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Abstract
A semi-automated chemiluminescent competitive immunosensor for the herbicide 2,4-dichlorophenoxyacetic acid
(2,4-D) is presented. Anti-2,4-D polyclonal antibodies are directly labelled with horseradish peroxidase allowing a
p-iodophenol enhanced chemiluminescent detection. Using antigen immobilised on UltraBind type pre-activated
membranes, the 2,4-D immunosensor exhibits low non-specific/specific binding ratio (maximum ratio: 5%) of the
labelled antibodies. The quantification of free 2,4-D in water is performed by co-injecting the sample and the labelled
antibodies in the flow system, incubating this solution with the antigen immobilised membrane and measuring the
amount of specifically bound labelled antibodies. Such an analytical system enables the detection of 4 mg l 1 of free
antigen in 20 min, and the 2,4-D detection is possible in the range 4 mg l 1 160 mg l 1. The immunosensor can be
regenerated by simply flowing a chaotropic solution (0.1 M HCl, 0.1 M NaCl, 0.1 M glycine) in the system. This
regeneration ability enables the achievement of more than 30 measurement cycles of free 2,4-D with the same antigen
immobilised membrane with a good reproducibility (RSD =12.5%). 2000 Elsevier Science B.V. All rights reserved.
Keywords: 2,4-Dichlorophenoxyacetic acid (2,4-D); Luminol chemiluminescence; Flow injection analysis; Immunosensor
1. Introduction
Enzyme linked immunosorbent assays (ELISA)
on microwell plates are the reference methods in
medical diagnostics for the detection of interesting
compounds. Conversely, the detection of small
toxic molecules in environmental and farm-produce safety, rarely involves such immunological
* Corresponding author. Tel.: +33-472-431397; fax: + 33472-442834.
E-mail address: loic.blum@univ-lyon1.fr (L.J. Blum)
0039-9140/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 2 9 8 - 2
396
2. Experimental
2.1. Reagents
Peroxidase (HRP, grade I, EC 1.11.1.7., from
horseradish, 250 IU mg 1) was supplied by
Boehringer Mannheim. Luminol (3-aminophthalhydrazide), 2,4-D, bovine serum albumin (BSA,
98% IgG free) were purchased from Sigma. Polyclonal sheep anti-2,4-D antibodies (5 mg ml 1)
were obtained from Europa Bioproducts (UK).
All other reagents were of analytical-reagent
grade. All buffers and aqueous solutions were
prepared with distilled demineralised water. For
chemiluminescence measurements, the reaction
medium was a 100 mM Tris buffer containing 30
mM KCl, 140 mM NaCl and adjusted to pH 8.5
with HCl 6 N. A luminol stock solution was made
as a 5.5 mM solution in 10 mM KOH and stored
at 4C.
397
398
Table 1
Sequence for a measurement cycle with the chemiluminescence-based 2,4-D immunosensor
Step
Regeneration
Sample mixing
Incubation
Washing
Light measurement
a
Washing
Reaction conditions
8
10
15
20
399
B
[B0 B]
(1)
400
performed over at least four decades of concentration, and was limited at high antigen concentration by the 2,4-D solubility in water. The mean B0
value for four replicates was 871 au with a standard deviation (SD) of 2 au giving a relative
standard deviation (RSD) equal to 0.2%. For a
2,4-D concentration of 4 mg l 1, the mean B value
of four replicates was 811 au (SD= 26 au, RSD=
3.2%).
In classical immunoassays, the limit of detection
is defined as the amount of free antigen generating
a signal variation equal to at least three times the
standard deviation of the B0 value. According to
this definition and taking into account the experimental values of B0 and of the related SD, a
theoretical detection limit of the order of 0.1 mg
l 1 could be calculated. However, as mentioned
above, a 0.4 mg l 1 concentration did not induced
a significant change in the measured chemiluminescent signal and it appeared more reasonable
to consider the 4 mg l 1 2,4-D concentration as the
true detection limit of the present system.
The current accepted level of free 2,4-D in water
is 0.1 mg l 1 (European Community). Consequently, the detection limit obtained with the
present system appeared insufficient. As mentioned above, the pre-incubation time is the main
parameter to be optimised to obtain low detection
limit. Previous works on 2,4-D immunodetection
The standard deviation for the first 33 measurements of the maximum signal (B0) was equal to
12.5%.
4. Conclusion
The present work demonstrates the efficiency of
the proposed membrane-based system for regenerable immunobiosensor developments. The approach developed in this study enables the easy
achievement of a rapid and sensitive immunosensor for the detection of the herbicide 2,4dichlorophenoxyacetic acid. Indeed, the detection
of 2,4-D was obtained within 20 min, which is a
low assay duration when considering immunochemical assays.
The performances of the present immunosensor
with a detection limit of 4 mg l 1, could be
considered as insufficient since the level actually
accepted by the European Community is 0.1 mg
l 1. Nevertheless, keeping in mind that the antibodies used are of polyclonal type, an immunosensor using monoclonal antibodies might
be able to reach the 0.1 mg l 1 critical detection
limit. Indeed, 2,4-D immunoprobes using monoclonal antibodies, usually exhibited lower detection limits (0.1 mg l 1 [5,10,11] and 0.01 mg l 1
[13]), than those involving polyclonal antibodies
(1 mg l 1 [7], 40 mg l 1 [12]).
Finally, the operational stability of the antigen
immobilised membranes and the reproducibility
of the method were demonstrated by the ability of
the sensor to perform, with the same sensing
layer, more than 30 measurement cycles with a
standard deviation of 12.5%. Most of the studies
concerning the imunodetection of 2,4-D did not
proposed a regeneration of the sensing layer (immobilised antibody or antigen) [5,7,11,13] and
401
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