The synthesized SNPs were characterized by UV-visible spectrophotometer, FT-IR, XRD, SEM and HRTEM and SAED. The absorbance measure for SNPs by UV-visible with Jascov 300 using a quartz cuvette. Fourier transform infrared spectra were recorded on Schimadzu IR-Affinity-1 using KBr pellets. The crystallinity of the SNPs was studied using XRD on Rigaku Miniflex 600. The surface morphology of the prepared SNPs was characterized by scanning electron microscopy (SEM) on Model JSM 6100 (Jeol). High resolution electron microscopy (HRTEM) were carried out on JEOLMODEL JEM 2100 plus. It revels structural morphology, polycrystalline nature and selected area electron diffraction (SAED).
UV-Visible Spectroscopy
Fig :1 UV-Visible spectrum for Sulphur Nanoparticle
The region of UV Visible spectrometer is 200 nm - 800 nm UV– visible spectroscopy is a crucial technique to ascertain the stability and formation of sulphur Nanoparticles visible absorption measurement of the wavelength ranging from 650-700 nm is utilized to characterize SNPs.
FT-IR Analysis
FT-IR spectrum of Psidium Gujava leaves aqueous extract is illustrated in Fig. 2 nanoparticles. The FT-IR analysis of aqueous leaf extract of was carried out to identify the possible biomolecules responsible for the reduction of sodium thiosulphate to sulphur NPs. The FTIR spectrum of the aqueous leaf extract (Fig) shows a broad peak at 3275 cm-1 which can be assigned to the stretching vibration of O–H of phenolic compound, while the weak band at 3059 cm-1 indicates the presence of C-H stretching vibration. The sharp peak at 2150 cm-1 is attributed to C-H stretching vibration of alkanes or [C=C] stretching vibration of aromatic ring. The characteristic peaks at 1438, 1118 and 613 cm-1 can be assigned to in plane OH bending of carboxylic acids, C–O stretch of phenol and C–H bend of aromatic compounds or alkynes, respectively. These peaks suggested the presence of flavonoid sand other polyphenolic compounds in the aqueous plant leaf extract of Psidium Guajava which could be responsible for the reduction of sulphur ions to their corresponding sulphur NPs. The FTIR spectrum of synthesized sulphur NPs shows peak at 466.77nm
Fig :2 FT-IR spectrum for Sulphur Nanoparticle
X-Ray Diffraction analysis:
XRD is characterization technique by which we confirm its particle size and crystanility. The XRD pattern of green synthesized sulphur nanoparticles by Psidium gujava leaves aqueous extract is as shown in fig 3. The XRD pattern shows sulphur nanoparticle in crystalline nature which gives sharp peak. The XRD pattern shows sulphur nanoparticles are crystalline in nature. There is formation of metallic sulphur nanoparticle. Which gives structure (JCPDS No.00-042-1278). The Average particle size of the synthesized sulphur nanoparticles was calculated by using Debye Scherrer formula D = (k λ/βcos θ). The average particle size was found to be 57.32 nm
Fig :3 X-ray diffraction for Sulphur Nanoparticle
Scanning Electron Microscopic:
The Scanning electron microscopy is a powerful technique to know the surface morphology of the particles. It was evident that synthesized NPs are crystalline in nature. These synthesised S NPs are stabilized by biomolecules present in the leaf extract; these molecules also act as a capping agent. The SEM micro photographs also evidence the polydispersity and purity of resulting sulphur nanoparticles. Fig.3 shows size, shape, morphology and distribution of biosythesized SNPs
Fig :4 Scanning electron microscopic for Sulphur Nanoparticle
Energy Dispersive X-ray Spectroscopic analysis:
Energy Dispersive X-ray (EDS) is technique to analyse elemental composition’s EDS spectra confirmed the presence of sulphur, oxygen in the synthesized SNPs.Narrow width of SNPs peaks indicated that the synthesized SNPs had crystalline nature.
Fig :5 Energy Dispersive X-ray Spectroscopic analysis for Sulphur Nanoparticle
HRTEM analysis:
HRTEM analysis is a technique used to determine of particle size, crystallinity and morphology of the sample. In particle size measurement microscopy is the only method in which the individual particles are directly observed and measured. Fig. 6 (a)represents the TEM image of SNPs were spherical like shape and average dimensions found to be 55 – 60 nm. Lattice fringe image Fig. 6 (b) exhibits the regular spacing of the lattice plane. The selected area electron diffraction (SAED) image in Fig. 6 (c) with single crystal nature being confirmed by ring pattern of the SAED.
Fig :6 High resolution transmission electronic microscopic analysis for Sulphur Nanoparticle
Antimicrobial Activity:
The Antimicrobial activity of pathogen was done by well diffusion method. At a different concentration of samples, the distinct zone of inhibition was formed around the wells. DMSO and Tetracycline were used as a negative and positive controls respectively to compare the zones. To cultivate the bacteria, nutrient agar media was used. About 20 ml of sterile molten agar was poured into the sterile petri dishes. After solidification of medium, the petri dishes were inoculated with the 24 hours old bacterial cultures by spread plate method and then the wells were prepared with 9 mm cork borer. Further the Sulphur nanoparticles with 0.05 g/ml concentration in DMSO were prepared and 100 μl volume was added into the well. Petri dishes were incubated for 24 hours at 37ºC. Antibacterial capacity of the sulphur
|
Bacteria
|
Zone of inhibition (Outer Zone-Inner zone) (in mm)
|
|
Escherichia coli
|
4
|
|
Bacillus subtilis
|
8
|
Table1: Observation table for Antibacterial activity
Fig:7 a: Zone of inhibition Antibacterial activity against E. coli
Fig:7 b: Zone of inhibition Antibacterial activity against Bacillus subtilis
Antifungal Activity:
In vitro study of anti-fungal activity of extract against was done by using Agar well diffusion method in which 18 hrs old growing Culture of Candida albicans was spread over agar plates. Four wells were prepared on it the by using micropipette tips. In positive control anti-fungal Fluconazole and Itraconazole(100ul) with concentrations …. was used. In negative control DMSO (100ul) was used. Sample extract(100ul) was added in another well and plates were kept in refrigerator for diffusion for 30 min and then plates were incubated at 37 for 24 hrs. After incubation, zone of inhibition was observed, and diameter of each zone was recorded.
|
Sample
|
mg
Sample
|
ml
DMSO
|
Candida albicans
(mm)
|
Mean (mm)
|
|
S NPs
(Fig.a)
|
10
|
1
|
16,16,15,17
|
16
|
|
Fluconazole (Fig.b)
|
10
|
1
|
18,19,21,20
|
19.5
|
|
Itraconazole (Fig:c)
|
10
|
1
|
18,16,17,16
|
16.75
|
Table2: Observation table for Antifungal activity
Fig:8 a: Zone of inhibition Antifungal activity of sulphur NPs
Fig:8 b: Zone of inhibition against Fluconazole
Fig:8 c: Zone of inhibition against Itraconazole
In present work a new green protocol for the synthesis of SNPs using extract of Psidium Gujava. leaves is developed, which is eco-friendly method and a promising, low cost and without using any toxic chemicals. Average size of synthesized SNPs 57.32nm.