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 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.

Fig. 1.

Nanocalorimeter detector. (a) Detector fabrication schematic: cross-section view. (b) Design principle of the nanocalorimeter detector (with electrical interconnects not shown). (c) Single detector set from a 96-format array, photographed and enlarged. The adjacent measurement and reference regions are in the center of the polyimide isolation membrane, surrounded by electrical contact pads supported by an underlying frame.

Fig. 2.

Electrostatic merging/mixing of two 500-nl droplets of water at three different times, starting when a voltage is applied across the gap. One drop has blue coloring to visualize mixing. The noncolored drop is placed asymmetrically across the gap between two electrodes. The mixing started within the first 33 msec, even before surface tension caused the drop to take its final shape.

Fig. 3.

RNase A–2′-CMP reaction. Data are plotted as the differential change in temperature versus time. The time at which the RNase A and 2′-CMP drops were merged and the expected peak height based on the enthalpy of the reaction are indicated. The rms noise at a 1 Hz bandwidth is 64 μK.

Fig. 4.

Streptavidin–biotin reaction. Data are plotted as the differential change in temperature versus time. The streptavidin and biotin drops were merged at t = 0. The expected peak height based on the enthalpy of the reaction and the concentration of active protein is indicated. The rms noise at a 1 Hz bandwidth is 96 μK.

Fig. 5.

Enzymatic phosphorylation of glucose by hexokinase. Data are plotted as the differential change in temperature versus time. The time at which the hexokinase and glucose drops were merged is indicated. The maximum peak height is indicated. The rms noise at a 1 Hz bandwidth is 50 μK.

Fig. 6.

Mitochondrial respiration in the presence of DNP uncoupler. Data are plotted as the differential change in temperature versus time. The time at which mitochondria in the presence of DNP were mixed with additional DNP is indicated. The rms noise at a 1 Hz bandwidth is 69 μK.

Fig. 7.

A four-array substrate undergoing outsourced processing.