Biomedical Engineering Reference
In-Depth Information
different phycobiliproteins are variants of open-chain tetrapyrroles coupled to specific
protein residues through thioether linkages, as is shown in Figure 1.2. As optical elements,
these phycobiliprotein chromophores possess distinct advantages. These include intense
fluorescence, nearly 20-fold greater than that of a fluorescein molecule, high quantum
yields, and large Stokes shifts—some 2.7-fold greater than that found in fluorescein (9).
Because of these desirable properties, some of the phycobiliproteins have found commer-
cial uses as biochemical and biotechnological probes (10). Here we describe experiments
that demonstrated they retained their desirable optical properties after being surface
immobilized using a number of different biosensor compatible strategies. These include
binding to Langmuir-Blodgett (LB) monolayer films, entrapment within optically accessi-
ble sol-gel glasses, and binding to conducting polymers immobilized upon optical fiber
surfaces. Therefore, the phycobiliproteins have potential for use in biomaterials and smart
biosensor applications.
One of the earliest phycobiliprotein immobilization strategies we investigated was to LB
monolayer films. The LB technique creates a monolayer of amphiphilic molecules at the
air-water interface above an aqueous subphase within an LB trough device. In the first sys-
tem we investigated, the monolayer was created using a biotinylated phospholipid,
N
-bioti-
noyl-
O
-dipalmitoyl-
L
-alpha-phosphatidyl ethanolamine, triethylammonium salt (B-DPPE).
In Figure 1.3, we show representative pressure-area isotherms for LB trough compression
experiments where monolayers have been created by close packing the
B-DPPE molecules (3,11-14). In all cases, the isotherms displayed a relatively steep slope
above a pressure of 15 mN/m, which corresponds to an area/molecule of just over 100 A
2
.
This is approximately the two-dimensional (2-D) cross-section area of the B-DPPE molecule
oriented vertically at the air-water interface. Phycoerythrin (PE) was chosen for this
study, since its absorption spectra matched the 496.5-nm output of the argon ion laser used
for excitation in the detection scheme. We immobilized the PE by derivatizing it with either
streptavidin (Str) or avidin (Av) proteins. Both of these proteins are tetrameric and each of
the four subunits binds biotin. The biotin-derivatized ends of the closely packed B-DPPE
molecules in the LB monomayer film are hydrophilic and are oriented downward into the
subphase of the LB trough. The biotin of each B-DPPE molecule can bind each subunit of
HN-Cys-CO
S
H-O
2
-C
C-O
2
-H
H
3
-C
H
3
-C
H
H
FIGURE 1.2
Chromophores of two of the phycobilipro-
teins: (a) phycocyanin; (b) phycoerythrin.
The highly conjugated tetrapyrrole ring
systems of these two phycobiliproteins are
shown as well as their covalent thioether
linkage to a cysteine residue of the protein
amino acid side chain. Reprinted from
Beladakere, N.N., Ravindran, T., Bihari, B.,
Sengupta, S., Marx, K.A., Kumar, J.,
Tripathy, S.K. (1993). Photovoltaic Effects
and Charge Transport Studies in
Phycobiliproteins. In: Viney, C., Case, S.T.,
Waite, J.H., eds. Biomolecular Materials,
Proc. Mat. Res. Soc.
, 292:193-198. With per-
mission from the Materials Research
Society.
O
O
N
N
N
H
N
H
H
(a)
HN-Cys-CO
S
H-O
2
-C
C-O
2
-H
H
3
-C
H
3
-C
H
H
H
H
O
O
N
H
N
N
N
H
H
(b)
