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One-step ultrasonic synthesis of graphene quantum dots with high quantum

yield and their application in sensing alkaline phosphatase

Article  in  Chemical Communications · December 2014

DOI: 10.1039/c4cc07449a · Source: PubMed

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Electronic Supplementary Material (ESI) for ChemComm.
This journal is © The Royal Society of Chemistry 2014

Electronic Supplementary Information

One-step ultrasonic synthesis of graphene quantum dots with high quantum yield and its

application in sensing of alkaline phosphatase

Yanhong Zhu, a Guangfeng Wang,a, b* Hong Jiang, a Ling Chen, a and Xiaojun Zhanga,b*

a Anhui Key Laboratory of Chem-Biosensing, College of Chemistry and Materials Science, Center

for Nanoscience and Nanotechnology, Anhui Normal University, Wuhu, 241000, P. R. China

b State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha,

410082, PR China

E-mail: xjzhang@mail.ahnu.edu.cn; wangyuz@mail.ahnu.edu.cn

*Corresponding author: E-mail: wanglun@mail.ahnu.edu.cn; Fax: +86-553-3869303; Tel: +86-

553-3869303

1 Experimental section
2 Quantum yield measurements
3 Comparison of reported methods for the detection ALP activity
4 Energy transfer between GQDs and Cu2+
5 Analysis performance on Cu2+
6 Charaterization of GQD-based ALP sensor
7 Reaction time of GQDs and Cu2+
8 The optimized concentration of PPi
9 The reaction time of ALP and PPi
10 The inhibition effect of Pi on ALP activity
11 The effect on the formation of luminescent GQDs by ultrasonication
12 The correlation between the pH and quantum yield
1 Experimental section
Reagents and Chemicals. Graphene oxide (GO) was purchased from Ningbo Institute of
Materials Technology & Engineering Chinese Academy of Sciences (China). CuCl2, KMnO4, and
other metal salt was obtained from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China)
and used without further purification. ALP from bovine intestinal mucosa, PPi, Pi, lysozyme from
chicken egg white, thrombin from human plasma, bovine serum albumin (BSA), glucose oxidase
from aspergillus niger (GOx), ATP, avidin from egg white, and PDGF-BB were purchased from
Sangon Biotechnology Co. Ltd. (Shanghai, China).
Apparatus. Fluorescence spectra were determined using a Hitachi F-4500 fluorescence
spectrometer (Hitachi Ltd., Japan) controlled by FL solution software. The optical path length of a
quartz fluorescence cell was 1.0 cm. Excitation and emission slits were all set for a 5.0 nm band-
pass. The mixtures in square quartz cuvettes were excited at 380 nm, and the emission spectra
were collected from 425 to 600 nm. The fluorescence intensity at 470 nm was used to evaluate the
performances of the proposed assay strategy. Ultraviolet-visible absorption spectra were measured
using UV-3010 spectrophotometer (Hitachi, Japan). High-resolution transmission electron
microscope (HRTEM) observations for the morphological measurements of GQDs were
performed using JEOL-2010F with an acceleration voltage of 200 KV. Dynamic light scattering
(DLS) measurement was conducted using a Compact Goniometer System (ALV/Laser
Vertriebsgesellschaft M.b.h, Germany) in air at room temperature. Prior to the DLS experiment,
the polydispersed nanomaterials were filtered through a filter film (The pore size: <0.22 μm) to
remove the impurities. All Atomic Force Microscope (AFM) measurements were performed in
liquid using an Asylum Research MFP-3D Stand-Alone AFM spectrometer. Decay curves
measurements were performed with the time correlated single photo counting technique on a
combined steady state and lifetime spectrometer (Edinburgh Analytical Instruments, FLS920).
Raman spectroscopy was performed using a LABRAM-HR spectrometer with a laser excitation
wavelength of 785 nm. Ultrapure water was obtained through PSDK2-10-C (Beijing, China).
Photos were taken using a digital camera (Canon, Japan). Fourier Transform Infrared
Spectroscopy (FTIR) were measured using an FTIR-8400S spectrometer (Japan, Shimadzu) in
500–4000 cm-1 region using powdered sample on a KBr plate. All pH measurements were
measured with a Model pHs-3c meter (Shanghai, China). All optical measurements were
performed with slit information that EX Slit=2.5 nm, EM Slit=5.0 nm at room temperature under
ambient conditions.
Reduction of Exfoliated GO with KMnO4 for the Preparation of GQDs. In a typical procedure,
1 mg/mL GO (50 mL) and 1 mol/L KMnO4 (50 mL) was mixed in a 250-mL round-bottom flask,
forming a homogeneous mixture. This mixture was sonicated using a sonicator (KH3200B,
Kunshan, China) for 4 hours, insuring the complete redox process. After the ultrasonic treatment,
the mixture was centrifuged for 10 min at 3000 rpm. The supernatant containing GQDs was
collected after being centrifuged for 30 min at 10000 rpm.
Procedures for Cu2+ Sensing. 400 µL of GQDs (0.02mg mL-1) was placed in a quartz cell of 1
cm path length. Then, 50 µL of CuCl2 (10-3 M) was added to the cell and incubated for 15 min at
room temperature before the spectral measurements. The final concentration of Cu2+ was ranging
from 0 to 200 μM. For comparison, different metal ions with the final concentration of 10 μM
were added to 400 µL of GQDs (0.02mg/mL), and incubated for 15 min at room temperature. The
fluorescence spectra were recorded under excitation at 380 nm.
Procedures for ALP Sensing. ALP at different concentrations was incubated with PPi (final
concentration of 250 µM) at 37 °C for 60 min in 25 µL Tris-HCl (10 mM, pH 7.4, 100 mM NaCl).
After incubation, Cu2+ (final concentration of 100 µM) was added into the reaction solution to
give a final volume of 50 µL and incubated for 10 min. Later, 50 µL GQDs (0.02mg mL-1) was
added into the abovementioned mixture containing ALP, PPi and Cu2+ for 30 min at room
temperature. In this research, the FL intensity ratio F1/F0 (where F0 and F1 was the FL intensity of
GQDs before and after the addition of ALP) was used to evaluate the performance of the present
method.
Inhibition Effect of Pi on ALP Activity. For the inhibition assay of ALP activity by Pi, 20 μL
ALP (final concentrations of 2.5 nM) was added into 25 μL 10 mM Tris-HCl (pH 7.4, 100 mM
NaCl) containing PPi (final concentration of 250 μM) and Pi of different final concentrations, and
then incubated at 37 °C for 60 min. The following detection procedure was the same as shown in
the aforementioned experiment for ALP detection.

2 Quantum yield measurements

Rhodamine B in water (literature S1 quantum yield 0.31) was chosen as a standard. The quantum
yield of GQDs in water was calculated according to the formula1:
Φ = Φr × (I / Ir) × (Ar / A) × (n2 /nr2)
Where Φ is the quantum yield, I is the measured integrated emission intensity, n is the refractive
index of the solvent (1.33 for water), and A is the optical density. The subscript “r” refers to the
reference standard with known quantum yield. To minimize reabsorption effects, absorbencies in
the 10 mm fluorescence cuvette were kept under 0.1 at the excitation wavelength (380 nm).

Sample Integated Abs at 350 nm Refractive index Quantum yield

emission Wavelength (A) of solvent (n) (%)
Intensity (I)
Rhodamine B 1793 0.137 1.33 0.31
GQDs 3542 0.302 1.33 0.278
Table S1. Quantum yield of the GQDs

Sample τ1 / ns (B1%) τ2 / ns (B2%) <τ>/ns

GQDs 1.4 (35.82) 11 (64.18) 10.3633
GQDs+Cu2+ 1.26 (56.52) 9.61 (43.48) 8.3941
Table S2. Fluorescence lifetimes of GQDs, and GQDs+Cu2+. The lifetimes were obtained by
double-exponential fitting1 and <τ> strands for the average lifetime2 (t is the time, τ is the lifetime,
and B is the pre-exponential factor).
1 F(t) = B1e-t/τ1 + B2e-t/τ2
2 <τ> = (B1τ12 + B2τ22 ) / (B1τ1 + B2τ2)

Sample τ1 / ns (B1%) τ2 / ns (B2%) <τ>/ns

GQDs 1.4 (35.82) 11 (64.18) 10.3633
GQDs+ALP 1.45 (33.65) 10.83 (66.35) 10.2336
GQDs+PPi+Cu2+ 1.44 (36.96) 10.79 (63.04) 10.1115
GQDs+ALP+PPi+Cu2+ 1.2 (32) 10 (68) 9.5296
Table S3. Fluorescence lifetimes of GQDs, GQDs+ALP, GQDs+PPi+Cu2+, and
GQDs+ALP+PPi+Cu2+. The lifetimes were obtained by double-exponential fitting1 and <τ>
strands for the average lifetime2 (t is the time, τ is the lifetime, and B is the pre-exponential factor).
1 F(t) = B1e-t/τ1 + B2e-t/τ2
2 <τ> = (B1τ12 + B2τ22 ) / (B1τ1 + B2τ2)

3 Comparison of reported methods for the detection ALP activity

Mehtod Characteristic Linear Range Detection Ref.

Limit
Fluorescence High sensitivity, laborious 0.171-30 nM 11.4 pM S1
procedures, synthesis of (0. 2 U/L)
mutiple compounds
Fluorescence Wide linear range, relatively 10-300 nM 5 nM S2
poor sensitivity, tedious
synthesizing process of
fluorophore
Electrochemstry Easy opeation, relative high 5−640 U/mL 0.02 U/mL S3
cost and low detection limit
Colorimetry Apparatus-free, wide linear 0.032-200 U/mL 0.032 U/mL S4
range ,easy operation, low
detection limit
Fluorescence High sensitivity, laborious 6.4×10-3-0.25 U/L 1.9×10-3 S5
procedures, seperation of U/L
reaction solution
Fluorescence High sensitivity and 0.05-10 nM 17 pM This
selectivity, environmental- Wor
friendly, easy operation, low k
cost
Table S4. Comparison of reported methods for the detection ALP activity.

S1. J. Liang, R. T. K. Kwok, H. Shi, B. Z. Tang, and B. Liu, ACS Appl. Mater. Interfaces 2013, 5,
8784−8789.
S2. Y. Liu, and K. S. Schanze, Anal. Chem. 2008, 80, 8605-8612.
S3. A. Hayat, and S. Andreescu, Anal. Chem. 2013, 85, 10028−10032
S4. H. P. Jiao, J. Chen, W. Y. Li, F. Y. Wang, H. P. Zhou, Y. X. Li, and C. Yu, ACS Appl. Mater.
Interfaces. 2014, 6, 1979−1985
S5. V. Román-Pizarro, J. M. Fernández-Romero, A. Gómez-Hens, J. Agric. Food Chem. 2014, 62,
1819−1825
Fig. S1 The AFM image of GQDs.

4 Energy transfer between GQDs and Cu2+

We investigated a series of experiments to support our concept. The FTIR spectroscopy was
studied to confirm this hypothesis (Figure S2A). The FTIR spectrum revealed the existence of OH
(3792-3871, 1081-1137 cm−1), and C=O functional groups in the GQDs (black curve). However,
the presence of Cu2+ ions (red curve) caused the OH peak to diminish, while a new peak in the
frequency bands (3792−3871 cm−1) appeared. This may be attributed to the formation of COO-
Cu2+-OH complex, confirming that COOH and OH groups of GQDs were used to form a GQDs-
Cu2+ conjugate. As shown in the UV-vis spectra (Figure S2B), no obvious absorption of the Cu2+
was observed at wavelengths longer than 250 nm (curve a). In contrast, the GQDs had strong
absorption peak at 350 nm (curve b). When Cu2+ was added into the GQDs solution, this strong
absorption peak at 350 nm decreased (curve c), further verifying the successful conjugation of
Cu2+ with GQDs. This result was consistent with the digital photograph of GQD dispersion in the
presence and absence of Cu2+ under UV (365 nm) irradiation (Figure S2C, inset). As can be seen
in Figure 2C, GQDs showed strong FL when excited at 380 nm, and the addition of Cu2+ induced
the energy transfer occurred from GQDs to Cu2+, resulting in a huge decrease of the FL intensity
of the GQDs. To verify our proposed quenching mechanism, we acquired fluorescence lifetime of
the GQDs before and after the addition of Cu2+ (Figure S2D). The FL intensity decay curve of the
GQDs could be fitted with a double exponential function, and two lifetimes (a fast component and
a slow component) were obtained as listed in Table S2. The slow component (τ2) was suggested
to be related to edge states of GQDs, and it increased from 35.82% to 56.52% after the addition of
Cu2+. The reason behind this phenomenon further proved that the binding of Cu2+ at the edge of
the GQDs facilitated the energy transfer from the edge states of the GQDs to Cu2+ through oxygen
containing groups (O-C=O, OH, and C=O groups), which were mostly functioned at the edge of
the GQDs, rather than the basal plane of the GQDs. On the other hand, the fast components (τ1)
were suggested to be related to intrinsic states of GQDs and decreased in the presence of Cu2+
from 64.18% to 43.48%. The AFM shown in Figure 2E revealed that the average thickness of
GQDs in the presence of Cu2+ was ≈5.025 nm while in the absence of Cu2+ it was about 0.479 nm,
as shown in Figure 1E. The AFM results also indicated that Cu2+ could attach itself to the surface
of the GODs and might induce the aggregation of GQDs.

(A) (B)
1.6
-OH Cu2+
1.2
Transmittance (a.u.)

carboxy(-C-O) GQDs

Absorbance / cm-1
-C=O -C=C GQDs+Cu2+

-OH 0.8
-OH
carboxy(-C-O)
-C=O
0.4
-C=C

-OH 0.0
4000 3000 2000 1000 0 300 400 500 600 700
Wave numbers (cm-1) Wavelength / nm

(C) 4000 (D)

3000
GQDs GQD
2500
Fluorescence Intensity

3000 GQDs+Cu2+ GQD+Cu2+

2000
2000
Counts

1500
1000
1000
500
0 0
400 450 500 550 600 0 20 40 60 80
Wavelength Time/ns

(E)

Fig. S2 (A) FTIR spectra of the GQDs before (black curve) and after (red curve) the addition of
Cu2+. (B) UV-vis absorption of sole GQDs (black curve) and the Cu2+ solution with (red curve)
and without (blue curve) GQDs. FL (C) and Lifetime (D) spectra of GQDs with (red curve) and
without (black) Cu2+. Inset shows photographs of the GQDs in the absence (a) and presence (b) of
Cu2+ under UV irradiation. (E) AFM image of the mixture containing GQDs and Cu2+.
5 Analysis performance on Cu2+
Based on the fact that Cu2+ can quench the FL intensity of GQDs, the as-prepared GQDs can be
directly used to detect Cu2+ in buffer condition. To demonstrate the quantification of Cu2+ using
GQDs, the fluorescence intensity of the probe incubated with Cu2+ at different concentration was
monitored. The FL quenching reached a stable value within 15 min after the addition of Cu2+
(Figure S4, ESI). Therefore, GQDs and Cu2+ with concentration of 0.02-200 μM were incubated
for 30 min and their FL intensity at emission maxima was recorded. Figure 4A depicted the
fluorescence spectra of GQDs after the addition of Cu2+ of various concentrations. It is clearly
observed that the intensity of GQDs at 470 nm decreased gradually as the concentration of Cu2+
increased, indicating our sensing principle. A plot of the F1/F0 (where F0 is the peak intensity of
GQDs, and F1 is the peak intensity of GQDs after introducing Cu2+) versus logarithmic Cu2+
concentration was shown in Figure 4B. As depicted in Figure 2C vial a, GQDs possessed bright
blue emission. However, the FL of GQDs was largely quenched upon the addition of Cu2+ (0.05,
0.4, 8, and 20 nM Cu2+ for vials 1, 2, 3, 4, respectively). Figure 4C showed a quasi-linear
correlation in the concentration of Cu2+ ranging from 0.02 to 8 µM. The calibration equation was
F = 0.668-0.155lg [Cu2+] (where R = 0.9889, F is the peak intensity, [Cu2+] is the concentration of
Cu2+). A detection limit of 13 nM can be obtained (Calculated by 3σ/m, where σ is the standard
deviation, m is the slope of the linear response region). To better understand the Cu2+-induced
quenching mechanism, the influence of some metal ions on FL quenching (Figure 4D) has been
studied. The experiment was carried out in which both 10 µM Cu2+ and competitive substances
were added. The results showed that the competitive substances had minor or no interference on
the FL quenching response to Cu2+, indicating the high selectivity for Cu2+ in the presence of these
interfering substances.

(A) 4000 (B) 1.0

0 M 0.8
3000
FL intensity

0.6
2000
F1 / F0

200 M

0.4
1000
0.2
0
400 450 500 550 600 -2 -1 0 1 2 3
Wavelength lg [Cu2+](M)
(C) (D)
0.9 1.0
0.9
0.8
0.8
F1 / F0

F1 / F0
0.7 0.7
0.6
0.6 Y = 0.668-0.155X
R = 0.9889 0.5
0.5 0.4
-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0

g 2+

+
+
Na +
+

+
Ag +

Ni 2+

+
+
K+

Fe 2
Co 2
Cu 2

Hg 2

Ca 2
k

Fe 3
Zn 2
ac

M
lg [Cu2+](M)

bl
Fig. S3 (A) FL quenching of GQDs in the presence of different concentrations of Cu2+. The
concentrations of Cu2+ were 0, 0.02, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 20, 40, 80, 100, and 200
µM, respectively. (B) Plots of emission intensity change (F1/F0) vs the logarithm of the Cu2+
concentration. The inset shows optical photos of solution of GQDs in the presence of 0.05, 0.4, 8,
and 20 nM Cu2+ from left to right. (C) Linear plots of emission intensity change (F1/F0) as a
function of the logarithm of the Cu2+ concentration. Linear relationship obtained over the range of
0.02-8 µM with the correlation coefficients of 0.9889. F0 and F1 is the peak intensity of GQDs
before and after the introducing of different concentrations of Cu2+. (D) Selectivity of the GQDs-
based sensing system for Cu2+ over other interfering metal ions.

6 Charaterization of GQD-based ALP sensor

(A) (B)
PPi+ALP 2.0
0.4 PPi 0.6
ALP 1.6

Absorbance / cm-1
Absorbance / cm-1

Absorbance / cm-1
0.5
d
0.4
1.2
0.2 0.3
0.8 a
0.2
280 300 320 340 360
0.4 Wavelength / nm

0.0 0.0
200 300 400 500 600 700 300 400 500 600 700
Wavelength / nm Wavelength / nm

(C) (D)
4000 GQDs
3000
GQD
GQDs+ALP
GQDs+PPi+Cu2+
2500 GQD+ALP
GQD+PPi+Cu2+
3000 GQDs+PPi+Cu2++ALP 2000 GQD+ALP+PPi+Cu2+
FL intensity

2000 Counts 1500

1000
1000
500
0 0
400 450 500 550 600 650 0 20 40 60 80
Wavelength Time/ns

Fig. S4 (A) UV-vis spectra of ALP (blue curve), PPi (red curve), ALP+PPi (black curve). UV-vis
spectra (B), FL emission spectra (C), and lifetime spectra (D) of GQDs (black), GQDs/ALP (blue
curve), GQDs/PPi+Cu2+ (red curve), and GQDs/PPi+Cu2++ALP (green curve). Inset shows the
photographs (from left to right) of GQDs, GQDs+ALP, and GQDs+PPi+Cu2+ (raw a), and the
solutions after incubation for 30 min (raw b).

7 Reaction time of GQDs and Cu2+

 3500
3000
2500
FL intensity

2000
1500
1000
500
0 200 400 600 800 1000
Time / s

Fig. S5 Reaction time of GQDs and Cu2+

8 The optimized concentration of PPi

1.0

0.8
F1 / F0

0.6

0.4

0.2
0 200 400 600 800
[PPi] (M)

Fig. S6 The optimized concentration of PPi.

9 The reaction time of ALP and PPi

 1.0

0.8

0.6
F1 / F0

0.4

0.2

0.0
0 10 20 30 40 50 60 70 80 90
Time / min

Fig. S7 The reaction time of ALP and PPi.

10 The inhibition effect of Pi on ALP activity
Morton has reported that the inhibitor of ALP may be the competitive inhibitors of hydrolysis,
such as phosphate due to the competitive binding to the ALP active site. 6 Thus, the inhibition of
ALP activity by Pi was investigated, as shown in Figure S8 in the supporting information. The
experiment was carried out in 10 mM Tris-HCl containing 2.5 nM ALP, 250 µM PPi and Pi of
different concentrations. Figure S4 shows that with increasing Pi concentration, the F1/F0 value
gradually increased. At about 3 mM Pi concentration, the F1/F0 reached a maximum value. The
results strongly suggest that Pi could inhibit the activity of ALP.
S6 R .K. Morton, Biochem. J. 1955, 61, 232−240.

0.9

0.8
F1/F0

0.7

0 1 2 3 4 5
[Pi] (mM)
Fig. S8 The inhibition effect of Pi on ALP activity.

11 The effect on the formation of luminescent GQDs by ultrasonication.

Ultrasonication is an important experimental step in the reduction of GO method to produce
GQDs. In order to prove the point, the effect on the formation of luminescent GQDs by
ultrasonication was also investigated. The ultrasonic processor was operated under the continuous
mode. In order to investigate the effect of ultrasonication on the fragmentized GQDs, control
experiment without ultrasonication under the same conditions has been tried. As the SEM showed,
without the ultrasonication treatment, the GO was still a monolithic structure but battered which
proved the role of ultrasonication in this work (Fig. S9). Besides we also investigated the effect of
ultrasonication conditions on the fragmentized GQDs, input power was set to the 25%, 50%, 75%,
100%, 150%, 200% of the power (700 W) and irradiation time to 0.5 h, 1.0 h, 1.5 h, 2 h, 4h, or 6 h,
respectively. The result in Fig. S10 showed that the ultrasonication process indeed played an
important role in the synthesis of GQDs.

Fig. S9 The SEM images before (left) and after (right) the addition of KMnO4 without
ultrasonication treatment.

(A) (B)
0.9 0.9

0.6 0.6
F1 / F0
F1 / F0

0.3 0.3

0.0 0.0
0 1 2 3 4 5 6 50 100 150 200
Irradiation Time (h) Input power (%)
Fig. S10 The irradiation time and input power used in the experiment.

12 The correlation between the pH and quantum yield

 We investigated the quantum yield in media with different pH conditions. (We used H2SO4 and
NaOH to adjust the pH value of the media.) The result was shown in the revised manuscript in
yellow color. As the result showed, the reaction media, whether under the acidic or alkaline
conditions, had some effect on the quantum yield of GQDs. It is found the QY is higher at pH 7.0
compared to that at acid or alkaline conditions. Specially, the QY decreased with the pH value
increasing in the range of 2 to 4. We assumed under this condition the concentration of H2SO4 is
high enough to play the role of the reactor as the literature reported.7 The alkaline condition
appeared to have more effect on the quantum yield than the acidic condition. An explanation
behind this may be that alkaline conditions can alter the functional group at the edge of GQDs
more easily.

30
25
20
QY (%)

15
10
5
0
2 4 6 8 10 12
pH
Fig. S11 The relationship between QY (%) and pH of reaction media.
S7. Y. Shin, J. Lee, J. Yang, J. Park, K. Lee, S. Kim, Y. Park, and H. Lee, Small 2014, 10, 866-
870.

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