BSA modified gold nanoclusters for sensi
Sensors and Actuators B 199 (2014) 42–46
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical
journal homepage: www.elsevier.com/locate/snb
BSA-modified gold nanoclusters for sensing of folic acid
Bahram Hemmateenejad a,∗ , Fatemeh Shakerizadeh-shirazi a , Fayezeh Samari b
a
b
Department of Chemistry, Shiraz University, Shiraz, Iran
Faculty of Science, Hormozgan University, Bandar Abbas, Iran
a r t i c l e
i n f o
Article history:
Received 23 December 2013
Received in revised form 17 March 2014
Accepted 18 March 2014
Available online 27 March 2014
Keywords:
Bovine serum albumin
Gold nanoclusters
Fluorescent sensor
Folic acid
a b s t r a c t
Gold nanoclusters have found increasing interests for fabricating of novel sensors and biosensors. This
paper presents a simple, rapid and novel method for determination of folic acid (FA) based on the quenching of the fluorescence of BSA-modified gold nanoclusters. By analysis of the fluorescence and absorbance
spectra of the gold nanoclusters in the presence and absence of FA, the probable mechanism of florescence quenching of BSA-AuNCs by FA was investigated. In the physiological pH of 7.4, the fluorescence
quenching was fitted to Stern–Volmer equation with a linear response in the concentration range of
120.0 ng mL−1 to 33.12 g mL−1 and the detection limit of 18.3 ng mL−1 . Furthermore, the feasibility
of the proposed method for determination of the contents of folic acid in pharmaceutical tablets was
demonstrated. The results were agreed with the claimed values.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Folic acid (FA) or pteroyl-l-glutamic acid, chemically known as
N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl) methyl] amino]
benzoyl]-l-glutamic acid (Fig. 1), is a well-known water-soluble
vitamin of B-complex group. It is an essential vitamin required
for the healthy functioning of all cells [1]. It is usually found
in the tissues of plants and animals as conjugates containing
one or more forms [2]. Nowadays, FA is routinely prescribed to
pregnant women and widely used in the treatment and prevention
of megaloblasticanemias [3]. Deficiency states lead to impaired cell
division manifested as megaloblasticanaemia and foetal development defects in humans. Folate deficiency has also been linked to
elevated levels of serum homocysteine, a condition implicated as
an independent risk factor for coronary artery disease and stroke
[4]. Therefore, the sensitive determination of FA is very important
for the clinical point of view.
There are various methods for the determination of FA, including flow injection chemiluminometry [3], liquid chromatography
[5–8], electroanalytical techniques [9] and spectrophotometric
methods [10]. The above-mentioned methods have their own
advantages, but at the same time they have certain drawbacks such
as for instance being laborious, time-consuming, nonspecific, less
safe, and too expensive and showing less sensitivity.
∗ Corresponding author. Tel.: +98 711 6460724; fax: +98 711 6460788.
E-mail addresses: hemmatb@shirazu.ac.ir, hemmatb@sums.ac.ir
(B. Hemmateenejad).
http://dx.doi.org/10.1016/j.snb.2014.03.075
0925-4005/© 2014 Elsevier B.V. All rights reserved.
Most of the spectrophotometric methods suffer from disadvantages such as narrow range of determination, heating or extraction,
long time for the reaction to be completed, and instability of the
colored product formed [11]. Fluorescence, in contrast, has proved
to be a more-powerful optical technique for the detection of low
concentration analytes, simplicity, cost-effective nature, and rapid
implementation [12]. In the literature, only few indirect fluorimetric methods for the determination of FA are described [13,14].
Manzoori et al. [15] reported a spectrofluorimetric method for
the determination of folic acid (FA), based on its quenching effect
on the fluorescence intensity of Tb3+ –1,10-phenanthroline complex as a fluorescent probe. Although these optical chemo sensors
have made great contributions in FA sensing, limitations, such as
environmentally harmful systems, poor photostability, easy interference from other species (especially by oxygen), still exist.
The coinage metal particles with well-defined nanostructure
have become one of the most active research areas in the past
decades. Gold nanoparticles (AuNPs) have received considerable attention in different areas such as chemical and biological
sensing, medical diagnostics and therapeutics and biological imaging, as these small particles exhibit a strong surface plasmon (SP)
absorption band [16,17]. Recently, noble metal nanoclusters (NCs)
are highly attractive for bio labeling and bio imaging applications because of their ultrafine size and nontoxicity [18,19]. Gold
nanoparticles exhibits size-tunable plasmon absorption widths by
confining conduction electrons in both ground and excited states
to dimensions smaller than the electron mean free path (∼20 nm),
but plasmon absorption disappears completely for nanoparticles
less than 2 nm which the size of nanoparticles are too small to
B. Hemmateenejad et al. / Sensors and Actuators B 199 (2014) 42–46
43
diluted with buffer. Working solution was prepared by appropriate
dilution of this sample solution, so that the final concentration was
within the linear range.
2.2. Apparatus
Fig. 1. The chemical structure of folic acid.
have the continuous density of states (DOS) necessary to support
a “plasmon” characteristic of larger free-electron metal nanoparticles and Mie’s theory no longer can be applied [20]. Au nanoclusters
have often been synthesized using a poly(amidoamine) dendrimer
[21], thiolate-protected [22] or bovine serum albumin (BSA) [23]
as a template. Compared with poly (amidoamine) dendrimer and
thiolate-AuNCs, BSA assisted synthesis of Au nanoclusters is just a
greener route, and even has higher quantum yield.
The application of AuNCs-based fluorescent sensors for detection and determination of important species have been reported in
the literature [24–33]. Although the recognition-based biosensors
capable of specifically detecting chemical and biological compounds in the environment are under active development using
semiconductor dots, biosensors constructed by using luminescent
Au/Ag NCs are very rare, especially for in vivo settings [34]. This
could be another very promising area for luminescent Au/Ag NCs
considering their ultra small size, which could endow NCs a better interaction with biological systems and subsequently a better
control in the cellular internalization, a more efficient renal clearance, and a better access to some organelles as compared with
large nanoparticles (NPs). The integration of these features into
future sensor design could further advance optical biosensors for
in vivo applications. It has been confirmed that the structure of
BSA-stabilized Au nanoclusters consisted of 25 gold atoms, and BSA
ligand plays an important role in the emission of Au nanoculsters
[19,24,35]. If the interaction of BSA and Au atoms is disrupted, the
core Au clusters will be isolated from the protection provided by
BSA and consequently, the fluorescence of Au nanoclusters will be
quenched [24]. It is reasonable to speculate that in the presence of
FA, the fluorescence of AuNCs will be quenched since the stabilizer
BSA molecules will cross link with each other. It is worthy to mention the non-toxic nature of the BSA-modified AuNCs, as reported
by Retnakumari [19].
Towards achieving the suitable optical sensor aim, herein, we
present a novel, BSA-stabilized gold nanocluster (AuNC)-based fluorescent sensor for the recognition and determination of FA, which
relies on the fluorescence quenching of Au NCs in the presence of
FA.
All fluorescence spectra were recorded on a LS-45 Spectrofluorimeter (Perkin-Elmer corporate, USA) equipped with a
thermostatic bath and a 10 mm quartz cuvette. The UV–vis
absorbance spectra were recorded on a BEL Gold spectrum Lab 53
UV–vis spectrophotometer using a 10 mm cuvette. The UV S53A
spectrophotometer software of the instrument was used to digitize the absorbance spectra in 1 nm intervals and then to collect
the absorbance data in spreadsheet. The pH value was measured
using a Metrohm827 pH-meter equipped with a combined glass
electrode.
2.3. Synthesis of BSA-modified AuNCs
The AuNCs were synthesized by chemical reduction of HAuCl4
with BSA according to the reported method [23]. In a typical experiment, all glass wares used in the experiments were cleaned in a
bath of freshly prepared aquaregia (HCl:HNO3 volume ratio = 3:1),
and rinsed thoroughly in water prior to use. Aqueous BSA solution (5.0 mL, 50.0 mg mL−1 , 37 ◦ C) was added to an equal volume of
10.0 mM HAuCl4 under vigorous stirring. After 2 min, NaOH solution (0.5 mL, 1.0 M) was introduced, and the mixture was incubated
at 37 ◦ C for 12 h. The role of BSA in the synthesis of AuNCs is to act
as both reducing agent and stabilizing ligand. The color of the solution changed from light yellow to deep brown. After synthesis of
BSA-AuNCs, the resulting solution was filtered through a 0.2 m
pore size Schleicher & Schtill filter.
2.4. Procedure
A 2.5 mL portion of the synthesized BSA-AuNC was transferred
to a 25 mL graduated tube with glass stopper and the solution
was diluted to the mark with phosphate buffer solution of pH 7.4
and mixed well. In order to have a linear calibration curve for the
determination of FA at buffer solutions, 2.5 mL of the above BSAAuNC solution was titrated manually by successive additions of L
amounts of a 100.0 g mL−1 solution of FA, followed by fluorescence intensity measurement of solution 5 min after each addition.
The excitation wavelength was set at 370 nm and the emission
spectra were recorded in the wavelength interval of 520–700 nm
(with 10 nm slit width for both excitation and emission). Fluorescent intensity (F) for the reaction product and that of the reagent
blank (F0 ) were measured at the maximum emission wavelength of
629 nm. All measurements were carried out at room temperature.
3. Results and discussion
2. Experimental
3.1. Quenching of BSA-Au nanoclusters fluorescence by FA
2.1. Reagents
Bovine serum albumin (BSA) and folic acid (FA) standards were
obtained from Sigma-Aldrich. HAuCl4 ·3H2 O salt was obtained from
Merck. A stock solution of FA was prepared by dissolving pure crystalline of salts in phosphate buffer solution (pH = 7.4) and it was
stored in the dark at 4 ◦ C. All other chemicals were of analytical
reagent grade, and doubly distilled water was used throughout.
For analysis of real samples, 10 FA containing tablets (Jalinus
Pharmaceutical Company) were ground into homogenized powder.
Then, an amount corresponding to one tablet was dissolved in phosphate buffer (pH = 7.4) in a small beaker. The solution was filtered
and the residue, was washed with water several times, and then
The aqueous solution of the Au nanoclusters was deep brown in
color and exhibited bright-red fluorescence upon excitation with
ultraviolet light. The absorbance and emission spectra of BSA-AuNC
in the absence and presence of FA are shown in Fig. 2. As seen,
the synthesized BSA-AuNCs exhibit a strong fluorescence emission at around 629 nm (with excitation wavelength of 370 nm).
A significant decreasing in fluorescence emission of BSA-AuNCs
was observed when FA was added to BSA-AuNC. In addition, the
absorbance of Au NCs increases in the presence of FA.
It is worthy to notice that the fundamental understanding of the
mechanism of protein-protected Au/Ag NCs luminescence properties and quenching mechanism is still lacking, mainly because of
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Fig. 2. (a, b) Absorbance spectra and (c, d) fluorescence emission spectra (with excitation wavelength of 370 nm) of BSA-AuNCs in the absence (a, c) and presence (b,
d) of 33.12 g mL−1 (75 M) FA. All solutions were prepared using phosphate buffer
solution, pH = 7.4. The spectrum b was recorded against a blank solution containing
FA with the same concentration as sample solution. So, the observed shoulder at
about 380 nm does not that of free FA.
the complications caused by the intrinsic complexity of the protein
templates and the lack of accurate structural information of the NCs
[34]. Several intrinsic properties of the proteins could affect the
formation and properties of the NCs. Different mechanisms have
been suggested for quenching of the fluorescence of Au-NCs by the
analytes [24–26,35–38]. For the ligand–shell AuNCs, in which BSA
takes part at ligand, analyte-induced NC aggregation [25], an enzymatic reaction [37] and alteration in chemical state of the ligand
[38] have been reported as three primary strategies to achieve a
specific interaction between the luminescent AuNCs and the analyte for recognition and signal generation. The enzymatic reaction
mechanism is not the case in this study. In addition, since AuNCs
are stabilized on the surface of BSA mainly by covalent interaction
between gold nanoclusters and the thiol groups of the cysteine [34],
FA is not able to substitute BSA and induce aggregation of AuNCs.
Moreover, we did not observe any aggregation even for a long time
after addition of FA to AuNCs, as it was evident by no changes in
the absorbance baseline at wavelengths longer than 500 nm (see
Fig. 1). So, the detaching mechanism also could not be considered
as the primary reason for fluorescence quenching.
On the other hand, interaction of FA with BSA has been reported
previously [39]. In addition, Retnakumari et al. [19] investigated
conjugation of FA to AuNCs that is proceeding by interaction of FA
with BSA. So, a probable reason for quenching effect of FA could be
attributed to its interaction with BSA and consequently changing
in the environment of the AuNCs. As an evident for this suggestion,
the effect of FA on the native fluorescence of BSA was investigated (see the Supplementary materials). The fluorescence of BSA,
excited at 290 nm, was quenched by FA. Moreover, the changes
in the absorbance spectrum of BSA-AuNCs in the presence of FA
shown in Fig. 2 are similar to those reported for the interaction of
FA with BSA alone [39].
The observed fluorescence quenching was best described by the
Stern–Volmer equation:
F0
= 1 + KSV [Q ]
F
(1)
where F0 and F are the fluorescence intensity of the AuNCs in the
absence and presence of FA (excitation wavelength of 370 nm and
emission wavelength of 629 nm), respectively, [Q] is the concentration of the quencher (i.e., FA), and KSV is the Stern–Volmer
constant. The Stern–Volmer constant calculated from the
Fig. 3. The effect of pH on the quenching of AgNCs fluorescence by FA.
equation is 0.0682 the unit of KSV is the inverse of concentration
(mL microg−1 ). It should be noted that the observed quenching
could not be attributed to the resonance energy transfer since
there is not any overlap between the absorbance spectrum of FA
and the fluorescence emission spectrum of BSA-AuNCs. Regardless of the mechanism of quenching, the significant quenching
of fluorescence intensity has been evaluated for developing a
sensitive analytical method for determination of FA based on
spectrofluorimetry.
The quantum yield (QY) of the synthesized BSA-AuNCs was
determined by measuring the integrated fluorescence intensities of
the AuNCs and the reference (fluorescein solution in basic ethanol,
QY = 97%) under 470-nm excitation [23]. A QY of about 6% was
obtained for the synthesized BSA-AuNC. These fluorescent AuNCs
showed a long-term stability over 10 month and only some shifts
(+10 nm) in the maximum wavelength of emission spectra were
observed.
3.2. Effect of pH and buffer solution
The effect of pH on the fluorescence response of AuNCs toward
FA (18.12 M or 8.0 g mL−1 ) is shown in Fig. 3. The results showed
a slight decreasing in fluorescence emission at higher pH (>9) that
should be related to unfavorable cross-linking of BSA with FA in
basic media. Investigating the effect type of buffer revealed that
amine-containing and Tris–HCl buffers should be avoided whereas
phosphate buffer and HEPES buffer are suitable. Thus, we chose pH
7.4 (phosphate buffer solution, 0.1 M) as suitable solvent media for
detection of FA.
3.3. Fluorescence detection of FA
The fluorescence spectra of the BSA-AuNC displayed a gradual decrease at 629 nm (excitation wavelength of 370 nm) with
increasing FA concentration (Fig. 4). Meanwhile, the maximum
fluorescence wavelength was weakly blue-shifted with an obvious change of fluorescence spectral profile, indicating the change
of surface state of the BSA–Au nanoclusters because of the crosslinking of BSA with FA. Plotting of (F0 /F) versus concentration of FA
resulted in a linear calibration graph in the range of 120.0 ng mL−1
to 33.12 g mL−1 (272.0 nM–75.00 M) with a correlation coefficient of R2 = 0.997 (Fig. 4B). The analytical appraisals of the
suggested method are listed in Table 1. As seen, the value for limit
of detection (LOD) was evaluated as 18.3 ng mL−1 . Besides to high
sensitivity, the method benefit from very low relative standard
deviation.
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B. Hemmateenejad et al. / Sensors and Actuators B 199 (2014) 42–46
Table 2
Results of determination of FA in pharmaceutical preparations.
Added
(g mL−1 )
Found
(g mL−1 )
Expected
(g mL−1 )
Recovery
(%)
RSD*
0.00
3.00
9.00
11.00
5.61
8.62
14.13
16.12
6.00
9.00
15.00
17.00
93.50
95.7
94.2
94.82
3.60
0.67
2.46
2.67
*
relative standard deviation for 3 measurements
3.5. Determination of folic acid in pharmaceutical preparations
The developed method was applied to the determination of FA
in the tablet formulations prepared according to the sample preparation procedure. The obtained recoveries (Table 2) were between
93.5 and 95.7%. As is obvious from Table 2, folic acid contents of
the tablets were in satisfactory agreement with the labeled values.
The labeled value of FA in the analyzed tablet formulations was
1.0 mg and the obtained value by the suggested method was 0.93
(±0.07) mg, where the value in the parenthesis is the confidence
limit at 95% confidence level. The averaged value of estimation is
attributed to 7% relative error. The reported RSDs for analysis of
FA in real samples are always lower than 4%, which suggests the
suggested method is reproducible.
4. Conclusions
Fig. 4. (A) Changing in the fluorescence spectra of the BSA-AuNCs in the presence of
increasing amounts of FA. The arrows indicate the signal changes by increasing analyte concentrations: (a) in the absence of FA and (s) in the presences of 33.12 g mL−1
of FA. (B) Plot of F0 /F at 629 nm versus FA concentration. The excitation wavelength
was set at 370 nm and the incubation time was 5 min.
We demonstrated a straightforward approach for the determination of FA in aqueous samples with the BSA-AuNCs-based
fluorescent sensor. It was based on the cross-linking of the BSA
with FA, which induced fluorescence quenching of Au nanoclusters.
The presented method has prominent advantages including relatively simple and convenient, without complex organic synthesis
and complicated instrumentation. This sensor can be used for sensitive determination of FA in pharmaceutical preparations. Further
development of NC-based optical sensors may integrate this interesting and powerful attribute into the sensor design, which could
open a new avenue for the construction of sensors for a variety of
analytes.
3.4. Effect of foreign substances
Acknowledgment
To evaluate the selectivity of the BSA–AuNC toward folic acid
and the possible analytical applications of the proposed method,
under the optimal conditions the effects of different vitamins,
especially those belonging to Group B (B1, B3, B6, B12) and
numerous minerals on the analysis of a standard solution of FA
22.7 M (10.0 g mL−1 ) were investigated. The tolerable concentration ratios with respect to FA at a 3% fluorescence level were
found to be: more than 200 for vitamin B3; more than 50 for vitamin B1 and B6; and more than 2 for vitamin B12. For Ca2+ , Zn2+ , Al3+ ,
Pb2+ and Cd2+ , no interference was observed until their sedimentation and the interfering effect of Fe3+ was observed at concentration
ratios of higher than 5. However, an equal amount of Ag+ , Cu2+ , and
Ni2+ ions was found to interfere with the determination of folic acid.
The interfering effects of the metal ions could be easily managed
by addition of EDTA solution.
Table 1
Analytical appraisal for determination of folic acid by AuNCs.
Figure of merit
Obtained value
Linear range
Limit of detection
Calibration sensitivity
Analytical sensitivity
RSD (%)a
120.0 ng mL−1 –33.12 g mL−1
18.3 ng mL−1
0.07 mL g−1.
72.31
1.49
a
RSD for FA concentration of 33.12 g mL−1 and five independent replicates.
This work has been done using a grant donation from National
Elites Foundation of Iran.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2014.03.075.
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Biographies
Bahram Hemmateenejad is a professor of Chemistry at Chemistry Department
of Shiraz University. He received his B.S. degree in pure chemistry from Shiraz
University in 1996, M.S. and Ph.D. in Analytical Chemistry from Isfahan University of Technology and Shiraz University in 1998 and 2002, respectively. He runs
a Lab on the development and applications of different chemometrics methods in
the chemical disciplines of bioanalytical chemistry, chemo-biological interactions,
computer-aided drug design and colorimetric sensors.
Fatemeh Shekerizadeh-Shirazi received her B.S. and M.S. degrees in Pure Chemistry and Analytical Chemistry from Persian Gulf University and Shiraz University in
2007 and 2009, respectively. Currently, she is doing her Ph.D. thesis to develop colorimetric sensors and sensor arrays mostly based on nanomaterials and multivariate
image analysis.
Fayezeh Samari has appointed an assistant professor position from Hormozgan
University since 2011. She received her B.S. degree from Arak University and then she
continued graduate studies in Shiraz University. Fayezeh received her M.S. and Ph.D.
degrees in Analytical Chemistry there in 2007 and 2011. Her research area includes
interaction of small molecules and nano-materials with biomacromolecules.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical
journal homepage: www.elsevier.com/locate/snb
BSA-modified gold nanoclusters for sensing of folic acid
Bahram Hemmateenejad a,∗ , Fatemeh Shakerizadeh-shirazi a , Fayezeh Samari b
a
b
Department of Chemistry, Shiraz University, Shiraz, Iran
Faculty of Science, Hormozgan University, Bandar Abbas, Iran
a r t i c l e
i n f o
Article history:
Received 23 December 2013
Received in revised form 17 March 2014
Accepted 18 March 2014
Available online 27 March 2014
Keywords:
Bovine serum albumin
Gold nanoclusters
Fluorescent sensor
Folic acid
a b s t r a c t
Gold nanoclusters have found increasing interests for fabricating of novel sensors and biosensors. This
paper presents a simple, rapid and novel method for determination of folic acid (FA) based on the quenching of the fluorescence of BSA-modified gold nanoclusters. By analysis of the fluorescence and absorbance
spectra of the gold nanoclusters in the presence and absence of FA, the probable mechanism of florescence quenching of BSA-AuNCs by FA was investigated. In the physiological pH of 7.4, the fluorescence
quenching was fitted to Stern–Volmer equation with a linear response in the concentration range of
120.0 ng mL−1 to 33.12 g mL−1 and the detection limit of 18.3 ng mL−1 . Furthermore, the feasibility
of the proposed method for determination of the contents of folic acid in pharmaceutical tablets was
demonstrated. The results were agreed with the claimed values.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Folic acid (FA) or pteroyl-l-glutamic acid, chemically known as
N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl) methyl] amino]
benzoyl]-l-glutamic acid (Fig. 1), is a well-known water-soluble
vitamin of B-complex group. It is an essential vitamin required
for the healthy functioning of all cells [1]. It is usually found
in the tissues of plants and animals as conjugates containing
one or more forms [2]. Nowadays, FA is routinely prescribed to
pregnant women and widely used in the treatment and prevention
of megaloblasticanemias [3]. Deficiency states lead to impaired cell
division manifested as megaloblasticanaemia and foetal development defects in humans. Folate deficiency has also been linked to
elevated levels of serum homocysteine, a condition implicated as
an independent risk factor for coronary artery disease and stroke
[4]. Therefore, the sensitive determination of FA is very important
for the clinical point of view.
There are various methods for the determination of FA, including flow injection chemiluminometry [3], liquid chromatography
[5–8], electroanalytical techniques [9] and spectrophotometric
methods [10]. The above-mentioned methods have their own
advantages, but at the same time they have certain drawbacks such
as for instance being laborious, time-consuming, nonspecific, less
safe, and too expensive and showing less sensitivity.
∗ Corresponding author. Tel.: +98 711 6460724; fax: +98 711 6460788.
E-mail addresses: hemmatb@shirazu.ac.ir, hemmatb@sums.ac.ir
(B. Hemmateenejad).
http://dx.doi.org/10.1016/j.snb.2014.03.075
0925-4005/© 2014 Elsevier B.V. All rights reserved.
Most of the spectrophotometric methods suffer from disadvantages such as narrow range of determination, heating or extraction,
long time for the reaction to be completed, and instability of the
colored product formed [11]. Fluorescence, in contrast, has proved
to be a more-powerful optical technique for the detection of low
concentration analytes, simplicity, cost-effective nature, and rapid
implementation [12]. In the literature, only few indirect fluorimetric methods for the determination of FA are described [13,14].
Manzoori et al. [15] reported a spectrofluorimetric method for
the determination of folic acid (FA), based on its quenching effect
on the fluorescence intensity of Tb3+ –1,10-phenanthroline complex as a fluorescent probe. Although these optical chemo sensors
have made great contributions in FA sensing, limitations, such as
environmentally harmful systems, poor photostability, easy interference from other species (especially by oxygen), still exist.
The coinage metal particles with well-defined nanostructure
have become one of the most active research areas in the past
decades. Gold nanoparticles (AuNPs) have received considerable attention in different areas such as chemical and biological
sensing, medical diagnostics and therapeutics and biological imaging, as these small particles exhibit a strong surface plasmon (SP)
absorption band [16,17]. Recently, noble metal nanoclusters (NCs)
are highly attractive for bio labeling and bio imaging applications because of their ultrafine size and nontoxicity [18,19]. Gold
nanoparticles exhibits size-tunable plasmon absorption widths by
confining conduction electrons in both ground and excited states
to dimensions smaller than the electron mean free path (∼20 nm),
but plasmon absorption disappears completely for nanoparticles
less than 2 nm which the size of nanoparticles are too small to
B. Hemmateenejad et al. / Sensors and Actuators B 199 (2014) 42–46
43
diluted with buffer. Working solution was prepared by appropriate
dilution of this sample solution, so that the final concentration was
within the linear range.
2.2. Apparatus
Fig. 1. The chemical structure of folic acid.
have the continuous density of states (DOS) necessary to support
a “plasmon” characteristic of larger free-electron metal nanoparticles and Mie’s theory no longer can be applied [20]. Au nanoclusters
have often been synthesized using a poly(amidoamine) dendrimer
[21], thiolate-protected [22] or bovine serum albumin (BSA) [23]
as a template. Compared with poly (amidoamine) dendrimer and
thiolate-AuNCs, BSA assisted synthesis of Au nanoclusters is just a
greener route, and even has higher quantum yield.
The application of AuNCs-based fluorescent sensors for detection and determination of important species have been reported in
the literature [24–33]. Although the recognition-based biosensors
capable of specifically detecting chemical and biological compounds in the environment are under active development using
semiconductor dots, biosensors constructed by using luminescent
Au/Ag NCs are very rare, especially for in vivo settings [34]. This
could be another very promising area for luminescent Au/Ag NCs
considering their ultra small size, which could endow NCs a better interaction with biological systems and subsequently a better
control in the cellular internalization, a more efficient renal clearance, and a better access to some organelles as compared with
large nanoparticles (NPs). The integration of these features into
future sensor design could further advance optical biosensors for
in vivo applications. It has been confirmed that the structure of
BSA-stabilized Au nanoclusters consisted of 25 gold atoms, and BSA
ligand plays an important role in the emission of Au nanoculsters
[19,24,35]. If the interaction of BSA and Au atoms is disrupted, the
core Au clusters will be isolated from the protection provided by
BSA and consequently, the fluorescence of Au nanoclusters will be
quenched [24]. It is reasonable to speculate that in the presence of
FA, the fluorescence of AuNCs will be quenched since the stabilizer
BSA molecules will cross link with each other. It is worthy to mention the non-toxic nature of the BSA-modified AuNCs, as reported
by Retnakumari [19].
Towards achieving the suitable optical sensor aim, herein, we
present a novel, BSA-stabilized gold nanocluster (AuNC)-based fluorescent sensor for the recognition and determination of FA, which
relies on the fluorescence quenching of Au NCs in the presence of
FA.
All fluorescence spectra were recorded on a LS-45 Spectrofluorimeter (Perkin-Elmer corporate, USA) equipped with a
thermostatic bath and a 10 mm quartz cuvette. The UV–vis
absorbance spectra were recorded on a BEL Gold spectrum Lab 53
UV–vis spectrophotometer using a 10 mm cuvette. The UV S53A
spectrophotometer software of the instrument was used to digitize the absorbance spectra in 1 nm intervals and then to collect
the absorbance data in spreadsheet. The pH value was measured
using a Metrohm827 pH-meter equipped with a combined glass
electrode.
2.3. Synthesis of BSA-modified AuNCs
The AuNCs were synthesized by chemical reduction of HAuCl4
with BSA according to the reported method [23]. In a typical experiment, all glass wares used in the experiments were cleaned in a
bath of freshly prepared aquaregia (HCl:HNO3 volume ratio = 3:1),
and rinsed thoroughly in water prior to use. Aqueous BSA solution (5.0 mL, 50.0 mg mL−1 , 37 ◦ C) was added to an equal volume of
10.0 mM HAuCl4 under vigorous stirring. After 2 min, NaOH solution (0.5 mL, 1.0 M) was introduced, and the mixture was incubated
at 37 ◦ C for 12 h. The role of BSA in the synthesis of AuNCs is to act
as both reducing agent and stabilizing ligand. The color of the solution changed from light yellow to deep brown. After synthesis of
BSA-AuNCs, the resulting solution was filtered through a 0.2 m
pore size Schleicher & Schtill filter.
2.4. Procedure
A 2.5 mL portion of the synthesized BSA-AuNC was transferred
to a 25 mL graduated tube with glass stopper and the solution
was diluted to the mark with phosphate buffer solution of pH 7.4
and mixed well. In order to have a linear calibration curve for the
determination of FA at buffer solutions, 2.5 mL of the above BSAAuNC solution was titrated manually by successive additions of L
amounts of a 100.0 g mL−1 solution of FA, followed by fluorescence intensity measurement of solution 5 min after each addition.
The excitation wavelength was set at 370 nm and the emission
spectra were recorded in the wavelength interval of 520–700 nm
(with 10 nm slit width for both excitation and emission). Fluorescent intensity (F) for the reaction product and that of the reagent
blank (F0 ) were measured at the maximum emission wavelength of
629 nm. All measurements were carried out at room temperature.
3. Results and discussion
2. Experimental
3.1. Quenching of BSA-Au nanoclusters fluorescence by FA
2.1. Reagents
Bovine serum albumin (BSA) and folic acid (FA) standards were
obtained from Sigma-Aldrich. HAuCl4 ·3H2 O salt was obtained from
Merck. A stock solution of FA was prepared by dissolving pure crystalline of salts in phosphate buffer solution (pH = 7.4) and it was
stored in the dark at 4 ◦ C. All other chemicals were of analytical
reagent grade, and doubly distilled water was used throughout.
For analysis of real samples, 10 FA containing tablets (Jalinus
Pharmaceutical Company) were ground into homogenized powder.
Then, an amount corresponding to one tablet was dissolved in phosphate buffer (pH = 7.4) in a small beaker. The solution was filtered
and the residue, was washed with water several times, and then
The aqueous solution of the Au nanoclusters was deep brown in
color and exhibited bright-red fluorescence upon excitation with
ultraviolet light. The absorbance and emission spectra of BSA-AuNC
in the absence and presence of FA are shown in Fig. 2. As seen,
the synthesized BSA-AuNCs exhibit a strong fluorescence emission at around 629 nm (with excitation wavelength of 370 nm).
A significant decreasing in fluorescence emission of BSA-AuNCs
was observed when FA was added to BSA-AuNC. In addition, the
absorbance of Au NCs increases in the presence of FA.
It is worthy to notice that the fundamental understanding of the
mechanism of protein-protected Au/Ag NCs luminescence properties and quenching mechanism is still lacking, mainly because of
44
B. Hemmateenejad et al. / Sensors and Actuators B 199 (2014) 42–46
Fig. 2. (a, b) Absorbance spectra and (c, d) fluorescence emission spectra (with excitation wavelength of 370 nm) of BSA-AuNCs in the absence (a, c) and presence (b,
d) of 33.12 g mL−1 (75 M) FA. All solutions were prepared using phosphate buffer
solution, pH = 7.4. The spectrum b was recorded against a blank solution containing
FA with the same concentration as sample solution. So, the observed shoulder at
about 380 nm does not that of free FA.
the complications caused by the intrinsic complexity of the protein
templates and the lack of accurate structural information of the NCs
[34]. Several intrinsic properties of the proteins could affect the
formation and properties of the NCs. Different mechanisms have
been suggested for quenching of the fluorescence of Au-NCs by the
analytes [24–26,35–38]. For the ligand–shell AuNCs, in which BSA
takes part at ligand, analyte-induced NC aggregation [25], an enzymatic reaction [37] and alteration in chemical state of the ligand
[38] have been reported as three primary strategies to achieve a
specific interaction between the luminescent AuNCs and the analyte for recognition and signal generation. The enzymatic reaction
mechanism is not the case in this study. In addition, since AuNCs
are stabilized on the surface of BSA mainly by covalent interaction
between gold nanoclusters and the thiol groups of the cysteine [34],
FA is not able to substitute BSA and induce aggregation of AuNCs.
Moreover, we did not observe any aggregation even for a long time
after addition of FA to AuNCs, as it was evident by no changes in
the absorbance baseline at wavelengths longer than 500 nm (see
Fig. 1). So, the detaching mechanism also could not be considered
as the primary reason for fluorescence quenching.
On the other hand, interaction of FA with BSA has been reported
previously [39]. In addition, Retnakumari et al. [19] investigated
conjugation of FA to AuNCs that is proceeding by interaction of FA
with BSA. So, a probable reason for quenching effect of FA could be
attributed to its interaction with BSA and consequently changing
in the environment of the AuNCs. As an evident for this suggestion,
the effect of FA on the native fluorescence of BSA was investigated (see the Supplementary materials). The fluorescence of BSA,
excited at 290 nm, was quenched by FA. Moreover, the changes
in the absorbance spectrum of BSA-AuNCs in the presence of FA
shown in Fig. 2 are similar to those reported for the interaction of
FA with BSA alone [39].
The observed fluorescence quenching was best described by the
Stern–Volmer equation:
F0
= 1 + KSV [Q ]
F
(1)
where F0 and F are the fluorescence intensity of the AuNCs in the
absence and presence of FA (excitation wavelength of 370 nm and
emission wavelength of 629 nm), respectively, [Q] is the concentration of the quencher (i.e., FA), and KSV is the Stern–Volmer
constant. The Stern–Volmer constant calculated from the
Fig. 3. The effect of pH on the quenching of AgNCs fluorescence by FA.
equation is 0.0682 the unit of KSV is the inverse of concentration
(mL microg−1 ). It should be noted that the observed quenching
could not be attributed to the resonance energy transfer since
there is not any overlap between the absorbance spectrum of FA
and the fluorescence emission spectrum of BSA-AuNCs. Regardless of the mechanism of quenching, the significant quenching
of fluorescence intensity has been evaluated for developing a
sensitive analytical method for determination of FA based on
spectrofluorimetry.
The quantum yield (QY) of the synthesized BSA-AuNCs was
determined by measuring the integrated fluorescence intensities of
the AuNCs and the reference (fluorescein solution in basic ethanol,
QY = 97%) under 470-nm excitation [23]. A QY of about 6% was
obtained for the synthesized BSA-AuNC. These fluorescent AuNCs
showed a long-term stability over 10 month and only some shifts
(+10 nm) in the maximum wavelength of emission spectra were
observed.
3.2. Effect of pH and buffer solution
The effect of pH on the fluorescence response of AuNCs toward
FA (18.12 M or 8.0 g mL−1 ) is shown in Fig. 3. The results showed
a slight decreasing in fluorescence emission at higher pH (>9) that
should be related to unfavorable cross-linking of BSA with FA in
basic media. Investigating the effect type of buffer revealed that
amine-containing and Tris–HCl buffers should be avoided whereas
phosphate buffer and HEPES buffer are suitable. Thus, we chose pH
7.4 (phosphate buffer solution, 0.1 M) as suitable solvent media for
detection of FA.
3.3. Fluorescence detection of FA
The fluorescence spectra of the BSA-AuNC displayed a gradual decrease at 629 nm (excitation wavelength of 370 nm) with
increasing FA concentration (Fig. 4). Meanwhile, the maximum
fluorescence wavelength was weakly blue-shifted with an obvious change of fluorescence spectral profile, indicating the change
of surface state of the BSA–Au nanoclusters because of the crosslinking of BSA with FA. Plotting of (F0 /F) versus concentration of FA
resulted in a linear calibration graph in the range of 120.0 ng mL−1
to 33.12 g mL−1 (272.0 nM–75.00 M) with a correlation coefficient of R2 = 0.997 (Fig. 4B). The analytical appraisals of the
suggested method are listed in Table 1. As seen, the value for limit
of detection (LOD) was evaluated as 18.3 ng mL−1 . Besides to high
sensitivity, the method benefit from very low relative standard
deviation.
45
B. Hemmateenejad et al. / Sensors and Actuators B 199 (2014) 42–46
Table 2
Results of determination of FA in pharmaceutical preparations.
Added
(g mL−1 )
Found
(g mL−1 )
Expected
(g mL−1 )
Recovery
(%)
RSD*
0.00
3.00
9.00
11.00
5.61
8.62
14.13
16.12
6.00
9.00
15.00
17.00
93.50
95.7
94.2
94.82
3.60
0.67
2.46
2.67
*
relative standard deviation for 3 measurements
3.5. Determination of folic acid in pharmaceutical preparations
The developed method was applied to the determination of FA
in the tablet formulations prepared according to the sample preparation procedure. The obtained recoveries (Table 2) were between
93.5 and 95.7%. As is obvious from Table 2, folic acid contents of
the tablets were in satisfactory agreement with the labeled values.
The labeled value of FA in the analyzed tablet formulations was
1.0 mg and the obtained value by the suggested method was 0.93
(±0.07) mg, where the value in the parenthesis is the confidence
limit at 95% confidence level. The averaged value of estimation is
attributed to 7% relative error. The reported RSDs for analysis of
FA in real samples are always lower than 4%, which suggests the
suggested method is reproducible.
4. Conclusions
Fig. 4. (A) Changing in the fluorescence spectra of the BSA-AuNCs in the presence of
increasing amounts of FA. The arrows indicate the signal changes by increasing analyte concentrations: (a) in the absence of FA and (s) in the presences of 33.12 g mL−1
of FA. (B) Plot of F0 /F at 629 nm versus FA concentration. The excitation wavelength
was set at 370 nm and the incubation time was 5 min.
We demonstrated a straightforward approach for the determination of FA in aqueous samples with the BSA-AuNCs-based
fluorescent sensor. It was based on the cross-linking of the BSA
with FA, which induced fluorescence quenching of Au nanoclusters.
The presented method has prominent advantages including relatively simple and convenient, without complex organic synthesis
and complicated instrumentation. This sensor can be used for sensitive determination of FA in pharmaceutical preparations. Further
development of NC-based optical sensors may integrate this interesting and powerful attribute into the sensor design, which could
open a new avenue for the construction of sensors for a variety of
analytes.
3.4. Effect of foreign substances
Acknowledgment
To evaluate the selectivity of the BSA–AuNC toward folic acid
and the possible analytical applications of the proposed method,
under the optimal conditions the effects of different vitamins,
especially those belonging to Group B (B1, B3, B6, B12) and
numerous minerals on the analysis of a standard solution of FA
22.7 M (10.0 g mL−1 ) were investigated. The tolerable concentration ratios with respect to FA at a 3% fluorescence level were
found to be: more than 200 for vitamin B3; more than 50 for vitamin B1 and B6; and more than 2 for vitamin B12. For Ca2+ , Zn2+ , Al3+ ,
Pb2+ and Cd2+ , no interference was observed until their sedimentation and the interfering effect of Fe3+ was observed at concentration
ratios of higher than 5. However, an equal amount of Ag+ , Cu2+ , and
Ni2+ ions was found to interfere with the determination of folic acid.
The interfering effects of the metal ions could be easily managed
by addition of EDTA solution.
Table 1
Analytical appraisal for determination of folic acid by AuNCs.
Figure of merit
Obtained value
Linear range
Limit of detection
Calibration sensitivity
Analytical sensitivity
RSD (%)a
120.0 ng mL−1 –33.12 g mL−1
18.3 ng mL−1
0.07 mL g−1.
72.31
1.49
a
RSD for FA concentration of 33.12 g mL−1 and five independent replicates.
This work has been done using a grant donation from National
Elites Foundation of Iran.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2014.03.075.
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Biographies
Bahram Hemmateenejad is a professor of Chemistry at Chemistry Department
of Shiraz University. He received his B.S. degree in pure chemistry from Shiraz
University in 1996, M.S. and Ph.D. in Analytical Chemistry from Isfahan University of Technology and Shiraz University in 1998 and 2002, respectively. He runs
a Lab on the development and applications of different chemometrics methods in
the chemical disciplines of bioanalytical chemistry, chemo-biological interactions,
computer-aided drug design and colorimetric sensors.
Fatemeh Shekerizadeh-Shirazi received her B.S. and M.S. degrees in Pure Chemistry and Analytical Chemistry from Persian Gulf University and Shiraz University in
2007 and 2009, respectively. Currently, she is doing her Ph.D. thesis to develop colorimetric sensors and sensor arrays mostly based on nanomaterials and multivariate
image analysis.
Fayezeh Samari has appointed an assistant professor position from Hormozgan
University since 2011. She received her B.S. degree from Arak University and then she
continued graduate studies in Shiraz University. Fayezeh received her M.S. and Ph.D.
degrees in Analytical Chemistry there in 2007 and 2011. Her research area includes
interaction of small molecules and nano-materials with biomacromolecules.