Inkjet printing technology based on the principle of piezoelectricity is one of the common non-contact printing techniques that can deliver small droplets with reproducible volumes. By applying this technology multi-analyte, microarray diagnostic assays can be produced. In many evaporation processes of practical importance such as the evaporation of solvents filled with protein molecules, the viscosity of the fluid increases substantially with solute concentration, also during the evaporation process of printed droplets.
This has a large influence on the distribution and the activity of (bio)molecules adsorbed onto the substrate, and, consequently, on the functioning of microarrays prepared by inkjet printing. One of the problems to solve is the so-called "coffee-stain effect", i.e. the clustering of biomolecules at the edges of the drying droplet. This results in a reduced overall signal and, therefore, applying printing conditions that result in an even distribution of active biomolecules is of utmost importance.
The STW project Bio-STIPS focused on the development of an extended physical model for the calculation of (Eindhoven University of Technology):
- the thickness of deposition that results after evaporation of a solvent from a droplet, and
- the distribution of (bio)molecules on/in the substrate
and on (Wageningen University and Research Centre):
- experimental techniques upon evaporating droplets, and
- the influence of the substrate surface and buffer composition on the orientation and conformation and, consequently, the functionality of biomolecules.
Some aspects of optimizing the inkjet printing of biomolecules were studied:
- Several substrates used in diagnostic assays were compared such as polystyrene, glass, hydrophobized glass and nitrocellulose.
The addition of surfactants / detergents: Additives appeared to play a positive role in reducing the "coffee-stain effect", provided the right concentration was used in the biomolecule solution. Pluronic F127 was investigated in more detail and the optimal concentration appeared to be substrate- and biomolecule-dependent.
- The distribution and orientation of the printed biomolecules and the final assay results following the various incubation steps with antigen, second antibody, etc., were analyzed by using a Confocal Laser Scanning Microscopy (CLSM). In the case of nitrocellulose the fluorescence in 1-μm-slices was recorded from top to bottom and the analysis was performed in a concentric-ring-format, thereby enabling the assessment of the distribution of biomolecules over the area of the spot, i.e., indicating whether the biomolecules were distributed evenly of at the edges of the spot.
- Atomic Force Microscopy was applied to measure the number of layers of the printed protein molecules and the surface topology of substrate materials.
- High-Speed Cameras enabled the accurate assessment of the fluid flow in the 3-dimensional nitrocellulose substrate
- Liyakat Hamid Mujawar, Aart van Amerongen, Willem Norde. Influence of buffer composition on the distribution of inkjet printed protein molecules and the resulting spot morphology. Talanta 98 (2012) 1–6.
- Liyakat Mujawar, Willem Norde and Aart van Amerongen. Spot morphology of non-contact printed protein molecules on non-porous substrates with a range of hydrophobicities. Analyst 138 (2013) 518-524.
- Liyakat Hamid Mujawar, Abid Aslam Maan, Muhammad Kashif Iqbal Khan, Willem Norde, and Aart van Amerongen. Distribution of Biomolecules in Porous Nitrocellulose Membrane Pads Using Confocal Laser Scanning Microscopy and High-Speed Cameras. Anal. Chem. 85 (2013) 3723-3729.
- Liyakat Hamid Mujawar, Antoine Moers, Willem Norde and Aart van Amerongen. Rapid mastitis detection assay on porous nitrocellulose membrane slides. Anal. Bioanal. Chem. (2013) 405:7469-7476.
- Liyakat Hamid Mujawar, J. G. M. Kuerten, D. P. Siregar, Aart van Amerongen and Willem Norde. Influence of the relative humidity on the morphology of inkjet printed spots of IgG on a non-porous substrate. RSC Advances 4 (37) (2014) 19380-19388.
- Liyakat Hamid Mujawar, Aart van Amerongen and Willem Norde. Influence of Pluronic F127 on the distribution and functionality of inkjet-printed biomolecules in porous nitrocellulose substrates. Talanta 131 (2015) 541-547.