Single Molecule Detection with an Aptamer-Integrated Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 61

fluorescence technique [ 41 ](Fig. 3.1b ). As studied in Sect. 2.1 , we have identified
the dramatic decrease in the number of TBA G-quadruplex signature blocks in the
nanopore in the presence of thrombin, compared with TBA alone without thrombin.
This sensitivity suggested the possibility of developing aptamer-based nanopore
sensors for protein detection. Actually this new generation of single molecule
biosensors has a vast breadth of applications because the selection of aptamer species
is unlimited.
3.3.1 Progress in Construction of Nanopore Sensors
For real-time applications in the future, such nanopore biosensors should be
portable for independent storage and free transportation, while they should be
independent, pluggable components that connect to other systems. The device
should work in the microarray platform for the high-throughput screenings, in
which each element contains a single responsive pore. To create a robust device
that functions with single pores, however, one of significant challenges is to
improve the fragility of the lipid bilayer membrane in which the protein pore is
embedded. There have been several strategies to stabilize the lipid membrane. For
example, reducing the size of the aperture over which the lipid bilayer is formed
may help to promote membrane stability. Through micro-fabrication, the aperture
in silicon can be made as small as several micrometers in diameter [ 25 , 92 ].
The lipid bilayers can also be covalently tethered to a solid surface to form a
solid-supported bilayer [ 17 , 68 ]. Peterson et al. has reported a sandwiched lipid
membrane generated by the painted method [ 83 ], in which the bilayer was formed
on a pre-cast gel slab and covered with another gel slab for double support [ 18 ].
With the sandwich structure, Schmidt et al. improved the lipid membrane stability
using an UV-triggered hydrogel in place of agarose [ 55 ], and demonstrated an
efficient system that conjugates the headgroups of lipids in the membrane to the
polyethylene glycol-based hydrogel to achieve extended lifetimes and resistance
to mechanical perturbation [ 75 ]. The White group and Cremer group generated a
long-lived suspended lipid bilayer over a 100 nm glass nanopore (GNP) membrane,
and incorporated single protein pores in this membrane as a sensor [ 120 ]. This
GNP-based bilayer can be fabricated using materials and instruments that are
commonly available in most laboratories and do not require microlithographic
fabrication techniques. While all of the methods described above form bilayers
in the aqueous phase, the Bayley group and Wallace group have pursued protein
pore research and applications on a bilayer that is formed in the oil phase, called the
droplet interface bilayer (DIB) [ 6 ]. The DIB demonstrates superior stability,
and many droplets can be assembled into a responsive DIB network
that functions as a microbattery [ 45 ] and a molecular device that simulates the
functions of semiconductor circuits, such as half-wave rectification and full-wave
rectification [ 74 ].