Sensing Single Protein Molecules with Solid-State Nanopores - Nanopores: Sensing and Fundamental Biological Interactions - page 140

Fig. 6.4 BSA protein in 0.5 M KCl solution measured as a function of pH at 120 mV. Panel

(a) shows a sketch of BSA translocation through a solid-state nanopore with BSA dimensions
shown. Panel (b) shows current drops due to BSA translocation at pH 7. Panel (c) shows the
current drops from BSA translocation at pH 4.5. Note that BSA is positively charged and the bias
across the nanopore has been reversed. Panels (d) and (e) are density plots of BSA translocation
through a solid-state nanopore at these two different pH values. (f) The most probable values of
DI b ,(g) the relative BSA electrical charge to pH 7. The open pore current was I 0 ~7.4 nA at
c¼ 120 mV for all pHs measured

c¼

positively biased. However, when the trans chamber was switched to negative bias,
current blockages appeared again as shown in Fig. 6.7c , indicating the net charge of
BSA protein had changed to positive at pH

<

5. This measurement is consistent
with the fact that BSA is positively charged when the pH is lower than its pI [ 38 ].
We studied the translocation of BSA through the same nanopore at three different
acidic pH values (4.5, 4.1 and 2.4). The BSA molecules proved to be positively
charged at all these pH values. When the pH was 4.5, near the pI of BSA, the most
probable values are s as shown in Fig. 6.7e . The same

measurement was performed at a higher pH value of 10.4. The most probable
values of

DI b ~71 pA and t d ~269 m

DI b (Cluster 2 events were used) varied as solution pH changed
(Fig. 6.4f ) indicating the shape of the BSA molecule was not the same. The open
pore current I 0 was approximately a constant at all the pH values measured.
A decrease in of BSA

molecules decreased at higher pH indicating a change in conformation or dimen-
sions of BSA molecules took place. These results are consistent with reports of BSA
denaturation at high pH, and formation of dimers at low pH [ 39 ] or BSA volume
expansion at acidic pH [ 37 ]. A change in the charge of BSA (Q) can be estimated by
the change in t d . The relative charges Q / Q pH7 estimated from Eq. (6.3) are shown in
Fig. 6.4g .ApI

DI b (Fig. 6.4f ) suggests that the local excluded volume L

5.3 was estimated for the BSA by this nanopore experiment.