Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 209

(m 2 v 1 s 1 ) is the electrophoretic mobility of a charged protein and where u e

m 1 ) is the strength of the electric field [ 14 ]. This distribution function
assumes a biased diffusion, first-passage process based on the one-dimensional
translocation of the protein through the length of the pore to an infinite sink. It should
also be noted that ( 9.2 ) assumes that the charges on the protein are distributed
uniformly and that electroosmotic flow does not contribute to transport [ 46 , 53 ].
Both of these assumptions are not always met in nanopore assays with proteins [ 46 ].
Furthermore, early studies have reported values for the drift velocity and the diffusion
constant that were one to three orders of magnitude lower than those measured in
the bulk solution [ 40 , 46 ]. These reduced values could be due to interactions of the
proteins with the walls of the pore or to a non-homogenous distribution of charges on
the proteins [ 40 , 46 ]. Nevertheless, ( 9.2 ) provides a good approximation.
Although accurate prediction of the effect of charge on the translocation time
of proteins through nanopores is still not possible, it is clear that the charge on
a protein affects t d . Stroeve and coworkers demonstrated the effects of protein
charge on translocation time by studying protein flux through nanopores [ 6 , 7 , 25 ].
Although not detecting single proteins, these investigations showed that the flux of
BSA and bovine hemoglobin through nanopores depended on the applied electric
potential difference and on the charge of the protein, which was changed by altering
the pH of the solution. These results indicated that protein charge influenced protein
translocation and that the polarity of the charge and isoelectric point (pI) of proteins
can be estimated from translocation experiments. A possible application could be to
separate similarly sized molecules of different charges through nanopores for
ultrafiltration [ 6 , 7 , 25 ].
Staufer's group provided clear evidence for the dependence of t d on charge by
sensing single proteins with nanopores and solutions of different pH (Fig. 9.2b )[ 15 ].
From these experiments, the translocation time of a protein was longest when the
pH of the solution was equal to the pI of a protein. This characteristic can be
beneficial since slowing the translocation speed of a protein (increasing t d ) improves
the accuracy of determining

e (V

DI . In addition, the pH also determined the polarity of
the potential difference at which proteins passed through the nanopore. For instance,
negatively charged proteins only passed through the pore when the solution on the
opposite side of the pore contained the positive anode. Hence, it is necessary to know
the pH of a solution and the pI of the protein under investigation.
Recently, Siwy's group took advantage of ion current rectification in nanopores
to estimate the pI of streptavidin [ 1 , 2 , 41 - 43 , 51 ]. 3 In this work, streptavidin
bound to biotin groups which were covalently attached to the nanopore walls.
Modifying the pH of the solution changed the charge of the bound streptavidin and
hence the charge on the walls of the nanopore. This change in the charge density on
the pore walls strongly affected the ion current rectification through the nanopore,
which could then be used to estimate the pI of streptavidin.

3 Ion current rectification refers to the condition where the current at one polarity of the electric
potential difference is significantly different than the current at the opposite polarity (i.e. the
system has non-ohmic behavior). For more information see refs. [ 40 - 42 ].