MATERIALS

10.2.1 EQUIPMENT

The equipment used are sterile 50 mL conical flasks, UV-vis spectrophotometer, lyophilizer, X-ray diffractometer, scanning electron microscope, 96-well flat-bottom culture plates, 12-well culture plates, six-well plates, ELISA plate reader, Illumina Eco™ Real-Time system, inverted fluorescence microscope, slides, and coverslips.

10.2.2 REAGENTS

The reagents used are silver nitrate, A. muricata fruits, HCT116 cell lines, Dulbecco’s modified Eagle’s medium (EhMedia), fetal bovine serum (FBS), streptomycin and penicillin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTT), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), fluorescence stains (AO, EtBr, Hoechst 33258, rhodamine 123, and dichloro-diliydro-fluorescein), glutaraldehyde, EhKaryo XL™ RPMI medium, crystal violet, ethanol, tiypsin, and SYBR Green PCR Master Mix.

METHODS

SYNTHESIS OF BIOSYNTHESIZED AMFAGNPS

  • 1. Fresh mature fruits were cut into pieces, shade-dried, and powdered finely.
  • 2. Ten grams of fruit powder was weighed, mixed with 200 mL of Milli-Q water, and boiled for 15 min.
  • 3. After cooling, the extract was filtered through muslin cloth and Whatman no. 1 filter paper and stored at 4 °C in a refrigerator (Mubarakali et al, 2011).
  • 4. For effective synthesis, different parameters such as the volume of fruit extract, concentration of metal ions, temperature, pH, and time were optimized (Shaniba et al., 2017).

5. The reaction mixture was periodically monitored in the range of 200-700 mn using a UV-vis spectrophotometer (PerkinElmer Lambda25) to detect the formation of silver NPs as indicated by the appearance of brown color.

The optimum conditions for the effective green synthesis of AMFAgNPs were 8.0 mL of AMF extract mixed with 92.0 rnL of 1.0 mM AgN03 (pH 7.0), at 90 °C for 30 min. Absorption spectra of AMFAgNPs formed in the reaction media, as evidenced by the formation of brown color (Figure 10.1), had an absorption maximum at 421 mn (Figure 10.2) due to surface plasmon resonance of AgNPs (Krishnaraj et al., 2010).

Aqueous solution of ImM AgN0 with AMF extract

FIGURE 10.1 Aqueous solution of ImM AgN03 with AMF extract: (a) before the addition of the leaf extract and (b) after the addition of leaf extract at 30 min.

UV-visible spectra of AMF extract-reduced AgNPs

FIGURE 10.2 UV-visible spectra of AMF extract-reduced AgNPs.

CHARACTERIZATION OF AMFAGNPS

  • 10.3.2.1 FTIR ANALYSIS
  • 1. The reaction mixture was subjected to centrifugation at 12,000 rpm for 20 min.
  • 2. The resultant pellet was resuspended in Milli-Q water and lyophi- lized (ScanVac CoolSafe).
  • 3. The freeze-dried powder was pelletized with potassium bromide (KBr) powder and subjected to FTIR analysis.
  • 4. The spectra were recorded using a JASCO 4100 at a wavelength ranging from 4000 to 400 cm'1.

FTIR analysis of AMFAgNPs showed an intensive peak at 3650 cm'1 corresponding to the O-H stretching of alcohols and phenols (Figure 10.3). The peaks at 3422 cm'1 indicated N-H groups. The peaks observed at 2923 cm'1,2853 cm"1,1457 cnr1, and 672 cm'1 represented C-H functional groups. Further, the peaks at 1747 cm'1 represented carbonyl groups (C=0) and the peaks at 1023 cnr1 indicated C-0 bonds. The results obtained depicted that the presence of these functional groups influenced the synthesis and stabilization of NPs. These results confirmed the encapsulation of AgNPs with the phytoconstituents from plant extracts.

FTIR spectrum of AMFAgNPs

FIGURE 10.3 FTIR spectrum of AMFAgNPs.

10.3.2.2 X-RAY DIFFRACTION ANALYSIS

The dried AMFAgNPs were coated on an XRD grid, and the spectrum was recorded using a Rigaku MiniFlex X-ray diffractometer with CuKa radiation (40 kV, 15 mA). The crystalline structure of silver NPs was identified by XRD analysis (Figure 10.4). XRD showed intense peaks in the whole spectrum of 26 values ranging from 20° to 70°. Four strong peaks observed at the 26 values 27.6°, 38°, 46°, and 63.6° corresponded to the (110), (111), (200), and (220) planes of face-centered cubic phase, obtained from JCPDS card 89-3722. Some of the unassigned peaks were observed at 26 values of 28°, 42.8°, and 53.2°, which are attributable to the biomolecules present in the plant extract impregnated on the surface of AgNPs. The average crystal size was found to be 19 mil, as calculated by the Debye-Scherrer equation D = 0.94/7/? cos/9. where D is the average crystallite domain size perpendicular to the reflecting planes, a is the X-ray wavelength, /6 is the full width at half-maximum, and 6 is the diffraction angle (Vivek et al., 2012).

Demonstration of the XRD analysis of AMFAgNPs

FIGURE 10.4 Demonstration of the XRD analysis of AMFAgNPs.

10.3,2.3 FIELD-EMISSION SCANNING ELECTRON MICROSCOPY

The average particle size was determined using FESEM, Horiba S46600, equipped with EDX. The morphology and size determination of the synthesized AMFAgNPs were based on FESEM images (Figure 10.5). The SEM image showed a spherical and relatively uniform shape of NPs with diameters varying from 30 to 100 mn. EDX analysis revealed the presence of elemental silver signal emanating from the silver NPs (Figure 10.6).

FESEM micrograph of AMFAgNPs

FIGURE 10.5 FESEM micrograph of AMFAgNPs.

 
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