Sensors and Actuators B 255 (2018) 2078–2085

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Research paper

Fluorescence turn-on sensing of thiamine based on Arginine –

functionalized graphene quantum dots (Arg-GQDs): Central
composite design for process optimization
Fatemeh Nemati a , Rouholah Zare-Dorabei a,∗ , Morteza Hosseini b,c ,
Mohammad Reza Ganjali d,e
a
Research Laboratory of Spectrometry & Micro and Nano Extraction, Department of Chemistry, Iran University of Science and Technology, Tehran, Iran
b
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran
c
Medical Biomaterials Research Center, Tehran University of Medical Sciences, Tehran, Iran
d
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran
e
Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: A low-cost, sensitive and convenient fluorescent probe of highly fluorescent Arginine – functionalized
Received 4 June 2017 graphene quantum dots (Arg-GQDs) was designed and synthesized by a facile one–pot hydrothermal
Received in revised form 27 August 2017 treatment of citric acid in the presence of Arginine. The fabricated Arg-GQDs with oxygen-rich functional
Accepted 2 September 2017
groups exhibit a strong blue emission with 28.3% quantum yield (QY). It is found that such Arg-GQDs
Available online 5 September 2017
with high QY can be used as efficient fluorescent probe for thiamine determination. In the detection,
photoluminescence (PL) intensity of the Arg-GQDs was reversibly quenched and recovered by Ag+ and
Keywords:
thiamine, respectively. The effect of different parameters were optimized by central composite design
Graphene quantum dots
Fluorescence probe (CCD) combined with response surface methodology (RSM). Furthermore, results of CCD were evaluated
Central composite design by standard analysis of variance (ANOVA) to assess the accuracy and suitability of the quadratic model.
Thiamine (Vitamin B1) Under optimal conditions, the fluorescence “Off-On” process showed a sensitive response to thiamine
Determination in the concentration range of 0.1–8.0 ␮M with a detection limit of 53 nM. Finally, the Arg-GQDs based
fluorescence method was applied to the analysis of thiamine in pharmaceuticals and foodstuff samples
with satisfying recoveries for the spiked samples.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction adequate dietary intakes are necessary [5]. An insufficiency of

Vitamins are critical organic substances that are required for
the normal growth, self-maintenance and functioning of human
and animal bodies [1]. Vitamins are mainly divided into in two
different types, water-soluble and fat-soluble vitamins [2]. Vita-
min B1 (thiamine), a member of water-soluble vitamins, is one
of the eight vitamins that make up the powerful group called
the vitamin B complex. This Vitamin plays a key role in the good
health of the body as well as in sound mental health [3]. The main
biological role of thiamine is to act as cofactor for carbohydrate
and amino acids metabolism in living system [4]. Furthermore,
thiamine is important for brain health, particularly in emotional
well-being, and also is useful for focus and concentration [3]. Bac-
teria, fungi and plants only synthesize this compound and thus,
∗ Corresponding author.
E-mail addresses: zaredorabei@iust.ac.ir, zaredoraby@gmail.com
(R. Zare-Dorabei).
thiamine cause nervous and cardiovascular problems because it
interferes with cellular metabolism [6]. Beriberi and Wernicke Kor-
sakoff syndrome also associated with lacked of thiamine [7]. There
have been suggestions that thiamine may have a beneficial effect
in treating Alzheimer’s disease [8]. For all these reasons, the devel-
opment of a sensitive Method for the determination of Vitamin
B1 is of much interest. A variety of methods have been applied to
vitamin B1 measurement including spectrophotometry [9,10] and,
spectrofluorimetry [11,12], chemiluminescence [13], amperomet-
ric [3], gas chromatography [14,15] capillary zone electrophoresis
[16] and high performance liquid chromatography [17–19]. Among
the methods for detection of thiamine, fluorometric methods have
received particular attention because of the high sensitivity, fast
response, and simple operation [20]. In the field of fluorescent
sensors, nanomaterials with different morphologies have received
widely attention for their unique optical performances [21–23]. In
recent years, more attention has been focused on semiconductor
quantum dots (SQDs) as novel luminescence indicators. However,

http://dx.doi.org/10.1016/j.snb.2017.09.009
0925-4005/© 2017 Elsevier B.V. All rights reserved.
 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085
the intrinsic defects of those QDs limit their widespread applica-
tion, such as complex synthesis procedures, heavy metals potential
toxicity and environmental hazards [24]. Graphene quantum dots
(GQDs) are generally defined as zero-dimensional carbon-based
material that are typically single or few-layer graphenes with lat-
eral dimensions of less than 100 nm [25,26]. Owing to the novel
properties, such as remarkable conductivity, high edge-to-area
ratio, stable luminescence, good biocompatibility, and low toxicity,
graphene quantum dots have recently emerged as promising alter-
natives to traditional quantum dots in some aspects [27]. However,
the photoluminescence quantum yield of the pristine GQDs is usu-
ally low and much lower than semiconductor quantum dots [28].
Recently demonstrated that high quantum yields of GQDs quantum
yield can be observed after either functionalization or doping [29].
Among foreign atoms, Nitrogen has been widely used for chem-
ical doping in carbon-based nanomaterials by virtue of it having
match able atomic size and five valence electrons for bonding with
carbon atoms [30,31]. The optimization in analytical chemistry is
generally carried out with a traditional single variable approach
(SVA) [32]. Its major disadvantage is that it ignores the combination
influence among the parameters studied. Due to above disadvan-
tage, multivariate statistic methods have been applied to optimize
experimental parameters. Among the most relevant multivari-
ate techniques, response surface methodology (RSM) has shown
considerable application in analytical optimization [33]. Response
surface methodology, a collection of mathematical and statistical
techniques, is a very useful tool for determining the effect of opera-
tional variables for process development and optimization [34,35].
The most frequent and efficient methods used in response surface
methodology is central composite design (CCD) [36]. The aim of
this work was to develop a simple, fast and non-toxic method, of
maximum possible sensitivity, to detection of thiamine in phar-
maceuticals and foodstuff samples. For this, we have synthesized
water-soluble Arg-GQDs by using citric acid and Arginine (Arg) as
carbon and nitrogen sources respectively. Based on the excellent
photoluminescent of the Arg–GQDs, a simple, potent, fast, non-
toxic fluorescent probe was designed. In this paper, the influence
of time, temperature and pH on fluorescence sensing was investi-
gated using response surface methodology (RSM) based on central
composite design (CCD). The range of linearity, the stability, the
response to interferents and the detection limit (LOD) of the sensor
have been evaluated. The optical sensor based on the Arg-GQDs has
been successfully applied in sensitive detection of thiamine in real
spiked samples.
2. Experimental section
2.1. Chemicals and materials
Citric acid (+99%), Arginine were purchased from Sigma–Aldrich
Co. Thiamine hydrochloride (98%) was purchased from Alfa
Aesar. All the other chemicals used were of analytical reagents
grade. Stock standard solution of thiamine hydrochloride
(1.0 × 10−3 mol L−1 ) was prepared in water and stable for at
least 1 month when kept refrigerated. Working solutions of lower
concentrations were prepared daily from the above stock solution
as required. All chemicals used were of analytical grade or of the
highest purity available. All solutions were prepared with doubly
deionized water (DDW).
2.2. Apparatus
UV–vis measurements were carried out on a PG Instruments
T80+ UV/Vis spectrophotometer and the spectra were collected
from 200 nm to 700 nm. All fluorescence measurements were
2079
carried out by a Perkin-Elmer LS50 luminescence spectrometer.
Fourier transform infrared (FT-IR) spectrum was recorded on a
Shimadzu-8400S spectrometer in the range of 400–4000 cm−1 at
room temperature using KBr pellets. Energy-dispersive X-ray (EDX)
spectra were obtained using the Philips XL30 scanning electron
microscope (SEM). The morphologies of Arg-GQDs samples were
characterized by transmission electron microscopy (TEM, Philips,
CM30, 300 Kv). The pH was adjusted and measured using pH meter
Metrohm. The X-ray diffraction patterns (XRD) were recorded on
a Philips diffractometer (model X’Pert MPD). Raman spectrum was
recorded using an Almega Thermo Nicolet Dispersive Raman Spec-
trometer with a 532 nm laser.
2.3. Synthesis of functionalized graphene quantum dots
In a typical synthesis, 0.21 g of citric acid and 0.10 g of Arginine
were mixed with 5 mL water, and stirred to form a clear solu-
tion. The solution was then transferred into a 20 mL Teflon-lined
stainless autoclave. The sealed autoclave was heated constantly
at 160 ◦ C for 4 h. The obtained mixture was filtered with a mem-
brane (0.22 ␮m) to remove the large dots. Finally, the as prepared
Arg-GQDs were stored at 4 ◦ C for further experiments.
2.4. General procedure
For thiamine detection, 25 ␮L of 40 mg mL−1 Arg-GQDs, 1 mL pH
6 phosphate buffer and 40 ␮L (0.01 M) of Ag+ solution were added
in a quartz cuvette and the mixture was stirred thoroughly. Then,
the mixture was finally diluted to 2 mL with ultrapure water. Next,
different amounts of thiamine added to the above mixed solution.
Finally, the fluorescence intensity of the mixture was recorded at
excitation wavelength of 350 nm, and the band-slits of both exci-
tation and emission were set as 10.0 and 15.0 nm, respectively.
2.5. Quantum yield measurements
The quantum yield of the as-synthesized GQDs were determined
by using quinine sulfate as the fluorescence standard and was cal-
culated according to the following Eq. (1):
Iu Ast n2u
Yu = Yst (1)
Ist Au n2st
Where Y is the quantum yield, I is the measured integrated emission
intensity, n is the refractive index, and A is the extinction. The sub-
script “st” refers to the standard and “u” for sample with unknown
QY.
2.6. Analysis of thiamine in real samples
Fluorescent detection of thiamine in food samples was per-
formed through the following steps: 1.0 g of samples that was
purchased from a local supermarket was grounded and mixed with
0.1 mol L−1 HCl solution. After integrating for 15 min, the mixture
was centrifuged for 15 min at 5000 rpm, and the supernatant was
filtered through a 0.45 mm cellulose acetate filter and diluted up to
10 mL with ultra-pure water. The pharmaceutical sample (vitamin
B1 tablet from Jalinous Pharmaceutical Co., Iran) was obtained from
the local drug store. For analysis, a piece of vitamin B1 tablet was
powdered in a mortar and dissolved in ultrapure water. The mixture
was sonicated for 10 min for fully dissolution. The insoluble com-
ponents were removed by filtration. After that, the solution was
transferred into 25 mL volumetric flask and stored in dark at 4 ◦ C.
Standard addition method was used for determination of thiamine
in the real samples.
2080 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085

Fig. 1. (a) TEM image of the Arg-GQDs (inset: size distribution of Arg-GQDs) (b) EDX spectrum of the Arg-GQDs (c) Raman spectra of as-prepared Arg-GQDs (d) XRD patterns
of the Arg-GQDs.

Table 1 Table 2
Experimental factors and levels in CCD. The design matrix for four variables with enhancement ratio (F- F0 )/F0 as response
(R).
Levels
Runs Block x1 x2 x3 (F − F0 )/F0
Independent variable -␣ -1(low) 0 (center) +1(High) +␣
1 1 6 15 35 2.73
(x1) pH 4 5 6 7 8 2 1 7 10 30 2.68
(x2 ) time(min) 5 10 15 20 25 3 1 5 10 30 2.54
◦
(x3) temperature ( C) 25 30 35 40 45 4 1 5 20 40 2.26
5 1 5 20 30 2.10
6 1 7 20 30 2.48
7 1 7 20 40 2.43
2.7. Optimization of factors affecting the fluorescence sensing of 8 1 7 10 40 2.28
thiamine using RSM approach 9 1 5 10 40 2.09
10 1 6 15 35 2.60
11 2 6 15 35 2.55
Central composite design was applied to investigate the effects 12 2 6 15 35 2.49
of the three operational factors on the response function. The 13 2 8 15 35 2.02
studied parameters were selected based on the preliminary experi- 14 2 6 15 45 2.39
ments and prior knowledge from literature. The high and low levels 15 2 6 15 35 2.53
16 2 4 15 35 1.37
defined for three independent variables are shown in Table 1. After
17 2 6 15 35 2.70
running the 20 trials, a second order polynomial model for predict- 18 2 6 25 35 2.51
ing the optimal point was explained by the following Eq. (2): 19 2 6 5 35 2.37
20 2 6 15 25 2.75


k 
k 
k

k
Y = ˇ0 + ˇi xi + ˇij xi xj + ˇii xi2 (2)
i=1 i=1 j=i+1 i=1

In the above equation, Y is the predicted response; xi and xj are

independent factors and ˇ0 is the intercept. ˇi and ˇii are the lin-
ear and quadratic coefficients, respectively. While ˇij represents
the interaction coefficients. Design Expert software version 7 was The design matrix and enhancement ratio (F − F0 )/F0 as response
employed to analyze the data and the design of the experiment. are presented in Table 2.
 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085
Fig. 2. (a) FT-IR spectrum (b) UV–vis absorption (c) fluorescence excitation and
emission spectra of Arg-GQDs.
3. Results and discussion
3.1. Characterization of GQDs
The Arg-GQDs were synthesized by the hydrothermal approach
described in the Experimental Section. TEM images gave the first
confirmation that nano-sized pieces formed during the synthesis
of NGQDs. Fig. 1a shows the TEM image of N-GQDs. Uniformly dis-
persed Arg-GQDs in a narrow size distribution were observed. And
the mean diameter of Arg-GQDs was estimated to be 5.5 nm.
Analysis of elemental composition of the prepared Arg-GQDs
was performed using EDX. The EDX spectrum of the N-GQD showed
the presence of elements O, N, and C (Fig. 1b).Raman spectrum of
the of Arg-GQD is shown in Fig. 1c. There are two closely peaks
at around 1600 cm−1 (G band) and 1400 cm−1 (D band), respec-
tively, which were assigned to in-plane vibrations of crystalline
graphite and disordered structures of the sp2 domains. For more
information about the structure, X-ray diffraction (XRD) analysis
was performed. There is a diffraction peak at range of 21.8–24.4◦
on the XRD pattern, which attributed to graphitic structure [37,38].
Fig. 2a showed the FT-IR spectrum of N-CQDs in the wavelength
range of 500–4000 cm−1 . The absorption band at 3420 cm−1 was
assigned to O H stretching vibration. The characteristic absorption
peaks for C = C at 1571 cm−1 and C C at 1234 cm−1 were observed,
indicating the existence of delocalized ␲-electrons in Arg-GQDs
molecules. The absorption peaks around 3172 cm−1 and 1451 cm−1
2081
were belonged to of N H and C N stretching vibration, respec-
tively [39]. This result further confirmed the doping of N in the
as-prepared sample. Moreover, three absorption peaks appeared
at 1451 cm−1 and 1109 cm−1 , corresponding to the stretching vibra-
tions of COO and C O groups, respectively. These functional groups
could stabilize Arg-GQDs in aqueous media. The as-prepared Arg-
GQDs solution remains homogeneous even after 2 months at room
temperature without any perceptible changes (e.g., aggregation or
color change), which could be further characterized by the almost
unchanged absorption and PL spectra (Fig. S1). To further explore
the optical properties of the N-GQDs, UV–vis absorption and FL
spectra were recorded. As shown in Fig. 2b, UV–vis absorption spec-
trum of the GQD shows a typical absorption peak at ca. 210 nm
was observed, which is assigned to the ␲–␲* transition of aro-
matic C C sp2 domains. Another strong absorption peak at 340 nm
can be regarded as the n–␲* transition of the C O bone, which
can result in strong fluorescence [40]. The GQD solution emitted
strong blue FL under excitation at 365 nm by a UV lamp (inset of
Fig. 2a). However, it was light yellow, transparent and clear under
daylight (inset of Fig. 2b). As shown in Fig. 2c, the Arg-GQDs exhib-
ited a maximum emission at 448 nm with an excitation wavelength
of 350 nm. Compared to the non-doped GQDs, the present Arg-
GQDs show blue-shifted emission, which could be ascribed to the
strong electron affinity of the doped nitrogen [41]. It should be
noted that the excitation peak is very close to the absorption peak,
suggesting the high-efficient emitting absorption of the Arg-GQDs.
The quantum yield of the Arg-GQDs was calculated to be 28.3%.
The photostability of as-synthesized Arg-GQDs under UV irradia-
tion was studied. As shown in Fig. S2, the fluorescence intensity of
Arg-GQDs remained constant within the studied range, indicating
excellent photo-stability of the as-prepared Arg-GQDs. The effects
of ion strength on the fluorescence of Arg-GQDs was also inves-
tigated in the presence of different concentrations of NaCl. The
signal intensity measured of the Arg-GQDs. When concentrations
of NaCl is 0.1–2 M, the measured fluorescence was nearly constant,
indicating the high stability of Arg-GQDs under high salt medium.
3.2. Optimization of sensing conditions
Based on CCD experiment, the effects of detection parame-
ters; incubation time, pH value and temperature were evaluated.
According to experimental data, using software the second-order
polynomial was used to describe the response variable (F − F0 /F0 )
and interaction among the variables as follows Eq. (3):
F − F0 /F0 = +2.60 + 0.14x1 − 0.003750x2
− 0.090x3 + 0.027x1 x2 − 0.020x1 x3
+ 0.12x2 x3 − 0.22x1 2 − 0.036x2 2 0.003542x3 2 (3)
Where x1 , x2 and x3 are pH, time and temperature, respectively. In
order to test the validity and adequacy of the model and the effect
of operational variables and their interactions, ANOVA was per-
formed. The ANOVA is a collection of statistical models, and their
associated procedures, in which the observed variance in a particu-
lar variable is partitioned into components attributable to different
sources of variation. It provides a statistical test of whether or not
the means of several groups are all equal [42]. The reliability of
the suggested model was assessed by the coefficient of determina-
tion (R2 and adjusted-R2 ). An R2 of 0.9532 and an adjusted-R2 of
0.9065 indicated excellent correlation between experimental val-
ues and fitted model, and showed a high- efficiency designed model
for prediction of response. Based on ANOVA data, the amounts of
F-value (20.38) and p-value (less than 0.0001) implies that defined
model is significant. The “lack-of-fit (LOF) P-value” of this model
(0.6243) is in significant because of its amount is more than 0.05
2082 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085
Fig. 3. The predicted response vs. the observed response.
Fig. 4. A plot of the internally studentized residuals vs. the predicted response.
critical value; thus, the non-significant lack-of-fit confirm the ade-
quacy of a model fit. Fig. 3 indicates the predicted responses versus
observed data. Most residuals were proximity close to the straight
line, which indicates a good relationship between actual data and
fitted model as a cause of large R2 values.
The plot of the residuals versus the predicted data (Fig. 4) con-
firms that the points were distributed randomly. In next step, to
observe the individual and interactive effect of the variables on
enhancement ratio (F − F0 )/F0 , 3-D surface plots and contour plots
were used. The responses plotted relative to the two significant
variables, while the other factor is fixed at its central level [43].
The goal of the response surface is to find the optimum values
of the factors, such that the response is maximized [44]. Fig. 5a
depicts the effect of pH, time and their mutual interaction on the
response. Thiamine has been reported to get oxidized at alkaline
medium to generate the thiochrome [45]. Hence, the pH effect was
studied in the 4–8 range. Result show that by increasing the pH of
solution, the enrichment ratio increases. This is related to protona-
tion of the carbonyl groups and hydroxyl groups on the surface of
Arg-GQDs that occur at the acidic pH. In other hand, at high con-
centration of H+ ions, the formation of hydrogen bond between
oxygen-containing functional groups on the surface of GQDs lead
to the aggregation of GQDs [46]. A pH of 6 was selected for the fur-
ther thiamine sensing. As can be seen from Fig. 5a–c, the higher
temperature leads to a decrease in the response surface and had an
inverse effect on enhancement ratio. The fluorescence signal of a
graphene quantum dot is very sensitive to temperature. The quan-
tum efficiency includes radiative and non-radiative decay of excited
states. The enhance in non-radiative decay rate at higher temper-
ature leads to decrease in quantum efficiency and corresponding
Fig. 5. Response using the central composite design obtained by plotting: (a) time
vs. pH, (b) temperature vs. pH, (c) time vs. temperature.
fluorescence intensity [47]. Effects of incubation time and temper-
ature on the enhancement ratio are shown in Fig. 5c. Incubation
time and temperature both had inverse effect on the enhancement
ratio, while the interaction between incubation time and temper-
ature was negatively correlated with response. The enhancement
ratio can be adjusted by changing the time because it decreases the
temperature. Therefore, the low temperature leads to an increase
in the initial intensity. The optimum predicted point of maximum
enhancement ratio obtained by Design-Expert software is about
2.77, and the corresponding optimal parameters of adsorption pro-
cess are listed as below: time 10 min, temperature 30 ◦ C, pH 6.
3.3. Possible mechanism
Upon addition of Ag+ ion to the dispersion of the Arg- GQDs, a
decrease in fluorescence intensity of Arg-GQDs is observed (Fig. 6).
The nature of quenching was attributed to the coordination of Ag+
and the functional groups of Arg-GQDs. According to HSAB (hard
and soft acids and bases) principle, the soft acids and soft bases
tend to have a strong interaction [48]. Thus, Ag+ ions (soft acids)
could have a high affinity with nitrogen atoms in Arg-GQDs surface
(soft bases) and form conjugation of the Arg-GQDs- Ag+ complex
[49]. Thus, the fluorescence of Arg-GQDs was quenched by the addi-
tion of Ag+ ions. The interaction between Arg-GQDs and Ag+ was
 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085 2083

Fig. 6. Fluorescence responses Arg-GQDs in the presence of several of Ag+ .

studied by FT-IR spectra (Fig. S4). When Ag+ is present in Arg-GQDs

solution, the peak position of N H stretching vibration band moves
from 3172 to 3170 cm−1 and the peak position of COO− stretch-
ing vibration band moves from 1451 to 1454 cm−1, together with
the decrease in the peak intensities of these two bands. The peak
intensities of 3420 also decreased, confirming the binding interac-
tion between Arg-GQDs and Ag+ . These results indicate that Ag+
interacted with functional groups present on the obtained Arg-
GQDs. According to the experimental result, we considered that
thiamine with thiazole ring would show strong binding ability
with Ag+ through an Ag S bond [50]. Obviously, the fluorescence
intensity of the quenched N-GQD recovered with increasing con-
centration of thiamine in the system. This enhancement could be
a result of interaction between Ag+ and the thiamine. This result Fig. 7. (a) Fluorescence response of Arg-GQDs containing Ag + to various con-
was further confirmed by the Zeta potential measurement of Arg- centrations of thiamine. (b) Plot of the enhancement ratio (F − F0 )/F0 vs thiamine
GQDs at different condition (Fig. S5). A significant shift of the zeta concentration. The Inset shows a linear relationship in the concentration range from
0.1 to 8 ␮M.
potential from −28.1 mV of free N-GQDs to −12.6 in the presence of
Ag+ shows the interaction between Arg-GQDs and Ag+ ions. On the
Table 3
other hand, the increase of the zeta potential to −22.3 mV resulted
Analytical results of thiamine in food samples and vitamin B1 tablet (n = 3).
from the reaction of the Ag+ with thiamine, which successfully
confirms the remove of Ag+ ions from the surface of the N-GQDs. Sample Added ␮(M) Found (␮M) Recovery (%, n = 3) RSD (%, n = 3)

Green pea 0.3 0.28 93 2.9

3.4. Analytical characteristics 2 1.95 97 3.1
Sunflower 0.3 0.31 103 1.4
2 2.05 102 3.7
To demonstrate the sensing performance of this proposed assay Wheat flour 0.3 0.29 96 4.5
for thiamine detection, we evaluated the response of this sensor 2 2.11 105 2.3
by adding varying concentrations of thiamine under experimen- VB1 tablet 0 2.34 – 4.8
tal optimal conditions (Fig. 7a). Fig. 7b presents the relationship of 0.3 2.61 90 3.6
2 4.41 103.5 2.8
the enhancement ratio (F − F0 )/F0 (F0 and F are the fluorescence
intensities of Arg-GQDs at 448 nm in the absence and presence
of thiamine, respectively) with the concentration of thiamine. The (vitamin B3), folic acid (vitamin B9), 100-fold glucose, sucrose, fruc-
inset of Fig. 7b shows that the (F − F0 )/F0 has a good linear correla- tose, lactose, cellulose and1000-fold Na+ , K+ , Ca2+ and Mg2+ . As
tion with the concentration of thiamine in the range of 0.1–8 ␮M, shown in Fig. 8a, various interference showed a negligible influence
and the linear regression equation is y = 0.4505 + 0.0556C, where C on the fluorescence intensity of the quenched Arg-GQDs, except for
is the concentration of thiamine (␮M). The corresponding regres- the fluorescence recovery induced by thiamine. In addition, a com-
sion coefficient was 0.995 and the detection limit of this method petitive experiment for the effect of interferences on the recovery
for thiamine was 53 nM (S/N = 3). Furthermore, the analytical per- fluorescence intensity of Arg-GQDs by thiamine was also operated
formance of the proposed sensing system for thiamine detection is using the current assay under the same conditions. The result shows
comparable to those reported (Table 4). that no change in the fluorescence response of probe towards thi-
amine was observed in the presence of interferences (Fig. 8b). These
3.5. Selectivity results indicate that the as-synthesized Arg-GQDs can be used as
effective fluorescence probe for thiamine detection.
In order to understand the selectivity of the reported assay
and interference to thiamine detection, the effect of some com- 3.6. Application of the fluorescent Arg-GQDs in real sample
pounds abundant in the selected real samples on the fluorescence
behavior of probe was examined. Under the optimal conditions, the To demonstrate the feasibility of the sensing system for detec-
developed method was used to including pyridoxine (vitamin B6), tion in real samples, the fluorescence assay was applied for
cyanocobalymine (vitamin B12), ascorbic acid (vitamin C), niacin thiamine sensing in pharmaceuticals and foodstuff samples by the
2084 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085

Table 4
Comparison of the present approach with other reported methods for the detection of thiamine.

Method Technique in detail Linearity LOD Ref.

Fluorescence Eu-doped Y2O3 nanoparticles 0–44 ␮M 0.144 ␮M [5]

Fluorescence carbon dots 10–50 ␮M 0.280 ␮M [45]
luminescence CdSe quantum dots 15–120 ␮M 0.207 ␮M [46]
Electrochemical luminescence Rhodamine B 0.3–5 ␮M 0.265 ␮M [47]
HPLC-CLD-UV – 0.07–27 ␮M 0.030 ␮M [48]
Fluorescence N-GQDs 0.1–8 ␮M 0.053 ␮M Present work

of fluorescence intensity of Arg-GQDs owing to the coordination

reaction between Ag+ and Arg-GQDs, while subsequent addition
of thiamine restores the fluorescence intensity caused the quench-
ing of fluorescence intensity. The effects of independent variables
were successfully optimized by the assessment of central compos-
ite design. The experimental results which were obtained during
the sensing of thiamine, were found to be fitted with model pre-
diction. The adequacy of regression model was tested by lack-of-fit
(LOF), F values and P values. Furthermore, the proposed method
has been utilized to accurately detect thiamine in practical samples.
Our present work provides the advantage of simplicity, sensitivity,
low cytotoxicity and selectivity.

Acknowledgements

The financial support of this study by Iran University of Science

and Technology, University of Tehran and Iran Nanotechnol-
ogy Initiative Council are gratefully acknowledged. The author
acknowledges financial support from the Iran National Science
Foundation (INSF).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.snb.2017.09.009

References

[1] K. Taguchi, E. Fukusaki, T. Bamba, Simultaneous analysis for water- and

Fig. 8. (a) The fluorescence enhancement factors [(F0 − F)/F0 ] of the Arg-GQDs con-
taining Ag+ with a series of interfering substance and thiamine. (b)The fluorescence
intensities of N-GQDs-Ag+ with thiamine in the presence of interfering substance.
standard addition technique. The samples were diluted to make
sure the thiamine concentration in the real samples were within the
linear range. The obtained results were listed in Table 3. The recov-
ery of thiamine for spiked samples were in the range of 90%–103.5%,
whereas the relative standard deviations range were no more than
4.8%. The obtained results were listed in Table 3. The results indi-
cated that the designed sensor was a reliable method for thiamine
analysis in real samples.
4. Conclusion
In summary, we have developed Arg-GQDs-based fluorescent
sensor for detection of trace amounts of thiamine in pharmaceutical
and foodstuff samples. Arg-GQDs, synthesized via a facile one-step
hydrothermal approach, were demonstrated to be a fluorescent
probe for label-free determination of thiamine with high sensi-
tivity and selectivity. The presence of Ag+ caused the quenching
fat-soluble vitamins by a novel single chromatography technique unifying
supercritical fluid chromatography and liquid chromatography, J.
Chromatogr. A 1362 (2014) 270–277.
[2] A.B. Tayade, P. Dhar, J. Kumar, M. Sharma, O.P. Chaurasia, R.B. Srivastava,
Sequential determination of fat- and water-soluble vitamins in Rhodiola
imbricata root from trans-himalaya with rapid resolution liquid
chromatography/tandem mass spectrometry, Anal. Chim. Acta 789 (2013)
65–73.
[3] E. Akyilmaz, İ. Yaşa, E. Dinçkaya, Whole cell immobilized amperometric
biosensor based on Saccharomyces cerevisiae for selective determination of
vitamin B1 (thiamine), Anal. Biochem. 354 (2006) 78–84.
[4] R.K. Gupta, S.K. Yadav, V.A. Saraswat, M. Rangan, A. Srivastava, A. Yadav, R.
Trivedi, S.K. Yachha, R.K.S. Rathore, Thiamine deficiency related
microstructural brain changes in acute and acute-on-chronic liver failure of
non-alcoholic etiology, Clin. Nutr. 31 (2012) 422–428.
[5] A. Bayandori Moghaddam, F. Gudarzy, Y. Ganjkhanlou, A fluorescent probe for
detecting thiamine using the luminescence intensity of nanoparticles, J.
Fluoresc. 24 (2014) 1025–1030.
[6] C.R. Gioda, L.S.A. Capettini, J.S. Cruz, V.S. Lemos, Thiamine deficiency leads to
reduced nitric oxide production and vascular dysfunction in rats, Nutr. Metab.
Cardiovasc. Dis. 24 (2014) 183–188.
[7] R.W. Körner, A. Vierzig, B. Roth, C. Müller, Determination of thiamin
diphosphate in whole blood samples by high-performance liquid
chromatography—a method suitable for pediatric diagnostics, J. Chromatogr.
B 877 (2009) 1882–1886.
[8] G.E. Gibson, J.A. Hirsch, R.T. Cirio, B.D. Jordan, P. Fonzetti, J. Elder, Abnormal
thiamine-dependent processes in Alzheimer’s Disease. Lessons from diabetes,
Mol. Cell. Neurosci. 55 (2013) 17–25.
[9] R.C. Barthus, L.H. Mazo, R.J. Poppi, UV spectrophotrometry and chemometrics
methods for simultaneous determinations of riboflavin (VB2) thiamine (VB1),
pyridoxine (VB6) and nicotinamide (VPP), Quim. Nova 30 (2007) 1638–1643.
[10] S. Liu, Z. Zhang, Q. Liu, H. Luo, W. Zheng, Spectrophotometric determination of
vitamin B1 in a pharmaceutical formulation using triphenylmethane acid
dyes, J. Pharm. Biomed. Anal. 30 (2002) 685–694.
 F. Nemati et al. / Sensors and Actuators B 255 (2018) 2078–2085
[11] T. Pérez-Ruiz, C. Martıı́nez-Lozano, V. Tomás, J. Martıı́n, Flow-injection
spectrofluorimetric determination of dissolved inorganic and organic
phosphorus in waters using on-line photo-oxidation, Anal. Chim. Acta 442
(2001) 147–153.
[12] A.B. Tabrizi, A cloud point extraction-spectrofluorimetric method for
determination of thiamine in urine, Bull. Korean Chem. Soc. 27 (2006)
1604–1608.
[13] T. Pérez-Ruiz, C. Martínez-Lozano, M.D. García-Martínez, Simultaneous
determination of thiamine and its phosphate esters by a liquid
chromatographic method based on post-column photolysis and
chemiluminescence detection, J. Pharm. Biomed. Anal. 50 (2009) 315–319.
[14] R.E. Echols, J. Harris, R.H. Miller, Modified procedure for determining vitamin
B1 by gas chromatography, J. Chromatogr. A 193 (1980) 470–475.
[15] R.E. Echols, R.H. Miller, W. Winzer, D.J. Carmen, Y.R. Ireland, Gas
chromatographic determination of thiamine in meats, vegetables and cereals
with a nitrogen-phosphorus detector, J. Chromatogr. A 262 (1983) 257–263.
[16] Y. Mrestani, R.H.H. Neubert, Thiamine analysis in biological media by capillary
zone electrophoresis with a high-sensitivity cell, J. Chromatogr. A 871 (2000)
351–356.
[17] S. Poongothai, R. Ilavarasan, C.M. Karrunakaran, Simultaneous and accurate
determination of vitamins B1, B6 B12 and alpha-lipoic acid in multivitamin
capsule by reverse-phase high performance liquid chromatographic method,
Int. J. Pharm. Pharm. Sci. 2 (2010) 133–139.
[18] X. Tang, D.A. Cronin, N.P. Brunton, A simplified approach to the determination
of thiamine and riboflavin in meats using reverse phase HPLC, J. Food Compos.
Anal. 19 (2006) 831–837.
[19] D. Bohrer, P.C. do Nascimento, A.G. Ramirez, J.K.A. Mendonça, L.M. de
Carvalho, S.C.G. Pomblum, Determination of thiamine in blood serum and
urine by high-performance liquid chromatography with direct injection and
post-column derivatization, Microchem. J. 78 (2004) 71–76.
[20] Z. Zhou, C. Tan, Y. Zheng, Q. Wang, Electrochemical signal response for
vitamin B1 using terbium luminescent nanoscale building blocks as optical
sensors, Sens. Actuators B 188 (2013) 1176–1182.
[21] Y. Ding, S. Wang, J. Li, L. Chen, Nanomaterial-based optical sensors for
mercury ions, TrAC Trends Anal. Chem. 82 (2016) 175–190.
[22] X. Fu, T. Lou, Z. Chen, M. Lin, W. Feng, L. Chen, Turn-on fluorescence detection
of lead ions based on accelerated leaching of gold nanoparticles on the surface
of graphene, ACS Appl. Mater. Interfaces 4 (2012) 1080–1086.
[23] D. Lin, T. Qin, Y. Wang, X. Sun, L. Chen, Graphene oxide wrapped SERS tags:
multifunctional platforms toward optical labeling, photothermal ablation of
bacteria, and the monitoring of killing effect, ACS Appl. Mater. Interfaces 6
(2014) 1320–1329.
[24] F.-e. Lin, C. Gui, W. Wen, T. Bao, X. Zhang, S. Wang, Dopamine assay based on
an aggregation-induced reversed inner filter effect of gold nanoparticles on
the fluorescence of graphene quantum dots, Talanta 158 (2016) 292–298.
[25] X.E. Zhao, C.H. Lei, Y.H. Wang, F. Qu, S.Y. Zhu, H. Wang, J.M. You, A
fluorometric assay for tyrosinase activity and its inhibitor screening based on
graphene quantum dots, RSC Adv. 6 (2016) 72670–72675.
[26] N. Yu, H. Peng, H. Xiong, X. Wu, X. Wang, Y. Li, L. Chen, Graphene quantum
dots combined with copper(II) ions as a fluorescent probe for turn-on
detection of sulfide ions, Microchim. Acta 182 (2015) 2139–2146.
[27] M. Arvand, S. Hemmati, Magnetic nanoparticles embedded with graphene
quantum dots and multiwalled carbon nanotubes as a sensing platform for
electrochemical detection of progesterone, Sens. Actuators B 238 (2017)
346–356.
[28] T. Yang, F. Cai, X. Zhang, Y. Huang, Nitrogen and sulfur codoped graphene
quantum dots as a new fluorescent probe for Au3+ ions in aqueous media,
RSC Adv. 5 (2015) 107340–107347.
[29] S. Kundu, R.M. Yadav, T.N. Narayanan, M.V. Shelke, R. Vajtai, P.M. Ajayan, V.K.
Pillai, Synthesis of N, F and S co-doped graphene quantum dots, Nanoscale 7
(2015) 11515–11519.
[30] X. Du, D. Jiang, Q. Liu, G. Zhu, H. Mao, K. Wang, Fabrication of graphene oxide
decorated with nitrogen-doped graphene quantum dots and its enhanced
electrochemiluminescence for ultrasensitive detection of pentachlorophenol,
Analyst 140 (2015) 1253–1259.
[31] S. Benítez-Martínez, M. Valcárcel, Graphene quantum dots in analytical
science, TrAC Trends Anal. Chem. 72 (2015) 93–113.
[32] H. Ebrahimzadeh, N. Tavassoli, O. Sadeghi, M.M. Amini, Optimization of
solid-phase extraction using artificial neural networks and response surface
methodology in combination with experimental design for determination of
gold by atomic absorption spectrometry in industrial wastewater samples,
Talanta 97 (2012) 211–217.
[33] M.A. Bezerra, R.E. Santelli, E.P. Oliveira, L.S. Villar, L.A. Escaleira, Response
surface methodology (RSM) as a tool for optimization in analytical chemistry,
Talanta 76 (2008) 965–977.
[34] R. Zare-Dorabei, S.M. Ferdowsi, A. Barzin, A. Tadjarodi, Highly efficient
simultaneous ultrasonic-assisted adsorption of Pb (II), Cd (II), Ni (II) and Cu
(II) ions from aqueous solutions by graphene oxide modified with 2,
2 -dipyridylamine: central composite design optimization, Ultrason.
Sonochem. 32 (2016) 265–276.
2085
[35] M.S. Tehrani, R. Zare-Dorabei, Competitive removal of hazardous dyes from
aqueous solution by MIL-68 (Al): Derivative spectrophotometric method and
response surface methodology approach, Spectrochim. Acta Part A Mol.
Biomol. Spectrosc. 160 (2016) 8–18.
[36] A. Keramat, R. Zare-Dorabei, Ultrasound-assisted dispersive magnetic solid
phase extraction for preconcentration and determination of trace amount of
Hg (II) ions from food samples and aqueous solution by magnetic graphene
oxide: central composite design optimization, Ultrason. Sonochem. 38 (2017)
421–429.
[37] T.-T. Xu, J.-X. Yang, J.-M. Song, J.-S. Chen, H.-L. Niu, C.-J. Mao, S.-Y. Zhang, Y.-H.
Shen, Synthesis of high fluorescence graphene quantum dots and their
selective detection for Fe3+ in aqueous solution, Sens. Actuators B 243 (2017)
863–872.
[38] C. Zhang, Y. Cui, L. Song, X. Liu, Z. Hu, Microwave assisted one-pot synthesis of
graphene quantum dots as highly sensitive fluorescent probes for detection of
iron ions and pH value, Talanta 150 (2016) 54–60.
[39] B. Shi, L. Zhang, C. Lan, J. Zhao, Y. Su, S. Zhao, One-pot green synthesis of
oxygen-rich nitrogen-doped graphene quantum dots and their potential
application in pH-sensitive photoluminescence and detection of mercury(II)
ions, Talanta 142 (2015) 131–139.
[40] J. Ju, R. Zhang, S. He, W. Chen, Nitrogen-doped graphene quantum dots-based
fluorescent probe for the sensitive turn-on detection of glutathione and its
cellular imaging, RSC Adv. 4 (2014) 52583–52589.
[41] J. Ju, W. Chen, Synthesis of highly fluorescent nitrogen-doped graphene
quantum dots for sensitive, label-free detection of Fe (III) in aqueous media,
Biosens. Bioelectron. 58 (2014) 219–225.
[42] H. Wang, Y. Liu, S. Wei, Z. Yan, Application of response surface methodology
to optimise supercritical carbon dioxide extraction of essential oil from
Cyperus rotundus Linn, Food Chem. 132 (2012) 582–587.
[43] K. Dashtian, R. Zare-Dorabei, Synthesis and characterization of functionalized
mesoprous SBA-15 decorated with Fe3O4 nanoparticles for removal of Ce(III)
ions from aqueous solution: ICP?OES detection and central composite design
optimization, J. Colloid Interface Sci. 494 (2017) 114–123.
[44] Kh. Dashtian, R. Zare-Dorabei, An easily organic-inorganic hybrid optical
sensor based on dithizone impregnation on mesoporous SBA-15 for
simultaneous detection and removal of Pb(II) ions from water samples:
response-surface methodology, Appl. Organomet. Chem. (2017), http://dx.
doi.org/10.1002/aoc.3842.
[45] G. Daneshvar Tarigh, F. Shemirani, Simultaneous in situ derivatization and
ultrasound-assisted dispersive magnetic solid phase extraction for thiamine
determination by spectrofluorimetry, Talanta 123 (2014) 71–77.
[46] M. Amjadi, R. Shokri, T. Hallaj, A new turn-off fluorescence probe based on
graphene quantum dots for detection of Au(III) ion, Spectrochim. Acta Part A
153 (2016) 619–624.
[47] C. Li, Y. Yue, Fluorescence spectroscopy of graphene quantum dots:
temperature effect at different excitation wavelengths, Nanotechnology 25
(2014) 435703.
[48] K. Hemelsoet, D. Lesthaeghe, V. Van Speybroeck, M. Waroquier, Bifunctional
acid-base catalyzed reactions in zeolites from the HSAB viewpoint, Chem.
Phys. Lett. 419 (2006) 10–15.
[49] R. Tabaraki, A. Nateghi, Nitrogen- doped graphene quantum dots: turn-off
fluorescent probe for detection of Ag+ ions, J. Fluoresc. 26 (2016) 297–305.
[50] H. Li, D. Chen, H. Wang, J. Li, W. Wang, Sub-picomole level
photoelectrochemical sensing of l-cysteine based on plasmonic silver
nanoparticles modified hierarchically structured zinc oxide, J. Electroanal.
Chem. 759 (Part 1) (2015) 21–26.
Biographies
Fatemeh Nemati is a Ph.D. student in Analytical Chemistry at Iran University of
Science and Technology, Tehran, Iran.
Rouholah Zare-Dorabei has a PhD in Analytical Chemistry obtained at University
of Tehran in 2010. He is currently Assistant Professor in Analytical Chemistry Iran
University of Science and Technology, Tehran, Iran. His research work has been
mainly focused on the design of new optical sensors.
Morteza Hosseini has a Ph.D. in analytical chemistry obtained at Tarbiat Modares
University in 2005. He is currently an associated professor in nanobiotechnology
group of Faculty of New Sciences and Technologies of University of Tehran. His
research work has been mainly focused on the design of new optical nanosensors
and nanobiosensors.
Mohammad Reza Ganjali has a Ph.D. in analytical chemistry obtained at University
of Tehran in 1997. He is currently a professor of analytical chemistry at University of
Tehran. His research work has been mainly focused on the design of new chemical
sensors.