The results of the equilibrium dialysis experiments are shown in Figure , where the percentage of COSAN (0.05 mM) bound (%bound ). Since COSAN and its derivatives showed a high affinity to proteins, COSAN might have bound to proteins in the DNA sample, thereby explaining its retention in the DNA-containing chamber. As a positive control, we conducted equilibrium dialysis of COSAN with human serum albumin (HSA). COSAN has a high affinity to serum albumin, as confirmed in the equilibrium dialysis experiment, where the percentage of COSAN bound to HSA was 100%. Lastly, we used NMR-based methods to study interactions between DNA and COSAN, i.e., diffusionordered spectroscopy (DOSY) and 1 H NMR. Ethidium bromide (EB), a model intercalator, was used as a control. Either COSAN or EB binding to ctDNA should decrease the diffusion rate of the ligand because ctDNA is significantly larger and slower. The measurements were conducted at concentration ratios of COSAN/ctDNA(pb) spanning from 1:1 to 1:15. The DOSY results show that the diffusion coefficients (𝐷) of COSAN remained virtually unchanged in all samples with and without ctDNA, but the diffusion coefficient of EB was an order of magnitude smaller with DNA than without DNA, indicating that EB bound to ctDNA (Table , Figure ). DOSY experiments cannot completely rule out binding because the lack of differences in COSAN diffusion coefficients might have resulted from the rapid exchange between bound and unbound fractions of COSAN. To exclude this possibility, 1 H NMR spectra were investigated using an internal standard. COSAN signals and their integrations in samples with and without DNA remained within the experimental error, with no significant 1 4E). EB samples showed clear differences between spectra with and without DNA. In the presence of DNA, most EB signals disappeared, except for the signal of the very mobile CH3 group, which was weaker and shifted. These results confirm the lack of binding between COSAN and DNA.