होम Food Analytical Methods A Simple and Sensitive Method for the Voltammetric Analysis of Theobromine in Food Samples Using...

A Simple and Sensitive Method for the Voltammetric Analysis of Theobromine in Food Samples Using Nanobiocomposite Sensor

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english
पत्रिका:
Food Analytical Methods
DOI:
10.1007/s12161-017-0867-5
Date:
April, 2017
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आप पुस्तक समीक्षा लिख सकते हैं और अपना अनुभव साझा कर सकते हैं. पढ़ूी हुई पुस्तकों के बारे में आपकी राय जानने में अन्य पाठकों को दिलचस्पी होगी. भले ही आपको किताब पसंद हो या न हो, अगर आप इसके बारे में ईमानदारी से और विस्तार से बताएँगे, तो लोग अपने लिए नई रुचिकर पुस्तकें खोज पाएँगे.
Food Anal. Methods
DOI 10.1007/s12161-017-0867-5

A Simple and Sensitive Method for the Voltammetric Analysis
of Theobromine in Food Samples Using Nanobiocomposite Sensor
Yingqiong Peng 1 & Wenjuan Zhang 1,2 & Juan Chang 2 & Yaoping Huang 1,2 & Li Chen 2,3 &
Hong Deng 1 & Zhong Huang 2 & Yangping Wen 2,3

Received: 16 December 2016 / Accepted: 9 March 2017
# Springer Science+Business Media New York 2017

Abstract Theobromine (TB) is one of important natural methylxanthine alkaloids in plants and their products, but there were
few reports on the electrochemistry of TB, especially electrochemical measurements using chemically modified electrode
owing to its poor detectability. In this work, a simple and sensitive method for the voltammetric analysis of TB in green tea,
chocolate, and coffee samples was successfully realized using
nanobiohybrid sensor based on glassy carbon electrode (GCE)
modified by both carboxyl-functionalized multiwalled carbon
nanotubes (fMWCNTs) and soluble biopolymer sodium salt of
carboxymethylcellulose (CMC). Water-dispersible
nanonbiohybrids with fMWCNTs were successfully prepared
using CMC assist, and CMC-fMWCNTs were characterized
by scanning electron microscopy, Fourier transform infrared
spectroscopy, transmission electron microscope, electrochemical impedance spectroscopy, and cyclic voltammetry. CMCfMWCNTs/GCE showed enlarged electrochemically active
surface area, good electrode stability, and enhanced electrocatalytic activity. The voltammetric behavior of TB demonstrated
Yingqiong Peng and Wenjuan Zhang contributed equally to this work.
* Hong Deng
jxaudh@aliyun.com
* Yangping Wen
wenyangping1980@gmail.com
1

Colleges and Uuniversities of Jiangxi Province for Key Laboratory of
Information Technology in Agriculture, Jiangxi Agriculture
University, Nanchang 330045, People’s Republic of China

2

Institute of Functional Materials and Agricultural Applied Chemistry,
Jiangxi Agricultural University, Nanchang 330045, People’s
Republic of China

3

Key La; boratory of Crop Physiology, Ecology and Genetic Breeding,
Ministry of Education, Jiangxi Agricultural University,
Nanchang 330045, People’s Republic of China

an irreversible electrochemical oxidation reaction involving
two electrons and two protons, which could detect TB in a
wider linear range from 0.5 to 80 μM with a lower limit of
detection (LOD) of 0.21 μM. The developed method displayed
a high sensitivity, low LOD, good sensing stability, remarkable
feasibility, and satisfactory practicality.
Keywords Voltammetric sensor . Nanobiocomposite .
Theobromine . Carbon nanotube . Carboxymethyl cellulose

Introduction
Theobromine (TB), a class of alkaloid molecules known as
xanthine derivatives, is an important methylxanthine found in
products of the cocoa tree, Theobroma cacao. TB also exists
in the tea and chocolate-related products. In general, chocolate
contains 5–7.5 mg g−1 TB (Baggott et al. 2013), and tea contains TB in concentration range of 1.2–4.4 mg cup−1 (Blauch
and Traka 1983). TB is a white crystalline powder with bitter
tasting, slightly soluble in water or ethanol, soluble in acids
and alkalis, and its chemical properties are similar to caffeine
(Vinjamuri 2008). The salts of TB are very easy to be
decomposed into free acid and base in aqueous solution, while
the free base is more stable (Scheller and Schubert 1991; Lind
et al. 1999). Moreover, TB is a weak phosphodiesterase inhibitor and adenosine receptor blocker (Sugimoto et al. 2016); it
has diuretic, cardiac excitation, vasodilatation, and smooth
muscle relaxation effects (Sharma et al. 2005). Therefore, it
is an important and effective component for cardiovascular
and renal diseases in clinical treatment.
To date, different methods have been exploited for the measurements of TB, such as ultraviolet spectrophotometry
(Schack and Waxler 1949), near-infrared spectroscopy
(Álvarez et al. 2012), high-performance liquid

Food Anal. Methods

chromatography (Thomas et al. 2004), and kinetic spectrophotometry (Xia et al. 2013). However, the sensitivity and
accuracy of these methods are not high. Although liquid chromatography with tandem mass spectrometry and amperometric measurements are generally accurate for measuring real
samples (Serra et al. 2011; Meyer et al. 1996), there are some
disadvantages such as the complicated sample pretreatment,
usage of expensive instruments, time-consuming and laborious process, and well-controlled experimental conditions. The
electrochemistry is a powerful analytical method due to its
merit of fast analysis, low cost, simple operation, and high
sensitivity. However, there are few reports for electrochemical
measurements of TB. Hansen and Dryhurst reported the electrochemistry of TB at the stationary pyrolytic graphite electrode (Hansen and Dryhurst 1971). Subsequently, Spataru
et al. studied the electrochemical oxidation of TB including
other xanthine derivatives using the boron-doped diamond
electrode and its analytical application in three commercially
available real products (Spataru et al. 2002). Vinjamuri prepared molecularly imprinted polypyrrole modified electrode
for the electrochemical detection of TB, but its sensing performance is very bad (the measurement is only in concentration
range from 1 to 20 mM) (Vinjamuri et al. 2008). However, the
electrochemical measurement of TB was the least using
chemo/biosensors based on chemically modified electrode in
comparison with other xanthine derivatives like hypoxanthine, caffeine, and theophylline owing to its bad detectability.
Carbon nanomaterials for the development of electrochemical bio/chemosensors is nowadays one of the most attractive
research fields because of their excellent detectability, superior
electrocatalytic ability, high adsorption capacity, tremendously small size, and specific surface (Llobet 2013; Serp and
Figueiredo 2009; Lukaszewicz 2006). Carbon nanotubes
(CNTs), an extraordinary carbon material with tubular nanostructure, displayed excellent properties including high conductivity, large active surface area, readily modifiable surface,
Scheme 1 The fabrication of
sensor based on CMCfMWCNTs/GCE and its
electrooxidation mechanism and
voltammetric measurement of TB
in food samples

high electrocatalytic activity, excellent chemical stability, and
good biocompatibility; this can forecast their prospective utilization in analytical chemistry for fabricating CNT-based
electrochemical sensors (Wang 2005; Jacobs et al. 2010;
Gooding 2005). Hydrophilic groups like carboxyl, hydroxy,
and amino-functionalized carbon nanotubes (fCNTs) have obtained much concern in recent years owing to their ability to
reinforce properties, enhance interfacial interactions, and improve the solubility (Gao et al. 2012). Biopolymers can enhance adhesion between the substrate electrode and the coating material, improve water processibility, enhance water stability, and more (Sahoo et al. 2010). Carboxymethyl cellulose (CMC), an excellent environment-friendly biopolymer
with water solubility, satisfactory biocompatibility, good
hydrophilicity, superior film-forming ability, synergistically electrocatalytic ability, and good adhesive property,
could well improve or solve problems mentioned above,
which were widely utilized in our previous work (Lu et al.
2015; Zhang et al. 2015a, b).
In this work, carboxyl-functionalized multiwalled carbon
nanotubes (fMWCNTs) nanobiohybrids with CMC-modified
glassy carbon electrode (GCE) have been successfully fabricated for the trace measurement of TB using linear sweep
voltammetry (LSV). The CMC-fMWCNTs/GCE for the
electrooxidation mechanism and voltammetric measurement
of TB in food samples was investigated in detail (Scheme 1).

Experimental
Chemicals
TB was bought from Aladd in Reagent Co., Ltd. CMC was
obtained from TCI Development Co., Ltd. fMWCNTs was
obtained from Chengdu Institute of Organic Chemistry,
Chinese Academy of Sciences. Phosphate buffer (0.1 M) with

Food Anal. Methods

various pH were prepared from aqueous solutions of 0.1 M
Na2HPO4, NaH2PO4, NaOH, and H3PO4. Other reagents
were of analytical reagent grade and employed without further
purification, and double-distilled water was used in all
experiments.

placed into phosphate buffer (pH 7.0) with the stirring time
of 60 s. The CV and DPV were used for the measurement of
TB, and experiments were performed to determine TB at a
scan rate of 50 mV s−1 in phosphate buffer solution (PH
7.0). All experiments were performed at room temperature.

Apparatus

Preparation of Real Samples

Cyclic voltammetry (CV) and LSV were implemented using
CHI660E electrochemical workstation (Chenhua Instrument
Co., Shanghai, China) in electrochemical cell with a threeelectrode system. Three-electrode system includes a saturated
calomel electrode (SCE) as the reference electrode, a GCE or
the modified GCE as the working electrode, and a platinum
(Pt) wire as the auxiliary electrode. The pH was measured by
CT-6023 portable pH meter (Shanghai Lin Yu Trading Co.,
Ltd.).

The green tea, chocolate, and coffee were purchased from the
local supermarket. The green tea sample was obtained by cutting them into small pieces, using a mortar and pestle to crush.
Sample sizes were 1 g tea, 6.5 g chocolate, and 1 g coffee, put
into 100 mL boiling water for 30 min, respectively. After
filtration, the green tea sample was diluted 1000-fold, the
chocolate sample was diluted 200-fold, and the coffee sample
was diluted 500-fold; all sample solutions were adjusted to
pH 7.5 with phosphate buffer.

Preparation of CMC-fMWCNTs/GCE

Results and Discussions
The GCE was carefully polished with chamois leather containing 0.05 μm alumina slurry until a mirror-shine surface
appeared and was ultrasonically cleaned with deionized distilled water, absolute ethanol, and deionized distilled water for
5 min, respectively, and afterwards dried in air. CMC was
ultrasonicated until completely dissolved in distilled water
(0.3 mg mL−1). fMWCNTs were dispersed by sonication in
CMC aqueous solution and ultrasonication until completely
dispersed. The CMC-fMWCNTs/GCE was fabricated by
drop-coating 5 μL water-dispersible CMC-fMWCNTs on
the GCE surface, and dried in infrared lamp.
Measurements of TB
Five microliters of phosphate buffer solution containing TB
was added into the sealed cell by Finnpipette, and then, the
three-electrode system was placed into the cell. Prior to electrochemical tests, the chemically modified electrode was

Fig. 1 The dispersibility in water
of both fMWCNTs (I) and CMCfMWCNTs (II), and the fresh
prepared sample (a) and the
sample after 60 days of stationary
cultivation (b)

Preparation of CMC-fMWCNTs
Solution-dispersible materials are often more favorable for the
advanced material processing; thus, the solution processability
of advanced materials is very necessary. Moreover, waterdispersible material can also reduce the cost. Figure 1 shows
the water dispersibility of fMWCNTs (I) and CMCfMWCNTs (II) with ultrasonic treatment after 20 min.
Obviously, fMWCNTs could not fully dispersed in aqueous
solution (Fig. 1a (I)), and they were deposited progressively in
the bottom of bottles owing to their bad water processability
(Fig. 1a (I)). After 1 h of static cultivate, there were massive
sediments into the bottom of bottles, and only a very small
amount of fMWCNTs was dispersed in water (Fig. 1b (I)),
which were attributable to their carboxyl group. While
CMC-fMWCNTs were thoroughly dispersed in aqueous solution after ultrasonic treatment for 20 min (Fig. 1a (II)), even

Food Anal. Methods

CMC-fMWCNTs still were dispersed stably after 60 days of
stationary cultivation (Fig. 1b (II)), indicating that CMC as an
extraordinary dispersant and stabilizing agent accelerated the
interaction with fMWCNTs.
Characterization of CMC-fMWCNTs
SEM
The surface morphological structures of advanced materials directly affect the performance of sensing devices. SEM was utilized to study surface morphological structures of original
fMWCNTs and CMC-fMWCNTs. Figure 2a presents the pristine CMC; it was a regular and homogeneous structure with
smoothness and compactness, which was in accordance with
previous reports (Lu et al. 2015; Zhang et al. 2015a, b), while
fMWCNTs were bended or entangled with each other and had
an inattentive filamentous structure (Fig. 2b). When fMWCNTs
were added into CMC aqueous solution, fMWCNTs were
embedded/incorporated into CMC (Fig. 2c); this resulted in the
formation of water-dispersible CMC-fMWCNTs.
TEM
TEM images of the water-dispersible CMC-fMWCNTs
with different magnifications are presented in Fig. 3. As
reported in previous reports, the pure MWCNTs tended to
form aggregates in bundles (Saleh et al. 2008). It could be
seen that CMC promoted the water dispersibility of
fMWCNTs, and the formation of fMWCNT network
embedded/incorporated by CMC, indicating that
fMWCNTs containing CMC possessed the ability to mutually enhance their own water dispersibility.
FTIR
FTIR spectra of raw fMWCNTs (a), CMC (b), and CMCfMWCNTs (c) are presented in Fig. 4. It could be seen from

Fig. 2 SEM images of CMC (a), fMWCNTs (b), and CMC-fMWCNTs (c)

the spectrum of CMC that there were two peaks around 1108
and 1030 cm−1 for the stretching vibrations of C–O–C, while
the bands around 1600 and 1427 cm−1 were attributed to
stretching vibrations of the carbonyl group. The existence of
the peak about 2900 cm−1 was ascribed to the stretching vibration of C–H, and a broad peak presented at 3100–
3600 cm−1 was assigned to the stretching frequency of the –
OH group. As for fMWCNTs, there was a stretch vibration of
C–O at 1086 cm−1 and the C–O stretching vibration around
1637 cm−1 was ascribed to the carbonyl group; the broad peak
at 3434 cm−1 belonged to O–H stretching vibrations. For the
spectrum of CMC-fMWCNT composite, the characteristic absorption peak of both CMC and fMWCNTs became weaker
and appeared a new peak, indicating that there was a strong
interaction between CMC and fMWCNTs, this interaction
was likely to promote the aqueous dispersion of fMWCNTs
in the presence of CMC.
EIS
EIS is a powerful tool to reveal the interface characteristic of
different modified electrodes; it was performed at the open
circuit potential of 0.17 V and the frequency extent from
0.1 Hz to 100 kHz in a redox probe of 5 mM [Fe(CN)6]3−/4−
containing 0.1 M KCl. It was well known that the semicircle
diameter of Nyquist plots was used to estimate the electron
transfer resistance (Rct) of different modified electrode. As can
be seen from Fig. 5, the semicircle diameter of Nyquist plots
increased when CMC (curve b) was modified onto the GCE
surface in comparison with the impedance of the GCE (curve
a); the main reason was that the electron transport was hindered by CMC owing to its nonconductivity, and made it
difficult to transfer the interfacial charge. Nevertheless, the
semicircle diameter of Nyquist plots decreased quickly when
CMC-fMWCNTs (curve c) was drop-coated onto the GCE
surface in comparison with the impedance of CMC/GCE, indicating that high conducting fMWCNTs accelerated the conduction path between the fMWCNTs/GCE and the supporting

Food Anal. Methods

Fig. 3 TEM images of aqueous-dispersible CMC-fMWCNTs

CVs
Cyclic voltammetric tests were implemented in 5 mM
[Fe(CN)6]3−/4− with 0.1 M KCl (Fig. 5 (inset)). A pair of
well-defined anodic and cathodic peaks was observed on different modified electrodes; the influence of the peak-to-peak
separation between cathodic peak potential and the anodic
peak potential of all modified electrodes was very close to
the bare GCE. Moreover, it could be seen that peak currents

of fMWCNTs/GCE and CMC/GCE were lower than the GCE,
but peak currents of fMWCNTs-CMC/GCE were higher than
the GCE, which was due to the fast electron transfer of high
conducting f-SWCNT and the poor effect of CMC, which was
in accordance with results of EIS.
Electrode Stability
The stability and adhesion of film electrode was very important
in the application in electrochemistry of chemically modified
electrode. Therefore, the electrode stability of CMCfMWCNTs/GCE was studied in 5 mM [Fe(CN)6]3−/4− with
0.1 M KCl by performing 50 cycles of CVs tests at a scan rate
of 50 mV s−1. The relative standard deviation (RSD) of redox
peak currents were 0.87 and 1.18%, respectively; this result revealed that the CMC-fMWCNTs/GCE has a better cycle stability
and a higher repetitive charge/discharge reversibility.
Electrochemically Active Surface Area
The electrochemically active surface area of CMCfMWCNTs/GCE was estimated by CVs using 5 mM
100
80

transmitance %

b

c

Zm / ohm

a

60
40

3000

2250

1500 750
-1
Wavenumber / cm

Fig. 4 FTIR spectra of fMWCNTs (a), CMC (b), and CMC-fMWCNTs (c)

a
b
c
d

250
0

d
a
b

-250
-500
-0.2 0.0 0.2 0.4 0.6
E / V (vs. SCE)

c

20
0
0

3750

500
I/ A

electrolyte, and enhanced the [Fe(CN)6]3−/4− diffusion towards the electrode surface due to high conductivity. More
interestingly, the introduction of CMC prominently decreased
the semicircle diameter of Nyquist plots of fMWCNTs/GCE;
this phenomenon was in accordance with previous reports
(Zhang et al. 2016; Chang et al. 2016). The main reason is
that CMC promoted good dispersion of fMWCNTs network
(Fig. 1), which increased the specific surface area or decreased
the aggregation of fMWCNTs network due to homogeneous
dispersion of fMWCNTs network (Figs. 1 and 2). They are
likely to improve the electron transfer and electroconductivity
(Zhang et al. 2016; Chang et al. 2016). In addition, CMC as a
surfactant also accelerated probably the electron transfer and
electroconductivity (Zhang et al. 2015a).

20

40
60
Zre / ohm

80

100

Fig. 5 Nyquist plots of different electrodes in 5 mM [Fe(CN)6]3−/4−
containing 0.1 M KCl: bare GCE (a), fSWCNT/GCE (b), CMC/GCE
(c), and CMC-fSWCNT/GCE (d). Inset: CVs of different electrodes in
5 mM [Fe(CN)6]3−/4− containing 0.1 M KCl. Scan rate, 100 mV s−1

Food Anal. Methods

280

K3[Fe(CN)6] containing 0.1 M KCl as a probe at different
scan rates (Fig. 6). For a reversible process,

Ip ¼ 2:69  105 n2=3 AD1=2 v1=2 C0

Bare/GCE
f MWCNTs/GCE
CMC-f MWCNTs/GCE

210

where A, C0, n, D, v, and Ip stand for the electrochemically
active surface area of electrode (cm−2), the concentration of
redox species (mol cm−3), the electron number of redox species, the diffusion coefficient of 5 mM K3[Fe(CN)6] with
0.1 M KCl (7.6 × 10−6 cm s−2), scan rates (V s−1), and the
redox peak current (A). The A of the CMC-fMWCNTs/GCE
was estimated to be approximately 0.22 cm−2, which significantly enlarged the A of GCE and provided a favorable platform for electrochemically sensing of analytes.
Electrochemical Behaviors of Theobromine
Figure 7 shows CVs of 80 μM TB at bare GCE, CMCfMWCNTs/GCE in phosphate buffer. There is no reduction
peak in the reverse scan (Fig. 8), indicating that the electrochemical response was an irreversible process. No obvious
oxidation peak for TB was observed at bare GCE. However,
when fMWCNTs-CMC was modified on the electrode surface, a remarkable oxidation peak of TB appeared at approximately 1.34 V. The result clearly demonstrated that CMCfMWCNTs had electrocatalytic ability towards the oxidation
of TB. Also, this result indicated that CMC-fMWCNT film
could provide more effective active sites than bare GCE.
Optimization of Parameters
Influence of Scan Rates
The influence of potential scan rates on voltammetric responses
of TB at CMC-fMWCNTs/GCE was given by CVs (Fig. 8a).
The anodic peak currents (Ipa) increased linearly with the increasing scan rates in ranges from 25 to 300 mV s−1 (Fig. 8b),

70
0
0.96

1.08 1.20 1.32
E / V (vs. SCE)

1.44

Fig. 7 CVs of 80 μM TB in phosphate buffer solution (pH 7.0) at bare
GCE fMWCNTs/GCE

suggesting that an irreversible electrode reaction for TB at
CMC-fMWCNTs/GCE was a typical adsorption-controlled process (Gosser 1993). From Fig. 8c, it could be also seen an upwards bending characteristic curve of Ipa vs. v1/2, indicating that
there was no linear relationship between Ipa and v1/2. However,
Ipa had a linear relationship with v (Fig. 8b), manifesting that the
electrochemical oxidation of TB at CMC-fMWCNTs/GCE was
an adsorption-controlled electrode process. There was a good
linear relationship between the anodic peak potential (Epa) and
lnv (Fig. 8d); this was obtained from the Laviron equation
(Laviron 1974):

E P ¼ E0 þ ðRT =αnF Þln RTk 0 =αnF −ðRT =αnF Þlnv
where α is the coefficient of charge transfer, E0 is the formal standard potential, n is the electron number, T, R, and F
have their convention meanings (T = 298 K,
R = 8.314 J mol−1 K−1, and F = 96,485 C mol−1). Thus,
the αn value was calculated from the slope (0.031) of Ep vs.
lnv. In most systems, α could usually be approximated 0.5.
The αn value was calculated as 1.24, and n was calculated
approximately to be 2.

a

b
k

40

12

a

y = 0.83( 0.01)x - 4.35( 0.16)
2
R = 0.998

6
I / µA

20

I / µA

Fig. 6 CV curves of CMCfMWCNTs/GCE in 5 mM
[Fe(CN)6]3− containing 0.1 M
KCl at scan rates from a to k with
0.025, 0.05, 0.075, 0.1, 0.15, 0.2,
0.25, 0.3, 0.35, 0.4, and
0.45 V s−1, respectively (a). Plots
of anodic and cathodic peak
currents vs. the square root of
scan rates (b)

I/ A

140

0

0

-20

-6

-40

-12
-0.2

0.0
0.2
0.4
E / V (vs. SCE)

0.6

y = -0.81( 0.006) + 3.95( 0.09)
2
R = 0.999

4

8
12
16
v1/2 / (mV s-1)1/2

20

Food Anal. Methods

a

b
320

200

h

240

150

a

160

I / µA

I / µA

Fig. 8 CVs of 80 μM TB at
CMC-fMWCNTs/GCE in
phosphate buffer (pH 7.5) at scan
rates from a to h with 0.025, 0.05,
0.075, 0.10, 0.15, 0.20, 0.25, and
0.3 V s−1, respectively (a). Plots
of anodic peak currents vs. scan
rates (b). Plots of anodic peak
currents vs. the square root of
scan rates (c). Plots of anodic
peak potentials vs. lnv for CMCfMWCNTs/GCE (d)

100

80

2
R = 0.994

50
0

y = 0.65(·0.018) + 10.61(·3.15)
0.91

1.04 1.17 1.30
E / V (vs. SCE)

0
0

1.43

d

c
220

140
210
-1
v / mV s

280

1.24 y = 0.031(·6.92)x + 1.27(·0.002)
2
R = 0.996
1.22

E / V (v s . S C E )

165

I / µA

70

1.20

110

1.18

55

1.16

0

0.12

0.24

0.36
0.48
v1/2 / (V s-1)1/2

Influence of pH
Figure 9a shows the influence of pH on electrochemical
behaviors of TB. The voltammetric response of 80 μM
TB was examined over the pH range from 5.5 to 8.5 by
CVs at a scan rate of 50 mV s −1. The relationship
between the anodic peak current (Ipa) and pH value is
shown in Fig. 9b. Ipa of TB at CMC-fMWCNTs/GCE
increased gradually with the increase of pH value until

a

0.60

220

240

1.44

200

110

160

0

120

0.98 1.12 1.26 1.40 1.54
E / V (vs. SCE)

1.50
E / V (vs. S C E )

I / µA

330

-2.60 -1.95
ln(v / V s-1)

-1.30

c
280

I / µA

440

-3.25

it acquired a maximal value of pH 7.5, then reduced
quickly with the increase of pH again. Considering the
sensitivity of the as-fabricated electrode for the detection of TB, the optimal pH of 7.5 was used for the
voltammetric detection of TB. Besides, the relationship
between anodic oxidation peak potentials (Epa) and pH
value was also presented in Fig. 9c, Epa shifted negatively with the increasing pH, suggesting that the proton
was participated in the electrochemical process of TB.

b
PH = 5.5
PH = 6
PH=6.5
PH=7
PH=7.5
PH=8
PH=8.5

-3.90

y = 1.74(·0.03)-0.05(·0.005)x
R2 = 0.95

1.38
1.32

5.6 6.3 7.0 7.7 8.4
pH

1.26

5.6 6.3 7.0 7.7 8.4
pH

Fig. 9 CVs of 80 μM TB at CMC-fMWCNTs/GCE with different pH values (a). Effect of pH on the anodic peak currents (b). The anodic peak
potentials for the adsorption of TB in phosphate buffer (pH 7.0) (c). Scan rate, 50 mV s−1

Food Anal. Methods

a

b
360

250

k

270
I / µA

200

180

I / µA

Fig. 10 LSVs of TB with
different concentrations at CMCfMWCNTs/GCE. The
concentrations of TB from a to k
with 0.5, 0.7, 0.9, 2, 4, 6, 8, 20,
40, 60, and 80 μM, respectively
(a). Linear relationship between
peak height and the square root of
concentration of TB (b)

150

a
90

100

0

50

0.96

1.08 1.20 1.32
E / V (vs. SCE)

The relationship between Epa and pH was given as follows:
dE p∂ =dpH ¼ −2:303mRT =n F
where m is the proton number taking part in the electrochemical process, which was obtained from slopes of Epa vs. pH. As
can be seen from Fig. 9c, the slope of dEpa/dpH plots was
0.054. So, the m/n ratio of TB was about 1, implying that
the number of electron and proton was the same. Thus, the
electrode reaction of TB at CMC-fMWCNTs/GCE involved a
two-proton and two-electron process (Spataru et al. 2002)
(Scheme 1).
Sensing Performance
Determination of TB
TB with different concentrations were obtained to study the
relationship between responsive peak currents and TB concentrations; we investigated it by LSVs at CMC-fMWCNTs/
GCE in phosphate buffer with pH 7.5. Responsive peak currents had good linear relationship in TB concentration ranges
from 0.5 to 80 μM (Fig. 10). In addition, the limit of detection
(LOD) and limit of quantitation (LOQ) were defined as 3 and
10 s/m, respectively, where m is the slope of the calibration
curve and s is the standard deviation for the replicate measurement of the sample in the absence of analytes under the same
conditions. In this work, the replication determination for ten
Table 1
reports

1.44

R2 = 0.9993
y = 22.938 (·0.19 )x + 38.174 (·0.85 )
0

2

4
6
1/2
C
/ µM

8

10

times was recorded in blank solution using CMC-fMWCNTs/
GCE. So, the calculated LOD and LOQ was 0.21 and 0.7 μM,
respectively. The sensitivity was defined as m/A, where A stand
for the electrochemically active surface area of electrode (cm−2)
and the sensitivity was 104.26 μA μM−1 cm−2. As listed in
Table 1, the as-developed CMC-fMWCNTs/GCE displayed the
better electrochemical sensing performance for the detection of
TB due to wider linear range. These also revealed that the developed CMC-fMWCNTs/GCE was a good platform for electrochemically sensing TB. In addition, the stability of successive
assays of the sensor based on CMC-fMWCNTs/GCE was evaluated by detecting the peak current of 80 μM TB in phosphate
buffer (pH 7.5), and a RSD of 2.21% was obtained for 30 successive assays. Indicating that the CMC-fMWCNTs modified
GCE had good repeatability. Six different modified electrodes
showed the RSD of 1.94%, revealing that the CMCfMWCNTs/GCE displayed high reproducibility.
Interferences
The anti-interference of the CMC-fMWCNTs/GCE was investigated by measurement of 8 μM of TB in the presence of different
common interferents. Under optimal experimental conditions,
different interferents like amino acid, organic acids, vitamins,
saccharides, and anion-cations were added. Many of them did
not produce any obvious interference towards the current response of CMC-fMWCNTs/GCE because of bad electroactivity,
poor interaction, or relatively far away from anodic peak potential of TB. Table 2 lists the LSVof FeCl3, glutamic acid, glucose,

The performance comparison of electrochemical sensor based on different chemically modified electrode for measurement of TB in previous

Modified materials

Buffer solution

Linear range(M)

Real samples

Ref.

PGE
BDD

Acetate buffer
BR buffer

–
1 × 10−6–4 × 10−4

–
Coffee, cola

Hansen and Dryhurst (1971)
Spataru et al. (2002)

MIPs-PPy

Phosphate buffer

1 × 10−3–2 × 10−2

Tea

Vinjamuri et al. (2008)

Green tea, chocolate, and coffee

This work

CMC-fMWCNTs

Phosphate buffer

−7

−4

5 × 10 –2 × 10

Food Anal. Methods
Table 2 The effects of different interferents containing 8 μM TB on
peak currents

Conclusions

Interferents

Concentration (μM)

FeCl3
FeCl3
FeCl3
Glutamic acid

8
40
80

0.62
1.38
2.93

8

-2.11

Glutamic acid
Glutamic acid

40
80

0.07
2.11

Glucose
Glucose

8
40

0.17
2.09

CMC-fMWCNT nanobiocomposites with water dispersibility
were successfully prepared, and their structure and properties
were characterized. The CMC-fMWCNTs/GCE displayed enlarged electrochemically active surface area, good electrode
stability, an irreversible electrochemical reaction towards TB
with a well-defined electrooxidation peak at approximately
1.34 V, and enhanced electrocatalytic activity for the
electrooxidation of TB, accompanying a two-proton and
two-electron process, which could detect TB in a linear range
from 0.5 to 80 μM with a low LOD of 0.21 μM, high sensitivity, and good sensing stability; the developed method was
employed to measure the content of TB in green tea, chocolate, and coffee samples with good feasibility and practicality.
Satisfactory results indicated that CMC-fMWCNT
nanobiocomposites will be good and attractive candidates
for practical applications in the cocoa, tea, and chocolaterelated products.

Signal change (%)

Glucose

80

4.02

Ascorbic acid
Ascorbic acid

8
40

0.8
3.96

Ascorbic acid

80

3.7

and ascorbic acid with different concentrations; they displayed
weak interference (the alteration of the anodic oxidation peak
currents of TB was not more than 5%), implying that the proposed method has good selectivity.
Practical Application
To evaluate the feasibility of the developed sensor, the CMCfMWCNTs/GCE was utilized to measure the concentration of
TB in chocolate, green tea, and coffee samples by using a
standard addition method, respectively. Three different concentrations of TB were added into three samples to measure
the recoveries of TB by LSV, respectively. The obtained results are listed in Table 3; the range of the recovery was 93.5–
109.7%, and the range of RSD was 0.18–2.11%, indicating
that the described method is adequate for practical application
in detecting TB in real samples.
Table 3 Nanobiocomposite sensor based on CMC-fMWCNTs/GCE
for the analysis of TB in green tea, chocolate, and coffee samples
Sample

Added (μM) Found (μM)

–
1 8
2 20
3 40
Chocolate
–
1 8
2 20
3 40
Coffee
–
1 8
2 20
3 40
Green tea

<DL
7.84 ± 0.13
18.70 ± 0.08
39.76 ± 0.12
<DL
7.88 ± 0.14
20.90 ± 0.06
41.04 ± 0.11
<DL
7.60 ± 0.16
21.93 ± 0.04
41.20 ± 0.1

RSD (%) Recovery (%)
–
1.66
0.43
0.31
–
1.78
0.29
0.27
–
2.11
1.18
0.24

–
98.0
93.5
99.4
–
98.5
104.5
102.6
–
97.0
109.7
103

Acknowledgements The authors would like to appreciative of financial
support from the National Science Foundation of China (31660492,
51662014), Jiangxi Provincial Department of Education (GJJ150428),
China Postdoctoral Science Foundation (2015M571987), Special Funds
for Jiangxi Province Postdoctoral Research Funds (2015KY44), and
Jiangxi Provincial Innovation Fund of Postgraduates (No. YC2016S183).
Compliance with Ethical Standards
Conflict of Interest Yingqiong Peng declares that she has no conflict of
interest. Wenjuan Zhang declares that she has no conflict of interest. Juan
Chang declares that she has no conflict of interest. Yaoping Huang declares that she has no conflict of interest. Li Chen declares that she has no
conflict of interest. Hong Deng declares that he has no conflict of interest.
Zhong Huang declares that he has no conflict of interest. Yangping Wen
declares that he has no conflict of interest.
Ethical Approval This article does not contain any studies with human
or animal subjects. This is an original research article that has neither been
published previously nor considered presently for publication elsewhere.
All authors named in the manuscript are entitled to the authorship and
have approved the final version of the submitted manuscript.
Informed Consent Not applicable.

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