Current applications of different type of aqueous two-phase systems

Abstract

In recent year, aqueous two-phase system (ATPS) has become a proven tool used in separation and purification technology. The application of ATPSs in clarification, partitioning and partial purification of biomolecules and bioproducts had showed the rapid development. This method is able to give high recovery yield and high purity in a single step. The ATPS shows characteristics of high selectivity and is easily to scale up. Therefore, ATPS offers an attractive alternative that meets the requirements of the high demand in industry processes and it is also beneficial in terms of economic and environmental protection. In the past, a lot of works and researches have been done in order to develop feasible separation processes using different types of ATPSs and their applications in numerous product separations. This paper aims to review on the recent literature works in the development of different type of ATPSs and their applications in novel separations and purifications of biomaterials.

Keywords

Aqueous two-phase system / Bioproducts recovery / Partitioning / Purification / Recovery yield

References

[1] Aksu Z, Tezer S. Biosorption of reactive dyes on the green alga Chlorella vulgaris. Process Biochem, 2005, 40(3): 1347-1361.

[2] Albertsson PA (1987) Partition of cell particles and macromolecules, Wiley-Interscience, New York, 346 pp. Analytical Biochemistry 161 (1):227

[3] Amid M, Shuhaimi M, Islam Sarker MZ, Abdul Manap MY. Purification of serine protease from mango (Mangifera Indica Cv. Chokanan) peel using an alcohol/salt aqueous two phase system. Food Chem, 2012, 132(3): 1382-1386.

[4] Anderson JL, Ding J, Welton T, Armstrong DW. Characterizing ionic liquids on the basis of multiple solvation interactions. JACS, 2002, 124(47): 14247-14254.

[5] Antov M, Anderson L, Andersson A, Tjerneld F, Stålbrand H. Affinity partitioning of a Cellulomonas fimi β-mannanase with a mannan-binding module in galactomannan/starch aqueous two-phase system. J Chromatogr A, 2006, 1123(1): 53-59.

[6] Ariff A, Anuar MS, Chen SK, Ling TC, Show PL, Tan CP. Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two phase systems. Bioresour Technol, 2011, 10(1): 10-16.

[7] Asenjo JA, Andrews BA. Aqueous two-phase systems for protein separation: a perspective. J Chromatogr A, 2011, 1218(49): 8826-8835.

[8] Azevedo AM, Rosa PAJ, Ferreira IF, de Vries J, Visser TJ, Aires-Barros MR. Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography. J Chromatogr B, 2009, 877(1–2): 50-58.

[9] Azevedo AM, Rosa PAJ, Ferreira IF, Aires-Barros MR. Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol, 2009, 27(4): 240-247.

[10] Azevedo AM, Rosa PAJ, Ferreira IF, Pisco AMMO, de Vries J, Korporaal R, Visser TJ, Aires-Barros MR. Affinity-enhanced purification of human antibodies by aqueous two-phase extraction. Sep Purif Technol, 2009, 65(1): 31-39.

[11] Bellot G, McClintock MA, Lin C, Shih WM. Recovery of intact DNA nanostructures after agarose gel-based separation. Nat Methods, 2011, 8(3): 192-194.

[12] Benavides J, Rito-Palomares M. Practical experiences from the development of aqueous two-phase processes for the recovery of high value biological products. J Chem Technol Biotechnol, 2008, 83(2): 133-142.

[13] Berggren K, Johansson HO, Yjerneld F. Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers. J Chromatogr A, 1995, 718(1): 67-79.

[14] Bhambure R, Sharma R, Gupta D, Rathore AS. A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products. J Chromatogr A, 2013, 1307: 49-57.

[15] Bolognese B, Nerli B, Pico G. Application of the aqueous two-phase systems of ethylene and propylene oxide copolymer-maltodextrin for protein purification. J Chromatogr B Analyt Technol Biomed Life Sci, 2005, 814(2): 347-353.

[16] Carda-Broch S, Berthod A, Armstrong DW. Solvent properties of the 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid. Anal Bioanal Chem, 2003, 375(2): 191-199.

[17] Chen CW, Thomas CA. Recovery of DNA segments from agarose gels. Anal Biochem, 1980, 101(2): 339-341.

[18] Chen J, Ma GX, Li DQ. HPCPC separation of proteins using polyethylene glycol-potassium phosphate aqueous two-phase. Prep Biochem Biotechnol, 1999, 29(4): 371-383.

[19] Chen J, Spear SK, Huddleston JG, Rogers RD. Polyethylene glycol and solutions of polyethylene glycol as green reaction media. Green Chem, 2005, 7(2): 64-82.

[20] Chen B, Han J, Wang Y, Sheng C, Liu Y, Zhang G, Yan Y. Separation, enrichment and determination of ciprofloxacin using thermoseparating polymer aqueous two-phase system combined with high performance liquid chromatography in milk, egg, and shrimp samples. Food Chem, 2014, 148: 105-111.

[21] Chow YH, Yap YJ, Anuar MS, Tejo BA, Ariff A, Show PL, Ng E-P, Ling TC. Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system. J Chromatogr B, 2013, 934: 71-78.

[22] Chow YH, Yap YJ, Tan CP, Anuar MS, Tejo BA, Show PL, Ariff AB, Ng E-P, Ling TC. Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems. J Biosci Bioeng, 2015, 120(1): 85-90.

[23] Cordes A, Flossdorf J, Kula MR. Affinity partitioning: development of mathematical model describing behavior of biomolecules in aqueous two-phase systems. Biotechnol Bioeng, 1987, 30(4): 514-520.

[24] da Silva LHM, da Silva MdCH, Júnior JA, Martins JP, Reis Coimbra JSd, Minim LA. Hydrophobic effect on the partitioning of [Fe(CN)₅(NO)]²⁻ and [Fe(CN)₆]³⁻ anions in aqueous two-phase systems formed by triblock copolymers and phosphate salts. Sep Purif Technol, 2008, 60(1): 103-112.

[25] Daugulis AJ. Integrated reaction and product recovery in bioreactor systems. Biotechnol Progr, 1988, 4(3): 113-122.

[26] de Brito Cardoso G, Mourão T, Pereira FM, Freire MG, Fricks AT, Soares CMF, Lima ÁS. Aqueous two-phase systems based on acetonitrile and carbohydrates and their application to the extraction of vanillin. Sep Purif Technol, 2013, 104: 106-113.

[27] de Lemos LR, Campos RA, Rodrigues GD, da Silva LHM, da Silva MCH. Green separation of copper and zinc using triblock copolymer aqueous two-phase systems. Sep Purif Technol, 2013, 115: 107-113.

[28] Dembczynski R, Bialas W, Jankowski T. Recycling of phase components during lysozyme extraction from hen egg white in the EO50PO50/K₂HPO₄ aqueous two-phase system. Biochem Eng J, 2010, 51(1–2): 24-31.

[29] Ding J, Welton T, Armstrong DW. Chiral ionic liquids as stationary phases in gas chromatography. Anal Chem, 2004, 76(22): 6819-6822.

[30] Duarte SP, Fortes AG, Prazeres DMF, Marcos JC. Preparation of plasmid DNA polyplexes from alkaline lysates by a two-step aqueous two-phase extraction process. J Chromatogr A, 2007, 1164(1–2): 105-112.

[31] Ferreira AM, Coutinho JAP, Fernandes AM, Freire MG. Complete removal of textile dyes from aqueous media using ionic-liquid-based aqueous two-phase systems. Sep Purif Technol, 2014, 128: 58-66.

[32] Flanagan SD, Barondes SH. Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems. J Biol Chem, 1975, 250(4): 1484-1489.

[33] Freeman A, Woodley JM, Lilly MD. In situ product removal as a tool for bioprocessing. Nat Biotechnol, 1993, 11(9): 1007-1012.

[34] Freire MG, Louros CLS, Rebelo LPN, Coutinho JAP. Aqueous biphasic systems composed of a water-stable ionic liquid + carbohydrates and their applications. Green Chem, 2011, 13(6): 1536-1545.

[35] Fukushima T, Kosaka A, Ishimura Y, Yamamoto T, Takigawa T, Ishii N, Aida T. Molecular ordering of organic molten salts triggered by single-walled carbon nanotubes. Science, 2003, 300(5628): 2072-2074.

[36] Guan Y, Lilley TH, Treffry TE, Zhou C-L, Wilkinson PB. Use of aqueous two-phase systems in the purification of human interferon-α1 from recombinant Escherichia coli. Enzyme Microb Technol, 1996, 19(6): 446-455.

[37] Hayenga KJ, Valex PP (2002) Methodes de purification de proteines par extraction biphasee aqueuse. Google Patents. EP1194445 A1, EP20000928866, 10 Apr 2002

[38] He C, Li S, Liu H, Li K, Liu F. Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt. J Chromatogr A, 2005, 1082(2): 143-149.

[39] Helfrich MR, El-Kouedi M, Etherton MR, Keating CD. Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems. Langmuir, 2005, 21(18): 8478-8486.

[40] Hessel C, Allegre C, Maisseu M, Charbit F, Moulin P. Guidelines and legislation for dye house effluents. J Environ Manage, 2007, 83(2): 171-180.

[41] Hinze WL, Pramauro E. A critical review of surfactant-mediated phase separations (cloud-point extractions): theory and applications. Crit Rev Anal Chem, 1993, 24(2): 133-177.

[42] J-h Ma, Guo C, Y-l Tang, Wang J, Zheng L, X-f Liang, Chen S, H-z Liu. Salt-induced micellization of a triblock copolymer in aqueous solution: a 1H nuclear magnetic resonance spectroscopy study. Langmuir, 2007, 23(6): 3075-3083.

[43] Jiang Y, Xia H, Yu J, Guo C, Liu H. Hydrophobic ionic liquids-assisted polymer recovery during penicillin extraction in aqueous two-phase system. Chem Eng J, 2009, 147(1): 22-26.

[44] Johansson HO, Karlström G, Tjerneld F. Temperature-induced phase partitioning of peptides in water solutions of ethylene oxide and propylene oxide random copolymers. Biochim Biophys Acta, 1997, 1335(3): 315-325.

[45] Johansson HO, Persson J, Tjerneld F. Thermoseparating water/polymer system: a novel one-polymer aqueous two- phase system for protein purification. Biotechnol Bioeng, 1999, 66(4): 247-257.

[46] Jönsson M, Johansson H-O. Protein partitioning in thermoseparating systems of a charged hydrophobically modified ethylene oxide polymer. J Chromatogr A, 2003, 983(1–2): 133-144.

[47] Kang KH, Kim HU, Lim KH. Effect of temperature on critical micelle concentration and thermodynamic potentials of micellization of anionic ammonium dodecyl sulfate and cationic octadecyl trimethyl ammonium chloride. Colloids Surf Physicochem Eng Aspects, 2001, 189(1): 113-121.

[48] Khan A, Marques EF. Synergism and polymorphism in mixed surfactant systems. Curr Opin Colloid Interface Sci, 1999, 4(6): 402-410.

[49] Kiran I, Akar T, Ozcan AS, Ozcan A, Tunali S. Biosorption kinetics and isotherm studies of acid red 57 by dried Cephalosporium aphidicola cells from aqueous solutions. Biochem Eng J, 2006, 31(3): 197-203.

[50] Kornmann H, Baer G (2008) Aqueous two-phase partitioning via polyethylene glycol phase and (Na₂)SO₄. Google Patents. US7439336 B2, US 11/630,845, 21 Oct 2008. http://www.google.com/patents/US7439336

[51] Lan JC-W, Yeh C-Y, Wang C-C, Yang Y-H, Wu H-S. Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system. J Biosci Bioeng, 2013, 116(4): 499-505.

[52] Lee SY, Khoiroh I, Ling TC, Show PL. Aqueous two-phase flotation for the recovery of biomolecules. Separation and purification reviews: null-null, 2015

[53] Lin YK, Ooi CW, Tan JS, Show PL, Ariff A, Ling TC. Recovery of human interferon α-2b from recombinant Escherichia coli using alcohol/salt-based aqueous two-phase systems. Sep Purif Technol, 2013, 120: 362-366.

[54] Lin YK, Show PL, Yap YJ, Tan CP, Ng E-P, Ariff AB, Mohamad Annuar MSB, Ling TC. Direct recovery of cyclodextringlycosyltransferase from Bacillus cereus using aqueous two-phase flotation. J Biosci Bioeng, 2015, 120(6): 684-689.

[55] Ling YQ, Nie HL, Su SN, Branford-White C, Zhu LM. Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology. Sep Purif Technol, 2010, 73(3): 343-348.

[56] Liu JF, Jiang GB, Chi YG, Cai YQ, Zhou QX, Hu JT. Use of ionic liquids for liquid-phase microextraction of polycyclic aromatic hydrocarbons. Anal Chem, 2003, 75(21): 5870-5876.

[57] Luechau F, Ling TC, Lyddiatt A. Partition of plasmid DNA in polymer–salt aqueous two-phase systems. Sep Purif Technol, 2009, 66(2): 397-404.

[58] Luechau F, Ling TC, Lyddiatt A. Selective partition of plasmid DNA and RNA in aqueous two-phase systems by the addition of neutral salt. Sep Purif Technol, 2009, 68(1): 114-118.

[59] Mank M, Stahl B, Boehm G. 2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules. Anal Chem, 2004, 76(10): 2938-2950.

[60] Matos T, Johansson HO, Queiroz JA, Bulow L. Isolation of PCR DNA fragments using aqueous two-phase systems. Sep Purif Technol, 2014, 122: 144-148.

[61] Mohamed Ali S, Ling TC, Muniandy S, Tan YS, Raman J, Sabaratnam V. Recovery and partial purification of fibrinolytic enzymes of Auricularia polytricha (Mont.) Sacc by an aqueous two-phase system. Sep Purif Technol, 2014, 122: 359-366.

[62] Ng HS, Tan CP, Mokhtar MN, Ibrahim S, Ariff A, Ooi CW, Ling TC. Recovery of Bacillus cereus cyclodextrin glycosyltransferase and recycling of phase components in an aqueous two-phase system using thermo-separating polymer. Sep Purif Technol, 2012, 89: 9-15.

[63] Ng HS, Ooi CW, Mokhtar MN, Show PL, Ariff A, Tan JS, Ng E-P, Ling TC. Extractive bioconversion of cyclodextrins by Bacillus cereus cyclodextrin glycosyltransferase in aqueous two-phase system. Bioresour Technol, 2013, 142: 723-726.

[64] Pei Y, Wang J, Wu K, Xuan X, Lu X. Ionic liquid-based aqueous two-phase extraction of selected proteins. Sep Purif Technol, 2009, 64(3): 288-295.

[65] Pereira M, Wu Y-T, Venâncio A, Teixeira J. Aqueous two-phase extraction using thermoseparating polymer: a new system for the separation of endo-polygalacturonase. Biochem Eng J, 2003, 15(2): 131-138.

[66] Rito-Palomares M. Practical application of aqueous two-phase partition to process development for the recovery of biological products. J Chromatogr B, 2004, 807(1): 3-11.

[67] Robinson T, McMullan G, Marchant R, Nigam P. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol, 2001, 77(3): 247-255.

[68] Rodrigues GD, da Silva MdCH, da Silva LHM, Paggioli FJ, Minim LA, Reis Coimbra JSd. Liquid–liquid extraction of metal ions without use of organic solvent. Sep Purif Technol, 2008, 62(3): 687-693.

[69] Rogers RD, Bauer CB. Partitioning behavior of group 1 and 2 cations in poly(ethylene glycol)-based aqueous biphasic systems. J Chromatogr B Biomed Sci Appl, 1996, 680(1–2): 237-241.

[70] Rogers RD, Zhang J. Effects of increasing polymer hydrophobicity on distribution ratios of TcO₄₋ in polyethylene/poly(propylene glycol)-based aqueous biphasic systems. J Chromatogr B Biomed Appl, 1996, 680(1–2): 231-236.

[71] Rogers RD, Bond AH, Bauer CB. Metal ion separations in polyethylene glycol-based aqueous biphasic systems. Sep Sci Technol, 1993, 28(5): 1091-1126.

[72] Rosa PAJ, Azevedo AM, Ferreira IF, de Vries J, Korporaal R, Verhoef HJ, Visser TJ, Aires-Barros MR. Affinity partitioning of human antibodies in aqueous two-phase systems. J Chromatogr A, 2007, 1162(1): 103-113.

[73] Rosa PAJ, Ferreira IF, Azevedo AM, Aires-Barros MR. Aqueous two-phase systems: a viable platform in the manufacturing of biopharmaceuticals. J Chromatogr A, 2010, 1217(16): 2296-2305.

[74] Ruiz-Ruiz F, Benavides J, Aguilar O, Rito-Palomares M. Aqueous two-phase affinity partitioning systems: current applications and trends. J Chromatogr A, 2012, 1244: 1-13.

[75] Sanchez-Ferrer A, Bru R, Garcia-Carmona F. Phase separation of biomolecules in polyoxyethylene glycol nonionic detergents. Crit Rev Biochem Mol Biol, 1994, 29(4): 275-313.

[76] Selber K, Tjerneld F, Collén A, Hyytiä T, Nakari-Setälä T, Bailey M, Fagerström R, Kan J, van der Laan J, Penttilä M, Kula M-R. Large-scale separation and production of engineered proteins, designed for facilitated recovery in detergent-based aqueous two-phase extraction systems. Process Biochem, 2004, 39(7): 889-896.

[77] Show PL, Tan CP, Anuar MS, Ariff A, Yusof YA, Chen SK, Ling TC. Direct recovery of lipase derived from Burkholderia cepacia in recycling aqueous two-phase flotation. Sep Purif Technol, 2011, 80(3): 577-584.

[78] Show PL, Tan CP, Anuar MS, Ariff A, Yusof YA, Chen SK, Ling TC. Primary recovery of lipase derived from Burkholderia cenocepacia strain ST8 and recycling of phase components in an aqueous two-phase system. Biochem Eng J, 2012, 60: 74-80.

[79] Show PL, Tan CP, Shamsul Anuar M, Ariff A, Yusof YA, Chen SK, Ling TC. Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two-phase systems. Bioresour Technol, 2012, 116: 226-233.

[80] Show PL, Ooi CW, Anuar MS, Ariff A, Yusof YA, Chen SK, Annuar MSM, Ling TC. Recovery of lipase derived from Burkholderia cenocepacia ST8 using sustainable aqueous two-phase flotation composed of recycling hydrophilic organic solvent and inorganic salt. Sep Purif Technol, 2013, 110: 112-118.

[81] Silva MdCHd, Silva LHMd, Paggioli FJ, Coimbra JSR, Minim LA. Sistema aquoso bifásico: uma alternativa eficiente para extração de íons. Quim Nova, 2006, 29: 1332-1339.

[82] Silva LH, Silva MC, Aquino RA, Francisco KR, Cardoso MV, Minim LA, Coimbra JS. Nitroprusside-PEO enthalpic interaction as a driving force for partitioning of the [Fe(CN)(5)NO](2−) anion in aqueous two-phase systems formed by poly(ethylene oxide) and sulfate salts. J Phys Chem B, 2006, 110(46): 23540-23546.

[83] Souza RL, Lima RA, Coutinho JAP, Soares CMF, Lima ÁS. Aqueous two-phase systems based on cholinium salts and tetrahydrofuran and their use for lipase purification. Sep Purif Technol, 2015, 155: 118-126.

[84] Takamuku T, Tabata M, Yamaguchi A, Nishimoto J, Kumamoto M, Wakita H, Yamaguchi T. Liquid structure of acetonitrile-water mixtures by x-ray diffraction and infrared spectroscopy. J Phys Chem B, 1998, 102(44): 8880-8888.

[85] Tan CH, Show PL, Ooi CW, Ng E-P, Lan JC-W, Ling TC. Novel lipase purification methods–a review of the latest developments. Biotechnol J, 2015, 10(1): 31-44.

[86] Tang MS, Whitcher TJ, Yeoh KH, Chua CL, Woon KL, Show PL, Lin YK, Ling TC. The removal of metallic single-walled carbon nanotubes using an aqueous two-phase system. J Nanosci Nanotechnol, 2014, 14(5): 3398-3402.

[87] Tang MSY, Show PL, Lin YK, Woon KL, Tan CP, Ling TC. Separation of single-walled carbon nanotubes using aqueous two-phase system. Sep Purif Technol, 2014, 125: 136-141.

[88] Teng SF, Sproule K, Hussain A, Lowe CR. A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand. J Mol Recognit, 1999, 12(1): 67-75.

[89] Tianwei T, Qing H, Qiang L. Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system. Biotechnol Lett, 2002, 24(17): 1417-1420.

[90] Tunnicliffe RB, Bolam DN, Pell G, Gilbert HJ, Williamson MP. Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding. J Mol Biol, 2005, 347(2): 287-296.

[91] Vaher M, Koel M. Specific background electrolytes for nonaqueous capillary electrophoresis. J Chromatogr A, 2005, 11(1): 83-88.

[92] Wang B, Ezejias T, Feng H, Blaschek H. Sugaring-out: a novel phase separation and extraction system. Chem Eng Sci, 2008, 63(9): 2595-2600.

[93] Welton T. Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chem Rev, 1999, 99(8): 2071-2084.

[94] Weschayanwiwat P, Kunanupap O, Scamehorn JF. Benzene removal from waste water using aqueous surfactant two-phase extraction with cationic and anionic surfactant mixtures. Chemosphere, 2008, 72(7): 1043-1048.

[95] Willauer HD, Huddleston JG, Rogers RD. Solute partitioning in aqueous biphasic systems composed of polyethylene glycol and salt: the partitioning of small neutral organic species. Ind Eng Chem Res, 2002, 41(7): 1892-1904.

[96] Wu B, Zhang Y, Wang H. Phase behavior for ternary systems composed of ionic liquid + saccharides + water. J Phys Chem B, 2008, 112(20): 6426-6429.

[97] Walter H (1986) Partitioning in aqueous two–phase system: theory, methods, uses Elsevier science and applications to biotechnology

[98] Yin H, Mao M, Huang J, Fu H. Two-phase region in the DTAB/SL mixed surfactant system. Langmuir, 2002, 18(24): 9198-9203.

[99] Zhang W, Zhu D, Fan H, Liu X, Wan Q, Wu X, Liu P, Tang JZ. Simultaneous extraction and purification of alkaloids from Sophora flavescens Ait. by microwave-assisted aqueous two-phase extraction with ethanol/ammonia sulfate system. Sep Purif Technol, 2015, 141: 113-123.

[100] Zhi W, Deng Q. Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography. J Chromatogr A, 2006, 1116(1–2): 149-152.

[101] Zijlstra GM, Gooijer CD, Tramper J. Extractive bioconversions in aqueous two-phase systems. Curr Opin Biotechnol, 1998, 9(2): 171-176.

Funding

Ministry of Science, Technology and Innovation, Malaysia(02-02-12-SF0256)

Fundamental Research Grant Scheme(FRGS/ 6901/2013/SG05/UNIM/02/1)