BR2 - ISCB - EPFL

Development of Enzymatic and Immuno-Bioreactor Based Biosensors for Analysis of
Pesticides and Herbicides in Water
Project ID:
BR2
Project
Duration:
1 April 2000 to 31 August 2004
Project
Coordinator:
Dr. K. Ravindranathan Thampi
Institute of Physical Chemistry (LPI-EPFL)
Swiss Federal Institute of Technology Lausanne (EPFL)
1015 Lausanne, SWITZERLAND
E-mail: ravindranathan.thampi@epfl.ch
Project
Partners:
Dr. C.R. Suri, Dr. Manoj Raje, Dr. G.C. Varshney
Institute of Microbial Technology (IMTECH)
Chandigarh – 160 036, INDIA
E-mail: raman@imtech.res.in
manojraje@yahoo.com
grish@imtech.res.in
Dr. N.G. Karanth
Fermentation Technology & Bioengineering Department
Central Food Technological Research Institute (CFTRI)
Mysore – 570 013, INDIA
E-mail: ferm@cscftri.ren.nic.in
ngkaranth@yahoo.com
CO-WORKERS:
Ms. Ashwini Hirlekar-Schmid, EPFL, Switzerland
E-mail: ashwini.hirlekar@epfl.ch
Ms. Jasdeep Kaur , IMTECH, India
E-mail: jasdeep@imtech.res.in
Mr. K.V.S. Rana, IMTECH, India
E-mail: kvsrana@imtech.res.in
Abstract
Organophosphorous and organochlorine pesticides are widely used in Indian agriculture.
Unfortunately, most of them are recalcitrant to biodegradation. As a consequence, pesticide
residues are found in the environment, in drinking water, and in crop food. Most of these
pesticides are toxic even at very low levels. Therefore, accurate monitoring of the hazardous
substances is essential for the health of the Indian population.
Consumption of selected insecticides in Indian agriculture (tonnes technical grade)
1993-94 1994-95 1995-96 1996-97 1997-98
Atrazine 200 300 400 325 400
2,4-D 850 850 850 800 800
Methyl Parathion 2'600 2'600 2'400 1'700 2'000
Monocrotophos 6'296 6'100 6'900 6'500 6'500
The currently available analytical methods are not suitable for quick and specific on-line
measurements in the field. Often, they require a complicated pretreatment of the samples.
Biosensors based on immunological principles are an attractive alternative to the
conventional technologies. In general, they are accurate, easy-to-use, specific, quick, and

available at a reasonable price.
Project Rationale
The overall goal of this project is to develop a simple, inexpensive immuno-biosensor, which
will be used in the form of a portable analyser for rapid field and on-line determination of
organophosphorous and organochlorine pesticides present in water at ng/l concentrations.
To achieve these goals, the following tasks will have to be tackled:
Development of two basic sample handling techniques, one for microreactor and one
for SPR.
Optimization of two detection techniques: fluorescence and SPR.
Research Plan
SUMMARY OF THE ACHIEVMENTS OF THE FIRST PROGRAM PHASE
adapted from the summary provided by the project partners

Synthetic pesticides and herbicides are widely used in agriculture. Many of these are
organochlorine and organophosphorous derivatives. Due to their recalcitrant nature, such
toxins enter the food chain, causing serious risk to humans and animals. Therefore, it is
essential to monitor their contamination in food, soil and water. The conventional analytical
methods are not suitable for quick and specific field analyses. They are also expensive and
laborious to apply. Biosensor based methods present an attractive alternative. In this
concluded project, the development of biosensors useful for monitoring pesticides and
herbicides was the prime objective. Biosensors based on enzymatic and immunological
principles and modern detection systems not only present an effective solution to this
vexing problem, but also a value-added product with good potential for commercialization
and gainful job creation. Manpower training, scientific exchange between India and
Switzerland and proliferation of knowledge between developed and developing countries
were other important benefits expected from this project. A joint project comprising four
different labs from India and Switzerland (IMTECH, Chandigarh; CFTRI, Mysore; EPFL,
Lausanne, institutes LPI and LCPPM; IMT, Neuchatel), having interdisciplinary and
complimentary expertise, was formed in order to develop sensitive, analyte specific, easy to
use and cost-effective biosensor devices for monitoring pesticide samples in aqueous media.
LPI-EPFL functioned as the overall project coordinator.
The partners chose 5 pesticides (insecticides and herbicides) for the biosensor development.
These were methyl and ethyl parathions, 2,4-dichlorophenoxy acetic acid (2,4-D), atrazine
and monochrotophos. The primary task was to develop good quality polyclonal antibodies
against these molecules. Secondly, appropriate assay techniques to individually detect up to
nano (ppb) or sub-nanogram (ppt) levels of these molecules in water were required. CFTRI
accepted the responsibility of developing polyclonal antibodies suitable for the 2 types of
parathions. Monochrotophos was kept as a molecule to be studied after completing the work
with parathions, since parathions are still widely used in India with serious harmful
consequences. IMTECH selected atrazine and 2,4-D as their target molecules, particularly
since the manufacture and use of these two compounds are rapidly increasing in India .
Apart from the development and characterization of antibodies, the Indian labs were also
involved in assay and transducer development. EPFL was involved in identifying appropriate
detection methods, antibody-antigen interactions and pre-concentration methods. The preselected
detection methods were surface plasmon resonance (SPR) and fluorescence. IMT ,
NE was involved in developing a Si (silicon) based micro-machined flow-device platform for
repeated multiple use. In order to select the best option, the efficacy of the two detection
methods was reviewed towards the end of the first two years, based on the actual
experience and performance data of the developed antibodies. SPR was abandoned at that
point, as this method was less sensitive with small molecules in comparison to fluorescence
techniques. However, the work in CFTRI and EPFL also showed that chemiluminescence
could be an equally competitive method for detecting pesticides in low concentration levels.
Therefore, fluorescence and chemiluminescence were finally chosen as the suitable detection
methods. Neverthless, at EPFL, SPR functioned throughout as an excellent workhorse in
characterising the generated antibodies and evaluating their purity and specificity.
IMTECH produced both polyclonal and monoclonal antibodies, which reached ppm limits of
detection. CFTRI clearly showed that large quantities of polyclonal antibodies against
parathions could be produced through egg-yolks of poultry. This method avoids the
traditional production of antibodies in rabbit blood. Both CFTRI and IMTECH used appropriate
conjugation chemistry for making haptens and optimized the conditions for obtaining best
quality antibodies. Hapten-protein conjugates with different hapten density were made by
chemical modification of selected organic toxins and subsequently linking covalently with
suitable carrier proteins (BSA and KLH). The developed conjugates were thoroughly
characterized by various methods such as UV, fluorescence, SDS-PAGE, MALDI etc., in order
to optimize the hapten-protein conjugation density. The suitable conjugates were used as
immunogens for the purpose of antibody generation. Specific antibodies were screened out
using various affinity purification techniques. Hapten columns were designed for getting
specific antibodies from the serum. Antibodies of different binding characteristics were eluted
using suitable elution buffers and characterized by ELISA, SPR and Western blot techniques.
These antibodies were used for different immunoassay formats.

In the case of 2,4-D and atrazine, highly sensitive hapten-fluorophore tracer molecules
using different fluorophores (FITC/RITC) and colloidal gold particles were made for their use
in fluorescence based immunoassay and dipstick applications. An on-line fluoroimmunosensor
was developed for the quantification of 2,4-D and atrazine in aqueous
samples. A noble hapten-protein-gold conjugate as detector molecule was also developed
for dipstick applications for the detection of atrazine and 2,4-D in water. The concentration
of antigen in test media was correlated with the change in intensity of the tracer signal. The
dipsticks were evaluated and tested under various field conditions. The transfer of this
technology to one USA company is under negotiation. Upon a request from the company, a
few dipstick samples and anti-atrazine antibodies were sent for evaluation purpose.
Chemiluminescence methods detected parathions with sensitivity up to sub-nanogram levels.
A critical factor in this case was the stabilization of the enzyme conjugate at nanomole
concentrations. Basic information on the production and immobilization of antibodies, and
data on several parametric optimizations of the bioreactor column and the resulting sensor
performance were generated. Several microreactors were designed and fabricated by IMT ,
NE. The microreactors' chambers were designed to obtain high packing homogeneity and
fluid distribution to prevent channelling effects. The final microreactor consisted in a flowthrough
chamber of approx 7 µl, at the end of which 6 interleaved rows of posts were
machined to retain the packing material. Prototypes were fabricated by Deep Reactive Ion
Etching (DRIE) to decrease the mesh size, to allow the use of Ø 25 µm beads and to further
improve the surface-to-volume ratio. The optical cell was integrated on a chip and consisted
in a simple flow through cell of 0.5 µl. Good packing/unpacking properties and flow
distribution were observed. Fluidic resistance was measured and was in good agreement
with the theoretical model. A number of cartridge prototypes were sent to the project
partners for evaluation. A generic fluid handling system was developed to interface the
chemiluminescent and fluorescent assays. External pumps and valves were selected to
control the incubation, the washing and the elution step. A user interface was developed
under a NI Labview platform to control the fluid motion and the data acquisition. An optical
system was also made to improve detection performances and robustness. This
configuration was evaluated against a bench top laser-induced fluorescence confocal
microscope, developed in-house. A detection limit of 0.2 ng/µL for fluorescein at pH 7.4 was
achieved with a simple light-emitting device. However, following the development of the
dipstick method, the microfluidic approach was suspended to better focus on the task and
achieve the target in the shortest time possible.
Besides the evaluation of detection methods and project coordination, investigations of
antibody-antigen interaction using SPR, AFM and microcalorimetry were carried out at EPFL.
IMTECH, CFTRI and EPFL jointly developed a method for the site-directed immobilization of
antibodies on gold substrates for SPR applications. A freshly prepared gold surface on a
glass carrier slide was modified with Protein A via a homobifunctional cross linker to achieve
uniform, stable and sterically accessible antibody coating. The modified gold surface was
found to be stable, repeatedly usable for several measurements and the experimental
reproducibility was good.
During the International Workshop on Biosensors held at CFTRI in 2003, the results of the
project were discussed and biosensor kits were demonstrated to international experts. The
progress of the project was well appreciated. It was felt that the results achieved so far
should be seriously pursued towards the construction of an integrated biosensor device for
immediate application and commercialization. In the light of the very positive results
obtained; a new project is therefore planned to fabricate integrated biosensor devices in
collaboration with suitable industrial partners.
In brief, at the close of the project, the partners have developed biosensor methods to
quantitatively detect parathions using chemiluminescent techniques up to sub-nanogram
levels of contamination. It must be mentioned that an immunobioreactor-based biosensor
for pesticides based on the chemiluminescence principle is being developed for the first time
to detect pesticides at ppt level. The high sensitivities of detection achieved in the project
promise excellent applications and exhibit very good potential for making future fieldapplicable
devices. For 2,4-D and atrazine, dipstick kits are now available for a Yes/No type

qualitative detection up to ppm levels. For parathions, polyclonal antibodies were produced
using egg yolk. As a result of this project, both polyclonal and monoclonal antibodies are
now available for atrazine and 2,4-D. There is a clear need for developing Yes/no type
dipsticks also for the detection of parathions at sub-nanogram level. Similarly, the existing
dipstick's sensitivity should be improved to sub-nanogram level. Further, field level
validation and industrial collaboration to make commercial prototypes are urgently required.
These issues and some more useful complexities will be addressed in the research plan of
the second phase, which is expected to run during the 2005-2007 period.