Development of a Capacitance based Immunodetection
Analyzer for the Assay of GMO’s in foods.
Contract Nr: QLK3-2002-02141
Period: Jan’03 – Jan’06
Project website address: http://users.uoi.gr/imagemo/
Coordination
T. Albanis
University of Ioannina (GR)
Partners
B. Hock
Technische Universität München (DE)
C. Martelet
Ecole Centrale de Lyon (FR)
P. Millner
University of Leeds (UK)
C. Efstathiou
University of Athens (GR)
T. Frelink
ECO CHEMIE B.V. (NL)
M. Pighini
TECHNOBIOCHIP S.c. a r.l. (IT)
Th. Michas
SKYLAB-MED S.A. (GR)
D. Koraki
ENGENE S.A. (GR)
Background and Objectives
The intensive public debate on the application of genetic engineering to the
production of food has led to a high degree of uncertainty, both among
consumers and within industry. There are two methods in general commercial use
today for detecting GMOs in field crops. One method involves the detection of
proteins that have been produced through biotechnology into the crop; the other
is based on the detection of DNA sequences inserted in the crop. ELISA and
Lateral Flow Strip methods are used for protein assay and PCR (Polymerase Chain
Reaction) and Southern Blot for DNA.
The aim of the project was to develop from concept a generic capacitance
based immunodetection analyzer for GMO related proteins. CP4 EPSPS (CP4synthase), and Cry1(A)b-protein are employed as relevant model targets, which
correspond to “Roundup Ready (RR)-Soy” and “Bt-Maize” GMOs, respectively. To
achieve the overall objective, the production of monoclonal antibodies specific to
the target proteins, the design and fabrication of a portable analyzer able to
monitor interactions between biomolecules, and the development of different
impedimetric electrode architectures were conducted in parallel.
Considering problems associated with the detection of the target proteins in
highly processed foods (e.g. extensive changes in conformation of the antigens),
the development of antibodies against characteristic peptide sequences as well
the development of an analyzer based on Quartz Crystal Microbalance (QCM) able
to monitor the hybridization between nucleic acids complementary strands were
also decided from the early meetings of the project already. In addition, studies
on PCR-based methods and the use of new primers were also included among the
objectives of the project.
Approach and methodology
To achieve the aforementioned objectives, different types of capacitive
immunosensors based on metal/metal oxide electrodes, on mixed self-assembled
monolayers onto gold electrodes and on antibody/amphiphile Langmuir–Blodgett
films deposited onto hydrophobic surface of 1-octadecanethiol (ODT) modified
silver electrodes were developed.
The development of MAbs specific to the target proteins was attempted by
following different immunization strategies: i) antigens were extracted and
purified from crude plant material containing CP4 EPSPS and Cry1Ab antigens, by
applying consecutive purification schemes consisting of ion exchange and size
exclusion chromatography ii) specific peptides, which described different families
of Cry-toxins and CP4-synthase were synthesized and coupled to neutravidin as
an immunogenic carrier protein. In addition we pursued the synthesis of
recombinant antibodies against GMO peptides. The rational behind the latter
approach was the availability of single pot libraries and that no immunization
procedures are required, which essentially saves time.
The design, construction and optimization of a stand-alone, low-cost electronic
device able to monitor interactions between biomolecules that may change the
capacitance of an electrode were also carried out. Particular attention was paid to
reduce or to eliminate the constant (dc) component of non-capacitive current
flowing through the cell, which is observed in electrodes characterized by a
dielectric layer of relatively low resistance.
For the development of a QCM-based method for probing DNA interaction, a
commercial QCM analyzer, μ-Libra was employed and the method set up was
performed using Cry1A(b) gene. Using the Bt-176 DNA sequence as template, a
200-bp fragment of the Cry1A(b) gene was amplified, and purified. The yield of
the PCR fragment was further cloned using the pGEM®–T Easy Vector System.
After the vector digestion (EcoRI) and fragment purification, the Cry 1A (b) gene
was labelled with biotin, and used as probe to functionalize the quartz surface.
Main findings and outcome
The so-produced capacitive immunosensors were tested with pilot antibodies
and found to be sensitivity with limit of detection of ng/mL of the pilot antigen. To
reduce the cost per sensor and in order to create a geometry suitable for in-field
measurements in combination with the palm-sized analyzer, the so called,
Multipulser, intedigitated screen-printed gold electrodes arrays were designed and
fabricated.
The applicability of the sensors to GMO specific proteins was restricted to the
low affinity of the antibodies, which have so far been produced. Screening of
hybridoma cell lines based on different peptide sequences is currently in progress.
However results are expected to be available after the completion of the project.
The operation of Multipulser is based on the repetitive charging of the
electrochemical cell capacitance by applying a predetermined number of shortduration, low-amplitude voltage pulses (perturbation pulses) (Figure 1). All
packets of charge are accumulated in an analog integrator whose output voltage
is proportional to the cell capacitance. Multipulser features three user-selectable
operating modes. Each of them is characterized by its own particular shape of the
applied perturbation pulses. “Mode 3” seems particular interesting for electrodes
characterized by a dielectric layer of relatively low resistance, as it effectively
reduces or eliminates the contribution of this dc current component to the
measured signal. Multipulser was successfully used to monitor the binding of
biotinylated molecules on two different avidin modified electrode assemblies; one
based on a thiol SAM's on gold and another based on Ti/TiO 2 semiconductor.
Measurements conducted in parallel with a commercial frequency response
analyzer gave similar reaction patterns.
Figure 1. Appearance and external controls and connectors of the
Multipulser analyzer.
The analytical performance of the QCM-based DNA analyzer was evaluated
using IRMM-413 certified reference materials. Total DNA extracted from IRMM
products of dried maize powder with different mass fractions (from 0.1% to 5%)
of genetically modified MON 810 maize flour have been analyzed by DNA-sensor.
The resonance frequency shift versus the different % of GMO maize flour DNA is
shown in Figure 2. The applicability of the analyzer in real samples was tested by
extracting total DNA from transgenic cookies (certified genetically modified MON
810 maize flour c.f. Figure 3).
Figure 2. Calibration curve for the QCM-based DNA sensor.
Figure
3.
Real-time
hybridization
between
biotinylated
Cry1A(b)
fragment and Transgenic Biscuit DNA.
In the frame of the project, a PCR based method for GM soy and maize was
also developed and validated. For this purpose a 35S primer set was designed,
and the successive procedures of sample extraction, preparation and analysis
were validated. Validation experiments were performed in various Certified
Reference Materials and commercial samples. Finally, commercial samples were
screened for the presence of GM Soya and GM Maize. Samples positive for the
presence of 35S were analyzed by REAL TIME PCR (TaqMan™) for quantification
of GM percentage.
Conclusions
This
project
provides
information
on
the
development
of
capacitive
immunosensors, novel instrumentation for the monitoring of interactions between
biomolecules and methods based on QCM and PCR for the detection of GMOs.
Publications
Y. Hou, C. Tlili, N. Jaffrezic-Renault, A. Zhang, C. Martelet, L. Ponsonnet, A.
Errachid, J. Samitier, J. Bausells “Study of mixed Langmuir-Blodgett films of
immunoglobulin G/amphiphile and their application for immunosensor engineering”
Biosensors and Bioelectronics, 20 (6) (2004) 1126-1133
T. Chaker, Y. Hou, H. K. Youssfi, L. Ponsonnet, C. Martelet, A. Errachid, N.
Jaffrezic-Renault «Impedancemetric probing of mixed amphiphile-antibody films
transferred onto silver electrodes” Sensors Letters 2 (2005) 1-6
A.G. Mantzila, M.I Prodromidis “Development and Study of Anodic and Thermal
Ti/TiO2 Electrodes. Potential Use as Impedimetric Immunosensors”, Electrochimica
Acta in press (2005)
A.G. Mantzila, M.I. Prodromidis “Performance of Anodically Formed Ti/TiO2
Based Architectures as Impedimetric Biosensors”, Electroanalysis 17 (20), 1878
(2005)
S.D Bolis, P.C Charalambous, C.E Efstathiou, A.G Mantzila, C.A Malamou, M.I
Prodromidis « Monitoring of the Avidin – Biotinylated Dextran Interaction on Au
and Ti/TiO2 Electrode Surfaces Using a Charge Integrating Device”, Sensors and
Actuators in press (2005)
H.C.W Hays, P.A Millner M.I Prodromidis “Development of Capacitance based
Immunosensors on mixed-seld-assembled monolayers” Sensors and Actuators in
press (2005)
M. Passamano, M. Pighini “QCM DNA sensor for GMOs detection”. (Submitted
to Sensor & Actuators B)
M.I.Prodromidis “Development of a kit for the construction of Voltammetric
Electrodes”, Patent GR200401001172