## 3 Biosensor Application of Nanostructures Electrodeposited onto a Template
### Electrodeposition onto Regularly Packed Thin Films of Colloidal or Nanoscale Spheres  
A popular and low-cost means of preparing an ordered template is the assembly of spherical colloidal particles of polystyrene or silica into hexagonal close-packed arrays. Such arrays can vary in the number of layers deposited on a substrate, and these layers can be produced by methods including vertical deposition, spin coating, and dip coating [39]. The degree of order in these arrays can be high, extending over centimeters with occasional defects. Following electrodeposition into the interstices between the particles, the template of spheres can be dissolved and washed away using solvent. The resulting macroporous metal structure has been achieved for gold and platinum [40] and also for platinum, palladium, and cobalt [41]. The polystyrene spheres are produced by emulsion polymerization, and are commercially available in a range of sizes. Through the colloidal template approach, the surface area of the electrode and its morphology can be controlled in a systematic way by varying the colloidal particle size, film thickness, and electrodeposition parameters. Then, such an approach allows for clearer study of the relationship between electrode structure and biosensor performance. Macroporous platinum films were formed by electrodeposition onto a self-assembled layer of polystyrene spheres, which is then followed by the use of solvent to remove the spheres [42]. A silicon wafer covered by a 200-nm layer of gold over a five-nm chromium adhesion layer was prepared by thermal evaporation. Polystyrene spheres of 460-nm diameter were carefully spread over a water-air interface, and then transferred by dip coating, with repeated transfers to build up multiple layers of close-packed spheres. Then, electrodeposition was carried out by cycling the potential from \(-\)0.18 V to 1.20 V (versus Ag/AgCl) at 175 mV sec\({}^{-1}\) in a solution of five mM of H\({}_{2}\)PtCl\({}_{6}\), 0.2 M of H\({}_{2}\)SO\({}_{4}\), and 35 mM of polyvinylpyrrolidone, which served to promote a uniformity of deposition. After deposition, the template of polystyrene spheres was removed by Soxhet extraction in toluene. The amount of Pt that was electrodeposited was used to define layers; for example, it was a 'three-fourths deposit' if the platinum was as high off the surface as three-fourths of the diameter of a polystyrene sphere or a seven-fourths deposit if the platinum was as high as 1.75 times the diameter of a Pt sphere. The first layer of the structure showed interconnected pores, and the second layer had pores connected to the first layer, which allowed for both horizontal and vertical fluid penetration. Using an underpotential deposition of H\({}_{2}\), a roughness factor of 30.3 \(\pm\) 1.0 was determined for the seven-fourths deposit. Figure 3 shows SEM (Strata DB235, FEI Co., Hillsboro, OR, USA) images of one-third, three-fourths, and seven-fourths deposits, along with an optical micrograph of an electrode. For these structures, the roughness factor as measured electrochemically steadily increased, being 18.3, 24.9, and 30.3, respectively. Electrodeposition onto a template was used to create a surface-enhanced Raman active (SERS) substrate [43]. ITO-coated glass was used as support for a 10-nm layer of sputter-coated gold. A monolayer of two-\(\upmu\)m diameter polystyrene spheres was formed on another glass slide, and then transferred onto the gold-coated ITO, which was then used as the substrate for electrodeposition from 0.5 g L\({}^{-1}\) of HAuCl\({}_{4}\) solution at a constant current of 0.04 mA cm\({}^{-2}\) for various fixed times from two to 30 h. Then, the polystyrene spheres were washed away using methylene chloride, leaving behind a hierarchical structure of the array of indentations previously occupied by the microspheres and electrodeposited gold structures resembling aggregated nanorods in what had been the gaps between the microspheres.