Isotachophoresis: Theory and Microfluidic Applications

Abstract

Isotachophoresis (ITP) is a versatile electrophoretic technique that can be used for sample preconcentration, separation, purification, and mixing, and to control and accelerate chemical reactions. Although the basic technique is nearly a century old and widely used, there is a persistent need for an easily approachable, succinct, and rigorous review of ITP theory and analysis. This is important because the interest and adoption of the technique has grown over the last two decades, especially with its implementation in microfluidics and integration with on-chip chemical and biochemical assays. We here provide a review of ITP theory starting from physicochemical first-principles, including conservation of species, conservation of current, approximation of charge neutrality, pH equilibrium of weak electrolytes, and so-called regulating functions that govern transport dynamics, with a strong emphasis on steady and unsteady transport. We combine these generally applicable (to all types of ITP) theoretical discussions with applications of ITP in the field of microfluidic systems, particularly on-chip biochemical analyses. Our discussion includes principles that govern the ITP focusing of weak and strong electrolytes; ITP dynamics in peak and plateau modes; a review of simulation tools, experimental tools, and detection methods; applications of ITP for on-chip separations and trace analyte manipulation; and design considerations and challenges for microfluidic ITP systems. We conclude with remarks on possible future research directions. The intent of this review is to help make ITP analysis and design principles more accessible to the scientific and engineering communities and to provide a rigorous basis for the increased adoption of ITP in microfluidics.

Continuous Molecular Concentration and Separation Using Pulsed-Field Conductive-Wall Single-Buffer Teı́chophoresis

“…If the TE concentration is too high, the platform will exhibit peak-mode TPE, and the sample zone will not accumulate to a great enough concentration to affect the overall local conductivity, rendering separation impossible. This experimental scenario is physically similar to that of peak-mode ITP …”

“…This experimental scenario is physically similar to that of peakmode ITP. 14 To measure the concentration of fluorescent dye during experiments, we measured the fluorescent intensity of known concentration standards and calibrated these measurements to a linear curve. Calibration and the experimental ITP concentration factor were calibrated fluorescently using an inverted swept field confocal microscope (Nikon Eclipse Ti, 70 μm confocal slit, Nikon/Prairie Technologies), an Andor iXon 897 EMCCD camera, a 50 mW solid-state laser for excitation, and a 20× objective.…”

“…The LE, sample, and TE are usually loaded into a capillary tube or a microfluidic channel, and an electric field is dropped down the axial length of the LE/sample/TE mixture. Upon application, the species of intermediate mobility focus at the interface between the LE/TE electrolytes due to the formation of a mismatched electric field, while species with higher or lower mobilities than the TE and LE are excluded from the concentration region . The charged analytes then continue to accumulate and focus into concentrated bands in order of decreasing electrophoretic mobility as they electrophoretically traverse down the channel length under capillary electrophoresis where they ultimately exit the channel and can be extracted in a concentrated/purified form.…”

“…Upon application, the species of intermediate mobility focus at the interface between the LE/ TE electrolytes due to the formation of a mismatched electric field, while species with higher or lower mobilities than the TE and LE are excluded from the concentration region. 14 The charged analytes then continue to accumulate and focus into concentrated bands in order of decreasing electrophoretic mobility as they electrophoretically traverse down the channel length under capillary electrophoresis where they ultimately exit the channel and can be extracted in a concentrated/ purified form. Due to its versatility and raw concentrating power, ITP has been used as a preconcentration method for a wide variety of ionic and molecular analytes, including concentrating potassium in explosive residues, bacteria in water, and rRNA indicative of urinary tract infections in urine.…”

Web-Based Open-Source Tool for Isotachophoresis

“…The dynamics show the development and motion of various ion interfaces and pH throughout the computational domain of interest. Note the two stationary “top hat” features in the HEPES distribution (centered near x = 20 and 40 mm) are stationary, expected, and due to the Jovin and Alberty regulating functions established by the initial distribution of ions . We present additional examples of CAFES simulations for cationic and for bidirectional ITP in the SI (and provide example input files for these computations).…”