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Microchip electrokinetic methods are capable of increasing the sensitivity of molecular assays by enriching and purifying target analytes. However, their use is currently limited to assays that can be performed under a high external electric field, as spatial separation and focusing is lost when the electric field is removed. We present a novel method that uses two-phase encapsulation to overcome this limitation. The method uses passive filling and pinning of an oil phase in hydrophobic channels to encapsulate electrokinetically separated and focused analytes with a brief pressure pulse. The resulting encapsulated sample droplet maintains its concentration over long periods of time without requiring an electric field and can be manipulated for further analysis, either on- or off-chip. We demonstrate the method by encapsulating DNA oligonucleotides in a 240 pL aqueous segment after isotachophoresis (ITP) focusing, and show that the concentration remains at 60% of the initial value for tens of minutes, a 22-fold increase over free diffusion after 20 minutes. Furthermore, we demonstrate manipulation of a single droplet by selectively encapsulating amplicon after ITP purification from a polymerase chain reaction (PCR) mix, and performing parallel off-chip detection reactions using the droplet. We provide geometrical design guidelines for devices implementing the encapsulation method, and show how the method can be scaled to multiple analyte zones. [ABSTRACT FROM AUTHOR]