Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

• Published in: Analytical chemistry (Washington) Vol. 77; no. 24; pp. 8013 - 8019
• Main Authors: Rupcich, Nicholas, Green, James R. A, Brennan, John D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.12.2005
• Subjects: Analytical chemistry; Arrays; Caseins - analysis; Chemistry; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - metabolism; Exact sciences and technology; General, instrumentation; Inhibitory Concentration 50; Microarray Analysis - methods; Nanotechnology; Oligopeptides - metabolism; Peptides; Phase Transition; Phosphotransferases - antagonists & inhibitors; Proteins; Catalytic reaction; Chemical analysis; Peptides; Microanalysis; Immobilization; Diamond; Chemical sensor; Protein; Multicomponent analysis; Sol gel process; Detection limit; Inhibitor; Inhibition; Sensor array; Monitoring
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac051405a

We report on the development of a new class of kinase microarrays based on the coimmobilization of both kinase and substrate components within a single pin-printed sol−gel microarray element and the use of such arrays for nanovolume inhibition assays. We successfully immobilized the α-catalytic subunit of cAMP-dependent protein kinase (PKA) and the peptide substrate kemptide within sol−gel-derived microarrays for the purpose of monitoring phosphorylation and inhibition. Using Pro-Q Diamond stain as an end-point indicator of phosphorylation, we demonstrate the selective detection of phosphoproteins over nonphosphorylated controls and the ability to detect phosphorylated proteins over a 500-fold concentration range. Limits of detection for the phosphoprotein β-casein were 7.5 pg, and the detectable signal remained linear up to 3.75 ng of protein per array spot. PKA is demonstrated to be active when coentrapped with two different substrates, and inhibition assays for PKA with the inhibitors H7 and H89 demonstrate the ability to detect kinase inhibition as well as derive IC50 plots from a single array using an overprinting method to deliver ∼0.6 nL of reagent per array element, or a total of 72 nL of reagents to generate a full, 12-point IC50 curve in pentuplicate.