Oxidation-triggered aggregation of gold nanoparticles for naked-eye detection of hydrogen peroxide

Article abstract of DOI:10.1039/c5cc09447j

Naked-eye detection of H₂O₂ was realized based on the color change of gold nanoparticles upon aggregation. The removal of polyethylene glycol chains from the nanoparticle surface under H₂O₂ treatment let to the exposure of inner hydrophobic ligands, causing the nanoparticle aggregation in aqueous medium. This detection system shows a wide dynamic range in the μM scale and a distinguishable limit of 10 μM.

Full text of DOI:10.1039/c5cc09447j

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DOI: 10.1039/C5CC09447J
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Oxidation-Triggered Aggregation of Gold Nanoparticles for Naked-
Eye Detection of Hydrogen Peroxide
Shaojue Wu,a,‡ Si Yu Tana,‡, Chung Yen Anga, Zhong Luo,a Yanli Zhao*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Naked-eye detecion of H2O2 was realized based on the color charged thiols,23 or positively charged biopolymers24 that are
change of gold nanoparticles upon aggregation. The removal of produced from enzyme catalyzed reactions. In addition, the
polyethylene glycol chains from the nanoparticle surface under aggregation of AuNPs can also occur during the NP growth in a
H2O2 treatment let to the exposure of inner hydrophobic ligands, specific solution involving H2O2 as a reducing agent9, without the
causing the nanoparticle aggregation in aqueous medium. This presence of cross-linking agents. Since H2O2-related enzymes like
detection system shows a wide dynamic range in the μM scale and GOx and HRP are among the most suitable enzymes employed in
ELISA25, developing H2O2-responsive signal generation systems
a distinguishable limit of 10 μM.
would be practically useful in the field of protein detection. In this
Hydrogen peroxide (H2O2) is an important biologically relevant small
molecule. It is the substrate or product of some intracellular
enzyme1,2 and antibody3,4 involved reactions. In the past years,
scientists have studied its complex roles in the mechanisms of some
diseases5-7. H2O2 can also serve as the intermediate object in the
detection of disease-related proteins by relying on analytical
work, we engineered the surface of AuNPs to be H2O2-responsive by
incorporating a phenylboronate structure in the surface-adsorbing
ligand, so that the surface property of AuNPs can be changed from
hydrophilic to hydrophobic upon H2O2 treatment, and the
accompanied color change in subsequent aggregation state can be
harnessed for naked-eye detection of H2O2 (Scheme 1).
techniques such as enzyme-linked immunosorbent assay (ELISA)8-10
.
In the ELISA technique, H2O2-generating/consuming enzymes like
glucose oxidase (GOx) and horseradish peroxidase (HRP) are
frequently selected as the label enzymes9, 10. The enzyme-induced
H2O2 concentration change could be measured by colorimetric
observation9-12
,
plasmonic absorption spectroscopy,13 or
electrochemical detection14-16
.
In particular, the colorimetric
method is instrument-free, and in some cases, can provide ultralow
sensitivity, which rivals the standard but costly methods9.
In recent years, gold nanoparticles (AuNPs) have been frequently
employed for applications in immunoassays17-21
, based on the
property that the frequency of their plasmonic absorption bands
shifts with the alteration of their compositions, shapes and
aggregation states. Particularly, the color change from red to blue
or from blue to red upon aggregation or redispersion of AuNPs can
be used as the signal to determine the presence or absence of
target proteins in the ELISA technique. Normally, the aggregation of
AuNPs could be induced in the presence of di-thiols22, positively
Scheme 1 Schematic representation of H2O2 triggered hydrophilicity
change and aggregation of AuNPs.
As illustrated in Scheme 1, AuNPs were modified with two ligands:
1-hexanethiol and lipoic-phenylboronate-mPEG5000. 1-Hexanethiol
is a hydrophobic ligand with a short length, and it forms a
hydrophobic layer (indicated as yellow layer in Scheme 1) around
AuNPs. Lipoic-phenylboronate-mPEG5000 is the ligand with a long
hydrophilic polyethylene glycol tail (indicated as cyan layer in
Scheme 1). Though the lipoyl and phenylboronate units are
hydrophobic, the long polyethylene glycol chain provides excellent
aDivision of Chemistry and Biological Chemistry, School of Physical and
Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link,
Singapore 637371. E-mail: zhaoyanli@ntu.edu.sg; Tel: +65 6316 8792
bSchool of Materials Science and Engineering, Nanyang Technological University, 50
Nanyang Avenue, Singapore 639798.
‡These authors contributed equally to this work.
†Electronic Supplementary Information (ESI) available: Additional synthesis and
characterization details. See DOI: 10.1039/x0xx00000x
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water solubility and steric hindrance to protect AuNPs from of hydrophobization, AuNPs did not aggregate when incubated with
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DOI: 10.1039/C5CC09447J
aggregation in aqueous medium. The middle phenylboronate entity H2O2. Since the lipoic-phenylboronate-mPEG5000 ligand has much
serves as the H2O2-responsive structure, since the phenylboronic stronger binding affinity toward AuNPs, it cannot be replaced
ester reacts with H2O2 to generate phenyl oxide that initiates significantly by 1-hexanethiol. Thus, the two ligand-modified AuNPs
quinone methide rearrangement around the aromatic structure and still possess enough solubility and stability in aqueous medium (Fig.
causes the fragmentation of the cresol structure26-30. As a result, the 2a). The aggregation of modified AuNPs started at the time of
polyethylene glycol chain could split from the lipoyl unit and leave around 4 h with higher concentrations of H2O2 (Fig. 2b). For those
the surface of AuNPs. The detachment of the polyethylene glycol samples with lower concentrations of H2O2, the AuNP aggregation
chains from AuNPs gradually decreases the water stability and and the color change of the AuNP solutions started after longer
eventually leads to the aggregation of AuNPs along with a red-to- hours. At the time of 24 h of incubation, the color change of the
blue color change of the solution. Since the rate of color change AuNP solution with 10 μM H2O2 could be clearly discriminated (Fig.
depends on the concentration of H2O2, different concentrations of 2e and its enlargement). In addition, the transmission electron
H2O2 can be discriminated by naked eye.
microscopy (TEM) images of the AuNP solutions before (Fig. 2g) and
after (Fig. 2h) the addition of H2O2 were recorded. The TEM image
shows a significant aggregation of the AuNPs after the treatment of
H2O2. The intrinsic stability of AuNPs without the addition of H2O2
was sufficiently high at the temperature of 37 °C. Only after 43 h of
incubation, the AuNP sample started to show some aggregation,
and the sedimentation of AuNPs took even longer time (Fig S2i,j in
the ESI). The high intrinsic stability of the as-prepared AuNPs
elevates the credibility of the experimental test.
Fig. 1 1H NMR spectra of compound 6 (a) before and (b) after
reaction with H2O2 at 37 °C for 70 h. (a) Compound 6 (9.76 mM) in
DMSO-d6 (456 μL) and deuterated PBS (pH 7.4, 130 μL). (b)
Compound 6 (9.76 mM) in DMSO-d6 (456 μL) and deuterated PBS
(pH 7.4, 124 μL) containing H2O2 (10 M in H2O, 6 μL, H2O2
concentration 96.8 mM). Compound 6 : H2O2 = 1 : 10.
The fact that the as-synthesized lipoic-phenylboronate-mPEG5000
ligand could undergo the fragmentation in the presence of H2O2
was substantiated by 1H NMR characterizations. To simplify the
spectrum, compound 6 (Fig. 1), a synthetic precursor of the final
lipoic-phenylboronate-mPEG5000 ligand, was employed to react with
H2O2. As shown in Fig. 1a, there are initially no peaks from 4.10 to
4.80 ppm that is the range of 1H NMR peaks for methylene protons
of benzyl alcohols. After reacting with H2O2 at 37 °C for 70 h, the
appearance of two singlet peaks at 4.52 ppm and 4.36 ppm (Fig. 1b)
was observed. These two peaks belong to methylene protons of
two benzyl alcohols generated from the fragmentation of the
middle phenylboronate and cresol units of compound 6.
Correspondently, the intensity of another two peaks at 4.90 ppm
and 5.04 ppm belonging respectively to methylene protons of the
benzyl ether and benzyl ester of compound 6 disappeared almost
g
h
Fig. 2 Gradual aggregation and color change of AuNP solutions
incubated with different concentrations of H2O2 at 37 °C. Column B:
AuNP solution without H2O2. Columns 1-14: 10 μM, 20 μM, 40 μM,
70 μM, 100 μM, 120 μM, 160 μM, 200 μM, 300 μM, 400 μM, 600
μM, 800 μM, 1 mM, and 2 mM of H2O2. Duration of incubation: a, 0
h; b, 3 h 45 min; c, 6 h 31 min; d, 16 h 24 min; e, 24 h; f, 28 h. TEM
images of AuNP solution (g) before and (h) after the treatment of
H2O2.
1
completely after the reaction. These changes of H NMR spectra
prove the hydrolysis of the benzyl ester bonds and the
fragmentation of the ligand in the presence of H2O2. It is
noteworthy to mention that the oxidation of phenylboronate by
H2O2 could complete within 30 min (Fig. S1a in the ESI), and the
hydrolysis of benzyl ether and benzyl ester began and progressed
slowly in the next 24 h (Fig. S1b-d in the ESI).
After being used to modify AuNPs, the lipoic-phenylboronate-
mPEG5000 ligand provides AuNPs with an excellent stability in
phosphate buffered saline (PBS, pH 7.4). Since the ligand could not
occupy all the surface sites of AuNPs, there are still substantial
citrate ligands remained on the AuNP surface. In order to remove
these citrate ligands and provide hydrophobicity, an excess amount
of 1-hexanethiol was added to the AuNP solution. Without this step
The progressing rate of AuNP aggregation in the samples with
high H2O2 concentration to low H2O2 concentration featured linear
characteristics. Upon the aggregation, the absorption intensity of
AuNPs exhibited a decrease at 527 nm and an increase at 650 nm
(Fig. S3 in the ESI). Thus, the ratio of absorption intensity at 650 nm
and 527 nm was employed to evaluate the extent of AuNP
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aggregation. At the time of 3 h 45 min, only samples with H2O2 of this detection system. It is very difficult to accuratVeielyw dAretitcelerOmnilninee
DOI: 10.1039/C5CC09447J
concentration above 400 μM showed observable color change (Fig. the ratio of two ligands on an AuNP surface, but this ratio should
2b). Fitting of the ratio data matched well with a straight line having depend on the initial ratio of the two ligands added to the reaction
a R2 of 95% (Fig. 3a). At the time of 6 h 31 min, only samples with solution. When increasing the amount of the lipoic-
H2O2 concentration above 100 μM showed observable color change phenylboronate-mPEG5000 ligand by 3 folds, the resulted AuNPs
(Fig. 2c). The data line of all these samples adopted an “S” shape could not aggregate under the conditions where the original AuNPs
(Fig. 3b) having a linear middle segment from 100 μM to 600 μM (R2 could aggregate (Fig. S5 in the ESI). Though reducing the amount of
99%, Fig. 3c). Finally, the sample with 10 μM of H2O2 showed the PEG ligand might in principle lead to faster response and lower
observable aggregation after 16 h 24 min (Fig. 2d). At this time, the limit of detection, it decreases the intrinsic stability of AuNPs and
samples with H2O2 concentration above 40 μM precipitated makes the color discrimination of blank AuNP sample and samples
completely. Only four data could be recorded and fitted to give a with lower concentrations of H2O2 unconvincing. Therefore, a
curve (Fig. 3d). Since the initial number of the ratio was 0.24 when balance between fast response/low detection limit and intrinsic
no aggregation occurred, 0.43 was selected as the threshold for stability of AuNPs should be taken into consideration.
high-confidence discrimination of the color change (Fig. 3e). In
summary, the rate of AuNP aggregation under a wide range of H2O2
concentrations follows a simple linear rule (or unitary quadratic
equation at the lowest concentration zone). This feature may
provide an access to quantitative detection of H2O2.
Fig. 4 Gradual aggregation and color change of AuNP solutions
incubated with different concentrations of H2O2 at 25 °C. Column B:
blank AuNP sample without H2O2. Columns 1-13: 10 μM, 20 μM, 40
μM, 70 μM, 100 μM, 120 μM, 160 μM, 200 μM, 300 μM, 400 μM,
Fig. 3 Fitting of the absorption intensity ratio between 650 nm and
527 nm in AuNP solutions. Duration of incubation: a, 3 h 45 min; b
600 μM, 800 μM, and 2 mM of H2O2. Duration of incubation: a, 0 h;
and c, 6 h 31 min; d, 16 h 24 min. Unit of the abscissa is μM. e,
b, 19 h; c, 34 h; d, 46 h; e, 62 h; f, 83 h; g, 140 h; h, 190 h.
Juxtaposition of sample color, H2O2 concentrations (μM), and
intensity ratios obtained from the samples at 6 h 31 min.
In conclusion, we have designed an AuNP-based platform
based on quinone methide rearrangement and hydrophilicity
Factors affecting the performance of this detection system may
change of colloidal AuNPs for naked-eye detection of H2O2.
include temperature of incubation and ratio between the lipoic-
The experimental results reveal a linear (or unitary quadratic)
phenylboronate-mPEG5000 ligand and 1-hexanethiol on AuNP
relationship between the AuNP aggregation rate and the
surface. Temperature of incubation influences the chemical
concentration of H2O2 added over a wide range (10 to 2000
reaction rate of the PEG ligand with H2O2 and the frequency of
μM, Fig. 3), indicating that this approach could be used for
AuNP collision. Lower temperature would slow down the oxidation
quantitative detection of H2O2. The lowest concentration of
and hydrolysis of the PEG ligand in H2O2 solution and reduce the
H2O2 that could be discriminated by naked eye using the
collision frequency of AuNPs. Hence, the color change rate of AuNPs
present system was 10 μM. In comparison, two recently
should be slower. The experimental results (Fig. 4, 25 °C) well
reported AuNP-based systems9,10 showed comparable
support this reasoning. Although the experiments exhibited a
detection limits (20 μM) but much narrower dynamic ranges
similar gradual progress of color change from high to low H2O2
(20-120 μM9 and 10-100 μM10). Since H2O2 is a very important
concentrations, the time it took to clearly discriminate the color
enzyme-related small molecule and these enzymes (i.e., GOx
change for 10 μM sample was much longer (5-8 days, Fig. 4g,h),
and HRP) are highly suitable to be used in ELISA25, this AuNP-
which is not suitable for practical use. Similarly, the rate of color
based detection system could be applied in ultrasensitive
change also followed a linear relationship (Fig. S4 in the ESI). In
detection of disease-related biomarkers, where the
addition to the incubation temperature, the ratio of the two ligands
concentration change of H2O2 that is numerically correlated to
on the AuNP surface is also a key element to affect the performance
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This research is supported by the National Research
Foundation (NRF), Prime Minister’s Office, Singapore under its
Campus for Research Excellence and Technological Enterprise
(CREATE) Programme–Singapore Peking University Research
Centre for a Sustainable Low-Carbon Future, as well as the
NTU-A*Star Silicon Technologies Centre of Excellence under
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