The development of DNA-based biosensors requires efficient immobilization of DNA probes on the sensor surfaces with optimum coverage and orientation. In this study, we have prepared thin films of a 718 base pair 5＇-thiol-labeled double-stranded DNA (dsDNA) on a gold surface by chemisorption and determined the quantity, surface coverage, and adsorption kinetics of dsDNA using cyclic voltammetry (CV), quartz crystal resonator (QCR), and radioactive labeling techniques. The adsorption and desorption rate constants of the 5＇-thiol-labeled dsDNA on a gold surface are estimated to be (1.9 +/-0.2) x 10(3) M-1 s(-1) and (1.3 +/- 1.1) x 10(-3) s(-1), respectively. The equilibrium constant is (1.5 +/- 1.3) x 10(6) M-1 and the fractional coverage is >75% for the DNA concentration range of 2-10 mu M. The ligand-binding properties of the immobilized dsDNA were investigated using doxorubicin, a DNA-intercalating anthracyclic antibiotic, as the probing molecule. The binding of doxorubicin to the immobilized dsDNA was examined by CV to verify the ligand-binding capacity and specificity of the immobilized dsDNA. The kinetics of the interaction between doxorubicin conjugated with a 200-kDa dextran polymer and the immobilized dsDNA was determined by QCR, which gives the association and dissociation rate constants of (1.8 +/- 0.5) x 10(3) M-1 s(-1) and (3.5 +/- 1.5) x 10(-3) s(-1), respectively, and an equilibrium binding constant of (5.1 +/- 2.6) x 10(5) M-1. The binding parameters for the conjugate-DNA interaction were also determined by using a surface plasmon resonance (SPR)-based biosensor, in which dsDNA was immobilized on the hydrogel matrix of the sensor chip through a biotin-streptavidin linkage. While the kinetic constants from SPR measurements, k(a) = (5.7 +/- 0.2) x 10(3) M-1 s(-1) and k(d) = (1.2 +/- 0.2) x 10(-2) s(-1), are different from-those determined by QCR, the thermodynamic constant K-A obtained by SPR, (4.8 +/- 0.8) x 10(5) M-1, is similar to that by QCR.