Improved Sample Preparation Method for Protein and Peptide Identification from Human Hair.

A fast and sensitive direct extraction (DE) method developed in our group can efficiently extract proteins in 30 min from a 5 cm-long hair strand. Previously, we coupled DE to downstream analysis using gel electrophoresis followed by in-gel digestion, which can be time-consuming. In searching for a better alternative, we found that a combination of DE with a bead-based method (SP3) can lead to significant improvements in protein discovery in human hair. Since SP3 is designed for general applications, we optimized it to process hair proteins following DE and compared it to several other in-solution digestion methods. Of particular concern are genetically variant peptides (GVPs), which can be used for human identification in forensic analysis. Here, we demonstrated improved GVP discovery with the DE and SP3 workflow, which was 3 times faster than the previous in-gel digestion method and required significantly less instrument time depending on the number of gel slices processed. Additionally, it led to an increased number of identified proteins and GVPs. Among the tested in-solution digestion methods, DE combined with SP3 showed the highest sequence coverage, with higher abundances of the identified peptides. This provides a significantly enhanced means for identifying proteins and GVPs in human hair.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Separating the Living from the Dead: An Electrophoretic Approach.

Cell viability studies are essential in numerous applications, including drug development, clinical analysis, bioanalytical assessments, food safety, and environmental monitoring. Microfluidic electrokinetic (EK) devices have been proven to be effective platforms to discriminate microorganisms by their viability status. Two decades ago, live and dead Escherichia coli (E. coli) cells were trapped at distinct locations in an insulator-based EK (iEK) device with cylindrical insulating posts. At that time, the discrimination between live and dead cells was attributed to dielectrophoretic effects. This study presents the continuous separation between the live and dead E. coli cells, which was achieved primarily by combining linear and nonlinear electrophoretic effects in an iEK device. First, live and dead E. coli cells were characterized in terms of their electrophoretic migration, and then the properties of both live and dead E. coli cells were input into a mathematical model built using COMSOL Multiphysics software to identify appropriate voltages for performing an iEK separation in a T-cross iEK channel. Subsequently, live and dead cells were successfully separated experimentally in the form of an electropherogram, achieving a separation resolution of 1.87. This study demonstrated that linear and nonlinear electrophoresis phenomena are responsible for the discrimination between live and dead cells under DC electric fields in iEK devices. Continuous electrophoretic assessments, such as the one presented here, can be used to discriminate between distinct types of microorganisms including live and dead cell assessments.

High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Electrophoretic Microplate Protein Identification Based on Gold Staining of Molybdenum Disulfide Hydrogels.

Numerous high-performance nanotechnologies have been developed, but their practical applications are largely restricted by the nanomaterials' low stabilities and high operation complexity in aqueous substrates. Herein, we develop a simple and high-reliability hydrogel-based nanotechnology based on the in situ formation of Au nanoparticles in molybdenum disulfide (MoS2)-doped agarose (MoS2/AG) hydrogels for electrophoresis-integrated microplate protein recognition. After the incubation of MoS2/AG hydrogels in HAuCl4 solutions, MoS2 nanosheets spontaneously reduce Au ions, and the hydrogels are remarkably stained with the color of as-synthetic plasmonic Au hybrid nanomaterials (Au staining). Proteins can precisely mediate the morphologies and optical properties of Au/MoS2 heterostructures in the hydrogels. Consequently, Au staining-based protein recognition is exhibited, and hydrogels ensure the comparable stabilities and sensitivities of protein analysis. In comparison to the fluorescence imaging and dye staining, enhanced sensitivity and recognition performances of proteins are implemented by Au staining. In Au staining, exfoliated MoS2 semiconductors directly guide the oriented growth of plasmonic Au nanostructures in the presence of formaldehyde, showing environment-friendly features. The Au-stained hydrogels merge the synthesis and recognition applications of plasmonic Au nanomaterials. Significantly, the one-step incubation of the electrophoretic hydrogels leads to high simplicity of operation, largely challenging those multiple-step Ag staining routes which were performed with high complexity and formaldehyde toxicity. Due to its toxic-free, simple, and sensitive merits, the Au staining integrated with electrophoresis-based separation and microplate-based high-throughput measurements exhibits highly promising and improved practicality of those developing nanotechnologies and largely facilitates in-depth understanding of biological information.

Dielectrophoresis: Measurement technologies and auxiliary sensing applications.

Dielectrophoresis (DEP), which arises from the interaction between dielectric particles and an aqueous solution in a nonuniform electric field, contributes to the manipulation of nano and microparticles in many fields, including colloid physics, analytical chemistry, molecular biology, clinical medicine, and pharmaceutics. The measurement of the DEP force could provide a more complete solution for verifying current classical DEP theories. This review reports various imaging, fluidic, optical, and mechanical approaches for measuring the DEP forces at different amplitudes and frequencies. The integration of DEP technology into sensors enables fast response, high sensitivity, precise discrimination, and label-free detection of proteins, bacteria, colloidal particles, and cells. Therefore, this review provides an in-depth overview of DEP-based fabrication and measurements. Depending on the measurement requirements, DEP manipulation can be classified into assistance and integration approaches to improve sensor performance. To this end, an overview is dedicated to developing the concept of trapping-on-sensing, improving its structure and performance, and realizing fully DEP-assisted lab-on-a-chip systems.

Proposal and performance evaluation of a new parallel plate continuous cell separation device using dielectrophoresis.

Along with the rapid development of cellular biological research in recent years, there has been an urgent need for a high-speed, high-precision method of separating target cells from a highly heterogeneous cell population. Among the various cell separation technologies proposed so far, dielectrophoresis (DEP)-based approaches have shown particular promise because they are noninvasive to cells. We have developed a new DEP-based device to separate large numbers of live and dead cells of the human mammary cell line MCF10A. In this study, we validated the separation performance of this device. The results showed the successful separation of a higher percentage of cells than in previous studies, with a separation efficiency higher than 90%. In the past, there have been no confirmed cases in which a separation rate of over 90% and high-speed processing of a large number of cells were simultaneously achieved. It was shown that the proposed device can process large numbers of cells at high speed and with high accuracy.

Commentary regarding "An entropy model for Carreau nanofluid ciliary flow with electroosmosis and thermal radiations: A numerical study".

A serious error exists in the paper: Alharbi KAM, Riaz A, Sikandar S. An entropy model for Carreau nanofluid ciliary flow with electroosmosis and thermal radiations: a numerical study. Electrophoresis. 2024;45:1112-1129.

Dielectric properties of human macrophages are altered by Mycobacterium tuberculosis infection.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.

On the behavior of sub-micrometer polystyrene particles subjected to AC insulator-based dielectrophoresis.

Polymer beads, especially polystyrene particles, have been extensively used as model species in insulator-based dielectrophoresis (iDEP) studies. Their use in alternating current iDEP (AC-iDEP) is less explored; however, an assessment in the low-frequency regime (≤10 kHz) allows to link surface conduction effects with the surface properties of polymer particles. Here, we provide a case study for various experimental conditions assessing sub-micrometer polystyrene particles with AC-iDEP and link to accepted surface conduction theory to predict and experimentally verify the observed AC-iDEP trapping behavior based on apparent zeta potential and solution conductivity. We find excellent agreement with the theoretical predictions, but also the occurrence of concentration polarization electroosmotic flow under the studied conditions, which have the potential to confound acting dielectrophoresis conditions. Furthermore, we study a case relevant to the assessment of microplastics in human and animal body fluids by mimicking the protein adsorption of high abundant proteins in blood by coating polystyrene beads with bovine serum albumin, a highly abundant protein in blood. Theoretical predictions and experimental observations confirm a difference in observed AC-iDEP behavior between coated and non-coated particles, which might be exploited for future studies of microplastics in blood to assess their exposure to humans and animals.

Numerical and experimental investigation of the deviation of microparticles inside the microchannel using the vortices caused by the ICEK phenomenon.

One field of study in microfluidics is the control, trapping, and separation of microparticles suspended in fluid. Some of its applications are related to cell handling, virus detection, and so on. One of the new methods in this field is using ICEK phenomena and dielectrophoresis forces. In the present study, considering the ICEK phenomena, the microparticles inside the fluid are deviated in the desired ratio using a novel ICEK microchip. The deviation is such that after the microparticles reach the floating electrode, they are trapped in the ICEK flow vortex and deviated through a secondary channel that was placed crosswise and noncoplanar above the main channel. For simulation verification, an experimental test is done. The method used for making two noncoplanar channels and separating the particles in the desired ratio with a simple ICEK microchip is an innovation of the present study. Moreover, the adjustment of the percentage of separation of microparticles by adjusting the parameters of the applied voltage and fluid inlet velocity is one of the other innovations of the present experimental study. We observed that for input velocities of 150-1200 µm/s with applied voltages of 10-33 V, 100% of the particles can be directed toward the secondary-channel.

Measurement and analysis of ionic leakage profiles in refrigerated human red blood cells using dielectrophoresis and inductively coupled mass spectroscopy.

Human red blood cells (RBCs) undergo ionic leakage through passive diffusion during refrigerated storage, affecting their quality and health. We investigated the dynamics of ionic leakage in human RBCs over a 20-day refrigerated storage period using extracellular ion quantification and dielectrophoresis (DEP). Four type O- human blood donors were examined to assess the relationship between extracellular ion concentrations (Na+, K+, Mg2+, Ca2+, and Fe2+), RBC cytoplasm conductivity, and membrane conductance. A consistent negative correlation between RBC cytoplasm conductivity and membrane conductance, termed the "ionic leakage profile" (ILP), was observed across the 20-day storage period. Specifically, we noted a gradual decline in DEP-measured RBC cytoplasm conductivity alongside an increase in membrane conductance. Further examination of the electrical origins of this ILP using inductively coupled plasma mass spectrometry revealed a relative decrease in extracellular Na+ concentration and an increase in K+ concentration over the storage period. Correlation of these extracellular ion concentrations with DEP-measured RBC electrical properties demonstrated a direct link between changes in the cytoplasmic and membrane domains and the leakage and transport of K+ and Na+ ions across the cell membrane. Our analysis suggests that the inverse correlation between RBC cytoplasm and membrane conductance is primarily driven by the passive diffusion of K+ from the cytoplasm and the concurrent diffusion of Na+ from the extracellular buffer into the membrane, resulting in a conductive reduction in the cytoplasmic domain and a subsequent increase in the membrane. The ILP's consistent negative trend across all donors suggests that it could serve as a metric for quantifying blood bank storage age, predicting the quality and health of refrigerated RBCs.

Enhancing the efficiency of lung cancer cell capture using microfluidic dielectrophoresis and aptamer-based surface modification.

Metastasis remains a significant cause to cancer-related mortality, underscoring the critical need for early detection and analysis of circulating tumor cells (CTCs). This study presents a novel microfluidic chip designed to efficiently capture A549 lung cancer cells by combining dielectrophoresis (DEP) and aptamer-based binding, thereby enhancing capture efficiency and specificity. The microchip features interdigitated electrodes made of indium-tin-oxide that generate a nonuniform electric field to manipulate CTCs. Following three chip design, scenarios were investigated: (A) bare glass surface, (B) glass modified with gold nanoparticles (AuNPs) only, and (C) glass modified with both AuNPs and aptamers. Experimental results demonstrate that AuNPs significantly enhance capture efficiency under DEP, with scenarios (B) and (C) exhibiting similar performance. Notably, scenario (C) stands out as aptamer-functionalized surfaces resisting fluid shear forces, achieving CTCs retention even after electric field deactivation. Additionally, an innovative reverse pumping method mitigates inlet clogging, enhancing experimental efficiency. This research offers valuable insights into optimizing surface modifications and understanding key factors influencing cell capture, contributing to the development of efficient cell manipulation techniques with potential applications in cancer research and personalized treatment options.

On the low-frequency dispersion observed in dielectrophoresis spectra.

The foundation of dielectrophoresis (DEP) as a tool for biological investigation is the use of the Clausius-Mossotti (C-M) factor to model the observed behaviour of cells experiencing DEP across a frequency range. Nevertheless, it is also the case that at lower frequencies, the DEP spectrum deviates from predictions; there exists a rise in DEP polarisability, which varies in frequency and magnitude with different cell types and medium conductivities. In order to evaluate the origin of this effect, we have studied DEP spectra from five cell types (erythrocytes, platelets, neurons, HeLa cancer cells and monocytes) in several conditions including medium conductivity and cell treatment. Our results suggest the effect manifests as a low-pass dispersion whose cut-off frequency varies with membrane conductance and capacitance as determined using the DEP spectrum; the effect also varies as a logarithm of medium conductivity and Debye length. These together suggest that the values of membrane capacitance and conductance depend not only on the impedance of the membrane itself, but also of the surrounding double layer. The amplitude of the effect in different cell types compared to the C-M factor was found to correlate with the depolarisation factors for the cells' shapes, suggesting that this ratio may be useful as an indicator of cell shape for DEP modelling.

Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237 nm. These aggregates experienced a dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8 mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68 mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

A facile double moving redox boundary model for visual electrophoresis titration of ascorbic acid.

In this work, we proposed a double moving redox boundary (MROB) model to realize the colorless analyte electrophoresis titration (ET) by the two steps of the redox reaction. Single MROB has been proposed for the development of ET sensing (Analyst, 2013, 138, 1137. ACS Sensor, 2019, 4, 126.), and faces great challenges in detecting the analyte without color change during redox reaction. Herein, a novel model of double-MROB electrophoresis, including its mechanisms, equations, and procedures, was developed for titration by using ascorbic acid as a model analyte. The first MROB was created with ferric iron (Fe3+) and iodide ion (I-) in which Fe3+ was reduced as Fe2+ and I- was oxidized as molecular iodine (I2) used as an indicator of visible MROB due to blue starch-iodine complex. The second boundary was then formed between the molecular iodine and model analyte of ascorbic acid. Under given conditions, there was a quantitative relationship between velocity of MROB (VMROB(ii)) and ascorbic acid concentration (CVit C) in the double-MROB system (1/VMROB(ii) = 0.6502CVit C + 4.5165, and R = 0.9939). The relevant relative standard deviation values of intraday and inter-day were less than ∼5.55% and ∼6.64%, respectively. Finally, the titration of ascorbic acid in chewable vitamin C tablets was performed by the developed method, the titration results agreed with those via the classic iodometric titration. All the results briefly demonstrated the validity of the double MROB model, in which Vit C was used as a model analyte. The developed method had potential use in quantitative analysis of redox-active species in biomedical samples.

Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Electrical signature of heterogeneous human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune-evasive, and multipotent differentiation potential. The inherent heterogeneity of hMSCs poses a challenge for therapeutic treatments and necessitates the identification of robust biomarkers to ensure reproducibility in both in vivo and in vitro experiments. In this study, we utilized dielectrophoresis (DEP), a label-free electrokinetic phenomenon, to investigate the heterogeneity of hMSCs derived from bone marrow (BM) and adipose tissue (AD). The electrical properties of BM-hMSCs were compared to homogeneous mouse fibroblasts (NIH-3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK-293). The DEP profile of BM-hMSCs differed most from HEK-293 cells. We compared the DEP profiles of BM-hMSCs and AD-hMSCs and found that they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteoblast cells revealed that they behave differently in response to differentiation-inducing cytokines. Histology and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses of the differentiation-related genes revealed differences in heterogeneity between BM-hMSCs and AD-hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate differences in the heterogeneity of BM-hMSCs and AD-hMSCs. Our results demonstrate that using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.

Prognostic significance of serum monoclonal proteins based on immunofixation electrophoresis in B-cell non-Hodgkin lymphoma.

The presence of serum monoclonal components has been associated with poor outcomes in various hematological malignancies. The current study focused on exploring its prognostic role in B-cell non-Hodgkin lymphoma. Our study represented 314 patients with information on serum immunofixation electrophoresis at diagnosis that were available with B-cell non-Hodgkin lymphoma. IFE was positive in 61 patients (19%). Baseline features were comparable between pairs of groups, poor ECOG PS, B symptoms, advanced stage, and high-risk IPI score were significantly more frequent in the + IFE group. Shorter PFS and OS of B-NHL patients were observed in patients who presented at diagnosis with a + IFE, and IFE was the independent predictor of PFS and OS in multivariate analysis. Moreover, integrating IFE into the IPI-M1, IPI-M2, and IPI-M3 models improved the area under the curve for more accurate survival prediction and prognosis. Serum monoclonal proteins are significant prognostic indicators for newly diagnosed B-cell non-Hodgkin lymphoma that can early identify patients with poor prognosis and guide clinical treatment decisions.

Enzymatic Dissociation of DNA-Histone Condensates in an Electrophoretic Setting: Modulating DNA Patterning and Hydrogel Viscoelasticity.

Development of an energy-driven self-assembly process is a matter of interest for understanding and mimicking diverse ranges of biological and environmental patterns in a synthetic system. In this article, first we demonstrate transient and temporally controlled self-assembly of a DNA-histone condensate where trypsin (already present in the system) hydrolyzes histone, resulting in disassembly. Upon performing this dynamic self-assembly process in a gel matrix under an electric field, we observe diverse kinds of DNA patterning across the gel matrix depending on the amount of trypsin, incubation time of the reaction mixture, and gel porosity. Notably, here, the micrometer-sized DNA-histone condensate does not move through the gel and only free DNA can pass; therefore, transport and accumulation of DNA at different zones depend on the release rate of DNA by trypsin. Furthermore, we show that the viscoelasticity of the native gel increases in the presence of DNA and a pattern over gel viscoelasticity at different zones can be achieved by tuning the amount of enzyme, i.e., the dissociation rate of the DNA-histone condensate. We believe enabling spatiotemporally controlled DNA patterning by applying an electric field will be potentially important in designing different kinds of spatiotemporally distinct dynamic materials.