Integrating technologies for the development of peanut allergen detection bio-assays

Peanut allergy is a common and often severe condition with no medical treatment available so far and the only existing therapy being avoidance of allergen-containing food. Because rigorous labeling of products is required, it is essential to improve the performance of current bio-assays for detecting potential allergens in food samples. In order to challenge this problem, we are employing magnetic detection technology combined with bioreceptors against one of the most important peanut allergens, Arah1 protein. Magnetic fields are used to increase the speed of the assay by transporting the on bead captured Arah1 proteins to the detection surface while magnetic forces discriminate specific from non-specific bonds between the particles and the surface. In this assay we used both antibodies against Arah1 protein as well as aptamers selected and characterized by our group 1 . An aptamer with high affinity (Kd 92.3 nM) and specificity for Arah1 was selected using capillary electrophoresis (CE)-SELEX approach. The performance of this bioreceptor is compared to already established biosensing technology with antibody coated superparamagnetic particles that capture Arah1 protein. In these model experiments we apply magnetic forces to the Arah1 proteinantibody bond and investigate dissociation properties of such a formed complex. For this purpose one of the biomolecules is immobilized on the surface of superparamagnetic particles while the complementary molecule is on a solid phase. The assay is performed using an instrumental set-up consisting of: (1) a sample holder that supports the fluid cell in which the beads are incubated, (2) a microscope-camera system for imaging the particles and (3) an electromagnet to apply a constant mechanical pulling force in picoNewton regime. When applying a force, a measurement of the time dependence dissociation reveals the rate constant (koff) and binding characteristics. In addition, the necessary applied force needed for the removal of unbound and non-specifically bound particles can be measured. We consider this a very promising technology as understanding the interaction between Arah1 protein and either of its bioreceptors will help in further optimization of novel assay technologies for achieving higher sensitivity and specificity. In a later phase, we will integrate the magnetic detection technology with a digital microfluidic chip. Digital microfluidics involves the individual droplet (300 nL) movement on a planar surface by means of the ‘electrowetting-on-dielectric’ principle. The integration allows the execution of this forcediscrimination assay in a highly automated and miniaturized way. Schematic images of the complete setup and images of chip design and holder are drawn in the figure below. The digital lab-on-a-chip will be biofunctionalized by the creation of spatially controlled micropatches 2 , allowing the covalent attachment of aptamers and antibodies against Arah1 protein on the hydrophobic chip surface.