## 6 Combining Active and Passive Antibiofilm Strategies
At the destructive interference zones where dark fringes are formed, the ATRP initiator stays unaffected by the UV light beam, whereas at the constructive interference zones where bright fringes are formed, the high intensity of the UV beam decomposes the ATRPinitiator as schematically illustrated in Scheme 17[143]. Consequently, using the IL nanopatterning technique, one gains a very precise spatial control of polymer brushes growth, endowing them with dual functionality. The periodicity of the patterns is easily tunable by varying the interference angle. Thermoresponsive poly(\(N\)-isopropylacrylamide) (PNIPAAm) polymer brushes were grown from the aforesaid nanopatterned surfaces via surface-initiated ARGET-ATRP polymerization of NIPAAm using 1,1,4,7,7-pentamethyldiethylclientamine (PMDETA) as a ligand, CuBr\({}_{2}\) as an oxidatively stable Cu(II) species, and ascorbic acid as a reducing agent. The troughs between the nanopatterned parallel PNIPAAm lines have been subsequently filled with the biocidal quaternary ammonium salt (QAS), [3-(trimethoxysily)]propyl]ammonium chloride. To this end, the nanopatterned PNIPAAm surfaces were incubated for 2 h in 1% aqueous QAS solution at 37 \({}^{\circ}\)C, a temperature that is higher than the lower critical solution temperature (LCST) of PNIPAAm. At this temperature, the polymer chains are hydrophobic and collapse, allowing the access of bacteria to the QAS layer covering the inter-spacing between the nanopatterned PNIPAAm lines. This state renders the nanopatterned PNIPAAm/QAS composite surface sticky and bactericidal. Hence, bacteria adhere to the surface being killed by the QAS. Lowering the temperature below the LCST, PNIPAAm becomes hydrated and swollen with an extended chain conformation, thereby hiding the biocidal QAS. In this state, the surface releases the attached dead bacteria, and a being cell repellent, prevents further bacterial adhesion. Increasing the temperature again above LCST, the initial bactericidal status of the nanopatterned hybrid surface can be restored. The authors tested the biocidal activity and bacterial release ability of these PNIPAAm/QAS composite surfaces against _E. coli_ K12 using QAS and nanopatterned PNIPAAm surfaces as controls. The sample surfaces were inoculated with a bacterial inoculum (\(1\times 10^{8}\) CFU/mL) and incubated at 37 \({}^{\circ}\)C for 2 h, and the viability of the attached cells was evaluated by a standard dead/live staining assay. When examined by fluorescence microscopy, most cells attached to the QAS control surface showed red fluorescence due to uptake of the membrane-impermeable propidium iodide dye, indicating that the cell envelope was badly damaged resulting in loss of the cell integrity and cell's death. In contrast, most cells attached to the patterned PNIPAAm surfaces were viable cells as indicated by the green fluorescence of the SYTO 9 dye, which means that these surfaces exhibit no intrinsic bactericidal activity. On the other hand, the nanopatterned PNIPAAm/QAS hybrid surfaces displayed bactericidal activity, although as compared to QAS surfaces, this activity was reduced from \(90\pm 2\%\) to \(73\pm 5\%\). However, cooling down the samples to 25 \({}^{\circ}\)C (below the LCST of PNIPAAm), the bactericidal activity of nanopatterned PNIPAAm/QAS composite surfaces drastically decreased [142]. The authors explained this behavior through the temperature-triggered reversible switch between the dehydrated collapsed state of the PNIPAAm polymer brush, which exposes the biocidal QAS moieties and the hydrated extended conformation of the PNIPAAm chains that hides the bactericidal compound. Regarding the capability to release the attached cells, this was poor for the control QAS surface. In contrast, \(81\pm 4\) and \(67\pm 7\%\) of the attached bacteria were released from the nanopatterned PNIPAAm surfaces and the nanopatterned PNIPAAm/QAS composite surfaces, respectively. Neither the bactericidal activity nor the releasing capability was significantly affected after several repeated switching cycles.