## 5 Biosensor Application of Electrodeposited Composite Nanostructures
### Composite Electrodes Containing Prussian Blue  
Prussian blue (PB) is the compound known as \((Fe_{4}^{III}[Fe^{II}(CN)_{6}]_{3})\), in which Fe(III) is coordinated to nitrogen, and Fe(II) is coordinated to carbon [131]. PB is known as an electrocatalyst for the oxidation of H\({}_{2}\)O\({}_{2}\). Layer-by-layer modification of a GCE with PB and gold was used to make an amperometric sensor for H\({}_{2}\)O\({}_{2}\)[132]. (PB-Au)\({}_{\mathrm{x}}\) multilayer films were made where x was varied from one to five. PB was electrodeposited first from a solution containing 0.1 M of K\({}_{2}\)SO\({}_{4}\), one mM of K\({}_{3}\)Fe(CN)\({}_{6}\), one mM of Fe\({}_{2}\)(SO\({}_{4}\))\({}_{3}\), and 0.05 M of H\({}_{2}\)SO\({}_{4}\) by scanning the potential from one V to \(-\)0.2 V (versus SCE) at 50 mV sec\({}^{-1}\). Gold was electrodeposited by scanning from 0.65 V to \(-\)0.2 V at 50 mV sec\({}^{-1}\) in a solution containing 0.1 M of K\({}_{2}\)SO\({}_{4}\), one mM of HAuCl\({}_{4}\), and 0.05 M of H\({}_{2}\)SO\({}_{4}\). The first step gave 80-nm cubic-shaped PB nanoparticles that were visible in SEM (JEM-6700F, JEOL Co. Ltd., Beijing, China) and the second step gave gold nanoparticles of 80 nm (first layer) or 100 nm (second layer). A solution of glucose oxidase and dopamine was combined with Na\({}_{2}\)PtCl\({}_{6}\) and allowed to undergo the polymerization of dopamine to yield a dispersion of platinum nanoparticles and polydopamine, entrapping the enzyme glucose oxidase. Then, the dispersion was cast onto the electrode surface covered by (PB-Au)\({}_{2}\). The current response to glucose arising from the oxidation of H\({}_{2}\)O\({}_{2}\), which was generated by the reaction, was monitored at \(-\)0.05 V. A detection limit of 0.5 \(\mu\)M and a linear range from 0.008 to 0.79 mM was found. Application to the determination of glucose in blood serum from healthy patients was successful. A biosensor for Cu\({}^{2+}\) based on a composite nanostructure containing a DNAzyme was formed by one-step electrodeposition [133]. The composite film was both three-dimensional and macroporous. Using vertical deposition, a layer of close-packed silica spheres was formed on a gold electrode. Then, single-walled carbon nanotubes were dispersed into a 0.05% (w/v) chitosan solution in 1.0% acetic acid. A second solution containing chitosan and the precursors to PB was prepared containing a solution with five mM of FeCl\({}_{3}\), five mM of K\({}_{3}\)[Fe(CN)\({}_{6}\)], 0.2 M of KCl, 0.2 M of HCl, and 10 mL of 0.05% chitosan (CS). After combining these two solutions, electrodeposition onto the silica sphere-modified gold electrode was carried out at 0.4 V (versus SCE) for 300 s. Then, the silica template was dissolved away by brief treatment with 5% HF, leaving behind a macroporous film. The film was then scanned by CV between \(-\)0.05 and 0.35 V at 50 mV sec\({}^{-1}\) for 25 cycles, which resulted in the film becoming conductive. A dispersion of gold nanorods associated with graphene oxide and conjugated to enzymes glucose oxidase and HRP was then prepared. Gold nanorods were also conjugated to DNAzyme against Cu\({}^{2+}\), which resulted in the cleavage of DNA, whose association with a complimentary strand would then increase the current response to the enzymatic reduction of glucose. The sensor was highly selective for Cu\({}^{2+}\) in the presence of other metal ions, and had a limit of detection of 10\({}^{-19}\) M.  
A biosensor for \(\beta\)-glucan was made by immobilizing \(\beta\)-glucanase and glucose oxidase into a composite electrode containing PB nanoparticles, gold nanoparticles, and chitosan [134].