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Biosynthesis, Characterization and Antibacterial Activity of Gold Nanoparticles (Au-NPs) using Black Lemon Extract
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 18 (2019) 5164–5169
www.materialstoday.com/proceedings
ICMPC-2019
Biosynthesis, Characterization and Antibacterial Activity of Gold Nanoparticles (Au-NPs) using Black Lemon Extract Hadeel Salih Mahdia and Azra Parveena* a
Department of Applied Physics, Z.H. College of Engineering & Technology, Aligarh Muslim University, Aligarh-202002, India.
Abstract The synthesis of gold nanoparticles (Au-NPs) have been performed by a biological method using HAuCl4, black lemon extract, and double distilled water (D.D.W). The structural analysis and average crystallite size have been investigated by XRD data and the average crystallite size came out to be 15 nm. The optical properties were recorded by UV-visible microscopy in the range of 400-800 nm. SEM and EDAX have been used to study the morphological and elemental structure of Au-NPs. The triangular and spherical shapes, verified by TEM play an important role in directing the shape evolution of nanoparticles. The diameter of inhibition zone, which was used to determine the antibacterial activities of the NPs, was obtained using the disk diffusion methods. The Au-NPs showed great ability to inhibit activities of various bacteria such as gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) by damaging their activity. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019 Keywords: HAuCl4, XRD, Uv-Visible, TEM, Black lemon Extract and antibacterial effect.
1. INTRODUCTION Metallic nanoparticles are extensively used for diagnosis, treatments, and drug delivery purposes. Nanoparticles have different excellent physical properties, such as large surface area to volume ratio, that differ from the bulk material [1]. Metallic nanoparticles show various optical properties that vary with their shape and size, which make them useful for several biomedical purposes, such as drug delivery, bio-sensing and catalytic processes. Gold
* Corresponding author. Tel.: +919411653414; E-mail address: [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019
H.S. Mahdia and A. Parveen / Materials Today: Proceedings 18 (2019) 5164–5169
5165
nanoparticles are the most important nanoparticles, and are widely used in industrial processes [2]. In the area of medicine, they are used in the treatment of cancer and for inhibiting the activities of bacterial [3, 4]. Several techniques, such as chemical, biological, and physical methods, are used to synthesize Au-NPs [5, 6]. The extracts of several plants, such as black lemon, fruit, stachys, have been used to synthesize Au-NPs by biological method. The synthesis of Au-NPs using black lemon extract as stabilizing agent is presented in this study. 2. Experimental 2.1. Preparation of Black lemon Extract 250 g of black lemon, which was collected from Iraq, was cut into bits with a mill. The black lemon was added to 500 ml of double distilled water (D.D.W) and boiled for 1hr. at a temperature of 100 0C. The mixture was separated into an aqueous extract, which was refrigerated at 6°C for further use in the production of Au-NPs. 2.2. Preparation of Au-NPs In this research, Au-NPs were produced by biological technique using black lemon extract. 100 ml of double distilled water was used to dissolve 1mM of HAuCl4, and the solution was stirred continuously for 20 min with magnetic stirrer at 140 rpm. Thereafter, 10 ml of black lemon extract was added to 20 ml of HAuCl4 solution, which was continuously stirred until the color of the solution changed to red. 3. Results And Discussion 3.1. X-Ray Diffraction The structure of Au-NPs was analysed using X-ray diffraction (XRD) as shown below in Fig. 1, with Cu -Kα radiations (λ=1.5406 Ǻ) and angle 2θ in the range of 30º-80º . The diffraction peaks occurring at (38.3), (44.3), (64.6) and (77.8) were indexed as (111), (200), (220) and (311) of lattice planes. The XRD plot of Au-NPs showed the face centered cubic (fcc) structure. The diffraction peaks obtained were similar to the standard of Au metal (JCPDS-card no. 04-0784) [7].
Fig. 1. XRD analysis of biological synthesis Au-NPs
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The average crystallite size of Au-NPs was determined to be 15 nm using the Debye- Scherrer’s formula [8, 9], which is given below: D = 0.9 λ/β cos θ Where, D is the average crystallite size, (λ) the XRD wavelength (1.54 nm), β is the (FWHM) and θ is the Bragg angle. 3.2. Scanning Electron Microscopy (SEM) The morphological structure of the synthesized Au-NPs was analyzed using the SEM device as shown below in Fig 2. The spherical and cubic shapes of Au-NPs were obtained of various sizes. Bar graph of EDAX analysis (Fig. 2) verified the presence of metallic gold nanoparticles and other elemental structure showing the presence of C, O, Na, Mg, Si, Ca that may be present in black lemon extract.
Fig. 2. SEM and EDAX of biological synthesis Au-NPs
3.3. Transmission Electron Microscopy (TEM) The TEM technique was used to analyse the surface composition of Au-NPs and this is shown below in Fig.3. From TEM image the triangular and spherical shapes of Au-NPs have been confirmed with a smooth and uniform particle composition. The average particles size was investigated by TEM histogram and found to lie between 15-20 nm which is comparative to the particles size as determined by Debye- Scherrer formula from XRD.
H.S. Mahdia and A. Parveen / Materials Today: Proceedings 18 (2019) 5164–5169
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Fig. 3. TEM image of biological synthesis Au-NPs
3.4. UV-Visible spectrophotometer The UV-Vis spectrophotometer (Perkin Elmer Lambda-35) was used to analyze the optical properties of biologically synthesized Au-NPs in the range of 400-800 nm, and is shown below in Fig.4. The change in the color from yellow to red of the reaction mixture confirmed successful reduction of HAuCl4 to Au-NPs. The absorption peak of Au-NPs was observed at 537 nm and there is no absorption peak for black lemon extract [10].
Fig. 4. UV-visible of biological synthesis Au-NPs
3.5. Antibacterial activities of Au-NPs The well diffusion method was used to study the antibacterial activity of Au-NPs. 100 μl of P. aeruginosa and S. aureus were evenly distributed over Luria Agar (LA) plates on regular basis. Soft agar was used to seal wells, which have been cut into 4 mm diameter. Test solution and different amounts of Au-NPs (25, 50 and 100 μl) were then
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placed in these wells. The plates were then incubated for 24hours at 37°C and the diameter of growth inhibition zones, which was used to obtain the size of the zones, was then determined [11]. This was used to evaluate the antimicrobial activity of Au-NPs against multi drug resistance P. aeruginosa and S. aureus. From Fig.5 (a, b), the zone of inhibition of Au-NPs at 3 different concentrations against of P. aeruginosa and S. aureus bacteria were exhibited. Table 1 shows the Au-NPs inhibit the growth of both bacteria P. aeruginosa and S. aureus. A substantial increase in the size of inhibition zones was observed when increase the concentration Au-NPs as shown in Fig.6. An increase in the concentration of Au-NPs (from 0 to 100 µg/mL) resulted in a corresponding increase in antibacterial activity against S. aureus (Gram-positive bacteria) and P. aeruginosa (Gram-Negative bacteria). This is attributable to increase in reactive oxygen species (ROS) which brings about oxidative stress and destruction of the cell.
Fig 5. Zone of inhibition of biosynthesis Au-NPs using Black lemon a) P. aeruginosa and b) S. aureus
Table 1. Diameter of the inhibition zone with the various concentration of biosynthesis Au-NPs using Black lemon S. No
Concentration of Au-NPs (µg/mL)
Diameter of inhibition (mm) P. aeruginosa
1
Control
S. aureus
0
0
2
25
12
14
3
50
16
20
4
100
18
22
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Fig 6. Zone of inhibition of biosynthesis Au-NPs using Black lemon a) P. aeruginosa and b) S. aureus
4. Conclusions Au-NPs were successfully synthesized in this study by biological method using black lemon extract. The absorption peak of Au-NPs was confirmed at 537 nm with the aid of UV-Vis spectroscopy. XRD data were used to analyze the structure of Au-NPs which showed the presence of all main peaks. Also, the presence of other elements, such as C, O, Na, Mg, Si, Ca, together with the main gold ions were observed from the structural analysis of the AuNPs using the EDAX. The TEM was used to analyze the spherical and triangular shapes of the Au-NPs. The average particle size, which lies between 15-20 nm, was obtained from the TEM analysis. The disk diffusion method was used to examine the activity of the Au-NPs to inhibit S. aureus and P. aeruginosa bacterial. It was observed that the Au-NPs greatly inhibited bacterial growth. References [1] Rodriguez P, Plana D, Fermin DJ, et al, Chinese J Catal 311 (2014) 182-189. [2] Sunita R. Boddu, Veera R. Gutti, Tushar K. Ghosh, Robert V. Tompson, Sudarshan K. Loyalka, Journal of Nanoparticle Research 13, 65916601. [3] Renat R Letfullin, Charles Joenathan, Thomas F George and Vladimir P Zharov, Future Medicine 1(4), (2006) 473-480. [4] T. V. M. Sreekanth • P. C. Nagajyothi • N. Supraja • T. N. V. K. V. Prasad, Appl Nanosci 5 (2015) 595–602. [5] Madu, A. N, Njoku, P. C, Iwuoha, G. N. and Agbasi, U. M, International Journal of Physical Sciences 6(4) (2011) 635-640. [6] O. Ya. Uryupina, V. V. Vysotskii, V. V. Matveev, A. V. Gusel’nikova, and V. I. RoldughinO, Colloid Journal 73 (2011) 551–556. [7] J. Anuradha, T. Abbasi, and S. A. Abbasi, Journal of Advanced Research. 6 (2015) 711–720. [8] H. S. Mahdi, A. Parveen, S. Agrawal, and A. Azam, AIP Conf. Proc. 1953 (2018) 030013. [9] A. Parveen, S. Agrawal, and A. Azam, Opt. Mater. (Amst) 76 (2018) 21–27. [10] Mohamed Anwar K Abdelhalim*, Mohsen M. Mady and Magdy M. Ghannam, J Nanomed Nanotechol, 3 (2012) 3. [11] M. Balouiri, M. Sadiki, S.K. Ibnsouda, Journal of Pharmaceutical Analysis, 6 (2016) 71-79.
ScienceDirect Materials Today: Proceedings 18 (2019) 5164–5169
www.materialstoday.com/proceedings
ICMPC-2019
Biosynthesis, Characterization and Antibacterial Activity of Gold Nanoparticles (Au-NPs) using Black Lemon Extract Hadeel Salih Mahdia and Azra Parveena* a
Department of Applied Physics, Z.H. College of Engineering & Technology, Aligarh Muslim University, Aligarh-202002, India.
Abstract The synthesis of gold nanoparticles (Au-NPs) have been performed by a biological method using HAuCl4, black lemon extract, and double distilled water (D.D.W). The structural analysis and average crystallite size have been investigated by XRD data and the average crystallite size came out to be 15 nm. The optical properties were recorded by UV-visible microscopy in the range of 400-800 nm. SEM and EDAX have been used to study the morphological and elemental structure of Au-NPs. The triangular and spherical shapes, verified by TEM play an important role in directing the shape evolution of nanoparticles. The diameter of inhibition zone, which was used to determine the antibacterial activities of the NPs, was obtained using the disk diffusion methods. The Au-NPs showed great ability to inhibit activities of various bacteria such as gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) by damaging their activity. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019 Keywords: HAuCl4, XRD, Uv-Visible, TEM, Black lemon Extract and antibacterial effect.
1. INTRODUCTION Metallic nanoparticles are extensively used for diagnosis, treatments, and drug delivery purposes. Nanoparticles have different excellent physical properties, such as large surface area to volume ratio, that differ from the bulk material [1]. Metallic nanoparticles show various optical properties that vary with their shape and size, which make them useful for several biomedical purposes, such as drug delivery, bio-sensing and catalytic processes. Gold
* Corresponding author. Tel.: +919411653414; E-mail address: [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019
H.S. Mahdia and A. Parveen / Materials Today: Proceedings 18 (2019) 5164–5169
5165
nanoparticles are the most important nanoparticles, and are widely used in industrial processes [2]. In the area of medicine, they are used in the treatment of cancer and for inhibiting the activities of bacterial [3, 4]. Several techniques, such as chemical, biological, and physical methods, are used to synthesize Au-NPs [5, 6]. The extracts of several plants, such as black lemon, fruit, stachys, have been used to synthesize Au-NPs by biological method. The synthesis of Au-NPs using black lemon extract as stabilizing agent is presented in this study. 2. Experimental 2.1. Preparation of Black lemon Extract 250 g of black lemon, which was collected from Iraq, was cut into bits with a mill. The black lemon was added to 500 ml of double distilled water (D.D.W) and boiled for 1hr. at a temperature of 100 0C. The mixture was separated into an aqueous extract, which was refrigerated at 6°C for further use in the production of Au-NPs. 2.2. Preparation of Au-NPs In this research, Au-NPs were produced by biological technique using black lemon extract. 100 ml of double distilled water was used to dissolve 1mM of HAuCl4, and the solution was stirred continuously for 20 min with magnetic stirrer at 140 rpm. Thereafter, 10 ml of black lemon extract was added to 20 ml of HAuCl4 solution, which was continuously stirred until the color of the solution changed to red. 3. Results And Discussion 3.1. X-Ray Diffraction The structure of Au-NPs was analysed using X-ray diffraction (XRD) as shown below in Fig. 1, with Cu -Kα radiations (λ=1.5406 Ǻ) and angle 2θ in the range of 30º-80º . The diffraction peaks occurring at (38.3), (44.3), (64.6) and (77.8) were indexed as (111), (200), (220) and (311) of lattice planes. The XRD plot of Au-NPs showed the face centered cubic (fcc) structure. The diffraction peaks obtained were similar to the standard of Au metal (JCPDS-card no. 04-0784) [7].
Fig. 1. XRD analysis of biological synthesis Au-NPs
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The average crystallite size of Au-NPs was determined to be 15 nm using the Debye- Scherrer’s formula [8, 9], which is given below: D = 0.9 λ/β cos θ Where, D is the average crystallite size, (λ) the XRD wavelength (1.54 nm), β is the (FWHM) and θ is the Bragg angle. 3.2. Scanning Electron Microscopy (SEM) The morphological structure of the synthesized Au-NPs was analyzed using the SEM device as shown below in Fig 2. The spherical and cubic shapes of Au-NPs were obtained of various sizes. Bar graph of EDAX analysis (Fig. 2) verified the presence of metallic gold nanoparticles and other elemental structure showing the presence of C, O, Na, Mg, Si, Ca that may be present in black lemon extract.
Fig. 2. SEM and EDAX of biological synthesis Au-NPs
3.3. Transmission Electron Microscopy (TEM) The TEM technique was used to analyse the surface composition of Au-NPs and this is shown below in Fig.3. From TEM image the triangular and spherical shapes of Au-NPs have been confirmed with a smooth and uniform particle composition. The average particles size was investigated by TEM histogram and found to lie between 15-20 nm which is comparative to the particles size as determined by Debye- Scherrer formula from XRD.
H.S. Mahdia and A. Parveen / Materials Today: Proceedings 18 (2019) 5164–5169
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Fig. 3. TEM image of biological synthesis Au-NPs
3.4. UV-Visible spectrophotometer The UV-Vis spectrophotometer (Perkin Elmer Lambda-35) was used to analyze the optical properties of biologically synthesized Au-NPs in the range of 400-800 nm, and is shown below in Fig.4. The change in the color from yellow to red of the reaction mixture confirmed successful reduction of HAuCl4 to Au-NPs. The absorption peak of Au-NPs was observed at 537 nm and there is no absorption peak for black lemon extract [10].
Fig. 4. UV-visible of biological synthesis Au-NPs
3.5. Antibacterial activities of Au-NPs The well diffusion method was used to study the antibacterial activity of Au-NPs. 100 μl of P. aeruginosa and S. aureus were evenly distributed over Luria Agar (LA) plates on regular basis. Soft agar was used to seal wells, which have been cut into 4 mm diameter. Test solution and different amounts of Au-NPs (25, 50 and 100 μl) were then
5168
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placed in these wells. The plates were then incubated for 24hours at 37°C and the diameter of growth inhibition zones, which was used to obtain the size of the zones, was then determined [11]. This was used to evaluate the antimicrobial activity of Au-NPs against multi drug resistance P. aeruginosa and S. aureus. From Fig.5 (a, b), the zone of inhibition of Au-NPs at 3 different concentrations against of P. aeruginosa and S. aureus bacteria were exhibited. Table 1 shows the Au-NPs inhibit the growth of both bacteria P. aeruginosa and S. aureus. A substantial increase in the size of inhibition zones was observed when increase the concentration Au-NPs as shown in Fig.6. An increase in the concentration of Au-NPs (from 0 to 100 µg/mL) resulted in a corresponding increase in antibacterial activity against S. aureus (Gram-positive bacteria) and P. aeruginosa (Gram-Negative bacteria). This is attributable to increase in reactive oxygen species (ROS) which brings about oxidative stress and destruction of the cell.
Fig 5. Zone of inhibition of biosynthesis Au-NPs using Black lemon a) P. aeruginosa and b) S. aureus
Table 1. Diameter of the inhibition zone with the various concentration of biosynthesis Au-NPs using Black lemon S. No
Concentration of Au-NPs (µg/mL)
Diameter of inhibition (mm) P. aeruginosa
1
Control
S. aureus
0
0
2
25
12
14
3
50
16
20
4
100
18
22
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Fig 6. Zone of inhibition of biosynthesis Au-NPs using Black lemon a) P. aeruginosa and b) S. aureus
4. Conclusions Au-NPs were successfully synthesized in this study by biological method using black lemon extract. The absorption peak of Au-NPs was confirmed at 537 nm with the aid of UV-Vis spectroscopy. XRD data were used to analyze the structure of Au-NPs which showed the presence of all main peaks. Also, the presence of other elements, such as C, O, Na, Mg, Si, Ca, together with the main gold ions were observed from the structural analysis of the AuNPs using the EDAX. The TEM was used to analyze the spherical and triangular shapes of the Au-NPs. The average particle size, which lies between 15-20 nm, was obtained from the TEM analysis. The disk diffusion method was used to examine the activity of the Au-NPs to inhibit S. aureus and P. aeruginosa bacterial. It was observed that the Au-NPs greatly inhibited bacterial growth. References [1] Rodriguez P, Plana D, Fermin DJ, et al, Chinese J Catal 311 (2014) 182-189. [2] Sunita R. Boddu, Veera R. Gutti, Tushar K. Ghosh, Robert V. Tompson, Sudarshan K. Loyalka, Journal of Nanoparticle Research 13, 65916601. [3] Renat R Letfullin, Charles Joenathan, Thomas F George and Vladimir P Zharov, Future Medicine 1(4), (2006) 473-480. [4] T. V. M. Sreekanth • P. C. Nagajyothi • N. Supraja • T. N. V. K. V. Prasad, Appl Nanosci 5 (2015) 595–602. [5] Madu, A. N, Njoku, P. C, Iwuoha, G. N. and Agbasi, U. M, International Journal of Physical Sciences 6(4) (2011) 635-640. [6] O. Ya. Uryupina, V. V. Vysotskii, V. V. Matveev, A. V. Gusel’nikova, and V. I. RoldughinO, Colloid Journal 73 (2011) 551–556. [7] J. Anuradha, T. Abbasi, and S. A. Abbasi, Journal of Advanced Research. 6 (2015) 711–720. [8] H. S. Mahdi, A. Parveen, S. Agrawal, and A. Azam, AIP Conf. Proc. 1953 (2018) 030013. [9] A. Parveen, S. Agrawal, and A. Azam, Opt. Mater. (Amst) 76 (2018) 21–27. [10] Mohamed Anwar K Abdelhalim*, Mohsen M. Mady and Magdy M. Ghannam, J Nanomed Nanotechol, 3 (2012) 3. [11] M. Balouiri, M. Sadiki, S.K. Ibnsouda, Journal of Pharmaceutical Analysis, 6 (2016) 71-79.