Molecular Detection and Force Spectroscopy in Solid-State Nanopores with Integrated Optical Tweezers - Nanopores: Sensing and Fundamental Biological Interactions - page 38

Fig. 2.2 (a) Typical I-V measurement on a 8 nm nanopore, demonstrating linear response. (b)
Measured conductance through the same pore as the trapping laser is rastered across the membrane
surface. Local heating increases the ionic mobility when the laser beam is at the pore opening,
making its location detectable as the bright peak near ( x , y ) ¼ (13, 18 mm)

tweezer focus, clean measurement solution is flowed into the chamber to prevent
additional beads from entering the trap during subsequent measurement. Since the
target molecule is mechanically attached to the bead, the membrane-bead separa-
tion must be minimized in order to ensure that the molecule is affected by the
applied electric field. Therefore, after rinsing, the membrane is lowered to within a
short distance (generally 1-3 mm) of the trapped bead; best results are found with a
separation roughly equal to the molecular radius of gyration.
Once positioned correctly, the application of a voltage with appropriate polarity
across the membrane will attract molecules toward the nanopore. The size of the
necessary voltage varies somewhat, but generally falls between 50 and 150 mV. Too
low voltage does not create a strong enough field to pull a molecule into the pore in a
practical amount of time, while too high voltage creates the risk of capturing
multiple molecules or pulling the bead out of the optical tweezer. Each system and
molecule will require finesse to identify the optimal conditions. The capture of a
molecule in the nanopore is manifested as a sudden change in the measured trans-
membrane ionic current together with a (roughly) simultaneous change in the PSD
signal, indicating bead motion away from its initial position (Fig. 2.3 ). When this
occurs, the applied voltage is immediately reduced to 10-20 mV; enough to keep the
captured molecule from diffusing out of the pore, but too weak to introduce others.
At this point, the bead position can be changed relative to the nanopore (by moving
the membrane in z ) without losing the molecule.
Force spectroscopy is performed by increasing the applied voltage in a step-wise
fashion while continuouslymonitoring the bead position via the PSD. Each increase in
voltage creates an increased electrophoretic force on the molecule, and thus pulls the
microbead farther from its initial position and closer to the membrane. Ameasurement
of the position can be done using a detector scheme in which the PSD signal scales
directly with the bead position [ 12 ], but the results we describe here utilize a calibra-
tion curve taken directly prior to incremental voltage stepping [ 9 , 13 ].