A New Design of Bio-Hybrid Nanopores for DNA/RNA Detection

A New Design of Bio-hybrid Nanopores for DNA/RNA Detection

Kwang Joo Kwak^1,2, Xuejin Wen^1,3, Wei-Ching Liao^1,4, Cherry Gupta², Bo Yu¹, Gintaras Valincius⁵, David J. Vanderah⁶, Wu Lu^1,3 and L. James Lee^1,2*

 

¹NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Device (NSEC-CANPBD)

²Department of Chemical and Biomolecular Engineering

³Department of Electrical and Computer Engineering

⁴Department of Mechanical Engineering

The Ohio State University, Columbus, Ohio 43210

⁵Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania

⁶Biomolecular Structure and Function Group at the Center for Advanced Research in Biotechnology (CARB), National Institute of Standards and Technology (NIST), Rockville, Maryland 20850

*Contact author- E-mail: lee.31@osu.edu

The dynamic nature of biomolecule transport across the native cell membranes is difficult to understand due to the complexity of the membranes and the transport phenomenon. Recent advances in tethered biomimetic lipid membranes provide opportunities to study protein adsorption, ion transport, and membrane mechanical properties. By selecting cell membrane lipids and adding proper protein, the tethered bilayer lipid membrane (tBLM) with well-defined nanopore array can serve as a useful tool for the investigation of various biological lipid structures and their interactions with other biomolecules including cell membrane-DNA interactions.

The tBLM with well-defined nanopores was demonstrated on the SiO₂ surface with pre-drilled nanochannels. The nanochannel array with from ~10,000 holes down to a single hole was prepared by E-beam lithography for electrochemical impedance spectroscopy (EIS) or electric current measurements. To expose dimensions of tens of nanometers, a beam current of 100 pA with an acceleration voltage of 100 kV was used. The spacing between two nanochannels in both x and y directions was 33 mm so that the number of nanochannels is 10,000 with a circular testing area with the diameter of 4 mm. 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 nanochannels with a diameter of from 100 nm down to 10 nm were drilled through the Au-coated SiO₂ by e-beam lithography. Since the lipid bilayers could also form on the sidewalls of the nanowells, the nanopore size was smaller than the nanochannel size as measured by AFM. The tBLMs with different pore size and pore density were used as DNA/RNA detection platforms.

The presence of nanochannels made the high frequency semicircle of the Cole-Cole 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. The Cole-Cole EIS spectra of SAMs and tBLMs with and without nanochannels were measured and compared to the finite element method (FEM) simulation. The results of our current work show that both the morphology and dielectric properties/conductivity of tBLM systems can be adjusted by varying experimental parameters.

This bio-hybrid nanopore system with a-hemolysin is used to investigate the DNA-pore interactions by measuring both EIS and electric conductance when large (e.g. vector pGFP) and small (e.g. microRNA) linear DNA/RNAs are used as model biomolecules.