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.

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.

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.

Designing with Iontronic Logic Gates─From a Single Polyelectrolyte Diode to an Integrated Ionic Circuit.

This article presents the implementation of on-chip iontronic circuits via small-scale integration of multiple ionic logic gates made of bipolar polyelectrolyte diodes. These ionic circuits are analogous to solid-state electronic circuits, with ions as the charge carriers instead of electrons/holes. We experimentally characterize the responses of a single fluidic diode made of a junction of oppositely charged polyelectrolytes (i.e., anion and cation exchange membranes), with a similar underlying mechanism as a solid-state p- and n-type junction. This served to carry out predesigned logical computations in various architectures by integrating multiple diode-based logic gates, where the electrical signal between the integrated gates was transmitted entirely through ions. The findings shed light on the limitations affecting the number of logic gates that can be integrated, the degradation of the electrical signal, their transient response, and the design rules that can improve the performance of iontronic circuits.

Optoelectronic Trajectory Reconfiguration and Directed Self-Assembly of Self-Propelling Electrically Powered Active Particles.

Self-propelling active particles are an exciting and interdisciplinary emerging area of research with projected biomedical and environmental applications. Due to their autonomous motion, control over these active particles that are free to travel along individual trajectories, is challenging. This work uses optically patterned electrodes on a photoconductive substrate using a digital micromirror device (DMD) to dynamically control the region of movement of self-propelling particles (i.e., metallo-dielectric Janus particles (JPs)). This extends previous studies where only a passive micromotor is optoelectronically manipulated with a translocating optical pattern that illuminates the particle. In contrast, the current system uses the optically patterned electrode merely to define the region within which the JPs moved autonomously. Interestingly, the JPs avoid crossing the optical region's edge, which enables constraint of the area of motion and to dynamically shape the JP trajectory. Using the DMD system to simultaneously manipulate several JPs enables to self-assemble the JPs into stable active structures (JPs ring) with precise control over the number of participating JPs and passive particles. Since the optoelectronic system is amenable to closed-loop operation using real-time image analysis, it enables exploitation of these active particles as active microrobots that can be operated in a programmable and parallelized manner.

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.

A Magnetically and Electrically Powered Hybrid Micromotor in Conductive Solutions: Synergistic Propulsion Effects and Label-Free Cargo Transport and Sensing.

Electrically powered micro- and nanomotors are promising tools for in vitro single-cell analysis. In particular, single cells can be trapped, transported, and electroporated by a Janus particle (JP) using an externally applied electric field. However, while dielectrophoretic (DEP)-based cargo manipulation can be achieved at high-solution conductivity, electrical propulsion of these micromotors becomes ineffective at solution conductivities exceeding ≈0.3 mS cm-1 . Here, JP cargo manipulation and transport capabilities to conductive near-physiological (<6 mS cm-1 ) solutions are extended successfully by combining magnetic field-based micromotor propulsion and navigation with DEP-based manipulation of various synthetic and biological cargos. Combination of a rotating magnetic field and electric field results in enhanced micromotor mobility and steering control through tuning of the electric field frequency. In addition, the micromotor's ability of identifying apoptotic cell among viable and necrotic cells based on their dielectrophoretic difference is demonstrated, thus, enabling to analyze the apoptotic status in the single-cell samples for drug discovery, cell therapeutics, and immunotherapy. The ability to trap and transport live cells towards regions containing doxorubicin-loaded liposomes is also demonstrated. This hybrid micromotor approach for label-free trapping, transporting, and sensing of selected cells within conductive solutions opens new opportunities in drug delivery and single-cell analysis, where close-to-physiological media conditions are necessary.

Switching from Chemical to Electrical Micromotor Propulsion across a Gradient of Gastric Fluid via Magnetic Rolling.

To address and extend the finite lifetime of Mg-based micromotors due to the depletion of the engine (Mg-core), we examine electric fields, along with previously studied magnetic fields, to create a triple-engine hybrid micromotor for driving these micromotors. Electric fields are a facile energy source that is not limited in its operation time and can dynamically tune the micromotor mobility by simply changing the frequency and amplitude of the field. Moreover, the same electrical fields can be used for cell trapping and transport as well as drug delivery. However, the limitations of these propulsion mechanisms are the low pH (and high conductivity) environment required for Mg dissolution, while the electrical propulsion is quenched at these conditions as it requires low conductivity mediums. In order to translate the micromotor between these two extreme medium conditions, we use magnetic rolling as means of self-propulsion along with magnetic steering. Interestingly, electrical propulsion also necessitates at least the partial consumption of the Mg, resulting in a sufficient geometrical asymmetry of the micromotor. We have successfully demonstrated the rapid propulsion switching capability of the micromotor, from chemical to electrical motions, via magnetic rolling within a microfluidic device with the concentration gradient of the simulated gastric fluid. Such triple-engine micromotor propulsion holds considerable promise for in vitro studies mimicking gastric conditions and performing various bioassay tasks.

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.

Dielectrophoretic Characterization of Dynamic Microcapsules and Their Magnetophoretic Manipulation.

In this work, we present dielectrophoresis (DEP) and in situ electrorotation (ROT) characterization of reversibly stimuli-responsive "dynamic" microcapsules that change the physicochemical properties of their shells under varying pH conditions and can encapsulate and release (macro)molecular cargo on demand. Specifically, these capsules are engineered to open (close) their shell under high (low) pH conditions and thus to release (retain) their encapsulated load or to capture and trap (macro)molecular samples from their environment. We show that the steady-state DEP and ROT spectra of these capsules can be modeled using a single-shell model and that the conductivity of their shells is influenced most by the pH. Furthermore, we measured the transient response of the angular velocity of the capsules under rotating electric field conditions, which allows us to directly determine the characteristic time scales of the underlying physical processes. In addition, we demonstrate the magnetic manipulation of microcapsules with embedded magnetic nanoparticles for lab-on-chip tasks such as encapsulation and release at designated locations and the in situ determination of their physicochemical state using on-chip ROT. The insight gained will enable the advanced design and operation of these dynamic drug delivery and smart lab-on-chip transport systems.

Testis on a chip-a microfluidic three-dimensional culture system for the development of spermatogenesisin-vitro.

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesisin-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

Digital microfluidics-like manipulation of electrokinetically preconcentrated bioparticle plugs in continuous-flow.

Herein, we demonstrate digital microfluidics-like manipulations of preconcentrated biomolecule plugs within a continuous flow that is different from the commonly known digital microfluidics involving discrete (i.e. droplets) media. This is realized using one- and two-dimensional arrays of individually addressable ion-permselective membranes with interconnecting microfluidic channels. The location of powered electrodes, dictates which of the membranes are active and generates either enrichment/depletion diffusion layers, which, in turn, control the location of the preconcentrated plug. An array of such powered membranes enables formation of multiple preconcentrated plugs of the same biosample as well as of preconcentrated plugs of multiple biosample types introduced via different inlets in a selective manner. Moreover, digital-microfluidics operations such as up-down and left-right translation, merging, and splitting, can be realized, but on preconcentrated biomolecule plugs instead of on discrete droplets. This technology, based on nanoscale electrokinetics of ion transport through permselective medium, opens future opportunities for smart and programmable digital-like manipulations of preconcentrated biological particle plugs for various on-chip biological applications.

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.

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.

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.