572
H.-S. Lee et al. / Chemosphere 46 32002) 571±576
ꢀ
1999) and amperometric -Palleschi et al., 1992; Skladal,
1992; Skladal and Mascini, 1992; Bernabei et al., 1993;
phthalate from Aldrich -Milwaukee, USA). Sodium
tetraphenylborate, acetylcholinestrase -EC3.1.1.7, V±S
type from electric eel, 1000±2000 units/mg protein),
acetylcholine chloride, glutaraldehyde -25% aqueous
solution), pyridine-2-aldoxime methiodide, HEPES
buer and aminopropyl CPG -200±400 mesh, average
ꢀ
Marty et al., 1993; Cagnini et al., 1995; Martorell et al.,
1997; Palchetti et al., 1997) biosensors were developed.
The ¯ow injection system oers the advantages of
highly reproducible timing and high sample throughput
and eliminates the need to attain a steady state -Hansen,
1993). However, ChE-based biosensors developed to
determine OPs were mostly of the probe-type and de-
velopment of the ¯ow injection-type has been rare
-Kumaran and Tran-Minh, 1992). In our previous work,
we developed a ¯ow injection-type ChE sensor that
ꢁ
pore size of 500 A, average amine content of 70 lmol=g)
were obtained from Sigma -St. Louis, USA). The 10 OPs
used -parathion-ethyl, parathion-methyl, fenthion, feni-
trothion, diazinon, EPN, chlorpyrifos, dichlofenthion,
bromophos-ethyl, and bromophos-methyl) were pur-
chased from Dr. Ehrenstorfer GmbH -Augsburg, Ger-
many). Bromine was obtained from Junsei Chemical
-Tokyo, Japan). Analytical TLC-silica gel F 254, ¯uo-
rescent) and preparative TLCplate -silica gel 60, ¯uo-
rescent, 1 mm) were from Merck. All other chemicals
that were used were of reagent grade quality or higher.
consists of a tubular H -selective membrane electrode
and a reactor with AChE immobilized on controlled
pore glass -CPG) -Chung et al., 1998). AChE catalyzes
the hydrolysis of acetylcholine to yield choline and acetic
acid. Thus, this potentiometric ChE-based biosensor can
determine ChE activity by measuring the pH change
that takes place with a H -selective electrode. In this
2.2. FIA manifold
paper, we describe the application of this sensor to the
analysis of ten OPs to examine the general applicability
of the sensor to OP analysis.
The con®guration of the FIA manifold is shown in
Fig. 1 -Chung et al., 1998). The carrier stream was
pumped through the FIA manifold by a multi-channel
peristaltic pump -Ismatec model IPC). Samples were in-
jected into the carrier stream using a four-way rotary in-
jection valve -Rheodyne). The decrease of pH in the
sample plug was sensed with a homemade membrane
electrode and a double junction reference electrode -Orion
90-02), in combination with a pH/mV meter -Mettler
model 340), and was recorded on a ¯atbed recorder
-Kipp & Zonen model BD 111). The carrier consisted of
a HEPES buer, pH 7.2, containing 20 mM MgCl2 and
100 mM NaCl. This buer solution will be referred to as
``HEPES working buer'' in the text. Reactors were as-
sembled by packing AChE-immobilized CPG in a 2.06
mm i.d. Tygon tubing. The components of the system
were connected with 0.89 mm i.d. Tygon tubing.
There have been several reports that OPs show
stronger inhibitory action when their thiophosphoryl
group, ꢀRO2Pꢀ@S±, is oxidized to a phosphoryl group,
ꢀRO2Pꢀ@O± -Fallscheer and Cook, 1956; Chambers
and Chambers, 1991). Therefore it was thought that
conversion of OPs to the oxidized form -oxons), prior to
analysis, would improve the sensitivity of ChE-based
sensor. Kumaran and Tran-Minh -1992) utilized this
observation to improve the sensitivity of their AChE
sensor, which they developed for the determination of
OPs. They used bromine as the in situ oxidant for the
aqueous sample solutions and injected the oxidized
sample solutions directly into the FIA system. In their
study, the eciency of the oxidation reactions was un-
known and the possibility of enzyme inhibition by cer-
tain undesirable products, including the bromine species,
was not addressed. Thus, it is uncertain whether or not
the enhanced sensitivity, observed in their study, resulted
purely from oxidation. In this paper, we describe the use
of our ChE-based sensor for the comparison of the in-
hibitory power of ten OPs with their corresponding ox-
ons. To examine the possibility of repeated use of the
proposed sensor by reactivation of the inhibited sensor
enzyme, the degree of reactivation of the inhibited en-
zyme by pyridine-2-aldoxime -2-PAM) was also studied.
2.3. Construction of the detector, H -selective membrane
electrode
Construction of the detector, a tubular H -selective
membrane electrode -Meyerho and Kovach, 1983;
Chung et al., 1998), was as follows. A casting solution
for the H -selective membrane was prepared by dis-
solving 20 mg of tridodecylamine, 132 mg of dioctyl
phthalate, 1.4 mg of sodium tetraphenylborate, and 51 mg
of PVCin 1 ml of THF. An 18 gauge syringe needle was
inserted into 50 mm Tygon tubing -i.d. 0.64 mm) and a 5
mm hole was cut out lengthwise with a razor blade.
Onto the cut-out region were poured 6 drops of the
casting solution with the THF evaporated after each
addition. After pulling out the needle, the membrane-
attached tubing was inserted through the two holes
drilled into the wall of a plastic centrifuge tube of 1.5 ml
capacity -outer jacket) and the holes were sealed with a
2. Materials and methods
2.1. Chemicals and instruments
Polyvinyl chloride -PVC, high molecular weight),
tridodecylamine and tetrahydrofuran -THF) were pur-
chased from Fluka -Buchs, Switzerland) and dioctyl