We have fabricated micro-channel with a periodic array of nano-gate with a width of 500 and 100 nm by electron beam lithography. The effect of nano-gates on the self-spreading of egg-PC bilayer doped with dye-labeled lipid TR-DHPE was investigated using fluorescence microscope. Compared with the control channel without the nano-gate, the self-spreading velocity was suppressed due to the presence of nano-gate. The quantitative analysis of the spreading behavior suggests that the self-spreading bilayer was compressed during passing through the gate. This structural deformation imposed an energetic barrier for the self-spreading, leading to reduction of the spreading velocity. Furthermore, the number of TR-DHPE molecule in the spreading bilayer was apparently decreased during spreading in the gate channels. This phenomenon was also closely related to the possible structural deformation at the gate. Formation of higher lipid density state at the gate region induced by the compression was thought to be responsible to the reduced penetration ability of bulky TR-DHPE molecules into the nano-gates, which resulted in the gradual decrease in TR-DHPE concentration in the spreading bilayer. (c) 2008 Elsevier Ltd. All rights reserved.