Microchimica Acta9 1:58 ) 102(

https://doi.org/10.1007/s00604-018-2737-2

ORIGINAL PAPER

Visual and colorimetric determination of H2O2 and glucose

based on citrate-promoted H2O2 sculpturing of silver nanoparticles
Chenghua Zong 1 & Bo Li 1 & Jing Wang 1 & Xiaojun Liu 1 & Wenfeng Zhao 1 & Qingquan Zhang 1 & Xinming Nie 2 &
Yang Yu 1

Received: 29 December 2017 / Accepted: 16 February 2018

# Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract
Isotropic silver nanoparticles (iAg NPs) can be easily prepared at low costs, have a low electrochemical potential and high
extinction coefficient. An effective colorimetric assay for H2O2 is reported here based on the finding that H2O2 can induce the
shape transformation of citrate-capped iAg NPs with the help of citrate. The substantial shape variation affords an apparent
surface plasmon resonance (SPR) shift, accompanied by a vivid color change from light yellow to mauve. The color change can
be observed visually if the concentration of H2O2 is 2 μM or higher. A good linear relationship was obtained over the concen-
tration range of 0.2–32 μM with a limit of detection of 90 nM. By making use of glucose oxidase, the method is further extended
to glucose detection. Glucose at a concentration as low as 10 μM can be well determined with bare eyes. Benefitting from the
high selectivity, the detection of glucose in human serum is realized, and the results are in good agreement with those provided by
a clinical analyzer.

Keywords Surface plasmon resonance . Absorption . Nanomaterials . Colorimetry . Shape transformation . Hydrogen peroxide .
Colorimetric and Ag nanoparticles

Introduction spectroscopy, mass spectrometry, electrochemistry and fluo-

Hydrogen peroxide (H2O2) is an essential mediator in food,
pharmaceutical, clinical and environmental analyses and be-
cause it is the final product of glucose oxidation. Therefore,
the glucose concentration can be indirectly detected by mon-
itoring the production of H2O2 [1–3]. Efforts have focused on
detecting H 2 O 2 by different strategies such as Raman
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00604-018-2737-2) contains supplementary
material, which is available to authorized users.
* Chenghua Zong
zongchenghua2008@163.com
* Yang Yu
yuyang@jsnu.edu.cn
1
School of Chemistry and Materials Science, Jiangsu Key Laboratory
of Green Synthesis for Functional Materials, Jiangsu Normal
University, Xuzhou, Jiangsu, People’s Republic of China 221116
2
School of Physics and Electronic Engineering, Jiangsu Normal
University, Xuzhou 221116, People’s Republic of China
rescence [4–8]. These methods made remarkable contribu-
tions in H2O2 and glucose detection. However, challenge still
exists when taking into account both the simplicity and accu-
racy of these methods. Owing to the simplicity and cheapness,
colorimetric methods have attracted special research interest.
Among the various colorimetric sensing approaches, enzy-
matic oxidation of peroxidase substrates has been one of the
most commonly used strategies [9, 10]. Recently, it was found
that some nanomaterials such as MoS2 nanosheets, RhNPs
possess intrinsic peroxidase-like activity, but are more stable
to biodegradation, and less vulnerable to denaturation
[11–13]. These nanozyme-based sensing platforms offer
promising new avenues in H2O2 detection, but in order to
obtain and maintain the high catalytic activity of the
nanozymes, strict control of the experiments and synthesis
conditions are typical required [14–16].
Plasmonic metal nanomaterials, such as silver and gold have
proved to be ideal alternative to construct colorimetric assays
[17–21]. The Au/Ag nanomaterial-based H2O2 colorimetric as-
says can be categorized into aggregation and non-aggregation
sensors. Although aggregation-based colorimetric methods are
merited with simplicity and high sensitivity [22–26], false
19 Page 2 of 7
positive signals from autoaggregation pose a significant chal-
lenge in complex system applications. Hence, non-aggregation
colorimetric sensors are gaining increasing attentions. In non-
aggregation colorimetric sensors, a nanomaterial solution re-
mains in a monodisperse state, but either the SPR intensity or
the SPR position is changed upon the addition of H2O2
[27–29]. Tuning the size of the Au/Ag nanoparticles (Au/Ag
NPs) to change their absorption intensity and altering the shape
of Au/Ag nanomaterials to induce SPR shifts are two of the
most effective design rationales [30–32]. Compared with the
intensity mode, the SPR shift assays are preferred because these
methods exhibit more vivid color variations, thereby offering
increased sensitivity and reliability. Currently available SPR
shift-based assays are primarily based on anisotropic Au/Ag
nanomaterials (ai-Au/Ag NMs) which possess some highly re-
active spots or faces. The aspect ratio of these anisotropic nano-
particles decreased upon reacting with H2O2, leading to a blue-
shifted LSPR. For instance, it has been reported that gold nano-
rods (Au NRs) can be selectively etched by H2O2 in the longi-
tudinal direction, giving rise to a visual color change; however,
because of the high electrochemical potential of Au, either high
concentrations of H2O2 or catalytic species are required in these
assays [33, 34]. Ag triangle nanoprisms (Ag TNPs) are another
kind of anisotropic nanomaterials that are commonly used in
H2O2 detection. Xia demonstrated that sharp tips and side faces
of Ag TNPs can be etched by H2O2, inducing a triangle to
round shape transformation, affording a significant LSPR blue
shift, accompanied by a blue to mauve color changes [35–37].
The highly reactive edges/tips of the Ag TNPs offer these
methods outstanding sensitivity. However, selectivity becomes
a major challenge because anions, such as Cl−, Br−, I−, H2PO4−,
and SCN−, can also react with these highly reactive spots and
faces [38–40].
Different from ai-Au/Ag NMs, isotropic Ag NPs (iAg
NPs) are not only easier in preparation, and exhibit higher
stability, but also, most importantly, exhibit moderate reac-
tivity that offers an improved selectivity, making them ide-
al candidates for colorimetric H 2O 2 detection. Despite
these favorable properties, non-aggregation colorimetric
sensor for H2O2 based on the SPR shift of mere iAg NPs
has rarely been reported. One of the main reasons is that it
is more difficulty in inducing apparent SPR shift of iAg
NPs than that of ai-Au/Ag NMs because of the scarcity of
specialized reactive spots in iAg NPs. In fact, current iAg
NPs-based H2O2 assay are generally operates on the size
change-induced absorption intensity variation [27, 30, 31].
Although previous reports have successfully realized the
anisotropic growth of Ag seeds, the requirement of high
concentration of H2O2, H2O2 injection rate, mixing effi-
ciency, and the initial Ag concentration-dependent shape
transformation efficiency prevent the applicability of these
strategies for H2O2 detection purpose [41]. We reported
herein for the first time a non-aggregation colorimetric
Microchim Acta9 1:58 ) 102(
assay for H2O2 based on citrate-promoted H2O2-mediated
shape transformation of iAg NPs. We discovered that cit-
rate can promote iAg NPs collapse into anisotropic shape
in the presence of trace amount of H2O2, resulting in evi-
dent red-shift SPR and color change. Notably, this process
varies from previous studies that smoothen anisotropic Au/
Ag nanomaterials, but directly roughen iAg NPs into an-
isotropic shape. The substantial variation in the shape of
iAg NPs in the presence of H2O2 affords an apparent SPR
shift, offering this method a high sensitivity, whereas the
moderate reactivity of iAg NPs ensured the selectivity of
the detection. We suppose that this study will offer a new
method for designing colorimetric assays and promoting
the application of i-Ag NPs in trace analysis.
Materials and methods
Materials
All reagents were of analytical grade and used without further
purification. Trisodium citrate was bought from Alfa Aesar.
Citrate functionalized isotropic silver nanoparticle (cit-iAg
NPs, 50 nm), polyvinyl pyrrolidone-protected iAg NPs
(pvp-iAg NPs, 50 nm), and glucose oxidase (GOx,
163,314 U/g) were purchased from Sigma-Aldrich
(Germany, https://www.sigmaaldrich.com/china-mainland.
html). Hydrogen peroxide (H2O2, 30 wt%), amino acids,
glucose, fructose, lactose, sucrose, maltose, mannose were
purchased from Aladdin Chemical Company (China, http://
www.aladdin-e.com/). The metal salt inducing NaF, NaCl,
NaH 2 PO 4 , KBr, KSCN and KI were purchased from
Sinopharm Chemical Reagent Co (China, http://www.
instrument.com.cn/netshow/SH101458/). Deionized water
was used in all experiments.
Instruments
UV − vis spectra were recorded on a Lambda 365 UV spec-
trometer (PerkinElmer, America). Scanning electron micro-
scope (SEM) images were taken with a S-8010 scanning elec-
tron microscope (Hitachi, Japan) at an accelerating voltage of
10kv. The hydrodynamic diameters and size distributions of
the Ag NPs suspensions at 25 °C were determined by dynamic
light scattering (DLS) (Brookhaven, New York). Origin 8.0
and excel 2010 is used to process the data. As for UV, DLS
and SEM analysis, the corresponding built-in software of the
instrument is used.
Detection of H2O2
A typical H2O2 detection procedure by using the cit-iAg NPs
was conducted as follows: 0.3 mL of the cit-iAg NPs solution
Microchim Acta9 1:58 ) 102(
was diluted with 2.7 mL water. Subsequently, citrate was
added to a final concentration of 6 mM. Afterward, 3 μL of
H2O2 solution with different concentrations was added to the
above mixture. The resulting mixture was incubated at room
temperature for 60 min. Finally, the absorbance spectra of the
solution were obtained by a UV–vis spectrometer.
Procedures for determination of glucose
The procedure for glucose detection was as follows: 200 μL of
glucose solutions with different concentrations were added into
1.8 mL of GOx solution (1.5 mg/mL, buffered with 50 mM
sodium acetate, pH 5.1) and incubated at 37 °C for 30 min.
Then 10 μL of the above mixture was added to the cit-iAg NPs
(3 ml) and incubated at room temperature (25 °C) for 60 min.
Finally, the absorbance of the solutions was measured at room
temperature. All absorbance detections were performed under
the same conditions. The selectivity toward glucose was eval-
uated by using potentially interfering substances that are com-
monly present in the serum. These substances include 16 vari-
eties of amino acids (cysteine, phenylalanine, glutamate, tryp-
tophan, valine, isoleucine, glycine, lysine, threonine, histidine,
asparagine, proline, alanine, serine, leucine, and arginine),
ascorbic acid, and dopamine. Moreover, the selectivity toward
glucose analogs was evaluated by using sucrose, mannose,
cellobiose, lactose, fructose, and maltose. Assay procedures
for the selectivity study were all the same as those described
in the glucose assay section except the presence of glucose.
Fig. 1 a) SEM image of the
original cit-iAg NPs; and cit-iAg
NPs after treated with 2 μM (b);
12 μM (c); 20 μM (D) H2O2,
respectively
Page 3 of 7 19
The SPR response toward F−, Cl−, H2PO4−, Br−, SCN− and I−
was tested by adding 20 μM these anions respectively in the
absence of H2O2. To detect glucose in human serum, human
serum samples were obtained from a local hospital and centri-
fuged using an Amicon Ultrafilter with a 1000 molecular
weight cutoff at 5000 rpm for 10 min. The samples were
spiked with different concentrations of glucose. The resulting
mixture (200 μL) were then incubated with 1.8 mL GOx so-
lution (1.5 mg/mL, buffered with 50 mM sodium acetate,
pH 5.1) and incubated at 37 °C for 30 min. Then 10 μL of
the above mixture were added to the colloidal cit-iAg NPs
(3 mL) and incubated at 37 °C for 60 min.
Results and discussion
H2O2 sensing mechanism
According to previous reports, H2O2 can oxidize Ag NPs and
decrease their size and corresponding absorbance intensity
through the following reactions: Ag NP + 2H2O2 → Ag++
O2·− + 2H2O; [27, 30]. Nevertheless, we discovered that when
20 μΜ H2O2 was added into the citrate-capped isotropic Ag
NPs (cit-iAg NPs) solution, not only the absorption intensity
of the cit-iAg NPs decreased, but the maximum absorption
peak shifted to the long wavelength (Fig. S1A). Importantly,
we found that this red shift can be further enhanced by exter-
nal free citrate molecules. As demonstrated in Fig. S1B, the
19 Page 4 of 7 Microchim Acta9 1:58 ) 102(

Glucose
Glucose acid
O2 H2O2
GOx
Citrate cit-iAg NPs
Scheme 1 Proposed sensing mechanism for the detection of H2O2 and
glucose
Δλ increased significantly with increasing citrate concentra-
tions from 0 to 6 mM, reached the maximum at 6 mM
(Δλ = 60 nm), and then slightly decreased at higher citrate
concentrations. However, no obvious SPR shift was observed
when treating cit-iAg NPs with 6 mM citrate alone (Fig. S1C),
suggesting that the SPR shift was caused by H2O2. Based on
the above results, the concentration of 6 mM citrate was se-
lected as an optimal condition in the following assay. Also, it
was noted that when the cit-iAg NPs were replaced with pvp-
iAg NPs, the shifted Δλ can be ignored, even when 6 mM
citrate was added (Fig. S1D). This result confirmed that both
the free and capping citrates play crucial roles in the H2O2
response process. We deduced that the SPR shift of the cit-
iAg NPs in response to H2O2 is a manifestation of morpho-
logical transformation occurred to cit-iAg NPs. To reveal in-
sight into this process, the morphologies of the cit-iAg NPs in
the absence and presence of different amount of H2O2 were
characterized by scanning electron microscopy (SEM). Fig.1
clearly shows that cit-iAg NPs were gradually sculptured by
H2O2, leading to anisotropic, flawed structure. Furthermore,
DLS analysis reveal that the cit-iAg NPs solution remain
monodispersed even after treating with 20 μΜ H2O2 (Fig.
S2). Therefore, the possibility that the red shift in SPR was
induced by cit-iAg NPs aggregation can be discounted.
According to previous reports, citrate can serve as a shape-
directing agent and play critical roles in the formation of an-
isotropic Au/Ag nanomaterials [42, 43]. We therefore ascribed
the effective SPR red shift of the cit-iAg NPs in the presence
of H2O2 to the citrate-promoted H2O2 anisotropic sculpturing
of the cit-iAg NPs (Scheme 1). The reduced diameters re-
vealed by the DLS analysis explained the decreased SPR
intensity.
Sensing performance to H2O2
To achieve the most remarkable performance for H2O2 detec-
tion, we examined the time-dependent SPR shift on exposure
to H2O2 (60 μM). Fig. S3 shows that the corresponding SPR

Fig. 2 a) Photographs and b) a

UV–vis absorption spectra of the
cit-iAg NPs upon addition of
various H2O2 concentrations at a
citrate concentration of 6 mM
(H2O2 concentrations from top to
bottom (μM): 0, 0.2, 0.4, 0.8, 2, 4,
8, 12, 16, 32, 48, 64 and 96). c)
The relationship between the
absorption change and the H2O2
concentration (n = 5). The inset is 0 2 4 8 16 24 32 48 64 96 (µM)
the linear plot in the range of 0–
32 μM
b c
Microchim Acta9 1:58 ) 102( Page 5 of 7 19

peak shifts considerably fast in the first 10 min and then grad- a µM: 0 10 20 40 80
ually decreases to a saturation value after 60 min. Thus, the
reaction time was set at 60 min in subsequent experiments.
Under optimized conditions, the sensing strategy was evalu-
ated with respect to its performance in detecting H2O2. A
distinct color change was observed with increased glucose
concentrations. The lowest H2O2 concentration observed by
the bare eye was low at approximately 2 μM (Fig. 2a). For the
quantitative detection of H2O2, the absorbance spectra in the
presence of various concentrations of H2O2 were monitored.
As shown in Fig. 2b, the maximum absorbance band shifted
gradually with increasing H2O2 concentrations. Plotting Δλ
as a function of H2O2 concentration showed a good linear
relationship over the concentration range of 0.2–32 μM with
a limit of detection (LOD) of 90 nM at a signal-to-noise ratio
of 3 (Fig. 2c).
b

Glucose detection based on the use of cit-iAg NPs

and glucose oxidase

It is well known that GOx can catalyze the oxidation of

glucose to produce H2O2, the successful sensitive detection
of H2O2 was, then, extended for the analysis of glucose. The
process of glucose detection includes two steps. First, H2O2 is
generated from the biocatalyzed reaction between varied
concentrations of glucose and excess amount of GOx. In
order to achieve the highly sensitive detection of glucose,
the effect of the catalytic reaction time was studied. The
results indicated that the catalytic reaction can be finished
within 30 min (Fig. S4). Second, a certain volume of the
resulting mixture is introduced into the mixture and then the
corresponding absorption spectra were measured.
Fig. 3a shows that the color gradually changes from light
yellow to mauve with increasing glucose concentrations from
0 to 80 μM. Glucose can be well-distinguished even at con-
centration as low as 10 μM. Controlled experiments showed
that no detectable spectral shift occurred for either the Ag NP/
citrate–GOx or Ag NP/citrate–glucose, even after 1 h of incu-
bation (Fig. S5). When the experimental parameters were
optimized, a linear calibration curve was achieved by
plotting Δλ versus glucose concentration in the range of
1–24 μM (Fig. 3b). The LOD for glucose was 0.4 μM, as
calculated from the equation (signal/noise = 3). A systematic
comparison of the present method with some other colorimet-
ric assays was listed in Table 1. Compared with the known
colorimetric strategies, the method we report herein possesses
some remarkable features: first, it is highly sensitive toward
both H2O2 and glucose; second, it exhibit an excellent selec-
tivity, thus having great advantage in real sample applications;
third, the cir-iAg NPs are among the most easily synthesized
nanoparticles, making this method relatively low technical
required and generalizable.
Fig. 3 a) Effect of glucose–GOx system with different glucose
concentrations on the absorption of cit-iAg NPs in the presence of
6 mM citrate; glucose concentration from top to bottom (μM): 0, 1, 2,
4, 8, 12, 16, 24, 32, 48, and 80. The inset is the representative color
evolution. b) Relationship between the SPR shift and the glucose
concentration (n = 5). The inset is the linear plot in the range of 0–24 μM
Selectivity
To investigate whether this assay is specific for glucose, the
response toward the potential interferents commonly present
in serum, including 16 varieties of amino acids, ascorbic acid
and dopamine were evaluated. Furthermore, the effects of
some glucose analogues (surcose, mannose, cellobiose, lac-
tose, fructose and maltose) on the SPR behaviors of the Ag
NPs were also evaluated at identical conditions. As illustrated
in Fig. 4 except the addition of glucose can induce a signifi-
cant SPR shift of the Ag NPs, while other substances exerted a
negligible effect. Reportedly, anions such as Cl−, Br−, I−,
H2PO4−, and SCN− can induce a blue shift in the SPR peak
of the Ag TNPs by reacting with the highly reactive tips and
side faces of Ag TNPs [38–40]. However, no evident SPR
shift was observed when those anions were added to the
19 Page 6 of 7 Microchim Acta9 1:58 ) 102(

Table 1 Comparison of different colorimetric methods for H2O2 and glucose determination

Response mode Sensing system Measured signal LOD LOD Ref

(H2O2) (glucose)

Nanozymes- based catalyzed oxidation of RhNPs/TMB Intensity increase 0.2 uM 0.75 uM 13

peroxidase substrates bimetallic (Co/2Fe) MOFs/TMB Intensity increase 5 uM × 16
Au/Ag nanomaterials-based Aggregation AuNPs/OPD/HRP Intensity decrease 0.6 uM × 22
GOD-AuNPs LSPR shift × 2.8 uM 23
Cysteine/AuNPs/I− Intensity ratio 2 uM 4 uM 24
AuNPs/ BDBA Intensity ratio 0.2 uM 0.3 uM 25
Size decrease/ increase AuNPs/HAuCl4 Intensity increase 30 uM 49 uM 28
PVA-AgNPs Intensity decrease 1 uM × 30
SiO2/AgNPs Intensity decrease × 128 uM 31
Smoothing anisotropic AuNRs/Fe2+ LSPR blue shift 0.1 uM 0.1 uM 33
Au/Ag nanomaterials AuNRs/ HRP Wavelength × 10 uM 34
AuNPs/ Ag TNPs LSPR blue shift × 0.3 uM 37
Ag TNPs LSPR blue shift 0.2 uM 35
Directly roughening AgNPs/Citrate LSPR red shift 0.09 uM 0.4 uM present
isotropic AgNPs

MOFs, metal organic framework; TMB, 3,3,5,5-tetramethylbenzidine; BDBA, benzene-1,4-diboronic acid; OPD, o-phenylenediamine; HRP, horseradish
peroxidase; PVA, poly(vinylalcohol); ×, not given

colloidal cit-iAg NPs (Fig. S6). This result is attributed to the
moderated reactivity of iAg NPs as compared with that of Ag
TNPs.
Determination of glucose in human serum
Blood glucose level is closely associated with diabetes and
hypoglycemia. Thus, frequent monitoring and strict control
of blood sugar levels are typically requisite for effective man-
agement of diabetes mellitus and reduction of associated
complications. To validate the practical application assay in
biological samples, the concentrations of glucose in human
serum samples were determined by the method of standard
addition. Considering the normal fasting blood glucose level
in the healthy human blood (3.9–6.1 mmol/L) and the high
sensitivity of our method, 200 μL of serum should well satisfy
the analysis need in each glucose measurement. Human serum
samples were obtained from a local hospital and diluted and
spiked with different concentrations of standard glucose solu-
tions after centrifugation treatment. Upon adding this mixture
to the solution containing cit-iAg NPs, a prolonged response
time was observed as compared with that of glucose alone

Fig. 5 Quantification of glucose concentration in human serum samples

Fig. 4 Relative SPR shift of the detection system in the presence of (samples 1 and 2). Final concentrations: citrate, 6 mM; GOx, 1.5 mg/mL;
various amino acid and carbohydrates (20 μM) glucose added 0, 5, 10, 15, and 20 μM
Microchim Acta9 1:58 ) 102(
Table 2 Analytical results of glucose detection in human serum (n = 3)
Sample No. Local Hospital Our method Relative
(mM) (mM) deviation (%)
1 6.36 6.59 3.7
2 5.62 5.89 4.8
(Fig. S7), which may probably due to the hampering effect of
the compounds present in serum. To solve this problem, the
reaction temperature was elevated to 37 °C. As demonstrate in
Fig. 5, a good linear relationship was obtained by potting the
concentration of glucose with Δλ. The serum glucose
concentration was obtained from the intercept of the linear
curve on the x-axis. Additionally, the glucose concentration
obtained by our method was in good agreement with that
provided by the hospital (Table 2). This agreement further
confirmed the general applicability of the present method in
the analysis of glucose in actual physiological clinical
samples.
Conclusion
In summary, a highly sensitive, selective, and cost-efficient
approach for H2O2 detection has been designed based on the
citrate-promoted and H2O2 mediated shape transformation of
cit-iAg NPs. The apparent shape changes of cit-iAg NPs in
response to H2O2, afford a large LSPR shift, which enable
visual detection of H2O2 and glucose at low concentrations
(2 and 10 μM, respectively). Furthermore, the method was
successfully applied to monitor glucose levels in human se-
rum with satisfactory results. We anticipate that the designed
strategy can also be extended to the detection of various H2O2-
involved analytes. Therefore, this approach is not only sensi-
tive and selective but also convenient and generalizable. We
expect that this strategy may open up a new avenue in devel-
oping low-cost and sensitive method for biological and clini-
cal diagnostics application.
Acknowledgements The authors are grateful to the Jiangsu Province
Natural Science Foundation of China (BK20150228), the Natural
Science Foundation of China (21601072), Natural Science Foundation
of the Jiangsu Higher Education Institutions of China (16KJB510009).
Compliance with ethical standards
The author state that the blood related experiments were performed in
strict accordance with the WHO guidelines on blood drawing (WHO
Publication ISBN-13: 978-92-4-159922-1, 2010) and was approved by
Jiangsu Normal University. The authors also state that informed consent
was obtained for any experimentation with human subjects and the
Jiangsu Normal University is committed to the protection and safety of
human subjects involved in research.
Page 7 of 7 19
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