Acceleration of surface-based hybridization reactions using isotachophoretic focusing.

We present a theoretical model and experimental demonstration of a novel method for acceleration of surface-based reactions using isotachophoresis (ITP). We use ITP to focus a sample of interest and deliver a high concentration target to a prefunctionalized surface, thus enabling rapid reaction at the sensor site. The concentration of the focused analyte is bound in space by the ITP interface and, upon reaction with the surface, continues electromigrating downstream, removing any contamination or reacted sample molecules from the surface. This constitutes a one-step react-and-wash assay which can be performed in a simple channel and does not require flow control elements or moving parts. We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field. Using this chip, we compared ITP-based surface hybridization to standard continuous flow-based hybridization and experimentally demonstrated a 2 orders of magnitude improvement in limit of detection (LoD) in a 3 min nucleic acid hybridization assay. The simple analytical model we present allows prediction of the rate of surface reaction under ITP and can be used to design and optimize such assays as a function of the physical properties of the system, including buffer chemistry, applied voltage, analyte mobility, analyte concentration, probe density, and surface length. The method, model, and experimental setup can be applied to various forms or surface reactions and may serve as the basis for highly genetic analysis and immunoassays.