Full Papers
Guangfu Reagent Company. 3,3’,5,5’-tetramethylbenzidine (TMB;
quality of the fit that included plots of surfaces (“carpets”) of the
weighted residuals versus the channel number. All curve fittings
presented here had minimizing the reduced chi-square, c , values
À1
ꢁ
98%), horseradish peroxidase (HRP; 250 Umg ), and 2-hydroxy-
terephthalic acid (HTA; 98%) were purchased from Energy Chemi-
2
cal. Terephthalic acid (TA; ꢁ98.5%) and methylene blue (MB;
of <1.1.
ꢁ
98.5%) were purchased from Chengdu cologne chemical reagent
factory. All reagents and solvents were of analytical grade and
used directly without further purification, and all aqueous solutions
were prepared with Milli-Q water (18.2 MWcm).
Synthesis of Co@C-dots
C-dots were prepared according to our previous report, and the
details are described in Section 1.1 of the Supporting Informa-
tion. The Co@C-dots hybrid material was prepared by a simple
chemical reduction route. Typically, an amount of cobalt chloride
(1.07 g) was dissolved directly in ethylene glycol (100 mL), and
Instruments
[24]
The morphology and microstructure of samples were analyzed by
transmission electron microscopy (TEM) on a JEM-2100 instrument
equipped with an energy-dispersive X-ray (EDX) spectrometer. The
samples were dispersed in ethanol and then dried on a micro grid.
X-ray diffraction (XRD) measurements were performed on an X-ray
diffractometer (D/max-2400pc, Rigaku, Japan) with CuKa radiation
CoCl ·6H O solution (45 mm; 10 mL) was mixed with an aqueous
2 2
suspension of C-dots (10 mL) in a 100 mL round-bottom flask with
a magnetic stirrer bar. After the mixture had been stirred for a few
minutes, hydrazine hydrate (1.125 g) was then added to the above
(
l=1.54178 ), by using an operating voltage and current of 40 kV
solution. The mixture was kept vigorously stirring at T=608C for
À1
and 60 mA, respectively. The 2q range was from 108 to 908 in steps
of 0.028. For XRD observations, the samples were dispersed in eth-
anol and then dried on a glass slide. X-ray photoelectron spectra
30 min. Subsequently, a 5 mgmL NaBH
4
aqueous solution was
added dropwise to trigger the reaction. The bright-red solution
became dark, which indicated the formation of Co NPs, and was
kept stirring for another 1 h at T=608C. Finally, the product was
washed with absolute alcohol and dissolved as an aqueous solu-
(
XPS) were measured on a PHI-550 spectrometer by using MgKa ra-
diation (hn=1253.6 eV) with a base vacuum operated at 300 W.
The Fourier transform infrared (FTIR) spectra were measured on
a Nicolet 360 FTIR spectrometer by using the KBr pellet technique.
À1
tion (5 mgmL ) to generate a colloidal suspension of Co@C-dots
nanoparticles.
Steady-state UV/Vis absorption and fluorescence
spectroscopy
Peroxidase-like activity of the Co@C-dots
The peroxidase-like activity of the Co@C-dots hybrid material was
tested through the catalytic oxidation of the peroxidase substrate
The absorbances of catalytic reaction processes were recorded on
a UV/visible spectrometer (Cary 100) under experimental condi-
tions. The steady-state excitation and emission spectra were ob-
tained on an FLS920 spectrofluorometer. 3D maps were collected
with an excitation range of l=300–500 nm and an emission range
of l=320–590 nm. Freshly prepared samples in 1 cm quartz cells
were used to perform all UV/Vis absorption and emission measure-
ments.
3
,3’,5,5’-tetramethylbenzidine (TMB) in the presence of H O . Typi-
2 2
cally, TMB (8 mm; 50 mL), H O2 (30 wt%; 20 mL), and colloidal
2
Co@C-dots nanoparticles (50 mL) were added into sodium acetate
buffer solution (2 mL; pH 3.0) at T=458C, unless otherwise stated.
The reactions were carried out for 10 min and then were moni-
tored by observing the absorbance at l=652 nm. The relative ac-
tivity was calculated by A /A , in which A was the maximum ab-
1
2
2
Quantum yields were determined by an absolute method with an
integrating sphere based upon that originally developed by de
Mello et al. Experiments were conducted on an FLS920 spectro-
fluorometer from Edinburgh Instruments.
sorbance and A was the sample absorbance measured under the
same conditions.
1
[27]
Kinetic analysis
Time-resolved fluorescence spectroscopy
The kinetic analysis was carried out by monitoring the absorbance
at various times with a spectrophotometer. The experiments were
performed at T=458C with enzyme mimetics (5 mgL ; 50 mL) in
Fluorescence lifetimes were measured on an Edinburgh Instru-
ments FLS920 spectrofluorometer equipped with different light-
emitting diodes (excitation wavelengths of l=300 and 330 nm),
À1
a 2 mL reaction volume of sodium acetate buffer solution (pH 3.0)
[28]
by using the time-correlated single-photon counting technique
with 8 mm TMB or 100 mm H O as a substrate, unless otherwise
2
2
in 2048 channels at room temperature. The sample concentrations
were adjusted to optical densities of <0.1 at the excitation wave-
length (l=300 or 330 nm). The monitored wavelengths were l=
stated. The Michaelis–Menten constant was calculated by using
the Lineweaver–Burk plot: 1/V =(K /V )(1/[S]+1/K ), in which V
0
0
m
m
m
is the initial velocity, Vm is the maximal reaction velocity, and [S] is
4
40, 460, and 480 nm.
the concentration of the substrate.
Histograms of the instrument response functions (by using LUDOX
scattering) and sample decays were recorded until they reached
typically 5.0103 counts in the peak channel. The obtained histo-
grams were fitted as sums of the exponentials, with Gaussian-
weighted nonlinear least squares fitting based on the Marquardt–
Levenberg minimization implemented in the software package of
the instrument. The fitting parameters (decay times and pre-expo-
nential factors) were determined by minimizing the reduced chi-
The measurement of the hydroxyl free radicals
The reaction was performed in a TA solution (1 mm; 2 mL) with
H O (30 wt%; 6 mL) and different volumes of Co@C-dots. The fluo-
2
2
rescence spectrum was then measured between l=350 and
600 nm with l=315 nm as the excitation wavelength. The same
experiments were also performed for C-dots and Co NPs.
2
square, c . An additional graphical method was used to judge the
ChemCatChem 2015, 7, 2467 – 2474
2473
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim