Label-Free Pyrophosphate Recognition with Functionalized Asymmetric Nanopores

Article abstract of DOI:10.1002/smll.201600160

The label-free detection of pyrophosphate (PPi) anions with a nanofluidic sensing device based on asymmetric nanopores is demonstrated. The pore surface is functionalized with zinc complexes based on two di(2-picolyl)amine [bis(DPA)] moieties using carbod

Full text of DOI:10.1002/smll.201600160

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Sensors
Label-Free Pyrophosphate Recognition with Functionalized
Asymmetric Nanopores
Mubarak Ali,* Ishtiaq Ahmed, Patricio Ramirez, Saima Nasir, Christof M. Niemeyer,
Salvador Mafe, and Wolfgang Ensinger
T he label-free detection of pyrophosphate (PPi) anions with a nanofluidic sensing
device based on asymmetric nanopores is demonstrated. The pore surface is
functionalized with zinc complexes based on two di(2-picolyl)amine [bis(DPA)]
moieties using carbodiimide coupling chemistry. The complexation of zinc (Zn2+)
ion is achieved by exposing the modified pore to a solution of zinc chloride to
form bis(Zn2+–DPA) complexes. The chemical functionalization is demonstrated
by recording the changes in the observed current–voltage (I–V) curves before and
after pore modification. The bis(Zn2+–DPA) complexes on the pore walls serve as
recognition sites for pyrophosphate anion. The experimental results show that the
proposed nanofluidic sensor has the ability to sense picomolar concentrations of
PPi anion in the surrounding environment. On the contrary, it does not respond to
other phosphate anions, including monohydrogen phosphate, dihydrogen phosphate,
adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate.
The experimental results are described theoretically by using a model based on the
Poisson–Nernst–Planck equations.
1. Introduction
Dr. M. Ali, Dr. S. Nasir, Prof. W. Ensinger
Department of Material- and Geo-Sciences
Recently, nanofluidic ion channels and pores have gained
remarkable attention as miniaturized sensing devices because
of their unique ion transport properties.[1] These nanosensors
have been used for the detection of a variety of analytes.[2]
The working principle is based on the modulation of trans-
membrane ion currents and electrical signals originated
either by the passage of an analyte through the pore under
an applied voltage or on the ligand–receptor interactions
occurring when the analyte is introduced in the environment
of the ligand-modified pore.
Protein ion channels (e.g., α-hemolysin) allow interfacial
chemistry processes controlled with high precision and have fre-
quently been employed for the sensing of analyte molecules.[3]
However, they are functional only if embedded in a fragile
bilayer. The lipid bilayer/protein nanopore system may be
mechanically unstable unless prepared in supported configura-
tions. Synthetic ion pores fabricated in solid-state materials dis-
play several advantages over protein pores concerning stability,
control over pore shape and size, possibility of integration into
nanofluidic devices, and tailored surface characteristics.[1b,4]
Materials Analysis
Technische Universität Darmstadt
D-64287, Darmstadt, Germany
E-mail: m.ali@gsi.de; m.ali@ca.tu-darmstadt.de
Dr. M. Ali, Dr. S. Nasir
Materials Research Department
GSI Helmholtzzentrum für Schwerionenforschung
D-64291, Darmstadt, Germany
Dr. I. Ahmed, Prof. C. M. Niemeyer
Karlsruhe Institute of Technology (KIT)
Institute for Biological Interfaces (IBG-1)
D-76344, Eggenstein-Leopoldshafen, Germany
Prof. P. Ramirez
Departamento de Física Aplicada
Universitat Politécnica de València
E-46022, Valencia, Spain
Prof. S. Mafe
Departamento de Física de la Terra i Termodinàmica
Universitat de València
E-46100, Burjassot, Spain
DOI: 10.1002/smll.201600160
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Asymmetric pores prepared through ion-track-technology[5]
show some transport properties reminiscent of those in
2. Results and Discussions
biological ion channels. Track-etched conical pores in Single conical nanopores were fabricated in polyethylene
polymer membranes exhibit transport characteristics such terephthalate (PET) membranes of thickness 12 µm via
as ion permselectivity and current rectification due to the asymmetric track-etching technique.[6d] Due to the chemical
carboxylic acid (COOH) groups on the pore surface.[6] etching of the latent tracks produced by the heavy ions, car-
Moreover, the pore surface can be easily decorated with boxylic acid (COOH) groups were generated on the pore
specific ligands for detection of particular analyte mol- surface because of the cleavage of polymeric chains. The
ecules, e.g., metal ions, small organic molecules, and exposed COOH groups were available for the covalent
macrobiomolecules (DNA and proteins).[1a,1c,2b,2c,2h,7] Pre- attachment of the desired ligands/functional moieties on the
vious related work using nanopore platforms has reported nanopore surface under mild conditions.
the detection of Zn,[8a] Pb,[8b] fl uorine,[8c] and carbonate[8d]
ions.
The chelating ligand bis(DPA)−NH2 was composed of
two di(2-picolyl)amine (DPA) moieties and a terminal amine
During the recent years, much attention has been group. The pore surface COOH groups were covalently
devoted to miniaturize the sensing devices for the selective attached with the amine group of the ligand. The attached
detection of biologically important anions.[9] Among the DPA moieties can then function as binding sites for Zn(II)
various phosphate anions, pyrophosphate (PPi) anion plays ion complexation which subsequently enables selective cap-
a vital role in different biological processes.[10] PPi is a ture of PPi anions. The amine terminated ligand bis(DPA)−
small negatively charged (P2O74–) analyte produced in the NH2 was synthesized by adopting the reported method with
living organisms as a by-product of adenosine triphosphate slight modifications (Figure 1A).[14] Briefly, the tosylation of
(ATP) hydrolysis in the cellular system. This anion partici- the 5-nitro-1,3-bishydroxymethylbenzene (1) was achieved
pates in a variety of enzymatic reactions controlling meta- by the reaction of p-toluene sulfonyl chloride to obtain the
bolic and energy transduction processes. The deficiency compound (2). The substitution of tosyl with DPA groups was
and an excess of PPi concentration in biological systems achieved by reacting the di-(2-picolyl)amine. This resulted in
results in the deposition of calcium in arteries, referred as a DPA-nitrobenzene derivative (3). The reduction of the nitro
Mönckeberg’s arteriosclerosis, and the calcium pyrophos- groups into an amine was carried out using Pearlman's cata-
phate deposition disease, respectively.[11] Moreover, the lyst (Pd(OH)2/C) to obtain bis(DPA)–NH2 molecules (4).
monitoring of the PPi concentration level is also important
Figure 1B represents the immobilization of ligand
in real-time DNA sequencing techniques.[12] The design of bis(DPA)–NH2) molecules on the pore surface. For this pur-
a sensing device for the selective detection of PPi under pose, the pore surface COOH groups were first activated into
aqueous conditions is therefore a challenge for the scien- amine-reactive PFP-esters via N-(3-dimethylaminopropyl)-
tific community.
N′-ethylcarbodiimide/pentafluorophenol (EDC/PFP) cou-
To date, a variety of fluorescent and colorimetric che- pling chemistry.[16] Subsequently, the PFP-intermediate was
mosensors have been developed for the detection of PPi
analyte.[9a–c] The majority of these techniques exploit
metal chelator ligands which transduce changes in the cor-
responding fluorescent or colorimetric signals on binding
with PPi anion. Among the various metal complexes, zinc
complexes based on two di(2-picolyl)amine [bis(DPA)]
moieties have proved effective binders for the PPi sensing.
[7d,9a,13] Recently, Varma and co-workers have reported label-
free electrical detections of PPi with a chelator-function-
alized field-effect transistor.[14] They have also successfully
employed this technique to detect PPi generated during the
DNA polymerase reactions.[15]
We demonstrate here the label-free detection of PPi with
a nanofluidic sensing device based on a single asymmetric
pore. To achieve this goal, the pore surface is chemically
functionalized with bis(DPA) ligands via carbodiimide cou-
pling chemistry. Subsequently, the complexation of zinc ions
is achieved by exposing the modified pore to a solution of
zinc(II). This procedure leads to the formation of bis(Zn2+–
DPA) complexes on the pore walls. The Zn(II)-complexes
act as recognition sites for the capturing of PPi anions. To
demonstrate the success of the chemical functionalization,
changes in the transmembrane ion flux are recorded in the
form of current–voltage (I–V) curves before and after pore
Figure 1. A) Reaction scheme for the synthesis of bis(DPA)–NH2.
B) The functionalization of the carboxylic acid groups on the surface of
modification. The single pore is employed for the highly sen-
sitive, specific detection of PPi anion.
asymmetric nanopore with amine-terminated bis(DPA)–NH2 molecules
via carbodiimide coupling chemistry.
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covalently coupled with the terminal amine group of the solution conditions and the electrolyte (100 × 10−3 m KCl)
ligand. Figure 2B shows the I–V characteristics of the single was prepared in 10 × 10−3 m tris-buffer (pH 8.0). In our elec-
conical pore before and after the immobilization of ligand on trode configuration, higher (positive) currents were recorded
the surface. The experiments were performed under symmetric at positive voltages while lower (negative) currents were
Figure 2. A) Cartoon representing the changes in pore surface chemistry. B) Experimental and theoretical characterizations: I) I–V curves of the
single conical nanopore measured in a 100 × 10−3 M KCl solution prepared in tris-buffer (10 × 10−3 M; pH 8.0) before and after the immobilization
of amine-terminated chelator [bis(DPA)–NH2] moieties. II) I–V curves of the chelator-modified nanopore prior to and after the formation of bis(Zn2+
DPA) complexes on the pore surface. III) and IV) theoretical I–V curves corresponding to the experimental results.
–
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obtained at negative voltages. The as-prepared (unmodi- interactions, and (iii) electrostatic interactions.[20] In the pre-
fied) conical pore preferentially transported cations from sent case, we focus on the electrostatic interaction, in which
the narrow opening to the wide opening of the cone due the detection process is based on the electrical characteris-
to the presence of ionized carboxylate (COO–) groups and tics of the pore walls.[2h,16] It is well-known that ion trans-
shows current rectification.[6c] The functionalization of ligand port through single conical pores is highly dependent on the
molecules resulted in the loss of pore surface charge due to surface charges: the direction of current rectification and the
the presence of uncharged DPA moieties. This caused the perm-selectivity both depend on the surface charges.[19a,21]
loss of current rectification (the pore behaved like an ohmic Changes in the sign and magnitude of the charge density on
resistor as evidenced from the linear I–V curve shown in the pore surface give significant effects on the ionic selec-
Figure 2B(I)). This clearly confirmed the successful modifica- tivity and rectification.
tion of the pore walls with ligand molecules.
Figure 3B shows the changes observed in the I–V curves
After functionalization of the ligand, the complexation of when the modified nanopore was in contact with different
Zn(II) ions with immobilized DPA moieties was achieved by PPi concentrations. The sensitivity is an important param-
treating the modified pore with an aqueous solution of zinc eter for designing nanofluidic sensing devices. To this end
chloride (Figure 2A). Figure 2B(II) shows the I–V curves of the the Zn2+-chelated pore was exposed to an electrolyte solu-
modified nanopore prior to and after the complexation of Zn2+ tion having low concentrations ranging from 10 × 10−12
m
ions with DPA moieties on the pore surface.The DPA unit with to 10 × 10−6 m of PPi analyte. The selective binding of the
three nitrogen donors acted as a tridentate ligand and had the PPi anion by the bis(Zn2+−DPA) moieties switched the pore
ability to form a stable complex with Zn(II) ions compared to polarity from positive to negative, thus reversing the pore
other metallic ions.[17] Upon complexation, the bis(Zn2+−DPA) permselectivity and rectification characteristics. The I–V
complexes imparted positive charges to the pore surface. As a curve corresponding to concentration 10 × 10−12 m showed
result the pore became anion-selective, leading to the inversion an increase in the positive current from 1.24 to 2.28 nA
of current rectification shown in the corresponding I–V curve.
and a decrease in the negative current from 2.63 to 1.35 nA
The ionic transport through conical nanopores can be at voltages +2 V and −2 V, respectively. Furthermore, the
described in terms of a Poisson–Nernst–Planck model previ- increase in the PPi concentration from 100 × 10−12 m to 100 ×
ously developed.[18] The input parameters of the model are 10−9 m led to a continuous increase in the pore rectification
the tip (aT) and base (aB) radii of the pore together with the because the number of PPi receptors bound to the immobi-
surface charge density σ.The values of these parameters were lized bis(Zn2+−DPA) increased significantly. This fact led to
determined separately using the following procedure. First, an increase in the magnitude of the (negative) charge den-
the radius of the pore basis was estimated by field emission sity on the pore surface (Figure 3A). Further increase in the
scanning electron microscopy techniques using a PET sample PPi concentration from 0.5 to 10 × 10−6 m did not induce sig-
containing 107 pores per cm2 etched simultaneously with the nificant changes in the electrical rectification, indicating the
sample containing the single pore. The average base opening saturation of the pore surface with PPi moieties. The theo-
radius obtained using this technique was aB = 240 nm. This retical curves of Figure 3B were calculated parametrically in
value is consistent with the pore base radius expected from the surface charge density using the pore radii obtained from
the etching time. Second, the radius of the pore tip was the experiments of Figure 2. The theory reproduces quanti-
determined from the experimental data of the pore with tatively the experimental data of PPi concentration by intro-
the amine-terminated chelator [bis(DPA)-NH2] moieties ducing surface charge densities ranging from −0.03 e nm−2
in Figure 2B(I) and (II). In these experiments, the surface to −0.12 e nm−2. Note the agreement between the changes
charge density is approximately zero and the only unknown of the charge sign in Figure 3B with the different chemical
model parameter is the radius of the pore tip. From the fit- functionalizations in Figures 2A and 3A.
ting of the linear I−V curve for the neutral pore, we obtained
Figure 4 shows the changes observed in the I–V curves
a tip opening radius aT = 9 nm. Once the pore radii have when the bis(Zn2+-DPA)-modified pore was exposed to an
been determined, the only free parameter available to fit electrolyte solution having different phosphates (sodium
the experiments of the as-prepared and the bis(Zn2+−DPA) salts). To this end, 1 × 10−6 m concentration of mono-
pores is the surface charge density. The theoretical curves of hydrogen phosphate (HPO42–), dihydrogen phosphate
–
Figure 2B(III) and (IV) reproduced the experimental data (H2PO4 ), adenosine monophosphate (AMP), adenosine
by introducing σ = −0.15 e nm−2 (as-prepared pore) and σ = diphosphate (ADP), ATP, and PPi was prepared in 0.1 m KCl
0.09 e nm−2 [bis(Zn2+−DPA) pore]. These values are in agree- (10 × 10−3 m tris-buffer; pH 8.0). From the I–V characteristics
ment with previous results obtained for asymmetric nano- in Figure 4A, we concluded that the bis(Zn2+−DPA)-modified
pores with different active groups.[19]
pore displayed a remarkable specificity toward PPi anion:
After the successful anchoring of bis(Zn2+−DPA) com- no significant changes were observed in the I–V character-
plexes, we studied the sensing capability of the sensor toward istics of the single pore exposed to HPO42–, H2PO4 , AMP,
–
phosphate anions, particularly PPi. Due to steric flexibility ADP, and ATP (phosphates concentrations of 1 × 10−6
m
in the immobilized ligand, the binding sites, i.e., Zn2+−DPA in the background electrolyte were not recognized by the
units, are organized in such a way that they can selectively bis(Zn2+−DPA) moieties). On the contrary, PPi selectively
capture PPi in contrast to other phosphates (Figure 3A). bound with the bis(Zn2+−DPA) moieties under these condi-
The mass transport through the pore is governed by three tions, imparting negative charge to the pore surface, with the
main mechanisms: (i) volume exclusion, (ii) hydrophobic resulting ionic current rectification.
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Figure 3. A) Cartoon representing the changes in pore surface chemistry before and after PPi interaction. B) Experimental I–V curves of the
bis(Zn2+–DPA)-modified nanopore measured in 0.1 M KCl (pH 8.0) at different PPi concentrations in the range of 10 × 10−12 M to 10 × 10−6 M. The
theoretical I–V curves are parametric in the surface charge density.
The above measurements proved sensing selectivity and changes in the current rectification shown in in Figure 3 were
subsequent transduction of specific events on exposure to only due to the selective binding of PPi with the Zn2+ ions in
a specific analyte. This fact clearly showed the validity of the chelator. For this purpose, an additional control experi-
our approach to miniaturize the sensing device. Indeed, the ment was conducted under the same experimental conditions
changes in the rectified current originated exclusively from as in Figure 3 with the bis(DPA)-modified pore (before Zn2+
the selective PPi binding with bis(Zn2+−DPA) moieties ion complexation). The I–V curves in Figure 5 did not show
(Figure 3A), and were not due to the adsorption of other any significant change in the ionic flux across the modified
anions onto the ligand modified pore.
nanopore on exposure to even at higher concentration of
As in Figures 2 and 3, the theoretical curves of Figure phosphate anions (10 × 10−6m). These experimental results
4B were able to reproduce quantitatively the experimental indicated again that PPi anion can only specifically bind with
data by introducing the surface charge density as the only chelated Zn2+ ion in the bis(Zn2+−DPA) moieties immobi-
parameter (the conical pore radii are those of Figure 2). Note lized on the pore surface.
again the agreement between the changes of the charge sign
in Figure 3B with the different chemical functionalizations in
Figures 2A and 3A.
3. Conclusions
Because PPi is a highly negative charged analyte, we
wanted to rule out the possibility of physical adsorption on The label-free specific detection of PPi anion using a nano-
the pore surface. To this end, it was necessary to demonstrate fluidic sensing device based on a single asymmetric pore was
that the switching of pore surface polarity and concomitant demonstrated experimentally and theoretically. The pore
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This indicated the sensitivity and speci-
ficity of the proposed nanofluidic sensor
toward PPi anion. Moreover, the experi-
mental results were also described theo-
retically by using a model based on the
Poisson–Nernst–Planck equations which
incorporated the geometrical and elec-
trical single pore characteristics. We envi-
sion that such nanofluidic sensors would
readily be used to monitor biological
important reactions, e.g., real-time DNA
sequencing.
4. Experimental Section
Materials: All the chemicals, including N-
(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC, 98%), PFP (99+ %),
5-nitro-m-xylene-α,α′-diol (96%), p-toluene-
sulfonyl chloride (TsCl, ≥ 99%), Di-(2-picolyl)
amine (DPA, 97%), disodium hydrogen phos-
Figure 4. A) I–V curves of the bis(Zn2+−DPA)-modified nanopore measured in a 100 × 10−3
M
KCl (pH 8.0) solution in the presence of 1.0 × 10−6 M concentration of various phosphates.
B) Theoretical I–V curves corresponding to the experimental results.
phate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), AMP,
ADP, ATP, and PPi, as well as all the solvents, were purchased from
Sigma-Aldrich, Taufkirchen, Germany, and were used without fur-
ther purification.
surface was chemically functionalized with zinc complexes
based on bis(DPA) moieties by means of carbodiimide
coupling chemistry and the feasibility of this functionaliza-
tion was evaluated by recording the I–V curves before and
after pore modification. The bis(Zn2+−DPA) complexes
formed on the pore walls acted as recognition sites, having
the ability to detect picomolar concentrations of the PPi
anion in the working electrolyte. In contrast, other phosphate
anions such as monohydrogen phosphate (HPO42–), dihy-
1H and 13C NMR spectra were recorded at 500 and 125 MHz in
CDCl3, respectively. High-resolution mass spectra were measured
using Finnigan MAT90 mass spectrometer. Analytical TLC (silica
gel, 60F-54, Merck) and spots were visualized under UV light and/
or phosphomolybdic acid-ethanol. Flash column chromatography
was performed with silica gel 60 (70-230 mesh, Merck) and basic
aluminum oxide (activated, basic, ≈150 mesh, 58 Å, Aldrich).
PET membranes (Hostaphan RN 12, Hoechst) of 12 µm thick-
ness were irradiated at the GSI Helmholtz Centre for Heavy Ion
Research (GSI, Darmstadt) with Au ions (energy: 11 MeV per
nucleon, ion fluence: either single or 107 ions cm−2). Subse-
quently, the ion tracked PET membranes were further irradiated
with UV light from each side for 30 min in order to sensitize the
latent tracks for the etching process.
–
drogen phosphate (H2PO4 ),AMP, ADP, and ATP could not
induce any significant change in the transmembrane ion flux.
Fabrication of Conical Nanopore: An asymmetric track-etching
technique was employed to fabricate the conical nanopores in
heavy ion tracked PET membranes.[6d] A custom-made conductivity
cell with three chambers was used for the fabrication of single-pore
and multipore membranes at the same time. A single-shot mem-
brane and a membrane irradiated with 107 ions cm−2 were placed
on both sides of the middle chamber of the conductivity cell and
clamped tight. The middle chamber, having apertures on both
sides, was filled with an etching solution (9 M NaOH). The other
two chambers were filled with a stopping solution (1 M KCl + 1 M
HCOOH). The etching process was carried out at room temperature.
During the etching process, a voltage of −1 V was applied across
the single ion tracked membrane in order to obs erve the cur-
rent flowing through the nascent pore. The current remained zero
as long as the pore was not yet etched through. After the break-
through, a point at which the etchant penetrated across the whole
length of membrane, an increase in ionic current was observed.
The etching process was terminated when the current had reached
a certain value. The etched membranes were then washed first
Figure 5. I–V curves of the bis(DPA)-modified nanopore measured in
a 100 × 10−3 M KCl (pH 8.0) solution in the presence of a 10 × 10−6
M
concentration of various phosphates.
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with stopping solution in order to neutralize the etchant, followed
1H NMR (500 MHz, CD3OD): δ (ppm) 3.52 (s, 4H), 3.73 (s, 8H),
by washing with deionized water. The etched membranes were fur- 6.71 (s, 2H), 6.82 (s, 1H), 7.23 (m, 4H), 7.62-7.78 (m, 8H), 8.41
ther immersed in deionized water overnight in order to remove the (m, 4H).
residual salts.
13C NMR (125 MHz, CD3OD): δ (ppm) 55.7, 57.5, 116.2, 119.9,
Synthesis of Amine-Terminated Bis(dipicolylamine) [bis(DPA)– 120.2, 120.3, 123.8, 123.9124.2, 138.7, 138.8, 140.1, 141.3,
NH2]: Preparation of compound (2): Aqueous sodium hydroxide 142.1, 149.0, 149.5, 149.9, 150.0, 160.5, 160.9, 161.2.
(0.5 N, 60 mL) was added to a solution of 5-nitro-m-xylene-α,α′-
diol 1 (1.0 g, 5.46 mmol) prepared in tetrahydrofuran (THF, 40 mL) [M+H]+ 516.6658.
HRMS-FAB: calcd. for C32H33N7 [M+H]+ 516.6650, found
at 0 °C. The mixture was stirred for 1 h at room temperature.
Functionalization of Nanopores: The above-described etching
The solution of p-toluenesulfonyl chloride (8.3 g, 43.7 mmol in procedure resulted in the generation of carboxyl groups on the sur-
50 mL of THF) was then added to reaction mixture dropwise at face and inner wall of the nanopore. These carboxyl groups were
room temperature. After the reaction mixture was stirred at room first converted into amine-reactive pentafluorophenyl esters via PFP
temperature overnight, ethyl acetate (100 mL) was added. The and carbodiimide (EDC) coupling chemistry. For activation, the track-
organic phase was washed with water (3 × 50 mL) and dried over etched single-pore membrane was exposed to an ethanolic solution
anhydrous sodium sulfate. The solvent was then removed under containing a mixture of 0.1 M EDC and 0.2 M PFP at room temper-
reduced pressure and the residue was purified by silica gel chro- ature for 1 h. After washing the activated membrane with ethanol
matography using a 2:1 to 1:1 mixture of hexane and ethyl acetate several times, it was further dipped in a solution of bis(DPA)–NH2
to afford pure tosylated compound 2 as colorless oil in 98% yield (40 × 10−3 M) prepared in anhydrous ethanol. The sample was left
(2.62 g, 5.35 mmol). The reaction products were characterized overnight in bis(DPA)-NH2 solution. During this reaction period,
with nuclear magnetic resonance (NMR) spectroscopy.
amine-reactive PFP-esters were covalently coupled with terminal
1H NMR (500 MHz, CDCl3): δ (ppm) 2.46 (s, 6H), 5.10 (s, 4H), amine group of the ligand. Then, the modified pore was washed
7.37 (d, J = 8.1Hz, 4H), 7.54 (s, 1H), 7.79 (d, J = 8.1Hz, 4H), thoroughly first with ethanol followed by careful rinsing with deion-
8.01 (bs, 2H).
ized water. After the successful immobilization of bis(DPA) moieties,
13C NMR (125 MHz, CDCl3): δ (ppm) 21.6, 69.5, 123.1, 127.9, the modified pore was treated with 0.1 × 10−3 M solution of zinc
130.1, 132.5, 133.2, 136.3, 145.6, 148.3.
HRMS-FAB: calcd. for C22H21NO8S2 [M+H]+ 492.5290, found
[M+H]+ 492.5293.
chloride for the complexation of Zn(II) ions with DPA moieties.
Current–Voltage Measurements: The as-prepared and modified
pores were characterized by measuring the current–voltage (I–V)
Preparation of Compound (3): Sodium carbonate (1.06 g, curves before and after the functionalization. To this end, the single-
10.18 mmol) and potassium iodide (1.69 g, 10.18 mmol) were pore membrane was fixed between the two halves of the conductivity
added to a solution of compound 2 dissolved in dried acetoni- cell. An electrolyte (0.1 M KCl,) prepared in 10 × 10−3 M tris-buffer (pH
trile (60 mL). The stirred was for 1 h at room temperature. Then 8.0), was filled on both sides of the membrane. An Ag/AgCl electrode
di-(2-picolyl)amine (DPA) (1.01 mL, 6.10 mmol) was added drop- was placed into each half-cell solution and the ionic current flowing
wise to the reaction mixture. The mixture was further stirred at through the single pore membrane was measured with a picoam-
room temperature for 5 h. The solvent was removed under reduced meter/voltage source (Keithley 6487, Keithley Instruments, Cleveland,
pressure to afford the yellow residue of compound (3). Ethyl ace- OH). The ground electrode was placed on the base opening side of
tate (150 mL) and water (150 mL) were then added to the yellow the asymmetric pore and the I–V curves were recorded by applying a
residue. The organic phase was washed with water (3 × 50 mL) scanning triangle voltage signal from −2 to +2 V across the membrane.
and dried over sodium sulfate. The solvent was removed under
Moreover, various concentrations of PPi and other phosphate
reduced pressure and the yellow residue was used in next step anions such as monohydrogen phosphate (HPO42–), dihydrogen
without further purification.
phosphate (H2PO4–),AMP, ADP, and ATP were prepared in a 0.1 M
1H NMR (500 MHz, CD3OD): δ (ppm) 3.78 (s, 4H), 3.82 (s, 8H), KCl solution with tris-buffer (10 × 10−3 M, pH 8.0) and the corre-
7.09 (m, 4H), 7.42 (m, 4H), 7.59 (m, 4H), 7.73 (s, 1H), 8.14 (s, 2H), sponding I–V curves were recorded under symmetric conditions.
8.54 (m, 4H).
13C NMR (125 MHz, CD3OD): δ (ppm) 57.6, 60.1, 121.9, 122.1,
122.2, 122.9, 135.0, 136.5, 141.3, 148.4, 149.0, 149.2, 158.9.
HRMS-FAB: calcd. for C32H31N7O2 [M+H]+ 546.6470, found
[M+H]+ 546.6477
Acknowledgements
Preparation of Compound (4): To a solution of compound 3
(200 mg, 0.37 mmol) in 30 mL of methanol, 20 wt% of carbon
Pd(OH)2 (500 mg) was added. The flask was purged with a vacuum
pump and back-filled with hydrogen using a hydrogen-filled bal-
loon. This was repeated three times and the mixture was stirred
under hydrogen atmosphere overnight. The carbon was then fil-
tered and the solvent was removed under reduced pressure. The
yellow residue was purified by flash chromatography on basic alu-
M.A., S.N., and W.E. acknowledge the funding from the Hessen
State Ministry of Higher Education, Research and the Arts, Ger-
many, under the LOEWE project iNAPO. P.R. and S.M. acknowl-
edge financial support by the Generalitat Valenciana (Program of
Excellence Prometeo/GV/0069), the Spanish Ministry of Economic
Affairs and Competitiveness (MAT2015-65011-P), and FEDER. I.A.
and C.M.N. acknowledge financial support through the Helmholtz
minum oxide column (activated, basic, ≈150 mesh, 58 Å, Aldrich)
programme BioInterfaces in Technology and Medicine. The authors
are also thankful to Prof. C. Trautmann, Department of Materials
Research from GSI, for support with irradiation experiments.
using dichloromethane to 5% methanol in dichloromethane to
afford compound (4), i.e., bis(DPA)–NH2 as a light yellow oil in
quantitative yield.
© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2020
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Received: January 18, 2016
Revised: January 29, 2016
Published online: March 3, 2016
© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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small 2016, 12, No. 15, 2014–2021