Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 222

9.6 Future of Protein Sensing with Nanopores
Nanopore-based sensing is a relatively new single molecule technique. Of all the
single molecule techniques, it is one of the simplest and most accessible methods.
With respect to the characterization of proteins, nanopores have the unique capa-
bility to determine the size of proteins in solution rapidly without requiring labels.
Furthermore, additional work on characterizing the state of folding of proteins
could perhaps lead to quantifying conformational changes of functional proteins.
Further developments may even make it possible to sequence amino acids in a
similar manner as researchers are trying to use nanopores for sequencing of DNA
and RNA based on nanopore recordings.
Nanopore-based techniques for detecting the binding of a protein and ligand do
not require a fluorescent label on the protein or ligand, and thus, the techniques have
a unique advantage over fluorescence-based methods. Similarly, nanopore-based
detection of enzyme activity can quantify the kinetics of enzymes without the use of
fluorescent labels or secondary enzymatic reactions. Detection of enzymes from
nanopore recordings can also employ nanopores that present a covalently-attached
substrate of an enzyme. These assays in particular exploit ion channel amplification, in
which the cleavage of onemolecule due to enzyme activitymay result in a large change
in the flow of ions through a pore [ 17 , 27 ]. This amplified sensitivity, coupled with the
small volumes used in nanopore-based assays, makes it possible to work with only
femtomoles of enzyme [ 27 , 28 ]. Together, these characteristic may permit the analysis
of filtered but otherwise untreated samples of biomolecules in physiologic solutions.
Although nanopore-based sensing has great potential to explore the function of
proteins, several challenges have to be met. For example, reliable quantification and
prediction of the translocation times of proteins through nanopores remains diffi-
cult. Values calculated to date for the diffusion constant and the electrophoretic
drift velocity from translocation times through nanopores are different from the
values found in bulk solution. These discrepancies may be due to interactions
between the protein and the nanopore walls. Hence minimizing these interactions
will be important. Furthermore, the surface charge on many proteins is not uniform,
and thus for proteins with a dipole moment, the electrophoretic force acting on the
protein may be a function of the position of the protein with respect to the electric
field in the nanopore [ 46 ]. In addition, proteins are entropically constrained in
nanopores with diameters on the order of the protein's dimensions [ 30 ]. Further
complications include that the electric field is not uniform near the narrowest
constriction of the nanopore and that electroosmotic flow (EOF) is typically not
considered in calculations based on the translocation time. We showed recently,
however, that EOF can be significant in nanopores under certain conditions [ 53 ].
In order to realize the full potential of quantitative nanopore recordings these
challenges will have to be addressed.
One strategy might be chemical modification of nanopore walls. Currently, the
ability to modify the surface of nanopores chemically in an efficient manner is
limited. Novel methods that alter the surface chemistry of nanopores with ease