(11b) 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.