## 4 Electrochemical Sandwich Assays Combining Aptamers and Antibodies  
A number of studies have used an aptamer together with an antibody. This can be an attractive approach when there are not two aptamers available that will bind to different regions of the target to create a sandwich. There are also established schemes for use of antibodies as detection agents such as when they are labeled with enzymes. Immobilization of either aptamer or antibody on the electrode surface may be utilized. For example, a sandwich assay for the viral protein of the H5N1 avian flu virus was designed using an H5N1 aptamer and an anti-H5N1 antibody that was conjugated to alkaline phosphatase (ALP) [74]. Gold nanoparticles were formed by electrodeposition from HAuCl\({}_{4}\) solution onto screen-printed carbon electrodes then modified with 3-mercaptoethanolpropionic acid to which the aptamer was conjugated using EDC/NHSS chemistry (NHSS = N-hydroxysulfosuccinimide). Incubation with H5N1 protein at different concentrations was done for 1 h followed by incubation with the antibody-enzyme conjugate for 1 h. The substrate p-aminophenylphosphate was converted to p-aminophenol by ALP that could be detected by its oxidation to p-quinoneimine. Detection was done using currents from cyclic voltammetry and from DPV and compared with DPV being found to be twice as sensitive. The response vs. protein concentration showed two linear regions, from 100 fM to 100 pM and from 25-100 pM. The detection limit was found to be 100 fM, comparing favorably with commercial ELISA. A biosensor for lysozyme was developed by first covalently immobilizing a 40-mer onto screen-printed carbon electrodes [75]. Sodium nitrite (NaNO\({}_{2}\)) and 4-aminobenzoic acid were reacted to form a 4-diazonium cation of benzoic acid that was then reacted with the carbon surface during a linear potential sweep from 0.6 to \(-\)0.8 V (vs. Ag/AgCl) to give covalently immobilized benzoic acid moieties on the carbon surface. The covalently immobilized benzoic acid was activated with EDC/NHS chemistry to covalently attach the aptamer through the 5\({}^{\prime}\)-end. Incubation of the lysozyme with the aptamer-modified electrode was done for 15 min, followed by incubation with the biotinylated anti-lysozyme antibody for 1 h. The next step was incubation with avidin labeled with alkaline phosphatase for 1 h. The substrate used was 1-naphthyl phosphate with product 1-naphth detected by DPV. The parameters of detection pH (7.4), concentration of anti-lysozyme antibody, concentration of alkaline phosphatase-avidin, and lysozyme incubation time were all optimized. The detection limit achieved of 4.3 fM was the lowest reported to date, and the linear range from 1 fM-5 nM was noted as broad compared to most of those previously reported. The selectivity was tested using BSA, cytochrome c, and casein and found to be very good, as was sample recovery for lysozyme spiked into wine samples. An aptamer sandwich-based assay was developed for protein tyrosine kinase-7 (PTK7), a biomarker for breast and possibly other cancers, using gold nanoparticle-modified screen-printed carbon electrodes (SPCE), as depicted in Figure 7[76]. PTK7 is a type of membrane protein that plays an important role in cell recognition, ion transport as well as cancer development [77]. The gold nanoparticles were modified with mercaptopropionic acid then conjugated to the amine-terminated aptamer (5\({}^{\prime}\)-H\({}_{2}\)N-ATC TTA CTG CTG CGC CGC GGA AAT ACT GTA CCGG TTA GAT TTT TTTTT-3\({}^{\prime}\)) using EDC/sulfo-NHS. The gold nanoparticles were electrodeposited onto SPCE from 0.5 mM HAuCl\({}_{4}\) in 0.5 M H\({}_{2}\)SO\({}_{4}\) while stirring at 0.18 V for 400 s. The sandwich structure consisted of the surface-bound aptamer, PTK7, and an anti-PK7 antibody labeled with alkaline phosphatase. The binding of PTK7 to the surface-bound aptamer and to the anti-PK7 antibody was evaluated on planar gold by SPR and the formation of the sandwich complex was also confirmed by SPR.