Magnetoresistive sensors for measurements of DNA hybridization kinetics - effect of TINA modifications

We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current. A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants k_on, and k_off. The effect of probe modifications with ortho-Twisted Intercalating Nucleic Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5' end (1 × TINA) or at both the 5' and 3' ends (2 × TINA) were compared. TINA modifications were found to provide a relative decrease of k_off by a factor of 6-20 at temperatures from 57.5 °C to 60 °C. The values of k_on were generally in the range between 0.5-2 × 10⁵ M^-1s^-1 and showed lower values for the unmodified probe than for the TINA modified probes. The observations correlated well with measured melting temperatures of the DNA hybrids.