## 3 Other Electrochemical Sandwich Assays Using Two Aptamers
Another dual aptamer-based sandwich biosensor was constructed to detect MCF-7 breast cancer cells using the MUC1 tumor marker [70]. MUC1 binding aptamer was immobilized on the surface of GCE modified with a multi-walled carbon nanotube/poly(glutamic acid) composite. Cancer cells were sandwiched between  
Figure 6: Schematic of the electrochemical sandwich assay for cardiac troponin I (cTnI) in which the capture aptamer is a thiolated DNA nanotetrahedron with one aptamer strand. The detection aptamer is bound to Au@Pt nanoparticles around magnetic metal organic framework (Fe3O4@UiO-66) particles bound to Tro4 and Tro6 aptamers along with G-quadruplex/hemin (GQH) DNAzyme and horseradish peroxidase (HRP). Pathway (**a**) shows the steps for the modification of the screen-printed gold electrodes with DNA nanotetrahedrons presenting the Tro4 aptamer, the formation of the sandwich structure with cTnI and the modified Fe3O4 nanoparticles presenting the Tro6 aptamer and enzymes. The sequences for Tro4 and Tro6 are shown at the bottom of the figure. Pathway (**b**) shows the steps for the production of the Tro6 modified aptamer-labeled nanoparticles and enzymes for detection. Reproduced with permission from [64]. Copyright Springer 2019.  
capture aptamer and AgNPs linked to detection aptamer by glutathione. The captured Ag nanoparticles were dissolved by 0.5 M HNO\({}_{3}\) and then detection of Ag\({}^{+}\) was carried out using differential pulse voltammetry. The concentration range of the detection was 1.0 \(\times\) 10\({}^{2}\) to 1.0 \(\times\) 10\({}^{7}\) cells mL\({}^{-1}\) with a detection limit of 25 cells. The detection of _S. aureus_ using a primary and secondary aptamer sandwich assay was reported [71]. The primary aptamer was immobilized on magnetic beads and the secondary aptamer was bound to silver nanoparticles. The capture bacteria and attached silver nanoparticles were treated in acid to dissolve the silver nanoparticles followed by detection of the Ag\({}^{+}\) ion by stripping voltammetry. A detection range from 10-1 \(\times\) 10\({}^{6}\) CFU mL\({}^{-1}\) was obtained with a detection limit of 10 CFU mL\({}^{-1}\). Results for real water samples containing _S. aureus_ were found to be consistent with those found using the assay. The detection of type 2 diabetes biomarker vaspin using an electrode modified by a high surface area natural biomineral structure (coccolith) from _Emiliania huxleyi_, an ocean unicell organism [72]. The coccoliths were drop-cast on a screen-printed gold electrode and then sputter-coated with gold. A pair of aptamers binding to different epitopes on vaspin was used in the sandwich assay, with the first aptamer drop-cast and then after vaspin binding, the second aptamer conjugated to enzyme HRP was added. The electrochemical signal was the oxidation of substrate TMB in the presence of H\({}_{2}\)O\({}_{2}\) achieving a detection limit of 298 pM. An electrochemical sandwich assay for lysozyme was developed using two aptamers [73]. Abnormal concentrations of lysozyme can serve as a biomarker for a range of diseases. A thiol terminated lysozyme binding aptamer (LBA, of sequence: 5\({}^{\prime}\)-SH-(CH\({}_{2}\))6-ATC-TAC-GAA-TTC-ATG-GGG-CCTAAA-GAG-TGG-AGA-GTT-ACT-TAG-3\({}^{\prime}\)) and horseradish peroxidase was loaded onto Au nanoparticles of average size 18 nm. LBA and dithiothreitol were drop-cast onto a flat gold electrode and then surface blocking was carried out using mercaptohexanol. EIS was used to confirm the binding of lysozyme to the aptamer-modified electrode and then the binding of the Au-LBA-HRP conjugates. The parameters of hydroquinone concentration, H\({}_{2}\)O\({}_{2}\) concentration, pH, immersion time and amount of HRP were all optimized to produce the largest DPV signal. Detection was performed using DPV in 150 mM H\({}_{2}\)O\({}_{2}\) and 30 mM hydroquinone. A linear dependence of the increase in DPV peak current with log[lysozyme] was found from 0.01 pg mL\({}^{-1}\) to 105 pg mL\({}^{-1}\) with a detection limit of 0.003 pg mL\({}^{-1}\).