Single-Cell-Derived Tumor-Sphere Formation and Drug-Resistance Assay Using an Integrated Microfluidics.

Considerable evidence points to cancer stem-like cells (CSCs) as responsible for promoting progression, metastasis, and drug resistance. Without damage to the cell biological properties, single-cell-derived tumor-sphere is encouraging options for CSCs identification and studies. Although several single cell-based microfluidic methods have been developed for CSCs studies, clarifying liaison between the biomechanics of cells (such as size and deformability) and stem (such as tumor-sphere formation and drug resistance) remains challenging. Herein, we present a platform of integrated microfluidics for the analysis of single-cell-derived tumor-sphere formation and drug resistance. Tumor-spheres derived from different biomechanics (size and/or deformation) single-cells could be formed efficiently using this device. To demonstrate the microfluidic-platform capability, a proof-of-concept experiment was implemented by evaluating single-cell-derived sphere formation of single glioblastoma cells with different biomechanics. Additionally, a course of chemotherapy to study these single-cell-derived spheres was determined by coculture with vincristine. The results indicate that tumor cell biomechanics is associated with single-cell-derived spheres formation; that is, smaller and/or more deformable tumor cells are more stem-like defined by the formation of single-cell-derived spheres than more prominent and/or lesser deformable tumor cells. Also, tumor-spheres derived from single small and/or more deformable tumor cell have higher drug resistance than more prominent and/or less deformable tumor cells. Our device offers a new approach for single-cell-derived sphere formation according to tumor cell different biomechanical properties. Furthermore, it offers a new method for CSC identification and downstream analysis on a single-cell level.

Determination of Aqueous Two-Phase System Binodals and Tie-Lines by Electrowetting-on-Dielectric Droplet Manipulation.

Handling the aqueous two-phase systems (ATPSs) formed by liquid-liquid phase separation (LLPS) relies on the accurate construction of binodal curves and tie-lines, which delineate the polymer concentrations required for phase separation and depict the properties of the resulting phases, respectively. Various techniques to determine the binodal curves and tie-lines of ATPSs exist, but most rely on manually pipetting relatively large volumes of fluids in a slow and tedious manner. We describe a method to determine ATPS binodals and tie-lines that overcomes these disadvantages: microscale droplet manipulation by electrowetting-on-dielectric (EWOD). EWOD enables automated handling of droplets in an optically transparent platform that allows for in situ droplet observation. Separated phases are clearly visible, and the volumes of each phase are readily determined. Additionally, in considering the molecular crowding present in living cells, this work examines the role of a macromolecule in prompting LLPS. These results show that EWOD-driven droplet manipulation effectively interrogates the phase dynamics of ATPSs and macromolecular crowding in LLPS.

Liver microsystems in vitro for drug response.

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Energy Harvester and Cell Proliferation from Biocompatible PMLG Nanofibers Prepared Using Near-Field Electrospinning and Electrospray Technology.

This paper describes the application of piezoelectric fibers and films formed using near-field electrospinning (NFES) and electrospray (ESP) technology. Poly(γ-methyl L-glutamate) (PMLG), a biocompatible material, was mixed with poly(ethylene oxide) (PEO) and surfactant to obtain a solution of appropriate viscosity and conductance. Because the orientation of the dipoles in PMLG was enhanced upon applying an electric field, we could use the NFES and ESP processes to align dipoles and enhance the piezoelectric properties of the resulting fibrous materials. The maximum peak voltage of a fiber-based harvester prepared using this approach was 0.056 V. Because the fibers and films were non-toxic biological materials displaying excellent piezoelectric characteristics, we deposited them on glass substrates coated with indium tin oxide to observe their effects on the proliferation of cells. The negative charge of PMLG decreased the proliferation of mouse fibroblast cells (NIH3T3); indeed, decreasing the interspacing between the fibers slightly decreased the proliferation of these cells. Moreover, the migration of the cells was inhibited significantly, or even halted, when the coverage of the ESP films increased, implying a growth inhibition effect.

Microfluidic Surface Plasmon Resonance Sensors: From Principles to Point-of-Care Applications.

Surface plasmon resonance (SPR) is a label-free, highly-sensitive, and real-time sensing technique. Conventional SPR sensors, which involve a planar thin gold film, have been widely exploited in biosensing; various miniaturized formats have been devised for portability purposes. Another type of SPR sensor which utilizes localized SPR (LSPR), is based on metal nanostructures with surface plasmon modes at the structural interface. The resonance condition is sensitive to the refractive index change of the local medium. The principles of these two types of SPR sensors are reviewed and their integration with microfluidic platforms is described. Further applications of microfluidic SPR sensors to point-of-care (POC) diagnostics are discussed.

Digital Microfluidics for Manipulation and Analysis of a Single Cell.

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

Digital Microfluidic Multiplex RT-qPCR for SARS-CoV-2 Detection and Variants Discrimination.

Continuous mutations have occurred in the genome of the SARS-CoV-2 virus since the onset of the COVID-19 pandemic. The increased transmissibility of the mutated viruses has not only imposed medical burdens but also prolonged the duration of the pandemic. A point-of-care (POC) platform that provides multitarget detection will help to track and reduce disease transmissions. Here we detected and discriminated three genotypes of SARS-CoV-2, including the wildtype and two variants of concern (VOCs), the Delta variant and Omicron variant, through reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a digital microfluidics (DMF)-based cartridge. Upon evaluating with the RNA samples of Omicron variant, the DMF RT-qPCR presented a sensitivity of 10 copies/µL and an amplification efficiency of 96.1%, capable for clinical diagnosis. When spiking with SARS-CoV-2 RNA (wildtype, Delta variant, or Omicron variant) and 18S rDNA, the clinical analog samples demonstrated accurate detection and discrimination of different SARS-CoV-2 strains in 49 min.

Determination of blood lithium-ion concentration using digital microfluidic whole-blood separation and preloaded paper sensors.

Existing microfluidic technologies for blood tests have several limitations, including difficulties in integrating the sample preparation steps, such as blood dilution, and precise metering of tiny samples (microliter) for accurate downstream analyses on a chip. Digital microfluidics (DMF) is a liquid manipulation technique that can provide precise volume control of micro or nano-liter liquid droplets. Without using sensitive but complex detection methods for tiny droplets involving fluorescence, luminescence, and electrochemistry, this article presents a DMF device with embedded paper-based sensors to detect blood lithium-ion (Li+) concentration by colorimetry. Dielectrophoresis on the DMF device between two parallel planar electrodes separates plasma droplets (from tens to hundreds of nanoliters in volume) from undiluted whole blood (a few microliters) within 4 min with an efficiency exceeding 90%. The embedded paper sensors contain a detection reagent to absorb the DMF-transported plasma droplets. These droplets change the color of the paper sensors in accordance with the Li+ concentration. Subsequently, colorimetry is used to reveal the Li+ concentration via image analysis. The proposed method meets the detection-sensitivity requirement for clinical diagnosis of bipolar disorder, making the DMF device a potential therapeutic tool for rapid whole-blood Li+ detection.

Digital Microfluidic qPCR Cartridge for SARS-CoV-2 Detection.

Point-of-care (POC) tests capable of individual health monitoring, transmission reduction, and contact tracing are especially important in a pandemic such as the coronavirus disease 2019 (COVID-19). We develop a disposable POC cartridge that can be mass produced to detect the SARS-CoV-2 N gene through real-time quantitative polymerase chain reaction (qPCR) based on digital microfluidics (DMF). Several critical parameters are studied and improved, including droplet volume consistency, temperature uniformity, and fluorescence intensity linearity on the designed DMF cartridge. The qPCR results showed high accuracy and efficiency for two primer-probe sets of N1 and N2 target regions of the SARS-CoV-2 N gene on the DMF cartridge. Having multiple droplet tracks for qPCR, the presented DMF cartridge can perform multiple tests and controls at once.

Pore-spanning lipid membrane under indentation by a probe tip: a molecular dynamics simulation study.

We study the indentation of a free-standing lipid membrane suspended over a nanopore on a hydrophobic substrate by means of molecular dynamics simulations. We find that in the course of indentation the membrane bends at the point of contact and the fringes of the membrane glide downward intermittently along the pore edges and stop gliding when the fringes reach the edge bottoms. The bending continues afterward, and the large strain eventually induces a phase transition in the membrane, transformed from a bilayered structure to an interdigitated structure. The membrane is finally ruptured when the indentation goes deep enough. Several local physical quantities in the pore regions are calculated, which include the tilt angle of lipid molecules, the nematic order, the included angle, and the distance between neighboring lipids. The variations of these quantities reveal many detailed, not-yet-specified local structural transitions of lipid molecules under indentation. The force-indentation curve is also studied and discussed. The results make a connection between the microscopic structure and the macroscopic properties and provide deep insight into the understanding of the stability of a lipid membrane spanning over nanopore.

Field-effect pump: liquid dielectrophoresis along a virtual microchannel with source-gate-drain electric fields.

We investigate liquid dielectrophoresis (LDEP) to implement field-effect pumps (FEPs) that drive liquids from source, via gate, toward drain electric fields between parallel plates without external pumps or the problem of dead volume. The appropriate gate electric field establishes a wall-less virtual microchannel to transfer the liquid from source to drain with an adjustable flow rate (Q) controlled by the difference of the square of the electric field strength (ΔE2DS). Analogous to field-effect transistors (FETs), the FEPs can operate in a "linear", "transition" or "saturation" region depending on ΔE2GD and ΔE2DS. With a sufficient ΔE2GD and a small ΔE2DS, the FEPs operated in the linear region where Q was linearly proportional to ΔE2DS and inversely proportional to the flow resistance R that was mainly determined by the length (L), width (W) and height (H) of a stable and fully-occupied virtual microchannel. With an insufficient ΔE2GD and a moderate to large ΔE2DS, narrowing, tapering and even pinch-off of virtual microchannels were observed, which increased R and changed the operation into the transition or saturation region. A field-effect stream merger regulating two streams was built based on two FEPs with shared gate and drain electrodes. The versatility of FEPs was demonstrated with preliminary studies on whole blood and particle solutions.

Microfluidics-Based Single-Cell Research for Intercellular Interaction.

Intercellular interaction between cell-cell and cell-ECM is critical to numerous biology and medical studies, such as stem cell differentiation, immunotherapy and tissue engineering. Traditional methods employed for delving into intercellular interaction are limited by expensive equipment and sophisticated procedures. Microfluidics technique is considered as one of the powerful measures capable of precisely capturing and manipulating cells and achieving low reagent consumption and high throughput with decidedly integrated functional components. Over the past few years, microfluidics-based systems for intercellular interaction study at a single-cell level have become frequently adopted. This review focuses on microfluidic single-cell studies for intercellular interaction in a 2D or 3D environment with a variety of cell manipulating techniques and applications. The challenges to be overcome are highlighted.

General digital microfluidic platform manipulating dielectric and conductive droplets by dielectrophoresis and electrowetting.

A general digital (droplet-based) microfluidic platform based on the study of dielectric droplet manipulation by dielectrophoresis (DEP) and the integration of DEP and electrowetting-on-dielectric (EWOD) is reported. Transporting, splitting, and merging dielectric droplets are achieved by DEP in a parallel-plate device, which expands the fluids of digital microfluidics from merely being conductive and aqueous to being non-conductive. In this work, decane, hexadecane, and silicone oil droplets were successfully transported in a 150 microm-high gap between two parallel plates by applying a DC voltage above threshold voltages. Non-volatile silicone oil droplets with viscosities of 20 and 50 cSt were studied in more detail in parallel-plate geometries with spacings of 75 microm, 150 microm, and 225 microm. The threshold voltages and the required driving voltages to achieve droplet velocities up to 4 mm/s in the different circumstances were measured. By adding a dielectric layer on the driving electrodes of the tested parallel-plate device, a general digital microfluidic platform capable of manipulating both dielectric and conductive droplets was demonstrated. DEP and EWOD, selectively generated by applying different signals on the same dielectric-covered electrodes, were used to drive silicone oil and water droplets, respectively. Concurrent transporting silicone oil and water droplets along an electrode loop, merging water and oil droplets, and transporting and separating the merged water-in-oil droplet were performed.

Reconfigurable liquid pumping in electric-field-defined virtual microchannels by dielectrophoresis.

Dielectrophoresis (DEP), widely used to generate body forces on suspended particles, is investigated to provide surface forces at the liquid-medium interfaces and pump a high-permittivity liquid in a low-permittivity medium along a virtual microchannel defined by an electric field between parallel plates. Because the pumping pressure is proportional to the square of the intensity of the electric field and independent of the channel width, DEP pumping is advantageous as the dimension of the microchannel shrinks down. The absence of the channel walls simplifies the fabrication processes and further increases its feasibility in nanofluidic applications. We demonstrate water pumping in an immiscible silicone oil medium at adjustable velocities by applying voltages above the threshold value whose square is linearly proportional to the cross-sectional aspect ratio (AR), i.e., the height to width ratio, of the microchannel. With a properly designed AR, liquid valve is achieved by appropriate voltage applications. Without the barriers of channel walls, merging multiple streams and capillary filling of the spacing between electric-field-defined virtual microchannels are observed and studied. Moreover, in situ reconfigurable liquid pumping is demonstrated by a four way switching valve on a programmable crossing electrode set.

Cross-scale electric manipulations of cells and droplets by frequency-modulated dielectrophoresis and electrowetting.

Two important electric forces, dielectrophoresis (DEP) and electrowetting-on-dielectric (EWOD), are demonstrated by dielectric-coated electrodes on a single chip to manipulate objects on different scales, which results in a dielectrophoretic concentrator in an EWOD-actuated droplet. By applying appropriate electric signals with different frequencies on identical electrodes, EWOD and DEP can be selectively generated on the proposed chip. At low frequencies, the applied voltage is consumed mostly in the dielectric layer and causes EWOD to pump liquid droplets on the millimetre scale. However, high frequency signals establish electric fields in the liquid and generate DEP forces to actuate cells or particles on the micrometre scale inside the droplet. For better performance of EWOD and DEP, square and strip electrodes are designed, respectively. Mammalian cells (Neuro-2a) and polystyrene beads are successfully actuated by a 2 MHz signal in a droplet by positive DEP and negative DEP, respectively. Droplet splitting is achieved by EWOD with a 1 kHz signal after moving cells or beads to one side of the droplet. Cell concentration, measured by a cell count chamber before and after experiments, increases 1.6 times from 8.6 x 10(5) cells ml(-1) to 1.4 x 10(6) cells ml(-1) with a single cycle of positive DEP attraction. By comparing the cutoff frequency of the voltage drop in the dielectric layer and the cross-over frequency of Re(fCM) of the suspended particles, we can estimate the frequency-modulated behaviors between EWOD, positive DEP, and negative DEP. A proposed weighted Re(fCM) facilitates analysis of the DEP phenomenon on dielectric-coated electrodes.

Asymmetric electrowetting--moving droplets by a square wave.

Here droplet oscillation and continuous pumping are demonstrated by asymmetric electrowetting on an open surface with embedded electrodes powered by a square wave electrical signal without control circuits. The polarity effect of electrowetting on an SU-8 and Teflon coated electrode is investigated, and it is found that the theta-V (contact angle-applied voltage) curve is asymmetric along the V = 0 axis by sessile drop and coplanar electrode experiments. A systematic deviation of measured contact angles from the theoretical ones is observed when the electrode beneath the droplet is negatively biased. In the sessile drop experiment, up to a 10 degrees increment of contact angle is measured on a negatively biased electrode. In addition, a coplanar electrode experiment is designed to examine the contact angles at the same applied potential but opposite polarities on two sides of one droplet at the same time. The design of the coplanar electrodes is then expanded to oscillate and transport droplets on square-wave-powered symmetric (square) and asymmetric (polygon) electrodes to demonstrate manipulation capability on an open surface. The frequency of oscillation and the speed of transportation are determined by the frequency of the applied square wave and the pitch of the electrodes. Droplets with different volumes are tested by square waves of varied frequencies and amplitudes. The 1.0 microl droplet is successfully transported on a device with a loop of 24 electrodes continuously at a speed up to 23.6 mm s(-1) when a 9 Hz square wave is applied.

Preface to Special Topic: Selected Papers from the 5th International Conference on Optofluidics.

The 5th International Conference on Optofluidics (Optofluidics 2015) was held in Taipei, Taiwan, July 26-29, 2015. The aim of this conference was to provide a forum to promote scientific exchange and to foster closer networks and collaborative ties between leading international researchers in optics and micro/nanofluidics across various disciplines. The scope of Optofluidics 2015 was deliberately broad and interdisciplinary, encompassing the latest advances and the most innovative developments in micro/nanoscale science and technology. Topics ranged from fundamental research to its applications in chemistry, physics, biology, materials, and medicine.

Opto-Microfluidic Immunosensors: From Colorimetric to Plasmonic.

Optical detection has long been the most popular technique in immunosensing. Recent developments in the synthesis of luminescent probes and the fabrication of novel nanostructures enable more sensitive and efficient optical detection, which can be miniaturized and integrated with microfluidics to realize compact lab-on-a-chip immunosensors. These immunosensors are portable, economical and automated, but their sensitivity is not compromised. This review focuses on the incorporation and implementation of optical detection and microfluidics in immunosensors; it introduces the working principles of each optical detection technique and how it can be exploited in immunosensing. The recent progress in various opto-microfluidic immunosensor designs is described. Instead of being comprehensive to include all opto-microfluidic platforms, the report centers on the designs that are promising for point-of-care immunosensing diagnostics, in which ease of use, stability and cost-effective fabrication are emphasized.

Reconfigurable liquid-core/liquid-cladding optical waveguides with dielectrophoresis-driven virtual microchannels on an electromicrofluidic platform.

An electrically reconfigurable liquid-core/liquid-cladding (L(2)) optical waveguide with core liquid γ-butyrolactone (GBL, ncore = 1.4341, εcore = 39) and silicone oil (ncladding = 1.401, εcladding = 2.5) as cladding liquid is accomplished using dielectrophoresis (DEP) that attracts and deforms the core liquid with the greater permittivity to occupy the region of strong electric field provided by Teflon-coated ITO electrodes between parallel glass plates. Instead of continuously flowing core and cladding liquids along a physical microchannel, the DEP-formed L(2) optical waveguide guides light in a stationary virtual microchannel that requires liquids of limited volume without constant supply and creates stable liquid/liquid interfaces for efficient light guidance in a simply fabricated microfluidic device. We designed and examined (1) stationary and (2) moving L(2) optical waveguides on the parallel-plate electromicrofluidic platform. In the stationary L-shaped waveguide, light was guided in a GBL virtual microchannel core for a total of 27.85 mm via a 90° bend (radius 5 mm) before exiting from the light outlet of cross-sectional area 100 µm × 100 µm. For the stationary spiral waveguide, light was guided in a GBL core containing Rhodamine 6G (R6G, 1 mM) and through a series of 90° bends with decreasing radii from 5 mm to 2.5 mm. With the stationary straight waveguide, the propagation loss was measured to be 2.09 dB cm(-1) in GBL with R6G (0.01 mM). The moving L-shaped waveguide was implemented on a versatile electromicrofluidic platform on which electrowetting and DEP were employed to generate a precise GBL droplet and form a waveguide core. On sequentially applying appropriate voltage to one of three parallel L-shaped driving electrodes, the GBL waveguide core was shifted; the guided light was switched at a speed of up to 0.929 mm s(-1) (switching period 70 ms, switching rate 14.3 Hz) when an adequate electric signal (173.1 VRMS, 100 kHz) was applied.

Constructing 3D heterogeneous hydrogels from electrically manipulated prepolymer droplets and crosslinked microgels.

Formation of multifunctional, heterogeneous, and encoded hydrogel building blocks, or microgels, by crosslinking and assembly of microgels are two essential steps in establishing hierarchical, complicated, and three-dimensional (3D) hydrogel architectures that recapitulate natural and biological structures or originate new materials by design. However, for the variety of the hydrogel materials crosslinked differently and for the varied scales of microgels and architectures, the formation and assembly processes are usually performed separately, which increases the manufacturing complexity of designed hydrogel materials. We show the construction of hydrogel architectures through programmable formation and assembly on an electromicrofluidic platform, adopting two reciprocal electric manipulations (electrowetting and dielectrophoresis) to manipulate varied objects (i) in multiple phases, including prepolymer liquid droplets and crosslinked microgels, (ii) on a wide range of scales from micrometer functional particles or cells to millimeter-assembled hydrogel architectures, and (iii) with diverse properties, such as conductive and dielectric droplets that are photocrosslinkable, chemically crosslinkable, or thermally crosslinkable. Prepolymer droplets, particles, and dissolved molecules are electrically addressable to adjust the properties of the microgel building blocks in liquid phase that subsequently undergo crosslinking and assembly in a flexible sequence to accomplish heterogeneous and seamless hydrogel architectures. We expect the electromicrofluidic platform to become a general technique to obtain 3D complex architectures.