Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 217

Due to the potential for large ligand-modified polymers to occlude the pore for
the duration of a protein-ligand interaction, this method could be used to deter-
mine k off by applying ( 9.7 )[ 24 ]. The authors pointed out, however, that due to the
high electric field within the nanopore, the dissociation rate of charged proteins
and polymers may be affected. In addition, when short polymers are used, this
technique could also determine the concentration of unbound ligand, [L], in
solution and consequently, the concentration of bound ligand [ 24 ]. If the mea-
surement of [L] was taken under equilibrium conditions, the association constant,
K a , could be determined using ( 9.6 ). This method requires a nanopore small
enough to prevent translocation of the protein and could therefore be implemented
with solid-state nanopores if their diameter is smaller than that of the proteins to be
detected.
Gu and coworkers reported a third technique that entails the use of ligand
immobilized on a nanopore wall [ 12 ]. Here the binding of a protein to a ligand
molecule in the pore resulted in a stepwise decrease in the current for each protein
bound. This information could be used to determine k on and potentially k off .
Figure 9.7 illustrates this sensing scheme and a representative current trace. The
authors first immobilized aptamers targeted toward immunoglobulin E, IgE, and
ricin, in glass nanopipettes. 4 In this method, the time to each binding event
(indicated by a stepwise current decrease) was fit with an exponential decay
equation with the time constant equal to t on (Fig. 9.7 inset). In combination with
( 9.8 ), the authors then determined k on (Table 9.1 ).
Due to the high affinity of aptamers toward their target ( K d <

10 8 M), the
authors did not observe dissociation events for the duration of their experiments.
Ligands with lower affinity may, however, yield measurement of k off as well [ 12 ].
In principle this method could be applicable to protein-ligand interactions if a ligand
of interest replaced the aptamer.

Fig. 9.7 Detecting the binding of individual IgE and Ricin proteins with aptamers immobilized on
the surface of a nanopore. Individual binding events resulted in stepwise decrease of the current
through the nanopore. ( Inset ) Number of binding events versus time was fit with an exponential
decay equation with the time constant equal to t on . Adopted from Ding et al . with permission [ 12 ]

4 Aptamers are short DNA or RNA segments that have been selected from a large pool (

10,000)
of molecules and enriched using the SELEX technology. They often have high affinity for their
target K d ~10 11 .

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