Biomedical Engineering Reference
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Fig. 9.8 Detecting protease activity with a
a
-hemolysin pore. Translocation of a ten amino acid
fragment of amyloid-
b
(residues 10-20) through the pore resulted in transient current blockages
(shown at time 0). Protease activity cleaved the amyloid-
b
into two smaller peptides, and
translocation of these peptides resulted in current fluctuations with a characteristic peak amplitude.
Adopted from Zhao et al
.
with permission [
55
]
with low ionic strength, this change in surface charge affected the local concentra-
tion of ions near the surface of the membrane and consequently the conductance
of gramicidin pores embedded in the lipid bilayer. Hence, the conductance of
gramicidin reported the enzyme activity of PLD and PLC. Due to the complex
nature of catalysis on a surface, the authors calculated a pseudo-first order rate
constant,
k
f
(s
1
) (Table
9.2
). This method permitted the detection of the two
phospolipases using picomolar to nanomolar amounts of enzyme.
In a third demonstration of quantifying enzyme activity with nanopores, Yang's
group and Mayer's group recently attached an enzyme substrate covalently to
gA pores and detected enzymatic hydrolysis by a change in the single-channel
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