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-

rescence [4–8]. These methods made remarkable contribu-
Hydrogen peroxide (H2O2) is an essential mediator in food, tions in H2O2 and glucose detection. However, challenge still
pharmaceutical, clinical and environmental analyses and be- exists when taking into account both the simplicity and accu-
cause it is the final product of glucose oxidation. Therefore, racy of these methods. Owing to the simplicity and cheapness,
the glucose concentration can be indirectly detected by mon- colorimetric methods have attracted special research interest.
itoring the production of H2O2 [1–3]. Efforts have focused on Among the various colorimetric sensing approaches, enzy-
detecting H 2 O 2 by different strategies such as Raman 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
Electronic supplementary material The online version of this article possess intrinsic peroxidase-like activity, but are more stable
(https://doi.org/10.1007/s00604-018-2737-2) contains supplementary to biodegradation, and less vulnerable to denaturation
material, which is available to authorized users. [11–13]. These nanozyme-based sensing platforms offer
promising new avenues in H2O2 detection, but in order to
* Chenghua Zong obtain and maintain the high catalytic activity of the
[email protected]
nanozymes, strict control of the experiments and synthesis
* Yang Yu conditions are typical required [14–16].
[email protected]
Plasmonic metal nanomaterials, such as silver and gold have
1
School of Chemistry and Materials Science, Jiangsu Key Laboratory
proved to be ideal alternative to construct colorimetric assays
of Green Synthesis for Functional Materials, Jiangsu Normal [17–21]. The Au/Ag nanomaterial-based H2O2 colorimetric as-
University, Xuzhou, Jiangsu, People’s Republic of China 221116 says can be categorized into aggregation and non-aggregation
2
School of Physics and Electronic Engineering, Jiangsu Normal sensors. Although aggregation-based colorimetric methods are
University, Xuzhou 221116, People’s Republic of China merited with simplicity and high sensitivity [22–26], false
19 Page 2 of 7 Microchim Acta9 1:58 ) 102(

positive signals from autoaggregation pose a significant chal- assay for H2O2 based on citrate-promoted H2O2-mediated
lenge in complex system applications. Hence, non-aggregation shape transformation of iAg NPs. We discovered that cit-
colorimetric sensors are gaining increasing attentions. In non- rate can promote iAg NPs collapse into anisotropic shape
aggregation colorimetric sensors, a nanomaterial solution re- in the presence of trace amount of H2O2, resulting in evi-
mains in a monodisperse state, but either the SPR intensity or dent red-shift SPR and color change. Notably, this process
the SPR position is changed upon the addition of H2O2 varies from previous studies that smoothen anisotropic Au/
[27–29]. Tuning the size of the Au/Ag nanoparticles (Au/Ag Ag nanomaterials, but directly roughen iAg NPs into an-
NPs) to change their absorption intensity and altering the shape isotropic shape. The substantial variation in the shape of
of Au/Ag nanomaterials to induce SPR shifts are two of the iAg NPs in the presence of H2O2 affords an apparent SPR
most effective design rationales [30–32]. Compared with the shift, offering this method a high sensitivity, whereas the
intensity mode, the SPR shift assays are preferred because these moderate reactivity of iAg NPs ensured the selectivity of
methods exhibit more vivid color variations, thereby offering the detection. We suppose that this study will offer a new
increased sensitivity and reliability. Currently available SPR method for designing colorimetric assays and promoting
shift-based assays are primarily based on anisotropic Au/Ag the application of i-Ag NPs in trace analysis.
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- Materials and methods
shifted LSPR. For instance, it has been reported that gold nano-
rods (Au NRs) can be selectively etched by H2O2 in the longi- Materials
tudinal direction, giving rise to a visual color change; however,
because of the high electrochemical potential of Au, either high All reagents were of analytical grade and used without further
concentrations of H2O2 or catalytic species are required in these purification. Trisodium citrate was bought from Alfa Aesar.
assays [33, 34]. Ag triangle nanoprisms (Ag TNPs) are another Citrate functionalized isotropic silver nanoparticle (cit-iAg
kind of anisotropic nanomaterials that are commonly used in NPs, 50 nm), polyvinyl pyrrolidone-protected iAg NPs
H2O2 detection. Xia demonstrated that sharp tips and side faces (pvp-iAg NPs, 50 nm), and glucose oxidase (GOx,
of Ag TNPs can be etched by H2O2, inducing a triangle to 163,314 U/g) were purchased from Sigma-Aldrich
round shape transformation, affording a significant LSPR blue (Germany, https://www.sigmaaldrich.com/china-mainland.
shift, accompanied by a blue to mauve color changes [35–37]. html). Hydrogen peroxide (H2O2, 30 wt%), amino acids,
The highly reactive edges/tips of the Ag TNPs offer these glucose, fructose, lactose, sucrose, maltose, mannose were
methods outstanding sensitivity. However, selectivity becomes purchased from Aladdin Chemical Company (China, http://
a major challenge because anions, such as Cl−, Br−, I−, H2PO4−, www.aladdin-e.com/). The metal salt inducing NaF, NaCl,
and SCN−, can also react with these highly reactive spots and NaH 2 PO 4 , KBr, KSCN and KI were purchased from
faces [38–40]. Sinopharm Chemical Reagent Co (China, http://www.
Different from ai-Au/Ag NMs, isotropic Ag NPs (iAg instrument.com.cn/netshow/SH101458/). Deionized water
NPs) are not only easier in preparation, and exhibit higher was used in all experiments.
stability, but also, most importantly, exhibit moderate reac-
tivity that offers an improved selectivity, making them ide- Instruments
al candidates for colorimetric H 2O 2 detection. Despite
these favorable properties, non-aggregation colorimetric UV − vis spectra were recorded on a Lambda 365 UV spec-
sensor for H2O2 based on the SPR shift of mere iAg NPs trometer (PerkinElmer, America). Scanning electron micro-
has rarely been reported. One of the main reasons is that it scope (SEM) images were taken with a S-8010 scanning elec-
is more difficulty in inducing apparent SPR shift of iAg tron microscope (Hitachi, Japan) at an accelerating voltage of
NPs than that of ai-Au/Ag NMs because of the scarcity of 10kv. The hydrodynamic diameters and size distributions of
specialized reactive spots in iAg NPs. In fact, current iAg the Ag NPs suspensions at 25 °C were determined by dynamic
NPs-based H2O2 assay are generally operates on the size light scattering (DLS) (Brookhaven, New York). Origin 8.0
change-induced absorption intensity variation [27, 30, 31]. and excel 2010 is used to process the data. As for UV, DLS
Although previous reports have successfully realized the and SEM analysis, the corresponding built-in software of the
anisotropic growth of Ag seeds, the requirement of high instrument is used.
concentration of H2O2, H2O2 injection rate, mixing effi-
ciency, and the initial Ag concentration-dependent shape Detection of H2O2
transformation efficiency prevent the applicability of these
strategies for H2O2 detection purpose [41]. We reported A typical H2O2 detection procedure by using the cit-iAg NPs
herein for the first time a non-aggregation colorimetric was conducted as follows: 0.3 mL of the cit-iAg NPs solution
Microchim Acta9 1:58 ) 102( Page 3 of 7 19

was diluted with 2.7 mL water. Subsequently, citrate was The SPR response toward F−, Cl−, H2PO4−, Br−, SCN− and I−
added to a final concentration of 6 mM. Afterward, 3 μL of was tested by adding 20 μM these anions respectively in the
H2O2 solution with different concentrations was added to the absence of H2O2. To detect glucose in human serum, human
above mixture. The resulting mixture was incubated at room serum samples were obtained from a local hospital and centri-
temperature for 60 min. Finally, the absorbance spectra of the fuged using an Amicon Ultrafilter with a 1000 molecular
solution were obtained by a UV–vis spectrometer. 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-
Procedures for determination of glucose 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 procedure for glucose detection was as follows: 200 μL of the above mixture were added to the colloidal cit-iAg NPs
glucose solutions with different concentrations were added into (3 mL) and incubated at 37 °C for 60 min.
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. Results and discussion
Finally, the absorbance of the solutions was measured at room
temperature. All absorbance detections were performed under H2O2 sensing mechanism
the same conditions. The selectivity toward glucose was eval-
uated by using potentially interfering substances that are com- According to previous reports, H2O2 can oxidize Ag NPs and
monly present in the serum. These substances include 16 vari- decrease their size and corresponding absorbance intensity
eties of amino acids (cysteine, phenylalanine, glutamate, tryp- through the following reactions: Ag NP + 2H2O2 → Ag++
tophan, valine, isoleucine, glycine, lysine, threonine, histidine, O2·− + 2H2O; [27, 30]. Nevertheless, we discovered that when
asparagine, proline, alanine, serine, leucine, and arginine), 20 μΜ H2O2 was added into the citrate-capped isotropic Ag
ascorbic acid, and dopamine. Moreover, the selectivity toward NPs (cit-iAg NPs) solution, not only the absorption intensity
glucose analogs was evaluated by using sucrose, mannose, of the cit-iAg NPs decreased, but the maximum absorption
cellobiose, lactose, fructose, and maltose. Assay procedures peak shifted to the long wavelength (Fig. S1A). Importantly,
for the selectivity study were all the same as those described we found that this red shift can be further enhanced by exter-
in the glucose assay section except the presence of glucose. nal free citrate molecules. As demonstrated in Fig. S1B, the

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
19 Page 4 of 7 Microchim Acta9 1:58 ) 102(

citrate was added (Fig. S1D). This result confirmed that both
Glucose the free and capping citrates play crucial roles in the H2O2
Glucose acid
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
O2 H2O2
GOx 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
Citrate cit-iAg NPs 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.
Scheme 1 Proposed sensing mechanism for the detection of H2O2 and
glucose 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
Δλ increased significantly with increasing citrate concentra- of H2O2 to the citrate-promoted H2O2 anisotropic sculpturing
tions from 0 to 6 mM, reached the maximum at 6 mM of the cit-iAg NPs (Scheme 1). The reduced diameters re-
(Δλ = 60 nm), and then slightly decreased at higher citrate vealed by the DLS analysis explained the decreased SPR
concentrations. However, no obvious SPR shift was observed intensity.
when treating cit-iAg NPs with 6 mM citrate alone (Fig. S1C),
suggesting that the SPR shift was caused by H2O2. Based on Sensing performance to H2O2
the above results, the concentration of 6 mM citrate was se-
lected as an optimal condition in the following assay. Also, it To achieve the most remarkable performance for H2O2 detec-
was noted that when the cit-iAg NPs were replaced with pvp- tion, we examined the time-dependent SPR shift on exposure
iAg NPs, the shifted Δλ can be ignored, even when 6 mM 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 Fig. 3 a) Effect of glucose–GOx system with different glucose
within 30 min (Fig. S4). Second, a certain volume of the concentrations on the absorption of cit-iAg NPs in the presence of
resulting mixture is introduced into the mixture and then the 6 mM citrate; glucose concentration from top to bottom (μM): 0, 1, 2,
corresponding absorption spectra were measured. 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
Fig. 3a shows that the color gradually changes from light concentration (n = 5). The inset is the linear plot in the range of 0–24 μM
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/ Selectivity
citrate–GOx or Ag NP/citrate–glucose, even after 1 h of incu-
bation (Fig. S5). When the experimental parameters were To investigate whether this assay is specific for glucose, the
optimized, a linear calibration curve was achieved by response toward the potential interferents commonly present
plotting Δλ versus glucose concentration in the range of in serum, including 16 varieties of amino acids, ascorbic acid
1–24 μM (Fig. 3b). The LOD for glucose was 0.4 μM, as and dopamine were evaluated. Furthermore, the effects of
calculated from the equation (signal/noise = 3). A systematic some glucose analogues (surcose, mannose, cellobiose, lac-
comparison of the present method with some other colorimet- tose, fructose and maltose) on the SPR behaviors of the Ag
ric assays was listed in Table 1. Compared with the known NPs were also evaluated at identical conditions. As illustrated
colorimetric strategies, the method we report herein possesses in Fig. 4 except the addition of glucose can induce a signifi-
some remarkable features: first, it is highly sensitive toward cant SPR shift of the Ag NPs, while other substances exerted a
both H2O2 and glucose; second, it exhibit an excellent selec- negligible effect. Reportedly, anions such as Cl−, Br−, I−,
tivity, thus having great advantage in real sample applications; H2PO4−, and SCN− can induce a blue shift in the SPR peak
third, the cir-iAg NPs are among the most easily synthesized of the Ag TNPs by reacting with the highly reactive tips and
nanoparticles, making this method relatively low technical side faces of Ag TNPs [38–40]. However, no evident SPR
required and generalizable. 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 complications. To validate the practical application assay in
moderated reactivity of iAg NPs as compared with that of Ag biological samples, the concentrations of glucose in human
TNPs. 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
Determination of glucose in human serum
sensitivity of our method, 200 μL of serum should well satisfy
the analysis need in each glucose measurement. Human serum
Blood glucose level is closely associated with diabetes and
samples were obtained from a local hospital and diluted and
hypoglycemia. Thus, frequent monitoring and strict control
spiked with different concentrations of standard glucose solu-
of blood sugar levels are typically requisite for effective man-
tions after centrifugation treatment. Upon adding this mixture
agement of diabetes mellitus and reduction of associated
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( Page 7 of 7 19

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