Automated and Integrated High Sensitivity Preconcentration and Separation with Isotachophoresis

Researchers in Prof. Juan Santiago's laboratory have developed a novel isotachophoresis (ITP) method to easily and seamlessly integrate various electrophoresis-based detection techniques with ITP preconcentration. The technique's variations include, but are not limited to, coupling multiple ITP processes, integrating ITP preconcentration with electrophoretic separation, and coupling the application of ITP to achieve fast chemical reactions with electrophoretic separation. The technique requires no manual or voltage control steps to initiate the transition from ITP to other electrophoretic processes. For example, the transition requires no exchange of buffers, switching of flows, or changing the state of power supplies. The technology is applicable to on-chip or off-chip electrophoresis systems (e.g., microfluidic chips or free-standing capillaries). Also, it requires low amounts of initial sample and can be easily integrated into conventional on- or off-chip capillary electrophoresis (CE) equipment.

Stage of Research
The inventors have demonstrated the technique with high resolution separation of ds-DNA fragments of a 1 kb DNA ladder. The figure below shows a separation of a 1 kb ds-DNA ladder using this technique. Each element of the ladder is fully resolved, including the 517 vs. 500 bp fragments not resolved by typical slab-gel electrophoresis (shown below the plot).

Preconcentration and subsequent separation of 1 kb ds-DNA ladder using this novel technique. (a) shows initial ITP-focused DNA fragments. (b) shows the transition from focusing to separation stage. (c) shows fully resolved DNA ladder, with 11 peaks. (d) shows visualization (inverted) of agarose gel electrophoresis separation (provided by the vendor, New England BioLabs, Ipswich, MA). Note that the agarose gel electrophoresis does not resolve peaks 10a and 10b corresponding to 517 and 500 bp fragments, while our on-chip ITP technique does resolve this.

The inventors have also demonstrated 20-fold increase in sensitivity of ITP detection of small ions in water, for example 2,4,6-tricholorophenol (a herbicide), by coupling two ITP processes with different electrolyte solutions.