1.9 Objectives of this thesis work................................................................. 11-12
4.3.3 Characterization of core-shell CS Cu@Ag NPs composite
UV-Visible spectra of the CS Cu@Ag NPs dispersion and Cu NPs seed particles, mixture of CS-Cu NPs and CS-Ag NPs were measured using a Hitachi U 2900 spectrophotometer. The bacterial growth was monitored by measuring optical density (OD) at 595 nm using a UV-visible spectrophotometer (Lambda 25; Perkin-Elmer, Fremont, CA, USA) of the sample at different times
4.3.3.2: Transmission electron microscopy (TEM) analysis
For TEM investigations, 5 µL of each sample was drop coated onto a carbon coated copper TEM grid (300 mesh) followed by air drying. The drop coated grid was then analyzed under TEM at a maximum acceleration voltage of 200 kV. High resolution transmission electron microscopic analysis was performed using the same equipment.
The interactions of the CS Cu@Ag NPs composite with bacterial cells were also examined using a high resolution transmission electron microscope operating at a maximum accelerating voltage of 80 KeV. For this, CS Cu@Ag NPs composite treated on E. coli samples were drop coated onto the TEM grid followed by air drying and the resulting grid was then analyzed under TEM and energy-dispersive X-ray (EDX)
analysis also carried out on composite treated samples at operating accelerating voltage 200 kV.
4.3.3.3: Scanning electron microscopic analysis
Scanning electron microscopy coupled with EDX and Field emission scanning electron microscopy coupled with EDX instruments were used for the study of surface morphology and elemental analysis for CS Cu@Ag NPs composite and composite treated on E. coli samples. Typically, 20 µL drop of each sample was deposited on a glass slide, dried and sputter-coated with gold film using a sputter coater and analyzed under the SEM and FESEM. The FESEM-EDX analysis of CS Cu@Ag NPs composite treated on E. coli samples was carried out on small area of the sample for the elemental analysis of the composite.
4.3.3.4: Powder XRD studies
We performed X-ray diffraction measurements to characterize the CS Cu@Ag NPs composite. We also studied and fate of the CS Cu@Ag NPs composite during antibacterial studies using XRD measurement. Firstly, vacuum dried composites were spread on glass microslides and XRD was recorded using Philips Diffrctometer (Model 1715) with Cu Kα1 radiation (λ= 1.54060 Å), operating at 55 kV and 250 mA. In order to confirm the presence of core-shell Cu@Ag NPs on the bacterial cell walls, again bacteria treated with CS Cu@Ag NPs composite for 12 h was centrifuged at 6000 rpm and vacuum dried and stored under vacuum before analysis. The dried cell pellet spread on a glass slide and XRD was recorded.
4.3.3.5: X-ray photoelectron spectra (XPS) analysis
We have performed XPS measurements to characterize the core-shell structure of the CS Cu@Ag NPs composites. The powdered CS Cu@Ag NP composite was pressed and made into pellets and placed in an ultra high vacuum chamber at 1×10−10 Torr for chemical analysis. XPS spectra were recorded on a PHI 5000 Versa Probe II (ULVAC- PHI, INC, Japan) equipment employing Al Ka X-rays (hν = 1486.6 eV). Charge
neutralization was used for each measurement using a combination of low energy Ar+ ions and electrons. The binding energy (eV) was corrected with the C1s (284.6 eV) as standard. Elemental compositions were determined from the spectra acquired at pass energy of 187.85 eV. High-resolution spectra were obtained with analyzer pass energy of 58.70 eV with a step of 0.125 eV and 50 ms time per step.
4.3.3.6: Atomic absorption spectrometry (AAS)
Quantitative analysis of the concentration of Cu and Ag present in CS Cu@Ag NPs composite was measured by using atomic absorption spectrophotometer (Model: AA240 Varian Inc) after dissolving the composite in dilute HCl solution. The supernatant solution obtained after centrifugation of the composites was also measured to determine concentration of Cu and Ag ions present in determining how much was being discarded during our synthesis.
4.3.3.7: Zeta potential (ζ)
Zeta potential (ζ) measurements were performed to determine the charge of CS Cu@Ag NPs composite in aqueous solution at pH 6.3. Zeta potential (ζ) was measured using Delsa™ Nano Submicron Particle Size and Zeta Potential Particle Analyzer.
4.3.3.8: Fourier Transform Infrared (FTIR) Spectroscopy
FT-IR spectra were recorded on a Perkin-Elmer-Spectrum One spectrometer with KBr disks in the range 4000-450 cm-1 for structural analysis of solid CS and solid CS Cu@Ag NPs composite. Both the CS and CS Cu@Ag NPs powders were mixed with KBr in order to make pellets for FTIR studies.
4.3.3.9: Bactericidal studies
For bactericidal activity, B. cereus was chosen as the Gram-positive bacteria and GFP-expressing E. coli as the Gram-negative bacteria. GFP-expressing E. coli was cultured in LB ampicillin media and B. cereus in NB media. Different concentrations of
CS Cu@Ag NPs composite were used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at pH 6.3. Bacteria (108 cfu/ml) were grown in media in the presence of different concentrations of CS Cu@Ag NPs composite for 12 h at 370C. The lowest concentration of composite at which there was no visual turbidity was taken as the MIC value of the composite.
Further, the cultures which lacked visual turbidity were reinoculated in fresh media. The lowest concentration of the composite that killed at least 99.9% of original inoculums was taken as MBC. The experiments were performed at least in triplicate to ensure reproducibility. The bacterial growth was monitored by measuring optical density (OD) at 595 nm using a UV-Visible spectrophotometer of the sample at different times.
4.3.3.10: Flow cytometric analysis for bacterial cell viability using GFP- propidium iodide combination
GFP-expressing recombinant bacterial cells viability and disruption of membrane integrity was measured using propidium iodide (PI), which enters only permeable cells, binds DNA, and fluoresces at 620 nm when stimulated by a laser at 488 nm. In intact cells, PI remains in the medium and does not fluoresce; in compromised cells, PI enters the cell through disrupted membrane and binds to DNA exhibiting fluorescence1, 2. Briefly, several tubes containing 500 µL (108 cells/mL) of the recombinant E. coli cells and 250 µL of the CS Cu@Ag NPs composite (at MIC, i.e. 63.4 µg/mL consisting 3.03 µg/mL of Cu and 1.47 µg/mL of Ag) in addition to 500 µL (108 cells/mL) of the recombinant E. coli cells and 375 µL of the composite (at MBC, i.e. 92.99 µg/mL consisting 4.44 µg/mL of Cu and 2.13 µg/mL of Ag) were incubated at 25°C for various time periods (2 h, 4 h, 6 h). After incubation, 1.5 µL of 0.2 mM PI was mixed into each tube and diluted with 150 mM NaCl. Samples were analyzed by a flow cytometer.
Samples were illuminated with a 15 mW argon ion laser (488 nm), and the fluorescence detected through standard filter configuration of 525±10 nm (green) and 620±10 nm (red) band pass filters. Signals were amplified in the logarithmic mode for side scattered light (SSC), forward scattered light (FSC) and fluorescence. In dot plots of fluorescence different cell populations i.e. live, compromised, dead and lysed were gated, based on the
4.3.3.11: DNA isolation and agarose gel electrophoresis analysis
Recombinant GFP plasmid (pGFP) isolated from control and CS Cu@Ag NPs treated E. coli cells by an alkaline lysis method and analyzed by agarose gel electrophoresis followed by visualization of EtBr stained DNA under UV- transilluminator.