Author’s Accepted Manuscript A novel composite of molecularly imprinted polymer-coated PdNPs for electrochemical sensing norepinephrine Jianrong Chen, Hong Huang, Yanbo Zeng, Huan Tang, Lei Li www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(14)00798-2 http://dx.doi.org/10.1016/j.bios.2014.10.011 BIOS7193
To appear in: Biosensors and Bioelectronic Received date: 1 July 2014 Revised date: 1 October 2014 Accepted date: 5 October 2014 Cite this article as: Jianrong Chen, Hong Huang, Yanbo Zeng, Huan Tang and Lei Li, A novel composite of molecularly imprinted polymer-coated PdNPs for electrochemical sensing norepinephrine, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.10.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
A novel composite of molecularly imprinted polymer-coated PdNPs
2
for electrochemical sensing norepinephrine
3
Jianrong Chena, Hong Huanga, Yanbo Zengb, *, Huan Tanga, Lei Li a, b,∗
4 5 6 7
a
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004,
P.R. China b
College of Biological, Chemical Sciences and Engineering, Jiaxing University,
Jiaxing 314001, P.R. China
8 9
Abstract: A novel composite of molecularly imprinted polymer-coated palladium
10
nanoparticles (MIP-coated PdNPs) was synthesized by sol-gel method using
11
norepinephrine as template, phenyl trimethoxysilane as functional monomer and
12
tetramethoxysilane as crosslinker. The combination of PdNPs and silica-based MIP
13
endowed the composite with good electrochemical catalytic property, large surface
14
area and template selectivity. MIP-coated PdNPs were characterized by Fourier
15
transform infrared spectroscopy and Transmission electron microscopy. Then
16
MIP-coated PdNPs composite was used as a recognition element in the construction
17
of an electrochemical sensor for norepinephrine. The properties of MIP-coated PdNPs
18
sensor such as special binding, adsorption dynamics and selective recognition ability
19
were evaluated by differential pulse voltammetry. The results demonstrated that
20
MIP-coated PdNPs sensor not only possessed a short response time, but also high
21
binding capacity for norepinephrine, which enabled the imprinted sensor with higher
22
current response than that of non-imprinted material and MIP without PdNPs. In
23
addition, the MIP-coated PdNPs sensor exhibited selectivity for norepinephrine in
24
comparison to other analogs. The MIP-coated PdNPs sensor had a wide linear range
25
over norepinephrine concentration from 0.5 to 80.0 μM with a detection limit of 0.1
26
μM. The MIP-coated PdNPs sensor was proved to be a suitable sensing tool for the ∗
Corresponding author. Tel: +86 573 83646203. E-mail: [email protected] (Y. Zeng),
[email protected] (L. Li). 1
1
fast, sensitive and selective determination of norepinephrine in injection and urine
2
samples.
3
Keywords: Palladium nanoparticles; molecularly imprinted polymer; electrochemical
4
sensor; norepinephrine.
5 6
1. Introduction
7
Norepinephrine (NE) is an important catecholamine neurotransmitter and is
8
secreted by the adrenal medulla (Nikolajsen and Hansen, 2001). It is released as a
9
metabotropic neurotransmitter from nerve endings in the sympathetic nervous system
10
and some areas of the cerebral cortex. It is used to treat myocardial infarction
11
hypertension, bronchial asthma and organic heart disease. Extremely abnormal
12
concentration levels of NE may lead to the occurrence of many diseases, such as
13
ganglion neuronal, ganglia neuroblastoma, paraganglioma and Parkinson’ disease
14
(Mazloum-Ardakani et al., 2011). In these cases, it is very necessary to develop fast,
15
accurate and sensitive methods for the quantitative determination of NE in biological
16
fluids including urine and blood samples. Many methods, such as spectrophotometry
17
(Sorouraddin et al., 1998), gas chromatography (Kuhlenbeck et al., 2000), capillary
18
electrophoresis (Liu et al., 2008) and liquid chromatography (Ji et al., 2010), have
19
been employed for the determination of NE. However, these methods suffer from
20
some disadvantages of low detection sensitivity, time-consuming procedure,
21
complicated process or expensive analysis settings. Therefore, electrochemical
22
methods (Beitollahi and Mohammadi, 2013; Li et al., 2012; Mazloum-Ardakani et al.,
23
2011; Mazloum-Ardakani and Khoshroo, 2014; Rosy et al., 2014; Salmanpour et al.,
24
2012) have attracted much attention owing to their fast response, relatively high
25
sensitivity and ability of miniaturization.
26
Molecular imprinting is used to create recognition sites that are chemically and
27
sterically complementary to the target molecules in a synthetic polymer (Huang and
28
Shen, 2014; Liu et al., 2013). Molecularly imprinted polymer (MIP) exhibits an
29
affinity for the template molecule over other structurally related compounds. However, 2
1
MIP prepared by the conventional technique has some disadvantages, such as
2
incomplete removal of template, low-affinity binding and slow mass transfer. Thus
3
surface imprinting technique has received extensive attention (Bi and Liu, 2014;
4
Lofgreen and Ozin, 2014), because it can provide the advantages of favorable
5
selectivity, fast association/dissociation kinetics, some unique physicochemical
6
properties and desired application. Some researchers have reported surface MIP
7
composites based on Fe3O4 nanoparticles (Zhao et al., 2014), Au nanoparticles (Yu et
8
al., 2012), carbon nanotubes (Gao et al., 2010), graphene (Mao et al., 2011), CdTe
9
quantum dots (Huy et al., 2014; Wang et al., 2013; Xu et al., 2013; Zhang et al., 2012),
10
carbon dots (Mao et al., 2012), graphene quantum dots (Zhou et al., 2014). Many
11
routes have been explored to develop these surface MIP composites, such as free
12
radical polymerization (Mao et al., 2011), reversible addition fragmentation chain
13
transfer polymerization (Li et al., 2011) and sol-gel method (Zhang et al., 2012). The
14
widely used sol-gel method has gained great attention for preparing these surface MIP
15
composites, which can be explained by the fact that imprinted silica films show fast
16
kinetic binding for the template due to their rigid structures and nanosized thickness.
17
Palladium nanoparticles (PdNPs) with good electrocatalytic activity (Gao et al.,
18
2013; Li et al., 2014; Uberman et al., 2014) should be a good candidate as a supported
19
material for preparing surface MIP composite. To the best of our knowledge, surface
20
MIP composite based on PdNPs with sol-gel method has not been reported. In this
21
paper, PdNPs were prepared via reduction of K2[PdCl4] by citric acid in the presence
22
of poly (vinylpyrrolidone) (PVP), which was served as a stabilizer for the formation
23
of PdNPs. Then a novel composite of MIP-coated PdNPs was synthesized by sol-gel
24
method using phenyl trimethoxysilane as functional monomer and tetramethoxysilane
25
as crosslinker. MIP-coated PdNPs was characterized by Fourier transform infrared
26
spectroscopy and Transmission electron microscopy. The MIP-coated PdNPs
27
composite was used as a recognition element in the construction of an electrochemical
28
sensor for NE. The properties such as special binding, adsorption dynamics and
29
selective recognition ability were evaluated through differential pulse voltammetry. 3
1
The sensor based on MIP-coated PdNPs was applied to the determination of NE in
2
injection and urine samples.
3 4
2. Experimental section
5
2.1. Reagents and materials
6
K2[PdCl4], citric acid, ascorbic acid (AA), uric acid (UA), folic acid (FA),
7
acetaminophen (AC), L-tryptophan (L-TRP), dopamine (DA), tetramethoxysilane
8
(TEOS) and Poly (vinylpyrrolidone) with average molar masses of 10 kg mol-1
9
(PVP-10) were purchased from Aladdin Industrial Corporation (Shanghai, China).
10
Norepinephrine hydrochloride (NE) and epinephrine hydrochloride (EP) were
11
purchased from Sigma-Aldrich Co. (USA). Phenyl trimethoxysilane (PTMOS) was
12
obtained from TCI Co. Ltd. (Japan). Norepinephrine bitartrate injection was obtained
13
from Shanghai Harvest pharmaceutical Co., Ltd. (Shanghai, China). Human urine
14
samples of 24 h were provided by six healthy adult volunteers. Other chemicals were
15
of analytical grade and were purchased from Aladdin Industrial Corporation
16
(Shanghai, China). Phosphate buffer solution (PBS, 0.1 M, pH=7.0) was prepared
17
from NaH2PO4 and Na2HPO4. Twice deionized water was used throughout the
18
experiments.
19
2.2. Apparatus and measurements
20
Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai
21
G2 T12 transmission electron microscope (TEM, USA) operating at 120 kV. All
22
Fourier transform infrared (FTIR) spectroscopic measurements were performed on an
23
Agilent
24
High-performance liquid chromatography (HPLC) analysis was performed using a
25
Agilent 1200 system (Agilent, USA). The injection volume was 20 μL. Separations
26
were carried out on a C18 column (5 μm particle size, 250 mm×4.6 mm). The mobile
27
phase was a mixture of KH2PO4 solution (0.02 M)-methanol (2:98, v/v), UV detector
28
was set at 275 nm and the flow rate was 0.8 mL min−1.
29
640
Fourier
transform
infrared
spectrometer
(Agilent,
USA).
All electrochemical experiments were performed on a CHI 660D electrochemical 4
1
workstation (CHI Instruments Co., Shanghai, China) with a conventional three
2
electrode system comprising platinum wire as auxiliary electrode, a saturated calomel
3
electrode (SCE) as reference electrode and the modified or unmodified glass carbon
4
electrode (3 mm diameter, GCE) as working electrode.
5
2.3. Preparation of PVP-capped PdNPs
6
Preparation of PVP-capped PdNPs was similar to the process reported by Corma
7
(Pérez et al., 2012). A 100 mL two-neck round bottom flask, equipped with a
8
condenser and a magnetic stirrer, was charged with a solution of PVP solution (0.142
9
g, 1.28 mmol) and citric acid (0.24 g, 1.25 mmol) in water (32.0 mL), and heated in
10
an oil bath at 90 oC under stirring. K2[PdCl4] (0.0816 g, 0.25 mmol) in water (12.0
11
mL) was added slowly to this solution. The mixture was stirred at 90 oC for 26 h to
12
obtain PVP-capped PdNPs.
13
2.4. Preparation of MIP-coated PdNPs
14
1 mL of the above PVP-capped PdNPs was dispersed in 1.7 mL ethanol. Then 0.3
15
mL ammonia and 300 μL 0.1 M NE were added into the above solution, respectively.
16
The resulting mixture was kept stirring for 2 hours at room temperature. Then 30 μL
17
of solution 1 (Table S1) was added and kept stirring for 2 hours at room temperature.
18
Finally 30 μL of solution 2 (Table S1) was added and kept stirring for 12 hours to
19
obtain MIP-coated PdNPs (Solution 1 and 2 were kept stirring for 2 hours before
20
being added into the mixture, respectively). The final MIP-coated PdNPs were
21
obtained by centrifuging, and washed thoroughly with ethanol to remove the excess
22
reactants. The non-imprinted polymer coated PdNPs (NIP-coated PdNPs) were
23
prepared by the same procedure, only without using the template molecule in the
24
polymerization process.
25
For comparison purposes, the MIP without PdNPs was also prepared by the same
26
procedure, only without using PVP-capped PdNPs in the polymerization process. The
27
NIP without PdNPs was prepared by the same procedure, only without using
28
PVP-capped PdNPs and template molecules in the polymerization process.
29 5
1
2.5. Electrochemical measurements
2
The GCE was polished with 0.05 mm alumina slurry, followed by rinsing with
3
twice deionized water, and then treated by ultrasonication in nitric acid (1:1, v/v), 1 M
4
NaOH, acetone, and twice-distilled water. MIP-coated PdNPs (or NIP-coated PdNPs)
5
were dispersed in 1 mL N,N-dimethyl formamide (DMF). The above suspensions (2.5
6
μL) were dropped on the clean GCE and dried at room temperature over night.
7
Removal of the template molecule was achieved by differential pulse voltammetry
8
(DPV) sweeping the modified electrodes. The DPV was carried out by potential
9
scanning repeatedly between -0.2 and 0.7V till there was no signal of NE. After
10
extracting the template, the electrodes were rinsed with twice deionized water and
11
then submitted to binding and selective recognition experiments. The modified
12
electrodes were incubated in 10 mL of NE solutions with different concentrations for
13
8 min, rinsed with twice deionized water and measured by DPV in NE-free PBS (pH
14
7.0, 0.1 M). The pulse amplitude, pulse period, and pulse width of DPV were 50 mV,
15
0.2 s, and 0.05 s, respectively.
16
2.6. Sample preparation
17
A NE injection was used without any pretreatment. The labeled concentration of a
18
NE injection was 2 mg mL-1 (calculated as 5.9×10-3 M). Forty microliter of initial NE
19
injection was added into 2.36 mL twice deionized water, which NE concentration of
20
the mixture solution could be calculated as 1.0×10-4 M. Fifty microliter of the mixture
21
solution was added into 5 mL PBS as the tested solution, which NE concentration of
22
the tested mixture solution could be calculated as 1.0×10-6 M. Then, the NE current of
23
the tested solution was measured by DPV for recovery testing.
24
Six human urine samples were provided by six healthy adult volunteers. Prior to
25
analysis, the urine samples were centrifuged for 10 min at 8000 rpm to remove
26
precipitated proteins and other particulate matters. Then 12 mL of 24-h urine samples
27
were evaporated to 4 mL urine samples under nitrogen stream by a organomation. NE
28
in urine samples was detected by the proposed method of MIP-coated PdNPs sensor.
29
In addition, recovery tests were conducted via the standard addition method. 6
1 2
3. Results and discussion
3
3.1. Preparation and characterization of MIP-coated PdNPs
4
Fig. 1 presented the illustration of the preparation procedure for MIP-coated
5
PdNPs and detection process for its electrochemical sensor. Details of the preparation
6
could be found in the experimental section. First, PdNPs were prepared via reduction
7
of K2[PdCl4] by citric acid in the presence of PVP, which served as a stabilizer for the
8
formation of PdNPs (Pérez et al., 2012; Xiong et al., 2007). Then a novel composite
9
of MIP-coated PdNPs was synthesized by sol-gel method using PVP-capped PdNPs
10
as supporter, NE as template, phenyl trimethoxysilane as functional monomer and
11
tetramethoxysilane as crosslinker. MIP-coated PdNPs was characterized by Fourier
12
transform infrared spectroscopy and Transmission electron microscopy.
13
As shown in Fig. 2A, PVP, PVP-capped PdNPs and MIP-coated PdNPs were
14
characterized by FTIR spectroscopy. As shown in Fig. 2A, PVP showed the typical
15
peak of 1652 cm-1 (curve a), which was attributed to the stretching vibration of C=O.
16
In addition, the peaks at 1431 cm-1 and 1288 cm-1 were attributed to the stretching
17
vibration peaks of C=C and C-N for PVP (Yang et al., 2009), respectively.
18
PVP-capped PdNPs (curve b) showed the similar absorption peaks. Compared with
19
PVP-capped PdNPs, MIP-coated PdNPs showed two new intensive peaks at 1098 and
20
478 cm-1 (curve c), which can be ascribed to the asymmetric vibration and bending
21
vibration of Si-O-Si respectively, while the peak at 763 cm-1 was related to Si-O-Si
22
symmetric vibrations (Zhu et al., 2012). In addition, the peak at 960 cm-1 for
23
MIP-caoted PdNPs was attributed to the stretching vibration of Si-O (Thiam et al.,
24
2013). According to the above results, the SiO2-based MIP film was successfully
25
imprinted onto the surface of the PVP-capped PdNPs. As shown in Fig. 2B, the
26
diameters of the PVP-capped PdNPs, MIP-coated PdNPs were investigated by TEM.
27
The diameter of PVP-capped PdNPs was about 6-9 nm (Xiong et al., 2007). After
28
being coated with the MIP film, its diameter increased to about 117-137 nm (Fig. 2C),
7
1
indicating that the SiO2-MIP was successfully introduced onto the surface of
2
PVP-capped PdNPs.
3
3.2. Template removal of MIP-coated PdNPs modified GCE using DPV
4
Before the rebinding adsorption of MIP-coated PdNPs, the template molecule
5
should be removed. In the traditional methods, organic reagents or buffer solution as
6
elution were used to extract the template in the imprinted polymers (Liang et al.,
7
2011). However, they are time-consuming and the template cannot be removed
8
completely (Gao et al., 2007). In this work, DPV was carried out to extract NE
9
molecules from the imprinted polymers, thus NE could be eluted rapidly and
10
thoroughly. The typical differential pulse voltammogram of MIP-coated PdNPs
11
modified GCE was recorded in Fig. S1A. The observed DPV peak for NE
12
corresponds to two-electron oxidation of NE to norepinephrine quinone (Kalimuthu
13
and Abraham, 2011). Fig. S1A showed that the peak current decreased sharply with
14
increasing number of scans, and then it gradually tended to be a steady state. It can be
15
seen that there was no electrochemical response observed after 29 scanning cycles,
16
and the disappearance of DPV signal confirmed that NE molecules had been removed
17
entirely from the MIP-coated PdNPs film matrix.
18
3.3. Electrochemical behavior of MIP-coated PdNPs and NIP-coated PdNPs
19
modified GCE
20
Cyclic voltammetry was carried out in 5 mM of K3 [Fe(CN)6] in 0.1 M KCl at bare
21
GCE, PVP-capped PdNPs, MIP-coated PdNPs after elution, NIP-coated PdNPs, MIP
22
and NIP without PdNPs modified GCE (Fig. 3A). The cyclic voltammogram at the
23
bare GCE showed a reversible redox reaction with a peak potential difference of 94
24
mV and a peak current ratio of about 1:1 (curve b). When the electrode was modified
25
with PVP-capped PdNPs, the peak currents of redox peaks decreased (curve c). This
26
might be attributed to blocking electron transfer of PVP. When the electrodes were
27
modified with NIP-coated PdNPs (curve d) and NIP without PdNPs (curve f) which
28
had less cavities, obvious decrease for peak currents of redox peaks were observed in
29
comparison to PVP-capped PdNPs modified GCE. When the electrode was modified 8
1
with MIP-coated PdNPs, the peak currents of redox peaks (curve a) increased
2
obviously compared with PVP-capped PdNPs modified GCE and bare GCE. These
3
results suggested that cavities produced in the imprinted membranes after the removal
4
of template NE molecules could enhance the diffusion of [Fe(CN)6]3-/4- through the
5
imprinted membranes and promote the redox reaction of [Fe(CN)6]3-/4- on the
6
electrode surface. In particular, when the electrode was modified with MIP without
7
PdNPs modified GCE, the peak currents of redox peaks (curve e) decreased
8
significantly compared with MIP-coated PdNPs modified GCE. This can be explained
9
by the fact that PdNPs with good electrochemical catalytic property in MIP promoted
10
the redox reaction of [Fe(CN)6]3-/4-.
11
The electrochemical effective surface area for MIP-coated PdNPs, NIP-coated
12
PdNPs, MIP and NIP without PdNPs modified GCE and bare GCE can be calculated
13
by the slope of the plot of Q vs. t1/2, which was obtained by chronocoulometry using
14
0.5 mM K3[Fe(CN)6] as model complex based on:
15
Q( t ) =
16
2nFACD1 / 2 t 1/ 2 + Q d l + Q ads π1/2
(1)
17
given by Anson (1), where n is the number of transfer electron (n of K3[Fe(CN)6] is 1),
18
A is the surface area of the working electrode, C is the concentration of substrate, D is
19
the diffusion coefficient (D of K3[Fe(CN)6] is 7.6×10−6 cm2 s−1), Qdl is double layer
20
charge which could be eliminated by background subtraction, Qads is Faradic charge.
21
As shown in Fig. 3B and C, the slope of the linear relationship between Q and t1/2 for
22
MIP-coated PdNPs, MIP without PdNPs, NIP-coated PdNPs, NIP without PdNPs
23
modified GCE and bare GCE can be obtained to be 1.02×10-5, 5.73×10-6, 4.44×10-6,
24
2.18×10-6 and 4.80×10-6, respectively. Thus, A can be calculated as 0.068 cm2, 0.038
25
cm2, 0.030 cm2, 0.015 cm2 and 0.032 cm2, correspondingly. The electrochemical
26
effective surface area of MIP without PdNPs modified GCE was smaller than that of
27
MIP-coated PdNPs, demonstrating the function of PdNPs can increase surface area of
28
MIP-coated PdNPs modified GCE. In addition, the electrochemical effective surface
29
area of the NIP-coated PdNPs modified GCE was smaller than that of bare GCE, 9
1
indicating the lack of recognition sites for NIP-coated PdNPs. The results
2
demonstrated that the largest electrochemical effective surface area was obtained after
3
modification of GCE with MIP-coated PdNPs, which could enhance the total
4
adsorption capacity of NE. The largest electrochemical effective surface area for
5
MIP-coated PdNPs modified GCE would lead to the increase of peak current for NE.
6
After incubating the modified electrodes into 5 mL of 5.0 µM NE solution,
7
MIP-coated PdNPs, MIP without PdNPs and NIP-coated PdNPs modified GCE in
8
NE-free PBS which had adsorbed NE were measured by DPV. Fig. 3D showed a
9
characteristic oxidation peak of NE at around 0.214 V at the MIP-coated PdNPs
10
modified GCE (curve a). However, NIP-coated PdNPs modified GCE nearly has no
11
peak current response. The results were ascribed to great quantities of recognition
12
sites for NE in the MIP-coated PdNPs, while NIP-coated PdNPs had no recognition
13
sites for binding NE. In addition, DPV current response to NE of MIP without PdNPs
14
modified GCE (curve b) was lower than MIP-coated PdNPs modified GCE (curve a).
15
The higher response of MIP-coated PdNPs modified GCE can be attributed to PdNPs
16
of good electrocatalytic activity and large surface area. Furthermore, we investigated
17
the cyclic voltammetry response for 50.0 µM NE of MIP-coated PdNPs, MIP without
18
PdNPs and NIP-coated PdNPs modified GCE in Fig. 3E. The cyclic voltammetry
19
response of MIP-coated PdNPs modified GCE was significantly higher than that of
20
MIP without PdNPs and NIP-coated PdNPs.
21
As shown in Fig. S1B, the response time of MIP-coated PdNPs modified GCE for
22
50.0 μM NE was investigated by varying the adsorption time from 0.5 to 12 min. An
23
adsorption equilibrium of 71.0% was achieved within a period of 4 min and the
24
adsorption equilibrium reached within 8 min. The result revealed rapid response
25
equilibrium of NE molecules to MIP-coated PdNPs.
26
3.4. Selectivity of the sensor
27
The selectivity experiments were investigated by using dopamine (DA),
28
epinephrine (EP), uric acid (UA), ascorbic acid (AA), acetaminophen (AC), folic acid
29
(FA) and L-tryptophan (L-TRP) as the analogs. Among these analogs, DA and EP 10
1
were tested due to their electrochemical activity and structural similarity. AA and UA
2
were measured because they largely coexisted with NE in urine samples (Goyal et al.,
3
2011). L-TRP was the essential amino acid that played an integral role in the
4
synthesis of serotonin (5-HT) and AC was known to increase 5-HT levels in brain. In
5
addition, 5-HT was known to play a role in NE release in the brain (Beitollahi and
6
Mohammadi, 2013). FA worked primarily in the brain and nervous system and was
7
necessary for the production of NE in the nervous system (Beitollahi et al., 2012).
8
Therefore, DA, EP, AA, UA, L-TRP, AC and FA were selected as the analogs.
9
Fig. 4A showed that the DPV peak current responses of MIP-coated PdNPs sensor
10
(modified GCE) toward NE were higher than other analogs, indicating a better
11
adsorption and binding capacity for the template molecules. Since the structures of
12
DA, EP, L-TRP, AC, FA, AA and UA are different with NE (Fig. 4B), the cavities
13
formed in the imprinting process could not bind them tightly, which resulted in lower
14
response. The specificity was probably due to the shape of the cavities in the
15
imprinted polymers just fitting for the unique molecular structure of NE, so they
16
cannot bind other analogs tightly. In particular, the chemical structures of FA, UA and
17
AA are very different from NE (Mao et al., 2011), so the MIP-coated PdNPs sensor
18
had good selectivity for NE compared with these three analogs. The above results
19
suggested that MIP-coated PdNPs showed potential applications as molecular
20
recognition element owing to its selectivity against the analogs.
21
3.5. Interference studies
22
In order to apply the proposed method in urine samples, it is vital to investigate
23
the effect of potential interference substances on NE determination, which was used
24
to evaluate the selectivity of MIP-coated PdNPs sensor for NE. Potential interference
25
substances involved some ions, urea, uric acid, ascorbic acid, amino acids and sugars.
26
The DPV determination of 10.0 μM NE was tested in presence of spiked known
27
amounts of interfering substances. The tolerance limit was defined as the amount and
28
fold of the interfering substances causing a change of ±5% in the peak current
29
intensity reading. The tolerable limits of interfering substances were given in Table S2. 11
1
The results showed that 1000-fold of K+, Na+, Ca2+, Mg2+, Al3+, SO42−, and Cl-- did
2
not interfere with NE determination. 500-fold of urea, 20-fold of uric acid, 50-fold of
3
ascorbic acid, 1000-fold of serine, alanine, histidine, threonine, 500-fold of glucose,
4
sucrose, lactose and maltose did not interfere with NE determination.
5
3.6. Linearity, reproducibility and stability of MIP-coated PdNPs sensor
6
DPV was used to investigate the linearity and detection limit of MIP-coated
7
PdNPs sensor for NE. As shown in Fig. 5A and B, the current responses increased
8
with successive addition of different NE concentrations in PBS. Fig. 5B illustrated the
9
corresponding plot showing a linear relation between current response and NE
10
concentration in the range of 0.5 to 80.0 μM. The regression equation was: I(μA)
11
=0.0235C(μM)-0.0160 (R2=0.9973). The detection limit was 0.1 μM (S/N=3, defined
12
as the concentration of NE corresponding to the three times the standard deviation for
13
11 replicate detections of the blank solution). It was worth to compare the
14
performance of the sensor based on MIP-coated PdNPs composite with other
15
electrochemical method reported previously. Comparison with other electrochemical
16
methods for determination of NE was summarized in Table 1. The comparison results
17
demonstrated low detection limit of the proposed sensor based on MIP-coated PdNPs.
18
In addition, in order to compare the response of MIP-coated PdNPs and NIP-coated
19
PdNPs sensors, we also investigated the linearity of NIP-coated PdNPs sensor for NE
20
(Fig. 5B). The regression equation was: I(μA) =0.00037C(μM)+0.00036 (R2=0.9639).
21
KMIP and KNIP were the linear slopes of MIP-coated PdNPs and NIP-coated PdNPs
22
sensor. So KMIP and KNIP were 0.0235 and 0.00037, respectively. The ratio of the KMIP
23
and KNIP was defined as imprinting factor (IF), which was used to evaluate the
24
specific recognition of the materials (Wang et al., 2009; Zhou et al., 2014). The IF (K
25
MIP/KNIP)
26
recognition sites for NE in the MIP-coated PdNPs, while NIP-coated PdNPs had no
27
recognition sites for binding NE. The higher current response efficiency of
28
MIP-coated PdNPs sensor suggested its specific imprint binding affinity for NE.
was 63.5, which indicated that MIP-coated PdNPs had great quantities of
12
1
So as to confirm the advantages of MIP-coated PdNPs sensor, we investigated the
2
linearity of MIP and NIP without PdNPs sensors (Fig. 5B and Fig. S2). The regression
3
equations were: I(μA)=0.00808C(μM)+0.0159 (R2=0.9740) (MIP without PdNPs
4
sensor) and I(μA)=0.000130C(μM)+0.000679 (R2=0.9186) (NIP without PdNPs
5
sensor), respectively. KMIP (without Pd) and KNIP (without Pd) were the linear slopes of MIP
6
without PdNPs sensor (Fig. 5B) and NIP without PdNPs sensor (Fig. S2). So KMIP
7
(without Pd)
8
(KMIP/KMIP (without Pd)) and IF (KNIP/KNIP (without Pd)) were 2.91 and 2.85, respectively. The
9
IF result showed that MIP with PdNPs sensor exhibited higher current response,
10
which could be attributed to the synergistic effect of good electrocatalytic property
11
and large surface area for PdNPs.
and KNIP
(without Pd)
were 0.00808 and 0.000130, respectively. The IF
12
The repeatability of MIP-coated PdNPs sensor was also evaluated by using the
13
same electrode for 8 repeated analyses of 50.0 μM NE, with a relative standard
14
deviation (RSD) of 3.56%. Good reproducibility was observed with a RSD of 3.68%
15
for 8 parallel detections. Moreover, the sensor retained a response of 95.6% of the
16
initial current after storage for 10 days at room temperature.
17
3.7. Analytical application
18
In order to evaluate the practical applications of the imprinted sensor,
19
norepinephrine injection and human urine samples were analyzed. The parallel
20
experiment was performed three times and recovery tests were conducted via the
21
standard addition method. The results were presented in Table S3 and Table S4.
22
According to the results, NE concentration found in 24 h-urine samples were 63.5 μg,
23
75.2 μg, 65.2 μg, 70.5 μg, 78.0 μg and 62.9 μg for the six people respectively, which
24
were in good agreement with NE normal values found in 24 h-urine (15-80 µg)
25
(Moyer et al., 1979; Ferreira et al., 2009). In addition, the obtained recoveries ranged
26
from 96.4% to 103.3%. Thus the proposed DPV method based on MIP-coated PdNPs
27
sensor can be used to detect NE in actual samples with good results.
28 29 13
1
3.8 Validation of the assay with HPLC
2
The MIP-coated PdNPs sensor for NE was validated by analysing the same
3
samples by a high performance liquid chromatography (HPLC) method (Anderson et
4
al., 1981; Yamazaki et al., 1995). Urine samples should be performed with a
5
pretreatment procedure prior to HPLC determination (Wu et al., 2000; Yamazaki et al.,
6
1995). The pretreatment procedure with activated aluminium oxide was added in
7
Supplementary Information. The results of NE determination with electrochemical
8
and HPLC methods were added in Table S3 and Table S4 of Supplementary
9
Information. It was worth emphasizing that the recoveries of HPLC reference method
10
for detecting NE in urine samples were all above 100% (Tables S4). There may be
11
some systematic error affecting the results of HPLC method. The systematic error
12
may be attributed to some interferences in urine samples, such as DA, EP, which may
13
increase the NE signal of HPLC and lead to the recoveries of above 100%. The
14
comparison of the proposed electrochemical method (EC) with HPLC for detecting
15
NE was shown in Fig. S3. A good correlation between HPLC (X) and EC (Y) was
16
obtained with the linear regression equation of Y=0.9875X+0.0108 (R=0.9991) (Cao
17
et al., 2013). These results suggested that the results of the proposed electrochemical
18
method for NE determination were agreeable with HPLC method, which further
19
confirmed that MIP-coated PdNPs sensor was suitable for detecting NE in actual
20
samples.
21 22
4. Conclusions
23
A novel composite of MIP-coated PdNPs was synthesized by sol-gel method
24
using NE as template, phenyl trimethoxysilane as functional monomer and
25
tetramethoxysilane as crosslinker. The combination of PdNPs and silica-based MIP
26
endowed the composite with good electrochemical catalytic property, large surface
27
area and template selectivity. The results demonstrated that MIP-coated PdNPs sensor
28
not only possessed a short response time, but also high binding capacity for NE,
29
which enabled the imprinted sensor with higher current response than that of 14
1
non-imprinted material and MIP without PdNPs. In addition, the MIP-coated PdNPs
2
sensor exhibited good selectivity for NE in comparison to FA, UA and AA. The
3
comparison results with other papers demonstrated low detection limit of MIP-coated
4
PdNPs sensor. The results of the proposed electrochemical method for NE
5
determination was agreeable with HPLC method, which confirmed that MIP-coated
6
PdNPs sensor was suitable for detecting NE in actual samples. This work may open a
7
new possibility for development of PdNPs-based imprinted polymers materials.
8 9
Acknowledgments
10
This work was supported by the National Natural Science Foundation of China
11
(No. 21177049), the Zhejiang Provincial Natural Science Foundation of China under
12
Grant No. LQ14B050002, and the Program for Science and Technology of Jiaxing
13
(No. 2013AY11017).
14 15
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14
Captions
15
Fig. 1. The illustration of the preparation procedure for MIP-coated PdNPs and
16
detection process for its electrochemical sensor.
17
Fig. 2. (A) The FTIR spectra of PVP(a), PVP-capped PdNPs (b), MIP-coated
18
PdNPs(c); (B) TEM images of PVP-capped PdNPs and (C) MIP-coated PdNPs.
19
Fig. 3. (A) Cyclic voltammograms of MIP-coated PdNPs modified GCE after elution
20
(a), bare GCE (b), PVP-capped PdNPs (c), NIP-coated PdNPs (d), MIP (e) and NIP
21
without PdNPs (f) modified GCE in a mix solution of 5 mM K3[Fe(CN))6] and 0.1 M
22
KCl detection conditions; (B) Plot of Q-t curves for MIPs-coated PdNPs (a), MIP
23
without PdNPs (b), NIPs-coated PdNPs (d) and NIP without PdNPs (e) modified GCE
24
and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (C) Plot of Q-t1/2 curves for MIPs-coated
25
PdNPs (a), MIP without PdNPs (b), NIPs-coated PdNPs (c) and NIP without PdNPs
26
(e) modified GCE and bare GCE (d) in 0.5 mM K3[Fe(CN)6]; (D) Differential pulse
27
voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated
28
PdNPs (c) modified GCE in PBS after incubating in 5.0 µM NE; (E) Cyclic
18
1
voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated
2
PdNPs (c) modified GCE in PBS after incubating in 50 µM NE.
3
Fig. 4. (A) The current responses of selective recognition for MIP-coated PdNPs
4
sensor of NE, DA, EP, L-TRP, AC, FA, AA and UA; (B) The chemical structures of
5
NE and other analogs.
6
Fig. 5. (A) DPV current response curves at MIP-coated PdNPs sensor with addition of
7
increasing concentration of NE (µM): 0 (a), 0.5 (b), 5.0 (c), 10.0 (d), 20.0 (e), 50.0 (f),
8
65.0 (g), 80.0 (h); (B) Calibration curves of NE detection obtained by MIP-coated
9
PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) sensors (n=5).
10
Table 1 Comparison with other electrochemical methods for determination of
11 12
NE. Detection methods
Linear range
Poly(cresol
Detecti
References
on limit
(μM)
(μM)
3– 30
0.2
(Chen et al. 2005)
0.3–
0.09
(Salmanpour et al. 2012)
8.7
(Goyal et al. 2011)
red)/GCE MWCNTs/CILE a
450.0 AuNPs/ITO b
0.1 –25
‐2
×10 Poly‐CCAc /GCE
0.63–62
0.1
(Liu et al. 2008)
0–10
0.131
(Huang et al. 2010)
4.0–110
0.5
(Mazloum‐Ardakani et al.
.5 PAAd‐MWCNTs/S e
PCE BHT/CPE f 0.0 Hematoxylin/GCE
2010) 0.5–274
0.14
.20 CACEg/GCE
(Nasirizadeh and Zare 2009)
0.55–23
0.28
(Zhang et al. 2009)
3.23 –
1.07
(Li et al. 2012)
0 WO3‐TiO2/ITO 388 19
MIP‐coated pdNPs /GCE 1
a
2
b
3
c
4
d
5
e
6
f
7 8
0.5 80.0
ITO : indium tin oxide electrode.
Poly‐CCA: poly‐Calconcarboxylic acid. PAA: polyacrylic acid‐coated.
SPCE: screen printed carbon electrode.
BHT/CPE:2,2‐[1,2‐buthanediylbis(nitriloethylidyne)]‐bis‐hydroquinone and TiO2 modified
carbon paste electrode. g
CACE: Calix[4]arene crown‐4 ether.
Highlights
10
z A novel composite of MIP‐coated PdNPs for electrochemical sensing of
11
norepinephrine was described. z The MIP‐coated PdNPs sensor possessed high current response for
13 14
norepinephrine. z Comparison results of MIP‐coated PdNPs and MIP without PdNPs
15 16
sensors showed the function of PdNPs. z The MIP‐coated PdNPs sensor could recognize norepinephrine from its
17 18
analogs. z Low detection limit of MIP‐coated PdNPs sensor for norepinephrine was
19 20
This work
CILE: carbon ionic liquid electrode.
9
12
0.1
–
obtained.
21
22
20
Fig.. 1. The illlustration of o the prepparation proocedure forr MIP-coatted PdNPs and deteection proceess for its ellectrochemiical sensor.
Fig.. 2. (A) The T FTIR spectra of PVP(a), PVP-capped P d PdNPs (b b), MIP-cooated PdN NPs(c); (B) TEM imagees of PVP-ccapped PdN NPs and (C) MIP-coatedd PdNPs.
Fig.. 3. (A) Cycclic voltamm mograms off MIP-coateed PdNPs modified m GC CE after eluution (a), bare GCE (b), ( PVP-caapped PdNP Ps (c), NIP--coated PdN NPs (d), MIP P (e) and NIP (f)
without PdNPs modified GCE in a mix solution of 5 mM K3[Fe(CN))6] and 0.1 M KCl detection conditions; (B) Plot of Q-t curves for MIPs-coated PdNPs (a), MIP without PdNPs (b), NIPs-coated PdNPs (d) and NIP without PdNPs (e) modified GCE and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (C) Plot of Q-t1/2 curves for MIP-coated PdNPs (a), MIP without PdNPs (b), NIP-coated PdNPs (d) and NIP without PdNPs (e) modified GCE and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (D) Differential pulse voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) modified GCE in PBS after incubating in 5.0 µM NE; (E) Cyclic voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) modified GCE in PBS after incubating in 50.0 µM NE.
B
OH NH2
HO
NH2
HO
HO
OH H N
HO
HO
HO
NE
DA
EP
OH
NH
O
N
O O
N
OH NH H2
N H
N
H2N
O
N
CH2NH N
C
OH H
OH H HO HO
O FA
AC O
O
O OH
AA
HN O
OH
CH2CH C 2 C OH
CH3 L-TRP
C NHCH
H N O
N H
N H
UA
Fig.. 4. (A) Thhe current responses of o selectivee recognitioon for MIP P-coated PddNPs senssor of NE, DA, EP, L--TRP, AC, FA, F AA andd UA; (B) The T chemiccal structurees of NE and other analogs. a
Fig.. 5. (A) DPV V current reesponse curves at MIP--coated PdN NPs sensor with w additioon of incrreasing conccentration of o NE (µM): 0 (a), 0.5 (b), ( 5.0 (c), 10.0 (d), 200.0 (e), 50.00 (f), 65.00 (g), 80.0 (h); (B) Caalibration curves c of NE N detection n obtained by MIP-cooated PdN NPs (a), MIP P without PdNPs (b) annd NIP-coatted PdNPs (c) ( sensors (n=5).
PII: DOI: Reference:
S0956-5663(14)00798-2 http://dx.doi.org/10.1016/j.bios.2014.10.011 BIOS7193
To appear in: Biosensors and Bioelectronic Received date: 1 July 2014 Revised date: 1 October 2014 Accepted date: 5 October 2014 Cite this article as: Jianrong Chen, Hong Huang, Yanbo Zeng, Huan Tang and Lei Li, A novel composite of molecularly imprinted polymer-coated PdNPs for electrochemical sensing norepinephrine, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.10.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
A novel composite of molecularly imprinted polymer-coated PdNPs
2
for electrochemical sensing norepinephrine
3
Jianrong Chena, Hong Huanga, Yanbo Zengb, *, Huan Tanga, Lei Li a, b,∗
4 5 6 7
a
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004,
P.R. China b
College of Biological, Chemical Sciences and Engineering, Jiaxing University,
Jiaxing 314001, P.R. China
8 9
Abstract: A novel composite of molecularly imprinted polymer-coated palladium
10
nanoparticles (MIP-coated PdNPs) was synthesized by sol-gel method using
11
norepinephrine as template, phenyl trimethoxysilane as functional monomer and
12
tetramethoxysilane as crosslinker. The combination of PdNPs and silica-based MIP
13
endowed the composite with good electrochemical catalytic property, large surface
14
area and template selectivity. MIP-coated PdNPs were characterized by Fourier
15
transform infrared spectroscopy and Transmission electron microscopy. Then
16
MIP-coated PdNPs composite was used as a recognition element in the construction
17
of an electrochemical sensor for norepinephrine. The properties of MIP-coated PdNPs
18
sensor such as special binding, adsorption dynamics and selective recognition ability
19
were evaluated by differential pulse voltammetry. The results demonstrated that
20
MIP-coated PdNPs sensor not only possessed a short response time, but also high
21
binding capacity for norepinephrine, which enabled the imprinted sensor with higher
22
current response than that of non-imprinted material and MIP without PdNPs. In
23
addition, the MIP-coated PdNPs sensor exhibited selectivity for norepinephrine in
24
comparison to other analogs. The MIP-coated PdNPs sensor had a wide linear range
25
over norepinephrine concentration from 0.5 to 80.0 μM with a detection limit of 0.1
26
μM. The MIP-coated PdNPs sensor was proved to be a suitable sensing tool for the ∗
Corresponding author. Tel: +86 573 83646203. E-mail: [email protected] (Y. Zeng),
[email protected] (L. Li). 1
1
fast, sensitive and selective determination of norepinephrine in injection and urine
2
samples.
3
Keywords: Palladium nanoparticles; molecularly imprinted polymer; electrochemical
4
sensor; norepinephrine.
5 6
1. Introduction
7
Norepinephrine (NE) is an important catecholamine neurotransmitter and is
8
secreted by the adrenal medulla (Nikolajsen and Hansen, 2001). It is released as a
9
metabotropic neurotransmitter from nerve endings in the sympathetic nervous system
10
and some areas of the cerebral cortex. It is used to treat myocardial infarction
11
hypertension, bronchial asthma and organic heart disease. Extremely abnormal
12
concentration levels of NE may lead to the occurrence of many diseases, such as
13
ganglion neuronal, ganglia neuroblastoma, paraganglioma and Parkinson’ disease
14
(Mazloum-Ardakani et al., 2011). In these cases, it is very necessary to develop fast,
15
accurate and sensitive methods for the quantitative determination of NE in biological
16
fluids including urine and blood samples. Many methods, such as spectrophotometry
17
(Sorouraddin et al., 1998), gas chromatography (Kuhlenbeck et al., 2000), capillary
18
electrophoresis (Liu et al., 2008) and liquid chromatography (Ji et al., 2010), have
19
been employed for the determination of NE. However, these methods suffer from
20
some disadvantages of low detection sensitivity, time-consuming procedure,
21
complicated process or expensive analysis settings. Therefore, electrochemical
22
methods (Beitollahi and Mohammadi, 2013; Li et al., 2012; Mazloum-Ardakani et al.,
23
2011; Mazloum-Ardakani and Khoshroo, 2014; Rosy et al., 2014; Salmanpour et al.,
24
2012) have attracted much attention owing to their fast response, relatively high
25
sensitivity and ability of miniaturization.
26
Molecular imprinting is used to create recognition sites that are chemically and
27
sterically complementary to the target molecules in a synthetic polymer (Huang and
28
Shen, 2014; Liu et al., 2013). Molecularly imprinted polymer (MIP) exhibits an
29
affinity for the template molecule over other structurally related compounds. However, 2
1
MIP prepared by the conventional technique has some disadvantages, such as
2
incomplete removal of template, low-affinity binding and slow mass transfer. Thus
3
surface imprinting technique has received extensive attention (Bi and Liu, 2014;
4
Lofgreen and Ozin, 2014), because it can provide the advantages of favorable
5
selectivity, fast association/dissociation kinetics, some unique physicochemical
6
properties and desired application. Some researchers have reported surface MIP
7
composites based on Fe3O4 nanoparticles (Zhao et al., 2014), Au nanoparticles (Yu et
8
al., 2012), carbon nanotubes (Gao et al., 2010), graphene (Mao et al., 2011), CdTe
9
quantum dots (Huy et al., 2014; Wang et al., 2013; Xu et al., 2013; Zhang et al., 2012),
10
carbon dots (Mao et al., 2012), graphene quantum dots (Zhou et al., 2014). Many
11
routes have been explored to develop these surface MIP composites, such as free
12
radical polymerization (Mao et al., 2011), reversible addition fragmentation chain
13
transfer polymerization (Li et al., 2011) and sol-gel method (Zhang et al., 2012). The
14
widely used sol-gel method has gained great attention for preparing these surface MIP
15
composites, which can be explained by the fact that imprinted silica films show fast
16
kinetic binding for the template due to their rigid structures and nanosized thickness.
17
Palladium nanoparticles (PdNPs) with good electrocatalytic activity (Gao et al.,
18
2013; Li et al., 2014; Uberman et al., 2014) should be a good candidate as a supported
19
material for preparing surface MIP composite. To the best of our knowledge, surface
20
MIP composite based on PdNPs with sol-gel method has not been reported. In this
21
paper, PdNPs were prepared via reduction of K2[PdCl4] by citric acid in the presence
22
of poly (vinylpyrrolidone) (PVP), which was served as a stabilizer for the formation
23
of PdNPs. Then a novel composite of MIP-coated PdNPs was synthesized by sol-gel
24
method using phenyl trimethoxysilane as functional monomer and tetramethoxysilane
25
as crosslinker. MIP-coated PdNPs was characterized by Fourier transform infrared
26
spectroscopy and Transmission electron microscopy. The MIP-coated PdNPs
27
composite was used as a recognition element in the construction of an electrochemical
28
sensor for NE. The properties such as special binding, adsorption dynamics and
29
selective recognition ability were evaluated through differential pulse voltammetry. 3
1
The sensor based on MIP-coated PdNPs was applied to the determination of NE in
2
injection and urine samples.
3 4
2. Experimental section
5
2.1. Reagents and materials
6
K2[PdCl4], citric acid, ascorbic acid (AA), uric acid (UA), folic acid (FA),
7
acetaminophen (AC), L-tryptophan (L-TRP), dopamine (DA), tetramethoxysilane
8
(TEOS) and Poly (vinylpyrrolidone) with average molar masses of 10 kg mol-1
9
(PVP-10) were purchased from Aladdin Industrial Corporation (Shanghai, China).
10
Norepinephrine hydrochloride (NE) and epinephrine hydrochloride (EP) were
11
purchased from Sigma-Aldrich Co. (USA). Phenyl trimethoxysilane (PTMOS) was
12
obtained from TCI Co. Ltd. (Japan). Norepinephrine bitartrate injection was obtained
13
from Shanghai Harvest pharmaceutical Co., Ltd. (Shanghai, China). Human urine
14
samples of 24 h were provided by six healthy adult volunteers. Other chemicals were
15
of analytical grade and were purchased from Aladdin Industrial Corporation
16
(Shanghai, China). Phosphate buffer solution (PBS, 0.1 M, pH=7.0) was prepared
17
from NaH2PO4 and Na2HPO4. Twice deionized water was used throughout the
18
experiments.
19
2.2. Apparatus and measurements
20
Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai
21
G2 T12 transmission electron microscope (TEM, USA) operating at 120 kV. All
22
Fourier transform infrared (FTIR) spectroscopic measurements were performed on an
23
Agilent
24
High-performance liquid chromatography (HPLC) analysis was performed using a
25
Agilent 1200 system (Agilent, USA). The injection volume was 20 μL. Separations
26
were carried out on a C18 column (5 μm particle size, 250 mm×4.6 mm). The mobile
27
phase was a mixture of KH2PO4 solution (0.02 M)-methanol (2:98, v/v), UV detector
28
was set at 275 nm and the flow rate was 0.8 mL min−1.
29
640
Fourier
transform
infrared
spectrometer
(Agilent,
USA).
All electrochemical experiments were performed on a CHI 660D electrochemical 4
1
workstation (CHI Instruments Co., Shanghai, China) with a conventional three
2
electrode system comprising platinum wire as auxiliary electrode, a saturated calomel
3
electrode (SCE) as reference electrode and the modified or unmodified glass carbon
4
electrode (3 mm diameter, GCE) as working electrode.
5
2.3. Preparation of PVP-capped PdNPs
6
Preparation of PVP-capped PdNPs was similar to the process reported by Corma
7
(Pérez et al., 2012). A 100 mL two-neck round bottom flask, equipped with a
8
condenser and a magnetic stirrer, was charged with a solution of PVP solution (0.142
9
g, 1.28 mmol) and citric acid (0.24 g, 1.25 mmol) in water (32.0 mL), and heated in
10
an oil bath at 90 oC under stirring. K2[PdCl4] (0.0816 g, 0.25 mmol) in water (12.0
11
mL) was added slowly to this solution. The mixture was stirred at 90 oC for 26 h to
12
obtain PVP-capped PdNPs.
13
2.4. Preparation of MIP-coated PdNPs
14
1 mL of the above PVP-capped PdNPs was dispersed in 1.7 mL ethanol. Then 0.3
15
mL ammonia and 300 μL 0.1 M NE were added into the above solution, respectively.
16
The resulting mixture was kept stirring for 2 hours at room temperature. Then 30 μL
17
of solution 1 (Table S1) was added and kept stirring for 2 hours at room temperature.
18
Finally 30 μL of solution 2 (Table S1) was added and kept stirring for 12 hours to
19
obtain MIP-coated PdNPs (Solution 1 and 2 were kept stirring for 2 hours before
20
being added into the mixture, respectively). The final MIP-coated PdNPs were
21
obtained by centrifuging, and washed thoroughly with ethanol to remove the excess
22
reactants. The non-imprinted polymer coated PdNPs (NIP-coated PdNPs) were
23
prepared by the same procedure, only without using the template molecule in the
24
polymerization process.
25
For comparison purposes, the MIP without PdNPs was also prepared by the same
26
procedure, only without using PVP-capped PdNPs in the polymerization process. The
27
NIP without PdNPs was prepared by the same procedure, only without using
28
PVP-capped PdNPs and template molecules in the polymerization process.
29 5
1
2.5. Electrochemical measurements
2
The GCE was polished with 0.05 mm alumina slurry, followed by rinsing with
3
twice deionized water, and then treated by ultrasonication in nitric acid (1:1, v/v), 1 M
4
NaOH, acetone, and twice-distilled water. MIP-coated PdNPs (or NIP-coated PdNPs)
5
were dispersed in 1 mL N,N-dimethyl formamide (DMF). The above suspensions (2.5
6
μL) were dropped on the clean GCE and dried at room temperature over night.
7
Removal of the template molecule was achieved by differential pulse voltammetry
8
(DPV) sweeping the modified electrodes. The DPV was carried out by potential
9
scanning repeatedly between -0.2 and 0.7V till there was no signal of NE. After
10
extracting the template, the electrodes were rinsed with twice deionized water and
11
then submitted to binding and selective recognition experiments. The modified
12
electrodes were incubated in 10 mL of NE solutions with different concentrations for
13
8 min, rinsed with twice deionized water and measured by DPV in NE-free PBS (pH
14
7.0, 0.1 M). The pulse amplitude, pulse period, and pulse width of DPV were 50 mV,
15
0.2 s, and 0.05 s, respectively.
16
2.6. Sample preparation
17
A NE injection was used without any pretreatment. The labeled concentration of a
18
NE injection was 2 mg mL-1 (calculated as 5.9×10-3 M). Forty microliter of initial NE
19
injection was added into 2.36 mL twice deionized water, which NE concentration of
20
the mixture solution could be calculated as 1.0×10-4 M. Fifty microliter of the mixture
21
solution was added into 5 mL PBS as the tested solution, which NE concentration of
22
the tested mixture solution could be calculated as 1.0×10-6 M. Then, the NE current of
23
the tested solution was measured by DPV for recovery testing.
24
Six human urine samples were provided by six healthy adult volunteers. Prior to
25
analysis, the urine samples were centrifuged for 10 min at 8000 rpm to remove
26
precipitated proteins and other particulate matters. Then 12 mL of 24-h urine samples
27
were evaporated to 4 mL urine samples under nitrogen stream by a organomation. NE
28
in urine samples was detected by the proposed method of MIP-coated PdNPs sensor.
29
In addition, recovery tests were conducted via the standard addition method. 6
1 2
3. Results and discussion
3
3.1. Preparation and characterization of MIP-coated PdNPs
4
Fig. 1 presented the illustration of the preparation procedure for MIP-coated
5
PdNPs and detection process for its electrochemical sensor. Details of the preparation
6
could be found in the experimental section. First, PdNPs were prepared via reduction
7
of K2[PdCl4] by citric acid in the presence of PVP, which served as a stabilizer for the
8
formation of PdNPs (Pérez et al., 2012; Xiong et al., 2007). Then a novel composite
9
of MIP-coated PdNPs was synthesized by sol-gel method using PVP-capped PdNPs
10
as supporter, NE as template, phenyl trimethoxysilane as functional monomer and
11
tetramethoxysilane as crosslinker. MIP-coated PdNPs was characterized by Fourier
12
transform infrared spectroscopy and Transmission electron microscopy.
13
As shown in Fig. 2A, PVP, PVP-capped PdNPs and MIP-coated PdNPs were
14
characterized by FTIR spectroscopy. As shown in Fig. 2A, PVP showed the typical
15
peak of 1652 cm-1 (curve a), which was attributed to the stretching vibration of C=O.
16
In addition, the peaks at 1431 cm-1 and 1288 cm-1 were attributed to the stretching
17
vibration peaks of C=C and C-N for PVP (Yang et al., 2009), respectively.
18
PVP-capped PdNPs (curve b) showed the similar absorption peaks. Compared with
19
PVP-capped PdNPs, MIP-coated PdNPs showed two new intensive peaks at 1098 and
20
478 cm-1 (curve c), which can be ascribed to the asymmetric vibration and bending
21
vibration of Si-O-Si respectively, while the peak at 763 cm-1 was related to Si-O-Si
22
symmetric vibrations (Zhu et al., 2012). In addition, the peak at 960 cm-1 for
23
MIP-caoted PdNPs was attributed to the stretching vibration of Si-O (Thiam et al.,
24
2013). According to the above results, the SiO2-based MIP film was successfully
25
imprinted onto the surface of the PVP-capped PdNPs. As shown in Fig. 2B, the
26
diameters of the PVP-capped PdNPs, MIP-coated PdNPs were investigated by TEM.
27
The diameter of PVP-capped PdNPs was about 6-9 nm (Xiong et al., 2007). After
28
being coated with the MIP film, its diameter increased to about 117-137 nm (Fig. 2C),
7
1
indicating that the SiO2-MIP was successfully introduced onto the surface of
2
PVP-capped PdNPs.
3
3.2. Template removal of MIP-coated PdNPs modified GCE using DPV
4
Before the rebinding adsorption of MIP-coated PdNPs, the template molecule
5
should be removed. In the traditional methods, organic reagents or buffer solution as
6
elution were used to extract the template in the imprinted polymers (Liang et al.,
7
2011). However, they are time-consuming and the template cannot be removed
8
completely (Gao et al., 2007). In this work, DPV was carried out to extract NE
9
molecules from the imprinted polymers, thus NE could be eluted rapidly and
10
thoroughly. The typical differential pulse voltammogram of MIP-coated PdNPs
11
modified GCE was recorded in Fig. S1A. The observed DPV peak for NE
12
corresponds to two-electron oxidation of NE to norepinephrine quinone (Kalimuthu
13
and Abraham, 2011). Fig. S1A showed that the peak current decreased sharply with
14
increasing number of scans, and then it gradually tended to be a steady state. It can be
15
seen that there was no electrochemical response observed after 29 scanning cycles,
16
and the disappearance of DPV signal confirmed that NE molecules had been removed
17
entirely from the MIP-coated PdNPs film matrix.
18
3.3. Electrochemical behavior of MIP-coated PdNPs and NIP-coated PdNPs
19
modified GCE
20
Cyclic voltammetry was carried out in 5 mM of K3 [Fe(CN)6] in 0.1 M KCl at bare
21
GCE, PVP-capped PdNPs, MIP-coated PdNPs after elution, NIP-coated PdNPs, MIP
22
and NIP without PdNPs modified GCE (Fig. 3A). The cyclic voltammogram at the
23
bare GCE showed a reversible redox reaction with a peak potential difference of 94
24
mV and a peak current ratio of about 1:1 (curve b). When the electrode was modified
25
with PVP-capped PdNPs, the peak currents of redox peaks decreased (curve c). This
26
might be attributed to blocking electron transfer of PVP. When the electrodes were
27
modified with NIP-coated PdNPs (curve d) and NIP without PdNPs (curve f) which
28
had less cavities, obvious decrease for peak currents of redox peaks were observed in
29
comparison to PVP-capped PdNPs modified GCE. When the electrode was modified 8
1
with MIP-coated PdNPs, the peak currents of redox peaks (curve a) increased
2
obviously compared with PVP-capped PdNPs modified GCE and bare GCE. These
3
results suggested that cavities produced in the imprinted membranes after the removal
4
of template NE molecules could enhance the diffusion of [Fe(CN)6]3-/4- through the
5
imprinted membranes and promote the redox reaction of [Fe(CN)6]3-/4- on the
6
electrode surface. In particular, when the electrode was modified with MIP without
7
PdNPs modified GCE, the peak currents of redox peaks (curve e) decreased
8
significantly compared with MIP-coated PdNPs modified GCE. This can be explained
9
by the fact that PdNPs with good electrochemical catalytic property in MIP promoted
10
the redox reaction of [Fe(CN)6]3-/4-.
11
The electrochemical effective surface area for MIP-coated PdNPs, NIP-coated
12
PdNPs, MIP and NIP without PdNPs modified GCE and bare GCE can be calculated
13
by the slope of the plot of Q vs. t1/2, which was obtained by chronocoulometry using
14
0.5 mM K3[Fe(CN)6] as model complex based on:
15
Q( t ) =
16
2nFACD1 / 2 t 1/ 2 + Q d l + Q ads π1/2
(1)
17
given by Anson (1), where n is the number of transfer electron (n of K3[Fe(CN)6] is 1),
18
A is the surface area of the working electrode, C is the concentration of substrate, D is
19
the diffusion coefficient (D of K3[Fe(CN)6] is 7.6×10−6 cm2 s−1), Qdl is double layer
20
charge which could be eliminated by background subtraction, Qads is Faradic charge.
21
As shown in Fig. 3B and C, the slope of the linear relationship between Q and t1/2 for
22
MIP-coated PdNPs, MIP without PdNPs, NIP-coated PdNPs, NIP without PdNPs
23
modified GCE and bare GCE can be obtained to be 1.02×10-5, 5.73×10-6, 4.44×10-6,
24
2.18×10-6 and 4.80×10-6, respectively. Thus, A can be calculated as 0.068 cm2, 0.038
25
cm2, 0.030 cm2, 0.015 cm2 and 0.032 cm2, correspondingly. The electrochemical
26
effective surface area of MIP without PdNPs modified GCE was smaller than that of
27
MIP-coated PdNPs, demonstrating the function of PdNPs can increase surface area of
28
MIP-coated PdNPs modified GCE. In addition, the electrochemical effective surface
29
area of the NIP-coated PdNPs modified GCE was smaller than that of bare GCE, 9
1
indicating the lack of recognition sites for NIP-coated PdNPs. The results
2
demonstrated that the largest electrochemical effective surface area was obtained after
3
modification of GCE with MIP-coated PdNPs, which could enhance the total
4
adsorption capacity of NE. The largest electrochemical effective surface area for
5
MIP-coated PdNPs modified GCE would lead to the increase of peak current for NE.
6
After incubating the modified electrodes into 5 mL of 5.0 µM NE solution,
7
MIP-coated PdNPs, MIP without PdNPs and NIP-coated PdNPs modified GCE in
8
NE-free PBS which had adsorbed NE were measured by DPV. Fig. 3D showed a
9
characteristic oxidation peak of NE at around 0.214 V at the MIP-coated PdNPs
10
modified GCE (curve a). However, NIP-coated PdNPs modified GCE nearly has no
11
peak current response. The results were ascribed to great quantities of recognition
12
sites for NE in the MIP-coated PdNPs, while NIP-coated PdNPs had no recognition
13
sites for binding NE. In addition, DPV current response to NE of MIP without PdNPs
14
modified GCE (curve b) was lower than MIP-coated PdNPs modified GCE (curve a).
15
The higher response of MIP-coated PdNPs modified GCE can be attributed to PdNPs
16
of good electrocatalytic activity and large surface area. Furthermore, we investigated
17
the cyclic voltammetry response for 50.0 µM NE of MIP-coated PdNPs, MIP without
18
PdNPs and NIP-coated PdNPs modified GCE in Fig. 3E. The cyclic voltammetry
19
response of MIP-coated PdNPs modified GCE was significantly higher than that of
20
MIP without PdNPs and NIP-coated PdNPs.
21
As shown in Fig. S1B, the response time of MIP-coated PdNPs modified GCE for
22
50.0 μM NE was investigated by varying the adsorption time from 0.5 to 12 min. An
23
adsorption equilibrium of 71.0% was achieved within a period of 4 min and the
24
adsorption equilibrium reached within 8 min. The result revealed rapid response
25
equilibrium of NE molecules to MIP-coated PdNPs.
26
3.4. Selectivity of the sensor
27
The selectivity experiments were investigated by using dopamine (DA),
28
epinephrine (EP), uric acid (UA), ascorbic acid (AA), acetaminophen (AC), folic acid
29
(FA) and L-tryptophan (L-TRP) as the analogs. Among these analogs, DA and EP 10
1
were tested due to their electrochemical activity and structural similarity. AA and UA
2
were measured because they largely coexisted with NE in urine samples (Goyal et al.,
3
2011). L-TRP was the essential amino acid that played an integral role in the
4
synthesis of serotonin (5-HT) and AC was known to increase 5-HT levels in brain. In
5
addition, 5-HT was known to play a role in NE release in the brain (Beitollahi and
6
Mohammadi, 2013). FA worked primarily in the brain and nervous system and was
7
necessary for the production of NE in the nervous system (Beitollahi et al., 2012).
8
Therefore, DA, EP, AA, UA, L-TRP, AC and FA were selected as the analogs.
9
Fig. 4A showed that the DPV peak current responses of MIP-coated PdNPs sensor
10
(modified GCE) toward NE were higher than other analogs, indicating a better
11
adsorption and binding capacity for the template molecules. Since the structures of
12
DA, EP, L-TRP, AC, FA, AA and UA are different with NE (Fig. 4B), the cavities
13
formed in the imprinting process could not bind them tightly, which resulted in lower
14
response. The specificity was probably due to the shape of the cavities in the
15
imprinted polymers just fitting for the unique molecular structure of NE, so they
16
cannot bind other analogs tightly. In particular, the chemical structures of FA, UA and
17
AA are very different from NE (Mao et al., 2011), so the MIP-coated PdNPs sensor
18
had good selectivity for NE compared with these three analogs. The above results
19
suggested that MIP-coated PdNPs showed potential applications as molecular
20
recognition element owing to its selectivity against the analogs.
21
3.5. Interference studies
22
In order to apply the proposed method in urine samples, it is vital to investigate
23
the effect of potential interference substances on NE determination, which was used
24
to evaluate the selectivity of MIP-coated PdNPs sensor for NE. Potential interference
25
substances involved some ions, urea, uric acid, ascorbic acid, amino acids and sugars.
26
The DPV determination of 10.0 μM NE was tested in presence of spiked known
27
amounts of interfering substances. The tolerance limit was defined as the amount and
28
fold of the interfering substances causing a change of ±5% in the peak current
29
intensity reading. The tolerable limits of interfering substances were given in Table S2. 11
1
The results showed that 1000-fold of K+, Na+, Ca2+, Mg2+, Al3+, SO42−, and Cl-- did
2
not interfere with NE determination. 500-fold of urea, 20-fold of uric acid, 50-fold of
3
ascorbic acid, 1000-fold of serine, alanine, histidine, threonine, 500-fold of glucose,
4
sucrose, lactose and maltose did not interfere with NE determination.
5
3.6. Linearity, reproducibility and stability of MIP-coated PdNPs sensor
6
DPV was used to investigate the linearity and detection limit of MIP-coated
7
PdNPs sensor for NE. As shown in Fig. 5A and B, the current responses increased
8
with successive addition of different NE concentrations in PBS. Fig. 5B illustrated the
9
corresponding plot showing a linear relation between current response and NE
10
concentration in the range of 0.5 to 80.0 μM. The regression equation was: I(μA)
11
=0.0235C(μM)-0.0160 (R2=0.9973). The detection limit was 0.1 μM (S/N=3, defined
12
as the concentration of NE corresponding to the three times the standard deviation for
13
11 replicate detections of the blank solution). It was worth to compare the
14
performance of the sensor based on MIP-coated PdNPs composite with other
15
electrochemical method reported previously. Comparison with other electrochemical
16
methods for determination of NE was summarized in Table 1. The comparison results
17
demonstrated low detection limit of the proposed sensor based on MIP-coated PdNPs.
18
In addition, in order to compare the response of MIP-coated PdNPs and NIP-coated
19
PdNPs sensors, we also investigated the linearity of NIP-coated PdNPs sensor for NE
20
(Fig. 5B). The regression equation was: I(μA) =0.00037C(μM)+0.00036 (R2=0.9639).
21
KMIP and KNIP were the linear slopes of MIP-coated PdNPs and NIP-coated PdNPs
22
sensor. So KMIP and KNIP were 0.0235 and 0.00037, respectively. The ratio of the KMIP
23
and KNIP was defined as imprinting factor (IF), which was used to evaluate the
24
specific recognition of the materials (Wang et al., 2009; Zhou et al., 2014). The IF (K
25
MIP/KNIP)
26
recognition sites for NE in the MIP-coated PdNPs, while NIP-coated PdNPs had no
27
recognition sites for binding NE. The higher current response efficiency of
28
MIP-coated PdNPs sensor suggested its specific imprint binding affinity for NE.
was 63.5, which indicated that MIP-coated PdNPs had great quantities of
12
1
So as to confirm the advantages of MIP-coated PdNPs sensor, we investigated the
2
linearity of MIP and NIP without PdNPs sensors (Fig. 5B and Fig. S2). The regression
3
equations were: I(μA)=0.00808C(μM)+0.0159 (R2=0.9740) (MIP without PdNPs
4
sensor) and I(μA)=0.000130C(μM)+0.000679 (R2=0.9186) (NIP without PdNPs
5
sensor), respectively. KMIP (without Pd) and KNIP (without Pd) were the linear slopes of MIP
6
without PdNPs sensor (Fig. 5B) and NIP without PdNPs sensor (Fig. S2). So KMIP
7
(without Pd)
8
(KMIP/KMIP (without Pd)) and IF (KNIP/KNIP (without Pd)) were 2.91 and 2.85, respectively. The
9
IF result showed that MIP with PdNPs sensor exhibited higher current response,
10
which could be attributed to the synergistic effect of good electrocatalytic property
11
and large surface area for PdNPs.
and KNIP
(without Pd)
were 0.00808 and 0.000130, respectively. The IF
12
The repeatability of MIP-coated PdNPs sensor was also evaluated by using the
13
same electrode for 8 repeated analyses of 50.0 μM NE, with a relative standard
14
deviation (RSD) of 3.56%. Good reproducibility was observed with a RSD of 3.68%
15
for 8 parallel detections. Moreover, the sensor retained a response of 95.6% of the
16
initial current after storage for 10 days at room temperature.
17
3.7. Analytical application
18
In order to evaluate the practical applications of the imprinted sensor,
19
norepinephrine injection and human urine samples were analyzed. The parallel
20
experiment was performed three times and recovery tests were conducted via the
21
standard addition method. The results were presented in Table S3 and Table S4.
22
According to the results, NE concentration found in 24 h-urine samples were 63.5 μg,
23
75.2 μg, 65.2 μg, 70.5 μg, 78.0 μg and 62.9 μg for the six people respectively, which
24
were in good agreement with NE normal values found in 24 h-urine (15-80 µg)
25
(Moyer et al., 1979; Ferreira et al., 2009). In addition, the obtained recoveries ranged
26
from 96.4% to 103.3%. Thus the proposed DPV method based on MIP-coated PdNPs
27
sensor can be used to detect NE in actual samples with good results.
28 29 13
1
3.8 Validation of the assay with HPLC
2
The MIP-coated PdNPs sensor for NE was validated by analysing the same
3
samples by a high performance liquid chromatography (HPLC) method (Anderson et
4
al., 1981; Yamazaki et al., 1995). Urine samples should be performed with a
5
pretreatment procedure prior to HPLC determination (Wu et al., 2000; Yamazaki et al.,
6
1995). The pretreatment procedure with activated aluminium oxide was added in
7
Supplementary Information. The results of NE determination with electrochemical
8
and HPLC methods were added in Table S3 and Table S4 of Supplementary
9
Information. It was worth emphasizing that the recoveries of HPLC reference method
10
for detecting NE in urine samples were all above 100% (Tables S4). There may be
11
some systematic error affecting the results of HPLC method. The systematic error
12
may be attributed to some interferences in urine samples, such as DA, EP, which may
13
increase the NE signal of HPLC and lead to the recoveries of above 100%. The
14
comparison of the proposed electrochemical method (EC) with HPLC for detecting
15
NE was shown in Fig. S3. A good correlation between HPLC (X) and EC (Y) was
16
obtained with the linear regression equation of Y=0.9875X+0.0108 (R=0.9991) (Cao
17
et al., 2013). These results suggested that the results of the proposed electrochemical
18
method for NE determination were agreeable with HPLC method, which further
19
confirmed that MIP-coated PdNPs sensor was suitable for detecting NE in actual
20
samples.
21 22
4. Conclusions
23
A novel composite of MIP-coated PdNPs was synthesized by sol-gel method
24
using NE as template, phenyl trimethoxysilane as functional monomer and
25
tetramethoxysilane as crosslinker. The combination of PdNPs and silica-based MIP
26
endowed the composite with good electrochemical catalytic property, large surface
27
area and template selectivity. The results demonstrated that MIP-coated PdNPs sensor
28
not only possessed a short response time, but also high binding capacity for NE,
29
which enabled the imprinted sensor with higher current response than that of 14
1
non-imprinted material and MIP without PdNPs. In addition, the MIP-coated PdNPs
2
sensor exhibited good selectivity for NE in comparison to FA, UA and AA. The
3
comparison results with other papers demonstrated low detection limit of MIP-coated
4
PdNPs sensor. The results of the proposed electrochemical method for NE
5
determination was agreeable with HPLC method, which confirmed that MIP-coated
6
PdNPs sensor was suitable for detecting NE in actual samples. This work may open a
7
new possibility for development of PdNPs-based imprinted polymers materials.
8 9
Acknowledgments
10
This work was supported by the National Natural Science Foundation of China
11
(No. 21177049), the Zhejiang Provincial Natural Science Foundation of China under
12
Grant No. LQ14B050002, and the Program for Science and Technology of Jiaxing
13
(No. 2013AY11017).
14 15
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Captions
15
Fig. 1. The illustration of the preparation procedure for MIP-coated PdNPs and
16
detection process for its electrochemical sensor.
17
Fig. 2. (A) The FTIR spectra of PVP(a), PVP-capped PdNPs (b), MIP-coated
18
PdNPs(c); (B) TEM images of PVP-capped PdNPs and (C) MIP-coated PdNPs.
19
Fig. 3. (A) Cyclic voltammograms of MIP-coated PdNPs modified GCE after elution
20
(a), bare GCE (b), PVP-capped PdNPs (c), NIP-coated PdNPs (d), MIP (e) and NIP
21
without PdNPs (f) modified GCE in a mix solution of 5 mM K3[Fe(CN))6] and 0.1 M
22
KCl detection conditions; (B) Plot of Q-t curves for MIPs-coated PdNPs (a), MIP
23
without PdNPs (b), NIPs-coated PdNPs (d) and NIP without PdNPs (e) modified GCE
24
and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (C) Plot of Q-t1/2 curves for MIPs-coated
25
PdNPs (a), MIP without PdNPs (b), NIPs-coated PdNPs (c) and NIP without PdNPs
26
(e) modified GCE and bare GCE (d) in 0.5 mM K3[Fe(CN)6]; (D) Differential pulse
27
voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated
28
PdNPs (c) modified GCE in PBS after incubating in 5.0 µM NE; (E) Cyclic
18
1
voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated
2
PdNPs (c) modified GCE in PBS after incubating in 50 µM NE.
3
Fig. 4. (A) The current responses of selective recognition for MIP-coated PdNPs
4
sensor of NE, DA, EP, L-TRP, AC, FA, AA and UA; (B) The chemical structures of
5
NE and other analogs.
6
Fig. 5. (A) DPV current response curves at MIP-coated PdNPs sensor with addition of
7
increasing concentration of NE (µM): 0 (a), 0.5 (b), 5.0 (c), 10.0 (d), 20.0 (e), 50.0 (f),
8
65.0 (g), 80.0 (h); (B) Calibration curves of NE detection obtained by MIP-coated
9
PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) sensors (n=5).
10
Table 1 Comparison with other electrochemical methods for determination of
11 12
NE. Detection methods
Linear range
Poly(cresol
Detecti
References
on limit
(μM)
(μM)
3– 30
0.2
(Chen et al. 2005)
0.3–
0.09
(Salmanpour et al. 2012)
8.7
(Goyal et al. 2011)
red)/GCE MWCNTs/CILE a
450.0 AuNPs/ITO b
0.1 –25
‐2
×10 Poly‐CCAc /GCE
0.63–62
0.1
(Liu et al. 2008)
0–10
0.131
(Huang et al. 2010)
4.0–110
0.5
(Mazloum‐Ardakani et al.
.5 PAAd‐MWCNTs/S e
PCE BHT/CPE f 0.0 Hematoxylin/GCE
2010) 0.5–274
0.14
.20 CACEg/GCE
(Nasirizadeh and Zare 2009)
0.55–23
0.28
(Zhang et al. 2009)
3.23 –
1.07
(Li et al. 2012)
0 WO3‐TiO2/ITO 388 19
MIP‐coated pdNPs /GCE 1
a
2
b
3
c
4
d
5
e
6
f
7 8
0.5 80.0
ITO : indium tin oxide electrode.
Poly‐CCA: poly‐Calconcarboxylic acid. PAA: polyacrylic acid‐coated.
SPCE: screen printed carbon electrode.
BHT/CPE:2,2‐[1,2‐buthanediylbis(nitriloethylidyne)]‐bis‐hydroquinone and TiO2 modified
carbon paste electrode. g
CACE: Calix[4]arene crown‐4 ether.
Highlights
10
z A novel composite of MIP‐coated PdNPs for electrochemical sensing of
11
norepinephrine was described. z The MIP‐coated PdNPs sensor possessed high current response for
13 14
norepinephrine. z Comparison results of MIP‐coated PdNPs and MIP without PdNPs
15 16
sensors showed the function of PdNPs. z The MIP‐coated PdNPs sensor could recognize norepinephrine from its
17 18
analogs. z Low detection limit of MIP‐coated PdNPs sensor for norepinephrine was
19 20
This work
CILE: carbon ionic liquid electrode.
9
12
0.1
–
obtained.
21
22
20
Fig.. 1. The illlustration of o the prepparation proocedure forr MIP-coatted PdNPs and deteection proceess for its ellectrochemiical sensor.
Fig.. 2. (A) The T FTIR spectra of PVP(a), PVP-capped P d PdNPs (b b), MIP-cooated PdN NPs(c); (B) TEM imagees of PVP-ccapped PdN NPs and (C) MIP-coatedd PdNPs.
Fig.. 3. (A) Cycclic voltamm mograms off MIP-coateed PdNPs modified m GC CE after eluution (a), bare GCE (b), ( PVP-caapped PdNP Ps (c), NIP--coated PdN NPs (d), MIP P (e) and NIP (f)
without PdNPs modified GCE in a mix solution of 5 mM K3[Fe(CN))6] and 0.1 M KCl detection conditions; (B) Plot of Q-t curves for MIPs-coated PdNPs (a), MIP without PdNPs (b), NIPs-coated PdNPs (d) and NIP without PdNPs (e) modified GCE and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (C) Plot of Q-t1/2 curves for MIP-coated PdNPs (a), MIP without PdNPs (b), NIP-coated PdNPs (d) and NIP without PdNPs (e) modified GCE and bare GCE (c) in 0.5 mM K3[Fe(CN)6]; (D) Differential pulse voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) modified GCE in PBS after incubating in 5.0 µM NE; (E) Cyclic voltammograms of MIP-coated PdNPs (a), MIP without PdNPs (b) and NIP-coated PdNPs (c) modified GCE in PBS after incubating in 50.0 µM NE.
B
OH NH2
HO
NH2
HO
HO
OH H N
HO
HO
HO
NE
DA
EP
OH
NH
O
N
O O
N
OH NH H2
N H
N
H2N
O
N
CH2NH N
C
OH H
OH H HO HO
O FA
AC O
O
O OH
AA
HN O
OH
CH2CH C 2 C OH
CH3 L-TRP
C NHCH
H N O
N H
N H
UA
Fig.. 4. (A) Thhe current responses of o selectivee recognitioon for MIP P-coated PddNPs senssor of NE, DA, EP, L--TRP, AC, FA, F AA andd UA; (B) The T chemiccal structurees of NE and other analogs. a
Fig.. 5. (A) DPV V current reesponse curves at MIP--coated PdN NPs sensor with w additioon of incrreasing conccentration of o NE (µM): 0 (a), 0.5 (b), ( 5.0 (c), 10.0 (d), 200.0 (e), 50.00 (f), 65.00 (g), 80.0 (h); (B) Caalibration curves c of NE N detection n obtained by MIP-cooated PdN NPs (a), MIP P without PdNPs (b) annd NIP-coatted PdNPs (c) ( sensors (n=5).
