Development of a Continuous Density Gradient of Immobilized Probes for Controlling the Stringency of DNA Hybridization

A new format for microfluidic based DNA biosensors is presented in which the biorecognition element (single stranded DNA probes) is immobilized as a continuous density gradient of probes along the length of a microfluidic channel instead of a standard array format commonly used in microarray technologies or DNA based biosensors. The development of continuous density gradients of immobilized probe was achieved by electrokinetically subjecting probes that were terminated with an appropriate functional group for a surface coupling reaction to increasing convective velocity along the length of the microfluidic channel. This gradient format was able to discriminate between a fully complementary target and one containing 3 BPM based on the spatial pattern of hybridization for picomole quantities of DNA targets. Temperature mediated destabilization of DNA hybrids demonstrated that the density of immobilized probes plays an important role in the thermodynamic stability of DNA hybrids. In addition, it was found that efficiency, selectivity and melt temperature of DNA hybrids for surface based hybridization is dependent on the density of the probe molecules.MAS