This document discusses immobilization of biomolecules on biomaterial surfaces. It begins by explaining that biomaterials must have suitable bulk and surface properties to function in biological environments. Common approaches involve fabricating materials with adequate bulk properties, then modifying the surface to enhance biocompatibility. The document then discusses various biomolecules that can be immobilized on surfaces like proteins, peptides, polysaccharides and describes techniques for covalent and non-covalent immobilization. Specific examples of immobilizing collagen, RGD peptide, hyaluronic acid and other biomolecules on scaffolds are provided to support tissue engineering applications.

1. Immobilization of
biomolecules on the
surface of biomaterials
By: Mohsen Norouzi
MSc Student of Tissue Engineering
Islamic Azad University of Najafabad (IAUN)
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2. Biomaterials must possess bulk properties that
permit its function in the bio‐environment, but
also the best surface properties.
It is difficult to design materials that fulfill both
needs.
A common approach is to fabricate with
adequate bulk properties followed by a
special treatment to enhance the surface
properties.
Preface
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3. Preface
 The broad interdisciplinary area where properties and
processes at this interface are investigated and biofunctional
surfaces are fabricated is called Biological Surface Science.
 Examples:
 medical implants in the human body (dental implants, artificial
hip and knee joints, artificial blood vessels and heart valves, etc.)
 tissue engineering
 biosensors and biochips for diagnosis (DNA‐chips, etc.)(clinical
diagnostics, environmental control, food production)
 Bioelectronics (systems to get information storage and processing
) and artificial photosynthesis (clean energy)
 biomimetic materials (mimic the functional properties of
biological materials/components in order to achieve new and
better materials; ow friction from the sharkskin or self‐cleaning
character like the lotus leaf )
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4. Preface
4

5. Approaches to improve biointerfaces:
reduction of unspecific protein adsorption
enhanced adsorption of specific proteins
material modification by immobilization of cell
recognition motives to obtain controlled
interaction between cells and synthetic substrate
• Using methods like selfassembly (SAMs),
surface modification, photochemical
immobilization or polymer chemistry,
complex surfaces with immobilized
peptides and proteins can be prepared
Preface
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6.  Biomolecules used in precision immobilization
strategies include proteins, lipids, polypeptides,
polynucleotides and polysaccharides
 Immobilization techniques range from relatively
low to extremely high specificity.
 characteristics of successful precision
engineered biorecognition surfaces:
 presence of one ligand site and the receptor‐ligand
affinity
 an appropriate surface density of those sites
 spatial distribution of the ligands
Preface
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7. The use of short peptides for surface
biorecognition has proved to be
advantageous over the use of the long
chain native ECM proteins, since the
latter tend to be randomly folded upon
adsorption, being the receptor binding
domains not always sterically
available.
Preface
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8. Immobilization
 Molecules may be immobilized either passively through;
o Hydrophobic
o Ionic interactions
o Covalently by attachment to activated surface groups.
 Non-covalent surfaces are effective for many applications;
however, passive adsorption fails in many cases.
 Covalent immobilization is often necessary for binding of
molecules that do not adsorb, adsorb very weakly, or adsorb
with improper orientation and conformation to non-covalent
surfaces.
 Covalent immobilization may result in better biomolecule
activity, reduced nonspecific adsorption, and greater stability.
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9. Immobilization
Immobilization reaction should have
several characteristics;
Firstly, the reaction should occur rapidly and
therefore allow the use of low concentrations of
reagents for immobilization.
The chemistry should require little, if any, post-
synthetic modification of ligands before
immobilization to maximize the number of
compounds that can be generated by solution
or solid-phase synthesis and minimize the cost
of these reagents.
Immobilized ligands must be in an oriented
and homogeneous manner.
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10. Immobilization
The immobilization process should occur
selectively in the presence of common
functional groups, including amines, thiols,
carboxylic acids, and alcohols.
Amino-NH2,
Carboxy-COOH,
Aldehyde-CHO,
Thiol-SH,
Hydroxyl-OH
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11. Immobilization
Surface density of the ligand should be
optimized.
Low density surface coverage will yield a
correspondingly low frequency.
High surface densities may result steric
interference between the covalently
immobilized ligand molecules, impending
access to the target molecules.
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12. 1) unhindered binding. 2) inaccessible binding site. 3) hindered
binding site when adjacent site is occupied. 4) restricted access
binding site.
Immobilization
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13. Immobilization
 Correct orientation of the ligand molecules on the surface, and using
a spacer arm are important and critical and makes the ligand
available for the target.
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14. Proteins are much more sensitive
to their physiological environments
and can easily be degraded or
denaturated by physical or
chemical effects. Protein`s 3-D
confirmation must not change
during immobilization procedure.
DNA molecules are much more
stable then proteins.
It is easier to immobilize DNA
molecules.
Immobilization
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15. Preparation of Surface for
Biomolecule Immobilization
Modification of the surface to create
functional groups.
Modification of biomolecules for
covalent attachment to the surface.
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16. General Route for Immobilization
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17. General Route for Immobilization
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18. General Route for Immobilization
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19. Surface Chemistry
Cross-linking Strategies for Protein Immobilization
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20. Surface Chemistry
Cross-linking Strategies for DNA Immobilization
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21. Surface engineered scaffolds
 Collagen:
 major structural component forming the natural ECM of
connective tissues and organs
 one of the most established methods for endowing cell
adhesive properties to the scaffolds
 Examples:
PLA and PLGA scaffolds chemically grafted with collagen by
plasma treatment have shown enhanced adhesion and
spreading of fibroblasts
Collagen modification by conjugation reactions onto PLA
scaffolds grafted with polymethacrylic acid also has improved
cell spreading and growth for use in cartilage tissue
engineering.
 its immunogenicity has limited its applications
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22. Gelatin:
a good alternative for collagen because of its
absence of antigenicity and ease of handling
at high concentrations
Example:
Gelatin immobilized onto porous scaffolds by
physical entrapment and chemical crosslinking
showed greatly enhanced surface properties on
attachment, proliferation, and ECM deposition of
osteoblasts
Surface engineered scaffolds
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23.  Cell adhesive peptides:
 Rather than immobilizing the whole protein, chemical
conjugation of short chain peptide moieties derived from
the cell adhesive proteins onto the polymer surface can be
a much more effective strategy
 Advantages of The surface immobilization of short peptides:
higher stability against conformational change
easy controllability of surface density,
orientation more favorable for ligand–receptor interaction and
cell adhesion
minimizing immune responses and infection
Surface engineered scaffolds
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24. peptide sequences involved in cellular
interactions by receptor binding:
RGD, IKVAV, and YIGSR
RGD sequence: one of the best known foruse in tissue
engineering applications
Examples:
 Immobilization of RGD onto 3-D matrices to improve cell
adhesive properties was previously demonstrated in collagen
gels, showing enhanced adherence of murine melanoma cells
 RGD, along with other short peptide sequences such as IKVAV,
YIGSR, RNAIAEIIKDI from laminin, and HAV from N-cadherin, was
also used for engineering of neural tissue.
 PLA scaffolds modified with RGD by plasma treatment not only
resulted in improved adhesion of the osteoblast-like cells, but
also supported its growth and differentiation
 osteoblasts seeded onto the RGD immobilized scaffolds greatly
enhanced mineralization and formation of bone-like tissues
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25. 25

26. Hyaluronic acid:
a non-sulfated glycosaminoglycan (GAG), is
a major substance of the gel-like component
in the extracellular matrix of connective
tissues
capable of specific cell interaction via the
CD44 receptor which promotes wound
healing and induces chondrogenesis
Examples:
Chitosan–gelatin composite scaffolds modified with
HA have been shown to increase the adhesion of
fibroblasts
PLGA scaffolds modified with HA supported the
growth of chondrocytes with maintenance of its
original phenotype, showing great potential for
cartilage tissue engineering
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27. Galactose:
utilized in scaffolds for liver tissue engineering
recognized by mammalian hepatocytes through
the asialoglyco protein receptor leading to
regulation of a degradative pathway I
glycoprotein homeostasis
Examples:
Porous scaffolds immobilized with galactose have been
fabricated to improve hepatocyte attachment, viability,
and metabolic functions. Gelatin sponges modified with
galactose were shown to support hepatocyte adhesion
and function such as release of lactate dehydrogenase
(LDH), albumin secretion, and urea synthesis. Perfusion
culture of hepatocytes with galactose-derivatized PLGA
scaffolds further improved viability and functional
activity of the cells
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28. Heparin:
intensively studied for growth factor releasing
matrices in tissue engineering.
a highly sulfated GAG constituting the
extracellular matrix, and is known for its specific
interactions with various angiogenic growth
factors
Examples:
Heparin binding has been shown to preserve the
stability and biological activity of the growth factors. A
wide variety of scaffold matrices, including nanofibers,
prepared from collagen, fibrin, chitosan, alginate, PLA
and PLGA, have been incorporated or immobilized with
heparin to achieve sustained release of growth factors
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29. 29

30. Examples from Literature (1)
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31. Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces
1- Surfaces based on PEG:
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32. 2- Surfaces based on anti-fouling comb-like polymers
Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces32

33. p-nitrophenyl chloroformate (NPC), Disuccinimidyl carbonate (DSC), 1,10-
Carbonyldiimidazole (CDI), succinic anhydride (SA)
Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces33

34. Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces34

35. 3- Surfaces based on co-polymers:
Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces35

36. Strategies for design and preparation of
anti-fouling, bioactive (AFB) surfaces36

37. Examples from Literature (2)
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38. Basement Material (Substrate):
 Synthetic polymer substrates, polystyrene (PS) and poly(lactic-co-
glycolic acid) (PLGA), polydimethylsiloxane (PDMS), silica (Si) and
titanium (Ti).
Linkage Material:
 Polydopamine
Chemical/Physical Method:
 Dipcoating a biomimetic polymer (PD) thin film onto the polymer
surface followed by conjugation of adhesion peptides and
neurotrophic growth factors to the biomimetic polymer film.
Because amine and thiol groups can be covalently conjugated to a
PD layer via the quinone group, PD coating exhibits latent reactivity
to various nucleophiles with those functional groups
Immobilized Material:
 ECM protein-derived adhesion peptides, fibronectin [Arg-Gly-Asp
(RGD)] and laminin [Try-Ile-Gly-Ser-Arg (YIGSR)], and neurotrophic
factors, NGF and GDNF
Goal:
 Modification of tissue engineering scaffolds for improving the
efficacy of stem cell therapy by generating physicochemical
stimulation promoting proliferation and differentiation of stem cells
surface modification for efficient and reliable manipulation of
human neural stem cell (NSC) differentiation and proliferation
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39. Result/Effectiveness:
 highly efficient, simple immobilization of neuro trophic growth factors
and adhesion peptides onto polymer substrates.
 greatly enhance differentiation and proliferation of human NSCs
(human fetal brain derived NSCs and human induced pluripotent stem
cell derived NSCs) at a level comparable or greater than currently
available animal derived coating materials (Matrigel) with safety issues.
 versatile platform technology for developing chemically defined, safe,
functional substrates and scaffolds for therapeutic applications of
human NSCs.
 efficient surface immobilization of proteins and peptides to a diverse
range of materials, including polymer scaffolds, ceramic substrates, and
metal devices, for stem cell culture and transplantation.
 versatile platform technology for efficient development of biomimetic
substrates and scaffolds that induce desirable stem cell behavior and
enhance stem cell function
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40. 40

41. Examples from Literature (3)
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42. Basement Material (Substrate):
 ZrO2, TiZr and Ti with its naturally occurring oxide layer TiO2
Linkage Material:
 Specific adsorbing peptides (Pep5 (SHKHGGHKHGGH KHGSSGKG)) are
used as anchor molecules to immobilize oligodesoxynucleotides (ODNs)
on the implant surface (anchor strand, AS)
Chemical/Physical Method:
 The BAM is conjugated to a complementary ODN strand (CS) which is
able to hybridize to the AS on the implant surface to immobilize the
BAM. The ODN double strand allows for a controlled release of the BAM
adjustable by the ODN sequence and length.
Immobilized Material:
 biologically active molecules (BAMs), e.g. antibiotics or growth
factors immobilize the parathyroid hormone (PTH) fragment 1-34
42

43. Result/Effectiveness:
 Successful immobilization of biologically active PTH (1-34)
 The high potential of the established surfaces to achieve an increased
osseointegration of variable implants, especially for patients with risk
factors. the development of bioinductive implant surfaces might
increase the healing capacity in the bone, especially for patients with
risk factors such as osteoporosis, where the healing of bone fractures is
disturbed.
 The ability of PTH (1-34) to induce the differentiation of osteoblast
precursor cells C2C12 was detected by the quantification of the ALP
activity.
 The conjugation of PTH with CS only slightly decreased the Alkaline
phosphatase(ALP) activity, indicating that the biological activity was
almost completely maintained. The application of the immobilization
system on the three materials allows for the modification of the surfaces
with PTH (1-34) as the ALP activity could be increased while
unspecifically bound PTH (1-34) itself showed no effect.
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44. 44

45. Examples from Literature (4)
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46. Basement Material (Substrate):
 gold, platinum, glass and titanium
Linkage Material:
 Peptide motifs
Chemical/Physical Method:
 We synthesized bifunctional quartz-binding peptide QBP1–RGD and
titanium-binding peptide TiBP1–RGD peptides via solid phase peptide
synthesis and immobilizes these peptide conjugates on the surface through
directed assembly in a single step
Immobilized Material:
 poly(ethylene glycol) anti-fouling polymer and the integrin-binding
RGD sequence
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47. Result/Effectiveness:
 We successfully imparted cell-resistant properties to gold and platinum
surfaces using gold- and platinum-binding peptides, respectively, in
conjunction with PEG.
 several-fold increase in the number and spreading of fibroblast cells on
glass and titanium surfaces using quartz and titanium-binding peptides
in conjunction with the integrin ligand RGD.
 Control over the extent of cell–material interactions by relatively simple
and biocompatible surface modification procedures using inorganic
binding peptides as linker molecules.
 Targeted assembly proved to be an efficient way of immobilizing large
molecules (i.e. PEG) through, first, coating the inorganic binding
peptides and then performing the conjugation reaction.
 Directed assembly, on the other hand, is preferred for the immobilization
of small molecules by synthesizing a single chimeric molecule with bi
functional domains.
 Control over the extent of cell–material interactions can be achieved
by relatively simple and biocompatible surface modification procedures
using GEPIs as linker molecules.
 QBP1 and the TiBP1 facilitate the immobilization of RGD on both
surfaces while preserving its functionality as a recognition site for cells.
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49. 49

50. References:
 Prof. Marco Mascini, Immobilization of Biomollecules, Grenoble,
2004.
 Laia Francesch de Castro , Surface modification of polymers by
plasma polymerization techniques for tissue engineering, doctorate
thesis, Universitat liull, Barcelona .
 Hyun Jung Chung, Tae Gwan Park, Surface engineered and drug
releasing pre-fabricated scaffolds for tissue engineering, Advanced
Drug Delivery Reviews 59 (2007) 249–262.
 Tina Micksch et al, A modular peptide-based immobilization system
for ZrO2, TiZr and TiO2 surfaces, Acta Biomaterialia (2014).
 Qian Yu et al, Anti-fouling bioactive surfaces, Acta Biomaterialia 7
(2011) 1550–1557.
 Dmitriy Khatayevich et al, Biofunctionalization of materials for
implants using engineered peptides, Acta Biomaterialia 6 (2010)
4634–4641.
 Kisuk Yang et al, Polydopamine-mediated surface modification of
scaffold materials for human neural stem cell engineering,
Biomaterials 33 (2012) 6952e6964.
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51. Thanks for your attention
Good luck
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