Advancing microarrays with acoustic dispensing technology and probing the kinome

We have developed a novel microarray platform for constructing enzyme bioassays. Utilizing acoustic dispensing technology, reactions were assembled on glass slides in a no-contact manner without cross-contamination, surface linkages, or wash steps. The method handles nanoliter reactions in the liquid phase (glycerol in the buffer and incubating under humidity minimized evaporation) with coefficients of variation under 10%, is compatible with radiolabel, fluorescence, or surface binding assays, and is suitable for enzyme-substrate, high-throughput, or compound library screening. Using proviral integration site-1 (Pim1) kinase, a three-point dose response at 0.75 muM, 1.5 muM, and 3 muM with a known Pim1 inhibitor (hb1217) and radiolabeled ATP showed the feasibility of multicomponent microarray assembly through acoustic dispensing as Pim1 was increasingly inhibited. In the detection step, the arrays were blotted to phosphocellulose membranes to capture the phosphorylated substrate, and phosphor imaging was used to quantify the washed membranes. Signal-to-background ratios were as high as 165 and average CVs were ∼20%. CVs from dispensing typical working buffers were ∼5%. While most microarray approaches use solid pins for contact spotting, acoustic dispensing avoids the drawbacks of undesired physical contact with biological samples, difficulties in assembling multicomponent reactions, rigid modes of printing operation that lacks flexibility, and time-consuming wash steps. We demonstrated the utility of acoustic dispensing over pins by delivering cathepsin L in a spot-on-spot fashion into individual 50 nL nanodroplets to rapidly activate reactions, generating variable volume spots from 2 to 50 nL with less spot size variations, and linearly dispensing suspensions of fluorescent beads from source wells containing 0.1 to 10 x 108 beads/mL. Acoustic dispensing meets the needs associated with spatially-addressed assembly of multicomponent enzyme reactions on a nanoliter scale, especially the need to re-address a previously spotted position. Finally, we have screened a series of ruthenium organometallic compounds whose structures are based on the indolocarbazole scaffold of staurosporine, a natural kinase inhibitor. Based on the information derived from profiling a library of 58 such compounds against a panel of 50 kinases, a series of structure activity relationship studies were performed, resulting in the development of selective, nanomolar inhibitors for TrkA and Pim1 kinase.