Biomedical Engineering Reference
In-Depth Information
impossible to rule out protein adsorption with luorescence without a calibration standard.)
Fibroblast-like baby hamster kidney (BHK) cells were also shown to attach selectively to the
aminosilane patterns in the presence of 10% calf serum.
What if the proteins themselves contained photoreactive groups, so as to be amenable to
selective photochemical immobilization directly? In principle, there are two ways one could
do this: either by cross-linking a photoreactive group in an existing protein, or by genetically
engineering the protein. David Tirrell and colleagues at Caltech were able to synthesize the non-
natural photosensitive amino acid
para
-azido-phenylalanine (
p
N
3
Phe in
Figure 2.9
) in the bac-
terium
E. coli
and incorporate it into extracellular proteins such as elastin and ibronectin. Upon
exposure to UV light (365 nm), the azidophenyl groups form a highly reactive nitrene group that
cross-links the protein to almost anything in proximity (so reactive that they undergo carbon-
hydrogen bond insertion, enabling patterning on virtually any surface). Essentially, Tirrell's
group has demonstrated a genetically engineered “protein photoresist” that can be used for pro-
ducing biosensors, cellular micropatterns (
Figure 2.9
), and others.
Tirrell's photoreactive proteins may ind their most useful application in the fabrication of pro-
tein gradients and 3-D protein structures for tissue engineering and cell biology. A collaborative
group led by Xiang Zhang from the University of California at Berkeley and Sarah Heilshorn (who
studied with Tirrell) at Stanford University has used a Digital Micromirror Device (see Section
1.3.6.1) to immobilize Tirrell's photoreactive proteins in gradients and 3-D structures (
Figure
2.10
). A 380 nm UV-LED light source was used, resulting in a light power density on the sample
plane of 0.2 to 1 mW/cm
2
; approximately 3 to 5 minutes of exposure time were required for a spun
(dried) ilm of protein and 5 to 20 minutes were required for immobilization from the liquid phase,
depending on the optical reduction used. Ater irradiation, samples were washed by agitation in
0.1% sodium dodecyl sulfate (a denaturant, to remove protein that is not covalently immobilized)
for at least 4 hours and thoroughly rinsed with water. he projections of neuronal-like PC12 cells
were observed to align with the topographical troughs and ridges of a linear 3-D protein pattern.
However, photoreactive proteins are not easy to produce and the one that you are inter-
ested in may not be commercially available for a while. In 2003, a clever chemist at Texas A&M
H
2
N
OH
O
Phe
N
3
H
2
N
100 µm
OH
O
ECM molecule
(human fibronectin)
pN
3
Phe
FIGURE 2.9
Micropatterning. of. genetically. engineered. photoreactive. proteins.. (Adapted. from.
Isaac.S..Carrico,.Stacey.A..Maskarinec,.Sarah.C..Heilshorn,.Marissa.L..Mock,.Julie.C..Liu,.Paul.J..
Nowatzki,.Christian.Franck,.Guruswami.Ravichandran,.and.David.A..Tirrell,.“Lithographic.patterning.
of.photoreactive.cell-adhesive.proteins,”.
J. Am. Chem. Soc.
.129,.4874-4875,.2007..The.ibronec-
tin.structure.is.from.the.Protein.Data.bank.archive.(www.wwpdb.org)..Reprinted.with.permission.of.
the.American.Chemical.Society.)
