Friday, 29 March, 2024

ISSN: 2456-7949

Ag and Au Nanoparticles: Green Synthesis, Catalytic and Bacterial Activity Studies

Saraschandra Naragintia,b and A. Sivakumara,*

aChemistry Division, School of Advanced Sciences, VIT University, Vellore - 632014, India.

bKey Laboratory of Integrated Regulation and Resource Development of Shallow Lakes, Ministry of Education, College of Environment, Hohai University, XiKang Road #1, Nanjing 210098, PR. China.

Published Online 05 01 2017

 


A rapid one step green synthetic method using kiwi fruit extract was employed for preparation of silver and gold nanoparticles. The synthesized nanoparticles were successfully used as green catalysts for the reduction of 4-nitrophenol (4-NP) and methylene blue (MB). They also exhibited excellent antimicrobial activity against clinically isolated Pseudomonas aeruginosa and Staphylococcus aureus. It was noticed that with increase in concentration of the aqueous silver and gold solutions, particle size of the silver and gold nanoparticles showed increase as evidenced from UV-Visible spectroscopy and TEM micrograph. The method employed for the synthesis required only a few minutes for more than 90% formation of nanoparticles when the temperature was raised to 80 °C. It was also noticed that the catalytic activity of nanoparticles depends upon the size of the particles. These nanoparticles were observed to be crystalline from the clear lattice fringes in the transmission electron microscopic (TEM) images, bright circular spots in the selected area electron diffraction (SAED) pattern and peaks in the X-ray diffraction (XRD) pattern. The Fourier-transform infrared (FTIR) spectrum indicated the presence of different functional groups in the biomolecule capping the nanoparticles.

INTRODUCTION

Nanoparticles are known to exhibit characteristic features depending on their size, morphology1 and these features make them good antibacterial and antifungal agents.2–5 It is expected that these unique features of nanoparticles would play a crucial role in biomedicine, energy science, catalysis, optics and other health care applications.6 Methods such as chemical reduction,7 electrochemical reduction,8 photochemical reduction9 and heat evaporation10 have all been employed for synthesis of nanoparticles by different researchers. All these methods do successfully produce the metal nanoparticles but have disadvantages such as high process cost and pollute the environmental because of usage of toxic organic solvents and reducing agent. To avoid these issues, green chemistry approaches have been employed for their production11–15 which are simple, convenient, less energy-intensive, eco-friendly and minimize the usage of unsafe materials and maximize the efficiency of the process.

4-nitrophenol (4-NP), listed by the US EPA as a major pollutant,16 has been used extensively as a raw material for the manufacture of pesticides, herbicides, synthetic dyes, pharmaceuticals, for treatment of leather and in several military applications.17 Since the nitro group in nitrophenols is comparatively static in biological systems, it could cause health hazards in humans and animals, making it necessary to develop efficient methods for its degradation in environmental samples. This would pave way for degrading hazardous phenolic compounds in water wastes, soil and air which are primary causes of serious health issues in human beings.18,19 MB is a thiazine dye, used for trace levels analysis of sulphide ions in aquatic samples. The cationic form of MB is used as an anti-malarial agent and chemotherapeutic agent in the aqua culture industry. Moreover, it is used in microbiology, surgery and diagnostic field.20,21

4-nitrophenol and methylene blue have been degraded using different physical and chemical methods employing adsorption, photocatalysis, UV irradiation, microwave, electro catalysis, and fenton reaction, which are energy consuming and require organic solvents. Biological methods have also shown poor degradation on 4-nitrophenol reduction.22,23 This made researchers to discover green catalysts for degradation of 4-nitrophenol and methylene, though few reports are available for green catalysis,24,25 it would be appropriate to develop more efficient and cost-effective methodology for their degradation via an eco-friendly approach. Few reports are also available for bactericidal activity of Ag, Au and other metal nanoparticles produced using various plant extracts.26–32

The present study reports a novel attempt of synthesizing gold and silver nanoparticles using kiwi fruit extract for catalytic reduction of two organic pollutants. Different reactions conditions such as temperature and volume of fruit extract on the formation of nanoparticles were also studied. Simultaneously, the bactericidal activity of the synthesized nanoparticles against clinically isolated pathogenic microorganisms was also investigated.

MATERIALS AND METHODS

Chemicals

Silver nitrate (AgNO3) and Chloroauric acid (HAuCl4) were purchased from Sigma-Aldrich chemicals and kiwi fruits were procured from local market. Millipore water was used in all experiments.

Bacterial Pathogens

Clinically isolated bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) from patient blood samples have been obtained from Santhiram Medical College & General Hospital, Nandyal, India.

Preparation of Fruit Extract

The kiwi fruit extract was prepared by taking 100 g of the peeled fruit which was ground well, filtered through Whatmann filter paper and centrifuged at 4000 rpm for 10 min. The extract was stored at 4 °C for further use.

Green Synthesis of Silver and Gold Nanoparticles

1 mL of fruit extract was added to 9 mL of silver nitrate or chloroauric acid (1 x 10-3M ) and the reaction mixture was placed at 80 °C till the color turned to characteristic yellow and ruby-red respectively indicating the formation of silver nanoparticles (Ag NPs) and gold nanoparticles (Au NPs). Both the reactions were carried out at five different concentrations in the range of 1 x 10-3 M to 3 x 10-3 M each of silver nitrate (S1 to S5)  and chloroauric acid  (G1 to G5) solutions. These reactions were also carried out at two other temperatures, 30 °C and 60 °C to study rate of the formation of the nanoparticles.

Characterization of Nanoparticles

The formation of  Ag NPs and Au NPs using fruit extract as reducing agent was monitored by periodically measuring the absorbance of the solution on the UV–Vis spectrophotometer (JASCO V – 670) till the solutions showed permanent yellow and ruby red colour respectively. TEM imaging, SAED pattern and EDAX analysis were carried out using JEOL JEM 2100 high resolution transmission electron microscope (HR-TEM) with an accelerating voltage of 200 KV. XRD patterns were recorded for the centrifuged and dried samples using X–ray BRUKER D8 Advance X-ray diffractometer with Cu Kα source (λ=1.5406 A°). FT-IR spectrum was recorded using SHIMADZU, IRAffinity 1 spectrometer. A differential light scattering Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., UK) instrument was used for zeta potential measurements.

Antibacterial Activity

The antibacterial activity of both nanoparticles against two pathogenic microorganisms, P. aeruginosa and S. aureus, which were clinically isolated, was studied by zone inhibition method using sterilized Muller Hinton agar. The inoculums from the isolates were spread on the plate using L-rod and wells were made in the agar using a cork borer. 10 µL, 20 µL and 30 µL of colloidal silver (S1) and gold (G1) nanoparticles were added into each one of these wells. The plates were incubated at 37 °C for 24 h in an aerobic incubation chamber and were visually analyzed for zones of inhibition.

Catalytic Reduction of 4-Nitrophenol and Methylene Blue

Catalytic reduction of 4-NP was studied by adding 100 µL of 1 x 10–2 M 4-NP aqueous solution to 1.5 mL of freshly prepared 3 x 10–2 M NaBH4 solution  in a quartz cell  (1.0 cm  path length and 3 mL volume). Then, 25 µL of the synthesized Ag NPs (S1 & S5) or Au NPs (G1 & G5) solution was added to each one of these solutions. A JASCO V–670 spectrophotometer was employed to monitor the progress of the conversion of 4-NP to 4-AP at ambient temperature in the wavelength range of 200–550 nm.

For studying the degradation process of MB, 1 mL 0.05 M NaBH4 solution was added to 5 mL of 1 x 10-4M Methylene blue while stirring the solution for five minutes. Then, 4 mL of Ag (S1 & S5) or 4 mL of Au (G1 & G5) colloidal solutions were added to each one of the solutions and the stirring continued for five more minutes. MB (blue colour) in an oxidizing environment became colorless in the presence of NaBH4 and catalyst indicating its reduction to Leucomethylene Blue.

RESULTS AND DISCUSSION

Effect of Concentration of Metal ion Solution and Reaction Temperature

Fig.1 shows the UV–Visible spectra recorded during the formation of Ag NPs and Au NPs from different concentrations of AgNO3 and HAuCl4. When the reaction went to completion, yellow and ruby-red colors were observed in Ag and Au solutions respectively, indicating the formation of nanoparticles. Reddish yellow and groovy pink-ruby colours for Ag and Au nanoparticles are known to emerge from surface plasmon vibrations.33,34 Extinction spectra of silver hydrosol synthesized from different concentrations of AgNO3 and HAuCl4 have shown characteristic surface plasmon absorption bands at 425 nm for silver and at 538 nm for gold nanoparticles synthesized from 1.0 x 10−3M of AgNO3 (S1) and HAuCl4 (G1) using a fixed volume (1 mL) of the fruit extract. The SPR band showed a shift to higher wavelength with increasing concentration of silver nitrate from 1.0 x 10−3M to 3.0 x 10−3M


Table 1. Effect of concentration and reaction temperature on synthesis of nanoparticles using 1 mL of fruit extract.

S. No.

Conc. of AgNO3

Time taken for formation of Ag NPs (Approx. in min)

Conc. of HAuCl4

Time taken for formation of Au NPs (Approx. in min)

 

 

30 °C

60 °C

80 °C

 

30 °C

60 °C

80 °C

1

1.0 mM

340

35

20

1.0 mM

100

15

05

2

1.5 mM

320

30

20

1.5 mM

90

10

04

3

2.0 mM

320

28

15

2.0 mM

80

10

03

4

2.5 mM

300

21

10

2.5 mM

50

08

03

5

3.0 mM

300

20

09

3.0 mM

40

08

01

 


and the corresponding colour changes are observed from reddish yellow to brown; similarly the corresponding colour of the gold NPs was also observed to change from ruby-red to purple colour. The broadening as well as shift SPR band from 425 to 442 nm for silver NPs and 538 to 549 nm for gold NPs could be attributed to the increase in particle size which is also confirmed by the TEM micrographs. This kind of phenomenon has earlier been reported in the case of silver nanoparticles synthesized using seed extract of Jatropha curcas.35

 

 

Figure 1. UV-Vis spectra of (a) silver and (b) gold nanoparticles at different concentration of AgNO3 and HAuCl4.

 

No shift in the wavelength of the absorption peaks was observed at fixed concentration of the metal salt solution during the reaction with extract at different temperatures (Fig. 2). When the reaction was carried out at 60 °C and 80 °C, the rate of formation of nanoparticles was observed to increase.

 

Figure 2. UV-Vis spectra of (a) silver and (b) gold nanoparticles at fixed concentration (1.0 X 10 -3M) during the reaction at different temperatures.

Table 1. gives data on time and temperature dependence of nanoparticle formation during the reaction. This illustrates that SPR band at 425 nm was observed for Ag NPs at 30 °C after 340 min but the same band appeared within 20 min when the reaction temperature was raised to 80 °C.

Similarly for Au NPs, the SPR band was observed at 538 nm at 30 °C in 100 min appeared within 5 min at 80 °C, which also indicating that the formation of gold nanoparticles at both the temperatures was faster than silver NPs, which could be attributed to the higher reduction potential of Au3+ ions than Ag+ ions,36 similar results have been reported during the synthesis of gold nanotriangles using lemongrass extract.37

 

Figure 3. XRD pattern of the dried (a) silver and (b) gold nanoparticles.

 

X-ray Diffraction Analysis

The XRD pattern (Fig. 3) of the dried nanoparticles indicates their spherical crystalline structure as can be seen from the diffraction pattern. The patter also indicates that the nanoparticles synthesized are polydispersed and have different particle size. Characteristic face-centered cubic phase (JCPDS File No.87-0720) is confirmed by the diffraction peaks corresponding to (1 1 1), (2 0 0), (2 2 2) and (3 1 1) planes. The average size of the formed silver and gold NPs, as calculated using Debye–Scherrer equation were found to be around 35 nm and 20 nm respectively. The Bragg reflections (2 0 0), (2 2 0) and (3 1 1) were weak and broadened relative to intense (1 1 1) reflection which indicates that the nanocrystals are primarily oriented along (1 1 1) plane and have small particle size as confirmed by TEM micrographs.

TEM, EDX, Zeta Potential Analysis

TEM image of Ag NPs synthesized by treating 1.0 x 10−3M (S1) and 3.0 x 10−3M (S5) AgNO3 solutions with 1 mL of fruit extract is shown in Fig. 4 (a) & (b). These micrographs indicate that the silver NPs formed were predominantly spherical having diameters ranging from 25 to 40 nm and 30 to 45 nm respectively for S1 and S5. Particle sizes of the silver NPs synthesized using two different concentrations of AgNO3 are in complete agreement with the observed SPR bands at 425 and 442 nm respectively. Similarly TEM images of Au NPs synthesized from 1.0 x 10−3M (G1) and 3.0 x 10−3M (G5) HAuCl4 shown in Fig. 5 (a) & (b), indicate that the average particle size has a range between 7 nm to 20 nm and 15 nm to 35 nm respectively for G1 and G5, which is in agreement with the observed SPR bands at 538 and 549 nm. Insets of TEM images show the selected area electron diffraction (SAED) pattern confirming the polycrystalline nature of the synthesized nanoparticles.

 

Figure 4. TEM micrograph of silver nanoparticles synthesized from (a) 1.0 X 10−3M AgNO3 (S1) and fruit extract (1 mL), (b) TEM image of larger particles synthesized from 3.0 X 10−3M (S5) AgNO3 solution and fruit extract (1 mL) (inset shows the SAED pattern of  nanocrystalline silver.

 

The TEM images taken after storing the nanoparticle solutions for 10 days showed very little agglomeration in silver NPs while no agglomeration was found in gold NPs. From this, it could be concluded that kiwi fruit extract might be acting as reducing as well as stabilizing agent. TEM images show that the small particle clusters are coated with a thin organic layer, which acts as a capping agent since the nanoparticles showed a very good distribution inside the bio-reduced aqueous solution, even in microscopic scale. EDAX results confirmed that silver and gold are the major elements. The optical absorption spectrum at 3 keV38 [Fig. S1(a), see SI] indicated the presence of metallic silver and the optical absorption spectrum at 2 keV39 [Fig. S1(b), see SI] indicated the presence of metallic gold.  The zeta potential values of S1 and G1 have been indicated as -1.46 and -22.3 mV (Fig. S2, see SI). It is worth to mention that the silver nanoparticles showed lower potential due to little agglomeration of the particles as indicated in TEM images while gold nanoparticles have shown high zeta potential value because of uniform distribution of particles.

 

Figure 5. TEM micrograph of gold nanoparticles synthesized from (a) 1.0 X 10−3M (G1) HAuCl4 and fruit extract (1 mL), (b) TEM image of larger particles synthesized from 3.0 X 10−3M (G5) HAuCl4 solution and fruit extract (1 mL) (inset shows the SAED pattern of  nanocrystalline gold).

 

FT-IR Analysis

The FT-IR spectra were analyzed for identification of possible functional groups which help in formation of nanoparticles. The FT-IR spectrum of fruit extract (Fig. S3, see SI) showed peaks at 3448 cm‑1 corresponding to free N–H (primary amine), at 1639 cm−1 corresponding to C=C vibration, which support the presence of some proteins. It has been reported that proteins present in the fruit extract might act as capping agents for the biosynthesized nanoparticles.40,41 Gole et al. have also reported that proteins could cap the gold NPs either through free amine groups or cysteine residues in the proteins.42

Antimicrobial Activity of Silver and Gold Nanoparticcles

To evaluate the antibacterial activity, the S1 and G1 particles have been utilized due to their smaller particle size.43 Two clinically isolated pathogens P. aeruginosa and S. aureus from the patient’s blood have been used for the antimicrobial activity study. Silver NPs showed excellent antimicrobial activity for the two replicates as presented in Fig. S4(a) (see SI). The antimicrobial activity of the nanoparticles has been observed to increase with increase in the concentration of nanoparticles (Table S1, see SI),44 which could be attributed to larger quantum of nanoparticles available to kill the microorganisms. The presence of thin peptidoglycon layer in its cell wall of Gram –ve bacterium facilitates easy penetration of silver and gold NPs making them strong antimicrobial agents against P. aeruginosa as seen by the maximum zone of inhibition. The smaller zone of inhibition noticed for Gram +ve bacteria may be because of the strong structure of linear polysaccharide making penetration of nanoparticles difficult.45 The significant bactericidal activity observed may be due to the released silver cations which change the membrane characteristics resulting in increased membrane permeability of the bacteria.46,47 Several reports indicate explicitly that silver ions released from silver NPs get attached to the negatively charged cell wall of the bacteria and rupture it leading to denaturing of the protein and finally their death.48 The attachment of either silver ions or nanoparticles to the cell wall causes accumulation of envelope protein precursors. Interestingly, Lok et al. have brought out that silver NPs exhibited destabilization of the outer membrane and rupture of the plasma membrane thereby causing depletion of intracellular ATP.49 Sarkar et al. have also reported that silver NPs showed greater bactericidal efficiency against S. aureus, compared to penicillin.50 The gold NPs synthesized showed excellent inhibition zone [Fig. S4(b), see SI] against all the test organisms but the activity was observed to be less compared to silver NPs. Furthermore, the nanoparticles synthesized by green route were found to be highly effective against Gram -ve bacteria, though the exact mechanism is not yet known.

Catalytic Degradation of 4-Nitrophenol

The catalytic reduction of 4-NP in the presence of silver NPs (S1 & S5) and gold NPs (G1 & G5) was monitored by UV-Vis spectroscopy. Though the reduction of 4-NP to 4-AP by aqueous NaBH4 is thermodynamically favorable, the presence of the kinetic barrier due to large potential difference between donor and acceptor molecules decreases the feasibility of this reaction.


Figure 6. Time-dependent UV-Visible spectra for the catalytic reduction of 4-NP by NaBH4 in the presence of (a) S5  (b) S1  (c) G5 and (d) G1 catalysts obtained from 1 mL of extract.

  

It is well-known that the metal nanoparticles catalyze this reaction by facilitating electron relay from the donor BH4- to acceptor 4-NP to overcome the kinetic barrier. The 4-NP shows an absorbance peak at around 317 nm, which shows a red shift to 400 nm in the presence of NaBH4 due to the formation of 4-nitrophenolate ion in the alkaline medium caused by NaBH4.51 The data in  Fig. 6(a) indicates that the reduction gets completed in 33 min in the presence of S5 nanoparticles, consistent with the disappearance of the yellow colour of 4-NP  while the reaction in the presence of S1 nanoparticles [Fig. 6(b)] takes 22 min for completion. Similarly the reduction of 4-NP in the presence of G5 nanoparticles [Fig. 6(c)] gets completed in 20 min while with G1 nanoparticles [Fig. 6(d)] it takes 14 min for completion. The reaction was carried out with water as control in place of nanoparticle solution and the peak at 400 nm remained unaltered even after 5 days, thus confirming the catalytic role of nanoparticles in the reduction process. Plots of  ln (A) vs time for the reduction of 4-NP by NaBH4 in the presence of both metal nanoparticles shown in Fig. S5 (a) & (b) (see SI) clearly indicates that after the induction time plot of ln (A) and time t shows a linear relation through 90% completion of the reaction. Decrease in reaction rate was observed with the increase in concentration of metal ion solution. Such decreasing rate of catalytic activity with increase in metal ion concentration can be attributed to an increase in the particle size, as suggested by TEM and DLS measurements. Esumi et al. have reported similar trend in the catalytic activity of dendrimer metal nanocomposites during the reduction of 4-NP with increase in the concentration of dendrimer.52

Catalytic Degradation of Methylene Blue

Kinetics of catalytic reduction of MB has been studied in the presence of NaBH4.  Relative absorbance at 664 nm and 614 nm is plotted as a function of time to evaluate the rate of reduction of MB.  It has been reported that the main absorption band at 664 nm is corresponding to n-π* transition of MB. During reduction reaction of MB it converts to its reduced form Leuco MB (LMB).53,54 Fig. 7 (a) shows the reduction of MB in the presence of S5 catalyst over a time period of 39 min, the observed decrease in absorbance indicating that the reduction of MB is a slow process. The reduction is found to get accelerated in the presence of S1 catalyst [Fig. 7(b)]


Figure 7. Time-dependent UV-Visible spectra for the catalytic reduction of MB by NaBH4 in the presence of (a) S5 (b) S1 (c) G5 t and (d) G1 catalyst obtained from 1 mL of extract.

  

and goes to completion in 24 min which is indicated by a strong decrease in the absorbance. Similarly particle size dependence on rate of catalytic reduction of MB to LMB was also investigated in the presence of G5 and G1 catalysts. From Fig. 7(c),  it can be seen that the time required for complete reduction of MB is recorded as 33 min in the presence of G5 catalyst, while G1 catalyst [Fig. 7(d)] helped completion of the reduction in 21 min. Figure S5 (c) & (d) (see SI) show Plots of ln (A) vs time for the reduction of MB to LMB in the presence of both metal nanoparticles showing a linear relation between ln (A) and time t till 90% completion of the reaction. The increase in reaction time with the increase in metal ion concentration might be because of increase in size of the particles as explained earlier in 4-NP reduction. For effective  catalysis, the redox potential of  silver  nanoparticles  needs to be  located between  the  redox  potential  of  donor (NaBH4)  and  the acceptor (MB)  system.55 Due to electron relay effect, the metal nanoparticles act as electron transfer mediators between MB and NaBH4 during their action as a redox catalyst.

Conclusions

Silver and gold nanoparticles have been successfully synthesized using kiwi fruit extract, an environmentally benign and renewable fruit which acts as reducing as well as stabilizing agent. UV-Vis spectroscopy and TEM micrographs of the synthesized silver and gold hydrosol suggest that with the increase of concentration of aqueous solutions, the particle size increases due to which broadening and shifting of SPR band takes place. The adopted method requires only a few minutes to achieve higher than 90% conversion when the reaction temperature was raised to 80 °C. The synthesized nanoparticles have shown good catalytic activity during the reduction of two organic pollutants 4-NP and MB. With the decrease in size of the nanoparticles, the reaction time is observed to decrease due to their higher surface area. In addition, the synthesized nanoparticles have also exhibited excellent bactericidal activity against clinically isolated P. aeruginosa and S. aureus.

Acknowledgements

The help extended by Dr. Balachandran Unni Nair, Chief Scientist and Head, Chemical Laboratory, Central Leather Research Institute, CSIR Chennai, India for instrumentation is gratefully acknowledged.

Notes and References

* Corresponding Author Details: A. Sivakumar, Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632014, India.

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†  Supporting Information (SI) available: [Spectral and Bacterial activity data].

 

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