## 4 Electrochemical Sandwich Assays Combining Aptamers and Antibodies
An antibody-aptamer sandwich assay for a platelet-derived growth factor (PDGF) was developed using a circular DNA probe for signal enhancement [85]. A gold electrode (d = 3 mm) was modified by mercaptopropionic acid and rabbit anti-human IgG for PDGF was conjugated to the surface using EDC/NHS chemistry followed by blocking of the remaining surface using bovine serum albumin (BSA). An aptamer binding PDGF was extended by a circular DNA probe then hybridized using DNA polymerase. The completion of the sandwich by binding of the aptamer-circular DNA probe was detected by binding of methylene blue whose reduction was detected by SWV. A linear range from 50-500 ng mL\({}^{-1}\) and a detection limit of 18 pg mL\({}^{-1}\) were found, and agreement of the assay with traditional ELISA was excellent. Another antibody-aptamer sandwich assay was designed for the cancer biomarker MUC1 [86]. Screen-printed electrodes with a graphite working electrode were used as a substrate on which to electropolymerize o-aminobenzoic acid. The conducting polymer was then conjugated to a primary antibody against MUC1 using EDC/NHS chemistry with unreacted sites capped using ethanolamine. An aptamer binding to another site on MUC1 completed the sandwich and binding of methylene blue was detected by its reduction during a DPV sweep. The binding of MUC1 to antibody was done for 90 min and the binding to aptamer was allowed to take place overnight. An increase in charge transfer resistance during each step of immobilization and sandwich formation was seen clearly using electrochemical impedance spectroscopy. A linear range of 3-10 ppb with a detection limit of 2.4 ppb was obtained. A related electrochemical sensor was developed for the detection of cancer biomarker glycoprotein MUC16 using indium tin oxide (ITO) electrodes  
Figure 8: Schematic representation of fabrication process of the CRP aptasensor. (**A**) Preparation of immunoprobe Zn\({}^{2+}\)/Ab/Au NPs@Si MSs, (**B**) Fabrication of MCH/RNA/Au NP-modified electrode. (**C**) Depiction of the components of the CRP aptasensor. Reprinted with permission from reference [82]. Copyright Elsevier 2017.  
onto which Au nanoparticles had been electrodeposited [87]. In this approach, the MUC16 polyclonal antibody was drop-cast onto the electrode surfaces. After binding of MUC16, an aptamer with a primer tail bound to complete the sandwich. Using two DNA probes, the hybridization chain reaction was used in multiple cycles to extend the DNA strand to yield long concatamers so that more of the methylene blue redox indicator could binding to the structure. Using DPV, a linear range of 0.39-200 units mL\({}^{-1}\) with a detection limit of 0.02 units mL\({}^{-1}\) was obtained. The inflammation marker tumor necrosis factor-alpha (TNF-\(\alpha\)) was detected using an electrochemical sandwich assay involving an aptamer and an antibody conjugated to HRP [88]. The electrode, a graphite screen-printed electrode, was covered by a film of cobalt hexacyanoferrate electrosynthesized from a solution of CoCl\({}_{2}\) and K\({}_{3}\)[Fe(CN)\({}_{6}\)] intended to provide superior electron transfer ability. Au nanoparticles (d = 15 nm) were adsorbed onto the thin film and then modified by a thiolated aptamer. After binding to TNF-\(\alpha\) for 20 min, the binding of an antibody conjugated to HRP for 60 min took place. The HRP activity against the substrate o-aminophenol in H\({}_{2}\)O\({}_{2}\) was detected using DPV yielding a linear range from 1-100 ng L\({}^{-1}\) with a detection limit of 0.52 ng L\({}^{-1}\). The step-by-step formation of the modified electrode and sandwich structure was followed by EIS. Agreement for TNF-\(\alpha\) three human serum samples with results from ELISA within error bars was found. Insulin-like growth factor 1 (IGF1) is a 70 amino acid hormone similar in structure to insulin and found to be a biomarker for abdominal aortic aneurysm. A sandwich assay on interdigitated electrodes (IDE) for IGF1 was created using a biotinylated aptamer for capture and an anti-IGF1 antibody [89].