Title Page
ABSTRACT
Contents
Abbrevations 13
CHAPTER I. Biosynthesized nanoparticles for biological applications 14
1.1. INTRODUCTION 15
1.2. BIOSYNTHESIS OF NANOPARTICLES 19
1.2.1. Bacteria 19
1.2.2. Yeast 22
1.2.3. Fungi 23
1.2.4. Actinomycetes 24
1.3. MOTIVATION AND CONCEPT 26
CHAPTER 2. Isolation and characterization of Streptomyces hygroscopicus 28
2.1. INTRODUCTION 29
2.1.1.Marine environment as a new source of bioactive metabolites 30
2.2. EXPERIMENTS 37
2.2.1. Isolation of actinomycetes 37
2.2.2. Molecular characterization 39
2.2.3. Instrumental analysis 41
2.3. RESULTS AND DISCUSSION 43
2.3.1. Morphological characterization 43
2.3.2. Molecular characterization of Streptomyces sp. 48
2.3.3. Instrumental ananlysis for extraction of Streptomyces hygroscopicus 50
2.4. CONCLUSION 56
CHAPTER 3. Biosynthesis of AgNPs by Streptomyces hygroscopicus and anti microbial activity against medically important microorganisms 57
3.1. INTRODUCTION 58
3.2. EXPERIMETS 61
3.2.1. Microbial synthesis of AgNPs 61
3.2.2. Instrumental analysis 62
3.2.3. Antimicrobial activity of AgNPs 64
3.3. RESULTS AND DISCUSSION 65
3.3.1. Spectral characterization 65
3.3.2. Morphological chacracterization by electron microscopic analysis 69
3.3.3. Antimicrobial activity 71
3.4. CONCLUSION 74
CHAPTER 4. Cytotoxic activity of biosynthesized Ag-NPs against A549 cell line 75
4.1. INTRODUCTION 76
4.2. EXPERIMENTS 78
4.2.1. Materials 78
4.2.2. XTT assay 78
4.2.3. Confocal laser scanning microscopy 79
4.3. RESULTS AND DISCUSSION 80
4.3.1. Effect on cell morphology 82
4.3.2. Instrumental characterization 83
4.4. CONCLUSION 85
CHAPTER 5. Synthesis of polygonal Au-NPs by Streptomyces hygroscopicus and antimicrobial, cytotoxic and electrochemical properties 86
5.1. INTRODUCTION 87
5.2. EXPERIMENTS 91
5.2.1. Culture and synthesis of AuNPs 91
5.2.2. Absorbance and analytical measurement 92
5.2.3. Morphological characterization of AuNPs 92
5.2.4. Electrochemical study: cyclic voltammetry 93
5.2.5. Minimum inhibitory concentration 94
5.2.6. Cell culture and cell viability rate 94
5.3. RESULTS AND DISCUSSION 96
5.3.1. Morphological analysis 97
5.3.2. Cyclic voltammetry analysis of AuNPs 105
5.3.3. Antimicrobial activity 108
5.3.4. Cytotoxicity of AuNPs on HeLa and 293T cells 109
5.4. CONCLUSION 116
CHAPTER 6. Summary and future directions 117
REFERENCES 121
Table 1. Merits and Demerits of Biological Vs Chemical synthesis of NPs. 20
Table 2. Literature reports on bioSynthesis of nanoparticles by different microorganisms. 25
Table 3. List of novel metabolites isolated from marine actinomycetes. 35
Table 4. Characterizations and identification of marine actinomycete (Morphological test). 43
Table 5. Cultural characteristics of S. hygroscopicus on different culture media. 46
Table 6. Evaluation of growth Conditions of S.hygroscopicus (I) Growth at temperature (II) Growth at pH. 47
Table 7. Diameter Zone of inhibition by Ag-NPs against various pathogenic microorganisms. 73
Table 8. Minimum inhibitory concentration (MIC) of Au-NPs against pathogenic bacterial strains. 109
Figure 1. Schematic representation of type's of nanoparticle synthesis method. 17
Figure 2. Schematic representation of biosource for nanoparticle synthesis. 18
Figure 3. Schematic representations of Structure of Prokaryotic and Eukaryotic cells. 21
Figure 4. Annual increase in the number of marine bacterial metabolites according to AntiBase of Laatsch (2005). 32
Figure 5. Isolated of streptomyces organisms on Agar Plate (a) early stage of organism from soil. (b,c,d) Sub culture of organisms. 44
Figure 6. Light Field Microscopic image shows spirally twisted structure of S.hygroscopicus (a) 40X (b) 400X. 44
Figure 7. FE-SEM images of tight spirals of spore chains of strain S. hygroscopicus (a) 10μm (b) 1μm (c, d, e, f) 100nm. 45
Figure 8. Agarose gel electrophoresis: Genomic DNA of S. hygroscopicus shows amplified Fragment at ~1500 bp; M: Marker DNA. 48
Figure 9. Evidence for Deposition of S. hygroscopicus strain (BDUS 49) for partial sequencing of 16S rRNA gene in NCBI gen Bank. 49
Figure 10. Neighbour-joining tree based on complete 16S rRNA gene sequence showing relationships between streptomycete isolate Streptomyces hygroscopicus BDUS 49 and related members of the genus Streptomyces sp.... 50
Figure 11. UV-Spectra for Extract of S. hygroscopicus. λmax at 210, 220 & 235 nm (n-σ* transition)λmax at 255, 265 & 295 nm (n-π* & π-π* transition).(이미지참조) 51
Figure 12. FTIR-Spectra for Extract of S. hygroscopicus shows the presence of hydroxyl, carbonyl, alkanes, alkenes and amine conjugated groups. 52
Figure 13. ¹H NMR-Spectra shows Proton resonance (-CH-, -OH, -CH₂-CH₃-O-) in the region of (δ1-5 ppm) for extract of S. hygroscopicus. 53
Figure 14. ¹³C NMR-Spectra shows carbon resonance for Extract of S. hygroscopicus 54
Figure 15. Mass Spectra showed that the expected [M+Na]+ molecular ion peak is at 936 m/z and a fragment ion m/z (M-H-Xyl-Glc)− at 931 [M-H] for Extract of S. hygroscopicus 54
Figure 16. Shematic representation of Nanoparticle synthesis, Bacterial enzyme substrate reaction with metal salt solution. 62
Figure 17. UV spectra of Ag-NPs by S. hygroscopicus. 67
Figure 18. EDX analysis shows the peak for presence of Ag. 68
Figure 19. 2θ peaks at ~38°, ~44°, ~64° shows the face-centered cubic planes of (fCC) silver. 68
Figure 20. TEM analysis of Ag-NPs (a) 50nm (b) 200nm. 69
Figure 21. SEM analysis of Ag-NPs (a) 1μm (b) 100nm. 70
Figure 22. Bio-AFM images of Ag-NPs (a) 1μm (b) 500nm and their 3D images. 70
Figure 23. Interaction of Ag-NPS with bacterial cell wall surface (a) Gram negative (b) Gram Positive. 72
Figure 24. Antimicrobial activity of Ag-NPs a) C. albicans b) B. subtilis c) E. coli d) E. faecalis e) S. typhimurium f) S. cerevisiae. 73
Figure 25. TAMRA coated Ag-NPS in optical Microscope (a) Dark Field (b) Light Field (c) Fluorescence image. 80
Figure 26. (a) Cell viability of XTT assay using A549 cells with different concentration of Ag-NPs (A)2 h, (B)4 h, (C)6h, (D)8 h. 81
Figure 26. (b) Cell viability of XTT assay using IMR90 cells with different concentration of Ag-NPs (A) 2 h, (B) 4 h, (C) 6h, (D) 8 h. 81
Figure 27. FE-SEM images of A549 cells. (a) Control A549 Cell (b) Particle Treated A549 Cell. 83
Figure 28. confocal laser scanning Microscope images of A549 cells after incubation with TAMRA labelled Ag-NPs. Blue-DAPI (nucleus) Red-particle (TAMRA) Green-membrane. 84
Figure 29. Schematic Representation of possible mechanisms of gold ions bioreduction. 97
Figure 30. UV-Vis absorption spectrum of Au-NPs after 48h of reaction (A) S. hygroscopicus in DI H₂O (B) UV-Vis absorption spectra of Au-NPs after the reaction of 10-3 M aqueous HAuCl₄ solution at neutral pH with the bacteria.(이미지참조) 98
Figure 31. XRD pattern recorded from fabricated Au-NPs. In spectrum (recorded from powder form of synthesized Au-NPs the diffractions at 38.5°, 44°, 64.5° 76.9°and 81.7° 2θ can be indexed to the (111), (200), (220) (311) and (222) planes of... 99
Figure 32. H-TEM images of polygonal Au-NPs after 48h of reaction (A) pH 7, (B,C,D) pH 4, (E,F,G) crystallite structure of Au-NPs. 102
Figure 33. FE-SEM images of Polygonal Au-NPs after 48h of reaction (A) pH 7, (B, C) pH 4. 103
Figure 34. EDXA spectra of synthesized Au-NPs recorded by FE-SEM-EDX. 104
Figure 35. Bio-AFM images of Polygonal Au-NPs after 48h reaction (a, b) pH 7, (c, d) pH 4. 104
Figure 36. CV studies of Au-NPs (a) Au-NPs with MB (b, c, d, e) Au-MB with 100, 200, 300, and 400 μl of DPBF, respectively. 107
Figure 37. FE-SEM image of Au-NPs-MB-modified Cu₂O-Pt substrate after CVs scan. 108
Figure 38. Cytotoxic effect on HeLa cells by different concentration of Polygonal (30-1.5μm) Au-NPs. Each value indicates mean ± SD. 111
Figure 39. Cytotoxic effect on HeLa cells by different concentration of spherical (2-20nm)Au-NPs. Each value indicates mean ± SD. 111
Figure 40. Cytotoxic effect on 293T cells by different concentration of Polygonal (30-1.5μm) Au-NPs. Each value indicates mean ± SD. 112
Figure 41. Cytotoxic effect on 293T cells by different concentration of spherical (2-20nm) Au-NPs. Each value indicates mean ± SD. 112
Figure 42. Time dependent confocal micrographs of AO/EB stained HeLa cells. 113
Figure 43. Time dependent confocal micrographs of AO/EB stained 293T cells. 114