In situ generation of self-enhanced luminophore by β-lactamase catalysis for highly sensitive electrochemiluminescent aptasensor

Article abstract of DOI:10.1021/ac500665b

This work described a new electrogenerated chemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin (TB) based on the in situ generating self-enhanced luminophore by β-lactamase catalysis for signal amplification. Briefly, a ruthenium complex (Ru-Amp), including two regions of [Ru(phen)₂(cpaphen)]²⁺ and ampicillin (Amp), was synthesized as a self-enhanced ECL luminophore, which can produce an ECL signal through intramolecular interactions. Then, carbon nanotubes (CNTs) were used for immobilization of Ru-Amp via π-π stacking interactions to form the Ru-Amp@CNTs nanocomposite. Using poly(ethylenimine) (PEI) as a linkage reagent, Au nanocages (AuNCs), owing to their electronic property and large surface areas, were decorated to the CNTs to form the Ru-Amp@CNTs-PEI-AuNCs nanocomposites, which were further used to immobilize thrombin binding aptamer II (TBA II) to form a signal probe (Ru-Amp@CNTs-PEI-AuNCs-TBA II). Through "sandwich" tactics, TBA II bioconjugates, TB and TBA I were immobilized onto the gold nanoparticles modified electrode. Then, with the enzyme catalysis of β-lactamase, a novel self-enhanced ECL luminophore (Ru-AmpA) was in situ produced, which could exhibit a significant enhancement of ECL signal, due to the structure transformation of an amide bond into a secondary amine. A sandwich ECL assay for TB detection was developed with excellent sensitivity of a concentration variation from 1.0 fM to 1.0 pM and a detection limit of 0.33 fM. Therefore, the self-enhanced ECL luminophore, combining the further enhancement by in situ enzymatic reaction, is expected to have potential applications in biotechnology and clinical diagnosis.

Full text of DOI:10.1021/ac500665b

Article
pubs.acs.org/ac
In Situ Generation of Self-Enhanced Luminophore by β‑Lactamase
Catalysis for Highly Sensitive Electrochemiluminescent Aptasensor
Guo-Feng Gui,†,‡ Ying Zhuo,*,† Ya-Qin Chai,† Yun Xiang,† and Ruo Yuan*,†
†Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China
‡College of Chemical Engineering, Bijie University, Bijie, Guizhou 551700, China
S
* Supporting Information
ABSTRACT: This work described a new electrogenerated
chemiluminescence (ECL) aptasensor for ultrasensitive
detection of thrombin (TB) based on the in situ generating
self-enhanced luminophore by β-lactamase catalysis for signal
amplification. Briefly, a ruthenium complex (Ru−Amp),
including two regions of [Ru(phen)2(cpaphen)]2+ and
ampicillin (Amp), was synthesized as a self-enhanced ECL
luminophore, which can produce an ECL signal through
intramolecular interactions. Then, carbon nanotubes (CNTs)
were used for immobilization of Ru−Amp via π−π stacking
interactions to form the Ru−Amp@CNTs nanocomposite.
Using poly(ethylenimine) (PEI) as a linkage reagent, Au nanocages (AuNCs), owing to their electronic property and large
surface areas, were decorated to the CNTs to form the Ru−Amp@CNTs−PEI−AuNCs nanocomposites, which were further
used to immobilize thrombin binding aptamer II (TBA II) to form a signal probe (Ru−Amp@CNTs−PEI−AuNCs−TBA II).
Through “sandwich” tactics, TBA II bioconjugates, TB and TBA I were immobilized onto the gold nanoparticles modified
electrode. Then, with the enzyme catalysis of β-lactamase, a novel self-enhanced ECL luminophore (Ru−AmpA) was in situ
produced, which could exhibit a significant enhancement of ECL signal, due to the structure transformation of an amide bond
into a secondary amine. A sandwich ECL assay for TB detection was developed with excellent sensitivity of a concentration
variation from 1.0 fM to 1.0 pM and a detection limit of 0.33 fM. Therefore, the self-enhanced ECL luminophore, combining the
further enhancement by in situ enzymatic reaction, is expected to have potential applications in biotechnology and clinical
diagnosis.
covalently linked with different amine reductants for an ECL
lectrogenerated chemiluminescence (ECL), a very power-
Eful analytical technique, is the process of producing an system and studied intra- and intermolecular interactions of
novel ruthenium complexes.12,13 In our previous work, we have
excited state in a photoactive molecule at the electrode surface,
designed a novel ECL immunosensor based on a Ru(II)
complex (Ru−PEI) for the detection of APE-1.14 They all
indicated that the ruthenium complex containing two regions of
ECL-active luminophore and coreactant, named self-enhanced
ECL luminophore, can generate an efficient ECL signal through
intramolecular interactions.
Ampicillin (Amp), one of β-lactamase antibiotics containing
the structure of amide and primary amine, could slightly
enhance the ECL efficiency of ruthenium.15 On the basis of this
property, a self-enhanced ECL luminophore (Ru−Amp) was
designed including two regions of [Ru(phen)2(cpaphen)]2+ and
Amp. More importantly, when they are hydrolyzed by β-
lactamase, the structure transformation of an amide bond into a
secondary amine in situ occurred to produce a novel self-
enhanced ECL luminophore (Ru−AmpA), which could
resulting in luminescence upon its return to the ground
state.1−3 Ruthenium(II)-based complexes, as one of the most
popular ECL luminophores, have received considerable
attention in clinical diagnosis and scientific research because
of their excellent stability and high efficiency in aqueous
phase.4,5 In a typical Ru(II) ECL process, the electron transfer
reaction occurs between electrochemically formed Ru(II)
species and coreactants, such as tripropylamine (TPA),6,7
peroxydisulfate8 and 2-(dibutylamino)ethanol (DBAE).9 How-
ever, the coreactants, owing to their water-solubility, have been
rarely used for the application of reagentless ECL sensors.
Considering all of this, the new superior ECL reagents,
designed by covalently linking the two parts of ECL-active
luminophore and coreactant, are expected to enhance both
ECL efficiency and electron transfer performances through
intramolecular interaction. Using this approach, Sun and co-
workers have patented an ECL reagent of ruthenium linked to
tripropylamine through an amide bond.10,11 Kehr’s group has
synthesized a series of ruthenium(II) tris-bipyridyl complexes
Received: February 18, 2014
Accepted: May 23, 2014
Published: May 23, 2014
© 2014 American Chemical Society
5873
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Scheme 1. Reaction Scheme for the Synthesis of Ru−Amp
significantly enhance the ECL efficiency due to the amine-to-
ruthenium electron transfer through intramolecular reactions
by the covalent approximation of the two reacting species.
Considering all of this, Ru−Amp was synthesized and first
applied to construct a highly sensitive ECL aptasensor coupling
with the in situ enzymatic reaction by β-lactamase as a signal
amplification strategy.
aptamer I (TBA I). Then the TBA II bioconjugates were linked
to the electrode surface via “sandwich” tactics. When β-
lactamase existed, the desirable Ru−AmpA was in situ
generated on the surface of electrode in high yields and
exhibited high ECL efficiency, which could improve the
sensitivity of the sensors. The in situ enzymatic reaction is an
especially promising method because it avoids complicated
labeling procedures and has the advantages of high efficiency,
simplicity and low toxicity. Thus, coupling with the attractive
self-enhanced ruthenium complex and the signal amplification
of in situ enzymatic reaction by β-lactamase, the tactics hold
promise as a great choice for sensitive and selective detection of
targets studied.
In this work, we developed an ECL aptasensor based on the
self-enhanced ECL luminophore of Ru−Amp and in situ
enzymatic reaction by β-lactamase for signal amplification to
detect the concentration of thrombin (TB). Here, Ru−Amp
wrapped on carbon nanotubes (CNTs) via π−π stacking
interactions to form the Ru−Amp@CNTs nanocomposites.16
Then, Au nanocages (AuNCs) with good biocompatibility,
electronic property and large surface areas,17 were conjuncted
on Ru−Amp@CNTs by the linkage reagent poly-
(ethylenimine) (PEI). The resultant AuNCs loaded Ru−
Amp@CNTs−PEI were further employed as carriers to
immobilize thrombin binding aptamer II (TBA II) to obtain
TBA II bioconjugates (Ru−Amp@CNTs−PEI−AuNCs−TBA
II). For constructing the aptasensor, graphene−Nafion (Gr−
Nf) film was fabricated by dropping a Gr−Nf homogeneous
suspension to enlarge the surface of the electrodes. Then gold
nanoparticles (AuNPs) were electrodeposited on the modified
electrode, which could provide a rough and stable surface that
was more favorable for immobilization of thrombin binding
EXPERIMENTAL METHODS
■
Reagents. Thrombin (TB, enzymatic activity of 40−300
NIH units/mg, lyophilized power), β-lactamase (1000 units/
mg), ampicillin (Amp), 1-hexanethiol (HT, 96%), chloroauric
acid (HAuCl4·3H2O), Nafion (Nf), N-hydroxy succinimide
(NHS) and N-(3-dimethyl aminopropyl)-N-ethylcarbodiimide-
hydrochloride (EDC) were received from Sigma-Aldrich Co.
(St. Louis, MO, USA). Poly(ethylenimine) (PEI, 50%) was
obtained from Fluka (Switzerland). Graphene (Gr) and the
carbon nanotubes (CNTs, 95% purity) were purchased from
Nanjing Xianfeng nano Co. (Nanjing, China). Prior to use, the
CNTs were pretreated with concentrated nitric acid in order to
5874
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Scheme 2. Schematic Diagrams of the Aptasensor
introduce carboxylic acid groups according to the previous
literature.18 γ-Glutamyltranspeptidase (γ-GTP) antigen and
alpha-L-fucosidase (AFU) antigen were bought from Biocell
Company (Zhengzhou, China). Polyvinylpyrrolidone (PVP),
RuCl3·3H2O and 1,10-phenanthroline (phen, 99%) were
purchased from Chengdu Chemical Reagent Company
(Chengdu, China). Thrombin binding aptamers (TBA) were
received from TaKaRa (Dalian, China), and the sequences of
the oligonucleotide were as follows:
TBA I: 5′-SH-(CH2)6-GGTTGGTGTGGTTGG-3′.
TBA II: 5′-NH2-(CH2)6-AGTCCGTGGTAGGGCAG-
GTTGGGGTGACT-3′.
Tris−HCl buffer, contaning 140 mM NaCl, 5 mM KCl and 1
mM MgCl2, was used to prepare the aptamer solution.
Phosphate buffer solution (PBS) (pH 7.4) was prepared
using 0.1 M Na2HPO4, 0.1 M KH2PO4, 0.1 M KCl. Double-
distilled water was used throughout every experiment. All other
reagents were of analytical grade and used as received without
further purification.
Apparatus. Cyclic voltammetry (CV) was monitored by a
CHI Instruments Model 660D electrochemical workstation
(Shanghai Chenhua Instrument Co., Ltd., China). The ECL
emission was carried out with an MPI-A electrochemilumi-
nescence analyzer (Xi′an Remax Electronic Science &
Technology Co. Ltd., Xi′an, China) in PBS (0.1 M, pH 7.4)
with the voltage of the photomultiplier tube (PMT) at 800 V in
the air. A conventional three-electrode system was used with a
glassy carbon electrode (GCE, φ = 4 mm) as the working
electrode, a platinum wire as the counter electrode and Ag/
AgCl (saturated KCl) as the reference electrode in the
experiment. The morphologies of various nanomaterials were
conducted by a scanning electron microscopy (SEM) system
(SEM, S-4800, Hitachi) with an acceleration voltage of 10 kV.
X-ray photoelectron spectroscopy (XPS) measurement was
performed by a VG Scientific ESCALAB 250 spectrometer
(Thermoelectricity Instruments, USA) with Al Kα X-ray
(1486.6 eV) as the light source.
Preparation of Ru−Amp Complex. The synthetic
procedure of the Ru−Amp complex is given in Scheme 1. 5-
(3-Carboxypropionyl)amino-1,10-phenanthroline (cpaphen)
(1) was prepared by reaction of the 1,10-phenanthrolin-5-
amine with succinic anhydride. cis-Ru(phen)2Cl2 (2) was
synthesized by an analogous route to that in the literature.19
[Ru(phen)2(cpaphen)](PF6)2 (3) was synthesized by a
reaction of 1 with 2. The Ru−Amp complex was synthesized
by reaction of the appropriate Amp with 3 using EDC and
NHS as the linkage reagents. Detailed information is shown in
the Supporting Information.
Preparation of Gold Nanocages (AuNCs). AuNCs were
prepared by the galvanic replacement reaction between Ag
nanocubes and HAuCl4 solution according to the literature20
with a little modification. The first step: synthesis of Ag
nanocubes (AgNCs). Ethylene glycol (EG) (30 mL) was
placed into a three-neck flask and the temperature approached
but did not exceed 160 °C. With magnetic stirring, 10 mL of
PVP/EG (0.2 g) solution was added at 160 °C. Then, 400 μL
of 3 mM Na2S/EG solution was added dropwise. Lastly, 3 mL
of Ag/EG (282 mM) was added dropwise. The color of the
solution changed from white to brown. Then the dispersion
was collected by centrifugation and washed several times by
acetone. The second step: synthesis of AuNCs. The obtained
AgNCs were suspended in 30 mL of double-distilled water and
refluxed for 10 min. Then, 100 μL of HAuCl4 (10 mg·mL−1)
was added slowly and the reflux continued for 30 min. The
color of the solution changed from brown to pink. Then the
pink dispersion was collected by centrifugation and washed
several times by double-distilled water. The resultant AuNCs
were dispersed in double-distilled water and used in subsequent
experiments.
Preparation of Ru−Amp@CNTs−PEI−AuNCs−TBA II
(TBA II Bioconjugates). First, the pretreated CNTs (0.5 mg/
5875
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Figure 1. SEM of AuNCs (A), Ru−Amp@CNTs−PEI (B) and Ru−Amp@CNTs−PEI−AuCNs (C). XPS analysis of Ru−Amp@CNTs−PEI−
AuCNs nanocomposite (D).
mL) were dispersed and sonicated in double-distilled water to
obtain a homogeneous dispersion. The nanocomposite Ru−
Amp@CNTs was achieved by simply stirring a solution of Ru−
Amp (400 μL, 0.5 mg mL−1) together with homogeneous
CNTs (2 mL) at 4 °C for about 16 h through noncovalent π−π
stacking interactions.21 Following this, the precipitates were
collected by centrifugation and washed several times with
double-distilled water. After that, Ru−Amp@CNTs were
dispersed in 1 mL 1% PEI with the aid of agitation for 6 h
to obtain more active −NH2 groups via electrostatic
interactions.22,23 AuNCs were attached on the Ru−Amp@
CNTs−PEI via Au−N junctions, which were further employed
as carriers for the immobilization of TBA II (2.5 μM) to form
Ru−Amp@CNTs−PEI−AuNCs−TBA II bioconjugates.
Fabrication of the Sensing Interface. Before modifica-
tion, the bare electrodes were pretreated according to the
previous literature.24 Then, 1 mg of Gr was added into 2 mL of
0.5% Nf ethanol solution and sonicated to obtain a
homogeneous suspension (Gr−Nf). Next 3 μL of Gr−Nf was
cast on the pretreated electrode and dried in the air to obtain a
Gr−Nf film. Afterward, electrodeposition was performed in the
mixture solution of 1% HAuCl4 to construct the AuNPs layer
on the electrode surface to get AuNPs/Gr−Nf modified
electrode with constant potential −0.2 V for 30 s. Subsequently,
the AuNPs/Gr−Nf modified electrode was incubated into 10
μL of 2.5 μM TBA I for about 16 h at 4 °C in a moist
environment. Finally, 10 μL of HT (1.0 mM) was drop-coated
onto the modified electrode surface and incubated for 40 min
to block the nonspecific binding sites to obtain HT/TBA I/
AuNPs/Gr−Nf modified electrode.
between TB and TBA, 10 μL of prepared Ru−Amp@CNTs−
PEI−AuNCs−TBA II homogeneous suspension was drop-
coated onto the modified electrode surface and incubated for
50 min. Then, 6 μL of β-lactamase (100 ng/mL), prepared by
Tris−HCl buffer, was drop-coated onto the modified electrode
surface and incubated for 1 h. The resultant electrodes were
rinsed with PBS each step. A schematic representation of the
fabrication of the sandwich ECL aptasensor is shown in Scheme
2. Finally, the ECL intensity of the electrode was performed in
PBS (0.1 M, pH 7.4) at room temperature in the process of
detection.
RESULTS AND DISCUSSION
■
Characteristics of Different Nonmaterials. The as-
synthesized nonmaterials were characterized by SEM (Figure
1). Figure 1A shows the SEM image of AuNCs, from which one
can easily observe the hollow and square structures, which is
similar to the previous description in the literature.20 And the
SEM profiles of CNTs and Ru−Amp@CNTs are shown in
Figure S1 (Supporting Information), which indicated that the
Ru−Amp@CNTs nanocomposites were successfully prepared.
When PEI was wrapped on Ru−Amp@CNTs, the structure of
Ru−Amp@CNTs−PEI was curved and bonded together due to
the glutinosity of PEI (Figure 1B). Then AuNCs were
decorated on the CNTs via Au−N junctions. As we can see
from Figure 1C, the bright slug structure of Ru−Amp@CNTs−
PEI−AuNCs was reserved. It suggested that the surface of
AuNCs was coated by the Ru−Amp@CNTs−PEI nanomaterial
matrix. Furthermore, the Ru−Amp@CNTs−PEI−AuNCs
nanomaterial was also characterized by means of XPS (Figure
1D). The characteristic peaks for Ru 3p, C 1s, Au 4p, N 1s, F
1p, N (A) and O 1s core level regions could be obviously
observed on the nanocomposite. The Ru 3p, C 1s, O 1s and F
Measurement Procedure. The proposed aptasensor was
incubated in 10 μL of TB with different concentrations for 50
min at room temperature. Based on the specific binding
5876
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Figure 2. (A) UV−vis absorption spectra of (a) CNTs, (b) Ru−Amp@CNTs, (c) Ru−Amp@CNTs−PEI composite and (d) Ru−Amp@CNTs−
PEI−AuCNs in double-distilled water. (B) Cyclic voltammograms of the electrode at different stages in 0.1 M PBS (pH 7.4) + 0.1 M KCl + 2.5 mM
[Fe(CN)6]3−/4− solution for (a) bare electrode, (b) Gr−Nf/GCE, (c) AuNPs/Gr−Nf/GCE, (d) TBA I/AuNPs/Gr−Nf/GCE, (e) HT/TBA I/
AuNPs/Gr−Nf/GCE and (f) TB/HT/TBA I/AuNPs/Gr−Nf/GCE.
Figure 3. (A) ECL curves of the aptasensor incubated without (a) and with (b) β-lactamase. (B) ECL dynamic curve of the aptasensor incubated
without (a) and with (b) β-lactamase. Applied potential: 0.2−1.25 V.
1p core level were mainly derived from the Ru−Amp@CNTs
and the Au 4p doublet indicated the presence of AuCNs. The
results all indicated that the nanocomposite was successfully
synthesized.
shown in Figure 2B, a couple of quasi-reversible, well-defined
redox peaks of [Fe(CN)6]3−/4− were observed on the bare
electrode (Figure 2B, curve a). When the Gr−Nf was first
casted onto the electrode, the redox peak current (Figure 2B,
curve b) obviously decreased, as the bulky Nf perturbed the
interfacial electrons transfer. After the electrodeposition of the
AuNPs onto the electrode, the redox peak current (Figure 2B,
curve c) obviously increased because the good conductivity of
the AuNPs monolayer. When the TBA I was drop-coated onto
the modified electrode, a decrease in the peak current was
noted (Figure 2B, curve d). The reason may be that the TBA 1
can hinder the electron transfer capability of the electrode.
After the electrode was blocked with HT, the redox peak
current was decreased continuatively (Figure 2B, curves e).
Finally, the modified electrode was incubated with TB and the
peak current of curve f decreased further, because the captured
TB could hinder the electron transfer.
Effect of β-Lactamase on the Response of the
Aptasensor. To evaluate the ECL efficiency of in situ
enzymatic reaction by β-lactamase, we conducted the contrast
experiment to compare the ECL responses of the aptasensor
incubated without and with β-lactamase. As illustrated in Figure
3A, when the potential reached higher than 1.13 V, an obvious
ECL emission was observed with the peak intensity of 1129 a.u.
(Figure 3A, curve a), indicating that the ECL was from Ru−
Amp light emission. After the incubation with β-lactamase, an
obvious ECL emission of Ru−AmpA was observed from the
potential of 1.07 V with the peak intensity of 6102 au (Figure
3A, curve b). The results indicated that the emission process of
UV−Vis Characterization of TBA II Tags. The absorption
spectrum of the as-formed composite of Ru−Amp@CNTs−
PEI−AuCNs has been measured, which was compared with the
spectra of pure CNTs, Ru−Amp@CNTs and Ru−Amp@
CNTs−PEI, respectively. As shown in Figure 2A, there was no
obvious absorption peaks for pure CNTs (Figure 2A, curve a).
However, the characteristic absorption peaks of the Ru−Amp@
CNTs composite (Figure 2A, curve b) appeared at 206 and 265
nm because the complex of Ru−Amp has several benzenes and
heterocyclic, which could prove the π−π interactions between
CNTs and Ru−Amp. After PEI was covered on the CNTs, the
characteristic absorption peak at 210 nm became higher and the
absorption band was red-shifted (Figure 2A, curve c), owing to
the amide bond between the CNTs and PEI. The result
indicated that the Ru−Amp@CNTs−PEI composite was
successfully synthesized. When AuCNs were conjuncted on
the Ru−Amp@CNTs−PEI composite, there a new absorption
peak appeared at 517 nm (Figure 2A, curve d), which was
attributed to the presence of AuCNs. These results revealed
that the Ru−Amp@CNTs−PEI−AuCNs composite was
successfully prepared.
Electrochemical Behaviors of the Aptasensor. Cyclic
voltammetry (CV) was employed to indicate the electro-
chemical behaviors of the modified electrode at different stages
using the redox probe of ferycyanide ([Fe(CN)6]3−/4−). As
5877
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Ru−AmpA was easier and more efficient than that of Ru−Amp.
Moreover, when β-lactamase was present, a faster ECL reaction
rate (Figure 3B, curve b) was achieved compared with that
without β-lactamase (Figure 3B, curve a). Thus, the comparison
results adequately indicate that the in situ enzymatic reaction by
β-lactamase could enhance the ECL efficiency of the proposed
aptasensor for the ultrasensitive detection of TB.
The mechanism of the ECL reaction of the self-enhanced
ECL luminophore of Ru−Amp or Ru−AmpA is thought to
involve a series of steps including electrode oxidation through
intramolecular electron transfer between the L (L on behalf of
Amp without β-lactamase or AmpA with β-lactamase) and
Ru(II). We propose the probable sensing ECL mechanism of
the self-enhanced luminophore as the following:
Figure 5. ECL-potential curves of the aptasensor with different
concentrations of TB in 2 mL of 0.1 M PBS (pH 7.4). TB
concentration: (a) 0 fM, (b) 1 fM, (c) 5 fM, (d) 10 fM, (e) 50fM, (f)
100fM, (g) 500fM and (h) 1 pM. The insert is a calibration curve for
TB determination.
•+
Ru(II)−L − 2e → Ru(III)−L
Ru(III)−L•+ → Ru(III)L + H+
•
•
*
Ru(III)−L → Ru(II) −L
*
Ru(II) −L → Ru(II)−L + hν
regression equation was I (a.u.) = 1544 log c + 1189 with a
correlation coefficient of 0.992. This suggests that this strategy
is highly sensitive and has great potential for early detection of
TB. Additionally, we also made a comparative study between
proposed aptasensor and previous reported aptasensor (Table
1). We can see that the present work for TB detection shows a
low detection limit over other approaches.
Optimization of the β-Lactamase Concentration. The
concentration of β-lactamase has an important role on the
analytical performance of the proposed ECL aptasensor. As
shown in Figure 4, the ECL intensity was enhanced with
Stability and Repeatability of the Aptasensor. Stability
is one of the important factors to measure the performance of
the aprasensor. It was evaluated by employing one aptasensor
for consecutive cyclic potential scans. As we can see from
Figure 6A, under continuous cyclic potential scans for 13 cycles,
the relative standard deviation (RSD) of ECL was 0.93%. The
result indicated that the proposed aptasensor had excellent
stability. The repeatability of the aptasensor was evaluated by
using five proposed aptasensors incubated with 1 pM TB. All
electrodes exhibited close ECL responses and relative standard
deviations of 1.24%, which was acceptable for detection of TB.
Selectivity of the Aptasensor. To investigate the
selectivity and specificity of the present aptasensor, the
aptasensor was incubated with different interfering substances
such as γ-GTP and AFU. As shown in Figure 6B, the control
experiments were performed by using γ-GTP (100 pg/mL) and
AFU (100 pg/mL) to replace TB (1 pM), respectively. We can
see that the ECL signal of AFU and γ-GTP did not exhibit any
obvious increase compared with the blank. The aptasensor was
also incubated with 1 pM TB containing 100 pg/mL γ-GTP
and 100 pg/mL AFU. When the ECL response of the mixture
was compared with that obtained from the 1 pM TB only, no
remarkable difference was found. They all indicated that the
ECL intensity in the presence of TB was much stronger than
those of the others, which meant that γ-GTP and AFU had no
obvious influence on the response to TB, and indicated that the
aptasensor displayed good selectivity and specificity for the
determination of TB.
Figure 4. ECL effects of aptasensor with the different concentrations
of β-lactamase. Applied potential: 0.2−1.25 V.
increases to the concentration of β-lactamase. When the
concentration of β-lactamase reached 100 ng/mL, the ECL
intensity increased slightly and then reached a constant value.
Thus, 100 ng/mL was chosen as the optimal concentration of
β-lactamase in this experiment.
Detection of TB with the Aptasensor. The relationship
between ECL signals and the concentrations of TB is shown in
Figure 5. As expected, the ECL intensity increased when the
concentration of TB was increased. (Figure 5, curves a−h).
Thus, TB could be quantitatively measured over a concen-
tration range from 1 fM to 1 pM, with an achieved detection
limit of 0.33 fM and the signal-to-noise ratio was 3. The
CONCLUSION
■
In the present study, a sensitive ECL aptasensor based on the
self-enhanced ECL luminophore and the in situ enzymatic
reaction for the signal amplification was developed for TB
detection. The self-enhanced ECL luminophore could over-
come the shortage of high concentration of coreactant needed
in the ECL system and enhance both the ECL efficiency and
5878
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
Table 1. Comparison of the TB Detection with Some Different ECL Emitters
method
fluoresce
label
linear range
LOD
ref
MCMP/FAM-ssDNA
FAM-labeled TA
0.25 nM−100 nM
1 nM−100 nM
1.74 nM−58 nM
4 fM−400 pM
1 pM−5 nM
0.25 nM
1 nM
25
26
27
28
29
fluoresce
SERS
AuNR−AuNP
1.74 nM
2 fM
chemiluminescence
luminol−H2O2−HRP
Fe3O4@CdSe/TPA
Ru@CNTs−AuCNs
ECL
ECL
0.12 pM
0.33 fM
1 fM−1 pM
present work
Figure 6. (A) Stability of the proposed ECL aptasensor incubated with 1 pM TB under consecutive cyclic potential scans for 13 cycles. (B)
Selectivity of the proposed ECL aptasensors with different targets: blank, γ-GTP (100 pg/mL), AFU (100 pg/mL) and TB (1 pM), a mixture (1 pM
TB + 100 pg/mL γ-GTP + 100 pg/mL AFU). (Error bars: SD, n = 3).
electron transfer performances through intermolecular inter-
actions. Moreover, the ECL signal of the Ru−Amp here could
be further enhanced by in situ enzymatic reaction with the aid
of β-lactamase, which could effectively improve the sensitivity
of the ECL aptasensor. On the basis of their excellent
performance, this aptasensor achieved a detection limit of
0.33 fM and exhibits excellent selectivity. The self-enhanced
ECL luminophore, coupled with the further enhancement by in
situ enzymatic reaction, is expected to have potential
applications in biotechnology and clinical diagnosis.
Central Universities (XDJK2012A004, XDJK2014C001,
XDJK2014A012), China.
REFERENCES
■
(1) Swanick, K. N.; Ladouceur, S.; Zysman-Colman, E.; Ding, Z. F.
Chem. Commun. 2012, 48, 3179−3181.
(2) Chen, Z.; Liu, Y.; Wang, Y.; Zhao, X.; Li, J. Anal. Chem. 2013, 85,
4431−4438.
(3) Ding, C.; Wei, S.; Liu, H. Chem.Eur. J. 2012, 18, 7263 −7268.
(4) Kurita, R.; Arai, K.; Nakamoto, K.; Kato, D.; Niwa, O. Anal.
Chem. 2012, 84, 1799−1803.
ASSOCIATED CONTENT
* Supporting Information
(5) Kim, Y.; Kim, J. Anal. Chem. 2014, 86 (3), 1654−1660.
(6) Kumar, S. S.; Bard, A. J. Anal. Chem. 2013, 85, 292−295.
(7) Wei, H.; Liu, J.; Zhou, L.; Li, J.; Jiang, X.; Kang, J.; Yang, X.;
Dong, S.; Wang, E. Chem.Eur. J. 2008, 14, 3687−3693.
(8) White, H. S.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 6891−6895.
(9) Liu, X.; Shi, L.; Niu, W.; Li, H.; Xu, G. Angew. Chem. 2007, 119,
425−428.
■
S
Details of the preparation of Ru−Amp complex and the SEM
profiles of CNTs and Ru−Amp@CNTs. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Y. Zhuo. Tel.: +86 23 68252277. Fax: +86 23 68253172. E-
mail: yingzhuo@swu.edu.cn.
(10) Sun, J.; Liang, P.; Martin, M. T.; Dong, L. US Patent
20050181443, August 18, 2005.
(11) Sun, J.; Liang, P.; Martin, M. T.; Dong, L. US Patent
■
20010003647, June 14, 2001.
(12) Sun, S. G.; Yang, Y.; Liu, F. Y.; Fan, J. L.; Peng, X. J.; Kehr, J.;
Sun, L. C. Dalton Trans. 2009, 38, 7969−7974.
(13) Sun, S. G.; Yang, Y.; Liu, F. Y.; Fan, J. L.; Peng, X. J.; Kehr, J.;
Sun, L. C. Dalton Trans. 2010, 39, 8626−8630.
(14) Zhuo, Y.; Liao, N.; Chai, Y. Q.; Gui, G. F.; Zhao, M.; Han, J.;
Xiang, Y.; Yuan, R. Anal. Chem. 2014, 86 (2), 1053−1060.
(15) Liang, P.; Dong, L. W.; Martin, M. T. J. Am. Chem. Soc. 1996,
118, 9198−9199.
*R. Yuan. Tel.: +86 23 68252277. Fax: +86 23 68253172. E-
mail: yuanruo@swu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the NNSF of China
(21275119, 21105081, 21075100), Research Fund for the
Doctoral Program of Higher Education (RFDP)
(20110182120010), Ministry of Education of China (Project
708073), Specialized Research Fund for the Doctoral Program
of Higher Education (20100182110015) and Natural Science
Foundation Project of Chongqing City (CSTC-2010BB4121,
CSTC-2009BA1003), the Fundamental Research Funds for the
(16) Ding, S. N.; Shan, D.; Cosnier, S.; Goff, A. L. Chem.Eur. J.
2012, 18, 11564−11568.
(17) Au, L.; Chen, Y.; Zhou, F.; Camargo, P. H. C.; Lim, B.; Li, Z. Y.;
Ginger, D. S.; Xia, Y. Nano Res. 2008, 1, 441−449.
(18) Wen, Z. H.; Ci, S. Q.; Li, J. H. J. Phys. Chem. C 2009, 113,
13482−13487.
(19) Ji, Z.; Huang, S. D.; Guadalupe, A. R. Inorg. Chim. Acta 2000,
305, 127−134.
5879
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880

Analytical Chemistry
Article
(20) Skrabalak, S. E.; Chen, J.; Sun, Y.; Lu, X.; Au, L.; Cobley, C. M.;
Xia, Y. Acc. Chem. Res. 2008, 41, 1587−1595.
(21) Tran, P. D.; Goff, A. L.; Heidkamp, J.; Jousselme, B.; Guillet, N.;
Palacin, S.; Dau, H.; Fontecave, M.; Artero, V. Angew. Chem. 2011,
123, 1407−1410.
(22) Jiang, L.; Gao, L. Carbon 2003, 41, 2923−2929.
(23) Liu, Y.; Wu, D. C.; Zhang, W. D.; Jiang, X.; He, C. B.; Chung, T.
S.; Goh, S. H.; Leong, K. W. Angew. Chem., Int. Ed. 2005, 44, 4782−
4785.
(24) Gui, G. F.; Zhuo, Y.; Chai, Y. Q.; Liao, N.; Zhao, M.; Han, J.;
Zhu, Q.; Yuan, R.; Xiang, Y. Biosens. Bioelectron. 2013, 47, 524−529.
(25) Zhang, Y.; Liu, S.; Sun, X. Biosens. Bioelectron. 2011, 26, 3876−
3880.
(26) Li, H.; Zhang, Y.; Luo, Y.; Sun, X. Small 2011, 7, 1562−1568.
(27) Wang, Y.; Lee, K.; Irudayaraj, J. Chem. Commun. 2010, 46, 613−
615.
(28) Huang, Y.; Zhao, S.; Chen, Z. F.; Shi, M.; Chen, J.; Liang, H.
Chem. Commun. 2012, 48, 11877−11879.
(29) Jie, G. F.; Yuan, J. X. Anal. Chem. 2012, 84, 2811−2817.
5880
dx.doi.org/10.1021/ac500665b | Anal. Chem. 2014, 86, 5873−5880