Tuning nanopore surface polarity and rectification properties through enzymatic hydrolysis inside nanoconfined geometries

Article abstract of DOI:10.1039/c3cc45318a

Enzyme-catalyzed reactions inside nanoconfined geometries promote sensitive changes in the surface charge polarity and the concomitant permselective transport properties of the single conical nanopores. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc45318a

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Tuning nanopore surface polarity and rectification
properties through enzymatic hydrolysis inside
nanoconfined geometries†
Mubarak Ali,*ab Saima Nasir,ab Ishtiaq Ahmed,c Ljiljana Frukc and
Wolfgang Ensingera
Cite this: Chem. Commun., 2013,
49, 8770
Received 14th July 2013,
Accepted 6th August 2013
DOI: 10.1039/c3cc45318a
www.rsc.org/chemcomm
Enzyme-catalyzed reactions inside nanoconfined geometries promote gating mechanism and current rectification, very similar to
sensitive changes in the surface charge polarity and the concomitant biological ion channels.5 The ionic transport through these
permselective transport properties of the single conical nanopores.
nanopores is controlled by the nature of the surface chemical
groups. Recently, several techniques have been reported for
In living organisms, ion channels and pores embedded in the modulation of pore surface chemistry to tune nanopore transport
cell membrane regulate and control the passage of ions or properties.6,7 These involve the covalent attachment of self-
particular analyte molecules across the membrane to facilitate assembled monolayers of functional molecules or polymer
the chemical and electrical communication with the extracellular brushes that respond to specific external stimuli, e.g., heat,7b
environment, e.g. for metabolic and signalling purposes.1 They solution pH,7c light irradiation,6a,c metal ions,6d,e biomolecules6b,7a
work as very sensitive and versatile nanodevices, e.g., (i) diodes or the modulation of both heat and pH.7d However, the inte-
for ionic current that restrict ion flow in one direction, (ii) ion gration of enzyme-sensitive nanopores still poses a challenge and
pumps, which control ion flow against concentration gradient addressing it would further broaden the scope and application
and (iii) ion sensitive elements, which are permselective, i.e., spectrum of nanoporous systems.
channels preferentially transport certain ions compared to
Enzymes are involved in almost all catalytic reactions,
others.2 Therefore, they are frequently used in the fabrication chemical interactions and signalling events occurring in living
of nanodevices for bioanalytical applications such as analysis organisms. They have also been considered as promising
and sequencing of DNA, biomolecular sensing and separa- triggering agents because of their highly selective and efficient
tion processes.2d These nano sized channels are precisely action towards specific substrates under physiological conditions.8
controlled structures with defined interfacial chemistry, and A variety of enzymes are involved in the catalytic breakdown of
have therefore proved to be very useful for a variety of inter- proteins, peptides and amino acid substrate molecules. Recently,
esting applications in nano/biotechnology such as sensing and the activity of some of these enzymes has been monitored through
manipulation of single molecules.3 However, the fragility and the changes in nanopore ionic transport properties.9 Mayer’s
sensitivity of the embedding lipid bilayer restrain their suit- group has reported the enzyme-induced modulation of electric
ability for more practical purposes. Conversely, synthetic nano- charge by the enzymatic cleavage of cationic choline, which led
pores have recently attracted a great deal of interest as their to detectable changes in the pore conductance of channel-
geometry, dimensions and surface chemistry can be tuned on forming peptide gramicidin A.9a
demand and they exhibit excellent chemical and mechanical
robustness.4
Herewith, we demonstrate a novel methodology to modulate
pore surface polarity and permselectivity through enzyme-catalyzed
Track-etched single conical nanopores display remarkable reactions. For this purpose, single conical nanopores are covalently
transport properties such as permselectivity, voltage-dependent functionalized with substrates for acetylcholinesterase (AChE)
and protease enzymes, and upon enzyme action carboxylic acid
groups are generated on the inner pore walls (Fig. 1). Consequently,
a
¨
Technische Universitat Darmstadt, Fachgebiet Materialanalytik, Petersenstr. 23,
the success of modification and enzyme-catalyzed reactions can be
monitored through the changes in current–voltage (I–V) characteri-
stics of these nanopores. To study the AChE enzymatic reaction
in a confined environment, a chemical compound O-(6-amino-
hexanoyl)-choline (AHCh), which acted as a possible AChE
substrate, was synthesized according to the published procedure
(ESI,† Scheme S1).10
D-64287 Darmstadt, Germany. E-mail: m.ali@gsi.de
b
¨
Matrialforschung, GSI Helmholtzzentrum fur Schwerionenforschung, Planckstr. 1,
D-64291 Darmstadt, Germany
c Karlsruher Institute of Technology, DFG-Center for Functional Nanostructures,
Wolfgang-Gaede-Str. 1, D-76131 Karlsruhe, Germany
† Electronic supplementary information (ESI) available: Experimental details of
the chemical synthesis, fabrication and modification of single conical polymeric
nanopores. See DOI: 10.1039/c3cc45318a
c
8770 Chem. Commun., 2013, 49, 8770--8772
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nanopore were arranged in such a way that high currents at
positive voltages and low currents at negative voltages were obtained.
Fig. 1a shows the I–V curves of single conical nanopores prior to
(black) and after (red) the immobilization of AHCh substrate
molecules. As-prepared single conical nanopores exhibit cation-
selectivity and rectify the ion current (i.e., cations preferentially flow
from the tip towards the base opening) passing through the nano-
pore due to the presence of ionized –COOꢀ groups on the pore walls
at physiological pH.5b The ion current rectification phenomenon in
these pores is a consequence of the combined effects of intrinsic
geometry and electrostatic asymmetries, which results in different
dependences of anion and cation concentrations inside the nano-
pore when a potential is applied across the membrane.5c
Fig. 1 (a) Functionalization of a single conical nanopore with (a) AChE substrate
O-(6-aminohexanoyl)-choline (AHCh) and subsequently, the enzymatic cleavage of
cationic choline from the inner pore walls; and (b) glycine p-nitroanilide (GNA) and
subsequently, protease enzyme hydrolyze and cut the uncharged p-nitroaniline
moieties, leading to the generation of carboxylate groups on the pore surface.
In conical nanopores, the direction of ion current rectifica-
tion is dictated by the electrically charged chemical groups on
the inner pore walls.6,7 Moreover, the degree of rectification
( frec) of the nanopore is directly related to the magnitude of the
surface charges. The frec value is calculated by dividing ionic
The commercially available N-t-butoxycarbonyl-6-aminohexanoic currents at positive voltages to that of negative voltages and vice
acid (1) was reacted with 2-chloro-N,N-dimethylethanamine (2) versa. As expected, the immobilized AHCh molecules onto the
in the presence of potassium carbonate using anhydrous ethyl pore surface resulted in the flipping of the I–V curve, i.e., the
acetate to yield N-dimethyl-O-(N-t-butoxycarbonyl-6-aminohexanoyl)- inversion of current rectification occurred (Fig. 2a). As it can be
cholamine (3). The crude 3 was refluxed with CH3I in acetone to seen from the I–V curves, the positive current at +2 V decreased
get O-(N-t-butoxycarbonyl-6-aminohexanoyl)-choline iodide (4). from 1190 pA to 137 pA and conversely, at ꢀ2 V applied bias the
Following the removal of the amine protecting Boc group with negative current increased from 220 pA to 811 pA. This clearly
dilute acetic HBr solution O-(6-aminohexanoyl)-choline bromide indicates the switching of pore surface polarity from negative to
hydrobromide (5) was obtained. Protease substrate glycine positive due to the presence of cationic trimethylammonium
p-nitroanilide (GNA) is commercially available and was used head groups, leading to the reversal of permselectivity and
without additional purification.
consequently, the modified-pore becomes anion-selective –
Single conical nanopores were prepared in polyethylene there is preferential transport of anions from the tip to the
terephthalate (PET) membranes of thickness 12 mm irradiated base opening. The rectification degree calculated from the I–V
with swift heavy ions through the well-established asymmetric curves (Fig. 1a) for as-prepared (|I (+2 V)|/|I (ꢀ2 V)|) and the
track-etching technique.5a The resulting conical nanopores modified nanopore (|I (ꢀ2 V)|/|I (+2 V)|) was B5.4 and B5.7,
have small (tip; d) and large (base; D) openings on the side of respectively. The observed similarity in the frec values suggested
the membrane facing neutralizing and etching solutions, respec- that modification reaction results only in the switching of pore
tively. The chemical etching of the ion tracks resulted in the surface charge polarity from negative to positive, while the
generation of native carboxylic acid (–COOH) groups on the magnitude of pore surface charges remained almost the same.
surface and inner pore walls.11 These groups serve as a starting
After successful functionalization, we proceeded to study the
point to change the chemical characteristics of the nanopore enzymatic cleavage of choline moieties from the immobilized
surface by attaching amine-terminated molecules through
carbodiimide coupling chemistry.6,7
The covalent attachment of amine-terminated enzyme sub-
strate molecules onto the pore surface was carried out in a two-
step reaction procedure (Fig. 1). In the first step, –COOH groups
were converted into amine-reactive PFP reactive-ester mole-
cules through N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC)/pentafluorophenol (PFP) coupling chemistry. Subsequently,
the PFP-reactive intermediate was covalently coupled with the
terminal-amine group of respective enzyme substrate molecules
as shown in Fig. 1.
The successful anchoring of the substrate molecules was
confirmed by the changes in the I–V characteristics of the
nanopore before and after chemical modification. To perform
the I–V recordings, the single-pore membrane was assembled
between the two chambers of the conductivity cell. The electro-
Fig. 2 The I–V curves of a single conical nanopore (d = B8 ꢁ 2 nm) in 100 mM
KCl solution (pH = 7.2) measured (a) before and after AHCh-substrate immobi-
lyte (100 mM aqueous KCl solution) was filled on both sides of
lization, and (b) before and after the AChE-enzymatic cleavage of cationic choline
the conical nanopore and the electrodes on each side of the moieties from the pore walls.
c
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responsible for the permselective ionic transport and rectifica-
tion properties of the nanopore, which can easily be followed by
I–V analysis. We believe that such enzyme-responsive nanoporous
systems have huge potential for biosensing, drug delivery and
design of controlled release platforms, especially when the
modulation of nanopore transport properties under biological
conditions is desired. Our further efforts will be focused on
exploring some of these applications.
S.N., M.A., and W.E. acknowledge financial support from the
Beilstein-Institut, Frankfurt/Main, Germany, within the research
collaboration NanoBiC, and L.F. and I.A. DFG-CFN Excellence
Initiative Project A5.7. We thank Prof. Christina Trautmann of
the GSI for help with the heavy ion irradiation experiments.
Fig. 3 The I–V curves of a single conical nanopore (d = B10 ꢁ 3 nm) in 100 mM
KCl solution (pH = 7.2) (a) before and after GNA-substrate immobilization, and
(b) before and after treatment with protease enzyme.
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substrate in the confined environment. To achieve this, the
AHCh-modified nanopore was exposed to 50 U mLꢀ1 solution
of AChE enzyme, which is known to specifically hydrolyze the
ester linkage in choline ester molecules such as acetylcholine.12
After treating the modified pore with AChE, the cationic choline
moieties were hydrolyzed and removed from the pore surface,
leading to the generation of carboxyl groups on the inner pore
walls (Fig. 1a). I–V curves shown in Fig. 2b reveal the recovery of
cation-selective and rectification behaviour of the nanopore
after AChE enzymatic hydrolysis reaction. The observed current
ratio (|I (+2 V)|/|I (ꢀ2 V)|) in the case of the AChE-treated pore
( frec = B5.2) was close to that of the as-prepared nanopore
( frec = B5.4), indicating almost complete cleavage and removal
of cationic moieties from the inner pore surface.
To further confirm the enzyme-triggered changes in nano-
pore surface polarity and permselectivity, we have also studied
the activity of protease enzyme inside confined geometries by
nanopore functionalization with a neutral substrate i.e., glycine
p-nitroanilide (GNA) as shown in Fig. 1b.
Fig. 3a shows the I–V curves of single conical nanopores
before (black) and after (olive) the functionalization with GNA
molecules. As expected, the nanopore rectification characteri-
stic was almost lost due to terminal uncharged p-nitrophenyl
moieties. The modified nanopore behaved like an ohmic resistor,
indicating the almost zero charge on the pore surface. Upon treat-
ment with protease enzyme, uncharged p-nitroaniline moieties were
removed from the inner pore walls due to the enzymatic cleavage of
the amide bond from the immobilized GNA-substrate molecules.13
The enzymatic-cleavage reaction resulted in the generation of
carboxylic acid groups, which were ionized to –COOꢀ under
physiological conditions (Fig. 1b). The I–V curves clearly indicated
that the protease catalyzed reaction switched the inner nanopore
environment from a hydrophobic (nonconducting) to a hydro-
philic (conducting) state and eventually, restored the cation-
selective and rectification characteristics of the nanopore (Fig. 3b).
In summary, we have demonstrated the functionalization of
single conical nanopores with enzyme substrate molecules. The
terminal moieties were removed from the immobilized sub-
strate by exposing the modified pore to respective enzyme. This
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