Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 21

High functionalization Dynamic functionalization Low Functionalization

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Figure 1.4 Three functionalization scenarios of fluorescently-labeled peptide in a
hydrogel scaffold. High functionalization indicates a high concentration
of peptide effectively bound to the hydrogel backbone. Dynamic functio-
nalization indicates that the peptide can be slowly released to the micro-
environment in a controlled manner over time. Low functionalization
indicates that the peptide is loosely bound to the hydrogel backbone due
to unspecific binding chemistry.

.

The fluorescently labeled probe can be highly functionalized to the
scaffold where a strong interaction between the probe and the scaf-
fold will take place. The probe could 'leach out' out of the scaffold
over time, a phenomenon that can be described as dynamic functio-
nalization. Finally, the interaction of the probe with the scaffold could
be very low, making it very dicult to detect the probe in the 3D
system.

A.3 A Protocol to Immobilize Growth Factors and Small
Peptides by 'Classic' Physisorption
1. Dissolve the biomolecule to be immobilized in coating buffer at a
pH near the biomolecule isoelectric point. Common buffers include:
(1) 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS); (2) 50 mM
sodiumborate, pH 8.5; (3) 50 mM sodium acetate, pH 3.6-5.6; (4) 25 mM
MES [2-(N-morpholino)ethane sulfonic acid], pH 6.1; and (5) 50 mM
sodium bicarbonate. The concentration of the biomolecule should be
calculated in order to have an excess of about 3-10 over the theoretical
monolayer density for that protein on the surface, depending on the
biomolecule size.

TIP: A protein titration study can be performed using a range of protein
concentrations and a fixed incubation time.