Isothermal titration calorimetry in a 3D-printed microdevice


Isothermal titration calorimetry (ITC) can benefit from operating in miniaturized devices as they enable quantitative, low-cost measurements with reduced analysis time and reagents consumption. However, most of the existing devices that offer ITC capabilities either do not yet allow proper control of reaction conditions or are limited by issues such as evaporation or surface adsorption caused inaccurate solution concentration information and unintended changes in biomolecular properties because of aggregation. In this paper, we present a microdevice that combines 3D-printed microfluidic structures with a polymer-based MEMS thermoelectric sensor to enable quantitative ITC measurements of biomolecular interactions. Benefitting from the geometric flexibility of 3D-printing, the microfluidic design features calorimetric chambers in a differential cantilever configuration that improves the thermal insulation and reduces the thermal mass of the implementing device. Also, 3D-printing microfluidic structures use non-permeable materials to avoid potential adsorption. Finally, the robustness of the polymeric MEMS sensor chip allows the device to be assembled reversibly and leak-free, and hence reusable. We demonstrate the utility of the device by quantitative ITC characterization of a biomolecular binding system, ribonuclease A (RNase A) bind with cytidine 2′-monophosphate (2’CMP) down to a practically useful sample concentration of 0.2 mM. The thermodynamic parameters of the binding system, including the stoichiometry, equilibrium binding constant, and enthalpy change are obtained and found to agree with values previously reported in the literature.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. N. Bhattacharjee, A. Urrios, S. Kanga, A. Folch, The upcoming 3D-printing revolution in microfluidics. Lab Chip 16(10), 1720–1742 (2016)

    Article  Google Scholar 

  2. C. Calderilla, F. Maya, V. Cerda, L.O. Leal, 3D printed device for the automated preconcentration and determination of chromium (VI). Talanta 184, 15–22 (2018)

    Article  Google Scholar 

  3. H.N. Chan et al., Simple, cost-effective 3D printed microfluidic components for disposable, point-of-care colorimetric analysis. Acs Sensors 1(3), 227–234 (2016)

    Article  Google Scholar 

  4. C.K. Chiang, A. Kurniawan, C.Y. Kao, M.J. Wang, Single step and mask-free 3D wax printing of microfluidic paper-based analytical devices for glucose and nitrite assays. Talanta 194, 837–845 (2019)

    Article  Google Scholar 

  5. J. P. Conde et al., Lab-on-chip systems for integrated bioanalyses, in Biosensor Technologies for Detection of Biomolecules, vol. 60, P. Estrela, Ed. (Essays in Biochemistry, no. 1), pp. 121–131, (2016)

  6. X. Feng, Y. Jia, H. Jiang, Q. Lin, Microfabrication-based isothermal titration calorimetry using a combined in-mixing and post-mixing titration approach. Anal. Methods 10(38), 4665–4670 (2018).

    Article  Google Scholar 

  7. E. Freire, O.L. Mayorga, M. Straume, Isothermal titration calorimetry. Anal. Chem. 62(18), 950A–959A (1990)

    Article  Google Scholar 

  8. Y. He, Y. Wu, J.Z. Fu, Q. Gao, J.J. Qiu, Developments of 3D printing microfluidics and applications in chemistry and biology: A review. Electroanalysis 28(8), 1658–1678 (2016)

    Article  Google Scholar 

  9. C.M.B. Ho, S.H. Ng, K.H.H. Li, Y.J. Yoon, 3D printed microfluidics for biological applications. Lab Chip 15(18), 3627–3637 (2015)

    Article  Google Scholar 

  10. I. Jelesarov, H.R. Bosshard, Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J. Mol. Recognit. 12(1), 3–18 (1999)

    Article  Google Scholar 

  11. W. Lee, W. Fon, B.W. Axelrod, M.L. Roukes, High-sensitivity microfluidic calorimeters for biological and chemical applications. Proc. Natl. Acad. Sci. 106(36), 15225–15230 (2009)

    Article  Google Scholar 

  12. B. Lubbers, F. Baudenbacher, Isothermal titration calorimetry in Nanoliter droplets with subsecond time constants. Anal. Chem. 83(20), 7955–7961 (2011)

    Article  Google Scholar 

  13. M. Michalec, L. Tymecki, 3D printed flow-through cuvette insert for UV-vis spectrophotometric and fluorescence measurements. Talanta 190, 423–428 (2018)

    Article  Google Scholar 

  14. L.S. Mizoue, J. Tellinghuisen, Calorimetric vs. van't Hoff binding enthalpies from isothermal titration calorimetry: Ba2+−crown ether complexation. Biophys. Chem. 110(1), 15–24 (2004)

    Article  Google Scholar 

  15. N.A. Plate et al., Gas and vapor permeation and sorption in poly(trimetylsilylpropyne). J. Membr. Sci. 60(1), 13–24 (1991)

    Article  Google Scholar 

  16. M.I. Recht, D. De Bruyker, A.G. Bell, M.V. Wolkin, E. Peeters, G.B. Anderson, et al., Enthalpy array analysis of enzymatic and binding reactions. Anal. Biochem. 377, 33–39 (2008)

    Article  Google Scholar 

  17. C. I. Rogers, K. Qaderi, A. T. Woolley, and G. P. Nordin, 3D printed microfluidic devices with integrated valves, Biomicrofluidics, vol. 9, no. 1, (2015)

  18. C. Sgarlata, V. Zito, G. Arena, Conditions for calibration of an isothermal titration calorimeter using chemical reactions. Anal. Bioanal. Chem. 405(2–3), 1085–1094 (2013)

    Article  Google Scholar 

  19. G.G. Tartaglia, S. Pechmann, C.M. Dobson, M. Vendruscolo, Life on the edge: A link between gene expression levels and aggregation rates of human proteins. Trends Biochem. Sci. 32(5), 204–206 (2007)

    Article  Google Scholar 

  20. W.B. Turnbull, A.H. Daranas, On the value of c: Can low affinity systems be studied by isothermal titration calorimetry? J. Am. Chem. Soc. 125(48), 14859–14866 (2003)

    Article  Google Scholar 

  21. S. Waheed et al., 3D printed microfluidic devices: Enablers and barriers. Lab Chip 16(11), 1993–2013 (2016)

    Article  Google Scholar 

  22. B. Wang, Y. Jia, Q. Lin, A microfabrication-based approach to quantitative isothermal titration calorimetry. Biosens. Bioelectron. 78, 438–446 (2016)

    Article  Google Scholar 

  23. T. Wiseman, S. Williston, J.F. Brandts, L.N. Lin, Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179(1), 131–137 (1989)

    Article  Google Scholar 

  24. A. A. Yazdi, A. Popma, W. Wong, T. Nguyen, Y. Y. Pan, and J. Xu, 3D printing: an emerging tool for novel microfluidics and lab-on-a-chip applications, Microfluidics and Nanofluidics, vol. 20, no. 3, (2016)

Download references


This work was jointly supported by the Natural Science Foundation of Jiangsu Province (Grant No. BK20180384); the National Natural Science Foundation of China (Grant No. 61604042); Fundamental Research for the Central Universities of China (Grant No. N160302001); the Natural Science Foundation of Fujian Province (Grant No. 2017 J01501).

Author information



Corresponding author

Correspondence to Qiao Lin.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(DOCX 701 kb)


(MP4 509 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jia, Y., Su, C., He, M. et al. Isothermal titration calorimetry in a 3D-printed microdevice. Biomed Microdevices 21, 96 (2019).

Download citation


  • 3D-printed microfluidic structures
  • Isothermal titration calorimeter
  • MEMS thermoelectric sensor
  • Polymer substrate