In DNA and protein microarrays the viscosity of the fluid increases substantially with solute concentration, and also during the evaporation process.
This has a large influence on the shape of the deposition and the distribution of (bio) molecules on the substrate, while the functioning of e.g. antibodies in microarrays is critically dependent on this distribution. Hence we intend to develop an extended physical model for the calculation of the thickness of deposition that results after evaporation of a solvent from a droplet and of the distribution of (bio) molecules on/in the substrate.
The aim of the project is to study spot formation and appearance after deposition of biomolecules by inkjet printing on various substrates (nitrocellulose, polystyrene, glass, epoxy, carboxyl, amine, hydrogel). The surface properties of the substrate play an important role in the orientation and conformation of the biomolecule on printed microarray. This orientation is of crucial importance for the functional properties of the biomolecule and, hence on the quality of the applications developed. Therefore we studied the analytical data of the bound biomolecules (antibodies) and we also tested the functional characteristics of the biomolecules. The influence of the spotting conditions (buffer pH, humidity) on these substrates will be studied. To study the distribution of antibody molecules on different substrates at various conditions, fluorescently labeled antibody will be used. Both the number of antibody molecules immobilized as well as the functionality (i.e., binding to their respective targets) of the immobilized antibodies will be assessed.
Mastitis model will be used to study various parameters in the production of antibody microarrays. Microarray for six different types of antibodies aCy5, aDIG, aDNP, aFITC, aTexas Red and IgG-biotin (control) was produced onto above mentioned substrates. These antibodies were specific for mastitis causing pathogens, by combining a rapid PCR method with a user-friendly and fast antibody microarray, the nucleic acid microarray immunoassay (NAMIA) was developed (Fig. 1). PCR was performed with tag-labelled primers; the forward primer with a discriminating tag and the reverse primer with biotin. The double-labelled amplicons were sandwiched in one-step incubation between these carbon nanoparticles conjugates and the immobilised antibodies resulting in black spots. The signal could be further enhanced by a short incubation with alkaline phosphatase substrate solution. (Fig. 1)
Spot morphology and distribution
The spot morphology of printed biomolecules was characterized by AFM. The distribution of the printed biomolecules on the spot was analyzed by confocal microscopy.