Single-molecule porphyrin-metal ion interaction and sensing application

Article abstract of DOI:10.1016/j.bios.2018.03.002

It remains a significant challenge to study the interactions between metal ions and porphyrin molecules at single ion level. Here, we constructed a nanopore-based sensing for label-free and real-time analysis of the interaction between Cu²⁺ and

Full text of DOI:10.1016/j.bios.2018.03.002

Author’s Accepted Manuscript
Single-molecule porphyrin-metal ion interaction
and sensing application
Keke Wei, Fujun Yao, Xiao-feng Kang
www.elsevier.com/locate/bios
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S0956-5663(18)30167-2
https://doi.org/10.1016/j.bios.2018.03.002
BIOS10328
DOI:
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Biosensors and Bioelectronic
Received date: 5 January 2018
Revised date: 19 February 2018
Accepted date: 1 March 2018
Cite this article as: Keke Wei, Fujun Yao and Xiao-feng Kang, Single-molecule
porphyrin-metal ion interaction and sensing application, Biosensors and
Bioelectronic, https://doi.org/10.1016/j.bios.2018.03.002
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Single-molecule porphyrin-metal ion interaction and sensing
application
Keke Wei1, Fujun Yao1, Xiao-feng Kang*
Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of
Chemistry & Materials Science, Northwest University, Xi’an 710069, P. R. China
*Corresponding author. Tel.: +86-029-88302604; fax: +86-029-88302604.
kangxf@nwu.edu.cn
Abstract
It remains a significant challenge to study the interactions between metal ions
and porphyrin molecules at single ion level. Here, we constructed a nanopore-based
sensing for label-free and real-time analysis of the interaction between Cu2+ and
5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin (TPPS). The results demonstrate
that emerging electronic signatures of the Cu2+-TPPS complex that is completely
different form the original free TPPS were observed in the α-hemolysin (α-HL)
nanopore. Based on the distinctive electronic signal patterns between TPPS and
Cu2+-TPPS complex, the unique nanopore sensor can achieve a highly sensitive
detection of Cu2+ in aqueous media. The frequency of signature events showed a
1
These authors contributed equally to this work.
1

linear response toward the concentration of Cu2+ in the range of 0.03 µM – 1.0 μM,
with a detection limit of 16 nM (S/N = 3). The sensing system also exhibited high
selectivity against other metal ions, and the feasibility of this approach for practical
applications was demonstrated with the determination of Cu2+ in running water.
Keywords: α-hemolysin nanopore; single-molecule detection; TPPS; copper ion;
biosensor
1. Introduction
Sensing of metal ions in aqueous solutions is of great importance in terms of
their widespread distribution in environmental systems and biological processes.
Among all the metal ions, copper is an essential trace metal that plays fundamental
roles in many physiological processes by serving as a cofactor for numerous proteins
and enzymes (Domaille et al., 2008). However, excess copper intake can cause
damage to liver, kidney and nervous system (Boal and Rosenzweig 2009).
Consequently, the development of facile, sensitive and reliable methods for
identification and quantification of copper ions is in high demand.
Conventional techniques for copper ions detection include atomic absorption or
emission spectroscopy (AAS/AES) (Kumar et al., 2011; Lima et al., 2012), and
inductively coupled plasma mass spectrometry (ICP-MS) (Dai et al., 2012). Although
these techniques can detect metal ions sensitively and accurately, and are considered
as standard methods, they usually require sophisticated instruments, tedious sample
2

preparation procedures and trained personnel. All of these shortcomings limit their
wide applications in routine copper ion detection. Alternatively, colorimetric and
fluorescent chemosensors based on small organic molecules have been intensively
developed for the detection of Cu2+ ions. Unfortunately, these chemosensors typically
suffer from low selectivity and poor water solubility, making it difficult to monitor
Cu2+ ions in practical applications. Recently, some highly sensitive methods for Cu2+
detection have been proposed by employing catalytic DNAzyme (He et al., 2014; Xu
et al., 2015; Yin et al., 2009), for examples, electrochemistry (Yang et al., 2016),
colorimetry (Sadollahkhani et al., 2014; Yuan et al., 2014; Z et al., 2014), quantum
dots (Dong et al., 2012) and molecular beacon (Brunner and Kraemer 2004) based on
transforming the target-specific activity of DNAzymes into detectable optical or
electric signals. These DNAzymes-based sensors have good specificity and sensitivity,
but most of them need labeling tags, which not only increase the cost and
complication of the sensing system, but also decrease its biocompatibility.
Accordingly, it is still a great challenge to develop a simple and label-free method for
Cu2+ ions detection with high sensitivity. In this work, we develop a new method for
the determination of Cu2+ ions by integrating a water-soluble small organic
molecules-based probe with the α-HL nanopore.
Nanopore is an emerging single-molecule analytical technology. Its inherent
merits such as label-free, ultrasensitivity, high signal-to-noise ratio and
single-molecule resolution, make it a powerful single-molecule identifier and detector
for DNAs (Venkatesan and Bashir 2011), RNAs (Zhang et al., 2015), peptides
3

(Asandei et al., 2013; Roozbahani et al., 2017; Yusko et al., 2012), proteins (Rotem et
al., 2012), biomarkers (Tian et al., 2017) as well as small molecules (Boersma et al.,
2012). With regard to metal ions detection, nanopore-based technologies have been
successfully used to detect a variety of metal ions including Cu2+ (Wang et al., 2017),
2+
Co2+, Cd2+, Hg2+ (Wen et al., 2011), Pb2+, Ba2+ (Yang et al., 2013) and UO2
(Roozbahani et al., 2017). Different sensing strategies including noncovalent or
covalent binding sites embedded within the pores, thymine-thymine mismatches in
DNA (Wen et al., 2011), G-quadruplex DNA (Yang et al., 2013), DNAzymes (Yin et
al., 2014) and peptides (Roozbahani et al., 2017) have been reported with high
sensitivity and selectivity. However, so far few studies have dealt with the detection of
metal ions by combining small organic molecule-based probes and nanopore sensor.
In this work, we employed TPPS as a recognition probe for highly selective Cu2+
detection based on α-HL nanopore sensor. Distinctive electronic signatures of
Cu2+-TPPS complexes were generated in the nanopore, which were different form the
original TPPS. By analyzing these new signatures, quantitative detection of Cu2+ ions
could be achieved. In comparison with previously reported Cu2+-specific detection
methods such as electrochemistry and fluorescence, the present nanopore sensor
provides a simple, label-free and amplification-free technique, which can be used to
study the interactions between TPPS and Cu2+ at single ion level. The whole detection
process does not need long time and tedious steps, making the sensing system simple
and cost-effective. In addition, the proposed method can also be successfully applied
to the determination of Cu2+ ions in running water.
4

2. Experimental section
2.1 Materials and regents
5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrin were purchased from J＆K
chemical. Tetramethylammonium chloride (TMA-Cl), CuCl2 were purchased from
Sigma-Aldrich. 2-Diphytanoylphosphatidylcholine (DPhPC) lipid was obtained from
Avanti Polar Lipids (USA). The thickness of Teflon film (Good fellow) was 25 µm.
The wild-type (WT) α-HL monomers were expressed in Escherichia coli BL-21 (DE3)
pLysS and purified by size exclusion chromatography. The assembly and purification
of heptametrical protein pores were carried out as reported previously (Cheley et al.,
1999).
2.2 Single-Channel Recording
A bilayer of DPhPC was formed over a 120-150 µm orifice in diameter in a
Teflon septum that divided a planar bilayer chamber into cis and trans compartments.
The formation of bilayer was achieved using the Montal-Mueller method (Montal and
Mueller 1972). The solutions in the compartments contained 1 M TMA-Cl, which was
buffered with 10 mM PB at pH 7.0 and 8.0, and 10 mM NaAc/HAc at pH 5.2 and 6.0,
respectively. α-HL proteins and TPPS were added to the cis compartment, which was
connected to a “grounded”. The final concentration of the α-HL proteins used for the
single-channel insertion was 0.05-0.2 ng/mL. Current were recorded with a
patch-clamp amplifer (Axopatch 200B, Molecular Devices, Sunnyvale, CA), filtered
with a built-in four-pole Bessel filler at 5 kHz, sampled at 20 kHz by a computer
5

equipped with a Digidata 1440AA/D converter (Molecular Devices).
2.3 Data Analysis
Single-channel event amplitude and duration were analyzed using Clampfit 10.5
(Molecular Devices) and origin 9.0 software (Microcal, Northampton, MA). Mean
dwell time values (τoff) and mean interevent interval values (τon) were obtained from
the dwell histograms, and the interevent histograms were fitted to single exponential
functions. The standard deviation of open pore current (I0) was obtained from single
channel current baseline histograms by fitting the distributions to Gaussian functions.
The values of mean signal amplitude and current blocking rate (∆I/I0) were obtained
from signal amplitude and I/I0 histograms by fitting the distributions to Gaussian
functions.
3. Results and discussion
3.1. The principle of Cu2+ ions detection using TPPS
The porphyrins and its derivatives are a class of heterocyclic macrocyclic
compounds, which play important roles in numerous fields such as catalysis, materials
and medicine (Ding et al., 2017; R et al., 2017). In these compounds, four pyrroline
subunits interconnected by their α carbon atoms through methine bridges (＝CH-).
The multiple pyrrolic N atoms in the planar tetrapyrrolic macrocyle lead to unique
metal ion binding affinity and selectivity (Ding et al., 2017). By taking advantage of
the photophysical characteristics such as quite different emission properties of the
metallo- and free base porphyrin, various porphyrin-based chemosensors have been
6

developed for the detection of heavy metal ions (Bill et al., 2015; He et al., 2006;
Pariyar et al., 2012; Santos et al., 2013).
The principle of nanopore assay for Cu2+ detection is shown in Scheme1. In the
absence of Cu2+, the interaction of TPPS with the α-HL protein nanopore only
generated short-lived and low amplitude events. In contrast, with the addition of Cu2+
to the solution, it can incorporate into the small-size coordination cavity of TPPS,
leading to the formation of stable Cu2+-TPPS complexes. The subsequent interaction
between Cu2+-TPPS and α-HL pore result in long-lived events with high amplitude.
Therefore, based on the different characteristic signatures, Cu2+ could readily be
determined.
3.2. Detection of Cu2+ ions using TPPS
To demonstrate the feasibility of this concept, we first conducted an analysis of
TPPS interaction with the α-HL pore. Recordings were made in solutions containing 1
M tetramethylammonium chloride (TMA-Cl), buffered with 10 mM NaAc, at pH 5.2.
We chose TMA-Cl, instead of KCl, as pore-filled electrolyte because it can improve
the stability of the planar lipid bilayer and reduce the noise of single-channel
recording (Wang et al., 2015). In a first set of experiments, we investigated the
binding of TPPS to the α-HL pore from the trans side. Since TPPS is a tetravalent
anion, negative potential was applied on the membrane so TPPS can be driven
electrophoretically into the β-barrel of α-HL pore. After adding 5 μM TPPS to the
trans chamber, no current blocking signal was observed at  140 mV (Fig. 1A). This
might be caused by that the diameter of the TPPS molecule (~2 nm) is comparable to
7

the diameter of the β-barrel (~2 nm), thus TPPS cannot enter into the pore and causes
ionic current modulation. Then, when TPPS was added to the cis chamber,
characteristic current blocking signals were generated at positive potential of + 140
mV, which resulted from the binding of TPPS with the inner wall of the pore. Typical
ionic current traces are shown in Fig. 1B and 1D. The binding events were
characterized by the dwell time (τoff) and the normalized blockage current (∆I/I0；∆I
represents magnitude of current drop, I0 is the open pore current). Fig. 1E shows a
scatter plot of the ∆I/I0 versus dwell time of a total of 929 binding events. Fitting the
∆I/I0 and dwell time histograms to a Gauss function and a single exponential decay
function (Fig. 1F and 1G), respectively, yielded mean values of ∆I/I0 = 0.38 ± 0.04
and τoff = 2.42 ± 0.17 ms (n = 5). Since the diameter of the TPPS molecule is larger
than the constriction of α-HL pore (~1.5 nm), it cannot translocate the pore to the
trans chamber. Therefore, the current blocks may result from trapped TPPS molecule
in the vestibule and then exit the pore from the cis entrance without translocation. The
voltage dependence experiments further supported the molecular mechanism. The
dwell time of TPPS was prolonged by a factor of 4.73 from 0.71 ms to 3.36 ms as the
voltage increased from + 100 mV to + 160 mV (Fig. S1).
Subsequently, when Cu2+ is being added in the cis side under identical condition,
new events with significantly different signatures were observed (Fig. 1C and 1H).
Since Cu2+ ions themselves could not produce any current blocking in the nanopore
(Fig. S2), the new generated signals should be ascribed to Cu2+-TPPS complexes. The
data analysis were shown in Fig. 1J and 1K. In the histogram of dwell time and ∆I/I0,
8

Cu2+-TPPS complex events displayed a higher mean ∆I/I0 (0.55 ± 0.03) (n = 5) and
increased mean dwell time (73.08 ± 2.42 ms) compared to those of free TPPS. In
addition, the scatter plot also displayed remarkably different distribution between free
TPPS and Cu2+-TPPS complex (Fig. 1I). The reason why the ∆I/I0 of Cu2+-TPPS
complex events was higher than that of uncomplex TPPS events may be due to
configuration distortion of the planar porphyrin molecular with the incorporation of
Cu2+, thus leading to the occupation of more space. A possible explanation for the
increased dwell time of Cu2+-TPPS complexes events is that the binding of Cu2+ to
TPPS changes the charge distribution of porphyrin molecules, thus resulting in an
enhanced interaction between the Cu2+-TPPS complexes and α-HL pore. The
discrimination between the free TPPS and Cu2+-TPPS complexes was readily
achieved based on the mean dwell time and the normalized blockage current. These
long-lived and deep current blockage events were observed only in the presence of
Cu2+ ions. The formation of Cu2+-TPPS complexes was also confirmed by UV-Vis
spectroscopy (Fig. S3).
3.3. pH effect on the sensitivity of the nanopore sensor
The pH values of the electrolyte solution usually show somewhat of an effect on
the properties of the protein pore such as ion selectivity, conductance, surface charge
and so on (Wang et al., 2014). It has been well documented that the entry and
residence of analytes in nanopore can be controlled by modulating the pH (Gu and
Bayley 2000). Therefore, at appropriate pH condition, the capture rate of analytes can
be enhanced and the transport rate can be slow down, which will improve the
9

sensitivity and resolution of the nanopore sensing systems (Merzlyak et al., 2005). To
achieve highly sensitive detection of Cu2+, the interactions between TPPS and the
α-HL pore in the presence of Cu2+ were carried out at different pH values of 5.2, 6.0,
7.0 and 8.0. Our results demonstrated that the entrance frequency and the mean dwell
time of Cu2+-TPPS signature events decreased as the pH increased. For example,
when the pH changed from 5.2 to 8.0, the signature events frequency decreased from
3.30 ± 0.14 s-1 to 0.19 ± 0.20 s-1, and the mean dwell time decreased from 73.08 ±
5.76 ms to 41.6 ± 4.04 ms. Among the four pH values, Cu2+-TPPS complexes showed
the largest entrance frequency and the mean dwell time at pH 5.2, with values of 3.30
± 0.14 s-1 and 73.08 ± 5.76 ms (Fig. 2B and 2C). When the pH value is less than 5, the
protonation of porphyrin macrocycle (pKa ≈ 4.9) is the competing reaction with
respect to the coordination of metal ion (Nam et al., 2015). Therefore, the electrolyte
buffer solution of pH 5.2 was used in the remaining experiments. In addition, the pH
effect on the event frequency and the mean dwell time for free TPPS showed a same
trend as that of Cu2+-TPPS complexes (Fig. 2D and 2E).
The pH effect on the detection of Cu2+ may be attributed to combined action of
electrophoresis (EP) and electroosmosis (EO), which dominated the transport of
charged molecules through the nanopore (Gu et al., 2003). As depicted in Fig. 2F, the
net charge of the α-HL inner surface is positive at pH 5.2, which contributes to an
enhanced anionic selectivity of the protein nanopore and electroosmotic flow (EOF)
of the fluid. For a positive voltage, the EOF is directed from the cis to the trans side of
the α-HL pore. As Cu2+-TPPS complexes are negative charge molecule, EOF and EP
10

are parallel and therefore enhance the entry of Cu2+-TPPS complexes in the pore. For
higher pH 7.0, the α-HL nanopore became weak anionic selectively, so the magnitude
of EOF is less significant than that at pH 5.2. Hence the enhancement ability for entry
in the pore was weakened. In contrast, the α-HL pore became weak cation selectivity
when the pH reached 8.0 (Fig. 2G). Therefore, EOF is directed form the trans to the
cis side, which is opposed to the EP movement (antiparallel) and consequently
suppress the entry of Cu2+-TPPS complexes in the pore. The pH effect on the dwell
time can be also explained by the EOF and EP interpretation. For positive voltage,
EOF helps the association of Cu2+-TPPS complexes with the α-HL pore with different
degrees for pH 5.2, 6.0 and 7.0. For pH 8.0, EOF resists the association to some
extent, and the mean dwell time is a little smaller than that at pH 7.0. Our
interpretations are in agreement with previous studies regarding peptide and protein
interactions with nanopore at various pH (Firnkes et al., 2010).
3.4 Effect of voltage and TPPS concentration on Cu2+ detection
It is well documented that the voltage has a profound effect on the sensitivity of
nanopore-based sensing system (Gu et al., 2001). To identify the optimum conditions
needed to achieve the maximum nanopore resolution for detection of Cu2+, we further
investigated the interactions of TPPS with α-HL nanopore in absence or presence of
Cu2+ at different voltages ranging from + 100 mV to +160 mV. Our experimental
results (Fig. 3B) showed that, in the absence of Cu2+, the frequency of the TPPS
events increase as the voltage changing from + 100 mV to + 160 mV. After addition
of Cu2+ to the TPPS solution, the frequency of characteristic signatures (Cu2+-TPPS
11

complexes events) (fsig) increased significantly with an increase in the voltage. As
shown in Fig. 3A, the +100 mV transmembrane voltage gave the lowest fsig at 0.88 ±
0.12 s-1. fsig increased by a factor of 3.75 to 3.3 ± 0.14 s-1 when the applied voltage
increased from + 100 mV to + 140 mV. As expected, the applied voltage of + 160 mV
achieved the highest fsig at 4.15 ± 0.17 s-1 (Fig. 3A). Although the fsig at + 140 mV was
slightly lower than that of + 160 mV, + 140 mV was chosen as the optimum applied
voltage, and the voltage was employed in the remaining experiments. This is because
that the lipid bilayer at + 160 mV was not as stable as that at +140 mV, meanwhile,
the spontaneous gating events generated in the α-HL nanopore at +160 mV in
TMA-Cl electrolyte would bring interference to Cu2+-TPPS signature events (Fig.
S4).
In addition, we also examined the effect of the TPPS concentration on the
nanopore sensor sensitivity. We found that the frequency of free TPPS events was
linearly proportional to the TPPS concentration, indicating that the concentration of
TPPS would not affect the sensitivity of the nanopore sensor. A concentration of 5 µM
TPPS was used in the remaining experiments since the single-channel recording
experiments demonstrated that TPPS could produce enough events for statistical
analysis within a relatively short time (Fig. S5).
3.5 Detection sensitivity and selectivity for Cu2+
To evaluate the sensitivity of the TPPS-based nanopore sensors, Cu2+ ions at
various concentrations were examined under the optimum conditions (1 M TMA-Cl
and 10 mM NaAc/HAc (pH = 5.2), with transmembrane potential of + 140 mV). As
12

shown in Fig. 4A and 4B, the frequency of Cu2+-TPPS signature events increased
with increased Cu2+ concentration. Linear regression analysis showed good linearity
between the signature event frequency and Cu2+ ion concentrations in the range of
0.03 µM -1.0 μM with a correlation coefficient of 0.995 (Fig. 4B, inset; linear
regression equation: fsig = 1.74[Cu2+] + 0.061). The limit of detection (LOD) was
estimated to be 16 nM at a signal-to-noise ratio of 3 in about 10 min of single-channel
recording, which is much lower than the US EPA-defined maximal contamination
level for copper (20 μM) in water (Yin et al., 2014). Table S1 summarizes other Cu2+
detection methods reported in the literatures. Although the LOD of our nanopore
sensor was slightly higher than those of sensitive fluorescent and electrochemical
sensor for Cu2+, it is still sensitive enough to monitor the Cu2+ in environmental
analysis.
Besides sensitivity, the selectivity of the sensing method was also evaluated by
using other metal ions in place of copper, including Mg2+, Al3+, Ca2+, Cr3+, Mn2+, Fe2+，
Fe3+，Co2+，Ni2+, Zn2+, Ag+，Cd2+，Ba2+，Pd2+，Hg2+, and Pb2+ at concentrations
of 100 μM. The single-channel recording experiments demonstrated that no new
signature events generated in the α-HL nanopore except the free TPPS signals. In
addition, our results also revealed that the coexistence of Cu2+ and other metal ions
did not affect detection by the sensor (Fig. S6). Hence the specificity in the detection
of Cu2+ is not compromised by complex mixtures of other metal ions, which is very
common in real samples analysis. All these results indicated that the other metal ions
could not interfere with this Cu2+ sensing system. The high selectivity of the
13

TPPS-based nanopore sensor may be due to the unique interactions between
Cu2+-TPPS complexes and the vestibule of α-HL nanopore.
3.6 Detection of Cu2+ in running water
The feasibility of this TPPS-based nanopore sensor for real-world applications
was demonstrated with the determination of Cu2+ in running water. In order to ensure
the reliability of the experiment, we employed tap water from our laboratory instead
of ultrapure water to prepare 1 M TMA-Cl and 10 mM NaAc/HAc (pH = 5.2) buffer
solution (real water samples without any pretreatment). The Cu2+ in the real water
were finally estimated to be 0.12 μM. Moreover, satisfactory recovery in the range of
95.0 to 101.7% was obtained as shown in table 1, and confirmed that this sensor is a
powerful tool for the detection of Cu2+ in real samples.
Conclusions
In conclusion, we have developed a biosensor for the determination of sub-nM
amount of Cu2+ by α-HL nanopore combined with a small molecule probe, TPPS.
Compared to conventional sensing approaches, the α-HL nanopore biosensor displays
several significant advantages, including simplicity, rapid response, label-free,
anti-interference and feasibility for practical applications. This promising sensing
platform enables rapid and accurate detection of Cu2+ with a low detection limit (16
nM). Experiments of running water demonstrated the present method could be
extended to Cu2+ detection in real samples. This investigation has also provided a
14

methodology of designing and constructing nanopore biosensors with excellent
selectivity and specificity for determination of biologically and environmentally
relevant analytes by using desirable molecular recognition probes.
Acknowledgment
This work was financially supported by the National Science Foundation of
China (Grants 20175105, 21375104, and 21327806), the Specialized Research Fund
for the Doctoral Program of Higher Education of China (Grant 20126101110015),
Natural Science Basic Research Plan in Shaanxi Province of China (Grant
2016JQ2021), Scientific Research Program Funded by Shaanxi Provincial Education
Department (Grant 16JK1780) and The Science Foundation of Northwest University
(No. 338020018).
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Fig. 1. Nanopore-based analysis of TPPS and Cu2+-TPPS binding events. (A) No
current modulation was observed with 5 μM TPPS in the trans chamber at the applied
voltage of  140 mV. (B) Representative single-channel current traces of TPPS
interactions with the α-HL pore. (C) Representative single-channel current traces of
TPPS after the addition of 10 μM Cu2+ to cis chamber. (D) Expanded view of a
typical event indicated in the panel B. (E) Scatter plots the events caused by TPPS. (F)
Histograms of normalized current blockade (∆I/I0). (G) Histograms of dwell times of
the TPPS. (H) Expanded view of a typical event indicated in the panel C. (I) Scatter
plots the events caused by TPPS (blue) and Cu2+-TPPS complexs (red). (J)
Histograms of normalized current blockage (∆I/I0). (K) Histograms of dwell times of
the Cu2+-TPPS. Experimental conditions: 1 M TMA-Cl and buffered with 10 mM
NaAc/HAc (pH = 5.2), with transmembrane potential of + 140 mV.
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Fig. 2. (A) Typical single-channel current traces in different electrolyte pH, in the
presence of 5 µM TPPS and 10 µM Cu2+ in the cis chamber. (B) The frequency of
Cu2+-TPPS complexes events at different electrolyte pH. (C) The dwell time of
Cu2+-TPPS complexes events at different electrolyte pH. (D) The frequency of TPPS
in absence of Cu2+ at different electrolyte pH. (E) The dwell time of TPPS at different
electrolyte pH. (F), (G) The combined action of EOF and EP on the interaction of
negative charge analytes with α-HL nanopore at pH ˃ 7.0 and pH ˂ 7.0 respectively.
Experimental conditions: 1 M TMA-Cl buffered with 10 mM NaAc/HAc (pH = 5.2,
6.0 ) or 10 mM PB (pH = 7.0 , 8.0 ), with transmembrane potential of + 140 mV.
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Fig. 3. (A) Effect of the applied potential bias on the Cu2+-TPPS TPPS frequency in
presence of 10 µM Cu2+. (B) Effect of the applied potential bias on the TPPS
frequency in absence of Cu2+. Experimental conditions: 1 M TMA-Cl buffered with
10 mM NaAc/HAc (pH = 5.2), TPPS concentration was 5.0 µM.
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Fig. 4. Detection of Cu2+ at various concentrations. (A) Representative single-channel
current traces showing various concentrations of copper ions added to the cis chamber
in the presence of 5 μM TPPS probe. The concentration of copper ions were ranging
from 0 nM to 30 μM, respectively (from up to down). (B) Dose-response curve for
coppper ions nanopore sensor system. The inset of panel B shows an enlarged portion
of the dose-response curve at a range 0.03-1.0 µM. (C) Selectivity for coppper ions
nanopore sensor system. Experimental conditions: 1 M TMA-Cl buffered with 10 mM
NaAc/HAc (pH = 5.2), with transmembrane potential of + 140 mV.
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Scheme1. Nanopore detection of Cu2+ using a TPPS as probe. The TPPS and
Cu2+-TPPS complexes with the pore produced events having significantly different
signatures, thus permitting them to be readily differentiated.
Table 1. Results of the detection of Cu2+ in real sample
Sample
Added Cu2+
(µM)
0
0.2
0.4
0.6
Measured
(µM)
0.12
0.31
0.51
Recovery
(100%)
—
95.0
97.5
Running water 1
Running water 2
Running water 3
Running water 4
0.73
101.7
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Highlights
 We report a label-free nanopore sensing approach that enables investigation of
metal ion-porphyrin interactions at single ion level.
 The unique nanopore sensor can provide a sensitive platform for detection of Cu2+
using TPPS as a recognition probe.
 The detection limit for Cu2+ is as low as 16 nM in aqueous solution.
 The proposed method has high selectivity and sensitivity to Cu2+
 The nanopore sensor showed a promising potential for the detection of Cu2+ in
real sample.
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