## 3 Other Electrochemical Sandwich Assays Using Two Aptamers
The ATP binding aptamer was split into two fragments and the first fragment was immobilized on the nanoporous gold surface and the second fragment was covalently bound to the activated carboxylic end of DABA using EDC/NHS chemistry. A 27-mer ATP binding aptamer was split into two fragments and these two fragments fold around the ATP. The electrochemical signal was generated by the oxidation of DABA and due to a nine-fold higher surface area of the nanoporous gold electrode compared to the geometric area of the electrode, the signal was amplified due to the loading of more DABA molecules. The nanoporous gold electrode was prepared by anodization by applying a step potential from open circuit potential to 4V followed by the reduction of gold oxide to metallic gold using ascorbic acid. The limit of detection of the sensor was 100 nM without significant interference from the other similar nucleotide analogs. Cardiac troponin (cTnI) is a protein found in heart muscle and when released into the blood it serves as a biomarker for myocardial infarction and can remain in the myocardial tissue for 10 days. An electrochemical dual-aptamer-based biosensor was proposed for the analysis of cTnI based on using DNA nanotetrahedron (NTH) capture probes and  
Figure 5: **(A)** Differential pulse voltammograms of SA-HRP-biotin aptamer/lgE/NH\({}_{2}\) aptamer/MWCNT/IL/Chit/GC electrode after incubation for 15 min with different IgG concentrations (from inner to outer: 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 2, 4, 6, 10, and 20 nM) in PBS (0.1 M, pH 7.4) containing 2.5 mM H\({}_{2}\)O\({}_{2}\). **(B)** Plot of current response vs. IgE concentration. **(C)** The linear portion of the current versus IgE concentration response. Reprinted with permission from reference [58]. Copyright, 2014 Elsevier. multifunctional hybrid probes [63]. NTTBs presenting the Tro4 aptamer were immobilized on a screen-printed gold electrode (SPGE). Magnetic Fe\({}_{3}\)O\({}_{4}\) nanoparticles were used as nanocarriers to load the larger amount of cTnI-specific Tro6 aptamer, HRP, HRP-mimicking Au@Pt nanoenzyme and G-quadruplex/hemin DNAzyme as the hybrid nanoprobes. The target cTnI was sandwiched between the two types of aptamers (Tro4 and Tro6) and the electrochemical signal from DPV was measured by immersing in the detection solution containing hydroquinone (HQ), which was oxidized to benzoquinone (BQ) with H\({}_{2}\)O\({}_{2}\) in the enzymatic reactions of the numerous HRP, HRP-like nanoenzymes and DNAzymes. Tro4 aptamer probes are built onto one of the NTH sequences, and the other 3 DNAs present thiol groups for bonding to the Au surface. The use of NTH results in well-spaced aptamer presentation on the electrode surface. The dynamic range of detection was 0.01-100 ng mL\({}^{-1}\) and the limit of detection was 7.5 pg mL\({}^{-1}\). In a related study, cTnI was analyzed using an aptasensor based on DNA NTH linked dual-aptamer and magnetic metal-organic frameworks of type Fe\({}_{3}\)O\({}_{4}\)@UiO-66 decorated by Au@Pt nanoparticles, HRP, G-quadruplex/hemin DNAzyme and Tro4 and Tro6 aptamers. The dynamic range of detection was 0.01-100 ng mL\({}^{-1}\) and the limit of detection was 5.7 pg mL\({}^{-1}\)[64]. The assembly of the components for this assay is shown in Figure 6. An electrochemical biosensor for cTnI was also created on SPCE coated with a carboxethylsilanetriol-modified graphene oxide and detection was achieved using an aptamer-biomarker-enzyme labeled aptamer sandwich scheme [65]. An amine-terminated capture aptamer was covalently attached to the modified graphene oxide sheets using EDC/NHS chemistry. Ethanolamine was used as a capping agent and casein was used as a blocking agent. The detection aptamer was conjugated to horseradish peroxidase. The amperometric current was recorded at \(-\)200 mV (vs. Ag/AgCl) in the presence of H\({}_{2}\)O\({}_{2}\) and hydroquinone in pH 7.6 buffer. For measurement in human serum spiked with cTnI, a detection limit of 3.4 pg mL\({}^{-1}\) was reported with a linear range extending up to 1.0 ug mL\({}^{-1}\).