Dielectrophoresis (DEP) bioparticle research has progressed from micro to nano levels. It has proven to be a promising and powerful cell manipulation method with an accurate, quick, inexpensive, and label-free technique for therapeutic purposes. DEP, an electrokinetic phenomenon, induces particle movement as a result of polarization effects in a nonuniform electrical field. This review focuses on current research in the biomedical field that demonstrates a practical approach to DEP in terms of cell separation, trapping, discrimination, and enrichment under the influence of the conductive medium in correlation with bioparticle viability. The current review aims to provide readers with an in-depth knowledge of the fundamental theory and principles of the DEP technique, which is influenced by conductive medium and to identify and demonstrate the biomedical application areas. The high conductivity of physiological fluids presents obstacles and opportunities, followed by bioparticle viability in an electric field elaborated in detail. Finally, the drawbacks of DEP-based systems and the outlook for the future are addressed. This article will aid in advancing technology by bridging the gap between bioscience and engineering. We hope the insights presented in this review will improve cell suspension medium and promote DEP-viable bioparticle manipulation for health-care diagnostics and therapeutics.

The complexity of biotherapeutic products implies an ever-increasing list of product quality attributes that need to be monitored and characterized. In addition, the growing interest in implementing process analytical technology in biopharmaceutical production has further increased the testing burden, together with the need for rapid testing that can facilitate real-time or near-real-time decision-making. Capillary electrophoresis (CE) has made a place in biopharmaceutical analysis but is regarded as a low-throughput method, with the instrument dead time constituting more than 80% of the total time of analysis. In this study, the dead time of CE was utilized to analyse 3 mAb samples in a single-CE run. This approach resulted in an up to 77% reduction in the total analysis time and increased the productivity by up to 300%, compared to traditional single CE-ultraviolet runs, without compromising resolution or relative peak areas. Additionally, good method reproducibility was observed. The compatibility of the method has been demonstrated with protein A eluate and cation exchange chromatography fractions. We, thus, propose that sequential injections can be applied for fast and robust CE analysis of biopharmaceuticals.

A number of microfluidic systems of interest essentially consist of micro-scaled channels/tubes, whose walls are inherently rough. The novelty of the current study lies in exploring the impact of the wall roughness on mass transfer in the case of flow through a microtube with porous wall. The current investigation is possibly the first attempt at exploring the effect of mass transfer for a porous-walled, rough microtube, as earlier studies were limited to the analysis of hydrodynamic and thermal effects only in an impervious microtube. In particular, the effects of the corrugation amplitude and the wavenumber on the mass transport have been assessed in detail in this work, via a combination of perturbation approximations and numerical analysis. Several interesting revelations are elicited regarding the effects of these pertinent parameters on the mass transfer coefficient, permeation flux, wall surface concentration, and delivery flux of the neutral solute. It has been unveiled that it is possible to enhance the solute mass flux by 10% by appropriate tuning of corrugation amplitude. The findings of the study can help in better understanding of mass transport for a porous-walled, rough microtube, which has critical relevance in several important applications such as micromixers, targeted drug delivery, etc. This article is protected by copyright. All rights reserved.

Tryptophan (TRP) is an essential amino acid catabolized mainly through the kynurenine pathway, and part of it is catabolized in the brain. The abnormal depletion of TRP and production of kynurenine (KYN) by two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), have been linked to various neurological diseases. The ratio of TRP/KYN in plasma is a valuable measure for IDO/TDO activity and the prognosis of disease conditions. The 4-vinylphenylboronic acid (4-VPBA) was evaluated as a novel stationary phase for OT-CEC-MS/MS. TRP, KYN, and 3-hydroxykynurenine were separated using optimum conditions of 15 mM (NH4 )2 CO3 at pH 8 as a background electrolyte and 25 kV separation voltage on a 90 cm column. The usefulness of the 4-VPBA column for simple, fast, repeatable, and sensitive CEC-ESI-MS/MS application was demonstrated for the quantitation of TRP and KYN in the plasma of healthy human subjects and neuroinflammation subjects. The plasma sample was extracted on a zirconia-based ion-exchange cartridge for simultaneous protein precipitation and phospholipid removal. The method of standard addition, in combination with the internal standards approach, was used to prepare the calibration curve to overcome matrix matching and eliminate procedural errors. The developed quantitation method was validated according to FDA guidelines for sensitivity, accuracy, precision, and extraction recovery. The measured plasma level of TRP and KYN in healthy humans is aligned with the human metabolome database for the same two metabolites.

The pressure due to electrophoretic motion of a charged colloidal sphere in a fluid-filled circular cylinder is determined in the limit in which the sphere radius is small compared with that of the cylinder. If the ends of the cylinder are open, pressure-driven Poiseuille flow occurs, but the magnitude of this flow is shown to be small when the cylinder is long compared to its radius. It is concluded that the flow has little effect upon electrophoretic velocities, unlike when the diameter of the sphere is comparable to that of the cylinder in which case the Poiseuille flow increases electrophoretic velocities and creates long-range interactions between spheres.

A procedure to determine albendazole and ivermectin in veterinary formulations, like tablet, bolus, oral suspensions, and injections by micellar liquid chromatography, has been developed. Sample preparation was a batch solid-to-liquid extraction in mobile phase, consisting of a stirring step (15 min), followed by ultrasonication (15 min) and filtration of the obtained supernatant, to reach a target concentration of 2 mg/L for both analytes. Using a mobile phase of 0.15 M sodium dodecyl sulfate-6% 1-pentanol buffered at pH 3 with a 0.01 M phosphate salt, running at 1 mL/min through a C18 column, both drugs were resolved in less than 10 min. Absorbance detection wavelength was 292 nm. Procedure was validated by the guidelines of the International Council on Harmonization in terms of specificity, calibration range (0.025-5 mg/L), trueness (97.8%-102.6%), precision (<2.2%), and system suitability. The method was found easy-to-handle, low cost, safe, green, and with high sample-throughput, thus useful for routine analysis. Therefore, it represents a valuable alternative for quality control of veterinary formulations. It was applied to samples of veterinary formulations purchased from local chemists and veterinarians, and label claims were inside the acceptance criteria (95%-105%).

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2 µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.

We reported a manually operated static droplet array (SDA)-based device for the synthesis of nonspherical microparticles with different shapes. The improved SDA structure and reversible bonding between poly(dimethylsiloxane) (PDMS) were used in the device for the large-scale synthesis and rapid extraction of nonspherical microparticles. To understand the device physics, the effects of flow rate, SDA well size, and shape on droplet generation performances were explored. The results indicated that droplet generation in SDA structures was insensitive to the flow rate, and monodisperse droplets were generated by the SDA-based device through manually pushing the syringe. Finally, we integrated four kinds of SDA structures in one device and successfully realized the synthesis and extraction of nonspherical microparticles with different shapes and materials. Our SDA-based device offers numerous advantages, such as simple manual operation, low equipment cost, controllable microparticle shapes and sizes, and large-scale production. Thus, it holds the potential to be used as a flexible tool for the production of nonspherical microparticles.

In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( μLeff(x) / κ(x) values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, x x x) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on. In this paper, we demonstrate some of the operational features of the tbdITP model using the new ITP/IEF version of Simul 6.

Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research.

The appearance of unexpected peaks in capillary electrophoresis (CE) is common and can lengthen the time of method development as assay conditions and experimental parameters are varied to understand and mitigate the effects of the additional peaks. Additional peaks can arise when a single-analyte zone is split into multiple zones. Understanding the underlying mechanism of these phenomena, recognizing conditions that favor its presence, and knowing how to confirm and eliminate the effect are important for efficient method optimization. In this study, we examine how the overlap of analyte zones with the sample plug can lead to peak splitting. This is explored experimentally using dual detection CE, which enables both the sample plug and analyte zones to be independently and simultaneously measured from the same detection volume. Simulations performed via COMSOL Multiphysics confirm the origin of the splitting and help guide experiments to reduce and eliminate the effect. Our findings show that this peak splitting mechanism can arise in separations of both small and large molecules but is, especially, prevalent in separations of slowly migrating macromolecules. This effect is also more prevalent when using a short length-to-detector, as is commonly found in microfluidic applications. A simple diffusion-less model is introduced to develop strategies for reducing peak splitting that avoids modifying the apparatus, such as by lengthening the separation length, which can be difficult. Decreasing the sample plug length and slowing the electroosmotic flow can both reduce this effect, which is confirmed experimentally.

Efficiently pumping fluids without moving parts in extremely miniaturized formats is challenging. Here, we propose and numerically explore a new type of fluid pump in which a series of electrodes driven at different phases produce a force directly on the molecules of the fluid. This effect is based on traveling-wave dielectrophoresis (twDEP), which has been observed to drive the motion of colloidal particles. Here, we leverage the time needed for fluid molecules with permanent dipoles to align with the applied field to maintain a phase lag between the applied field and the molecular polarization. While requiring operation in the GHz range, this effect is predicted to be efficient due to its ability to directly drive bulk fluid motion. We begin by establishing the foundational equations for this effect and performing finite element simulations to determine its magnitude in a model geometry. By combining theory and a systematic series of calculations, we validate that twDEP pumps should exhibit a fluid flow that scales as the voltage squared divided by the electrode period and that it should increase with the complex permittivity of the fluid and decrease with increasing viscosity. This results in a general equation that predicts the performance of twDEP pumps. Collectively, these computations provide a blueprint for producing twDEP pumps of polar fluids such as water and ethanol. We conclude by noting that the growing interest in high power microwave technology along with metasurfaces to locally tailor phase could provide a path to realizing twDEP pumps in practice.

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization-mass spectrometry (ESI-MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor-stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization-mass spectrometry (CE-ESI-MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE-ESI-MS setup.

Statins are cholesterol-lowering medications which are widely prescribed as first-line treatment for hyperlipidemia, against high blood cholesterol aimed at reducing the risk of atherosclerotic diseases. Notwithstanding their undoubted efficacy, the needed long-term treatment with these drugs is characterized by a high percentage of dropout. Consequently, an effective tool to verify the patients' compliance to statin therapy is needed. In this context, the analysis for drugs and drug metabolites in the hair may represent an almost ideal tool because, according to a sound body of forensic toxicological literature, concentrations in the hair matrix reflect the chronic intake of drugs and pharmaceuticals. In this light, in the present study, a novel, specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method has been developed to determine six statins and their metabolites (namely atorvastatin, (p)α-OH-atorvastatin-lactone, (o)α-OH-atorvastatin-lactone, rosuvastatin, N-desmethyl rosuvastatin and pravastatin) in human hair. After optimization, the method was successfully validated in terms of selectivity, linearity, sensitivity, precision, accuracy, stability and matrix effect. Moreover, the practical applicability of this method for verifying adherence to statin therapy was assessed by testing samples of hair collected from subjects under long-term therapy with statins.

In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.

The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H3 O+ and OH- ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pKa values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.