Rapid hybridization of nucleic acids using isotachophoresis

Abstract

We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants.hybridization kinetics | DNA | RNA | molecular beacons | electrophoresis N ucleic acid hybridization is ubiquitous in molecular biology, biotechnology, and biophysics, and has been instrumental in the development of numerous important techniques including genetic profiling (1, 2), pathogen identification (3, 4) sequencing reactions (5), and single-nucleotide polymorphism typing (6). In nucleic acid hybridization, two single-stranded nucleic acid molecules with complementary sequences bind and form more stable double-stranded molecules. Diffusion, transport, and reaction rates limit hybridization of nucleic acids at low concentrations (7). While diffusion and transport limitations can be effectively overcome using mixing and flow control methods (8-10), reaction rates still limit assay times and sensitivity (7,11).Nucleic acid amplification techniques (e.g., polymerase chain reaction, PCR) are often used as an initial step to improve sensitivity and accelerate hybridization. However, amplifications such as PCR can suffer from as much as 10,000-fold amplification bias (1, 4), require significant sample preparation (12), can be difficult to reproduce quantitatively across laboratories (13), and require a well-controlled environment (12). Amplification-free hybridization avoids amplification-associated sequence bias and may be particularly important for applications beyond the conventional laboratory settings.Temperature, monovalent salt concentration, and divalent cation concentration (in particular, magnesium ion) are most commonly used to control the rate of hybridization. The hybridization of short oligonucleotides in the presence of 50 mM MgCl 2 has been reported as fivefold faster than at 1 mM MgCl 2 (14). Similarly, higher salt concentration and higher temperatures can accelerate reactions (14-16). However, these approaches reduce the energy of binding events and strongly affect specificity. Adjustment of these hybridization parameters is therefore often a trade-off between acceleration and specificity (12, 17). Other methods such as volume exclusion by inert polymers (e.g., dext...

Oxidized Porous Silicon Nanostructures Enabling Electrokinetic Transport for Enhanced DNA Detection

“…[ 42,43 ] Isotachophoresis (ITP) is a simple and robust electrokinetic technique that can achieve as much as a million-fold preconcentration, effi cient separation, and extraction based on ionic mobility. [44][45][46] The analyte of interest is focused at the interface of two distinct electrolyte solutions resulting in a fi nite zone of highly concentrated analyte. Typically, ITP is performed on nonconductive planar substrates (e.g., glass and plastics), [ 44,[47][48][49] while recently successful ITP focusing was also demonstrated in porous media such as paper.…”

“…[44][45][46] The analyte of interest is focused at the interface of two distinct electrolyte solutions resulting in a fi nite zone of highly concentrated analyte. Typically, ITP is performed on nonconductive planar substrates (e.g., glass and plastics), [ 44,[47][48][49] while recently successful ITP focusing was also demonstrated in porous media such as paper. [ 50,51 ] In this work, we design a novel proof-of-concept assay that interfaces PSi Fabry-Pérot interferometry with ITP and demonstrates its applicability for highly sensitive optical detection of DNA oligonucleotides.…”

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

“…Similarly, the product c R c T is volume-averaged over the ITP zone (see Bercovici et al. 17 ). The single fitting parameter, F, represents the flux (per concentration) of molecules from the TE into the focused sample zone.…”

“…16 We use ITP preconcentration to enhance hybridization of target microRNA molecules with fluorescently labeled oligos we refer to as reporters ( Figure 1A). 17 We then migrate the ITP zone through a DNA-functionalized hydrogel where unreacted reporters are removed through affinity interactions ( Figure 1B). Following the functional gel region, the concentration of unreacted fluorescent probes is mimimized, significantly reducing background signal.…”

“…ITP increases the concentration of both target and reporter, thereby significantly enhancing forward reaction rates. 17 Additionally, we use a relatively high initial reporter concentration (2 nM in the TE reservoir) to further promote fast kinetics. We note that the sample and reporter injection is robust and repeatable (see the Supporting Information for details on the repeatability of the sample injection signal).…”