Phosphoprotein Detection with a Single Nanofluidic Diode Decorated with Zinc Chelates

Article abstract of DOI:10.1002/cplu.202000045

We report a nanofluidic device for the label-free detection of phosphoprotein (PPn) analytes. To achieve this goal, a metal ion chelator, namely 4-[bis(2-pyridylmethyl)aminomethyl]aniline (DPA?NH₂) compound was synthesized. Single asymmetric nanofluidic channels were fabricated in polyethylene terephthalate (PET) membranes. The chelator (DPA?NH₂) molecules are subsequently immobilized on the nanochannel surface, followed by the zinc ion complexation to afford DPA?Zn²⁺ chelates, which act as ligand moieties for the specific binding of phosphoproteins. The success of the chemical reaction and biomolecular recognition process that occur in a confined geometry can be monitored from the changes in electrical readout of the nanochannel. The nanofluidic sensor has the ability to sensitively and specifically detect lower concentrations (≥1 nM) of phosphoprotein (albumin and α-casein) in the surrounding environment as evidenced from the significant decrease in ion current flowing through the nanochannels. However, dephosphoproteins such as lysozyme and dephospho-α-casein even at higher concentration (>1 μM) could not induce any significant change in the transmembrane ion flux. This observation indicated the sensitivity and specificity of the proposed nanofluidic sensor towards PPn proteins, and has potential for use in differentiating between phosphoproteins and dephosphoproteins.

Full text of DOI:10.1002/cplu.202000045

Accepted Article
Title: Biomolecular Detection with a Single Nanofluidic Diode
Decorated with Metal Chelates
Authors: Mubarak Ali, Saima Nasir, Ishtiaq Ahmed, Christof M.
Niemeyer, and Wolfgang Ensinger
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemPlusChem 10.1002/cplu.202000045
Link to VoR: http://dx.doi.org/10.1002/cplu.202000045
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ChemPlusChem
Biomolecular Detection with a Single Nanofluidic Diode
Decorated with Metal Chelates
Dr. Saima Nasir,a,b Dr. Mubarak Ali,a,b* Dr. Ishtiaq Ahmed,c,d Prof. Christof M. Niemeyer,c and
Prof. Wolfgang Ensingera
aTechnische Universität Darmstadt, Fachbereich Material- u. Geowissenschaften, Fachgebiet
Materialanalytik, Alarich-Weiss-Str. 2, D-64287 Darmstadt, Germany
bGSI Helmholtzzentrum für Schwerionenforschung, Planckstr. 1, D-64291 Darmstadt, Germany
cKarlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG-1), Hermann-von-
Helmholtz-Platz, D-76344 Eggenstein-Leopoldshafen, Germany
dUniversity of Cambridge, Department of Chemical Engineering and Biotechnology, Philippa Fawcett
Drive, Cambridge, CB3 0AS, United Kingdom
*Corresponding author:
E-mail address: M.Ali@gsi.de ; M.Ali@ma.tu-darmstadt.de
Abstract
Here we demonstrate a nanofluidic device for the label-free detection of
phosphoprotein (PPn) analytes. To achieve this goal, a metal ion chelator, i.e., the 4-
[bis(2-pyridylmethyl)aminomethyl]aniline (DPA–NH2) compound is synthesized.
Single asymmetric nanofluidic channels are fabricated in polyethylene terephthalate
(PET) membranes. Then the chelator (DPA–NH2) molecules are immobilized on the
nanochannel surface followed by the zinc ion complexation to afford DPA–Zn2+
chelates which act as ligand moieties for the specific binding of phosphoproteins. The
success of chemical reaction and biomolecular recognition process occurring in
confined geometry is monitored from the changes in electrical readout of the
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nanochannel. The designed nanofluidic sensor has the ability to sensitively and
specifically detect lower concentrations (≥1 nM) of phosphoprotein (albumin and α-
casein) in the surrounding environment as evidenced from the significant decrease in
ion current flowing through the nanochannels. While, dephosphoproteins such as
lysozyme and dephospho-α-casein even at higher concentration (˃1 µM) could not
induce any significant change in the transmembrane ion flux. This indicated the
sensitivity and specificity of the proposed nanofluidic sensor towards PPn proteins. In
this context, we believe that metal affinity-based nanofluidic sensor would readily be
used to differentiate in between phosphoproteins and dephosphoproteins.
Keywords: biomolecules; ion current rectification; ligand–receptor interactions;
sensors; synthetic nanochannels;
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Introduction
The solid-state nanochannels/ nanopores exhibit higher stability (chemical or
mechanical) compared to biological ion channels. The synthetic nanochannels have
been successfully employed to mimic the functionality of ion channels.[1] Moreover,
they also act as sensors. In this context, much attention has been devoted to miniaturize
the nanofluidic based sensing devices during the recent years.[2] In the nanofluidic
systems, the analyte sensing/recognition mainly depends on the nature of chemical
groups (ligands) immobilized on the inner channel walls. These groups act as binding
sites, and interact also with ionic species passing through the channel.[3] The
biorecognition events are visualized from the changes in ion currents originating either
from the passage of an analyte through the channel under an applied voltage or from
the ligand-receptor interactions occurring inside the confined channel. Various methods
have already been developed to introduce recognition units inside confined geometries
for the detection of different (bio)molecules.[4] The biomolecular recognition process
relies on the traditional ligand–receptor interactions such as protein–protein, biotin–
streptavidin/avidin, antigen–antibody, and peptide nucleic acid–DNA complexes.[3a-
g] [4d-g]
,
Moreover, synthetic nanochannels also have the ability to selectively detect a
specific ionic moiety by the functionalization of desired ion responsive molecules on
the channel surface.[5]
Among the various proteins/ enzymes, phosphoprotein (PPn) plays a vital role in
different biological processes including signal transduction, metabolic pathways, gene
transcription, membrane transport, and others occurring in living organisms.[6] In both
prokaryotic and eukaryotic organisms, phosphorylation/dephosphorylation of protein
takes place in response to extracellular signals to regulate the enzymatic functions via
conformational changes in their structures.[7] The process of phosphorylation/
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dephosphorylation of protein is frequently studied in bioanalytical and medicinal
chemistry. It is foremost important to design molecular probes/ligands and develop
versatile technique to recognise phosphoprotein of interest.
To date, the detection of phosphorylated (bio)molecules is mainly achieved by using
antibodies and fluorescent techniques.[8] Although the antibody based method has been
successfully employed in flow cytometry and immunohistochemistry in the cellular
system of living organisms.[9] But, the use of antibody approach is limited because of
the large size of the antibody which sometimes interfere in the structure and function
of phosphoprotein. For the case of fluorescent/ colorimetric technique, Zn(II)-
coordinated dipicolylamine (DPA–Zn2+) chelates have been proved effective receptors
for phosphoprotein recognition.[10] But in this approach, the presence of a
chromophore/fluorophore moiety (signalling unit) directly linked with the receptor
(binding unit) is required to monitor the changes in light absorption or emission (or
colour change/ fluorescence change) on the binding of specific phosphorylated
(bio)molecule.[8c] To overcome the above mentioned restrictions, the design and
synthesis of alternative receptors which can be used for the antibody- and label-free
phosphoprotein recognition are still at preliminary stages. Recently, we have
demonstrated the detection of a small negative charged pyrophosphate (PPi) molecule
with synthetic nanochannels functionalized with bis(Zn2+-DPA) complexes.[11] Later
on, Jiang and his co-workers also demonstrated the detection of PPi anion by using the
terpyridine-zinc (TPYD–Zn2+) complexes immobilized on the single channel
surface.[12]
Compared to the antibody and optical detection methods, our sensing technique
relies on confining the sensing reaction into a volume of single channel which offers a
simple method to recognize protein analyte through host guest interactions. Moreover,
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the electronic readout is very easy to follow by using simple experimental setup
(picoammeter and voltage source).
In this study, we demonstrate a nanofluidic diode sensor for specific phosphoprotein
(PPn) recognition in confined geometries. To this end, the interior of the nanochannel
is tailored with dipicolylamine–zinc (DPA–Zn2+) chelates. The metal complexes act as
recognition elements for the capturing of PPn molecules (Figure1). The sensing device
has the ability to specifically recognize phosphoproteins (albumin and α-casien). On
the contrary, it did not show significant response to other control proteins (lysozyme
and dephospho-α-casien). The success of chemical reactions and biorecognition events
occurring in confined environment are monitored from the changes in ion current
flowing through the nanochannel.
Figure 1. (A) Synthesis of the amine-terminated DPA chelator (DPA–NH2). (B)
Nanochannel surface functionalization with amine-terminated DPA molecules, and subsequent
Zn2+ ion complexation.
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Results and discussion
Single asymmetric nanochannels are prepared in heavy ion irradiated 12 µm thick
PET foils through asymmetric chemical etching of the damage trails caused by the
energetic ions along their trajectories.[13] The irradiation and chemical track-etching
process resulted in the generation of carboxyl (-COO‾) groups on the channel surface.
Under physiological condition, the ionized carboxylate (-COO‾) groups import
negative charges to the channel surface. When negatively charged asymmetric channel
is in contact with an electrolyte solution, the cavity of the cone is mainly filled with the
cations (counter-ions). Under positive potential (V > 0) the electric field drag the cations
toward narrow channel region and subsequently, the cations preferentially transported
from tip to base side of the cone. While anions electrostatically prohibited to enter the
nanochannel. For the case of negative potential (V < 0), the electric field derive the
cations away from the small opening of the cone.[14] Therefore, under applied bias
higher value of ionic current is noticed at positive voltages compared to the negative
one. This non-ohmic behavior is termed as ionic current rectification – a unique
characteristic of asymmetric nanochannels.[5e, 14-15] The polarity of the fixed chemical
groups on the channel surface decides the direction of ion current rectification.
Moreover, the carboxylic acid groups on the channel surface serve as sites for the
linkage of desired ligand molecules having primary amine in their backbone. For the
capturing of (bio)molecule, first step is to design and synthesize a suitable ligand
molecule. In this study, we have synthesized a dipicolylamine (DPA–NH2) based metal
ion chelator by following the reaction scheme schown in Figure 1A. To this end, the 4-
nitrobenzyl bromide is reacted with di-(2-picolyl)amine to obtain the DPA nitrobenzene
(DPA–NO3) derivative.[16] Then the reduction of nitro group yields the DPA–NH2
chelator.
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After the synthesis of ligand, we proceeded to the immobilization of DPA–NH2
molecules on the channel surface. The DPA–NH2 molecule is first covalently attached
on the channel surface by using a single step reaction process through HATU
(Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) coupling chemistry.
The HATU reagent in the presence of DIEA base converts the channel carboxylic acid
groups into activated esters which immediately react with amine groups of the DPA–
NH2 chelator. Then the zinc (Zn2+) ion complexation with the DPA moieties is carried
out by exposing the DPA-modified membrane to Zn(NO3)2 solution.
Figure 2A shows the mechanism of phosphoprotein recognition and bioconjugation
on the channel surface. The I–V characteristics of the single nanofluidic channel prior
to and after the immobilization of DPA–Zn2+ chelates on the channel surface are shown
in Figure 2B. For this purpose, the single-channel membrane is assembled between the
two compartments of conductivity cell. The electrolyte (0.1 M KCl, pH 7.2) solution is
filled on both sides of the membrane. For I–V measurements, the ground electrode is
facing the base opening and working electrode on the tip opening side of the channel.
With this electrode configuration higher value of ionic current is obtained at positive
voltages when a potential applied across the membrane. Due to the presence of native
carboxylate groups, as-prepared channel exhibits current rectification. For the case of
modified channel, it is clearly evidenced from the current response that DPA–Zn2+
complexes impart positive charges to the channel surface, leading to the inversion of
current rectification due to the switching of channel permselectivity from cation to
anion.
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Figure 2. (A) Selective recognition and bioconjugation of phosphoprotein in confined
geometries. (B) I–V characteristics of as-prepared and DPA–Zn2+-modified single conical
nanochannel (d ~ 15 nm and D ~ 600 nm). (C) I–V characteristics of the modified channel upon
exposing to electrolyte solutions containing lysozyme and dephospho-α-casein proteins,
separately. (D) I–V curves of DPA–Zn2+-modified channel on exposure to electrolyte solution
containing various concentrations of albumin protein. (E) The changes in ionic current
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measured at a potential of -2V prior to (blank) and after exposure to various protein solutions,
i.e., lysozyme (1 µM), dephospho- -casein (1 µM) and albumin (0.5 µM). Inset shows the
voltages vs changes in rectification ratio (frec) calculated as I(-V) I(+V) for blank and 1 nM
albumin concentration, while for other albumin concentrations (10, 100 and 500 nM), the frec
values are obtained as I(+V) I(-V)
α
ǀ
ǀ
/
ǀ
ǀ
ǀ
ǀ
/
ǀ
ǀ.
The immobilized DPA–Zn2+ chelates served as recognition elements for the
detection of phosphoproteins.[10d] The sensitivity and the selectivity (specificity) are the
main analytical parameters which should be taken into account when designing a
biosensing device. The sensing capability of the nanofluidic sensor is studied by
exposing the modified channel to an electrolyte solution containing dephospho- and
phosphor-protein analyte. To verify the specificity, the nanofluidic diode sensor is
exposed to an electrolyte solution containing nonphosphorylated protein, i.e., lysozyme
and dephospho-α-casein, respectively. From the recordings of I–V curves as provided
in Figure 2C clearly shows that the presence of these proteins in the background
electrolyte could not induce any significant change in the ionic flux across the
membrane. This confirmed the lack of binding capability of nonphosphorylated
proteins toward the DPA–Zn2+ chelate. Thus the original surface remains undisturbed
with the free coordination sites of metal cations in the DPA–Zn2+ chelate for binding
with analyte of interest. Subsequently, when the same channel is exposed to albumin
protein, bioconjugation occurred in the confined geometry due to the specific
coordination of Zn2+ ion with the phosphoryl moieties in protein analyte. This
biorecognition process leads to significant change in the ion current flowing through
the modified nanofluidic channel (Figure 2D).
The sensitivity of the nanofluidic sensor is also tested against various albumin
concentrations. As expected, even a very low albumin concentration resulted in a drastic
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decrease in the channel ion current, indicating the binding of albumin to the chelated
zinc ion inside confined geometry as seen in Figure 2D. The observed decrease in the
ionic current is attributed to the formation of bioconjugates onto the inner channel
surface (Figure 2A). By increasing the albumin concentration, a further decrease in
ionic current is noticed. It is evident from Figure 2D that the channel surface becomes
saturated with bioconjugates at ~100 nM albumin concentration in the surrounding
electrolyte. Further increase in protein concentration did not induce significant change
in the ionic current flowing through the channel. Moreover, bioconjugation process
occurring on the channel surface also modulate the rectification degree (frec) which is
obtained from the ratio of higher currents to that of lower currents at given potential.
Inset in Figure 2D shows the changes in rectification ratio (frec) values versus voltages
calculated as ǀI(-V)ǀ / ǀI(+V)ǀ for blank and 1 nM albumin concentration (because the
current at negative potential is higher compared to that of positive one). Although, a
significant decrease in ion current is noticed on exposure to 1 nM albumin concentration
(Figure 2D) but we did not observe any significant change in the frec values when
compare to blank solution. While, when the channel is exposed to electrolyte solution
containing 10 nM albumin concentration, the direction of ion current rectification and
frec values obtained as ǀI(+V)ǀ / ǀI(-V)ǀ) are switched from negative to positive which
shows that the bioconjugation process import negative charges on the channel surface.
Further increase in albumin concentrations to 100 nM and then 500 nM lead to almost
blockage of channel and therefore, the frec values (ǀI(+V)ǀ / ǀI(-V)ǀ) are almost ~1 due to
hindered flow of ions across the nanochannel. Note that from the I-V response and frec
values shown in Figure 2D, it is very hard to find a linear relation in the change of
current or frec versus different albumin protein concentrations due to current fluctuation/
instabilities. The previously reported results also showed that bioconjugation in
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confined geometries clogged the tip opening, leading to the partial/ permanent blockage
of the ion current.[3a, 4d, 4e, 4g, 17] Figure 2E showed significant decrease in ion current
measured at a potential of -2 V on exposure to electrolyte solution containing
phosphoprotein (albumin) and nonbinding (lysozyme and dephospho-α-casein)
proteins. From the experimental data provided in Figure 2, we can infer that the
presented nanofluidic diode sensor exhibits a remarkable sensitivity and specificity
towards albumin because of possession of phosphate groups in the protein molecules.
To evaluate the reproducibility of the nanofluidic sensor, another nanochannel is
modified with DPA–Zn2+ chelates and then exposed to phospho-α-casein under similar
experimental parameters. Figure 3A shows the changes in current response of the
nanofluidic sensor in the presence of various concentrations of phospho-α-casein
protein in electrolyte solution.
Note that at lower protein concentrations, a decrease in ion current together with the
current instabilities is observed, indicating the interaction of proteins with the binding
sites on channel walls. Figure 3B clearly shows the reversible change in current vs time
curves under applied voltages in the range (+2V, -2V). The hysteric behavior of ion
current in one complete cycle of I–V curve is provided in Figure 3C. At lower protein
concentrations (10 and 100 nM), the hysteric effect appeared at higher voltages. The
arrows show the direction of the I–V recordings with increasing time. It was reported
that transient changes in the channel fixed charges are responsible for ion current
fluctuations.[18] Higher protein concentrations (≥ 100 nM) lead to the origination of
more bioconjugates, which ultimately cause steric obstruction of the channel tip
opening and consequently, a reduction/blockage of the ionic current was noticed.
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(A)
(B)
4
2
4
2
α-Casein (nM)
α-Casein (nM)
0
0
1
10
1
10
100
500
1000
100
500
1000
0
0
-2
-2
-2
-1
0
1
2
0
100
200
300
Voltage (V)
Time (s)
(D)
(C)
4
2
1.0
0.8
0.6
0.4
0.2
0.0
α-Casein (nM)
0
1
10
100
500
1000
0
-2
blank
Casein
-2
-1
0
1
2
Protein analyte
Voltage (V)
Figure 3. (A) I–V curves and (B) current vs time response and (C) ion current hysteretic
behaviour of modified channel (d ~ 13 nm and D ~ 750 nm) on exposure to an electrolyte
solution having various concentrations of phospho- -casein protein. (D) The channel
α
conductance normalized to maximum conductance prior to (blank) and after exposure to
casein protein solution (1 µM).
α-
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The experimental results (Figures 2 and 3) proved that the proteins having different
molecular weights / chemical composition can be identified based on the changes in
electrical readout of the nanochannel. Although in both cases, nanofluidic sensor
exhibits current instabilities and fluctuations might be due to the adsorption/desorption
of protein analyte on the channel surface or passage of protein molecule through the
nanochannel interrupt the ion flow under applied potential. But for the case of albumin,
the current instabilities noticed even at lower concentration as can be seen from the
error bar provided in Figure 2D. While in the case of casein, current
blockage/instabilities observed at higher concentration (Figure 3). This difference in
electrical signal is mainly due to different molecular sizes of both proteins. Note that
the molecular weight of the albumin (~44.3 kDa)[19] is almost double compared to
phospho-α-casein (~22 kDa).[20]
(A)
(B)
9
6
α-Casein (nM)
9
6
3
0
1
10
100
250
500
1000
3
y = a + b*x
No Weighting
0.91
Equation
Weight
0
Residual
Sum of
Squares
0.991
0.975
Pearson's r
Adj. R-Squar
Value Standard Erro
Intercept
Slope
2.39
0.335
0.001
B
-3
0.017
0
100 200 300 400 500
-2
-1
0
1
2
Voltage (V)
phospho-α-casein (nM)
Figure 4. (A) I–V characteristics of asymmetric nanochannel (d ~ 30 nm and D ~ 486 nm)
modified DPA–Zn2+ on exposure to electrolyte solutions containing various concentrations of
phospho-α-casein only on the tip side of single asymmetric nanochannel. (B) Changes in the
ion current versus phospho-α-casein concentrations (1 – 500 nM) at a potential of +2V.
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We have also performed the experiment under asymmetric addition of protein
analyte in the electrolyte solution. For this purpose, DPA–Zn2+-modified channel is
fixed in a conductivity cell. An electrolyte solution containing phospho-α-casein (100
mM KCl + protein) is filled on the tip side while the base side of modified channel
remain exposed to blank electrolyte (100 mM KCl) solution. Figure 4A shows the I–V
characteristics of the modified nanochannel whose tip region is exposed to various
concentration of phospho-α-casein. It can be seen that at lower protein concentration (1
to 10 nM), we did not notice any significant change in the I–V curve, indicating the
presence of insufficient protein molecules required to induce significant change in the
ion flow. By increasing the phospho-α-casein concentration to 100 nM, bioconjugation
occurred on the tip side which lead to current rectification much similar to negatively
charged asymmetric channel. Note that the isoelectric point (pI ~4.44) of α-casein is
lower than the pH (7.2) conditions used in the present study. Therefore, the binding of
α-casein import negative charge to the tip region, resulting the rectification of ion
current flowing through the nanochannel. Upon increasing the α-casein concentration
from 100 to 500 nM, a further increase in rectified ion flux is noticed. At higher α-
casein concentration (1 µM), a decrease in rectified ion flux along with fluctuation is
observed due to accumulation of protein at the tip regions, obstructing the flow of ions
across the membrane. On exposure to more concentrated solution (results not shown
here), the bioconjugation process clogged the tip opening, leading to almost blockage
of the ion current. Previously, Siwy and her co-workers have also reported that the
asymmetric binding of protein molecules induce change in the channel polarity which
in turn switched the current rectification phenomenon of the nanochannel.[21] Moreover,
Karnik et al. have also demonstrated that the analyte molecular charge dominate at
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lower concentrations while at higher concentrations volume exclusion effect dominate
due to the formation of bioconjugates in confined geometries.[22] Figure 4B shows the
changes in the ion current versus protein concentrations obtained at the positive
potential (+2V). The rectified ion current increased almost in a linear fashion up to 500
nM concentration of α-casein. Thus the preseneted nanofluidic sensor also provides
quantitative information on the protein concentrations down to nanomolar range.
It is worth to mention that the data presented in Figure 2 and Figure 3 exhibits
instabilities and fluctuation in the electrical signal of the nanofluidic sensor because of
very small opening (≤ 15 nm) of the asymmetric nanochannel. Moreover, the modified
channel exposed to symmetric electrolyte condition, i.e., protein is added on both sides
of the channel. While for the case of Figure 4, the tip opening diameter (~ 30 nm) is
almost double and the protein is added only on one side , i.e., cone tip side of the conical
nanochannel. Therefore, the ionic transport through the channel is now dictated mainly
by the charge of bioconjugates formed on the channel surface.
On the contrary, a nanofluidic channel functionalized with zinc complexes
comprised of two di(2-picolyl)amine [bis(Zn2+−DPA)] has the ability to sense
picomolar concentration of pyrophosphate anion (PPi) in the surrounding electrolyte as
evidenced from the change in permeselectivity of the nanochannel.[11] While, the
nanofluidic sensor could not show significant reponse on exposure to other phosphate
anions including such as monohydrogen phosphate, dihydrogen phosphate, adenosine
monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate
(ATP) even at highre concentrations.
Finally, we have also perfomed a negative control experiment under the same set of
experimental conditions by using an as-prepared (carboxylated) and DPA-modified
nanochannel. The as-prepared (unmodified) conical channel exhibits ion current
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rectification due to the presence of fixed negative charges (COO–) on the channel
surface (Figure 5A). After chemical modification, the channel surface charge is reduced
due to the presence of uncharged DPA moieties on the channel walls. This lead to
significant decrease in rectified ion flux as evidenced from the I–V curve (Figure 5B).
It is evident from the I–V response shown in Figure 5 that even higher protein
concentrations could not induce any significant change in rectified ion flux across the
membrane. These experimental results further supported our finding that the
phosphoproteins can only specifically bind with recognition moieties (DPA–Zn2+) on
the channel surface.
12
(A)
(B)
Unmodified channel
DPA-modified channel
3
2
blank
9
6
blank
α-casein
DP-α-casein
Albumin
α-casein
DP-α-casein
Albumin
Lysozyme
Lysozyme
1
3
0
0
-1
-3
-2
-1
0
1
2
-2
-1
0
1
2
Voltage (V)
Voltage (V)
Figure 5. I–V curves of the single asymmetric channel before (A) and after DPA-
modification (B) measured in presence of a 10 µM concentration of various proteins in
electrolyte solution.
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The nanofluidic sensor demonstrated here are not reusable because of irreversible
binding in between DPA-Zn2+ and phosphoprotein. For reuse of such sensor, exposure
to strongly acidic solution (pH ≤ 3) is required to dissociate bioconjugates from the
nanofluidic channel. But under these condition, the metal complex (DPA-Zn2+) can also
be dissociated due to protonation of nitrogen groups on the DPA molecule, leaving
DPA moieties on the channel surface. There would be a possibility to recomplex the
metal ion to regenerate the DPA-Zn2+ chelates on the channel surface for the detection
of phosphoprotein analytes. But there would be some limitation in the reuse of such
nanofluidic sensor: a) it is not sure that all the bioconjuagtes will be dissociated and
washed out from the channel surface. b) on exposure to strong acidic solution and re-
complexation of metal ion, the tip opening (sensing zone) of the conical nanochannels
could be damaged or deformed.
Conclusions
In summary, we have demonstrated the phosphoprotein recognition/conjugation in
confined geometries. To this end, DPA–NH2 was synthesized and immobilized on the
channel surface followed by the complexation of zinc ion to generate DPA–Zn2+
complexes which acted as an artificial ligand for the binding of phosphoproteins. The
nanofluidic channel modification and biomolecular recognition processes occurring in
confined environment were monitored through I–V measurements. Significant changes
in the ion flux occurred when the nanofluidic sensor was exposed to an electrolyte
solution containing phosphoprotein even in the nanomolar (nM) concentration range.
In contrast, control proteins such as lysozyme and dephospho-α-casein could not induce
any significant change in the transmembrane ion flux. This indicated the sensitivity and
specificity of the proposed nanofluidic sensor towards almost all biomolecules having
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phosphate moieties in their backbone. In this context, we believe that metal affinity-
based nanofluidic biosensors would readily be used to differentiate between phospho-
and dephospho-proteins based on the changes in the electronic read-out originated by
the ionic transport through the channel.
Materials and methods
Materials
All the chemicals, including di-(2-picolyl)amine (DPA, 97 %), 4-
nitrobenzylbromide (99%), lysozyme from chicken egg white (product number L6876),
albumin from chicken egg white (product number A5503), α-casein from bovine milk
(product number C6780), α-casein dephosphorylated from bovine milk (DP-α-casein,
product number C8032)
diisopropylethylamine (DIEA, 99.5%), 1-[bis(dimethylamino)methylene]-1
triazolo[4,5- ]pyridinium-3-oxid hexafluorophosphate (HATU, 97 %),
hydroxyethyl)piperazine- ′-(2-ethanesulfonic acid) (I M HEPES, pH 7.0 – 7.6)
N,N
-dimethylformamide (DMF, 99.8%),
N,N-
H-1,2,3-
b
N
-(2-
N
solution and zinc nitrate hexahydrate (Zn(NO3)2·6H2O) as well as all the solvents were
purchased from Sigma-Aldrich, Taufkirchen, Germany, and were used without further
purification.
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).
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Polyethyleneterephthalate (PET) membranes (Hostaphan RN 12, Hoechst) of 12 μm
thickness were irradiated at the GSI Helmholtz Centre for Heavy Ion Research
(Darmstadt) with Au ions (energy: 11 MeV/u, ion fluence: either single or 107 ions cm-
2). Subsequently, the ion tracked PET membranes were further exposed to UV light
from each side for 1 hour to sensitize the latent tracks for the etching process.
Fabrication of single asymmetric nanochannels
The heavy ion tracked PET membranes were chemical etched using an asymmetric
track-etching technique developed by Apel and his co-workers.[13] A detailed procedure
for the conversion of ion tracks into asymmetric channels was described previously.
Briefly, ion tracked PET membrane was chemically etched from one side with an
etching solution (9M NaOH) at room temperature. While, the other side of the
membranes was exposed to stopping solution to neutralize the etchant after the
breakthrough. During the etching process, a voltage of 1 V was applied across the single
ion tracked membrane to monitor the current flowing through the nascent channel. The
etching process was terminated when the current had reached a certain value. After
etching, etched membranes were immersed in deionized water to remove the residual
salts.
Synthesis di-(2-picolyl)amine based chelator DPA–NH2 (4)
The numbers of the different compounds in the synthesis of DPA chelator are
identified in Figure 1A.[16]
Synthesis of compound (3)
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10.1002/cplu.202000045
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Potassium carbonate (3.0 g, 21.73 mmol) was added to a solution of 4-
nitrobenzylbromide (1.65 g, 7.63 mmol) dissolved in anhydrous CH3CN (45 mL) and
stirred for 20 minutes. Di-(2-picolyl)amine (1.0 g, 7.53 mmol) solution in CH3CN (10
mL) was then added drop wise. The reaction mixture was stirred overnight under argon.
The volatiles were removed under reduced pressure to obtain dark brown crude product.
The residue was purified by silica gel flash column chromatography (CH2Cl2/MeOH,
50:1, v/v) to afford pure 4-[bis(2-pyridylmethyl)aminomethyl]nitrobenzene (3) as an
orange oil (2.2 g, 6.58 mmol, 87% yield).
1HNMR (500 MHz, CDCl3): δ (ppm) 3.81 (s, 2H), 3.83 (s, 4H), 7.17 (m, 2H), 7.52
(d,
= 8.7 Hz 2H), 8.54 (m, 2H).
13CNMR (125 MHz, CDCl3): δ (ppm) 57.7, 60.1, 122.2, 123.0, 123.5, 129.4, 136.8,
J = 7.8 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.68 (td, J = 7.8 Hz, 1.7 Hz, 2H), 8.15 (d,
J
147.0, 149.1, 158.8.
HRMS-FAB: calcd. for C19H19N4O2 [M+H]+ 335.2451, found 335.2489
Synthesis of compound (4)
To a solution of 4-[bis(2-pyridylmethyl)aminomethyl]nitrobenzene (3) (1.53 g, 4.58
mmol) in ethanol (60 mL) was added tin chloride dihydrate (5.25 g, 22.55 mmol). The
reaction mixture was refluxed for 16 hrs. The solvent was evaporated under reduced
pressure to give the crude product. The residue was purified by silica gel column
chromatography using 10% MeOH in dichloromethane to afford pure 4-[bis(2-
pyridylmethyl)aminomethyl]aniline (4) as an orange oil (0.93 g, 3.98 mmol, 86%
yield).
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10.1002/cplu.202000045
ChemPlusChem
1HNMR (500 MHz, CDCl3): δ (ppm) 3.55 (s, 2H), 3.59 (bs, 2H), 3.73 (s, 4H), 6.61
= 7.9 Hz, 2H), 7.12 (m, 2H), 7.15 (d, = 7.9 Hz, 2H), 7.55 (d, = 7.8 Hz,2H),
7.62 (m, 2H), 8.44 (d, = 8.6 Hz, 2H).
(d,
J
J
J
J
13CNMR (125 MHz, CDCl3): δ (ppm) 57.4, 60.1, 115.2, 122.0, 122.9, 128.7, 130.1,
136.4, 148.9, 160.2.
HRMS-FAB: calcd. for C19H21N4 [M+H]+ 305.2591, found 305.2537
Chemical functionalization of channel surface with DPA–Zn2+ complexes
Carboxylic acid groups were generated on the channel surface because of the heavy
ion irradiation and chemical etching. These functional moieties can be exploited for the
covalent linkage of DPA–NH2 molecule through HATU (Hexafluorophosphate
Azabenzotriazole Tetramethyl Uronium) coupling chemistry. It is a single step reaction
employed to functionalize the channel surface. To accomplish this goal, the DPA–NH2
(10 mM), HATU (10 mM) and DIEA (5 mM) were dissolved in anhydrous DMF
solvent. Then the polymer foil with single asymmetric channel was dipped in this
solution under argon atmosphere for overnight at room temperature. The modified
membrane was washed thoroughly first with DMF followed by careful rinsing with
deionized water. The zinc ion complexation on channel surface was archived by
exposing the DPA-modified membrane to an aqueous solution of Zn(NO3)2 (1 mM) for
2 hrs at room temperature.
Current-voltage measurements
As-prepared and modified single-channel membrane were fixed between the two
halves of the conductivity cell, separately. Then an electrolyte solution (100 mM KCl)
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10.1002/cplu.202000045
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was filled on both sides of the membrane. The ionic current flowing through the single
channel membrane was measured with a picoammeter/voltage source (Keithley 6487,
Keithley Instruments, Cleveland, OH) through Ag/AgCl electrode placed into each
half-cell solution. A scanning triangle voltage from –2 to +2 V was applied across the
single-channel membrane to record the I–V curves.
Moreover, various concentrations of phosphoproteins (ablumin and α-casien) and
control proteins (lysozyme and DP-α-casien) were prepared in the electrolyte (0.1 M
KCl) solution (pH 7.2) and the corresponding I–V curves were recorded under
symmetric and also asymmetric electrolyte conditions.
Acknowledgements
M.A., S.N. and W.E. acknowledge the funding from the Hessen State Ministry of
Higher Education, Research and the Arts, Germany, under the LOEWE project iNAPO.
I.A. and C.M.N. acknowledge financial support through the Helmholtz programme
BioInterfaces in Technology and Medicine. The authors are thankful to Prof. C.
Trautmann from GSI (Department of Material Research) for support with the heavy ion
irradiation experiments.
22
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Table of contents entry
A nanofluidic biosensing device based on a single asymmetric nanochannel
functionalized with metal ion chelates (DPA-Zn2+) can selectively recognize
phosphoproteins via specific binding interactions between metal ion and phosphate
moieties in the protein molecule. The bioconjugation process occurring inside
nanoconfined geometries lead to significant changes in the electronic read-out
originated due to hindered flow of ionic species passing through the nanofluidic
channel.
ionic
blockage
ionic transport
phosphoprotein
25
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