Photoluminescent C-dots@RGO probe for sensitive and selective detection of acetylcholine

Article abstract of DOI:10.1021/ac303613d

We have developed a sensitive and selective photoluminescence (PL) quenching assay for the detection of acetylcholine (ACh) that uses reduced graphene oxide decorated with carbon dots (C-dots@RGO). The highly stable C-dots@RGO synthesized from catechin and graphene oxide through a hydrothermal reaction displays excitation-wavelength dependence of PL. Acetylcholinesterase (AChE) converts ACh to choline, which in turn is oxidized by choline oxidase (ChOx) to produce betaine and H₂O₂ that generates the reactive oxygen species (ROS). The as-produced ROS induces PL quenching of the C-dots@RGO through an etching process. With respect to sensitivity, the optimal reaction/sensing temperature and pH are 37 C and 9.0, respectively, using C-dots@RGO (0.4 mg·mL^-1) and AChE and ChOx at the activities of 0.5 and 0.1 unit·mL^-1, respectively. The PL intensity (excitation/emission wavelengths 365/440 nm) of the C-dots@RGO is inversely proportional to the concentration of ACh over a range of 0.05-10 nM (r = 0.997), with a limit of detection (signal-to-noise ratio 3) of 30 pM. We have validated this assay by determination of concentrations of ACh in plasma and blood samples, with results of 2.6 ± 0.8 nM (n = 5) and 6.8 ± 0.4 nM (n = 5), respectively. Our study opens an avenue for the detection of various analytes by use of C-dots@RGO in conjunction with different enzymes, substrates, and/or inhibitors.

Full text of DOI:10.1021/ac303613d

Article
pubs.acs.org/ac
Photoluminescent C‑dots@RGO Probe for Sensitive and Selective
Detection of Acetylcholine
Chen-I Wang, Arun Prakash Periasamy, and Huan-Tsung Chang*
Department of Chemistry, National Taiwan University, Taipei, Taiwan
S
* Supporting Information
ABSTRACT: We have developed a sensitive and selective photoluminescence
(PL) quenching assay for the detection of acetylcholine (ACh) that uses reduced
graphene oxide decorated with carbon dots (C-dots@RGO). The highly stable
C-dots@RGO synthesized from catechin and graphene oxide through a
hydrothermal reaction displays excitation-wavelength dependence of PL.
Acetylcholinesterase (AChE) converts ACh to choline, which in turn is oxidized
by choline oxidase (ChOx) to produce betaine and H2O2 that generates the
reactive oxygen species (ROS). The as-produced ROS induces PL quenching of
the C-dots@RGO through an etching process. With respect to sensitivity, the
optimal reaction/sensing temperature and pH are 37 °C and 9.0, respectively,
using C-dots@RGO (0.4 mg·mL−1) and AChE and ChOx at the activities of 0.5
and 0.1 unit·mL−1, respectively. The PL intensity (excitation/emission
wavelengths 365/440 nm) of the C-dots@RGO is inversely proportional to
the concentration of ACh over a range of 0.05−10 nM (r = 0.997), with a limit
of detection (signal-to-noise ratio 3) of 30 pM. We have validated this assay by determination of concentrations of ACh in
plasma and blood samples, with results of 2.6 0.8 nM (n = 5) and 6.8 0.4 nM (n = 5), respectively. Our study opens an
avenue for the detection of various analytes by use of C-dots@RGO in conjunction with different enzymes, substrates, and/or
inhibitors.
cetylcholine (ACh) is a neurotransmitter that can be
found in the peripheral nervous system and central
(TMB), and luminol,4−8 for the H2O2-mediated reaction are
used in highly sensitive and selective optical sensors for the
detection of ACh.4−8 Although these sensing systems are
sensitive, interference, high cost, short shelf life, and/or tedious
processes of preparation of the ACh biosensors are problem-
atic.
A
nervous system.1 In cholinergic neurons, ACh is synthesized
from choline by choline acetyltransferase and acetyl-coenzyme
A.1 Increases in the level of ACh cause a decreased heart rate
and increased production of saliva. On the other hand,
decreases in the level of ACh are associated with motor
dysfunction, as well as Parkinsonʼs and Alzheimerʼs diseases.1
Thus, determination of the concentration of ACh in biological
samples such as blood is important for cholinergic research.
This is a difficult task, mainly because its normal concentration
in human blood is quite low (8.66 1.02 nM) and the matrix is
rather complicated.1
Many nanoparticle-based approaches have been developed
for determining the activity of AChE and its inhibitors, based
on the enzymatic conversion of acetylthiocholine to thiocho-
line, which induces the growth of gold nanoparticles (Au NPs)
and subsequent optical changes.9−11 Although the above-stated
approaches are sensitive and selective for the tested analytes,
they cannot be used for the detection of ACh, mainly because
the product (choline) cannot induce the growth of Au NPs.
Recently we have demonstrated Au/Ag bimetallic NPs
possessing peroxidase-like activity for the fluorescence
detection of ACh, using AUR as a substrate.4 AChE converts
ACh to choline, which in turn is oxidized by ChOx to produce
betaine and H2O2 that reacts with AUR to form a fluorescent
product catalyzed by the Au/Ag NPs. Although the sensing
system is selective and sensitive (with a limit of detection,
LOD, of 0.21 nM), stability and loss in the catalytic activity of
Au/Ag NPs due to adsorption of matrices are problematic.
There is no electro-active, chromophore, or fluorophore
group in ACh, which makes it difficult to detect. Although gas
chromatography and high-performance liquid chromatography
have been applied for the detection of ACh, tedious sample
pretreatment, long separation time, use of large volumes of
sample and eluate, and an experienced operator are required.2
To overcome these disadvantages, many sensitive sensing
systems have been demonstrated.1,2 ACh biosensors are
fabricated by taking advantage of high catalytic activities of
acetylcholinesterase (AChE) and choline oxidase (ChOx) for
converting nonelectroactive or nonfluorescent substrates to an
electrochemically active product (H2O2) or fluorescent
products.3 Common substrates, including Amplex UltraRed
(AUR), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), 3,3,5,5-tetramethylbenzidine
Received: December 12, 2012
Accepted: February 11, 2013
Published: February 11, 2013
© 2013 American Chemical Society
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Recently, reduced graphene oxide (RGO), with its flat
network of sp2-hybridized carbon atoms and two-dimensional
(2D) honeycomb lattices, has become a very popular sensing
material for the detection of DNA, proteins, and small
molecules, mainly because it has large planar surface and high
photoluminescence (PL) quenching efficiency of fluorophores
(e.g., organic dyes, quantum dots).12,13 Although these
detection strategies can be sensitive, expensive fluorophores
and tedious processes are required for preparation of the
sensing probes. In addition, as-prepared RGO is usually
hydrophobic and nonphotoluminescent, leading to irreversible
agglomeration and precipitation, which limit its direct use for
biological application (e.g., biosensing and cell imaging).14
Herein, we developed a simple, green, and cost-effective one-
pot synthetic strategy for the synthesis of hydrophilic,
photoluminescent carbon dots on RGO (C-dots@RGO)
from graphene oxide (GO) through a hydrothermal reduction
route using catechin as a reductant, based on our previous
study.15,16 On the basis of reactive oxygen species (ROS)
-induced PL quenching of C-dots@RGO, we demonstrated a
sensitive and selective sensing system for the detection of ACh.
The ROS was generated through the formation of H2O2 from
ACh in an AChE/ChOx system. We evaluated several
important factors such as the concentrations of C-dots@
RGO, AChE/ChOx, and NaCl, temperature, reaction time, and
solution pH values to optimize the sensing conditions. The
practicality of the H2O2−C-dots@RGO sensing system was
validated through the detection of ACh in blood samples. To
the best of our knowledge, C-dots@RGO probes have not been
demonstrated. In addition, no tradition fluorophores are
required in our sensing system.
the vial was capped well. The mixture was heated in an oil bath
at 95 2 °C for 3 h. The yellow color of the GO solution
gradually changed to black, indicating the formation of RGO.
The resulting RGO solution was washed with ultrapure water
(ca. 50 mL) to remove excess hydrazine monohydrate and
ammonium hydroxide after filtration through an inorganic
membrane filter paper (pore size 0.02 μm). Washing was
continued until the pH of the filtrate reached approximately
7.0. The product was carefully collected from the filter paper
and dried overnight in an oven at 50 °C. The RGO was then
dispersed in N,N-dimethylformamide (DMF). Experimental
details can be found in the Supporting Information. By
controlling reaction time (2−12 h), we prepared different sizes
of C-dots on the surfaces of RGO. Using the same amount of
catechin, we separately prepared C-dots@RGO with three
different sizes of RGO from GO sheets (0.3−0.5, 0.7−0.9, and
1−2 μm) that had been prepared according to the method of
Hummers and Richard.18 Each of the as-prepared C-dots@
RGO solutions were subjected to centrifugation at different
rotation speeds (5000g and 45000g for the first two and the
last, respectively) for 30 min.
We extracted the C-dots from the surface of RGO by
ultrasonicating the C-dots@RGO sample for 1 h and further
subjecting it for centrifugation at 45000g for 30 min. A similar
approach has also been demonstrated for determining the
concentration of single-walled carbon nanotubes (SWCNTs) in
aqueous dispersions containing both surfactants and
SWCNTs.19 On the basis of the fact that C-dots provided a
defined PL peak at 440 nm, but RGO did not, we determined
the concentration of as-extracted C-dots using a calibration plot
of the PL intensities at 440 nm as a function of pure C-dots
concentration. There was about 0.32 mg of C-dots in 1 mg of
C-dots@RGO.
Detection of H2O2 and ACh by Use of Photo-
luminescent C-dots@RGO. To detect H2O2, aliquots (300
μL) of 25 mM Tris-HCl buffer (pH 9.0) containing C-dots@
RGO (0.4 mg·mL−1), and H2O2 (10 pM−15 μM) were
equilibrated and reacted under gentle shaking at 25−70 °C for
1 h. In order to compare the sensitivity of pure C-dots and C-
dots@RGO toward H2O2, they were used to detect H2O2
under the same condition (pH 9.0, 37 °C). Pure C-dots (0.13
mg·mL−1) and C-dots@RGO (0.4 mg·mL−1, about 0.13
mg·mL−1 C-dots) were separately mixed with H2O2 (0.1
nM−15 μM), which were reacted under gentle shaking for 1 h.
The slope of (IPL0 − IPL)/IPL0 ratios against H2O2
concentration were used to compare the sensitivity of the
pure C-dots and C-dots@RGO in 25 mM Tris-HCl buffers for
H2O2, where IPL0 and IPL represent the PL intensities of carbon
nanomaterials (pure C-dots and C-dots@RGO) in the absence
and presence of H2O2, respectively. For the detection of ACh,
aliquots (300 μL) of 25 mM Tris-HCl buffer (pH 9.0)
containing C-dots@RGO (0.4 mg·mL−1), AChE (0.5
unit·mL−1), ChOx (0.1 unit·mL−1), and ACh (0.05−50 nM)
were equilibrated and reacted under gentle shaking at 37 °C for
2 h. To optimize sensitivity in a short analysis time, the enzyme
reaction and sensing were conducted at temperatures of 25−50
and 25−70 °C, respectively, and over a pH range 4.0−10.0,
both for 10 min. Prior to PL measurement, aliquots (200 μL)
of the product solutions were transferred separately into 96-
well microtiter plates. Their fluorescence spectra were recorded
on a microplate reader (μ-Quant Biotek Instruments, Winooski,
VT). The excitation and emission wavelengths were set at 365
EXPERIMENTAL SECTION
■
Synthesis of Photoluminescent C-dots@RGO and
RGO. Catechin-mediated reduction of GO was applied to
prepare photoluminescent C-dots@RGO. Catechin (21.6 mg)
was added to a diluted GO solution (0.34 mg·mL−1, 5 mL).
The resulting mixture was gently stirred for 10 min before it
was transferred to a Teflon-lined stainless steel autoclave, which
was then placed in a muffle furnace and subjected to
hydrothermal treatment at 300 °C for 2 h. During the
hydrothermal reaction process, C-dots were produced and
deposited on the RGO sheets. Upon completion of the
reaction, the resulting black solution of C-dots@RGO was
cooled to room temperature. It was then subjected to filtration
through an inorganic membrane filter paper (pore size 0.02
μm) and the filtrate was washed with ultrapure water (ca. 50
mL) to remove the unreacted excess catechin. The product was
dried overnight at 50 °C in an oven and then carefully collected
from the filter paper. The as-purified C-dots@RGO product
was redispersed in distilled water (4 mg·mL−1) with the aid of
ultrasonication for 1 h, and then filtered again to remove the
large chunks and other graphitic impurities by use of cotton
cellulose filter paper (pore size <5 μm). The as-purified C-
dots@RGO was then dispersed in aqueous solution (5 mL),
which was then transferred to a polypropylene test tube for
storage at 4 °C prior to use.
As a control, RGO was synthesized from a commercial GO
solution (5 mg·mL−1) through conventional hydrazine-
mediated chemical reduction.17 The GO solution was diluted
with water (5 mL) in a borosilicate glass scintillation vial.
Hydrazine monohydrate (5 μL, 65%) and ammonium
hydroxide (35 μL, 30%) were added sequentially and then
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Figure 1. (A) TEM and (B) SEM images of C-dots@RGO. (Insets) Magnified views of the corresponding TEM and SEM images. (C) PL emission
and UV−vis absorption (inset) spectra of C-dots@RGO. Excitation wavelength varied from 365 to 465 nm, with 20-nm increments. (D) PL images
of C-dots@RGO excited by (a) bright field and at (b) 360−380, (c) 410−430, and (d) 510−530 nm. PL intensities and absorbance are plotted in
arbitrary units (a.u.). Concentration of C-dots@RGO: 0.4 mg·mL−1.
and 440 nm, respectively. The measurements were conducted
in triplicate for each sample.
of RGO through the redox reaction of catechin and GO;20 (2)
formation of C-dots on the RGO through dehydration,
polymerization, pyrolysis, and surface passivation.15,16 Under
alkaline conditions, the interaction of catechin (or its reduced
form) with RGO is strong,21 assisting the formation of C-dots
that were well distributed on the surfaces of the RGO. In
addition, the reducing ability of catechin is also strong under
alkaline conditions, leading to the formation of RGO from GO.
On the other hand, the OH groups of catechin allow the
formation of intermolecular hydrogen bonds, which play an
important role in the formation of photoluminescent C-dots on
RGO surfaces.22
Figure S-1A (Supporting Information) displays the UV−vis
absorption spectra of GO, RGO that had been synthesized
from the reaction of GO and hydrazine, and C-dots@RGO.
GO exhibited a well-defined absorption peak at 230 nm; RGO
provided a characteristic peak at 263 nm.23 Because this RGO
had poor solubility in aqueous (phosphate) solution, we
dispersed it in DMF. In contrast, the C-dots@RGO was highly
soluble in aqueous solution and exhibited a blunted absorption
peak over the wavelength range from 200 to 260 nm, ascribable
to the π−π* transitions of both the C-dots and RGO.24 After
the reaction, the color of the GO solution changed from
yellowish brown to brownish black (inset to Figure S-1A,
Supporting Information), mainly due to restoration of the
electronic conjugation network within the carbon structure
upon catechin-mediated reduction of GO.21,25 Figure S-1B
(Supporting Information) reveals that only the C-dots@RGO
exhibited PL at a wavelength of 440 nm upon excitation at 365
nm. Notably, we observed no PL for the solution containing
GO and catechin that had not been subjected to the
hydrothermal reaction. In addition, RGO did not fluoresce
Detection of ACh in Blood Samples. Prior to analysis,
aliquots (200 μL) of the blood samples were treated with
acetonitrile (300 μL, 99.5% w/w) for 30 min to denature the
proteins and release ACh from the protein complexes. After
centrifugation (9056g, 10 min), the supernatant was evaporated
by gentle heating and finally cooled down to room temperature.
The remaining sample solution was then passed through a filter
having a cutoff of 3 kDa (membrane nominal pore size ca. 0.3
nm). The purified samples were diluted with Tris-HCl buffer (2
mL, 250 mM, pH 9.0). Aliquots (100 μL) of the diluted
samples were spiked with standard ACh solution (50 μL, final
concentrations 0−100 nM). The spiked sample solutions (30
μL) were separately mixed with aliquots of Tris-HCl buffer
(270 μL, 25 mM, pH 9.0) containing C-dots@RGO (0.4
mg·mL−1), AChE (0.5 unit·mL−1), ChOx (0.1 unit·mL−1), and
NaCl (100 μM). The mixtures were equilibrated under gentle
shaking at 37 °C for 2 h, and the product solutions (200 μL)
were transferred separately into 96-well microtiter plates. The
PL spectrum of each solution was recorded at 440 nm
(excitation wavelength 365 nm).
RESULTS AND DISCUSSION
■
Formation Mechanism and Spectroscopic Properties.
The transmission electron microscopy (TEM) and scanning
electron microscopy (SEM) images in Figure 1A,B reveal that
C-dots@RGO featured thin, flat sheets with fine, curled edges
having the morphology of RGO. From the magnified TEM and
SEM images (insets), it is apparent that the RGO sheets were
decorated with C-dots having sizes ranging from 5 to 10 nm.
The C-dots@RGO was synthesized in two steps: (1) formation
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after it alone was subjected to the hydrothermal reaction.
However, C-dots@RGO exhibited excitation wavelength
dependence of their PL properties (Figure 1C). To assess the
detailed PL properties of the C-dots@RGO, we also acquired
PL images. Upon irradiation at 360−380, 410−430, and 510−
530 nm, the C-dots@RGO film exhibited photoluminescence
with blue, green, and red colors, respectively, revealing the
excitation wavelength dependence of its PL properties (Figure
1D).16 C-dots@RGO in liquid solution also exhibited
excitation wavelength dependence of its PL properties. The
C-dots prepared from catechin (in the absence of GO)
exhibited excitation wavelength dependence of their PL
properties (Figure S-2, Supporting Information), mainly due
to their wide size distribution and surface properties.26,27
Together, these observations support the successful formation
of C-dots@RGO. Further characterization studies [Fourier
transform infrared (FT-IR), Raman spectra, X-ray diffraction
(XRD), and C 1s X-ray photoelectron spectroscopy (XPS)
profiles] of GO, RGO, and C-dots@RGO are provided in
Supporting Information (Figure S-3), supporting the formation
of C-dots@RGO.
Scheme 1. Schematic Representation of Sensing Strategy of
PL-Quenched Assay That Uses C-dots@RGO for Detection
of ACh
Stability of C-dots@RGO. We investigated the effects of
pH and salt concentration on the PL intensity of the as-
prepared C-dots@RGO. The PL intensity of the C-dots@RGO
increased upon increasing the pH of the solution from 4.0 to
9.0, revealing a wide operating pH range (Figure S-4A,
Supporting Information). The pH dependence of the PL
indicates a potential application of the C-dots@RGO as a pH
sensor. Under acidic conditions, the free sites were protonated,
leading to lower PL through breaking of the emissive triple
carbene state.28 The free sites were restored under alkaline
conditions, leading to a return of the PL. We observed no
notable change in the PL intensity at values of pH higher than
9.0. The PL intensity of the C-dots@RGO increased upon
increasing the NaCl concentration from 0 to 150 mM, partially
due to greater stability of the C-dots@RGO, but remained
almost constant at concentrations greater than 150 mM (Figure
S-4B, Supporting Information). The pH and salt dependence of
the PL of the C-dots@RGO was similar to that of the C-dots
synthesized from catechin. The C-dots and C-dots@RGO
exhibited great photostability (decreases in PL intensity <0.1%)
under continuous irradiation with a Xe lamp for 6 h (Figure S-
4C, Supporting Information). With such high chemical and
photostability, the C-dots@RGO appears to have great
potential for use in the labeling of analytes and in cell imaging.
Sensing Strategy. Scheme 1 displays the detection of ACh
by use of photoluminescent C-dots@RGO at pH 9.0. AChE
converts ACh to choline, which in turn is oxidized by ChOx to
produce betaine and H2O2. We noted that the activities of the
two enzymes are optimal at 37 °C. In addition, decomposition
of H2O2 to form ROS occurs more rapidly at higher
temperature than that at ambient temperature. The ROS
induces PL quenching of the C-dots@RGO significantly
through an etching process.29 It has been demonstrated that
RGO can generate reactive oxygen species (ROS).30−33 Under
the optimal experimental conditions, strong oxygen-containing
substrates to form substrates that have different optical
properties (e.g., absorption and fluorescence).35−38 The
quenching efficiency induced by H2O2 relative to ROS is
lower (about 5-fold); therefore, the sensing temperature is set
at higher temperature (e.g., 37 °C) for the sake of sensitivity
and simplicity of the sensing system. The amount of ROS
generated is proportional to the concentration of ACh, thus the
PL of C-dots@RGO at the emission wavelength of 440 nm
decreases upon increasing the concentration of ACh.
Detection of H2O2 by Use of C-dots@RGO. To test the
possibility of using C-dots@RGO probe for the detection of
H2O2 (100 nM), we investigated the effects of solution pH,
temperature, and salt concentration on the PL quenched
efficiency of C-dots@RGO. In the absence of H2O2, PL
intensity of the C-dots@RGO increased upon increasing the
pH (from 4.0 to 9.0) and the NaCl concentration (from 0 to
150 mM) of the solution as shown in Figure S-5A,B
(Supporting Information), mainly due to more stability of the
C-dots@RGO. H2O2 induced PL quenching of the C-dots@
RGO increased upon increasing pH values from 4.0−9.0 as
displayed in Figure S-5A (Supporting Information). Decom-
position rate of H2O2 to form ROS increased upon increasing
the pH values, leading to increased PL quenching efficiency
induced by ROS. Above pH 5.0, decomposition of H2O2
increased sharply.39 NaCl concentrations up to 150 mM did
not affect the H2O2-induced PL quenching of C-dots@RGO as
displayed in Figure S-5B (Supporting Information). Figure S-
5C (Supporting Information) displays that in the absence of
H2O2 the PL of C-dots@RGO remained almost constant over a
temperature range of 25−70 °C. However, the PL decreased
upon increasing temperature in the presence of H2O2 (100
nM), which revealed the generation of ROS. The generation of
ROS is related to the peripheral carboxylic/carbonyl moieties of
RGO, which promotes the adsorption of H2O2 and thus the
transfer of electrons from H2O2 to the surface of C-dots@
32,33
•
•
•
free radicals (such as OH, O, HO2 , etc.) were produced on
the surface of C-dots@RGO that oxidized the sp2-hybridized
graphite-like carbons (−C=C−) and sp3-hybridized carbons
(−C−C−),34 leading to the formation of oxygen-containing
functional groups and PL quenching. We note that most
nanoparticle (NP) -based sensors are sensitive to H2O2, based
on the formation of radicals that catalyze the conversion of
RGO, leading to efficient oxidation of H2O2.
It has been
demonstrated that generation of ROS from H2O2 increased
upon increasing temperature.39 The response time of this
sensor toward H2O2 at 37 °C was further investigated by
recording the PL intensity of C-dots@RGO over a sensing time
ranging from 0 to 120 min (Figure S-5D, Supporting
Information). Upon increasing the sensing time, the PL
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Figure 2. Effect of sizes of (A) C-dots (reaction time 2, 8, and 12 h) and (B) RGO (1−2, 0.7−0.9, and 0.3−0.5 μm) on PL quenching efficiencies
induced by H2O2 (100 nM) in Tris-HCl buffer (25 mM, pH 9.0).
Figure 3. Effects of (A) pH (4.0−10.0), (B) NaCl concentration (0−150 mM), (C) temperature (25, 37, and 50 °C), and (D) activities of AChE/
ChOx on the PL intensity of C-dots@RGO in the presence of ACh (10 nM). C-dots@RGO was prepared in (A) Tris-HCl solutions (25 mM, pH
4.0−10.0), (B) Tris-HCl buffer (25 mM, pH 9.0) containing various concentrations of NaCl, or (C, D) Tris-HCl buffer (25 mM, pH 9.0) containing
100 mM NaCl. IPL0 and IPL are the PL intensities of solutions in the absence and presence of ACh (10 nM), respectively. Other conditions are as
described in the text.
decreased from 0 to 60 min and reached saturation after 60
min. Given the PL quenching efficiency induced by H2O2,
further studies were performed in Tris-HCl buffer solution (25
mM, pH 9.0) containing 100 mM NaCl at 37 °C for 1 h.
The sizes of C-dots and GO had a significant effect on the
sensitivity of this PL quenching assay. Figure 2 shows the effect
of sizes of C-dots and GO on their PL quenching efficiencies.
The PL intensity of C-dots decreased (changed from 105 to 6 ×
103 a.u.) upon increasing the reaction time from 2 to 12 h,
mainly because of the formation of larger and less soluble C-
dots.40 The C-dots with smaller size exhibited higher catalytic
activity, mainly due to their higher surfaces energies. Upon
increasing the size of C-dots, aggregation of the particles
occurred, leading to decreases in the photon flux penetration
and thus decreases in PL quenching efficiency. Figure 2B shows
the ROS quenching efficiency increased with increases in the
diameter of RGO sheets, mainly ascribed to their larger surface
area and edge−plane-like defective sites on RGO. The TEM
images of various sizes of RGO are shown in Figure S-6A−C
(Supporting Information), revealing that their sizes were 0.3−
0.5, 0.7−0.9, and 1−2 μm, respectively.
Figure S-7A (Supporting Information) reveals that the values
of IPL0 − IPL of the mixtures increased linearly (r = 0.986) upon
increasing the concentration of H2O2 over the range 0.1 nM−
15 μM, where IPL0 and IPL represent the PL intensities of the
mixtures in the absence and presence of H2O2. Based on a
signal-to-noise ratio (S/N) of 3, the limit of detection (LOD)
for H2O2 was 36 pM, which is lower than those provided by
other sensors.41,42 The good sensitivity and wide linearity for
the detection of H2O2 indicates that the C-dots@RGO probe is
useful for the determination of the concentration of ACh.
We further compared the PL quenching efficiencies of C-
dots@RGO and pure C-dots (prepared from catechin) in the
presence of H2O2. We tested the PL quenching efficiencies of
C-dots@RGO and pure C-dots in the presence of H2O2. Under
optimal conditions, the PL intensity increased upon increasing
C-dots concentrations, with a linear response (r = 0.997) over
the concentration range 0.15−16.5 mg/mL (Figure S-7B,
Supporting Information). As shown in Figure S-7C (Support-
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ing Information), C-dots@RGO exhibits higher sensitivity for
H2O2 than pure C-dots, mainly because of its higher ROS
generation efficiency. The linear ranges of C-dots@RGO and
pure C-dots for H2O2 were 0.1−10 and 15−430 nM,
respectively. The corresponding LOD values were 36 pM and
7.6 nM, respectively. Our results reveal that RGO played a
significant effect on the sensitivity of this PL quenching assay.
We also measured the PL quenching efficiencies of H2O2
toward C-dots@RGO at various RGO concentrations. The PL
quenching efficiencies toward C-dots@RGO increased with
increases in the concentrations of RGO (0−0.5 mg·mL−1) as
shown in Figure S-8 (Supporting Information). Being an
effective oxidant, RGO enhanced the ROS generation rate with
increases in its concentrations, resulting in greater quenching
efficiencies. On the basis of these results, we report an ROS-
induced oxidative mechanism for the PL quenching and
chemical degradation of C-dots@RGO. The highly reactive
ROS oxidized the C-dots@RGO surface, which in turn
generated the lattice defects and caused the PL quenching,29
as supported by the UV−vis absorption spectra results (Figure
S-7D, Supporting Information). The absorbance of C-dots@
RGO decreased upon increasing the H2O2 concentration from
0.1 to 10 μM. A slight blue shift was also observed in the
wavelength range 225−250 nm, suggesting that C-dots@RGO
surface was etched by the ROS.
Under optimal conditions, the PL intensity decreased upon
increasing the concentration of ACh, with a linear response (r =
0.997) over an ACh concentration range 0.05−10 nM, where
IPL0 and IPL represent the fluorescence intensities of the
mixtures in the absence and presence of ACh (Figure 4A). The
In order to demonstrate that ROS plays an important role in
this sensing strategy, we pretreated the C-dots@RGO with a
ROS scavenger (ascorbic acid). We found that the pretreatment
abolished the PL quenching of C-dots@RGO induced by ROS
(Figure S-9, Supporting Information). The PL of C-dots@
RGO treated with ascorbic acid (0.1−10 μM) remained almost
constant in the presence of H2O2 (100 nM), supporting our
proposed PL quenching mechanism.
Detection of ACh by Use of C-dots@RGO. We
investigated the effect of pH (Tris-HCl buffer solution) and
the concentrations of AChE/ChOx on the detection of ACh by
use of C-dots@RGO. On the basis of the effects of pH, salt, and
temperature on the H2O2-induced PL quenching of C-dots@
RGO, pH (4.0−10.0), temperature (25, 37, 50 °C), and NaCl
(0−150 mM) were selected to optimize experimental
conditions. With consideration of the time required for
completing enzyme reactions and sensing, the equilibrium
Figure 4. (A) PL spectra of C-dots@RGO (0.4 mg·mL−1) at various
ACh concentrations. (Inset) Linearity of the expression IPL0 − IPL
against the concentrations of ACh. (B) Selectivity of the PL-quenched
assay using C-dots@RGO for ACh. The concentration of ACh is 10
nM, while those for other solutes are all 100 nM. The activities of
AChE/ChOx are 0.5/0.1 unit·mL−1. Reaction time for the enzyme and
PL-quenched reactions at 37 °C is 2 h. IPL0 and IPL are the PL
intensities of the solutions in the absence and presence of ACh,
respectively. Other conditions are as described in the text.
LOD for ACh (S/N 3) was 30 pM, which was lower than those
provided by other optical sensors.46,47 We investigated the
selectivity of the assay toward ACh (10 nM) over some
possible interferences (each 100 nM), including dopamine,
serotonin, glutamic acid, 4-aminobutyric acid, glycine, and
aspartic acid. Figure 4B reveals that this sensing system was
quite selective toward ACh over the tested interferences. In
comparison with reported optical and electrochemical methods
(Table 1), our assay is the most sensitive assay for ACh, mainly
because of the high stability, high surface area, and efficient PL
quenching induced by ROS of C-dots@RGO. The sensing time
required for our simple and cost-effective assay is 2 h, which is
slightly longer than that reported in the literature.48−54
Nonetheless, the sensing time can be further shortened if the
sensitivity is not a major issue. When we conducted the assay at
pH 9.0 and 37 °C for 10 min (for the reaction of ACh with
AChE and ChOx) and at 50 °C for another 10 min (to speed
up generation of ROS), the C-dots@RGO probe provided an
LOD of 5.2 nM for ACh.
time was set at 2 h. Figure 3A−C displays the values of IPL0
−
IPL for C-dots@RGO against pH, NaCl concentration, and
temperature, in which IPL0 and IPL represent the PL intensities
of C-dots@RGO in the absence and presence of ACh (10 nM).
The optimal conditions of pH value, temperature, and salt
concentration were found to be 9.0, 37 °C, and 100 mM NaCl,
respectively. These conditions are highly correlated with the
enzyme kinetics and decomposition rate of H2O2.43−45
Although decomposition rate of H2O2 at high temperature is
faster, 37 °C was selected for the sake of simplicity of the
sensing system. At pH 9.0 and 37 °C, we further investigated
the optimal activity ratio of AChE/ChOx enzymes. Figure 3D
shows that PL quenching increased upon increasing the
concentrations of AChE and ChOx ranging from 0 to 0.5
unit·mL−1 and from 0 to 0.1 unit·mL−1, respectively, and then it
decreased thereafter. Although the reaction rate increased at
only high concentrations of AChE and ChOx, adsorption of the
enzymes on the surfaces of C-dots@RGO was found to be
problematic, leading to decreased PL quenching efficiency.
Therefore, the optimal activities of the two enzymes were
selected as 0.5 and 0.1 unit·mL−1, respectively.
Detection of ACh in Blood Samples. To validate the
practicality of the present assay, we determined the
concentrations of ACh in five blood samples by applying a
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Analytical Chemistry
Article
Table 1. Comparison of Various Biosensors for Detection of ACh
method
amperometry
LOD (nM)
linearity (nM)
ref
1000
63
(5 × 103)−(4 × 105)
103−(4 × 105)
1−105
49
54
1
amperometry
amperometry
0.1
electrochemiluminescence
potentiometry
4700
10 000
50
104−(2.5 × 105)
104−108
52
48
50
51
53
4
chemiluminescence
fluorescence
50−104
10 000
1000
0.2
104−(5 × 106)
(5 × 103)−(9 × 106)
1−102
fluorescence
fluorescence
photoluminescence
0.03
0.05−10
this study
standard addition method. The PL responses against the
concentrations of ACh spiked into representative samples of
whole blood and plasma were both linear (r > 0.98) over the
concentration ranges 0−10 nM (Figure 5). The ACh
that used photoluminescent C-dots@RGO through PL
quenching induced by ROS was demonstrated. Since many
substrates react with enzymes to form H2O2 and then ROS, C-
dots@RGO can theoretically be combined with many enzymes
for developing sensitive assays for the detection of various
substrates (such as glucose, cholesterol, and uric acid) and
inhibitors, as well as for determining the activity of enzymes.
The C-dots@RGO in conjunction with AChE and ChOx was
applied to the detection of ACh, with an LOD of 30 pM,
showing that this assay relative to most reported ones is more
sensitive. The study shows that this assay can be further applied
to the detection of AChE inhibitors such as pesticides
(organophosphates and carbamates) and holds great potential
for neuronal study.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text describing chemicals, characterization of
nanomaterials, and blood sampling, and nine figures as
described in the text. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel/fax 011-886-2-33661171; e-mail changht@ntu.edu.tw.
■
Figure 5. Analyses of (A) blood and (B) plasma samples by the PL-
quenched assay using C-dots@RGO. Each of the samples was spiked
with ACh (final concentrations 0−10 nM). Other conditions are as
described in the text.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the National Science Council of
Taiwan under Contract NSC 101-2627-M-002-007. A.P.P.
thanks the National Taiwan University for the award of a
postdoctoral fellowship in the Department of Chemistry,
National Taiwan University.
concentrations in the plasma and blood samples were
determined to be 2.6
0.8 nM (n = 5) and 6.8
0.4 nM
(n = 5), respectively. The ACh concentrations determined by
use of a commercial assay kit were 2.5 0.3 nM (460 50
pg·mL−1) and 7.0
0.8 nM (1260
150 pg·mL−1) in the
plasma and whole blood samples, respectively. Student’s t-test
performed at the 95% confidence level demonstrated that the
two approaches did not provide significantly different results,
revealing that the C-dots@RGO probe holds great potential for
neuronal study.
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