Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent detection of dopamine

Article abstract of DOI:10.1021/ac401894w

A stepwise chemical reaction strategy based on the specific recognition of boronic acid to diol, and N-hydroxysuccinimide (NHS) ester to amine group, was designed to construct a signal on electrochemiluminescence (ECL) platform for highly sensitive detection of dopamine. A boronic acid-functionalized pyrene probe was synthesized and was self-assembled on the sidewalls of carbon nanotubes via π-π stacking interactions as capture probes on a glassy carbon electrode. Meanwhile, 3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP)-functionalized CdTe quantum dots (QDs) were designed as signal probes and characterized with transmission electron microscopy and spectroscopic techniques. Upon stepwise chemical reaction of dopamine with boronic acid and then DSP-QDs, the QDs were captured on the electrode as ECL emitters for signal readout, leading to an ultralow background signal. By using O₂ as an endogenous coreactant, the signal on ECL method was employed to quantify the concentration of dopamine from 50 pM to 10 nM with a detection limit of 26 pM. Moreover, the stepwise chemical reaction-based biosensor showed high specificity against cerebral interference and was successfully applied in the detection of dopamine in cerebrospinal fluid samples. The stepwise chemical reaction strategy should be a new concept for the design of highly selective analytical methods for the detection of small biomolecules.

Full text of DOI:10.1021/ac401894w

Article
pubs.acs.org/ac
Stepwise Chemical Reaction Strategy for Highly Sensitive
Electrochemiluminescent Detection of Dopamine
Lei Zhang, Yan Cheng, Jianping Lei,* Yueting Liu, Qing Hao, and Huangxian Ju
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing 210093, P. R. China
*
S Supporting Information
ABSTRACT: A stepwise chemical reaction strategy based on the
specific recognition of boronic acid to diol, and N-hydroxysucci-
nimide (NHS) ester to amine group, was designed to construct a
“
signal on” electrochemiluminescence (ECL) platform for highly
sensitive detection of dopamine. A boronic acid-functionalized
pyrene probe was synthesized and was self-assembled on the
sidewalls of carbon nanotubes via π−π stacking interactions as
capture probes on a glassy carbon electrode. Meanwhile, 3,3′-
dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP)-
functionalized CdTe quantum dots (QDs) were designed as signal
probes and characterized with transmission electron microscopy
and spectroscopic techniques. Upon stepwise chemical reaction of
dopamine with boronic acid and then DSP-QDs, the QDs were
captured on the electrode as ECL emitters for signal readout, leading to an ultralow background signal. By using O as an
2
endogenous coreactant, the “signal on” ECL method was employed to quantify the concentration of dopamine from 50 pM to 10
nM with a detection limit of 26 pM. Moreover, the stepwise chemical reaction-based biosensor showed high specificity against
cerebral interference and was successfully applied in the detection of dopamine in cerebrospinal fluid samples. The stepwise
chemical reaction strategy should be a new concept for the design of highly selective analytical methods for the detection of small
biomolecules.
lectrochemiluminescence (ECL) as a sensitive technique
is sequentially recognized by other molecules at different sites
to improve specificity. A stepwise molecular recognition
strategy has been applied in the preparation of supramolecular
E
has attracted continuous interest owing to the integrated
1
advantages of electrochemistry and chemiluminescence.
Especially, semiconductor quantum dot (QD)-based ECL has
been widely applied in bioanalysis because QDs own several
unique merits such as size-controlled luminescence and good
12−17
structures, sandwich immunoassays, and chiral sensing.
For example, by using a dual recognition linker, β-cyclodextrin/
α-mannopyranoside, which can act as a host for chemical
molecular recognition of the adamantane and as a ligand for
biological recognition of concanavalin A simultaneously, self-
2
−4
stability against photobleaching.
QD-based ECL biosensing
strategies are usually divided into four methodologies: the
inhibition/enhancement effect of the analyte, generation/
consumption of coreactant, steric hindrance from the5
biorecognition reaction, and ECL resonance energy transfer.
Because QDs in these strategies are immobilized directly on the
electrode surface as a sensing element, QD-based ECL
analytical methods suffer from a high background, which
precludes their application in practice. To reduce the
background, it is preferred to introduce labeled QDs to the
12
assembled protein−polymer conjugates were attained. On the
basis of target versatility (amines and diols) with ferrocene-
based chiral boronic acids, a chiral sensor was constructed for
the determination of enantiomeric excess of binol by using an
13
electrochemical method. The method based on stepwise
chemical reaction was especially favorable for selective
detection of small molecules due to the sequential recognition
processes. Here, on the basis of stepwise chemical reaction
between a boronic acid and a diol, and an amine group and N-
hydroxysuccinimide (NHS) ester, an ECL biosensor was
designed for highly sensitive and selective detection of
dopamine.
6
electrode via molecular recognition for signal readout. In fact,
many ECL detection methods using QDs as detection probes
have been developed via biomacromolecular recognition such
7
−11
as antibody−antigen and aptamer−protein.
However, ECL
sensing of small biomolecules is still a great challenge due to
the lack of specific recognition sites. Therefore, it was desirable
to seek a new binding approach to enhance specificity in the
QD-based ECL detection of small biomolecules.
Received: June 24, 2013
Accepted: July 31, 2013
Published: July 31, 2013
Fortunately, this issue can be addressed through a stepwise
chemical or biological recognition strategy, in which a molecule
©
2013 American Chemical Society
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Scheme 1. (A) Synthesis of the Recognition Probe, (B) the Functionalization of QDs with DSP, and (C) the Stepwise Chemical
Reaction Strategy for ECL Detection of Dopamine
Dopamine (DA) as an important neurotransmitter plays a
very important role in the renal, hormonal, and cardiovascular
systems, and abnormal levels of DA are related to neurological
chemical reaction and extends the applications of ECL in the
selective detection of small biomolecules.
18
EXPERIMENTAL SECTION
disorders such as Parkinson’s disease. Many elegant methods
■
have been reported for DA determination, including
Materials and Reagents. Cadmium chloride (CdCl2·
19
chromatography combined with mass spectrometry, UV−vis
2.5H O), meso-2,3-dimercaptosuccinic acid (DMSA), and
2
2
0
21
spectrophotometry, fluorometry, and electrochemical tech-
niques.
dopamine hydrochloride were purchased from Alfa Aesar
China Ltd. Uric acid (UA), D-(+)-glucose, lactate, 3,4-
dihydroxyphenylacetic acid (DOPAC), homovanillic acid
2
2,23
Although those methods for the detection of
dopamine can reach a low detection limit, most of them suffer
from low selectivity in the presence of other catechol
derivatives. Meanwhile, a single molecular recognition method
was also imperfect in terms of specificity. In the case of ECL
detection of DA, ECL was conventionally quenched by the
oxidation products of DA and charge/energy-transfer between
(
HVA), epinephrine hydrochloride, catechol, L-tyrosine, and
glutamic acid were purchased from Shanghai J&K Chemical
Ltd. (China). 3-Aminophenylboronic acid, 1-pyrenebutanoic
acid succinimidyl ester, and 3,3′-dithiodipropionic acid di(N-
hydroxysuccinimide ester) were purchased from Sigma-Aldrich
and used as supplied. L-Ascorbic acid (AA) was purchased from
Sinopharm Chemical Reagent Co., Ltd. Noradrenaline
bitartrate was purchased from Chengdu Xiya Chemical
Technology Co., Ltd. Carbon nanotubes (CNTs, CVD
method, purity 98%, multiwalled, diameter 40−60 nm, and
length 1−2 μm) were purchased from Shenzhen Nanotech Port
Co., Ltd. (Shenzhen, China). Phosphate buffer (PBS) (0.1 M,
pH 9.0) was prepared by mixing stock solutions of NaH PO
24−29
the analytes and ECL emitters on the electrode.
In this
work, by using labeled QDs as signal probes, a “signal on” ECL
detection strategy was proposed by coupling with stepwise
chemical reaction for selective and sensitive detection of DA
(
(
Scheme 1). First, a boronic acid-functionalized pyrene probe
P1) was synthesized, which was self-assembled on the
sidewalls of carbon nanotubes (CNTs) via π−π stacking
interactions as a capture probe on a glassy carbon electrode
2
4
and Na HPO with 0.1 M KNO as the supporting electrolyte.
2
4
3
(
GCE). Then the immobilized P1 would recognize DA via
specific interaction between boronic acid and diol. Sequentially,
,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester)
DSP)-functionalized CdTe QDs were covalently bound with
Other reagents were of analytical grade and used as received.
Ultrapure water (≥18 MΩ, Milli-Q, Millipore) was used
throughout the work.
3
(
Multiwalled CNTs were first sonicated with 3:1 (v/v)
H SO /HNO for 4 h to obtain carboxylic group-functionalized
DA via amidation. Therefore, the QDs were captured on the
electrode as ECL emitters, and then the “signal on” ECL
emission was observed by using O2 as an endogenous
coreactant. The proposed method shows a detection limit
down to picomoles and high selectivity for the determination of
DA in real samples. This work is the first report on the “signal
on” ECL sensor for the detection of DA based on stepwise
2
4
3
CNTs. The resulting dispersion was filtered and washed
repeatedly with water until the pH was about 7.0. The collected
functionalized CNTs were redispersed in water to a
−1
concentration of 0.1 mg mL .
For the determination of DA in practice, two real
cerebrospinal fluid (CSF) samples were kindly obtained from
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volunteer patients. Before utilization, CSF samples were
centrifuged at 14 000 rcf for 15 min and the supernates were
collected. After precipitation of the proteins in the samples by
acetonitrile, the resulting supernates were further centrifuged at
low modulated frequency for over 0.5 h and then incubated for
4−6 h at room temperature. Successively, the unreacted DSP
was removed from the DSP-QDs by filtration in a Millipore
Microcon (10 000 MW) at 8000 rcf for 8 min. The product was
washed with acetone twice. The final product was redissolved in
deionized water and stored at 4 °C in the dark prior to use.
Construction of the ECL Biosensor. The GCE was
polished successively with 1.0, 0.3, and 0.05 mm of alumina
slurry (Beuhler). After successive sonication in ethanol and
1
2 000 rcf for 15 min to get the final samples for assay.
Apparatus. X-ray photoelectron spectroscopy (XPS)
experiments were carried out on an ESCALAB 250
spectrometer (Thermo-VG Scientific Co., Waltham, MA)
with an ultrahigh vacuum generator. Infrared (IR) spectra
were recorded on a Nicolet NEXUS870 Fourier transform
infrared (FT-IR) spectrometer (Madison, WI). The UV−vis
absorption spectra were obtained with a UV-3600 UV−vis-NIR
spectrophotometer (Shimadzu Co., Kyoto, Japan). The trans-
mission electron micrograph (TEM) was obtained using a
JEM-2100 TEM instrument (JEOL, Japan). ESI-MS was
recorded on an Agilent G6540Q-TOF LC/MS equipped with
an electrospray ionization (ESI) probe operating in positive ion
deionized water, the electrode was dried under N . To
2
accelerate electron transfer and provide a large surface for
−1
immobilization of P1, 10 μL of a 0.1 mg mL multiwalled
CNT dispersion was spread on a pretreated bare GCE and
dried at room temperature. Ten microliters of 0.5 mmol L− P1
solution was then dropped onto the CNT film. After removal of
excess P1 with water, 10 μL of the appropriate concentration of
DA solution was applied to the CNT−P1 layer in the dark for 3
h. The resulting surface was incubated with 10 μL of DSP-QDs
for 4 h after washing with deionized water to remove the
unreacted DA. The whole assay time including the construction
of the biosensor was around 7 h. Finally, the as-prepared
biosensor was rinsed three times with deionized water to
eliminate the noncovalently absorbed DSP-QDs, and then ECL
measurements for signal readout were carried out by using
1
1
13
mode. H and C NMR spectra were recorded on a Bruker
APX-400 spectrometer with chemical shifts referenced to SiMe4
as internal standard. Electrochemical impedance spectroscopic
(EIS) measurements were carried out on a PGSTAT30/FRA2
system (Autolab, The Netherlands) in 0.1 M KCl containing 5
mM K Fe(CN) /K Fe(CN) . Electrochemiluminescent meas-
3
6
4
6
urements were carried out on a MPI-E multifunctional
electrochemical and chemiluminescent analytical system
dissolved O
, without introduction of exogenous coreactants
2
2
−
(
(
Xi’an, China), with a modified glassy carbon electrode
GCE, 5 mm in diameter, China) as a working, a platinum
such as S
2
O
8
, which was beneficial to the biosensor.
RESULTS AND DISCUSSION
Characterization of DSP-QDs. The UV−vis spectrum of
QDs showed a wide absorption band with an absorption
wire as a counter, and a Ag/AgCl (saturated KCl) as a reference
electrode. The ECL emission window was placed in front of the
photomultiplier tube (PMT, detection range from 300 to 650
nm) biased at 1000 V with scan rate of 100 mV/s.
■
inflection point at 460 nm that is consistent with the data
31
Synthesis of P1. A mixture of 3-aminophenylboronic acid
described previously (Figure 1A, curve a). After modification
(
30.9 mg, 0.2 mmol) and 1-pyrenebutanoic acid succinimidyl
ester (77.1 mg, 0.2 mmol) in methanol (10 mL) was sonicated
for 2 h. The crude product was directly purified by flash
chromatography in 1/1 (v/v) ethyl acetate/petroleum ether to
afford the product as a pale yellow solid. Yield: 21.3 mg, 26.4%.
+
1
ESI-MS: m/z 440.42, [M + CH OH + H] . H NMR (400
3
MHz, C D ): δ (ppm) 8.49 (s, 1H), 8.17−7.66 (m, 13H), 3.26
6
6
(
s, 2H), 3.11 (t, J = 7.6 Hz, 2H), 2.35 (t, J = 6.8 Hz, 2H), 2.0−
.94 (m, 2H). 1 C NMR (400 MHz, DMSO-d ): δ (ppm)
3
1
1
1
1
6
72.3, 171.1, 135.9, 130.8, 130.4, 130.3, 129.6, 129.4, 128.1,
28.0, 127.5, 127.4, 127.3, 126.5, 126.1, 126.0, 125.0, 124.9,
24.8, 124.2, 124.1, 123.4, 123.3, 31.6, 30.7, 26.7 (Figures S1−
S3 in Supporting Information).
Synthesis of QDs and DSP-QDs. The DMSA-stabilized
CdTe (DMSA-CdTe) QDs were prepared according to the
30
reported electrolysis method. Briefly, 7.8 mg of DMSA was
dissolved by stirring in 20 mL of ultrapure water. After
regulation of the pH to around 9.0, 120 μL of 0.1 M CdCl was
2
next added dropwise to obtain a homogeneous solution. When
a terminal charge quantity of 0.5 °C was reached upon the
applied potential of about −1.0 V at a polished tellurium
electrode, the resulting solution was air-proofed and immersed
in a constant temperature bath at 80 °C for 20 h to harvest
DMSA-CdTe QDs and then stored at 4 °C in the dark. Before
usage, the as-prepared QD solution was purified, sedimented in
Figure 1. (A) UV−vis absorption spectra. (B) ECL emission curves
with PMT biased at 900 V. (C) FT-IR spectra and (D) Cd3d XPS of
QDs (a) and DSP-QDs (b). Curve c is the FT-IR spectrum of DSP.
with DSP, the absorption peak of DSP-QDs significantly blue-
shifted to 437 nm (Figure 1A, curve b), which indentified that
DSP was assembled on the surface of QDs. According to Peng’s
empirical equation, the concentration of the redispersed DSP-
1:1 (v/v) isopropyl alcohol/water, and centrifuged at 8000 rcf
for 5 min. The precipitate was then dissolved in deionized
water.
DSP-QDs were prepared by mixing 0.50 mL of the
redissolved QDs with 0.45 mL of 2 mM DSP via ligand
exchange. The mixture was allowed to react ultrasonically under
32
QD solution was estimated to be ∼23 μM. The ECL emission
peak of DSP-QDs occurred at a potential more positive than
that of QDs (Figure 1B), suggesting surface-state ECL emission
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3
3
in this designed system. The IR spectra also confirmed that
appeared on the CNTs (Figure 2D), which became blurry with
the increase in their average diameter, indicating successful
assembly of the DSP-QDs on the biosensor surface.
Furthermore, DSP-QDs appeared only on the surface of
CNTs, suggesting specific recognition in the ECL detection.
These results suggested that the designed ECL platform has the
capacity for sensitive detection with an ultralow background.
On the other hand, electrochemical impedance spectra were
utilized to monitor the sequential fabrication of the ECL sensor
the ligand of DSP was successfully linked with QDs (Figure
C). Compared with QDs (curve a), DSP-QDs showed the
1
characteristic vibrational frequencies of DSP at 1710.6, 1421.7,
1
1
363.2, 1235.3, and 1095.0 cm− corresponding to CO
stretching vibration, two C−H bending vibrations of
methylene, C−O asymmetric stretching vibration, and C−O
symmetric stretching vibration, respectively (curve b). DSP-
QDs were as well validated from XPS by the alteration of the
binding energy (Figure 1D). It displayed a mild movement of
the Cd 3d peak from 404.2 to 403.9 eV in which Cd 3d
somewhat coincided with N1s (402.9 ev) of DSP. In other
words, these results successfully verified the assembly of DSP to
the surface of QDs.
(
Figure 3A). The bare GCE showed a relatively small electron-
transfer resistance (R ) (curve a). After CNTs were
et
immobilized on the electrode, Ret decreased (curve b),
indicating that the high electrical conductivity of the CNTs
facilitated the electron transfer. The modification of the CNTs
Characterization of Stepwise Assembly of ECL
Biosensor. TEM images showed the morphology change
during the construction of the sensor. First, the TEM image of
the acid-pretreated CNTs displayed a uniform structure in the
form of single tubes or small bundles with diameters of 40−60
nm (Figure 2A). The size of the DSP-QDs was estimated to be
with P1 by π−π stacking led to a much larger R (curve c)
et
because it blocked the electron transfer in the CNTs. Finally,
the presence of QDs greatly enhanced the R (curve e) due to
et
34
the unique properties of this semiconductor. The impedances
of the biosensor in the absence and presence of DA were
similar (curves c and d), indicating that the binding of DA as a
small organic molecule did not greatly affect the electron
transfer. On the whole, the tendency for increased impedances
further testified to the immobilization of these substances on
the electrode surface.
Feasibility of the ECL Biosensor. In the present sensor,
DSP-QDs as a signal probe were finally introduced to the
electrode by covalent reaction with DA instead of direct
physical absorption as in most ECL detections. IR spectra were
employed to prove the successful covalent linkage between
DSP-QDs and DA (Figure 3B). After the reaction of DSP-QDs
with DA, the peaks at 3009.9, 2976.6, 2931.4, and 950.1 cm−
disappeared, which correspond to three stretching vibrations of
C−H, and one stretching vibration of N−O in the NHS group
of DSP, respectively, because NHS esters as a good leaving
group can react with amine via amidation. Meanwhile, the
stretching vibration around 1650 cm− was significantly
enhanced due to the formation of amide I via reaction between
the amine group of DA and the NHS ester of DSP-QDs.
The feasibility of the ECL biosensor was further investigated
by measuring the ECL signal in the presence or absence of the
target (Figure 4). In the absence of the target, no ECL signal
was observed at the constructed sensor (curve a) because no
DSP-QDs were absorbed on the electrode after rinsing with
water. In contrast, in the presence of the target, a sharp ECL
emission peak appeared at −1.16 V (curve b). To exclude that
the ECL signal was caused by the nonspecific absorption of
1
1
Figure 2. TEM images of (A) CNTs, (B) DSP-QDs, (C) CNTs/P1/
DA, and (D) CNTs/P1/DA/DSP-QDs.
4
.0−6.5 nm by TEM (Figure 2B). When P1 was assembled on
the surface of CNTs, no obvious change was observed
compared with that of pure CNTs (Figure 2C). Sequentially,
DSP-QDs were assembled on the CNTs, and many black dots
Figure 3. (A) EIS plots of bare GCE (a), GCE/CNTs (b), GCE/CNTs/P1 (c), GCE/CNTs/P1/DA (d), and GCE/CNTs/P1/DA/DSP-QDs (e).
B) FT-IR spectra of DSP-QDs (a) and DSP-QDs/DA (b).
(
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QDs. Conversely, no obvious signal change was observed for
DSP-QDs in the absence of DA (curve a). Therefore, 4 h of
incubation time was selected for the ECL biosensor.
ECL Response to DA. Under optimal conditions, the
stepwise chemical reaction strategy was performed by the
reaction of DA at a known concentration with P1 as the
recognition probe and then with DSP-QDs as the signal probe.
The captured DSP-QDs on the sensor surface gave ECL
emission at the applied potential to produce a detectable signal
at different electrodes for different concentrations of the target.
As shown in Figure 6A, the ECL signal increased as the
concentration of DA increased. The calibration plot shows a
good linear relationship between the ECL intensity and the
logarithmic value of DA concentration in a range of 50 pM to
1
2
0 nM (inset in Figure 6A). The regression equation was I =
872.3 + 257.5 × log c with a correlation coefficient of 0.992,
Figure 4. ECL responses of the GCE/CNTs/P1 in air-saturated 0.1 M
pH 9.0 PBS in the presence of DSP-QDs (a), DA + DSP-QDs (b),
and DA + DMSA-QDs (c).
where I is the ECL intensity of the biosensor in the presence of
DA, and c is the concentration of DA. The detection limit at a
signal-to-noise ratio of 3 was 26 pM, which was much lower
than 0.1 μM of the near-infrared Ag Se QD-based ECL
2
2
6
method, 30 nM of the graphene oxide-based electrochemical
method, and 50 nM of the mesoporous silica particle-based
fluorescent sensing method. The low detection limit of the
QDs, the response of the sensor was investigated in the
presence of DA by using no DSP-functionalized QDs as a signal
probe. As shown in curve c, no obvious ECL peak was
observed. Therefore, a highly sensitive strategy for “signal on”
ECL sensing of DA was developed by stepwise chemical
reaction between boronic acid and diol, and then between the
amine group and NHS ester, among the P1, DA, and DSP-
QDs. Moreover, the proposed method of QD immobilization
on GCE by stepwise chemical reaction greatly decreased the
background for highly sensitive detection.
35
36
proposed biosensor is attributed to the ultralow background
signal resulting from dual molecular recognition and the “signal
on” detection strategy.
Selectivity of the ECL Biosensor. Selective electro-
chemical determination of DA is usually limited by the lack
of the resolution between DA and other electroactive
compounds coexisting in cerebral systems, because they can
37−39
be electrochemically oxidized at similar potentials.
In the
Optimization of Detection Conditions. The pH of the
electrolyte is a significant factor relevant to the ECL response.
To obtain sufficient ECL intensity, the influence of the
detection solution pH on the ECL response of DSP-QDs was
investigated as shown in Figure 5A. In the examined pH range,
the ECL intensity increased as the pH changed from 6.0 to 9.0
proposed ECL sensor, a high selectivity for DA was achieved by
a stepwise chemical reaction strategy, which was required both
for boronic acid recognition of the catechol group of DA, and
for DSP recognition of amine group of DA. Therefore, possible
interference from physiological components, such as glucose,
•−
3
,4-dihydroxyphenylacetic acid, tyrosine, ascorbic acid, cat-
because the electrogenerated intermediates (e.g., O2 and
•
echol, uric acid, homovanillic acid, lactate, glutamic acid, and
the catecholamines epinephrine and norepinephrine, were
examined (Figure 7A). Although norepinephrine has reactive
groups similar to that of DA, further reaction may be blocked
by steric hindrance from the hydroxyl group near the amine
group. Thus, no obvious response was observed in the present
biosensor, indicating excellent selectivity of the designed
biosensor. Moreover, the antiinterference experiment was
performed by measuring a solution containing 2 nM DA and
HO ) of dissolved O as well as the resulting H O as the
2
2
2
coreactant were more stable at high pH. Taking into account
further application under physiological conditions, pH 9.0 PBS
was used as the detection solution.
The incubation time is another important parameter that
influences the ECL signal (Figure 5B). At room temperature,
the ECL signal was enhanced as the incubation time increased
and then approached a constant value after 4 h (curve b), which
was limited by the saturated binding site between DA and DSP-
Figure 5. Effects of (A) pH of detection solution and (B) incubation time of DSP-QDs in the absence (a) and presence (b) of 10 nM DA on the
ECL response. When one parameter is changed, the others are at their optimal value.
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Figure 6. (A) ECL responses of the optimized sensor to different concentrations of DA: 0, 0.04, 0.045, 0.05, 0.1, 0.2, 0.4, 0.8, 2, 6, and 10 nM (from
a to k). Inset: plot of ECL intensity vs logarithmic value of DA concentration. (B) Continuous cyclic scans at GCE/CNTs/P1/DA/DSP-QDs in the
air-saturated detection solution.
Figure 7. ECL responses of the biosensor toward 10 nM glutamic acid, glucose, 3,4-dihydroxyphenylacetic acid, tyrosine, ascorbic acid, catechol, uric
acid, homovanillic acid, lactate, epinephrine, and norepinephrine (from a to k) in the absence (A) and in the presence (B) of 2 nM DA. Column l is
ECL response of the sensor toward 2 nM DA.
1
0 nM interfering reagent as listed above. Similarly, no obvious
CSF of patients, the low detection limit at the picomolar level
in the present report was especially appropriate for the analysis
of the CSF samples. After pretreatment, the DA concentrations
in the samples were 39.8 and 60 pM by the standard addition
method, which is consistent with the value reported in ref 40.
The average recoveries ranged from 101.9% ± 6.9% to 107.7%
change was observed (Figure 7B), indicating acceptable
selectivity for DA detection.
Reproducibility and Precision of the ECL Biosensor.
The interassay precision of five ECL sensors was examined at
1.0 nM DA. The relative standard deviation (RSD) of the five
measurements was 4.7%, showing good precision and accept-
able fabrication reproducibility. Seven measurements of ECL
emission from continuous cyclic scans of the ECL sensor at 1.0
nM DA showed a coincident signal with an RSD of 1.7%
±
3.6% for three determinations, indicating good accuracy and
acceptable precision. Thus, the present method is satisfactory
for practical application in the detection of DA for clinical
diagnosis.
(
Figure 6B), indicating acceptable reliability and stability of the
detection signal. The stability of the sensor for DA was also
examined by measuring the ECL responses after storage at 4 °C
for 10 days. The ECL response had no obvious decline with an
RSD of 7.6%, demonstrating good stability of the sensor.
Application in Cerebrospinal Fluid Samples. To
evaluate the applicability and reliability of the present ECL
system, two real cerebrospinal fluid (CSF) samples were
examined (Table 1). Due to low concentrations of DA in the
CONCLUSIONS
■
A universal electrochemiluminescent platform was designed
with a stepwise chemical reaction strategy for the highly
sensitive detection of small biomolecules. Because it required
two chemical recognitions, both boronic acid−diol and NHS
ester−amine, the present approach showed excellent selectivity
for the target. Moreover, compared with the direct physical
absorption of QDs in conventional ECL methods, the present
approach captured the QDs as a signal probe to the electrode
by covalent chemical reaction. Therefore, the “signal on” ECL
sensing strategy was achieved and further decreased the
background to allow for highly sensitive detection of DA. By
Table 1. Detection Results and Recoveries of DA in CSF
Samples
sample no.
1
addition, pM
0
found, pM
recovery, %
39.8
66.7
89.5
60.0
96.9
124.4
using O as an endogenous coreactant for the ECL signal
2
2
4
8
0
0
0
104.6 ± 4.8
101.9 ± 6.9
output, this biosensor showed wide linear response range, low
detection limit in the picomole range, and extremely high
selectivity for DA and was successfully applied for the detection
of dopamine in real samples. This stepwise chemical reaction
strategy provides a powerful tool to design biosensing
4
2
3
6
107.7 ± 3.6
103.7 ± 6.0
8
006
dx.doi.org/10.1021/ac401894w | Anal. Chem. 2013, 85, 8001−8007

Analytical Chemistry
Article
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Corresponding Author
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Notes
(
2
(
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
2
(
1
This work was financially supported by the National Basic
Research Program of China (2010CB732400), National
Natural Science Foundation of China (21075060, 21135002,
2
1121091), and the program for New Century Excellent
(
Talents in University (NCET100479).
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2
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