Fusion of biomimetic stealth probes into lipid bilayer cores

Abstract

Many biomaterials are designed to regulate the interactions between artificial and natural surfaces. However, when materials are inserted through the cell membrane itself the interface formed between the interior edge of the membrane and the material surface is not well understood and poorly controlled. Here we demonstrate that by replicating the nanometer-scale hydrophilic-hydrophobic-hydrophilic architecture of transmembrane proteins, artificial "stealth" probes spontaneously insert and anchor within the lipid bilayer core, forming a high-strength interface. These nanometer-scale hydrophobic bands are readily fabricated on metallic probes by functionalizing the exposed sidewall of an ultrathin evaporated Au metal layer rather than by lithography. Penetration and adhesion forces for butanethiol and dodecanethiol functionalized probes were directly measured using atomic force microscopy (AFM) on thick stacks of lipid bilayers to eliminate substrate effects. The penetration dynamics were starkly different for hydrophobic versus hydrophilic probes. Both 5- and 10 nm thick hydrophobically functionalized probes naturally resided within the lipid core, while hydrophilic probes remained in the aqueous region. Surprisingly, the barrier to probe penetration with short butanethiol chains (E(o,5 nm) = 21.8k(b)T, E(o,10 nm) = 15.3k(b)T) was dramatically higher than longer dodecanethiol chains (E(o,5 nm) = 14.0k(b)T, E(o,10 nm) = 10.9k(b)T), indicating that molecular mobility and orientation also play a role in addition to hydrophobicity in determining interface stability. These results highlight a new strategy for designing artificial cell interfaces that can nondestructively penetrate the lipid bilayer.

Fig. 1.

Diagram of stealth probe integration with a lipid bilayer. (A). Surface-surface interactions regulated by tethering molecules to the substrate. (B) The “stealth probe” structure with a hydrophobic domain designed to interact specifically with the hydrophobic membrane core through selective surface functionalization. (C) A functionalized band several nanometers thick is defined by selective self-assembly of molecules. (D) A hydrophobic functionalized band interacts specifically with the hydrophobic core of the lipid bilayer, similar to the behavior of membrane proteins.

Fig. 2.

AFM post probe fabrication. (A) A Si AFM tip is used to fabricate stealth probes. (B) Using the FIB, the tip was shaped into a post ∼500 nm in diameter. (C) Cr-Au-Cr metal films were evaporated onto the entire cantilever and post, covering the top and sidewalls of the milled post. (D) Tips were remilled in the FIB to trim the excess metal from the sidewalls of the post and expose the edge of the Au layer. Final tip diameter is ∼200 nm. (E) Fabricated tips were subsequently functionalized using alkanethiol self-assembly. (F–H) SEM images of corresponding fabrication step (A–C). (I) TEM image of final stealth probe. (J) TEM image of layered metal stack at tip of stealth probe. The 10 nm Au band (Dark, Central Band) is visible between the two Cr layers, with a clean edge profile.

Fig. 3.

(A) Representative AFM curve of penetration through a bilayer stack with an unfunctionalized probe (Red: approach, Black: withdrawal). Each of the 39 vertical drops corresponds to breaking through the hydrophobic core of a single lipid bilayer. (B) Histograms of stacked bilayer penetration distances for unfunctionalized, 5 nm Au/butanethiol, and 5 nm Au/dodecanethiol functionalized probes. (C) Schematic of breakthrough regions for each membrane probe. Hydrophilic, unfunctionalized probes breakthrough the hydrophobic bilayer core only, while hydrophobic functionalized probes jump from bilayer center to center. (D) Tip penetration behavior as a function of z-piezo displacement. Unfunctionalized probes have sharp jumps corresponding to breaking through the hydrophobic core, followed by relaxation through the hydrophilic region. Hydrophobic functionalized probes jump from hydrophobic core to hydrophobic core, with breakthrough distances corresponding to the lamellar repeat distance.

Fig. 4.

Representative AFM force curves for 3 different stealth probes (Red: approach, Black: withdrawal). (A) Unfunctionalized probe. (B) 10 nm Au/butanethiol functionalized probe. (C) 10 nm Au/dodecanethiol functionalized probe. (Insets) Timescale of interface rupture for the tear-off event.

Fig. 5.

Adhesion force as a function of pull-off distance. Unfunctionalized probes show behavior consistent with tether formation, namely a constant adhesion force with increasing pull-off distance. Butanethiol functionalized probes exhibit and increase in force with increasing pull-off distance. Dodecanethiol functionalized probes display reduced adhesion compared to both butanthiol and unfunctionalized probes. Dotted lines to guide eye.

Fig. 6.

(A) Force-clamp testing. The force is ramped to a high force load (60 nN) on top of the bilayer stack, then the position of the z-piezo is fixed, and the probe relaxes by breaking though the bilayers. Each stair step corresponds to a single bilayer breakthrough. The breakthrough rate is then measured as a function of the applied force. (B and C) Linear fits to ln(k) for the 5 nm (B) and 10 nm (C) band thicknesses for dynamic force spectroscopy reveal butanethiol has a higher adhesion energy than dodecanethiol.