When developing a biosensor it is important to know the affinity of your biorecognition element to the analyte of interest. Should the affinity be too low the interaction will be weak and transient, and the biosensor will not function in a satisfactory way. It is therefore important to employ some method to characterise the interaction between your analyte and biorecognition element. 

Isothermal titration calorimetry (ITC) is one technique frequently used to investigate such biomolecular interactions. ITC monitors these interactions by measuring the heat change from the interaction (1). This can be either heat uptake or heat generation depending on if the reaction is endo- or exothermic (2). An ITC instrument contains two cells, one reference and one sample cell. Both cells are kept at a constant temperature during the experiment. The reference cell will not be subject to any heat change whereas the sample cell will, however, this heat change will be compensated by the instrument to keep both cells at a constant temperature. The heat is thus indirectly measured from the power used by the instrument to keep the temperatures equal (3). Through ITC a plethora of different information can be obtained. The dissociation constant (KD) & stoichiometry (n) of the binding can be obtained as well as thermodynamic data (ΔH, ΔS & ΔG) (4). 

The protein is kept in the sample cell and the ligand is injected several times over the course of the experiment. Initially the heat change will be at its highest but it will decrease with each subsequent injection until an equilibrium is reached. The resulting thermogram, with heat change plotted against time, can be used to extract a binding curve. The binding curve is obtained by integrating the peaks from the ligand injections and plotting it against the molar ratio between protein and ligand. From this binding curve the stoichiometry, dissociation constant and enthalpy can be extracted (5). From the enthalpy, dissociation constant and the following equations entropy and binding free energy can be obtained: 

∆G=-RTln(KD) ∆G=∆H-T∆S

An advantage of ITC is that it requires no modification or immobilisation. Setting it apart from immobilisation-based techniques like surface plasmon resonance (SPR) (6) and modification-based techniques like fluorescence polarization (FP) (7). One notable downside of ITC is that it requires high amounts of samples as compared to competing techniques (6).

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Figure 1. Overview of an ITC machine, resulting thermogram and the extracted binding curve (8).


1. Wiseman T, Williston S, Brandts JF, Lin L-N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Analytical Biochemistry. 1989 May 15;179(1):131–7. 

2. Indyk L, Fisher HF. [17] Theoretical aspects of isothermal titration calorimetry. In: Methods in Enzymology [Internet]. Academic Press; 1998 [cited 2022 Mar 24]. p. 350–64. (Energetics of Biological Macromolecules Part B; vol. 295). Available from: https://www.sciencedirect.com/science/article/pii/S0076687998950480

3. Leavitt S, Freire E. Direct measurement of protein binding energetics by isothermal titration calorimetry. Current Opinion in Structural Biology. 2001 Sep 1;11(5):560–6. 

4. Pierce MM, Raman CS, Nall BT. Isothermal Titration Calorimetry of Protein–Protein Interactions. Methods. 1999 Oct 1;19(2):213–21. 

5. Duff, MR, Grubbs J, Howell EE. Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity. J Vis Exp. 2011 Sep 7;(55):2796. 

6. Hevener KE, Pesavento R, Ren J, Lee H, Ratia K, Johnson ME. Chapter Twelve – Hit-to-Lead: Hit Validation and Assessment. In: Lesburg CA, editor. Methods in Enzymology [Internet]. Academic Press; 2018 [cited 2022 Mar 24]. p. 265–309. (Modern Approaches in Drug Discovery; vol. 610). Available from: https://www.sciencedirect.com/science/article/pii/S0076687918303859

7. Moerke NJ. Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding. Current Protocols in Chemical Biology. 2009;1(1):1–15. 

8. Using Isothermal Titration Calorimetry for Biophysical Characterization of Chromatin-Binding Proteins [Internet]. News-Medical.net. 2016 [cited 2022 Mar 28]. Available from: https://www.news-medical.net/whitepaper/20161027/Using-Isothermal-Titration-Calorimetry-for-Biophysical-Characterization-of-Chromatin-Binding-Proteins.aspx

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