Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 596–604
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Surface plasmon resonance optical sensor and antibacterial activities of biosynthesized silver nanoparticles M.R. Bindhu, M. Umadevi ⇑ Department of Physics, Mother Teresa Women’s University, Kodaikanal 624101, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Green synthesis of Ag nanoparticles
using Ananas comosus fruit extract as reducing agent. Stable and spherical Ag nanoparticles were prepared. Shows size dependent antimicrobial activity. Act as very good copper and zinc sensors.
a r t i c l e
i n f o
Article history: Received 18 September 2013 Received in revised form 3 November 2013 Accepted 5 November 2013 Available online 13 November 2013 Keywords: Silver nanoparticles Ananas comosus Surface plasmon resonance Antibacterial activity Zinc sensor Copper sensor
a b s t r a c t Silver nanoparticles were prepared using aqueous fruit extract of Ananas comosus as reducing agent. These silver nanoparticles showed surface plasmon peak at 439 nm. They were monodispersed and spherical in shape with an average particle size of 10 nm. The crystallinity of these nanoparticles was evident from clear lattice fringes in the HRTEM images and bright circular spots in the SAED pattern. The antibacterial activities of prepared nanoparticles were found to be size-dependent, the smaller nanoparticles showing more bactericidal effect. Aqueous Zn2+ and Cu4+ selectivity and sensitivity study of this green synthesized nanoparticle was performed by optical sensor based surface plasmon resonance (SPR) at room temperature. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Noble metal nanoparticles are used for the purification of water which is one of the essential enablers of life on earth [1]. Water is one of the purest symbols of wealth, health, tranquility, beauty and originality. Pure water, which is free of toxic chemicals and pathogenic bacteria, is necessary for human health. Water and environment get contaminated by the heavy metals due to industrial and
⇑ Corresponding author. Tel.: +91 04542241685; fax: +91 04542 241122. E-mail address: [email protected] (M. Umadevi). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.019
agricultural pollution. Heavy metals such as cadmium (Cd), Zinc (Zn), mercury (Hg), arsenic (As), silver (Ag), chromium (Cr), copper (Cu), iron (Fe), and the platinum group elements [2] in water causes dangerous toxic effects on human beings. In particular the detection of Cu4+ and Zn2+ in water, air and soil has been of great research interest due to the important role of Zn and Cu plays in the biological processes within the human body [3–5]. Zinc and copper are essential at trace concentrations as heavy metal ions or nutrients to maintain the metabolism of human body [5,6]. Drinking water can be a source of Zn and Cu to humans as the result of water treatment, usage of galvanized pipes, copper pipes and tanks in distribution systems. The beverages stored in metal
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containers, which are coated with zinc in order to resist rust, contain high levels of Zn. So, detection and control of Cu and Zn ions in various media such as water, biological, environmental, medical and industrial samples is very important. Keeping this in mind, detection of Zn and Cu concentration in water is explained in the present study. A number of techniques have been developed for heavy metal ions analysis, including Atomic absorption spectroscopy (AAS), Inductively coupled plasma mass spectrometry (ICPMS), Anodic stripping voltammetry, X-ray fluorescence spectrometry and microprobes. However, these techniques generally require expensive equipment, sample pretreatment, and/or a long measuring period. Thus, a simple, rapid, inexpensive, sensitive and selective method is strongly needed. Optical sensor based on surface plasmon resonance for detection of heavy metals in water is one of the most sensitive methods which will be advantageous over other earlier techniques as it will be a simple, inexpensive and fast. In the past years a number of nanoparticles based sensor have been reported [7–10]. Recently green synthesized silver nanoparticles used in an optical sensor based on localized SPR for ammonia and mercury detection was studied [11,12]. In this study, we have designed a silver nanoparticles based optical sensor for detection of concentration of Cu4+ and Zn2+ ions in water. Waterborne pathogens including helminthes, protozoa, fungi, bacteria, rickettsiae, viruses and prions, can cause many diseases [13]. In India 80% of the diseases are due to bacterial contamination of drinking water. To protect the water purity, the removal or deactivation of pathogenic bacteria in water is very important. Silver is being used as a bactericide for water purification and also to prevent the buildup of bacteria and algae in water filters since more than a decade. Antibacterial activities of silver nanoparticles against various pathogens have also been established [14–16]. In the present study, the antibacterial assay was done on various pathogenic bacteria like Escherichia coli and Proteus Mirabilis, which are commonly found in water. The physicochemical and optoelectronic properties of metal nanoparticles are based on specific characteristics such as size, distribution and morphology [17]. Among the known nanoparticles, silver has been widely studied for its optical, spectroscopic, catalytic, antimicrobial and SERS properties. Due to these properties, silver nanoparticles have been broadly applied in consumer products and industrial fields. In recent years, green synthesis approaches of metal nanoparticles, using microorganisms and plants have received great attention to chemical and physical methods. Generally the reduction of silver nitrate using plant extract was slow, but it had some advantage of producing stable and uniform size nanoparticles without using any additional chemical stabilizers [18]. Recently, green synthesis of silver nanoparticles using Hibiscus cannabinus, Solanum lycopersicums, Moringa oleifera, Murraya koenigii leaf, Citrus limon and Daucus carota have been reported [19–24]. Ananas comosus is a readily available fruit and it is a good source of water, carbohydrates, sugars, vitamins A, C and carotene, beta [25]. It is one of the fruits with highest in the flavonoid antioxidant Vitamin C. This antioxidant reduces the oxidative damage such as that caused by free radicals and chelating metals [26]. It contains low amount of protein, fat, ash and fiber. There are three types of amino acids in A. comosus that promote exceptional health benefits through essential, semiessential, and non-essential amino acids. Along with this, A. comosus also contain bromelain, a protein-digesting enzyme that reduces inflammation. Modified pineapple peel fiber was used to remove heavy metal ions in water through the reaction with succinic acid anhydride [27,28]. Bhosale et al. has reported the synthesis of silver and gold nanoparticles using A. comosus extract with kanamycin A, and neomycin as stabilizing agents [29]. They prepared larger nanoparticles with agglomeration. In the present study,
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the synthesis and characterization of monodispersed smaller silver nanoparticles using fruit extract of A. comosus has been described. Here the size and aggregation of the nanoparticles were controlled without additional stabilizing agents. In the present study, the synthesis and characterization of biosynthesized silver nanoparticles for sensor and antibacterial activities have been described. Experimental details Material and methods A. comosus fruit was collected from the local supermarket in Kodaikanal, Tamilnadu, India. Silver nitrate, copper sulphate, ferric chloride, nickel nitrate, potassium chloride, cadmium acetate, manganous acetate, mercuric iodide, lithium hydroxide and zinc acetate were obtained from Sigma Aldrich Chemicals. All glasswares were properly washed with distilled water and dried in hot air oven before use. Preparation of A. comosus extract Fully riped A. comosus fruit, weighing 50 g was taken and cut into fine pieces and were crushed into 100 ml distilled water in a mixer grinder for extraction. The extract was then separated by centrifugation at 1000 rpm for 10 min to remove insoluble fractions and macromolecules. The extract thus obtained was filtered and finally a light yellow extract was collected for further experiments. Synthesis of silver nanoparticles 5 ml of A. comosus extract was added to aqueous solution of AgNO3 (3 mM) and stirred continuously for 5 min. The reaction completed slowly and it showed stable reddish brown color of the silver colloid (S1). Similarly by adding 10 and 15 ml of fruit extract two more set of samples, henceforth called (S2) and (S3) respectively, were prepared. UV–vis spectra of these solutions were recorded. Here the formation of silver nanoparticles started within 20 min, and increased up to for 2 h. After 2 h, no color variation was observed up to 1 month, showing that the silver nanoparticles prepared by this green synthesis method were very stable. Then the solutions were dried. The dried powders were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Radiation (FTIR), Transmission Electron Microscope (TEM) and Energy Dispersive X-ray Spectroscopy (EDX). Characterization methods and instruments The absorption spectra of the prepared nanoparticles were measured using a Shimadzu spectrophotometer (UV 1700) in 300– 800 nm range. X-Ray Diffraction analysis of the prepared nanoparticles was done using PANalytical X’pert – PRO diffractometer with Cu Ka radiation operated at 40 kV/30 mA. FTIR measurements were obtained on a Nexus 670 FTIR instrument with the sample as KBr pellets. Transmission Electron Microscopic (TEM) analysis was done using a JEOL JEM 2100 High Resolution Transmission Electron Microscope equipped with an EDX attachment, operating at 200 kV. For the detection of concentration of zinc in water using optical characteristics of silver nanoparticles, different concentration of aqueous solution of zinc acetate dehydrate salt were prepared. This analyte solution was added to the prepared silver nanoparticle solution from 3 104 M to 12 104 M and stirred continuously for 1 min at room temperature. For the detection of concentration of copper in water, different concentration of aqueous solution copper sulphate (2 mM to 7 mM) and of the same conditions were added into the silver nanoparticles. The antibacterial
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assay was done on various pathogenic bacteria like E. coli and P. Mirabilis. Nutrient agar medium was used to cultivate bacteria. Sterile paper disc of 3 mm diameter containing freshly prepared 50 lg/ml silver nanoparticles were prepared. These plates were incubated at 37 °C for 24 h. After 24 h the diameter of the growth inhibition zones were measured. Results and discussion Optical studies The optical properties of prepared silver nanoparticles were characterized by UV–vis spectroscopy. Fig. 1 represents the optical absorption spectra of silver colloids S1, S2 and S3 obtained at different concentration of fruit extract. Silver nanoparticles exhibit an intense absorption peak in the visible region due to the surface plasmon excitation. Surface plasmon resonance (SPR) absorption band is observed due to the combined oscillation of free conduction electrons of metal nanoparticles in resonance with light wave. The absorption spectrum of isolated spherical particles is characterized by the Mie resonance occurring at a frequency x0 such that: es (x0) = 2em, where es(x0) is the dielectric function of the silver spherical particles and em is the dielectric function of the surrounding medium [30]. The color variation of the S1, S2 and S3 has been shown in Fig. 1 (inset). When the frequency of the electromagnetic field becomes resonant with the coherent electron motion, a strong absorption takes place, which is the origin of the observed color. The observed characteristic color variations of the prepared silver nanoparticles is changed from light orange to dark reddish brown as the concentration of fruit extract increases. This absorption strongly depends on the particle size, dielectric medium and chemical surroundings [31]. S1 was showing the formation of SPR band at 446 nm with broad band confirming the formation of varied size and shape nanoparticles and silver colloid S3 showed SPR band at 439 nm with narrow peak indicating the formation of spherical nanoparticles. As the concentration of fruit extract increased, the SPR bands of the prepared colloids exhibited blue shift in the reaction medium. This result represents that the diameter of the prepared silver nanoparticles decrease with increasing concentration of the fruit extract, when electrons are donated to the particles [32]. The position of the plasmon absorption peak depends on the particle size and shape and the
Fig. 1. Optical absorption spectra of silver nanoparticles at different concentration of Ananas comosus fruit extract (inset: the Colour changes of the solution at different concentration of Ananas comosus fruit extract in reaction system) (a, b, and c versus S1, S2 and S3 respectively).
adsorption of nucleophile or electrophile to the particle surface. In the case of aqueous silver nanoparticles, the Fermi level can float upon chemisorption, depending on whether the substrate is nucleophilic and donates electron density into the particles or is electrophilic and withdraws electron density. Usually, a blue shift is associated with a decrease in particle size or with the donation of electron density from the surface. It is well-known that adsorption of the nucleophile to the particle surface bind the silver particles and increases the Fermi level of the silver particle due to its donation of electron density to the particles [32].This directly corresponds to a shift towards the blue end or red end, whereby small silver nanoparticle sizes would cause an absorption peak shift to smaller wavelengths, higher frequency and energies [30]. At the same time, the observed decrease of full-width at half-maximum (FWHM) value from 234 nm to 220 nm with increasing concentration of fruit extract indicates that the size of the particle decreases [33]. The FWHM is reported to be helpful in understanding the particle size and their distribution within the medium. The plasmon peak and full width at half maxima depends on the extent of colloid aggregation [34]. In the present case, the particle size of silver nanoparticles decreases with decreasing FWHM value. Thus from the results it can be concluded that the concentration of fruit extract plays an important role in the formation of silver nanoparticles. The symmetric nature of the SPR and the absence of peaks in the longer wavelength region indicated the absence of nanoparticle aggregation. This was also confirmed by the TEM results. These nanoparticle solutions were observed to be stable for a time period of one month. In the present case, the prepared silver colloidal nanoparticle introduced negative charge due to the biomolecules and thus repels the particles away from each other, preventing them from aggregation. FTIR studies FTIR analysis was carried out to identify the possible reducing biomolecules in the fruit extract responsible for the formation of silver nanoparticles and to identify the chemical change of the functional groups involved in bioreduction. Fig. 2(a and b) shows that the FTIR spectrum of A. comosus fruit extract and the silver nanoparticles, respectively. The peaks at 3417, 1640, 1019 and 801 cm1 are assigned to OAH stretching, CH stretching, C@C ring stretching and CAC ring stretching of Vitamin C (ascorbic acid), respectively [35]. The broad and asymmetric feature of the C@C ring stretching of
Fig. 2. FTIR spectra of (a) Ananas comosus fruit extract and (b) S3.
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Fig. 3. TEM micrograph of the S3 (a) the scale bar corresponds to 50 nm, (b) the scale bar corresponds to 20 nm, (c) the scale bar corresponds to 10 nm and (d) SAED pattern.
Fig. 4. TEM micrograph of the S1(a) the scale bar corresponds to 50 nm, (b) the scale bar corresponds to 20 nm, (c) the scale bar corresponds to 10 nm and (d) SAED pattern.
Vitamin C mode at 1640 cm1 in the spectrum of extract appeared at a well-defined peak of 1621 cm1 in the spectrum of silver nanoparticles (S3). An asymmetric band at 1411 cm1 in the extract was observed in S3 at 1394 cm1 assigned to the
ACAO stretching vibration modes of phytochemicals like water soluble components such as phenolic compounds including flavonoids and antioxidant vitamins [22,36]. It was possible that the antioxidant vitamin C (ascorbic acid) present in the extract,
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formed by the adsorption of various layers of phytochemicals around this electrostatic double layer. Steric or electrostatic barriers take place around the silver nanoparticle surface due to the variability of the molecule structures of phytochemicals in the extract. This may have thereby helped to cap the obtained silver nanoparticles, restrict the agglomeration and enhance the stability. Morphological studies
Fig. 5. X-ray diffraction pattern of (a) Ananas comosus fruit extract and (b) S1 and (c) S3 (*due to Ananas comosus fruit extract).
adsorbed on the surface of silver nanoparticles, could have led to the reduction of Ag+ to Ag0 state. The peak at 1098 cm1 corresponds to CAO, CAN or CAC stretching of amino acids. This region indicates the presence of proteins [37]. A peak at 2369 cm1 in the spectrum of extract was assigned to NH stretching of amines. This vibrational mode might be due to the presence of bromelain in the extract. This enzyme has the ability to separate all important amino acid bonds in protein. This represents that release of some protein components into the reaction medium may bind the nanoparticles through cysteine residues in the proteins through hydrogen bond or it may cap the silver nanoparticles through electrostatic attraction preventing from agglomeration and enhance the stability of the silver nanoparticles. In this green synthesis method, silver nanoparticles were formed by self assembling of the phytochemicals present in the extract. The nucleation of silver nanoparticles might have formed by transferring charge from ascorbic acid to Ag+ ions. Due to the Coulomb force, molecules of other phytochemicals, possibly of proteins got adsorbed towards the silver nanoparticles and form electrostatic double layers and particle size was controlled. Due to the Van der walls force of attraction, diffuse double layer was
Transmission electron microscopy (TEM) has been used to characterize the size, shape and morphologies of formed silver nanoparticles. The size dependent morphology of the silver nanoparticles prepared using different fruit extract concentration was studied. The TEM images of the silver colloid S3 and S1 are shown in Figs. 3 and 4 respectively. The TEM image of S3 showing the presence of mono dispersed and isotropic spherical nanoparticles with average size of 10 nm ranging from 7 to 13 nm size were observed at Fig. 3(a–d). This effect was in agreement with the shape of SPR bands of silver colloid S3. The present case shows that the presence of a large quantity of biomolecules in the extract, strong interaction between biomolecules in the fruit extract and surface of nanoparticles was sufficient to the formation of spherical nanoparticles preventing them from sintering. The presence of twining in silver nanoparticles observed in Fig. 3(b). The twinned particles were identified by showing brightness in part of the particles as compared to the other parts. Generally, twinning, the planar defect is observed for face-centered cubic (fcc) structured metallic nanocrystals. Sharing of a common crystallographic plane by two subgrains give rise to twinning. The twinned silver nanoparticles were further evidenced by the presence of (1 1 1) plane in selected-area electron diffraction (SAED) pattern shown in Fig. 3(d) (inset). Face-centered cubic (fcc) structured metallic nanocrystals have a tendency to nucleate and grow into twinned particles with their surfaces bounded by lowest energy facets (1 1 1) [38]. TEM image of S1 was shown in Fig. 4(a–d). It shows the formation of anisotropic nanocrystals and broad size distribution in the range of 12–40 nm. This result was also confirmed by UV–vis spectra of S1. The anisotropic nanostructures grow by a process involving rapid reduction, assembly and room temperature sintering of spherical nanoparticles. As the concentration of fruit extract decreases less number of ascorbate ions are available to reduce silver ions and thus forms large nanoparticles. The less specific surface area and surface area to volume ratio, and polycrystalline nature of the S1 also tends to larger particles. The formation of nonpolyhedral shapes such as silver nanotriangles, hexagonal and rods,
Fig. 6. EDX graph of S3.
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which are a result in stable morphologies, might be due to the minimization of surface energy by the low-index crystal planes [39]. A face-centered cubic (fcc) lattice of noble metals possesses different surface energies for different crystal planes. The excess free energy per unit area for a particular crystallographic face is surface energy. It determines the faceting and crystal growth for nanoparticles. The selected–area electron diffraction (SAED) pattern of S1was shown in Fig. 4(d). The observed SAED pattern with bright circular rings corresponding to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes show the crystalline (fcc) nature of the nanoparticles. Structural studies The crystalline nature of the prepared silver nanoparticles was confirmed with X-ray diffraction (XRD) analysis. The XRD pattern for the dried powder of A. comosus was shown in Fig. 5(a). The observed peaks at 2h values 28.5°, 40.8° and 50.9° indicates the presence of ascorbic acid (JCPDS 22-1560) in the A. comosus extract. Fig. 5(b and c) shows the XRD patterns of the prepared silver nanoparticles. The diffraction peaks were observed at 38.3°, 44.4°. 64.6° and 77.4° in the 2h range 20–80° can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflection planes of face centered cubic (fcc) structure of metallic silver, respectively (JCPDS 04-0783). No peaks of crystallographic impurities in the sample have been found. Generally, the breadth of a specific phase of material is directly proportional to the mean crystallite size of that material. The sharper peaks indicating the crystallite size was large whereas broader peaks are indicative of small crystallite materials. Based on our XRD data, broadening of diffraction peaks was obtained with increasing fruit extract concentration. This reveals that as the concentration of fruit extract increases, the average crystallite size decreases. The average size of the prepared silver nanoparticles was determined from the Debye–Scherrer Eq. (1) by using the width of the (1 1 1) Bragg’s reflection.
D¼
kk b cos h
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Crystallinity was evaluated by comparing the crystalline size obtained by XRD to TEM particle size determination. Crystallinity index of the prepared silver nanoparticles was evaluated by the following equation:
Icry ¼
DpðSEM; TEMÞ ðIcry P 1:00Þ DcryðXRDÞ
ð3Þ
where Icry is the crystallinity index, Dp is the obtained particle size from either TEM or SEM analysis and Dcry is the particle size obtained from Scherrer equation. If Icry is close to 1, then it is monocrystalline whereas if it is greater than 1, it is a polycrystalline in nature [40]. The calculated values of crystallinity index of S1 and S3 were 2.25 and 1.11 respectively. This indicates that S3 shows monocrystalline whereas S1 shows polycrystalline nature.
ð1Þ
k is the Scherrer constant (k = 0.94), k is the wavelength of the Xray, b is the FWHM of the peak and h is the half of the Bragg angle. The calculated average particle size was found to be 16 nm for S1 and 9 nm for S3.This indicates that the particle size decreased with the increase in concentration of the fruit extract. The calculated cell volume for S1 and S3 was 68.02 Å3 and 67.69 Å3, respectively. The lattice parameter for S1 and S3 has been found out to be 4.082 Å and 4.075 Å, respectively, It was in very good agreement with the standard value 4.086 Å (JCPDS 04-0783). The ratio between the intensity of the (2 0 0) and (1 1 1) diffraction peaks was 0.28 for S1 and 0.27 for S3, which was lower than the conventional bulk intensity ratio 0.52, suggesting that the (1 1 1) plane was the predominant orientation as confirmed by high-resolution TEM measurements. In order to determine the type and property of a material, surface area to volume ratio (SA:V) or Specific Surface Area (SSA) can be calculated. Each material has its own SSA. The SSA is of particular importance in reactivity. It gives the rate at which the reaction will proceed. SSA can be calculated using the following equation:
SSA ¼
SApart Vpart density
ð2Þ
where SApart is particle surface area, Vpart is particle volume and density of silver is 10.5 g/cm3. The values of SA: V ratio and SSA of S1 was 0.38 and 37 m2/g respectively. For S3, the values of SA: V ratio and SSA was 0.66 and 64 m2/g respectively. The SA: V ratio and SSA was high for the silver nanoparticles synthesized using higher concentration of fruit extract.
Fig. 7. Zone of inhibition of (a) S3 and (b) S1 for (i) E. coli and (ii) Proteus Mirabilis, respectively and (c) antibacterial activity of S3 and S1.
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Fig. 6 shows the energy dispersive X-ray analysis (EDX) of S3. It confirms the formation of silver nanoparticles and reveals higher counts at 3 keV due to silver nanoparticles. It was also due to surface plasmon resonance of metallic silver nanocrystals as shown in optical absorption spectra. Antibacterial activity In this present case, the antibacterial activity of prepared silver nanoparticles was deliberated against Gram negative pathogens E. coli and P. Mirabilis. The antibacterial effect of silver nanoparticles on microorganisms may be held through the electrostatic attraction of positive charged silver and negative charged cell surface of microorganism. Fig. 7(a and b) shows the zone of inhibition of silver nanoparticles S3 and S1 against E. coli and P. Mirabilis. Fig. 7(c) shows the antibacterial activity of silver nanoparticles produced from (S3) and (S1) against pathogens. The antibacterial activity of S3 was more than S1 because of its larger specific surface area, smaller size and spherical shape. Depending on the surface area available for interaction, silver nanoparticles can bind bacteria and disturb its permeability and respiration function. Smaller particles having larger surface area can increase the ability to penetrate cell membrane and give more bactericidal effect than the larger particles [41]. Thus, the antibacterial activity of prepared silver nanoparticles are influenced by the size and morphology of the particles, and the effect of antibacterial activity increases with decreasing size of silver nanoparticles. The prepared silver nanoparticles function as a good antibacterial agent was significant in biomedical applications as well as in the removal or inactivation of pathogenic bacteria in water.
was observed that the intensity of the SPR bands got reduced and red shifted for all metal ions as compared to that of the AgNPs. It was observed that there was no prominent SPR peak for Zn2+ and a secondary peak for Cu4+, indicating the prepared AgNPs were sensitive and selective towards Zn2+ and Cu4+. The activity of S3 in the presence of Zn2+ was studied by adding various concentration of heavy metal ion (Zn2+) from 3 104 to 12 104 M in water. Fig. 9(a) shows the UV–vis absorption spectrum for activity of S3 as zinc sensor. The prepared AgNPs (S3) solution did not exhibit color change from reddish brown as well as change in absorption spectra up to the addition of 2 104 M Zn2+. This indicates that no aggregation effect has taken place. The addition of 3 104 to 12 104 M Zn2+ to the silver nanoparticles solution causes color changes from reddish brown to colorless, were observed as shown in Fig. 9(a) inset. The addition of 3 104 M Zn2+ causes immediate reduction in the intensity of surface plasmon peak at 444 nm. When zinc acetate added to the prepared nanoparticles, newly produced zinc atoms strongly bonded on the silver surface and moved the capping biomolecules in the extract, away from the surface, which could be accounted for the broadening and slight blue shift of the SPR band of silver nanoparticles. The optical absorbance intensity reduces and band
Detection of zinc and copper ions in water To investigate the interaction of prepared AgNPs (S3) with various alkali metal (Li+, K+, Fe3+) and transition metal ions (Ni2+, Mn2+, Cu4+, Zn2+, Hg2+, Cd2+), 0.3 ml of (3 mM) salts of these metals were added into 3 ml of AgNPs by drop by drop and stirred for 2 min. The photographs (Fig. 8 (inset)) and UV–vis spectra (Fig. 8) of AgNPs were taken immediately after addition of metal ions, after 2 min of interaction. Upon addition of Zn2+, the reddish brown color of the solution changed to colorless. Similarly the reddish brown color of the AgNPs solution changed to light gray. Based on Fig. 8, it
Fig. 8. UV–vis absorption spectrum and photographs (inset) of S3 with various heavy metal ions.
Fig. 9. (a) UV–vis spectra and color changes (inset) of S3 as a function of various concentrations of Zn2+ ions (3 104 to 12 104 M) and (b) plot of absorbance at 444 nm versus Zn2+ concentration (3 104 to 12 104 M) with a correlation factor R2 = 0.959.
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broadens gradually with the increase of concentration of Zn2+, suggesting that aggregation had taken place due to the reduction of interparticle distance in the aggregates less than about the average particle diameter. Upon addition of the 3 104 M to 12 104 M Zn2+ to S3, color changes from reddish brown to colorless were observed, and after the addition of 12 104 M Zn2+ absorbance changes were negligible. This shows that the formation of stable aggregates. So the concentration of zinc was limited to 10 104 M with notable color and surface plasmon resonance band. Fig. 9(b) shows a good linear correlation (y = 0.036x + 0.965, R2 = 0.959) between the absorbance (DA) versus concentration of Zn2+, and the sensitivity of the system towards analyte concentration was found to be 0.036/104 M as measured from the plot of absorbance (DA) versus concentration of Zn2+. Fig. 10(a) shows the UV–vis absorption spectrum for activity of silver nanoparticles as copper sensor. When 1 mM copper sulphate added to S3, the color of the nanoparticle solution did not change, only the addition of 2 mM copper sulphate causes color changes. This indicates that the analyte directed aggregation of nanoparticles taken place only after the addition of 2 mM Cu4+. With increasing analyte concentration from 2 mM to 7 mM, the
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intensity of the SPR peak centered at 478 nm decreases gradually with band broadening and a new SPR band appears at 780 nm with increasing intensity. The main reason for SPR broadening is electron surface scattering which may be enhanced for very small clusters [42]. The observed band broadening of the surface plasmon band reveals that the copper atoms may bind on the silver surface. The presence of new SPR peak was due to the adsorption of molecules causing changes in dielectric environment around a nanoparticle or agglomerated particles. When copper sulphate is added to S3, Cu4+ ions interact with the biomolecules in the extract on the surface of the nanoparticles, forming bonds among nanoparticles with Cu4+ ions performing as link for binding sites of biomolecules and eliminating it away from the surface of the nanoparticle surface, in that way aggregation of nanoparticles has taken place. The addition of 2 mM to 7 mM Cu4+ to S3 causes color changes from reddish brown to light blue shown in Fig. 10(a) inset. The observed color changes and absorbance changes after the addition of 7 mM Cu4+ were negligible, suggesting the formation of stable aggregates. So the concentration of copper was limited to 7 mM with notable color and surface plasmon resonance band. The linear variation of absorbance (A780 nm/A478 nm) changes and the concentration of Cu4+ over the range from 2 mM to 7 mM are shown in Fig. 10(b). This plot can be fit by a linear equation y = 0.605x0.202, R2 = 0.967 and the sensitivity of the system towards analyte concentration was found to be 0.605/mM. Applications of nanoparticle sensors by the aggregation of small particles were useful because aggregates with multiple particles yield large enhancements due to the enormous electromagnetic field that coherently interfere at the junction site between the particles. This zinc and copper sensor based on surface plasmon optical sensor can be used in environmental monitoring especially in water purification. Conclusion An ecofriendly method of obtaining spherical silver nanoparticles with average size of 10 nm has been synthesized, using A. comosus as reducing agent. The prepared silver nanoparticles are stable for one month without aggregation. Crystalline nature of the nanoparticles was evident from bright spots in the SAED pattern, and peaks in the XRD pattern. The prepared silver nanoparticles reveal good antimicrobial activity against Gram negative pathogens E. coli and P. Mirabilis, which are found in water. So the prepared nanoparticles have found applications in biomedical and water purification processes for inhibiting the growth of bacteria. The prepared nanoparticles were used for sensing ions of heavy metals like Zn2+ and Cu4+ in water using a SPR optical sensor. A strategy explained in the present study is detectable by using naked eye or UV–vis spectrophotometer. The optical absorption spectrum of the colloidal suspension before and after addition of metal ions is a good pointer to detect the concentration of the heavy metal ions. For continuous monitoring and real time information, this Zn and Cu sensor based on surface plasmon optical sensor is preferred, and it can be used in environmental monitoring, especially in water purification. Acknowledgements The authors are thankful to DST-CURIE New Delhi, UGC-DAE CSR Indore for financial assistance. References
Fig. 10. (a) UV–vis spectra and color changes (inset) of S3 with Cu4+ ions (2–7 mM) and (b) plot of absorbance changes (A780 nm/A478 nm) versus Cu4+ concentration (2– 7 mM) with a correlation factor R2 = 0.967.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Surface plasmon resonance optical sensor and antibacterial activities of biosynthesized silver nanoparticles M.R. Bindhu, M. Umadevi ⇑ Department of Physics, Mother Teresa Women’s University, Kodaikanal 624101, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Green synthesis of Ag nanoparticles
using Ananas comosus fruit extract as reducing agent. Stable and spherical Ag nanoparticles were prepared. Shows size dependent antimicrobial activity. Act as very good copper and zinc sensors.
a r t i c l e
i n f o
Article history: Received 18 September 2013 Received in revised form 3 November 2013 Accepted 5 November 2013 Available online 13 November 2013 Keywords: Silver nanoparticles Ananas comosus Surface plasmon resonance Antibacterial activity Zinc sensor Copper sensor
a b s t r a c t Silver nanoparticles were prepared using aqueous fruit extract of Ananas comosus as reducing agent. These silver nanoparticles showed surface plasmon peak at 439 nm. They were monodispersed and spherical in shape with an average particle size of 10 nm. The crystallinity of these nanoparticles was evident from clear lattice fringes in the HRTEM images and bright circular spots in the SAED pattern. The antibacterial activities of prepared nanoparticles were found to be size-dependent, the smaller nanoparticles showing more bactericidal effect. Aqueous Zn2+ and Cu4+ selectivity and sensitivity study of this green synthesized nanoparticle was performed by optical sensor based surface plasmon resonance (SPR) at room temperature. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Noble metal nanoparticles are used for the purification of water which is one of the essential enablers of life on earth [1]. Water is one of the purest symbols of wealth, health, tranquility, beauty and originality. Pure water, which is free of toxic chemicals and pathogenic bacteria, is necessary for human health. Water and environment get contaminated by the heavy metals due to industrial and
⇑ Corresponding author. Tel.: +91 04542241685; fax: +91 04542 241122. E-mail address: [email protected] (M. Umadevi). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.11.019
agricultural pollution. Heavy metals such as cadmium (Cd), Zinc (Zn), mercury (Hg), arsenic (As), silver (Ag), chromium (Cr), copper (Cu), iron (Fe), and the platinum group elements [2] in water causes dangerous toxic effects on human beings. In particular the detection of Cu4+ and Zn2+ in water, air and soil has been of great research interest due to the important role of Zn and Cu plays in the biological processes within the human body [3–5]. Zinc and copper are essential at trace concentrations as heavy metal ions or nutrients to maintain the metabolism of human body [5,6]. Drinking water can be a source of Zn and Cu to humans as the result of water treatment, usage of galvanized pipes, copper pipes and tanks in distribution systems. The beverages stored in metal
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containers, which are coated with zinc in order to resist rust, contain high levels of Zn. So, detection and control of Cu and Zn ions in various media such as water, biological, environmental, medical and industrial samples is very important. Keeping this in mind, detection of Zn and Cu concentration in water is explained in the present study. A number of techniques have been developed for heavy metal ions analysis, including Atomic absorption spectroscopy (AAS), Inductively coupled plasma mass spectrometry (ICPMS), Anodic stripping voltammetry, X-ray fluorescence spectrometry and microprobes. However, these techniques generally require expensive equipment, sample pretreatment, and/or a long measuring period. Thus, a simple, rapid, inexpensive, sensitive and selective method is strongly needed. Optical sensor based on surface plasmon resonance for detection of heavy metals in water is one of the most sensitive methods which will be advantageous over other earlier techniques as it will be a simple, inexpensive and fast. In the past years a number of nanoparticles based sensor have been reported [7–10]. Recently green synthesized silver nanoparticles used in an optical sensor based on localized SPR for ammonia and mercury detection was studied [11,12]. In this study, we have designed a silver nanoparticles based optical sensor for detection of concentration of Cu4+ and Zn2+ ions in water. Waterborne pathogens including helminthes, protozoa, fungi, bacteria, rickettsiae, viruses and prions, can cause many diseases [13]. In India 80% of the diseases are due to bacterial contamination of drinking water. To protect the water purity, the removal or deactivation of pathogenic bacteria in water is very important. Silver is being used as a bactericide for water purification and also to prevent the buildup of bacteria and algae in water filters since more than a decade. Antibacterial activities of silver nanoparticles against various pathogens have also been established [14–16]. In the present study, the antibacterial assay was done on various pathogenic bacteria like Escherichia coli and Proteus Mirabilis, which are commonly found in water. The physicochemical and optoelectronic properties of metal nanoparticles are based on specific characteristics such as size, distribution and morphology [17]. Among the known nanoparticles, silver has been widely studied for its optical, spectroscopic, catalytic, antimicrobial and SERS properties. Due to these properties, silver nanoparticles have been broadly applied in consumer products and industrial fields. In recent years, green synthesis approaches of metal nanoparticles, using microorganisms and plants have received great attention to chemical and physical methods. Generally the reduction of silver nitrate using plant extract was slow, but it had some advantage of producing stable and uniform size nanoparticles without using any additional chemical stabilizers [18]. Recently, green synthesis of silver nanoparticles using Hibiscus cannabinus, Solanum lycopersicums, Moringa oleifera, Murraya koenigii leaf, Citrus limon and Daucus carota have been reported [19–24]. Ananas comosus is a readily available fruit and it is a good source of water, carbohydrates, sugars, vitamins A, C and carotene, beta [25]. It is one of the fruits with highest in the flavonoid antioxidant Vitamin C. This antioxidant reduces the oxidative damage such as that caused by free radicals and chelating metals [26]. It contains low amount of protein, fat, ash and fiber. There are three types of amino acids in A. comosus that promote exceptional health benefits through essential, semiessential, and non-essential amino acids. Along with this, A. comosus also contain bromelain, a protein-digesting enzyme that reduces inflammation. Modified pineapple peel fiber was used to remove heavy metal ions in water through the reaction with succinic acid anhydride [27,28]. Bhosale et al. has reported the synthesis of silver and gold nanoparticles using A. comosus extract with kanamycin A, and neomycin as stabilizing agents [29]. They prepared larger nanoparticles with agglomeration. In the present study,
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the synthesis and characterization of monodispersed smaller silver nanoparticles using fruit extract of A. comosus has been described. Here the size and aggregation of the nanoparticles were controlled without additional stabilizing agents. In the present study, the synthesis and characterization of biosynthesized silver nanoparticles for sensor and antibacterial activities have been described. Experimental details Material and methods A. comosus fruit was collected from the local supermarket in Kodaikanal, Tamilnadu, India. Silver nitrate, copper sulphate, ferric chloride, nickel nitrate, potassium chloride, cadmium acetate, manganous acetate, mercuric iodide, lithium hydroxide and zinc acetate were obtained from Sigma Aldrich Chemicals. All glasswares were properly washed with distilled water and dried in hot air oven before use. Preparation of A. comosus extract Fully riped A. comosus fruit, weighing 50 g was taken and cut into fine pieces and were crushed into 100 ml distilled water in a mixer grinder for extraction. The extract was then separated by centrifugation at 1000 rpm for 10 min to remove insoluble fractions and macromolecules. The extract thus obtained was filtered and finally a light yellow extract was collected for further experiments. Synthesis of silver nanoparticles 5 ml of A. comosus extract was added to aqueous solution of AgNO3 (3 mM) and stirred continuously for 5 min. The reaction completed slowly and it showed stable reddish brown color of the silver colloid (S1). Similarly by adding 10 and 15 ml of fruit extract two more set of samples, henceforth called (S2) and (S3) respectively, were prepared. UV–vis spectra of these solutions were recorded. Here the formation of silver nanoparticles started within 20 min, and increased up to for 2 h. After 2 h, no color variation was observed up to 1 month, showing that the silver nanoparticles prepared by this green synthesis method were very stable. Then the solutions were dried. The dried powders were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Radiation (FTIR), Transmission Electron Microscope (TEM) and Energy Dispersive X-ray Spectroscopy (EDX). Characterization methods and instruments The absorption spectra of the prepared nanoparticles were measured using a Shimadzu spectrophotometer (UV 1700) in 300– 800 nm range. X-Ray Diffraction analysis of the prepared nanoparticles was done using PANalytical X’pert – PRO diffractometer with Cu Ka radiation operated at 40 kV/30 mA. FTIR measurements were obtained on a Nexus 670 FTIR instrument with the sample as KBr pellets. Transmission Electron Microscopic (TEM) analysis was done using a JEOL JEM 2100 High Resolution Transmission Electron Microscope equipped with an EDX attachment, operating at 200 kV. For the detection of concentration of zinc in water using optical characteristics of silver nanoparticles, different concentration of aqueous solution of zinc acetate dehydrate salt were prepared. This analyte solution was added to the prepared silver nanoparticle solution from 3 104 M to 12 104 M and stirred continuously for 1 min at room temperature. For the detection of concentration of copper in water, different concentration of aqueous solution copper sulphate (2 mM to 7 mM) and of the same conditions were added into the silver nanoparticles. The antibacterial
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assay was done on various pathogenic bacteria like E. coli and P. Mirabilis. Nutrient agar medium was used to cultivate bacteria. Sterile paper disc of 3 mm diameter containing freshly prepared 50 lg/ml silver nanoparticles were prepared. These plates were incubated at 37 °C for 24 h. After 24 h the diameter of the growth inhibition zones were measured. Results and discussion Optical studies The optical properties of prepared silver nanoparticles were characterized by UV–vis spectroscopy. Fig. 1 represents the optical absorption spectra of silver colloids S1, S2 and S3 obtained at different concentration of fruit extract. Silver nanoparticles exhibit an intense absorption peak in the visible region due to the surface plasmon excitation. Surface plasmon resonance (SPR) absorption band is observed due to the combined oscillation of free conduction electrons of metal nanoparticles in resonance with light wave. The absorption spectrum of isolated spherical particles is characterized by the Mie resonance occurring at a frequency x0 such that: es (x0) = 2em, where es(x0) is the dielectric function of the silver spherical particles and em is the dielectric function of the surrounding medium [30]. The color variation of the S1, S2 and S3 has been shown in Fig. 1 (inset). When the frequency of the electromagnetic field becomes resonant with the coherent electron motion, a strong absorption takes place, which is the origin of the observed color. The observed characteristic color variations of the prepared silver nanoparticles is changed from light orange to dark reddish brown as the concentration of fruit extract increases. This absorption strongly depends on the particle size, dielectric medium and chemical surroundings [31]. S1 was showing the formation of SPR band at 446 nm with broad band confirming the formation of varied size and shape nanoparticles and silver colloid S3 showed SPR band at 439 nm with narrow peak indicating the formation of spherical nanoparticles. As the concentration of fruit extract increased, the SPR bands of the prepared colloids exhibited blue shift in the reaction medium. This result represents that the diameter of the prepared silver nanoparticles decrease with increasing concentration of the fruit extract, when electrons are donated to the particles [32]. The position of the plasmon absorption peak depends on the particle size and shape and the
Fig. 1. Optical absorption spectra of silver nanoparticles at different concentration of Ananas comosus fruit extract (inset: the Colour changes of the solution at different concentration of Ananas comosus fruit extract in reaction system) (a, b, and c versus S1, S2 and S3 respectively).
adsorption of nucleophile or electrophile to the particle surface. In the case of aqueous silver nanoparticles, the Fermi level can float upon chemisorption, depending on whether the substrate is nucleophilic and donates electron density into the particles or is electrophilic and withdraws electron density. Usually, a blue shift is associated with a decrease in particle size or with the donation of electron density from the surface. It is well-known that adsorption of the nucleophile to the particle surface bind the silver particles and increases the Fermi level of the silver particle due to its donation of electron density to the particles [32].This directly corresponds to a shift towards the blue end or red end, whereby small silver nanoparticle sizes would cause an absorption peak shift to smaller wavelengths, higher frequency and energies [30]. At the same time, the observed decrease of full-width at half-maximum (FWHM) value from 234 nm to 220 nm with increasing concentration of fruit extract indicates that the size of the particle decreases [33]. The FWHM is reported to be helpful in understanding the particle size and their distribution within the medium. The plasmon peak and full width at half maxima depends on the extent of colloid aggregation [34]. In the present case, the particle size of silver nanoparticles decreases with decreasing FWHM value. Thus from the results it can be concluded that the concentration of fruit extract plays an important role in the formation of silver nanoparticles. The symmetric nature of the SPR and the absence of peaks in the longer wavelength region indicated the absence of nanoparticle aggregation. This was also confirmed by the TEM results. These nanoparticle solutions were observed to be stable for a time period of one month. In the present case, the prepared silver colloidal nanoparticle introduced negative charge due to the biomolecules and thus repels the particles away from each other, preventing them from aggregation. FTIR studies FTIR analysis was carried out to identify the possible reducing biomolecules in the fruit extract responsible for the formation of silver nanoparticles and to identify the chemical change of the functional groups involved in bioreduction. Fig. 2(a and b) shows that the FTIR spectrum of A. comosus fruit extract and the silver nanoparticles, respectively. The peaks at 3417, 1640, 1019 and 801 cm1 are assigned to OAH stretching, CH stretching, C@C ring stretching and CAC ring stretching of Vitamin C (ascorbic acid), respectively [35]. The broad and asymmetric feature of the C@C ring stretching of
Fig. 2. FTIR spectra of (a) Ananas comosus fruit extract and (b) S3.
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Fig. 3. TEM micrograph of the S3 (a) the scale bar corresponds to 50 nm, (b) the scale bar corresponds to 20 nm, (c) the scale bar corresponds to 10 nm and (d) SAED pattern.
Fig. 4. TEM micrograph of the S1(a) the scale bar corresponds to 50 nm, (b) the scale bar corresponds to 20 nm, (c) the scale bar corresponds to 10 nm and (d) SAED pattern.
Vitamin C mode at 1640 cm1 in the spectrum of extract appeared at a well-defined peak of 1621 cm1 in the spectrum of silver nanoparticles (S3). An asymmetric band at 1411 cm1 in the extract was observed in S3 at 1394 cm1 assigned to the
ACAO stretching vibration modes of phytochemicals like water soluble components such as phenolic compounds including flavonoids and antioxidant vitamins [22,36]. It was possible that the antioxidant vitamin C (ascorbic acid) present in the extract,
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formed by the adsorption of various layers of phytochemicals around this electrostatic double layer. Steric or electrostatic barriers take place around the silver nanoparticle surface due to the variability of the molecule structures of phytochemicals in the extract. This may have thereby helped to cap the obtained silver nanoparticles, restrict the agglomeration and enhance the stability. Morphological studies
Fig. 5. X-ray diffraction pattern of (a) Ananas comosus fruit extract and (b) S1 and (c) S3 (*due to Ananas comosus fruit extract).
adsorbed on the surface of silver nanoparticles, could have led to the reduction of Ag+ to Ag0 state. The peak at 1098 cm1 corresponds to CAO, CAN or CAC stretching of amino acids. This region indicates the presence of proteins [37]. A peak at 2369 cm1 in the spectrum of extract was assigned to NH stretching of amines. This vibrational mode might be due to the presence of bromelain in the extract. This enzyme has the ability to separate all important amino acid bonds in protein. This represents that release of some protein components into the reaction medium may bind the nanoparticles through cysteine residues in the proteins through hydrogen bond or it may cap the silver nanoparticles through electrostatic attraction preventing from agglomeration and enhance the stability of the silver nanoparticles. In this green synthesis method, silver nanoparticles were formed by self assembling of the phytochemicals present in the extract. The nucleation of silver nanoparticles might have formed by transferring charge from ascorbic acid to Ag+ ions. Due to the Coulomb force, molecules of other phytochemicals, possibly of proteins got adsorbed towards the silver nanoparticles and form electrostatic double layers and particle size was controlled. Due to the Van der walls force of attraction, diffuse double layer was
Transmission electron microscopy (TEM) has been used to characterize the size, shape and morphologies of formed silver nanoparticles. The size dependent morphology of the silver nanoparticles prepared using different fruit extract concentration was studied. The TEM images of the silver colloid S3 and S1 are shown in Figs. 3 and 4 respectively. The TEM image of S3 showing the presence of mono dispersed and isotropic spherical nanoparticles with average size of 10 nm ranging from 7 to 13 nm size were observed at Fig. 3(a–d). This effect was in agreement with the shape of SPR bands of silver colloid S3. The present case shows that the presence of a large quantity of biomolecules in the extract, strong interaction between biomolecules in the fruit extract and surface of nanoparticles was sufficient to the formation of spherical nanoparticles preventing them from sintering. The presence of twining in silver nanoparticles observed in Fig. 3(b). The twinned particles were identified by showing brightness in part of the particles as compared to the other parts. Generally, twinning, the planar defect is observed for face-centered cubic (fcc) structured metallic nanocrystals. Sharing of a common crystallographic plane by two subgrains give rise to twinning. The twinned silver nanoparticles were further evidenced by the presence of (1 1 1) plane in selected-area electron diffraction (SAED) pattern shown in Fig. 3(d) (inset). Face-centered cubic (fcc) structured metallic nanocrystals have a tendency to nucleate and grow into twinned particles with their surfaces bounded by lowest energy facets (1 1 1) [38]. TEM image of S1 was shown in Fig. 4(a–d). It shows the formation of anisotropic nanocrystals and broad size distribution in the range of 12–40 nm. This result was also confirmed by UV–vis spectra of S1. The anisotropic nanostructures grow by a process involving rapid reduction, assembly and room temperature sintering of spherical nanoparticles. As the concentration of fruit extract decreases less number of ascorbate ions are available to reduce silver ions and thus forms large nanoparticles. The less specific surface area and surface area to volume ratio, and polycrystalline nature of the S1 also tends to larger particles. The formation of nonpolyhedral shapes such as silver nanotriangles, hexagonal and rods,
Fig. 6. EDX graph of S3.
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which are a result in stable morphologies, might be due to the minimization of surface energy by the low-index crystal planes [39]. A face-centered cubic (fcc) lattice of noble metals possesses different surface energies for different crystal planes. The excess free energy per unit area for a particular crystallographic face is surface energy. It determines the faceting and crystal growth for nanoparticles. The selected–area electron diffraction (SAED) pattern of S1was shown in Fig. 4(d). The observed SAED pattern with bright circular rings corresponding to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes show the crystalline (fcc) nature of the nanoparticles. Structural studies The crystalline nature of the prepared silver nanoparticles was confirmed with X-ray diffraction (XRD) analysis. The XRD pattern for the dried powder of A. comosus was shown in Fig. 5(a). The observed peaks at 2h values 28.5°, 40.8° and 50.9° indicates the presence of ascorbic acid (JCPDS 22-1560) in the A. comosus extract. Fig. 5(b and c) shows the XRD patterns of the prepared silver nanoparticles. The diffraction peaks were observed at 38.3°, 44.4°. 64.6° and 77.4° in the 2h range 20–80° can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflection planes of face centered cubic (fcc) structure of metallic silver, respectively (JCPDS 04-0783). No peaks of crystallographic impurities in the sample have been found. Generally, the breadth of a specific phase of material is directly proportional to the mean crystallite size of that material. The sharper peaks indicating the crystallite size was large whereas broader peaks are indicative of small crystallite materials. Based on our XRD data, broadening of diffraction peaks was obtained with increasing fruit extract concentration. This reveals that as the concentration of fruit extract increases, the average crystallite size decreases. The average size of the prepared silver nanoparticles was determined from the Debye–Scherrer Eq. (1) by using the width of the (1 1 1) Bragg’s reflection.
D¼
kk b cos h
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Crystallinity was evaluated by comparing the crystalline size obtained by XRD to TEM particle size determination. Crystallinity index of the prepared silver nanoparticles was evaluated by the following equation:
Icry ¼
DpðSEM; TEMÞ ðIcry P 1:00Þ DcryðXRDÞ
ð3Þ
where Icry is the crystallinity index, Dp is the obtained particle size from either TEM or SEM analysis and Dcry is the particle size obtained from Scherrer equation. If Icry is close to 1, then it is monocrystalline whereas if it is greater than 1, it is a polycrystalline in nature [40]. The calculated values of crystallinity index of S1 and S3 were 2.25 and 1.11 respectively. This indicates that S3 shows monocrystalline whereas S1 shows polycrystalline nature.
ð1Þ
k is the Scherrer constant (k = 0.94), k is the wavelength of the Xray, b is the FWHM of the peak and h is the half of the Bragg angle. The calculated average particle size was found to be 16 nm for S1 and 9 nm for S3.This indicates that the particle size decreased with the increase in concentration of the fruit extract. The calculated cell volume for S1 and S3 was 68.02 Å3 and 67.69 Å3, respectively. The lattice parameter for S1 and S3 has been found out to be 4.082 Å and 4.075 Å, respectively, It was in very good agreement with the standard value 4.086 Å (JCPDS 04-0783). The ratio between the intensity of the (2 0 0) and (1 1 1) diffraction peaks was 0.28 for S1 and 0.27 for S3, which was lower than the conventional bulk intensity ratio 0.52, suggesting that the (1 1 1) plane was the predominant orientation as confirmed by high-resolution TEM measurements. In order to determine the type and property of a material, surface area to volume ratio (SA:V) or Specific Surface Area (SSA) can be calculated. Each material has its own SSA. The SSA is of particular importance in reactivity. It gives the rate at which the reaction will proceed. SSA can be calculated using the following equation:
SSA ¼
SApart Vpart density
ð2Þ
where SApart is particle surface area, Vpart is particle volume and density of silver is 10.5 g/cm3. The values of SA: V ratio and SSA of S1 was 0.38 and 37 m2/g respectively. For S3, the values of SA: V ratio and SSA was 0.66 and 64 m2/g respectively. The SA: V ratio and SSA was high for the silver nanoparticles synthesized using higher concentration of fruit extract.
Fig. 7. Zone of inhibition of (a) S3 and (b) S1 for (i) E. coli and (ii) Proteus Mirabilis, respectively and (c) antibacterial activity of S3 and S1.
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Fig. 6 shows the energy dispersive X-ray analysis (EDX) of S3. It confirms the formation of silver nanoparticles and reveals higher counts at 3 keV due to silver nanoparticles. It was also due to surface plasmon resonance of metallic silver nanocrystals as shown in optical absorption spectra. Antibacterial activity In this present case, the antibacterial activity of prepared silver nanoparticles was deliberated against Gram negative pathogens E. coli and P. Mirabilis. The antibacterial effect of silver nanoparticles on microorganisms may be held through the electrostatic attraction of positive charged silver and negative charged cell surface of microorganism. Fig. 7(a and b) shows the zone of inhibition of silver nanoparticles S3 and S1 against E. coli and P. Mirabilis. Fig. 7(c) shows the antibacterial activity of silver nanoparticles produced from (S3) and (S1) against pathogens. The antibacterial activity of S3 was more than S1 because of its larger specific surface area, smaller size and spherical shape. Depending on the surface area available for interaction, silver nanoparticles can bind bacteria and disturb its permeability and respiration function. Smaller particles having larger surface area can increase the ability to penetrate cell membrane and give more bactericidal effect than the larger particles [41]. Thus, the antibacterial activity of prepared silver nanoparticles are influenced by the size and morphology of the particles, and the effect of antibacterial activity increases with decreasing size of silver nanoparticles. The prepared silver nanoparticles function as a good antibacterial agent was significant in biomedical applications as well as in the removal or inactivation of pathogenic bacteria in water.
was observed that the intensity of the SPR bands got reduced and red shifted for all metal ions as compared to that of the AgNPs. It was observed that there was no prominent SPR peak for Zn2+ and a secondary peak for Cu4+, indicating the prepared AgNPs were sensitive and selective towards Zn2+ and Cu4+. The activity of S3 in the presence of Zn2+ was studied by adding various concentration of heavy metal ion (Zn2+) from 3 104 to 12 104 M in water. Fig. 9(a) shows the UV–vis absorption spectrum for activity of S3 as zinc sensor. The prepared AgNPs (S3) solution did not exhibit color change from reddish brown as well as change in absorption spectra up to the addition of 2 104 M Zn2+. This indicates that no aggregation effect has taken place. The addition of 3 104 to 12 104 M Zn2+ to the silver nanoparticles solution causes color changes from reddish brown to colorless, were observed as shown in Fig. 9(a) inset. The addition of 3 104 M Zn2+ causes immediate reduction in the intensity of surface plasmon peak at 444 nm. When zinc acetate added to the prepared nanoparticles, newly produced zinc atoms strongly bonded on the silver surface and moved the capping biomolecules in the extract, away from the surface, which could be accounted for the broadening and slight blue shift of the SPR band of silver nanoparticles. The optical absorbance intensity reduces and band
Detection of zinc and copper ions in water To investigate the interaction of prepared AgNPs (S3) with various alkali metal (Li+, K+, Fe3+) and transition metal ions (Ni2+, Mn2+, Cu4+, Zn2+, Hg2+, Cd2+), 0.3 ml of (3 mM) salts of these metals were added into 3 ml of AgNPs by drop by drop and stirred for 2 min. The photographs (Fig. 8 (inset)) and UV–vis spectra (Fig. 8) of AgNPs were taken immediately after addition of metal ions, after 2 min of interaction. Upon addition of Zn2+, the reddish brown color of the solution changed to colorless. Similarly the reddish brown color of the AgNPs solution changed to light gray. Based on Fig. 8, it
Fig. 8. UV–vis absorption spectrum and photographs (inset) of S3 with various heavy metal ions.
Fig. 9. (a) UV–vis spectra and color changes (inset) of S3 as a function of various concentrations of Zn2+ ions (3 104 to 12 104 M) and (b) plot of absorbance at 444 nm versus Zn2+ concentration (3 104 to 12 104 M) with a correlation factor R2 = 0.959.
M.R. Bindhu, M. Umadevi / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 596–604
broadens gradually with the increase of concentration of Zn2+, suggesting that aggregation had taken place due to the reduction of interparticle distance in the aggregates less than about the average particle diameter. Upon addition of the 3 104 M to 12 104 M Zn2+ to S3, color changes from reddish brown to colorless were observed, and after the addition of 12 104 M Zn2+ absorbance changes were negligible. This shows that the formation of stable aggregates. So the concentration of zinc was limited to 10 104 M with notable color and surface plasmon resonance band. Fig. 9(b) shows a good linear correlation (y = 0.036x + 0.965, R2 = 0.959) between the absorbance (DA) versus concentration of Zn2+, and the sensitivity of the system towards analyte concentration was found to be 0.036/104 M as measured from the plot of absorbance (DA) versus concentration of Zn2+. Fig. 10(a) shows the UV–vis absorption spectrum for activity of silver nanoparticles as copper sensor. When 1 mM copper sulphate added to S3, the color of the nanoparticle solution did not change, only the addition of 2 mM copper sulphate causes color changes. This indicates that the analyte directed aggregation of nanoparticles taken place only after the addition of 2 mM Cu4+. With increasing analyte concentration from 2 mM to 7 mM, the
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intensity of the SPR peak centered at 478 nm decreases gradually with band broadening and a new SPR band appears at 780 nm with increasing intensity. The main reason for SPR broadening is electron surface scattering which may be enhanced for very small clusters [42]. The observed band broadening of the surface plasmon band reveals that the copper atoms may bind on the silver surface. The presence of new SPR peak was due to the adsorption of molecules causing changes in dielectric environment around a nanoparticle or agglomerated particles. When copper sulphate is added to S3, Cu4+ ions interact with the biomolecules in the extract on the surface of the nanoparticles, forming bonds among nanoparticles with Cu4+ ions performing as link for binding sites of biomolecules and eliminating it away from the surface of the nanoparticle surface, in that way aggregation of nanoparticles has taken place. The addition of 2 mM to 7 mM Cu4+ to S3 causes color changes from reddish brown to light blue shown in Fig. 10(a) inset. The observed color changes and absorbance changes after the addition of 7 mM Cu4+ were negligible, suggesting the formation of stable aggregates. So the concentration of copper was limited to 7 mM with notable color and surface plasmon resonance band. The linear variation of absorbance (A780 nm/A478 nm) changes and the concentration of Cu4+ over the range from 2 mM to 7 mM are shown in Fig. 10(b). This plot can be fit by a linear equation y = 0.605x0.202, R2 = 0.967 and the sensitivity of the system towards analyte concentration was found to be 0.605/mM. Applications of nanoparticle sensors by the aggregation of small particles were useful because aggregates with multiple particles yield large enhancements due to the enormous electromagnetic field that coherently interfere at the junction site between the particles. This zinc and copper sensor based on surface plasmon optical sensor can be used in environmental monitoring especially in water purification. Conclusion An ecofriendly method of obtaining spherical silver nanoparticles with average size of 10 nm has been synthesized, using A. comosus as reducing agent. The prepared silver nanoparticles are stable for one month without aggregation. Crystalline nature of the nanoparticles was evident from bright spots in the SAED pattern, and peaks in the XRD pattern. The prepared silver nanoparticles reveal good antimicrobial activity against Gram negative pathogens E. coli and P. Mirabilis, which are found in water. So the prepared nanoparticles have found applications in biomedical and water purification processes for inhibiting the growth of bacteria. The prepared nanoparticles were used for sensing ions of heavy metals like Zn2+ and Cu4+ in water using a SPR optical sensor. A strategy explained in the present study is detectable by using naked eye or UV–vis spectrophotometer. The optical absorption spectrum of the colloidal suspension before and after addition of metal ions is a good pointer to detect the concentration of the heavy metal ions. For continuous monitoring and real time information, this Zn and Cu sensor based on surface plasmon optical sensor is preferred, and it can be used in environmental monitoring, especially in water purification. Acknowledgements The authors are thankful to DST-CURIE New Delhi, UGC-DAE CSR Indore for financial assistance. References
Fig. 10. (a) UV–vis spectra and color changes (inset) of S3 with Cu4+ ions (2–7 mM) and (b) plot of absorbance changes (A780 nm/A478 nm) versus Cu4+ concentration (2– 7 mM) with a correlation factor R2 = 0.967.
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