On-chip microfluidic buffer swap of biological samples in-line with downstream dielectrophoresis.

Microfluidic cell enrichment by dielectrophoresis, based on biophysical and electrophysiology phenotypes, requires that cells be resuspended from their physiological media into a lower conductivity buffer for enhancing force fields and enabling the dielectric contrast needed for separation. To ensure that sensitive cells are not subject to centrifugation for resuspension and spend minimal time outside of their culture media, we present an on-chip microfluidic strategy for swapping cells into media tailored for dielectrophoresis. This strategy transfers cells from physiological media into a 100-fold lower conductivity media by using tangential flows of low media conductivity at 200-fold higher flow rate versus sample flow to promote ion diffusion over the length of a straight channel architecture that maintains laminarity of the flow-focused sample and minimizes cell dispersion across streamlines. Serpentine channels are used downstream from the flow-focusing region to modulate hydrodynamic resistance of the central sample outlet versus flanking outlets that remove excess buffer, so that cell streamlines are collected in the exchanged buffer with minimal dilution in cell numbers and at flow rates that support dielectrophoresis. We envision integration of this on-chip sample preparation platform prior to or post-dielectrophoresis, in-line with on-chip monitoring of the outlet sample for metrics of media conductivity, cell velocity, cell viability, cell position, and collected cell numbers, so that the cell flow rate and streamlines can be tailored for enabling dielectrophoretic separations from heterogeneous samples.

Biophysical quantification of reorganization dynamics of human pancreatic islets during co-culture with adipose-derived stem cells.

Islet transplantation is a potential therapy for type 1 diabetes, but it is expensive due to limited pancreas donor numbers and the variability in islet quality. The latter is often addressed by co-culture of harvested islets with stem cells to promote in vitro remodeling of their basement membrane and enable expression of angiogenic factors for enhancing vascularization. However, given the heterogeneity in islet size, shape and function, there is a need for metrics to assess the reorganization dynamics of single islets over the co-culture period. Based on shape-evolution of individual multi-cell aggregates formed during co-culture of human islets with adipose derived stem cells and the pressures required for their bypass through microfluidic constrictions, we present size-normalized biomechanical metrics for monitoring the reorganization. Aggregates below a threshold size exhibit faster reorganization, as evident from rise in their biomechanical opacity and tightening of their size distribution, but this size threshold increases over culture time to include a greater proportion of the aggregates. Such biomechanical metrics can quantify the subpopulation of reorganized aggregates by distinguishing them versus those with incomplete reorganization, over various timepoints during the co-culture.

High-throughput dynamical analysis of dielectrophoretic frequency dispersion of single cells based on deflected flow streamlines.

Phenotypic quantification of cells based on their plasma membrane capacitance and cytoplasmic conductivity, as determined by their dielectrophoretic frequency dispersion, is often used as a marker for their biological function. However, due to the prevalence of phenotypic heterogeneity in many biological systems of interest, there is a need for methods capable of determining the dielectrophoretic dispersion of single cells at high throughput and without the need for sample dilution. We present a microfluidic device methodology wherein localized constrictions in the microchannel are used to enhance the field delivered by adjoining planar electrodes, so that the dielectrophoresis level and direction on flow-focused cells can be determined on each traversing cell in a high-throughput manner based on their deflected flow streamlines. Using a sample of human red blood cells diluted to 2.25 × 108 cells/mL, the dielectrophoretic translation of single cells traversing at a flow rate of 1.68 µL/min is measured at a throughput of 1.1 × 105 cells/min, to distinguish positive versus negative dielectrophoresis and determine their crossover frequency in media of differing conductivity for validation of the computed membrane capacitance to that from prior methods. We envision application of this dynamic dielectrophoresis (Dy-DEP) method towards high-throughput measurement of the dielectric dispersion of single cells to stratify phenotypic heterogeneity of a particular sample based on their DEP crossover frequency, without the need for significant sample dilution. Grapical abstract.

Self-aligned microfluidic contactless dielectrophoresis device fabricated by single-layer imprinting on cyclic olefin copolymer.

The trapping and deflection of biological cells by dielectrophoresis (DEP) at field non-uniformities in a microfluidic device is often conducted in a contactless dielectrophoresis (cDEP) mode, wherein the electrode channel is in a different layer than the sample channel, so that field penetration through the interceding barrier causes DEP above critical cut-off frequencies. In this manner, through physical separation of the electrode and sample channels, it is possible to spatially modulate electric fields with no electrode-induced damage to biological cells in the sample channel. However, since this device requires interlayer alignment of the electrode to sample channel and needs to maintain a thin interceding barrier (~ 15 µm) over the entire length over which DEP is needed (~ 1 cm), variations in alignment and microstructure fidelity cause wide variations in cDEP trapping level and frequency response across devices. We present a strategy to eliminate interlayer alignment by fabricating self-aligned electrode and sample channels, simultaneously with the interceding barrier layer (14-µm width and 50-µm depth), using a single-layer imprint and bond process on cyclic olefin copolymer. Specifically, by designing support structures, we preserve fidelity of the high aspect ratio insulating posts in the sample channel and the interceding barrier between the sample and electrode channels over the entire device footprint (~ 1 cm). The device operation is validated based on impedance measurements to quantify field penetration through the interceding barrier and by DEP trapping measurements. The presented fabrication strategy can eventually improve cDEP device manufacturing protocols to enable more reproducible DEP performance. Graphical abstract.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Electrokinetic preconcentration and detection of neuropeptides at patterned graphene-modified electrodes in a nanochannel.

Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions.

Electrical tweezer for highly parallelized electrorotation measurements over a wide frequency bandwidth.

Electrorotation (ROT) is a powerful tool for characterizing the dielectric properties of cells and bioparticles. However, its application has been somewhat limited by the need to mitigate disruptions to particle rotation by translation under positive DEP and by frictional interactions with the substrate. While these disruptions may be overcome by implementing particle positioning schemes or field cages, these methods restrict the frequency bandwidth to the negative DEP range and permit only single particle measurements within a limited spatial extent of the device geometry away from field nonuniformities. Herein, we present an electrical tweezer methodology based on a sequence of electrical signals, composed of negative DEP using 180-degree phase-shifted fields for trapping and levitation of the particles, followed by 90-degree phase-shifted fields over a wide frequency bandwidth for highly parallelized electrorotation measurements. Through field simulations of the rotating electrical field under this wave-sequence, we illustrate the enhanced spatial extent for electrorotation measurements, with no limitations to frequency bandwidth. We apply this methodology to characterize subtle modifications in morphology and electrophysiology of Cryptosporidium parvum with varying degrees of heat treatment, in terms of shifts in the electrorotation spectra over the 0.05-40 MHz region. Given the single particle sensitivity and the ability for highly parallelized electrorotation measurements, we envision its application toward characterizing heterogeneous subpopulations of microbial and stem cells.

Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of Cryptosporidium parvum.

Microbial persistence to antibiotics is attributed to subpopulations with phenotypic variations that cause a spread of susceptibility levels, leading to the recurrence of infections and stability of biofilms. Herein, persistent oocyst subpopulations identified by animal infectivity and excystation assays during the disinfection of Cryptosporidium parvum, a water-borne pathogen capable of causing enteric infections at ultra-low doses, are separated and characterized by quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz). To enable the simultaneous and facile dielectrophoretic tracking of individual oocysts, insulator constrictions in a microfluidic channel are utilized to spatially modulate the localized field over the extent needed for defining oocyst trajectories and for obtaining high-resolution displacement versus time measurements under both, positive and negative dielectrophoresis. In this manner, by obviating the need for averaging dielectrophoretic data over a large collection region, the force response is more sensitive to differences in electrophysiology from sub-population fractions. Hence, the electrophysiology of sensitive and persistent oocysts after heat and silver nanoparticle treatments can be quantified by correlating the force response at low frequencies (<100 kHz) to the integrity of the oocyst wall and at high frequencies (0.4-1 MHz) to the sporozoites in the oocyst. This label-free method can characterize heterogeneous microbial samples with subpopulations of phenotypically different alterations, for quantifying the intensity of alteration and fraction with a particular alteration type.

Perfusable cell-laden micropatterned hydrogels for delivery of spatiotemporal vascular-like cues to tissues.

The integration of vasculature at physiological scales within 3D cultures of cell-laden hydrogels for the delivery of spatiotemporal mass transport, chemical and mechanical cues, is a stepping-stone towards building in vitro tissue models that recapitulate in vivo cues. To address this challenge, we present a versatile method to micropattern adjoining hydrogel shells with a perfusable channel or lumen core, for enabling facile integration with fluidic control systems, on one hand, and to cell-laden biomaterial interfaces, on the other hand. This microfluidic imprint lithography methodology utilizes the high tolerance and reversible nature of the bond alignment process to lithographically position multiple layers of imprints within a microfluidic device for sequential filling and patterning of hydrogel lumen structures with single or multiple shells. Through fluidic interfacing of the structures, the ability to deliver physiologically relevant mechanical cues for recapitulating cyclical stretch on the hydrogel shell and shear stress on endothelial cells in the lumen are validated. We envision application of this platform for recapitulation of the bio-functionality and topology of micro-vasculatures, alongside the ability to deliver transport and mechanical cues, as needed for 3D culture to construct in vitro tissue models.

RuO2 Nanorods as an Electrocatalyst for Proton Exchange Membrane Water Electrolysis.

A custom-built PEM electrolyzer cell was assembled using 6" stainless-steel ConFlat flanges which were fitted with a RuO2 nanorod-decorated, mixed metal oxide (MMO) ribbon mesh anode catalyst. The current density-voltage characteristics were measured for the RuO2 nanorod electrocatalyst while under constant water feed operation. The electrocatalytic behavior was investigated by making a series of physical modifications to the anode catalyst material. These experiments showed an improved activity due to the RuO2 nanorod electrocatalyst, resulting in a corresponding decrease in the electrochemical overpotential. These overpotentials were identified by collecting experimental data from various electrolyzer cell configurations, resulting in an improved understanding of the enhanced catalytic behavior. The micro-to-nano surface structure of the anode electrocatalyst layer is a critical factor determining the overall operation of the PEM electrolyzer. The improvement was determined to be due to the lowering of the potential barrier to electron escape in an electric field generated in the vicinity of a nanorod.

Self-aligned sequential lateral field non-uniformities over channel depth for high throughput dielectrophoretic cell deflection.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-µL min-1 flow rates and <105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 µm) that are created by metal patterned over the entire depth (50 µm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (>µL min-1 flow and >106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

On-Chip Impedance for Quantifying Parasitic Voltages During AC Electrokinetic Trapping.

OBJECTIVE: Assessing the effectiveness of microfluidic device structures for enabling electrokinetic or acoustic trapping requires imaging of model particles within each device under the requisite force fields. To avoid the need for extensive microscopy, the use of valuable biological samples, and reliance on a trained operator to assess efficacy of trapping, we explore electrical means to identify device geometry variations that are responsible for the poor trapping. RESULTS: Using the example of AC electrokinetic trapping over an insulated channel in a contact-less dielectrophoresis mode, we present an on-chip method to acquire impedance spectra on the microfluidic device for quantifying the parasitic voltage drops. CONCLUSION: Based on the parasitic voltage drops, device geometries can be designed to maximize fraction of the applied voltage that is available for dielectrophoretic manipulation and the measured on-chip impedance can rapidly inform downstream decisions on particle manipulation.

Crystallization of high aspect ratio HKUST-1 thin films in nanoconfined channels for selective small molecule uptake.

We present the ability to create unique morphologies of a prototypical metal organic framework (MOF), HKUST-1, by carrying out its crystallization within a set of nano-confined fluidic channels. These channels are fabricated on cyclic olefin copolymer by the high-fidelity hot embossing imprinting method. The picoliter volume synthesis in the nanochannels is hypothesized to bias the balance between nucleation and growth rates to obtain high aspect ratio large-crystalline domains of HKUST-1, which are grown in defined morphologies due to the patterned nanochannels. Confined crystal growth is achieved in nanofluidic channels as shallow as 50 nm. HKUST-1 crystalline domains with aspect ratios greater than 2500, and lengths up to 144 µm are obtained using the nanochannels, exceeding values obtained using chemical modulation and other confinement methods. HKUST-1 crystals are characterized using optical microscopy and scanning electron microscopy with energy dispersive spectroscopy. Porosity of the MOF and selective molecular uptake is demonstrated through inclusion of anthracene and methylene blue within the HKUST-1 framework, and with exclusion of rhodamine B and riboflavin, characterized using a confocal fluorescence microscope. We attribute this selectivity to the analyte size and electrostatic characteristics. Nanoconfined crystallization of MOFs can thus yield control over crystalline morphology to create ideal MOF crystals for enabling selective molecular enrinchment and sensing.

Deformability-based microfluidic separation of pancreatic islets from exocrine acinar tissue for transplant applications.

The long-term management of type-1 diabetes (T1D) is currently achieved through lifelong exogenous insulin injections. Although there is no cure for T1D, transplantation of pancreatic islets of Langerhans has the potential to restore normal endocrine function versus the morbidity of hypoglycemic unawareness that is commonly associated with sudden death among fragile diabetics. However, since endocrine islet tissues form a small proportion of the pancreas, sufficient islet numbers can be reached only by combining islets from multiple organ donors and the transplant plug contains significantly high levels of exocrine acinar tissue, thereby exacerbating immune responses. Hence, lifelong administration of immunosuppressants is required after transplantation, which can stress islet cells. The density gradient method that is currently used to separate islets from acinar tissue causes islets to be sparsely distributed over the centrifuged bins, so that the transplant sample obtained by combining multiple bins also contains significant acinar tissue levels. We show that in comparison to the significant size and density overlaps between the islet and acinar tissue populations post-organ digestion, their deformability overlaps are minimal. This feature is utilized to design a microfluidic separation strategy, wherein tangential flows enable selective deformation of acinar populations towards the bifurcating waste stream and sequential switching of hydrodynamic resistance enables the collection of rigid islets. Using 25 bifurcating daughter channels, a throughput of ∼300 islets per hour per device is obtained for enabling islet enrichment from relatively dilute starting levels to purity levels that meet the transplant criteria, as well as to further enhance islet purity from samples following density gradient enrichment. Based on confirmation of viability and functionality of the microfluidic-isolated islets using insulin secretion analysis and an angiogenesis assay, we envision utilizing this strategy to generate small-volume transplant plugs with high islet purity and significantly reduced acinar levels for minimizing immune responses after transplantation.

Nanoslit design for ion conductivity gradient enhanced dielectrophoresis for ultrafast biomarker enrichment in physiological media.

Selective and rapid enrichment of biomolecules is of great interest for biomarker discovery, protein crystallization, and in biosensing for speeding assay kinetics and reducing signal interferences. The current state of the art is based on DC electrokinetics, wherein localized ion depletion at the microchannel to nanochannel interface is used to enhance electric fields, and the resulting biomarker electromigration is balanced against electro-osmosis in the microchannel to cause high degrees of biomarker enrichment. However, biomarker enrichment is not selective, and the levels fall off within physiological media of high conductivity, due to a reduction in ion concentration polarization and electro-osmosis effects. Herein, we present a methodology for coupling AC electrokinetics with ion concentration polarization effects in nanoslits under DC fields, for enabling ultrafast biomarker enrichment in physiological media. Using AC fields at the critical frequency necessary for negative dielectrophoresis of the biomarker of interest, along with a critical offset DC field to create proximal ion accumulation and depletion regions along the perm-selective region inside a nanoslit, we enhance the localized field and field gradient to enable biomarker enrichment over a wide spatial extent along the nanoslit length. While enrichment under DC electrokinetics relies solely on ion depletion to enhance fields, this AC electrokinetic mechanism utilizes ion depletion as well as ion accumulation regions to enhance the field and its gradient. Hence, biomarker enrichment continues to be substantial in spite of the steady drop in nanostructure perm-selectivity within physiological media.

Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels.

Correction: Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Correction for 'Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene' by Bankim J. Sanghavi et al., Lab Chip, 2015, DOI: 10.1039/c5lc00840a.

Quantifying spatio-temporal dynamics of biomarker pre-concentration and depletion in microfluidic systems by intensity threshold analysis.

Microfluidic systems are commonly applied towards pre-concentration of biomarkers for enhancing detection sensitivity. Quantitative information on the spatial and temporal dynamics of pre-concentration, such as its position, extent, and time evolution are essential towards sensor design for coupling pre-concentration to detection. Current quantification methodologies are based on the time evolution of fluorescence signals from biomarkers within a statically defined region of interest, which does not offer information on the spatial dynamics of pre-concentration and leads to significant errors when the pre-concentration zone is delocalized or exhibits wide variations in size, shape, and position over time under the force field. We present a dynamic methodology for quantifying the region of interest by using a statistical description of particle distribution across the device geometry to determine the intensity thresholds for particle pre-concentration. This method is applied to study the delocalized pre-concentration dynamics under an electrokinetic force balance driven by negative dielectrophoresis, for aligning the pre-concentration and detection regions of neuropeptide Y, and for quantifying the polarizability dispersion of silica nano-colloids with frequency of the force field. We envision the application of this automated methodology on data from 2D images and 3D Z-stacks for quantifying pre-concentration dynamics over delocalized regions as a function of the force field.

Control of ruthenium oxide nanorod length in reactive sputtering.

Ruthenium oxide nanorods have been grown on Si wafer substrates under a variety of pre-existing surface conditions by reactive radio frequency sputtering in an electron cyclotron resonant plasma process. Nanorod formation by this method is fast relative to that observed in other processes reported in the literature, with nucleation being the rate determining step. Growth in the axial direction is limited by the availability of ruthenium precursors which competes with their consumption in the lateral growth of the nanorods. The availability of Ru precursors at the top of the nanorods can be controlled by surface diffusion and therefore substrate temperature. The ultimate length of the nanorods is determined by the mole fraction of oxygen used in the reactor ambient through the production of mobile Ru hyperoxide precursors. The results of this investigation show the way to develop a process for producing a high density field of nanorods with a specified length.