Biomedical Engineering Reference
In-Depth Information
in the presence of varying concentrations of either inhibitor—paraoxon or methyl
parathion. Without attempting serious optimization of the system, the lower limits of
detection in solution via this inhibition approach was determined to be 50 ppb for
paraoxon and 80 ppb for methyl parathion.
Next, we studied this system after carrying out immobilization of the AP onto the inte-
rior walls of small-bore and small-volume glass capillaries using a biotinylated conducting
polymer attachment mechanism. This method had the advantage of being technically sim-
ple, involving a few dipping steps of the optical surface into a succession of solutions
(17,44,45). We show a schematic of the self-assembly process system in Figure 1.15. The
interior wall glass surfaces were first silanized with chlorodimethyloctadecyl-silane and
then stable hydrophobic interactions were formed in a second step with the surface bind-
ing of the conducting copolymer poly(3-undecylthiophene-co-3-thiophenecarboxalde-
hyde)6-biotinamidohexanohydrazone (B-PUHT). Within this immobilized polymer, the
biotin ligand is pendant in solution because of a long hydrophilic spacer group attaching it
to the polymer backbone, making the ligand readily available for efficient streptavidin
binding. Following the streptavidin derivatization of AP to form Str-AP, this complex was
then bound to the immobilized copolymer in a third step, completing the core of the biosen-
sor. In a variation of the polythiophene polymer attachment method just discussed, we first
silanized the glass surface with 3-aminopropyl trimethoxysilane, which created a pendant
amino group. Polythiophene was then grown in situ by chemical polymerization upon the
pendant amino group, before a final biotinylation step (46). In all of these experiments, the
small-bore capillary was used for convenience since it provided a high surface to volume
ratio. The long length presented a large surface area for immobilizing a significant number
of Str-AP molecules and the small bore and interior volume (100
L) brought the CSPD
everywhere in solution into close proximity to the surface-immobilized AP for enzymatic
catalysis to form the product that emitted chemiluminescent photons. Using this simple
set-up, we detected the activity of as little as 0.1 fmol of AP. In Figure 1.16, we present data
S
CH=N-NH-CO-(CH
2
)
5
-NH-CO-(CH
2
)
4
S
CH
3
Si
HN
NH
O
S
CH
3
O
S
CH=N-NH-CO-(CH
2
)
5
-NH-CO-(CH
2
)
4
S
CH
3
Si
O
HN
NH
S
CH
3
O
n
Silanized glass
FIGURE 1.15
Schematic representation of the hydrophobic interaction-based surface modification of alkyl-chain-silanized
glass by the undecyl chain of the biotinylated 3-thiophene copolymer, B-PUHT. Reprinted with permission from
Pande, R., Kamtekar, S., Ayyagari, M.S., Kamath, M., Marx, K.A., Kumar, J., Tripathy, S.K., Kaplan, D.L. (1996). A
Biotinylated Undecylthiophene Copolymer Bioconjugate for Surface Immobilization: Creating an Alkaline
Phosphatase Chemileuminescence-Based Biosensor.
Bioconjugate Chem.
7:159-164. Copyright (1996) American
Chemical Society.
