Talanta 131 (2015) 136141

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Application of curcumin nanoparticles in a lab-on-paper device

as a simple and green pH probe
Nahid Pourreza n, Hamed Golmohammadi
Department of Chemistry, College of Science, Shahid Chamran University, Ahvaz, Iran

article info abstract

Article history: This article describes the design and fabrication of a novel lab-on-paper device for pH sensing using
Received 19 May 2014 curcumin nanoparticles (CURNs). In order to fabricate the lab-on-paper, the wax dipping method was
Received in revised form used. The color of loaded paper with CURNs changed from yellow to orange and red to brown in the pH
20 July 2014 range of 713. The image of the lab-on-paper was taken by a digital camera and the picture was
Accepted 21 July 2014
processed and analyzed using Adobe Photoshop software. The change in mean color intensity with pH
Available online 30 July 2014
was recorded and employed as an analytical signal for quantitative sensing of pH. The parameters
Keywords: affecting the pH sensor were optimized to enhance the selectivity and sensitivity of the method. Under
Lab-on-paper optimum conditions, the mean color intensity was linearly proportional to the pH in the range of 813.
Curcumin nanoparticle
The relative standard deviations of 10 replicate measurements of pH 9 and pH 12 were 2.3% and 1.5%,
pH Sensor
respectively. The developed sensor was successfully applied to the determination of pH in different
Mean color intensity
water samples with satisfactory results.
& 2014 Elsevier B.V. All rights reserved.

1. Introduction
Paper-based devices have been developed for providing portable,
low-cost and disposable platforms for detecting various analytes and
clinical diagnosis [13]. The pioneering work on Lab-on-paper was
carried out by Whitesides's group at Harvard University in 2007 [4].
This method presents a wide range of applications including biochem-
ical analysis, health diagnostics, food quality control, environmental
monitoring and forensic analysis [58]. Colorimetric [916], electro-
chemical [1719], chemiluminescence [20,21], electrochemilumines-
cence [22], and electrical conductivity [23] detections are main
quantifying approaches in the lab-on-paper method although other
techniques such as mass spectrometry [24] and surface enhanced
Raman spectroscopy [25] have also been applied. Among these
methods, colorimetric detection is the most useful method because
of its simplicity and easy performance by applying inexpensive and
non-sophisticated image analysis software such as Photoshop and also
instruments such as a digital camera, scanner or even a mobile phone
camera [14,15]. Lab-on-paper devices have many advantages including
cost-effective, portability, ease of fabrication and using minimum
required reagent and sample [5,14,26]. Paper as a exible and porous
matrix is composed of hydrophilic cellulose bers with an intrinsic
capillary property which causes the ow of liquid without any external
n
Corresponding author. Tel.: 98 611 3331044; fax: 98 611 3337009.
E-mail address: npourreza@scu.ac.ir (N. Pourreza).
forcing power [7,13]. On the other hand, having the advantages of low
cost, high availability, biodegradability, portability, porous matrix and
hydrophilic surface are making paper a suitable candidate for loading
and absorbing the reagents such as suspensions of nanoparticles
[12,27,28]. A variety of techniques have been developed to design
and fabricate the lab-on-paper devices including photolithography,
inkjet printing, plasma etching, paper cutting, wax printing, analog
plotting, screen printing, and laser treatment [2,4,26].
The pH sensing has received great attention because of the
importance of pH measurement in different scientic research
elds and practical applications [2931]. Many examples of pH
sensors have been reported [2936] in literature but only a few
attempts have been made to pH sensing using paper devices [37
39]. Abe et al. have reported the simultaneous sensing of pH, total
protein and glucose in urine samples using an inkjet printed
microuidic multianalyte chemical sensing paper device. They
used a mixture of four different colored pH indicators comprising
of thymol blue, methyl red, bromothymol blue and phenolphtha-
lein in order to monitor pH in the range of 59 in urine samples
[38,39]. In a different approach, Lei et al. proposed a carbon
nanotube (CNT) based sensor on paper for the determination of
pH in the range of 59. In the research of this group, pH
measurement was based on decreasing of the resistance change
with increasing the pH of the solution [37]. Various types of
nanoparticles have been used in paper-based devices [12].
In particular, gold and silver nanoparticles as the two metal
nanoparticles with surface plasmon resonance (SPR) characteristic

http://dx.doi.org/10.1016/j.talanta.2014.07.063 0039-
9140/& 2014 Elsevier B.V. All rights reserved.
 N. Pourreza, H. Golmohammadi / Talanta 131 (2015) 136141
Fig. 1. Bis-keto (top) and enolate (bottom) tautomeric forms of curcumin.
have attracted much attention in paper based devices for colori-
metric sensing of several chemical and biological compounds [40
42]. Hydroxy pyrene trisulfonate encapsulated as a microcapsule
prepared by assembly of silica nanoparticles has also been used for
uorescence sensing of pH in the range of 5.88.0 [43].
Curcumin [(E, E)-1,7-bis (4-hydroxy-3-methoxy-phenyl)-1,6-
heptadiene-3,5-dione] is a hydrophobic polyphenol compound
which has been recognized as a naturally occurring yellow
pigment and component of the spice, turmeric. It is extracted
from the dried root of the rhizome Curcuma longa. As shown in
Fig. 1 Curcumin demonstrates ketoenol tautomerism [4446].
At basic mediums; the enolate form is prevailed form of curcumin
and in the pH range of 37, curcumin demonstrates the H-atom
donor characteristic because of its keto form. Curcumin bears
properties such as antioxidant, anti-inammatory, anticarcino-
genic, antibacterial, wound healing and can readily chelate the
metal ions to form the complexes [44]. Apart from these benets,
curcumin has low solubility in aqueous solutions and nanonization
can help to overcome this drawback [45,46]. An alternative
approach to improve the aqueous solubility is encapsulation of
curcumin in micelle or surfactant solutions. Patra and Barakat
studied the role of ionic liquid on solubilization of curcumin in the
micellar system using various surfactants and determined the
critical micelle concentration (cmc) values of aqueous CTAB, SDS
and TX100 solutions in the presence and absence of ionic liquid
[47]. Curcumin has been also applied as a uorescence probe for
sensing o-nitrophenol [48], DNA [49], protein [50] and estimation
of phase transition temperature of liposomes [51]. The application
of CUR-NPs as a probe for detection of analytes was reported by
Pendya et al. [52]. They demonstrated that CURNs aggregate in the
presence of trinitrotoluene (TNT) as an explosive compound and
surface plasmon intensity of CURNs is changed. They have found
that this property can be employed for selective, ultra-sensitive
and simple recognition of TNT in aqueous media. We have recently
reported the application of curcumin nanoparticle in a micelle
mediated system for colorimetric sensing of sulde [53].
The glass electrode which is the most widely used pH sensor
suffers from alkaline error in basic solutions. This limitation of
glass electrode is due to its sensitivity towards alkali metal ions at
pH values above 9 [54]. However, determination of pH in strong
alkaline industrial solutions and wastewater samples is of sig-
nicant importance [55].
This research work presents the application of CURNs in fabrication
of a novel lab-on-paper device for pH measurement in alkali solutions.
137
The CURNs were spotted into the lab-on-paper device followed by the
addition of solutions with different pH values in the test zone, which
subsequently changed the color of the test zone. An image of the test
zone was recorded by a digital camera and the picture was processed
and analyzed using Adobe Photoshop software and the mean color
intensity in gray mode for each test zone was determined. The mean
color intensity was decreased upon increasing the pH. The decrease in
mean color intensity was proportional to the pH of solutions and was
applied as an analytical signal for pH monitoring.
2. Experimental
2.1. Apparatus
Absorption spectra were recorded by a GBC UVvisible spectro-
photometer model Cintra 101 (Sidney, Australia) using 1 cm glass cells.
TEM image was performed with a transmission electron microscope
(Zeiss-EM10C-80 KV, Germany). SEM images were obtained by a
scanning electron microscope (Leo 1455 VP, Germany). A Metrohm
digital pH-meter model 632 (Switzerland) with a combined glass
electrode was used for pH measurements. An ultrasound bath (DSA
100-SK2, 100 W power, 40 kHz Frequency, China) was used for
fabrication of the nanoparticles ultrasonically. A rotary evaporator
(Labrota 4000, Heidolph, Germany) was used for removing the solvent.
Photographic images were taken with a digital camera (Power Shot
SX230 HS, 12.1 megapixels, Canon, Japan).
2.2. Reagents and materials
Analytical grade chemicals and doubled distilled water was
used. BrittonRobinson buffer solutions in the pH range of 712
were prepared from a mixture of phosphoric acid (Merck), boric
acid (Merck) and citric acid (Merck) (0.04 mol L 1 each). Buffer
solutions in the pH range of 1213 were prepared from
0.2 mol L 1 KCl (Merck). The pH of the buffer solutions was
adjusted to the exact value by the addition of 0.2 mol L 1 NaOH
(Merck) and using a pH meter. 1.0 mol L 1 of NaCl was prepared
by dissolving 5.844 g of sodium chloride (Merck) in water and
diluting to 100 mL in a volumetric ask. 5% (v/v) of Triton X-100
(Merck) was prepared by diluting 5 mL of the reagent to 100 mL.
Filter paper (Whatman No.1) was purchased from Whatman
International, Ltd (England). Solid white beeswax was purchased
from an herbaceous pharmacy (Ahvaz, Iran). Permanent magnets
(circle shaped with 6 mm diameter and 1 mm thickness) were
purchased from a local magnet shop (Ahvaz, Iran).
2.3. Synthesis of curcumin nanoparticles
The CURNs were fabricated by our previously reported method
for the modication of the technique described by Bhawana et al.
[45,53]. The fabrication procedure was performed as follows: the
organic phase was prepared by dissolving 125 mg of curcumin in
25 mL of dichloromethane. For preparation of aqueous phase,
10 mL of Triton X-100 5% (v/v) was added to 90 mL of boiling
water. Then 2 mL of organic phase was added dropwise to the
aqueous phase (about 10 drops/min) under ultrasonic conditions,
with an ultrasonic power of 100 W and a frequency of 40 kHz. The
sonication was continued for 20 min followed by stirring on a
magnetic stirrer at room temperature for 20 min until a yellow
color was obtained. This solution was placed in a rotary evaporator
in order to ensure the complete removal of dichloromethane. This
solution was then stored in a brown bottle. The total concentration
of CURNs solution was 0.271 mmol L 1. The prepared CURNs are
stable for more than 6 months. The TEM image of the CURNs after
fabrication using the above approach can be observed in Fig. 2.
138 N. Pourreza, H. Golmohammadi / Talanta 131 (2015)
2.4. Fabrication of lab-on-paper
The wax dipping method recommended by Songjaroen et al. was
used for fabrication of lab-on-paper device with some modication
[2]. A typical fabrication procedure was depicted as follows: the solid
beeswax was placed in a Pyrex beaker and melted on a hot plate. The
temperature was maintained in the range of 10075 1C throughout
the experiment using a thermometer. A piece of lter paper Whatman
No.1 (2.5 cm ~ 7.5 cm) was cut and placed on a glass slide. Then 10
circle shaped permanent magnets (6 mm diameter and 1 mm thick-
ness) were put onto the paper and temporarily attached by means of
magnetic force using an iron piece (2.5 cm ~ 10 cm) which was placed
on the backside of the glass slide. The assembly was soaked in melted
wax for 1 s to allow molten wax to penetrate into the depth of the
paper to form the hydrophobic walls and allowed to cool at room
temperature. It was then removed from the glass slide, and the
magnets were separated out from the paper.
2.5. Recommended procedure
The colorimetric assays were accomplished by the following
steps: 1.0 mL volumes of CURNs solution were spotted into the
middle of hydrophilic test zones on the lab-on-paper and allowed
to dry for 15 min at room temperature. Then 1 mL volumes of
different pH solutions were added to each test zone separately.
After 30 min, an image of each test zone was captured by a digital
camera. The picture was then processed and analyzed using Adobe
Photoshop CS8 and the mean color intensity in gray mode for each
test zone was determined. The color of the loaded lab-on-paper
using the CURNs changed from yellow to orange and red to brown
upon changing of pH from 8 to 13. The mean color intensity
change with pH was recorded and employed as an analytical signal
for pH sensing.
Fig. 2. TEM image of synthesized CURNs.
Fig. 3. Photographical images of the CURNs solutions with various pH values in the pH range of 713. The pH values from left to right are 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,
11.0, 11.5, 12.0, 12.5 and 13.0. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
136141
3. Results and discussion
3.1. Characteristics of CURNs at different pH values
Curcumin acts as a potent H-atom donor at pH 37, because of
its keto form and above pH 8, the enolate form predominates, and
curcumin acts mainly as an electron donor [44]. It has been shown
that curcumin is completely deprotonated at pH 13 to form the
highly negatively charged species Cur3 . At pH 13, Cur3 in water
shows absorption maxima at 468 nm that is shifted to 457 nm in
the presence of liposomes. Studies have also shown that the
degradation of curcumin in aqueous solution is linked to the
hydrolysis that occurs rapidly above neutral pH [56].
Based on the above discussions the pH dependence of the CURNs
color was also investigated by changing the pH of solution from
neutral to high alkaline solutions. The color changing and absorption
spectra of the CURNs at different pH values in aqueous medium are
shown in Fig. 3 and Fig. 4, respectively. As shown in Fig. 4, the
absorption intensity of CURNs, initially decreased at 423 nm in the
pH range of 7.09.5 with a change of color from yellow to red, and
new red shifted absorption bands appeared at 460 nm in the pH
range of 1013 and absorption intensity at this wavelength increased
from pH 10 to 13 with a change of color from red to brown.
However the behavior was different on the paper and the obtained
mean color intensity for lab-on-paper test zones was decreased upon
increasing of the pH values. Therefore, this decrease in mean color
intensity which is proportional to the pH of the solutions was applied
as an analytical signal for pH monitoring. In order to evaluate the
change in optical properties of CURNs by increasing the pH values
from neutral to alkali, the test zones of lab-on-paper at different pH
values were characterized by SEM. The resulting SEM images pre-
sented in Fig. 5 indicate that there is a decrease in the number of
CURNs and yet an increase the particle size of CURNs by changing the
Fig. 4. UVvis spectra of the CURNs solutions with various pH values in the range
of 713 after 30 min. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)
 N. Pourreza, H. Golmohammadi / Talanta 131 (2015) 136141
Fig. 5. SEM images (20,000 ~ magnication) of the test zone after (A) dropping the 1 mL solution with pH 7 and (B) dropping the 1 mL solution of CURNs followed by
dropping 1 mL solutions with pH 7, (C) pH 10 and (D) pH 13, respectively.
pH from 7 to 13. On the other hand studies have shown that
curcumin molecules are unstable and suffer rapid hydrolytic degra-
dation at neutralbasic pH conditions [57]. It has also been suggested
that micelles might suppress alkaline hydrolysis to some extent [58].
Thus, by increasing pH from 7 to 13 it is possible that both
aggregation and degradation of CURNs are taking place and these
phenomena could probably be responsible for the color change of
CURNs under these conditions.
3.2. Inuence of CURNs concentration and multiple addition
of CURNs
In lab-on-paper devices the color intensity can be increased
using multiple additions of the reagents on the test zone. Hence, to
Fig. 6. (A) Color changes of the test zones after (I) one time dropping the 1 mL of
the CURNs (1.35 ~ 10 3 mol L 1) and (II) one (III) two (IV) three (V) four and (VI)
ve times dropping of the 1 mL of the CURNs (2.71 ~ 10 3 mol L 1) followed by
dropping of 1 mL of the solutions with pH values of 7, 8, 9, 10, 11, 12, 13 respectively,
(B) Mean color intensity changes corresponding to observed color changes in part
(A) of this gure. (For interpretation of the references to color in this gure legend,
the reader is referred to the web version of this article.)
139
obtain the maximum analytical signal, the effect of CURNs con-
centration and multiple additions of CURNs as reagent were
investigated. To accomplish this goal the dropped reagent (1 mL
of CURNs at each addition) was allowed to air dry (about 15 min)
before the next addition. Then, after the nal addition of the
reagent, 1 mL of the solutions with specied pH values were placed
on the test zone. As can be ascertained from Fig. 6, the maximum
analytical signal (decrease in mean color intensity) was achieved
when four times addition of CURNs was applied for all pH values.
3.3. Inuence of electrolytes
High concentrations of salts often affect the response of the pH
sensor. In order to appraise the electrolyte impact on the current
sensor, the experiments were carried out by adding different salts such
as NaCl, KCl, NaNO3, KNO3, and Na2SO4. The results indicate that the
addition of up to 0.25 mol L 1 of NaCl, KCl and Na2SO4 and
0.1 mol L 1 of NaNO3 and KNO3 did not have signicant effect on
the performance of the pH probe.
3.4. Analytical performance
The analytical gures of merit for the developed sensor were
evaluated using the recommended procedure under the optimum
conditions. As shown in Fig. 7 the calibration graph for pH sensing
was linear in the range of 813 with a correlation coefcient (r) of
0.9991 and the linear regression equation was A 14.075C282.64.
Fig. 7. Color changes of the test zones (A) and the corresponding calibration graph (B)
for pH sensing using CURNs incorporated lab-on-paper. (For interpretation of the
references to color in this gure legend, the reader is referred to the web version of this
article.)
140 N. Pourreza, H. Golmohammadi / Talanta 131 (2015) 136141

Table 1
The inuence of interfering ions on the measurement of pH 10.

Interfering ion Tolerable concentration (mg L 1)

Na, Cl, Ca2 , K, PO3 , SO2 1000

4 4
PO3
4 , borate, NO3 , acetate,
2 NH4 500
CO2
3 , HCO3 , citrate, Mg
Tartarate, Ba2 250
Br , C2O24 , F , I 50
Pb2 , Co2 , Zn2 25
Cd2 , Al3 , Mn2 , Ni2 10
Ag , Bi3 5
Cr3 , Fe3 2
Cu2 , Hg2 1

Table 2
Determination of pH in various water samples.

Water samples pH Meter Lab-on-papera Relative error (%) Color

Mahshahr renery wastewater 12.3170.36 12.7270.36 3.33

Karoon River 10.0270.30 10.2270.15 1.99

Ahvaz tap water 9.5270.26 9.7570.27 2.41

a
x 7ts/n at 95% condence (n 3).

The relative standard deviations for 10 replicate measurements of pH
9 and pH 12 were 2.3% and 1.5%, respectively.
3.5. Interferences
In this study, the competitive experiments were carried out by
adding different anions and cations and measuring the pH under
the optimum experimental conditions. Any relative error equal or
greater than 75% from the analytical signal value was considered
as interference. The pH of the solutions was adjusted at 10
(midpoint of linear range) and then measuring the analytical
signal before and after adding the interfering ion. The results of
this study summarized in Table 1 show the good selectivity of the
sensor.
3.6. Application for pH sensing in water samples
absorption spectra and in the paper medium by SEM. The color change
of CURNs at different pH values could probably be as a consequence of
degradation and aggregation of CURNs by increasing the pH of the
solution. The developed pH sensor offers advantageous features of
being entirely environmental friendly due to utilizing the CURNs as a
safe and green nanoparticle, paper and beeswax for fabrication of
sensor, minimal required reagent and sample and not requiring
sophisticated instrumentation.
Acknowledgments
The authors are sincerely grateful to Shahid Chamran Univer-
sity, Research Council for the nancial support of this project
(Grant 1392). The nancial support of the Iranian Nanotechnology
Initiative Council is also appreciated.

The performance of the developed lab-on-paper sensor was References

validated by determining the pH values in water samples from
various sources (Mahshahr renery wastewater, Karoon River and
Ahvaz tap water samples). The collected water samples were
ltered to eliminate impurities and suspended solids and the pH
of the samples was measured using the recommended procedure.
Three measurements were performed at each pH. The results were
compared with those obtained by a pH meter. The results of these
assays presented in Table 2 indicate that there was a good
agreement between the obtained pH values of both methods.
The results also reveal that the matrices of the analyzed water
samples have no signicant inuence on the performance of the
method.
4. Conclusion
The above results illustrate the novel and effective application of
CURNs in a lab-on-paper device for pH sensing. The change in optical
characteristics of the CURNs was followed by changing the pH in the
range of 813 and monitoring this phenomenon in solution by UVvis
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