We describe a cantilever-based method for measuring the self-assembly of alkanethiols on a gold surface in a flow system that permits easy step changes in concentration and acquire a continuous in situ measure of the resulting chemisorption through the change in resonance frequency. A gold-coated (2.2 mm(2)), piezoelectric-excited, millimeter-sized cantilever (PEMC) sensor was exposed to 1-hexadecanethiol (HDT) in ethanol at concentrations ranging from 1 fM to 1 mM, sequentially and separately. A high-order flexural mode at similar to 850 kHz was monitored during the self-assembly. The resonance frequency decreases as a result of increased mass as chemisorption occurs on the surface. We show for the first time that the chemisorption of HDT at 1 fM is readily measurable and gave a response of 220 +/- 13 Hz (n = 4). At higher concentrations (10 and 100 fM; 1, 10, and 100 pM; 1, 10, and 100 nM; 1 mu M; and 1 mM), the responses were proportionately, but nonlinearly, higher. At high concentrations (1 mM), the responses to C-4, C-8, C-11, C-16, and C-18 alkanethiols were linearly proportional and were complete in similar to 25 min. We report for the first time that, once the Au surface is equilibrated at 1 pM, further chemisorption at a lower HDT concentration does not take place, even though over 99% of surface adsorption sites are available. At 1 fM, the overall chemisorption rate did not increase with a 2-fold increase in the HDT flow rate, suggesting that chemisorption at 1 fM is not transport-limited. The measured overall chemisorption rate constant at 1 fM was more rapid than 0.1 min(-1).