(316g) Tethered Bilayer Lipid Membranes with Pre-Designed Nanopores for DNA-Membrane Interaction Analysis

By selecting cell membrane lipids and adding proper protein and sugar molecules, the tethered bilayer lipid membrane (tBLM) with well-defined nanopore array can serve as a useful model for the investigation of various biological lipid structures and their interactions with other biomolecules. Examples include cell membrane-DNA interactions, cell membrane-lipoplex interactions, cell membrane-exosomes interactions and gene delivery by electroporation. The tBLM with well-defined nanopores was demonstrated on the SiO2 surface with pre-drilled nanoholes. The nanohole array with ~10,000 holes was prepared by e-beam lithography for electrochemical impedance spectroscopy (EIS) measurements. A thin self-assembled monolayer (SAM) was then formed on the Au layer. The tethered BLM was prepared by incubation with a diphytanoylphosphatidylcholine (DPhyPC) solution of 10 mM concentration in ethanol for ~10 min and subsequently injection of PBS buffer within ~10 s. The atomic force microscopy (AFM) image with the nanopores on a tBLM was observed in the liquid environment. The nanoholes with a diameter of 150 nm were drilled through the Au-coated SiO2 by e-beam lithography, while the observed nanopore size was around 100 nm. Since the lipid bilayers could also form on the sidewalls of the nanowells, the nanopore size was smaller than the nanohole size as measured by AFM. The tBLMs with different pore size and pore density were also prepared. The Cole-Cole EIS spectra of SAMs and tBLMs with and without nanoholes were measured and compared to the finite element method (FEM) simulation based on a 3-layer circuit model. For the SAM with nanoholes, the top gold surface and the sidewalls of nanoholes were modified with the mixed SAMs. Although the SAM layer was essentially defect free, the EI spectra showed some difference. For the tBLMs, the presence of nanoholes made the high frequency semicircle of the EI spectrum incomplete, similar to that observed when the densely-packed tBLM turned into a loosely-packed tBLM (lp)tBLM because of the reduced DPhyPC concentration. Unlike the (lp)tBLMs, this tBLM with well-defined nanopore array showed very stable and repeatable EI spectra. Several plasmid and linear DNAs used in gene therapy such as vector pGFP and pCAG2LMKOSimO are placed on top of the porous tBLM. The AFM is used to measure the morphology of DNA-pore interaction, while EIS is used to quantify the DNA-pore dynamics. We have designed a nanopore electrophoresis set-up to carry out these experiments.