Biomedical Engineering Reference
In-Depth Information
(a)
(b)
Protruding octadecy1
alkyl chain of CDOS
Biotin
Phycoerythrin
Silanization
B-PUMT
attachment
n
Streptavidin
Alkyl chain of the
biotinylated polymer
A glass surface
(slide/fiber/capillary)
Protein
attachmen
t
Washing, etc.
B
B
B
B
PE
PE
PE
PE
B
B
B
PE
PE
PE
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
FIGURE 1.7
(a) Schematic of the steps involved in B-PUMT self-assembly on the surface of declad optical fibers, where X-PE
is Str-PE protein conjugate. On the right is a diagram (b) of the structure built up on the optical fiber surface as
a result of the assembly process. Reprinted from Ayyagari, M.S., Pande, R., Kamtekar, S., Gao, H., Marx, K.A.,
Kumar, J., Tripathy, S.K., Akkara, J., Kaplan, D.L. (1995). Molecular Assembly of Proteins and Conjugated
Polymers: Toward Development of Biosensors.
Biotech. Bioeng.
45:116-121. With permission of Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.
spectrum of phycoerythrin was then measured via the optical coupling between evanescent
waves extending from the optical fiber and the immobilized phycoerythrin chomophores
(17,18). One of the advantages of the attachment system involving the biotin-streptavidin
interaction is the fact that these derivatizations can be carried out for all biological molecules
and polymers. It results in a very useful and versatile cassette methodology for surface
immobilization of any photodynamic protein, as well as other proteins, and nucleic acids.
Another surface immobilization procedure we successfully investigated was the use of
sol-gels for the direct entrapment of phycobiliproteins. The sol-gel formation technique we
used involved the reactions shown in Figure 1.8: the hydrolysis and polycondensation of
silicon alkoxides (19,20). The sol-gel technique produced transparent glass possessing
excellent optical, mechanical, and thermal properties, without any necessity for high-tem-
perature processing. PE, entrapped physically within this sol-gel, possessed absorption
and fluorescence properties of the native protein. However, in a more extensive study of
all three phycobiliproteins entrapped in the sol-gel matrices, the optical properties of the
phycocyanin and allophycocyanin species were found to undergo minor changes upon
sol-gel entrapment that we associated with changes in aggregation state (21). In the same
study, we also showed that phycoerythrin could be entrapped in a thin film of the sol-gel
matrix on the surface of an optical fiber, allowing retention of its native optical properties.
The normal fluorescence spectrum was measured through the optical coupling between
the protein and the evanescent wave of the optical fiber.
We have also presented evidence for two-photon absorption-induced fluorescence from
phycoerythrin both in solution and entrapped in sol-gels (19,22). In this environment, the
protein's fluorescence, following two-photon absorption, is nearly identical to the fluores-
cence resulting from single photon absorption, as shown in Figure 1.9. Moreover, it is
