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;
[email protected]
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 photoirradiation)is needed. 24
Bionanomaterials are synthesized by bioreductionmethodswhere 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 UVvisible 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
NSNMRN 2012 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, Schizosac25
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 photoirradiation) 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 crossdisciplinary nanoscience research involvingChemists', 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
J
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 was addedto 10mL of leaf extractwith continuous homogeneouschemical composition(s).
Biogensis Preparation of cell free microbial extract: Differentculturemediumwere prepared, sterilized and inoculatedwith freshculture of the test strains. The culturedflaskswere incubatedat 37°Cfor 24h and at 30°C for 24 h for Candida albicans. The temperature range and time of incubation is differingfrom speciesto species.After incubation time the cultures were centrifuged at 12000 rpm and their supernatants were used for further experiments. Preparation of plant extract: Differentparts of the plants were cut, thoroughly washed with water to removedebris,and oven dried at 90°Cfor a week (differ from species to species), followed by grinding and sieving through a 100-mesh screen to achieve a homogeneous powder. A portionof the powderwas used to prepare extracts with metal-reducingcapabilities,and the rest was stored for later experiments. In order to prepare plant extract, 1 g of finely grinded and meshed plant powder (user can select any parts of the plant) was mixedwith 100mL of deionised water and heated at 90re%Con temperature controlled water bath for 1h and cooled, passed through 0.2 m cellulosenitratemembranefilter paper. Freshly prepared aqueous extract was used immediately after filtration. No old extract was used for this study at any stage.Another method described was 1 mM silver nitrate was added to plant extract to make up a final solution 200 ml and centrifuged at 18.000 rpm for 25 min. The collected pellet stored at -4°c.The supernatantwas heated at SOOe to 95°C.A change in the color of solution was observedduringthe heating process. Fresh leaves or any plant parts can also be used for the biogenesis. Leaves or any part of the p1ant was washed thoroughly with sterile distilled water, 26
stirrIng, at 40°C or so. Within 30 min, a yellow colorationappeared, indicatingthe onset of AgNps fonnation. The synthesis ofAgNps was monitored by UV-visible spectroscopy operating at the wavelength of 280-700 nm at different time intervals. Preparations of metal solutions for biogenesis Pd(II) solutions: For the preparation of Pd(O)-coatedcells (bioPd), an aqueous Pd(II) solution(2 mM, to pH 2.3 with 0.0I M HNO3)was made by dissolving an appropriateamount of sodium tetrachloropalladate(NaldCIJ Au(III) solutions: AqueousAu(III) solutions (1 mM, to pH 2.3 with 0.01 M HNO3)were made by dissolving hydrogen tetrachloroaurate (HAuCI4'nH2O)in pre-acidifieddistilledwater. In another methodology,trivalent gold in the form of potassium tetrachloroaurate salt (KAuCI4) was purchased,and a 3 mM stock solution in 0.01 mM HCI was freshly prepared prior to the experiments and used in the gold nanoparticles biosynthesis. Indium: lindium (III) acetylacetonatehaving 99.99 % purity can be used as starting chemical material for In203. In the preparation of In203 nanoparticles,3 g of indium (III) acetylacetonate was first dissolved in 30 ml plant extract solution under vigorous stir at 60°C (for example only) for several hours until dried. Initially, the optical absorption spectra were measured in the range of 200-800 Dmusing a UV-VIS. The dried precursor was crushed into powder using mortar and pestle and used for the characterization. Ag NPs solution: 2 mL of 0.01 M Ag]SO4 solution was added to 10 mL aqueous extract of plant and mixed thoroughly by manual shaking. The colourchangefromyellowto reddishbrown indicatedthe formationof AgNPs.
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.
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.
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.
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
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
oxidation-reductionpotentialof Ag metal in broth during the conversion of Ag-salt to Ag J\rpswas studied with time usinga potentiometerwhich was equipped with platinum electrode and saturated calomel electrodes. EDAX measurements: For EDAX analysis, the plant extracts reduced/ microbial reduced metal nanoparticleswere dried and drop coated on to carbon film and performedon SEM instrument equipped with a Thermo EDAX attachments.
hospitals to prevent or to minimize infection with pathogenicbacteria such as S. aureus (Jianrong et aI., 2004). Trichoderma viride-based Ag has synergistic effect with antibiotics (Fayaj et al., 2009) 3. Pseudomonas aeruginosa (Duran et al., 2007), E. coli (Mullen et al., 1989) and CitrobacterSF. Metal sulphide microcrystallites were formulated by using S. pombe, which can' function as quantum semiconductor crystallite. These crystallites also have properties like optical absorption, photosynthetic and electron transfer. Small interfering RNA (siRNA) delivery can be monitored by a novel method based on nano device that combines unmodified siRNA with semiconductorquantum dots (QDs) as multicolor biological probes.
Drawback of microorganism and advantages of botanicals Microbiological methods generate nanoparticles at a much slower rate than that observedwhen plant extractsare used. This is one of the major drawbacksof biological synthesis of 4. Silver bionanoparticles have also been nanoparticles using microorganismsand must be used in non linear optics, spectrally selective corrected if it must compete with other methods. 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 6. Gold nanoparticles synthesized using E. advance of bioscience, high- yielding, low cost coli has been used for realizing the direct technologyand non-toxic to vertebrate animals. electrochemistryof haemoglobin(Du et al., 2007). Applications 7. Gold nanoparticles of barbated skullcap Nanotechnology has a wide range of extract have been modified to the glass electrode applications in the fields of biology, medicine, and this has been used to enhance the electronic optical,electrical,mechanical,optoelectronicsetc. transmission rate between the electrode and p1. The ability of microorganismsto extract nitrophenol (Wang et al., 2009). and/or accumulate metals is employed in 8.. Ulva fasciata crude ethyl acetate'extract commercial biotechnological processes such as based silvernanoparticles has been utilized for the bioleachingand bioremediation. 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
9. SHver nanoparticles (Ag NPs) were ofmaterialssuchas cloths.Theseclothswithsilver synthesizedby using aqueousleavesextractsof nanopartic1esare sterile and can be useful in
28
NSNMRN 2012
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 13iopestesticides, 5 (Supplementary):
95
- 102.
Daizy Philip, 2009. Biosynthesis of Au, Ag and Au-Ag nanoparticles using edible mushroom extract Spectrochimica 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 produt;ed by fungal process on textile fabrics and their effluenttreatment. Journal of Biomedical Nanotechnolngy 3: 203-208.
Duran, N., P.D.Marcato, S.De, I.H.Gabriel, O.L. Alves, E.Esposito, 2007. Antibacterial effect of silver nanopartic1es produced by fungal process on textile fabrics and their effluent treatment Journbal ofBiomed Nanotechnol, 3:203. Jianrong, c., M.Yuqing, H.Nongyue,w.Xiaohua, LSijiao,2004. Biotechnol Adv,2004,22,505. Li, S., Y Shen, A Xie, X. Yu,L. Qui, L. Zhang, Q. Zhang, 2007 Green synthesis of silver nanoparticles using Capsicum annum L. extract. Green Chemistry. 9: 852-858 .
biosensing and electronic applications (Daizy Philip, 2009). 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 shape.
Mullen, M.D., D.C. Wolf, F.G.Ferris, T.1.Beveridge, C.A. Flemming, G.W. Bailey, Appl Environ Microbiol, 1989, 55,3143. Rajesh, S., D. Patrie Rajal, 1.M. Rathi and K. Sahayaraj. 2012. Biosynthesis of Ag nanoparticles using Ulvafasciata (Delile) ethyl acetate extract and its activity against Xanthomonas campestris pv. Malvacearum. Journal of Biopesticides 5 (suppl): 119-128. Raveendran, P., 1. Fu and S.L Wallen,2003. Completely 'Green' synthesis and stabilization of metal nanoparticles. 1. Am. Chern. Soc., 125: 13940-13941. Parashar, U. K., S. P. Saxena, A Srivastava, 2009 Bioinspired synthesis of silver nanoparticles. DigestJjournal of Nanomaterials and Biostructures 4(1); 159 166 . Sahayaraj,K. and Rajesh, S. 20II. Bionanoparticles:synthesisand antimicrobial applications. In: Science against microbial pathogens: communicating current research and technological advances (Antonio Mendez-Vilas ed.), FormatexResearchCenter,Spain(VolumeI ISBN(13):97884-939843-1-1),pp. 228-244. Singaravelu, G., 1. S. Arockiamary,V.G.Kumar, K. Govindaraju, 2007 A novel extracellular synthesis of monodisperse gold nanoparticles using marine alga, Sargassum wightii Greville. ColloidsandsurfacesB: Biointerfaces 57: 97-101 . Wang, Y, X. He, K. Wang, X. Zhang, W. Tan, 2009 Barbated Skullcap herb extract- mediated biosynthesis of gold nanoparticles and its primary application in electrochemistry. Colloids and Surfaces B: Biointerfaces, 73(1): 75-9.
.'
