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In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.

We present a technology for the highly parallel dispensing of a multitude of reagents. It allows one to dispense up to 96 different reagents simultaneously in a fixed array, in a volume range of 100 pL up to several nL. The pitch of the dispensed droplets can be as small as 500 microm. All channels are fired simultaneously, giving an unprecedented throughput. The system was originally developed for the high-throughput fabrication of microarrays, but can easily be adopted for other applications such as highly parallel filling of nanotiterplates. Based on our standard configuration we achieved droplets with 125- micro m in-flight diameter (1.2 nL) with a CV of <1%.