Nernst-Planck model of Photo-triggered, pH–Tunable Ionic Transport
through Nanopores Functionalized with “Caged” Lysine Chains
Saima Nasir,1,2 Patricio Ramirez,3 Mubarak Ali,1,2,a) Ishtiaq Ahmed,4 Ljiljana Fruk,4
Salvador Mafe,5 and Wolfgang Ensinger1
1
Department of Material- and Geo-Sciences, Materials Analysis, Technische Universität
Darmstadt, D-64287 Darmstadt, Germany
2
Materials Research Department, GSI Helmholtzzentrum für Schwerionenforschung,
Planckstrasse 1, D-64291, Darmstadt, Germany
3
Dept. de Física Aplicada. Univ. Politècnica de València. Camino de Vera s/n, E-46022
Valencia, Spain.
4
Karlsruher
Institute
of
Technology,
DFG-Center
for
Functional
Nanostructures
Wolfgang-Gaede-Strasse 1, D-76131 Karlsruhe, Germany
5
Dept. de Física de la Terra i Termodinàmica, Universitat de València, E-46100 Burjassot,
Spain
a)
Author to whom correspondence should be addressed.
Electronic mail: m.ali@gsi.de; m.ali@ca.tu-darmstadt.de
MATERIALS AND METHODS
Polyethyleneterephthalate (PET) membranes (Hostaphan RN 12, Hoechst) of 12 μm
thickness were irradiated at the Helmholtz Centre for Heavy Ion Research (GSI, Darmstadt)
with Au ions (energy: 11 MeV/u; ion fluence is either single or 5 × 107 ions cm-2).
Subsequently, ion tracked PET membrane were further irradiated with UV light (50 W m 2)
from each side for 15 minutes in order to sensitize the latent tracks for etching process.
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride
(EDC,
98%,),
N,N′-
dicyclohexylcarbodiimide (DCC, 98 %,) pentafluorophenol (PFP, 99+ %,) and 6-nitroveratryl
1
alcohol,
(99
%,),
4,5-dimethoxy-2-nitrobenzyl
chloroformate
(97
%,),
4-
dimethylaminopyridine, N,N-Diisopropylethylamine, and piperidine were purchased from
Sigma-Aldrich, Taufkirchen, Germany, and used without further purification. The solution
1
H-NMR spectra were measured on a Bruker Spectrospin 300 (300 MHz).
Fabrication of single conical and arrays of conical nanopores
An asymmetric track-etching technique developed by Apel et al is used to fabricate the
conical nanopores in heavy ion tracked PET membranes.1 A custom-made conductivity cell
with three chambers is used for the fabrication of single conical nanopore and arrays of
conical nanopores at the same time. A single-shot membrane and a membrane irradiated with
5 × 107 ions/cm2 are placed on both sides of the middle chamber of the conductivity cell and
clamped tight. The middle chamber, having holes on both sides, is filled with the aqueous
chemical etchant (9 M NaOH) while the other two chambers are filled with a stopping
solution (1 M KCl + 1 M HCOOH). The etching process is carried out at room temperature.
During the etching process, a voltage of -1 V is applied across the membrane in order to
observe the current flowing through the nascent nanopore. The current remains zero as long
as the pore is not yet etched through, and after the breakthrough an increase in ionic current is
observed. The etching process is stopped when the current has reached a certain value. The
pore is then washed first with stopping solution in order to neutralize the etchant, followed by
with deionized water. The etched membranes are further immersed in deionized water for
overnight in order to remove the residual salts
Synthesis of “caged” amino acid lysine (7):
2
Synthesis of compound (3): Fmoc-Lys(Boc)-OH (1) (2.0 g, 4.27 mmoles) is added to an
oven dried round bottom flask and the flask is purged with argon. The sample is diluted with
CH2Cl2 (20 mL), N,N-dicyclohexyl carbodiimide (DCC, 1.77 g, 8.54 mmoles), 4-(dimethyl
amino)-pyridine (DMAP, 1.04 g, 8.54 mmoles), and 6-nitroveratryl alcohol (2) (1.4 g, 6.83
mmoles) are added to the flask. The reaction is allowed to proceed overnight. The reaction
mixture is rinsed with 1 M HCl (25 mL). The organic layer is dried over sodium sulfate,
concentrated to give yellow residue which is purified by silica gel column chromatography
eluting with cyclohexane to cyclohexan/ ethyl aceate (5:1) to afford product 3 in 73% yield
(2.06 g, 3.11 mmol) as yellow solid.
1
H-NMR (300 MHz, CDCl3): δ ppm = 1.35 (s, 9 H), 1.40-1.42 (m, 2 H, overlapped), 1.52-
1.63 (m, 1 H), 1.65-1.73 (m, 1 H), 1.82-1.85 (m, 2 H), 2.99-3.05 (br 2 H), 3.84 (s, 6 H), 4.09
(t, 1 H, J = 6.8 Hz ), 4.23-4.36 (m, 3 H), 4.59 (br, 1 H), 5.48 (m, 2 H, CH2-Ph), 6.90 (br, 1 H,
Ar-H), 7.16-7.22 (m. 2 H, Ar-H), 7.24-7.32 (m, 2 H, Ar-H), 7.48 (d, J = 7.3 Hz, 2 H, Ar-H),
7.61 (s, 1 H, Ar-H), 7.65 (d, J = 7.4 Hz, 2 H, Ar-H)
C-NMR (75 MHz, CDCl3): δ = 22.4, 26.9, 28.4, 29.7, 33.9, 39.4, 47.1, 54.1, 56.3, 56.7,
13
64.1, 67.0, 79.2, 108.2, 110.4, 119.2, 125.0, 127.0, 127.7, 139.7, 141.2, 143.6, 148.3, 153.7,
156.2, 172.2
FT-IR (ATR) ύmax: 3315, 2934, 2857, 1748, 1694, 1677, 1618, 1580, 1519, 1449, 1365, 1324,
1273, 1221, 1196, 1086, 931, 731 cm-1.
3
MS (+FAB): m/z (%) = 686.3 (10) [M+Na], 664.3 (28) [M+H], 564.3 (35), 336 (20), 195.9
(40), 177.9 (62), 165 (21), 153.9 (100)
HRMS (FAB) calcd for C35H41N3O10 [M + H]+: 664.2870; found: 664.2872
Synthesis of compound (6): Lysine derivative 3 (2.0 g, 3.01 mmol) is dissolved in
acetonitrile (30 mL) and piperidine (10 mL) is then added. The mixture is stirred at room
temperature for 2 h. The solvent (acetonitrile) and piperidine are removed at reduced pressure
to give fmoc-deprotected lysine 4 as brown sticky residue which is used in the next step
without further purification.
To a stirred solution of fmoc-deprotected lysine 4 (1.50 g, 3.40 mmol) in anhydrous
THF (50 mL) is added N,N-diisopropylethylamine (DIPEA) (1.18 mL, 6.80 mmol, 2 eq.). The
mixture is stirred at room temperature for 10 min. The reaction mixture is cooled to 0oC and
4,5-dimethoxy-2-nirobenzyl chloroformate (5) (0.935 g, 3.40 mmol) is added at 0oC. The
reaction mixture is allowed to warm at room temperature and stirred for overnight. The
mixture is neutralized by dilute HCl and extracted three times by ethyl acetate (3 x 60 mL).
The organic layers are combined, washed with water (50 mL) and dried over anhydrous
sodium sulfate. After filtration, the solvent is evaporated under reduced pressure to give
brown residue which is purified by silica gel chromatography eluting with cyclohexane to
4
cyclohexan/ ethyl aceate (5:1) to afford 6 in 71% yield (1.64 g, 2.41 mmol) as pale yellow
solid.
1
H-NMR (300 MHz, CDCl3): δ ppm = 1.16 (m, 2 H), 1.33 (s, 9 H), 1.41 (m, 2 H, overlapped),
1.57-1.71 (m, 1 H), 1.74-1.83 (m, 1 H), 3.03 (br 2 H), 3.84 (s, 12 H), 4.33 (m, 1 H), 4.82 (m, 3
H), 5.30-5.52 (m, 4 H), 6.90 (br, 2 H), 7.59 (br. 2 H).
C-NMR (75 MHz, CDCl3): δ = 22.4, 28.2, 29.5, 31.0, 39.6, 54.2, 56.2, 56.3, 56.4, 60.3,
13
63.6, 64.0, 79.1, 107.9, 108.1, 109.4, 110.1, 126.5, 128.1, 139.3, 139.5, 147.9, 148.2, 153.6,
155.8, 172.2.
FT-IR (ATR) ύmax: 3332, 2932, 2854, 1752, 1692, 1674, 1618, 1579, 1516, 1323, 1273, 1219,
1167, 981, 868, 795, 754, 599 cm-1.
MS (+FAB): m/z (%) = 703.1 (3) [M+Na], 581.1 (10) [M-Boc], 520.4 (5), 415 (22), 316.2
(38), 95 (100)
HRMS (FAB) calcd for C30H40N4O14 [M + H]+: 681,2541; found: 681.2536
Synthesis of compound (7): Trifluoroacetic acid (5 mL) is added to a solution of the tertboc protected lysine 6 (520 mg, 0.76 mmol) in dry CH2Cl2 (15 mL). The reaction mixture is
5
stirred for 2 h at room temperature and the volatiles are removed on rotary evaporator.
Dichloromethane (15 mL) is again added and the solvent is removed under reduced pressure.
The procedure is repeated thrice to remove traces of trifluoroacetic acid to afford amine 7 in
88% yield (390 mg, 0.67 mmol) as yellow semi-solid.
1
H-NMR (300 MHz, CDCl3): δ ppm = 1.17 (m, 2 H), 1.44 (m, 2 H), 1.66 (m, 2 H), 2.97 (br 2
H), 3.81 (s, 12 H), 4.28 (m, 1 H ), 5.25-5.48 (m, 4 H), 6.88 (br, 2 H), 7.51 (br. 2 H), 7.60 (br.
2 H), 8.06 (br. 1 H).
C-NMR (75 MHz, CDCl3): δ = 22.1, 26.6, 29.6, 31.0, 39.7, 54.0, 56.2, 56.2, 56.3, 56.4,
13
63.9, 64.4, 108.0, 108.1, 109.8, 110.9, 125.9, 127.6, 139.2, 139.7, 148.0, 148.4, 153.7, 153.7,
156.1, 160.9, 161.4, 172.1.
FT-IR (ATR) ύmax: 3307, 2923, 2851, 1738, 1680, 1581, 1518, 1457, 1436, 1324, 1268,
1063, 983, 877, 794, 720 cm-1.
MS (+FAB): m/z (%) = 581.1 (10) [M+H], 551.6 (5), 503 (22), 383.4 (38), 109.4 (62) 95
(100)
HRMS (FAB) calcd for C25H32N4O12 [M + H]+: 581,2016; found: 581.2093
Functionalization of “caged” lysine chains on nanopore surface
The above-described etching procedure results in the generation of carboxyl groups on
the surface and inner wall of the nanopore. These carboxyl groups are converted into aminereactive pentafluorophenyl esters via pentafluorophenol (PFP) and carbodiimide (EDC)
coupling chemistry. For activation, the track-etched membranes are immersed in an ethanolic
solution containing a mixture of 0.1 M EDC and 0.2 M PFP at room temperature for one hour.
After washing the activated membrane with ethanol several times, the samples are further
6
dipped in a solution of “caged” lysine 7 (10 mM) prepared in anhydrous ethanol and left for
overnight. During this reaction period, amine-reactive PFP-esters are covalently coupled with
terminal amine group of lysine 7. Then, the modified membranes are washed thoroughly first
with ethanol followed by careful rinsing with deionized water and kept in dark until further
measurements.
UV light irradiation
The membranes modified “caged” lysine chains are irradiated with UV light of
wavelength 365 nm for 15 minutes on each side in dry state. For UV light irradiation, an Oriel
Instruments 500 W mercury lamp is used with a 365 nm filter. After irradiation, the
membranes are washed first with ethanol followed by deionized water in order to remove the
photocleaved groups from the interior of the nanopores.
Current–voltage measurements
The single-pore membrane is characterized by measuring the I–V curves. To this end,
the membrane is clamped between the two halves of the conductivity cell. An electrolyte
(0.1M KCl,) prepared in a phosphate buffer (10 mM) solution is filled on both sides of the
membrane. The pH of the solution is adjusted with dilute HCl or NaOH to the desired values:
3.0, 5.0 and 9.5. An Ag/AgCl electrode is placed into each half-cell solution and the ionic
current flowing through the single pore membrane is measured with a picoammeter/voltage
source (Keithley 6487, Keithley Instruments, Cleveland, OH). The ground electrode is placed
on the base opening side of the conical pore and the I–V curves are recorded by applying a
scanning triangle voltage signal from -2 to +2 V across the membrane.
Mass transport experiments
The membrane with conical nanopore arrays is fixed between the two halves of the
conductivity cell. Each cell volume is 3.4 ml with an effective membrane permeation area of
7
1.15 cm2. The analyte methyl viologen (MV2+) and 1,5-naphthalenedisulfonic acid (NDS2)
solutions of known concentration (10 mM) are prepared in the phosphate buffer at different
pH values (3.0, 5.0, and 9.5). The feed half-cell facing the tip opening of the conical
nanopores is filled with analyte solution. The permeate half-cell on the side facing to the base
opening of the conical pores is filled with a pure buffer solution. Both solutions are
continuously stirred during the experiment. After fixed time periods, the concentrations of
respective analyte in the permeate half-cell are estimated from the UV absorbance values. The
mass transport experiments are performed separately for each analyte at specific pH
conditions. Similar transport experiments are performed after UV light irradiation of the
modified membrane.
References:
1. P. Y. Apel, Y. E. Korchev, Z. Siwy, R. Spohr, and M. Yoshida, Nucl. Instrum.
Methods Phys. Res., Sect. B 184, 337 (2001).
8