Nanofabrication for the analysis and manipulation of membranes

Abstract

Recent advancements and applications of nanofabrication have enabled the characterization and control of biological membranes at submicron scales. This review focuses on the application of nanofabrication towards the nanoscale observing, patterning, sorting, and concentrating membrane components. Membranes on living cells are a necessary component of many fundamental cellular processes that naturally incorporate nanoscale rearrangement of the membrane lipids and proteins. Nanofabrication has advanced these understandings, for example, by providing 30 nm resolution of membrane proteins with metal-enhanced fluorescence at the tip of a scanning probe on fixed cells. Naturally diffusing single molecules at high concentrations on live cells have been observed at 60 nm resolution by confining the fluorescence excitation light through nanoscale metallic apertures. The lateral reorganization on the plasma membrane during membrane-mediated signaling processes has been examined in response to nanoscale variations in the patterning and mobility of the signal-triggering molecules. Further, membrane components have been separated, concentrated, and extracted through on-chip electrophoretic and microfluidic methods. Nanofabrication provides numerous methods for examining and manipulating membranes for both greater understandings of membrane processes as well as for the application of membranes to other biophysical methods.

Figure 1

(A) Zero-mode waveguides (ZMWs) and (B) planarized apertures utilize nanofabricated metallic structures to limit the illumination for fluorescence spectroscopy to sub-diffraction-limited areas. ZMWs illuminate the membrane at the bottom of the aperture while planarized apertures provide 60 nm resolution of planar membranes. (Adapted with permission from (A) Refs. and and (B) Ref. 50)

Figure 2

Scanning probes have been used both for nanoscale imaging and patterning of membranes. (A) Fluorescence enhancement at the tip of nano-antenna on a scanning probe provides 30 nm resolution of membrane-bound integrin LFA-1; scale bar = 1 µm. A SEM image of the nano-antenna is shown in the inset of (A). (B) A line scan of fluorophore emission intensity vs. distance demonstrates 29 nm resolution. (C) SLBs of varying composition have been simultaneously patterned with dip-pen lithography at 200 nm resolution. (D) Multiplexed “pens” with varying composition of lipid-containing inks allow for simultaneous writing of many patterns and combinatorial mixing of the membrane components. (Reprinted with permission from (A,B) Ref. and (C,D) Ref. .)

Figure 3

Membrane reorganization is induced upon the binding of T cells to antigen presenting cells, forming an immunological synapse. (A) Key elements of antigen presenting cells (i.e. the peptide-major histocompatiability complex (pMHC) and the inter-cellular adhesion molecule (ICAM)) have been incorporated into a SLB and reorganized upon binding to T cell receptors (TCR) and to lymphocyte function-associated antigen 1 (LFA-1), respectively. The (B) presence or (C) absence of the metallic barriers to diffusion results in distinctly different morphology of the resulting immunological synapse as the T cell attempts to concentrate the TCRs within a ring of ICAMs. (Reprinted with permission from Ref. .)

Figure 4

Supported lipid bilayers and nanofabricated metallic barriers have been used for high-throughput single-molecule analysis of DNA. DNA molecules are bound to lipid molecules and analyzed with total-internal reflection fluorescence microscopy, as shown schematically (A) with and (B) without a hydrodynamic force confining them to metallic barriers. Isolated molecules 48.5 kb λ DNA stained with YOYO1 can be seen in a single image (C) with and (D) without flow. (Reprinted with permission from Ref. . Copyright 2008 American Chemical Society.)

Figure 5

Membranes and biomolecules have been patterned with polymer lift-off. (A) Micron scale patterns of antigen-incorporating SLBs recruit IgE on live cell membranes for spatial control of membrane-mediated cell signaling; scale bar = 10 µm. (B) Biomolecules, such as fluorescently-labeled antibodies, have been patterned with a resolution of 90 nm via polymer lift-off and electron beam lithography in nanoscale dots and lines. Polymer lift-off holds promise for nanoscale patterning of membranes with high-throughput and high-resolution. ((A) Reprinted with permission from Ref. . Copyright 2004 National Academy of Sciences, U.S.A. (B) Reprinted with permission from Ref. . Copyright 2010 American Chemical Society.)

Figure 6

The incorporation of electrophoresis and metallic barriers provide a mechanism of concentrating, separating, and purifying membrane components. (A) Initially mixed SLB components can be (B) separated by electrophoresis within the corral. (C) Controlled laminar flow can expose only a portion of the SLB to a stripping solution to remove the exposed SLB from the surface. (D) The partially stripped SLB can then be refilled via vesicle fusion to reform a complete SLB with potentially new components. SLBs are composed 97.5% zwitterionic egg phosphocholine lipids, 0.5% negatively charged Texas Red-phosphoethanolamine lipids (red), and 2% neutral NBD-phosphocholine lipids (green). (Reprinted with permission from Ref. . Copyright 2003 American Chemical Society.)

Figure 7

Incorporating electric fields with complex metallic structures provides a means of concentrating membrane components. (A) Initially homogeneous SLBs diffuse uniformly around the metallic structures, but application of alternating electric fields may concentrate select components (B). SLBs are 99.8% zwitterionic phosphocholine lipids and 0.2% negatively charged Texas Red-phosphoethanolamine lipids. (Reprinted with permission from Ref. . Copyright 2011 American Chemical Society.)