## 4 Electrochemical Sandwich Assays Combining Aptamers and Antibodies
The aptamer immobilized Au NPs can bind with human IgE at different binding sites and this specific binding of the aptamer and human IgE on the electrode surface enables the accumulation of the MB on the electrode surface by the interaction of MB with G bases in the aptamer and therefore an amplified signal was observed in the electrochemical sensing system. The range of the detection of human IgE was 1-10,000 ng mL\({}^{-1}\) with a limit of detection of 0.52 ng mL\({}^{-1}\). C-reactive protein (CRP) is one of the cytokine-induced acute phase proteins and its normal concentration in serum is below 5.0 mg L\({}^{-1}\) with a clinical range within 1-500 mg L\({}^{-1}\), and is widely accepted as one of the biomarkers for cardiovascular diseases and inflammation. An RNA aptamer-based electrochemical aptasensor was designed to detect C-reactive protein using functionalized silica microspheres as immunoprobes, and is depicted in Figure 8[82]. Silica microspheres were synthesized and then functionalized with Au nanoparticles to provide a large surface area for immobilizing Zn\({}^{2+}\) and antibodies. The CRP aptamer used was a 44-mer RNA aptamer of sequence: 5\({}^{\prime}\)-SH-(CH\({}_{2}\))\({}_{6}\)-GCCUGUAAGGUGUGUGGGGGAGUGUGUGUGAGGAGAUUGC-3\({}^{\prime}\). The aptamer was immobilized on the surface of Au nanoparticles deposited onto GCE for 5 h. An immunoprobe was prepared by the incubation of the anti-CRP (Ab) on the AuNP-modified silica microspheres followed by re-dispersion in a solution of Zn(NO\({}_{3}\))\({}_{2}\) to load the Zn\({}^{2+}\) on the surface. Various concentrations of the CRP samples were sandwiched between the aptamer immobilized electrode and immunoprobe and the response was recorded by SWV detecting reduction of Zn\({}^{2+}\). Under optimized conditions, the linear range of the detection was 0.005-125 ng mL\({}^{-1}\), and the detection limit was 0.0017 ng mL\({}^{-1}\). An electrochemical aptamer-antibody sandwich assay for CRP was developed with the aid of magnetic nanoparticles [83]. Streptavidin-coated magnetic beads (1.05 \(\upmu\)m size) were modified with a CRP 44-mer RNA aptamer that was 5\({}^{\prime}\)-biotinylated. The incubation period was 15 min followed by washing for 2 min which was aided by collecting the particles using a magnetic field and then re-suspending them. After binding of CRP, a biotinylated monoclonal antibody to CRP was then bound, again for 15 min followed by a magnetically aided washing step. Finally, a conjugate of streptavidin and alkaline phosphatase was added and after washing the particles were magnetically collected onto SPCE. Using the substrate 1-naphthylphate and DPV detection of the 1-naphth project a dose-response curve was obtained with a measuring range of 1-1000 mg L\({}^{-1}\), a dynamic range from 0.1-50 mg L\({}^{-1}\) and a limit of detection of 0.054 mg L\({}^{-1}\). An aptamer-antibody sandwich assay was developed for \(\upalpha\)-1 antitrypsin (AAT), a biomarker for Alzheimer's disease [84]. Carbon nanotubes (l-5 \(\upmu\)m, d-20 nm) were modified by adsorption of 3, 4, 9, 10-perylene tetracarboxylic acid and then cast onto screen-printed carbon electrodes (SPCE). The carboxylic groups were then activated by EDC/NHS chemistry and conjugated to the 37-mer AAT aptamer. The other part of the sandwich was composed of silver nanoparticles that were modified by mercaptopropionic acid followed by application of EDC/NHS chemistry and conjugation to an anti-AAT monoclonal antibody conjugated to alkaline phosphatase. Incubation of the aptamer-modified electrode with AAT was for 1 h and detection was done using DPV with p-aminophenylphosphate (p-APP) at 100 \(\upmu\)M as higher p-APP concentration did not increase the response further. A linear range from 0.05-20 pM was found with a detection limit of 0.01 pM. The assay was found to be selective against IgG or IgE interfering proteins, and recovery from serum samples with AAT was 95-102%.