Chemical Grafting of Poly(ethylene glycol) Methyl Ether Methacrylate ontoPolymer Surfaces by Atmospheric Pressure Plasma Processing

R D'Sa, BJ Meenan

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

This article reports the use of atmospheric pressure plasma processing to induce chemical grafting of poly(ethylene glycol) methyl ether methacrylate (PEGMA) onto polystyrene (PS) and poly(methyl methacrylate) (PMMA) surfaces with the aim of attaining an adlayer conformation which is resistant to protein adsorption. The plasma treatment was carried out using a dielectric barrier discharge (DBD) reactor with PEGMA of molecular weights (MW) 1000 and 2000, PEGMA1000 and PEGMA2000, being grafted in a two step procedure: (1) reactive groups are generated on the polymer surface followed by (2) radical addition reactions with the PEGMA. The surface chemistry, coherency, and topography of the resulting PEGMA grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM), respectively. The most coherently grafted PEGMA layers were observed for the 2000 MW PEGMA macromolecule, DBD processed at an energy dose of 105.0 J/cm2 as indicated by ToF-SIMS images. The effect of the chemisorbed PEGMA layer on protein adsorption was assessed by evaluating the surface response to bovine serum albumin (BSA) using XPS. BSA was used as a model protein to determine the grafted macromolecular conformation of the PEGMA layer. Whereas the PEGMA1000 surfaces showed some protein adsorption, the PEGMA2000 surfaces appeared to absorb no measurable amount of protein, confirming the optimum surface conformation for a nonfouling surface.
LanguageEnglish
Pages1894-1903
JournalLangmuir
Volume26
Issue number3
DOIs
Publication statusPublished - 2010

Fingerprint

Methyl Ethers
Plasma applications
Methacrylates
Polyethylene glycols
Atmospheric pressure
glycols
Ethers
ethers
atmospheric pressure
ethylene
Proteins
proteins
Conformations
Bovine Serum Albumin
Secondary ion mass spectrometry
Adsorption
albumins
serums
secondary ion mass spectrometry
adsorption

Cite this

@article{504a13b659754e81a9bda0744d415227,
title = "Chemical Grafting of Poly(ethylene glycol) Methyl Ether Methacrylate ontoPolymer Surfaces by Atmospheric Pressure Plasma Processing",
abstract = "This article reports the use of atmospheric pressure plasma processing to induce chemical grafting of poly(ethylene glycol) methyl ether methacrylate (PEGMA) onto polystyrene (PS) and poly(methyl methacrylate) (PMMA) surfaces with the aim of attaining an adlayer conformation which is resistant to protein adsorption. The plasma treatment was carried out using a dielectric barrier discharge (DBD) reactor with PEGMA of molecular weights (MW) 1000 and 2000, PEGMA1000 and PEGMA2000, being grafted in a two step procedure: (1) reactive groups are generated on the polymer surface followed by (2) radical addition reactions with the PEGMA. The surface chemistry, coherency, and topography of the resulting PEGMA grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM), respectively. The most coherently grafted PEGMA layers were observed for the 2000 MW PEGMA macromolecule, DBD processed at an energy dose of 105.0 J/cm2 as indicated by ToF-SIMS images. The effect of the chemisorbed PEGMA layer on protein adsorption was assessed by evaluating the surface response to bovine serum albumin (BSA) using XPS. BSA was used as a model protein to determine the grafted macromolecular conformation of the PEGMA layer. Whereas the PEGMA1000 surfaces showed some protein adsorption, the PEGMA2000 surfaces appeared to absorb no measurable amount of protein, confirming the optimum surface conformation for a nonfouling surface.",
author = "R D'Sa and BJ Meenan",
note = "Reference text: *Corresponding author. Address: Nanotechnology and Integrated Bio- Engineering Centre (NIBEC), University of Ulster, Shore Road, Newtownabbey, Co Antrim, BT37 0QB, Northern Ireland. Telephone: {\th}44(0) 28 90368939. E-mail: bj.meenan@ulster.ac.uk. (1) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17, 1225–1233. (2) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. A. Langmuir 2001, 17, 5605–5620. (3) Kingshott, P.; Griesser, H. J. Curr. Opin. Solid State Mater. Sci. 1999, 4, 403– 412. (4) Ratner, B. D.; Bryant, S. J. Annu. Rev. Biomed. Eng. 2004, 6, 41–75. (5) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (6) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2008, 20, 86– 100. (7) Anderson, J. M. Annu. Rev. Mater. Res. 2001, 31, 81–110. (8) Morra, M. J. Biomater. Sci., Polym. Ed. 2000, 11, 547–569. (9) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043–1079. (10) Harris, J. M. In Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. (11) Emoto, K.; Harris, J. M.; Van Alstine, J. M. Anal. Chem. 1996, 68, 3751– 3757. (12) Bergstrom, K.; Osterberg, E.; Holmberg, K.; Hoffman, A. S.; Schuman, T. P.; Kozlowski, A.; Harris, J. H. J. Biomater. Sci., Polym. Ed. 1994, 6, 123–132. (13) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059– 5070. (14) Irvine, D. J.; Mayes, A. M.; Satija, S. K.; Barker, J. G.; Sofia-Allgor, S. J.; Griffith, L. G. J. Biomed. Mater. Res. 1998, 40, 498–509. (15) Michel, R.; Pasche, S.; Textor, M.; Castner, D. G. Langmuir 2005, 21, 12327–12332. (16) Borcia, G.; Anderson, C. A.; Brown, N. M. D. Appl. Surf. Sci. 2004, 221, 203–214. (17) Borcia, G.; Anderson, C. A.; Brown, N. M. D. Appl. Surf. Sci. 2004, 225, 186–197. (18) Liu, C.; Brown, N. M. D.; Meenan, B. J. Surf. Sci. 2005, 575, 273–286. (19) Liu, C.; Cui, N.; Brown, N. M. D.; Meenan, B. J. Surf. Coat. Technol. 2004, 185, 311–320. (20) Liu, C.; Brown, N. M. D.; Meenan, B. J. Surf. Coat. Technol. 2006, 201, 2341–2350. (21) Goddard, J. M.; Hotchkiss, J. H. Prog. Polym. Sci. 2007, 32, 698–725. (22) Uyama, Y.; Kato, K.; Ikada, Y. Adv. Polym. Sci. 1998, 137, 1–39. (23) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1994, 10, 481–485. (24) Kato, K.; Uchida, E.; Kang, E.; Uyama, Y.; Ikada, Y. Prog. Polym. Sci. 2003, 28, 209–259. (25) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677–710. (26) Wang, P.; Tan, K. L.; Kang, E. T.; Neoh, K. G. J. Membr. Sci. 2002, 195, 103–114. (27) Zou, X. P.; Kang, E. T.; Neoh, K. G. Surf. Coat. Technol. 2002, 149, 119– 128. (28) Upadhyay, D. J.; Cui, N.; Anderson, C. A.; Brown, N. M. D. App. Surf. Sci. 2004, 229, 352–364. (29) Upadhyay, D. J.; Cui, N.; Anderson, C. A.; Brown, N. M. D. Colloids Surf., A 2004, 248, 47–56. (30) Cui, N.; Upadhyay, D. J.; Anderson, C. A.; Brown, N. M. D. Surf. Coat. Technol. 2005, 192, 94–100. (31) Chastain, J., Ed. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer Corporation: Eden Prairie, MN, 1992. (32) Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 1399–1414. (33) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043–2056. (34) Wagner, M. S.; Castner, D. G. Appl. Surf. Sci. 2003, 203-204, 698–703. (35) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23, 4775–4785. (36) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649–4660. (37) Yasuda, H. Plasma Polymerization; Academic Press: Orlando, 1985. (38) Johnston, E. E.; Ratner, B. D. Surface modification of Polymeric Biomaterials. In XPS and SSIMS characterization of surface modified by plasma deposited Oligo(Glyme) films; Ratner, B. D., Castner, D. G., Eds.; Plenum Press: New York, 1996; pp 35-43. (39) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912–6921. (40) Kingshott, P.; St. John, H. A. W.; Chatelier, R. C.; Caruso, F.; Griesser, H. J. Polym. Prepr. 1997, 38, 1008. (41) Wagner, M. S.; McArthur, S. L.; Shen, M.; Horbett, T. A.; Castner, D. G. J. Biomater. Sci., Polym. Ed. 2002, 13, 407–428. (42) Ma, H.; Wells, M.; Beebe, T., Jr.; Chilkoti, A. Adv. Funct. Mater. 2006, 16, 640–648. (43) Chen, H.; Zhang, Z.; Chen, Y.; Brook, M. A.; Sheardown, H. Biomaterials 2005, 26, 2391–2399. (44) Dong, B.; Jiang, H.; Manolache, S.; Wong, A. C.; Denes, F. S. Langmuir 2007, 23, 7306–7313. (45) Chen, H.; Hu, X.; Zhang, Y.; Li, D.; Wu, Z.; Zhang, T. Colloids Surf., B 2008, 61, 237–243. (46) McPherson, T. B.; Shim, H. S.; Dewanjee, M. K.; Park, K. J. Biomater. Sci., Polym. Ed. 2000, 11, 1121–34. (47) McPherson, T. B.; Shim, H. S.; Park, K. J. Biomed. Mater. Res. 1997, 38, 289–302. (48) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. J. Biomed. Mater. Res. 1998, 42, 165–171. (49) Malmsten, M.; Muller, D. J. Biomater. Sci., Polym. Ed. 1999, 10, 1075– 1087. (50) Dorai, R.; Kushner, M. J. J. Phys. D: Appl. Phys. 2003, 36, 666–685. (51) Rinsch, C. L.; Chen, X.; Panchalingam, V.; Eberhart, R. C.; Wang, J.-H.; Timmons, R. B. Langmuir 1996, 12, 2995–3002. (52) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Langmuir 2005, 21, 1036– 1041. (53) Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547–1562. (54) Freij-Larsson, C.; Jannasch, P.; Wesslen, B. Biomaterials 2000, 21, 307–315. (55) Han, D. K.; Ryu, G. H.; Park, K. D.; Jeong, S. Y.; Kim, Y. H.; Min, B. G. J. Biomater. Sci., Polym. Ed. 1993, 4, 401–413.",
year = "2010",
doi = "10.1021/la902654y",
language = "English",
volume = "26",
pages = "1894--1903",
journal = "Langmuir",
issn = "0743-7463",
number = "3",

}

Chemical Grafting of Poly(ethylene glycol) Methyl Ether Methacrylate ontoPolymer Surfaces by Atmospheric Pressure Plasma Processing. / D'Sa, R; Meenan, BJ.

In: Langmuir, Vol. 26, No. 3, 2010, p. 1894-1903.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Chemical Grafting of Poly(ethylene glycol) Methyl Ether Methacrylate ontoPolymer Surfaces by Atmospheric Pressure Plasma Processing

AU - D'Sa, R

AU - Meenan, BJ

N1 - Reference text: *Corresponding author. Address: Nanotechnology and Integrated Bio- Engineering Centre (NIBEC), University of Ulster, Shore Road, Newtownabbey, Co Antrim, BT37 0QB, Northern Ireland. Telephone: þ44(0) 28 90368939. E-mail: bj.meenan@ulster.ac.uk. (1) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17, 1225–1233. (2) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. A. Langmuir 2001, 17, 5605–5620. (3) Kingshott, P.; Griesser, H. J. Curr. Opin. Solid State Mater. Sci. 1999, 4, 403– 412. (4) Ratner, B. D.; Bryant, S. J. Annu. Rev. Biomed. Eng. 2004, 6, 41–75. (5) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (6) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2008, 20, 86– 100. (7) Anderson, J. M. Annu. Rev. Mater. Res. 2001, 31, 81–110. (8) Morra, M. J. Biomater. Sci., Polym. Ed. 2000, 11, 547–569. (9) Lee, J. H.; Lee, H. B.; Andrade, J. D. Prog. Polym. Sci. 1995, 20, 1043–1079. (10) Harris, J. M. In Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. (11) Emoto, K.; Harris, J. M.; Van Alstine, J. M. Anal. Chem. 1996, 68, 3751– 3757. (12) Bergstrom, K.; Osterberg, E.; Holmberg, K.; Hoffman, A. S.; Schuman, T. P.; Kozlowski, A.; Harris, J. H. J. Biomater. Sci., Polym. Ed. 1994, 6, 123–132. (13) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059– 5070. (14) Irvine, D. J.; Mayes, A. M.; Satija, S. K.; Barker, J. G.; Sofia-Allgor, S. J.; Griffith, L. G. J. Biomed. Mater. Res. 1998, 40, 498–509. (15) Michel, R.; Pasche, S.; Textor, M.; Castner, D. G. Langmuir 2005, 21, 12327–12332. (16) Borcia, G.; Anderson, C. A.; Brown, N. M. D. Appl. Surf. Sci. 2004, 221, 203–214. (17) Borcia, G.; Anderson, C. A.; Brown, N. M. D. Appl. Surf. Sci. 2004, 225, 186–197. (18) Liu, C.; Brown, N. M. D.; Meenan, B. J. Surf. Sci. 2005, 575, 273–286. (19) Liu, C.; Cui, N.; Brown, N. M. D.; Meenan, B. J. Surf. Coat. Technol. 2004, 185, 311–320. (20) Liu, C.; Brown, N. M. D.; Meenan, B. J. Surf. Coat. Technol. 2006, 201, 2341–2350. (21) Goddard, J. M.; Hotchkiss, J. H. Prog. Polym. Sci. 2007, 32, 698–725. (22) Uyama, Y.; Kato, K.; Ikada, Y. Adv. Polym. Sci. 1998, 137, 1–39. (23) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1994, 10, 481–485. (24) Kato, K.; Uchida, E.; Kang, E.; Uyama, Y.; Ikada, Y. Prog. Polym. Sci. 2003, 28, 209–259. (25) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677–710. (26) Wang, P.; Tan, K. L.; Kang, E. T.; Neoh, K. G. J. Membr. Sci. 2002, 195, 103–114. (27) Zou, X. P.; Kang, E. T.; Neoh, K. G. Surf. Coat. Technol. 2002, 149, 119– 128. (28) Upadhyay, D. J.; Cui, N.; Anderson, C. A.; Brown, N. M. D. App. Surf. Sci. 2004, 229, 352–364. (29) Upadhyay, D. J.; Cui, N.; Anderson, C. A.; Brown, N. M. D. Colloids Surf., A 2004, 248, 47–56. (30) Cui, N.; Upadhyay, D. J.; Anderson, C. A.; Brown, N. M. D. Surf. Coat. Technol. 2005, 192, 94–100. (31) Chastain, J., Ed. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer Corporation: Eden Prairie, MN, 1992. (32) Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 1399–1414. (33) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002, 23, 2043–2056. (34) Wagner, M. S.; Castner, D. G. Appl. Surf. Sci. 2003, 203-204, 698–703. (35) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23, 4775–4785. (36) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649–4660. (37) Yasuda, H. Plasma Polymerization; Academic Press: Orlando, 1985. (38) Johnston, E. E.; Ratner, B. D. Surface modification of Polymeric Biomaterials. In XPS and SSIMS characterization of surface modified by plasma deposited Oligo(Glyme) films; Ratner, B. D., Castner, D. G., Eds.; Plenum Press: New York, 1996; pp 35-43. (39) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912–6921. (40) Kingshott, P.; St. John, H. A. W.; Chatelier, R. C.; Caruso, F.; Griesser, H. J. Polym. Prepr. 1997, 38, 1008. (41) Wagner, M. S.; McArthur, S. L.; Shen, M.; Horbett, T. A.; Castner, D. G. J. Biomater. Sci., Polym. Ed. 2002, 13, 407–428. (42) Ma, H.; Wells, M.; Beebe, T., Jr.; Chilkoti, A. Adv. Funct. Mater. 2006, 16, 640–648. (43) Chen, H.; Zhang, Z.; Chen, Y.; Brook, M. A.; Sheardown, H. Biomaterials 2005, 26, 2391–2399. (44) Dong, B.; Jiang, H.; Manolache, S.; Wong, A. C.; Denes, F. S. Langmuir 2007, 23, 7306–7313. (45) Chen, H.; Hu, X.; Zhang, Y.; Li, D.; Wu, Z.; Zhang, T. Colloids Surf., B 2008, 61, 237–243. (46) McPherson, T. B.; Shim, H. S.; Dewanjee, M. K.; Park, K. J. Biomater. Sci., Polym. Ed. 2000, 11, 1121–34. (47) McPherson, T. B.; Shim, H. S.; Park, K. J. Biomed. Mater. Res. 1997, 38, 289–302. (48) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. J. Biomed. Mater. Res. 1998, 42, 165–171. (49) Malmsten, M.; Muller, D. J. Biomater. Sci., Polym. Ed. 1999, 10, 1075– 1087. (50) Dorai, R.; Kushner, M. J. J. Phys. D: Appl. Phys. 2003, 36, 666–685. (51) Rinsch, C. L.; Chen, X.; Panchalingam, V.; Eberhart, R. C.; Wang, J.-H.; Timmons, R. B. Langmuir 1996, 12, 2995–3002. (52) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Langmuir 2005, 21, 1036– 1041. (53) Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547–1562. (54) Freij-Larsson, C.; Jannasch, P.; Wesslen, B. Biomaterials 2000, 21, 307–315. (55) Han, D. K.; Ryu, G. H.; Park, K. D.; Jeong, S. Y.; Kim, Y. H.; Min, B. G. J. Biomater. Sci., Polym. Ed. 1993, 4, 401–413.

PY - 2010

Y1 - 2010

N2 - This article reports the use of atmospheric pressure plasma processing to induce chemical grafting of poly(ethylene glycol) methyl ether methacrylate (PEGMA) onto polystyrene (PS) and poly(methyl methacrylate) (PMMA) surfaces with the aim of attaining an adlayer conformation which is resistant to protein adsorption. The plasma treatment was carried out using a dielectric barrier discharge (DBD) reactor with PEGMA of molecular weights (MW) 1000 and 2000, PEGMA1000 and PEGMA2000, being grafted in a two step procedure: (1) reactive groups are generated on the polymer surface followed by (2) radical addition reactions with the PEGMA. The surface chemistry, coherency, and topography of the resulting PEGMA grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM), respectively. The most coherently grafted PEGMA layers were observed for the 2000 MW PEGMA macromolecule, DBD processed at an energy dose of 105.0 J/cm2 as indicated by ToF-SIMS images. The effect of the chemisorbed PEGMA layer on protein adsorption was assessed by evaluating the surface response to bovine serum albumin (BSA) using XPS. BSA was used as a model protein to determine the grafted macromolecular conformation of the PEGMA layer. Whereas the PEGMA1000 surfaces showed some protein adsorption, the PEGMA2000 surfaces appeared to absorb no measurable amount of protein, confirming the optimum surface conformation for a nonfouling surface.

AB - This article reports the use of atmospheric pressure plasma processing to induce chemical grafting of poly(ethylene glycol) methyl ether methacrylate (PEGMA) onto polystyrene (PS) and poly(methyl methacrylate) (PMMA) surfaces with the aim of attaining an adlayer conformation which is resistant to protein adsorption. The plasma treatment was carried out using a dielectric barrier discharge (DBD) reactor with PEGMA of molecular weights (MW) 1000 and 2000, PEGMA1000 and PEGMA2000, being grafted in a two step procedure: (1) reactive groups are generated on the polymer surface followed by (2) radical addition reactions with the PEGMA. The surface chemistry, coherency, and topography of the resulting PEGMA grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM), respectively. The most coherently grafted PEGMA layers were observed for the 2000 MW PEGMA macromolecule, DBD processed at an energy dose of 105.0 J/cm2 as indicated by ToF-SIMS images. The effect of the chemisorbed PEGMA layer on protein adsorption was assessed by evaluating the surface response to bovine serum albumin (BSA) using XPS. BSA was used as a model protein to determine the grafted macromolecular conformation of the PEGMA layer. Whereas the PEGMA1000 surfaces showed some protein adsorption, the PEGMA2000 surfaces appeared to absorb no measurable amount of protein, confirming the optimum surface conformation for a nonfouling surface.

U2 - 10.1021/la902654y

DO - 10.1021/la902654y

M3 - Article

VL - 26

SP - 1894

EP - 1903

JO - Langmuir

T2 - Langmuir

JF - Langmuir

SN - 0743-7463

IS - 3

ER -