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Protein microarrays are used various research areas including drug discovery, diagnosis, and analysis of protein-ligand interactions. Their efficacy depends on a well-defined pattern of immobilized proteins that also have retained their bioactivity. Protein microarrays are classically fabricated using the robotic spotting drop method (“pin printing”), which can lead to spots with uneven protein concentration within the spotted area, leading to difficult to quantify readings. Among the alternative techniques, microcontact printing (μCP) with a poly(dimethylsiloxane) (PDMS) stamp appears to deliver more defined protein patterns on surfaces, while maintaining bioactivity for a wide range of proteins. Here we have quantitatively compared the distribution of fluorescently labeled proteins deposited using direct pipetting, pin printing and μCP printing with flat stamps onto various functionalized glass surfaces of different contact angles through fluorescent microscopy. The uniformity of the deposited protein spots across deposition techniques was also qualitatively analyzed. It was found that with the use of either the direct pipetting or pin printing techniques that protein concentration on surfaces varied largely across surfaces with different contact angles, whereas adsorption did not vary significantly when using the μCP printing Furthermore, when μCP printing was performed with flat relief structures the spot inhomogeneity was lower than when classical methods were used, and even less so when a pyramid relief structure was used. This suggests that μCP printing with pyramid relief structures could produce protein patterns on various surfaces and with increased spot uniformity to enable more reliable protein microarrays.
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