1

Biological Methods
Synthesis of nanomaterials

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

2 Introduction
 Inorganic material inside organic matter or organisms are
known as biocomposites.
 These materials exhibit a rich variety in their functions like
providing support to body, allow body movements and general
function.
 Many of the materials synthesized by microorganisms, animals
and plants in nature can be synthesized using them in
laboratories, even on large scale.
 Eco-friendly- green synthesis
 A variety of metal, semiconductors and insulator nanoparticles
have been made using enzymes, DNA, membranes, etc.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

3

Green synthesis
3 types:
1. Use of microorganisms like fungi, yeast(eukaryotes) or bacteria,
actinomycetes (prokaryotes)
2. Use of plant extracts or enzymes
3. Use of templates like DNA, membranes, viruses and diatoms

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

4 Biology: The inspiration for nanomaterials
synthesis

Radiolarians Sponge Spicule Diatoms Rice leaf blade

Nanotechnology, Dr. Neeru Bhagat 2/13/2014
5 Green synthesis
 Rapid synthesis, controlled toxicity, controlling on
 size characteristics, economical and eco-friendly approach
Crucial point before going for biological method
 What biological system can be used to (bacteria, plant and fungi)
synthesis nanostructure ?
 Can we do it extracellularly ?
 What is the biochemical process leading to nanoparticle formation ?
 Is size control possible ? Better monodispersity ?
 How about nanoparticle shape modulation ?
 Can we use extracts of plants in nanobiosynthesis ?

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

6 WHY GREEN SYNTHESIS ?
 EASY
 EFFICIENT
 ECO-FRIENDLY
 ELIMINATES USE OF TOXIC CHEMICALS
 CONSUME LESS ENERGY
 PRODUCE SAFER PRODUCTS & BY PRODUCTS
 DOES NOT IMPART ANY HAZARDOUS EFFECT ON
ENVIORNMENT

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

7 Biological systems

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

8 Synthesis of Nanoparticles from
Bacteria

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

9 Why do organisms synthesize
nanoparticles?
 Bacteria have been most extensively researched for synthesis of
nanoparticles because of their fast growth and relative ease of
genetic manipulation
 Major reasons involved in such synthesis is detoxification which
can be carried out by
1. Modifications in the cellular transport mechanisms to restrict
entry of the toxicions into the cell
2. Sequestration of the toxic species within the cell (intracellular
sequestration) or outside the cell(extracellular sequestration)
3. Activation of energy-dependent efflux pathways to
eliminate the toxic species
4. Enzyme catalyzed oxidation or reduction of the toxic species to a
less toxic form
Nanotechnology, Dr. Neeru Bhagat 2/13/2014
10
 Microorganisms are capable of interacting with metals
coming in contact with them through their cells and form
nanoparticles.
 The cell-metal interactions are quite complex
 Certain microorganisms are capable of separating metal ions.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

11 METHODS OF SYNTHESIS

 Intracellular:
 Inside the cell, in cytoplasm or cytosol.
 Extracellular :
 Outside the cell on the surface or between the cells inside a colony.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

12 INTRACELLULAR SYNTHESIS
OF
NANOPARTICLES BY
BACTERIA
Peptides
Proteins
Polyols
Heterocyclic compounds

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

13
 Pseudomonas stuzeri Ag259 bacteria are commonly found in silver mines.
 Capable of accumulating silver inside or outside their cell walls
 Numerous types of silver nanoparticles of different shapes can be produced
having size <200nm intracellularly
 Low concentrations of metal ions (Au⁺,Ag⁺ etc) can be converted to metal
nanoparticles by Lactobacillus strain present in butter milk.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

14 INTRACELLULAR SYNTHESIS
cont……
 Bioaccumulation
 In order to release the intracellularly synthesized
nanoparticles, additional processing steps such as
ultrasound treatment or reaction with suitable
detergents are required.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

15 Cont….

 Synthesis of Ag NPs by bacteria

 Culture supernatants of bacteria can be used for synthesis of Ag
NPs.
 Such as culture supernatants of E. coli, Klebsiella pneumonia, B.
subtilis,
 Enterobacter cloacae and Bacillus licheniformis.
 Also, Lactobacillus strains can be used for synthesis of Ag NPs.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

 Cont….
16

Crystal topologies by P. stutzeri AG259. (a,

b) Triangular, hexagonal, and
spheroidal Ag-NPs found at different
cellular binding sites (National Academy of
Sciences, U.S.A)

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

17 Bacterial biosynthesis

 Mainly extracellular
 Silver nanoparticle
 Gold nanoparticle
 Cadmium

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

18 Fungi – Fusarium oxysporum challenged with gold or silver salt for app. 3
days produces gold or silver nanoparticles extracellularly.
 Extremophilic actinomycete Thermomonospora sp. Produces gold
nanoparticles extracellularly.
 Semiconductor nanoparticles like CdS, ZnS, PbS etc., can be produced using
different microbial routes.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

19
 Sulphate reducing bacteria of the family Desulfobacteriaceae can form 2-5nm
ZnS nanoparticle.
 Klebsiella pneumoniae can be used to synthesize CdS nanoparticles.
 when [Cd(NO₃)₂] salt is mixed in a solution containing bacteria and solution is
shaken for about 1 day at ~38ºC ,CdS nanoparticle in the size range ~5 to 200 nm
can be formed.
 Titanium nanoparticles of spherical aggregates of 40–60 nm were produced
extracellularly using the culture filtrate of Lactobacillus sp. at room
temperature. Titanium salt is mixed in a solution containing the bacteria and
the solution is shaken for 24 hours at low temperature. The Ti nanoparticles
are thus synthesized. These titanium nanoparticles were lighter in weight and
high resistance to corrosion

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

20 SYNTHESIS USING DNA

 CdS or other sulfide nanoparticles can be synthesized using DNA.

 DNA can bind to the surface of growing nanoparticles.
 ds Salmon sperm DNA can be sheared to an average size of 500bp
(base pair).
 Cadmium acetate is added to a desired medium like water,
ethanol, propanol etc.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

21 Reaction is carried out in a glass flask- facility to purge the solution and flow
with an inert gas like N₂.
 Addition of DNA should be made and then Na₂S can be added dropwise.
 Depending on the concentrations of cadmium acetate, sodium chloride and
DNA ,nanoparticles of CdS with sizes less than ~10 nm can be obtained.
 DNA bonds through its negatively charged PO₄ group to positively charged
(Cd⁺) nanoparticle surface.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

22 USE OF PROTEINS,
TEMPLATES LIKE DNA , S-
LAYERS ETC
 Various inorganic materials such as carbonates, phosphates,
silicates etc are found in parts of bones, teeth, shells etc.
 Biological systems are capable of integrating with inorganic
materials
 Widely used to synthesize nanoparticles

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

23 FERRITIN
 Ferritin is a colloidal protein of nanosize.
 Stored iron in metabolic process and is abundant in animals.
 Capable of forming 3 dimensional hierarchical structure.
 24 peptide subunits – arranged in such a way that they create a central cavity of
~6 nm.
 Diameter of polypeptide shell is 12 nm.
 Ferritin can accommodate 4500 Fe atoms.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

24

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

  Ferritin without inorganic matter in its cavity is called apoferritin and can be
25 used to entrap desired nanomaterial inside the protein cage.

Remove iron from ferritin to form apoferritin

Introduce metal ions to form metal nanoparticles inside the cavity

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

 PROCEDURE TO CONVERT
26 FERRITIN TO APOFERRITIN
Horse spleen ferritin

diluted with sodium acetate buffer (placed in

dialysis bag)

sodium+ thioglycolic
acetate acid

dialysis bag kept under

N₂ gas flow for 2-3 hrs

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

 solution needs to be
27 replaced from time to time
for 4-5 hrs.

saline for 1 hr

refreshed saline
for 15-20 hrs

APOFERRITIN

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

28
APOFERRITIN

mixed with NaCl and N-tris

methyl-2-aminoethanosulphonic
acid (TES)

aq. Cadmium acetate added and

stirred with constant N₂ spurging

aq. Solution of Na₂S is added twice

with 1 hr interval.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

29 Synthesis using Plant extracts
 To date various toxic chemicals are being used either in the form of
reducing agent or stabilizing agent for Nano particle synthesis.
 Green synthesis of metal nanoparticles has been an emerging
highlight of the research in the field of Nano biotechnology and
chemistry due to growing need for the development of the
environmentally benign technologies especially in material science
and medicine.
 A great deal of effort has been invested into the synthesis of
inorganic material especially metal nanoparticles using
microorganism and plants .
 The rate of reduction of metal ions using plant extracts has been
observed to be much faster as compared to that with
microorganisms besides microorganisms being bio hazardous.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

30
Plants and Plant extracts as a
tool for nanoparticles
 Provides single step biosynthesis process
 Protocols involving free from toxicants and natural capping
agents
 Can generate bimetallic silver and gold shell nanoparticles
 Excellent stability and size control
 cost-effective large-scale production of metallic,
 semiconductor and metal oxide nanoparticles

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

31
Process involved in nanoparticles
synthesis

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

32

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

33

EXTRACTION
PLANT FROM PLANT
PLANT
EXTRACT

NANOPARTICL EXTRACT AND

E RECOVERY STIRRING METAL
Nanotechnology, Dr. Neeru Bhagat
SOLUTION2/13/2014
34 SYNTHESIS USING PLANT
EXTRACTS
 Leaves of geranium plant ( Pelargonium graveolens) have been
used to synthesize
gold nanoparticles
 Plant associated fungus- produce compounds such as taxol and
gibberellins
 Exchange of intergenic genetics between fungus and plant.
 Nanoparticles produced by fungus and leaves have different
shapes and sizes.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

35 Nanoparticles obtained using Colletotrichum sp., fungus is mostly spherical
while those obtained from geranium leaves are rod and disk shaped.

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

36finely crushed leaves
(Erlenmeyer flask)

boiled in water ( 1 min)

cooled and decanted

added to HAuCl₄ aq. Solution

gold nanoparticles within a minute

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

37
Hybrid Routes

 Chemical + Biological

Nanotechnology, Dr. Neeru Bhagat 2/13/2014

 Flow chart of synthesis-chemical route
38
CuSO4.5H2O
(5 moles/ litre)

Base solution
(0.5 moles/ litre)

Stirring at 60 0C

Precipitate filtered

Dried at 80 0C for 24 hours

Calcination at different temperatures for

2 hrs
 Flow chart of synthesis - hybrid route
39
CuSO4.5H2O
(5 moles/ litre)

Base solution Plant extract

(0.5 moles/ litre)

Stirring at 60 0C

Precipitate
filtered

Dried at 80 0C for 24 hours

Calcination at different
temperatures for 2 hrs
 Scanning Electron Micrographs
40
Calcined at 200 °C

without MO with MO

The most striking observation is the growth of particle in one and

two dimension when only chemicals are used while in the
presence of MO the growth is three dimensional.
 SEM
41

CNa-200

CNa-400

Initial growth is 2 dimensional then grows to a

monoclinic structure
CNa-600
 42
SEM
Synthesized with MO

200 °C

400 °C

Phase change observed from 200 to 400 °C

Gradual growth to monoclinic structure 600 °C
 Selected Area Electron Diffraction
43
Calcined at 400 °C

without plant extract with plant extract

The images clearly indicate that the introduction of plant extract in the
process promotes smaller crystallite size and uniformity in the lattice
 Observations
44

● Introduction of Plant extract during the synthesis process

leads to

•smaller and relatively uniform particle size

• growth is uniform in all directions