Biomedical Engineering Reference
In-Depth Information
harsh chemical and thermal environments useful for denaturing the DNA, as
well as allowing for easier integration with other electrical or microfluidic compo-
nents. Solid-state membrane materials range from polymers [
36
], solid-state
dielectrics [
37
,
38
], and semiconductors [
35
,
39
] to metal films [
40
,
41
]. Different
membrane materials allow for tailoring different electrical properties, such as
surface charge density and capacitance, and they are susceptible to a wide variety
of surface treatments useful for controlling the pore charge and hydrophilicity. Most
importantly, a solid-state membrane can be reduced to sub-micrometer scale using
conventional semiconductor nanofabrication techniques, mitigating parasitic capa-
citance effects and improving electrical performance [
38
,
39
,
44
]. There is also a
panoply of methods for fabricating pores - ion-beam milling [
42
], ion-track etching
[
43
], reflow [
45
] or electron-beam ablation [
37
] to name a few. In particular, we were
the first to report the use of a tightly focused, high energy electron beam to sputter a
nanopore [
37
]. Figure
12.2a-d
are examples of the devices we have produced: i.e. a
0.7 nm nanopore sputtered into a 4.5 nm thick silicon oxide membrane - this is the
smallest synthetic pore in the thinnest membrane ever reported. Leveraging this
capability and the phenomenal electron beam stability and brightness below 2 nm,
we can use a scanning TEM to tailor the shape of the pore making slits like that
shown in Fig.
12.2e
or even irregular patterns like the “L” in (f).
12.2.2 DNA Conveyance to the Nanopore
While a nanopore is the ultimate analytical tool with single molecule sensitivity,
there is a shortcoming in its application to sequencing DNA that is related to the
diffusion equivalent capacitance [
46
,
47
]. When an electric field is applied across a
membrane with a
d <
3 nm bi-conical pore in it that is immersed in electrolyte, the
voltage is effectively focused near the center of the membrane over a region about
1-3 nm wide [
48
,
49
]. This means that
dsDNA
has to first diffuse within range of the
pore to be driven through it by the electric field. The rate of DNA capture is roughly
given by
R ¼
2
pCDr
, with
R
the capture rate,
C
the concentration of DNA,
D
the
DNA diffusion coefficient in free solution, and
r
the radius of probable capture by
the pore, which is on the scale of microns at high voltage [
50
]. The diffusion
capacitance governs the time required to capture a molecule, which is about 1 s for
the 10
9
molecules/
L concentration used routinely to test nanopore performance.
This capacitance leads to a trade-off between response time and detectable concen-
tration. So, the key to single molecule operation of a pore is conveying a small
volume of material within the capture radius.
To maintain a response time of ~1 s while utilizing single molecule fragments,
we have to focus the molecules into the capture volume over the pore. We can
accomplish this one of two ways: (1) by adapting a technique used prevalently in
flow cytometry to concentrate cells [
51
,
52
] - hydrodynamic focusing or (2) by
trapping the molecule in a small volume over the pore. Figure
12.3
demonstrates the
principle of hydrodynamic focusing using fluorescent molecules in a laminar flow
m
Search WWH ::
Custom Search