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"DEVELOPMENT OF AN AUTOMATED, NOVEL BIOSENSOR PLATFORM FOR PESTICIDE RESIDUE DETECTION"

Final Report Summary - FOODSCAN (DEVELOPMENT OF AN AUTOMATED, NOVEL BIOSENSOR PLATFORM FOR PESTICIDE RESIDUE DETECTION)

Executive Summary:
The objective of the FOODSCAN project is to develop a novel and automated biosensor platform for pesticide and other chemical residue detection incorporating membrane-engineered cells with pesticide-specific antibodies. The system is primarily based on the Bioelectric Recognition Assay (BERA) technology. Furthermore a specially designed electronic interface is realized in order to acquire and manipulate the corresponding signals from the real time analysis.
The pre-production prototype developed during this project is a Bioelectric Recognition Assay (BERA) sensor aimed at the detection of organophosphate and carbamate pesticides, 2-methyl-4-chlorophenoxyalcanoic (MCPA), other phenoxyalcanoic herbicides, or other small organic contaminants, e.g. 2,4,6-trichloroanisole (TCA) (in cork and wine). The product is developed by an SME consortium based in Cyprus, Spain, Germany, Greece and Portugal with the support of RTD performers from the UK and Greece.
In the framework of the FOODSCAN project, a fully operational platform has been developed, allowing for automated residue screening in approximately three minutes. The novel biosensor was rigidly validated and found to outperform conventional systems in terms of high speed, low cost, portability, user friendliness and high throughput. Each system component was fully assessed in terms of commercial manufacturing and cost considerations.
The European farming community is under a number of threats not least due to the increasing demands to produce higher quality food to increasingly stringent standards. They lack the technology to test their produce for the presence of pesticide residues at the site of production. If a system was developed that gave farmers, food companies and distributors the flexibility to test for a range of analytes regularly and in a cost effective way it would allow them to offer remedial solutions quicker than is currently possible. The conventional analysis of pesticide residues in food commodities is a labour intensive procedure. Standard analysis methods include extensive sample pre-treatment and determination by gas chromatography and high performance liquid chromatography to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. Therefore, rapid pesticide residue testing is necessary.
Based on the project results so far, we can claim, in confidence, the availability of both the General and Specific Toxicity Sensor as a novel, breakthrough system for the detection and classification of pesticide residues in food commodities. The ultimate goal is to establish FOODSCAN as the standard tool for mass-screening food samples at a scale never before realized, possibly reaching a hundredfold testing volume than currently feasible. In this case, FOODSCAN could enjoy shares of 1-4% of the total three billion € market for food safety control.

Project Context and Objectives:
The objective of the FOODSCAN project is to develop a novel and automated, biosensor platform for pesticide residue detection incorporating membrane-engineered cells. The autonomous automated system will be primarily based on the Bioelectric Recognition Assay (BERA) technology, combined with sophisticated membrane engineering and artificial intelligence.
The BERA is a biosensor method based on a unique combination of living, physiologically active cells immobilized in a matrix with an electrical sensor system. Cellular interactions with bioactive substances are measured by the sensor, which consists of an electro conductive probe containing the immobilized cells. Although a highly sensitive cellular biosensor has been already developed based on the BERA working principle for detecting organophosphate and carbamate pesticide residues in food commodities, the system could not be applied on a commercial and practically exploitable manner due to the following drawbacks:
- The use of lab-based, non - user friendly bioelectric measurement devices.
- The broad responsiveness of cells to structurally different analyses, leading to poor selectivity.
- The employment of an empirical way to determine the presence of a pesticide in a sample, by examining the biosensor’s response data curve.

These needs are addressed by the FOODSCAN project.

In order for the project objectives to be realized, it was necessary to optimize, in a customized fashion, the individual components of the final biosensor platform and the technology associated with each of these components, such as:
- The cellular biorecognition element, i.e. the core consumable part of the biosensor system, upon which is based the operating principle of the FOODSCAN system.
- Target-specific antibodies, such that enable the cellular biorecognition element to selectively detect the desired pesticides or other target compounds in a screened sample.
- The prototype device for measuring electric signals from the cellular biorecognition elements, designed in order to satisfy both low/high throughput screening capabilities, high speed of assay and portability.
- Dedicated software for the automated and successful classification of target compounds or groups in the assayed sample.
The coordinated development of the four individual FOODSCAN technological components is schematically described in the following Figure 1.
Since the FOODSCAN Project is addressed to the specific needs of the participating SMEs, it was first required to define these needs, expressed as targets for the novel screening system. In order to achieve this goal, the project partners conducted an extensive market and literature survey, as well as consultation with national and European food safety authorities, always considering the needs of the program and the conditions laid out in the DoW. The Project Consortium decided to divert the R&D effort in the following two directions:
- The development of a General Toxicity Sensor (GTS) , capable of screening food samples for the presence of pesticides belonging to certain chemical groups, though not individual pesticidal compounds. The selected groups were organophosphates, carbamates (including benzimidazole carbamates) and pyrethroids.
- The development of a Specific Toxicity Sensor (STS), capable of identifying individual pesticides in screened samples. A number of five different substances, i.e. 2,4,6-trichloroanisole (TCA), 2-methyl-4-chloro-phenoxyacetic acid (MCPA), chlorpyrifos/chlorpyrifos methyl, orthophenylphenol and Trinexapac-ethyl (or, alternatively, carbendazim) was decided to be specifically targeted through the development of target-specific antibodies, which were incorporated to membrane-engineered cells

The final deliverables of the projects were achieved on-schedule and are detailed in following:

1. The pre-production prototype, elegant in its simplicity, was developed during the project. It os essentially a potentiometer, receiving measurements from sensor strips. A host PC with Microsoft Windows™ software application via USB is connected to ensure the power and communications for the instrument. Depending on the desired application single or 8 disposable electrode strips can be used.It is characterized by reduced size (i.e. full portability) and high cost-efficiency (Figure 2).
2. The design and training of the FOODSCAN Artificial Neural Network (F-ANN) which was essential for the development of the General Toxicity Sensor (GST), allowing for the automated classification of the test results.
3. The customized antibodies, leading to the development of the Specific Toxicity Sensor (STS).
4. A cell immobilization system as an alternative to the cell attachment method used as the main option for incorporating the cellular biorecognition elements on disposable electrode sensor strips. Cells were being immobilized in a 3D gel matrix in that way it protected the cells for an extended period of time without interfering with the bioelectric measurements using the FOODSCAN prototype.
5. An advanced user interface (read-out software: BERA-Classifier™) developed outside the framework of the project, enabling the execution of quantitative determinations using the STS-FOODSCAN system and irrespective of the target residue, only requiring a preliminary system calibration.
6. A fully colour-illustrated End User’s Manual and associated instructive video.
7. An extensive Validation phase, which included double-blind trials based on samples received by different sources (over 200 samples). Samples were screened for either the general presence of pesticide residues, particular residues and TCA by using the GST and STS version of the biosensor. All samples were also analysed according to standard reference methods by both Project Partners and independent laboratories. Depending on the sample type (matrix), 93-100% success rates were recorded and summarized in the Validation Report.
8. A Techno Economical Study demonstrating a considerable market potential for FOODSCAN. Key steps and cost components of the manufacturing process have been identified. A base (pessimistic) marketing scenario has been modelled for forecasting perspective sales and sustainability of business developed as a consequence of FOODSCAN commercialization

Supporting actions involved:

• Setting-up a User-friendly website (www.foodscan.net).
• Publishing a series of scientific articles in international, peer-reviewed, high impact journals as well as oral and poster presentations in various national and international conferences (Eurosensors 2012, CEST).
• Preparing a flyer and presenting it at several conferences such as the International Baking Trade in Munich, the Practical Training Course for Baking Technologists, Sovata Romania, Eursosensors, Fruit Logistica etc.
• The Consortium performed a suite of press releases, radio interviews and on site presentations to potential end-users.
• The video was recorded to reach visual understanding of importance in practice and wide target audience.
• FOODSCAN presentation as part of the EURONEWS special documentary on novel analytical technologies (http://euronews.com/2013/11/25/invisible-at-first-sight/)

It has been estimated that the total food quality sector has a market value of 3.6 billion €, 1.7 billion € of which are dedicated to quality tests of exported food and other agricultural commodities. A major part of the initiative for reduced use of pesticides belongs to the European food industry and retail trade. In particular, various business-to-business systems have been developed to control and certify Integrated Crop Management (ICM) products and their supply chains on a European-wide scale, some of them with considerable success (e.g. EUREPGAP). Therefore, companies, which will adopt FOODSCAN for their regular internal monitoring system for pesticide residues, will be able to deliver ICM products and advertise them as such. In turn, and based on studies concerning organic food production in the EU, we anticipate that implementation of FOODSCAN by food companies can lead to a market efficiency increase at least above the 1,5% threshold value. This increase is reflected in both, sales and market share, at individual, national or European level. In the latter case, should FOODSCAN eventually be applied throughout the EU, a theoretical increase of exported value in agricultural trade in the height of 33 million Euros would be expected.
Based on the project results so far, we can claim, in confidence, the availability of both the General and Specific Toxicity Sensor as a novel, breakthrough system for the detection and classification of pesticide residues in food commodities. The system is able to simultaneously assay eight samples in three minutes and can classify correctly the presence of the investigated pesticide groups with overall very high success rate.
The ultimate goal is to establish FOODSCAN as the standard tool for mass-screening food samples at a scale never before realized, possibly reaching a hundredfold testing volume than currently feasible. In this case, FOODSCAN could enjoy shares of 1-4% of the total three billion € market for food safety control. It is demonstrated that, even a minimum investment in a manufacturing unit and its basic operation is sufficient for the annual production of 110,000 test kits, in actuality exceeding the needs for screening the current volume of samples tested on a European level.

Project Results:
WP1

Task 1.1. Obtain ethical clearance: Careful consideration has been given to ethical issues. The number of the experimental animals used for immunization was kept to a minimum, in accordance with the European legislation. The work necessitating the use of animals and the relevant experimental details were presented to the Board in charge (Committee of Ethics and Deontology, NCSRD) and its execution has been officially permitted through written approval
Deliverable 1.1. Ethical clearance (Month 1)

Task 1.2. Selection of target pesticide groups and compounds: The survey was realized in four basic axes:
• Official reports
• Official authorities
• Market research
• Literature
Preliminary Selection of Pesticide Groups and Compounds
General classification of food was in the following categories: cereals; fruits/vegetables; baby food; meat/animal products; grapes
24 compounds and 4 groups were preliminary identified and a target suggestion for membrane-engineering was proposed to all partners:
• 2,4,6-Tricholoroanisole (TCA)
• 2,4-dichlorophenoxyacetic acid or 2-methyl-4-chloro-phenoxyacetic acid (MCPA)
• Triclopyr - Chlorpyrifos, chlorpyrifos-methyl
• Diphenylamine
As for General Toxicity the groups were:
• Organophosphates
• Carbamates
• Pyrethroids

Deliverable 1.2. Specification of individual pesticides to be used as test materials (Month 2)
The following target-molecules were finally selected:
TCA, MCPA, triclopyr / chlorpyrifos / chlorpyrifos methyl, orthophenylphenol (OPP), trinexapac (or, alternatively, carbendazim)

Task 1.3. Development of synthetic derivatives of the pesticides; conjugation of the pesticide derivatives to carrier Proteins: We obtained suitable starting chemicals, i.e. reagents that are necessary to prepare immunizing haptens (antigens) for each pesticide. One of these chemicals (namely, trinexapac - free acid) was offered by few Companies worldwide and, at least to our knowledge, by just one Company in Europe (Dr. Ehrenstorfer GmbH). Trinexapac free-acid was supplied as a solution in acetonitrile, at a concentration too low (10 ng/µL) to meet the needs of our coupling protocol. As revealed by e-mail communication, the supplier was not able to offer us any Trinexapac free-acid solution of higher concentration. All the other chemicals fit exactly the specifications necessary to accomplish our experimental schedule. More specifically, we have designed and completed the chemical synthesis of special derivatives of TCA, MCPA, chlorpyrifos / chlorpyrifos methyl, OPP and carbendazim.

Task 1.4. Immunization of New Zealand white rabbits with the pesticide-protein conjugates; collection of the antisera and isolation of the antibodies:
- Immunizations by using specific, in-house prepared KLH-conjugates of the above mentioned synthetic derivatives as immunogenic material -properly emulsified (w/o) with Complete Freund’s Adjuvant (Neokosmidi et al., 2004). Different combinations of immunizing haptens were used per each host-animal (New Zealand white rabbits).
- Five animal groups, each group corresponding to each of the pesticides TCA, MCPA, chlorpyrifos / chlorpyrifos methyl, OPP and carbendazim, were immunized.
- Two to four host animals were used per each pesticide group. Immunizing injections were given on the animal’s back, as first described by Vaitukaitis (Vaitukaitis, 1981).
- First booster injection was given five-six weeks after the first immunization. Further boosters, at four-week intervals, were given to each host animal.
- Two/three consecutive bleedings of then different antisera (two different antisera for each pesticide) were collected. The anti-pesticide antibodies (gamma - immunoglobulins or IgGs, i.e. the IgG-fraction of the corresponding antiserum) were isolated with well-established methodology, i.e. through sequential precipitation with caprylic acid and ammonium sulphate (Perosa et al., 1990).

Task 1.5. Immunochemical evaluation of the anti-pesticide antibodies with ELISA.
- Antisera were evaluated in an in-house developed ELISA system.
- Specific pesticide haptens were coupled onto various synthetic lysine-dendrimers (in-house prepared, according to the method described by Papasarantos et al., 2010, slightly modified) and the pesticide-dendrimers were used as immobilizing haptens in the ELISA system.
- Various control-negative microwells were included in each ELISA assay run.
- The anti-pesticide antisera showing best ELISA-immunochemical characteristics underwent suitable treatment (see above) for antibody isolation.
- The corresponding antibodies (IgGs) were provided to partner AUA in order to be further evaluated as specific cell-membrane biorecognition elements and used in the construction of the Specific Toxicity Sensor platform.
- Moreover, the NCSRD group purified anti-biotin antibodies from a rabbit antiserum previously developed at our lab and evaluated in a similar to the above mentioned ELISA system (Papasarantos et al., 2010). The anti-biotin antibodies, along with in-house prepared biotinylated bovine serum albumin (biotinylated BSA) were provided to the AUA Partner as a model “anti-pesticide / pesticide” pair of molecules for facilitating early execution of membrane-engineering experiments.

Deliverable 1.3. Development of pesticide-specific antibodies (Month 9)
Summary of Results
- Specification of target pesticides
- Development of synthetic pesticide/TCA derivatives
- Development of ELISA-high titer anti-pesticide/anti-TCA antisera/antibodies
WP2
Task 2.1. Development of cellular biorecognition elements. (Month 12)

Task 2.1.1. Development of sensors based on cells (derived from cell culture) having a natural selective response against target pesticide groups (Month 15).
- A first, proof-of-principle approach to the development of selective biorecognition elements was realized by membrane-engineering fibroblast cells with anti-biotin antibodies. It was proven, with the aid of fluorescence microscopy, that (1) engineered cells incorporated the specific antibodies in the correct orientation and that (2) the inserted antibodies are selectively interacting with the homologous target molecules.
- General Toxicity Sensor Calibration: Four different cell lines (N2a, SK-N-SH, Vero, HAK) have been used for the calibration of the biosensor against three different pesticide groups (organophosphates, carbamates, pyrethroids) at different concentrations – dilutions of the Minimum Residue Level for each group (2XMRL, MRL, MRL/2, MRL/10 and MRL/20). Each concentration was tested three times and each experiment was replicated at least three times at three distinct time periods.
D 2.1. Membrane-engineered cell arrays with differential recognition pattern against target pesticides (Month 12).

Task 2.2: Cell immobilization (Month 15)

Task 2.3. Design of receptacle-prototype interface (Month 15)
The alternative 3D immobilization system did not interfere with the expected pattern of the bioelectric measurements, as a result of the exposure of Vero cells to carbamate pesticides. Indeed, we observed a considerable difference in response compared to control, while the response was concentration-dependent in a linear fashion until the concentration of MRL/2. Thus, the overall pattern of response was similar to the one observed with attached, though non-gel immobilized cells which has been already described in D2.1 (with the only difference being the sign of the pattern compared to control). Therefore, we can safely consider the alternative immobilization system as absolutely successful (Fig. 3).
D 2.2. Immobilization system for manufacturing consumable, storable sensors (Month 15).
D 2.3. Production of consumable sensor array (Month 15).
Summary of Results
- Proof-of-concept of the application of Molecular Identification through Membrane Engineering for the specific detection of target pesticides (Specific Toxicity Sensor) (Fig. 4).
- Calibration of the cell-based sensor against different pesticide groups (General Toxicity Sensor) (Fig. 5).
- Definition of various biosensor operational parameters (cell lines, electrode material, mode of electrophysiology measurement).
WP3

Task 3.1. Consumable biosensor-electrode interface.The User Requirement Specification (URS) was finalized, which details the requirements of the scientific instrumentation for the first prototype within the FOODSCAN project. It was decided that the device will be a portable potentiometer, having a replaceable guide bearing eight pairs of electrodes connecting on the underside. The sensor strip was also specified. The URS was the essential step for the development of the first version of the prototype (V1).
D 3.1. User Interface hardware and software modules developed (Month 15).

Task 3.2. Biosensor data acquisition and processing Hardware The first version of the prototype (V1) was developed able to measure electric signals from the cellular biorecognition elements, designed in order to satisfy both low/high throughput screening capabilities, high speed of assay and portability. V1 has advanced measurement capabilities, going beyond the actual end-user defined needs: For example, it enables the measurement of different electric properties of the cellular biorecognition elements, such as potentiometry (which is the only measurement described in the DoW), amperometry and cyclic voltametry. In this way, the prototype allowed for testing a considerable number of different measurement options as well as different combinations of device-cell interfaces (e.g. type of metal used for the screen-printed electrodes). Many different factors were considered in this process, involving not only scientific but also practical and economical parameters, such as the user-friendliness of the system and the reduction of the final cost per sample.
D 3.2 Low-powered Microcontroller based processing unit developed (Month 15).

Task 3.3. Pesticide classification and results registration software System: An Artificial Neural Network (ANN) was developed to correctly classify a specific time-series of data as positive or negative as far as the existence of individual pesticides or of pesticides belonging to the pyrethroid, the organophosphate or the carbamate group is concerned. The results from the high throughput testing of the General Toxicity Sensor (Task 2.1.1) were used for training the FOODSCAN Artificial Neural Network (F-ANN), in other words the computational classifier system able to interact with the biosensors as a pesticide classification software, able to learn during use and therefore to improve its classification accuracy. More than 4000 different software architectures were designed and tested during this period and the best models were incorporated in the GTS platform. This coordinated work lead to the successful completion of the General Toxicity Sensor. Upon consequent validation, the GTS system performed exceptionally well, with a diagnostic ability (correct pesticide classification rate) of 85-87%, depending on the target pesticide group.
D 3.3. Pre-production prototype (Month 15).

Task 3.4. Main control electronics of the device.In its final version, the prototype integrates the housing and the corresponding electrodes, signal processing, control and user interface electronics of the portable device. This includes the biosensor data acquisition and processing hardware, the pesticide classification and results registration software system, the development of the main control electronics of the device and the PC based user interface software.
- The specification architecture of the complete system was finalized after the validation of the first prototype
- The draft specification for the Final Prototype (V2) much more compact, easier to operate and more cost effective to manufacture.
Task 3.5 Design of pre-production prototype: The final FOODSCAN prototype was specified and built to fully operational capacity.
Summary of Results
1. Conclusion of the User Requirement Specification.
2. Completion of the cell-device interface.
3. Development of the first version of the Prototype (for experimental use, calibration studies and training of the classifier software).
4. Development of the FOODSCAN Artificial Neural Network (F-ANN), the Pesticide Classification Software.
5. Training of the Pesticide Classification Software (F-ANN).
6. Completion of the General Toxicity Sensor (GST).
7. Development of the second version of the Prototype (V2) (Fig. 6)
• Provides a reduced size and a more cost-effective instrument compared to the prototype by focusing the specification and scope of the requirements.
• Two types of sensor strip, a single channel and an 8 channels
• Interface with sensor strips as specified in the sensor specification section.
• Provide potentiometric measurements from sensor strips with respect to time.
• Connect to a host PC via USB, which provide power and communications for the instrument.
• A host Microsoft Windows™ software application provides the user control of the instrument and the facilities to display and analyze the data.
The V2 instrument combined with the development and training of the FOODSCAN Artificial Neural Network (F-ANN), the Pesticide Classification Software, completes the General Toxicity Sensor (GST) (Fig. 7). For the Specific Toxicity Sensor (STS) the BERA Classifier™ software (developed outside the framework of the project) was incorporated in the biosensor platform (Fig. 8).
WP4
Task 4.1 Manufacture of the Demonstrators: The final prototype was installed in the testing lab at AUA for further validation. Compatibility of the results’ format with further data processing software (e.g. BERA Reader™) was tested at this stage
Task 4.2 Testing of the pre-production prototypes:Validation of the final FOODSCAN prototype was done using, as a core model, the screening for TCA. The target-specific sensor was based on membrane-engineered cells with the anti-TCA (Ab 55) antibodies provided by partner NCSRD. It was determined that the final pre-production prototype (V2) performed as expected, in fact providing more reproducible results than V1, in terms of the characteristic sample-associated curve of sensor response against time.
Task 4.3 Technical and economic comparison study:The performance of the final prototype was compared with the first, desktop prototype. Results were assessed both by means of biosensor response pattern analysis and statistical analysis. In addition, results were evaluated by five independent researchers. The feasibility of using FOODSCAN vs. conventional/existing analytical systems for the detection of pesticide residues and/or TCA in food commodities and cork was briefly evaluated.
D4.1 Techno-economic comparison of FOODSCAN-like
Summary of Results
• The results of the validation using TCA screening as a model have clearly demonstrated that the prototype is ready to be released to SME partners for validation trials on their own.
• Minor problems concerning the correct installation and operation of the user-device interface have been identified and solved
• A base (pessimistic) marketing scenario has been modelled for forecasting perspective sales and sustainability of business developed as a consequence of FOODSCAN commercialization.
• According to a realistic business scenario, FOODSCAN could be easily adopted by a starting 5% of the existing European food control laboratories, corresponding to 12-13 customers
• an initially limited market penetration would create a sustainable business, even without necessitating the considerable increase of sample testing allowable by the adoption of the FOODSCAN technology
• It was demonstrated that, even a minimum investment in a manufacturing unit and its basic operation is sufficient for the annual production of 110,000 test kits, in actuality exceeding the needs for screening the current volume of samples tested on a European level.
WP5
Task 5.1 Biosensor distribution to SMEs and End Users (Month 19): - In total, 20 FOODSCAN prototypes were produced and distributed to SME Partners together with all necessary documentation
- The freshly isolated antibodies (3rd bleeding - antisera, whole IgG fraction) have been provided to the AUA partner to implement WP5.
- Analytical, fully colour- illustrated user manuals were prepared and included, both for system operation and software installation.
- In addition, one-on-one, hands-on training was provided by AUA (with the collaboration of the other two RTD performers) directly to SME partners during separate events in Athens and Lisbon (Figs. 10, 11).
Task 5.2:Design of double-blind experiments (Month 19):In order to validate the FOODSCAN pre-prototype, AUA received a series of known and unknown samples to be tested at their own facilities. All samples were also analysed according to standard reference methods by both Project Partners and independent laboratories. Although not required by the original FOODSCAN Project DoW, we also developed an assay for testing the in vitro toxicity of the target pesticide residue groups (carbamates, organophosphates and pyrethroids) according to the official OECD-GD129 protocol .
Task 5.3: Screening of the samples (Month 22):Following the receipt of the samples, AUA screened them for either the general presence of pesticide residues, particular residues and TCA. In other words, both the GST and STS version of the biosensor were employed. The validation experiments were so thorough to additionally investigate differential food matrix effects and the impact of the human end-user.
Task 5.4: Validation Report(Month 23): The results of the validation trials clearly demonstrated the successful applicability of the FOODSCAN biosensor as a reliable tool for residue screening at a pre-commercial level. As a general conclusion, the FOODSCAN system performed quite satisfactorily, demonstrating a high level of positive correlation with the conventional assay methods. In most cases, success rates (positive identification of residues) exceeded 90% and actually reached 100% in half of the sample groups.An analytical Validation Report was prepared accordingly.
D5.1 Validation report and consolidated Biosensor User Guide (Month 23)
Summary of Results
- Colour-illustrated End User’s Manual was prepared and distributed
- End user training was optimized by a dual system (detailed Biosensor User’s Manual and instructive video/on-site, hands-on training)
- Both the results of the validation trials and the successful end user training/guidance program provide a clear justification for a perspective successful applicability of the FOODSCAN biosensor as a reliable tool for residue screening at a pre-commercial level
- The experimental results of the comparative ELISA-evaluation have shown that the isolated antibodies retain their immunological functionality after lyophilisation; this finding is considered of great importance for the future application of the specific toxicity sensor (STS), since the antibodies are easier and safer to transport and store for long-term periods as lyophilized powders (Fig. 12).
WP6
Results achieved
- Consortium Agreement was drawn based on the DESCA model and concluded by each Beneficiary with its signature. CA regulates composition of Boards (Management, Scientific and Dissemination) and is a competitive internal document for overall management actions.
- All meetings of the consortium have been held regularly as planned in order to monitor the progress of the project.
- All foreseen tasks have been accomplished entirely within schedule.
- The project is proceeding largely according to plan, without consortium changes and without payment delays.
- The deliverables due in both reporting periods, as indicated in Annex I to the Grant Agreement, have been delivered by Partners, and then were approved and submitted by Coordinator IGV.
- A secure webpage was established with an internal communication platform set up for exchange of project information, documentation and results.
WP7
The deliverables describe in details the main instruments used for press communication and provides an overview of the most important designated publication, the developed website and other (leaflet/ posters/press releases/video) dissemination materials which were already applied and will have an on-going character.
Significant results:
- Website:
• with easy to access design with exact element and menu points where the visitor and the user may obtain a clear view of the project objectives, current status of project advancement as well as expected steps.
• Online communication platform where the project beneficiaries can exchange information on-line
- Press releases- presents the same release material propagated on different websites, suggesting the project’s main concept and expected results and to make the first
- Publication- is used in order to extensively spread our results
• Publication Abstracts- participation in conferences, seminars and workshops provide several opportunities to discuss the project’s results
• Leaflet- have been designed to facilitate the dissemination during public events
• Posters- in form of a flyer presenting key features of FOODSCAN project
- Video- to make FOODSCAN more visible to present the project objectives
- Conferences, seminars and workshops- participation of International or European major events to increase project exposure to the public.
- TV documentaries and radio interviews – in EuroNews and national networks
- Cooperation-
• Coordination with other relevant EU and national projects where identified as relevant to FOODSCAN
• Project meetings to share information and to strengthen the cooperation among the partners in the Consortium.
- Training- Development of appropriate training material to support the SMEs

Potential Impact:
1.4.1 Potential Impact

During the last decade, European governmental policies seek to drastically reduce the amount of chemical inputs used in agriculture (Struik and Kropff 2003) . It has been already estimated that high pesticide usage is counter-productive, due to its uncontrolled external effects on the quality of the environment, therefore considerably reducing the benefit/cost ratio (Zadoks and Waibel 2000) . By enabling high throughput, cost-efficient residue screening at the site of production and/or processing, FOODSCAN has the potential to become the tool of choice in order to optimize pesticide input in intensive agriculture. In other words, implementation of FOODSCAN fully complies with the regulatory drive towards minimum lethal pesticide doses (MLPDs). The feasibility of such an approach has been previously demonstrated with measurements of chlorophyll fluorescence shortly after the application of herbicides, albeit at a very limited scale of agricultural practice. In the context of the core Directive 91/414/EEC, pesticide use is “sustainable” not only when the product meets certain requirements but also when the application is limited to a strict minimum and priority is given to methods that are the least harmful from an environmental point of view (Vogelezang-Stoute 2003) . This is also reflected in the increasing demand for innovative techniques in the so-called Green Analytical Chemistry , which typically use less solvent and energy .
In addition, the availability of new, rapid assays for TCA is an imminent requirement of the cork and wine industry. In particular, performance characteristics such as high sensitivity, speed and low cost are highly desired. TCA gives a musty, mould and/or earthy off-odor to affected wines, therefore masking the natural wine aroma and diminishing the quality of the final product. Consequently, its presence results into an economic loss and occasional damage of the profile and the reputation of the wine industry. It has been estimated that the portion of tainted bottles can be higher than 30% (Sefton and Simpson 2005) . The EU is the leading producer of wine. Producing some 175m hl every year, it accounts for 45% of wine-growing areas, 65% of production, 57% of global consumption and 70% of exports in global terms. Also the main region of cork production (Portugal and Spain) from the region’s 2.7 million hectares of cork forests, belong to EU countries. These forests have been the world’s wine-bottle stopper of choice. 1999 reform of the CMO for wine, strengthened the goal of achieving a better balance between supply and demand on the Community market, giving producers the chance to bring production into line with a market demanding higher quality and to allow the sector to become competitive in the long term - especially in the face of increased global competition following GATT - by financing the restructuring of a large part of present vineyards. After 2015, current EU restrictions on planting vines will be lifted, enabling competitive producers to increase production. While 70 percent of wine bottles contain still natural cork stoppers, in recent years, alternative closures like metal screw caps and synthetic stoppers have boosted their market share and, more important, convinced wine drinkers that a screw top was not the mark of plonk. Lower costs helped the upstarts. So too did industry complacency regarding cork’s responsibility for “taint,” a malodorous malady caused by the contaminant TCA that can result in ruinous wine spoilage rates. Cork alternatives make up about a third of the 18 billion or so closures used worldwide. Australia’s industry is now 85% screw cap, and close to half of U.S. wineries have opted for cork alternatives. The problem of wine taint has been tackled in many ways, the most obvious and widespread of which is the use of screw caps or synthetic stoppers. But while this is one way of significantly reducing the problem (although evidence suggests that TCA from sources other than cork - such as wooden barrels or even the drainage system of the winery where it is made - can still contaminate wine in screw cap bottles) consumer acceptance of such closures remains mixed. The good news for cork is that many wine drinkers — notably in the U.S. which is set to become the world’s largest wine market — still equate cork with quality. People also like the cork-pulling ritual. It’s a ritual that can help save the planet, say cork’s proponents. Natural cork, it’s claimed, has a much smaller carbon footprint than its synthetic competitors, while cork forests sequester an estimated 10 million tons of CO2 annually. Cork fits right in with the wine industry’s growing interest in sustainability. Natural cork wine stoppers are the best choice, primarily because harvesting the real stuff is an age-old practice that keeps the world’s relatively small population of cork oak trees, which can live for hundreds of years, alive. These scattered pockets of cork oaks, mostly in Portugal and Spain, thrive in the hot, arid conditions of the southern Mediterranean, sheltering a wide array of biodiversity and helping to protect the soil from drying out. In addition, some wildlife depends upon cork oak forests for their survival, including the Iberian lynx, the Barbary deer and the Egyptian mongoose, as well as rare birds such as the Imperial Iberian eagle and the black stork. As wine producers switch to other types of wine stoppers, the cork oak forests could be abandoned and the trees and the myriad plants and animals that depend on them could die out. The vast majority of the global wine-drinking public is enthusiastic amateurs or absolute beginners - in other words, they are highly unlikely to realize that corked wine is a widespread problem, which can affect any product or brand. They are in fact much more likely to link it directly to the specific wine affected by the taint, making the chances of them buying that wine again extremely remote, with obvious implications for the winemaker. Research carried out recently in the UK suggests that while British consumers are the most accepting of a wide variety of closures, most still prefer to hear the pop of a natural cork when they open a bottle of wine. Asian consumers are notoriously sensitive to perceived status and, while they are not yet sophisticated wine consumers, they are prepared to spend extravagantly on wine that looks top class. Unfortunately for New Zealand's winemakers, prestige international wines do not have screw caps. If consumers want quality, they will see the closures as an indicator of what to buy. Corks mean quality.

Reduced supply remains the major bottleneck in the distribution of ICM products in several European countries. FOODSCAN contributes to the increase of the supply of ICM products due to the availability of a locally (“site of production”) applicable residue monitoring system. This is particularly important for the SMEs in Spain and Greece, since these two countries already belong to the top ten with organic horticultural area (0,38 million hectares) (FIBL/IFOAM 2010). Both countries also belong to the top ten leaders in organic grape production (36,000 hectares).

Food safety control is a major economic activity with a volume of $2 billion and a growth rate of 12 % (BCC Research, 2007). According to sector surveys, food companies dedicate up to 2% of their revenue to food quality control, although the market is segmented and, in part, immature (Alocijla and Radke, 2003). The food sector represents the largest market segment within the Industrial Microbiology Market and represents almost half of the total market (Strategic Consulting 2005). Given that the volume of the combined food and beverage global microbiology market in 2005 is approximately $2 billion the FOODSCAN project represents a significant opportunity.

Together, these markets represent a significant and realistic opportunity for the SME partners to make a real impact in terms of job creation and maintenance and in terms of turnover creation and maintenance. Food safety and complete food identification through traceability are at present two of the major characterizing issues of the EU policy about Health and Consumers protection. The demand for higher standards in food safety and traceability implies more onerous and cost-effective procedures for the food industry. There is a growing market demand for point of care/use diagnostic devices that provide quick, consistently accurate, high quality results in a number of market sectors (e.g. environmental, infectious diseases detection, chemical detection). The potential to operate at the portable scale with a very flexible supply chain enables the SME beneficiaries to satisfy demand and generate economic growth for the region.

The FOODSCAN project is fully compatible with overarching objectives of FP7 Research for SMEs call to strengthen the industrial competitiveness of the EU SME community and to increase European competitiveness through the creation of employment opportunities via the development of innovative products and services.

The main aim of the Capacities program for SMEs is to enhance the research and innovation capacities of European SMEs, which have little or no research capacities themselves and have to outsource research to specialized RTD-performers. The FOODSCAN project develops a key point of care/use diagnostic tool that subsequently enables the manufacture of a range of high added-value products and thus supports the achievement of another key objective of the Capacities Work Program, which is to enhance the SME consortium’s capacity to interact with the EU research community. Over this project, the focus was Europe based, to develop a fully integrated process drawing on the knowledge and skills of the RTD performers in the collaboration with and to the specific benefit of the SME performers. In the longer term this makes a significant contribution to the creation of a true knowledge based industry and provide a strong platform on which to engage with the wider global manufacturing community.
Providing novel solutions for food quality assurance can be a very critical issue, especially in view of the new EU and international regulations for minimal residue concentration in marketed food. Conventional techniques for the detection and identification of pesticide residues are very elaborate, time-consuming and expensive. The already existing food quality control laboratories use conventional techniques, providing a limited range of analyses. Standard analysis methods (e.g. according to the ISO Standard 4389) include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by GC and HPLC to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. As a consequence, current methods of analysis provide a limited sample capacity resulting in a general lack of resources for implementation and enforcement of lab analysis capability. For that reason they are unable to cover the needs of the domestic market, thus the offer to demand ratio is extremely low. Consequently, there is a major requirement for simple, rapid, specific, sensitive and economically feasible methods.

Food safety control in Europe is implemented by 282 certified public and private laboratories, analyzing just 77,000 samples/year, which is translated to a total of 14 million individual tests. The considerably large number of tests per sample is due to the fact that, on average, 180 different chemical compounds/sample are targeted, requiring different extraction and/or analysis protocols. There is considerable variability in the number of samples tested on a population basis, ranging from 84 (Cyprus) to 6 (UK) per 100,000 people.
It is quite obvious, therefore, that conventional methods for monitoring food safety are largely inadequate for meeting large-scale testing, and such that would secure a more accurate determination of the level of residue detection in food commodities, in turn safeguarding the public from food crisis events. A key element of this inadequacy is the capacity factor, i.e. the combined effect of high throughput, cost, speed and ease of use. In other words, conventional methods are characterized by low capacity, in spite of their undisputed resolution power (up to 800+ individual compounds in German laboratories).
FOODSCAN addresses this niche, yet extremely important requirement for a high capacity screening system, able to inform the end-user (primary, an analytical lab) on the presence or not of target residues in a sample, prior to its further processing by means of conventional methods (such as gas/liquid chromatography and mass spectrometry).
As it stands, FOODSCAN can compete a sample screening within three minutes, with a capacity of 90-100 samples/hour. In other words, the total of 77,000 samples annually tested in Europe (as already mentioned above) could be screened with the aid of FOODSCAN in just 770 hours or approximately 96 days (considering an 8-hr working day). On average, this would require less than three hours of lab operation per year per laboratory in order to satisfy the screening needs corresponding to the annual sample quota of an average laboratory. Certainly, this calculation does not reflect the actual needs and activities of every lab (due to the aforementioned variability in individual testing capacities) but it provides a fairly true picture of the potential use and advantages of the FOODSCAN system.
In the same line of thought, an average analytical laboratory would use FOODSCAN to screen 273 samples (the average sample quota per lab) for general toxicity (i.e. screening for the presence of pesticide groups rather than individual compounds with the aid of the General Toxicity Sensor) and, depending on their customized demands, also screen these samples for the presence of one or more of the 6+ individual residues currently detectable by the Specific Toxicity Sensor. That means that, should a laboratory opt for taking full advantage of the FOODSCAN capabilities (i.e. utilizing both the General and the Specific Toxicity Sensor), a total of 273 x 7 = 1911 tests would be required, translated into less than three working days per year, an extremely minor fraction of the annual workload.
In this context, technical implementation of tools and procedures for the food control and traceability can greatly help to simplify analysis and reduce costs, thus making the labeling procedures more “producers’ friendly”. It also provides decision-making tools and quick solutions for all of the HACCP, IFS-FOOD and SQMS food safety and quality standards related to predictive parameters to be set. The laboratory and industrial scale-up validation of our cell-based devices provides a serious alternative to be taken into account when considering that the most quick, stable and feasible tool is needed to explicitly provide preventive actions to be taken before processing steps within the food industry. Our model offers a strong basis for the initiation and development of R&D capabilities of the SMEs involved together with the strengthening of quality claims available for end-user SMEs, which altogether lead to the exponential market positioning increase due to the anticipated favorable response given by the food consumers. Handheld, reliable sensors designed for performing food quality and toxicity analysis can have a significant social, economic and commercial impact. Such sensing units can be of invaluable use by individuals (people with species allergies, food and wines tasters, immuno-sensitive), public authorities (custom offices, forensic services, ministries of commerce, public health controllers) or private bodies (food production industry, transportation companies, biological and infant food stores) for the “in-situ” monitoring of food quality. Such units provide reliable information on the food quality, eliminating dangers emerging from adulteration, chemical or biological contamination, and improper storage conditions and fertilizers/insecticides residues. The broad use and public spread of such probing devices applies pressure on industry and growers for producing food and food products of improved quality, while reducing chemical or other harmful agents during the growing or processing level. The application of such sensors during all levels of production and processing assures the overall quality and reveal quality-breaching attempts. The “in-situ” monitoring capability is being further exploited for the fair control of the food price according to the quality provided, helping the adjustment of the markets in regional or continental level per specific item and quality.
The scope for FOODSCAN to help sustain existing SMEs and create new SME business opportunities is significant. The BERA-based FOODSCAN technology is a very flexible platform enabling the possible creation of multi-sensors that all operate using a common readout device. For example, the diagnostic system could be carried onto a farm and, using separate disposable cartridges, an official could test crops for the presence of a wide range of pesticides. The manufacturing processes for the readout device and for the disposable cartridges were designed generic. The development of the portable FOODSCAN system combines several knowledge intensive technologies including digital signal processing, electronic design and implementation, sensor optimization and production, prototype design and development and system integration. The combination, managed by the SME partners, exploits the convergence of a number of technologies and will rely on networked and cooperative research and development for the specific benefit of the SME members of the consortium. The development of this project creates a knowledge intensive system applicable to a wide range of industrial sectors.
The businesses to benefit most from this project are the European SMEs who are at the forefront of implementing the new knowledge but have a limited capacity for product focused research. The smaller companies often have the capacity to high growth rates in terms of job creation and increase in business opportunity. The benefit of the FOODSCAN project to the SME partners in the consortium in terms of competitiveness and business improvements will be substantial compared to the scale of their investment, typically a ratio of 1:50. The multi-disciplinary aspect of this project brought together an SME consortium including IGV, EMBIO, ALVES MANUEL, BODEGAS CONTRERAS, STOLZENBERGER BACKEREI, NESTOR SERRA LARANJEIRA and world-renowned research institutions and companies such as UNISCAN in the UK, Demokritos and the Agricultural University of Athens in Greece.
All of the contributors to the project are based in Europe so combining this knowledge and developing it into a diagnostic platform technology will be a real boost to European competitiveness.

The FOODSCAN platform enables the development of products with improved performance over currently used testing procedures. This is particularly important for applications requiring the highest level of speed, performance and reliability in challenging conditions essential in applications ranging such as on-farm pesticide detection or point of care medical diagnostic testing. Higher performance products will provide the SME beneficiaries with an additional source of competitive advantage.
We expect 50-55% interest from the baking and wining industries due to the wide relation-network in which the SMEs of the projects possess. This anticipation is mainly correlated with the fact that from the total European population 35- 40% of convenience food consumers are still anxious to experience the characteristics of super-sure foods, which were screened against potentially harmful intrusion of pesticide residues. Thus the industrial manufacturers are being “forced” to follow the end consumer trends and modern habits in order to keep and/or to increase their market position.
The newly developed technology represents the first pre-production prototype of a totally new generation of organism-based portable biosensors, which has no alternative or comparison scale on the international market, thus we believe that the major impact leads to a completely new biosensor device segment on the market.
The predictability of the new segment’s growth is hard to be anticipated mainly due to fluctuations of the market, but according to the continuous growth experienced within the past 5 years, we are counting with a direct increase of 15% each year of the sales at the SMEs involved in the project and a minimum of 10% indirect sales increase each year induced by the added value occurring at outside SME parties revealed by the proved safety claims of their products assured by the daily use of the FOODSCAN validated technology.
The example of organic (and, to a lesser extent, ICM) agriculture has demonstrated that very high net monetary returns (up to 90%) (Seavert et al. 2007) and increase of exports (up to 2200%) (Martinez 2010) can be realized by increasing the traceability and chemical safety of food. Therefore, FOODSCAN represents one serious approach to moving agriculture in a more sustainable direction, one that can provide added value to producers in niche markets, enhance food security in developing countries, and deliver a range of ecosystem services to the public at large.
It should be finally mentioned that, according to the FOODSCAN Technical and Economical Comparison Study (Deliverable 4.1) an initially limited market penetration would create a sustainable business from Year 1, even without necessitating the considerable increase of sample testing allowable by the adoption of the FOODSCAN technology. Furthermore, by simply exploiting the minimum capacity of the manufacturing plan (110,000 test kits/year, as described in the previous section), the FOODSCAN consortium can satisfy the current needs of more than 25% of the analytical community, translated into a revenue of ~1,7 million €.

1.4.2 Main dissemination activities

The FOODSCAN project has the following dissemination strategy:

To widely promote the FOODSCAN project with its European dimension to a wide community of industry and the science base, followed by targeted dissemination to identified user groups to aid rapid exploitation of project outputs.

In order to ensure the widest dissemination of project results, a comprehensive set of materials was designed, printed and/or electronically posted. Dissemination materials are used as reference in order to market the FOODSCAN technological solutions to potential customers and to build the project identity.

The dissemination plan was divided into the following parts:
- Dissemination materials- several dissemination materials were produced (website, logo, leaflet, videos etc.) with graphical items to illustrate the project’s concept and expected results and to make more attractive for the interested parties.
- Publications of project results and innovations
- Conferences, seminars and workshops- participation of International or European major events to increase project exposure to the public.
- TV documentaries and radio interviews – in EuroNews and national networks
- Cooperation-
- Coordination with other relevant EU and national projects where identified as relevant to FOODSCAN
- Project meetings to share information and to strength the cooperation among the partners in the Consortium.
- Training- Development of appropriate training material to support the SMEs

See the dissemination activities in details in Chapter 2 (Section A).
The FOODSCAN logo has created which is included on all materials and other documents concerning the project.
Flyer has been designed during the project runtime to give general information about the project aims and vision, and the list of contacts.
1.4.3 Exploitation of results

The project management includes specific preparatory measures for the exploitation and dissemination of results, and identified a dedicated Exploitation and Dissemination Manager (Mr. János-István Petrusán, IGV) who has the responsibility to prepare and update the Exploitation Plan and to co-ordinate any action needed to ensure protection of the foreground and side ground IP generated within the scope of the FOODSCAN project, specifically for the benefit of the SMEs.

The Final Exploitation Plan includes:
- Implementation strategies to be followed, including industrial scale-up and validation
- IPR methods of evaluation for the prototype and other results of the project (copyrights)
- Acquisition of IPRs and patents for the developed technologies and for products
- Definition of target groups, organizations (mainly SMEs) and the means of reaching them

1. As described in the Annex I of the Grant Agreement (Description of Work), clause 4.1.1.§3, EMBIO, as the owner of the basic BERA and membrane-engineering technologies, will be the focal point of promoting FOODSCAN platform beyond the Project lifetime and beyond Project, i.e. the default Partner with exclusive jurisdiction over the use of these technologies in the FOODSCAN platform and the manufacturing/assembly of these technologies into the platform. However, all the other participating SMEs hold an exclusive license, including rights to use and commercially distribute the technology on a privileged basis, which is specified in the Exploitation Agreement. In order to implement the joint FOODSCAN commercialization plan, the following framework is suggested:
- The SMEs participating in the consortium will sign an MoU with a mutually binding duration of five (5) years. After the expiration of this period, SMEs have no legal obligation to the consortium unless a new MoU is signed.
- In the beginning of the first year of the agreement (exact date should be agreed upon and may differ from beginning of calendar year), EMBIO invites negotiations with each of the SMEs with the intent of concluding a sales- and representation agreement for the duration of the current (1st) year. It is not required that all partners will sign agreements with EMBIO, so this settlement allows for a considerable flexibility.
- In the agreement, exact terms related to expected sales volume and other quantitative commercialization targets are agreed upon, along with exclusivity agreements regarding sales territory and list of food products to be analyzed.
- In the beginning of the second year, the fulfillment of the targets of each bilateral agreement will be evaluated and the agreement re-negotiated for the 2nd year. This process will be iterated each year for the duration of the 5-year period.
- Should any partner fail to meet the targets set for a specific year, then the other party has the right to withdraw from the agreement unless otherwise negotiated between the partners. Termination of a bilateral agreement between an SME and EMBIO will not affect the rest of the bilateral agreements.

2. The Foreground generated by all SME comprises patentable items regarding (i) the UNISCAN device (as a utility model and/or industrial design patent) and, (ii) the method for manufacturing the target-specific antibody. IP protection per se may not be sought by all SME partners; therefore, the initiative for patent application should involve by default EMBIO, as the administrator of the original BERA-based IP and any other interested partner. In such case, the joint owners of the Foreground shall establish a separate Agreement regarding the allocation and terms of exercising joint ownership rights for jointly applying for a patent.

3. Since EMBIO is the owner of the major background (BERA system and associated membrane-engineered cells) and supplied this background on a royalty-free basis for the successful generation of the foreground, it will initiate negotiations with all Consortium SMEs interested in the patenting of FOODSCAN system. Such protection plan will have to include an initial exploration of the possibility to submit a patent application with the necessary assistance of the relevant experts (e.g. patent lawyers). EMBIO, as the owner of both the background IP and major part of foreground IP (i.e. BERA system and associated membrane-engineered cells) will initiate such exploration and the relevant costs will be shared among all SMEs interested to protect the foreground knowledge.

As agreed in the Annex I of the Grant Agreement (Description of Work), the SMEs will have the following rights regarding the FOODSCAN technology and system: See in attachments

List of Websites:

The public project web site is up and running and can be found at: www.foodscan.net
It is one of the main media for the dissemination of FOODSCAN results and ensures a vision of the project to the general public.
The website contains the general objectives of the project, all partners involved with contact details, platform for the visitors for messaging and available information, including the advancement and partial scientific results achieved along the project.
Furthermore various internal share platforms have been designed, where the project beneficiaries can exchange information on-line. The internal communication platform has all elements for quick share of documents and communications between FOODSCAN Partners.

The project members can be contacted through the following emails:
• Project Coordinator: János- István Petrusán: j_petrusán@igv-gmbh.de
• Scientific Manager: Prof. Spyridon Kintzios: skin@aua.gr
• Administrative and Financial Manager: Erika Fábik: e_fabik@igv-gmbh.de