Molecular Detection and Force Spectroscopy in Solid-State Nanopores with Integrated Optical Tweezers - Nanopores: Sensing and Fundamental Biological Interactions - page 39

Fig. 2.3 Capturing scheme. (a) A microbead (center indicated by dot ) with target molecules
attached is delivered near the nanopore opening with an optical tweezer. (b) Upon application of
an appropriate voltage, a molecule is pulled in to the pore and held there statically by the optically
trapped bead. This action both blocks the nanopore current and displaces the bead from its initial
position relative to the membrane ( horizontal lines )
2.3 DNA Detection
dsDNA (l-phage, 48.5 kb) is end-labeled through hybridization to a biotin-linked
primer with the complimentary 12-base overhang. The resulting material is then
attached to streptavidin-coated microbeads with a 1-h incubation at 37 C under
buffer conditions of 1 M KCl and 10 mM Tris-HCl (pH 8.0). The concentration of
DNA is controlled relative to the beads in order to attach only 1-10 molecules per
bead on average. This acts to further reduce the chance of having multiple mole-
cules enter the pore simultaneously.
Captures are performed by applying a voltage across the nanopore membrane
while monitoring nanopore current (conductance) and bead position (PSD signal).
In order to confirm that target molecules are indeed the cause of such signal
changes, a range of applied voltages can be used, as shown in Fig. 2.4a . In this
case, the solvent contains 1 M KCl. As increasing positive voltage is applied,
dsDNA capture events are eventually observed in both the conductance and the
PSD signal ( I, II ). However, by reversing the polarity ( III ), an opposing force is
applied which ejects the molecules from the nanopore. Thus, when a positive
voltage is applied ( IV ), the original open-pore conductance is again measured
until another molecule is eventually captured ( V ) when one returns to negative
voltages again.
A closer look at a typical dsDNA capture event is shown in Fig. 2.4b . Two
aspects of this measurement bare further mentioning here. First, the amount of
conductance change measured (referred to as conductance blockade in high ionic
strength conditions [ 14 ]) is found to be very consistent at ~1 nS. This is in