Hydroelectric actuator for 3-dimensional analysis of electrophoretic and dielectrophoretic behavior of cancer cells; suitable in diagnosis and invasion studies.

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).

Development and clinical application of hydrogel formulations containing papain and urea for wound healing

Abstract Hydrogels are used for wound treatment, as they may contain one or more active components and protect the wound bed. Papain is one of the active substances that have been used with this purpose, alongside urea. In this paper, carboxypolymethylene hydrogels containing papain (2% and 10% concentrations) and urea (5% concentration) were produced. Physical-chemical stability was performed at 0, 7, 15 and 30 days at 2-8ºC, 25ºC and 40ºC, as well as the rheological aspects and proteolytic activity of papain by gel electrophoresis. Clinical efficacy of the formulations in patients with lower limb ulcers was also evaluated in a prospective, single-center, randomized, double-blind and comparative clinical trial. The results showed 7-day stability for the formulations under 25ºC, in addition to approximately 100% and 15% of protein activity for 10% and 2% papain hydrogel, respectively. The rheological profile was non-Newtonian for the 10% papain hydrogel tested. There were no significant differences regarding the mean time for healing of the lesions, although 10% papain presented a better approach to be used in all types of tissue present in the wound bed.

Novel electroosmotic micromixer configuration based on ion-selective microsphere.

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.

Rapid detection and enumeration of aerobic mesophiles in raw foods using dielectrophoresis.

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 (R2 > 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.

Rapid isolation method of Saccharomyces cerevisiae based on optically induced dielectrophoresis technique for fungal infection diagnosis.

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.

Miniaturized free-flow electrophoresis: production, optimization, and application using 3D printing technology.

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.

3D-printed assistive pipetting system for gel electrophoresis for technicians with low acuity vision.

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.

Joule heating-enabled electrothermal enrichment of nanoparticles in insulator-based dielectrophoretic microdevices.

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.

Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor.

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-1b 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-1b 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.

Toward low-voltage dielectrophoresis-based microfluidic systems: A review.

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.

Quantitative analysis of the three-dimensional trap stiffness of a dielectrophoretic corral trap.

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 VP-P to 10 VP-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 VP-P to 10 VP-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.

Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot.

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 105 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.

Simultaneous Determination of Linear and Nonlinear Electrophoretic Mobilities of Cells and Microparticles.

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 (EEEC) is a recently introduced fundamental concept that has radically shifted the perspective behind the analysis of particle manipulation in these microfluidic devices. The EEEC 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 (µEP(1)), the particle zeta potential (ζP), the EEEC, and the electrophoretic mobility of the second kind (µEP(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 influence of electrolyte concentration on nanofractures fabricated in a 3D-printed microfluidic device by controlled dielectric breakdown.

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.

Review: Dielectrophoresis in cell characterization.

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.

Inexpensive and nonconventional fabrication of microfluidic devices in PMMA based on a soft-embossing protocol.

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.

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

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

Grayscale surface patterning using electrophoretic motion through a heterogeneous hydrogel material.

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.

Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device.

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.

Electrochemical attack and corrosion of platinum electrodes in dielectrophoretic diagnostic devices.

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.