Green synthesis of gold nanoparticles using aqueous Aegle marmelos leaf extract and their application for thiamine detection

Article abstract of DOI:10.1039/c4ra03883e

Nanoparticles of noble metals, especially gold nanoparticles are studied extensively because of their new and amazing properties. Among several synthesis techniques, green synthesis of nanoparticles is promising in recent years. In this study, we investigated the potential of Aegle marmelos leaf extract (LE) in reduction of HAuCl₄ to form ～38.2 ± 10.5 nm spherical shape polyphenol capped gold nanoparticles. The stoichiometric proportion of the LE to HAuCl₄ and the equilibrium time to complete the reduction process for the nanoparticle formation were also identified. The total reaction time was observed to be within ～30 min from the particle formation kinetics study. The as-synthesized gold nanoparticles capped with polyphenols of leaf extract were shown to be very effective for the detection of vitamin B or thiamine to a minimum concentration of ～0.5 μM.

Full text of DOI:10.1039/c4ra03883e

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Green synthesis of gold nanoparticles using
aqueous Aegle marmelos leaf extract and their
application for thiamine detection†
Cite this: RSC Adv., 2014, 4, 28645
*
K. Jagajjanani Rao and Santanu Paria
Nanoparticles of noble metals, especially gold nanoparticles are studied extensively because of their new
and amazing properties. Among several synthesis techniques, green synthesis of nanoparticles is
promising in recent years. In this study, we investigated the potential of Aegle marmelos leaf extract (LE)
in reduction of HAuCl4 to form ꢀ38.2 ꢁ 10.5 nm spherical shape polyphenol capped gold nanoparticles.
The stoichiometric proportion of the LE to HAuCl4 and the equilibrium time to complete the reduction
process for the nanoparticle formation were also identified. The total reaction time was observed to be
within ꢀ30 min from the particle formation kinetics study. The as-synthesized gold nanoparticles capped
with polyphenols of leaf extract were shown to be very effective for the detection of vitamin B or
thiamine to a minimum concentration of ꢀ0.5 mM.
Received 28th April 2014
Accepted 18th June 2014
DOI: 10.1039/c4ra03883e
www.rsc.org/advances
potential use of the medicinal plants for the synthesis of AuNPs.
For example, Citrus limon extract was employed to have a 10 nm
AuNPs at ambient temperature and neutral pH.12 Similarly,
1 Introduction
Synthesis of nanoparticles (NPs) using green processes is
receiving immense attention in recent years over the conven-
tional bottom-up chemical routes, because of several advan-
tages of green routes such as environmental friendliness, use of
renewable natural resources, more bio-compatible nature for
biological applications.1,2 Among the available green method-
ologies, biosynthesis of nanoparticles has attracted greater
attention for its mild reaction conditions, eco-friendly residual
products, and in situ capping abilities.2–4 Unlike microbial
based synthesis processes, utilization of plant based natural
compounds has some advantages such as user friendliness,
cost-effectiveness, easy separation and purication processes.5–7
As a result, various novel nanoparticles have been synthesized
by this technique.6,7
Among the different NPs, gold (Au) NPs have attracted great
attention for their technological and biomedical applications
such as diagnostics, biosensors, molecular imaging, drug
delivery.8,9 Numerous studies have also been documented for
the phytochemical based synthesis of AuNPs, where the gold
ion is reduced by the electron donating plant components or
phytochemicals.10–13 For example, AuNPs from Chenopodium
album (10–30 nm), geranium leaf (16–40 nm), Memecylon edule
(20–50 nm), banana peel extract (300 nm), etc. are reported
before.14,15 Additionally, few recent studies have also shown the
plant extract from cypress leaves resulted in AuNPs of ꢀ15 nm
particles within 10 min,13 active polyphenols and avonoids of
black tea leaf broth gives ꢀ20 nm mixed shape AuNPs.16 Some
other therapeutic plant extracts such as Syzygium aromaticum,
Azadirachta indica, Emblica officinalis, Cinnamoum camphora,
Avena sativa, etc. were also able to produce AuNPs.11,14,15,17,18
Although widespread studies have been reported for AuNPs
synthesis, systematic studies with applications are limited. In
this study, we report a single step quick preparation route of
AuNPs of ꢀ38.2 ꢁ 10.5 nm spherical size and stabilized in situ
without any external capping agent using Aegle marmelos LE.
Furthermore, these as-synthesized caped AuNPs were also used
for facile detection of thiamine in aqueous solution. Thiamine
or vitamin B1 is a sulphur containing water soluble vitamin
naturally produced in bacteria, fungi, and plants.19 This vitamin
enters into human or animal body from food, which is an
important cofactor for many cellular processes. Thiamine
deciency causes various diseases in the human body such as
beriberi, and neurological disorders, optic neuropathy, etc.19
Additionally, it is also an essential vitamin to avoid various
neurological and muscular disorders in poultry, ruminants,
birds, sh, and mammals.20
A positive diagnosis of this vital vitamin in blood and tissues
will help to avoid many diseases in advance, especially neuro-
logical disorders. Additionally, an easy and reliable method of
detection of this essential vitamin in food and pharmaceutical
industries is highly essential for different product formula-
tions.21 The widely used chemical techniques for thiamine
detection are based on the reaction between chemical oxidising
Interfaces and Nanomaterials Laboratory, Department of Chemical Engineering,
National Institute of Technology, Rourkela–769 008, Orissa, India. E-mail:
santanuparia@yahoo.com; sparia@nitrkl.ac.in; Fax: +91 661 246 2999
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra03883e
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 28645–28652 | 28645

View Article Online
RSC Advances
Paper
agents and alkali, followed by the detection of uorescent active
To elucidate thiamine detection efficiency of the capped
compound thiochrome formed by oxidation reaction.22 The AuNPs, 12 mM thiamine was exposed to various concentrations
uorescent measurement technique is useful because of its of AuNPs (9.25 to 370 mg mLꢃ1) in water : methanol (1 : 2)
high sensitivity, even at low concentration with less noise in the solutions. Then these solutions were mixed thoroughly, incu-
biological system; nevertheless, in other techniques such as UV- bated for 20 min, the uorescence spectra were analyzed at 425
visible spectroscopy various biomolecules interfere during the nm wavelength, while excited at 356 nm wavelength. Further-
analysis. Finally, numerous methods have been developed and more, to check the particle size effect on thiamine detection
still developing to trace out the thiamine content especially in efficiency, smaller size particles (ꢀ10 nm) were synthesized
solution phases.21,23 Some research groups even used transition chemically by NaBH4 reduction using a published protocol31
metal (ruthenium), and metal (cerium) mediated oxidation of and then LE was added externally to adsorb on the particle
thiamine, which required ꢀ8 h incubation time with the metal surface.
catalyst for the completion of reaction.24 Similarly, different
chemical methodologies were reported for thiamine detection,
2.2 Characterization
where different special reaction conditions such as long incu-
UV-visible spectra were recorded using a UV-visible-NIR spec-
trophotometer (Shimadzu, UV-3600). High-resolution trans-
bation time (ꢀ16 h), elevated temperature,25 and alkalinity26 are
required to be maintained. Nanoparticles based biosensors
mission electron microscopy (HR-TEM, JEOL-2100F) and
having good optical properties (absorption or emission)
scanning electron microscopy (SEM, JEOL JSM 6480 LV) were
drawing signicant interest in recent years.27,28 However, there
used to identify the particle sizes and shapes wherever
is no reported study available on thiamine detection using
necessary. Participation of the active functional groups in the
reduction process of gold precursor was identied by FT-IR
spectroscopy (Bruker, USA), equipped with a horizontal
attenuated total reectance (ATR) device with zinc selenide
(ZnSe) crystal. Zeta (z) potentials of LE and AuNPs were
measured by Malvern zeta size analyser, (Nano ZS). X-ray
diffraction measurements were done by Philips (PW1830
HT) X-ray diffractometer, in the range of 20–90ꢂ (2q) at a scan
rate of 0.05ꢂ sꢃ1. Thermal gravimetric analysis (TGA) was
AuNPs based sensor. This study reveals that polyphenols cap-
ped AuNPs have quick oxidation ability towards thiamine to
generate thiochrome (a uorescent active compound). This
sensing property of capped AuNPs towards thiamine has a huge
scope to design novel sensor in bio-chemical and medical
applications.
2 Experimental
performed using Shimadzu, DTG-60H. The changes in
ꢂ
weight in TGA were recorded from 50 to 600 C at a heating
2.1 Materials and methods
rate of 5 K minꢃ1
.
The chloroauric acid (HAuCl4$3.5H2O) was purchased from
Loba Chemie. Potassium bromide (KBr), Sodium hydroxide
(NaOH) from Merck. Ultrapure water of 18.2 mU cm resistivity 3 Results and discussion
was used throughout the study. Aegle marmelos LE was prepared
3.1 Effect of leaf extract concentration on particle size
according to our previous study.29 The aqueous extract was then
freeze dried to powder form (Labconco, FreeZone 2.5). A 5%
stock solution of LE was prepared in deionized water and was
used for the experiments.
In general, identication of the stoichiometric ratio for green
route synthesis is equally important as that of the wet chemical
route to minimise the excess precursor during the reaction
process. To identify that, the prepared LE (5.0%) was diluted to
0.1, 0.2, 0.3, 0.5, 0.7, and 1.0% solutions and exposed to 1 mM
HAuCl4 for 2 h to complete the reaction. The spectral patterns
because of the surface plasmon resonance (SPR) effect of AuNPs
in the presence of different LE concentrations are shown in
Fig. 1(a). At 0.1% LE, one can clearly see that there is no
prominent peak of AuNPs. This could be because of low LE
concentration which is insufficient to produce NPs. As the LE
concentration increases to 0.2%, a broad peak pattern of AuNPs
is observed. The presence of a shoulder type hump at 700–750
The AuNPs were synthesized from 1 mM HAuCl4 in the
presence of different concentrations of LE from 0.1–1.5% for 2 h
at room temperature (ꢀ25 ꢂC). The stoichiometric amount of LE
required for the reduction reaction was determined using gold
bromide complexation reaction, where the excess HAuCl4
present in solution was react with externally added KBr solution
as per reaction mentioned below.30
HAuCl4$3.5H2O + 4KBr / KAuBr4$2H2O + 3KCl + HCl (1)
Aer the completion of the reduction reaction for NPs nm along with a peak at 550 nm may be because of the presence
formation, particles were separated by centrifugation and then of various sizes and non-spherical shape NPs. While the
the supernatant (1 mL) was treated with 0.3 mL of 1 N KBr concentration of LE increases to 0.3% and above, the charac-
solution. The gold bromide complex (KAuBr4$2H2O), was teristic peaks with lmax value at ꢀ543 nm are observed which
conrmed by UV-visible spectroscopy peak at 380 nm wave- indicates the absorption of AuNPs in the visible region of the
length. The absence of peak indicates that the excess HAuCl4 is electromagnetic spectrum.32
absent aer the reaction. Additionally, kinetics of particle
To nd the presence of excess HAuCl4 in the reaction
formation was studied by UV-visible spectroscopy which was mixture, KBr solution was added to the supernatant aer the
recorded as a function of reaction time of using 1.5% LE.
separation of particles to form a gold complex (KAuBr4$2H2O).
28646 | RSC Adv., 2014, 4, 28645–28652
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Fig. 1 (a) UV spectra of the AuNPs after reaction with different LE concentrations (0.1–1.0%). (b) UV-Vis spectra of KBr treated supernatents after
reaction with 1 mM HAuCl4 and different concentrations of LE (0.1 to 1.0%). (c) Phorographs of pure LE before reaction (left to right increasing
concentration, 0.1 to 1.0%). (d) Colour changes of respective solutions after AuNPs formation. (e) Photographs of respective supernatents (after
completion of particle formation) treated with 1 N KBr.
The complex formation was conrmed by the characteristic UV- The self-assembled pattern is formed mainly at the centre of the
visible peak (lmax) at 380 nm shown in Fig. 1(b). The physical drop (Fig. S1a†). The SEM images show that the presence of
appearances because of the colour change of different reaction excess HAuCl4 helps to form fractal type self-assembly, where
systems are shown in photographs of Fig. 1(c)–(e). The absence branches are consist of already formed AuNPs. Thus AuNPs
of positive absorbance peak at 380 nm for 0.3% of LE and above alone cannot form the pattern, but presence of metal salt and
in UV-visible spectra indicates no excess HAuCl4 present in the NPs are required for the formation of self assembly as shown by
reaction mixture.30 Therefore, 1.0 mM/0.3% (HAuCl4/LE) some reported study.34
concentration was taken as the stoichiometric ratio for
complete reduction.
3.2 Kinetics of particle formation
The role of LE concentration below the stoichiometric ratio
To know the equilibrium reaction time of AuNPs formation, the
on particle formation was studied further. The UV-Visible
progress of reaction using 1.0 mM aqueous HAuCl4 in the
spectral pattern of AuNPs synthesized from non-stoichiometric
presence of 1.5% LE was monitored with time by UV-Vis spec-
proportion of 1.0 mM/0.2% (HAuCl4/LE) is shown in Fig. 1(a).
troscopy. The absorption spectra at 543 nm wavelength (lmax
)
The less intense broad spectral pattern extended towards NIR
region may be because of the presence of either larger sized or
non-homogenous nanoparticles. The possibility of formation of
larger size particles was mainly because of the insufficient
capping effect.33 The molecules of LE responsible for capping
effect are discussed in later sections. The as-synthesized NPs in
the presence of excess HAuCl4 and water washed AuNPs aer
removing excess HAuCl4 was observed under SEM (Fig. S1†).
Microscopic analysis of as-synthesized AuNPs from Fig. S1a†
displays the self-assembled pattern having branches at 90ꢂ
when the drop was dried on a glass surface. Interestingly, the
patterned behaviour was absent when the mentioned AuNPs
were separated by centrifugation and are subjected to water
wash as shown in Fig. S1b.†
during the reaction at different time intervals are shown in
Fig. 2(a). The rise in the maximum absorbance value is initially
very fast because of the formation of AuNPs and then nally
saturated aer 30 min. This result indicates, ꢀ30 min time is
required for the completion of the reduction reaction. The
change in absorbance as a function of time at a constant
wavelength of 543 nm is shown in Fig. 2(b) and the colour
change due to nanoparticles was shown in Fig. 2(c). This spec-
tral behaviour indicates the reduction reaction and subsequent
nuclei generation processes are completed within ꢀ25 min and
followed by growth.
3.3 XRD and electron microscopic characterization of
nanoparticles
Self-assembled structures of AuNPs on the glass surface,
shown in Fig. S1a† are because of excess residual HAuCl4 upon
evaporation on glass substrate under a 60 W incandescent bulb.
The temperature of ꢀ125 ꢂC might assist in the assembled
pattern of residual HAuCl4 in the presence of low strength LE.
The TEM micrographs of AuNPs prepared from 1 mM HAuCl4
and 1.5% LE is shown in Fig. 3(a)–(c). The particles formed
in this green route are mostly spherical to little irregular
shapes having almost homogeneous size distribution with
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 28645–28652 | 28647

View Article Online
RSC Advances
Paper
3.4 FT-IR analysis for functional groups identication
The FT-IR spectroscopy was done in ATR mode to identify the
responsible functional groups of LE in the Au3+ ions reduction
and capping of AuNPs. Fig. 4 shows the spectra of pure LE
(curve I), supernatant aer reduction of Au salt (curve II), water
washed AuNPs (curve III), and NaOH washed AuNPs (curve IV).
The absorbance in the range of 1500–900 cmꢃ1 from the IR
spectra of curves I and II, reveal that the characteristic of
polyphenolic tannin molecules present in LE.35 The pure LE
shows (curve I) the peak bands at 1521, 1458, 1397, 1340, 1320,
1273, 1235, 1163, and 1034 cmꢃ1 which are exclusively for the
presence of tannins.36 Additionally, the presence of broader
peaks in the regions of ꢀ1350 cmꢃ1 and between 1290 and
1150 cmꢃ1 indicate that tannins of LE are ‘hydrolysable’ type.
These two peak regions can be assigned to C–O stretching (at
1370 cmꢃ1) and OH deformation vibrations (with absorptions at
1273, 1235 and 1163 cmꢃ1).36 The presence of these molecules
was also conrmed before in our previous study.29
The LE aer the reduction (curve II) of HAuCl4 shows almost
similar peak pattern that of curve I with few changes are as
follows: a new peak appears at 637 cmꢃ1 and peak splitting
patterns, and multiple absorptions (indicated with a star) at the
peak regions of 1541, 1652 and 1700 cmꢃ1. These peaks of
ꢀ1500 and 1600 cmꢃ1 are because of aromatic C]C bond of the
aromatic ring and peak at ꢀ1700 cmꢃ1 is because of isolated
and exposed carbonyl groups (C]O) of LE aer the reduction
reaction.37
Fig. 2 (a) UV-visible spectra of AuNPs recorded with the progress of
reaction time using 1.0 mM aqueous HAuCl4 and 1.5% LE (taking LE as a
reference). (b) Particle growth kinetics (absorbance vs. time) at 543 nm
wavelength. (c) The colour change of the LE before (left) and after the
reaction (right) due to AuNPs formation.
Furthermore, IR spectra of the supernatants of water and
NaOH wash AuNPs are shown in curves III and IV respectively.
While comparing curves I and III, signicant changes in peaks
are not observed, especially in the ngerprint region (peaks
1517, 1466, 1398, 1340, 1320, 1273, 1031 cmꢃ1, etc.). This can be
attributed to the adsorption of phenolic compounds such as
tannic acid product on the surface of the water washed AuNPs.
Fig. 3 (a) Low magnification TEM image of AuNPs using 1.0 mM/1.5%
(HAuCl4/LE). (b) and (c) HR-TEM images of AuNPs. Inset of (b), HRTEM-
EDS analysis of the AuNPs. Inset of (c), powder XRD pattern. (d) SAED
pattern of the obtained AuNPs.
an average diameter of ꢀ38.2 ꢁ 10.5 nm. Particle histograms
of TEM images are given in Fig. S2.† The presence of gold was
conrmed by the EDX analysis, as shown in the inset of
Fig. 3(b), and the SAED pattern of AuNPs from HR-TEM
(Fig. 3(d)) shows the rings corresponding to the (111), (200),
(220), and (311) reections of the face-centered-cubic (fcc)
structure of Au. The crystallinity of puried AuNPs was also
conrmed by the powder XRD analysis. The XRD pattern of
AuNPs with 2q peaks at 38.20ꢂ, 44.50ꢂ, 64.68ꢂ, 77.61ꢂ matching
with JCPDS le no: 04-0784 corresponds to the above
mentioned planes and fcc structure, shown in the insert of
Fig. 3(c).
Fig. 4 ATR-FT-IR spectra of the LE (1.5%) before reduction (curve I)
and after reduction (curve II) of Au salt (1.0 mM). AuNPs subjected to
water wash (curve III) and NaOH wash (curve IV) also shown. Curves III,
IV, represent spectrums of centrifuged, repeated water washed, dried
and suspended AuNPs in deionized water.
28648 | RSC Adv., 2014, 4, 28645–28652
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
And curve IV shows no signicant peaks in the ngerprint LE. Between 355 and 500 ꢂC is the second pyrolysis step and
region, which indicates the total removal of capping agent aer contributes to 22.78% weight loss. Finally, above ꢀ500 ꢂC
NaOH treatment of AuNPs.
weight remains constant (42.23%) because of carbonized
residue.38
The TGA pattern of water washed AuNPs (curve II) also
shows three thermal zones similar to that of pure LE. These
transitions are between 65 to 110 ꢂC, 110 to 345 ꢂC, and 346 to
513 ꢂC with weight losses of 3.6, 19.6, and 26.43% respectively.
However, the weight remained above ꢀ515 ꢂC is 50.37%
because of the presence of residual carbon (due to capping
agent) and AuNPs.
The TGA analysis of naked AuNPs obtained aer NaOH wash
(curve III) displayed no distinguished transition steps, unlike to
that of curve I and II. The thermal zone appreciated between 65
to 477 ꢂC with a mass loss of 62.11% attributes to moisture loss
and/or oxidation step to eliminate the adhered volatile frac-
tions. Finally, aer heating at 477 ꢂC the residue was 37.89% of
naked AuNPs. The total weight loss calculated from curve II and
III at 112 to 513 ꢂC is 9.05%, which contributes to the weight of
capping agent present in the total weight of water washed
capped AuNPs.
3.5 Tests for capping effect
FT-IR analysis indicates the presence of polyphenols in LE,
which has a signicant role in the gold ion reduction as well as
stabilization of AuNPs because of capping. To further probe the
capping ability of these molecules, synthesized AuNPs were
washed with water and subsequently treated with NaOH solu-
tion, and then the supernatant solutions were analyzed by UV-
visible spectroscopy. Fig. 5(a) clearly shows the absence of any
distinct peak for the water wash supernatant, whereas NaOH
wash supernatant shows a maximum absorbance at 276 nm;
which indicates the presence of polyphenols released from
AuNPs surface.
The strong adsorption ability of polyphenols onto AuNPs can
also be supported by TGA analysis. The TGA analysis of pure LE,
AuNPs (water and NaOH washed) are presented in Fig. 5(b). The
thermal decomposition of LE – (curve I) shows majorly three
ꢂ
zones. The rst step of the transition is between 65 to 115 C
with a mass loss of 4.56%, which can be attributed to the loss of
moisture and preliminary oxidation steps and elimination of
3.6 Zeta potential (z) studies
volatile fractions. The second step of the transition where a The z potentials of the LE solutions (before and aer the reac-
weight loss of 30.43% was occurred between 115 to 354 ꢂC, tion) and AuNPs (as-synthesized, water washed, and NaOH
attributed to rst pyrolysis step of the adsorbed molecules of treated) were measured to know the presence of capping agent
adsorbed on the particle surface. One can see from the Fig. S3†
that all samples show negative z potential. While comparing z
potentials, LEtreat shows a slight lower potential than LEfresh
because of the absence of some active tannin molecules and the
presence of extra H+ ions from HAuCl4. Further, the negative z
potential values of AuNPs show the following order: within LE <
water wash < NaOH wash. The pattern observed is mainly
because of capping effect of tannin molecules on the surface of
the AuNPs. Repeated water wash NPs show more negative value
than that in pure LE because of the removal of loosely bound
tannin molecules, and NaOH wash particles show highest z
potential value because of complete removal of the adsorbed
molecules from the particle surface. The observed z potential
value aer NaOH wash (ꢃ42.9 ꢁ 2.45 mV) is close to the
reported value of pure AuNPs which are around ꢀꢃ41 to 44 mV
at neutral pH.39,40 Moreover, it has also been found that the z
potential value of repeated methanol washed AuNPs is ꢃ42.4 ꢁ
1.9 mV is close to that of NaOH wash (Fig. S3†). So the adsorbed
phenolic compounds can also be removed by repeated meth-
anol wash too.
3.7 Capped AuNPs as thiamine sensors
A solution of 12 mM of thiamine was added to different
concentrations polyphenols capped AuNP suspensions
(9.25, 27.75, 46.25, 92.50 and 185 mg mLꢃ1) in a mixture of
water : methanol (1 : 2) solutions and mixed thoroughly. The
nal mixtures were then excited at 356 nm wavelength (excita-
tion spectra shown in Fig. S4a†), and the respective emission
spectra were measured as shown in Fig. 6. From the gure, it is
very clear that the emission spectra are very prominent for all
Fig. 5 (a) UV-visible absorbance spectra of supernatants of water
washed and NaOH (0.5 M) treated AuNPs. (b) TGA of pure LE, AuNPs
after water wash and NaOH wash.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 28645–28652 | 28649

View Article Online
RSC Advances
Paper
Fig. 6 (a) Fluorescence spectra of thiamine (Thi) 12 mM with tannin capped AuNPs from ꢀ9.25 to 185.0 mg mLꢃ1 concentrations. (b) Spectra of
thiamine (0.5 to 3.0 mM) exposed to 92.5 mg mLꢃ1 capped AuNPs and insert of (b) displays the fluorescence signal at fixed lemi of 425 nm with
respect to increase in thiamine concentration. Fluorescence signal spectrum devoid of AuNPs with only capping agent (10.0 mg mLꢃ1) exposed to
thiamine (0.5 mM) is also shown in (b).
different doses of AuNPs and the intensity increases linearly at stronger for in situ synthesis, and which is also essential for the
lmax of 425 nm with increasing concentration of AuNPs dose.
detection of thiamine. The enhancement of uorescence
Furthermore, Fig. 6(b) also shows intense uorescent peaks intensity in the presence of capped AuNPs is attributed to the
with varying thiamine concentrations (0.5 to 3.0 mM) for a behavior of distance dependent uorescence resonance elec-
constant dose of AuNPs (92.50 mg mLꢃ1). Form the gure it can tron transfer (FRET).41 In general; AuNPs are excellent FRET-
be observed that a constant spectral pattern for 2.0 and 3.0 mM based quenchers because of their extraordinary high extinction
thiamine concentrations, and this infers that there is a coefficient and broad energy absorption bandwidth in the
maximum level of the detection limit (2 mM) for a particular visible region.42 Usually in quenching effect, initially a donor
dose of capped AuNPs (92.50 mg mLꢃ1). This behaviour may be uorophore absorbs the energy due to the excitation of incident
because of the unavailability of the capping agent for thiamine light and transfer the excitation energy to a nearby acceptor
oxidation. The uorescence signal pattern at lmax (425 nm) chromophore (here AuNPs).
clearly indicates the threshold level of thiamine detection is
about ꢀ1.5 mM by 92.50 mg mLꢃ1 of AuNPs dose (Fig. 6(b)
insert). It is also interesting to observe that the uorescence
signal was enhanced by the use of AuNPs in combination
with capping agent which is responsible for the thiamine
oxidation. When the only LE equivalent to that associated with
92.50 mg mLꢃ1 AuNPs i.e. ꢀ10.0 mg mLꢃ1 (revealed from TGA
analysis) was individually exposed to thiamine (0.5 mM), an
emission intensity of 4.2 at 425 nm wavelength was observed;
which is ꢀ3.5 times lower than the capped AuNPs of
92.50 mg mLꢃ1 (Fig. 6(b)). The uorescence signal detection by
pure LE devoid of AuNPs also followed a linear pattern as shown
in Fig. S4b.†
D (donor) + hn / D*
D* + A (acceptor) / D + A*
A* / A + hn0
(2)
(3)
(4)
In this case, generally two uorophores (donor and acceptor)
must be in the close proximity to one another. The intensi-
cation in uorescence signal is observed aer thiamine oxida-
tion by capped AuNPs, which is a reverse behaviour of
quenching effect. The signal enhancement is acceptable as the
particle and uorophore are separated by capping agent which
in turn reduced the quenching effect and probably enhanced
the rate of uorescence decay.41
As the AuNPs surface is well capped by polyphenols of LE, it
is possible to maintain this distance easily to get FRET. The
signal enhancement is highly advantageous for the develop-
ment of high sensitive thiamine sensors. It is also worthy to
note that only pure thiamine or naked AuNPs in the presence of
thiamine (12 mM) exhibited no signicant uorescent peak at
425 nm (Fig. S6a†) indicates the absence of thiochrome gener-
ation. The process of thiamine oxidation by capped AuNPs is
illustrated in Scheme 1.
To see the importance of in situ capping by LE polyphenols
and effect of particles size on thiamine detection, ꢀ10 nm
AuNPs were synthesized chemically and tested. The AuNPs were
synthesized by NaBH4 reduction method31 and incubated with
LE for the adsorption of polyphenols. The particles were sepa-
rated, used for thiamine detection, and emission spectra are
shown in Fig. S5.† It has been found that the detection signal is
signicantly lower compared to that of in situ synthesized
particles. In this case, although the particle size is low, poly-
phenol adsorption or capping effect is also low, which in turn
reduce the detection efficiency. Less adsorption of polyphenols
was also conrmed by the z potential measurements. The
observed z potential values for AuNPs prepared by borohydrate
method are ꢃ26 mV (as-prepared) and ꢃ30.9 mV (aer water
wash). From the z potential and uorescence results, we can say
that the stabilization effect by LE molecules towards AuNPs is
The detection procedure is novel because of the simplicity of
the process, detection at neutral pH without long incubation
time. This simple procedure of thiamine detection at ambient
condition was not possible by the reported chemical
routes,20,21,24 and our study shows better detection efficiency
28650 | RSC Adv., 2014, 4, 28645–28652
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Scheme 1 Schematic illustration of thiamine oxidation by capped AuNPs.
than the reported studies, which are able to detect minimum AuNPs (with or without capping) also be used in other appli-
thiamine concentration in the range of 100,22 ꢀ86.4,21 and 50 cations, as well.
mM24 effectively.
Further, as a supporting evidence, we also investigated the
structural changes of thiamine (20 mM) by UV-visible spectro-
References
scopic study (Fig. S6b†) aer the addition of capped AuNPs (2.32
mg mLꢃ1). The UV spectrum of thiamine usually exhibits two
1 G. J. Hutchings, A golden future for green chemistry, Catal.
Today, 2007, 122, 196–200.
distinct absorption maxima at neutral pH and a merged
appearance at acidic pH.23 Fig. S6b† shows UV absorption
spectra before exposure to capped AuNPs with two distinct
absorption peaks characteristic of pure thiamine at 241 and 259
nm wavelengths at pH ꢀ6.0 which in accordance with a repor-
ted study.23 These lmax values shied to 234 and 267 nm
wavelengths aer addition of capped AuNPs, attributed to the
oxidation of the thiazole ring of thiamine. It has been also
found that the reaction time for the conversion of thiamine in
the presence of capped AuNPs to its oxidized product thio-
chrome (specied in uorescent results) is instantaneous. The
structural changes in amine functional group of thiamine are
clearly evident aer oxidation process and visible shis in the
IR absorption region of ꢀ3400 cmꢃ1 was also observed (ESI,
Fig. S7† dotted region). From the gure, peaks at 3489 and 3423
cmꢃ1 (because of absorption by primary amine) were replaced
by a single peak at 3427 cmꢃ1 (because of the secondary amine)
aer oxidation of thiamine.
2 R. Luque, R. S. Varma, J. H. Clark and G. A. Kraus,
Sustainable Preparation of Metal Nanoparticles: Methods and
Applications, Royal Society of Chemistry, 2012.
3 K. N. Thakkar, S. S. Mhatre and R. Y. Parikh, Biological
synthesis of metallic nanoparticles, Nanomedicine, 2010, 6,
257–262.
4 J. A. Dahl, B. L. S. Maddux and J. E. Hutchison, Toward
greener nanosynthesis, Chem. Rev., 2007, 107, 2228–2269.
5 J. H. Clark, Green chemistry: challenges and opportunities,
Green Chem., 1999, 1, 1–8.
6 A. K. Jha, K. Prasad, K. Prasad and A. R. Kulkarni, Plant
system: nature's nanofactory, Colloids Surf., B, 2009, 73,
219–223.
7 S. Girija, Y. L. Balachandran and J. Kandakumar, Plants as
green nanofactories: application of plant biotechnology in
nanoparticle synthesis – A review, Plant Cell Biotechnol.
Mol. Biol., 2009, 10, 79–86.
8 D. A. Giljohann, D. S. Seferos, W. L. Daniel, M. D. Massich,
P. C. Patel and C. A. Mirkin, Gold nanoparticles for biology
and medicine, Angew. Chem., Int. Ed., 2010, 49, 3280–3294.
9 M. C. Daniel and D. Astruc, Gold nanoparticles: assembly,
4 Conclusions
supramolecular
chemistry,
quantum-size-related
In conclusion, we demonstrated a simple, one-pot, economical
properties, and applications toward biology, catalysis, and
and fast green approach to synthesize AuNPs of ꢀ38 nm size
nanotechnology, Chem. Rev., 2004, 104, 293–346.
within 30 min. A stoichiometric proportion of LE (0.3%) to 10 N. A. Begum, S. Mondal, S. Basu, R. A. Laskar and D. Mandal,
HAuCl4 (1 mM) was established for complete reduction process.
The HR-TEM and XRD analysis shows the spherical shape and
crystalline fcc structure of the formed AuNPs. Apart from
Biogenic synthesis of au and ag nanoparticles using aqueous
solutions of black tea leaf extracts, Colloids Surf., B, 2009, 71,
113–118.
reduction process, in situ capping effect of polyphenolic mole- 11 D. Raghunandan, M. D. Bedre, S. Basavaraja, B. Sawle,
cules (tannins) of LE on AuNPs was also supported by FT-IR
spectroscopy, UV-vis spectroscopy, TGA, and zeta potential
studies. The sensing capability of capped AuNPs towards thio-
chrome (an oxidative product of thiamine) by uorescence
S. Manjunath and A. Venkataraman, Rapid biosynthesis of
irregular shaped gold nanoparticles from macerated
aqueous extracellular dried clove buds (Syzygium
aromaticum) solution, Colloids Surf., B, 2010, 79, 235–240.
spectroscopy shows ꢀ0.5 mM or below thiamine concentration 12 W. Liang, T. L. Church and A. T. Harris, Biogenic synthesis of
can be easily detected by a minimum nanoparticle dose of 92.50
photocatalytically active Ag/TiO2 and Au/TiO2 composites,
Green Chem., 2012, 14, 968.
mg mLꢃ1. We strongly believe these results are useful for the
synthesis of bio-compatible polyphenols caped AuNPs, which 13 M. Noruzi, D. Zare and D. Davoodi, A rapid biosynthesis
can be further applicable for sensing of thiamine. The produced
route for the preparation of gold nanoparticles by aqueous
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 28645–28652 | 28651

View Article Online
RSC Advances
Paper
extract of cypress leaves at room temperature, Spectrochim. 28 M. Mandal, N. R. Jana, S. Kundu, S. K. Ghosh,
Acta, Part A, 2012, 94, 84–88.
M. Panigrahi and T. Pal, Synthesis of Au core–Ag shell
type bimetallic nanoparticles for single molecule
detection in solution by SERS method, J. Nanopart. Res.,
2004, 6, 53–61.
14 A. K. Mittal, Y. Chisti and U. C. Banerjee, Synthesis of
metallic nanoparticles using plant extracts, Biotechnol.
Adv., 2013, 31, 346–356.
´
15 O. V. Kharissova, H. V. R. Dias, B. I. Kharisov, B. O. Perez and 29 K. Jagajjanani Rao and S. Paria, Green synthesis of silver
´
V. M. J. Perez, The greener synthesis of nanoparticles, Trends
nanoparticles from aqueous Aegle marmelos leaf extract,
Mater. Res. Bull., 2013, 48, 628–634.
Biotechnol., 2013, 31, 240–248.
16 N. A. Begum, S. Mondal, S. Basu, R. A. Laskar and D. Mandal, 30 A. K. Gangopadhayay and A. Chakravorty, Charge transfer
Biogenic synthesis of Au and Ag nanoparticles using
aqueous solutions of black tea leaf extracts, Colloids Surf.,
B, 2009, 71, 113–118.
spectra of some gold(III) complexes, J. Chem. Phys., 1961,
35, 2206–2209.
31 M. P. Mallin and C. J. Murphy, Solution-phase synthesis of
sub-10 nm Au–Ag alloy nanoparticles, Nano Lett., 2002, 11,
1235–1237.
17 S. S. Shankar, A. Rai, A. Ahmad and M. Sastry, Rapid
synthesis of Au, Ag, and bimetallic Au core–Ag shell
nanoparticles using neem (Azadirachta indica) leaf broth, J. 32 T. Sakai and P. Alexandridis, Single-step synthesis and
Colloid Interface Sci., 2004, 275, 496–502.
18 V. Armendariz, I. Herrera, J. R. Peralta-videa, M. Jose-
yacaman, H. Troiani, P. Santiago and J. L. Gardea-
stabilization of metal nanoparticles in aqueous pluronic
block copolymer solutions at ambient temperature,
Langmuir, 2004, 20, 8426–8430.
Torresdey, Size controlled gold nanoparticle formation by 33 P. R. Sajanlal, T. S. Sreeprasad, A. K. Samal and T. Pradeep,
Avena sativa biomass: use of plants in nanobiotechnology,
J. Nanopart. Res., 2004, 6, 377–382.
Anisotropic nanomaterials: structure, growth, assembly, and
functions, Nano Rev., 2011, 2, 5883.
19 G. F. Combs Jr, The Vitamins, Academic Press, 2007.
20 A. F. Makarchikov, B. Lakaye, I. E. Gulyai, J. Czerniecki,
B. Coumans, P. Wins, T. Grisar and L. Bettendorff,
Thiamine triphosphate and thiamine triphosphatase
34 N. N. Jason, R. Ghosh Chaudhuri and S. Paria, Selfassembly
of colloidal sulfur particles inuenced by sodium oxalate salt
on glass surface from evaporating drops, So Matter, 2012, 8,
3771–3780.
˜
´
activities: from bacteria to mammals, Cell. Mol. Life Sci., 35 L. Falcao and M. E. M. Araujo, Tannins characterization in
2003, 60, 1477–1488.
historic leathers by complementary analytical techniques
ATR-FTIR, UV-vis and chemical tests, J. Cult. Herit., 2013,
14, 499–508.
´
´
´
´
21 P. L. Lopez-de-Alba, L. Lopez-Martınez, V. Cerda and
´
J. Amador-Hernandez, Simultaneous determination and
classication of riboavin, thiamine, nicotinamide and 36 L. Laghi, G. P. Parpinello, D. D. Rio, L. Calani, A. U. Mattioli
pyridoxine in pharmaceutical formulations, by UV-visible
spectrophotometry and multivariate analysis, J. Braz. Chem.
Soc., 2006, 17, 715–722.
and A. Versari, Fingerprint of enological tannins by multiple
techniques approach, Food Chem., 2010, 121, 783–788.
37 J. Coates, Interpretation of Infrared Spectra, a Practical
Approach, Wiley Online Library, 2000.
22 M. Fujiwara and K. Matsui, Determination of thiamine by
thiochrome reaction, Anal. Chem., 1953, 25, 810–812.
23 S. Y. Reddy, R. Pullakhandam and B. Dinesh Kumar,
Thiamine reduces tissue lead levels in rats: mechanism of
interaction, BioMetals, 2009, 23, 247–253.
´
´
38 M. A. Pantoja-Castro and H. Gonzalez-Rodrıguez, Study by
infrared spectroscopy and thermogravimetric analysis of
tannins and tannic acid, Rev. Latinoam. Quim., 2011, 39,
107–112.
24 K. S. Byadagi, D. V. Naik, A. P. Savanur, S. T. Nandibewoor 39 S. Park, M. Park, P. Han and S. Lee, The effect of pH-adjusted
and S. A. Chimatadar, Ruthenium(III) mediated oxidation
of thiamine hydrochloride by cerium(IV) in perchloric acid
medium: a kinetic and mechanistic approach, React. Kinet.,
Mech. Catal., 2010, 99, 53–61.
gold colloids on the formation of gold clusters over APTMS-
coated silica cores, Bull. Korean Chem. Soc., 2006, 27, 1341–
1345.
40 T. Kim, C.-H. Lee, S.-W. Joo and K. Lee, Kinetics of gold
nanoparticle aggregation: experiments and modeling, J.
Colloid Interface Sci., 2008, 318, 238–243.
25 K. Gold, Some factors affecting the stability of thiamine,
Limnol. Oceanogr., 1968, 13, 185–188.
26 H. K. Penttinen, Differences in thiochrome uorescence 41 K. A. Kang, J. Wang, J. B. Jasinski and S. Achilefu,
produced by thiamine and its mono-, di-, and triphosphate
esters, Acta Chem. Scand., 1976, 30, 659–663.
27 K. L. Kelly, E. Coronado, L. L. Zhao and G. C. Schatz, The
Fluorescence manipulation by gold nanoparticles: from
complete quenching to extensive enhancement, J.
Nanobiotechnol., 2011, 9, 16.
optical properties of metal nanoparticles: the inuence of 42 P. C. Ray, A. Fortner and G. K. Darbha, Gold nanoparticle
size, shape, and dielectric environment, J. Phys. Chem. B,
2002, 107, 668–677.
based FRET assay for the detection of DNA cleavage, J.
Phys. Chem. B, 2006, 110, 20745–20748.
28652 | RSC Adv., 2014, 4, 28645–28652
This journal is © The Royal Society of Chemistry 2014