Solid State Nanopores for Selective Sensing of DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 120

ethanolic solution containing 1% (v/v) -aminopropyltriethoxysilane (APTES) [ 46 ].

Due to APTES functionalization, the nanopore walls had positive charge [ 47 , 48 ],
which would slow down the speed of negatively charged DNA translocating through
it. Kim et al. reported 56.7

g

s for 539 bp and 910 bp
ds-DNA without sacrificing the current blockade magnitudes. They reported that
cation selectivity of the nanopore would be decreased after modification with
APTES due to excess positive charge inside the nanopore.
Similar types of silanization have been reported by other groups [ 49 , 50 ].
Umehara et al. reported ion current rectification with a quartz nanopipette coated
with positively charged poly-L-lysine (PLL). Adsorption of PLL molecules on
the negatively-charged surface was followed by baking to achieve a stable coating
layer. Wanunu et al. reported that the modification of the surface of nanopore below
5 nm tended to clog upon simple immersion of the chip in saline solution [ 51 ].
They suggested conductive cell salination for this problem. In conductive
cell salination, organic electrolyte is mixed with saline chemistries and certain
voltage is applied. The flow of ions disturbs any clogging of unbound saline
molecules. They verified their results by ionic current measurements, which were
in agreement with the actual nanopore diameter measurements.
There have been other chemistries proposed for nanopore functionalization.
For example, the amide terminal can be functionalized with homo-bifunctional
agent whose one active side can be attached to the NH 2 terminal of the chip.
The other side can be attached to an amine-terminated DNA probe. The DNA
attachment can be verified by hybridization with complementary florescent tagged
ss-DNA followed by imaging. Ellipsometry or contact angle measurements can
be used for verifying the functionalization of the chip with DNA probes. The SiO 2
surface without functionalization is more hydrophilic as compared to the chip
functionalizedwithDNA. A lesser number of OH groups will be present on function-
alized chips, which results in reduced hydrophilicity. In one recent report, Jang et al.
showed that the ratio of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro-
chloride (EDC) and N-hydroxysuccinimide (NHS) for functionalization is very
critical [ 52 ]. They suggested that for protein immobilization a ratio of 3:1 worked
best for EDC:NHS. They showed that EDC diluted with ethanol instead of DI
water and NHS diluted with PBS gave the best fluorescent intensity after protein
attachment. One other important factor was the time duration of the reaction. They
got the best results when the chips were immersed for 4 h in functionalizing mixture.
When a nanopore device is functionalized, a SAM layer is formed all over
the chip surface and inside the nanopore. After DNA probe attachment, some of
the probes would attach to the outer walls of the nanopores, which would also bind
to specific target molecules but do not participate in signal yield. This reduces the
sensitivity of the selective nanopore. In one report, it has been suggested that a
soft lithography technique using polydimethylsiloxane (PDMS) can be used for
selective functionalization of the outer nanopre membrane with hexaethyleneglycol
thiol derivative [ 53 ]. Selective functionalization would thus block the non-specific
attachment of the DNA molecules on the outer membrane while only the inner
walls of the nanopore are functionalized. During detection the target molecules

2.6 m s and 104.6 28.6 m