Enthalpy arrays

Abstract

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 proteinligand 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.U nderstanding the thermodynamics of molecular interactions is central to biology and chemistry. Although a number of methods are available, calorimetry is the only universal assay for the complete thermodynamic characterization of these interactions. Under favorable circumstances, the enthalpy, entropy, free energy, and stoichiometry of a reaction can be determined (1, 2). In addition, calorimetry does not require any labeling or immobilization of the reactants and hence offers a completely generic method for characterizing the interactions. Indeed, titration calorimetry is widely used in both drug discovery and basic science, but its use is severely constrained to a small number of very high-value measurements by the large sample requirements and long measurement times. No currently available methods for calorimetric measurements lend themselves to modern approaches in which large libraries of compounds, ranging from small molecules in combinatorial libraries to proteins and other macromolecules, are studied.Here we report a low-cost nanocalorimetry detector that can be used as a high-throughput assay tool to detect enthalpies of binding interactions, enzymatic turnover, and other chemical reactions. The detectors are made by using microscale fabrication technology, resulting in a nearly 3 orders of magnitude reduction in both the sample quantity and the measurement time over conventional microcalorimetry. The fabrication technology is low-cost and enables fabrication of 96-detector arrays, which we call enthalpy arrays, on large substrates. Accordingly, the technology will scale to high-volume production of disposable arrays. This increase in performance and reduction in cost promises to enable calorimetry to be used to investigate a substantial number of samples. Nanocalorimetry in the enthalpy array format has valuable applications in proteomics for protein interaction and protein chemistry research and in high-throughput screening and lead optimization for drug discovery. Materials and MethodsDevice Fabrication. The schematic cross section of a nanocalorimeter detector is shown in Fig. 1a. The d...

Enthalpy array analysis of enzymatic and binding reactions

“…We previously presented data in which the measurements were performed one at a time with manual deposition of the sample drops using a syringe [1]. We now typically perform measurements with automated deposition and data capture.…”

“…Figure 2A). We need to convert this information into an enthalpy, and we do so by deconvoluting Q (τ) from the equation [1] In this equation, T(t) is the temperature change (T) as a function of time (t) relative to the baseline temperature, Q(τ) is the rate of heat generation (e.g. due to a biochemical reaction) as a function of time, and T(t-τ), the "impulse response", is the temperature rise that would occur at a time t given a Dirac delta function impulse of heat at time τ ( Figure 2C).…”

“…Previously, we demonstrated that enthalpy arrays could be used to measure enthalpies of binding reactions at a single ligand concentration [1]. In addition, we demonstrated an enzymesubstrate interaction at a substrate concentration that allowed a very short reaction.…”

“…Fabrication of devices with n+ amorphous silicon thermistors on a polyimide membrane was described previously [1]. The device improvements we describe here were achieved by changing the thermistor material to vanadium oxide (VO x ) and fabricating the detectors on a polyethylene naphthalate (PEN) membrane.…”

“…Enthalpy arrays, which are arrays of nanocalorimeters, allow scientists to measure thermodynamics and kinetics of molecular interactions using small sample volumes and short measurement times [1]. The measurements do not require immobilization or labeling of reactants, an attractive feature compared with surface-based methods such as Surface Plasmon Resonance and photometric methods requiring tagging.…”