On-Chip Electrochemical Sensing with an Enhanced Detecting Signal Due to Concentration Polarization-Based Analyte Preconcentration.

Here, we integrated two key technologies within a microfluidic system, an electrokinetic preconcentration of analytes by ion Concentration Polarization (CP) and local electrochemical sensors to detect the analytes, which can synergistically act to significantly enhance the detection signal. This synergistic combination, offering both decoupled and coupled operation modes for continuous monitoring, was validated by the intensified fluorescent intensities of CP-preconcentrated analytes and the associated enhanced electrochemical response using differential pulse voltammetry and chronoamperometry. The system performance was evaluated by varying the location of the active electrochemical sensor, target analyte concentrations, and electrolyte concentration using fluorescein molecules as the model analyte and Homovanillic acid (HVA) as the target bioanalyte within both phosphate-buffered saline (PBS) and artificial sweat solution. The combination of on-chip electrochemical sensing with CP-based preconcentration renders this generic approach adaptable to various analytes. This advanced system shows remarkable promise for enhancing biosensing detection in practical applications while bridging the gap between fundamental research and practical implementation.

The influence of frequency and gravity on the orientation of active metallo-dielectric Janus particles translating under a uniform applied alternating-current electric field.

Theoretical and numerical models of active Janus particles commonly assume that the metallo-dielectric interface is parallel to the driving applied electric field. However, our experimental observations indicate that the equilibrium angle of orientation of electrokinetically driven Janus particles varies as a function of the frequency and voltage of the applied electric field. Here, we quantify the variation of the orientation with respect to the electric field and demonstrate that the equilibrium position represents the interplay between gravitational, electrostatic and electrohydrodynamic torques. The latter two categories are functions of the applied field (frequency, voltage) as well as the height of the particle above the substrate. Maximum departure from the alignment with the electric field occurs at low frequencies characteristic of induced-charge electrophoresis and at low voltages where gravity dominates the electrostatic and electrohydrodynamic torques. The departure of the interface from alignment with the electric field is shown to decrease particle mobility through comparison of freely suspended Janus particles subject only to electrical forcing and magnetized Janus particles in which magnetic torque is used to align the interface with the electric field. Consideration of the role of gravitational torque and particle-wall interactions could account for some discrepancies between theory, numerics and experiment in active matter systems.

Micromotor-based localized electroporation and gene transfection of mammalian cells.

Herein, we studied localized electroporation and gene transfection of mammalian cells using a metallodielectric hybrid micromotor that is magnetically and electrically powered. Much like nanochannel-based, local electroporation of single cells, the presented micromotor was expected to increase reversible electroporation yield, relative to standard electroporation, as only a small portion of the cell's membrane (in contact with the micromotor) is affected. In contrast to methods in which the entire membrane of all cells within the sample are electroporated, the presented micromotor can perform, via magnetic steering, localized, spatially precise electroporation of the target cells that it traps and transports. In order to minimize nonselective electrical lysis of all cells within the chamber, resulting from extended exposure to an electrical field, magnetic propulsion was used to approach the immediate vicinity of the targeted cell, after which short-duration, electric-driven propulsion was activated to enable contact with the cell, followed by electroporation. In addition to local injection of fluorescent dye molecules, we demonstrated that the micromotor can enhance the introduction of plasmids into the suspension cells because of the dielectrophoretic accumulation of the plasmids in between the Janus particle and the attached cell prior to the electroporation step. Here, we chose a different strategy involving the simultaneous operation of many micromotors that are self-propelling, without external steering, and pair with cells in an autonomic manner. The locally electroporated suspension cells that are considered to be very difficult to transfect were shown to express the transfected gene, which is of significant importance for molecular biology research.

Microvalve-Based Tunability of Electrically Driven Ion Transport through a Microfluidic System with an Ion-Exchange Membrane.

Microfluidic channels with an embedded ion permselective medium under the application of electric current are commonly used for electrokinetic processes as on-chip ion concentration polarization (ICP) and bioparticle preconcentration to enhance biosensing. Herein, we demonstrate the ability to dynamically control the electrically driven ion transport by integrating individually addressable microvalves. The microvalves are located along a main microchannel that is uniformly coated with a thin layer of an ion-exchange membrane (IEM). The interplay of ionic transport between the solution within the microchannel and the thin IEM, under an applied electric current, can be locally tuned by the deformation of the microvalve. This tunability provides a robust and simple means of implementing new functionalities into lab-on-a-chip devices, e.g., dynamic control over multiple ICP layers and their associated preconcentrated molecule plugs, multiplex sensing, suppression of biofouling, and plug dispersion, while maintaining the well-known application of microvalves as steric filtration.

Electro-Orientation and Electro-Rotation of Metallodielectric Janus Particles.

The electro-rotation (EROT) and electro-orientation (EOR) behavior of metallodielectric spherical Janus particles (JP) are studied analytically and verified experimentally. This stands in contrast to previous either heuristic or numerically computed models of JP dipoles. First, we obtain frequency-dependent analytic expressions for the corresponding dipole terms for a JP composed of dielectric and metallic hemispheres by applying the "standard" (weak-field) electrokinetic model and using a Fourier-Legendre collocation method for solving two sets of linear equations. EROT and EOR spectra, describing the variation of the JP's angular velocity on the forcing frequency of a rotating and nonrotating spatially uniform electric field, respectively, are explicitly determined and compared against experiments (different JP sizes and solution conductivities). While a favorably good agreement between theory and experimental measurements was found for the frequency response (∼8% difference), there is still a factor of ∼2 difference in the amplitude of the angular velocity, which necessitates further future improvements to the model.

Logic gating of low-abundance molecules using polyelectrolyte-based diodes.

Bioinspired artificial ionic components are extensively utilized to mimic biological systems, as the vast majority of biological signaling is mediated by ions and molecules. Particular attention is given to nanoscale fluidic components where the ion transport can be regulated by the induced ion permselectivity. As a step from fundamentals toward ion-controlled devices, this study presents the use of ionic diodes made of oppositely charged polyelectrolytes, as a gate for low-abundance molecules. The use of ionic diodes that exhibited nonlinear current-voltage responses enabled realization of a basic Boolean operation of an ionic OR logic gate. Aside from the electrical response, the asymmetric ion transport through the diode was shown to affect the transport of low-abundance molecules across the diode, only allowing crossing when the diode was forward-biased. Integration of multiple diodes enabled implementation of an OR logic operation on both the voltage and the molecule transport, while obtaining electrical and optical output readouts that were associated with low and high logic levels. Similarly to electronics, implementation of logic gates opens up new functionalities of on-chip ionic computation via integrated circuits consisting of multiple basic logic gates.

Electrical Propulsion and Cargo Transport of Microbowl Shaped Janus Particles.

Herein the effective electrical propulsion, cargo trapping, and transport capabilities of microbowl-shaped Janus particles (JPs) are demonstrated and evaluated. These active JPs are made by deposition of Au and Ti layers onto sacrificial spherical polystyrene particles, followed by oxidation of the Ti to TiO2 . In contrast to the commonly studied spherical JP, the dual broken symmetry of both geometrical and electrical properties of the microbowl renders a strong dependence of its mobility and cargo loading on the order of the layering of Au and TiO2 . Specifically, an opposite direction of motion is obtained for interchanged layers of Au and TiO2 , using only electrical propulsion as the sole mechanism of motion. The concave side of the microbowl exhibits a negative dielectrophoretic trap of large size wherein trapped cargo is protected from hydrodynamic shearing, leading to an enhanced cargo loading capacity compared to that obtained using common spherical JP. Such enhanced cargo capability of the microbowl along with the ease of engineering it by interchanging the order of the layers are very attractive for future in vitro biological and biomedical applications.

Perfusion in Organ-on-Chip Models and Its Applicability to the Replication of Spermatogenesis In Vitro.

Organ/organoid-on-a-chip (OoC) technologies aim to replicate aspects of the in vivo environment in vitro, at the scale of microns. Mimicking the spatial in vivo structure is important and can provide a deeper understanding of the cell-cell interactions and the mechanisms that lead to normal/abnormal function of a given organ. It is also important for disease models and drug/toxin testing. Incorporating active fluid flow in chip models enables many more possibilities. Active flow can provide physical cues, improve intercellular communication, and allow for the dynamic control of the environment, by enabling the efficient introduction of biological factors, drugs, or toxins. All of this is in addition to the fundamental role of flow in supplying nutrition and removing waste metabolites. This review presents an overview of the different types of fluid flow and how they are incorporated in various OoC models. The review then describes various methods and techniques of incorporating perfusion networks into OoC models, including self-assembly, bioprinting techniques, and utilizing sacrificial gels. The second part of the review focuses on the replication of spermatogenesis in vitro; the complex process whereby spermatogonial stem cells differentiate into mature sperm. A general overview is given of the various approaches that have been used. The few studies that incorporated microfluidics or vasculature are also described. Finally, a future perspective is given on elements from perfusion-based models that are currently used in models of other organs and can be applied to the field of in vitro spermatogenesis. For example, adopting tubular blood vessel models to mimic the morphology of the seminiferous tubules and incorporating vasculature in testis-on-a-chip models. Improving these models would improve our understanding of the process of spermatogenesis. It may also potentially provide novel therapeutic strategies for pre-pubertal cancer patients who need aggressive chemotherapy that can render them sterile, as well asfor a subset of non-obstructive azoospermic patients with maturation arrest, whose testes do not produce sperm but still contain some of the progenitor cells.

Rational Design of Self-Propelling Particles for Unified Cargo Loading and Transportation.

Recent studies on electrically powered active particles that can both self-propel and manipulate cargo load and release, have focused on both spherically shaped Janus particles (JP) and on a parallel electrically conducting plates setup. Yet, spherically shaped JPs set a geometrical limitation on the ability to smartly design multiple dielectrophoretic traps on a single active particle. Herein, these active carriers are extended to accommodate any desired shape and selective metallic coating, using a standard photolithography method. The resulting designed positive and negative dielectrophoretic traps of controlled size, location, and intensity, performed as sophisticated active carriers with a high level of control over their mobility and cargo loading. In addition to cargo loading, the engineered particles exhibit interesting motion in an electrically insulating substrate setup, with in-plane electric field, and, in particular, a tilt angle, and even flipping, that strongly depended on the field frequency and amplitude, hence, exhibiting a much more diverse and rich behavior than spherical JP. The engineered self-propelling carriers are expected to open up new possibilities for unified, label-free and selective cargo loading, transport, and delivery of complex multi-particles.

Tunable Nanochannels Connected in Series for Dynamic Control of Multiple Concentration-Polarization Layers and Preconcentrated Molecule Plugs.

Integration of ionic permselective medium (e.g., nanochannels, membranes) within microfluidic channels has been shown to enable on-chip desalination, sample purification, bioparticle sorting, and biomolecule concentration for enhanced detection sensitivity. However, the ion-permselective mediums are generally of fixed properties and cannot be dynamically tuned. Here we study a microfluidic device consisting of an array of individually addressable elastic membranes connected in series on top of a single microfluidic channel that can be deformed to locally reduce the channel cross-section into a nanochannel. Dynamic tunability of the ion-permselective medium, as well as controllability of its location and ionic permselectivity, introduces a new functionality to microfluidics-based lab-on-a-chip devices, for example, dynamic localization of preconcentrated biomolecule plugs at different sensing regions for multiplex detection. Moreover, the ability to simultaneously form a series of preconcentrated plugs at desired locations increases parallelization of the system and the trapping efficiency of target analytes.

Electrothermal Active Control of Preconcentrated Biomolecule Plugs.

Concentration-polarization (CP)-based biomolecule preconcentration is highly effective in enhancing the detection sensitivity yet fails to precisely and dynamically control the location of the preconcentrated biomolecule plug to ensure overlap with the sensing region (e.g., immobilized molecular probes). Here, we used electrothermal (ET) stirring as a means of controlling the location of a preconcentrated biomolecule plug. The applied microfluidic device consisted of a Nafion membrane to induce the CP and an array of individually addressable microscale heaters for active local ET stirring. The experimental results demonstrated that such a novel platform enabled active control of the location of the preconcentrated plug of target biomolecules, ensuring its overlap with the functionalized microparticles and ultimately yielding enhanced detection sensitivity and binding kinetics. This was demonstrated using avidin-biotin particles as a simple bead-based bioassay model.

Active Particle Based Selective Transport and Release of Cell Organelles and Mechanical Probing of a Single Nucleus.

Self-propelling micromotors are emerging as a promising microscale tool for single-cell analysis. The authors have recently shown that the field gradients necessary to manipulate matter via dielectrophoresis can be induced at the surface of a polarizable active ("self-propelling") metallo-dielectric Janus particle (JP) under an externally applied electric field, acting essentially as a mobile floating microelectrode. Here, the application of the mobile floating microelectrode to trap and transport cell organelles in a selective and releasable manner is successfully extended. This selectivity is driven by the different dielectrophoretic (DEP) potential wells on the JP surface that is controlled by the frequency of the electric field, along with the hydrodynamic shearing and size of the trapped organelles. Such selective and directed loading enables purification of targeted organelles of interest from a mixed biological sample while their dynamic release enables their harvesting for further analysis such as gene/RNA sequencing or proteomics. Moreover, the electro-deformation of the trapped nucleus is shown to be in correlation with the DEP force and hence, can act as a promising label-free biomechanical marker. Hence, the active carrier constitutes an important and novel ex vivo platform for manipulation and mechanical probing of subcellular components of potential for single cell analysis.

Distinct and independent dielectrophoretic behavior of the head and tail of sperm and its potential for the safe sorting and isolation of rare spermatozoa.

Often, in semen samples with minute amounts of sperm, even the single spermatozoon required to fertilize an oocyte cannot be found in the ejaculate. This is primarily because currently, sperm is generally searched for manually under a microscope. In this study, dielectrophoresis (DEP) was investigated as an alternative automated technique for sorting sperm cells. Using a quadrupolar electrode array it was shown that the head and tail of the sperm had independent and unique crossover frequencies corresponding to the transition of the DEP force from repulsive (negative) to attractive (positive). These surprising results were further analyzed, showing that the head and tail have their own distinct electrical properties. This significant result allows for the sperm's head, which contains the DNA, to be distanced from potentially damaging high electric fields using negative DEP while simultaneously manipulating and sorting the sperm using the positive DEP response of the tail. A proof of concept sorting chip was designed and tested. The low crossover frequency of the tail also allows for the use of a higher conductivity, and thus more physiological, medium than the conventional DEP solutions. Although more research is required to design and optimize an efficient, user-friendly, and high-throughput device, this research is a proof of concept that DEP has the potential to automate and improve the processing of semen samples, especially those containing only rare spermatozoa.

Analysis of Cargo Loading Modes and Capacity of an Electrically-Powered Active Carrier.

The use of active colloids for cargo transport offers unique potential for applications ranging from targeted drug delivery to lab-on-a-chip systems. Previously, Janus particles (JPs), acting as mobile microelectrodes, have been shown to transport cargo which is trapped at the JP surface by a dielectrophoretic mechanism. Herein, we aim to characterize the cargo loading properties of mobile Janus carriers, across a broad range of frequencies and voltages. In expanding the frequency range of the carrier, we are able to compare the influences of different modes of carrier transport on the loading capacity as well as highlight the differences between cargo trapped by positive and negative dielectrophoresis. Specifically, it is shown that cargo trapping results in a reduction in carrier velocities with this effect more pronounced at low frequencies where cargo is trapped close to the substrate. Interestingly, we observe the existence of a maximum cargo loading capacity which decreases at large voltages suggesting a strong interplay between trapping and hydrodynamic shear. Finally, we demonstrate that the control of the frequency can enable different assemblies of binary colloidal solutions on the JP. The resultant findings enable the optimization of electrokinetic cargo transport and its selective application to a broad range of targets.

Dielectrophoretic characterization and isolation of jellyfish stinging capsules.

Jellyfish stinging capsules known as nematocysts are explosive, natural-injection systems with high potential as a natural drug-delivery system. These organelles consist of a capsule containing a highly folded thin needle-like tubule and a matrix highly concentrated with charged constituents that enable the tubule to fire and penetrate a target. For the purpose of using these nematocysts as drug delivery system it is first required to purify subpopulations from heterogeneous population of capsules and to investigate each subpopulation's distinct function and characteristics. Here, the nematocysts' dielectric properties were experimentally investigated using dielectrophoretic and electrorotational spectra with best fits derived from theoretical models. The dielectric characterization adds to our understanding of the nematocysts' structure and function and is necessary for the dielectrophoretic isolation and manipulation of populations. As expected, the effect of monovalent and divalent exchange cations resulted in higher inner conductivity for the NaCl treated capsules; this result stands in agreement with their relative higher osmotic pressure. In addition, an efficient dielectrophoretic isolation of different nematocyst subpopulations was demonstrated, paving the way to an understanding of nematocysts' functional diversity and the development of an efficient drug delivery platform.

Interplay between Nanochannel and Microchannel Resistances.

Current nanochannel system paradigm commonly neglects the role of the interfacing microchannels and assumes that the ohmic electrical response of a microchannel-nanochannel system is solely determined by the geometric properties of the nanochannel. In this work, we demonstrate that the overall response is determined by the interplay between the nanochannel resistance and various microchannel attributed resistances. Our experiments confirm a recent theoretical prediction that in contrast to what was previously assumed at very low concentrations the role of the interfacing microchannels on the overall resistance becomes increasingly important. We argue that the current nanochannel-dominated conductance paradigm can be replaced with a more correct and intuitive microchannel-nanochannel-resistance-model-based paradigm.