Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 23

TIP: Step 4 has been proved to effective both with and without the use of
cross-linking agents (EDC; Thermo Fisher) for coupling the biomolecules
to the polydopamine layer. For experimental instructions on the use of
EDC, refer to the manufacture's protocol.

d n 3 r 4 n g | 1

5. Wash with 10 mM Tris-HCl (pH 8.5) solution.

A.5 Protocol to Immobilize Growth Factors and Small
Peptides by Silanization-based Physisorption
1. Prepare a 95% ethanol solution in water and adjust the pH to 4.5-5.5
with acetic acid.
2. Dissolve 3-aminopropyltriethoxysilane (APTS) at 5% vol in the above-
described solution, allowing hydrolysis to occur for 5 min at room
temperature.

TIP: In Step 2, APTS will introduce amine groups to the target surface;
other silane molecules can be considered to introduce different func-
tional groups (e.g., carboxyethylsilanetriol for carboxylic groups).

3. Incubate the scaffolds with the silane solution for 2 h (shorter or longer
times change the APTS content and its layer thickness).
4. Wash in ethanol or ethanol-water and cure at 110 1C for 30 min.
5. Incubate the silanized scaffolds in the biomolecule solution (for the
optimal concentration, preliminary studies using a range of concen-
tration should be considered) for 2 h at room temperature, under gentle
stirring. As cross-linking agent, EDC is one of the most effective to co-
valently immobilize the biomolecule to the introduced functional groups
of the surface. Cross-linking alternatives are glutaraldehyde and genipin.
6. Wash in PBS or water.
7. Determine the amount of adsorbed proteins on the surface by
bifunctional chelating agents (BCA Protein Assay; Thermo Fisher). For
peptides, it is convenient to use fluorescent tags, as mentioned above,
and calculate the amount adsorbed on the surface by depletion (i.e.,
analyzing the coating solution before and after the incubation step). As
an alternative technique, FTIR in an ATR configuration can be used,
although it requires a strong background to use this technique for
quantification purposes.

.

References
1. E. Mohseni, E. Zalnezhad and A. R. Bushroa, Comparative investigation
on the adhesion of hydroxyapatite coating on Ti-6Al-4V implant: A
review paper, Int. J. Adhes. Adhes., 2014, 48, 238-257.