Free-flow biomolecular concentration and separation of proteins and nucleic acids using techophoresis.
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abstract
The ability to preconcentrate, separate, and purify biomolecules, such as proteins and nucleic acids, is an important requirement for the next generation of portable diagnostic tools for environmental monitoring and disease detection. Traditionally, such pretreatment has been accomplished using large, centralized liquid- or solid-phase extraction equipment, which can be time-consuming and requires many processing steps. Here, we present a newly developed electrokinetic concentration technique, techophoresis (TPE), to concentrate and separate proteins, and to concentrate nucleic acids. In TPE, a free-flowing sample is exposed to a perpendicular electric field in the vicinity of a mass-impermeable conductive wall and a conductive terminating electrolyte (TE), which creates a high electric field strength zone between the lower mobility sample and the no-flux barrier. Unlike a similar electrokinetic concentration method, isotachophoresis (ITP), TPE does not require a leading electrolyte (LE), yet still enables a continuous field-driven electrophoretic ion migration across the channel and a free-flowing biomolecular concentration at the conductive wall. Here, we demonstrate the use of free-flow TPE (FFTPE) to manipulate biomolecular samples containing proteins or nucleic acids. We first use TPE to drive a 6.6-fold concentration increase of avidin-FITC, and also demonstrate protein separation and stacking between ovalbumin-fluorescein and BSA-AlexaFluor 555, both without the use of a conventional LE. Further, we utilize TPE to perform a 21-fold concentration increase of nucleic acids. Our results show that TPE is biocompatible with both proteins and nucleic acids, requires only 10V DC, produces no significant sample pH changes during operation, and demonstrates that this method can be used as an effective sample pretreatment to prepare biological samples for downstream analysis in a continuous free-flowing microfluidic channel.
