Biomedical Engineering Reference
In-Depth Information
in understanding many fundamental biological interactions. For example, knowledge
of the mechanical properties of DNA gives insight into how packaging, transcription,
and replication of genetic material occur.
Mechanical probing of single biomolecules has been accomplished using a variety
of techniques [
1
] including optical tweezers [
2
,
3
], magnetic tweezers [
4
,
5
], atomic
force microscopy [
6
], biomembrane probe [
7
,
8
], and subjection to flow [
9
]. These
techniques require the attachment of molecules by linkers to some larger object, such
as an Atomic Force Microscope (AFM) tip/cantilever, or a micrometer-scale bead as
required in optical and magnetic tweezers experiments. Attachment of linkers can
change the behavior of the system, and reloading the apparatus with a new biomole-
cule after each measurement can be time-consuming and require prodigious effort.
Nanopore Force Spectroscopy (NFS), on the other hand, is a technique in which
mechanical forces can be applied to single biomolecules without chemical modi-
fication or linker attachment. Instead, NFS works with biomolecular assemblies
having a relatively thin portion, which can thread through the pore, and a second
bulkier portion, which cannot. The basic design of a typical NFS apparatus is
illustrated in Fig.
14.1a
. A small chamber is separated into two sections by a
membrane no more than a few tens of nanometers thick. Both sides of the chamber
Fig. 14.1 Nanopore force spectroscopy. (a) Cartoon of a typical nanopore force spectroscopy
experiment. Force is applied to double-stranded DNA threaded through the pore. An enzyme
bound to the DNA is too large to pass through the pore. Therefore, the force on the DNA stresses
the intermolecular bond between the enzyme and DNA, allowing the interaction between the two
to be observed under the application of force. The top and bottom chambers (not to scale) are
separated by a membrane having a thickness of several nanometers. The entire system is filled with
electrolyte. Electrodes in each chamber are used to apply a voltage across the membrane. The
transmembrane voltage determines the force on the analyte and also causes a flow of ions through
the pore, which is measured using a sensitive ammeter. (b) Focusing of electrostatic force by the
nanopore. The component of the electric field directed along the pore axis (
E
z
) is plotted as a
function of position along this axis. An image of the pore faithfully overlays the plot, although the
electric field was derived from a simulation in which no analyte was present in the pore. Because
the solution filling the system is a good conductor, the magnitude of electric field near the pore
constriction is many orders of magnitude larger than outside the pore. The data plotted here was
derived from an MD simulation of a pore in the absence of an analyte
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