Immunobinding-induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis.

The objective of this study is to explore an approach for analyzing negatively charged proteins using paper-based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen-antibody immunobinding. Cationic ITP was performed on a paper-based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3-aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA-only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine-rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP-based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost-effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.

Design and Finite Element Model of a Microfluidic Platform with Removable Electrodes for Electrochemical Analysis.

A microfluidic platform for hydrodynamic electrochemical analysis was developed, consisting of a poly(methyl methacrylate) chip and three removable electrodes, each housed in 1/16" OD polyether ether ketone tube which can be removed independently for polishing or replacement. The working electrode was a 100-µm diameter Pt microdisk, located flush with the upper face of a 150 µm × 20 µm × 3 cm microchannel, smaller than previously reported for these types of removable electrodes. A commercial leak-less reference electrode was utilized, and a coiled platinum wire was the counter electrode. The platform was evaluated electrochemically by oxidizing a potassium ferrocyanide solution at the working electrode, and a typical limiting current behavior was observed after running linear sweep voltammetry and chronoamperometry, with flow rates 1-6 µL/min. While microdisk channel electrodes have been simulated before using a finite difference method in an ideal 3D geometry, here we predict the limiting current using finite elements in COMSOL Multiphysics 5.3a, which allowed us to easily explore variations in the microchannel geometry that have not previously been considered in the literature. Experimental and simulated currents showed the same trend but differed by 41% in simulations of the ideal geometry, which improved when channel and electrode imperfections were included.

Taylor dispersion in equilibrium gradient focusing at steady state.

An analytic expression is presented for the effective dispersion coefficient in the case where a solute is focused in a parabolic flow against a linear gradient in a restoring force. This expression was derived by employing a minor variation on the method of moments used by Aris in his development of the dispersion coefficients for a time-dependent, isocratic system. In the present case, dispersion is controlled by two dimensionless groups, a Peclet number which is proportional to the parabolic component of the flow, and a gradient number which is proportional to the slope of the restoring force. These results confirm that the Aris-Taylor expression for the dispersion coefficient should not be applied in cases where a solute is focused to a stationary steady state.

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 C(4) D 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.

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.

Counterflow isotachophoresis in a monolithic column.

This study describes stationary counterflow isotachophoresis (ITP) in a poly(acrylamide-co-N,N'-methylenebisacrylamide) monolithic column as a means for improving ITP processing capacity and reducing dispersion. The flow profile in the monolith was predicted using COMSOL's Brinkman Equation application mode, which revealed that the flow profile was mainly determined by monolith permeability. As monolith permeability decreases, the flow profile changes from a parabolic shape to a plug shape. An experimental monolithic column was prepared in a fused-silica capillary using an ultraviolet-initiated polymerization method. A monolithic column made from 8% (wt.) monomer was chosen for the stationary counterflow ITP experiments. Counterflow ITP in the monolithic column showed undistorted analyte zones with significantly reduced dispersion compared to the severe dispersion observed in an open capillary. Particularly, for r-phycoerythrin focused by counterflow ITP, its zone width in the monolithic column was only one-third that observed in an open capillary. These experiments demonstrate that stationary counterflow ITP in monoliths can be a robust and practical electrofocusing method.

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.

Isotachophoresis with counterflow in an open capillary: computer simulation and experimental validation.

The purpose of applying a countercurrent flow to isotachophoretic migration is to increase the effective separation channel length during ITP. However, severe dispersion induced by applying a counterflow can be detrimental to ITP. This paper uses numerical simulations in a 2D axisymmetric domain to investigate the dispersion caused by a parabolic counterflow in open-capillary ITP. Counterflow in these simulations was generated by applying a back pressure to stop the isotachophoretic stack, i.e., forming stationary ITP zones. It is found that dispersion is strongly related to analyte molecular diffusivity: R-phycoerythrin, due to its small diffusivity, showed ~20-fold increase in zone width in stationary counterflow ITP, compared to ITP in the absence of counterflow, while fluorescein only had ~10% increase in zone width under similar operating conditions. Applying the Taylor-Aris dispersion formula in counterflow ITP simulations provided only a rough estimate of the dispersion, e.g., overestimation of analyte zone widths. Experiments on counterflow ITP were conducted in a silica capillary that was covalently and dynamically coated to exclude electroosmosis effect. The counterflow was generated by adjusting the relative height of the fluids in the two reservoirs at the capillary ends. Good qualitative agreement between simulations and experiments was found.

One-dimensional simulation of lanthanide isotachophoresis using COMSOL.

Electrokinetic separations can be used to quickly separate rare earth metals to determine their forensic signature. In this work, we simulate the concentration and separation of trivalent lanthanide cations by isotachophoresis. A one-dimensional simulation is developed using COMSOL v4.0a, a commercial finite element simulator, to represent the isotachophoretic separation of three lanthanides: lanthanum, terbium, and lutetium. The binding ligand chosen for complexation with the lanthanides is α-hydroxyisobutyric acid (HIBA) and the buffer system includes acetate, which also complexes with the lanthanides. The complexes formed between the three lanthanides, HIBA, and acetate are all considered in the simulation. We observe that the presence of only lanthanide:HIBA complexes in a buffer system with 10 mM HIBA causes the slowest lanthanide peak (lutetium) to split from the other analytes. The addition of lanthanide:acetate complexes into the simulation of the same buffer system eliminates this splitting. Decreasing the concentration of HIBA in the buffer to 7 mM causes the analyte stack to migrate faster through the capillary.

Direct current dielectrophoretic simulation of proteins using an array of circular insulating posts.

This paper presents a mathematical model for the manipulation of proteins using insulator-based dielectrophoresis (iDEP) and direct current (DC) electric fields. Simulations via COMSOL v4.1 Multiphysics software are implemented to study the response of moderately sized proteins on a lab-on-a-chip platform. The geometry of the device is incorporated in a model that solves current and mass conservation equations within an array of circular insulating silicon posts embedded in a channel. Both micro- and nano-scale geometries are utilized to investigate the protein concentration distributions in the iDEP device. Our results indicate that the trapping of proteins is independent of the scale with respect to the geometry of a device as long as the applied electric field remains constant. DC voltage dependency on concentration distributions has also been explored in both micro- and nano-scale device geometries. To achieve DEP trapping of the proteins, nano-scale geometry is a better selection, as the voltage necessary to generate the required electric field (2.5 MV/cm) is 10(5) × lower compared with the voltage required to generate the same field in the micro-scale device.

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.

Enhanced fluorescence anisotropy assay for human cardiac troponin I and T detection.

Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5-40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15-500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing.

Unveiling the Interfacial and Structural Heterogeneity of Ti3C2Tx MXene Etched with CoF2/HCl by Integrated in Situ Thermal Analysis.

Ti3C2Tx MXene is a member of the recently discovered two-dimensional early transition metal carbide and nitride family of MXenes with potential applications in energy storage and heterogeneous catalysis at elevated temperatures. Here, we apply a suite of in situ techniques to probe Ti3C2Tx MXene's thermal evolutions, including in situ X-ray diffraction (XRD), in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and integrated thermogravimetry-differential scanning calorimetry-mass spectrometry (TG-DSC-MS). In light of this set of in situ investigations, we find heterogeneity in the layering of Ti3C2Tx MXene revealed only at higher temperatures. Our findings present behavior up to 600 °C, particularly interlayer water and -OH surface end-capping groups. In one group of layers, their interlayer spacing shrinks as water deintercalates, but the other group of layers unexpectedly shows no change in the interlayer spacing. This is strong evidence that intercalants act as guest pillaring agents in the latter layering group, which stabilize these layers at higher temperatures while keeping the interlayer space accessible.

Influence of the semi-permeable membrane on the performance of dynamic field gradient focusing.

This paper is part of our continued effort to understand the underlying principles of dynamic field gradient focusing. In this investigation, we examined three problems associated with the use of a semi-permeable membrane. First, the influence of steric and ionic exclusion of current carrying ions through the membrane was examined. It was found that resistance to the transport of ions across the membrane resulted in a shallowing of the electric field profile and an increase in the size of the defocusing zone, which is where the slope of the electric field is reversed so that it disperses rather than concentrates solutes. These problems could be reduced by using a membrane with large pores relative to the size of the buffering ions and completely void of fixed charges. Next, a numerical simulation was used to investigate concentration polarization of protein onto the surface of the membrane. Due to the presence of a transverse electric field, species were pulled toward the membrane. If the membrane is restrictive to those species, a concentrated, polarized layer will form on the surface. The simulation showed that by decreasing the channel to a depth of 20 microm, the concentrated region next to the membrane could be reduced. Finally, it was found that changes in column volume due to loss of membrane structural integrity could be mitigated by including a porous ceramic support. The variation in peak elution times was decreased from greater than 20% to less than 3%.

Development of a membrane-less dynamic field gradient focusing device for the separation of low-molecular-weight molecules.

Dynamic field gradient focusing uses an electric field gradient generated by controlling the voltage profile of an electrode array to separate and concentrate charged analytes according to their individual electrophoretic mobilities. This study describes a new instrument in which the electrodes have been placed within the separation channel. The major challenge faced with this device is that when applied voltages to the electrodes are larger than the redox potential of water, electrolysis will occur, producing hydrogen ions (H+) plus oxygen gas on the anodes and hydroxide (OH(-)) plus hydrogen gas on the cathodes. The resulting gas bubbles and pH excursions can cause problems with system performance and reproducibility. An on-column, degassing system that can remove gas bubbles "on-the-fly" is described. In addition, the use of a high capacity, low-conductivity buffer to address the problem of the pH shift that occurs due to the production of H+ on the anodes is illustrated. Finally, the successful separation of three, low-molecular-weight dyes (amaranth, bromophenol blue and methyl red) is described.

Preconcentration mechanism of trivalent lanthanum on eQCM electrodes in the presence of α-hydroxy isobutyric acid.

Electroprecipitation can be used to preconcentrate lanthanum on carbon electrode surfaces. The use of complexing ligands is expected to improve the electroprecipitation of lanthanum by protecting La ions in solution from the alkaline region near the electrode surface. However, the electroprecipitation mechanism of La in the presence of a complexing ligand is not known. The goal of this work is to 1) determine the effect of the complexing ligand, α-hydroxy isobutyric acid (HIBA), on the electroprecipitation of La onto the gold electrodes, and 2) identify the changes in the mechanism of accumulation when preconcentrating in the presence of HIBA. We used an electrochemical quartz crystal microbalance (eQCM) and needle type pH microelectrodes to determine pH near the electrode surface in combination with cyclic voltammetry to understand the electroprecipitation mechanism. We used the bi-dentate ligand HIBA as a ligand and found that lanthanum electroprecipitation is hindered in the presence of HIBA. The presence of HIBA also delayed the onset of film formation during a cyclic voltammetric experiment by ~100 mV compared to experiments performed without HIBA. The shift in onset potential is attributed to the buffering action of HIBA (pKa = 3.7) since the shift is not present in subsequent scans. The precipitated film was characterized by scanning electron microscopy, X-ray photoelectron spectrometry, and Auger nanoprobe spectrometry. While we found that La(OH)3 was the predominant chemical state of the film on electrodes in the absence of HIBA, La2O3 was found for films created in the presence of HIBA. Our finding demonstrates that La(OH)3 can be electrodeposited at room temperature.

Paper-based ITP technology: An application to specific cancer-derived exosome detection and analysis.

Exosomes derived from cancer cells/tissues have great potential for early cancer diagnostic use, but their clinical potential has not been fully explored because of a lack of cost-effective multiplex approaches capable of effectively isolating and identifying specific exosome populations and analyzing their content biomarkers. This study was aimed at overcoming the technical barrier by developing a paper-based isotachophoresis (ITP) technology capable of 1) rapid isolation and identification of exosomes from both malignant and healthy cells and 2) multiplex detection of selected exosomal protein biomarkers of the target exosomes. The technology integrates the focusing power of ITP and the multiplex capability of paper-based lateral flow to achieve on-board separation of target exosomes from large extracellular vesicles, followed by electrokinetic enrichment of the targets, leading to an ultrasensitive platform for comprehensive exosome analysis. For a proof of concept, the technology platform was tested with human serum samples spiked with exosomes derived from healthy human serum and a prostate cancer cell line. Under an anionic ITP condition, the device showed superior performance in simultaneous detection of the cancer exosomes and normal exosomes at concentrations as low as 1.2-2.0 × 106 exosomes/mL, which is equivalent to 2.0-3.0 × 10-18 M. The observed limit of detection was more than 30-fold better than that of enhanced ELISA. More importantly, in a subsequent step the technology was capable of the rapid profiling of a selected protein biomarker panel associated with the target exosomes. The results represent a significant step toward translating the detection of tumor-derived exosomes to a medical use at a point of care.

Electrochemical precipitation of neptunium with a micro electrochemical quartz crystal microbalance.

Microliter volumes are used in electrochemical detection and preconcentration of radionuclides to reduce the dose received by researchers and equipment. Unfortunately, there is a lack of analysis of radionuclides with coupled electrochemical techniques and microliter volume reactors. The goals of this work are 1) to develop a miniaturized micro-electrochemical quartz crystal microbalance (µeQCM) reactor for use in small volume (50-200 µL) electrogravimetric experiments and 2) to use this reactor to characterize the preconcentration of neptunium on carbon electrodes via electroprecipitation. We successfully deposited neptunium in the new µeQCM reactor and verified its operation. We found that preconcentration of neptunium on carbon coated electrodes was possible by chronoamperometry at -1.6 VAg/AgCl. The mass shift of the resulting precipitate was indicative of the amount of neptunium on the electrode, although the correlation between the mass increase and activity of the preconcentrated material was not linear. Neptunium precipitate reduced electron transfer to the solution as evidenced by the increase in charge transfer resistance compared to bare electrodes.

Correlating inter-particle forces and particle shape to shear-induced aggregation/fragmentation and rheology for dilute anisotropic particle suspensions: A complementary study via capillary rheometry and in-situ small and ultra-small angle X-ray scattering.

HYPOTHESIS: Understanding the stability and rheological behavior of suspensions composed of anisotropic particles is challenging due to the complex interplay of hydrodynamic and colloidal forces. We propose that orientationally-dependent interactions resulting from the anisotropic nature of non-spherical sub-units strongly influences shear-induced particle aggregation/fragmentation and suspension rheological behavior. EXPERIMENTS: Wide-, small-, and ultra-small-angle X-ray scattering experiments were used to simultaneously monitor changes in size and fractal dimensions of boehmite aggregates from 6 to 10,000 Å as the sample was recirculated through an in-situ capillary rheometer. The latter also provided simultaneous suspension viscosity data. Computational fluid dynamics modeling of the apparatus provided a more rigorous analysis of the fluid flow. FINDINGS: Shear-induced aggregation/fragmentation was correlated with a complicated balance between hydrodynamic and colloidal forces. Multi-scale fractal aggregates formed in solution but the largest could be fragmented by shear. Orientationally-dependent interactions lead to a relatively large experimental suspension viscosity when the hydrodynamic force was small compared to colloidal forces. This manifests even at low boehmite mass fractions.