ABSTRACT
We demonstrate rapid mixing of sub-microlitre droplets (250 nl) using miniaturized magnetic stir bars (400 µm × 200 µm × 15 µm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 s. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.
Subject(s)
Magnetics , Microfluidic Analytical Techniques/methods , Fluorescence Resonance Energy Transfer , Microfluidic Analytical Techniques/instrumentation , Miniaturization , Physical Phenomena , ThermodynamicsABSTRACT
Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. Compared with conventional calorimetry, enthalpy arrays achieve a significant reduction of sample volume and measurement time through the combination of the small size of the detectors and ability to perform measurements in parallel. The current capabilities of the technology for studying enzyme-catalyzed reactions are demonstrated by determining the kinetic parameters for reactions with three model enzymes. In addition, the technology has been used with two classes of enzymes to determine accurate inhibitor constants for competitive inhibitors from measurements at a single inhibitor concentration.
Subject(s)
Calorimetry/methods , Enzymes/metabolism , Nanotechnology/methods , KineticsABSTRACT
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.
