Printed protein microarrays on unmodified plastic substrates

A key challenge for the generation of protein microarrays is the immobilization of functional capture probe proteins at the chip surfaces. Here, a new concept for a single step production of protein microarrays to unmodified plastic substrates is presented. It is based on the printing of polymer/protein mixtures and the photochemical attachment of the obtained microstructures to the plastic chip surfaces. In the photochemical process three reactions occur simultaneously: transformation of the polymer into hydrogel dots, covalent binding of the forming gel to the substrate, and covalent immobilization of the proteins to the three-dimensional hydrogel scaffold. As an example we use anti-bovine serum albumin as a protein (anti-BSA) and a water swellable polymer network based on polydimethylacrylamide as a scaffold, which is photochemically crosslinked using benzophenone as a crosslinking agent. In one series of microarray experiments the probe density of the immobilized proteins was determined by incorporating fluorescence-labeled anti-BSA in the hydrogels. In a typical experiment, the number of immobilized probes was determined to 4 × 10 9 protein molecules per spot. In other experiments, the microarrays were brought into contact with fluorescently labeled BSA. In such analyses signal-to-noise values of more than 200 were obtained and about 9 × 10 7 antigen molecules were bound per spot. This demonstrates that in a very simple way microarrays with large amount of probes per spot can be realized and that antibodies immobilized in the printed hydrogels remain accessible and retain their functionality.