Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 22

2. Soak the scaffold in the biomolecule solution and mix gently for 1 h at
room temperature (unless the biomolecule is extremely sensitive to this
temperature).

TIP: A range of incubation times and a fixed biomolecule concentration
can be used to optimize Step 2.

d n 3 r 4 n g | 1

3. Remove the solution; wash the scaffold with coating solution.
4. 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 de-
pletion (i.e., analyzing the coating solution before and after the in-
cubation step). As alternative technique, FTIR in an attenuated
total reflectance (ATR) configuration can be used, although it re-
quires a strong background to use this technique for quantification
purposes.

A.4 A Protocol to Immobilize Growth Factors and
Small Peptides by Polydopamine Coating-based
Physisorption
A.4.1 One-step Protocol
1. Soak the scaffold in a 2 mg mL 1 dopamine containing the biomole-
cules to be immobilized (for the optimal concentration, preliminary
studies using a range of concentration should be considered) in 10 mM
Tris-HCl (pH 8.5) solution.
2. Stir gently for 18 h at room temperature. The coating layer formation
will turn the sample dark.
3. Wash with 10 mM Tris-HCl (pH 8.5) solution. To quantify the amount
of biomolecules adhered on the surface, the use of fluorescent labels is
the best choice as the polydopamine molecules can give false positive
results by other techniques.

.

A.4.2 Two-step Protocol
1. Soak the scaffold in a 2 mg mL 1 dopamine in 10 mM Tris-HCl (pH 8.5)
solution.
2. Stir gently for 18 h at room temperature. The coating layer formation
will turn the sample dark.
3. Wash with 10 mM Tris-HCl (pH 8.5) solution.
4. Incubate the sample with the biomolecule solution (for the optimal
concentration, preliminary studies using a range of concentration
should be considered) for 12 h.