Research and development of innovative molecular sensor technologies for the monitoring of volatile organic compounds in wines

The scientific and social increasing demand for quantitative/qualitative determination of natural and artificial chemical species in different sector of human life (health, living environment, food, etc.) highlights the need for new strategies and new measurement methods. In particular, the agro-food industries need quick and sensitive tools to identify molecules in its final products or to monitor them during production processes. This need is due to the increase of food frauds. Indeed, in last decades, adulteration and falsification of food become a very serious problems also as regard human health. Biosensors are cutting-edge and the cheapest tools that can satisfy the monitoring of food quality because they represent an intelligent combination of biological components, such as enzymes or bacteria, and technological components that detect physical and chemical changes and transmit them in the form of data. Among agro-food chain products, wine plays an important role in world trade. Wine is one of the most appreciated beverages and it is a complex mixture of sugars, acid and odours that concourse to give it characteristic flavour. Concerning flavours, specific volatile organic compounds (VOCs) in wine are able to give it fruity o floral aroma. These VOCs belong to terpenes class (monoterpenes and sesquiterpenes) and they all together contribute to wine aroma. Analysis of wine aroma is at the base of quality assessment and physiochemical parameters are important to be monitored in order to limit frauds or adulterations. In an automated food production system, devices as biosensors are important to monitor and maintain product quality and uniformity based on aroma characteristics. The nature is the principal source to discover new molecules that could be used to realize biosensors. In this context, insects represent the main source from which to be inspired because they constitute the largest animal phyla. Insects have an important part in all ecosystems considering their role in pollination, biological control and bioconversion. They are a resource of genes, molecules and processes that can inspire several technological innovations. Considerably sophisticated in insect is the “chemoreception” that is a process by which organisms respond to chemical stimuli in their environments that depends primarily on the senses of taste and smell. Chemoreception plays an essential role throughout the life cycle of insects that respond to different arrays of chemical, biological and environmental signals to locate and select food, mates, oviposition sites and avoid predators. To interpret these signals, insects use a range of molecular components that are located in specialized structures called chemosensilla. The last ones are characterized by a variety of forms and by one or more pores on their surface, that allow the access of molecules to the internal aqueous phase, the sensillar lymph, which surrounds the dendrites of sensory neurons. In the sensillar lymph, a large amount of small soluble proteins, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) are present. Some aphids OBPs are able to bind the terpene (E)-β-Farnesene that it is used as alarm pheromone. In Acyrthosiphon pisum, OBP3 and OBP7 are able to bind (E)-β-Farnesene and they are responsible of alarm response. Recently, Acyrthosiphon pisum OBP9 was found to be involved in perception of this compound. Even Megoura viciae use (E)-β-Farnesene as alarm pheromone and its perception is mediate by OBP3. Acyrthosiphon pisum OBP3 are able to bind also farnesol, structurally related to (E)-β-Farnesene and Megoura viciae OBP3 is able to bind limonene. Farnesol and limonene are listed as wine terpenes. Starting from this consideration, aphids OBPs were produced through recombinant procedures and used to develop a biosensor able to bind terpenes in wine. Beside farnesol and limonene, already tested with aphids recombinant proteins, others wine terpenes were tested with Acyrthosiphon pisum OBP3 and Megoura viciae OBP3. A preliminary in silico analysis of aphid OBPs, including also Acyrthosiphon pisum OBP9 and OBP7, was conducted to select other terpenes useful for a rational design of the biosensor. Terpenes selected were geraniol, nerol and citronellol. Concerning biosensor assembly, the choice was to produce optical silicon biosensors. Optical sensors are more versatile than others because they can be made from different materials (silicon, glass, polymers, etc.) and among materials, silicon has gained attention and popularity in recent years because of its ease fabrication, special optical properties and its versatile surface chemistry. Moreover, silicon is readily available, low cost and biocompatible. Acyrthosiphon pisum OBP3 was the first protein used to optimize the optical biosensor assembly protocol because of its previous functional characterization. For biosensor development, Porous Silicon (PSi) surfaces were used and the flow sensing was used as a strategy to verify the behavior of sensing in real time. OBP was adsorbed onto oxidized surface simply flowing the protein on surface and farnesol was tested in different concentration (range: 10 μM – 400 μM). Even if results are not conclusive, they are encouraging considering that terpenes are very small molecules that give a small change in the refractive index.