Highly chemiluminescent gold nanopopcorns functionalized by N-(aminobutyl)-N-(ethylisoluminol) with lipoic acid as a co-stabilizing reagent

Article abstract of DOI:10.1039/c2tb00375a

Herein we report a novel facile synthesis of Au nanopopcorns (AuNPCs) with high chemiluminescent yield by reducing (±)-α-lipoic acid (LA) and HAuCl₄ with N-(aminobutyl)-N-(ethylisoluminol) (ABEI) in ethyl alcohol solution at room temperature through a seed growth method. The morphologies of AuNPCs could be changed by adjusting the mole ratios of ABEI, LA and HAuCl₄. The characterization results demonstrated that ABEI and the reduction product of LA as stabilizers were coated on the surface of AuNPCs during the synthesis. The AuNPCs, functionalized with ABEI, could react with various oxidants (H₂O₂, KMnO₄, NaClO, Fe(CN)₆^3- and AgNO₃) to produce chemiluminescence (CL). The special morphology of AuNPCs maintains the large specific surface area, inherited from its shape and demonstrated excellent catalytic property when involved in CL reaction with H₂O₂. Thus the CL efficiency of AuNPCs was more than two orders of magnitude higher than that of ABEI functionalized gold nanoparticles in our previous work. The AuNPCs are capable of direct conjugation with protein and DNA while maintaining their luminescent properties, thus they could be potentially useful as a bio-probe for immunoassays and DNA assays. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00375a

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Materials Chemistry B
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Highly chemiluminescent gold nanopopcorns
functionalized by N-(aminobutyl)-N-(ethylisoluminol)
Cite this: J. Mater. Chem. B, 2013, 1,
970
with lipoic acid as a co-stabilizing reagent
*
Hongli Zhang, Mengxiao Liu, Guangming Huang, Yuqi Yu, Wen Shen and Hua Cui
Herein we report a novel facile synthesis of Au nanopopcorns (AuNPCs) with high chemiluminescent yield
by reducing (ꢀ)-a-lipoic acid (LA) and HAuCl4 with N-(aminobutyl)-N-(ethylisoluminol) (ABEI) in ethyl
alcohol solution at room temperature through a seed growth method. The morphologies of AuNPCs
could be changed by adjusting the mole ratios of ABEI, LA and HAuCl4. The characterization results
demonstrated that ABEI and the reduction product of LA as stabilizers were coated on the surface of
AuNPCs during the synthesis. The AuNPCs, functionalized with ABEI, could react with various oxidants
3ꢁ
(H2O2, KMnO4, NaClO, Fe(CN)6
and AgNO3) to produce chemiluminescence (CL). The special
morphology of AuNPCs maintains the large specific surface area, inherited from its shape and
demonstrated excellent catalytic property when involved in CL reaction with H2O2. Thus the CL
efficiency of AuNPCs was more than two orders of magnitude higher than that of ABEI functionalized
gold nanoparticles in our previous work. The AuNPCs are capable of direct conjugation with protein
and DNA while maintaining their luminescent properties, thus they could be potentially useful as a bio-
probe for immunoassays and DNA assays.
Received 7th November 2012
Accepted 22nd November 2012
DOI: 10.1039/c2tb00375a
www.rsc.org/MaterialsB
In our previous work, a direct synthesis strategy was devel-
oped for the preparation of CF-GNMs through a one-pot method
1 Introduction
or seed growth method.17–19 CL reagents, such as luminol and
N-(aminobutyl)-N-(ethylisoluminol) (ABEI), were directly coated
on the surface of GNMs as stabilizers during the synthesis and
these functionalized gold nanoparticles were successfully used
for immunoassays and DNA assays.20,21 However, the CL signal
still needs to be further improved to achieve excellent sensi-
tivity. It is well known that GNMs' properties are both size- and
shape-dependent. Earlier studies showed that nanomaterials
with novel shapes exhibited unique optical, electrical and
catalytic properties.22,23 Thus we aim to prepare novel CF-GNMs
with high CL yield by controlling their size and shape. Accord-
ing to previous studies, there are three principal strategies for
generating complex nanostructures: template-based synthesis,
oriented attachment of primary nanoparticles, and anisotropic
growth induced by specic capping agents.24–28 For the seeding
approach carried out in aqueous solution, the third method is
commonly used. One approach is to introduce surfactants.
Preferential bonding of surfactants such as hexadecyl-
trimethylammonium bromide (CTAB) on a given facet of
nanomaterials promotes the growth of the uncovered surface,
resulting in the anisotropic growth. However, it is not suitable
for bioassays such as immunoassays and DNA analysis due to
the toxicity of CTAB. As for another capping agent, (ꢀ)-a-lipoic
acid (LA) is widely used in preparing metal nanoparticles, gold
monolayers and nanoclusters.29–33 The presence of both a
Nanomaterials have shown great promise due to their high
surface area, catalytic property and chemical reactivity. They
possess unique properties different from those of bulk systems
that make them useful in a number of elds.1–6 Particular
interests have been generated about nanostructured noble
metal particles because of their low toxicity, high affinity with
biomolecules and suitability for surface immobilization of a
wide range of biomolecules.7,8 Recently, attention has been paid
to chemiluminescence (CL) functionalized gold nanomaterials
(CF-GNMs) for bio-labeling.9,10 The used CL reagents included
Ru-complexes, luminol and its analogues.11,12 Most synthetic
methods for obtaining CF-GNMs follow indirect strategies, i.e.,
to gra the CL reagents on the surface of GNMs by virtue of
bridging molecules. The obtained CF-GNMs could be used for
bioassays with high sensitivity.13,14 However, this strategy is
complicated and time-consuming. Moreover, for some ultra-
sensitive detections, the CL intensity cannot satisfy the
requirements and needs further amplication, which would
increase the complexity of the experiments.15,16 Therefore, it is
highly desired to exploit a facile synthesis strategy for the
preparation of CF-GNMs with improved CL property.
CAS Key Laboratory of So Matter Chemistry, Department of Chemistry, University of
Science and Technology of China, Hefei, Anhui 230026, P. R. China. E-mail: hcui@
ustc.edu.cn; Fax: +86-0551-3600730; Tel: +86-0551-3600730
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disulde (in its oxidized form) and the ring structure leads to a and the reaction was kept on for another 12 h, during which the
rigid conformation for the coating on the GNMs and confor- color of the solution changed from yellow to green, or purple,
mation stability is enhanced aer association with the amine depending on the volume of HAuCl4 added. All the prepared
groups. Thus, LA may serve as a suitable stabilizing reagent, colloidal gold dispersions were stored at 4 ꢂC for future use. In
together with ABEI as a co-stabilizing reagent in the reaction to the synthesis of various morphologies of GNMs, the mole ratios
obtain CF-GNMs with new morphologies.
Herein we present a facile method for the synthesis of When the mole ratio was 1 : 3 : 5.25, AuNPCs could be obtained.
popcorn-shaped GNMs with high CL yield and good mono- The as-synthesized colloidal gold dispersions were subse-
of ABEI, LA and HAuCl4 were in the range 1 : 3 : 4.5 to 1 : 3 : 6.
dispersity by reducing LA and HAuCl4 with ABEI in ethyl alcohol quently characterized by TEM (JEOL Ltd, JEOL-2010, Japan),
solution at room temperature through a seed growth method. SEM (JEOL JSM-670F, Japan), EDS (JEOL Ltd, JEOL-2010, Japan),
The morphologies of GNMs were characterized by transmission FT-IR (Bruker Instruments, Billerica, MA), and UV-visible
electron microscopy (TEM), scanning electron microscopy spectroscopy (Agilent 8453 UV-visible spectrophotometer, USA).
(SEM) and energy dispersive spectroscopy (EDS). The effect of The histograms were obtained by measuring the diameters of
the mole ratios of ABEI, LA and HAuCl4 on the morphologies of nanoparticles (n > 100) in TEM photomicrographs. AuNPCs
GNMs was examined. The surface composition of AuNPCs was were chosen for UV-visible spectroscopy, SEM, FT-IR and XPS
studied by UV-visible spectroscopy, X-ray photoelectron spec- characterization. Through a centrifugation–ultrasonic disper-
troscopy (XPS) and Fourier transform infrared spectroscopy sion process, the inorganic or organic impurities were removed
(FT-IR). The results strongly supported that ABEI, the oxidation completely. The precipitates were redispersed in the ultrapure
product of ABEI (ABEI-ox) and reduction product of LA (LA-H) water for UV-visible detection and redispersed in ethyl alcohol
were attached to a gold core by covalent bonds. A possible solution for SEM detection. The puried AuNPCs were dried
mechanism of shape evolution is proposed. Furthermore, since under vacuum at room temperature for XPS and FT-IR detec-
ABEI was coated on the surface of AuNPCs, the CL property of tions. XPS was performed on an ESCALABMK II electron spec-
AuNPCs was studied by static injection and this material trograph (VG Scientic, UK) with Al Ka radiation as the X-ray
exhibited good CL property. Finally, the applicability of AuNPCs source. The XPS data were tted by XPS Peak and further plotted
as labels for protein and DNA to construct bio-probes was by Origin Pro 7.0.
explored. The bio-probes still maintained good CL activities,
which make them suitable for bioassays with high sensitivity in
the future.
2.3 CL detection of AuNPCs
The static injection CL detection was conducted on a BPCL
Luminescence Analyzer (Beijing, China) with a xed voltage of
ꢁ800 V. For a typical CL measurement, 300 mL of the centri-
fuged AuNPCs dissolved in NaOH solution of different pH
values was added to a cylindrical cell, then 100 mL of various
oxidants with different concentrations were injected into the
cell to initiate the CL reaction, respectively. The CL intensity
during the reaction along with time was recorded by the
Luminescence Analyzer.
2 Experimental section
2.1 Materials and chemicals
HAuCl4 stock solution (2% HAuCl4, w/w) was prepared by dis-
solving 1.0 g of HAuCl4$4H2O (Shanghai Reagent, China) in
412 mL of puried water and stored at 4 ꢂC. ABEI and LA were
purchased from TCI (Japan). Protein was obtained from Solar-
bio (Beijing, China). DNA used in this work was synthesized and
HPLC-puried by Sangon Inc. (Shanghai, China). A 4.0 mM
stock solution of ABEI was prepared by dissolving ABEI in 0.1 M
NaOH solution and was kept at 4 ꢂC. A 6.0 mM stock solution of
LA was prepared by dissolving LA in ethyl alcohol and kept at
4 ꢂC. All other reagents were of analytical grade. Ultrapure water
was prepared by a Milli-Q system (Millipore, France) and used
throughout.
2.4 Fabrication of the bio-probe and its CL measurements
The AuNPCs were used as tags to label protein and DNA to
obtain bio-probes. The bio-probe, AuNPCs labeled lysozyme,
was prepared as follows: 300 mL (pH 7.4) of the post-centrifu-
gation and resuspension colloidal gold solution was added to
10 mL of lysozyme (1.0 mg mLꢁ1). Aer incubation at 25 C for
ꢂ
1 h, AuNPCs labeled lysozyme bio-probes were obtained. The
fabrication of AuNPCs labeled DNA bio-probes was carried out
as follows: 300 mL (pH-adjusted; pH 7.4) of the post-centrifu-
2.2 Synthesis and characterization of GNMs
All glassware used in the following procedures was cleaned in a gation and resuspension AuNPCs solution was added to 10 mL of
bath of freshly prepared HNO3–HCl (3 : 1, v/v), rinsed thor- 1 ꢃ 10ꢁ6 M DNA solution that was dissolved in 0.05 M Tris–HCl
ꢂ
oughly in ultrapure water, and dried prior to use. Gold colloids buffer (pH 7.4, containing 0.1 M NaCl) and incubated at 37 C
with various morphologies were prepared by the reduction of LA for 1 h. A thermoshaker (Biosan, Latvia) was used to control the
and HAuCl4 by ABEI in ethyl alcohol solution at room temper- temperature of the reactions.
ature. 1 mL of ABEI solution (4 mM) and 2 mL of LA (6 mM)
The static injection CL measurement of AuNPCs labeled
solution were mixed in 5 mL of ethyl alcohol. While stirring lysozyme or DNA bio-probes was conducted on a Luminescence
vigorously, 3 mL of HAuCl4 (6 mM) solution was added rapidly Analyzer with a xed voltage of ꢁ800 V. For a typical CL
and the solution was maintained at room temperature for 12 h. measurement, 300 mL of AuNPCs labeled lysozyme or DNA bio-
Then another portion of HAuCl4 solution was secondly added probes was added to a cylindrical cell, then 100 mL of 0.1 M H2O2
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containing 0.1 M NaOH was injected into the cell to initiate the
CL reaction. The CL intensity during the reaction along with
time was recorded by the Luminescence Analyzer.
3 Results and discussion
3.1 Synthesis and characterization of GNMs
The GNMs were prepared by reducing LA and HAuCl4 with ABEI
at room temperature through a seed growth method. The
reaction pathways and the molecular formulas of reagents are
shown in Scheme 1. In a typical experiment, in the rst step, 1
mL of ABEI (4 mM) was mixed with 2 mL of LA (6 mM) solution
in ethyl alcohol solution. In the second step, 3 mL of HAuCl4
(6 mM) was added to the mixed solution as mentioned above
and stirred strongly for 12 hours at room temperature. A light
yellow solution containing gold nanodots was obtained
(Fig. 1A). In the third step, another portion of HAuCl4 was added
and the mixed solution stirred until the color changed from
Fig. 1 TEM images and UV-visible spectra of GNMs synthesized by different mole
ratios of ABEI, LA and HAuCl4: (A) 1 : 3 : 4.5, (B) 1 : 3 : 5.25, (C) 1 : 3 : 5.5, and (D)
1 : 3 : 6.
light yellow to green (MABEI : MLA : MHAuCl ¼ 1 : 3 : 5.25), dark
4
green (MABEI : MLA : MHAuCl
¼
1 : 3 : 5.5) and purple
4
(MABEI : MLA : MHAuCl ¼ 1 : 3 : 6) (Fig. 1A–D). Fig. 1 shows TEM
4
When the mole ratio of ABEI, LA and HAuCl4 was 1 : 3 : 5.25,
AuNPCs could be obtained. Since the as-prepared AuNPCs
could provide a larger active surface than other morphologies
due to the irregular bumps on the surface of AuNPCs34 and
might produce strong CL, they were further characterized. Fig. 2
shows the TEM images and EDS results of AuNPCs. The average
overall dimension of AuNPCs was 48 ꢀ 6 nm (see the inset in
Fig. 2 for the histogram of the size distribution). The peaks of
EDS were labeled with the lines of the corresponding elements,
and revealed that the main elements that AuNPCs contained
were Au and S apart from C, H, O and N. Fig. 3 shows the SEM
result of AuNPCs. The image shows plenty of AuNPCs distrib-
uted on the surface of the substrate, which was very consistent
with the TEM results. Furthermore, the images indicate that the
morphology of AuNPCs was different from the regular spherical
gold nanoparticles in our previous study.17
images and UV-visible spectra of various morphologies of
GNMs. With the increased amount of HAuCl4 (second addition),
the morphologies of the as-synthesized GNMs evolved from
gold nanodots (Fig. 1A), nanopopcorns (Fig. 1B), quasi-spher-
ical (Fig. 1C), to irregular GNMs (Fig. 1D). Corresponding to the
morphology evolution, a blue shi of the maximum absorption
wavelength from around 600 nm to 545 nm was observed and
the shape of the absorption peaks also changed, which was
accompanied by a color change from green, dark green to
purple. Therefore, various morphologies of CF-GNMs can be
synthesized by the proposed method. Moreover, this synthetic
method is also reproducible.
The stability of AuNPCs was examined, which was found to
be strongly dependent on the pH value. When stored at 4 ꢂC, the
colloids were stable for at least 3 months if the pH was $8,
whereas coagulation and precipitation occurred rapidly if the
Scheme 1 Schematic illustration for the synthesis of GNMs with different
Fig. 2 TEM images and the EDS result of AuNPCs. The inset is the histogram of
the size distribution of AuNPCs and the average overall dimension was 48 ꢀ 6 nm.
morphologies.
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Fig. 4 UV-visible absorption spectra of pure ABEI (black line), pure LA (red line)
and AuNPCs (blue line). Inset: partial magnification of the blue line.
Fig. 3 SEM image of AuNPCs after centrifugation and redispersion.
molecules existed on the surface of AuNPCs and LA also existed
probably.
pH was #3. This observation implied that the surface of
AuNPCs was negatively charged and the electrostatic repulsion
was the main factor for the stabilization.18 At low pH values,
most of the negatively charged groups were protonated, and
consequently the coulombic repulsive interaction became weak
and aggregation and precipitation occurred. This pH-depen-
dent stability was similar to the citrate-capping gold
nanoparticles.35
Fig. 5A and D show the Au 4f and S 2p X-ray photoelectron
spectra of pure LA and as-prepared AuNPCs aer centrifugation,
washing and drying under vacuum. The Au 4f spectrum of
AuNPCs was curve-tted into two components at 83.6 and
87.3 eV, and the spin–orbit splitting of doublet components for
Au 4f7/2 and Au 4f5/2 was measured to be 3.7 eV. It was in good
agreement with our previous report.37 This result conrmed the
presence of Au0 in AuNPCs. As shown in Fig. 5D, the S 2p
spectrum of AuNPCs indicated the existence of S element,
supporting that LA-H molecules were adsorbed on the surface
of AuNPCs through the Au–S bond.
The N 1s X-ray photoelectron spectra of pure ABEI and the as-
prepared AuNPCs are shown in Fig. 5B. All binding energies
(BEs) were calibrated with respect to the C 1s BEs at 284.6 eV.
The analysis of the N 1s spectrum was consistent with
the discussion above. The N 1s spectrum of pure ABEI was
3.2 Clarication of the surface state of AuNPCs
It has been reported that LA is readily reduced by reducing
agents to LA-H,32,36 and ABEI could be oxidized by HAuCl4 to
ABEI-ox.19 Accordingly, in this reaction, LA was rst reduced by
ABEI to form LA-H probably. Then HAuCl4 was reduced by ABEI
to form GNMs and ABEI was probably oxidized to ABEI-ox. Since
LA-H contains thiol groups, LA-H is prone to be bonded on the
surface of GNMs due to the Au–S bonds. Considering that both
ABEI and ABEI-ox contain an aliphatic amine group, they might
be co-adsorbed onto the surface of AuNPCs through covalent
interaction between gold and nitrogen atoms in their amino
groups.17 Thus it can be deduced that ABEI, ABE-ox and LA-H
might co-exist on the surface of AuNPCs.
Accordingly, the surface composition of AuNPCs was exam-
ined by UV-visible spectroscopy, XPS and FT-IR. Before the
characterizations, free ABEI and other molecules from colloids
were removed by appropriate methods as described in the
Experimental section so that the true information about the
surface of AuNPCs could be obtained. The UV-visible absorption
spectrum of AuNPCs is shown in Fig. 4. The characteristic
absorption peaks of pure ABEI (black line) appeared at around
290 nm and 320 nm, respectively. The characteristic absorption
peak of pure LA (red line) appeared around 320 nm. The
maximal surface plasmon absorption wavelength of AuNPCs
colloid treated by centrifugation and redispersion showed two
broad peaks around 290–390 nm and 500–800 nm. The peak
around 500–800 nm was attributed to the special morphology of
Fig. 5 X-ray photoelectron spectra of (A) Au 4f, (B) N 1s of pure ABEI and
AuNPCs, (C) C 1s of pure ABEI, pure LA and AuNPCs, and (D) S 2p of pure LA and
AuNPCs. The peak around 300 nm demonstrated that ABEI AuNPCs.
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curve-tted into two components at 399.5 and 400.8 eV attrib-
uted to the nitrogen atoms in the C–NH2 and –N–CO– group,
respectively. The area ratio (1.04 : 1) matched the theoretical
value with the same full width at half-maximum (fwhm) 1.60 eV.
The peaks of the N 1s spectrum of AuNPCs had a negative shi
and the area ratio was no longer 1 : 1, implying that the –N–CO–
group of ABEI was partially oxidized to the –COO– group. This
result revealed that both ABEI and ABEI-ox co-existed on the
surface of AuNPCs. Notably, the N 1s spectrum of AuNPCs also
+
conrmed the absence of the protonated state (–NH3 ) reported
to be at approximately 402.3 eV. Consequently, the electrostatic
interaction between the protonated amino group and negatively
charged AuNPCs did not exist, which was reported to occur in
the other fatty-amine-protected GNMs in aqueous solution.38
Therefore, the Au–N covalent interaction was the only force
between the gold core and ABEI or ABEI-ox. In addition, the
FT-IR spectra below also provided the evidence for the existence
of Au–N covalent interaction.
Fig. 6 FTIR spectra of pure LA (blue line), pure ABEI (red line) and AuNPCs (black
line).
In the rst step of this synthesis, xed amounts of ABEI and
LA were mixed in ethyl alcohol solution and an oxidation–
reduction reaction occurred. ABEI was oxidized to ABEI-ox and
LA was reduced to LA-H. In the second step, 3.0 mL of HAuCl4
was added to the mixture solution and reduced by ABEI to
generate gold nanodots. Meanwhile, ABEI, ABEI-ox and LA-H
acted as stabilizing reagents, being coated on the surface of gold
nanodots. In the third step, another portion of HAuCl4 was
added to the reaction mixture, and further reduced by the
excess ABEI in the mixture. When adding 0.5 mL of HAuCl4 in
the solution, AuNPCs were obtained. This might be due to the
formation of more gold nanodots and the aggregation of gold
nanodots, which seems to be a reasonable pathway for mini-
mizing the surface energy of nanomaterials.39 When adding
0.7 mL of HAuCl4 in the solution, quasi-spherical GNMs were
obtained. In this case, AuNPCs were rst formed, which may
grow into quasi-spherical GNMs via a second-growth process in
order to further reduce the surface energy of GNMs.39 When
adding 1 mL of HAuCl4 in the solution, irregular nanomaterials
were obtained. This might be due to the formation of a high
concentration of gold nanodots with a large volume of HAuCl4,
resulting in the anisotropic growth to produce faceted irregular
GNMs.40,41 However, the mechanism still needs to be further
investigated and related work is underway.
Fig. 5C demonstrates the C 1s X-ray photoelectron spectra of
pure ABEI, pure LA and the as-prepared AuNPCs. BEs were also
calibrated with respect to the C 1s BE at 284.6 eV. As can be seen
from Fig. 5C, the C 1s spectrum of pure ABEI was curve-tted
into three components at 284.9, 286.0, 287.3 eV with the same
fwhm 1.25 eV. The maximal peak centered at 284.9 eV was
attributed to the carbon atom in the –C–C– group. The
component at 286.0 eV was associated with the carbon atom in
the –C–NH2 group. The component at 287.3 eV was due to the
carbon atom in the –CO–NH– group. The C 1s spectrum of pure
LA was curve-tted into two components at 284.6 and 288.9 eV
with the same fwhm. The maximal peak centered at 284.6 eV
was attributed to the carbon atom in the –C–C– group. The
component at 288.9 eV was due to the carbon atom in the
–COO– group. For the AuNPCs we synthesized in this work,
the C 1s spectrum showed that –C–C–, –C–NH2, and –COO–
groups existed. Compared with the C 1s spectrum of pure ABEI,
peaks shied a bit respectively, toward lower binding energies.
Moreover, the C 1s peak of –C–NH2 indicated that ABEI existed
on the surface of AuNPCs.
The results above revealed that LA-H, ABEI and ABEI-ox
coexisted on the surface of AuNPCs. Moreover, XPS results of
the N 1s spectrum revealed the existence of the weak interaction
between the amino group and AuNPCs. Fig. 6 showed the FTIR
spectra of pure LA, pure ABEI and AuNPCs. The typical doublets
3.4 CL property of AuNPCs
of the amino group in the ABEI standard IR spectra around 3400 Since the prepared AuNPCs contain a large amount of ABEI
cmꢁ1 (nN–H) disappeared and only one broad band appeared in molecules, it should be highly CL active. Its CL performance was
the range of 3500 cmꢁ1, which also implied the existence of the investigated by static injection. We examined the CL properties
weak Au–N covalent interaction between the amino group and of AuNPCs that reacted with various oxidants, including H2O2,
AuNPCs. Therefore, the gold core and its capping molecules KMnO4, NaClO, Fe(CN)63ꢁ and AgNO3. As shown in Fig. 7, when
were connected by Au–N covalent interaction and Au–S covalent 100 mL of oxidants and 300 mL of centrifuged AuNPCs were
interaction.
injected into a cylindrical cell, obvious CL emission was
observed. The results demonstrated that all the oxidants
studied in this work could react with ABEI coated on the surface
of AuNPCs to produce CL. The light emission was due to the
3.3 Mechanism for the formation of GNMs
Various morphologies of GNMs were observed in this study and reaction between ABEI and the oxidant as shown in Scheme 2.
the schematic illustration of synthetic reactions is summarized During the reaction, ABEI was oxidized by the oxidant to the
in Scheme 1. Herein, we proposed a possible mechanism for the excited ABEI-ox. The light emission was observed when the
formation of various morphologies of GNMs.
excited state decayed to the ground state.43–46 Among these
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properties of AuNPCs in various pH conditions were similar to
the properties of luminol-type compounds.42 Moreover, a
comparison of the CL behaviors of AuNPCs with the ABEI
functionalized Au nanoparticles obtained in our previous work
when both reacted with 0.1 M H2O2 was carried out by static
injection. As shown in Fig. 9, the results revealed that the CL
intensity of AuNPCs was almost two orders of magnitude higher
than ABEI functionalized gold nanoparticles at pH 13 (the
optimized condition for ABEI functionalized spherical gold
nanoparticles); further improvement could be obtained at pH
12 (the optimized condition for the popcorn-shaped GNMs).
The CL intensity of AuNPCs reached its peak value in a few
seconds, and then kept fairly stable for over half an hour. This
feature is good for bioassays. As an antioxidant, LA and its
reduced form, LA-H, are known to be capable of quenching
reactive oxygen and nitrogen species,47 thus LA is not the reason
for the improved CL property. In addition, the mole ratio of
ABEI–HAuCl4 (1 : 5.25) was less than that of our previous work
(1 : 2.8), which implies that excess ABEI was also not the reason
for the increased CL intensity. It is well known that gold
nanoparticles with a size range from 6 to 99 nm could enhance
the CL of the luminol–H2O2 system; the rate of heterogeneous
catalysis increased with the available active surface area of the
catalyst.42 Obviously, the unusual morphology of the as-
prepared AuNPCs provided a larger active surface than the
spherical nanoparticles in our previous work due to the irreg-
ular bumps on the surface of AuNPCs.34 That might be the
reason for the almost two orders of magnitude higher CL
intensity over the ABEI functionalized gold spherical
nanoparticles.18
Fig. 7 CL kinetic curves of different oxidants on reaction with AuNPCs using
static injection. Inset: magnification of the black line, red line and blue line.
Scheme 2 The mechanism of CL reaction between ABEI and oxidants.
oxidants, H2O2 exhibited stronger CL intensity and the CL
kinetic curve had a platform, which is good for bioassays.
Therefore, the CL reaction of AuNPCs with H2O2 was further
studied. The ABEI molecules coated on the surface of AuNPCs
were oxidized by H2O2 under the alkaline condition to produce
CL. As shown in Fig. 8, the effect of pH value on the CL emission
was tested in the range of pH 9–13. The optimized pH condition
for the AuNPCs–H2O2 system was pH 12. When the pH of
AuNPCs solution was lower than 12, the CL intensity increased
with increasing pH. The CL intensity decreased with increasing
pH when the pH of AuNPCs solution was higher than 12. The CL
The applicability of AuNPCs with CL activity to bio-probes
was also explored. The AuNPCs were directly conjugated with
protein and DNA in 0.05 M Tris–HCl buffer (pH 7.4, containing
0.1 M NaCl) to form the bio-probes and the CL property was
studied by a static injection CL detection system. Fig. 10 illus-
trated that both AuNPCs labeled protein and DNA maintained
high CL activity. Due to its high CL efficiency, AuNPCs are
suitable for bioassays in vitro with high sensitivity. Moreover,
Fig. 9 CL kinetic curves of ABEI functional gold nanoparticles in our previous
work and AuNPCs of this work with static injection. Full scale of the CL kinetic
curve when 100 mL of 0.1 M H2O2 was injected into 300 mL of the centrifuged
GNMs (dissolved in NaOH solutions of pH 13 and pH 12).
Fig. 8 CL kinetic curves of AuNPCs with different pHs with static injection. Full
scale of the CL kinetic curve when 100 mL of 0.1 M H2O2 was injected into 300 mL
of the centrifuged AuNPCs (dissolved in NaOH solution with different pHs).
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