A numerical study on microfluidic devices to maintain the concentration and purity of dielectrophoresis-induced separated fractions of analyte.

Determining the physical and chemical properties of biologically important particles such as cells, organelles, viruses, exosomes, complexes, nucleotides, and proteins is needed to understand their function. These properties are determined with common analytical tools (mass spectrometry, cryo-EM, NMR, various spectroscopies, nucleotide sequencing, etc.) whose function can be improved when samples are pure and concentrated. Separations science plays a central role in conditioning samples, ranging from low-resolution benchtop operations like precipitations or extractions to higher-resolution chromatography and electrophoresis. In the last two decades, gradient insulator-based dielectrophoresis (g-iDEP) has emerged as a high-resolution separation technique capable of highly selective enrichment of cells, viruses, exosomes, and proteins. Specific evidence has been shown that pure homogeneous and concentrated fractions of cells and exosomes can be generated from complex mixtures. However, recovering those fractions for analysis has not been developed, limiting the technique to an analytical rather than a preparative one. Here, a finite element analysis was undertaken to identify geometries and operational parameters to efficiently remove the enriched fraction while retaining maximum concentration and providing total mass transfer. Geometric factors (e.g., side channel width and distance from the gradient-inducing gap) were studied, along with the addition of a second inlet side channel. Two flow-generating mechanisms-electroosmosis and hydrostatic pressure-were evaluated for semi-optimized device designs, including a comparison of the one- and two-inlet designs. Simulations indicate effectively one hundred percent mass transfer and a concentration increase by an order of magnitude for several device configurations and operational parameters.

Interfacing microfluidics with information-rich detection systems for cells, bioparticles, and molecules.

The development of elegant and numerous microfluidic manipulations has enabled significant advances in the processing of small volume samples and the detection of minute amounts of biomaterials. Effective isolation of single cells in a defined volume as well as manipulations of complex bioparticle or biomolecule mixtures allows for the utilization of information-rich detection methods including mass spectrometry, electron microscopy imaging, and amplification/sequencing. The art and science of translating biosamples from microfluidic platforms to highly advanced, information-rich detection system is the focus of this review, where we term the translation between the microfluidics elements to the external world "off-chipping." When presented with the challenge of presenting sub-nanoliter volumes of manipulated sample to a detection scheme, several delivery techniques have been developed for effective analysis. These techniques include spraying (electrospray, nano-electrospray, pneumatic), meniscus-defined volumes (droplets, plugs), constrained volumes (narrow channels, containers), and phase changes (deposition, freezing). Each technique has been proven effective in delivering highly defined samples from microfluidic systems to the detection elements. This review organizes and presents selective publications that illustrate the advancements of these delivery techniques with respect to the type of sample analyzed, while introducing each strategy and providing historical perspective. The publications highlighted in this review were chosen due to their significance and relevance in the development of their respective off-chip technique. This review highlights advancements of delivery methods off a microfluidic chip for additional information rich detection schemes.

Orders-of-Magnitude Larger Force Demonstrated for Dielectrophoresis of Proteins Enabling High-Resolution Separations Based on New Mechanisms.

Proteins are perhaps the most important yet frustratingly complicated and difficult class of compounds to analyze, manipulate, and use. One very attractive option to characterize and differentially concentrate proteins is dielectrophoresis, but according to accepted theory, the force on smaller particles the size of proteins is too low to overcome diffusive action. Here, three model proteins, immunoglobulin G, α-chymotrypsinogen A, and lysozyme, are shown to generate forces much larger than predicted by established theory are more consistent with new theoretical constructs, which include the dipole moment and interfacial polarizability. The forces exerted on the proteins are quantitatively measured against well-established electrophoretic and diffusive processes and differ for each. These forces are orders of magnitude larger than previously predicted and enable the selective isolation and concentration of proteins consistent with an extremely high-resolution separation and concentration system based on the higher-order electric properties. The separations occur over a small footprint, happen quickly, and can be made in series or parallel (and in any order) on simple devices.

Biophysical differentiation of susceptibility and chemical differences in Staphylococcus aureus.

Differentiating bacteria strains using biophysical forces has been the focus of recent studies using dielectrophoresis (DEP). The refinement of these studies has created high-resolution separations such that very subtle properties of the cells are enough to induce significant differences in measurable biophysical properties. These high-resolution capabilities build upon the advantages of DEP which include small sample sizes and fast analysis times. Studies focusing on differentiating antimicrobial resistant and susceptible bacteria potentially have significant impact on human health and medical care. A prime example is Staphylococcus aureus, which commonly colonizes adults without ill effects. However, the pathogen is an important cause of infections, including surgical site infections. Treatment of S. aureus infections is generally possible with antimicrobials, but antimicrobial resistance has emerged. Of special importance is resistance to methicillin, an antimicrobial created in response to resistance to penicillin. Here, dielectrophoresis is used to study methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA) strains, both with and without the addition of a fluorescent label. The capture onset potential of fluorescently-labeled MRSA (865 ± 71 V) and thus the ratio of electrokinetic to dielectrophoretic mobility, was found to be higher than that of fluorescently-labeled MSSA (685 ± 61 V). This may be attributable to the PBP2a enzyme present in the MRSA strain and not in the MSSA bacteria. Further, unlabeled MRSA was found to have a capture onset potential of 732 ± 44 V, while unlabeled MSSA was found to have a capture onset potential of 562 ± 59 V. This shows that the fluorescently-labeled bacteria require a higher applied potential, and thus ratio of mobilities, to capture than the unlabeled bacteria.

Dielectrophoresis of proteins: experimental data and evolving theory.

The ability to selectively move and trap proteins is core to their effective use as building blocks and for their characterization. Analytical and preparative strategies for proteins have been pursued and modeled for nearly a hundred years, with great advances and success. Core to all of these studies is the separation, isolation, purification, and concentration of pure homogeneous fractions of a specific protein in solution. Processes to accomplish this useful solution include biphasic equilibrium (chromatographies, extractions), mechanical, bulk property, chemical equilibria, and molecular recognition. Ultimately, the goal of all of these is to physically remove all non-like protein molecules-to the finest detail: all atoms in the full three-dimensional structure being identical down the chemical bond and bulk structure chirality. One strategy which has not been effectively pursued is exploiting the higher order subtle electrical properties of the protein-solvent system. The advent of microfluidic systems has enabled the use of very high electric fields and well-defined gradients such that extremely high resolution separations of protein mixtures are possible. These advances and recognition of these capabilities have caused a re-evaluation of the underlying theoretical models and they were found to be inadequate. New theoretical descriptions are being considered which align more closely to the total forces present and the subtlety of differences between similar proteins. These are focused on the interfacial area between the protein and hydrating solvent molecules, as opposed to the macroscale assumptions of homogeneous solutions and particles. This critical review examines all data which has been published that place proteins in electric field gradients which induce collection of those proteins, demonstrating a force greater than dispersive effects or countering forces. Evolving theoretical constructs are presented and discussed, and a general estimate of future capabilities using the higher order effects and the high fields and precise gradients of microfluidic systems is discussed. Graphical abstract.

Electrochemically Modulated Liquid Chromatography in Fused Silica Capillary Columns.

Electrochemically modulated liquid chromatography (EMLC) uses electrical potentials, applied to a conductive chromatographic stationary phase (e.g., porous graphitic carbon [PGC]), to manipulate analyte retention. This paper reports the design of a capillary EMLC column with a smaller internal diameter (ID; 250 µm) than that of the standard bore predecessor (3.3 mm ID). The new capillary EMLC columns are configured so that the PGC stationary phase serves as the working electrode in a two-electrode electrochemical cell and simplifies electrode placement by obviating the need for a counter electrode. This configuration also eliminates the internal Nafion sleeve that is critical to operation for the standard bore columns, thereby avoiding Nafion deformation as a source of chromatographic band broadening and rupturing as a mode of column failure. Indeed, values for chromatographic efficiency obtained on the capillary columns meet or exceed those measured for the standard columns (20 000-40 000 vs 14 000 plates/m, respectively) with near symmetric elution bands (asymmetry factors of 1.1-1.4 for well-packed capillaries) that surpass band symmetries observed in all prior studies. A test suite of aromatic sulfonates was used to characterize the chromatographic performance of the capillary EMLC columns. Separations of this test mixture showed that retention factors for individual analytes could be manipulated by as much as 21× by changing the applied potential at the PGC stationary phase. Changes in retention behavior at different potential ranges, hypothesized to result from differences in adsorption orientation, were also observed and are consistent with past work. Collectively, the retention behavior unique to EMLC is operative in this new capillary configuration and promises to open new avenues in tuning LC separations.

Simulation and experiment of asymmetric electrode placement for electrophoretic exclusion in a microdevice.

Electrophoretic exclusion (EE) is a counterflow gradient technique that exploits hydrodynamic flow and electrophoretic forces to exclude, enrich, and separate analytes. Resolution for this technique has been theoretically examined and the smallest difference in electrophoretic mobilities that can be completely separated is estimated to be 10-13  cm2 /Vs. Traditional and mesoscale systems have been used, whereas microfluidics offers a greater range of geometries and configurations towards approaching this theoretical limit. To begin to understand the impact of seemingly subtle changes to the entrance flow and the electric field configurations, three closely related microfluidic interfaces were modeled, fabricated, and tested. These interfaces consisted of systematically varying placement of an asymmetric electrode relative to a channel entrance: leading electrode placed outside the channel entrance, leading electrode aligned with the channel, and leading electrode placed within the channel. A charged fluorescent dye is used as a sensitive and accurate probe for the model and to test the concentration variation at these interfaces. Models and experiments focused on visualizing the concentration profile in areas of high temporal dynamics, thus providing a severe test of the models. Experimental data and simulation results showed strong qualitative agreement. The complexity of the electric and flow fields about this interface and the agreement between models and testing suggests the theoretical assessment capabilities can be used to faithfully design novel and more efficient interfaces.

A smart curtailment approach for reducing bat fatalities and curtailment time at wind energy facilities.

The development and expansion of wind energy is considered a key global threat to bat populations. Bat carcasses are being found underneath wind turbines across North and South America, Eurasia, Africa, and the Austro-Pacific. However, relatively little is known about the comparative impacts of techniques designed to modify turbine operations in ways that reduce bat fatalities associated with wind energy facilities. This study tests a novel approach for reducing bat fatalities and curtailment time at a wind energy facility in the United States, then compares these results to operational mitigation techniques used at other study sites in North America and Europe. The study was conducted in Wisconsin during 2015 using a new system of tools for analyzing bat activity and wind speed data to make near real-time curtailment decisions when bats are detected in the area at control turbines (N = 10) vs. treatment turbines (N = 10). The results show that this smart curtailment approach (referred to as Turbine Integrated Mortality Reduction, TIMR) significantly reduced fatality estimates for treatment turbines relative to control turbines for pooled species data, and for each of five species observed at the study site: pooled data (-84.5%); eastern red bat (Lasiurus borealis, -82.5%); hoary bat (Lasiurus cinereus, -81.4%); silver-haired bat (Lasionycteris noctivagans, -90.9%); big brown bat (Eptesicus fuscus, -74.2%); and little brown bat (Myotis lucifugus, -91.4%). The approach reduced power generation and estimated annual revenue at the wind energy facility by ≤ 3.2% for treatment turbines relative to control turbines, and we estimate that the approach would have reduced curtailment time by 48% relative to turbines operated under a standard curtailment rule used in North America. This approach significantly reduced fatalities associated with all species evaluated, each of which has broad distributions in North America and different ecological affinities, several of which represent species most affected by wind development in North America. While we recognize that this approach needs to be validated in other areas experiencing rapid wind energy development, we anticipate that this approach has the potential to significantly reduce bat fatalities in other ecoregions and with other bat species assemblages in North America and beyond.

Effects of surface treatments on trapping with DC insulator-based dielectrophoresis.

A central challenge in measuring the biophysical properties of cells with electrokinetic approaches is the assignment of these biophysical properties to specific biological characteristics. Changes in the electrokinetic behavior of cells may come from mutations, altered gene expression levels, post-translation modifications, or environmental effects. Here we assess the electrokinetic behavior of chemically surface-modified bacterial cells in order to gain insight into the biophysical properties that are specifically affected by changes in surface chemistry. Using E. coli as a scaffold, an amine coupling reaction was used to covalently attach glycine, spermine, bovine serum albumin (protein), or 7-amino-4-methyl-3-coumarinylacetic acid (fluorescent dye) to the free carboxylic acid groups on the surface of the cells. These populations, along with unlabeled control cells, were subject to electrokinetic and dielectrophoretic measurements to quantify any changes in the biophysical properties upon alteration. The properties associated with each electrokinetic force are discussed relative to the specific reactant used. We conclude that relatively modest and superficial changes to cell surfaces can cause measurable changes in their biophysical properties.

Identification of neural stem and progenitor cell subpopulations using DC insulator-based dielectrophoresis.

Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.

A mathematical model of dielectrophoretic data to connect measurements with cell properties.

Dielectrophoresis (DEP) brings about the high-resolution separations of cells and other bioparticles arising from very subtle differences in their properties. However, an unanticipated limitation has arisen: difficulty in assignment of specific biological features which vary between two cell populations. This hampers the ability to interpret the significance of the variations. To realize the opportunities made possible by dielectrophoresis, the data and the diversity of structures found in cells and bioparticles must be linked. While the crossover frequency in DEP has been studied in-depth and exploited in applications using AC fields, less attention has been given when a DC field is present. Here, a new mathematical model of dielectrophoretic data is introduced which connects the physical properties of cells to specific elements of the data from potential- or time-varied DEP experiments. The slope of the data in either analysis is related to the electrokinetic mobility, while the potential at which capture initiates in potential-based analysis is related to both the electrokinetic and dielectrophoretic mobilities. These mobilities can be assigned to cellular properties for which values appear in the literature. Representative examples of high and low values of properties such as conductivity, zeta potential, and surface charge density for bacteria including Streptococcus mutans, Rhodococcus erythropolis, Pasteurella multocida, Escherichia coli, and Staphylococcus aureus are considered. While the many properties of a cell collapse into one or two features of data, for a well-vetted system the model can indicate the extent of dissimilarity. The influence of individual properties on the features of dielectrophoretic data is summarized, allowing for further interpretation of data. Graphical abstract.

Refinement of insulator-based dielectrophoresis.

The ability to separate analytes with increasingly similar properties drives the field of separation science. One way to achieve such separations is using trapping and streaming dielectrophoresis (DEP), which directly exploits the subtle differences in the electrophysical properties of analytes. The non-uniform fields necessary for DEP can be formed using various insulator shapes in microchannels. Current insulator shapes include triangles, diamonds, circles, and rectangles. However, all of these insulators pose problems for trapping, streaming, and sorting (deflection) as the induced fields/gradients are not behaviorally consistent across the lateral dimension. This leads to analytes experiencing different forces depending on their pathline in the microchannel and result in low resolution separations. Based on an iterative process that explored approximately 40 different insulator shapes, a design was chosen that indicated improved particle streamlines, better trapping efficiency, and consistent electrical environments across the lateral dimension. The design was assessed by simulations where the electric field, gradient of the electric field squared, and the ratio of the two were plotted. The improved design includes a unique new multi-length scale element. The multi-length scale structure streamlines the analyte(s) and improves homogeneity in the lateral dimension, while still achieving high gradients necessary for analyte separation using DEP. The design is calculated to keep analytes on the centerline which should improve resolution, and eliminate extraneous trapping zones. Behaviors consistent with the features of the simulations were observed in proof of principle experiments using representative test probes.

Biofluid pretreatment using gradient insulator-based dielectrophoresis: separating cells from biomarkers.

Blood is one of the most important biofluids used for clinical diagnostics. Cells and proteins in the blood can provide a rich source of information for the evaluation of human health. Efficient separation of blood components is a necessary process in order to minimize the interference of unwanted components during sensing, separation, and detection. In this paper, an insulator-based gradient dielectrophoretic device has been applied to separate red blood cells from model protein biomarkers for myocardial infarction in buffer. Within one min, red blood cells are largely depleted regardless of the minimum adherence on the channel wall. Considering the adhered red blood cells will not be transported further, a purified protein solution can be delivered for potential downstream processing or detection. Graphical Abstract ᅟ.

Correction: Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance.

Correction for 'Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance' by Paul V. Jones et al., Analyst, 2015, 140, 5152-5161.

Concentration of Sindbis virus with optimized gradient insulator-based dielectrophoresis.

Biotechnology, separation science, and clinical research are impacted by microfluidic devices. Separation and manipulation of bioparticles such as DNA, protein and viruses are performed on these platforms. Microfluidic systems provide many attractive features, including small sample size, rapid detection, high sensitivity and short processing time. Dielectrophoresis (DEP) and electrophoresis are especially well suited to microscale bioparticle control and have been demonstrated in many formats. In this work, an optimized gradient insulator-based DEP device was utilized for concentration of Sindbis virus, an animal virus with a diameter of 68 nm. Within only a few seconds, the concentration of Sindbis virus can be increased by two to six times in the channel under easily accessible voltages as low as about 70 V. Compared with traditional diagnostic methods used in virology, DEP-based microfluidics can enable faster isolation, detection and concentration of viruses in a single step within a short time.

Development of the resolution theory for gradient insulator-based dielectrophoresis.

New and important separations capabilities are being enabled by utilizing other electric field-induced forces besides electrophoresis, among these is dielectrophoresis. Recent works have used experimentally simple insulator-based systems that induce field gradients creating dielectrophoretic force in useful formats. Among these, juxtaposing forces can generate gradient-based steady-state separations schemes globally similar to isoelectric focusing. The system of interest is termed gradient insulator-based dielectrophoresis and can create extremely high resolution steady-state separations for particles four nanometers to ten micrometers in diameter, including nearly all important bioparticles (large proteins, protein aggregates, polynucleotides viruses, organelles, cells, bacteria, etc.). A theoretical underpinning is developed here to understand the relationship between experimental parameters and resolution and to identify the best expected resolution possible. According to the results, differences in particles (and bioparticles) as small as one part in 10(4) for diameter (subnanometer resolution for a one micrometer particle), one part in 10(8) for dielectrophoretic parameters (dielectrophoretic mobility, Clausius-Mossotti factor), and one part in 10(5) for electrophoretic mobility can be resolved. These figures of merit are generally better than any competing technique, in some cases by orders of magnitude. This performance is enabled by very strong focusing forces associated with localized gradients.

Biophysical separation of Staphylococcus epidermidis strains based on antibiotic resistance.

Electrophoretic and dielectrophoretic approaches to separations can provide unique capabilities. In the past, capillary and microchip-based approaches to electrophoresis have demonstrated extremely high-resolution separations. More recently, dielectrophoretic systems have shown excellent results for the separation of bioparticles. Here we demonstrate resolution of a difficult pair of targets: gentamicin resistant and susceptible strains of Staphylococcus epidermidis. This separation has significant potential implications for healthcare. This establishes a foundation for biophysical separations as a direct diagnostic tool, potentially improving nearly every figure of merit for diagnostics and antibiotic stewardship. The separations are performed on a modified gradient insulator-based dielectrophoresis (g-iDEP) system and demonstrate that the presence of antibiotic resistance enzymes (or secondary effects) produces a sufficient degree of electrophysical difference to allow separation. The differentiating factor is the ratio of electrophoretic to dielectrophoretic mobilities. This factor is 4.6 ± 0.6 × 10(9) V m(-2) for the resistant strain, versus 9.2 ± 0.4 × 10(9) V m(-2) for the susceptible strain. Using g-iDEP separation, this difference produces clear and easily discerned differentiation of the two strains.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.