Optical Extinction Combined with Phase Measurements for Probing DNA−Small-Molecule Interactions Using an Evanescent Waveguide Biosensor

We demonstrate the use of both optical extinction and phase measurements for probing the interactions between DNA and small molecules by dual polarization interferometry. On binding to DNA at the interface, mitoxantrone (MTX) and methylene blue (MB) induced reversible concentration-dependent optical extinction due to light absorption, which clearly revealed the association and dissociation of small molecules with DNA in real time. The binding constants of MTX-DNA and MB-DNA determined from the masses derived from optical extinction are 1.8 x 10(5) and 4.2 x 10(4) M(-1), respectively, and shown to be buffer salt concentration-dependent. Apart from optical extinction, phase measurements reflected the overall change of the interaction; namely, a combined result of the binding of small molecules and any changes in DNA structure. The masses derived from phase could be very different from those derived from optical extinction. The structural changes detected by phase measurements showed a contraction and densification of DNA upon intercalation by MTX or MB. The combination of optical extinction and phase measurements allows a detailed understanding of the interaction process.