## 4 Electrochemical Sandwich Assays Combining Aptamers and Antibodies
The substrate used for ALP was p-aminophenol phosphate and oxidation of the product p-aminophenol was detected electrochemically using both CV and DPV in 50 mM TRIS buffer of pH 9. The current responses showed two distinct linear ranges as a function of PTK7 concentration, one from 0.1-10 pM and a second from 20-300 pM, and the limit of detection was 100 fM. A capacitive-based aptamer-based immunosensor was developed for the detection of vascular endothelial growth factor-165 (VEGF) in human serum [78]. The aptamer-based immunosensor utilized a highly specific and selective VEGF aptamer functionalized on the aptasensor. The assay design involves capturing of VEGF protein followed by sandwiching with antibody-conjugated magnetic beads and then the change in capacitance was measured using non-faradaic electrochemical impedance spectroscopy. Interdigitated gold electrode arrays were used which were modified with mercaptopropionic acid followed by conjugation to the 5\({}^{\prime}\)-NH\({}_{2}\)-(CH\({}_{2}\))\({}_{6}\)-modified aptamer using EDC/NHS chemistry. The signal generated using this aptasensor was greatly enhanced by the use of the magnetic beads with the detection range of 5 pg mL\({}^{-1}\) to 1 ng mL\({}^{-1}\) of VEGF protein in human serum achieved with no additional reagents or redox species. The \(\beta\)-amyloid oligomers associated with Alzheimer's disease were detected by an antibody-aptamer electrochemical sandwich assay. Amyloid \(\beta\)-peptide (A\(\beta\)) in its oligomeric form is often considered as the most toxic species in Alzheimer's disease (AD) [79]. Antibodies for A\(\beta\) oligomers were used as the recognition elements for specifically binding to A\(\beta\) oligomers. A nanocomposite of aptamer-Au-thionine prepared by in situ modification of gold nanoparticles (AuNPs) with DNA aptamer and thionine (Th) was utilized as the detection probe. A\(\beta\) oligomer-specific DNA aptamers were isolated by the combination of a gel-shift assay and a competitive screening method. The electrochemical signal was generated and amplified by high loading of Th on AuNPs, and a limit of detection of 100 pM was achieved with a linear range of 0.5-30 nM. The physiological level of A\(\beta\) in normal human cerebrospinal fluid (CSF) is about 1-2 nM. The level of A\(\beta\) oligomers in CSF is efficacious for predicting the severity and the progression of the early preclinical stage of the AD disease. The level of the protein tau in cerebrospinal fluid is also thought to be clinically significant for the prognosis of [PERSON]'s disease and an aptamer-antibody electrochemical sandwich assay has been developed for the \(\tau\)-381 form of the protein [80]. In this assay, an Au electrode was modified by mercaptopropionic acid and the anti-tau antibody was linked to the surface using EDC/NHS chemistry. Au nanoparticles were modified with cysteamine and the tau aptamer of the sequence 5\({}^{\prime}\)-H\({}_{2}\)N-GCGAGCGTGGCAGG-3\({}^{\prime}\). Differential pulse voltammetry was used with [Fe(CN)\({}_{6}\)]\({}^{3-4-}\) used as a redox probe. A current response linear in log[tau] was found for the concentrations studied ranging from 0.5-100 pM \(\tau\)-381 in 0.1 M PBS. Recovery in the detection of \(\tau\)-381 from human cerebrospinal fluid samples diluted 100-fold in 0.1 M PBS was found to be excellent. Figure 7: Schematic of the electrochemical sandwich assay for protein tyrosine kinase-7 that uses and aptamer and an antibody labeled with the enzyme alkaline phosphatase. Reproduced with permission from reference [76]. Copyright Elsevier 2021. Human immunoglobulin E (IgE) was detected by using methylene blue (MB) as a redox probe and using the combined advantages of the aptamer, nanomaterial and antibody to design the electrochemical sandwich biosensor [81]. Goat anti-human IgE was immobilized on the surface of the gold electrode followed by exposure to different concentrations of human IgE.