Hydroxyl radical-induced etching of glutathione-capped gold nanoparticles to oligomeric AuI-thiolate complexes

Article abstract of DOI:10.1039/c5ra05159b

Thiol-induced core etching of gold nanoparticles is a general method for the production of gold nanoclusters (AuNCs) of various sizes. This paper is the first report on the efficient reaction of glutathione-capped gold nanoparticles (GSH-AuNPs) with hydroxyl radicals to produce oligomeric Au^I-thiolate complexes at ambient temperature. Also, hydroxyl radicals can etch commercially available gold nanoparticles (100 nm); this strategy can be applied for the removal of gold from scrap electronics. Additionally, proteins can trigger the aggregation of oligomeric Au^I-thiolate complexes under neutral conditions resulting in the formation of fluorescent AuNCs. For example, the reaction of trypsin, lysozyme, and glucose oxidase with oligomeric Au^I-thiolate complexes produces Au₅, Au₈, and Au₁₃ clusters with emission maxima at 415, 460, and 535 nm, respectively. Interestingly, trypsin- and glucose oxidase-stabilized AuNCs could sense GSH and glucose via GSH-induced etching of AuNCs and H₂O₂-mediated oxidation of AuNCs, respectively. This journal is

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ARTICLE
Hydroxyl Radical-Induced Etching of Glutathione-
Capped Gold Nanoparticles to Oligomeric AuI-
Thiolate Complexes
Cite this: DOI: 10.1039/x0xx00000x
TzuꢀHeng Chen,a ChihꢀChun Nieh,a YaꢀChen Shin,a ChenꢀYi Kea and WeiꢀLung
Tseng*a,b
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
Thiolꢀinduced core etching of gold nanoparticles is a general method for the production of gold
nanoclusters (AuNCs) of various sizes. This paper is the first report on the efficient reaction of
glutathioneꢀcapped gold nanoparticles (GSHꢀAuNPs) with hydroxyl radicals to produce
oligomeric AuIꢀthiolate complexes at ambient temperature. Also, hydroxyl radicals can etch
commercially available gold nanoparticles (100 nm); this strategy can be applied for the
removal of gold from scrap electronics. Additionally, proteins can trigger the aggregation of
oligomeric AuIꢀthiolate complexes under neutral conditions resulting in the formation of
fluorescent AuNCs. For example, the reaction of trypsin, lysozyme, and glucose oxidase with
oligomeric AuIꢀthiolate complexes produces Au5, Au8, and Au13 clusters with emission
maxima at 415, 460, and 535 nm, respectively. Interestingly, trypsinꢀ and glucose oxidaseꢀ
stabilized AuNCs could sense GSH and glucose via GSHꢀinduced etching of AuNCs and H2O2ꢀ
mediated oxidation of AuNCs, respectively.
www.rsc.org/
Hydroxylꢀterminated poly(amidoamine) dendrimer and bovine
serum albumin were utilized as templates for preparing blueꢀ
emitting Au8 and redꢀemitting Au25 clusters, respectively.9, 10
1. Introduction
Gold nanoclusters (AuNCs), which consist of a couple of
atoms up to a few hundred atoms, have received significant
interest over the past decade because of their good optical,
electrical, and catalytic properties.1 Because the sizes of AuNCs
range between those of isolated gold atoms and nanoparticles,
their discrete energy levels result in moleculeꢀlike properties
such as sizeꢀdependent fluorescence. For example,
poly(amidoamine) dendrimer–encapsulated Au5, Au8, Au13,
Au25, and Au31 clusters emit UV, blue, green, red, and nearꢀIR
light, respectively.2 Compared to organic fluorophores, AuNCs
provide the distinct advantages of a oneꢀpot synthesis, large
Stokes shift, long fluorescence lifetime, good photostability,
and low toxicity. Because of their intrinsic fluorescence,
AuNCs are emerging as optical probes for use in the fields of
Alternatively, AuNCs can be produced by ligandꢀinduced
removal of gold atoms from the surface of nanometerꢀsized
gold particles (>2 nm). The mechanism of ligandꢀinduced
etching of gold particles involves electron injection from the
ligand to the gold particles.11ꢀ13 The type of ligand is one of the
most critical factors in determining the size and quantum yield
of AuNCs. Longꢀchain thiols, such as 11ꢀmercatoundecanoic
acid,14 dihydrolipoic acid,15 11ꢀmercaptoꢀ3,6,9ꢀtrioxaundecylꢀ
αꢀDꢀmannopyranoside,16 and glutathione,17 are the most
commonly used ligands for the topꢀdown approach. However,
thiolꢀcontaining ligands suffer from low water solubility, strong
odors, and air oxidation. Moreover, the topꢀdown approach
requires an additional purification process to remove the excess
thiols.
The Fenton reaction involves the production of strongly
oxidizing hydroxyl radicals via FeIIꢀmediated reduction of
hydrogen peroxide.18, 19 The formed hydroxyl radicals, which
are one of the most powerful oxidizing species, are effective for
the degradation of organic pollutants in soils and waters.20ꢀ22
Also, the Fenton reaction has been demonstrated to be capable
of removing metastable surface gold atoms from a Au surface23,
24 and preparing graphene quantum dots from graphene oxide.25
These results suggest that the Fenton reaction could be used to
etch goldꢀbased nanomaterials in the absence of thiolꢀ
containing ligands.
sensors,
molecular
imaging,
flow
cytometry,
and
bioconjugation.3, 4
Numerous synthetic methods have been developed to produce
different sized AuNCs with fluorescent emission ranging from
the UV to nearꢀIR regions. Generally, the formation of AuNCs
can be achieved by reducing gold ions to their zeroꢀvalent state
in the presence of thiolꢀterminated small molecules and a
cavityꢀcontaining template. Choosing a suitable capping ligand
and template is of immense importance to the production of
highly stable, fluorescent AuNCs with specific emission
wavelengths. For example, nearꢀIR–emitting AuNCs have been
prepared via NaBH4 reduction of a gold precursor in the
presence of lipoic acid5, 6 or glutathione,7 while blueꢀemitting
AuNCs have been synthesized by reducing a mixture of gold
precursor and 3ꢀmercaptopropionic acid with NaBH4.8
Herein, we disclose that the hydroxyl radicals in Fenton’s
reagent quickly etch glutathione (GSH)ꢀcapped gold
nanoparticles (GSHꢀAuNPs) to form oligomeric AuIꢀthiolate
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complexes. Hydroxyl radical–induced dissolution of GSHꢀ obtained solution was gently stirred at ambient temperature for
AuNPs was applied to recycle gold from scrap electronics. 12 h. To remove excess GSH and HAuCl4, a solution of GSHꢀ
Also, the addition of trypsin, lysozyme, and glucose oxidase to AuNPs was centrifuged at 175 000 rpm (34 230 g) for 10 min.
the oligomeric AuIꢀthiolate complexes directly produced Au5ꢀ, To obtain oligomeric AuIꢀthiolate complexes, the asꢀprepared
Au8ꢀ, and Au13ꢀthiolate clusters, respectively. Trypsinꢀ and GSHꢀAuNPs reacted with Fenton’s solution (5 mM FeII and
glucose oxidaseꢀstabilized Au8 and Au13 clusters were utilized
to sense GSH and glucose via thiolꢀinduced coreꢀetching of Au8
clusters and H2O2ꢀmediated oxidation of Au13 clusters,
respectively. Scheme 1 illustrates an overview of the disclosed
methods.
100 mM H2O2.) at ambient temperature for 0
−3 h.
Synthesis of Gold Nanorods
.
A seed solution was prepared by adding 120 ꢁL of iceꢀcold 1
mM NaBH4 to 2 mL of a solution containing HAuCl4 (0.05
mM, l mL) and CTAB (200 mM, l mL). The mixture was
stirred until a brownish yellow solution was formed. The
formed seed solution (12 ꢁL) was incubated with the growth
solution consisted of CTAB (0.2 M, 5 mL), AgNO3 (0.4 mM,
0.1 mL), HAuCl4 (0.1 mM, 5 mL), and ascorbic acid (80 mM,
70 ꢁL) at ambient temperature, resulting in the production of
gold nanorods.
Synthesis of Octahedral Gold Nanocrystals.
A seed solution was prepared by adding 450 ꢁL of iceꢀcold
0.02 M NaBH4 to 10 mL of a solution containing HAuCl4 (0.05
mM, 5 mL) and CTAB (200 mM, 5 mL). The mixture was left
to continue under gentle stirring at ambient temperature for 1 h.
Two growth solutions were prepared. For the preparation of the
first growth solution (denoted as
A), 220 ꢁL of 4 mM ascorbic
acid was thoroughly mixed with a solution containing CTAB
(0.1 M, 20 mL), KI (0.1 mM, 55 ꢁL), and HAuCl4 (10 mM,
0.25 mL). Similarly, the second growth solution (denoted as B)
was obtained by mixing 880 ꢁL of 4 mM ascorbic acid with a
solution containing CTAB (0.1 M, 20 mL), KI (0.1 mM, 220
ꢁL), and HAuCl4 (10 mM, 1 mL). Next, 0.1 mL of the seed
Scheme 1: Schematic illustration for the process of the
synthesis of GSHꢀAuNPs, hydroxyl radicalꢀtriggered etching of
GSHꢀAuNPs, the formation of oligomeric AuIꢀthiolate
complexes, the production of proteinꢀstabilized AuNCs, and the
use of proteinꢀstabilized AuNCs as a sensor.
solution was added to
A with gentle shaking until the solution
color turned light pink. The obtained solution (0. 5 mL) was
then transferred to B. After 2 h, the mixture was purified via
centrifugation (3000 rpm, 10 min).
Synthesis of Protein-Stabilized AuNCs.
2. Materials and Methods
A solution of 25 mg mLꢀ1 protein (trypsin, bovine serum
albumin, lysozyme, glucose oxidase, and choline oxidase) was
added to an equal volume of the asꢀsynthesized oligomeric AuIꢀ
thiolate complexes. The resulting mixture was incubated in 5
mM phosphate (pH 7.0) at ambient temperature for 8 h. To
remove excess protein, a solution of proteinꢀstabilized AuNCs
was dialysed with 5 mM phosphate (pH 7.0). The purified
proteinꢀstabilized AuNCs were prepared in 400 mM NaCl and
Chemicals.
Hydrogen tetrachloroaurate (III) dehydrate was purchased from
AlfaꢀAesar (Ward Hill, MD, USA). Bovine serum albumin
(from bovine serum), NaOH, HCl, NaCl, Na2HPO4, NaH2PO4,
H3PO4, Na3PO4, KI, FeCl2, NaBH4, AgNO3, CTAB, GSH,
quinine sulfide, ascorbic acid, glucose, glucose oxidase (from
Aspergillus niger, 117200 U gꢀ1), choline oxidase (from
Alcaligenes species, 10 U mgꢀ1), αꢀcyanoꢀ4ꢀhydroxyꢀcinnamic
0−5 mM GSH to test their stability.
acid,
tris(hydroxymethyl)aminomethane)
(TRIS),
and
Sensing of GSH and Glucose.
ammonium persulfate were ordered from SigmaꢀAldrich (St.
Louis, MO, USA). A solution of H2O2 (30 %) was obtained
from Showa (Tokyo, Japan). Lysozyme (from chicken egg
white) and trypsin (from bovine pancreas) were obtained from
MP Biomedicals (Irvine, CA). Premixed 30
acrylamide/bisacrylamide solution (29:1), sodium salt of
N,N,N′,N′ꢀTetramethylethylenediamine (TEMED),
bromophenol blue, and Coomassie brilliant blue Rꢀ250 were
obtained from BioꢀRad. MilliꢀQ ultrapure water (MilliꢀPore,
Hamburg, Germany) was used in all of the experiments.
Stock solutions of GSH and glucose were prepared in 10 mM
phosphate buffer at pH 6. A solution of proteinꢀstabilized
AuNCs was diluted to 5ꢀfold with 10 mM phosphate (pH 6.0).
Different concentrations of GSH (0−10 mM, 250 ꢁL) were
%
incubated with a solution of trypsinꢀstabilized AuNCs (250 ꢁL)
at ambient temperature for 30 min. After centrifugation at 175
000 rpm (34 230 g) for 3 min, the obtained supernatant was
transferred into a 1 mL quartz cuvette. Their fluorescence
spectra were recorded by operating the fluorescence
spectrophotometer at an excitation wavelength of 350 nm. For
sensing glucose, glucose oxidaseꢀstabilized AuNCs (250 ꢁL)
Synthesis of GSH-AuNPs and Oligomeric AuI-Thiolate
Complexes.
were incubated with various concentrations of glucose (0−200
ꢁM, 250 ꢁL) at ambient temperature for 30 min. The
fluorescence spectra of the resulting solutions were collected by
operating the fluorescence spectrophotometer at an excitation
An aqueous solution of HAuCl4 (2.5 mM, 2 mL) was mixed
with GSH (0.1 mM, 16 mL) under gentle stirring at ambient
temperature for 30 min. Subsequently, freshly prepared NaBH4
(1 mg mLꢀ1, 2 mL) was rapidly added to the mixture. The
wavelength of 380 nm
.
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Reaction of Fenton reagent with GSH-AuNPs
GSHꢀAuNPs were produced by reducing HAuCl4 with NaBH4
Instrumentation.
The absorption and fluorescence spectra were collected using a in the presence of GSH. The asꢀprepared solution exhibited a
doubleꢀbeam UVꢀvis spectrophotometer (Cintra 10e; GBC, surface plasmon resonance (SPR) at 525 nm, which is
Victoria, Australia) and a Hitachi Fꢀ7000 fluorometer (Hitachi, characteristic of AuNPs (Fig. 1A). The Fenton reaction of the
Tokyo, Japan), respectively. Highꢀresolution transmission GSHꢀAuNPs proceeded in an aqueous solution containing 100
electron microscopy (HRTEM, FEI Tecnai G2 F20 SꢀTwin mM H2O2 and 5 mM FeII. H2O2 can be dissociated into
working at 200kV) was used to take TEM images. The size and hydroxyl radicals via oneꢀelectron reduction by FeII.26 The
size distribution of particles were measured using dynamic light reaction of the asꢀgenerated hydroxyl radicals and GSHꢀAuNPs
scattering (DLS) (N5 submicrometer particle size analyzer, was monitored over 60 min using absorption spectroscopy,
Beckman Coulter Inc., U.S.A.). The labꢀmade darkꢀfield TEM, and DLS. Fig. 1A reveals the gradual change in the
microscopy (DFM) system consisted of an Olympus IX71 intensity of the SPR with time, which accompanies a color
inverted microscope (Tokyo, Japan), a 100ꢀW halogen lamp, a change from purple to yellow. As a control experiment, the
condenser (IXꢀULWCD, Olympus, Japan), an objective (40X; extinction spectra and solution color of GSHꢀAuNPs remained
numerical apertures = 0.75), and a digital camera (DP70, almost constant over time with the sole addition of FeII or H2O2
Olympus, Tokyo, Japan). Timeꢀresolved fluorescence
(Figs. S1 and S2, ESI). The TEM image revealed that the
measurements of proteinꢀstabilized AuNCs were performed average diameters of the GSHꢀAuNPs were 5(±2) nm (Fig.
using a timeꢀcorrelated single photon counting system (Timeꢀ S3A, ESI). After treatment of GSHꢀAuNPs with Fenton reagent
Harp 200, PicoQuant GmbH, Berlin, Germany) equipped with a for 1 h, the size of the resulting products was smaller than 5 nm
pulsed diode laser at 390 nm (tens of ps pulseꢀwidth). The
(Figs. S3B, ESI). The analysis of GSHꢀAuNPs and the
elemental compositions of proteinꢀstabilized AuNCs were resulting products by DLS showed that the hydrodynamic size
measured by JAMPꢀ9500F Auger Electron Spectroscopy of the resulting products was obviously smaller than that of
(JEOL, Japan). The quantification of gold was performed by GSHꢀAuNPs (Fig. S4, ESI); this provides evidence that the
Inductively coupled plasma mass spectrometry (ICPꢀMS) hydroxyl radicals indeed etch the AuNPs at ambient
(PerkinꢀElmerꢀSCIEX, Thornhill, ON, Canada).
A mass temperature. Note that oligomeric Auꢀthiolate complexes did
spectrometer MicrOTOFꢀQII from Bruker Daltonics (Billerica, not form when GSHꢀAuNPs reacted with Fenton reagent for 1
MA, USA) was applied to obtain positiveꢀion electrospray h.
ionization mass spectra. Raman spectra were measured using a
confocal Raman microscope (CRM200, WITec, Ulm,
Germany) equipped with a pulsed 532 nm laser. Native PAGE
(MiniꢀPROTEAN Tetra Cell, BioRad, Richmond, CA)
containing 7.5% separating gel and 4% (m/v) stacking gel was
conducted at 120 V for 90 min. Gels were stained with a
solution containing 0.04% w/v Coomassie brilliant blue Rꢀ250,
41% v/v methanol, and 9% v/v acetic acid for 1 h. After that,
gels were deꢀstained with a solution containing 20% v/v
methanol and 10% v/v acetic acid.
Fig. 2. (A) Absorption spectrum and (D) ESIꢀMS spectrum of
oligomeric AuIꢀthiolate complexes. (B) Raman spectra and (C)
XPS spectra of (a) GSHꢀAuNPs and (b) oligomeric AuIꢀthiolate
complexes. (C): (a) The original spectrum is in black, the fitted
spectrum is in red, and the Au0 4f spectrum is in blue, and the
AuI 4f spectrum is in orange. (b) The original spectrum is in
black, the fitted spectrum is in red, and the AuI 4f7/2 spectrum is
in blue, and the AuI 4f5/2 spectrum is in orange.
When the reaction continued to 3 h, the final products
displayed a broad absorption and no SPR band (Fig. 2A). This
Fig. 1. Timeꢀevolution of visible spectra of GSHꢀAuNPs in the
presence of in the presence of 5 mM FeII and 100 mM H2O2.
result is similar to that observed in oligomeric Auꢀthiolate
GSHꢀAuNPs were incubated with Fenton’s solution at ambient
complexes, such as sodium aurothiomalate (Myocrisin) and
temperature.
aurothioglucose (Solganal).27 Comparison of the Raman spectra
of the GSHꢀAuNPs and final products revealed the appearance
of characteristic peaks for the Au–S bonds at 225 and 286 cm−1;
3. Results and discussion
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these peaks are associated with goldꢀsulfur vibrations, as red spots in the DFM images of the single gold nanoparticle to
reported by Varnholt et al.28
Fig. 2B). The valence states of the dim green spots, which vanished over time (Fig. S5, ESI).
(
GSHꢀAuNPs and final products were determined from their Xꢀ Taken together, the scattering images of particles at the
ray photoelectron spectroscopy (XPS) spectra. The XPS different etching stages provide further evidence of hydroxyl
spectrum of the GSHꢀAuNPs exhibited two peaks at 87.6 and radical–induced etching of GSHꢀAuNPs.
83.9 eV, corresponding to Au 4f5/2 and Au 4f7/2, respectively.
The successful etching of GSHꢀAuNSs by hydroxyl radicals
Deconvolution of the Au 4f7/2 spectrum resulted in two distinct suggests that the same strategy may be implemented to etch
components at 83.9 and 84.8 eV, which correspond to Au0 and other goldꢀbased nanomaterials. Short gold nanorods and
AuI, respectively. The best fit of the data indicates that the octahedral gold nanocrystals, for example, were used to test this
GSHꢀAuNPs contained 4.6% AuI and 95.4% Au0 (curve a in hypothesis. Compared to the treatment of GSHꢀAuNSs with
Fig. 2C). In contrast, the binding energies of the Au 4f5/2 and hydroxyl radicals, a similar transition in the absorption spectra
Au 4f7/2 electrons in the final products were 88.5 and 84.8 eV, was observed in the treatment of short gold nanorods and
respectively, signifying that only AuI was present in the final octahedral nanocrystals with hydroxyl radicals (Fig. 3). TEM
products (curve
b in Fig. 2C). Positiveꢀion ESI mass images show that the size of short gold nanorods and octahedral
spectroscopy with the soft ionization technique was used to nanocrystals became extremely small (~ 2 nm) after the
identify the composition of the final products. The peaks addition of hydroxyl radicals, reflecting that hydroxyl radicals
observed at
m/z 770.4, 778.1 and 780.3 Da are consistent with indeed etch gold nanorods and octahedral nanocrystals (Fig. S6
[3Au197 + 5S32 + H2O16]+, [3Au197 + S32 + 4S34 + H2O16], and and S7, ESI).
[3Au197 + 5S34 + H2O16] fragments, respectively (Fig. 2D).
Encouraged by these results, we envisaged that hydroxyl
However, there is no peak in the ESIꢀMS spectrum that radicals could be used in place of cyanide for the removal of a
corresponds to the GSHꢀprotected gold clusters (AunGSHm). layer of gold from scrap electronics. Importantly, the use of
Because GSH was prone to decomposition under hydroxylꢀ cyanide as a stripping solution has negative environmental
radical treatment, we suggest that most of the GSH decomposed implications. The goldꢀplated scrap was immersed into a
and lost its thiol groups in the presence of the hydroxyl radicals. solution of 100 mM H2O2 and 5 mM FeII at ambient
As a result, the final products contained sulfur instead of GSH. temperature. ICPꢀMS was utilized to quantitatively determine
Similarly, previous studies demonstrated that GSH decomposed the concentration of liberated gold ions in the solution after
under thermal treatment, and the produced sulfur was used as a varied incubation times. The concentration of gold liberated
source for the synthesis of CdS, ZnS, and Ag2S quantum from the scrap gradually increased over time (Fig. S8, ESI),
dots.29ꢀ33 According to previously reported results and our signifying that the hydroxyl radicals effectively remove gold
findings, we strongly believe that hydroxyl radical–induced from scrap electronics. We suggest that the efficiency of the
etching of GSHꢀAuNCs produces oligomeric AuIꢀthiolate hydroxyl radical–induced removal of gold from scrap
complexes rather than oligomeric Au0ꢀthiolate complexes and electronics can be further improved by increasing the
GSHꢀprotected Au clusters.
concentration of the Fenton's solution.
Fig. 3. Time evolution of visible spectra and photo images of
(A) gold nanorods and (B) octahedral gold nanocrystals in the
presence of 5 mM FeII and 100 mM H2O2. Goldꢀbased
nanomaterials were incubated with Fenton’s solution at ambient
temperature. The arrow indicates the direction of time evolution
(A: 0, 1, 3, 5, 10, 20, 30, 40, 50, and 60 min; B: 0, 1, 3, 5, 10,
20, 30, 40, 50, and 60 min)
Fig. 4. (A) Fluorescence spectra of (a) trypsinꢀ, (b) lysozymeꢀ,
and (c) glucose oxidaseꢀstabilized AuNCs under the excitation
wavelength at (a) 350, (b) 350, and (c) 380 nm. Digital photos
of proteinꢀstabilized AuNCs under visible and UV light. (B)
XPS spectra of (a) trypsinꢀ, (b) lysozymeꢀ, and (c) glucose
oxidaseꢀstabilized AuNCs. (aꢀc) The original spectrum is in
black, the Au0 4f7/2 spectrum is in red, and the Au0 4f5/2
spectrum is in blue, the AuI 4f7/2 spectrum is in orange, and the
AuI 4f5/2 spectrum is in green. (C, E, and G) PAGE gels of
protein (lane 1) and proteinꢀstabilized AuNCs (lane II): (C)
Reaction of Fenton reagent with gold-based nanomaterials
DFM was used to monitor the hydroxyl radical–induced etching
of a single gold nanoparticle with a diameter of 100 nm.
Previous studies have demonstrated that 100, 50, and 10 nm
single gold nanoparticles scatter red, green, and no light,
respectively.34 Incubation of 100 nm gold nanoparticles with
hydroxyl radicals at ambient temperature changed the intense
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trypsin, (E) lysozyme, and (G) glucose oxidase. (D, F, H) from Au5ꢀ, Au8ꢀ, and Au13ꢀthiolate clusters. The quantum yields
Fluorescence spectra of band 1, 2, and 3.
of the Au5ꢀ, Au8ꢀ, and Au13ꢀthiolate clusters calibrated with
quinine sulfate were 2.9, 4.7, and 2.1%, respectively.
Native polyacrylamide gel electrophoresis (PAGE) was used
to confirm the formation of AuNCs from proteinꢀinduced
aggregation of the oligomeric AuIꢀthiolate complexes. In
contrast to the nativeꢀPAGE electropherograms of the three
Protein-induced the aggregation of AuI-thiolate complexes
Many proteins, such as BSA, lysozyme, transferrinꢀfamily
proteins, horseradish peroxidase, ribonuclease A, insulin, and
trypsin, have been successfully used as templates for the
proteins (Figs. 4C, 4E, and 4F, lane I), the nativeꢀPAGE
electropherograms of the trypsinꢀ, lysozymeꢀ, and glucose
oxidaseꢀstabilized AuNCs all exhibited multiple bands as a
result of protein oligomerization during the synthetic process
preparation of redꢀemitting Au25 clusters under alkaline
35
conditions.10,
However, proteins are unable to retain their
activity in proteinꢀstabilized AuNCs because the synthetic
process is conducted under a high concentration of NaOH.
Although pepsinꢀstabilized AuNCs with blue (Au5 and Au8),
green (Au13), and red (Au25) emissions were successfully
prepared at pH values of 9.0, 1.0, and 12.0, respectively,36 there
have been no reports on the synthesis of proteinꢀstabilized
AuNCs with different emissions under neutral conditions.
Accordingly, a simple, oneꢀpot, green synthetic route for
preparing proteinꢀstabilized AuNCs with different emissions is
proposed in this study. The asꢀgenerated oligomeric AuIꢀ
thiolate complexes were directly mixed with different kinds of
proteins at ambient temperature for ~8 h. Because oligomeric
AuIꢀthiolate complexes are relatively insoluble in aqueous
solutions, they could move into hydrophobic pockets in the
presence of proteins. This behavior could promote aggregation
of the oligomeric AuIꢀthiolate complexes through intraꢀ and
intercomplex aurophilic interactions between AuI ions, leading
to the formation of either Au0ꢀthiolate clusters or AuI@Au0ꢀ
thiolate coreꢀshell clusters.
(
Figs. 4C, 4E, and 4F, lane II). The multiple bands in the
PAGE electropherograms were consistent with the analysis of
human serum albumin–stabilized AuNCs by native PAGE.41
Because the molecular weight of trypsin resembles that of
trypsinꢀstabilized Au8 clusters, we proposed that trypsin and
trypsinꢀstabilized Au8 clusters could have similar chargeꢀtoꢀ
mass ratios. Accordingly, band
that of trypsinꢀstabilized Au8 clusters. To prove this, band
was cut from the gel (Fig. 4C) and analyzed by fluorescence
spectroscopy. The fluorescence spectrum of band was almost
the same as that of a solution of trypsinꢀstabilized Au5 clusters
Fig. 4D). Similarly, the fluorescence spectra of bands and
1 in lane II could be identical to
1
1
(
2
3
resembled those of lysozymeꢀ and glucose oxidaseꢀstabilized
AuNCs, respectively (Figs. 4F and 4H). These results support
that the oligomeric AuIꢀthiolate complexes were assembled and
converted to AuNCs inside the proteins. The stability of the
proteinꢀstabilized AuNCs was examined by monitoring their
fluorescence under various conditions. The three proteinꢀ
stabilized AuNCs exhibited excellent stability after storage in
As a proofꢀofꢀconcept, three model proteins, including
trypsin, lysozyme, and glucose oxidase, were selected to induce
aggregation of the oligomeric AuIꢀthiolate complexes. The
absorption spectra of the three asꢀsynthesized samples are
featureless (Fig. S9, ESI), indicating that the proteins did not
cause the formation of gold nanoparticles. After incubating the
oligomeric AuIꢀthiolate complexes with trypsin, lysozyme, and
glucose oxidase, the resulting products exhibited emission
maxima at 415, 460, and 535 nm with excitation wavelengths
of 350, 350, and 380 nm, respectively (Fig. 4A). The trypsinꢀ,
lysozymeꢀ, and glucose oxidaseꢀstabilized products emitted
intense violet, blue, and green light, respectively. Fig. S10
(ESI) demonstrates that the strong emissions arose from the
final products rather than from the native fluorescence of the
protein. The XPS spectra of trypsinꢀ, lysozymeꢀ, and glucose
oxidaseꢀstabilized products showed Au0 4f7/2 binding energies
at 84.3, 84.0, and 83.9 eV, respectively (Fig. 4B). This shift is
attributed to the fact that the binding energy of AuNCs
increases with decreasing cluster size.36, 37 Deconvolution of the
Au 4f7/2 spectrum revealed that the trypsinꢀ and lysozymeꢀ
stabilized products had very little AuI on the surface of the Au
core, while the glucose oxidaseꢀstabilized products contained
approximately 70% Au0 and 30% AuI. A previous study
reported that HgII efficiently quenched the fluorescence of
10 mM phosphate (pH 6.0) at ambient temperature (Fig. S12
ESI) and in the presence of a high salt concentration (Fig. S13
ESI). This strategy is also suitable for the preparation of
fluorescent AuNCs using other proteins, including bovine
serum albumin (Fig. S14, ESI) and choline oxidase (Fig. S15
ESI).
,
,
,
proteinꢀstabilized Au25 clusters through metallophilic bonding
39
between HgII and AuI in Au25 clusters.38,
Therefore, the
presence of AuI in glucose oxidase–stabilized AuNCs could be
confirmed by the addition of HgII. Fig. S11 (ESI) shows that
the fluorescence of glucose oxidaseꢀstabilized AuNCs was
quenched upon the addition of HgII, indicating the presence of
AuI in the glucose oxidase–stabilized AuNCs. Based on the
simple relationship (EFermi/N1/3) of the spherical Jellium model40
and the aforementioned results, the emissions at 415, 460, and
535 nm of the trypsinꢀ, lysozymeꢀ, and glucose oxidaseꢀ
stabilized products, respectively, were identified to be derived
Fig. 5. (A) Fluorescence intensity at 415 nm of trypsinꢀ
stabilized AuNCs in the presence of different concentrations of
GSH. Trypsinꢀstabilized AuNCs were incubated with GSH in
10 mM phosphate (pH 6.0) at ambient temperature for 15 min.
(B) A plot of the fluorescence intensity at 415 nm versus the
GSH concentration. The error bars represent standard
deviations based on three independent measurements. (C)
Fluorescence intensity at 535 nm of glucose oxidaseꢀstabilized
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

RSC Advances
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VJieowuArrtnicalelONnlainme e
ARTICLE
DOI: 10.1039/C5RA05159B
AuNCs in the presence of different concentrations of glucose. biosensors. Interestingly, it was found that the oligomeric AuIꢀ
Glucose oxidaseꢀstabilized AuNCs were incubated with glucose thiolate complexes can efficiently catalyze NaBH4ꢀmediated
in 10 mM phosphate (pH 6.0) at ambient temperature for 30 reduction of 4ꢀnitrophenol to 4ꢀaminophenol (Fig. S18, ESI).
min. (D) A plot of the fluorescence intensity at 535 nm versus Further investigation of these results will follow.
the glucose concentration. The error bars represent standard
deviations based on three independent measurements.
Acknowledgements
We would like to thank the Ministry of Science and
Technology (NSC 100ꢀ2628ꢀMꢀ110ꢀ001ꢀ MY4) and for the
financial support of this study.
Sensing of GSH and glucose
Shichibu et al. reported that GSH can etch AuNC clusters
smaller than Au25.42 Thus, it is assumed that GSH could switch
off the fluorescence of trypsinꢀ, lysozymeꢀ, and glucose
oxidaseꢀstabilized AuNCs through GSHꢀinduced coreꢀetching
Notes and references
aDepartment of Chemistry, National Sun Yatꢀsen University, Taiwan.
bSchool of Pharmacy, College of Pharmacy, Kaohsiung Medical
University, Taiwan.\
of AuNCs. Incubation of trypsinꢀstabilized AuNCs with 0−5
mM GSH at ambient temperature for 15 min resulted in a
progressive decrease in the fluorescence of trypsinꢀstabilized
AuNCs with increasing GSH concentration (Fig. 5A). The
linear relationship (R2 = 0.9846) of the fluorescence intensity at
415 nm versus the GSH concentration ranged from 0.2 to 1 mM
Electronic Supplementary Information (ESI) available: Experimental
procedures for prepared compounds and Fig. S1ꢀS15.
See DOI: 10.1039/c000000x/
(
Fig. 5B). The limit of detection (LOD) at a signalꢀtoꢀnoise
ratio of three for GSH was estimated to be 60 ꢁM, which is
lower than the normal level (0.5 10 mM) of GSH in
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