The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!! Mayur D. Chauhan

1. Synthesis of Nanomaterials
Biological Methods
M.Sc. Biotechnology
Part II (Semester IV)
Paper I, Unit I
Mumbai University
By: Mayur D. Chauhan
1

2. Introduction
• Nobel Laureate Richard Feynman asked the
scientists to derive the inspiration from
Mother Nature to make the things smaller and
see the advantages of making things smaller.
• Indeed the biological world, animal kingdom
and plants make optimum use of materials
and space.
2

3. • Nature uses insoluble, complex organic
molecules as a reactor in which nucleation
and growth of complex and hierarchical
structures of inorganic materials takes place
by reactions of organic soluble molecules.
• Inorganic materials inside organic matter or
organisms are known as Biocomposites /
Biominerals.
3

4. • Many of the materials synthesized by
microorganisms, animals and plants in nature
can indeed be synthesized using them in
laboratories even on large scale.
• This is considered to be a very attractive
possibility so as to have eco-friendly or so-
called Green synthesis.
4

5. Challenges
• Ability of bio-world to synthesize materials of
variety of shapes and sizes is quite unique and
often difficult to mimic because many processes
are still not understood inside the living cells.
• Solution composition, ionic activities, their
transport, availability of suitable growth sites, ion
accumulation and transportation are all
governing factors in the nucleation and growth of
particles.
5

6. Applications
• Nanomaterials should be biocompatible,
bioactive or biopassive specially for different
medical applications like body implants, drug
delivery, cancer therapy and so on.
6

7. Methods of Synthesis
Use of Microorganisms
Use of Plant extracts / Enzymes
Use of DNA, Viruses, Diatoms
7

8. Vital Terms
• Essential Elements
• Trace Elements
• Proteins
• Nucleic Acids
• Carbohydrates
• Molecular Recognition
8

9. Diatoms
9

10. • Diatoms are algae found in marine or any
moist environment.
• They are unicellular, forming amorphous silica
shell with living part inside.
• Diatoms can be 20–200 μm long.
• Silicic acid [Si(OH)4] is absorbed inside the cell,
which is bonded to cofactor so that
polycondensation is avoided.
10

11. • Silica transport vesicles: Vesicles are
phospholipid bilayers which can trap some
solution or water and combine to form silica
deposition vesicles (SDV), mineralized part of
organism.
• Silicic acid condense in SDV and grow rapidly.
11

12. Synthesis Using Microorganisms
• Some bacteria are quite useful and are used in
the processing of cheese, curds, bread, alcohol
and vaccines.
• Some are harmful and responsible for spoiling
food or causing diseases.
12

13. 13

14. • Microorganisms are capable of interacting
with metals coming in contact with them
through their cells and form nanoparticles.
• Certain microorganisms are capable of
separating metal ions. This is widely used to
either recover precious metals or detoxify
water.
14

15. Cell – Metal Interactions
• Some microorganisms produce hydrogen
sulphide (H2S). It can oxidize organic matter
forming sulphate, which in turn acts like an
electron acceptor for metabolism.
• This H2S can, in presence of metal salt, convert
metal ions into metal sulphide, which deposits
extracellularly.
15

16. • The metal ions are converted into a non-toxic or
less toxic form and covered with certain proteins
in order to protect the remainder of the cell from
toxic environment.
• Certain microorganisms are capable of secreting
some polymeric materials like polysaccharides.
They have some phosphate, hydroxyl and
carboxyl anionic groups which complex with
metal ions and bind extracellularly.
16

17. 17

18. 01. Metal Nanoparticles (Bacteria)
• Pseudomonas stutzeri Ag259 bacteria are usually
found in silver mines and are capable of
accumulating silver inside or outside of their cell
walls.
• Using this fact, these bacterial strains can be
challenged with high concentration of silver salt
like AgNO3.
• Numerous silver nanoparticles of different shapes
can be intracellularly produced having sizes
<200 nm.
18

19. 02. Alloys (Bacteria)
• Low concentrations of metal ions can be
converted to metal nanoparticles by
Lactobacillus strain present in butter milk.
• By exposing the mixture of two different metal
salts to bacteria, it is indeed possible to obtain
alloys under certain conditions.
19

20. 03. Metal Nanoparticles (Fungi)
• Extracellular Synthesis: Fusarium Oxysporum
challenged with gold or silver salt for
approximately three days produces gold or
silver particles extracellularly.
• Extremophilic actinomycete
Thermomonospora sp. produces gold
nanoparticles extracellularly.
20

21. Intracellular Synthesis (Fungi)
• When silver metal salt is treated with another
fungus Verticillium sp., the nanoparticles can
be produced intracellularly.
• Process of Synthesis is as follows:
21

22. Verticillium sp. extracted from Taxus plant
should be placed at 250C in Potato–Dextrose
Agar slant.
Fungus should be grown in Erlenmeyer flask
containing MGYP medium composed of 3 %
Malt extract, 1 % Glucose, 0.3 % Yeast extract
and 0.5 % Peptone maintained at 250C
The flask needs to be shaked for 4 days.
After fermentation mycelia is separated from
culture broth by centrifugation at 10 0C.
22

23. Harvested Mycelial mass needs to be
washed with water for 20 mins and
then exposed to a metal salt soultion -
AgNO3 (5.5–6.0 pH)
After 3 days on a shaker, Verticillium
biomass can be fixed in Glutaraldehyde
in distilled water by maintaining it at
room temperature for 2 h.
After fixation, the centrifugation leads
to cell sedimentation rich in silver
nanoparticles.
23

24. • Changes in biomass color from initial yellow to
final brown, after exposure to silver salt, is a
visual indication of silver nanoparticles
formation.
• Particles can be recovered by washing with
some suitable Detergent or Ultrasonication.
• Verticillium does not die if exposed to AgNO3
solution.
24

25. Mechanism of Formation
• Silver ions from a silver salt possibly get
trapped on the surface of the fungal cells
perhaps due to electrostatic interaction
between positively charged silver ions and
negatively charged carboxylic groups in the
enzymes present in cell walls of mycelia.
• The ions after nucleation can grow by further
accumulation of silver ions to form
nanoparticles.
25

26. • In a similar way gold nanoparticles can be
produced using Verticillium sp.
• However the color of biomass is from pink to
blue depending upon the particle size.
26

27. 04. Semiconductor Nanoparticles
(Bacteria)
• Semiconductor nanoparticles like CdS, ZnS,
PbS and many others can be produced using
different microbial routes.
• There are some sulphate reducing bacteria of
the family Desulfobacteriaceae which can
form 2–5 nm ZnS nanoparticles.
27

28. • Bacteria Klebsilla pneumoniae, which is a
pathogen, can be used to synthesize CdS
nanoparticles.
• When [Cd(NO3)2] salt is mixed in a solution
containing bacteria and solution is shaked for
about one day at 38oC, the CdS nanoparticles
in the size range 5–200 nm can be formed.
28

29. 05. Semiconductor Nanoparticles
(Yeasts)
• CdS nanoparticles with narrow size
distribution can be synthesized using the
yeasts like Candida glabrata and
Schizosaccharomyces pombe.
• A nitrogen-rich medium containing 2%
Tryptone, 1% Yeast extract and 2% Glucose
(pH 5.6) is used to grow Schizosaccharomyces
pombe.
29

30. When challenged with cadmium sulphate
solution after about 12–13 of growth, after 36h
cells rich with CdS get formed.
Solution can be centrifuged and frozen at 200C.
The frozen cells can be thawed at 40C for 2–4 h.
The thawed solution can be centrifuged again so
that the cell debris precipitates and supernatant
contains CdS nanoparticles.
30

31. • The particles are covered by proteins which
can be removed by heating at 800C for few
minutes.
• Process of CdS nanoparticles formation by
Yeast can be understood as follows:
31

32. Mechanism
• When the cells are exposed to cadmium salt, a
series of reactions takes place.
• Cadmium ions are toxic to the cells and in
order to reduce this effect they need to coat
them with some protective coating of proteins
available to them.
32

33. • To begin, an enzyme Phytochelatin synthase
gets released to synthesize phytochelatin with
structure (Glu-Cys)n–Gly with n = 2–6.
• A low molecular weight phytochelatin–Cd
complex is formed.
• An ATP binding cassette (ABC) type vacuolar
membrane protein HMT1 transports Cd
complex across the membrane.
33

34. • When inside the vacuole, Cd ions receive
sulphur ions forming large molecular weight
phytochelatin–CdS complex.
• These are nothing but the CdS nanoparticles.
• Similarly it is possible to synthesize PbS by
challenging Torulopsis species with lead salt
like PbNO3.
34

35. Synthesis Using Plant Extracts
• Compared to the use of microorganisms to
produce nanoparticles, use of plant extracts is
relatively less investigated.
• Alfalfa plants are found to produce gold
nanoparticles from solids.
35

36. • Leaves of Geranium plant (Pelargonium
graveolens) have also been used to synthesize
nanoparticles of gold.
• It should be mentioned that there is also a plant
associated fungus which can produce compounds
such as taxol and gibberellins.
• There is an exchange of intergenic genetics
between fungus and plant.
• However, the nanoparticles produced by fungus
and leaves have quite different shapes and size
distributions.
36

37. • Nanoparticles obtained using Colletotrichum
sp. fungus related to geranium plant has a
wide distribution of sizes and particles are
mostly spherical.
• On the other hand geranium leaves produce
rod and disc shaped nanoparticles.
37

38. Synthesis Protocol
Finely crushed leaves are put in
Erlenmeyer flask and boiled in water just
for a minute.
Leaves get ruptured and cells release
intracellular material. Solution is cooled
and decanted.
This solution is added to HAuCl4
aqueous solution, when nanoparticles
of gold start forming within a minute.
38

39. Use of Proteins, Templates Like DNA,
S-Layers
• Biocomposites have been widely used not
only to synthesize nanoparticles but obtain
organized arrays, super lattices or hierarchical
structures of inorganic materials using
biological systems.
• DNA, S-layers (i.e. surface layers of cell walls of
some bacteria) or some membranes have long
range periodic order in terms of some
molecular groups of their constituents.
39

40. • Therefore on some periodic active sites
preformed nanoparticles can be anchored.
40

41. Ferritin (Protein)
• Ferritin is a colloidal protein of nanosize. It
stores iron in metabolic process and is
abundant in animals.
• Ferritin proteins in different animals have only
upto 14 % difference in the amino acid
sequences.
41

42. Structure
• There are 24 protein (peptides) subunits in a
ferritin molecule, which are arranged in such a
way that they create a central cavity of 6 nm.
• Diameter of polypeptide shell is 12 nm.
• Ferritin can accommodate 4,500 Fe iron
atoms.
42

43. 43

44. • They are in Fe3+ state as hydrated iron oxide
mineral called as Ferrihydrite.
• The protein subunits are composed of light as
well as heavy chains having dinuclear
ferroxide centres.
• These centres are catalysts for in vitro
oxidation of Fe2+ ions.
44

45. Aim
• The ferritin without inorganic matter in its
cavity is known as Apoferritin and can be used
to entrap desired nanomaterial inside the
protein cage.
• Ions can be removed or introduced inside the
ferritin, through some available channels
45

46. Synthesis of CdS using Spleen Ferritin
46

47. Self Assembly
• Self assembly is a gathering or collection of
certain entities without any external
influence.
• It relates to spontaneously formed, reversible,
locally ordered and thermodynamically stable
assemblies.
• Self assemblies are very sensitive and can
transform back to the state of disorder.
47

48. • In disorder state there are some building
blocks or motifs which are uniform in the
shape and size which assemble to form
ordered structure by some weak interactions
spontaneously.
• Self assembly helps in understanding the type
of formation (Ordered / Random) in some
smaller motifs or units.
48

49. • Major bonds prevailing in the inorganic solids
are ionic, covalent or metallic. They have
rather large energies of formation or
dissociation.
• Self assemblies are formed spontaneously by
weak interactions like – π - π , van der Waals,
colloidal, electrical, optical, shear or capillary
forces.
49

50. Applications
• Fabricate self assembled organic, inorganic
materials to obtain novel electrical,
mechanical, magnetic or optical properties.
• Langmuir-Blodgett films, micelles, liquid
crystals, layers obtained by dip-pen
lithography, deposition of materials in the
voids of the self assembled spheres are some
of the methods to realize self assembled
structures with desired materials.
50

51. • Such structures are useful to create photonic
band gap materials, novel sensors, lasers,
electroluminescent devices, photovoltaic solar
cells to name a few.
• Nanofabrications using DNA have potential
applications in nanoelectronics,
nanomechanical devices as well as computers.
51

52. Mechanism of Self Assembly
• Self assembly involves weak to strong forces
and nanoscopic to gigantic structures in one,
two or three dimensions.
• Self assembly can be a result of very weak
forces like van der Waals interaction, hydrogen
bonds, static charges, magnetic interactions
and so on.
52

53. Low Energy
State
Molecules
Self
Assembly
Formation
53

54. • Ability of a system to go to a well ordered low
energy state depends upon the availability of
units of same size and shape.
• Molecules with definite shape, number of atoms
and therefore size, already in the state of low
energy are good candidates for the self assembly.
• But Atoms in such molecules or Building blocks of
self assembly themselves are held together by
stronger forces.
54

55. • When the building blocks are available for self
assembly, state of minimum energy may be
attained spontaneously in the absence of any
external force.
• However self assembly may occur in the
presence of an external driving force like
temperature, pressure, magnetic field and so
on.
55

56. Types of Self -Assemblies
•Absence of external
driving forcesStatic
•Presence of
external driving
forces
Dynamic
56

57. 57

58. • Static assembly is realized when system achieves
minimum energy state and can stay there unless
it is subjected to strong external forces.
• On the other hand, dynamic self assembly
involves constant influence of external force from
the ambient. If the energy intake from the
ambient stops, the self assembly can leave the
state of organized structure and de-assemble.
58

59. Examples
• Static Self-Assembly: Formation of ordered
crystalline structure from a melt, thin films
formed by L-B technique, dip pen lithography etc.
• Dynamic Self-Assembly: The living animals or
plants form good examples of dynamic self
assembly. As soon as the supply of food, proper
temperature and air pressure discontinue, the
animals and plants disintegrate.
59

60. 60

61. • Hierarchical self assembly is characterized by
small range, medium range and long range
interactions of one type of a building block.
• Directed self assembly occurs when the building
blocks occupy the pre-designed places like some
portions of a lithographically patterned substrate,
pores in membranes or spaces between ordered
particles.
• Co-assembly, as the name suggests, can be
formed with two or more types of blocks which
can fit into each other.
61

62. Self Assembly of Nanoparticles by
Organic Molecules
• Preformed inorganic nanoparticles can be
assembled on solid substrates through some
organic molecules adsorbed on their surfaces.
• Examples: (i) CdS nanoparticles functionalized
with carboxylic (COO-) group can be
transferred to aluminium thin films.
62

63. 63

64. • (ii) Dithiols adsorbed on metal surfaces also could
adsorb CdS nanoparticles to form layers of them.
• (iii) Silver particles have been adsorbed on
oxidized aluminium layers using bi-functional
molecule such as 4-aluminium layers
carboxylthiophenol.
• Molecules bind to aluminium oxide layer through
carboxylic group and thiol attaches to silver
particles.
64

65. Self Assembly in Biological Systems
• When organized arrays of inorganic crystals
are embedded in biological systems they are
often referred to as biomineralized systems.
• Magnetotactic bacteria are small bacteria, 35–
120 nm sized with permanent magnets inside
them.
• The magnets are of either iron sulphide (Fe3S4-
greigite) or iron oxide (Fe3O4-magnetite). Such
magnets make a chain of nanomagnets.
65

66. • The size 35–120 nm is quite critical and smaller or
bigger magnets would not have served the
purpose for which these magnets are used.
• The magnets smaller than 35 nm cannot have
permanent magnetism at ambient temperature
and those larger than 120 nm would have
reduced magnetism due to multidomain
formation above this size.
• Only those between 35 and 120 nm size are
single domain permanent magnets, the chain of
which is useful for navigation of bacteria.
66

67. • These bacteria live in mud, marshy areas,
ponds or sea and prefer anaerobic condition.
• If by any chance they come to the surface of
water or soil, they navigate downwards
making use of their magnets aligning in the
earth’s magnetic field direction.
67

68. Self Assembly by S- Layers
• They are two dimensional, crystalline single
proteins or glycoprotein monomers organized
in hexagonal, oblique or square lattices.
68

69. • Such S-layers after extraction from bacterial
cells have been transferred on some metallic
substrates (or grids).
• In general, S-layers extracted from the
biological cells can be directly used to deposit
nanoparticles from liquid or vapour phase.
69

70. 70

71. Self Assembly in Inorganic Materials
• It is possible to spontaneously create the
quantum dots for example of germanium (Ge)
on silicon (Si) or Indium arsenide (InAs) on
gallium aresenide (GaAs).
• The origin of self assembly is strain induced.
• Quantum Dot is a semiconductor crystal of
nanometre dimension with distinctive
conductive properties determined by its size.
71

72. • Germanium and silicon have only 4 % lattice
mismatch. Therefore Ge can be deposited
epitaxially on Si single crystal upto 3–4
monolayers.
• Although grown (hetero)epitaxially, the layers of
deposited Ge are highly strained (coherently i.e.
without any defects or dislocations).
• When further deposition takes place, the lattice
strain caused by depositing Ge on Si with
different lattice constants cannot be
accommodated.
72

73. • This results in spontaneous formation of
nanosized islands or quantum dots.
• However the temperature of the substrate has
to be >350o C during deposition or post-
deposition annealing is required.
73

74. 74

75. 75