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Bionanomaterials: Synthesis and applications

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 Proceedings of the frist National Seminar on New Materials Research and Nanotechnology
-
(NSNMRN 2012) 12th 14th Sept. 2012 at Govt. Arts College, Doty. TamiiNadu.

BIONANOMATERIALS: SYNTHESIS AND APPLICATIONS

K. Sahayaraj

Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St. Xavier s
College (Autonomous), Palayamkottai - 627 002, Tamil Nadu, India.
Tel: + 91 4624264376, Fax: + 91 4622561765, E mail: ksra;42@gmail.com

Cellulose, chitin, and starch are abundant,
natural, renewable and biodegradable molecules
(or whiskers).In most cases, aqueous suspensions
of these nano crystallites are prepared by acid
hydrolysis process naturally and also in the
laboratory.However, synthetic nanomaterials are
prepared by physical and chemical methods by
both strong and weak chemical reducing agents
and protective agents (sodium borohydride,
sodium citrate and alcohols). They are harmful to
the nature, human beings, domestic animals, and
also to wild animals. These impacts can be
minimized by biosynthesis or green methods.
Green synthesisof nanoparticles can be done by
using five methods:a) Polysaccharidemethod, b)
Tollens method, c) Irradiation method, d)
biological methods, and e) Polyoxometalates
method. The biosynthesis of nanoparticles
employs use of biological agents including
microbes,plantsand animals.The most effectively
studied nanoparticlestoday are those made from
noble metals, in particular silver (Ag), platinum
(Pt), gold (Au), cadmium sulfide (CdS), lnS,
bariumtitanate(BT), In203Copper and Palladium
(Pd). Microbes,plants, animal products (egg) and
animal by-products have been used for the
bionanoparticlessynthesis. It is well known that
many organismscan provide inorganic materials
either intra-or extracellularmanner.For example,
unicellular organisms like magnetotactic bacteria
produce magnetite nanoparticles, and diatoms
synthesize siliceous materials. Secondary
metabolites of the plants such as geranial In
addition, reducing agent and stabilizing agent,
while no additional reagent (surfactant, template,
and capping agent) or treatment (heat and photo-
irradiation)is needed.
Bionanomaterials are synthesized by bio-
reductionmethodswhere reducingsugar, proteins,
enzymes and phenolic compounds are suggested
to cause the reduction. Synthesisprocess has been
controlled by pH, concentrations (reaction
mixture), source materials nature ect. After
synthesisthe materials are characterizedwith UV-
visible spectroscopy, X-ray diffraction (XRD)
technique, Energy-dispersiveX-ray spectroscopy
(EDS), X-ray photoelectron spectroscopy (XPS),
Fourier transforms infrared spectroscopy (FTlR),
Coupled plasma spectrometry (ICP),
Transmissions electron microscope (TEM),
Scanning electron microscopy (SEM), Atomic
force microscopy (AFM) ect. Though, metal
nanoparticleshave tremendousapplications in the
area of catalysis, sensor, optoelectronics,optically
functionalthin-filmcoatings,diagnosticbiological
probes and display devices, bionanopartic1esplay
a significantrole in the field of agriculture,.biology
and medicine.Removalof pollutantshas also been
carrying out using nanomaterials. Since the
increasinguse of bionanomaterialsmany fieldsand
in consumerproducts will increasetheir release to
the environment and any advancement in
nanotechnologywould thus require assessment of
environmentalrisks associatedwith these particles.
Keywords:Biogensis,metals, nanomaterials,
microbes, plants, animals, synthesis, application
Introduction
Nanopartic1es can be broadly grouped into
two kinds: nameiy organic and inorganic
nanoparticles. Organic nanoparticles may include
carbonnanopartic1es(ful!erenes)while some of the
inorganic nanoparticles may include magnetic

24

nanoparticles, noble metal nanoparticles and
semiconductornanoparticles(like titaniumdioxide
and zinc oxide). Noble metals particularly, silver
(Ag), platinum (Pt), gold (Au), cadmium sulfide
(CdS), ZnS, barium titanate (BT), In2O3Copper
and Palladium (Pd) have been routinely used for
the synthesisof metal nanoparticles. Traditionally
nanoparticleswere produced only by physical and
chemical methods (ion sputtering, solvothermal
synthesis, reduction and sol gel technique).
Basicallythereare two approachesfor nanopartic1e
synthesisnamely the Bottom up approach (BUA)
and the Top down approach (TUA). Former
approach is a process that builds towards larger
and more complex systems by starting at the
molecularleveland maintainingprecise control of
molecularstructure.Whereasin latter,scientiststry
to formulate nanoparticles using larger ones to
directtheir assembly.Biosynthesisof nanoparticles
is a kind of BUA where the main reaction
occurring is reduction/oxidation. The need for
biosynthesh.of nanoparticles rose as the physical
and chemical processes were costly. So in the
search of for cheaper pathways for nanoparticle
synthesis,scientistsused microorganismsand then
plant extracts for synthesis. Nature has devised
various processes for the synthesis of nano- and
micro- length scaled inorganic materials which
have contributedto the development of relatively
new and largelyunexploredarea of research based
on the biosynthesisof nanomaterials(Mohanpuria
et al., 2007; Sahayaraj and Rajesh, 2011).
Microbes,plants, animalcell, animalproducts
and animal by-products have been used for the
biogenesis of nanoparticles either intra- or
extracellular manner. For example, unicellular
organisms like magnetotactic bacteria produce
magnetite nanoparticles, and diatoms synthesize
siliceousmaterials.Moreover,microbesbelongsto
the Verticiliium, Fusarium, Aspergillus,
Pseudomonas, Streptomyces, Lactobacillus,
Corynebacterium, Aeromonas, Bacillus,
Desulfovibrio, Plectonema, Rhodopseudomonas,
Rhodopseudomonas capsulate, Schizosac-
25
NSNMRN 2012 J
charomyces etc. have been used for the synthesis
of microbes-based bionanoparticles. In addition,
either plants [eg:Aloe, Avena sativa,Azadirachta,
Capsicum, Boswellia ovalifoliolataBal & Henry,
Calotropis gigantean, Cinnammum, lemongrass
(Cymbopogon flexuosus), Diopyros kaki,
Eucalyptus, Pelargonium, Jatropha, Ipomoea,
Medicago, alfa alfa Magnoliakobus, Parthenium
hysterophorus; Mentha ,piperita, Sesbania
drummondii) or its bioactive compounds of the
plants (eg: geranial, citric acid, azadirachtin)have
been used for the biogensis of nanomaterials. In
addition, reducing agent and stabilizing agent,
while no additional reagent (surfactant, template,
and capping agent) or treatment (heat and photo-
irradiation) is needed. The aim of this manuscript
is.to highlight the various synthesis methods of
bionanomaterials and their utility value.
Biogenesis methodology
Biogensis is an environmentally benign
procedure, sustainable method, but needs cross-
disciplinary nanoscience research involving-
Chemists', Physicists, biologists and Engineers.
The three main steps in the preparation of
nanoparticles that shouid be evaluated from a
green chemistry perspective are: 1. the choice of
the solvent medium used for the synthesis, 2. the
choice of an environmentally benign reducing
agent and 3. the choice of a non toxic material for
the stabilization of the nanopartic1es.Most of the
synthetic methods reported to date rely heavily on
organic solvents. This is mainly due to the
hydrophobicity of the capping agents used
(Raveendranet aI., 2002). Li et ai. (2007) view of
synthesis of nanomateriais using bio-organismsis
compatiblewith the greenchemistryprinciples:the
bio-organism is: (i) eco-friendly as are, (ii) the
reducing agent employed and (Hi) the capping
agent in the reaction. Often chemical synthesis
methods lead to the presence of some toxic
chemical species adsorbed on the surfacethat may
have adverse effects in medical applications
(Parashar et aI., 2009). This is not an issue when
it comes to biosynthesized nanoparticles as they
NSNMRN 2012

are eco- friendly and biocompatible for finely cut and boiled with desiered quantity of
pharmaceuticalapplications.A distinct advantage sterile, distilled water.After boiling, the broth was
of the bottom-up approach is the enhanced cooled at room temperatureand filteredthrough
possibility of obtainingmetallic nanoparticleswith Whatman filter paper to obtain a clear, leaf broth.
comparatively lesser defects and more In a typical synthesis, 75 mL of 0.01 M AgNO3
homogeneouschemical composition(s). was addedto 10mL of leaf extractwith continuous
stirrIng, at 40°C or so. Within 30 min, a yellow
Biogensis
colorationappeared, indicatingthe onset of AgNps
Preparation of cell free microbial extract:
fonnation. The synthesis ofAgNps was monitored
Differentculturemediumwere prepared, sterilized
and inoculatedwith freshculture of the test strains. by UV-visible spectroscopy operating at the
The culturedflaskswere incubatedat 37°Cfor 24h wavelength of 280-700 nm at different time
intervals.
and at 30°C for 24 h for Candida albicans. The
temperature range and time of incubation is Preparations of metal solutions for biogenesis
differingfrom speciesto species.After incubation Pd(II) solutions: For the preparation of
time the cultures were centrifuged at 12000 rpm Pd(O)-coatedcells (bioPd), an aqueous Pd(II)
and their supernatants were used for further solution(2 mM, to pH 2.3 with 0.0I M HNO3)was
experiments. made by dissolving an appropriateamount of
Preparation of plant extract: Differentparts sodium tetrachloropalladate(NaldCIJ
of the plants were cut, thoroughly washed with Au(III) solutions: AqueousAu(III) solutions
water to removedebris,and oven dried at 90°Cfor (1 mM, to pH 2.3 with 0.01 M HNO3)were made
a week (differ from species to species), followed by dissolving hydrogen tetrachloroaurate
by grinding and sieving through a 100-mesh (HAuCI4'nH2O)in pre-acidifieddistilledwater. In
screen to achieve a homogeneous powder. A another methodology,trivalent gold in the form of
portionof the powderwas used to prepare extracts potassium tetrachloroaurate salt (KAuCI4) was
with metal-reducingcapabilities,and the rest was purchased,and a 3 mM stock solution in 0.01 mM
stored for later experiments. In order to prepare HCI was freshly prepared prior to the experiments
plant extract, 1 g of finely grinded and meshed and used in the gold nanoparticles biosynthesis.
plant powder (user can select any parts of the
Indium: lindium (III) acetylacetonatehaving
plant) was mixedwith 100mL of deionised water
99.99 % purity can be used as starting chemical
and heated at 90re%Con temperature controlled
material for In203. In the preparation of In203
water bath for 1h and cooled, passed through 0.2
nanoparticles,3 g of indium (III) acetylacetonate
m cellulosenitratemembranefilter paper. Freshly
was first dissolved in 30 ml plant extract solution
prepared aqueous extract was used immediately
under vigorous stir at 60°C (for example only) for
after filtration. No old extract was used for this
several hours until dried. Initially, the optical
study at any stage.Another method described was
absorption spectra were measured in the range of
1 mM silver nitrate was added to plant extract to
200-800 Dmusing a UV-VIS. The dried precursor
make up a final solution 200 ml and centrifuged
was crushed into powder using mortar and pestle
at 18.000 rpm for 25 min. The collected pellet and used for the characterization.
stored at -4°c.The supernatantwas heated at SOOe
to 95°C.A change in the color of solution was Ag NPs solution: 2 mL of 0.01 M Ag]SO4
observedduringthe heating process. Fresh leaves solution was added to 10 mL aqueous extract of
or any plant parts can also be used for the plant and mixed thoroughly by manual shaking.
biogenesis. Leaves or any part of the p1ant was The colourchangefromyellowto reddishbrown
washed thoroughly with sterile distilled water, indicatedthe formationof AgNPs.

26

Synthesis methodologyfor plant extracts
In a 250-mLErlenmeyer flask equipped with
a condenser and a magnetic stir bar, 15 g (user
desire) of plant powder was suspended in 75 mL
of Millipore water or 75 mL of isopropanol,
refluxed for 1 h under constant heating and
stirring. The extractionwas then set on cold water
bath,decanted in 50 mL centrifugetubes, and spun
down at 3000 rpm for 10 min in a centrifuge.The
deep green supernatant(not commonfor all plants)
was filtered through a medium pore Buchner
funnel with a fritted disk and used directly in the
biosynthesis of gold nanoparticles. The
biosynthesis was carried out in 5 mL centrifuge
tubes,at a 4: 1 ratio of 3 mM Au solution: plant
extracts (triplicate samples are mandatory), under
constantlymixing in a mechanicalrocker for 1-12
h. Initial pH was recorded at the beginning of the
reactions. The pH of the extracts was determined.
The water extracts used for this experiment had a
pH of 3.5 and the isopropyl alcohol extract had a
pH of 3.0. At the end of the reaction, the samples
were washed with ethanol for 3-15 min under
ultrasonicmotionfollowedby centrifugation,three
times at 40000 rpm.A drop of sample can be used
for SEM and TEM characterization.
Synthesis methodology for microbes
Silvernitrateat concentrationof 10-3M was
separatelyadded to the reactionvessels containing
different supernatants (1% v/v). The reaction
betweendifferentsupernatantsandAg+ ionswas
carried out in the dark or bright condition.
Periodically,aliquotsof the reaction
solution were removed and the absorptions
were measuredusing a UV-Vis spectrophotometer.
What is responsible for nanoparticles
synthesis?
C-S lyase, Phytochelatin synthase /
phytochelatins, Oxidoreuctases / quinines,
Chitosan,heparins,NADHdependantreductase;
P-d-glucose;Amino,carboxyls,andthiolmoieties
found in plant metabolitesand other organisms;
ionizedcarboxylgroupof aminoacidresiduesand
27
NSNMRN 2012
the amide of peptide chains; enzymes present in
the cell wall and on the cytoplasmicmembraneof
microorganism; terpenoids and polyphenols such
as flavonoids,flavonols,flavanones,anthocyanins,
and isoflavonesof plants in generaland terpenoids
in neem, apigenin and luteolin glycosides
(flavonoids) of alfalfa extracts and Curcain,
curacycline A, curacycline B are responsible for
biogensis of nanoparticles synthesis.
Characterization
UV-visible spectroscopy: The initial
characterisation of synthesised Ag nanocolloids
was carried out using UV-visible spectroscopy.
The reduction of silver ions was monitored from
400 to 1200 nm by lasco V-670 UV-Vis double
beam spectrophotometerafter 10-folddilutingthe
sample with deionised water against deionised
water as blank. The spectral data recorded were
then plotted using Origin 6.1.
X-ray diffraction: After mixing of extract
and Ag-salt solution the mixture was allowed to
complete conversion and Ag NPs (only example)
were collected after centrifugation followed by
filtration. The obtained purified Ag NPs were
subjected to X-ray diffraction anaiysis.
Fourier transformed infrared spectroscopy
(FTIR): Purified Ag NPs in the form of powder
were analysedusing FT-IRspectral measurements.
The measurements were carried out on a FT-IR
instrument in the diffuse reflectance mode at a
resolution of 4 cm"l in KBr pellets. For
comparison, plant or microbes samples were
pelletized and used as control.
Transmission electron microscope: The size
and morphology of the synthesised Ag NPs
(exampie only) was determined by High
Resolution Transmission Electron Microscope.
The sample for TEM studies was prepared as
follows. 1 mL of reaction mixture containing Ag
NPs was diluted to 10 mL, sonicated using
ultrasonic bath and a drop of i1was placed on Cu
grid with Ultrathin Cu on holey C film and
allowed to drYit
'" in vacuum.
!;'SNMRN 2012

Potentiometric study: Variation in hospitals to prevent or to minimize infection with

oxidation-reductionpotentialof Ag metal in broth pathogenicbacteria such as S. aureus (Jianrong et
during the conversion of Ag-salt to Ag J\rpswas aI., 2004). Trichoderma viride-based Ag has
studied with time usinga potentiometerwhich was synergistic effect with antibiotics (Fayaj et al.,
equipped with platinum electrode and saturated 2009)
calomel electrodes.
3. Pseudomonas aeruginosa (Duran et al.,
EDAX measurements: For EDAX analysis, 2007), E. coli (Mullen et al., 1989) and
the plant extracts reduced/ microbial reduced CitrobacterSF. Metal sulphide microcrystallites
metal nanoparticleswere dried and drop coated on were formulated by using S. pombe, which can'
to carbon film and performedon SEM instrument function as quantum semiconductor crystallite.
equipped with a Thermo EDAX attachments. These crystallites also have properties like optical
absorption, photosynthetic and electron transfer.
Drawback of microorganism and advantages
of botanicals Small interfering RNA (siRNA) delivery can be
monitored by a novel method based on nano
Microbiological methods generate device that combines unmodified siRNA with
nanoparticles at a much slower rate than that
semiconductorquantum dots (QDs) as multicolor
observedwhen plant extractsare used. This is one
biological probes.
of the major drawbacksof biological synthesis of
nanoparticles using microorganismsand must be 4. Silver bionanoparticles have also been
corrected if it must compete with other methods. used in non linear optics, spectrally selective
The advantageof using plants for the synthesis of coating for solar energy absorption, biolabelling
nanoparticlesis that they are easily and commonly and antibacterial activities.
available; most of them are having medicinal 5. Magnetosome particles isolated from
value, safe to handle and possess a broad , magnetotacticbacteria have been used as a carrier
variabilityof metabolitesthat may aid in reduction. for the immobilization of bioactive substances
Nanoparticles synthesized using marine algal such as enzymes, DNA, RNA and antibodies
seaweedwas quite stable in solution (Singaravelu (Mohanpuriaet aI., 2007).
et aI.. 2007).Moreover,the proposedmethod is an
advance of bioscience, high- yielding, low cost 6. Gold nanoparticles synthesized using E.
technologyand non-toxic to vertebrate animals. coli has been used for realizing the direct
electrochemistryof haemoglobin(Du et al., 2007).
Applications
Nanotechnology has a wide range of 7. Gold nanoparticles of barbated skullcap
applications in the fields of biology, medicine, extract have been modified to the glass electrode
and this has been used to enhance the electronic
optical,electrical,mechanical,optoelectronicsetc.
transmission rate between the electrode and p-
1. The ability of microorganismsto extract nitrophenol (Wang et al., 2009).
and/or accumulate metals is employed in
commercial biotechnological processes such as 8.. Ulva fasciata crude ethyl acetate'extract
bioleachingand bioremediation. based silvernanoparticles has been utilized for the
management of Xanthomonas campestris pv.
2. It has been shown that extracellularly Malvacearum,phytopathogenof cotton (Rajesh et
produced silver or gold nanopartic1es using al.,2012)
F oxysporum, can be incorporated in several kinds
ofmaterialssuchas cloths.Theseclothswithsilver 9. SHver nanoparticles (Ag NPs) were
nanopartic1esare sterile and can be useful in synthesizedby using aqueousleavesextractsof

28

Euphorbia prostrate was suggested for the
management of Sitophilus oryzae L. (AbduzZahir
et al., 2012)
10.bactericidal, wound healing and other
medical
II. Rapid developments are taking place in
the synthesis of metallic and bimetallic
nanomaterials and their surface modification for
References
Abduz Zahir, A, A. Bagavan, C. Kamaraj, G Elango, A. Abdul
Rahuman.2012. Efficacy ofplailt-mediated synthesizedsilver
nanopartic1es against Sitophilus oryzae. Journal of
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Acta Part A 73 : 374-381.
Du, L, H. Jiang, X. Liu, E. Wang, 2007 Biosynthesis of gold
nanopartic1es assisted by Escherichia coli DH5a and its
application on direct electrochemistry of haemoglobin.
Eleclrochemistry Communications. 9: 1165-1170 .
Duran, N., P. D. Marcato, S. De , 1. H. Gabriel, O. L. Alves, E.
Esposito, 2007 Antibacterial effect of silver nanoparticles
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.'
NSNMRN 2012
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Conclusion
Biogenic synthesis of nanoparticles has
opened its doors to a world of nanoparticles with
easy preparationprotocols,lesstoxicity and a wide
range of applications according to their size and
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