Supported biomimetic membranes hold potential for applications such as biosensors and water purification by filtration. The current paper reports on the preparation of a supported bolaamphiphile membrane on two polymeric nanofiltration membranes: NF-270 made of polyamide with carboxylic surface charges and NTR-7450 made of sulfonated polyethersulfone with sulfonic surface charges. The results, obtained using a proprietary microfluidic device, confirmed that the supported membrane coverage on the polymeric membranes was governed by the double-layer interactions, in agreement with previous studies on silica, mica, and gold. Remarkably, the formation of the biomimetic membrane was more favorable on the sulfonated polyethersulfone than on the polyamide surface, although both surfaces exhibit a similar surface charge density. It is suggested that the higher dissociation constant of sulfonic groups was the main reason for the higher coverage of NTR-7450. Finally, spinach aquaporins, which are trans-membrane proteins that facilitate the water transport, were incorporated into a supported membrane on NTR-7450. The incorporated aquaporin resulted in enhanced pressure-driven water transport through the membrane, however, for still unclear reasons, the transport was not selective. Despite this failure, the results provide new insights into formation of biomimetic membrane on water permeable polymeric substrates, as a generic approach towards biomimetic water filters. (C) 2014 Elsevier B.V. All rights reserved.