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1 Fondazione Bruno Kessler April 2015 Newsletter, Issue 1 SYMPHONY Newsletter Integrated SYsteM based on PHOtonic microresonators and Microfluidic Components for rapid detection of toxins in milk and dairy products IN THIS ISSUE The project SYMPHONY is a research and innovation project, funded by the European Commission to find a rapid, cheap and effective solution to detect toxins in milk. More infos about the project and the consortium at pag. 2-3 Achievements First prototypes of Photonic sensors demonstrated to achieve Limit Of Detection (LOD) of 0.8*10-6 Refractive Index Units (RIU). Find more results after 18 months of the project at page 4. The Milk Day Experts in milk analysis and quality, scientists in microsystem technologies and biosensors and end users gathered in a workshop organized in Trento in February 2015, to talk about milk. More details from pag. 6. This project has received funding from the European Union s Seventh Framework Programme FP7-ICT under grant agreement No Project duration: 1 st November st October 2016.

2 SYMPHONY in a nutshell Integrated SYsteM based on PHOtonic microresonators and Microfluidic Components for rapid detection of toxins in milk and dairy products I n the dairy industry, one of the most pressing needs for food quality and safety is the timely detection of aflatoxin M1 contaminations that originate from animal feed and are secreted into milk. This issue represents a hazard for human health and an economic loss for the dairy industry. The available technology for aflatoxin detection is laboratory-based, in most cases requiring sample preparation and does not provide timely identification of contaminants, thus fails to deliver cost-effective management of milk quality. The entire supply chain relies on the fact that the dairy products delivered to consumers are safe. Current best practices for Aflatoxin management in milk in the modern dairy industry recommend aflatoxin M1 analysis to be carried out routinely for quality assurance and quality control purposes as well as for feed-back mechanisms to farm and animal health management, as requested by European Commission regulation (EC) No. 1881/2006. The current procedure involves the collection of samples from collection tanks. The milk samples are analysed in a laboratory with screening tests such as Enzyme-Linked Immunosorbent Assay (ELISA). Contaminated milk that exceeds the 50 ng/kg (ppt) aflatoxin limit is not allowed in the market and has to be disposed. T he SYMPHONY STREP project (FP7-ICT ) addresses the needs of the dairy industry with novel microfluidic technologies and biochemistry for sample preparation, photonic integrated sensors and compact hardware for integration in the production chain of the dairy industry, leading toward precision process management. Interview with the coordinator Dr. Leandro Lorenzelli, Head of the MST research unit at Fondazione Bruno Kessler, Trento Could you explain what SYM- PHONY is about? SYMPHONY is proposing a new generataion of high-tech devices allowing for fast and reliable analysis of the dairy products quality. These would lead to a more precise and effective management of production and distribution processes. What is SYMPHONY main objective? The objective is to integrate different technologies like photonics, microfluidics and microbiology to create a portable and quick tool to detect aflatoxin in milk. This project has received funding from the European Union s Seventh Framework Programme FP7-ICT under grant agreement No Project duration: 1 st November st October 2016.

3 SYMPHONY Consortium SYMPHONY objectives Symphony facts EU STREP project 7 partners in 5 country 3 years project 3 M Funding 3 private companies 3 research and academic centers 1 dairy cooperative Follow us on 3 SYMPHONY is a demand-driven project pursuing: Advancement of MNBS devices toward real-world applications by providing: Integrated sample preparation for analytical tools in dairy industry, Advancement in high sensitivity photonics biosensors, by using aptamers to provide selectivity to targets and improved sensor stability and hybrid integration of sources and detectors. Improvement of milk quality assurance by making available new technologies and procedures for the detection of aflatoxin and other contamination Improvement of dairy industry competitiveness by early detection and effective corrective action against aflatoxin outbreaks and contaminants: On field simultaneous detection of various contaminants, thanks to arrays of photonic sensors; Analysis of raw milk and other liquid dairy products, such as yogurt, butter milk, kefir, whey; Quality assurance at various HACCP Improvement of food quality with positive impact on human health

4 First year scientific achievements Stakeholders needs, system specifications & architecture Stakeholder needs were surveyed by interviews and questionnaires for the definition of user requirements, specifications and the general architecture of the system. Antibiotics were selected as best additional target to be evaluated in addition to Aflatoxin for multianalyte system. Development of photonic sensors SiN asymmetric Mach-Zehnder interferometers (amzi) were developed by LioniX SiON microring resonators (mrr) were jointly developed by FBK and UniTN The achieved Limit Of Detection (LOD) was 0.8*10-6 Refractive Index Units (RIU) and 1.6*10-6 RIU in the case of amzi and mrr, respectively. VCSEL sources and photodiodes are being integrated by means of heterogeneous assembly Functional surfaces and analytical methods Aflatoxin purification is based on functional surfaces based on surface coating with mussel foot protein. Anti-aflatoxin aptamers are immobilized by FBK on the detector surface for the selective detection of aflatoxin, with a demonstrated layer density around aflatoxin molecules per cm 2 The sensor regeneration and reusability was demonstrated up to 17 regeneration cycles. Development of microfluidic components Microfluidic modules for fat separation and milk fractionation produced and tested showing a fat reduction comparable with laboratory techniques. Reversible Sensor packaging with integral flow cells, has been designed and fabricated. Micro Ring Resonators Watch the video on: node/3 Aflatoxin response of functionalized microring resonator Hybrid integration of microfluidics and silicon chip Success criteria for SYMPHONY project 4

5 SYMPHONY dissemination Other events Workshop on biosensing, Trento 30th March evento/dphys/4244/workshop -on-biosensing Workshop on Smart systems for food industries, Trento 15th November Researcher s Night 2013, Trento Upcoming events 21th World micromachine summit, Berlin May Recent Advance in Food Analysis (RAFA 2015), Prague 3-6 November index.html 3rd EOS Conference on Optofluidics (EOSOF 2015) held under the umbrella of the World of Photonics Congress 2015, Munich June

6 The Milk Day rationale The Milk Day was organized in Trento on February 4th in the framework of the AISEM conference to discuss relevant open issues in the application of MST in the analysis of milk contaminations. Can the use of Micro System Technologies and biosensors provide a solution to overcome these limitations? What are the major factors that may jeopardize the integration and automation of current analysis techniques? Milk chemistry and composition, strict regulations for milk quality, dairy industry requirements, the large diffusion of milk consumers and producers pose substantial challenges, but, in the meanwhile, offer a number of opportunities for exploiting the huge potential of miniaturized systems and sensors. Presentations available on: / TheMilkDay Milk quality testing and regulations Ulrich Leist, DRRR Germany Dr. Leist provides an overview of DRRR activities as an accredited body for proficiency testing services. Milk is principally tested at food producers for two purposes: 1) Testing for milk payment, where main quality parameters are: Fat Protein Freezing point Somatic cell count below Total count of bacteria below This is typically performed off-line in large laboratories with very large number of test per day (25000 sample/day) with high throughput instruments like IR spectroscopy 2) Testing for chemical residues, pathogens and contaminants for safety. Since food manufacturer are responsible for their products, they are in charge for safety enforcement; tests Milk quality testing in Trentino Nicola Cologna, CONCAST, Trento are performed for legal limits of: Chloramphenicol Mycotoxins Aflatoxin M1 Trichlormethan Radionuclides Residues Others While reference lab only apply reference methods, since testing campaigns are limited and time is not a constraint, reference tests cannot be performed on all samples at the manufacturer site. It is therefore possible to accredit screening test to have a fast feedback on all samples (or a larger number) if properly calibrated. For this purpose, calibration solutions are required, which is an art by itself since the challenge for test kit producers is to assure sufficient selectivity, sensitivity, accuracy. CONCAST structure, includes more than 800 associated farmers, 17 dairies and the centralized testing lab. Dr. Cologna presented the tests performed at Concast laboratory, which, in addition to common milk quality parameters like fat and protein content, antibiotics, etc. also measures technical properties of milk related to cheese making (e.g. milk coagulation properties, whey starter analysis, presence of spores, which are particularly important in hard cheese making). A specific case of introduction of a new accredited test for quality is presented, where FTMIR was correlated to milk coagulation properties in order to process the test with fast and high-throughput procedure with respect to traditional method, which requires 30 minutes to run. This study was conducted in collaboration with the Department of Agronomy Food Natural resources Animals Environment of the University of Padova (Italy). 6

7 Milk and health in the province of Trento Alberto Mattivi, Director of the Department of Hygiene and Veterinary Public Health, Health Services Provider of the Autonomous Province of Trento. Statistics of dairies in Trentino region highlight the particular production structure in the region, where farms are very small and mountains huts are used as feeding grounds in the summer. Primary production in Trentino region is the high quality traditional cheeses, which represent an interesting point for safety enforcing with respect to large companies found in Europe and other Italian regions. In Trentino, the official tests are performed by audits and inspections on all production aspects (veterinary, product safety, HACCP, etc.) and according to both regulations and internal rules agreed by producers and competent authority, which are often more restrictive than general rules. For instance screening test for aflatoxin are required to be confirmed by test on single producers by ELISA and eventually by reference HPLC testing if a value of aflatoxin higher than 35ng/kg is found. In the Department experience, the majority of non-conformances are related to non-observance of internal rules of the production plant. Interestingly, there may be local issues with particular, new contaminants as in a recent case in Trentino region related to steel manufacturing plant, which resulted in local contamination with dioxins and an intense testing campaign to identify possible contaminated products before they reached the market. This would open the field for new screening test if technically available. The cost per test to identify dioxins is quite high ( /sample for reference test, /test for screening) with reduced number of molecules identified. Other similar examples of specific needs may be found. Focus on Aflatoxin Aflatoxins are naturally occurring mycotoxins that are produced by Aspergillus flavus and Aspergillus parasiticus, species of fungi. The name, aflatoxin, was created around 1960 after the discovery that the source of "Turkey 'X' disease" was Aspergillus flavus toxins. Aflatoxins are toxic and among the most carcinogenic substances known. After entering the body, aflatoxins may be metabolized by the liver to a reactive epoxide intermediate or hydroxylated to become the less harmful aflatoxin M 1 (Wikipedia) A SEM image of an Aspergillus 7

8 Rapid Detection of Aflatoxin M1 in Milk Veronica Lattanzio, Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR). The Institute of Sciences of Food Production (ISPA-CNR) is a centre of excellence, worldwide renowned, acting in the fields of scientific research, innovation and technology transfer aimed to improve safety and quality of agro-food products Dr. Lattanzio presents the work of ISPA institute on rapid detection of aflatoxin M1 in milk, and an overview of analytical challenges and validation aspects. Rapid test methods for measuring Aflatoxin M1 (AFM1) in milk are available either as commercial kits or as research methods. Enzyme-linked immunosorbent assays (ELISA), lateral flow tests, immunoaffinity columns coupled with fluorimetric assay are common formats in the current market. The main purpose of screening methods is to detect the presence of a contaminant at level of interest allowing rapid decision making, i.e. in 5 to 20 minutes with respect to about 4 hours/samples in reference methods. European Union has set a maximum permitted limit of µg/kg for AFM1 in milk. Besides the high sensitivity required for AFM1 detection in milk, the major analytical challenge when developing screening tests is to make them reliable and robust, in particular by reducing the false negatives, for laboratory and in-field use. This means to cope with differences from matrix to matrix, environmental conditions, operator skills, lot-to-lot reproducibility. Recent efforts of the European Union, for establishing practical guidelines for the generation of fit-for-purposes performance parameters for screening methods for mycotoxins in foods, resulted in the Regulation 519/2014/EC. The EC validation scheme and its practical application to evaluate performances of a commercial kit for AFM1 detection in milk were presented and discussed. It is common experience that fat removal can change the calibration curve of a method and sample treatment/clean-up is needed in order to improve the analytical accuracy of the system. Dilution is already a commonly used sample preparation method. 8

9 Research & Business: Transitioning Innovation to Commercial Success Mark Whatton, Technical Projects Manager at QuadraChem Laboratories Ltd, UK QCL provide rapid analytical testing solutions for a wide range of industries across the UK. Using extensive experience QCL applies advanced technologies to the improvement of industrial processing, quality control and environmental monitoring. Dr. Whatton discussed in his presentation an opinion on why Micro Systems Technologies, while promising exciting and novel ways to monitor the quality of food and beverages, are difficult to translate in commercial reality. There are many reasons why promising systems fail to reach the market, but is it possible to understand and mitigate these risks early on? The presentation described the target needs and wants required to hit the market, as evaluated in the development of the Symphony project: Innovation aims need to work with commercialisation aims, since commercial successes are those that provide clear benefits to those who will use them. These benefits can only be known by understanding the target market. Selection of technology and design should target specific requirements from end-users. Development phase should go from a proof-of-concept into a pre-production prototype. An industrial partner focusing on instrument pre-production prototype will understand and work to maximize the major attraction of the innovation, and bring it to final users in such a way it is clearly perceived. Working out what benefits are for a project AFTER the research and innovation is too late MST for food quality and safety Andrea Adami, FBK-Technical manager of the SYMPHONY project. Dr. Adami presents a critical review of advantages of microfluidic devices in food analysis. Among different peculiarities of microfluidics, the major points of interest are mainly the integration of multiple functions in an integrated system, the faster mass and heat transfer in small channels, the possibility to create a laminar flow, multiple parallel testing and high surface interaction. On the other hand, small samples typically used in microfluidics are not a key issue in food analysis, since sampling large volumes are often required for sample representativeness and typically low cost of the sample. Techniques, concepts and devices of interest in food analysis include capillary electrophoresis, micro PCR, immunoassay, particle and drop separation and multiplexing techniques. 9

10 Project Coordinator: Dr. Leandro Lorenzelli Tel Technical Manager: Dr. Andrea Adami Tel FBK - "Bruno Kessler" Foundation Center for Materials and Microsystems Via Sommarive, Trento Project Website: To subscribe/unsuscribe write an to: morganti@fbk.eu