ABSTRACT
Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. The combination of the small size of the detectors and the ability to perform measurements in parallel results in a significant reduction of sample volume and measurement time compared with conventional calorimetry. We have made significant improvements in the technology by reducing the temperature noise of the detectors and improving the fabrication materials and methods. In combination with an automated measurement system, the advances in device performance and data analysis have allowed us to develop basic enzyme assays for substrate specificity and inhibitor activity. We have also performed a full titration of 18-crown-6 with barium chloride. These results point to future applications for enthalpy array technology, including fragment-based screening, secondary assays, and thermodynamic characterization of leads in drug discovery.
Subject(s)
Calorimetry/methods , Enzymes/metabolism , Automation/instrumentation , Barium Compounds/metabolism , Calorimetry/instrumentation , Chlorides/metabolism , Crown Ethers/metabolism , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hexokinase/metabolism , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Protein Binding , Substrate Specificity , Thermodynamics , Titrimetry , Trypsin Inhibitors/metabolism , Trypsin Inhibitors/pharmacologyABSTRACT
We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.
