Multiplexed Lipid Dip‐Pen Nanolithography on Subcellular Scales for the Templating of Functional Proteins and Cell Culture

Abstract

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.

Lipid-nanostructure hybrids and their applications in nanobiotechnology

“…The massively parallel and direct delivery with DPN of multi-component lipids and lipophilic materials to a solid surface on multiple length scales enables the mimicking of heterogeneous micro-and nanodomains of native cell membranes that are similar to lipid rafts (Figure 2b). 32 An atomic force microscopy tip can locally remove a preexisting thin film from a solid support; this method is known as nanoshaving lithography (Figure 2c). 33 When a shaved region is backfilled with another lipid layer, a lipid pattern as narrow as 50 nm can be formed.…”

“…Representative brightfield, RICM, and fluorescence microscopy images of three cells that are engaged with a nanopatterned supported membrane. 32 (d) The fluorescence image of phospholipid nanopatterns deposited on a glass surface. A three-channel image shows the T-cells adhering to the corners of lipid-protein dip-pen nanolithography (DPN) patterns and activated by anti-CD3/anti-CD28 (green ¼ lipid pattern, blue ¼ the nucleus of cells by 4,6-diamidino-2-phenylindole (DAPI), red ¼ CD69 by anti-CD69-phosphatidylethanolamine (PE) and anti-PE-tetramethylrhodamine-5(and-6)-isothiocyanate (TRITC).…”

“…In another example, DPN-generated protein-tethered lipid nanopatterns on a glass substrate were used to activate Jurkat T cells. 32 The multilayered lipid patterns were stable under cell culture conditions, and the biomimetic function of the protein-bound lipid patterns was demonstrated by testing the ability of the nanopatterns to activate T cells. This group modified two different types of activating antibodies specific for the CD3 e chain of the TCRs (a-CD3e), the co-stimulatory receptor (a-CD28).…”

Nanoscale Biomolecular Structures on Self‐Assembled Monolayers Generated from Modular Pegylated Disulfides

“…For example, SAMs have been utilised for studies involving enzymatic processing of DNA, [21,22] DNA computing, [23] protein assays [24,25] and studies of cellular responses towards sur-A C H T U N G T R E N N U N G faces. [26][27][28] Following the success of DNA functionalised sur-A C H T U N G T R E N N U N G faces in genomic analysis, significant efforts have been made to develop protein and oligosaccharide functionalised chips for proteomics-and glycomics-based research. [29][30][31] Also, with the ever increasing drive towards smaller sample sizes and higher throughput for whole-organism analysis, has come the need to develop nanometer-scale, biomolecular, patterned surfaces.…”

Nano-bioelectronics via dip-pen nanolithography

“…92 Lenhert and Fuchs have printed lipids by DPN with the goal of generating biomimetic membrane patterns as model substrates for cell culture. 143 They demonstrated the multi-plexed printing of lipids with lateral resolution down to 100 nm. By binding functional proteins to lipids containing either a nickel chelating headgroup or a biotinylated headgroup, they could demonstrate the selective adhesion and activation of T-cells.…”