BR2 - ISCB - EPFL

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