Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells

In this paper, a new dielectrophoresis (DEP) method based on capture voltage spectrum is proposed for measuring dielectric properties of biological cells. The capture voltage spectrum can be obtained from the balance of dielectrophoretic force and Stokes drag force acting on the cell in a microfluidic device with fluid flow and strip electrodes. The method was demonstrated with the measurement of dielectric properties of human colon cancer cells (HT-29 cells). From the capture voltage spectrum, the real part of Clausius-Mossotti factor of HT-29 cells for different frequencies of applied electric field was obtained. The dielectric properties of cell interior and plasma membrane were then estimated by using single-shell dielectric model. The cell interior permittivity and conductivity were found to be insensitive to changes in the conductivity of the medium in which the cells are suspended, but the measured permittivity and conductivity of cell membrane were found to increase with the increase of medium conductivity. In addition, the measurement of capture voltage spectrum was found to be useful in providing the optimum operating conditions for separating HT-29 cells from other cells (such as red blood cells) using dielectrophoresis.     

Applications of electrowetting-on-dielectric (EWOD) technology for droplet digital PCR

Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade.     

Study of flow rate induced measurement error in flow-through nano-hole plasmonic sensor

Flow-through gold film perforated with periodically arrayed sub-wavelength nano-holes can cause extraordinary optical transmission (EOT), which has recently emerged as a label-free surface plasmon resonance sensor in biochemical detection by measuring the transmission spectral shift. This paper describes a systematic study of the effect of microfluidic field on the spectrum of EOT associated with the porous gold film. To detect biochemical molecules, the sub-micron-thick film is free-standing in a microfluidic field and thus subject to hydrodynamic deformation. The film deformation alone may cause spectral shift as measurement error, which is coupled with the spectral shift as real signal associated with the molecules. However, this microfluid-induced measurement error has long been overlooked in the field and needs to be identified in order to improve the measurement accuracy. Therefore, we have conducted simulation and analytic analysis to investigate how the microfluidic flow rate affects the EOT spectrum and verified the effect through experiment with a sandwiched device combining Au/Cr/Si₃N₄ nano-hole film and polydimethylsiloxane microchannels. We found significant spectral blue shift associated with even small flow rates, for example, 12.60 nm for 4.2 μl/min. This measurement error corresponds to 90 times the optical resolution of the current state-of-the-art commercially available spectrometer or 8400 times the limit of detection. This really severe measurement error suggests that we should pay attention to the microfluidic parameter setting for EOT-based flow-through nano-hole sensors and adopt right scheme to improve the measurement accuracy.     

Spatial concentration distribution analysis of cells in electrode-multilayered microchannel by dielectric property measurement

The spatial concentration distribution of cells in a microchannel is measured by combining the dielectric properties of cells with the specific structure of the electrode-multilayered microchannel. The dielectric properties of cells obtained with the impedance spectroscopy method includes the cell permittivity and dielectric relaxation, which corresponds to the cell concentration and structure. The electrode-multilayered microchannel is constructed by 5 cross-sections, and each cross-section contains 5 electrode-layers embedded with 16 micro electrodes. In the experiment, the dielectric properties of cell suspensions with different volume concentrations are measured with different electrode-combinations corresponding to different electric field distributions. The dielectric relaxations of different cell concentrations are compared and discussed with the Maxwell-Wagner dispersion theory, and the relaxation frequencies are analysed by a cell polarization model established based on the Hanai cell model. Moreover, a significant linear relationship with AC frequency dependency between relative permittivity and cell concentration was found, which provides a promising way to on-line estimate cell concentration in microchannel. Finally, cell distribution in 1 cross-section of the microchannel (X and Y directions) was measured with different electrode-combinations using the dielectric properties of cell suspensions, and cell concentration distribution along the microchannel (Z direction) was visualized at flowing state. The present cell spatial sensing study provides a new approach for 3 dimensional non-invasive online cell sensing for biological industry.     

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).     

Microwave frequency sensor for detection of biological cells in microfluidic channels

We present details of an apparatus for capacitive detection of biomaterials in microfluidic channels operating at microwave frequencies where dielectric effects due to interfacial polarization are minimal. A circuit model is presented, which can be used to adapt this detection system for use in other microfluidic applications and to identify ones where it would not be suitable. The detection system is based on a microwave coupled transmission line resonator integrated into an interferometer. At 1.5 GHz the system is capable of detecting changes in capacitance of 650 zF with a 50 Hz bandwidth. This system is well suited to the detection of biomaterials in a variety of suspending fluids, including phosphate-buffered saline. Applications involving both model particles (polystyrene microspheres) and living cells—baker’s yeast (Saccharomyces cerevisiae) and Chinese hamster ovary cells—are presented.     

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

A combination of a microfluidic device with a light modulation system was developed to detect the oxygen consumption rate (OCR) of a single developing zebrafish embryo via phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of glass microwells containing Pt(II) octaethylporphyrin as an oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest. The total basal respiration (OCR, in pmol O₂/min/embryo) of a single developing zebrafish embryo inside a sealed microwell has been successfully measured from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage (48 hpf). The total basal respiration increased in a linear and reproducible fashion with embryonic age. Sequentially adding pharmacological inhibitors of bioenergetic pathways allows us to perform respiratory measurements of a single zebrafish embryo at key developmental stages and thus monitor changes in mitochondrial function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf inside a microfluidic device. The total basal respiration is partitioned into the non-mitochondrial respiration, mitochondrial respiration, respiration due to adenosine triphosphate (ATP) turnover, and respiration due to proton leak. The changes in these respirations are correlated with zebrafish embryonic development stages. Our proposed platform provides the potential for studying bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate mitochondrial physiology and disease.     

Sealing SU-8 microfluidic channels using PDMS

A simple method of irreversibly sealing SU-8 microfluidic channels using PDMS is reported in this paper. The method is based on inducing a chemical reaction between PDMS and SU-8 by first generating amino groups on PDMS surface using N₂ plasma treatment, then allowing the amino groups to react with the residual epoxy groups on SU-8 surface at an elevated temperature. The N₂ plasma treatment of PDMS can be conducted using an ordinary plasma chamber and high purity N₂, while the residual epoxy groups on SU-8 surface can be preserved by post-exposure baking SU-8 at a temperature no higher than 95 °C. The resultant chemical bonding between PDMS and SU-8 using the method create an interface that can withstand a stress that is greater than the bulk strength of PDMS. The bond is permanent and is long-term resistant to water. The method was applied in fabricating SU-8 microfluidi-photonic integrated devices, and the obtained devices were tested to show desirable performance.     

Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

Human pancreatic islets are seldom assessed for dynamic responses to external stimuli. Thus, the elucidation of human islet functionality would provide insights into the progression of diabetes mellitus, evaluation of preparations for clinical transplantation, as well as for the development of novel therapeutics. The objective of this study was to develop a microfluidic platform for in vitro islet culture, allowing the multi-parametric investigation of islet response to chemical and biochemical stimuli. This was accomplished through the fabrication and implementation of a microfluidic platform that allowed the perifusion of islet culture while integrating real-time monitoring using impedance spectroscopy, through microfabricated, interdigitated electrodes located along the microchamber arrays. Real-time impedance measurements provide important dielectric parameters, such as cell membrane capacitance and cytoplasmic conductivity, representing proliferation, differentiation, viability, and functionality. The perifusion of varying glucose concentrations and monitoring of the resulting impedance of pancreatic islets were performed as proof-of-concept validation of the lab-on-chip platform. This novel technique to elucidate the underlying mechanisms that dictate islet functionality is presented, providing new information regarding islet function that could improve the evaluation of islet preparations for transplantation. In addition, it will lead to a better understanding of fundamental diabetes-related islet dysfunction and the development of therapeutics through evaluation of potential drug effects.     

Implementation of tetra-poly(ethylene glycol) hydrogel with high mechanical strength into microfluidic device technology

Hydrogels have several excellent characteristics suitable for biomedical use such as softness, biological inertness and solute permeability. Hence, integrating hydrogels into microfluidic devices is a promising approach for providing additional functions such as biocompatibility and porosity, to microfluidic devices. However, the poor mechanical strength of hydrogels has severely limited device design and fabrication. A tetra-poly(ethylene glycol) (tetra-PEG) hydrogel synthesized recently has high mechanical strength and is expected to overcome such a limitation. In this research, we have comprehensively studied the implementation of tetra-PEG gel into microfluidic device technology. First, the fabrication of tetra-PEG gel/PDMS hybrid microchannels was established by developing a simple and robust bonding technique. Second, some fundamental features of tetra-PEG gel/PDMS hybrid microchannels, particularly fluid flow and mass transfer, were studied. Finally, to demonstrate the unique application of tetra-PEG-gel-integrated microfluidic devices, the generation of patterned chemical modulation with the maximum concentration gradient: 10% per 20 μm in a hydrogel was performed. The techniques developed in this study are expected to provide fundamental and beneficial methods of developing various microfluidic devices for life science and biomedical applications.     

Light-driven directional ion transport for enhanced osmotic energy harvesting

Light-driven ion (proton) transport is a crucial process both for photosynthesis of green plants and solar energy harvesting of some archaea. Here, we describe use of a TiO₂/C₃N₄ semiconductor heterojunction nanotube membrane to realize similar light-driven directional ion transport performance to that of biological systems. This heterojunction system can be fabricated by two simple deposition steps. Under unilateral illumination, the TiO₂/C₃N₄ heterojunction nanotube membrane can generate a photocurrent of about 9 μA/cm², corresponding to a pumping stream of ∼5500 ions per second per nanotube. By changing the position of TiO₂ and C₃N₄, a reverse equivalent ionic current can also be realized. Directional transport of photogenerated electrons and holes results in a transmembrane potential, which is the basis of the light-driven ion transport phenomenon. As a proof of concept, we also show that this system can be used for enhanced osmotic energy generation. The artificial light-driven ion transport system proposed here offers a further step forward on the roadmap for development of ionic photoelectric conversion and integration into other applications, for example water desalination.     

An integrated actuating and sensing system for light-addressable potentiometric sensor (LAPS) and light-actuated AC electroosmosis (LACE) operation

To develop a lab on a chip (LOC) integrated with both sensor and actuator functions, a novel two-in-one system based on optical-driven manipulation and sensing in a microfluidics setup based on a hydrogenated amorphous silicon (a-Si:H) layer on an indium tin oxide/glass is first realized. A high-intensity discharge xenon lamp functioned as the light source, a chopper functioned as the modulated illumination for a certain frequency, and a self-designed optical path projected on the digital micromirror device controlled by the digital light processing module was established as the illumination input signal with the ability of dynamic movement of projected patterns. For light-addressable potentiometric sensor (LAPS) operation, alternating current (AC)-modulated illumination with a frequency of 800 Hz can be generated by the rotation speed of the chopper for photocurrent vs bias voltage characterization. The pH sensitivity, drift coefficient, and hysteresis width of the Si₃N₄ LAPS are 52.8 mV/pH, −3.2 mV/h, and 10.5 mV, respectively, which are comparable to the results from the conventional setup. With an identical two-in-one system, direct current illumination without chopper rotation and an AC bias voltage can be provided to an a-Si:H chip with a manipulation speed of 20 μm/s for magnetic beads with a diameter of 1 μm. The collection of magnetic beads by this light-actuated AC electroosmosis (LACE) operation at a frequency of 10 kHz can be easily realized. A fully customized design of an illumination path with less decay can be suggested to obtain a high efficiency of manipulation and a high signal-to-noise ratio of sensing. With this proposed setup, a potential LOC system based on LACE and LAPS is verified with the integration of a sensor and an actuator in a microfluidics setup for future point-of-care testing applications.     

Characterizing the dielectric properties of human mesenchymal stem cells and the effects of charged elastin-like polypeptide copolymer treatment

Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.     

Electrodes on a budget: Micropatterned electrode fabrication by wet chemical deposition

Precise patterning of metals is required for diverse microfluidic and microelectromechanical system (MEMS) applications ranging from the separation of proteins to the manipulation of single cells and drops of water-in-oil emulsions. Here we present a very simple, inexpensive method for fabricating micropatterned electrodes. We deposit a thin metal layer of controlled thickness using wet chemistry, thus eliminating the need for expensive equipment typically required for metal deposition. We demonstrate that the resulting deposited metal can be used to fabricate functional electrodes: The wet-deposited metal film can sustain patterning by photolithography down to micron-sized features required for MEMS and microfluidic applications, and its properties are suitable for operative electrodes used in a wide range of microfluidic applications for biological studies.     

A novel actuation method of transporting droplets by using electrical charging of droplet in a dielectric fluid

We evaluate the feasibility of manipulating droplets in two dimensions by exploiting Coulombic forces acting on conductive droplets immersed in a dielectric fluid. When a droplet suspended in an immiscible fluid is located near an electrode under a dc voltage, the droplet can be charged by direct contact, by charge transfer along an electrically conducting path, or by both mechanisms. This process is called electrical charging of droplet (ECOD). This charged droplet may then be transported rapidly by exploiting Coulombic forces. We experimentally demonstrate electrical actuation of a charged droplet by applying voltage sequences. A charged droplet is two dimensionally actuated by following the direction of the electrical field signal. The droplet does not contact the surface of the microfluidic chip when it moves. This characteristic is very advantageous because treatments of the substrate surfaces of microfluidic chip become simpler. In order to test the feasibility of using ECOD in a droplet-based microreactor, electrocoalescence of two oppositely charged droplets is also studied. When two droplets approach each other due to Coulombic attraction, a liquid bridge is formed between them. We postulate that if the applied electric field is weaker than a certain critical level, the two droplets coalesce instantaneously when the charges are exchanged and redistributed through this liquid bridge.     

Superconductivity in chromium nitrides Pr3Cr10-xN11 with strong electron correlations

Exploration of superconductivity in Cr-based compounds has attracted considerable interest because only a few Cr-based superconductors (CrAs, A₂Cr₃As₃ and ACr₃As₃ (A = K, Rb, Cs, Na)) have been discovered so far and they show an unconventional pairing mechanism. We report the discovery of bulk superconductivity at 5.25 K in chromium nitride in Pr₃Cr_10-xN₁₁ with a cubic lattice structure. A relatively large upper critical field of H_c2(0) ∼ 12.6 T is determined, which is larger than the estimated Pauli-paramagnetic pair-breaking magnetic field. The material has a large electronic specific-heat coefficient of 170 mJ K⁻² mol⁻¹—about 10 times larger than that estimated by the electronic structure calculation, which suggests that correlations between 3d electrons are very strong in Pr₃Cr_10-xN₁₁, and thus quantum fluctuations might be involved. Electronic structure calculations show that the density of states at the Fermi energy are contributed predominantly by Cr 3d electrons, implying that the superconductivity results mainly from the condensation of Cr 3d electrons. Pr₃Cr_10-xN₁₁ represents a rare example of possible unconventional superconductivity emerging in a 3D system with strong electron correlations. Nevertheless, clarification of the specific pairing symmetry needs more investigation.     

Electric field assisted manipulation of microdroplets on a superhydrophobic surface

The efficient manipulation of low-volume droplets offers many potential applications in relation to chemical and biomedical tests and protocols. A novel approach to the manipulation of a microdroplet on a superhydrophobic surface is introduced in the present communication. The microdroplet was first picked up onto a hydrophilic needle, transported from one location to another, and finally released under the action of an electric field force. Three key parameters in this process, the radius of the droplet, the distance between the two electrodes, and the required voltage, were investigated. This study should be helpful for the design of microfluidic devices.     

Ultrafast growth of large single crystals of monolayer WS2 and WSe2

Monolayer transition metal dichalcogenides (TMDs) have attracted considerable attention as atomically thin semiconductors for the ultimate transistor scaling. For practical applications in integrated electronics, large monolayer single crystals are essential for ensuring consistent electronic properties and high device yield. The TMDs available today are generally obtained by mechanical exfoliation or chemical vapor deposition (CVD) growth, but are often of mixed layer thickness, limited single crystal domain size or have very slow growth rate. Scalable and rapid growth of large single crystals of monolayer TMDs requires maximization of lateral growth rate while completely suppressing the vertical growth, which represents a fundamental synthetic challenge and has motivated considerable efforts. Herein we report a modified CVD approach with controllable reverse flow for rapid growth of large domain single crystals of monolayer TMDs. With the use of reverse flow to precisely control the chemical vapor supply in the thermal CVD process, we can effectively prevent undesired nucleation before reaching optimum growth temperature and enable rapid nucleation and growth of monolayer TMD single crystals at a high temperature that is difficult to attain with use of a typical thermal CVD process. We show that monolayer single crystals of 450 μm lateral size can be prepared in 10 s, with the highest lateral growth rate up to 45 μm/s. Electronic characterization shows that the resulting monolayer WSe₂ material exhibits excellent electronic properties with carrier mobility up to 90 cm² V⁻¹ s⁻¹, comparable to that of the best exfoliated monolayers. Our study provides a robust pathway for rapid growth of high-quality TMD single crystals.     

Solution pH change in non-uniform alternating current electric fields at frequencies above the electrode charging frequency

AC Faradaic reactions have been reported as a mechanism inducing non-ideal phenomena such as flow reversal and cell deformation in electrokinetic microfluidic systems. Prior published work described experiments in parallel electrode arrays below the electrode charging frequency (f_c), the frequency for electrical double layer charging at the electrode. However, 2D spatially non-uniform AC electric fields are required for applications such as in plane AC electroosmosis, AC electrothermal pumps, and dielectrophoresis. Many microscale experimental applications utilize AC frequencies around or above f_c. In this work, a pH sensitive fluorescein sodium salt dye was used to detect [H⁺] as an indicator of Faradaic reactions in aqueous solutions within non-uniform AC electric fields. Comparison experiments with (a) parallel (2D uniform fields) electrodes and (b) organic media were employed to deduce the electrode charging mechanism at 5 kHz (1.5f_c). Time dependency analysis illustrated that Faradaic reactions exist above the theoretically predicted electrode charging frequency. Spatial analysis showed [H⁺] varied spatially due to electric field non-uniformities and local pH changed at length scales greater than 50 μm away from the electrode surface. Thus, non-uniform AC fields yielded spatially varied pH gradients as a direct consequence of ion path length differences while uniform fields did not yield pH gradients; the latter is consistent with prior published data. Frequency dependence was examined from 5 kHz to 12 kHz at 5.5 V_pp potential, and voltage dependency was explored from 3.5 to 7.5 V_pp at 5 kHz. Results suggest that Faradaic reactions can still proceed within electrochemical systems in the absence of well-established electrical double layers. This work also illustrates that in microfluidic systems, spatial medium variations must be considered as a function of experiment time, initial medium conditions, electric signal potential, frequency, and spatial position.     

Continuous sheath-free magnetic separation of particles in a U-shaped microchannel

Particle separation is important to many chemical and biomedical applications. Magnetic field-induced particle separation is simple, cheap, and free of fluid heating issues that accompany electric, acoustic, and optical methods. We develop herein a novel microfluidic approach to continuous sheath-free magnetic separation of particles. This approach exploits the negative or positive magnetophoretic deflection to focus and separate particles in the two branches of a U-shaped microchannel, respectively. It is applicable to both magnetic and diamagnetic particle separations, and is demonstrated through the sorting of 5 μm and 15 μm polystyrene particles suspended in a dilute ferrofluid.