Danny Bottenus

Microfluidic isotachophoresis: a review.

Electromigration methods including CE and ITP are attractive for incorporation in microfluidic devices because they are relatively easily adaptable to miniaturization. After its popularity in the 1970s, ITP has made a comeback in microfluidic format (μ‐ITP, micro‐ITP) driven by the advantages of the steady‐state boundary, the self‐focusing effect, and the ability to aid in preconcentrating analytes in the sample while removing matrix components. In this review, we provide an overview of the developments in the area of μ‐ITP in a context of the historic developments with a focus on recent developments in experimental and computational ITP and discuss possible future trends. The chip‑ITP areas and topics discussed in this review and the corresponding sections include: PC simulations and modeling, analytical μ‐ITP, preconcentration ITP, transient ITP, peak mode ITP, gradient elution ITP, and free‐flow ITP, while the conclusions provide a critical summary and outlook. The review also contains experimental conditions for μ‐ITP applications to real‐world samples from over 50 original journal publications.

10 000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the authors knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results.

This paper describes both the experimental application and 3‐D numerical simulation of isotachophoresis (ITP) in a 3.2 cm long “cascade” poly(methyl methacrylate) (PMMA) microfluidic chip. The microchip includes 10× reductions in both the width and depth of the microchannel, which decreases the overall cross‐sectional area by a factor of 100 between the inlet (cathode) and outlet (anode). A 3‐D numerical simulation of ITP is outlined and is a first example of an ITP simulation in three dimensions. The 3‐D numerical simulation uses COMSOL Multiphysics v4.0a to concentrate two generic proteins and monitor protein migration through the microchannel. In performing an ITP simulation on this microchip platform, we observe an increase in concentration by over a factor of more than 10 000 due to the combination of ITP stacking and the reduction in cross‐sectional area. Two fluorescent proteins, green fluorescent protein and R‐phycoerythrin, were used to experimentally visualize ITP through the fabricated microfluidic chip. The initial concentration of each protein in the sample was 1.995 μg/mL and, after preconcentration by ITP, the final concentrations of the two fluorescent proteins were 32.57±3.63 and 22.81±4.61 mg/mL, respectively. Thus, experimentally the two fluorescent proteins were concentrated by over a factor of 10 000 and show good qualitative agreement with our simulation results.

Impact of leakage current and electrolysis on FET flow control and pH changes in nanofluidic channels

We have fabricated multiple-internal-reflection Si infrared waveguides integrated with an array of nanochannels sealed with an optically transparent top cover. The channel walls consist of a thin layer of SiO2 for electrical insulation, and gate electrodes surround the channel sidewalls and bottom to manipulate their surface charge and zeta-potential in a fluidic field effect transistor (FET) configuration. This nanofluidic device is used to probe the transport of charged molecules (Alexa 488) and to measure the pH shift in nanochannels in response to an electrical potential applied to the gate. During gate biasing for FET operation, laser-scanning confocal fluorescence microscopy (LS-CFM) is used to visualize the flow of fluorescent dye molecules (Alexa 488), and multiple internal reflection-Fourier transform infrared spectroscopy (MIR-FTIRS) is used to probe the characteristic vibrational modes of fluorescein pH indicator and measure the pH shift. The electroosmotic flow of Alexa 488 is accelerated in response to a negative gate bias, whereas its flow direction is reversed in response to a positive gate bias. We also measure that the pH of buffered electrolyte solutions shifts by as much as a pH unit upon applying the gate bias. With prolonged application of gate bias, however, we observe that the initial response in flow speed and direction as well as pH shift becomes reversed. We attribute these anomalous flow and pH shift characteristics to a leakage current that flows from the Si gate through the thermally grown SiO2 to the electrolyte solution.

A new fabrication technique to form complex polymethylmethacrylate microchannel for bioseparation

Recent studies show that reduction in cross-sectional area can be used to improve the concentration factor in microscale bioseparations. Due to simplicity in fabrication process, a step reduction in cross-sectional area is generally implemented in microchip to increase the concentration factor. But the sudden change in cross-sectional area can introduce significant band dispersion and distortion. This paper reports a new fabrication technique to form a gradual reduction in cross-sectional area in polymethylmethacrylate (PMMA) microchannel for both anionic and cationic isotachophoresis (ITP). The fabrication technique is based on hot embossing and surface modification assisted bonding method. Both one-dimensional and two-dimensional gradual reduction in cross-sectional area microchannels were formed on PMMA with high fidelity using proposed techniques. ITP experiments were conducted to separate and preconcentrate fluorescent proteins in these microchips. Thousand fold and ten thousand fold increase in concentrations were obtained when 10 × and 100 × gradual reduction in cross-sectional area microchannels were used for ITP.

Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high-abundance contaminant, serum albumin

Cationic ITP was used to separate and concentrate fluorescently tagged cardiac troponin I (cTnI) from two proteins with similar isoelectric properties in a PMMA straight‐channel microfluidic chip. In an initial set of experiments, cTnI was effectively separated from R‐Phycoerythrin using cationic ITP in a pH 8 buffer system. Then, a second set of experiments was conducted in which cTnI was separated from a serum contaminant, albumin. Each experiment took ∼10 min or less at low electric field strengths (34 V/cm) and demonstrated that cationic ITP could be used as an on‐chip removal technique to isolate cTnI from albumin. In addition to the experimental work, a 1D numerical simulation of our cationic ITP experiments has been included to qualitatively validate experimental observations.

ITP of lanthanides in microfluidic PMMA chip

An ITP separation of eight lanthanides on a serpentine PMMA microchip with a tee junction and a 230‐mm‐long serpentine channel is described. The cover of the PMMA chip is 175 μm thick so that a C4D in microchip mode can be used to detect the lanthanides as they migrate through the microchannel. Acetate and α‐hydroxyisobutyric acid are used as complexing agents to increase the electrophoretic mobility difference between the lanthanides. Eight lanthanides are concentrated within ∼ 6 min by ITP in the microchip using 10 mM ammonium acetate at pH 4.5 as the leading electrolyte and 10 mM acetic acid at ∼ pH 3.0 as the terminating electrolyte. In addition, a 2D numerical simulation of the lanthanides undergoing ITP in the microchip is compared with experimental results using COMSOL Multiphysics v4.3a.

On-Line Optical Fiber Detection in a Preparative Free-Flow Electrofocusing Apparatus

Many analytical isoelectric focusing (IEF) instruments are equipped with on‐line detection, e.g., conductivity or UV‐vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor‐intensive off‐line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on‐line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free‐flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex‐stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.

Preconcentration of Cardiac Proteins in a Cascade Microchip

Concentration of bio-molecules prior to detection is very critical in the development of an integrated, multifunctional lab-on-a chip device for detection of ultra trace molecules from complex biological fluids such as serum, urine, or saliva. In this work, the preconcentration of a clinically relevant biomarker, cardiac troponin I (cTnI), is demonstrated in a cascade microfluidic channel using cationic isotachophoresis (ITP). The cascade chip is formed on PMMA (poly methyl methacrylate) with gradual changes in size both in width and depth direction to achieve a 100× reduction in overall cross sectional area between inlet (anode) and outlet (cathode) sections. The ITP experiments were conducted with two fluorescent proteins, FITC (Fluorescein isothiocyanate-conjugated) albumin and cTnI labeled with Pacific Blue. Potassium ions were used as the leader and hydronium ions were used as the terminator for these cationic ITP experiments. The microchip ITP demonstrates that it is possible to increase the concentration of cTnI by 10,000 folds using a potential drop of 400 V across a 3.5 cm long microchannel. The reduction in cross sectional area facilitates additional concentration gain, as the proteins migrate through cascade microchannel under discontinuous electric field and stacked into nearly pure zones.Copyright

Preconcentration of Cardiac Proteins in a Microfluidic Device

Preconcentration of cardiac proteins was demonstrated using isotachophoresis (ITP) on a polymethyl methacrylate (PMMA) microchip. PMMA microchip was formed using solvent imprinting and temperature-assisted solvent bonding. ITP experiments were performed on two types of microchip: one containing straight microchannel and the other one with 10X step reducing microchannel. In ITP experiments, Hydrochloric Acid (HCl) was used as leading electrolyte, while Aminocaproic Acid (EACA) was used as terminating electrolyte. Three fluorescent proteins, cTnI Labeled w/ Pacific Blue, Green Fluorescent Protein (GFP) and R-Phycoerythrin (PE), were allowed to separate and concentrate in presence of a constant electric field. Microchip ITP experiments show that the sample proteins were concentrated and stacked into adjacent zones. The final concentration of protein zones were calculated from the microchannel dimensions and initial volume of proteins. In straight microchannel, the concentration factors for PE, GFP, and cTnI proteins were 80, 40, and 30, respectively. The concentration factors were 10 fold higher in the step reducing microchannel.Copyright