## 2 Biosensor Application of Electrodeposited Nanoparticles
### Deposition at a Constant Potential for a Fixed Time Period  
The formation of metal nanoparticles by electrodeposition at a constant applied potential for a fixed period of time is a popular strategy. The electrodeposition potential for HAuCl\({}_{4}\) to Au in aqueous solution is 0.853 V (versus standard hydrogen electrode, SHE) [11]. Glassy carbon, which has the advantage of a wide potential window [12], is a popular choice for the supporting electrode. For example, a biosensor for the anti-cancer agent vinblastine was constructed by first electrodeposition of gold nanoparticles onto a glassy carbon electrode (GCE) [13]. Electrodeposition was carried out in 3 mM of HAuCl\({}_{4}\) and 0.5 M of H\({}_{2}\)SO\({}_{4}\) at \(-\)0.20 V (versus Ag/AgCl) for 220 s. Scanning electron microscopy (SEM, MIRA3, TESCAN, Brno-Kohoutovice, Czech Republic) revealed a layer of gold nanoparticles of fairly uniform coverage; some of the nanoparticles were irregular in shape or in contact with their neighbors, and each of them was about 20 nm in size, as shown in Figure 1.  
The gold nanoparticles were then surface modified by 3-mercaptoethanolpropionic acid, which was then covalently conjugated to tubulin using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) coupling reagents. Using Faradaic electrochemical impedance spectroscopy in phosphate buffer of pH 7.4 containing the redox probe 2.0 mM [Fe(CN)\({}_{6}\)]\({}^{3-/4-}\), the shift in charge transfer resistance versus vinblastine concentration was found to show two linear regions, one from 0.4 to 12.0 nM, and another from 12.0 to 65.0 nM. The detection limit for vinblastine was found to be 0.084 nM. Enzymatic biosensors based on gold nanoparticles directly electrodeposited on GCE or related substrates have been prepared. Gold nanoparticles were electrodeposited onto GCE and used as a support for the enzyme horseradish peroxidase (HRP), both in native form and in a form that had been chemically conjugated to lactose [14]. The gold nanoparticles were electrodeposited at \(-0.2\) V (versus Ag/AgCl) for 60 s onto a GCE from 100 mg L\({}^{-1}\) of HAuCl\({}_{4}\) solution. SEM (JSM-7500F; JEOL USA Inc., Peabody, MA, USA) showed the gold nanoparticles with an average diameter of 100 nm covering much of the electrode surface. The lactsylated form of the enzyme was found to show greater electrocatalytic activity for hydroquinone in the presence of 200 uM of H\({}_{2}\)O\({}_{2}\), having a detection limit of 74 nM versus 83 nM for the native enzyme. Hydroquinone is more commonly used as a redox mediator. A significant feature was that the immobilized lactsylated enzyme was more stable, retaining 60% of its activity over four months of storage versus only 10% retained activity for the native enzyme. In another example, gold nanoparticles electrodeposited onto graphite electrodes were used as a support for acetylcholinesterase esterase and to make an amperometric assay for thiocholine [15]. The Au nanoparticles were electrodeposited from 50 mM of tetrachloroauric(III) acid (HAuCl\({}_{4}\)) in 0.1 M of HCl at \(-0.155\) V (versus Ag/AgCl) for 10 s. Acetylcholinesteraseerase converted acetylcholinesterase to acetic acid and thiocholine, and thiocholine was oxidized at 0.8 V. The detection limit for thiocholine was 2.5 uM, and the response was linear up to 600 uM. The activity of the immobilized enzyme was inhibited by the insecticide monocrotophos. Using a one-step electrodeposition, both gold nanoparticles and HRP were deposited on a GCE [16]. Electrodeposition was conducted from a solution that was 1 mM of HAuCl\({}_{4}\), 10 mM of cetyltrimethylammonium bromide (CTAB), and 3 mg L\({}^{-1}\) of HRP in pH 7.5 phosphate buffer at \(-0.5\) V (versus Ag/AgCl) for 50 mins. In the absence of CTAB and HRP, the deposited nanoparticles appeared aggregated under SEM (JSM-6701F, JEOL Ltd., Tokyo, Japan) while the addition of CTAB produced a deposit of uniformly dispersed nanoparticles that were about 10 nm in diameter.