Sensing Single Protein Molecules with Solid-State Nanopores - Nanopores: Sensing and Fundamental Biological Interactions - page 139

possibly due to an increased drag force when the molecular volume is larger.
The integrated area histogram (Fig. 6.3f ) is a measure of total charge of ions in

solution displaced by the protein, A ecd ¼ R event DI b ðtÞdt , and clearly shows that the
nanopore measurement differentiated these three proteins. Even though BSA and
Fibrinogen had similar total charges ( 7.0), the difference in

their molecular weights (and therefore volumes) allowed the current drop signals of
fibrinogen to be clearly differentiated from those of BSA [ 12 ].
Advantages of using solid-state nanopores to characterize native state proteins
include that the measurement is performed in aqueous salt solution and no marker
or modifications of proteins are required. However, the data also show resolution
in sizing different native state proteins is relatively low. One possible reason for
this is that the distribution of the peaks including both

16 e vs 18 e at pH ¼

DI b and t d are broad, limiting
the precision of the excluded volume measurement. Another possible limitation
in application to native state proteins is that most proteins have positive and
negative charged residues. In the strong electric field in a nanopore, positive
and negative charges will be driven to opposite directions possibly inducing
structural changes in native state proteins. Both transient and long-lived structural
changes could occur and the contribution of these phenomena to the nanopore
events has yet to be fully characterized. When present, these nanopore-induced
conformational changes could lead to systematic bias in the estimation of the
volume L when using Eq. (6.1).

6.4.2 Measuring a Protein's Relative Charge at Different pH

Native state proteins, depending on their structure and charge distribution, can be
partially or fully unfolded by the strong electrical field strength (~10 5 V/cm) in a
nanopore [ 35 ]. However, proteins with many disulfide bonds are the most likely to
maintain their native state form during electrophoretic translocation. Native state
BSA has 17 disulfide bonds, which should make it relatively stable in a nanopore;
it is expected to behave like a simple charged particle during translocation and not
experience large long-lived conformational changes [ 36 ]. Therefore BSA is a good
model system for measuring the electrical charge change as a function of pH.
BSA (Fig. 6.4a ) has an isoelectric point (pI) ranging from pH 5.1-5.5 [ 37 ]. The
protein has an overall negative charge ( 18 e) at pH 7. The charge of BSA can be

altered by varying the pH of the solution. Using a ~16 nm diameter pore in a
solution of 0.5 M KCl at pH 7.0, with 120 mV, I 0 ~ 7.4 nA was measured. After

addition of BSA to the negatively biased cis chamber, downward blockage events
occurred (Fig. 6.7a ) indicating that BSA molecules were negatively charged. When
the cis chamber was positively biased, no blockage events were observed at the
beginning of the experiment. The cumulative results are presented in an event
number distribution plot (Fig. 6.7d ).
When the solution pH of the chambers was lowered to acidic conditions (pH

c¼

5)
below the pI of BSA, current blockages disappeared if the trans chamber remained

<