A new signal-on photoelectrochemical biosensor based on a graphene/quantum-dot nanocomposite amplified by the dual-quenched effect of bipyridinium relay and AuNPs

Article abstract of DOI:10.1002/chem.201202213

A new photoelectrochemical (PEC) biosensor was developed by using carboxyl-functionalized graphene and CdSe nanoparticles. This sensitive interface was then successfully applied to detection of thrombin based on the dual-quenched effect of PEC nanoparticle, which relied on the electron transfer of a bipyridinium relay and energy transfer of AuNPs. After recognition with an aptamer, the PEC nanoparticle was removed and a signal-on PEC biosensor was obtained. Moreover, the bio-barcode technique used in the preparation of PEC nanoparticle could avoid cross-reaction and enhances the sensitivity. Taking advantages of the various methods mentioned above, the sensitivity could be easily enhanced. In addition, in this work we also investigated graphene that was modified with different functional groups and AuNPs of different particle sizes. Under optimal conditions, a detection limit of 5.9×10^-15 M was achieved. With its simplicity, selectivity, and sensitivity, this strategy shows great promise for the fabrication of highly efficient PEC biosensors. PECking order! A new photoelectrochemical (PEC) biosensor was developed by using carboxyl-functionalized graphene and CdSe nanoparticles (NPs). This sensitive interface was successfully applied to detection of thrombin based on the dual-quenched effect of a PEC nanoparticle, which relied on the electron transfer of a bipyridinium relay and energy transfer of AuNPs (see figure). After recognition with an aptamer, the PEC nanoparticle was removed and a signal-on PEC biosensor was obtained. (TEOA=triethanolamine.).

Full text of DOI:10.1002/chem.201202213

FULL PAPER
DOI: 10.1002/chem.201202213
A New Signal-On Photoelectrochemical Biosensor Based on a Graphene/
Quantum-Dot Nanocomposite Amplified by the Dual-Quenched Effect of
Bipyridinium Relay and AuNPs
Xiaoru Zhang,[a] Yunpeng Xu,[a] Yanqiang Yang,[b] Xia Jin,[a] Sujuan Ye,[a]
Shusheng Zhang,*[a] and Lilin Jiang[b]
Abstract: A new photoelectrochemical
(PEC) biosensor was developed by
using carboxyl-functionalized graphene
and CdSe nanoparticles. This sensitive
interface was then successfully applied
to detection of thrombin based on the
dual-quenched effect of PEC nanopar-
ticle, which relied on the electron
transfer of a bipyridinium relay and
energy transfer of AuNPs. After recog-
nition with an aptamer, the PEC nano-
particle was removed and a signal-on
PEC biosensor was obtained. More-
over, the bio-barcode technique used
in the preparation of PEC nanoparticle
could avoid cross-reaction and enhan-
ces the sensitivity. Taking advantages
of the various methods mentioned
above, the sensitivity could be easily
enhanced. In addition, in this work we
also investigated graphene that was
modified with different functional
groups and AuNPs of different particle
sizes. Under optimal conditions, a de-
tection limit of 5.9ꢀ10ꢀ15 m was ach-
ieved. With its simplicity, selectivity,
and sensitivity, this strategy shows
great promise for the fabrication of
highly efficient PEC biosensors.
Keywords: biosensors
transfer · energy transfer · graph-
ene · nanoparticles
·
electron
AHCTUNGTRENNUNG
Introduction
process. Hence, some strategies have been attempted includ-
ing the introduction of nanomaterials with a suitable band
energy as efficient acceptors.[8,9]
The photoelectrochemical detection method is a newly de-
veloped and promising analytical method for a biological
assay.[1] By using photoirradiation coupled with electrochem-
ical detection, the photoelectrochemical sensor is very sensi-
tive with low background signals. Photoelectrochemically
active species that are usually used are rutheniumbipyridine
derivatives,[2,3] semiconductor nanostructures (such as CdSe/
Graphene, as an atomic-layer-thick 2D system, has trig-
gered considerable research interests owing to its unique op-
tical, electronic, thermal, and mechanical properties. These
fascinating properties of graphene hold promising potential
for electronic devices, sensitive chemical sensors, thermal
management, and composite materials.[10,11] Recently, Chen
et al.[11] has reviewed the applications of graphene in organic
photovoltaic (OPV) cells, including transparent electrodes,
active layers, and interface layers. As a good candidate for
the acceptor material in OPV applications, graphene has
large donor/acceptor interfaces for charge generation and a
continuous pathway for electron transfer. The groups of
Lin[12] and Li[13] have reported quantum dot (QD)-sensitized
graphene heterostructures prepared by in situ growth of
QDs on graphene, and applied them to construct QD-sensi-
tized photoelectrochemical cells. Geng et al.[10] has fabricat-
ed composite films through the noncovalent coupling of
CdSe QDs to graphene for flexible and transparent opto-
[5]
CdS,[4] TiO2 and ZnO[6]), and dyes.[7] Among them, semi-
conductor nanoparticles (NPs) have attracted much atten-
tion, due to the unique size- and shape-dependent optical
and electronic properties. The photochemical excitation of
semiconductor NPs to form an electron–hole pair is the pri-
mary event for the photocurrent generation. However, the
transfer of conduction-band electrons to the electrode is
hampered by the competing electron–hole recombination
[a] Dr. X. Zhang, Y. Xu, X. Jin, S. Ye, Prof. S. Zhang
Key Laboratory of Biochemical Analysis
Ministry of Education, College of Chemistry and
Molecular Engineering
AHCTUNGTRENNUNG
Qingdao University of Science and Technology
Qingdao 266042 (P.R. China)
AHCTUNGTRENNUNG
workers[15] have prepared a graphene–CdS composite in a
one-step synthesis, which was then used for the fabrication
of a photoelectrochemical cytosensor by using a layer-by-
layer assembly process. Our group has prepared a photo-
electrochemical biosensor based on functionalized graphene
and CdSe nanoparticles multilayers.[16] In all these studies,
QD–graphene composites were prepared by in situ growth,
Fax : (+86)532-84022750
E-mail: shushzhang@126.com
[b] Prof. Y. Yang, Dr. L. Jiang
Center for Condensed Matter Science and Technology
Department of Physics, Harbin Institute of Technology
Harbin 150001 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202213.
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physical adsorption and chemical modification. It is expect-
ed that the chemical modification could not only enhance its
solubility and processability, but also render new properties
to graphene and even greatly widen its application scope.
Thus, developing chemical methods to functionalize graph-
widely utilized in bioaffinity sensors due to their physical
properties. It is well known that when CdS QDs and AuNPs
are in close proximity under certain conditions, a unique
phenomenon of interparticle energy-transfer will occur.[26–28]
Recently, Xu and co-workers[29] have reported the energy
transfer between CdS QDs and AuNPs in a PEC detection
system and a sensitive biosensing of DNA was realized
based on this mechanism.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
with azide groups at one end and carboxyl group at the
other end was utilized to prepare carboxyl-functionalized
graphene by using nitrene cycloaddition.[17] The resulting
functionalized graphene nanosheets are electrically conduc-
tive, readily dispersible in solvents and easily processable,
making them excellent candidates for further modification
with QD.
A graphene–QD nanocomposite was assembled for the
construction of the photoelectrochemical biosensor. Photo-
current variation was produced prior to and after the biorec-
In this study, we report an efficient method to fabricate a
signal-on PEC sensor. Carboxyl-functionalized graphene
was synthesized to construct a CdSe QD-sensitized graph-
AHCTUNGTREGeNNNU ne PEC-sensing interface. The dual-quenched PEC film,
achieved by electron transfer of a bipyridinium relay and
energy transfer of AuNPs, largely lowered the initial signal.
After removal of bipyridinium relay and AuNPs through a
biorecognition event, significant signal restore could be ob-
tained. By integrating the signal amplification effect of func-
tionalized graphene and the dual-quenched effect of bipyri-
dinium relay and AuNPs in a conventional analytical strat-
egy, thrombin could be sensitively detected. Furthermore,
this work opens up the possibility for designing more effi-
cient PEC biosensors.
ACHTUNGTRENNUNGognition event. In terms of signal changes, sensors could be
classified into two types: signal-off and signal-on. In the
case of a signal-off photoelectrochemical biosensor, the rec-
ognition of proteins on the modified electrode generate a
hydrophobic layer and thus partly hinder the diffusion of a
sacrificial electron-donor to the surface of CdS or CdSe
QDs, which lead to a decrease of photocurrent. So far, most
photoelectrochemical biosensors developed belong to this
kind.[15,18–20] However, these signal-off sensors suffer from
false positives when photoactive species become degraded
and are limited in their signal gain because the target can
suppress no more than 100%
Results and Discussion
Principle of the assay for the detection of thrombin: The
fabrication of the sensing design is shown in Scheme 1. First-
of the original signal.[21,22] In
this regard, signal-on sensors
are highly deserved.
The photoinduced electron-
transfer between semiconduc-
tor nanoparticle and N,N’-di-
methyl-4,4’-bipyridinium (V2+
)
salts has attracted a great deal
of attention by the group of
Willner.[23,24,25] The concept of
vectorial electron-transfer was
adopted in semiconductor
nanoparticle systems. When
the CdS NPs are located close
to the electrode and positioned
between the relay units V2+
and electrode through the hy-
bridization of template DNA,
the photocurrent that is meas-
ured is substantially lower than
the photocurrent measured in
the presence of the CdS NPs
alone. This implies that the bi-
pyridinium acceptor units can
act as conduction-band elec-
tron traps. Besides photoin-
duced electron-transfer of bi-
pyridinium, AuNPs have been
Scheme 1. Schematic diagram for preparation of carboxyl-functionalized graphene and the fabrication of am-
plified signal-on thrombin biosensor.
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Graphene–Quantum Dot Photoelectrochemical Biosensor
FULL PAPER
ly, the carboxyl-functionalized graphene was synthesized
based on nitrene chemistry.[16,30] 4-(2-azidoethoxy)-4-oxobu-
tanoic acid, which contains both azido and carboxyl groups
in one molecule, was prepared by the acylation of succinic
anhydride with 2-azidoethanol. Then, the azido group was
anchored onto graphene by an electrophilic [2+1] cycloaddi-
tion to afford the aziridine rings. The left carboxyl group ex-
tended into the surrounding solvent to solubilize graphene
and could be used for further chemical modification. Sec-
ondly, the indium tin oxide (ITO) electrode was silanized
with aminopropyltriethoxysilane (APS) and then the car-
boxyl-functionalized graphene was assembled onto an
amino-functioned electrode through an amide bond. Then,
CdSe-NH2 NPs was also assembled on the surface graphene
through an amide bond to give the photoactive film. The
PO3H groups that were used to modify the capture DNA
were covalently bound to the CdSe NPs by using the classic
ꢀ
coupling reactions between NH2 groups on the surfaces of
ꢀ
CdSe NPs and PO3H groups of DNA. Finally, the capture
DNA underwent hybridization with the thrombin aptamer
on PEC nanoprobes, which contained V2+ and the aptamer
on the bio-barcode of AuNPs. The photocurrent was re-
duced due to the introduction of AuNPs and V2+ to the sur-
face of electrode. After recognition of thrombin by the ap-
tamer, the PEC nanoprobes were removed and the photo-
current was enhanced accordingly. Thus, a signal-on PEC
bioACHTUNGTRENNUNGsensor for the detection of thrombin was obtained.
Characterization of graphene and the CdSe NP-modified
ITO electrode and the performance of composite material:
To confirm that the successful assembly of graphene and
CdSe NPs on ITO electrode, SEM was performed and the
images were generated. Figure 1A is a typical SEM image
of G-COOH assembled on an APS-modified ITO electrode,
showing that the functionalized graphene has a sheet-like
morphology with a clear, smooth surface. Figure 1B shows
Figure 1. SEM images of A) G-COOH and B) G-COOH/CdSe composite
film on the ITO electrode.
ꢀ
the SEM image of the composite film, in which the CdSe
NH2 NPs were attached to the graphene. The NPs uniformly
distributed on graphene, evidencing the well-behaved as-
sembly process.
To verify the amplification effect of graphene on the pho-
tocurrent, the photocurrents generated by graphene, CdSe-
QDs and QD-sensitized graphene on ITO electrodes were
measured under the same conditions. As demonstrated in
curve (c) of Figure 2, the QD-sensitized graphene photo-
electrode generated photocurrent at about 1.65 mA, whereas
the control experiments showed no significant photocurrent
generation by graphene-modified ITO (curve a) and only
about 520 nA for the pure CdSe-QD-modified ITO elec-
trode (curve b), which indicated the enhancing ability of
graphene. This could be explained by two aspects: Firstly,
the two-dimensional sheet morphology of graphene provid-
ed a large surface area for QD-loading, which facilitated the
carrying of more photoactive QDs and capturing more light.
Secondly, the electron-accepting ability of graphene might
contribute to the enhancement of electron transport and
thus impede the charge recombination of excited CdSe.[8,15]
Figure 2. Photocurrent responses for a) G-COOH; b) CdSe-NH2 QDs;
c) G-COOH and CdSe-NH2 QD-modified ITO electrodes.
The photoluminescence (PL) spectra were also used to il-
ꢀ
lustrate the role of graphene. The PL spectra of pure CdSe
NH2 NPs solution and the graphene-CdSe NPs mixture were
measured. As can be seen in Figure 3, a dominant emission
peak at 549 nm could be clearly observed for the pure
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16413

S. Zhang et al.
Figure 4. Photocurrent value for a) CdSe-NH2; b) CdSe-NH2/G-COOH;
c) CdSe-COOH; d) CdSe-COOH/G-NH2-modified Au electrode. Inset
from left to right: Photographs of G-COOH, G-NH2, and GO dispersed
in water.
Figure 3. Absorption spectrum of a) AuNPs as well as the corresponding
PL spectra (excited at 400 nm) of b) pure CdSe NPs and c) graphene-
CdSe NPs mixture.
CdSe þ hv ! CdSe þ ðh þ eÞ
CdSe ðh þ eÞ ! CdSe þ hv0
ð1Þ
ð2Þ
ð3Þ
ð4Þ
CdSe-NH2 NPs (red), whereas the PL of graphene-CdSe
NPs mixture is too weak to be identified (blue). The signifi-
cantly quenched PL can be attributed to the fact that the
photoinduced electrons and holes in the CdSe NPs prefer
separately transferring to the graphene and electron–hole
pairs recombination of CdSe NPs is hampered.[12]
Chemical modification could not only enhance its solubili-
ty and processability, but also render new properties to
graphACHTUNGTRENNUNGene. The properties of graphene may be different
CdSe ðh þ eÞ þ MV2þ ! CdSe ðhtÞ þ MV
þC
þC
2þ
CdSe ðhtÞ þ MV ! CdSe þ MV
when it is modified through various methods. Here we com-
pared the photoelectrochemical interface made from car-
boxyl-modified graphene (G-COOH, inset of Figure 4A)
with amino-modified graphene (G-NH2, insert of Fig-
ure 4B), which was assembled with an amino- or carboxyl
group-modified CdSe QD, respectively. This contrast experi-
ment was carried out on planar Au electrode for easy assem-
bly (for the preparation process see the Supporting Informa-
The study of photoinduced electron-transfer effect of viol-
ogen and its usage in the detection of thrombin was describ-
ed in Scheme 2 and the change of photocurrent during each
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
step was described in Figure 5A. For G-COOH/CdSe-NH2
nanocomposite film on ITO, as described earlier, a large
photocurrent was observed (1.65 mA, curve c). When cap-
ture DNA was covalently attached on CdSe NPs, the photo-
current was increased further (1.98 mA, Figure 5A; curve d).
Then, the photocurrent decreased after the bipyridinium-
functionalized aptamer nucleic acid hybridized with the cap-
ture DNA template (Figure 5A; curve a, 1.28 mA). This
result could be explained by the fact that in addition to the
ejection of the conduction-band electrons to the electrode,
the other competitive electron-transfer path that exists in-
volves the ejection of the photoexcited conduction-band
electrons to the bipyridinium relay (see Figure 5B). The net
photocurrent in the system is the result of the oppositely di-
rected photocurrents.[23,24] Finally, after the recognition of
aptamer DNA with 1.0ꢀ10ꢀ11 m thrombin, the photocurrent
was restored to about 1.54 mA for the removal of bipyridini-
um (Figure 5A; curve b).
ꢀ
tion). As shown from Figure 4, the photocurrent of CdSe
NH2 QD alone was 364 nA. After assembly with G-COOH,
the photocurrent increased to 1.54 mA. Nevertheless, the en-
hancement effect for G-NH2 to CdSe-COOH was not as dis-
tinct as the former. The excellent solubility of G-COOH
compared with G-NH2 is an important factor for the im-
proved performance of G-COOH.
Photoinduced electron-transfer effect of viologen: MV2+ is
widely used as an electron acceptor in molecular and nano-
particle systems. From previous work,[31,32] the electrons ac-
cumulated within the conduction band of the CdSe (ECB
=
ꢀ0.6 V vs NHE)[33] are energetic enough to transfer to
MV2+
+C
(E0(MV2+/MV )=
ꢀ0.445 V vs. NHE).[34,35] The
photoinduced reactions be-
tween CdSe and MV2+ are
summarized in Equations (1)–
(4) in which hv is photon
energy, h and e represent holes
and electrons, respectively, and
ht represents shallow and
deep-trap holes..[31]
Scheme 2. Schematic diagram for the detection of thrombin based on the photoinduced electron-transfer
effect of viologen alone.
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Graphene–Quantum Dot Photoelectrochemical Biosensor
FULL PAPER
Figure 6. TEM images of A) AuNPs: 5 nm, B) AuNPs: 18 nm, and
C) AuNPs: 45 nm.
Figure 5. A) Photocurrent responses for a) ITO/G-COOH/CdSe/capture
DNA/V2+; b) ITO/G-COOH/CdSe/capture DNA/V2+/1.0ꢀ10ꢀ11 m throm-
bin; c) ITO/G-COOH/CdSe; d) ITO/G-COOH/CdSe/capture DNA.
B) Schematic diagram for the direction of photocurrent under the action
of V2+
.
Energy transfer between CdSe QDs and AuNPs: Recently,
Xu and co-workers have reported that when CdS QDs and
AuNPs have a larger interparticle distance in a PEC system,
a
unique phenomenon of interparticle energy transfer
occurs.[29] Herein, the PL- and UV/Vis absorption spectra
were analyzed to verify this phenomenon. As shown in
Figure 3, the PL spectrum of the prepared CdSe QDs,
whose main peak centered at 549 nm and shoulder peak
centered at 537 nm, was observed with excitation wave-
length of 400 nm (red). However, the UV/Vis absorption
spectrum of AuNPs observed at 520 nm (black) has a large
spectral overlap with the emission spectrum of the CdSe
QDs. So, energy transfer between CdSe QDs and AuNPs
could lead to the reduction of photocurrents produced by
the film of CdSe QDs.
Figure 7. A) Photocurrent value of different particle sizes. 1, 2, and 3 rep-
resent the photocurrent response of particles of sizes 5, 18, and 45 nm, re-
spectively. White columns represent the original photocurrent without
AuNPs and V2+. The black columns indicate the value of the photocur-
rent after introducing AuNPs alone. The gray columns represent the pho-
tocurrent after introducing PEC nanoprobes, which contained both
AuNPs and V2+. B) Schematic diagram for the direction of photocurrent
under the action of PEC nanoprobes.
The role of AuNPs in this design is two-fold: First, it can
serve as carrier, supporting relay viologen through cystea-
mine. Second, the presence of AuNPs can cause energy
transfer, which leads to a further decrease of the photocur-
rent. Here we prepared the AuNPs in three different sizes
(5, 18, and 45 nm; for preparation see the Supporting Infor-
mation). TEM images of prepared AuNPs showed regular
shape and homogeneous distribution (Figure 6). The influ-
ence of different particle size on the depression of the pho-
tocurrent was investigated in Figure 7A. Comparing the
white and black columns, the decrease in the photocurrent
was 43.5, 29, and 19.2%, respectively, for the differently
sized AuNPs (5, 18, and 45 nm); the optimal energy-transfer
efficiency was produced by 5 nm AuNPs. After incorporat-
ing V2+, the decrease of photocurrents produced by PEC
nanoprobes was 72, 79 and 51%, respectively; however, this
might be caused by the high-loading capacity of bigger
AuNPs. Taking these factors into account, 18 nm AuNPs
were used in following experiment. The influence of PEC
nanoprobes to photocurrent is illustrated in Figure 7B. By
incorporating the electron-transfer effect of the bipyridini-
um relay and energy-transfer effect of AuNPs, the photocur-
rent was reduced significantly.
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S. Zhang et al.
Sensitivity: An aptamer can bind tightly and specifically to
its target molecules with a binding constant greater than
that of an ordinary double-stranded DNA. So, when the as-
sembled electrode ITO/graphene/CdSe/AuNPs/V2+ was
added to a solution containing thrombin, the double helixes
were opened and the PEC nanoprobes were detached from
the surface of electrode, which led to the increase of photo-
currents to produce a signal-on photoelectrochemical bio-
sensor. Figure 8A showed the photocurrent signals that
Selectivity: Control experiments were conducted to reveal
the specificity of the recognition reaction for thrombin. As
seen in Figure 9, when thrombin (1.0ꢀ10ꢀ13 m) was added,
Figure 9. The selectivity of thrombin toward different analytes.
the photocurrent increased by 270 nA. However, no obvious
photocurrent change was detected when the aptamer was
treated with foreign proteins, such as bovine serum albumin
(BSA), hemoglobin (BHb), lysozyme, and glucose oxidase
(GOD), even with concentrations of 104-fold excess. This
may be explained by the specific binding between the target
and the aptamer.
Conclusion
We have prepared a new composite material interface
through a simple approach and developed a signal-on PEC
biosensor based on dual-quenched mechanism. Several ad-
vantages of our method have been demonstrated: 1) Car-
boxyl-functionalized graphene was prepared and used as a
good candidate for the collection and transport of photogen-
erated charges of CdSe NPs for the first time. Such compo-
site material interface possesses higher sensitivity and is
promising as a PEC biosensor. 2) Compared with the con-
ventional signal quenching PEC biosensors, a signal-on PEC
assay was developed that could avoid false-positive results
and enhance the accuracy of the result. 3) The dual-
quenched PEC nanoparticle, achieved by electron transfer
of bipyridinium relay and energy transfer of AuNPs, could
largely reduce the initial signal before recognition of throm-
bin and improve the detection sensitivity greatly. 4) The spe-
cificity of a bio-barcode and the recognition of an aptamer
were integrated in photoelectrochemical biosensor. In this
way, improvement of selectivity and sensitivity could be
easily achieved. So, with its simplicity, selectivity, and sensi-
tivity, the present work details a promising new approach
for highly efficient photoelectrochemical biosensors, which
can expand the application scope of photoelectrochemistry,
and has extensive potential in analytical applications.
Figure 8. A) Photocurrent responses for different concentrations of
thrombin (in m): a) 0, b) 2.0ꢀ10ꢀ14, c) 4.0ꢀ10ꢀ14, d) 6.0ꢀ10ꢀ14, e) 8.0ꢀ
10ꢀ14, f) 1.0ꢀ10ꢀ13, g) 2.0ꢀ10ꢀ13, h) 4.0ꢀ10ꢀ13, i) 6.0ꢀ10ꢀ13, j) 8.0ꢀ10ꢀ13
,
k) 1.0ꢀ10ꢀ12 l) 2.0ꢀ10ꢀ12
, . B) Calibration curve corresponding to the
analysis of different concentration of thrombin from 2.0ꢀ10ꢀ14 to 2.0ꢀ
10ꢀ12 m. Inset: the linear range from 2.0ꢀ10ꢀ14 to 2.0ꢀ10ꢀ13 m.
were responsive to the changing concentrations of thrombin
(a–l). The photocurrent intensity gradually increased with
increasing thrombin concentrations. As shown in Figure 8B,
the increase of photocurrent intensity was linearly related to
the concentration of thrombin (cthrombin) in the range from
2.0ꢀ10ꢀ14 to ꢁ2.0ꢀ10ꢀ13 m. The regression equation was DI
(nA)=ꢀ6.25+26.39c
(10ꢀ14 m) with a regression coeffi-
thrombinACTHUNGTRENNNUG
cient of 0.9982. The relative standard deviation for 11 repeti-
tive measurements of 6.0ꢀ10ꢀ14 m was 4.3%, and the limit
of quantification (LOQ) was 2.0ꢀ10ꢀ14 m (10s) under the
optimum conditions. With the amplification by AuNPs, the
sensitivity of the proposed method was enhanced about 100-
fold compared with a simple photoelectrochemical detection
based on V2+ (see Figure S3 in the Supporting Information).
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Graphene–Quantum Dot Photoelectrochemical Biosensor
FULL PAPER
incubated at room temperature for 12 h. After rinsing once with PBS and
twice with distilled water, the electrode was dried under N2 atmosphere,
before CdSe-NH2 NPs (10 mL; synthesis detailed in the Supporting Infor-
mation) was casted on and incubated at room temperature for 2 h. The
electrode was then washed twice with distilled water and dried under N2
atmosphere to give the photoelectrochemically active interface.
Experimental Section
Materials: Sodium azide, iodomethane, succinic anhydride, triethylamine
(Et3N), 3-chloropropylamine hydrochloride, N,N-dimethyl formamide
(DMF), N-methyl-2-pyrrolidinone (NMP), N,N-(dimethylamino)-pyridine
(DMAP), 2-chloroethanol, and 4,4’-bipyridine were obtained from Sino-
pharm Chemical Reagent Co., Ltd. 3-aminopropyltriethoxysilane (APS),
HAuCl4· 4H2O (48%, w/w), ascorbic acid (AA), N-hydroxysuccinimide
(NHS) and 1-(3-dimethylamino- propyl)-3-ethylcarbodiimide hydrochlor-
ide (EDC) were purchased from Aladdinꢂs reagent (Shanghai) Co., Ltd.
Glucose oxidase (GOD), bovine serum albumin (BSA), hemoglobin
(BHb), and lysozyme were obtained from Sigma–Aldrich. Thrombin was
obtained from Merck-chemicals. 2-Aminothanethiol (AET) and mercap-
toacetic acid were purchased from ACROS Organics (Japan) and used as
received.
Conjugation of capture DNA onto a CdSe nanoparticle-modified elec-
trode was also achieved by using the classic EDC coupling reactions be-
tween NH2 groups on the surfaces of the AET-capped CdSe NPs and the
-PO3H groups of capture DNA. Briefly, phosphate-modified capture
DNA (20 mL, 10ꢀ5 m) was activated in imidazole buffer (pH 6.8; 40 mL,
0.1m) containing EDC (40 mL, 0.2m). Next, activated capture DNA
(10 mL) was casted onto the surface of amino group modified electrode
and incubated at room temperature overnight. After incubation, the elec-
trode was rinsed with 0.4m NaOH and 0.25% SDS solution to remove
the free DNA.
All of synthetic oligonucleotides were purchased from SBS Genetech.
Co. Ltd. (China). Their base sequences are as follows: capture DNA: 5’-
PO3H -TTT TTC CAA CCA CAC CAA CC-3’; thrombin aptamer: 5’-
SH-TTT TTT GGT TGG TGT GGT TGG-3’.
PEC nanoprobes were assembled on electrode by hybridization between
capture DNA and aptamer on nanoprobes. The nanoprobes (10 mL) ob-
tained above were casted on the electrode and incubated at 378C for 2 h.
Finally, the thrombin solutions (10 mL) were casted onto the surface of
the modified electrode and incubated at room temperature for 0.5 h. The
electrode surface was washed with distilled water twice and dried under
N2 atmosphere after each step of fabrication process.
Apparatus: The photocurrent was measured on an electrochemical work-
station (Zahner Zennium, Germany). A three-electrode system was em-
ployed with Pt wire as an auxiliary electrode, Ag/AgCl as a reference
electrode, and ITO conductive glass supplied by Weiguang Corp. (Shen-
PEC detection: Photoelectrochemical detection was carried out in PBS
(pH 7.4, 0.1m) containing 0.1m ascorbic acid (AA) which was served as a
sacrificial electron donor during the photocurrent measurement. Light
excitation of 430 nm was switched every 10 s. The applied potential was
0.1 V.
zhen, Peopleꢂs Republic of China, ITO coating ((180ꢂ25) nm, sheet re-
sistance ꢃ 10 W/square) as a working electrode. The planar Au electrode
was supplied by Nanjing Research Institute of the fifty-fifth (2000 ꢃ).
Scanning electron microscopy (SEM) (JSM-6700F, JEOL, Japan) was
used to examine the morphology of modified electrodes. Photolumines-
cence (PL) spectra were obtained on an RF-540 spectrophotometer
(ShimACHTUNGTRENNUNGadzu). UV/Vis spectra were carried out on a Cary 50 UV/Vis-NIR
spectrophotometer (Varian).
Synthesis of PEC nanoprobes:[36] Tris-HCl (pH 8.2; 1.5 mL of 500 mM),
TCEP (6 mL of 10 mm), -SH-modified thrombin aptamer (7.2 mL of
100 mm), and cysteamine (7.2 mL of 100 mm) were mixed and incubated
for 30 min at room temperature. AuNPs (1 mL) (synthesized in Support-
ing Information) was added to the mixture and incubated for 6 h at room
temperature. Then sodium boracic acid buffer (pH 9.0; 120 mL 0.1m) and
synthesized V-NHS (8 mL; see the Supporting Information) were added.
The tube was placed on a shaking table for 10 h in dark. Then, the solu-
tion was centrifuged at 48C for 30 min at 10000 rpm and resuspended in
buffer (800 mL of 100 mm NaCl, 25 mm Tris acetate, pH 8.2).
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (Nos. 21275003, 21025523, 20973050), the Excellent Young Scien-
tists Encouragement Foundation of Shandong Province (No.
BS2011L015), and the Program for Changjiang Scholars and Innovative
Research Team in University (PCSIRT).
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Revised: September 18, 2012
Published online: November 5, 2012
16418
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 16411 – 16418