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Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study
journal contribution
posted on 2015-12-17, 02:57 authored by Viktor Shkolnikov, Juan G. SantiagoWe present an experimental study of coupling of isotachophoresis
(ITP) and affinity chromatography (AC) to effect rapid, selective
purification with high column utilization and high resolution. We
provide a detailed protocol for performing ITP-AC and describe the
design of a buffer system to perform sequence specific separation
of nucleic acids. We describe the synthesis and functionalization
of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM).
This substrate allows easy immobilization of affinity probes, is nonsieving
(even to macromolecules), and exhibits negligible nonspecific binding.
We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA
probe and study the spatiotemporal dynamics using epifluorescence
imaging. We make qualitative and quantitative comparisons between
these data and the model presented in the first part of this two-paper
series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the
range of 10–50 μm s–1, and thereby
explore over 4 orders of magnitude of scaled target amount. We observe
very good agreement between predictions and experimental data for
the spatiotemporal behavior of the coupled ITP and affinity process,
and for key figures of merit, including scaled capture length and
maximum capture efficiency. Lastly, we demonstrate that the resolution
of ITP-AC increases linearly with time and purify 25 nt target DNA
from 10 000-fold higher abundance background (contaminating)
genomic fish sperm DNA. We perform this capture from 200 μL
of sample in under 1 mm column length and within <10 min.
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spatiotemporal behaviorgenomic fish sperm DNAaffinity chromatographyabundance backgroundbuffer systemspatiotemporal dynamics4 ordersSelective Purification25 ntepifluorescence imagingHigh Column UtilizationDNA probepolymer monolithcolumn utilizationaffinity processpgCy 5DNA targetITP velocitytarget amount1 mm column length25 nt target DNAaffinity probesPPMtarget concentrationAC200 μ LdataAffinity Chromatographyaffinity substrate
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