammonium chloride) were prepared of size 100 nm and decorated with 40 nm Pd@Pt nanoflowers functioning as nanozymes followed by assembly of the detection aptamer and horseradish peroxidase onto these particles. The three-component nanocatalyst was designed to very effectively catalyze the reaction of hydroquinone with H\({}_{2}\)O\({}_{2}\) to form benzoquinone. Following the formation of the sandwich structure and additional complementary DNA strand terminated in Pd@Pt nanoflower was hybridized to the detection aptamer to further enhance the signal. A detection limit of 0.08 ng mL\({}^{-1}\), the lowest yet reported for HER2, and a linear range of 0.1-100 ng mL\({}^{-1}\) was reported. Agreement with ELISA kits was within 5-8% for HER2 spiked into diluted human serum samples. The relevant diagnostic range for HER2 was noted as 4-75 ng mL\({}^{-1}\). An aptamer sandwich assay for cocaine was developed that included enzymatic redox-cycling for signal amplification [68]. SPCE were drop coated with graphene oxide that was then reduced to graphene followed by electrodeposition of gold nanoparticles from a solution of HAuCl4 precursor. A thiolated cocaine binding aptamer was then self-assembled onto the gold nanoparticles. The electrodes were incubated for 40 min with concentrations of cocaine and a second cocaine binding aptamer that was biotinylated. Alkaline phosphatase (ALP) conjugated to streptavidin was then bound to the sandwich complex and the enzyme substrate p-aminophenyl phosphate (p-APP) and NADH were introduced. ALP catalyzed the conversion of p-APP to p-aminophenol that was then oxidized on the electrode surface to p-quinoneminine. The presence of NADH caused conversion of p-quinoneminine to p-aminophenol resulting in enhanced current response during a DPV sweep. A linear range from 1-500 nM was found with a detection limit of 1 nM. An aptamer sandwich approach was used to detect MCF-7 human breast cancer cells [69]. A thiolated MUC1 (mucin glycoprotein) aptamer was assembled on gold electrodes. The cell suspension was incubated with the electrode for 2 h and after non-specifically bound cells were washed away incubation with MUC1 aptamer conjugated to horseradish peroxidase was done. In the presence of thionine as an electron transfer mediator, the catalytic current observed by cyclic voltammetry arising from the reduction of H2O2 was linear with respect to log(cell count) with a detection limit of 100 cells and a range from 100-10\({}^{7}\) cells.