Acceleration of surface-based hybridization reactions using isotachophoretic focusing
Merav Karsenty, Shimon Rubin, and Moran Bercovici
- We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field.
- Demonstrated a two orders of magnitude improvement in limit of detection (LoD), in a 3 min assay, of ITP-based surface hybridization assay compared to standard continuous flow-based hybridization.
- A simple analytical model that allows prediction of the rate of surface reaction under ITP, and can be used to design, and optimize such assays as a function of the physical properties of the system, including buffer chemistry, applied voltage, analyte mobility, analyte concentration, probe density, and surface length.
Video 1. Experimental results showing fluorescently labeled DNA delivered via ITP focusing to a surface containing immobilized probes. Due to the locally high concentration, rapid reaction occurs as the ITP interface passes over the surface. The sample continues electromigrating downstream in a tight band, leaving the surface in pure buffer and eliminating the need for a wash step. Here, the sample is fluorescently labeled for illustration purposes only. Other experimental results in this work make use of unlabeled sample.
Surface based biosensors
Surface-based biosensors are some of the most common type of sensors for biological targets such as nucleic acids and proteins. In most implementations, they are based on a “capturing probe” (e.g. an antibody or synthetic DNA sequence) which is immobilized on a surface, and to which targets specifically bind. Detection of the binding events can then be obtained in various ways, however, regardless of the binding or transduction mechanism, the sensitivity of all surface biosensors is fundamentally limited by the rate at which target molecules bind to the surface. Reaction rates remain a major bottleneck toward achieving rapid binding of biomolecules at low concentrations. This is because hybridization and binding typically take the form of second order reactions, with reaction time inversely proportional to the concentration of the reactants. Therefore there is a growing need for methods that significantly accelerate reaction rates and lower detection time.
We developed a detailed, yet simple, analytical model which can be used to predict the rate of surface reaction under ITP as a function of the physical properties of the system, such as ITP chemistry, applied voltage, analyte mobility, probe density, and length of the surface.
Fig 1. Analytical model results showing the ratio of surface hybridization fractions between ITP-based and standard flow hybridization, as a function of the initial target concentration. ITP-based reactions provide significant signal enhancement, where, in addition to a higher total number of target molecules delivered, sample focusing accelerates the characteristic reaction rate. At higher concentrations the initial concentration in the well (i.e. before focusing) is sufficient to saturate the sensor, and no significant gain is obtained from ITP.
Creating microchannel-embedded Surface biosensors
The common methods for patterning of capture probes on a surface require multiple well controlled chemical steps, and strongly depend on the substrate used. To overcome these diffoculties we designed a microfluidic chip in which reaction surfaces are formed by streptavidin-coated paramagnetic beads, immobilized at desired sites by an external magnetic field. The beads are pre-labeled with capture-probes (e.g. biotinilated oligonucleotides, or biotinilated antibodies), eliminating the need for surface modifications, and enables reusability of the chip. Our microfluidic chip includes a 5 μm deep “trench” of dimensions 100 μm x 30 μm, in which magnetic beads are concentrated and trapped creating a uniform and well defined surface.
Figure 2. Raw fluorescence images of 2.8 magnetic beads immobilized in a microchannel by an external cylindrical NdFeB magnet (1/8”X3/8” grade N52) (a) beads trapped in a standard flat bottom micro-channel. The beads are successfully immobilized, but create a non-uniform and non-repeatable distribution. (b) The same magnet is used to capture the same beads within a microfluidic trench, creating a uniform and well defined surface.
Figure 3. Schematic illustration of the assay. (a) A microfluidic channel connecting two reservoirs is initially filled with LE and pre-labeled paramagnetic beads. The solution is flown through the channel by applying pressure driven flow, and beads are trapped in the designated trench under the magnetic force of a permanent magnet placed on top of the chip. (b) A mixture of TE and sample is injected in the West reservoir, and an electric field is applied between the two reservoirs to initiate ITP. (c) Highly focused target molecules are delivered to the reactive surface by ITP, and rapidly react with the probes on the surface. (d) Unbound target molecules leave the reaction site and continue electromigrating with the ITP interface, leaving the surface embedded in TE buffer.
Two orders of magnitude improvement in limit of detection (LOD)
Figure 4. (a) Analytical and experimental results comparing ITP-based hybridization with standard flow through hybridization for initial concentrations between 1 and 100 nM. In both hybridization techniques the total assay time was 3 min. Each measurement corresponds to the difference in the average fluorescence intensity between a post- and pre- hybridization image, across a 80 x 20 pixels square at the center or the reactive surface shown in figure 5 shown in figure 5. The height of each bar represents the average of at least 3 realizations, with range bars representing 95% confidence on the mean. Results show a 107 fold and 12 fold increase in signal at concentrations of 10 and 100 nM, respectively. At 1 nM, the improved signal in ITP is clearly evident, though direct estimation of the improvement in signal is difficult since the standard hybridization case did not result in significant signal above the baseline. Consistent with our theoretical predictions, using ITP hybridization, we obtain a two orders of magnitude improvement in LoD. (b) Kinetic experiment, measuring the signal as a function of time for the case of a standard pressure driven flow reaction, using 100 nM target concentration. Based on this measurement, we estimate the on-rate of the reaction to be 6E3 .
- Karsenty M., Rubin S., and Bercovici M., Accelerated surface hybridization reactions using isotachophoretic focusing, Analytical Chemistry 86 (6), 3028–3036 (2014).