Biomedical Engineering Reference
In-Depth Information
O - O
O - O
OCH
3
OCH
3
Alkaline
Phosphatase
Cl
Cl
O
−
OPO
=
3
CSP
O
D
CSPD
HPO
4
=
OCH
3
O
O
−
O
+
Cl
h
FIGURE 1.14
Reaction sequence starting with chloro 3-(4-methoxy spiro [1,2-dioxetane-3-2
-tricyclo-[3.3.1.1.]-decan]-4-yl)
phenyl phosphate (CSPD) that results in emission of a chemiluminescent photon from the unstable product
species. The reaction is initiated by dephosphorylation of the CSPD substrate by the enzyme alkaline phos-
phatase. Reprinted from Ayyagari, M.S., Kamtekar, S., Pande, R., Marx, K.A., Kumar, J., Tripathy, S.K., Kaplan,
D.L. (1994). Chemileuminescence-Based Pesticide Biosensor Utilizing the Intelligent Evolved Properties of the
Enzyme Alkaline Phosphatase. In: Rogers, C., ed.
Proceedings Second International Conference on Intelligent
Materials.
Technomic Press, pp. 85-96.
dioxetane ring that undergoes subsequent ring cleavage at the weak -O-O- bond in a sec-
ond step to form two separate product molecules. The unstable phenolate species then
emits a chemiluminescent photon over a broad wavelength range. Chemileuminescence, as
an optical process for biosensor use, has distinct advantages over absorption or fluores-
cence. To begin with, no light source is needed because the chemiluminescent molecule
already contains a photon energy equivalent to the free energy stored in its internal elec-
tronic structure. There is a concomitant advantage that no optical background source noise
can interfere with the chemiluminescent signal being detected, as is often the case with
widely used fluorescence detection schemes.
1.2.1.2.1 Organophosphorus Pesticide Detection
The organophosphorus pesticides were developed in the 1950s as an alternative to the
chlorinated pesticides (DDT and lindane) that were available first, but suffered from their
ecological persistence in soil. As a class, organophosporus molecules are considerably
more water soluble than the chlorinated compounds (41). Consequently, their continued
use poses a current threat to aquatic life. Given this situation, it is essential to be able to
monitor the concentrations of these compounds in various environments to determine
compliance with EPA regulations as well as the efficacy of remediation treatments (42).
With this in mind, we developed an enzyme-based biosensor that utilizes chemileumi-
nescence output for the quantitation of organophosphorus-based pesticides. Detection
was carried out in our biosensor design by the pesticide's inhibition of alkaline phos-
phatase's (AP) enzymatic catalysis of the CSPD substrate to its chemiluminescent reac-
tion product. Our initial studies characterized the solution kinetics of the
chemileuminescence output of CSPD catalysis by AP and then its inhibition by varying
concentrations of two representative organophosphorus pesticides—paraoxon and
methyl parathion (18,41,43). Using a Michaelis-Menten analysis of the kinetics, we
showed that a mixed competitive and noncompetitive inhibition was exhibited by the AP
