In this paper, we tested the hypothesis that there is an increased sensitivity for detecting and measuring disease biomarkers (such as human cardiac troponin I, cTnI) by a decrease of nonspecific adsorption in diluted self-assembled monolayers (SAMs) on planar sputtered gold films. Combining grazing angle Fourier-transform infrared spectroscopy (FT-IR) and antibody-antigen-antibody (sandwich) fluorescence-based immunoassay, we examined the relationship of sensitivity, specificity of detection of cTnI and the level of nonspecific protein adsorption in the following SAMs: pure MHA (16-mercaptohexadecanoic acid, 1 mM, with head COO-, x = 1.0), a mixed SAM comprising MHA (0.1 mM) and UDT (1-undecane thiol, 0.9 mM, with hydrophobic head CH3, x = 0.1(UDT)), and a mixed SAM comprising MHA (0.1 mM) and MUD (11-mercapto-1-undecanol, 0.9 mM, with hydrophilic head OH, x = 0.1(MUD)). Our data revealed that nonspecific binding to SAMs is favored in the following order: CH3 > COO- > OH, consistent with previous studies. Compared with pure SAMs, diluting MHA SAMs with MUD increases the sensitivity of cTnI, whereas diluted MHA SAMs with UDT has the same sensitivity of detection of cTnI, suggesting it is the nature of the second diluting thiol that plays an important role on the amount of adsorbed protein on the surface. We obtained a 10-fold increase in the limit of detection of cTnI to 10 ng/ml using x = 0.1(MUD) due to a decrease of nonspecific binding. Further, specific binding between the antigen cTnI and its antibody is unaltered on pure and diluted SAMs. (c) 2012 Elsevier B.V. All rights reserved.