Rationally Designed Aquatic Receptors integrated in label-free biosensor platforms for remote surveillance of toxins and pollutants

Projektinformationen

RADAR

ID Finanzhilfevereinbarung: 265721

Projekt abgeschlossen

Startdatum 1 Januar 2011

Enddatum 31 Dezember 2014

Finanziert unter

• Specific Programme "Cooperation": Food, Agriculture and Biotechnology

Gesamtkosten € 3 896 412,00

EU-Beitrag € 2 926 127,00

Koordiniert durch

CSEM CENTRE SUISSE D'ELECTRONIQUE ET DE MICROTECHNIQUE SA - RECHERCHE ET DEVELOPPEMENT
Switzerland

Final Report Summary - RADAR (Rationally Designed Aquatic Receptors integrated in label-free biosensor platforms for remote surveillance of toxins and pollutants)

Executive Summary:
RADAR is a 7-member consortium that has developed in the past four years a robust, sensitive, and versatile biochemical sensor platform for spot measurements and on-line monitoring of toxins and pollutants, with a focus on Endocrine Disrupting Compounds (EDCs) and Polycyclic Aromatic Hydrocarbons (PAHs), in food production processes and in the aquatic environment.
One originality of the project was the design and production of aquatic organisms derived receptors i.e. proteins, derived from aquatic organisms, that recognize and respond to a specific class of pollutants and toxins. These receptors were designed on the basis of the in-vivo occurring receptors affected by the presence of the class of pollutants targeted. The targeted receptors, namely the Estrogen Receptor (ER) and the Aryl Hydrocarbon Receptor (AhR), are sensitive not only to a single toxic molecule, but to an entire class of potentially hazardous molecules. To the best of our knowledge, RADAR has produced the first
• Aryl Hydrocarbon Receptor from aquatic organisms with highest affinity towards Polycyclic Aromatic Hydrocarbons (PAHs), e.g. epigallocatechin gallate, indole 3,2 b-carbazole (I32bC), or 6-formylindole-3,2 b-carbazole (FICZ).
• Estrogen Receptor and genetically engineered mutants from aquatic organisms with an increase of their specificity (from uM to nM IC50), sensitivity (more than a factor of 5x) and robustness (shelf life longer than 9 months at -20C).
Another originality of the project was the unique combination of an automated filtration/ separation/ pre-concentration module with a novel detection module. The resulting platform has allowed attaining unsurpassed sensitivity in a continuous label-free monitoring platform towards toxins and pollutants, allowing for early detection of class-specific compounds thanks to
• The pre-concentration factor of up to 1000x as achieved for E2 (17- estradiol) in the automated filtration/separation/pre-concentration module in fresh and marine water samples.
• The integrated compact detection module based on a combination of WaveGuide Grating (WGG) biochips and the ARGOS reader. This module has reached comparable performance to best-in-class commercial label-free platform at a fraction of the cost.
• The novel assay format based on peptides specific to ER yielding conformational change upon ligand binding. Amplification of the signal lowered the limit of quantification of ER-ligand complexes down to the low ppb level (working range between1-10 ppb). For 17β-estradiol (E2), the LOD is 5 ppt with 200 times pre-concentration.
Finally, to the best of our knowledge, when combined with its wireless communication module, the RADAR platform is the first automated compact platform designed
• to perform label-free, robust, specific and sensitive detection of toxins and pollutants, and
• to send an alarm signal to a remote control station.
Several dozens of samples ranging from fresh to marine water originating from various locations such as sludge tank, input and output filters of fish tanks, sewage, were successfully processed and measured (with positive samples for E2 found in fish farm at the filter output and in the sludge tank), proving the versatility of the module for different real-life applications and proving the validity of the method and the platform for the detection of EDCs in food production processes and in the aquatic environment.

Project Context and Objectives:
RADAR stands for Rationally Designed Aquatic Receptors integrated in label-free biosensor platforms for remote surveillance of toxins and pollutants.

Project context
Environmental monitoring is becoming increasingly important as the number of compounds in use continues to increase. Within the last ten years, some of the 85,000 chemicals just in the United States have been found to coincidentally have hormonal effects, where the most common effect is to mimic estrogen (e.g. alkylphenols). Although the European Community has achieved major strides in environmental performance during the last two decades, threats of environmental damage and depletion still exist. It is estimated that more than 150,000 contaminated sites exist in the EU.
Furthermore, increased intensity of water use, discharge of untreated domestic and industrial wastes, excessive application of fertilisers, pesticides and insecticides in agriculture, and accidental spills of harmful substances have led to increased pollution of water-bodies throughout Europe. The importance of monitoring activities at polluted sites has gained attention from national authorities due to increased evidence of contamination from past improper waste management, industrial, and agricultural practices. Today, both, monitoring and characterization activities require water and food samples to be taken on-site and then analyzed at a laboratory using conventional analytical methods such as LC-MS, which is costly and time consuming.
Developing timely and cost-effective monitoring and characterisation technologies is critical, both to reduce costs to the European economy in addressing contamination problems, and to promote European competitiveness in global environmental and food markets. RADAR novel technology can contribute to the health and safety of the European population by reducing risks associated with contaminants in the environment and food.

Project objectives
The RADAR project aimed at developing biosensor platforms for the monitoring of toxins and pollutants in the environment and for the surveillance of production processes using aquatic organisms’ derived biological recognition elements. To achieve the goals, the RADAR project integrated various state-of-the-art technologies that advanced biosensors beyond the current state-of-the-art. It implemented sensitive, specific, and versatile biological recognition elements based on recombinant receptors derived from aquatic organisms on robust, label-free, multiplexed, remotely-controlled, and portable biosensor platforms with an integrated automated in-line sample preparation unit.
In this context, the RADAR consortium defined the project’s main objectives as:
Objective 1: To increase the sensitivity, specificity and versatility of biosensors using nanostructured surfaces and genetically engineered recombinant bio-receptors derived from aquatic organisms.
Objective 2: To provide a robust, label-free, remotely-controlled, and portable biosensor platform for cost-effective spot measurements and on-line monitoring with integrated fully automated sample preparation for non-experts.
Objective 3: To validate the RADAR biosensor and demonstrate its application for cost-effective spot measurements and on-line monitoring of toxins and pollutants in food processes and in the aquatic environment.

Objective 1: Increase the sensitivity, specificity and versatility of biosensors
Aquatic organisms have proteins capable of binding endocrine disruptive compounds (EDCs) such as estrogenic substances, polychlorinated compounds, polycylic aromatic hydrocarbons, steroids, pesticides, etc. Such proteins are ideal candidates as biological recognition elements for biosensors, since they will bind any EDC present in the analyzed sample. Rather than selecting a single protein and use it as is as biosensor, RADAR identified the particular structural and functional features required to bind EDCs through the use of genetic, structural and functional information, complemented by computational modelling tools and mutational experiments.
RADAR succeeded in the design, production, purification, and testing of the Estrogen Receptor (ER) and the Aryl Hydrocarbon Receptor (AhR). Both receptors have been successfully produced in E.Coli a low cost bacterial system with minimal requirements in terms of safety and containment of manufacturing processes. RADAR also succeeded in the production of rationally designed mutated receptors for the Estrogen receptor. Protein mutants have been designed by structural and genetic analysis with the intent of increasing the specificity and selectivity of the receptors so that they could be used to recognize and differentiate different classes of pollutants.
The characterization of expressed proteins was performed mainly in terms of i) purity, thorough SDS-PAGE; ii) structure stability, by means of Circular Dichroism Spectroscopy; and iii) ligand binding, using a fluorescence competitive binding assay. The in vitro testing and selection of best receptors by affinity binding measurements to selected toxins and pollutants was achieved, for ER, towards six selected compounds thanks to the commercially available PolarScreen™ ER Alpha Competitor Assay, Green.
In summary, RADAR has achieved the following results towards objective 1:
• Receptors for both Estrogen (ER) and Aryl Hydrocarbon (AhR) were successfully derived from aquatic organisms and genetically engineered to increase their specificity (from uM to nM IC50), sensitivity (more than a factor of 5x) and robustness (shelf life longer than 9 months at -20C). Moreover, they were expressed and purified in quantities compatible for application testing during the last 12 months of the project.
• Nanostructured surface were implemented with enhanced sensitivity through higher activity of receptors at the surface. Detection of 17β-estradiol (E2) binding to ER and EPGC binding to AhR was detected with the WGG sensors by direct detection for EPGC binding and indirect detection by peptide enhancement for E2 binding.
• Generic binding of the receptors to the surface was achieved via amine coupling as well as Ni(II)-NTA surface chemistries on several 2D and 3D surfaces (Optodex C, XC200, Surfix, CMD50) showing the versatility of the biochips.

Objective 2: Provide a robust, label-free, remotely-controlled, and portable biosensor platform
This objective combines multiple objectives providing a best-in-class biosensor platform for the versatile, specific and sensitive detection of toxins and pollutants:
1. Optimized Label-Free detection with nanostructured, chemically modified biochip
For the label-free biosensor, RADAR used an evanescent field based sensors (waveguide grating: WGG), transparent and non-metallic (tantalum pentoxide) tuned for a penetration depth of the evanescent wave matching surface chemistry and assay. The optical biochip is read with a novel Angle Interrogating Optical Sensor (ARGOS) that provides best-in-class sensitivity while keeping the expected versatility and cost effectiveness of a platform instrument.
WGG sensors were nanostructured in order to achieve best-in-class specificity and sensitivity to EDCs and therefore to improve the performance of the platform compared to state of the art ones. The physico-chemical properties of the surface were engineered at the nanoscale to control the spatial distribution, density and conformation of immobilized bio-receptors. The surface nanostructuring had two effects: Bio-receptors activity improvement thanks to proteins confinement on the nanostructure & Detection sensitivity improvement by creating nano-grating enhancing the evanescent wave coupling.
2. Easy to use sample preparation with integrated sample preparation unit (SPU)
The samples were collected and automatically prepared on a microfluidic device to increase the accuracy of the biosensors. The integrated sample preparation unit (SPU) had three functions, filtration/separation/pre-concentration integrated in an automated module for delivery to the ARGOS detection unit.
3. Remotely controlled capability via Wireless Connectivity
Our biosensor was equipped with a wireless connectivity module to inform users as quickly as possible about any contamination. Mobile telephony (GSM) challenge was to allow operation on batteries for very long period of time while being able to send alarms within minutes. New protocols with wake-up functions were implemented to achieve ultra-low power function.
In summary, RADAR has achieved the following results towards objective 2:
• Thanks to an automated filtration/separation/pre-concentration module, a pre-concentration factor of up to 1000x was achieved for E2 (17 estradiol). The module was tested with fresh and marine water samples showing the versatility of the module next to its sensitivity.
• An integrated version of the detector was successfully designed, constructed and tested that, by performing angle scanning, can be applied as SPR and WGG readers. The sensor module showed superior performance than best-in-class commercial WGG systems. Label-free was achieved by design of the WaveGuide Grating (WGG) based sensor biochip and the ARGOS reader. Cost-effectiveness was achieved both at the biochip level (by developing and testing biological regeneration procedures) and at the instrument level (by developing a compact platform with COGs at a fraction of the cost of the competitive products).
• Several dozens of samples ranging from fresh to marine water originating from various locations such as sludge tank, input and output filters of fish tanks, sewage, were successfully filtered, separated, and pre-concentrated, proving the versatility of the module for different real-life applications. Sensitivity and specificity to E2 were achieved down to 5ppt proving the validity of the platform for monitoring food production processes and in the aquatic environment.

Objective 3: Validate the RADAR biosensor and demonstrate its application
To demonstrate the full potential and applicability of the developed RADAR platform, fresh and marine water, fish feeds, urine, were collected and measured for EDCs.
In summary, RADAR has achieved the following results towards objective 3:
• Selected visits were organized with potential end-users and testing sites were identified for the project. Sample harvesting was accomplished by application partners to build a collection of samples that were subsequently analyzed.
• Several matrices (fresh and marine water, fish feeds, sewage) were successfully processed and measured, proving the versatility of the module for different real-life matrices.
• Most importantly, the platform was tested for several matrices for E2 (17 estradiol) allowing the identification of positive and negative samples confirmed by standard methods, validating the overall method and platform for monitoring of toxins and pollutants in food processes and in the aquatic environment.

Project Results:
RADAR aimed at developing a biosensor platform for the monitoring of complex biomolecules such as toxins and pollutants in the environment and for the surveillance of production processes by integrating the following advances:
• Specific and sensitive biological recognition elements based on recombinant receptors derived from aquatic organisms,
• Organized nanostructured, chemically modified substrates,
• Efficient sample preparation and pre-concentration modules,
• Compact and cost-effective label-free detection modules,
• Deployable remotely-controlled robust biosensor platforms
The complete scientific and technological development was designed around four poles of activity, corresponding to WP1-4 () which are centered to the system testing WP5 (Figure 4). The pole WPs can operate simultaneously, and are strongly interlinked and iterative. Each pole WP is in strong connection and collaboration with the other three pole WPs, and once the two prototypes based on WGG and SPR have been realized, they will finally be tested in WP5.

Hereafter the main S&T results and foregrounds are reported with a brief description of main activities. Further details are reported in the referenced deliverable documents. Deliverables and milestone description are also reported.



Work Package 1
Rationally designed recombinant receptors derived from aquatic organisms

WP Leader: JRC-IES

Executive Summary
WP1 was the key-starting step of the project which objectives were focused on
1. Production of two recombinant receptors namely the Estrogen Receptor (ER) and the Aryl Hydrocarbon Receptor (AhR) for the detection of chemical pollutants including toxin in complex matrices e.g. water, and food;
2. Rational design of modified ER and AhR receptors (mutants) in order to increase specificity and sensitivity towards class of pollutants.
The final goal of the WP1 was to provide rationally designed battery of recombinant ERs (wild type + mutants) and wild type AhR to be immobilized on surfaces (WP2), to be tested for their binding, sensitivity and specificity (WP4, WP5).
Production of Estrogen Receptor (ER) and Aryl Hydrocarbon Receptor (AhR) as recombinant receptors
This objective has been successfully achieved. RADAR could express, purify and produce high yield of the two recombinant receptors in Escherichia coli, a very cheap heterologous system (see D1.1 D1.2 and D1.2 D1.4 for ER and AhR, respectively). Characterization of the two recombinant receptors was performed to test the ligand binding activity and stability, two key parameters required for the immobilization on surface.
Estrogen receptor characterization has been quite easy since an in vitro binding assay is commercially available which confirmed the ability of binding, first to the natural ligand binding (17β-estradiol) and then to several class of compounds (see D1.5). The recombinant ER also has been also tested for complex matrices such as chemical mixture showing its binding ability.
For the AhR, a setup of the binding assay has been required and therefore took more time, the first approach has been the development of an assay similar to the ER assay based on fluorescence ligand molecule as described in D1.6. Since among the four designed molecules, no positive results could be seen, an assay was setup based on radio-ligand [3H] dioxin (TCDD), which has a strong binding affinity for the AhR to verify the activity of AhR. The results, shown in the amended D1.7 confirmed the ability of the recombinant AhR to bind to the ligands.
Rational design of modified ER and AhR (mutants)
To generate rationally modified recombinant receptors, computational analysis was carried out to identify the amino acids to be mutated in order to modify the affinity and therefore the specificity toward class of pollutants.
As predicted in the project, this approach has been considered as high risk for the AhR since no crystal structure is available, comparing to ER foreseen as low risk due to well-known crystal structure which provides which amino acids interact with the ligand. The outcome of this objective has confirmed the prediction, indeed RADAR could succeed for ER and only partially for AhR.
Based on computational analysis three mutants for ER have been identified which were experimentally expressed and purified (D1.4). Their ability to actively bind to the ligands have been tested as described in deliverable D1.5 and in the peer-reviewed publication (Ferrero et al., PloS One 2014). Particularly one ER mutant showed an increased affinity for some class of pollutants designated as the best mutant receptor.
A more complex analysis has been performed for AhR, due to the lack of crystal structure, models have been considered to predict potential amino acids involved into the ligand binding (D1.7). Some potential mutations were identified and the mutants generated; however the expression did not work, probably an expression protocol needs to be set up.
Under the WP1, Milestone 1.1 1.2 1.3 (partially) and M6.1 have been achieved.

Main Tasks
Identification of ligand binding domain for the EDCs through genetic, structural and functional comparison: Identification of ligand bind domain was achieved for both, estrogen receptor (ER) and Aryl Hydrocarbon receptors (AhR). They are the recombinant receptors in RADAR (D1.1).
Selection of amino acids to be mutated based on structural analysis and cloning in suitable expressing vector: Expression and purification of ER and AhR2 as well as identification of amino acids to be mutated in ER were completed. Regarding the mutant construct generation for ER, three mutants were generated. Identification of AhR2 amino acids to be mutated was not possible due to a lack of crystal structure (D1.2/ D1.3/D1.4).
Expression and purification of wild type and mutant constructs in suitable organism: The expression and purification of ER and AhR2 wild type recombinant receptor was completed. Purification of mutants constructs for ER were achieved (D1.2/D1.4).
Characterization of expressed protein: The expressed recombinant receptors were characterized in term of stability, storage and binding activity. It was already demonstrated their binding affinity towards natural ligands end EDCs: 17β-Estradiol, 17α-EthynylEstradiol, Tamoxifen, BisPhenol-A, 4-NonylPhenol and 4-tert-OctylPhenol (D1.6).
In vitro testing and selection of best receptors by affinity binding measurements to selected toxins and pollutants: The affinity binding measurements allowed the selection of wild type ER, mutants ER_M421F, ER_M421L, -ER_ M421I, wild type AHR2. AHR2 mutants were designed and a mutant containing only the ligand binding domain (AHR2LBD, generated by deletion of 515 aminoacids) was successfully expressed in E.coli. D1.7 was focused on AhR activity analysis. (D 1.7)




Deliverable Number Due Date Actual Date Deliverable Description
D 1.1 5 5 Progress report on first sequence alignments identifying the common motif for ER and AhR receptors in aquatic organisms: Identification of the conserved domain among the aquatic organism and synthesis of the ligand binding domain (LBD) encoding gene for ER and DNA binding and ligand bind domains for AhR.
D 1.2 9 9 Progress report on expression and purification of wild type ER and AhR: The encoding genes for ER and AhR were expressed and purified. The purification method was based on Ni-affinity system.
D 1.3 13 13 Progress report on expression and purification of a representative, rationally designed mutant: Structural conformational analysis allowed identifying a set of residues to be mutated. One mutant was successfully tested for the expression and purification.
D 1.4 17 17 Progress report on cloning and generation of mutants: Other mutants (a set of three) have been subsequently cloned and expressed.
D 1.5 29 29 Data report on expression and purification protocols of recombinant receptors: High levels of pure wild type and four mutants of ER were obtained. We also expressed and purified the wild type Aryl Hydrocarbon Receptor. The structure stability was confirmed through circular dichroism spectroscopy.
D 1.6 32 32 Data report on characterization of recombinant receptors: Recombinant receptors of ER were characterized through fluorescence competitive ligand binding assay. The binding activities of wild type and mutant ERs were demonstrated towards six compounds: 17α-EthynylEstradiol (EE2), 17β-Estradiol (E2), BisPhenol-A (BPA), 4-NonylPhenol (4NP), 4-tert-OctylPhenol (4TOP) and Tamoxifen (TAM). A ligand binding assay for AhR is under development.
D 1.7 34 40 Data report on in vitro test for binding activities of wild type and mutant receptors: A mutant of AhR containing only the ligand binding domain (AHR2LBD) was successfully expressed in E.coli and activity verified by a radiolabeled assay using [3H]TCDD as marker. Mutants in single amino acid positions of AhR were designed and expressed but not active.


Milestone N. Due Date Actual Date Milestone Description
MS3 12 12 First set of pilot recombinant receptors, as starting point for the testing of engineered biosensor substrates: Wild type recombinant receptors have been successfully produced. The recombinant receptors were tested for their stability and be further used as substrate for the immobilization on biosensor.
MS5 18 18 Second set of recombinant receptors, as validation of our approach: Identification of the first set of estrogen receptor mutants has been achieved. These receptors have been expressed and purified. Characterization of one of these mutants e.g. in vitro binding assay has been performed.
MS8 34 40 Final set of recombinant receptors, as best-in-class receptors: ER-wt, AhR-wt and four mutants were produced with different affinities for toxicant classes



Work Package 2
WP 2: Engineered biosensor substrate with optimized receptor immobilization

WP Leader: JRC-IHCP

Executive Summary
The main objective of work package 2 was to engineer the biosensor chip surfaces for optimizing the immobilization of the Estrogen and Aryl hydrocarbon Receptors developed in the frame of work package 1. The final goal was to maximize the performances of the biosensor in particular to enable the detection of estrogenic compounds.
The main difficulty of this task was related to the small size of the compounds to be detected which have size lower than 500 Daltons representing the limit of detection of most label free biosensors in particular of surface plasmon resonance (SPR).
In order to control their orientations, Estrogen (ER) and Aryl receptors (AhR) with poly-histidine tags were immobilized via specific metal (Ni)-protein interactions. Two sensing platforms were used i.e. surface plasmon resonance (SPR) and wavelength interrogated optical sensors (WIOS). Several surface chemistries were tested in parallel i.e. Nitrilotriacetic acid derivatised carboxymethyldextran hydrogel (NTA-CMD) on SPR and WIOS and NTA derivatized Mercapto-hexadecanoic acid Self Assembled Monolayer (MHDA-SAMs) on SPR. Above mentioned functionalized surfaces were nanostructured for evaluating further potential detection signal enhancement. The chosen protein model to test the performance of the surfaces was the His tagged-Transthyretin (TTR) and Tyroxine hormone (T4, size 770 Da). The surface nano-structuring methods were optimized, fully characterized and tested with the TTR/T4 model.
The best results were obtained with the NTA-MHDA nanostructured surfaces using the TTR/T4 model. We observed an increase of the signal of about 20% even if the active area of nanostructured surface is only 15% of the uniform surface. The results obtained with nanostructured NTA-CMD chemistry did not give any significant enhancement. Next to the nanostructuration, the buffer composition was optimized for ERs immobilization. The goal was to find the conditions giving the highest immobilization level of receptors. HBS buffer containing 10mM Hepes, 150mM NaCl, 0.003% Tween 20 has been selected giving a good compromise between the binding capacity and compatibility with circular dichroism (Triton free). The ER-wt and mutants ER M421L, ER M421F and ER L346M were successfully immobilized on both platforms (SPR and WIOS) functionalized with NTA-MHDA and NTA-CMD chemistries nanostructured or uniforms. The immobilization experiments gave similar results on both nanostructured and uniform chemistries (> 1000 pg/mm2). -Estradiol detection was monitored at different concentrations. Only a slight detectable signal has been observed on the nanostructured surfaces (see Figure 3 of deliverable 2.4). Nevertheless, the obtained signal was found too low to develop a robust assay for real samples.
WIOS sensor has been used to determine the best running buffer enabling AhRs receptors immobilization. Two buffers have been tested namely NaP and NaP/NaCl buffers. The presence of NaCl in the buffer decreases the immobilization efficiency of the receptors. Receptor surface coverage is respectively 1400 and 770 pg/mm2 for NaP and Nap/NaCl buffer. Experiments of Epigallocatechin (EPGC) detection have been performed. Noticeably, whereas no EPGC binding is detected on AhRs immobilized in NaP buffer, binding was slightly detected for concentration from 1 to 10 M with AhRs immobilized in NaP/NaCl buffer. NaCl seems to have a positive effect of AhRs folding that allows a better exposition of binding site of immobilized receptors. Nevertheless the reproducibility of the results was very poor.


Complementary experiments have been performed using Biacore which is a one of the most sensitive SPR biosensor instrument. Several methods of receptors immobilization have been tested i.e. random immobilization with amine coupling or oriented immobilizations using NTA chips and chips coated with an antibody against the His-tag. However, the interaction of the immobilized receptors with high concentrations of the estrogenic compounds in solution resulted in low or no response. Similar results were obtained with an alternative reversed system in which Estrogen derivatives were immobilized on the surface and the interaction with the ER in solution was investigated.
As conclusion, Due to the inherent limit of detection of the used sensors and the small size of the analyte, detection of chemical compounds such as estradiol and tamoxifen by using direct assay has not been possible. A New assay format has been successfully developed. Peptides specifics to ER conformational change upon ligand binding have been used as amplification agents. By immobilizing these peptides on the sensor surface, limit of quantification of ER-ligand complex was found lower than 10 nM. The optimization of the assays is described in WP4.


Main Tasks
Optimization of the chemical composition and geometry of the nanostructures: Two different nanostructured surfaces have been tested: Carboxy-methyldextran hydrogel (CMD) and Mercapto-hexadecanoic acid Self Assembled Monolayer (MHDA-SAMs). NI(II)-NTA derivatization protocol has been optimized. Nano-structuring protocols have been optimized for both chemistries. (D2.1 and D2.3)
Physical-chemical characterization: Developed surfaces have characterized at each step of fabrication by XPS, AFM, SEM. (D2.2)
Immobilization and activity test of nanostructured surfaces: TTR/T4 model tested on SPR with and without nano-structures. Signal enhancement observed with SPR detection method.
Array patterning and immobilization of the novel receptors: Binding of wild type ER on nanostructured surfaces SPR. Binding of wild type AhR on non-nanostructured surfaces SPR. Binding of wild type AhR on nanostructured surfaces SPR. SPR detection with wild type ER. Binding of wild type ER on non -nanostructured surfaces WGG. WGG detection of pollutants with wild type AhR. Satisfying receptor loading on both SPR and WGG platforms.
Initial assay development / performance testing: Bioaffinity Mass Spectrometry (BioMS) competitive inhibition assay was developed with LC-QqQ-MS. Direct assay with ER immobilized on the sensor surface. Direct assay with ER immobilized on the sensor surface. Due to the inherent limit of detection of the used sensors and the small size of the analyte to detect, detection of chemical compound such as estradiol and tamoxifen by using direct assay has not been possible.



Deliverable Number Due Date Actual Date Deliverable Description
D 2.1 5 5 Preliminary description of nano-engineered surfaces and preparation protocols: Protocols for carboxy-methyldextran hydrogel (CMD) and Mercapto-hexadecanoic acid Self Assembled Monolayer (MHDA-SAMs). NI(II)-NTA derivatization protocol has been optimized. Nano-structuring protocols have been optimized for both chemistries.
D 2.2 9 9 Data report on the extended characterization of the nanostructure surfaces: AFM, XPS and SEM analysis have been performed. Chemical characterisation by XPS is limited due to the limit of detection of this method.
D 2.3 13 13 Data report on performances of the model test on the developed nanostructure surfaces: Immobilized His-tag TTR density close to those obtained with the CMD surface. NTA derivatized carboxymethyldextran hydrogel surfaces give promising results for both SPR and WGG platforms.SPR signal is 5 times higher than for uniform Dextran for normalized area.
D 2.4 17 17 Sensor surfaces with immobilized AhR, ER and mutant receptors against toxin/ pathogens in array format: All the receors developed in the frame of WP1 have been immobilized successfully with the desired surface density.
D 2.5 29 29 Data report on initial assay development and biophysical properties of AhR and ER protein constructs against toxins/pathogens: Due to the inherent limit of detection of the used sensors and the small size of the analyte to detect, detection of chemical compound such as estradiol and tamoxifen by using direct assay has not been possible. Another strategy has been implemented.


Milestone N. Due Date Actual Date Milestone Description
MS4 12 12 Nanostructured and chemically treated surfaces available with array-like patterned AhR and ER receptors for SPR and WGG: The sensitivity enhancement by using structured surfaces has been observed with SPR and Model proteins(TTR/T4).
No enhancement has been observed with the WIOS and ER / estradiol.





Work Package 3
WP 3: Detection platform design, fabrication and validation

WP Leader: Optics Balzers

Executive Summary
WP3 aimed at the development of a robust and highly sensitive label-free detection platform. These criteria were the main drivers during the development of all system components involved, meaning the sensor chip itself, the cartridge and its microfluidics, the optical read-out system and the related electronics. During the entire development, the future integration with the subsystem developed in WP4 and additional factors like production compatibility at reasonable costs for sensor and consumables were considered.
In Task 3.1 the sensor cartridge was developed. The cartridge is used to apply the concentrated analyte dissolved in aqueous solution on the sensitive part of the optical transducer chip. In addition to the transport of the fluid onto the chip, the cartridge needs to satisfy other requirements like leak-tightness, low dead-volume, easy replacement and it should preferably be mass producible at a wafer level. After the development of some computer assisted design (CAD) models and finite element calculations of the flow, several cartridges have been produced and tested. The current cartridge supports eight measurement channels in parallel, whereas six are used as measurement and two as reference channels. In contrast to the reference channel, the measurement channel is split into two channels, just before the actual sensor part of the chip. This T-junction allows guiding the liquid either over the sensor or to a waste channel. This approach allows switching from one liquid to the next within a few seconds, without mixing of the two liquids as in the previous systems: the two liquids are being separated by an air bubble, hence the two liquids are unable to mix.
Since air bubbles cannot be flown over the sensor part due to loss of a stable baseline, the bubble needs to be by-passed. To do so, an internally developed, low-cost, non-invasive bubble detector at the inlet of the measurement channel detects an incoming bubble, which will trigger a valve at the outlet to close the measurement channel but open the waste channel and due to the hydrostatic back-pressure the bubble will exit via the latter. A second bubble detector at the outlet inverts the process and toggles the valve and the second liquid will directly be applied on the sensor surface. As this switching directly takes place on the sensor chip itself, the dead- as well as the mixing volume is marginal and leads therefore to very fast switching times. Additionally, together with an automated selector valve, this switching principle allows “walk-away-operation-mode”, since the new liquid is detected automatically and so is the switching of the valve. An entire assay can be preprogrammed and autonomously been run on the system.
As the system’s sensitive element, the optical transducer chip and its performance is of utmost importance to achieve a stable and sensitive measurement. The sensor optimizations were part of Task 3.2. Extensive computational simulations on various parameters have been performed to gain sensitivity and stability. Various test structures have been produced and tested. Simulations and experiments indicate a rather high potential to optimize the previous sensor design regarding both, sensitivity and stability. One of the benefits of the current system with the MEMS micro-mirror (as described) is its independency of the selected laser wavelength. As the simulations suggest, a shorter wavelength leads to a higher sensitivity regarding surface related binding processes but lower sensitivity to bulk effects of the cover liquid. The current system is equipped with a 532nm green DPSS laser, compared to 762nm in the previous wavelength scanning system. These simulations have independently been confirmed experimentally in T3.6 at DLO and CSEM by comparing the sensor sensitivities towards refractive index changes and the adsorption of molecules to the sensor surface. Whereas the measurements at DLO have been performed directly with the ARGOS sensor and compared to the corresponding sensitivities of a commercial SPR system (Biacore 3000), the measurements at CSEM have previously been collected ex-situ with different light sources.
The transduced signal, generated by the sensor chip, is being read by the portable sensor module developed in Tasks 3.3 and relies on an angular interrogation scheme to measure the change of the effective refractive index induced by the adsorption of the analyte (here EDC) onto the waveguide grating transducer surface. The heart of the optical module consists of so-called opto-mechanical cage structure, housing the laser source, diaphragm, the MEMS mirror and plano-convex lenses. This sturdy structure can be mounted in the reader platform as a whole at its predefined position and can also easily be removed for maintenance if needed, as well. It is decoupled from the related electronics needed to drive the system and acquires, processes and stores the sensor data. The (mechanical) decoupling of the optical module from the rest of the reader has an additional advantage, since it’s less prone to external effects like vibrations, thermal drift etc. The system design again aims at highest performance regarding both sensitivity and stability, with additional constraints like size, manufacturability, cost and power-consumption. The newly developed optical reader system performs at a superior performance level compared to commercial WGG sensor systems on the market (BR8, Dynetix) and comparable to expensive and bulky lab systems (e.g. Biacore 3000). The performance has been analyzed by refractometric (glycerol) measurements as well as biosensing via IgG immunoassays before the systems have been shipped to the partners.

Main Tasks
Cartridge design and manufacturing for on-line EDC detection for SPR and WGG: Standardized cartridge based on simple plastic design (suitable for molding) including 6 measurement and 2 reference channels was designed and manufactured. Working cartridge. 100 times faster switching times.
WGG Sensor substrate optimization: Extensive in-silico simulations finalized. New structures manufactured tested in newly developed reader module. Better sensor design (stability and sensitivity) New theoretical models.
Design and adaption of portable sensor module for WGG and SPR: Transferred to WP4 (system integration).Running prototype.
Multiplex assay development on portable SPR and WGG biosensors: Testing of integrated system done with glycerol and IgG demonstrating state of the art sensitivity level of the final ARGOS V2 version. k-casein anti-k-casein interaction assay performed on many surface chemistries
Comparison of performance of the developed assays in commercial SPR and WGG machines and the portable SPR and WGG instruments: Transferred to WP5. ARGOS about 5x less sensitive compared to SPR Biacore but portable and low cost


Deliverable Number Due Date Actual Date Deliverable Description
D 3.1 11 11 Report on the cartridge design for on-line detection compatible with SPR and WGG: After computer assisted design (CAD) models and finite element calculations of the flow, several cartridges have been produced and tested. The final design (mainly outer shape) highly depends on the integration of the subsystems, which is an upcoming task in WP4 and can easily be adapted if necessary. UPDATE: Final cartridge/holder delivered in RP3.
D 3.2 13 13 Report on the optimized WGG sensor substrate: Extensive computational simulations on various parameters have been performed to gain sensitivity and stability. Various test structures have been produced and tested. Simulations and experiments indicate a rather high potential to optimize the previous sensor design regarding both, sensitivity and stability.
D 3.3 21 21 Report on the portable sensor module for WGG: Working prototype with high sensitivity and stability (electronics, optics, MEMS actuator, software) delivered for WP4.
D 3.4 21 21 Report on the portable sensor module for SPR: Working prototype with high sensitivity and stability (electronics, optics, MEMS actuator, software) delivered for WP4.


Milestone N. Due Date Actual Date Milestone Description
MS2 21 21 Portable SPR and WGG Detection Platform ready: Portable WGG Detection Platform delivered and transferred to WP4, overall sensor integration. Compact, low-cost but robust system proves best in class performance.
MS6 36 44 Portable SPR and WGG Detection Platform and specific assays for EDC detection ready: Three ARGOS V2 systems available for use at partners.





Work Package 4
WP 4: System integration and validation with sample preparation module

WP Leader: CSEM

Executive Summary
The main goal of Work Package 4 was the integration of the biosensor platform, including development of the sample preparation unit and of the wireless communication module.
Prior to any measurement, sample preparation is the key step for a sensitive and selective detection of target analytes. If the selectivity of the analysis is provided by the use of biological receptors, immunoassays used for the detection are sensitive to matrix effects and pre-treatment is crucial to prevent denaturation of the biomolecules. Moreover EDCs are present at the ng/L level in the environment, when limit of detections of optical biosensors reach the 1 μg/L level. A pre-concentration is therefore needed before analysis. In this work package, solid phase extraction (SPE) was chosen. For the development of the sample preparation unit, the natural hormone 17β-estradiol (E2) was chosen as the model compound due to its affinity with the estrogen receptor use for the biosensor assay. Prototypes were built that enable to perform fully automated SPE, with 500 fold pre-concentration and full recovery of E2, starting with 1 to 100 ng/L in only 100 ml sample volume. The output of the sample preparation unit is a 20-200 μl volume of methanol 5% or 10% v/v, totally compatible with analysis by immunoassay. This unit was successfully applied to the extraction and pre-concentration of E2 from real sea water samples with analysis by enzyme-linked immuno-sorbent assay (ELISA) on a well-plate format.
A prototype for wireless communication was developed and further integrated. This device enables transfer of data from the system to a database accessible by the user, and for remote control of the biosensor platform. The mobile telecommunication technology, global system for mobile telecommunication (GSM), was chosen as it provides a world-wide network coverage and high bandwidth requirements. The communication unit is a gateway between the biosensor platform and the GSM network, capable of transporting the information collected on the sensor to a central location for controlling, monitoring and data storage. The gateway is using GSM as radio interface and establishes a point of connection to Internet. An on-field monitoring application implies limited power resources for the overall platform. Therefore a high concern was the power consumption of the communication module. In order to spare energy, the wireless module is kept in sleep mode most of the time. It then periodically wakes up to establish a link to the controlling server. Remote monitoring on the field is then possible for the user.
Finally, the optical biosensor that was developed in Work Package 3 was further integrated to provide a compact, robust, user friendly and performant sensor for the analysis of EDCs by receptor immunoassay. The electronics was developed with a modular approach. Both sample preparation and biosensor units have independent electronic controls that enable to work with two stand-alone instruments. On the other hand, there were made compatible so that the two system units can be used in-line for full assay, from sample preparation to immunoassay and data analysis. A receptor immunoassay was transferred from gold standard surface plasmon resonance biosensor to the optical biosensor developed in the project.
In conclusion, the collaboration of the partners within WP4 lead to the development and the release of a modular biosensor platform allowing for remote monitoring of EDCs in the environment. The device includes sample preparation, with extraction and pre-concentration of analytes, sample analysis by immunoassay on an optical biosensor, and the possibility for remote control. The biggest challenge in monitoring programs is to obtain a result as close as possible as the sample upon collection. The overall platform offers an alternative to standard analytical methods with this regard. The different units allow to reduce the costs associated to monitoring programs, by proposing high quality low-cost instruments for either in-laboratory or on-field analysis. The complete automation of the process steps helps reducing human-related errors, and provides a high sensitivity throughout the process.

Main Tasks
Specification gathering of overall biosensor platform based on WGG and SPR: List of potential compounds for the receptors defined. Two model ligands for ER and AhR: respectively E2 and BaP. First priority is given to water samples.
Elaboration of extraction procedure and module: Solid phase extraction has been chosen instead of isotachophoresis. A transfer of knowledge from DLO to CSN has been done, and CSN is developing a new method for the extraction, separation and preconcentration of the EDCs from water samples. 500 to 1000 fold preconcentration of 17β-estradiol from di-water demonstrated.
Platform design and fabrication of module for bringing EDCs from solid into liquid phase: Replaced by extended T4.2 / As priority is given to liquid samples, this module might be developed in a later phase of the project.
Sieving element to remove solid particles and gas bubbles: Working prototype for macro-filtration ready and successfully tested. Proof of concept for micro-filtration done. Device available for the removal of gas bubbles.
Isotachophoresis for EDC pre-concentration, separation and extraction: Isotachophoresis replaced by Solid Phase extraction. 10-fold and 500- fold concentrations on water samples
Development of global wireless connectivity module: Prototype of the wireless module ready & tested. Characterization of its power consumption done. Integrated on the electronic boards’ stack.
Biosensor platform design, construction and assembly including microcontroller and electronic instrument control unit: A first set of prototypes was tested with real samples and ER assay. A second set of improved systems were delivered.
System control firmware and software: -
Integration of all subsystems in biosensor: Two separate prototypes compatible for use in line.
Optimization and Validation of system with final receptors: Optimized assay developed for ER, and transferred to SPR platform. AhR-based assay not optimized (not quantitative)


Deliverable Number Due Date Actual Date Deliverable Description
D 4.1 5 5 Report on the four-month specifications gathering for the instrument from the final users: List of potential compounds for the receptors defined, as well as two model ligands for ER and AhR. First priority is given to water samples. The frequency of analysis will depend on the application.
D 4.2 14 14 Report on wireless connectivity module performance: Prototype of the wireless module ready and tested. Characterization of its power consumption done.
D 4.3 26 26 Report on design and performances of sieving, preconcentration, separation and extraction modules of sample preparation platform for food and water: Development and validation of separate elements of the sample preparation module: sample collection, filtration, gas removal and pre-concentration by solid phase extraction.
D 4.4 27 27 Report on biosensor platform design: Description of the opto-electronic and fluidic components of the WGG biosensor and characterization of the first prototype by injections of glycerol.
D 4.5 29 29 (+48) Report on integrated WGG and SPR biosensor platform prototypes: Optimization of the solid phase extraction process and system. Update on the opto-electronics for the biosensor. Choice of materials for the wetted parts of the system.
D 4.6 36 36 (+48) Data report on WGG and SPR biosensor platforms performances with final receptors: Tests done with the WGG biosensor prototype, with antibodies, on different sensor chips (different surface chemistries). No tests with the receptors yet.


Milestone N. Due Date Actual Date Milestone Description
MS1 5 5 Specifications of overall biosensor platform based on WGG and SPR: List of potential compounds for the receptors defined, as well as two model ligands for ER and AhR. First priority is given to water samples. The frequency of analysis will depend on the application.
MS7 26 26 Sample-preparation platform for food and water ready: Two automated prototypes for pre-concentration of filtered water samples have been developed and validated. 10-fold and 500- fold concentrations on water samples with E2 were demonstrated.
MS10 36 40 THREE portable biosensor prototypes based on WGG and SPR ready: Transfer of the prototypes to the application partners


Work Package 5
WP 5: Biosensor platform system testing

WP Leader: RIKILT

Executive Summary
The main objectives of the Work Package 5 can be stated as follows:
1. To demonstrate the application of the label-free WGG and SPR biosensors, to detect EDCs with high precision and specificity in the following testing sites: sea water, fresh water, fish farming, dairies, fish lines and fruit juice processes.
2. To demonstrate the use of WGG and SPR biosensors as an alternative low cost monitoring strategy that may document the impact of pollution.
3. To carry out a series of field sampling campaigns that will generate a database of information that will enable evaluation of the biosensors and guidelines for the extension of the results to other targets of the same family.
The biosensor estrogen receptor (ER) assay works very well in the SPR-based biosensor. The peptide-coated CM5 chips coated with biotinylated peptide (via streptevidin) or with amine-peptide, are very stable for months and for hundreds of injections. The diluted ER is also usable during a working week. The assay is fast (ca. 10 min) and works in 10 % methanol which is good for the combination with the SPU. The sensitivity for E2 and EE2 is similar and in the low ppb level (working range between1-10 ppb) and can be improved to the low ppt level by SPE extraction and concentration.
However, the test does not work properly in the WGG (ARGOS) system. Successful tests performed with Surfix chips could not be reproduced consistently. We see binding of the receptor to the peptide-coated surfaces but non reproducible influence of E2. This needs to be investigated to get this application working in the ARGOS. The overall conclusion is that it is too early to switch to the ARGOS to get good results with the receptor test and additional research is necessary.
The Biacore SPR assay was tested with blank MilliQ water and drinking water and with “off-line” SPE and resulted in a limit of detection of 5 ppt after a 200 times preconcentration and with an average recovery of 75% of E2 spiked at 50 ppt and these data were confirmed by GCMS and ELISA. All samples obtained from fish farms from the UK and Slovenia and from sea and river water samples near and in Slovenia were found negative for E2 equivalents with the biosensor assay (< 5 ppt in 200 times concentrated samples and < 1 ppt in 1000 times concentrated samples). This was confirmed by the E2 and EE2 ELISAs. In two fish farm water samples from the UK, low levels of EE2 of 0.27 and 0.44 ppt were found with ELISA and one of these sample contained E2 (2.7 ppt). One sewage sample from Slovenia showed low levels of E2 (1.4 ppt) and 0.23 ppt EE2.The spiked samples at 50 ppt gave recoveries of 60 to 82 %. The SPU gave varying recoveries (6-100%) with an average of 53 ± 31% when samples were spiked at 50 ppt and concentrated (87 to 147 times) and an improvement of the system is required to provide better results.
For the application in food, the EU inspection programs for meat control focus on sample materials that are more suitable for testing for banned substances, especially if the animals are still on the farm, such as urine and faeces or hair. To test the assay with positive samples, urine samples from horses (n=20) were used and they gave varying concentrations of E2 in the biosensor ranging from 0.3 to >10 ng/ml (ppb) and this was confirmed by ELISA.


Main Tasks
Technical support from all RADAR partners: Support was given in the supply of reagents, instruments, samples and know-how.
SPU tested with fish farming and fresh water: EPL and MBS sampled both salt and freshwater from fish farms and sea and river water samples.
SPU tested with sea water and fish farming water: See above
WGG tested Fish, fruit juices, milk testing: Drinking water, beer and urine samples were taken.
Fish, fruit juices, milk testing: Samples were tested
Fish farming and fresh water testing: Testing was done on the benchtop SPR biosensor in RIKILT
Sea water and fish farming testing: See above


Deliverable Number Due Date Actual Date Deliverable Description
D 5.1 48 48 Report on the performance of the WGG sensor for laboratory use and remote-controlled in situ monitoring of several pollutants and toxins in fish farm and fresh water: The SPU was tested with fish farm water samples and gave varying recoveries (6-100%) with an average of 53 ± 31% when samples were spiked at 50 ppt and concentrated (87 to 147 times).
D 5.2 48 48 Report on the performance of the WGG sensor for remote-controlled in situ monitoring of several pollutants and toxins in sea water and fish farms: The SPU was tested with water samples from sea water, a fish farm and river water and gave varying recoveries (10 to 118%) and an improvement of the system is required to provide better results.
D 5.3 48 48 Report on the performance of the WGG sensor for laboratory use and monitoring of several pollutants and toxins in food processing, specifically in fish, fruit juices and milk: The overall conclusion is that it is too early to switch to the ARGOS to get results with the receptor test for food.
D 5.4 44 48 Report on the performance of the SPR based and/or other benchmarking: sensor for laboratory use and monitoring of several pollutants and toxins in food processing, specifically in fish, fruit juices and milk: The biosensor estrogen receptor assay works well in the Biacore SPR-based biosensor with a working range between1-10 ppb of E2.
D 5.5 44 48 Report on the performance of the SPR based and/or other benchmarking sensor for laboratory use and remote-controlled in situ monitoring of several pollutants and toxins in fish farm and fresh water: Levels of E2 were found to be below 5 ng/L (ppt) and all spiked samples at 50 ppt resulted in levels between 35 and 41 ppt of E2 (70 to 82% recovery). Trace levels of other hormones (testosterone (approx. 17ppt) and progesterone (<5ppt) were found in one of the samples.
D 5.6 44 48 Report on the performance of the SPR based and/or other benchmarking sensor for remote-controlled in situ monitoring of several pollutants and toxins in sea water and fish farms: All samples obtained from a fish farm from Slovenia and from sea and river water samples near and in Slovenia were found negative for E2 equivalents with the biosensor assay (< 5 ppt in 200 times concentrated samples and < 1 ppt in 1000 times concentrated samples).


Milestone N. Due Date Actual Date Milestone Description
MS12 48 48 WGG and SPR based biosensor platforms testing completed in water and food processes: The ER assay in the SPR based platform was applied during the End user week.



Potential Impact:
1. Policy impact
We expect the scientific evidence gathered using the new platforms and technologies resulted from the project to be translated into guidelines and recommendations for control of endocrine disrupting compounds in food and water. Our dissemination strategy views the policy impact as a key factor in the subsequent success of raising public awareness and commercial development of the outputs from the project. We engaged with the policy makers, for example via the Symposium “Small solutions for big water-related problems”, held in Rome, through the project partner JRC and through our external advisor Prof. I. Werner, of EAWAG, Switzerland, to inform governmental environment, food and health agencies at an EU-wide and national level, and NGOs in these areas.
The feasibility of developing a technically sound, cost-effective control strategy is an important part of developing effective guidelines for controlling pollutants such as endocrine disruptors and RADAR has been and will continue to be active in promoting its outputs for policy consideration.

2. Societal impact
Outside the pure scientific and technical impact, this is the first area where the RADAR project is expected to have an impact. We have used the outputs of the consortium work to raise awareness of the issues around endocrine disrupting compounds to the general public and also within the context of trade and its impact on the businesses producing food and water. This has been achieved through use of the website which has pointed interested parties to the science base behind the news stories on these compounds, for example referring to the EU roadmap on EDCs and topical news from reliable source such as Food Manufacture; through consortium members’ open days for schools and through face to face discussions with individual contacts at meetings. This first area of societal influence will be continued over the coming year as the outputs from the programme are collected and publicised in trade journals and magazines.
Socio-economic impacts as well as wider societal implications of the project include a better monitoring of water supply world-wide, monitoring from the source to the waste throughout all distribution channels. Water is a scarce supply, so preventing pollution of water supply by monitoring toxin and pollutants is critical and the RADAR platform just allows for this to happen.

3. Commercial impact
The commercial area is the second main area of impact benefitting the consortium members and creating added value from the support provided by the EU FP7 programme. This impact arises from the solution to the problem of how to monitor the endocrine disrupting compounds in non-laboratory conditions making it feasible to consider implement a monitoring programme at an industrial scale. The individual components of the RADAR instrument is designed to perform this monitoring function are valuable intellectual assets for exploitation in their own right and may be used for detecting compounds in the case of the receptor assays in different market areas such as within the pharmaceutical, veterinary or medical sectors and the instrumentation is likely to be used for monitoring of a range of other chemical compounds of environmental interest by pairing with different assay types. The RADAR consortium has now entered active commercialisation phase post-project with a number of confidential discussions underway for the partners with a view to progressing the development of commercial outputs from the research.



4. Scientific and technical impact
The expected final scientific impact of the project is manifold. At the end of the project, i.e. Month 48, we can proudly cite three technical results of the project with high impact much beyond the project itself:
To the best of our knowledge, RADAR has produced the first Aryl Hydrocarbon Receptor from aquatic organisms with highest affinity towards Polycyclic Aromatic Hydrocarbons (PAHs), e.g. epigallocatechin gallate.
For the first time to the best of our knowledge, an Estrogen Receptor and several mutants were produced from aquatic organisms with a successful increase of the specificity and the selectivity of the receptors to recognize and differentiate different classes of pollutants. This technical progress beyond the state-of-the-art is opening new routes towards class specific robust capture biomolecules for EDCs compound detection.
To the best of our knowledge, the ARGOS compact label-free platform with the ER coated biochips linked to the automated filtration/separation/pre-concentration module is the first combined label-free system successfully able to detect EDCs at relevant concentration levels in the aquatic environment and in food processes.

The potential use of the ARGOS compact label-free platform with the ER coated biochips linked to the automated filtration/separation/pre-concentration module is the field of monitoring EDCs in the aquatic environment and in food processes. Knowing that the number of EDCs is increasing, such a platform approach is without doubt promised to be a success as we have now proved that its sensitivity and specificity is matching the regulatory values as shown for the specific case of E2. Moreover, its potential use is greatly extended as, thanks to the use of ER or the AhR receptors, it can detect all molecules that interact with the receptors and not only specific toxins and pollutants (class specific and not molecule specific).
The ARGOS compact label-free platform with the ER coated biochips linked to the automated filtration/separation/pre-concentration module can have a significant impact on the way water treatment and monitoring is performed today and will be performed tomorrow. Even without regulatory incentives, the platform, due to its limited cost of ownership, should be able to penetrate the market by providing a direct feedback (monitoring) of level of toxins and pollutants, thus allowing the significant reduction of chemicals (costly !) used in the water treatment cycle. Further applications will target the field of monitoring: With the integration of the system with a wireless module for on-line detection of toxins and pollutants in diverse matrices, the final system has shown to have a huge potential impact for monitoring of class specific toxins and pollutants in the aquatic environment.

5. Main dissemination activities

The website was then used as the primary dissemination vehicle for information on our general activities. It has been regularly updated throughout the course of the project with information on scientific progress, consortium meetings and as a way of advertising future events of general and technical interest. Links were provided to relevant websites for both general and specialist resources and news of consortium members speaking activities at conferences etc. highlighted as well as news articles of interest.

An important part of the dissemination process has been reporting scientific progress via publication of learned articles in scientific journals and presentation of work in progress at international and national conferences, technical meetings and congresses. To date the consortium have published 5 original articles, 8 oral presentations at conferences across the EU and USA, 9 poster presentations including the best poster prize winner at the Conference on Bio-Sensing Technology in Sitges, Spain in 2013.

The RADAR consortium joined forces with another EU FP7-funded project μAQUA to organise a joint scientific symposium entitled “Small solutions for big water-related problems” on sensors for water quality and food security in Rome in October 2014. The meeting had 145 delegates, 30 speakers and a similar number of poster presentations. A short film showing a tree planting ceremony during the conference and clips from the meeting is available on YouTube RADAR members provided oral presentations for the meeting provided a scientific overview of progress made in the project to date and we have guest edited a special edition of the International Journal of Environmental Research and Public Health associated with the consortium as well as providing articles due for publication in 2015.
The educational aspects of the dissemination process have also been considered by the consortium with posters presented at open days for the general public and school children, at least three open days and general meetings including the Food Matters Live event in London in November 2014. The distribution list for general dissemination of the final summary factsheet, now in production, includes all the major water companies or authorities within the EU, a wide range of food industry companies including the retail, primary production and manufacturing sectors.

The technological advances made by the RADAR consortium have generated opportunities for commercialisation of knowledge gained. The options for further development the components of the RADAR instrument for on-line monitoring of endocrine disrupting compounds in environmental samples which is the focus of the project, including specific types of proteins, biosensor assays, sample preparation units and the detection and reporting hardware and software modules have been considered within the work package by all the consortium members. The potential for commercial development of the RADAR outputs is indicated by the filing of a patent by CSEM and Optics Balzers with another patent filing under consideration for the receptor work at JIC Ispra. Technology offering factsheets have been prepared or are in preparation to aid the dissemination of knowledge and provide an easy to understand description of the innovation or product prototype for future development.
The commercial background and likely areas of interest and concern for technology developers have been considered within the consortium’s commercialisation plan and will provide a starting point for future commercial exploitation. RADAR partners will continue to work on the commercial and dissemination activities to maximise the social and financial impact of the EU FP7 funding throughout 2015.

List of Websites:
Project website: www.fp7-radar.eu

RADAR project main contact persons

CSEM CENTRE SUISSE D’ELECTRONIQUE ET DE MICROTECHNIQUE SA
Scientific
Dr. Stéphane Follonier
CSEM AG
Bahnhofstrasse 1
CH-7302 Landquart
[e-mail]
[phone]

JRC in Ispra, IHCP and JRC in Ispra, IES,
Scientific
Dr. Pascal Colpo (IHCP)
Via E. Fermi 2749
I-21027 Ispra/Italy
[e-mail]
[phone]
Dr. Luigi Calzolai (IHCP)
Via E. Fermi 2749
I-21027 Ispra/Italy
[e-mail]
[phone]
Dr Teresa Lettieri (IES)
European Commission, DG Joint Research Centre
Institute for Environment and Sustainability
Water Resources Unit TP 121
Via E. Fermi 2749
I-21027 Ispra/Italy
[e-mail]
[phone]

OPTICS BALZERS AG
Scientific
Florian Kehl
Optics Balzers AG
Neugrüt 35
LI-9496 Balzers
[e-mail]
T [phone]
F [phone]

INSTITUTE FOR RESEARCH IN BIOMEDICINE
Scientific
Dr. Luca Varani
Via Vincenzo Vela 6
6500 Bellinzona
Switzerland
[e-mail]
[phone]

STICHTING DIENST LANDBOUWKUNDIG ONDERZOEK (RIKILT INSTITUTE OF FOOD SAFETY, WAGENINGEN
Scientific
Dr. Willem Haasnoot
Veterinary drugs (DGM)
RIKILT-Institute of Food Safety
Akkermaalsbos 2,
6708WB, WAGENINGEN
The Netherlands
[e-mail]
T [phone]
F [phone]

ELYSIUM PROJECTS LTD
Scientific
Mr. John Bostock
Deiniol Road
Unit 2, Mentec
LL57 2UP Bangor
United Kingdom
[e-mail]
[e-mail]
[phone]

NATIONAL INSTITUTE OF BIOLOGY/MARINE BIOLOGY STATION
Scientific
Ass. Prof. Valentina Turk
Marine Biology Station Piran
National Institute for Biology
Fornače 41
Sl-6330 Piran
[e-mail]
[phone]




Work package leaders

Coordinator
Dr. Stéphane Follonier
CSEM AG
Bahnhofstrasse 1
CH-7302 Landquart
[e-mail]
[phone]

WP1 Rationally designed recombinant receptors derived from aquatic organisms
Dr Teresa Lettieri (IES)
European Commission, DG Joint Research Centre
Institute for Environment and Sustainability
Water Resources Unit TP 121
Via E. Fermi 2749
I-21027 Ispra/Italy
[e-mail]
[phone]

WP2 Engineered biosensor substrate with optimized receptors immobilization
Dr. Pascal Colpo (IHCP)
Via E. Fermi 2749
I-21027 Ispra/Italy
[e-mail]
[phone]

WP3 Detection platform design, fabrication and validation
Florian Kehl
Optics Balzers AG
Neugrüt 35
LI-9496 Balzers
[e-mail]
T [phone]
F [phone]

WP4 System integration and validation with sample preparation module
Sarah Heub
CSEM AG
Bahnhofstrasse 1
CH-7302 Landquart
[e-mail]
T [phone]
F [phone]

WP5 Biosensor platform system testing
Dr. Willem Haasnoot
Veterinary drugs (DGM)
RIKILT-Institute of Food Safety
Akkermaalsbos 2,
6708WB, WAGENINGEN
The Netherlands
[e-mail]
T [phone]
F [phone]

WP6 Dissemination and exploitation
Mr. John Bostock
Deiniol Road
Unit 2, Mentec
LL57 2UP Bangor
United Kingdom
[e-mail]
[phone]

WP7 Overall management and consortium coordination
Dr. Silvia Generelli
CSEM AG
Bahnhofstrasse 1
CH-7302 Landquart
[e-mail]
T [phone]
F [phone]

Verwandte Dokumente

• final1-radar-final-report-final.pdf