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Mobile wireless Device microcantilever-based biosensor to identify and measure the aflatoxin B1 in animal food and M1 in the milk-chain

Periodic Reporting for period 1 - MEDIuM (Mobile wireless Device microcantilever-based biosensor to identify and measure the aflatoxin B1 inanimal food and M1 in the milk-chain)

Reporting period: 2015-12-01 to 2016-05-31

The aim of the project was a dramatic innovative solution to solve all the problems related to the high mycotoxins (B1, M1) contamination levels checked in animal feed and milk and more in general in the food-chain. This study defines process and product innovation.
Mycotoxins are secondary metabolites generated by fungus, produced on vegetable food for human and animal use. They are antinutritional extrinsic factors, xenobiotic contaminating the food, toxicity charged, and the cause of mycotoxicosis pathologies. Currently only laboratory instruments are available, but it is needs controls in a capillary way, providing tools which will not only be used by the skilled-people, but also by the producers themselves.
This feasibility study represents the first step to the development and marketing of a new product: a portable, mobile wireless device, cantilever -based nano-biosensor, to measure in field the B1, M1-aflatoxins to very low level values in animal feedstock and in the milk-chain, with a dramatic improvement of the food quality and safety. This product is a milestone in product and process innovation.
The feasibility study had also the objective to define a new validation method, defining a new process to identify and measure the B1 and M1 aflatoxins in the primary sources and in the milk-chain. This study has defined an integrated system to control and to implement all the processes to guarantee the correct B1 and M1 aflatoxins quantity measurement extending the control to the total live stock and milk chain.
The objectives performed have been:
o A mobile device to do the measurements everywhere
o A new method to obtain more precision and accuracy
o Cost reduction, the sensitive element is very cheap
o Increase of security and quality of the measured materials
o increase the productivity and the competitiveness
o Easy use of the device, usable also by non-skilled staff and increase of the measurements number
In comparison with the current measurements methods:
• cost reduction up to 90%
o easy to use, no specialized people or laboratory instruments
o precision and accuracy, the nano- biosensors sensitivity is in the order of the zeptogram (10-21 g).
o measurement everywhere, in the production plants and in field
o affordable price, list price defined for a large SME’s composed market
The main tasks of the work (feasibility study) and the related time frame have been:
T1. In depth analysis and user needs assessment (M1-M3)
T2. Market analysis and commercial forecast (M1-M6)
T3. Definition of support material/methods of integration/sensitive element (M2-M6)
T4. Definition of the system of detection/ electronic control/sensor package (M4-M6)
T5. Definition of the communication system/ user-interface and data management (M5-M6)
T6. Business plan (M4-M6)

The development of the feasibility study, as described in the follow, has defined the features for a mobile wireless device microcantilever biosensor based and Lab On Chip Micro Elisa based system with these functional parameters:
• Mycotoxins: aflatoxins B1 e M1
• Measurement system: micro-cantilever biosensor based
• Resolution: 0,1 ppb (B1)
10 ppt (M1)
• Solutions (for B1 measurement): water-based
• Measurement time: less 1 hour
• Wireless mobile device for “in-field” use
• User interface: non skilled people like
• Well defined use-procedures

The macro-result is represented by the device definition and feasibility to measure in a quantitative method the level of the B1 and M1 aflatoxins to improve the food security level.

This device can be supported by two alternative technology bases:
• Microcantilever Biosensor
• Lab On Chip Micro Elisa
A more accuracy quantitative method measurement means more efficiency in control process, cost reductions and an improvement for the competitiveness.
In addition an innovative aspect is represented by the use of water-based solvents(B1) to improve the security of the people involved in measurement process both outdoor then indoor.
This device has a dramatic positive effect on the B1 and M1 aflatoxins contaminations reduction.
As reported in the feasibility study proposal, Mycotoxins represent a considerable world-wide problem, as recent (2012) European Commission evaluations estimated: 25% of cultivation in Europe is contaminated and more than 35% of imported raw material from the rest of the world, particularly from China.
The toxic effects caused by the ingestion of mycotoxins can include productive performance decrease (reduced growth and fertility), immunosuppression and increasing sensitivity to infections, gastroenteric disorders, pulmonary aedema, bleedings and neoplasia, even though the concentrations we usually find in food seldom lead to high toxicity symptoms.
Considering these preliminary remarks, it is clear the high social cost represented by the mycotoxins contamination and the strong negative impact for the European health system.
The detection of the right value of micoxtoxins contamination in feedstock and in the milk chain improve in a dramatic way the food quality and safety, in specific all the milk -derived aliments.
The above highlighted needs have been identified thanks to a continuous cooperation with important end-users which felt the necessity of such an equipment for a better control of their products. The SME proposer is based in Cuneo area (Piedmont- Italy) where are present the 45% of Italian farmers and the 60% of animal-feed producers.

The new MEDIuM device brings the following main advantages:
 Quantity measurement
 Fast and easy: -80% time consuming
 Safety of all operators (both in the lab and in “the field”). The system is in fact compatible from the point of view of company workplace safety (for example, it limits the use of organic solvents harmful to human health)
 Measurement in field
 Increase food safety: from 70% to 100%
 Increase health safety
 Cost reduction: -60% (average)
Today the qualitative measurements have done through the WOOD lamp using a fluorescent method.
The ELISA method (Life Technologies, R&D Systems, Sigma-Aldrich,etc), is fast but it is sensible to the different interfering and to the environment conditions in which the measures are done. Furthermore it needs laboratory instruments.
The qualitative methods (Gipsa, Vicam, Diachemix, etc), fast and easy, detect only the fungus presence, but there is not a linear proportion between the fungus detection, in field, and the mycotoxin presence.
This situation underlines the need to implement new and innovative monitoring methods and the set-up of new measurement devices and instruments to fill real user needs and monitoring lacks.
Quantitative analysis of M1 can be performed by High-performance liquid chromatography method (HPLC). This technique jointly with techniques such as UV absorption, fluorescence and mass spectrometry offers good sensitivities but they frequently require skilled operators, extensive sample pretreatment with long sample preparation time, expensive and not portable equipments.

In particular, the rapid and accurate identification of AM1 in the field is important for prompt prevention of spreading. Therefore, a method employing a portable detection system and real-time analysis of B1 and M1 in the field is needed.

The instrument proposed in the project represents a real dramatic process and product innovation to solve the B1 and M1 contamination problems.
The expected improvements are (see attached image)

It is clear the impact on the mixotoxins contamination problem, also considering the high level of contaminations detected in the last years in the European market. A technological advanced device, as described in this project, can represent a dramatic improvement in the control of milk chain reducing costs, improvement of profits and facilitating the Health Control Authority activities.
In summary, the presence of aflatoxin B1 in animal food and M1 in dairy food is a significant problem for human health and its identification and quantification in milk is a crucial issue which has to be carefully managed.