Sensing Single Protein Molecules with Solid-State Nanopores - Nanopores: Sensing and Fundamental Biological Interactions - page 131

6.1.2 Proteins Measured by Solid-State Nanopores

Solid-state nanopores are capable of measuring proteins of any conformation or
size due to their tunable dimensions. Using 30-55 nm diameter, 20 nm thick
nanopores formed by e -beam lithography in a free standing silicon nitride mem-
brane, Han et al. measured bovine serum albumin (BSA), ovalbumin,
avidin, streptavidin, human chorionic gonadotropin

b ( b -HCG), and monoclonal

anti- -HCG proteins [ 10 , 11 ]. Using ~15 nm diameter silicon nitride nanopores,

Fologea et al. compared the current blockage signal of BSA with fibrinogen, and
measured the pH dependence of the BSA current blockage signal. In this work we
confirmed that BSA indeed translocated through a nanopore using a chemilumines-
cent method [ 12 ]. Talaga and Li have studied unfolding of bovine

b

b -lactoglobulin

variant a ( LGa) and Histidine-containing phosphocarrier protein (HPr) [ 13 ].

Recently, Firnkes et al. reported on translocation of avidin [ 14 ], and Niedzwiecki
et al. have reported on the adsorption of BSA in silicon nitride nanopores [ 15 ].

b

6.1.3 Parameters to Be Measured in a Nanopore Experiment

As illustrated in Fig. 6.1a , the main component of a nanopore sensing system is a
single nanopore in a silicon nitride membrane separating two chambers connected
electrically only by the electrolyte solution inside the nanopore. When a voltage is
applied across the membrane, negatively (or positively) charged protein molecules
added to the cis chamber near the nanopore are captured by the electric field, and
driven through the nanopore to the positively (or negatively) biased trans chamber.
The translocation process of a protein molecule in a nanopore can be quantita-
tively described in terms of the nanopore and protein geometries. The geometric
parameters required are illustrated in Fig. 6.1a and are: the average diameter ( d m )
and the length ( l m ) of a protein molecule, the mean diameter ( D p ) or area ( A p ) and
the effective thickness ( H eff ) of a nanopore.
At the macroscopic level, a nanopore's electrical behavior in an electrolyte
solution of conductivity obeys Ohm's Law for an electrolytic resistor to good

approximation. For an approximately cylindrical nanopore under applied voltage
C

s

, the open pore current measured when the nanopore is not occupied by a protein
molecule is I o ¼C / R 0 ¼CsA p / H eff . When a protein molecule is in the nanopore, it

partially blocks the flow of ions [Fig. 6.1a, b (right)] producing a transient decrease
in the open pore current. The current trace in Fig. 6.1c was recorded when a laminin
(L6274, Sigma-Aldrich) protein sample was added to the cis chamber.
The pH 7 electrolyte solution contained 1 M KCl and 3 M guanidine. The laminin
protein was partially denatured in 3 M guanidine. The trans chamber was nega-
tively biased for the recording; laminin is positively charged at pH 7.
Each current blockage event in Fig. 6.1c represents a laminin protein molecule
interacting with or translocating through the nanopore. The transient decrease in