Talanta 51 (2000) 395 401

www.elsevier.com/locate/talanta

Regenerable immunobiosensor for the chemiluminescent

flow injection analysis of the herbicide 2,4-D
Christophe A. Marquette, Loc J. Blum *
Laboratoire de Genie Enzymatique, UPRES-A CNRS 5013 -Uni6ersite Claude Bernard Lyon 1, Bat. 308 -43,
Bd du 11 no6embre 1918, 69622 Villeurbanne Cedex, France
Received 20 July 1999; received in revised form 30 September 1999; accepted 5 October 1999

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)

measurement but preferred the classical and well

known chromatographic (HPLC) analysis.
Nevertheless, these two methods are more and
more considered as time-consuming. Consequently, an increasing number of immunosensors
are described. Based on the antibody/antigen
recognition, rapid, simple and sensitive methods
are then developed for the measurement of a wide
type of target compounds such as bacteria
(Yersinia pestis), alphatoxin, ricin, brevetoxin [1]
and okadaic acid [2], pesticide such as atrazine [3]
and cocaine [4].

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

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C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

In classical ELISA tests, the problem of the

duration of the assays is overcome by the
concomitant measurement of multiple assays,
and the poor repeatability is compensate for by
performing a calibration within each series of
assays. On the contrary, the aim of immunosensor development is the achievement of a
system able to perform a single point determination without calibration between each measurement. The challenge is then to obtain an
immunosensor which on the one hand
exhibits good reproducibility and repeatability
and on the other hand could be regenerated in
order to minimise the consumption of expensive
reactants.
Various transduction systems, such as electrochemistry [1,4 6], potentiometry [7], capacitance [8], electrochemiluminescence [9] and
chemiluminescence [2,9], have been used
successfully in term of sensitivity and time consumed
for
immunosensor
developments.
However, the immunobiosensor stability, which
is for a great extent governed by the regeneration process, is often a critical point that
may impairs the reproducibility and the repeatability.
Recently, the use of new pre-activated
polyethersulfone membranes (UltraBind) for
immunosensor development was described [2].
These membranes allowed to minimise the nonspecific binding of antibodies on their surface.
They were then used for the development of a
chemiluminescent flow injection competitive immunosensor for the detection of the planktonic
toxin, okadaic acid. The performances of this
immunosensor was promising and thus, we investigated the possibility to develop with a similar
approach an analytical system for the detection of
another small target analyte, the herbicide 2,4dichlorophenoxyacetic (2,4-D) acid. Such a compound needs to be detected in drinking water at a
very low concentration (0.1 mg l 1 according to
the European Community specification) [10]
and, in order to avoid population intoxication,
the analytical procedure must be as fast as possible.
Several 2,4-D immunosensors have been described, involving different immobilisation sup-

ports such as glass capillary [10], nitro-cellulose

membranes [11], screen-printed electrodes [5],
photoactivated collagen membranes [7], glassy
carbon [9] or graphite electrodes [12] and different
transduction systems such as flow amperometry,
batch amperometry, pH-sensitive field effect transistor and electrochemiluminescence, respectively.
In every case, the quantification of the free 2,4-D
was performed by the determination of the label
enzyme activity. Depending on whether the antibodies involved were monoclonal [5,10,11,13] or
polyclonal [7,12], the detection limits obtained
were below or above the critical value of 0.1 mg
l 1. Nevertheless, only two of these works gave
the possibility to regenerate the immobilised
sensing element [9,10,12] and only the glass capillary-based sensor [10] was integrated in a flow
system.
In the work presented here, the flow injection
system developed, integrating a chemiluminescence-based fiberoptic sensor, enables the sensitive
and rapid competitive immunodetection of 2,4-D
in semi-automated conditions. Each step of the
immunoassay procedure, including the regeneration of the sensing element was realised in the
flow system.

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.

C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

2.2. Instrumentation and sensor assembly

The chemiluminescence measurements were
performed in a flow system and recorded on a
graphic recorder (Servotrace, Sefram). The flow
system consisted of a one-channel peristaltic
pump (model P-1, Pharmacia) connected to a
six-way connection valve (model 5011, Rheodyne), two injection valves (model 5020, Rheodyne) on which a 40 ml and a 200 ml sample loops
were fitted, and a specially designed flow cell with
a 250 ml inner volume (Fig. 1A) containing a
stirring bar (length 8 mm). A liquid core single
optical fibre from LOT Oriel, France (core diameter 5 mm, overall diameter 7 mm) was connected
to the photomultiplier tube of a luminometer
(Biocounter M 2500, Lumac). The light intensity
was expressed in arbitrary unit (au). In all measurements, the immobilised antigen membrane

397

was placed in close contact with the plexiglass

window (Fig. 1B).

2.3. Immobilisation of 2,4 -D on UltraBind

membrane
In order to covalently immobilise the 2,4-D on
pre-activated membranes, the antigen was first
conjugated to BSA, after a carbodiimide activation reaction. The 2,4-D was activated via its
carboxylic acid function by a pre-treatment in
1,4-dioxane at the concentration of 1.36 mg ml 1
in the presence of 3.9 mg ml 1 N-hydroxysuccinimide and 14.8 mg ml 1 N,N%-dicyclohexylcarbodiimide. After an incubation time of 15 min,
the dicyclourea precipitate was eliminated by centrifugation and 20 ml of the supernatant was
added to 500 ml of a 10 mg ml 1 BSA solution in
0.1 M carbonate buffer, pH 11. The obtained
solution was then incubated under stirring for 3 h
at room temperature for the coupling process to
be completed. The formed BSA-2,4-D conjugate
was then separated from the non-reacted species
on a desalting chromatography column (Sephadex
G-25 M). The conjugate was stored in PBS buffer
containing 0.1% sodium azide (w/v) at 4C.
The BSA-2,4-D conjugate was covalently immobilised on a 11 mm diameter preactivated UltraBind disc by dipping the disc for 10 s in a 1.7
mg ml 1 conjugate solution in PBS and drying it
15 min at room temperature. The discs were then
washed, first 10 min in PBS then, 20 min in PBS
containing 4% BSA (w/v) (PBSA), and finally 10
min in PBS. The immobilised BSA-2,4-D membranes were stored dry at 4C.

2.4. Labelling of the anti-2,4 -D polyclonal

antibodies with horseradish peroxidase

Fig. 1. Scheme of the immunochemiluminescent set up. A:

flow system; CL, chemiluminescent substrate solution; FC,
flow cell; FO, optical fibre; M, measurement buffer; R, regeneration solution; W, waste. B optical fibre/flow cell interface.

Horseradish peroxidase undergone a periodate

activation process of its carbohydrate moiety before reacting with the anti-2,4-D polyclonal antibodies. One point four milligrams of peroxidase
was dissolved in 350 ml of distilled water and
incubated for 20 min with stirring with 70 ml of
21.5 mg ml 1 sodium periodate in water.
One hundred and fifty microlitres of the activated peroxidase solution was then added to 200

C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

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

Additional duration (min)

0.1 M glycine, 0.1 M HCl, 0.1 M NaCl

0.28 ml min1, 500 rpm
Buffera, 0.28 ml min1, 500 rpm
Stop flow, 500 rpm
Buffera, 2.8 ml min1, 1000 rpm
Buffera, 0.11 ml min1, 1000 rpm

8
10
15
20

100 mM Tris-buffer, pH 8.5 containing 30 mM KCl and 100 mM NaCl.

ml of the polyclonal anti-2,4-D antibodies stock

solution (5 mg ml 1 in PBS). One hundred microlitres of a 0.1 M carbonate buffer pH 11 were
added and the solution allowed to react 90 min
under stirring at room temperature. After that
time, the labelling process was complete and the
antibody/peroxidase solution was desalted by
chromatography (Sephadex G-25 M).
The peroxidase-labelled antibodies were then
separated from the unbound enzyme with a
freezyme purification kit (Pierce). The antibodyenzyme bonds were then stabilised by the addition
of 0.2 mg ml 1 sodium borohydride and stored in
PBS, pH 7.4 at 4C.
The peroxidase-labelled antibodies were characterised by spectrophotometric absorbance at 278
1 1
nm (total protein: oAb
]
cm 1,
278 =[1.4 mg ml
1 1
1
Pod
o278 = 0.61 [mg ml ]
cm ) and 402 nm (per1 1
oxidase specific wavelength: oPod
]
402 =2[mg ml
1
cm ). A ratio of about 1:1 in mole of peroxidase
per mole of antibody was found.

rate of 2.8 ml min 1 and the stirring fixed at 1000

rpm during 5 min. A 0.11 ml min 1 flow rate was
then applied, the substrate solution (final concentration: 0.22 mM luminol, 0.5 mM H2O2, 0.4 mM
p-iodophenol) was injected with the 40 ml injection loop and the chemiluminescent signal was
recorded.
At that time, the regeneration solution (0.1 M
glycine, 0.1 M HCl and 0.1 M NaCl) was circulated at a rate of 0.28 ml min 1 during 8 min,
with the stirring fixed to 500 rpm. Afterwards, the
cell was washed 2 min by switching on the measurement buffer flowing stream at 0.28 ml min 1.
The immunosensor was then ready for a new
measurement cycle.

2.5. Flow injection immunoassay procedure

The non-specific binding of antibodies is the

major source of problems in the immunosensor
development. As a matter of fact, an efficient
washing is generally time consuming, increasing
the total duration of the measurement cycle. In a
recent work, we showed that non-specific binding
could be easily overcome, without extensive washing and saturation steps, by injecting the antibodies in a complex matrix solution [2]. In the present
study, a 4% BSA (w/v) solution in PBS (PBSA
4%) is used as a non-specific binding blocker. Fig.
2 presents the signal intensities obtained after the
injection in the flow system of labelled antibody
solutions at different concentrations, from 0.2 to

All free 2,4-D measurements were performed

using the same procedure (Table 1). Peroxidase
labelled anti-2,4-D antibodies were diluted in the
sample mixing cell, filled with 250 ml of PBSA and
10 ml of a free 2,4-D solution in PBS. This solution was mixed 5 min at room temperature and
injected in the flow cell by the 200 ml injection
loop. The flowing stream, composed of a 100 mM
Tris buffer at pH 8.5 and containing 30 mM KCl
and 140 mM NaCl, was stopped 5 min and the
stirring was then fixed at 500 rpm. After this
incubation time, the flow was switched on, at a

3. Results and discussion

3.1. Specific and non-specific binding of the

labelled antibodies

C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

399

concentrations, from 0.2 to 2.4 mg ml 1. This plot

enables the selection of an antibody concentration
value compatible with immunosensor requirements, i.e. an antibody concentration as low as
possible giving a measured signal as high as possible. Thus, an anti-2,4-D labelled antibody concentration of 1.2 mg ml 1 was chosen because the
signal obtained at this value, equal to 780 au,
corresponded to a signal to noise ratio of 280.
This allowed to obtain a high sensitivity for the
measurement of the signal variations as required
for the competition reaction quantification.
Fig. 2. Specific and non-specific binding of the anti-2,4-D
peroxidase labelled antibodies in the flow cell. Specific ( ) and
non-specific () signal. Insert: specific and non-specific signal
at low antibody concentrations.

48 mg ml 1 in PBSA 4%, using the immunosensor

assembled either with an immobilised BSA-2,4-D
membrane or with a membrane bearing only
BSA. As it can be seen, the differences between
the corresponding specific and non-specific signals
are large, even in the presence of high antibody
concentrations. The higher non-specific/specific
signal ratio obtained was approximately 5%.
Moreover, such a low non-specific binding was
obtained without the need of a saturation step of
the membrane which should increase the duration
of the measurement cycle.
The inset in Fig. 2 shows the variation of the
specific and non-specific signals at low antibody

3.2. Competiti6e measurement of 2,4 -D

The competitive measurements of free 2,4-D in
water are performed according to the procedure
presented in Table 1. The length and the flow rate
of the different steps were determined previously
[2]. More particularly, it was shown that the
duration of the sample pre-incubation step with
labelled antibodies and that of the incubation of
this mixed solution with the immobilised antigen
membrane was long enough for acceptable immunosensor performances to be obtained. Indeed,
the longer the pre-incubation time, the lower the
detection limit. This lapse of time was thus an
important parameter to be optimised.
Samples containing free 2,4-D at different concentrations, from 0.4 mg l 1 to 160 mg l 1, were
injected in the sample mixing cell, pre-incubated
during 5 min with labelled antibodies at a concentration of 1.2 mg ml 1 and then injected in the
flow cell. For each concentration tested, four
replicates were performed. The calibration curve
obtained and its logit linearisation are presented
in Fig. 3. The logit representation is calculated as
described by Eq. (1), where B0 is the maximum
signal obtained in the absence of free 2,4-D, and
B the signal obtained in the presence of 2,4-D.
logit= ln

Fig. 3. Free 2,4-D calibration curve. Insert: logit linearisation.

Each point is the result of four replicate assays. Error bars
represent the standard deviation values.

B
[B0 B]

(1)

The results show that the presence of free 2,4-D

in water could be detected at a concentration of 4
mg l 1 and that a 0.4 mg l 1 concentration does
not induced a significant change in the measured
chemiluminescent signal. The detection could be

400

C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

[9] showed that increasing the pre-incubation time

from 5 to 30 min allowed to lower the detection
limit from 200 to 0.2 mg l 1.
The pre-incubation time of the free 2,4-D with
the labelled antibodies in the sample mixing cell
have been then increased to 30 min. Unfortunately, such a long pre-incubation time at 30C
induces a lost of the antibodies ability to bind
specifically the immobilised antigen, and no interpretable results could be obtained.

3.3. Regeneration and stability of the immobilised

antigen membranes
Fig. 4. Operational stability of the chemiluminescent 2,4-D
immunosensor. Maximum signal B0 ( ), assay signal B ()
([2,4-D]=40 mg l 1). The arrows show the storage over night
at 4C. The solid line represents the mean value and the dotted
lines represent the mean value 9SD.

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 regeneration of the sensing element is a

critical point when considering the use of immunobiosensors, contrary to the classical ELISA on
microwell plates with which the reactant immobilised on the solid phase is used only once. The
achievement of a regenerable antigen support appears then as an absolute requirement for the
development of a reliable immunosensor. It has
been shown previously that the anti-2,4-D antibodies/immobilised antigen complex dissociated
only with high difficulty and required then a 7 min
sonication treatment in HCl 0.1 M [9]. With the
present membrane-based system, the regeneration
of the immobilised antigen was obtained by simply
flowing through the cell a chaotropic solution
composed of 0.1 M HCl, 0.1 M Glycine and 0.1 M
NaCl.
The stability of the 2,4-D immunosensor was
studied by performing more than 35 successive
measurements and corresponding regenerations,
over a three-day period. For that purpose, the
maximum specific binding (B0) of the anti-2,4-D
labelled antibody was measured, i.e. in the absence
of free 2,4-D, and assays of free 2,4-D at the
concentration of 40 mg l 1 (B) was performed
approximately each ten measurement cycles.
As shown in Fig. 4, the 2,4-D immobilised
support appeared to be reusable at least 30 times.
No lost of specific binding ability was observed
before the 33rd cycle and a stable assay signal (B)
was obtained throughout this period. Moreover,
the two nights of dry storage of the immobilised
antigen membrane have had no effect on the
sensor stability.

C.A. Marquette, L.J. Blum / Talanta 51 (2000) 395401

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

only few realizations provide this possibility

[9,12]. The present work which enables the integration of the immunosensor in a flow injection
system and the easy regeneration of the immobilised compound appears then to be an interesting development.
The use of pre-activated polyethersulfone membranes for the sensing layer elaboration in association with a chemiluminescent detection system
appears to be powerful for the development of
regenerable immunobiosensors. The total automation of the system, in order to obtain a better
reproducibility, and the extent to other analytes
and other test formats, such as sandwich tests, are
now under investigation.

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