Selectivity enhancements in gel‐based DNA‐nanoparticle assays by membrane‐induced isotachophoresis: thermodynamics versus kinetics. Issue 20 (30th August 2017)

• Record Type: Journal Article
• Authors: Marczak, Steven; Smith, Elaine; Senapati, Satyajyoti; Chang, Hsueh‐Chia
• Other Names: Flanagan Lisa A. guestEditor.; Labeed Fatima H. guestEditor.; Lapizco‐Encinas Blanca H. guestEditor.
• Is Part Of: Electrophoresis. Volume 38:Issue 20(2017)
• Page Start: 2592
• Page End: 2602
• Publication Date: 2017-08-30
• Subjects: Depletion isotachophoresis -- DNA selectivity -- Ion‐selective membrane -- Irreversible dehybridization; Electrophoresis -- Periodicals; Electrophoresis -- Periodicals; 541.372
• DOI: 10.1002/elps.201700146
• Languages: English
• ISSNs: 0173-0835

Abstract

Abstract: Selectivity against mutant nontargets with a few mismatches remains challenging in nucleic acid sensing. Sensitivity enhancement by analyte concentration does not improve selectivity because it affects targets and nontargets equally. Hydrodynamic or electrical shear enhanced selectivity is often accompanied by substantial losses in target signals, thereby leading to poor limits of detection. We introduce a platform based on depletion isotachophoresis in agarose gel generated by an ion‐selective membrane that allows both selectivity and sensitivity enhancement with a two‐step assay involving concentration polarization at an ion‐selective membrane. By concentrating both the targets and probe‐functionalized nanoparticles by ion enrichment at the membrane, the effective thermodynamic dissociation constant is lowered from 40 nM to below 500 pM, and the detection limit is 10 pM as reported previously. A dynamically optimized ion depletion front is then generated from the membrane with a high electrical shear force to selectively and irreversibly dehybridize nontargets. The optimized selectivity against a two‐mismatch nontarget (in a 35‐base pairing sequence) is shown to be better than the thermodynamic equilibrium selectivity by more than a hundred‐fold, such that there is no detectable signal from the two‐mismatch nontarget. We offer empirical evidence that irreversible cooperative dehybridization plays an important role in this kinetic selectivity enhancement and that mismatch location controls the optimum selectivity even when there is little change in the corresponding thermodynamic dissociation constant.