The sensitivity of surface-enhanced Raman spectroscopy (SERS) highly depends on experimental factors including aggregating agents and pH. Using silver nanoparticles as the substrate, the effect of five cationic (K+, Na+, Mg2+, Li+, Ca2+) and three anionic (Cl-, Br-, I-) aggregating agents was examined on the SERS detection of tobacco-related biomarkers, namely cotinine (COT) and trans-3'-hydroxycotinine (3HC). The optimal concentrations of the aggregating agents with respect to highest SERS intensity varied widely (from 1.5 mM for MgCl2 to 150 mM for LiCl). Both cations and anions strongly influenced the SERS enhancement. When Cl- was used as the anion, Mg2+ and Na+ exhibited the highest SERS intensities for COT and 3HC, respectively. When Mg2+ was used as the cation, Cl- and Br- generated the highest SERS enhancement for COT and 3HC, respectively. Clearly, SERS enhancement also depended on the target molecule. Among the 11 aggregating agent combinations tested, the highest SERS enhancement is obtained using 1.5 mM MgCl2 for COT at pH 7.0 and 50 mM NaBr for 3HC at pH 3.0. (C) 2013 Elsevier Inc. All rights reserved.