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When exposed to an alternating current (AC) electric field, a polarized microparticle is moved by the interaction between the voltage-induced dipoles and the AC electric field under dielectrophoresis (DEP). The DEP force is widely used for manipulation of microparticles in diverse practical applications such as 3D manipulation, sorting, transfer, and separation of various particles such as living cells. In this study, we propose the integration of surface-enhanced Raman spectroscopy (SERS), an extremely sensitive and versatile technique based on the Raman scattering of molecules supported by nanostructured materials, with DEP using a microfluidic device. The microfluidic device combines microelectrodes with gold nanohole arrays to characterize the electrophysiological and biochemical properties of biological cells. The movement of particles, which varies depending on the electrical properties such as conductivity and permittivity of particles, can be manipulated by the cross-frequency change. For proof of concept, Raman spectroscopy using the DEP-SERS integration was performed for polystyrene beads and biological cells and resulted in an improved signal-to-noise ratio by determining the direction of the DEP force applied to the cells with respect to the applied AC power and collecting them on the nanohole arrays. The result illustrates the potential of the concept for simultaneously examining the electrical and biochemical properties of diverse chemical and biological microparticles in the microfluidic environment.

Circulating tumor cell separation has been the focus of numerous studies owing to its importance in the diagnosis, prognosis, and therapy of cancer. This study reports a highly efficient microfluidic device that integrates a specialized dielectrophoresis configuration, namely the facing-electrode configuration dielectrophoresis (FEC-DEP) structure, to isolate circulating tumor cells (CTCs) from various blood components, including red blood cells, white blood cells, and platelets. The FEC-DEP design features a bottom-slanted electrode array positioned parallel to a basic rectangular top electrode. A non-homogeneous electric field is produced between these parallel electrodes, generating dielectrophoretic forces acting on cells. Consequently, when the FEC-DEP is integrated into a flow, it can direct various biological objects in the flow along separate trajectories. As a result, cells with comparable characteristics might move together within a similar path. This configuration may simplify the microfabrication process and lessen dependency on particle position within the microchannel. The separation process was numerically analyzed using the finite element method, and device parameters were optimized to obtain high-efficiency and high-purity cell separation. The simulations show that the microfluidic device may effectively enrich tumor cells in a label-free and non-invasive manner, with a high-efficiency rate of almost 80%., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval Not applicable. Consent for publication All authors have given consent to this publication. Consent to participate Not applicable., (© 2024. The Author(s).)

Technologies for rapid and high-throughput separation of rare cells from large populations of other types of cells have recently attracted much attention in the field of bioengineering. Among the various cell separation technologies proposed in the past, dielectrophoresis has shown particular promise because of its preciseness of manipulation and noninvasiveness to cells. However, one drawback of dielectrophoresis devices is that their application of high voltage generates Joule heat that exposes the cells within the device to high temperatures. To further explore this problem, this study investigated the temperature field in a previously developed cell separation device in detail. The temperature rise at the bottom of the microfluidic channel in the device was measured using a micro-LIF method. Moreover, the thermofluidic behavior of the cell separation device was numerically investigated by adopting a heat generation model that takes the electric-field-dependent heat generation term into account in the energy equation. Under the operating conditions of the previously developed cell separation device, the experimentally obtained temperature rise in the device was approximately 20 °C, and the numerical simulation results generally agreed well. Next, parametric calculations were performed with changes in the flow rate of the cell sample solution and the solution conductivity, and a temperature increase of more than 40 °C was predicted. The results demonstrated that an increase in temperature within the cell separation device may have a significant impact on the physiological functions of the cells, depending on the operating conditions of the device.

Along with the rapid development of cellular biological research in recent years, there has been an urgent need for a high-speed, high-precision method of separating target cells from a highly heterogeneous cell population. Among the various cell separation technologies proposed so far, dielectrophoresis (DEP)-based approaches have shown particular promise because they are noninvasive to cells. We have developed a new DEP-based device to separate large numbers of live and dead cells of the human mammary cell line MCF10A. In this study, we validated the separation performance of this device. The results showed the successful separation of a higher percentage of cells than in previous studies, with a separation efficiency higher than 90%. In the past, there have been no confirmed cases in which a separation rate of over 90% and high-speed processing of a large number of cells were simultaneously achieved. It was shown that the proposed device can process large numbers of cells at high speed and with high accuracy., (© 2024 Wiley‐VCH GmbH.)

This study explores the frontiers of microparticle manipulation by introducing an actuator platform for the three-dimensional positioning of microparticles using dielectrophoresis (DEP), a technique known for its selectivity and ease of integration with microtechnology. Leveraging advancements in carbon-based devices due to their biocompatibility and electrochemical stability, our work extends the application of DEP from two-dimensional constraints to precise 3D positioning within microvolumes, employing a photolithography-based fabrication process known as Carbon-MEMS technology (C-MEMS). We present the design, finite element simulation, fabrication, and testing of this platform, which utilizes a unique combination of planar and 3D carbon microelectrodes individually addressable on a transparent substrate. This setup enables the application of DEP forces, allowing for high-throughput manipulation of multiple microparticles simultaneously, as well as displacement of individual microparticles in any desired direction. Demonstrated with spherical 1μm and 10μm diameter polystyrene microparticles, this platform features straightforward fabrication and is suitable for batch industrial production. The study concludes with a discussion of the platform's advantages and limitations, marking a significant step toward a valuable tool for studying complex biological systems., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pilloni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The spread of metastatic cancer cells poses a significant challenge in cancer treatment, making innovative approaches for early detection and diagnosis essential. Dielectrophoretic impedance spectroscopy (DEPIS), a powerful tool for cell analysis, combines dielectrophoresis (DEP) and impedance spectroscopy (IS) to separate, sort, cells and analyze their dielectric properties. In this study, we developed and built out-of-plane inkjet-printed castellated arrays to map the dielectric properties of MDA-MB-231 breast cancer cell subtypes across their metastatic potential. This was realized via modulating the expression of connexin 43 (Cx43), a marker associated with poor breast cancer prognosis and increased metastasis. We employed DEP-based trapping, followed by EIS measurements on bulk cell population, for rapid capture and differentiation of the cancer cells according to their metastatic state. Our results revealed a significant correlation between the various MDA-MB-231 metastatic subtypes and their respective dielectrophoretic and dielectric properties. Notably, cells with the highest metastatic potential exhibited the highest membrane capacitance 16.88 ± 3.24 mF m -2 , followed by the less metastatic cell subtypes with membrane capacitances below 14.3 ± 2.54 mF m -2 . In addition, highly metastatic cells exhibited lower crossover frequency (25 ± 1 kHz) compared to the less metastatic subtypes (≥27 ± 1 kHz), an important characteristic for cell sorting. Finally, EIS measurements showed distinct double layer capacitance ( C DL ) values at 1 kHz between the metastatic subgroups, confirming unique dielectric and dielectrophoretic properties correlated with the metastatic state of the cell. Our findings underscore the potential of DEPIS as a non-invasive and rapid analytical tool, offering insights into cancer biology and facilitating the development of personalized therapeutic interventions tailored to distinct metastatic stages.

Dielectrophoresis (DEP), which arises from the interaction between dielectric particles and an aqueous solution in a nonuniform electric field, contributes to the manipulation of nano and microparticles in many fields, including colloid physics, analytical chemistry, molecular biology, clinical medicine, and pharmaceutics. The measurement of the DEP force could provide a more complete solution for verifying current classical DEP theories. This review reports various imaging, fluidic, optical, and mechanical approaches for measuring the DEP forces at different amplitudes and frequencies. The integration of DEP technology into sensors enables fast response, high sensitivity, precise discrimination, and label-free detection of proteins, bacteria, colloidal particles, and cells. Therefore, this review provides an in-depth overview of DEP-based fabrication and measurements. Depending on the measurement requirements, DEP manipulation can be classified into assistance and integration approaches to improve sensor performance. To this end, an overview is dedicated to developing the concept of trapping-on-sensing, improving its structure and performance, and realizing fully DEP-assisted lab-on-a-chip systems., (© 2024 Wiley‐VCH GmbH.)

Small extracellular vesicles (sEVs) reflect the genotype and phenotype of original cells and are biomarkers for early diagnosis and treatment monitoring of tumors. Yet, their small size and low density make them difficult to isolate and detect in body fluid samples. This study proposes a novel acDEP-Exo chip filled with transparent micro-beads, which formed a non-uniform electrical field, and finally achieved rapid, sensitive, and tunable sEVs capture and detection. The method requires only 20-50 μL of sample, achieved a limit of detection (LOD) of 161 particles/μL, and can detect biomarkers within 13 min. We applied the chip to analyze the two markers of sEV's EpCAM and MUC1 in clinical plasma samples from breast cancer (BC) patients and healthy volunteers and found that the combined evaluation of sEV's biomarkers has extremely high sensitivity, specificity and accuracy. The present study introduces an alternative approach to sEVs isolation and detection, has a great potential in real-time sEVs-based liquid biopsy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a class of point-of-care (POC) assays with visible distance readout (thermometer style), the electrophoresis titration (ET) biosensor affords high robustness, versatility, and simplicity for point-of-care quantification. However, naked-eye observation of the distance readout is unreliable in POC settings and manual processing of distance readout is time-consuming. Herein, we developed a smartphone-deployable and all-in-one machine vision for four ET biosensors (bovine serum albumin, melamine, uric acid, glutathione) to classify and quantify the samples simultaneously. To ensure accurate and rapid quantification on the smartphone, we customized the decolorization methods and edge detection operators to balance the region of interest (ROI) extraction performance and processing speed. We then established a dataset of 180 distance readout images to endow our machine vision with the ability to classify four sample types. Consequently, our machine vision demonstrated high accuracy in determining the sample type (>97.2%) and concentration (>97.3%). Moreover, expanding its applications to other targets was readily achieved by including distance readout images of other ET biosensors (e.g., hemoglobin A1c) in the dataset. Therefore, our strategy of constructing machine vision is compatible with the versatile ET biosensor technique, suggesting that the same strategy can be used for other thermometer-style POC assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metastasis remains a significant cause to cancer-related mortality, underscoring the critical need for early detection and analysis of circulating tumor cells (CTCs). This study presents a novel microfluidic chip designed to efficiently capture A549 lung cancer cells by combining dielectrophoresis (DEP) and aptamer-based binding, thereby enhancing capture efficiency and specificity. The microchip features interdigitated electrodes made of indium-tin-oxide that generate a nonuniform electric field to manipulate CTCs. Following three chip design, scenarios were investigated: (A) bare glass surface, (B) glass modified with gold nanoparticles (AuNPs) only, and (C) glass modified with both AuNPs and aptamers. Experimental results demonstrate that AuNPs significantly enhance capture efficiency under DEP, with scenarios (B) and (C) exhibiting similar performance. Notably, scenario (C) stands out as aptamer-functionalized surfaces resisting fluid shear forces, achieving CTCs retention even after electric field deactivation. Additionally, an innovative reverse pumping method mitigates inlet clogging, enhancing experimental efficiency. This research offers valuable insights into optimizing surface modifications and understanding key factors influencing cell capture, contributing to the development of efficient cell manipulation techniques with potential applications in cancer research and personalized treatment options., (© 2024 Wiley‐VCH GmbH.)

Type 2 diabetes mellitus (T2DM) is a prevalent and debilitating disease with numerous health risks, including cardiovascular diseases, kidney dysfunction, and nerve damage. One important aspect of T2DM is its association with the abnormal morphology of red blood cells (RBCs), which leads to increased blood viscosity and impaired blood flow. Therefore, evaluating the mechanical properties of RBCs is crucial for understanding the role of T2DM in cellular deformability. This provides valuable insights into disease progression and potential diagnostic applications. In this study, we developed an open micro-electro-fluidic (OMEF) biochip technology based on dielectrophoresis (DEP) to assess the deformability of RBCs in T2DM. The biochip facilitates high-throughput single-cell RBC stretching experiments, enabling quantitative measurements of the cell size, strain, stretch factor, and post-stretching relaxation time. Our results confirm the significant impact of T2DM on the deformability of RBCs. Compared to their healthy counterparts, diabetic RBCs exhibit ∼27% increased size and ∼29% reduced stretch factor, suggesting potential biomarkers for monitoring T2DM. The observed dynamic behaviors emphasize the contrast between the mechanical characteristics, where healthy RBCs demonstrate notable elasticity and diabetic RBCs exhibit plastic behavior. These differences highlight the significance of mechanical characteristics in understanding the implications for RBCs in T2DM. With its ∼90% sensitivity and rapid readout (ultimately within a few minutes), the OMEF biochip holds potential as an effective point-of-care diagnostic tool for evaluating the deformability of RBCs in individuals with T2DM and tracking disease progression.

Reliable separation of circulating tumor cells from blood cells is crucial for early cancer diagnosis and prognosis. Many conventional microfluidic platforms take advantage of the size difference between particles for their separation, which renders them impractical for sorting overlapping-sized cells. To address this concern, a hybrid inertial-dielectrophoretic microfluidic chip is proposed in this work for continuous and single-stage separation of lung cancer cell line A549 cells from white blood cells of overlapping size. The working mechanism of the proposed spiral microchannel embedded with planar interdigitated electrodes is validated against the experimental results. A numerical investigation is carried out over a range of flow conditions and electric field intensity to determine the separation efficiency and migration characteristics of the cell mixture. The results demonstrate the unique capability of the proposed microchannel to achieve high-throughput separation of cells at low applied voltages in both vertical and lateral directions. A significant lateral separation distance between the CTCs and the WBCs has been achieved, which allows for high-resolution and effective separation of cells. The separation resolution can be controlled by adjusting the strength of the applied electric field. Furthermore, the results demonstrate that the lateral separation distance is maximum at a voltage termed the critical voltage, which increases with the increase in the flow rate. The proposed microchannel and the developed technique can provide valuable insight into the development of a tunable and robust medical device for effective and high-throughput separation of cancer cells from the WBCs., (© 2024 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Non-spherical flagellate algae play an increasingly significant role in handling problematic issues as versatile biological micro/nanorobots and resources of valuable bioproducts. However, the commensalism of flagellate algae with distinct structures and constituents causes considerable difficulties in their further biological utilization. Therefore, it is imperative to develop a novel method to realize high-efficiency selection of non-spherical flagellate algae in a non-invasive manner. Enthused by these, we proposed a novel method to accomplish the selection of flagellate algae based on the numerical and experimental investigation of dielectrophoretic characterizations of flagellate algae. Firstly, an arbitrary Lagrangian-Eulerian method was utilized to study the electro-orientation and dielectrophoretic assembly process of spindle-shaped and ellipsoid-shaped cells in a uniform electric field. Secondly, we studied the equilibrium state of spherical, ellipsoid-shaped, and spindle-shaped cells under positive DEP forces actuated by right-angle bipolar electrodes. Thirdly, we investigated the dielectrophoretic assembly and escape processes of the non-spherical flagellate algae in continuous flow to explore their influences on the selection. Fourthly, freshwater flagellate algae ( Euglena , H. pluvialis , and C. reinhardtii ) and marine ones ( Euglena , Dunaliella salina , and Platymonas ) were separated to validate the feasibility and adaptability of this method. Finally, this approach was engineered in the selection of Euglena cells with high viability and motility. This method presents immense prospects in the selection of pure non-spherical flagellate algae with high motility for chronic wound healing, bio-micromotor construction, and decontamination with advantages of no sheath, strong reliability, and shape-insensitivity.

One field of study in microfluidics is the control, trapping, and separation of microparticles suspended in fluid. Some of its applications are related to cell handling, virus detection, and so on. One of the new methods in this field is using ICEK phenomena and dielectrophoresis forces. In the present study, considering the ICEK phenomena, the microparticles inside the fluid are deviated in the desired ratio using a novel ICEK microchip. The deviation is such that after the microparticles reach the floating electrode, they are trapped in the ICEK flow vortex and deviated through a secondary channel that was placed crosswise and noncoplanar above the main channel. For simulation verification, an experimental test is done. The method used for making two noncoplanar channels and separating the particles in the desired ratio with a simple ICEK microchip is an innovation of the present study. Moreover, the adjustment of the percentage of separation of microparticles by adjusting the parameters of the applied voltage and fluid inlet velocity is one of the other innovations of the present experimental study. We observed that for input velocities of 150-1200 µm/s with applied voltages of 10-33 V, 100% of the particles can be directed toward the secondary-channel., (© 2023 Wiley‐VCH GmbH.)

Developing paper-based electrophoretic methods involve dealing with significant uncertainty levels when compared to their capillary counterparts. Critical information for developing these kinds of methods are the electrophoretic mobility of background electrolytes and samples. This work presents the design and characterization of a device for measuring the electrophoretic mobilities of dyes in porous media. The device was developed with the aim of validating a previously presented model and also proposing a protocol for the straightforward determination of electrophoretic mobilities in porous media when open-channel values are already known. Whatman #1 paper was used as a model substrate as far as it is the most common porous medium substrate for paper-based electrophoresis. The device was designed using a numerical simulation-assisted approach, utilizing OpenFOAM® and specific solvers for capillary transport and electromigration, namely porousMicroTransport and electroMicroTransport, respectively. The electrophoretic mobilities of five dyes were analyzed experimentally with the proposed device. To establish appropriate comparative values at different pHs, experiments in fused silica capillaries were also performed. An effective parameter model for describing the electrophoretic behavior of dyes in porous media, that is, the constriction factor, was found consistent with previous reports for the Whatman #1 paper. This consistency was found after considering (via direct measurements) the chromatographic effect of the medium over each dye. Consequently, the recorded values hold significant worth due to their potential for direct application in designing new experiments or devices in Whatman #1 paper. With the validation of the model through the experiments with the proposed device, those researchers interested on developing electrophoretic methods in porous substrates can make use of the open-channel electrophoretic mobilities reported in the literature, or in the well-known software databases, and correct them for the media of interest just by performing two simple characterization steps., (© 2023 Wiley‐VCH GmbH.)

Electrophoretic tissue clearing has been a commonly used laboratory method since the early twentieth century. Infrastructure for standard procedures has yet to be formed. In particular, control of the heat produced by electrophoresis, the voltage applied to the electrodes, the resistance, and the speed of liquid circulation create difficulty for researchers. We aimed to develop a compact organ electrophoresis system that enables the researcher to have easy, rapid, and inexpensive working opportunities. The system includes an electronic control unit, a liquid tank, a temperature control unit, and an electrophoresis chamber. The control unit software can keep the system stable by using information on temperature and circulation rate received through the sensors using the feedback principle. Corrosion and particle collection are reduced to a minimum as platinum wires are used for electrophoresis electrodes. A temperature control unit can heat and cool via a liquid tank base. The CORES is an all-in-one, easy-to-use solution for electrophoretic tissue clearing. It assures efficient, rapid, and consistent tissue clearing. The system was stable with 72 h of continuous operation. Patent applications and trial version studies for introducing the system to researchers are still in progress.

Cancer is a cellular-based disease, so cytological diagnosis is one of the main challenges for its early detection. An extensive number of diagnostic methods have been developed to separate cancerous cells from normal ones, in electrical methods attract progressive attention. Identifying and specifying different cells requires understanding their dielectric and electric properties. This study evaluated MDA-MB-231, HUVEC, and MCF-10A cell lines, WBCs isolated from blood, and patient-derived cell samples with a cylindrical body with two transparent FTO (fluorine-doped tin oxide) plate electrodes. Cell mobility rates were recorded in response to these stimuli. It was observed that cancer cells demonstrate drastic changes in their motility in the presence and absence of an electric field (DC/AC). Also, solution viscosity's effect on cancer cells' capturing efficacy was evaluated. This research's main distinguished specification uses a non-microfluidic platform to detect and pathologically evaluate cytological samples with a simple, cheap, and repeatable platform. The capturing procedure was carried out on a cytological slide without any complicated electrode patterning with the ability of cytological staining. Moreover, this platform successfully designed and experimented with the invasion assay (the ability of captured cancer cells to invade normal cells)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Single-cell analysis of cell phenotypic information such as surface protein expression and nucleic acid content is essential for understanding heterogeneity within cell populations. Here the design and use of a dielectrophoresis-assisted self-digitization (SD) microfluidics chip is described; it captures single cells in isolated microchambers with high efficiency for single-cell analysis. The self-digitization chip spontaneously partitions aqueous solution into microchambers through a combination of fluidic forces, interfacial tension, and channel geometry. Single cells are guided to and trapped at the entrances of microchambers by dielectrophoresis (DEP) due to local electric field maxima created by an externally applied AC voltage. Excess cells are flushed away, and trapped cells are released into the chambers and prepared for in situ analysis by turning off the external voltage, by running reaction buffer through the chip, and by sealing the chambers with a flow of an immiscible oil phase through the surrounding channels. The use of this device in single-cell analysis is demonstrated by performing single-cell nucleic acid quantitation based on loop-mediated isothermal amplification (LAMP). This platform provides a powerful new tool for single-cell research pertaining to drug discovery. For example, the single-cell genotyping of cancer-related mutant gene observed from the digital chip could be useful biomarker for targeted therapy., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this paper, a micromixer of a new configuration is presented, consisting of a spherical chamber in the center of which an ion-selective microsphere is placed. Stratified liquid is introduced through the chamber via inlet and outlet holes under an external pressure gradient and an external electric field is directed in such a way that the resulting electroosmotic flow is directed against the pressure-driven flow, resulting in mixing. The investigation is carried out by direct numerical simulation on a super-computer. Optimal values of the applied electric field are determined to yield strong mixing. Above this optimal mixing regime, a number of instabilities and bifurcations are realized, which qualitatively coincide with those occurring during electrophoresis of an ion-selective microgranule. As shown by our calculation, these instabilities do not lead to an enhanced mixing. The resulting electroconvective vortices remain confined near the surface of the microgranule, and do not sufficiently perturb the stratified fluid flow further from the granule. On the other hand, another type of instability caused by the salt concentration gradient can generate sufficiently strong oscillations to enhance mixing. However, this only occurs when the external electric field is sufficiently high that the electroosmotic flow is comparable to the pressure-driven flow. This ultimately leads to creation of reverse flows of the liquid and cessation of the device operation. Thus, it was shown that the best mixing occurs in the absence of electrokinetic instability. Based on the data obtained, it is possible to select the necessary geometric characteristics of the micromixer to achieve the optimal mixing mode for a given set of liquids, which may be ten times more effective than passive mixers at the same flow rates. A comparison with the experimental data of the other authors confirms the effectiveness of this device and its other capabilities. Furthermore, the basic device design can be operated in other modes, for example, an electrohydrodynamic pump, a streaming current generator, or even a micro-reactor, depending on the system parameters and choice of an ion-selective granule., (© 2021 Wiley-VCH GmbH.)

The concept of dielectrophoresis (DEP), which involves the movement of neutral particles by induced polarization in nonuniform electric fields, has been exploited in various biological applications. However, only a few studies have investigated the use of DEP for detecting and enumerating microorganisms in foodstuffs. Therefore, we aimed to evaluate the accuracy and efficiency of a DEP-based method for enumerating viable bacteria in three raw foods: freshly cut lettuce, chicken breast, and minced pork. The DEP separation of bacterial cells was conducted at 20 V of output voltage and 6000 to 9000 kHZ of frequency with sample conductivity of 30-70 μS/cm. The accuracy and validity of the DEP method for enumerating viable bacteria were compared with those of the conventional culture method; no significant variation was observed. We found a high correlation between the data obtained using DEP and the conventional aerobic plate count culture method, with a high coefficient of determination (R 2  > 0.90) regardless of the food product; the difference in cell count data between both methods was within 1.0 log CFU/mL. Moreover, we evaluated the efficiency of the DEP method for enumerating bacterial cells in chicken breasts subjected to either freezing or heat treatment. After thermal treatment at 55 °C and 60 °C, the viable cell counts determined via the DEP method were found to be lower than those obtained using the conventional culture method, which implies that the DEP method may not be suitable for the direct detection of injured cells. In addition to its high accuracy and efficiency, the DEP method enables the determination of viable cell counts within 30 min, compared to 48 h required for the conventional culture method. In conclusion, the DEP method may be a potential alternative tool for rapid determination of viable bacteria in a variety of foodstuffs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Saccharomyces cerevisiae(S. cerevisiae) has been classically used as a treatment for diarrhea and diarrhea-related diseases. However, cases of the fungal infections caused by S. cerevisiae have been increasing in the last two decades among immunocompromised patients, while a long time was spent on S. cerevisiae isolation clinically so it was difficult to achieve timely diagnosis the diseases. Here, a novel approach for isolation and selection of S. cerevisiae is proposed by designing a microfluidic chip with an optically induced dielectrophoresis (ODEP) system. S. cerevisiae was isolated from the surroundings by ODEP due to different dielectrophoretic forces. Two special light images were designed and used to block and separate S. cerevisiae, respectively, and several manipulation parameters of ODEP were experimentally optimized to acquire the maximum isolation efficiency of S. cerevisiae. The results on the S. cerevisiae isolation declared that the purity of the S. cerevisiae selected by the method was up to 99.5 % ±0.05, and the capture efficiency was up to 65.0 % ±2.5 within 10 min. This work provides a general method to isolate S. cerevisiae as well as other microbial cells with high accuracy and efficiency and paves a road for biological research in which the isolation of high-purity cells is required.

Dielectrophoretically driven microfluidic devices have demonstrated great applicability in biomedical engineering, diagnostic medicine, and biological research. One of the potential fields of application for this technology is in point-of-care (POC) devices, ideally allowing for portable, fully integrated, easy to use, low-cost diagnostic platforms. Two main approaches exist to induce dielectrophoresis (DEP) on suspended particles, that is, electrode-based DEP and insulator-based DEP, each featuring different advantages and disadvantages. However, a shared concern lies in the input voltage used to generate the electric field necessary for DEP to take place. Therefore, input voltage can determine portability of a microfluidic device. This review outlines the recent advances in reducing stimulation voltage requirements in DEP-driven microfluidics., (© 2020 Wiley-VCH GmbH.)

Insulator-based dielectrophoresis (iDEP) exploits the electric field gradients formed around insulating structures to manipulate particles for diverse microfluidic applications. Compared to the traditional electrode-based dielectrophoresis, iDEP microdevices have the advantages of easy fabrication, free of water electrolysis, and robust structure, etc. However, the presence of in-channel insulators may cause thermal effects because of the locally amplified Joule heating of the fluid. The resulting electrothermal flow circulations are exploited in this work to trap and concentrate nanoscale particles (of 100 nm diameter and less) in a ratchet-based iDEP microdevice. Such Joule heating-enabled electrothermal enrichment of nanoparticles are found to grow with the increase of alternating current or direct current electric field. It also becomes more effective for larger particles and in a microchannel with symmetric ratchets. Moreover, a depth-averaged numerical model is developed to understand and simulate the various parametric effects, which is found to predict the experimental observations with a good agreement., (© 2020 Wiley-VCH GmbH.)

Dielectrophoresis is a robust approach for the manipulation and separation of (bio)particles using microfluidic platforms. We developed a dielectrophoretic corral trap in a microfluidic device that utilizes negative dielectrophoresis to capture single spherical polystyrene particles. Circular-shaped micron-size traps were employed inside the device and the three-dimensional trap stiffness (restoring trapping force from equilibrium trapping location) was analyzed using 4.42 μm particles and 1 MHz of an alternating electric field from 6 V P-P to 10 V P-P . The trap stiffness increased exponentially in the x- and y-direction, and linearly in the z-direction. Image analysis of the trapped particle movements revealed that the trap stiffness is increased 608.4, 539.3, and 79.7% by increasing the voltage from 6 V P-P to 10 V P-P in the x-, y-, and z-direction, respectively. The trap stiffness calculated from a finite element simulation of the device confirmed the experimental results. This analysis provides important insights to predict the trapping location, strength of the trapping, and optimum geometry for single particle trapping and its applications such as single-molecule analysis and drug discovery., (© 2020 Wiley-VCH GmbH.)

The increasing resolution of three-dimensional (3D) printing offers simplified access to, and development of, microfluidic devices with complex 3D structures. Therefore, this technology is increasingly used for rapid prototyping in laboratories and industry. Microfluidic free flow electrophoresis (μFFE) is a versatile tool to separate and concentrate different samples (such as DNA, proteins, and cells) to different outlets in a time range measured in mere tens of seconds and offers great potential for use in downstream processing, for example. However, the production of μFFE devices is usually rather elaborate. Many designs are based on chemical pretreatment or manual alignment for the setup. Especially for the separation chamber of a μFFE device, this is a crucial step which should be automatized. We have developed a smart 3D design of a μFFE to pave the way for a simpler production. This study presents (1) a robust and reproducible way to build up critical parts of a μFFE device based on high-resolution MultiJet 3D printing; (2) a simplified insertion of commercial polycarbonate membranes to segregate separation and electrode chambers; and (3) integrated, 3D-printed wells that enable a defined sample fractionation (chip-to-world interface). In proof of concept experiments both a mixture of fluorescence dyes and a mixture of amino acids were successfully separated in our 3D-printed μFFE device., (© 2020 The Authors. Electrophoresis published by Wiley-VCH GmbH.)

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1 b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1 b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off ) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

In molecular biology laboratories, many tasks require fine motor control and high acuity vision. For example, lab technicians with visual impairment experience difficulty loading samples into the small wells of a horizontal agarose gel. We have developed a 3D-printable gel loading system which allows technicians with low-contrast vision to load gels correctly. It includes a casting tray, a bridge, and a modified comb. The system provides a high-contrast visual field to improve visibility, and the bridge allows pipette tips to be inserted at the correct location and only to the correct depth. The necessary computer files for printing this device are freely available to increase the accessibility of molecular biology laboratories to people with visual impairment.

A three-dimensional-printed microfluidic device made of a thermoplastic material was used to study the creation of molecular filters by controlled dielectric breakdown. The device was made from acrylonitrile butadiene styrene by a fused deposition modeling three-dimensional printer and consisted of two V-shaped sample compartments separated by 750 µm of extruded plastic gap. Nanofractures were formed in the thin piece of acrylonitrile butadiene styrene by controlled dielectric breakdown by application voltage of 15-20 kV with the voltage terminated when reaching a defined current threshold. Variation of the size of the nanofractures was achieved by both variation of the current threshold and by variation of the ionic strength of the electrolyte used for breakdown. Electrophoretic transport of two proteins, R-phycoerythrin (RPE; <10 nm in size) and fluorescamine-labeled BSA (f-BSA; 2-4 nm), was used to monitor the size and transport properties of the nanofractures. Using 1 mM phosphate buffer, both RPE and f-BSA passed through the nanofractures when the current threshold was set to 25 µA. However, when the threshold was lowered to 10 µA or lower, RPE was restricted from moving through the nanofractures. When we increased the electrolyte concentration during breakdown from 1 to 10 mM phosphate buffer, BSA passed but RPE was blocked when the threshold was equal to, or lower than, 25 µA. This demonstrates that nanofracture size (pore area) is directly related to the breakdown current threshold but inversely related to the concentration of the electrolyte used for the breakdown process., (© 2020 Wiley-VCH GmbH.)

Direct-current insulator-based electrokinetics (DC-iEK) is a branch of microfluidics that has demonstrated to be an attractive and efficient technique for manipulating micro- and nano- particles, including microorganisms. A unique feature of DC-iEK devices is that nonlinear EK effects are enhanced by the presence of regions of higher field intensity between the insulating structures. Accurate computational models, describing particle and cell behavior, are crucial to optimize the design and improve the performance of DC-iEK devices. The electrokinetic equilibrium condition ( E EEC ) is a recently introduced fundamental concept that has radically shifted the perspective behind the analysis of particle manipulation in these microfluidic devices. The E EEC takes into consideration previously neglected nonlinear effects on particle migration and indicates that these effects are central to control particle motion in DC-iEK devices. In this study, we present a simultaneous experimental characterization of linear and nonlinear electrokinetic (EK) parameters, that is, the electrophoretic mobility (μ EP (1) ), the particle zeta potential (ζ P ), the E EEC , and the electrophoretic mobility of the second kind (μ EP (3) ), for four types of polystyrene microparticles and four cell strains. For this, we studied the electromigration of polystyrene microparticles ranging in size from 2 to 6.8 μm, three bacteria strains ( B. cereus , E. coli , and S. enterica ) and a yeast cell ( S. cerevisiae ), ranging in size from 1 to 6.3 μm, in a polydimethylsiloxane (PDMS) microfluidic channel with a rectangular cross-section. The results illustrated that electrokinetic particle trapping can occur by linear and nonlinear electrophoresis and electroosmosis reaching an equilibrium, without the presence of insulating posts. The experimentally measured parameters reported herein will allow optimizing the design of future DC-iEK devices for a wide range of applications (e.g., to separate multiple kinds of particles and microorganisms) and for developing computational models that better represent reality.

The growing demand for rapid, portable, and economical detection methods for environmental analysis has resulted in increasing demands on the portability and miniaturization of analytical instruments. The miniaturization of scientific instruments facilitates analysis in the field of medicine, food, and environment, especially for the under-resourced areas. The gel electrophoresis devices currently available for protein separation are primarily used in laboratories. Miniaturized instruments that can be used for on-site and rapid separation of protein have not yet been reported. In this study, a portable gel electrophoresis device for rapid separation and detection of proteins was developed and manufactured by 3D printing in a laboratory, which was economical, convenient, and quick. First, four kinds of portable gel electrophoresis devices that included three kinds of columnar gel and one slab gel electrophoresis device were designed with computer-aided design software SolidWorks 2017 (Dassault Systemes SE, France); the components including gel tubes, gel plates, and gel electrophoresis tanks were then printed using a 3D printer after optimization of the printing parameters. Then, the performance of the four kinds of gel electrophoresis devices was investigated using prestained protein molecular weight standards. The results showed that the single-channel slab gel electrophoresis design can quickly separate proteins with the best separation efficiency. Moreover, the effect of different separation gel lengths (5, 10, 15, and 20 mm) on protein separation was studied and it was found that 10% separation gels with a length of 5 mm could effectively separate prestained protein molecular weight standards (in the range of 15-250 kD) in 20 minutes. Next, the battery was optimized for the portable GE device and a 25 V lithium battery (70 mm×60 mm×40 mm) was used as the power supply, which could provide a constant voltage of 25 V for 100 hours during gel electrophoresis. Then, the One-Step Blue TM reagent (Biotium, USA) was used to color the separation results of the five standard proteins (carbonic anhydrase, ovalbumin, bovine serum albumin, conalbumin, ribonuclease A), and the results were recorded by mobile phone. Finally, the proposed gel electrophoresis device was compared with the commercial device. The results showed that the two devices are comparable; however, the slab gel electrophoresis was faster, portable, and economical. In summary, this research designed and manufactured a portable gel electrophoresis device using 3D printing technique, which can be used for on-site analysis and detection of proteins. The device presents the following advantages compared with the commercial devices:1) small and portable:the size of the electrophoresis tank of the device is only 15 mm×20 mm×17 mm and the 25 V lithium battery has a working time of approximately 100 hours; 2) low cost:it can be processed in 5 hours using 3D printing technology, with 10 mL of printing material while the total cost is less than 400 RMB; 3) fast separation:this device can quickly achieve protein separation compared with commercial devices and can further use multiple electrophoresis tanks in parallel to analyze more samples at the same time. Besides, this research also highlights the advantages of 3D printing for the development of miniaturized analytical equipment. Though this study has achieved preliminary results for rapid separation of proteins using gel electrophoresis devices, the quantitative analysis of proteins following protein detection and the application of more samples need further research. Meanwhile, the continued application of 3D printing technology will promote the development of miniaturized and portable experimental equipment.

Many cellular functions are affected by and thus can be characterized by a cell's electrophysiology. This has also been found to correspond to other biophysical parameters such as cell morphology and mechanical properties. Dielectrophoresis (DEP) is an electrostatic technique which can be used to examine cellular biophysical parameters through the measuring of single or multiple cell response to electric field induced forces. This label-free method offers many advantages in characterizing a cell population over conventional electrophysiology methods such as patch clamping; however, it has yet to see mainstream pharmacological application. Challenges such as the transdisciplinary nature of the field bridging engineering and the biological sciences, throughput, specificity as well as standardization are being addressed in current literature. This review focuses on the developments of DEP-based cell electrophysiological characterization where determining cellular properties such as membrane conductance and capacitance, and cytoplasmic conductivity are the primary motivation. A brief theoretical review, techniques for obtaining these cell parameters, as well as the resulting cell parameters and their applications are included in this review. This review aims to further support the development of DEP-based cell characterization as an important part of the future of DEP and electrophysiology research., (© 2020 Wiley-VCH GmbH.)

The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 10 5 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte.

This study describes an inexpensive and nonconventional soft-embossing protocol to produce microfluidic devices in poly(methyl methacrylate) (PMMA). The desirable microfluidic structure was photo-patterned in a poly(vinyl acetate) (PVAc) film deposited on glass substrate to produce a low-relief master. Then, this template was used to generate a high-relief pattern in stiffened PDMS by increasing of curing agent /monomer ratio (1:5) followed by thermal aging in a laboratory oven (200°C for 24 h). The stiffened PDMS masters were used to replicate microfluidic devices in PMMA based on soft embossing at 220-230°C and thermal sealing at 140°C. Both embossing and sealing stages were performed by using binder clips. The proposed protocol has ensured the replication of microfluidic devices in PMMA with great fidelity (>94%). Examples of MCE devices, droplet generator devices and spot test array were successfully demonstrated. For testing MCE devices, a mixture containing inorganic cations was selected as model and the achieved analytical performance did not reveal significant difference from commercial PMMA devices. Water droplets were successfully generated in an oil phase at rate of ca. 60 droplets/min (fixing the continuous phase flow rate at 100 μL/h) with size of ca. 322 ± 6 μm. Glucose colorimetric assay was performed on spot test devices and good detectability level (5 μmol/L) was achieved. The obtained results for two artificial serum samples revealed good agreement with the certified concentrations. Based on the fabrication simplicity and great analytical performance, the proposed soft-embossing protocol may emerge as promising approach for manufacturing PMMA devices., (© 2020 Wiley-VCH GmbH.)

Chemical surface patterning can be an incredibly powerful tool in a variety of applications, as it enables precise spatial control over surface properties. But the equipment required to create functional surface patterns-especially "grayscale" patterns where independent control over species placement and density are needed-is often expensive and inaccessible. In this work, we leveraged equipment and methods readily available to many research labs, namely 3D printing and electroblotting, to generate controlled grayscale surface patterns. Three-dimensional-printed molds were used to cast polyacrylamide hydrogels with regions of variable polymer density; regions of low polymer density within the hydrogels served as reservoirs for proteins that were later driven onto a target surface using electrophoresis. This mechanism was used to deposit grayscale patterns of fluorescently labeled proteins, and the fluorescent intensity of these patterns was measured and compared to a theoretical analysis of the deposition mechanism., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Though the advances in microelectronic device fabrication have realized new capabilities in integrated analytical and diagnostic platforms, there are still notable limitations in point-of-care sample preparation. AC electrokinetic devices, especially those leveraging dielectrophoresis (DEP), have shown potential to solve these limitations and allow for sample-to-answer in a single point-of-care device. However, when working directly with whole blood or other high conductance (~ 1 S/m) biological fluids, the aggressive electrochemical conditions created by the electrode can fundamentally limit the device operation. In this study, platinum wire-based electrode devices spanning circular polytetrafluorethylene (PTFE) wells and a planar microarray device with sputtered platinum electrodes were tested in plasma and PBS buffers of differing concentration across a wide range of frequencies and electric field intensities (AC voltages) to determine their respective safe regions of operation and to gain an understanding about the failure mechanisms of this class of device. At frequencies of 10 kHz and below, the upper bound of operation is the degradation of electrodes due to electrochemical attack by chlorine overcoming the native platinum oxide passivation. At higher frequencies, 100 kHz and above, the dielectric loss and subsequent heating of the buffer will boil before the electrodes suffer observable damage, due to the slow irreversible reaction kinetics. Effective dielectrophoretic capture of small biological particles at these frequencies is limited, and heat/oxidative denaturation of target material are a major concern. A new class of smaller devices, ones capable of high throughput at voltages low enough to maintain the integrity of the platinum passivation layer, is needed to mitigate these fundamental limitations.

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.

This paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In an open microfluidic device, we investigate protein polyacrylamide gel electrophoresis (PAGE) separation performance on single-cell lysate. Single-cell protein electrophoresis is performed in a thin layer of polyacrylamide (PA) gel into which microwells are molded. Individual cells are isolated in these open microwells, then lysed on-chip with a dual lysis and electrophoresis sodium dodecyl sulfate (SDS) buffer. We scrutinize the effect of sieving gel composition on electromigration of protein targets, using a wide range of cellular protein standards (36 kDa to 289 kDa). We find that as PA concentration increases, protein electromigration deviates from the empirical log-linear relationship predicted between migration distance and molecular mass. We perform Ferguson analysis to calculate retardation coefficients and free solution mobilities of nine cellular protein standards and observe that the largest-molecular-mass protein, mTOR (289 kDa), does not behave as predicted by established linear-fit models for SDS-denatured proteins, indicating that mTOR is beyond the linear range of this assay. Lastly, we performed in-gel immunoprobing on the single-cell electrophoretic separations and observed that smaller pore-size gels (higher gel concentration) reduce protein diffusion out of the gel, which does not notably impact the measured immunoprobed protein expression. Compared to larger pore-size gels, the smaller pore-size gels lead to higher local concentrations of the target protein in each protein band, resulting in an increase in the signal-to-noise ratio (SNR) for each protein. Understanding the separation and immunoprobing performance at different gel concentrations improves assay design and optimization for target proteins.

In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius-Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)