A new method to study substrate-mediated gene delivery was developed using a hexahistidine-tagged polymeric gene delivery vehicle (His-tagged polyplex) and nickel nitrilotriacetic acid (Ni-NTA) self-assembled monolayers (SAMs) on gold surfaces. The His-tagged polyplexes showed specific interaction with Ni-NTA surfaces compared to control surfaces, with increasing NTA content in SAM formulations corresponding to increasing amounts of immobilized His-tagged polypexes on the surface. Cells seeded on NTA SAMs demonstrated uptake of His-tagged polyplexes in the presence of imidazole and EDTA with low cytotoxicity. Cells seeded on NTA SAMs without hnidazole and EDTA showed minimal a amount of His-tagged polyplex uptake. This showed that the release of polyplexes from the surface by imidazole and EDTA was necessary for cell uptake. Thus, this system provides potential spatial specificity for polyplex delivery controlled by location of NTA surfaces and temporal specificity for polyplex delivery controlled by the addition of imidazole and EDTA.