Single Molecule Detection with an Aptamer-Integrated Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 66

surface engineering; and the ability to function as a probe platform for in situ and
high throughput applications.
The pore sizes have been verified by translocation of molecules of known sizes,
including dsDNA (2 nm), gold nanoparticles (10 nm) and ring-shaped cyclodextrin
(1.5 nm) [ 28 ]. Particularly when the pore size is comparable to dsDNA, the DNA
translocation speed is slowed down and translocation steps can be revealed from the
block type (Fig. 3.6b left), which is different from that for DNA translocation in a
wider pore (Fig. 3.6b right). Strikingly, the glass nanopore can be fabricated at
1 nm, such that the pore can trap a single cyclodextrin in the lumen. The trapped
cyclodextrin functions in a similar way to that in the protein pore: acting as a
molecular adapter to identify small chemicals in the mixture [ 33 ]. We found
the glass nanopore with a trapped

-cyclodextrin is able to discriminate chiral
enantiomers on the basis of their characteristic block signatures [ 28 ] (Fig. 3.6c ).
This finding supports that it is possible to observe the signature signal for single
molecule binding in the nanopore, a phenomenon that may apply to the aptamer-
encoded nanopore for protein detection.

b

3.4.4 Capture of Single IgE Molecules
in the Aptamer-Modified Nanopore

Immunoglobulin E (IgE) is consistently a representative biomarker for clinical
detection methods. Abnormal levels of IgE are associated with allergy-mediated
disorders and immune deficiency-related diseases, such as AIDS. We first devel-
oped aptamer-encoded nanopore for IgE [ 22 ]. To test the single molecule detec-
tion ability of aptamer-encoded nanopore, the aptamer for IgE was immobilized
on the inner surface of nanopore, then low concentration IgE was applied to the
nanopore from the wide opening of the pipette, to observe the block signal. Unlike
the typical rectangle shape blocks, we can observe a series of stepwise current
blocks (Fig. 3.7a ). After confirmation with a series control tests, we conclude that
the stepwise blocks are associated with single IgE molecules that sequentially
bind to the immobilized aptamers in the nanopore, one molecule per block level.
It is clear that the stepwise blocks are also distinguished from those produced
by protein molecule translocation, as protein molecules traversing a nanopore
typically generate short-lived blocks with a duration ranging from 10 2 m sto

10 2 ms [ 93 , 97 , 111 ].
We observed a number of stepwise current blocks, but rarely observed characteris-
tic current increase for releasing IgE from immobilized aptamers [ 22 ]. This suggests a
long binding duration for IgE￿aptamer in hours, which is equivalent to a dissociation
rate constant of ~10 5 s 1 . The slow dissociation rate in the glass nanopore suggests
that the confinement in the nanopore could enhance the bonding strength between the
target and its aptamer. The apparent association rate constant is evaluated by analyzing
the frequency of the stepwise block occurrence was calculated as 1.9

10 5 M 1 s 1 .