In the quest for environmental remediation which involves eco-friendly synthetic routes, we herein report synthesis and modeling of silver nanoparticles (Ag NPs) and silver/nickel allied bimetallic nanoparticles (Ag/Ni NPs) using plant-extract reduction method. Secondary metabolites in the leaf extract of
Recently, paradigm shift in technology has led to the synthetic protocols involving application of green chemistry, part of environmental remediation, encompassing use of biomaterials because of their eco-friendliness. The use of biological materials as sensors, information storage devices, and bimolecular array is on the increase. No doubt, novel characteristics are possessed by materials on nanometre scale, as this creates special interest which is applicable virtually to every aspect of life including medicine, agriculture, and polymer industry among others [
Biosynthesized conjugated bimetallic nanoparticles are now used in biomedical field, imaging, luminescence tagging, labeling, and drug delivery due to their compatibility in
From survey, biological syntheses of nanomaterials make use of bacteria, yeasts, fungi, and algae (microorganisms). The use of plants or plant extracts for metal and metal hybrid nanoparticles synthesis is currently a new research focus that has gained wide acceptance [
Quite a number of literatures have reported syntheses of allied silver-nickel nanoparticles using various chemical methods. Adekoya et al. [
Freshly cultured clinical isolates of
Indian shot plant was collected from a garden at Atan-Iju, Ogun State, Nigeria. Plant identification and authentication were carried out at Forest Research Institute of Nigeria (FRIN); voucher specimen FHI 109928 was deposited at the herbarium headquarters, Ibadan, Nigeria. Fresh leafy part of the plant was washed with distilled water, finely cut, and ground using mortar and pestle. It was then extracted at a ratio of 1 : 5 wt/v using distilled deionized water, filtered with Whatman number 1 filter paper, and then kept at 4°C. The filtrate was used for phytochemical screening and nanoparticle synthesis. The procedure is modified from previous work [
Plant extract was screened to identify the phytochemicals present according to literature [
Metallic nanoparticles were prepared by plant-extract reduction method with modification to previous work [
The biosynthesized nanoparticles were collected by centrifugation using centrifuge model 0508-1, operated at 5000 rpm for 30 minutes. For purification, the nanoparticles suspension was redispersed in distilled deionized water so as to remove the unbounded organics and finally centrifuged at 5,000 rpm for 10 minutes. The suspension was oven dried and kept in Eppendorf tubes for further characterizations.
Optical properties of the prepared metallic nanoparticles were determined using a double beam Thermo Scientific GENESYS 10S UV-Vis spectrophotometer between 200 and 800 nm wavelength ranges. Absorbance measurement was carried out by placing each aliquot sample taken at time intervals in quartz cuvette (1 cm path length), operated at a resolution of 1 nm, using distilled deionized water as blank.
Structural, morphological characteristics and size determination of the particles were verified with Technai G2 transmission electron microscope (TEM) coupled with an energy-dispersive X-ray spectrometer (EDX), operated at an accelerating voltage of 200 KeV and 20
McFarland standard on laboratory guidance was used for the standardization of organisms for susceptibility testing, using a modified method by British Society for Antimicrobial Chemotherapy. BaSO4 turbidity standard equivalent to 0.5 McFarland standards or its optical equivalent was used. The 0.5 McFarland standard was prepared by adding 0.5 mL of 0.048 M BaCl2 of (1.175% w/v) BaCl2 in 2H2O to 99.5 mL of 0.18 M H2SO4 with constant stirring to maintain a suspension. The correct density of the turbidity standard was verified by a pg instrument UV-Vis spectrophotometer model T90+, with 1 cm light path, and matched cuvette to determine the optical density at a wavelength of 625 nm. The acceptable range for the standard is 0.08–0.13 for 0.5 McFarland standard which is equivalent to 1.5 × 108 bacterial cells per mL. The standard was distributed into screw cap tubes of the same size and volume, similar to method of growing or diluting the bacterial inocula. The tubes were tightly sealed to prevent loss by evaporation. They were then stored in the dark at room temperature. The turbidity standard was vigorously agitated on a vortex mixer before use. The standard remains potent for six months; appearance of large particles in the standard is an indication of expiration [
The microbial strains were propagated in Mueller Hinton broth, prepared by dispersing 5 mL of the prepared broth medium into each screw capped test tube, sterilized by autoclaving at 12°C for 15 minutes. The test tubes were cooled and kept in an incubator for 24 hours at 37°C in order to determine the sterility. The isolates were inoculated into the sterilized test tubes containing the medium and placed in an incubator overnight at 37°C. Appearance of turbidity in broth culture was adjusted equivalent to 0.5 McFarland standards. This was done to obtain standardized suspension. Sterile normal saline was added in order to obtain turbidity optically comparable to that of the 0.5 McFarland standards or against a white background with contrasting black line. The McFarland 0.5 standard provided turbidity comparable to bacterial suspension containing 1.5 × 108 cfu/mL [
Antimicrobial properties of the biosynthesized nanoparticles were investigated in the form of sensitivity testing, using modified version of the method described by Aida [
Antibacterial activity of synthesized nanoparticles was evaluated by the well plate agar diffusion method as described in the Aida modified method [
Serial dilution method was employed according to CLSI guidelines. Sterile test tubes (12) were arranged in a rack. 1 mL of sterile nutrient broth was added to tube labeled 2 to 10. 1 mL of known nutrients broth concentration was added to tubes 1 and 2. Afterwards, serial doubling dilution from tube 2 to tube 10 was made, while the remaining 1 mL was discarded. 1 mL of ciprofloxacin was added to tube 11 (positive control) and water to tube 12 (negative control). 1 mL of 0.5 McFarland was added overnight and broth culture to all the tubes and then covered. The experiment was incubated overnight at 37°C and observed for the highest dilution showing no turbidity. The zone of inhibition was then verified and interpreted according to CLSI guidelines [
MBC, the lowest concentration of antibiotic agent that kills at least 99.9% of the organisms, was determined by using Doughari et al. method. 0.5 mL of the sample was removed from those tubes from MIC which did not show any visible sign of growth and inoculated on sterile Mueller Hinton agar by streaking. The plates were then incubated at 37°C for 24 hours. The concentration at which no visible growth was seen was recorded as the minimum bactericidal concentration (MBC). For MFC, 0.5 mL of the sample which showed no visible sign of growth during MIC screening was taken from the test tubes and then inoculated on sterile potato dextrose agar by streaking. The plates were then incubated at 37°C for 24 hours. The concentration at which no visible growth was seen was recorded as the minimum fungicidal concentration [
UV/Visible spectra of the biosynthesized silver nanoparticles (Ag NPs) and silver-nickel (Ag/Ni) bimetallic nanoparticles (Ag/Ni NPs) as a result of photon absorption by their solutions are displayed in Figures
(a) Colour dispersion before and after nanoparticles formation, UV-Vis spectra of Ag NPs prepared by reducing (b) 0.5 mM, (c) 1.0 mM, and (d) 2.0 mM precursor solutions using the extract of
UV-Vis spectra of Ag/Ni bimetallic nanoparticles prepared by reducing (a) 0.5 mM, (b) 1.0 mM, (c) 2.0 mM, and (d) 3.0 mM solutions using the extract of
Growth comparison in
Growth comparison in Ag-Ni bimetallic nanoparticles using the extract of
Ag NPs formation had maximum absorption in the visible region with absorption wavelength of 416 nm and maximum intensity of 0.312 a.u. There was electron confinement effect when 1.0 mM AgNO3 was reduced, and this culminated in sharp peaks and strong intensity observed. Intensity of absorption in the hybrid nanoparticles prepared from 2.0 mM precursor solution was in contrast to the maximum intensity of absorption noticed in the corresponding monometallic Ag NPs. In the bimetallic Ag/Ni NPs, there were narrow absorption spectra which increased in peak intensity without any shift in wavelength (421 nm). Hence, this signified presence of spherically shaped nanoparticles. Moreover, surface of the hybrid nanoparticles in Figure
However, presence of nickel (Ni) in the hybrid synthesis of course led to a red shift in the absorbance wavelength from 416.0 to 421 nm, as observed in the reduced 2.0 mM precursor solution of Ag/Ni NPs. There was an obvious increase in intensity of absorption when compared with the corresponding Ag NPs. Growth comparison and optimum concentration for Ag NPs and Ag/Ni bimetallic synthesized at 70°C are displayed in Figures
Biomolecules which acted as the reducing and capping/stabilizing agents for the newly formed nanoparticles were considered to be adequate as a result of unprecedented fast and successful bioreduction [
Phytochemical analysis of
Phytochemical | ||||||||||
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Proteins | Carbohydrates | Phenols | Tannins | Flavonoids | Saponins | Glycoside | Steroids | Terpenoids | Alkaloids | |
Aqueous extract |
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+ | +++ |
Methanolic extract |
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+++ | ++ | + | ++ | − | ++ | +++ |
Weak presence +; strong presence ++; stronger presence +++; absent −.
Bioreduction of silver ion to silver nanoparticles by glycosides.
Bioreduction of silver/nickel ions to silver/nickel nanoparticles by glycosides.
Bioreduction of silver ion to silver nanoparticles by alkaloids.
Bioreduction of silver/nickel ions to silver/nickel nanoparticles by alkaloids.
Bioreduction of silver ion to silver nanoparticles by terpenoids.
Bioreduction of silver/nickel ions to silver/nickel nanoparticles by terpenoids.
See Schemes
Particle size distribution histogram and TEM image of the biosynthesized Ag/Ni bimetallic nanoparticles are depicted in Figures
(a) Particle size distribution histogram of Ag/Ni determined from TEM images. (b) Representative TEM image of the bimetallic Ag/Ni NPs under
Representative TEM images of Ag/Ni bimetallic NPs derived from
EDX showing atomic composition of elements present in Ag NPs
EDX showing atomic composition of elements present in Ag/Ni bimetallic NPs
This finding is unique in biosynthesis of nanoparticles. Related finding was reported by Mntungwa et al. [
Activity of the biosynthesized Ag NPs and Ag/Ni bimetallic nanoparticles based on size of zones of inhibition in millimetre (mm) is shown in Figure
Sensitivity testing of organisms with standard deviation in zones of inhibition (agar diffusion test).
NPs | Organisms/mean zone diameter (mm) ± SD | |||||
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Ag 0.5 |
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Nil |
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Nil |
Ag 1.0 |
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Nil |
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Ag 2.0 |
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Ag 3.0 |
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Ag-Ni 0.5 |
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Ag-Ni 1.0 |
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Ag-Ni 2.0 |
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Ag-Ni 3.0 |
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Control |
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Stat |
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Control: ciprofloxacin (bacteria) and fluconazole (fungi); mean zone inhibition (mm) ± standard deviation of triplicate measurements. Ag = silver nanoparticles of specified precursor concentration using
Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC).
Nanoparticles |
Organisms/MIC, MBC & MFC (mg/mL) | |||||
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MIC, MBC | MIC, MBC | MIC, MBC | MIC, MBC | MIC, MFC | MIC, MFC | |
Ag 0.5 | 100, 100 | 50, 100 | 100, 100 | 100, 100 | 50, 50 | 100, 100 |
Ag 1.0 | 100, 100 | 25, 50 | 50, 100 | 100, 100 | 25, 25 | 100, 100 |
Ag 2.0 | 50, 100 | 12.5, 25 | 12.5, 25 | 100, 100 | 12.5, 25 | 100, 100 |
Ag 3.0 | 12.5, 25 | 12.5, 25 | 12.5, 12.5 | 100, 100 | 12.5, 12.5 | 50, 100 |
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Ag/Ni 0.5 | 50, 100 | 25, 50 | 12.5, 25 | 100, 100 | 50, 100 | 50, 100 |
Ag/Ni 1.0 | 12.5, 25 | 12.5, 12.5 | 25, 50 | 50, 100 | 25, 50 | 100, 100 |
Ag/Ni 2.0 | 12.5, 25 | 12.5, 12.5 | 12.5, 12.5 | 100, 100 | 12.5, 25 | 12.5, 12.5 |
Ag/Ni 3.0 | 12.5, 12.5 | 6.25, 12.5 | 6.25, 12.5 | 100, 100 | 12.5, 25 | 12.5, 25 |
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Control |
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Comparison of inhibition zones between Ag NPs and Ag-Ni bimetallic nanoparticles synthesized using
Zones of inhibition recorded in agar well diffusion test led to the conduction of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) tests. Results of MIC, MBC, and MFC tests are presented in Table
In hybrid Ag/Ni nanoparticles, analysis of variance (ANOVA) using SPSS statistical tool indicated no significant difference in the concentrations as
From the above, there is a need to affirm if the penetrations of the nanoparticle through the microbial samples influence its experimental result. Tan et al. [
Kleinstreuer and Xu [
The basic condition for the metallic nanoparticle to go through the polymer matrix of the thin peptidoglycan layer is
The dimensionless dispersion of the metallic nanoparticle under varying conditions (a) when Bragg’s angle of the nanoparticle is strictly 45°; (b) when Bragg’s angle of the nanoparticle ranges between −30° and 30°; (c) when Bragg’s angle of the nanoparticle ranges between −45° and 45°; (d) when Bragg’s angle of the nanoparticle ranges between −60° and 60°.
The dimensionless dispersion of the metallic nanoparticle under varying conditions at diameter greater than 10−9 nm (a) when Bragg’s angle of the nanoparticle is strictly 45°; (b) when Bragg’s angle of the nanoparticle ranges between −30° and 30°; (c) when Bragg’s angle of the nanoparticle ranges between −45° and 45°; (d) when Bragg’s angle of the nanoparticle ranges between −60° and 60°.
The first case (i.e., (
The second case (i.e., (
Rapid, facile, and environmental-friendly syntheses of monometallic Ag NPs and Ag/Ni bimetallic nanoparticles using
The authors declare no conflicts of interest in this research work.
The authors are grateful to Mr. Olusola Rotimi of the University of Western Cape, Bellville Campus, Cape-Town, Mr. Shitole Joseph of iThemba Labs, and Mr. Olufemi Olaofe in South Africa for the TEM and EDX characterizations. The authors also acknowledge Covenant University for funding this publication.