Electroneutral Layer Dynamics Drive Ion Migration in Low Frequency AC Electrophoresis Below the Water Electrolysis Threshold.

Low-frequency AC electrophoresis lies in a regime between DC microchannel electrophoresis and dielectrophoresis, which typically utilizes frequencies above 1000 Hz. Although few electrophoretic methods have been reported in this ≤100 Hz range, traveling wave electrophoresis (TWE) and transverse AC electrophoresis (TrACE) operate in this frequency range, and use low voltages to avoid bubble formation from water electrolysis. TWE provides molecular separations with enhanced control and TrACE provides multiplexed, multiparameter particle characterization. However, two related fundamental questions remain about the mechanisms of electrophoretic migration in these systems. First, particle electrophoresis in TrACE is largely captured by a simple model that combines the alternating electric field with DC electrokinetics, but a deviation from the model is observed for applied square electric field waves that increases with decreasing frequency. Second, although electrode charging is believed to drive ion migration in TWE, the estimated electrode charging time is about 2-3 orders of magnitude faster than the wave period. In this study, a 1D finite numerical model that excludes Faradaic reactions simulates ion and particle migration across the microchannel width in TrACE. The 1D model results show good agreement with both particle and ion migration in TrACE systems. Furthermore, although ion migration between the pair of electrodes slows during each excursion of a 1 Hz square wave, there is substantial ion migration throughout the 0.5 s half-period. This modeling result agrees with experimental observations in TWE. Therefore, the clarification of the mechanisms of ion migration in these low-frequency and low-voltage AC electrophoresis is expected to expand their applications.

Measuring the electrophoretic mobility and size of single particles using microfluidic transverse AC electrophoresis (TrACE).

The ability to measure the charge and size of single particles is essential to understanding particle adhesion and interaction with their environment. Characterizing the physical properties of biological particles, like cells, can be a powerful tool in studying the association between the changes in physical properties and disease development. Currently, measuring charge via the electrophoretic mobility (µep) of individual particles remains challenging, and there is only one prior report of simultaneously measuring µep and size. We introduce microfluidic transverse AC electrophoresis (TrACE), a novel technique that combines particle tracking velocimetry (PTV) and AC electrophoresis. In TrACE, electric waves with 0.75 to 1.5 V amplitude are applied transversely to the bulk flow and cause the particles to oscillate. PTV records the particles' oscillating trajectories as pressure drives bulk flow through the microchannel. A simple quasi-equilibrium model agrees well with experimental measurements of frequency, amplitude, and phase, indicating that particle motion is largely described by DC electrophoresis. The measured µep of polystyrene particles (0.53, 0.84, 1, and 2 µm diameter) are consistent with ELS measurements, and precision is enhanced by averaging ∼100 measurements per particle. Particle size is simultaneously measured from Brownian motion quantified from the trajectory for particles <2 µm or image analysis for particles ≥2 µm. Lastly, the ability to analyze intact mammalian cells is demonstrated with B cells. TrACE systems are expected to be highly suitable as fieldable tools to measure the µep and size of a broad range of individual particles.

Correction: Measuring the electrophoretic mobility and size of single particles using microfluidic transverse AC electrophoresis (TrACE).

Correction for 'Measuring the electrophoretic mobility and size of single particles using microfluidic transverse AC electrophoresis (TrACE)' by M. Hannah Choi et al., Lab Chip, 2023, https://doi.org/10.1039/D3LC00413A.

Analyte Enrichment via Ion Concentration Polarization with Hydrogel Plugs Polymerized in PDMS Microchannels by a Facile and Comprehensive Method for Improved Polymerization.

Hydrogels are incorporated into microfluidic devices to provide enhanced functionality by enabling processes such as enzyme immobilization, sample enrichment, and ionic current rectification. However, in the microfluidic devices with the commonly used material poly(dimethylsiloxane) (PDMS), hydrogels are very difficult to polymerize in situ in an ambient atmosphere because of the high oxygen concentration in PDMS. Even with very high (1.8%) photoinitiator concentrations, the polymerized hydrogel does not completely fill the microchannel. Here, we report a facile and broadly applicable protocol that utilizes microchannel pretreatment with 20% benzophenone in acetone to provide a hydrogel plug that completely fills the microchannel cross section by consuming the oxygen in the PDMS substrate near the microchannel wall. Both negatively charged and neutral hydrogels are polymerized from monomer solutions that utilize the photoinitiator/solvent combinations of VA-086 in water and benzophenone or IRG in DMSO. The photoinitiators were tested at different concentrations and in devices with different levels of oxidation. The hydrogel morphology is characterized using phase contrast microscopy and is related to the hydrogel's performance for concentration enrichment and ionic current rectification. A novel method is employed to confine the precursor solution in desired locations so that a photomask is not required for the spatial control of the plug location. Among six hydrogel formulations, at 100 V, the best current rectification factor obtained is ∼600 and the best analyte enrichment achieved is ∼120-fold in 5 min. This method provides a rapid and simple approach to increase the capabilities of PDMS microfluidic devices through improved polymerization of nanoporous hydrogels.

Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture.

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, », and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose-response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

Exceeding ohmic scaling by more than one order of magnitude with a 3D ion concentration polarization system.

We report an ion concentration polarization (CP) system that exceeds ohmic scaling, a barrier that has stood for more than four decades, by more than one order of magnitude. CP is used in many important applications, including the enrichment of trace analytes in microfluidic systems and water purification by electrodialysis. The mechanisms that control the current through these systems have been largely discovered, but the reduced currents and loss of efficiency imparted by the high resistance of the CP ion depleted zone have not been overcome. To obtain high currents, an ion permselective element with a microscale cross-section is interfaced with a macroscale reservoir. Confocal fluorescence microscopy and microparticle tracking velocimetry (µ-PTV) are used to characterize the depleted zone that emanates vertically from the CP inducing nanoporous gel into the macroscale reservoir. The shape and growth of the depleted zone and velocity in the surrounding bulk solution are consistent with natural convection being the driver of the depleted zone morphology and eliminating the high resistance created by the depleted zone in 1D and 2D systems. Once the resistance of the depleted zone is negated, the high currents are hypothesized to result from enhancement of counter-ion concentration in the nanoporous gel-filled microchannel. In contrast with conventional systems, the current increases monotonically and remains stable at a high quasi-steady level in the reported systems. These results may be used to increase the efficiency and performance of future devices that utilize CP, while the ability to collect purified water with this geometry is demonstrated.

Asymmetric-Fluidic-Reservoirs Induced High Rectification Nanofluidic Diode.

We demonstrate a novel nanofluidic diode that produces rectification factors in excess of 1000. The nanofluidic diode consists of ion permselective nanopores that connect two reservoirs of different diameters- a micropore reservoir and a macropore reservoir. On the application of +100 V to the micropore, a low OFF state current is observed. The OFF state is caused by formation of the ion depleted zone in the micropore because the anions are prevented from entering the nanopores from the micropore and the cations are depleted in this region to maintain charge neutrality. On the application of -100 V, we observe a high ON state current. The ON state is caused by formation of the ion enriched zone in the microchannel because the anions cannot pass through the nanopores and accumulate in the microchannel. To maintain charge neutrality the cations also become enriched in the microchannel. The ratio of ON state current to the OFF state current gives the rectification of current. Here, plasma oxidation is used to achieve a nanopore with a large wall surface charge density of σn = -55 mC/m2 which yields a rectification of current on the order of 3500 that is nearly two orders of magnitude higher than those reported thus far. In contrast to the other nanofluidic diodes, this nanofluidic diode does not introduce asymmetry to the nanopore, but asymmetry is produced by having the nanopores join a micropore and a macropore. Introduction of asymmetry into the fluidic reservoirs which the nanopores connect is quite simple. Hence, the nanofluidic diode is easy to scale up to industrial level.

Diffusion layer formation drives zone migration in travelling wave electrophoresis.

COMSOL finite element modeling software is used to simulate 2D traveling-wave electrophoresis for microfluidic separations and sample concentration. A four-phase AC potential is applied to a periodic interdigitated four-electrode array to produce a longitudinal electric wave that travels through the channel. Charged particles are carried along with the electric wave or left behind, depending on their mobilities. A simplified model of asymmetric electrode reactions resolves the issue of electric double layer shielding at the electrodes. Selective reactions allow for the formation of diffusion layers of charged particles which follow the traveling electric wave. These diffusion layers determine the transport of charged species through the system. Our model reproduces experimental separations of charged species based on mobility. With easy control over the frequency and direction, one may employ this method for concentrating and/or separating charged particles.

Microfluidic immunosensor based on mesoporous silica platform and CMK-3/poly-acrylamide-co-methacrylate of dihydrolipoic acid modified gold electrode for cancer biomarker detection.

We report a hybrid glass-poly (dimethylsiloxane) microfluidic immunosensor for epidermal growth factor receptor (EGFR) determination, based on the covalent immobilization of anti-EGFR antibody (anti-EGFR) on amino-functionalized mesoporous silica (AMS) retained in the central channel of a microfluidic device. The synthetized AMS was characterized by N2 adsorption-desorption isotherm, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and infrared spectroscopy. The cancer biomarker was quantified in human serum samples by a direct sandwich immunoassay measuring through a horseradish peroxidase-conjugated anti-EGFR. The enzymatic product was detected at -100 mV by amperometry on a sputtering gold electrode, modified with an ordered mesoporous carbon (CMK-3) in a matrix of poly-acrylamide-co-methacrylate of dihydrolipoic acid (poly(AC-co-MDHLA)) through in situ copolymerization. CMK-3/poly(AC-co-MDHLA)/gold was characterized by cyclic voltammetry, EDS and SEM. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0.01 ng mL-1 to 50 ng mL-1. The detection limit was 3.03 pg mL-1, and the within- and between-assay coefficients of variation were below 5.20%. The microfluidic immunosensor is a very promising device for the diagnosis of several kinds of epithelial origin carcinomas.

Controlling the ionic current rectification factor of a nanofluidic/microfluidic interface with symmetric nanocapillary interconnects.

The current rectification factor can be tailored by changing the degree of asymmetry between the fluid baths on opposite sides of a nanocapillary membrane (NCM). A symmetric device with symmetric fluid baths connected to opposite sides of the NCM did not rectify ionic current; while a NCM connected between fluid baths with a 32-fold difference in cross-sectional area produced a rectification factor of 75. The data suggests that the primary mechanism for the current rectification is the change in cross-sectional area of the fluid baths and the polarity dependent propagation of the enriched and depleted concentration polarization (CP) zones into these regions. An additional contribution to the increasing rectification factor with increasing bath asymmetry appears to be a result of electroconvection in the macropore, with inside diameters (IDs) of 625 and 850-µm. Power spectral density (PSD) analysis reveals chaotic oscillations that are consistent with electroconvection in the I-t data of the 625 and 850-µm ID macropore devices. In the ON state, current rectification keeps ionic transport toward the NCM high, increasing the speed of processes like sample enrichment. A simple means is provided to fabricate fluidic diodes with tailored current rectification factors.

Fabrication and performance of a microfluidic traveling-wave electrophoresis system.

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ∼25 µm, provides high electric fields of ∼200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 µm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.

A study of electrospray ionization emitters with differing geometries with respect to flow rate and electrospray voltage.

The performance of several electrospray ionization emitters with different orifice inside diameters (i.d.s), geometries, and materials are compared. The sample solution is delivered by pressure driven flow, and the electrospray ionization voltage and flow rate are varied systematically for each emitter investigated, while the signal intensity of a standard is measured. The emitters investigated include a series of emitters with a tapered outside diameters (o.d.) and unaltered i.d.s, a series of emitters with tapered o.d.s and i.d.s, an emitter with a monolithic frit and a tapered o.d., and an emitter fabricated from polypropylene. The results show that for the externally etched emitters, signal was nearly independent of i.d. and better ion utilization was achieved at lower flow rates. Furthermore, emitters with a 50 µm i.d. and an etched o.d. produced about 1.5 times more signal than etched emitters with smaller i.d.s and about 3.5 times more signal than emitters with tapered inner and outer dimensions. Additionally, the work presented here has important implications for applications in which maximizing signal intensity and reducing frictional resistance to flow are necessary. Overall, the work provides an initial assessment of the critical parameters that contribute to maximizing the signal for electrospray ionization sources interfaced with pressure driven flows.

Analyte transport past a nanofluidic intermediate electrode junction in a microfluidic device.

A glass microfluidic device is presented in which a microchannel is split into two regions with different electric fields by a nanochannel intermediate electrode junction formed by dielectric breakdown. The objective is to sink current through the nanochannel junction without sample loss or broadening of the band as it passes the junction. This type of performance is desired in many microfluidic applications, including the coupling of microchannel/CE with ESI-MS, electrochemical detection, and electric field gradient focusing. The voltage offsets in this study are suitable for microchannel/CE-ESI-MS. Imaging of the transport of model anions and cations through the junction indicates that the junction exhibits nanofluidic behavior and the mean depth of the nanochannel is estimated to be approximately 105 nm. The ion permselectivity of the nanochannel induces concentration polarization and enriched and depleted concentration polarization zones form on opposite sides of the nanochannel, altering the current and electric field distributions along the main microchannel. Anion transport efficiency past the junction was high, 96.0%, and varied little over the pH range of 4.0-8.0. In contrast, cation transport is much lower, and decreases from 72 to 11% from pH 4.0 to 8.0. Band broadening increases with increasing pH less than 70% over the pH range of 4.0-8.0. It is anticipated that this characterization will aid in the understanding and optimization of such junctions made from permselective membranes and porous glass.

Simultaneous separation and detection of cations and anions on a microfluidic device with suppressed electroosmotic flow and a single injection point.

A rapid and simultaneous separation of cationic and anionic peptides and proteins in a glass microfluidic device that has been covalently modified with a neutral poly(ethylene glycol) (PEG) coating to minimize protein adsorption is presented. The features of the device allow samples that contain both anions and cations to be introduced from a central flow stream and separated in different channels with different outlets-all in the presence of low electroosmotic flow (EOF) imparted by the PEG coating. The analytes are electrophoretically extracted from a central hydrodynamic stream and electrophoretically separated in two different channels, in which pressure driven flow has been suppressed through the use of hydrodynamic restrictors. Having different outlets for the electrophoretic separation channels that are spatially separated from the injection enables coupling with further downstream functionalities or off-chip detection, such as mass spectrometry. A plug of charged analyte is hydrodynamically pumped to the sampling intersection and anions from the plug migrate electrophoretically toward the anode in one channel while cations migrate toward the cathode in the other channel due to suppressed EOF from the PEG coating. The separations presented here required less than a minute to complete and produced average separation efficiencies of up to about 3,500 plates from a separation length of 2 cm. The extraction efficiency of both cations and anions from the hydrodynamic stream is determined experimentally and compared with a previously reported model that was used to determine anion extraction efficiency. The extraction efficiency is determined to be 87% and 98% for the two sample mixtures analyzed, and the values predicted by the model are within 3.5% of the experimental data. It is anticipated that this basic approach for simultaneous separation of anions and cations with reduced EOF will be integrated into larger microfluidic systems because the design provides separate outlets that can feed downstream processes or linked to off-chip detection.

A theoretical and experimental study of the electrophoretic extraction of ions from a pressure driven flow in a microfluidic device.

The electrophoretic extraction of ions from a hydrodynamic flow stream is investigated at an intersection between two microfluidic channels. A pressure gradient is used to drive samples through the main channel, while ions are electrophoretically extracted into the side channels. Hydrodynamic restrictors and a neutral coating are used to suppress bulk flow through the side channels. A theoretical model that assumes Poiseuille flow in the main channel and neglects molecular diffusion is used to calculate the extraction efficiency, eta, as a function of the ratio, R, of the average hydrodynamic velocity to the electrophoretic velocity. The model predicts complete extraction of ions (eta=1) for R<2/3 and a monotonic decrease in eta as R becomes greater than 2/3, which agrees well with the experimental results. Additionally, the model predicts that the aspect ratio of the microfluidic channel has little effect on the extraction efficiency. It is anticipated that this device can be used for on-line process monitoring, sample injection, and 2D separations for proteomics and other fields.

Traveling-wave electrophoresis for microfluidic separations.

Models and microfluidic experiments are presented of an electrophoretic separation technique in which charged particles whose mobilities exceed a tunable threshold are trapped between the crests of a longitudinal electric wave traveling through a stationary viscous fluid. The wave is created by applying periodic potentials to electrode arrays above and below a microchannel. Predicted average velocities agree with experiments and feature chaotic attractors for intermediate mobilities.

Investigation of zone migration in a current rectifying nanofluidic/microfluidic analyte concentrator.

A simple microfluidic device that uses a nanocapillary membrane (NCM) to connect a microfluidic channel and solution reservoir is capable of rectifying ionic current and enrichment of ionic species. Application of a potential induces concentration polarization (CP), which creates ion-depleted and ion-enriched zones on opposite sides of the permselective NCM. A force balanced (FB) enriched zone forms at the interface of the bulk buffer solution and depleted CP zone in the off state or the low-current case. After polarity reversal, the migration of a FB enriched zone of anionic tracer is imaged. By decreasing the solution volume at the microchannel and NCM interface, the response time of the current rectifier is decreased and elution of the zone of anionic tracer is achieved. The decrease in response time is most dramatic for the on to off state transition. For this transition, the response time decreases from approximately 50 to approximately 1 s. The decrease in response time for the off to on state is not as dramatic and is characterized by the time from polarity reversal to current peak, which decreased from 84 to 21 s. The features in the I-t plots can be accounted for with schematics of the zone migration that show the migration of depleted CP and enriched CP zones. Together, the fluorescent images and the I-t plots provide the foundation for schematics that describe the zone elution following polarity reversal. These results provide an improved understanding of the zone migration and current rectification in nanofluidic-microfluidic interfaces with symmetric nanochannels.

Ionic current rectification at a nanofluidic/microfluidic interface with an asymmetric microfluidic system.

A nanofluidic-microfluidic interface is reported that rectifies ionic current using uncoated symmetric nanocapillaries. Previously, ionic current rectification has been achieved by other groups with nanochannels with differential coatings and in nanopores that are conical in shape. This simple device uses nanocapillary membranes (NCMs) with uncoated symmetric channels to connect a microfluidic channel and a larger solution reservoir. The conductivity of the solution in the microchannel appears to be critical in the formation of the low "off" state current and the high "on" state current. It is hypothesized that the "off" state current is low due to the formation of an ion depletion zone in the microchannel while the higher "on" state currents are produced by a zone of enhanced ionic concentration in the microchannel.

Adsorption and desorption of stearic acid self-assembled monolayers on aluminum oxide.

Development of coatings to minimize unwanted surface adsorption is extremely important for their use in applications, such as sensors and medical implants. Self-assembled monolayers (SAMs) are an excellent choice for coatings that minimize nonspecific adsorption because they can be uniform and have a very high surface coverage. Another equally important characteristic of such coatings is their stability. In the present study, both the bonding mechanism and the stability of stearic acid SAMs on two aluminum oxides (single-crystal C-plane aluminum oxide (sapphire) and amorphous aluminum oxide (alumina)) are investigated. The adsorption mechanism is investigated by ex situ X-ray photoelectron spectroscopy and infrared (IR) spectroscopy. The results revealed that stearic acid binds to sapphire surfaces via a bidentate interaction of carboxylate with two oxygen atoms while it binds to alumina surfaces via both bidentate and monodentate interactions. Desorption kinetics of stearic acid self-organized on both aluminum oxide surfaces into water is explored by ex situ tapping mode atomic force microscopy, IR spectroscopy, and contact angle measurements. The results exhibit that the SAMs of stearic acid formed on sapphire are not stable in water and are continuously lost through desorption. Water contact angle measurements of SAMs that are immersed in water further indicate that the desorption rate of adsorbates from atomically smooth terrace sites is substantially faster than that of adsorbates from the sites of surface defects due to weaker molecular interaction with the smooth surface. A time-dependent desorption profile of SAMs grown on amorphous alumina reveals that contact angles decrease monotonically without any regional distinction, providing further evidence for the presence of adsorption sites with different types of affinity on the amorphous alumina surface.