Picoliter-Volume Isothermal Titration Calorimetry Using Parylene Chip Calorimeter Integrated with on-Demand Droplet Microfluidics

Abstract

Isothermal titration calorimetry (ITC) is a gold-standard technique for directly quantifying biomolecular interactions, but its broader applicability is limited by large sample consumption and low throughput. To address these challenges, considerable efforts are made to develop chip calorimeter systems. Here, a high-sensitivity chip calorimeter integrated with on-demand droplet microfluidics is presented, capable of performing ITC with picoliter-volume samples. The device combines vanadium pentoxide thermistors, vacuum-insulated parylene microfluidics, and multilayer Polydimethylsiloxane microfluidics to achieve precise thermal measurement and fluidic control. On-demand generation and merging of titrant and titrand droplets enable accurate control of molar ratios for droplet-based titration. The chip calorimeter achieves a temperature resolution of 14.9 µK and a power resolution of 2.31 nW. The platform is validated by measuring the binding interaction between 18-crown-6 and barium chloride, with extracted thermodynamic parameters in good agreement with conventional ITC. This work advances miniaturized ITC technology by providing a scalable and efficient platform for quantitative biochemical analysis, particularly in sample-limited and high-throughput applications.

Keywords: chip calorimeter; droplet microfluidics; isothermal titration calorimetry; parylene microfluidics; vanadium oxide.

Figure 1

Droplet‐based chip calorimeter for ITC. a) Schematic comparison of conventional ITC and droplet‐based ITC. In the droplet‐based system, the heat of the reaction is measured from the merging of droplets with varying volumes. b) Cross‐sectional structure of the parylene chip calorimeter (not to scale). c) Images of parylene chip calorimeter devices, showing a diced device (left) and a fully assembled device (right) with on‐chip vacuum chambers and PDMS microfluidics. The magnified microscopic image of the membrane shows the calorimetric chamber and the thermistor. d) Schematic of the electrical measurement setup used for differential temperature sensing and thermal conductance calibration. e) Two‐level thermostat system designed to provide a highly stable thermal reservoir.

Figure 2

Fabrication and assembly of the chip calorimeter. a) Fabrication steps. Parylene is deposited on both the PDMS channel mold and the sensor wafer containing V₂O₅ thermistors. The parylene layers are bonded using the iCVD bonding technique. b) Images of the wafer and the calorimetric chamber during key fabrication steps. c) Assembly of the PDMS microfluidic system and the on‐chip vacuum chamber.

Figure 3

Structure and operation of the droplet microfluidic system. a) Schematic of the integrated microfluidic system. The PDMS microfluidics, which includes the on‐demand droplet generator and flow control valves, are combined with the parylene microfluidics containing the calorimetric chamber. b) Droplet generation by alternating sample and oil flows into the main channel. Droplet volume is controlled by the sample injection pressure. (n = 9) c) Titrand and titrant droplet capture and merging by sequential generation and transport of droplets.

Figure 4

Characterization of the chip calorimeter. a) TCR of the V₂O₅ thermistor. b) Typical output voltage signal from the bridge circuit at V_B = 0.45 V. Three representative signals are overlaid. The RMS noise voltage is 24.3 ± 5.57 nV Hz^−1/2. c) RMS noise voltage and corresponding NETD at varying bias voltages. The black dotted line indicates the Johnson–Nyquist noise level of the thermistor. d) Calibration of the thermal conductance. e) Temperature change of the inner thermostat with PID control and the chip calorimeter response. f) Baseline temperature drift of the inner thermostat after stabilization.

Figure 5

ITC measurement of the binding reaction between 18‐C‐6 and BaCl₂. a) Calorimetric power signal measured from droplet mixing with various molar ratios. b) Total heat of binding plotted as a function of the titrant‐to‐titrand molar ratio R.