EuroBioSAS_NANOIOBIP_Insect Odorant-Binding Proteins on Biosensor to Diagnose Crop Disease

The area of plant-insect interaction and detection of plant diseases has been rather descriptive, and perhaps less inclined to use bioanalytical research tools. More specifically, even as biosensors found a wide use and acceptance as reliable measuring tools, their application in the above-mentioned areas seems to lag behind the mainstream, as objectively evidenced by the available publications. This CRP aims at developing a nano-biosensor system to diagnose crop disease. This consists of either an array of insect Odorant-Binding Proteins (OBPs) or, alternatively, Pheromone Binding Proteins (PBP) -modified Conductive Polymer (CP) nanofibers placed on Inter-Digitated microelectrodes Array (IDA) of a Liquid Ion-Gate Field Effect Transistor (LIGFET) or of an array for organic field-effect transistors (OFET) with a protective coating deposited by plasma polymerization allowing, e.g., for antibody titration. Specific binding of odor molecules to OBPs or pheromones to PBPs will generate conformational changes that will affect the source-drain current of the transistors. This combined to the CP`s inherent charge transport properties yield a direct and label-free readout. At the conclusion of this CRP, our best nano-biosensor system prototype is expected to achieve LoDs down to very few femtoMolar (10-14 M) or better, for low-cost, reliable operation. This makes it by far the best usable bio-tool designed to date for crop disease diagnosis. Furthermore, this outcome has a strong potential for applications in other fields, such as detecting odor molecules in conjunction with applications in healthcare or defense. Some of the specific goals of this CRP include: Determine selectivity/specificity of insect olfactory cells toward odor molecule using whole insect antennae- based microbiosensor (ABB), a well characterized system. Insect olfactory cells are compartmentalized in cuticular structures, the sensillas, on the antennae`s surface. Micro-fabricate several prototypes of IDA LIGFET and OFETs as microelectronic readout devices. Synthesize CP nanofibers and semiconducting nanowires using template method, deposit nanofibers attached onto IDA LIGFET. Functionalize nanostructures with OBPs/PBPs, resulting in a nanobiosensor, assemble these in array. Test nano-biosensor systems with odorants as analytes, compare with ABB.

For the sensing of light, e.g., in optical communication, we have extremely powerful devices with the ability to detect even single photons. The monitoring of sound in acoustic communication is also technically no problem: microphones are available with amazing performance parameters. Only for chemical communication, for smell or taste detection on a technical level we have (nearly) nothing. Despite the fact that the monitoring of chemicals in chemotaxis, i.e., in the search for food of many organisms or the exchange of chemicals between species as a way to communicate with each other is the oldest of our sensory repertoire, we have essentially no technical device that offers the sensitivity and the bandwidth needed to sense and to differentiate many different odors. There are many needs for air analysis and the detection of smells, e.g., in food quality control, health diagnostics, crop disease detection, indoor air management, military and security applications and so on. However, earlier attempts to fill this gap by artificial noses failed (with the only notable exception being the alcohol breath analyzer used by police) mostly because of lack of sufficient sensitivity. What was developed in this project is a bio-mimetic approach, a bio-electronic nose. We merged the world of ultrasensitive microelectronic devices like transistors as the transducer platform and coupled it with the living world of bio-functional (building blocks/) modules as a way to synergize the sensitivity and selectivity of certain protein structures - here in our case odorant binding proteins - towards recognizing and binding their ligands, the odorants we want to smell, with the versatility of microelectronics: we now can begin to smell electronically. We demonstrated this by fabricating a field-effect transistor with reduced graphene oxide as the gate material which we functionalized by the covalent attachment of Odorant Binding Proteins (OBPS) from various species, mostly from the honey bee Apis mellifera. This biosensor, when mounted to a flow cell and exposed to aqueous solutions of a variety of odorants, all more or less relevant of the behaviour of bees, generated a quantitative signal form the transistor, a change in its source-drain current at a constant gate voltage, that we could analyse quantitatively in terms of a Langmuir binding model. Thus we could discriminate between strongly and weakly binding odorants and could compare the results with physiological measurements of the binding of odorants by the antenna of the honey bee.