## 3 Other Electrochemical Sandwich Assays Using Two Aptamers
First, an amide-terminated capture aptamer and thionine (redox mediator) were covalently attached to the amine functional group of chitosan via amide bond formation using phthalolyl chloride as linker on glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNT)/ionic liquid (IL)/chitosan nanocomposite. The biotin-streptavidin interaction was used to attach another IgE aptamer with biotin and horseradish peroxidase (HRP). HRP has been used as a label for signal amplification and catalyzing the oxidation of thionine by hydrogen peroxide. The presence of IgE induces the formation of a sandwich structure on the electrode and was monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) and differential pulse voltammetry as shown in Figure 5. The limit of detection found was 6 pM with a sensitivity of 1.88 \(\mu\)A nM\({}^{-1}\) at a range up to 20 nM. The purposed aptasensor was used to detect IgE in human serum as other proteins such as lysosome and bovine serum albumin (BSA) did not interfere with the detection of IgE. An electrochemical aptamer-based assay for the detection of both total and glycated hemoglobin in blood has been reported [59]. A blood concentration of the glycated form of hemoglobin (HbA1c) of over 6.5% is one of the criteria for the diagnosis of diabetes. A higher amount of HbA1c is associated with other diseases including cardiovascular disease, nephropathy, and retinopathy. Thiol-modified forms of the aptamers were immobilized on gold nanoparticle-modified array electrodes. A new DNA aptamer against HbA1c and tHb was identified and characterized from a 60-mer library with dissociation constants of 2.8 nM and 2.7 nM for HbA1c and tHb, respectively. The aptasensor voltammetry response towards tHb and HbA1c was measured in the concentration range of 100 pg mL\({}^{-1}\)-10 \(\mu\)g mL\({}^{-1}\). Detection limits for tHb of 0.34 ng mL\({}^{-1}\) and 0.2 ng mL\({}^{-1}\) for HbA1c were found. The detection of the HbA1c in whole blood samples was also carried out showing its potential application to discriminate between diabetic and healthy individuals. Recently, an electrochemical assay capable of detecting the glycosylation of prostate-specific antigen (PSA) using two aptamers in a sandwich format was developed [60]. Biotinylated aptamer was immobilized on a gold electrode by conjugation to a self-assembled monolayer followed by incubation with serial dilutions of a standard PSA solution followed by binding of the detection aptamer, which was labeled with 6-carboxyl fluorescein that would bind to a Fab anti-fluorescein-peroxidase conjugate. The enzyme substrate used to generate the detection current was 3, 3\({}^{\prime}\), 5, 5\({}^{\prime}\)-tetramethylbenzamide (TMB). A novel DNA aptamer was isolated targeting human PSA (hPSA) by a counter-selection process using recombinant PSA (rPSA) that does not carry the glycan moiety, thus selecting for an aptamer that bound both to regions of the protein and the glycan. The selected aptamerwas tested using an electrochemical binding assay with chronoamperometric detection. The range of detection for hPSA was 0.66-62.5 ng mL\({}^{-1}\) in TBS buffer and 0.66-25 ng mL\({}^{-1}\) in diluted serum with a detection limit of 0.66 ng mL\({}^{-1}\). The response of the assay to rPSA was very low as the glycan was absent. The sensor supported measurement in serum samples with minimum dilution which allows evaluation of the PSA levels in the diagnosis of prostate cancer. A subsequent study presented a computational prediction of the structure of the aptamer and of its complex with the glycoprotein [61]. The detection of adenosine triphosphate was performed on a nanoporous gold electrode using 3,4-diaminobenzoic acid (DABA) as a molecular reporter in the aptamer-based electrochemical biosensor [62].