A method for chemically differentiating the surface of a set of small, closely-spaced, lithographically-defined Au features on a die, from another set of similar features intimately inter-dispersed, is described. The key enabler of the method is a standard electronics probe array adapted to carry out electrochemistry on the features. The probe array is used first to verify the electrical integrity of features and the quality of electrical contacts by measuring electrical resistance, then, in the presence of the electrolyte, simultaneously maintain one potential on one set of Au features and another potential on the other set in order to carry out desired electrochemical reactions. The technique was demonstrated on dies bearing 40 electrically isolated Au features (based on 5 μm wide stripes) accessed via 64 contact pads each 100×100 μm 2 in area. The array had 64 probes, of which 16 were maintained at a desorbing potential (-1.6 V vs. Ag/AgCl) and 48 at a stability potential (-0.3 V). The surface compositions were analyzed with time-of-flight secondary ion mass spectrometry by imaging ion fragments characteristic to the thiols forming SAMs, thereby validating the process.

Several biosensing techniques like SPR and QCM use glass as the substrate, bearing a thin Au layer which serves as the sensing surface. Other substrates of interest include Si due to processing knowledge and the potential integration of electronics, and Cytop which has a refractive index close to that of water. Au on glass is highly crystalline, with a large 111 character, especially after annealing, whereas on Si and on Cytop, Au is non-textured (“polycrystalline”). These structural differences result in distinct voltammetric behavior, both faradaic and capacitive, of SAM- and protein-coated Au on different substrate types. A contact angle and double-layer capacitance examination of protein adsorption on SAM-coated Au on these substrates, and electrochemical desorption of the resulting molecular stacks, indicates that the usual assumption that hydrophobic SAMs promote adsorption and hydrophilic ones prevent adsorption is not universally true, and depends both on the protein type and the substrate. Thus, caution is advised against applying results obtained on one substrate to another. On the other hand, systems utilizing bioconjugation seem to be much less sensitive to Au type, and thus the underlying substrate. Electrochemical desorption of proteins from Au does occur but with limited efficiency.