Retracted Article: Shape-specific silver nanoparticles prepared by microwave-assisted green synthesis using pomegranate juice for bacterial inactivation and removal

Abstract

Herein, we have synthesized four different shapes (spherical, oval, rod and flower shape) of silver nanoparticles (AgNPs) using a green synthesis approach via microwave-assisted synthesis. The nanoparticles were synthesized using pomegranate juice as a novel reducing agent. The synthesized AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and tunnelling electron microscopic (TEM) analysis. The as-prepared AgNPs show a very rapid, effective, shape-specific and dose-dependent bacteriostatic/bactericidal effect towards four different bacterial strains (two Gram negative and two Gram positive). The analysis was determined by different methods such as a disc diffusion study, growth curve analysis, minimum inhibitory concentration and minimum bactericidal concentration determinations. Amongst the four different shaped AgNPs, the flower shaped AgNPs demonstrated the best results and mediated the fastest bactericidal activity against all the tested strains at similar bacterial concentrations. The results were also confirmed by confocal microscopic analysis. Additionally, the flower shaped AgNPs have the ability to non-specifically remove various bacterial strains from a real water sample with ∼98% removal efficiency, as compared to other synthesized different shaped nanoparticles.

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Introduction

A big challenge in the field of pharmaceuticals and biomedicines is resistant of human beings towards pathogens or bacteria's. In this, the anti-biotic resistance has acquired a large range of fear for emergence and re-emergence of multidrug-resistant (MDR) pathogens and parasites.^1,2 When a person gets infected with MDR bacteria, their cure becomes very difficult, resulting in loss of money as well as time in hospitals. The MDR bacteria infected patient requires multiple treatments of broad-spectrum antibiotics, which are less effective, more toxic and more expensive.^3,4 Therefore, some modifications and development in anti-bacterial compounds with improved bacteriostatic and bactericidal effects are now a high priority in the field of research.^5,6

From past decades, silver has been used as an antimicrobial compound to deal with infections, burns and chronic wounds due to its low toxicity to the human body. Many scientists have reported that silver nanoparticles (AgNPs) with a size in the range of 10–100 nm showed strong bactericidal potential against both Gram-positive and Gram-negative bacteria.⁷ Though the mechanism of antibacterial killing or prevention is not very clear to date, some of the authors have discussed the mechanism based on their own obtained results. Accordingly, AgNPs may penetrate the bacterial cell wall and modulate cellular signaling by dephosphorylating peptide substrates on tyrosine residues.^8–10 During the study, it was also found that the antibacterial property of AgNPs depends up on the size, morphology, stability and (chemical and physical) properties of the nanoparticles. The shape, size and other factors are strongly influenced by the experimental conditions viz., kinetics of the interaction of metal ions with reducing agents, and adsorption processes of stabilizing agents with metal nanoparticles.¹¹ Generally, specific control of the shape, size and distribution of the produced nanoparticles could be achieved by changing the methods of synthesis, the reducing agents and stabilizers.^12–15

During the last three decades, synthesis of metal nanoparticles using natural resources i.e. green technology and/or synthesis has been increased a lot. Using natural precursors or natural compounds as intermediate chemicals in nanoparticle synthesis is not only an environmental friendly step, it is a step towards less-harmful and more cost-effective chemical synthesis.¹⁶ In the literature, several works have been reported for the synthesis of AgNPs using a green synthesis approach. Vinod et al. have reported the synthesis of Ag, Au and Pt nanoparticles using a natural hydrocolloid gum kondagogu (Cochlospermum gossypium) as a precursor molecule.¹⁷ Logeswari et al. reported the synthesis of AgNPs from commercially available plant powders i.e. Solanum trilobatum, Syzygium cumini, Centella asiatica and Citrus sinensis.¹⁸ AgNPs have also been synthesised from three medicinal leaf extracts, Musa balbisiana (banana), Azadirachta indica (neem) and Ocimum tenuiflorum (black tulsi), by Banerjee et al.¹⁹

Although, several studies have been reported the synthesis of AgNPs using chemical routes and their applications in antibacterial studies, the synthesis of shape-specific nanoparticles using a green synthesis approach is rarely reported. The effect of shape specific nanoparticles on bacterial growth was first explored by Pal et al.,²⁰ where three different shaped AgNPs (spherical, rod shaped and truncated) were reported. It was found that the truncated AgNPs had a better response for antibacterial purposes (Sodium borohydride). Similarly, Dong et al. have reported the synthesis of triangular nanoprism and spherical AgNPs and it was reported that the triangular-shape nanoparticle had a better antibacterial property than the spherical one due to its geometric structure and geometric plane.²¹ To the best of our knowledge, a green chemistry approach for the synthesis of shape-specific AgNPs as antimicrobial agents has not been reported yet.

The present work has focused on the development of an easy and one-pot synthesis of AgNPs by an environmentally friendly procedure (microwave assisted method) with pomegranate fruit juice as the reducing agent. Microwave irradiation has several advantages in comparison to the traditional heating methods. It produced uniform heating of the solution (short thermal induction period) and as a result, more homogeneous nucleation (less-time) with a short crystallization time is obtained at a much lower cost.²² In addition, herein, pomegranate fruit juice was used as a reducing agent which contains mainly gallic acid, ellagic acid and flavonol glycosides that are able to reduce silver nitrate into AgNPs very efficiently.²³ The nanoparticles were prepared as four different shapes, namely, spherical, oval, rod and flower. The synthesized AgNPs have been characterized by field emission scanning electronic microscopy (FE-SEM), transmission electron microscopy (TEM), UV-visible spectroscopy and powder X-ray diffraction (XRD) techniques. The bactericidal activity of the as prepared different shaped AgNPs against the pathogenic, MDR as well as multi-drug susceptible strains of bacteria such as Pseudomonas aeruginosa (Gram negative), ampicillin-resistant Escherichia coli (Gram negative), Bacillus subtilis (Gram positive), and methicillin-resistant Staphylococcus aureus (Gram positive) were studied. It was found that the shape of the nanoparticle plays a very important role in the killing of the bacterial strain. In addition, some real water samples were also used to explore the water purification impact of the synthesized nanoparticles.

Experimental section

Reagents and instruments

Silver nitrate (AgNO₃) and cetyltrimethylammonium bromide (CTAB) were purchased from Fluka. All chemicals and reagent used here were of analytical grade or higher.

The synthesized nanoparticles were characterized by Field emission scanning electron microscope (FE-SEM, Zeiss, model Super 55). The powder X-ray diffraction (XRD) study was carried out on a Bruker D8 Focus X-ray diffractometer instrument using a Cu target radiation source. Transmission electron microscopy (TEM) studies were performed on a Tecnai 30 G2 S-Twin electron microscope operated at 300 kV accelerating voltage. UV-visible spectroscopic characterization was done on a Perkin-Elmer Lambda 35 (Singapore) spectrophotometer.

Preparation of the pomegranate extract

The pomegranate was collected from a local market on the basis of cost effectiveness and ease of availability. Fresh and healthy pomegranate were collected and rinsed thoroughly first with tap water followed by distilled water to remove all the dust and unwanted visible particles. Seeds were separated from the fruit, crushed and juice was collected in a beaker (250 mL). To this, 100 mL distilled water was added and the mixture was boiled for about 20 min. After that, the juice was filtered and stored at 4 °C, before use.

Synthesis of the different shaped silver nanoparticles (AgNPs)

For the synthesis of the different shaped AgNPs, AgNO₃ was used as the precursor compound with CTAB as the stabilizer and pomegranate juice as the reducing agent. The amount of constituents and reaction conditions were varied to synthesize the different shaped AgNPs. The procedures are given below and shown in Scheme 1.

Scheme 1 Graphical representation for synthesis of shape-specific silver nanoparticle using green synthesis approach.

Spherical shaped AgNPs (S-AgNPs). For this, an aqueous solution of AgNO₃ (0.01% by weight) was prepared in a 250 mL volumetric flask. 50 mL was then taken into a conical flask into which 1.82 g of CTAB was added. To the mixture, 3.0 mL of freshly prepared pomegranate juice was added and placed inside a microwave oven for complete bioreduction at 300 W for 5 min. The color of the solution changed to brown, which indicated the formation of AgNPs. The solution was kept under stirring at room temperature for a further 12 h. The resulting nanoparticles were collected, centrifuged and stored in vacuum desiccators.

Oval shaped AgNPs (O-AgNPs). Oval shaped silver nanoparticles were synthesized by a seed-mediated growth method.²⁴ Firstly, a seed solution for O-AgNPs was prepared by addition of 1.0 mL cold aqueous solution of pomegranate seed extract into a mixture of 0.25 mM AgNO₃ and 0.25 mM trisodium citrate (10.0 mL each). The resultant mixture was stirred for 2 hours and stored as a seed solution. Similarly, a growth solution was also prepared by mixing 7.0 mL of 0.020 M CTAB, 0.50 mL of 10.0 mM AgNO₃ and 0.50 mL of pomegranate juice. To the as prepared growth solution, 0.50 mL of the seed solution was injected and incubated at 27 °C for 12 h. The resulting nanoparticles (O-AgNPs) were collected by centrifugation, re-dispersed in deionised water, washed, dried and stored in vacuum desiccators.

Rod shaped AgNPs (R-AgNPs). For the preparation of the rod shaped AgNP growth solution, 0.2 M CTAB (10 mL) and 4.0 mM AgNO₃ (1.2 mL) were added to 25 mL distilled water. To the mixture, 0.5 mL pomegranate juice and 20 mL oval shaped nanoparticle solution was added and the mixture was kept in a water bath at 30 °C for 24 h. The resulting nanoparticles (R-AgNPs) were collected by centrifugation, re-dispersed in deionised water, washed, dried and stored in vacuum desiccators.

Flower shaped AgNPs (F-AgNPs). For the synthesis of the flower shaped AgNPs, 0.2 M CTAB (5 mL) and 4.0 mM AgNO₃ (3 mL) were added to 33.0 mL distilled water. To the mixture, 8.0 mL pomegranate juice and 10.0 mL of rod shaped nanoparticle solution was added and the solution was kept at 30 °C for 24 h. The resulting nanoparticles (F-AgNPs) were collected by centrifugation, re-dispersed in deionised water, washed, dried and stored in vacuum desiccators.

Preparation of bacterial samples and antibacterial study

The various shaped AgNPs synthesized using pomegranate juice were applied to explore their shape-specific properties towards different bacterial strains i.e. Pseudomonas aeruginosa (Gram negative), Escherichia coli (Gram negative), Bacillus subtilis (Gram positive), and Staphylococcus aureus (Gram positive). The study was performed by different analysis methods: (1) a disc diffusion method, (2) minimum inhibition concentration (MIC), (3) minimum bactericidal concentration (MBC) and (4) growth kinetics.

Disc diffusion study (DDS). The disc diffusion study (DDS) was carried out with a bacterial strain (10⁵–10⁶ CFU mL⁻¹) cultured separately on Luria–Bertani (LB) agar plates. Separately, 6.0 mm filter paper disks were impregnated with 1.0 mL of the different shaped AgNP sample solutions and gently placed on the center of the bacteria growth on the LB agar plates and incubated overnight at 37 °C. The inhibition zone was monitored and the diameter of the inhibition zone was calculated by visualizing the blank space around the filter paper to evaluate the shape-specific antibacterial performance of the AgNPs.

Growth curve. For the growth kinetic test, the bacterial strains of all four bacteria [Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), and Staphylococcus aureus (S. aureus)] were prepared in LB medium and incubated overnight at 37 °C. After that, the incubated bacteria were injected into fresh media and grown at 37 °C with shaking at 200 rpm. To the solution, various concentrations of the different shaped AgNPs were added and optical density (O.D.) was measured at different time intervals. Bacterial growth rates were measured by monitoring the optical density at 600 nm (O.D₆₀₀) using a UV-visible spectrophotometer.

Determination of minimum inhibitory concentration (MIC). To examine the minimum inhibitory concentration (MIC) for all four bacterial strains, silver nanoparticles of various concentrations (0.05–25.00 μg L⁻¹) were introduced into sterile flasks containing 50.0 mL nutrient broth. The mixture was sonicated for 15 minutes to avoid any type of aggregation in the solution. To the flask, 5.0 mL of freshly prepared bacterial suspension (1 × 10⁵ to 1 × 10⁶ CFU mL⁻¹) was added and the culture media was incubated for 24 hours (37 °C) in an orbital shaker at high rotation speed (120 rpm). After 24 h incubation, 5.0 mL of fresh bacterial solution was further added to the flask and incubated at similar conditions for the next 24 h. The growth or no growth of the bacterial colony was determined by visual observation. The lowest concentration that is the highest dilution required to arrest the growth of bacteria was regarded as the minimum inhibitory concentration (MIC). For accuracy of result, all of the concentration values were analyzed three times and the relative standard deviation was less than 1%. The aqueous solution of AgNO₃ was used as a control or standard.

Estimation of minimum bactericidal concentration (MBC). The minimum bactericidal concentration (MBC) can be defined as the lowest concentration of AgNPs that kills 99.9% of the bacteria and it was determined from the batch culture studies done for the measurement of MIC values. The sample flasks that appeared to have little or no bacterial growth were selected and a loop full from each flask was plated on fresh solid media (2% agar in nutrient broth) and incubated at 37 °C for 24 hours. The nanoparticle concentration causing a bactericidal effect was selected based on the absence of colonies on the agar plate and the lowest concentration causing a bactericidal effect was reported as the MBC. For accuracy, each experiment was done thrice to assess the MBC values.

Cell death or % killing. To study the cell death or percentage killing of bacteria, firstly, the bacterial strain was spread on the agar plates and incubated at 37 °C, followed by addition of 100.0 μL of the nanoparticle (20 μg L⁻¹). The plate was finally incubated at 37 °C for 24 h and the bacterial colony was counted at regular time intervals of 2 hours. The antibacterial drug (streptomycin sulphate) was used as a positive control and each result was presented as an average of three independent experiments. The death rate of the bacteria was calculated using the following eqn (1).⁷

Death rate (%) = [(counts in the control) − (counts upon incubation with the nanoparticle)]/(counts in the control) (1)

Removal of bacteria by flower shaped AgNPs

Nowadays, the water purification system including filtration membranes etc. are modified with nanoparticles to remove bacterial contaminations more efficiently. Herein, we have also tried to explore the water purification aspect of the flower shaped AgNPs for removal of bacterial contamination from water samples collected from different areas of our state. For the analysis, the diluted bacterial solution (1 × 10³ CFU mL⁻¹) was added in a glass vial containing 10.0 mL of F-AgNPs (20.0 μg L⁻¹). The mixed solution was placed in a rotary shaker for 15 min to ensure the effective binding between the bacteria and nanoparticles. After that, the nanoparticles were filtered and separated from the mixture solution and the remaining supernatant was used to analyze the bacterial count by a conventional surface plate count method. The bacteria removal efficiency of the F-AgNPs was calculated using eqn (2):

Removal efficiency (%) = (CFU₀ − CFU_t)/CFU₀ × 100 (2)

where CFU₀ and CFU_t are the initial and residual numbers of bacterial colonies in the samples.²⁵

Result and discussion

Characterization of AgNPs

UV-visible spectroscopy. Fig. S1,† shows the UV-vis spectra of the silver nanoparticles synthesized with the help of pomegranate juice as a reducing agent. The UV-vis spectrum of pomegranate juice solution was taken as a reference and no absorption spectrum was observed. UV-visible spectroscopy is one of the basic techniques for structural characterization of metal nanoparticles due to their surface plasmon resonance (SPR) behavior.²⁶ As shown in Fig. S1,† all the spectra possess the characteristic SPR band of AgNPs at 420 nm, which confirms the successful synthesis of nanoparticles.²⁷ According to the literature, the absorption spectra of metal nanoparticles, governed by SPR, show a shift to a longer wavelength with an increase in the particle size.²¹ However, according to Mie’s theory, only a single SPR band is expected in the absorption spectra of the spherical shaped nanoparticles, whereas other nanoparticles could give rise to two or more SPR bands depending on their shape, due to the decrease in the symmetry of the nanoparticles.²⁸ Herein, the spherical and oval shaped nanoparticles show one adsorption band at 420 and 430 nm, respectively, whereas the rod and flower shaped nanoparticles show two adsorption bands at 437 and 660 nm and 450 and 680 nm, respectively. This confirms the large size and multi-plane structure of the synthesized nanoparticles. Herein, the various shapes of the AgNPs were developed due to the agglomeration of the spherical shaped nanoparticles, which first forms a rod shaped nanoparticle and then these rod shaped nanoparticles agglomerate and lead to the flower shaped structure. It may be possible that during this structural transformation, some amount of spherical as well as rod-shaped nanoparticles may be present along with nanoflowers, which leads to weak UV adsorption peaks at 660 and 680 nm, respectively. However, most of the spherical/rod shaped particles are transformed to flower shaped nanoparticles, which gives an additional peak in the UV spectra and is later on also supported by SEM images. In addition, the red-shift in wavelength of the nanoparticle also supports the mechanism shown for preparation of different shaped nanoparticles i.e. multi-nanoparticle aggregation (Scheme 1).

XRD spectra. Crystalline information of the synthesized nanoparticles was obtained by XRD measurement. Fig. 1 shows the XRD patterns of the spherical (curve 1), oval (curve 2), rod (curve 3), and flower shaped nanoparticles (curve 4). All the synthesized nanoparticles show five distinct diffraction peaks at 38.5°, 44.4°, 64.6°, 77.4° and 81.7°, which correspond to the (111), (200), (220), (311) and (222) planes of face centered cubic silver, respectively. The resultant data was matched with the JCPDS file number 87-0720. The XRD patterns clearly suggest that the AgNPs synthesized from the pomegranate seed extract were crystalline in nature and were well matched with a previous report.²⁹

Fig. 1 Powder X-ray diffraction patterns of (1) spherical, (2) oval, (3) rod and (4) flower shaped silver nanoparticles.

SEM analysis. An SEM technique was employed to visualize the size and shape of the synthesized silver nanoparticles. The corresponding SEM images of the synthesized AgNPs (various shapes) are shown in Fig. 2. Fig. 2A shows the SEM image of the spherical shaped AgNPs. The S-AgNPs are round in shape and have an average size of ∼50 nm. As shown in Fig. 2B, the O-AgNPs have a slightly larger size and an elongated shape in comparison to the S-AgNPs. However, the rod shaped AgNPs have a diameter of ∼100 nm and length of ∼250 nm (Fig. 2C). The rod shaped AgNPs get agglomerated to form a flower shaped nanoparticle, which is clearly shown in the FE-SEM image (Fig. 2D). To get a closer view and a more magnified image of the F-AgNPs, their high magnification image was also recorded and is shown in Fig. 2E. The SEM images also support the weak UV peaks obtained at 660 and 680 nm for the R-AgNPs and F-AgNPs, respectively. Therefore, the SEM study clearly supports the formation of different shaped silver nanoparticles with their morphological dimensions, as shown in Scheme 1.

Fig. 2 FE-SEM images of the (A) spherical, (B) oval, (C) rod and (D) flower shaped silver nanoparticles at low and (E) high magnifications.

TEM analysis. The TEM images of the different shaped AgNPs (spherical, oval, rod and flower) are illustrated in Fig. 3, where it can be easily visualized that well-defined shapes of AgNPs are formed. As shown in the Fig. 3A and B, the spherical and oval shape nanoparticles have small size in which approximately 85–90% of nanoparticles possess the spherical and/or oval shape. A single TEM image of the rod shape is presented in Fig. 3C, and reveals that the diameter of the rod shape is ∼100 nm. Fig. 3D, shows a monodispersed large flower shaped AgNP with an average size of 550 nm. The flower shaped AgNP has a very dark contrast image, which indicates the rich three-dimensional (3D) structures of the F-AgNPs, likely to be formed by self-assembly of two-dimensional (2D) rod shaped AgNPs.³⁰ The single-crystalline nature of the synthesized AgNPs was also confirmed by a selected area electron diffraction (SAED) pattern shown in Fig. S2.† It also indicates that the pure face-centered cubic (fcc) silver structure were oriented with (1 1 1) planes as the basal plane.³¹ The obtained TEM images are in good agreement with the earlier synthesized AgNPs by other research groups like Bakshi,³² Lou et al.,³³ Zaheer and Rafiuddin,³¹ and Mirkin et al.³⁴ This suggests that natural resources, either in the form of a precursor or reducing agent, have much potential in the field of nanomaterial synthesis.

Fig. 3 HR-TEM images of (A) spherical, (B) oval, (C) rod and (D) flower shape silver nanoparticles.

Antibacterial study

Disc diffusion method. The antibacterial activity of different shaped AgNPs were studied by both disc diffusion or the inhibition zone method. The nanoparticles were impregnated in a small disc, placed on the plates occupied by different bacterial strains and the zone of inhibition around the disk (the clear zone around the disc) was monitored, calculated and portrayed in Fig. 4A–D. The different nanoparticles are depicted by a number i.e. (1) represents S-AgNPs, (2) O-AgNPs, (3) R-AgNPs and (4) F-AgNPs. From the figure, it is clear that in the case of all four bacteria, the bacterial growth inhibition was lowest in the case of the spherical shaped AgNPs, for which the zones of inhibition were 8 mm (E. coli), 10 mm (Pseudomonas aeruginosa), 11 mm (B. subtilis) and 6 mm (S. aureus), and highest with the flower shaped AgNPs, for which the zones of inhibition were 15 mm (E. coli), 15 mm (Pseudomonas aeruginosa), 17 mm (B. subtilis) and 13 mm (S. aureus). The maximum antibacterial activity of nanoparticles can be ordered in the following sequence:

S-AgNPs < O-AgNPs < R-AgNPs < F-AgNPs

Here, the maximum zone of inhibition (17 mm) was observed for F-AgNPs and the minimum for S-AgNPs, which may be attributed to the rounded edges of the spherical nanoparticles, which had poor interaction with bacterial cells, resulting in low bactericidal activity.³⁵ Similarly, amongst all the four bacterial strains, the best performance was observed with B. subtilis.

Fig. 4 Disk diffusion test of different shaped silver nanoparticles against different bacterial strains: (A) E. coli, (B) Pseudomonas aeruginosa, (C) B. subtilis and (D) S. aureus. Different shaped-AgNPs were used in each plate and they were represented by numbers: (1) spherical, (2) oval, (3) rod and (4) flower shaped AgNPs.

Growth curve analysis. A bacterial inhibition growth curve was used to study the growth kinetics of E. coli, P. aeruginosa, B. subtilis and S. aureus with prepared bacterial samples (Fig. S3†). The optical density at 600 nm (OD₆₀₀) was measured to monitor bacterial growth. As shown in Fig. S3A–D,† the bacterial growth (for all four strains) was delayed when the different shaped AgNPs were added at the same concentration as compared to the control. The growth of all the bacterial strains was completely inhibited by the F-AgNPs, however, the least effect was observed with S-AgNPs. The results confirm that the F-AgNPs possessed a much more enhanced antimicrobial property than the S-AgNPs. This may be due to the large surface area provided by the F-AgNPs, which results in better interaction with bacterial membranes.

MIC and MBC test. For the evaluation of the minimum concentration of all the AgNPs required for inhibition of bacterial growth, a MIC test was performed. For such a study, an aqueous solution of AgNO₃ was used as a standard or reference. The results are portrayed in Table 1, in terms of positive (+) and negative (−) symbols, where “−” represents no growth and “+” represents the growth of bacteria. As shown in the table, the Gram-negative bacterial strain of E. coli and P. aeruginosa show a 0.6 μg L⁻¹ MIC value for F-AgNPs, 1.0 μg L⁻¹ for R-AgNPs, 2.0 μg L⁻¹ for O-AgNPs and 3.0 μg L⁻¹ for S-AgNPs in comparison to an 8.0 μg L⁻¹ MIC for AgNO₃ after 24 h incubation (Table 1), which is approximately, 16, 8, 4, and 3 times lower than the standard solution of silver. It was also found that MIC values at 48 h were higher than those at 24 h because of the addition of a fresh bacterial strain as well as more time for growth. Even at 48 h, a low MIC value of 1.0 μg L⁻¹ was observed for F-AgNPs, to inhibit the growth of Gram negative bacteria, which suggests the very good antibacterial properties of F-AgNPs.

Table 1 Minimum inhibitory concentration tests of various shape silver nanoparticles on Gram negative and Gram positive bacteria

Concentration (μg L^−1)	Gram negative bacterial strain
	E. coli										P. aeruginosa
	S-AgNPs		O-AgNPs		R-AgNPs		F-AgNPs		AgNO3		S-AgNPs		O-AgNPs		R-AgNPs		F-AgNPs		AgNO3
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
25.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
20.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
15.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
12.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
10.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
8.0	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	+
6.0	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	−	−	+	+
3.0	−	+	−	−	−	−	−	−	+	+	−	+	−	−	−	−	−	−	+	+
2.0	+	+	−	+	−	−	−	−	+	+	+	+	−	+	−	−	−	−	+	+
1.0	+	+	+	+	−	+	−	−	+	+	+	+	+	+	−	+	−	−	+	+
0.60	+	+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	−	+	+	+
0.50	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
0.40	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
0.30	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
0.10	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
0.05	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+

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Concentration (μg L^−1)	Gram positive bacterial strain
	B. subtilis										S. aureus
25.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
20.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
15.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
12.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+
10.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	+
8.0	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	+
6.0	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	+	+
3.0	−	−	−	−	−	−	−	−	−	+	+	+	−	+	−	−	−	−	+	+
2.0	−	−	−	−	−	−	−	−	−	+	+	+	+	+	−	+	−	−	+	+
1.0	−	−	−	−	−	−	−	−	−	+	+	+	+	+	+	+	−	+	+	+
0.60	−	+	−	−	−	−	−	−	+	+	+	+	+	+	+	+	+	+	+	+
0.50	+	+	−	−	−	−	−	−	+	+	+	+	+	+	+	+	+	+	+	+
0.40	+	+	−	+	−	−	−	−	+	+	+	+	+	+	+	+	+	+	+	+
0.30	+	+	+	+	−	+	−	−	+	+	+	+	+	+	+	+	+	+	+	+
0.10	+	+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	+	+	+	+
0.05	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+

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Similarly, for the Gram positive bacterial strains (B. subtilis and S. aureus), the lowest MIC values were found for F-AgNPs, 0.1 μg L⁻¹ and 1.0 μg L⁻¹, respectively, and an increase in the value was obtained with same trend as presented for Gram negative bacteria i.e. F-AgNPs < R-AgNPs < O-AgNPs < S-AgNPs (Table 1). Therefore, it is clearly demonstrated that the shape of nanoparticles plays a very important role on their antibacterial activity and herein, the multi-plane structure i.e. F-AgNPs shows the maximum response towards all the four bacterial strains.

As discussed in the UV and SEM analysis, during the structural transformation, the rod-shaped or flower-shaped AgNPs may contain some amount of oval or spherical nanoparticles. Their separation is not possible. Therefore, to study their coordinated effect on the antibacterial activity, we have conducted a MIC study with different mixtures of AgNPs. The corresponding data are portrayed in Table S1.† As shown in the table, a mixture containing the same concentration of S-AgNPs and F-AgNPs was unable to inhibit the growth of bacteria (1.0 μg L⁻¹ for Gram negative and 0.1 μg L⁻¹ for Gram positive), however, a similar concentration of F-AgNPs could effectively inhibit the growth (Table S1† and Table 1). The study suggests that the mixing of nanoparticles was unable to show a strong antibacterial activity, due to the presence of different surface areas and shapes. However, the individual nanoparticles, with large surface areas, have more potential towards inhibiting the growth of bacterial strains.

Comparative study for antibacterial potential of different shaped silver nanoparticles. It was already demonstrated by disc diffusion and a growth kinetics study that bactericidal efficacy of AgNPs is significantly enhanced as the shape of nanoparticles is changes from spherical to flower shaped. To further explore the shape-dependent antibacterial efficacy of the synthesized AgNPs, a test was performed against four bacterial strains. For comparison, the MBC value of individual nanoparticles was considered for a particular bacterial strain, which could be sufficient to not only resist the growth but kill the bacteria. The initial bacterial concentration was adjusted to 10⁵ to 10⁶ CFU mL⁻¹ in 50.0 mL nutrient broth, while the time period for the antibacterial activity was estimated up to 120 minutes, as the minimum time necessary to achieve a 99% bacteriostatic effect and 99.9% bacterial killing (bactericidal effect) is expected to fall within this duration.⁷

Fig. 5, represents the size dependent killing kinetics of S-AgNPs, O-AgNPs, R-AgNPs and F-AgNPs against the E. coli (A), P. aeruginosa (B), B. subtilis (C) and S. aureus (D) bacterial strains used in the earlier studies. In the case of Gram negative bacteria, F-AgNPs and R-AgNPs showed bacteriostatic (99.36% and 99.00% killing) and bactericidal effects (99.90% and 99.00% killing) in 60 and 90 minutes respectively, while, O-AgNPs and S-AgNPs displayed almost similar bactericidal efficacy and requires 120 minutes to achieve ∼99.9% bacterial reduction. This clearly indicates that the flower shaped AgNPs mediate the fastest bactericidal action by first quickly inhibiting bacterial proliferation within 60 minutes and thereafter achieving bacterial reduction over the next half an hour.

Fig. 5 Bar diagrams representing the killing kinetics of bacterial strains exposed to different silver nanoparticles at their MBC values. (A) E. coli, (B) P. aeruginosa, (C) B. subtilis and (D) S. aureus.

Similarly, for the Gram positive bacteria, F-AgNPs and R-AgNPs show antibacterial activity by achieving bacteriostatic (99.42% and 99.21% killing) and bactericidal effects (99.93% and 99.30% killing) within 90 and 120 minutes, respectively. Although, the similar efficacy for all bacterial strains was achieved in 120 minutes using O-AgNPs and S-AgNPs, out of the four bacterial strains tested, S. aureus was found to be the least sensitive against AgNPs. This can be explained on the basis of the time taken to achieve the bacteriostatic effect, which was extended from 60 minutes for F-AgNPs and R-AgNPs to 90 and 120 minutes for O-AgNPs and S-AgNPs, respectively.

Mechanism for antibacterial activity of different shaped nanoparticles. During the various antibacterial studies in this work, we have found that different shaped nanoparticles show different results and on that basis we have concluded that the F-AgNPs have the best activity, in comparison to the other AgNPs. The observed trend can be explained on the basis of the percent active facets present in the different shapes AgNPs. According to Pal et al., the reactivity of silver is favored by high-atom-density facets such as (111).²² Morones et al. have proved the faceting of nanoparticles as well as the direct interaction of the (111) facets with the bacterial surface.⁸ Herein, the flower shaped AgNPs shows the maximum intensity of the (111) facet i.e. more facets, in comparison to other nanoparticles that contain a low intensity for the (111) facet, like spherical or oval shaped AgNPs. Therefore, the trend of antibacterial activity (obtained by experimental data): S-AgNPs < O-AgNPs < R-AgNPs < F-AgNPs, could be attributed to the presence of different effective surface areas in terms of active facets.

Confocal microscopic analysis. The antibacterial efficiency of the synthesized AgNPs was further confirmed by confocal microscopy analysis using back light fluorescent staining (Fig. 6). Green fluorescence indicates live bacterial cells (E. coli), however, the dead ones are depicted by a red colour. As shown in the figure, initially the all the bacterial cells are alive and green in colour in the absence of nanoparticles (Fig. 6A). But, when the cells were treated with F-AgNPs, after just 5 minutes, the cells started dying, represented by the presence of a red colour (Fig. 6B). After increasing the incubation time to 15 minutes, more red colour appears and after 30 minutes of incubation the samples demonstrated an almost complete eradication of the bacterial cells (Fig. 6C and D). The analysis fully supports the high and rapid antibacterial efficacy of the synthesized flower shaped AgNPs.

Fig. 6 Confocal images of live (green) and dead (red) E. coli bacterial cells: (A) without F-AgNPs, (B) after 5 min, (C) 15 min and (D) 30 min incubation with F-AgNPs.

Non-specific removal of bacteria from aqueous and real water samples. According to the standard of water, a limit of less than 100 CFU E. coli per mL of water was employed for general use. The water sample, containing an E. coli concentration more than the limit, is taken as polluted or contaminated water. We also aware of the fact that E. coli is an enterohemorrhagic strain responsible for mortality in the whole world.³⁶ Therefore, in this work, we have also tried to remove the bacterial contamination from aqueous as well as real water samples. Herein, various bacteria were removed using different shaped AgNPs (concentration 1.0 mg mL⁻¹). As shown in the Fig. S3,† the F-AgNPs have the ability to non-selectively remove all four of the bacterial strains with ∼98% removal efficiency.

We have also employed a bacterial colony count method to explore the real world applicability of the synthesized F-AgNPs. The water samples used in the experiment were collected from the local pond and river (Dhanbad, Jharkhand). The F-AgNPs (different concentrations) was incubated with 5.0 mL of the collected water sample for 15 min, then the nanoparticles were filtered from the solution and the remaining water sample was cultured on a LB plate for 24 h at 37 °C. The number of colonies grown on the surface of LB plate was used to monitor the bactericidal efficiency of the F-AgNPs. As expected, we found that the F-AgNPs have a very high bacterial capture ability and the bacterial cells were removed with 90–95% efficiency.

Conclusions

In summary, we represent an environmental friendly method to synthesize different shaped AgNPs with the help of pomegranate juice as a reducing agent and a microwave assisted method. No toxic reagents were used to synthesize the different shaped AgNPs (spherical, oval, rod and flower). All the AgNPs were found to be highly effective as antibacterial agents to the all studied bacterial strains and their antibacterial efficacy increased when the shapes of the AgNPs were changed (F-AgNPs > R-AgNPs > O-AgNPs > S-AgNPs). The bacteria killing efficiency was also visualized, herein, using confocal microscopic analysis. Additionally, the synthesized F-AgNPs were also applied to the capture and rapid removal of pathogenic bacteria from real water samples.

Acknowledgements

The authors are thankful to the Department of Science and Technology, Government of India for the sanction of the Fast Track Research Project for Young Scientists to Dr Rashmi Madhuri (Ref. No.: SB/FT/CS-155/2012) and Dr Prashant K. Sharma (Ref. No.: SR/FTP/PS-157/2011). Dr Sharma (FRS/34/2012-2013/APH) and Dr Madhuri (FRS/43/2013-2014/AC) are also thankful to the Indian School of Mines, Dhanbad for a Major Research Project grant under the Faculty Research Scheme. We are also thankful to the Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy, Government of India, for the major research project (34/14/21/2014-BRNS).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18575k

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