Accepted Manuscript Title: Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia Author: R. Emmanuel Chelladurai Karuppiah Shen-Ming Chen Selvakumar Palanisamy S. Padmavathy P. Prakash PII: DOI: Reference:
S0304-3894(14)00547-0 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.06.066 HAZMAT 16075
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
22-5-2014 27-6-2014 28-6-2014
Please cite this article as: R. Emmanuel, C. Karuppiah, S.-M. Chen, S. Palanisamy, S. Padmavathy, P. Prakash, Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.06.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia R. Emmanuelb, Chelladurai Karuppiaha, Shen-Ming Chena,* Selvakumar Palanisamya,
ip t
S. Padmavathyc and P. Prakashb, ** a
Department of Chemical Engineering and Biotechnology, National Taipei University of
Post Graduate and Research Department of Chemistry, Thiagarajar College, Madurai-625009,
us
b
cr
Technology, Taipei 106, Taiwan, ROC
Tamilnadu, India.
Department of Zoology and Microbiology, Thiagarajar College, Madurai-625009, Tamilnadu,
an
c
Corresponding Authors:
te
d
M
India.
Ac ce p
*SM. Chen, Fax: +886227025238, Tel: +886227017147, E-mail: [email protected] **P. Prakash, Fax: +914522312375, Tel: +919842993931, E mail: [email protected]
1
Page 1 of 30
Abstract The present study involves a green synthesis of gold nanoparticles (Au-NPs) using Acacia Nilotica twig bark extract at room temperature and trace level detection of one of the
ip t
hazardous materials, viz. nitrobenzene (NB) that causes Methemoglobinaemia. The synthesis protocol demonstrates that the bioreduction of chloroauric acid leads to the formation of gold
cr
nanoparticles within 10 min, suggesting a higher reaction rate than any other chemical methods
us
involved. The obtained Au-NPs have been characterized by UV–visible spectroscopy, X-ray diffraction, transmission electron microscopy, Energy-Dispersive X-Ray Spectroscopy and
an
Fourier Transform Infrared Spectroscopy. The electrochemical detection of NB has been investigated at the green synthesized Au-NPs modified glassy carbon electrode by using
M
differential pulse voltammetry (DPV). The Au-NPs modified electrode exhibits excellent
d
reduction ability towards NB compared to unmodified electrode. The developed NB sensor at
te
Au-NPs modified electrode displays a wide linear response from 0.1 – 600 µM with high sensitivity of 1.01 µAµM-1 cm-2 and low limit of detection of 0.016 µM. The modified electrode
Ac ce p
shows exceptional selectivity in the presence of ions, phenolic and biologically coactive compounds. In addition, the Au-NPs modified common metal electrode exhibits an outstanding recovery results towards NB in various real water samples. Keywords: Gold nanoparticles, green synthesis, nitrobenzene, electrocatalysis, differential pulse voltammetry
2
Page 2 of 30
1. Introduction In recent times nanoparticles are widely applied in electronic and optical displays, super computers, fabrication of advanced materials, energy storage devices, chemical and biosensors
ip t
and biomedical devices [1]. Particularly gold nanoparticles (Au-NPs) find applications in
cr
biological and photo-thermal therapeutic contexts due to their broad spectrum of physical, optical and chemical properties which have been well documented in selected reviews [2, 3].
us
There are various physical and chemical methods which have been adopted for the synthesis of gold and other metal nanoparticles. In particular the chemical synthesis protocol cannot avoid the
an
use of toxic chemicals [4, 5]. Therefore, there is an urgent need to develop green procedures
M
devoid of toxic chemicals procedure.
On the other hand, nitroaromatic compounds are suspected as carcinogenic compounds
d
and they have been widely used for the fabrication of dyes, pharmaceuticals, explosives and
te
pesticides [6]. Particularly nitrobenzene (NB) has been certified as one of the priority pollutants by United States Environmental Protection Agency [7]. In the toxological profile for
Ac ce p
nitrobenzene released by the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service, NB has been categorized as one of the hazardous pollutants, exposure to which can result in a blood condition called Methemoglobinaemia [8]. This condition affects the ability of the blood to carry oxygen. Following such an exposure, the skin may turn a bluish color. This may be accompanied by nausea, vomiting and shortness of breath. Drinking alcoholic beverages may result in nitrobenzene entering our body at a faster rate, no matter how we are exposed, which can cause a wide variety of harmful health effects to exposed persons. If the exposure level is extremely high, NB can cause coma and possibly death unless prompt medical treatment is received [9]. The strong electron-withdrawing nature of nitro group keeps the NB more stable
3
Page 3 of 30
resulting in poor biodegradable nature in the waste water [10]. Therefore, the trace level detection of NB in environmental and biological samples is imperative. Notwithstanding various analytical methods have been used for the sensitive
ip t
determination of NB, including fluorescence, high-performance liquid chromatography, gas chromatography and luminescence [11-15], the electrochemical methods have more advantages
cr
such as low-cost, highly sensitive and user friendly than that of aforementioned traditional
us
methods [16]. More recently our group has found that green synthesized metal nanoparticles have excellent electrocatalytic ability towards nitro group substituted aromatic compounds owing
an
to its high stability, conductivity, biocompatibility, low cytotoxicity and size-related electrochemical properties [17]. The same strategy has been used here to synthesize the shape
M
and size controlled Au-NPs using the twig bark extract of Acacia Nilotica. To the best of our
te
Au-NPs.
d
knowledge, there are no reports available for the determination of NB using green synthesized
In the present work, we describe the green synthesis of Au-NPs by using twig bark
Ac ce p
extract of Acacia Nilotica as a reducing as well as stabilizing agent for the first time. The green synthesized Au-NPs modified electrode exhibits excellent electrochemical detection ability towards NB compared to unmodified electrode. The modified electrode shows a wide linear range, low detection limit and good stability along with good selectivity in pH 7.0. In addition, the modified electrode has been successfully employed for the determination of nitroaromatics in various real water samples.
2. Experimental 2.1. Materials and methods
4
Page 4 of 30
The Chloroauric acid (HAuCl4.3H2O) was purchased from Sigma–Aldrich and used as received. The fresh twig bark of Acacia nilotica was collected in Thiagarajar College campus, Madurai, India. Nitrobenzene was purchased from Aldrich and used without purification. The 0.1
ip t
M phosphate buffer pH 7.0 solution (PBS) was prepared by using NaH2PO4 and Na2HPO4 with doubly distilled water. All other chemicals were of analytical grade and used as received. spectrum
was
recorded
by
using
JascoV-560
double-beam
cr
UV–visible
UV
us
spectrophotometer. The XRD spectra was recorded and analyzed for the purified, air-dried gold nanoparticles with X-ray diffraction analysis using a PANalytical X’Pert PRO, PW3050/60 X-
an
ray diffractometer. The size and shape of gold nanoparticles were inspected using transmission electron microscopy (TEM) using JEOL JEM 2100 instrument. The elemental analysis (EDX)
M
was carried out using the JEOL JEM 2100 TEM instrument attached with Energy Dispersive X-
d
ray Analyzer. The IR spectrum was recorded using Shimadzu FT- IR-8201PC instrument.
te
Electrochemical experiments such as cyclic voltammetry and differential pulse voltammetry were performed by using CH instruments model CHI-750A. A conventional three-electrode
Ac ce p
setup consisting of a glassy carbon electrode (GCE) as a working electrode (active surface area = 0.079 cm2), an Ag/AgCl electrode (Sat. KCl) as a reference electrode and a platinum wire with 0.5 mm diameter as a counter electrode was used for electrochemical experiments. 2.2. Preparation of bark extract
The fresh twig bark was finely cut into small pieces and washed several times with deionized water to remove any dust. The 0.250 g of twig bark of Acacia nilotica was boiled with 100 mL of deionized water at 900 C for 15 min at room temperature and the extract was twice filtered using Whatmann filter paper. The filtered twig bark extract was pale yellow in color and
5
Page 5 of 30
it was used as reducing as well as stabilizing agent for the synthesis of Au-NPs. The filtrate was stored in the refrigerator at 40 C for further experiments. 2.3. Green synthesis of Au-NPs
ip t
In a typical experimental method, 30 mL aqueous solution of 1 mM HAuCl4 was mixed with different volume ratio (10 mL, 20 mL and 30 mL) of Acacia nilotica bark extract in 250 mL
cr
flask at room temperature. The change in the color of the solution from pale yellow to violet
us
confirms the successful formation of Au-NPs. The formation of Au-NPs was further confirmed by excitation of Surface Plasmon vibration (See Fig. 3A). The entire reduction process is
an
completed within 10 min and there is no further color change in aqueous solution which indicates that the formation of Au-NPs is completed within 10 min. The effect of bark extract
M
concentration on gold nanoparticles was recorded by UV-Vis spectra. The stable Au-NPs
d
aqueous solutions were prepared by dispersing 3 mg mL-1 Au-NPs (optimum concentration) in
te
doubly distilled water and sonicated for 45 min. 2.4. Fabrication of Au-NPs modified electrode
Ac ce p
The pre-cleaned GCE surface was dried at room temperature. About 10 µL (optimized concentration) of the prepared Au-NPs dispersion was drop-casted on GCE surface and dried at room temperature. The fabricated Au-NPs modified GCE was gently washed with water to remove the loosely attached Au-NPs at the electrode surface. The Au-NPs modified electrode was used for further experiments and stored at room temperature under dry condition when not in use.
6
Page 6 of 30
3. Results and discussion 3.1. Characterization of green synthesized Au-NPs The surface morphology and size of the green synthesized Au-NPs were characterized by
ip t
TEM. As shown in Fig. 1A, the green synthesized Au-NPs using optimum concentration (30 mL) of bark extract with the clear lattice fringes having a spacing of 0.23 nm reveals that the
cr
growth of Au-NPs occurs preferentially on the (1 1 1) plane. The inter-planar distance of the Au-
us
NPs (1 1 1) plane is in agreement with the (1 1 1) d-spacing of bulk gold (0.2355 nm) as reported previously [18]. The formation of Au-NPs was further confirmed by using EDX analysis. The
an
corresponding EDX profile of Au-NPs is shown in Fig. 1B. A strong Au metallic peak appears at 2 keV, which is attributed to the presence of Au-NPs. Fig. 1C shows the selected area electron
M
diffraction pattern (SAED) of the green synthesized Au-NPs. The SAED pattern exhibits a bright
d
circular rings corresponding to the (1 1 1) (2 0 0) (2 2 0) (3 1 1) (2 2 2) planes, which further
te
confirms that the Au-NPs are highly crystalline. Fig. 1D shows the XRD pattern of green synthesized Au-NPs. The five distinct diffraction peaks are observed at 38.10, 39.350, 44.40, 640
Ac ce p
and 770, which can be indexed as (1 1 1) (2 0 0) (2 2 0) (3 1 1) and (2 2 2) reflections of fcc structured metallic gold respectively (JCPDS, file no PDF#04-0784). Similar results have been documented in the earlier report on the synthesis of gold nanoparticles using leaf extracts of Cassia auriculata, Coriander, Piper betle and Cinnamomum zeylanicum [19-22]. Furthermore the size of synthesized Au-NPs was calculated using Debye–Scherrer equation which shows that the nanoparticles are in the average size of 30 nm. Fig. 2 shows the TEM image of Au-NPs synthesized from different concentrations of bark extract (A= 10 mL, B= 20 mL, C = 30 mL and D= 40 mL) with 1 mM chloroauric acid. The difference in sizes and shapes is possibly due to the fact that the nanoparticles are formed with
7
Page 7 of 30
different bark extract concentrations. It can be seen that the formation of stable gold nanoparticles takes place predominantly with unshaped/quasi-spherical and anisotropic particles with an average size of 10 to 50 nm (Fig. 2A). When the concentration of bark extract is
ip t
increased to 20 mL, the Au-NPs exhibit unshaped/quasi-spherical particles which are more abundant than the anisotropic shapes (Fig. 2B). Whereas using somewhat higher extract
cr
concentration of bark (30 mL), the formed Au-NPs are predominantly hexagonal in morphology
us
with the diameter of 20 - 50 nm (Fig. 2C). This shows that an excess amount of the extract reduces the chloroaurate ion effectively. However, the extract concentration more than 30 mL
an
does not have any further effect on the reduction of metal ions (Fig. 2D). The sufficient biomolecules in extract act as capping agent and stabilizing agents. Lower quantities of the
M
extract reduce the chloroaurate ions, but do not protect most of the quasi-spherical nanoparticles
d
from aggregating because of the deficiency of biomolecules to act as protecting agents. Similar
te
result to ours shows that the comparatively excess of the extract in volume is responsible for the synthesis of quasi-spherical nanoparticles [23].
Ac ce p
UV–visible spectroscopy is an important technique to ascertain the formation and stability of metal nanoparticles in aqueous solution and it is sensitive to several factors like particle size, shape and particle–particle interaction with the medium and local refractive index. After the addition of bark extract, the color of chloroauric acid turns from pale yellow to violet and it is an obvious result that chloroaurate ions are completely reduced to form Au-NPs. The color change arises because of the surface plasmon vibrations with Au-NPs which is absent in bulk material [24]. Fig. 3A shows the UV–visible spectra of the gold colloids obtained after 10 min of bioreduction using 10 mL (a), 20 mL (b) and 30 mL (c) bark extract. The surface Plasmon resonance (SPR) band of colloid gold with 10 mL bark extract appearing around 585 nm is
8
Page 8 of 30
attributed to the formation of large anisotropic like spherical / hexagonal shaped nanoparticles. The Plasmon bands of Au-NPs are broad with an absorption tail in the longer wavelength region that extends well into the near infrared region for colloids gold (a). The broad absorption peak
ip t
between 500 nm to 600 nm in case of Au-NPs is due to transverse interaction of radiance. The band becomes narrower with a shift towards lower wavelength as the volume of extract is
cr
increased (10 to 30 mL). The fairly sharp SPR is observed at 549 nm which is indicative of
us
almost hexagonal nano particles (c). It is evident that as the quantity of extract is increased, the bandwidth of SPR decreases with a shift of the peak towards lower wavelength. Similar results
an
have been reported in Au-NPs synthesis using coriander leaf, apiin, edible mushroom, honey, Hibiscus rosa sinensis and Cinnamomum zeylanicum [25-28].
M
The FTIR measurements are carried out to find the possible molecules present in the bark
d
of Acacia Nilotica which are responsible for the reduction of chloroaurate ion and stabilization of
te
the synthesized nanoparticles. Fig. 3B shows the typical FTIR spectra of Acacia nilotica bark (a) after and (b) before encapsulation of gold. It can be clearly seen in curve b that the broad and
Ac ce p
intense peak at 3432 cm−1 is due to the O–H stretching vibration of hydroxyl functional groups of poly phenols and alcohols and -NH stretching vibrations of amide (II) (or) amine, C-H stretching vibrations of aldehydic amine group appear at around 2917 and 2851 cm−1, C=O stretching vibrations of esters appear at 1745 cm−1, C=O stretching vibration of carbonyl and carboxylic group of amide-I appear at 1633 cm−1, -N-H stretching vibrations of the amide-II linkage of proteins appear around 1521 cm−1, methylene scissoring vibrations of the proteins around 1452 cm−1, C–O stretching vibration of polyols around 1219 cm−1 and C-N stretching vibration of primary amine at 1018 cm−1. All these data reveal the presence of high concentrated alcohol or phenol, aromatic amines, amide (I) and (II) of proteins, aldehyde, polyol and
9
Page 9 of 30
methylene groups along with the Au-NPs. The curve (a) illustrates the peak of treated crushed bark in which a peak at 3432 cm−1 is due to the O–H stretching vibration of phenols or high concentrated alcohols. After the encapsulation of chloroaurate ion, the peak intensity decreases
ip t
suggesting the possible involvement of above mentioned groups. The sharp and narrow peaks occurring at 2917 and 2851 cm−1 are due to C-H stretching vibrations of aldehyde. After the
cr
encapsulation of chloroaurate ion, the intensities of these two peaks become weaker and shift
us
from 2917 to 2928 cm−1, 2851 to 2844 cm−1, illustrating the involvement of aldehydic groups in the nanoparticles synthesis. The sharp peak appearing at 1745 cm−1 is due to C=O stretching of
an
ester, the peak at 1633 cm−1 is due to C=O stretching vibration of carbonyl and carboxylic groups in amide-I and the peak at 1521 cm−1 is due to –N-H stretching vibration of amide-II
M
linkage of proteins. The peak appearing at 1452 cm−1 is due to methylene scissoring vibrations of proteins. After the encapsulation of chloroaurate ion, the intensities of these three peaks become
te
d
weaker and shift from 1745 to 1782 cm−1, 1633 to 1638 cm−1 and 1018 to 908 cm−1, suggesting that the C=O, –N-H and methylene groups are sensitive indicators to conformational changes in
Ac ce p
the secondary structure of the protein and these groups are involving in the synthesis of nanoparticles. The peak at around 1219 cm−1 is due to C–O stretching vibration of polyols. After the encapsulation of chloroaurate ion, the intensity of the peak shifts from 1219 to 1236 cm−1 indicating that polyols also involve in the synthesis. The peak at around 1018 cm−1 is due to the C-N stretching vibration of primary amines [29] which after bio reduction shifts from 1018 to 986 cm−1, indicating possible involvement of primary amines during nanoparticles synthesis. As indicated by the FTIR data, the plant Acacia nilotica bark extract has been reported to contain phytochemicals such as tannins, terpenoids, alkaloids, saponins and glycosides and the functional groups like high concentrated alcohol or phenol, amine, amide (I) and (II), aldehydes,
10
Page 10 of 30
polyol and methylene groups. The above mentioned functional groups are mainly derived from water-soluble heterocyclic compounds, which are responsible for reduction and stabilization of gold nanoparticles. The tannins are active constituents in Acacia Nilotica bark and it is rich in
ip t
phenolics with sufficient number of hydroxyl and carboxyl groups, which possesses oxidationreduction abilities to bind metals and inactivate them from chelation. The general chelating
cr
ability of phenolic compounds is probably related to the high nucleophilic character of the
us
aromatic rings rather than to specific chelating groups within the molecule [30]. From the above results, it can be suggested that mostly the hydroxyl groups are involving in the reduction of Au
M
AuCl4 − + 3R–OH → Au0 + 3R–O + 3H+ + 4Cl−
an
(III) to Au (0) through oxidation of hydroxyl to carbonyl group, which can be represented as:
d
3.2. Electrochemical behavior of NB at Au-NPs modified electrode
te
In order to evaluate the electrochemical behavior of NB, the cyclic voltamograms were performed in the presence and absence of NB at different modified electrodes. Fig. 4A shows the
Ac ce p
cyclic voltammetric response of bare (a) and Au-NPs (b) modified electrodes in the presence of 300 µM NB in PBS at the scan rate of 50 mV s-1. The Au-NPs modified electrode does not show any obvious reduction peak response in the absence of NB (curve b), clearly suggesting that the Au-NPs modified electrode is electrochemically inactive at this potential window in PBS. A sharp irreversible reduction peak is observed at -0.647 V in the presence of 300 µM NB, which is attributed to the direct reduction of NB to phenylhydroxylamine with four electrons and protons transfer process. On the reverse scan, a less intense oxidation peak appears at 0.274, which is attributed to the irreversible oxidation of phenylhydroxylamine to nitrazobenzene. The electrochemical reduction mechanism of NB is well studied and reported early that
11
Page 11 of 30
electroreduction of NB to aniline occurs in acidic medium and phenylhydroxylamine in neutral and basic medium [31]. The overall electrochemical mechanism of reduction of NB is similar to that of previously reported literatures [31, 32]. Fig. 4B shows the DPVs of bare (a) and Au-NPs
ip t
modified electrodes (b) in the presence of 50 µM NB in PBS. It can be seen that the modified electrode shows 3 fold enhanced current response with -0.08 V negative shifts for NB than that
cr
of bare GCE. The results clearly indicate that Au-NPs modified electrode is more suitable for the
us
determination of NB compared to unmodified electrode. The good electrocatalytic ability of the modified electrode towards NB is attributed to the excellent conductivity and large active sites of
an
Au-NPs. The schematic representation for the fabrication of Au-NPs from bark extract and its mechanism of NB reduction is shown in Fig. 5.
M
The effect of scan rate also was studied by using cyclic voltammetry by varying different
d
sweeping rates toward the reduction of NB. Fig. 6A shows the cyclic voltammetry response of
te
Au-NPs modified electrode in PBS containing 300 µM NB at different scan rates (10 to 500 mVs-1). The reduction peak current response of NB has a linear dependence with the square root
Ac ce p
of scan rate from 10–500 mV s-1 (inset). The result indicates that the electrochemical reduction of NB at Au-NPs modified electrode is a typical diffusion-controlled process. The pH is another important parameter that affects the analytical parameters of Au-NPs modified electrode towards the reduction of NB. Hence, the cyclic voltammograms were performed at Au-NPs modified electrode in the presence of 300 µM containing different pH solutions (3 to 11) at the scan rate of 50 mV s-1. Fig. 6B shows the calibration plot of effect of different pH vs reduction peak current response of NB. It can be seen that the maximum reduction peak current response of NB is observed at pH 7 when compared to other pH solutions. The change of the solution little above
12
Page 12 of 30
and below pH 7 decreases the cathodic peak current, which is due to the effect of hydrogen ion on the reduction of NB. Hence, pH 7 is found to be an optimum pH for the detection of NB.
ip t
3.3 Electrochemical determination of NB
To verify the feasibility of the determination of NB at Au-NPs modified electrode, the
cr
DPV was performed for the different concentrations of NB. Fig. 7 shows the DPV of Au-NPs
us
modified electrode toward different concentrations of NB in PBS. Under optimized conditions, the reduction peak current of NB increases at Au-NPs modified electrode with the increase of
an
NB concentration. It can be seen from Fig. 7 inset that the reduction peak current of NB linearly increases over the NB concentrations ranging from 0.1 to 600 µM with the correlation
M
coefficients of 0.9983. The sensitivity is calculated from the obtained linear regression equation
d
as 1.01 ± 0.065 µAµM-1 cm-2. The limit of detection (LOD) is estimated as 0.016 µM [(3 * Sd of
te
the blank response)/slope of the calibration plot (Sd = 0.214 nA and slope = 0.0803]. The obtained LOD and sensitivity are more comparable than that of previously reported literature for
Ac ce p
the detection of NB using electrochemical methods. The analytical performance of proposed AuNPs modified electrode (3 successive measurements) has been compared with previously reported modified electrodes for NB and the comparative results are shown in Table. ST1. From the Table. ST1, it is evident that the Au-NPs modified electrode exhibits a better analytical performance towards NB in terms of sensitivity and LOD than that of previously reported modified electrodes for NB determination. The excellent electrocatalytic activity of Au-NPs modified electrode towards NB is attributed to the good conductivity and large active sites of the Au-NPs, which increase the electrocatalytic activity for NB and prevent fouling of oxidized products on the electrode surface.
13
Page 13 of 30
3.4 Effect of potentially interfering substances The selectivity of the modified electrode is more important and plays a key role for further practical applications. In order to investigate the selectivity of the Au-NPs modified
ip t
electrode towards the electrochemical reduction of NB in the presence of phenol derivatives, nitro aromatic compounds and inorganic metal ions, the Au-NPs modified electrode was
cr
evaluated by DPV for the detection of 10 µM NB in the presence of 100 fold concentration of
us
inorganic metal ions and biologically coactive species, 20 fold concentrations of phenolic derivatives and nitroaromatic compounds. The obtained results are shown in Fig. 8. It can be
an
seen from Fig. 8 that common metal ions and biologically coactive compounds almost have no effect (
S0304-3894(14)00547-0 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.06.066 HAZMAT 16075
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
22-5-2014 27-6-2014 28-6-2014
Please cite this article as: R. Emmanuel, C. Karuppiah, S.-M. Chen, S. Palanisamy, S. Padmavathy, P. Prakash, Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.06.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Green synthesis of gold nanoparticles for trace level detection of a hazardous pollutant (nitrobenzene) causing Methemoglobinaemia R. Emmanuelb, Chelladurai Karuppiaha, Shen-Ming Chena,* Selvakumar Palanisamya,
ip t
S. Padmavathyc and P. Prakashb, ** a
Department of Chemical Engineering and Biotechnology, National Taipei University of
Post Graduate and Research Department of Chemistry, Thiagarajar College, Madurai-625009,
us
b
cr
Technology, Taipei 106, Taiwan, ROC
Tamilnadu, India.
Department of Zoology and Microbiology, Thiagarajar College, Madurai-625009, Tamilnadu,
an
c
Corresponding Authors:
te
d
M
India.
Ac ce p
*SM. Chen, Fax: +886227025238, Tel: +886227017147, E-mail: [email protected] **P. Prakash, Fax: +914522312375, Tel: +919842993931, E mail: [email protected]
1
Page 1 of 30
Abstract The present study involves a green synthesis of gold nanoparticles (Au-NPs) using Acacia Nilotica twig bark extract at room temperature and trace level detection of one of the
ip t
hazardous materials, viz. nitrobenzene (NB) that causes Methemoglobinaemia. The synthesis protocol demonstrates that the bioreduction of chloroauric acid leads to the formation of gold
cr
nanoparticles within 10 min, suggesting a higher reaction rate than any other chemical methods
us
involved. The obtained Au-NPs have been characterized by UV–visible spectroscopy, X-ray diffraction, transmission electron microscopy, Energy-Dispersive X-Ray Spectroscopy and
an
Fourier Transform Infrared Spectroscopy. The electrochemical detection of NB has been investigated at the green synthesized Au-NPs modified glassy carbon electrode by using
M
differential pulse voltammetry (DPV). The Au-NPs modified electrode exhibits excellent
d
reduction ability towards NB compared to unmodified electrode. The developed NB sensor at
te
Au-NPs modified electrode displays a wide linear response from 0.1 – 600 µM with high sensitivity of 1.01 µAµM-1 cm-2 and low limit of detection of 0.016 µM. The modified electrode
Ac ce p
shows exceptional selectivity in the presence of ions, phenolic and biologically coactive compounds. In addition, the Au-NPs modified common metal electrode exhibits an outstanding recovery results towards NB in various real water samples. Keywords: Gold nanoparticles, green synthesis, nitrobenzene, electrocatalysis, differential pulse voltammetry
2
Page 2 of 30
1. Introduction In recent times nanoparticles are widely applied in electronic and optical displays, super computers, fabrication of advanced materials, energy storage devices, chemical and biosensors
ip t
and biomedical devices [1]. Particularly gold nanoparticles (Au-NPs) find applications in
cr
biological and photo-thermal therapeutic contexts due to their broad spectrum of physical, optical and chemical properties which have been well documented in selected reviews [2, 3].
us
There are various physical and chemical methods which have been adopted for the synthesis of gold and other metal nanoparticles. In particular the chemical synthesis protocol cannot avoid the
an
use of toxic chemicals [4, 5]. Therefore, there is an urgent need to develop green procedures
M
devoid of toxic chemicals procedure.
On the other hand, nitroaromatic compounds are suspected as carcinogenic compounds
d
and they have been widely used for the fabrication of dyes, pharmaceuticals, explosives and
te
pesticides [6]. Particularly nitrobenzene (NB) has been certified as one of the priority pollutants by United States Environmental Protection Agency [7]. In the toxological profile for
Ac ce p
nitrobenzene released by the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service, NB has been categorized as one of the hazardous pollutants, exposure to which can result in a blood condition called Methemoglobinaemia [8]. This condition affects the ability of the blood to carry oxygen. Following such an exposure, the skin may turn a bluish color. This may be accompanied by nausea, vomiting and shortness of breath. Drinking alcoholic beverages may result in nitrobenzene entering our body at a faster rate, no matter how we are exposed, which can cause a wide variety of harmful health effects to exposed persons. If the exposure level is extremely high, NB can cause coma and possibly death unless prompt medical treatment is received [9]. The strong electron-withdrawing nature of nitro group keeps the NB more stable
3
Page 3 of 30
resulting in poor biodegradable nature in the waste water [10]. Therefore, the trace level detection of NB in environmental and biological samples is imperative. Notwithstanding various analytical methods have been used for the sensitive
ip t
determination of NB, including fluorescence, high-performance liquid chromatography, gas chromatography and luminescence [11-15], the electrochemical methods have more advantages
cr
such as low-cost, highly sensitive and user friendly than that of aforementioned traditional
us
methods [16]. More recently our group has found that green synthesized metal nanoparticles have excellent electrocatalytic ability towards nitro group substituted aromatic compounds owing
an
to its high stability, conductivity, biocompatibility, low cytotoxicity and size-related electrochemical properties [17]. The same strategy has been used here to synthesize the shape
M
and size controlled Au-NPs using the twig bark extract of Acacia Nilotica. To the best of our
te
Au-NPs.
d
knowledge, there are no reports available for the determination of NB using green synthesized
In the present work, we describe the green synthesis of Au-NPs by using twig bark
Ac ce p
extract of Acacia Nilotica as a reducing as well as stabilizing agent for the first time. The green synthesized Au-NPs modified electrode exhibits excellent electrochemical detection ability towards NB compared to unmodified electrode. The modified electrode shows a wide linear range, low detection limit and good stability along with good selectivity in pH 7.0. In addition, the modified electrode has been successfully employed for the determination of nitroaromatics in various real water samples.
2. Experimental 2.1. Materials and methods
4
Page 4 of 30
The Chloroauric acid (HAuCl4.3H2O) was purchased from Sigma–Aldrich and used as received. The fresh twig bark of Acacia nilotica was collected in Thiagarajar College campus, Madurai, India. Nitrobenzene was purchased from Aldrich and used without purification. The 0.1
ip t
M phosphate buffer pH 7.0 solution (PBS) was prepared by using NaH2PO4 and Na2HPO4 with doubly distilled water. All other chemicals were of analytical grade and used as received. spectrum
was
recorded
by
using
JascoV-560
double-beam
cr
UV–visible
UV
us
spectrophotometer. The XRD spectra was recorded and analyzed for the purified, air-dried gold nanoparticles with X-ray diffraction analysis using a PANalytical X’Pert PRO, PW3050/60 X-
an
ray diffractometer. The size and shape of gold nanoparticles were inspected using transmission electron microscopy (TEM) using JEOL JEM 2100 instrument. The elemental analysis (EDX)
M
was carried out using the JEOL JEM 2100 TEM instrument attached with Energy Dispersive X-
d
ray Analyzer. The IR spectrum was recorded using Shimadzu FT- IR-8201PC instrument.
te
Electrochemical experiments such as cyclic voltammetry and differential pulse voltammetry were performed by using CH instruments model CHI-750A. A conventional three-electrode
Ac ce p
setup consisting of a glassy carbon electrode (GCE) as a working electrode (active surface area = 0.079 cm2), an Ag/AgCl electrode (Sat. KCl) as a reference electrode and a platinum wire with 0.5 mm diameter as a counter electrode was used for electrochemical experiments. 2.2. Preparation of bark extract
The fresh twig bark was finely cut into small pieces and washed several times with deionized water to remove any dust. The 0.250 g of twig bark of Acacia nilotica was boiled with 100 mL of deionized water at 900 C for 15 min at room temperature and the extract was twice filtered using Whatmann filter paper. The filtered twig bark extract was pale yellow in color and
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it was used as reducing as well as stabilizing agent for the synthesis of Au-NPs. The filtrate was stored in the refrigerator at 40 C for further experiments. 2.3. Green synthesis of Au-NPs
ip t
In a typical experimental method, 30 mL aqueous solution of 1 mM HAuCl4 was mixed with different volume ratio (10 mL, 20 mL and 30 mL) of Acacia nilotica bark extract in 250 mL
cr
flask at room temperature. The change in the color of the solution from pale yellow to violet
us
confirms the successful formation of Au-NPs. The formation of Au-NPs was further confirmed by excitation of Surface Plasmon vibration (See Fig. 3A). The entire reduction process is
an
completed within 10 min and there is no further color change in aqueous solution which indicates that the formation of Au-NPs is completed within 10 min. The effect of bark extract
M
concentration on gold nanoparticles was recorded by UV-Vis spectra. The stable Au-NPs
d
aqueous solutions were prepared by dispersing 3 mg mL-1 Au-NPs (optimum concentration) in
te
doubly distilled water and sonicated for 45 min. 2.4. Fabrication of Au-NPs modified electrode
Ac ce p
The pre-cleaned GCE surface was dried at room temperature. About 10 µL (optimized concentration) of the prepared Au-NPs dispersion was drop-casted on GCE surface and dried at room temperature. The fabricated Au-NPs modified GCE was gently washed with water to remove the loosely attached Au-NPs at the electrode surface. The Au-NPs modified electrode was used for further experiments and stored at room temperature under dry condition when not in use.
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3. Results and discussion 3.1. Characterization of green synthesized Au-NPs The surface morphology and size of the green synthesized Au-NPs were characterized by
ip t
TEM. As shown in Fig. 1A, the green synthesized Au-NPs using optimum concentration (30 mL) of bark extract with the clear lattice fringes having a spacing of 0.23 nm reveals that the
cr
growth of Au-NPs occurs preferentially on the (1 1 1) plane. The inter-planar distance of the Au-
us
NPs (1 1 1) plane is in agreement with the (1 1 1) d-spacing of bulk gold (0.2355 nm) as reported previously [18]. The formation of Au-NPs was further confirmed by using EDX analysis. The
an
corresponding EDX profile of Au-NPs is shown in Fig. 1B. A strong Au metallic peak appears at 2 keV, which is attributed to the presence of Au-NPs. Fig. 1C shows the selected area electron
M
diffraction pattern (SAED) of the green synthesized Au-NPs. The SAED pattern exhibits a bright
d
circular rings corresponding to the (1 1 1) (2 0 0) (2 2 0) (3 1 1) (2 2 2) planes, which further
te
confirms that the Au-NPs are highly crystalline. Fig. 1D shows the XRD pattern of green synthesized Au-NPs. The five distinct diffraction peaks are observed at 38.10, 39.350, 44.40, 640
Ac ce p
and 770, which can be indexed as (1 1 1) (2 0 0) (2 2 0) (3 1 1) and (2 2 2) reflections of fcc structured metallic gold respectively (JCPDS, file no PDF#04-0784). Similar results have been documented in the earlier report on the synthesis of gold nanoparticles using leaf extracts of Cassia auriculata, Coriander, Piper betle and Cinnamomum zeylanicum [19-22]. Furthermore the size of synthesized Au-NPs was calculated using Debye–Scherrer equation which shows that the nanoparticles are in the average size of 30 nm. Fig. 2 shows the TEM image of Au-NPs synthesized from different concentrations of bark extract (A= 10 mL, B= 20 mL, C = 30 mL and D= 40 mL) with 1 mM chloroauric acid. The difference in sizes and shapes is possibly due to the fact that the nanoparticles are formed with
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different bark extract concentrations. It can be seen that the formation of stable gold nanoparticles takes place predominantly with unshaped/quasi-spherical and anisotropic particles with an average size of 10 to 50 nm (Fig. 2A). When the concentration of bark extract is
ip t
increased to 20 mL, the Au-NPs exhibit unshaped/quasi-spherical particles which are more abundant than the anisotropic shapes (Fig. 2B). Whereas using somewhat higher extract
cr
concentration of bark (30 mL), the formed Au-NPs are predominantly hexagonal in morphology
us
with the diameter of 20 - 50 nm (Fig. 2C). This shows that an excess amount of the extract reduces the chloroaurate ion effectively. However, the extract concentration more than 30 mL
an
does not have any further effect on the reduction of metal ions (Fig. 2D). The sufficient biomolecules in extract act as capping agent and stabilizing agents. Lower quantities of the
M
extract reduce the chloroaurate ions, but do not protect most of the quasi-spherical nanoparticles
d
from aggregating because of the deficiency of biomolecules to act as protecting agents. Similar
te
result to ours shows that the comparatively excess of the extract in volume is responsible for the synthesis of quasi-spherical nanoparticles [23].
Ac ce p
UV–visible spectroscopy is an important technique to ascertain the formation and stability of metal nanoparticles in aqueous solution and it is sensitive to several factors like particle size, shape and particle–particle interaction with the medium and local refractive index. After the addition of bark extract, the color of chloroauric acid turns from pale yellow to violet and it is an obvious result that chloroaurate ions are completely reduced to form Au-NPs. The color change arises because of the surface plasmon vibrations with Au-NPs which is absent in bulk material [24]. Fig. 3A shows the UV–visible spectra of the gold colloids obtained after 10 min of bioreduction using 10 mL (a), 20 mL (b) and 30 mL (c) bark extract. The surface Plasmon resonance (SPR) band of colloid gold with 10 mL bark extract appearing around 585 nm is
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attributed to the formation of large anisotropic like spherical / hexagonal shaped nanoparticles. The Plasmon bands of Au-NPs are broad with an absorption tail in the longer wavelength region that extends well into the near infrared region for colloids gold (a). The broad absorption peak
ip t
between 500 nm to 600 nm in case of Au-NPs is due to transverse interaction of radiance. The band becomes narrower with a shift towards lower wavelength as the volume of extract is
cr
increased (10 to 30 mL). The fairly sharp SPR is observed at 549 nm which is indicative of
us
almost hexagonal nano particles (c). It is evident that as the quantity of extract is increased, the bandwidth of SPR decreases with a shift of the peak towards lower wavelength. Similar results
an
have been reported in Au-NPs synthesis using coriander leaf, apiin, edible mushroom, honey, Hibiscus rosa sinensis and Cinnamomum zeylanicum [25-28].
M
The FTIR measurements are carried out to find the possible molecules present in the bark
d
of Acacia Nilotica which are responsible for the reduction of chloroaurate ion and stabilization of
te
the synthesized nanoparticles. Fig. 3B shows the typical FTIR spectra of Acacia nilotica bark (a) after and (b) before encapsulation of gold. It can be clearly seen in curve b that the broad and
Ac ce p
intense peak at 3432 cm−1 is due to the O–H stretching vibration of hydroxyl functional groups of poly phenols and alcohols and -NH stretching vibrations of amide (II) (or) amine, C-H stretching vibrations of aldehydic amine group appear at around 2917 and 2851 cm−1, C=O stretching vibrations of esters appear at 1745 cm−1, C=O stretching vibration of carbonyl and carboxylic group of amide-I appear at 1633 cm−1, -N-H stretching vibrations of the amide-II linkage of proteins appear around 1521 cm−1, methylene scissoring vibrations of the proteins around 1452 cm−1, C–O stretching vibration of polyols around 1219 cm−1 and C-N stretching vibration of primary amine at 1018 cm−1. All these data reveal the presence of high concentrated alcohol or phenol, aromatic amines, amide (I) and (II) of proteins, aldehyde, polyol and
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methylene groups along with the Au-NPs. The curve (a) illustrates the peak of treated crushed bark in which a peak at 3432 cm−1 is due to the O–H stretching vibration of phenols or high concentrated alcohols. After the encapsulation of chloroaurate ion, the peak intensity decreases
ip t
suggesting the possible involvement of above mentioned groups. The sharp and narrow peaks occurring at 2917 and 2851 cm−1 are due to C-H stretching vibrations of aldehyde. After the
cr
encapsulation of chloroaurate ion, the intensities of these two peaks become weaker and shift
us
from 2917 to 2928 cm−1, 2851 to 2844 cm−1, illustrating the involvement of aldehydic groups in the nanoparticles synthesis. The sharp peak appearing at 1745 cm−1 is due to C=O stretching of
an
ester, the peak at 1633 cm−1 is due to C=O stretching vibration of carbonyl and carboxylic groups in amide-I and the peak at 1521 cm−1 is due to –N-H stretching vibration of amide-II
M
linkage of proteins. The peak appearing at 1452 cm−1 is due to methylene scissoring vibrations of proteins. After the encapsulation of chloroaurate ion, the intensities of these three peaks become
te
d
weaker and shift from 1745 to 1782 cm−1, 1633 to 1638 cm−1 and 1018 to 908 cm−1, suggesting that the C=O, –N-H and methylene groups are sensitive indicators to conformational changes in
Ac ce p
the secondary structure of the protein and these groups are involving in the synthesis of nanoparticles. The peak at around 1219 cm−1 is due to C–O stretching vibration of polyols. After the encapsulation of chloroaurate ion, the intensity of the peak shifts from 1219 to 1236 cm−1 indicating that polyols also involve in the synthesis. The peak at around 1018 cm−1 is due to the C-N stretching vibration of primary amines [29] which after bio reduction shifts from 1018 to 986 cm−1, indicating possible involvement of primary amines during nanoparticles synthesis. As indicated by the FTIR data, the plant Acacia nilotica bark extract has been reported to contain phytochemicals such as tannins, terpenoids, alkaloids, saponins and glycosides and the functional groups like high concentrated alcohol or phenol, amine, amide (I) and (II), aldehydes,
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polyol and methylene groups. The above mentioned functional groups are mainly derived from water-soluble heterocyclic compounds, which are responsible for reduction and stabilization of gold nanoparticles. The tannins are active constituents in Acacia Nilotica bark and it is rich in
ip t
phenolics with sufficient number of hydroxyl and carboxyl groups, which possesses oxidationreduction abilities to bind metals and inactivate them from chelation. The general chelating
cr
ability of phenolic compounds is probably related to the high nucleophilic character of the
us
aromatic rings rather than to specific chelating groups within the molecule [30]. From the above results, it can be suggested that mostly the hydroxyl groups are involving in the reduction of Au
M
AuCl4 − + 3R–OH → Au0 + 3R–O + 3H+ + 4Cl−
an
(III) to Au (0) through oxidation of hydroxyl to carbonyl group, which can be represented as:
d
3.2. Electrochemical behavior of NB at Au-NPs modified electrode
te
In order to evaluate the electrochemical behavior of NB, the cyclic voltamograms were performed in the presence and absence of NB at different modified electrodes. Fig. 4A shows the
Ac ce p
cyclic voltammetric response of bare (a) and Au-NPs (b) modified electrodes in the presence of 300 µM NB in PBS at the scan rate of 50 mV s-1. The Au-NPs modified electrode does not show any obvious reduction peak response in the absence of NB (curve b), clearly suggesting that the Au-NPs modified electrode is electrochemically inactive at this potential window in PBS. A sharp irreversible reduction peak is observed at -0.647 V in the presence of 300 µM NB, which is attributed to the direct reduction of NB to phenylhydroxylamine with four electrons and protons transfer process. On the reverse scan, a less intense oxidation peak appears at 0.274, which is attributed to the irreversible oxidation of phenylhydroxylamine to nitrazobenzene. The electrochemical reduction mechanism of NB is well studied and reported early that
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electroreduction of NB to aniline occurs in acidic medium and phenylhydroxylamine in neutral and basic medium [31]. The overall electrochemical mechanism of reduction of NB is similar to that of previously reported literatures [31, 32]. Fig. 4B shows the DPVs of bare (a) and Au-NPs
ip t
modified electrodes (b) in the presence of 50 µM NB in PBS. It can be seen that the modified electrode shows 3 fold enhanced current response with -0.08 V negative shifts for NB than that
cr
of bare GCE. The results clearly indicate that Au-NPs modified electrode is more suitable for the
us
determination of NB compared to unmodified electrode. The good electrocatalytic ability of the modified electrode towards NB is attributed to the excellent conductivity and large active sites of
an
Au-NPs. The schematic representation for the fabrication of Au-NPs from bark extract and its mechanism of NB reduction is shown in Fig. 5.
M
The effect of scan rate also was studied by using cyclic voltammetry by varying different
d
sweeping rates toward the reduction of NB. Fig. 6A shows the cyclic voltammetry response of
te
Au-NPs modified electrode in PBS containing 300 µM NB at different scan rates (10 to 500 mVs-1). The reduction peak current response of NB has a linear dependence with the square root
Ac ce p
of scan rate from 10–500 mV s-1 (inset). The result indicates that the electrochemical reduction of NB at Au-NPs modified electrode is a typical diffusion-controlled process. The pH is another important parameter that affects the analytical parameters of Au-NPs modified electrode towards the reduction of NB. Hence, the cyclic voltammograms were performed at Au-NPs modified electrode in the presence of 300 µM containing different pH solutions (3 to 11) at the scan rate of 50 mV s-1. Fig. 6B shows the calibration plot of effect of different pH vs reduction peak current response of NB. It can be seen that the maximum reduction peak current response of NB is observed at pH 7 when compared to other pH solutions. The change of the solution little above
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and below pH 7 decreases the cathodic peak current, which is due to the effect of hydrogen ion on the reduction of NB. Hence, pH 7 is found to be an optimum pH for the detection of NB.
ip t
3.3 Electrochemical determination of NB
To verify the feasibility of the determination of NB at Au-NPs modified electrode, the
cr
DPV was performed for the different concentrations of NB. Fig. 7 shows the DPV of Au-NPs
us
modified electrode toward different concentrations of NB in PBS. Under optimized conditions, the reduction peak current of NB increases at Au-NPs modified electrode with the increase of
an
NB concentration. It can be seen from Fig. 7 inset that the reduction peak current of NB linearly increases over the NB concentrations ranging from 0.1 to 600 µM with the correlation
M
coefficients of 0.9983. The sensitivity is calculated from the obtained linear regression equation
d
as 1.01 ± 0.065 µAµM-1 cm-2. The limit of detection (LOD) is estimated as 0.016 µM [(3 * Sd of
te
the blank response)/slope of the calibration plot (Sd = 0.214 nA and slope = 0.0803]. The obtained LOD and sensitivity are more comparable than that of previously reported literature for
Ac ce p
the detection of NB using electrochemical methods. The analytical performance of proposed AuNPs modified electrode (3 successive measurements) has been compared with previously reported modified electrodes for NB and the comparative results are shown in Table. ST1. From the Table. ST1, it is evident that the Au-NPs modified electrode exhibits a better analytical performance towards NB in terms of sensitivity and LOD than that of previously reported modified electrodes for NB determination. The excellent electrocatalytic activity of Au-NPs modified electrode towards NB is attributed to the good conductivity and large active sites of the Au-NPs, which increase the electrocatalytic activity for NB and prevent fouling of oxidized products on the electrode surface.
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3.4 Effect of potentially interfering substances The selectivity of the modified electrode is more important and plays a key role for further practical applications. In order to investigate the selectivity of the Au-NPs modified
ip t
electrode towards the electrochemical reduction of NB in the presence of phenol derivatives, nitro aromatic compounds and inorganic metal ions, the Au-NPs modified electrode was
cr
evaluated by DPV for the detection of 10 µM NB in the presence of 100 fold concentration of
us
inorganic metal ions and biologically coactive species, 20 fold concentrations of phenolic derivatives and nitroaromatic compounds. The obtained results are shown in Fig. 8. It can be
an
seen from Fig. 8 that common metal ions and biologically coactive compounds almost have no effect (
