Bio-directed synthesis and

assembly of nanomaterials
“Biological synthesis of nano particles is a “green
method” (for the reaction, microbial enzymes or plant
phytochemicals are used)”
Biological synthesis of Nanoparticles

The enzymes act as reducing agent, or stabilising agent,or capping agent

Why to use biological methods?
• Reduce toxic chemical concentration

• Eco-friendly nanoparticles

• Economically viable

• Easier to tailor size, shape, and nature just by modifying culture, pH, temperature,
and nutrient medium
Nanoscale structures and nano particles in nature

Magnetotactic bacteria that

synthesised nano sized particles to
help them navigate
SEM of silica produced by living diatoms
Multicellular organisms produce hard inorganic-organic composite materials
such as bones, shells, and spicules

Radiolarians

Rice leaf blade Sponge spicules

Use of bacteria
• Bacteria are considered as potential biofactories for the synthesis of nanoparticles,
such as gold, silver, platinum, lead, and cadmium
• Bacteria are known to produce inorganic nanoparticles, either intracellularly or
extracellularly
• Pseudomonas stutzeri isolated from silver mines has been shown to produce silver
nanoparticles within periplasmic space.
• Morganella spp isolated from insect midgut when exposed to a AgNO3 produced
extracellular crystalline silver nanoparticles.
Use of yeast
• Candida glabrata and S. pombe were used for the first time in the biosynthesis of
cadmium sulphide (CDS) nano crystals.

• Recently, yeast strains have been identified for their ability to produce gold nano
particles, whereby controlling growth and other cellular activities, controlled size
and shape of the nano particles can be achieved.
Use of fungi
• Bioreduction of aqueous AuCl4 ions was carried out using the fungus Verticillium
species that led to the formation of gold nanoparticles with fairly well-defined
dimensions and good mono dispersity.

• Fungi are known to secrete much higher amounts of proteins, thus might have
significantly higher productivity of nanoparticles in biosynthetic approach.
Use of plants
• Quantum dots have huge application in nano biotechnology and plants have been
observed to be good source for the synthesis of quantum dots.
• Ex. Alfalfa roots have capability for absorbing Ag (0) from agar medium and
transferring them to shoot of the plant in the same oxidation state.
• Using Geranium leaf extract, silver ions can be reduced to silver nano particles.
• In addition to individual pure metallic Ag and Au, bimetallic Ag/Au nanoparticles
(50-100 nm), using Azadirachta indica leaf broth have also been synthesised
extracellularly.
Biosynthesis
Magnetotactic bacteria

Examples for template

• Magnetotactic bacteria are a heterogeneous group of prokaryotes.
• They orient and migrate along geomagnetic field lines (it doesn’t have flagella).
• Migration based on intracellular magnetic structure, so called magnetosomes.
• Magnetosomes: membrane-bound magnetite particles
Proposed model for magnetite biomineralisation in Magnetospirillum species.
Fe (III) is actively taken up by the cell , possibly via a reductive step. Iron is then thought to be
reoxidised to form a low density hydrous oxide which dehydrates to form a high-density Fe(III)
oxide. Finally, one third of the Fe(III) ions are reduced, and with further dehydration magnetite is
produced within the magnetosome vesicles. The magnetosome membrane contains specific
proteins, which helps in the accumulation of iron, nucleation of minerals, and redox and pH
Electron Micrographs of Magnetosomes Crystal morphologies and intracellular organization
of magnetosomes found in various magnetotactic bacteria. Shapes of magnetic crystals
include cubo-octahedral (a), bullet-shaped (b, c) elongated prismatic (d, e, f, g, h, i, j, k) and
rectangular morphologies (l).
The magnetosome particles can be arranged in one (a, b, c, e), two (f, i) or multiple chains (g,
h) or irregularly (j, k, l). The bar is equivalent to 0.1 μ m.
• Magnetic nanoparticles can be assembled into ordered structures when the motion
of the magnetic bacteria M. magnetotacticum is controlled by applying a magnetic
field.
• After assembling the bacteria with micro electromagnets, the cellular membranes
of the bacteria can be removed by cell lysis to leave the biogenic magnetic nano
particles at desired locations.
Scanning electron micro graphs of assembled magnetic structures after removing the
cellular membrane of trapped bacteria.
(a) Single chain of magnetic nano particles is shown along with cellular debris. Two
small nanoparticles indicated by an arrow are still attached to the chain due to the
magnetic field from the adjacent large particle.
Long chain from single bacterium (b). A ring of magnetic nano particles was formed
by trapping and lysing two bacteria (c)
Mechanism of synthesis of silver Nanoparticles
• A widely accepted mechanism for the synthesis of silver nanoparticles is the
presence of enzyme “nitrate reductase”.
• Nitrate reductase is an enzyme in the nitrogen cycle, responsible for the
conversion of nitrate to nitrite.
• During the catalysis, nitrate is converted to nitrite, and an electron will be shuttled
to the incoming silver ions.
• This has been excellently described in the organism B. Licheniformis. B.
Licheniformis is known to secrete the cofactor NADH and NADH-dependent
enzymes, especially nitrate reductase, that might be responsible for the bio
reduction of Ag+ to Ag0 and the subsequent formation of silver nanoparticles.
Mechanism of synthesis of silver nanoparticles

Likely mechanism for

synthesis of silver
nanoparticles by
Bacillus licheniformis
including Nitrate
Reductase enzyme
depending on NADH
which might transform
Ag+ to Ag°
Viral nanotechnology
• Viral nanotechnology is an emerging and highly interdisciplinary field in which
viral nanoparticles (VNPs) are applied in diverse areas such as electronics, energy,
and next generation medical devices.

• VNPs have been developed as candidates for novel materials, and are often
described as “programmable” because they can be modified and functionalised
using a number of techniques including:
1. Bioconjugation chemistries
2. Encapsulation techniques
3. Mineralization strategies, and
4. Film and hydrogel development
Nano container and protein cages
• Viral capsids devoid of their nucleic acid genomes can be thought of as nano
containers.

• Inspired by nature – a biomimetic approach to nanomaterial’s synthesis is bio

assisted.

• We can utilise protein cage architectures to serve as size-constrained reaction

vessels and chemical building blocks.
Schematic representation of protein cage functionalisation
Why plant viruses?
• Scientists have engineered viral nano particles from plant viruses, insect viruses,
and animal viruses.
• They avoid using human viruses in order to minimise the chances of the virus
interacting with human proteins and causing toxic side-effects, infection, and
immune response.
• Plant viruses are easiest to produce in large quantities.
• Plant viruses are also ideal, because they can self assemble around a nano particle
in vitro and hold approximately 10 cubic nm of particles. Therefore, many
molecules of cancer drugs can fit in plant viral nanoparticles.
Viral nanoparticles
Viral nanotechnology- the assembly line
• VNP can be produced in their natural
host – plants when using plant viruses,
bacteria when using bacteriophages,
mammalian cells when using
mammalian viruses. Heterologous
expression of virus like particles (VLP)
in bacteria and yeast is also a common
production technique.
• Once purified, chemical tuning and
design are carried out to attach and
encapsulate molecules that confer
different functionalities.
• The hybrid and functionalised VNP is
then evaluated in vitro and in vivo.
Protein cages for inorganic nano particle synthesis
• The exterior and interior of the cowpea chlorotic mottle virus (CCMV) viral
capsid are chemically distinct environments.
• The interiors surface is more positively charged than the exterior, thus allowing it
to serve as a nucleation site for crystal growth.
• After nucleation, the size and shape of the mineral are defined by the interior of
the virion.
• The virion’s interior cavity constraints mineral growth, resulting in a spherical
nano particle with maximum diameter of approximately 24 nm.
Protein cages for inorganic nano particle synthesis
• In addition to the endogenous properties of size, shape, and delineation of charge
on the interior and exterior surfaces, the CCMV viral capsid undergoes a pH
dependent structural transition (gating).

• This pH-dependent reversible gating or swelling is a structural change of the virus

capacity that results in a 10% increase of viral dimension at pH greater than 6.5 in
the absence of metal cations (at pH less than 6.5 the virion is in its closed
confirmation).
Encapsulation of artificial cargos within VNPs
• Viral capsids have many functions, one of which is to form a shell or tube to
protect the virus genome (which can be considered a natural cargo), and another is
to deliver that cargo to cells, fulfilling the viral replication cycle.
• Researchers, developing VNP seek to adapt the natural properties of virus coat
proteins to maintain their ability to self assemble into shells and tubes, but at the
same time to allow the encapsulation of artificial cargos, such as synthetic
polymers, drugs, imaging reagents, other proteins, and inorganic particles.
• Pores in the capsid structure allow small molecules to diffuse between the external
medium and the capsids interior.
Encapsulation of materials during particle cell assembly
• Schematic illustration of the preparation
of a doxorubicin-loaded hibiscus
chlorotic ringspot virus (HCSRV)
protein cage with and without folic acid
conjugation (fPC-Dox, PC-Dox).
• Steps A1 and B2 indicate the removal of
viral RNA from the plant virus and
purification of coat proteins.
• Steps A2 and B3 involves the
encapsulation of polyacid and
doxorubicin during the assembly of the
protein cage.
• Step B1 refers to the conjugation of
folic acid on to the viral coat protein
Size dependence
• Different core particle sizes result in the formation of virus like particles (VLP),
with different sizes and distinct symmetries.
• VLP assembled from smaller nanoparticle cores are smaller than those assembled
from larger gold cores
• Templating allows many different sized hybrid structures to be assembled
Size dependence
Deposition of materials on the external and internal surfaces
of viral rods and filaments
TMV is a highly versatile scaffold because both the external surface and the 4 nm
diameter interior channel can be mineralised, and spatially controlled materials
synthesis can take place because each surface has a distinct amino acid composition.

A broad range of materials have been deposited on TMV using different methods:
i. Semi conductor nanocrystals such as CdS and iron oxide can be nucleated under
benign conditions by exposure to precursor salts.
ii. Noble metal coatings, such as gold, silver, platinum, Palladium, nickel, cobalt,
and copper, can be achieved by electroless deposition, in which the VNPs are
exposed to a metallisation bath containing metal irons and a reductant.
Deposition of materials on the external and internal surfaces
of viral rods and filamentS
• Electroless Deposition:
transmission electron micro
graphs of metallised TMV
particles produced by electroless
deposition

• TMV after Pd (II) activation,

followed by electroless deposition
of Ni. TMV is filled with a nickel
wire (3 nm diameter).
• TMV metallised with nickel on the
external surface.
Biotemplating using genetically engineered viruses
• Biomimetic approaches to materials
synthesis have explored the interaction
between proteins and minerals at their
interface.
• Genetic incorporation of this CoPT,
finding peptide into the heat shock
protein (HSP) cage interior CP-hsp
enabled phase specific nucleation and
size constrained formation of COPT
nano particles with an average
diameter of 6.5 nm
VNPs as a scaffold for 3D cell culture
• TMV (plant virus) displaying Arginine- Glycine-Aspartic Acid (RGD) peptides
promote the adherence of cells more effectively.

• By incorporating non-toxic magnetic Nanoparticles within the virus in order to

create a system of cellular levitation whereby cells can be grown in three
dimensions through the application of a magnetic field.
VNPs as a scaffold for 3D cell culture