Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 17

using traditional analytical tools such as 1 H nuclear magnetic resonance
( 1 H NMR) or Fourier transform infrared spectroscopy (FTIR). Thus, the
functionalization of fluorescent moieties to growth factors and small pep-
tides is a practical approach to determine the extent of covalent functiona-
lization of small molecules in a (hard) scaffold or hydrogel. When choosing
the fluorescent tag, the researcher has to take into consideration the mo-
lecular mass of the fluorescent tag to attach to the growth factor or peptide.
Fluorescent tags such as FITC (5,6-fluorescein isothiocyanate, MW = 389.38)
or N-hydroxysuccinimide (NHS)-fluorescein (MW = 473.39) could be used as
they are small compared to proteins, peptides, and growth factors, thus
minimizing the impact in the biological function of the growth factor or
peptide. 102
A brief description of the process of determining the functionalization of
growth factors and peptides in naturally derived hydrogels or scaffolds is
outlined below. We need to clarify that no covalent functionalization is 100%
effective and part of the growth factor or peptide will eventually leach out of
the hydrogel or scaffolds. Thus, we present a simple and straightforward
method to characterize the diffusivity of the growth factor or peptide out of
the matrix. We will use FITC as the model fluorescent tag attached to a short
peptide such as the QK peptide, mentioned previously, functionalized to a
type I collagen hydrogel.
The pros and cons of the fluorescence method are:

d n 3 r 4 n g | 1

.

Pros
J Applicable for 3D structures
J Applicable to the broad range of natural or synthetic hydrogels
J Applicable for all types of polymers
Cons
J The use of large fluorescent tags is not recommended
J Activity of the functionalized 3D structure may decrease with time
J Functionalization will be limited by diffusivity of species into
hydrogels

A Appendix: Protocols for the Visualization and
Immobilization of Proteins

A.1 Protocol for the Visualization of Protein on a Surface
Using Atomic Force Microscopy
Visualization of the presence of proteins on a surface and the evaluation of
their density is a significant challenge due to the small size of the proteins
(a few nanometers to tens of nanometers). Typically, surfaces have complex
topography with roughness often much greater than a few nanometers, and
small protein are dicult to be distinguished from surface features. Hence
to visualize proteins on the surface a very flat substrate such as a silicon
wafer or a mirror-finished surface are required.