Affibody-functionalized bacterial cellulose tubes for biofiltration applications Hannes Orelma a , Luis O. Morales a , Leena- Sisko Johansson a , Ingrid C. Hoeger b , Ilari Filpponen a , Cristina Castro c , Janne Laine a , Orlando J. Rojas a a Aalto University, Biobased Colloids and Materials group (BiCMat), Finland. b North Carolina State University, Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, Raleigh, USA c Universidad Pontificia Bolivariana, School of Engineering, Medellenin, Colombia E-mail: [email protected]/ [email protected]1
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Affibody-functionalized bacterial cellulose tubes for biofiltration applications Hannes Orelma a, Luis O. Morales a, Leena-Sisko Johansson a, Ingrid C.
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Affibody-functionalized bacterial cellulose tubes for biofiltration applicationsHannes Orelmaa, Luis O. Moralesa, Leena-Sisko Johanssona, Ingrid C. Hoegerb, Ilari Filpponena, Cristina Castroc, Janne Lainea, Orlando J. Rojasa
a Aalto University, Biobased Colloids and Materials group (BiCMat), Finland. b North Carolina State University, Departments of Forest Biomaterials and Chemicaland Biomolecular Engineering, Raleigh, USAc Universidad Pontificia Bolivariana, School of Engineering, Medellenin, Colombia
Objective: Develop a nanocellulose-based biofiltration system for
affinity separation of HSA
Feed
Filtrate
Filtrate
Filtr
ate
Filtrate
Residue
66.5 kDa
Human serum albumin (HSA): the most abundant protein in human blood plasma
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Microbial cellulose / bacterial cellulose
• Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium or Sarcina are able to produce cellulose extra-cellularly– Acetobacter xylinum/Gluconacetobacter xylinus is considered to be
the most efficient strain– A continuous source of air and carbon is required – Cellulose yield of 35-40% in relation to glucose in culture medium
• Acetobacter microfibrils usually have large crystal structures and thickness in the range of 6-10 nm
• Bacterial cellulose has a high degree of polymerization (4000-10000 anhydroglucose units) and it forms a pellicle
– It can contain more than 99 % water– Strong in wet state!
Klemm et al. 2011, Angewandte Chemie International Edition,50, 5438
P. Gatenholm, D. Klemm, MRS Bull. 2010, 35, 208
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Applications of bacterial cellulose
Skin therapy
Artificial blood vessels
Scaffolds for tissue engineering
Nanopaper & paper additives
Food
W. Czaja, A. Krystynowicz, S. Bielecki, R. J. Brown, 2006 Biomaterials, 27 2
Kowalska-Ludwicka, Karolina et al. Archives of Medical Science :
AMS 9.3 (2013): 527.
Pure bacterial cellulose
Nata de Coco
de Olyveira et al. Bacterial Nanocellulose for Medicine Regenerative J. Nanotechnol.
Molecular cut-off• Never dried membrane up to 66 kDA (BSA)
Sokolnicki et al. 2006. J. Membr. Sci. 272. 15-27.
• Dried membrane 20 kDa (PEG) Shibazaki et al. 1993. J. Appl. Pol. Sci. 50. 965-969.
Pressure resistance• Up to 880 mmHg (1.17 bar)
Bodin et al. 2006. Biotechnol. Bioeng. 97(2). 425-434.
Chemistry• No electrostatic interactions with proteins• Highly stable due to high crystallinity degree
vanderhart et al. 1984. Macromolecules. 17. 1465-1472.
Biofiltration of fluorescence stained HAS with affibody-functionalized BC tubes
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1E-3 0.01 0.10
5
10
15
20
25
30
35
Nor
mal
ized
fluo
resc
ence
inte
nsity
HSA concentration (mg/ml)
Anti-HSA-CMC-BC
Anti-HSA-TEMPO-BC
• High fluorescence when anti-HSA was conjugated to CMC-BC• CMC modification decreases the background noise compared
to TEMPO-oxidation.
Dansylated HSA on…
CMC-BC with anti-HSA
CMC-BC without anti-HSA
Background CMC-BC
Dan
syla
ted
-HSA
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Conclusions
• Bacterial tubes were incubated in the presence of CMC
• CMC lowers irreversible changes in BC upon drying
• Affibodies were covalently conjugated to BC tubes via EDC/NHS chemistry
• BC tubes were utilized for specific binding of HSA
• Orelma et al., Affibody conjugation onto bacterial cellulose tubes and bioseparation of human serum albumin, RSC Advances, 4, 51440-51450 (2014).
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Funding and acknowledgements
• Lignocell project of the Finnish Centre for Nanocellulosic Technologies financially supported by the Finnish Funding Agency for Technology and Innovation (TEKES) and UPM
• Academy of Finland’s Centres of Excellence Programme (2014-2019)
• Dr Joseph M. Campbell for performing XPS; Anu Anttila, Ritva Kivela, and Marja Karkkainen for technical assistance.