Biodegradable Polymers for Micro Elastofluidics.

Micro elastofluidics is an emerging research field that encompasses characteristics of conventional microfluidics and fluid-structure interactions. Micro elastofluidics is expected to enable practical applications, for instance, where direct contact between biological samples and fluid handling systems is required. Besides design optimization, choosing a proper material is critical to the practical use of micro elastofluidics upon interaction with biological interface and after its functional lifetime. Biodegradable polymers are one of the most studied materials for this purpose. Micro elastofluidic devices made of biodegradable polymers possess exceptional mechanical elasticity, excellent bio compatibility, and structural degradability into non-toxic products. This article provides an insightful and systematic review of the utilization of biodegradable polymers in digital and continuous-flow micro elastofluidics.

Investigation of viscoelastic focusing of particles and cells in a zigzag microchannel.

Microfluidic particle focusing has been a vital prerequisite step in sample preparation for downstream particle separation, counting, detection, or analysis, and has attracted broad applications in biomedical and chemical areas. Besides all the active and passive focusing methods in Newtonian fluids, particle focusing in viscoelastic fluids has been attracting increasing interest because of its advantages induced by intrinsic fluid property. However, to achieve a well-defined focusing position, there is a need to extend channel lengths when focusing micrometer-sized or sub-microsized particles, which would result in the size increase of the microfluidic devices. This work investigated the sheathless viscoelastic focusing of particles and cells in a zigzag microfluidic channel. Benefit from the zigzag structure of the channel, the channel length and the footprint of the device can be reduced without sacrificing the focusing performance. In this work, the viscoelastic focusing, including the focusing of 10 µm polystyrene particles, 5 µm polystyrene particles, 5 µm magnetic particles, white blood cells (WBCs), red blood cells (RBCs), and cancer cells, were all demonstrated. Moreover, magnetophoretic separation of magnetic and nonmagnetic particles after viscoelastic pre-focusing was shown. This focusing technique has the potential to be used in a range of biomedical applications.

Digital Imaging-based Colourimetry for Enzymatic Processes in Transparent Liquid Marbles.

Liquid marbles are a promising microreactor platform that recently attracts significant research interest owing to their ability to accommodate a wide range of micro reactions. However, the use of destructive and ex-situ methods to monitor reactions impairs the potential of liquid-marble-based microreactors. This paper proposes a non-destructive, in situ, and cost-effective digital-imaging-based colourimetric monitoring method for transparent liquid marbles, using the enzymatic hydrolysis of starch as an illustrative example. The colourimetric reaction between starch and iodine produces a complex that exhibits a dark blue colour. We found that the absorbance of red channel of digital images showed a linear relationship with starch concentration with high sensitivity and repeatability. This digital-imaging-based colourimetric method was used to study the hydrolysis of starch by α-amylase. The results show high accuracy and applicability of first-order kinetics for this reaction. The demonstration of digital-imaging-based colourimetry indicates the potential of liquid marble-based microreactors.

Measuring the effective surface tension of a floating liquid marble using X-ray imaging.

A liquid marble (LM) is a droplet coated with microparticles that isolate the liquid interior from its surroundings, making it perfectly non-wetting. This attractive feature allows the LM to perform useful tasks such as coalescence, targeted delivery, and controlled release. The non-wetting characteristic also allows the LM to float on a carrier liquid. The growing number of applications in digital microfluidics requires further insights into the fundamental properties of a LM such as its effective surface tension. Although the coating provides the LM with various desirable characteristics, its random construction presents a major obstacle to accurate optical analysis. This paper presents a novel method to measure the effective surface tension of a floating LM using X-ray imaging and curve fitting procedures. X-ray imaging reveals the true LM liquid-air interface hidden by the coating particles. Analysis of this interface showed that the effective surface tension of a LM is not significantly different from that of its liquid content. This indicates that the particle coating might not have significantly altered the behaviour of the liquid interface. We also found that our method is sensitive enough to detect the variations across individual LMs.

Direct Measurement of the Contents, Thickness, and Internal Pressure of Molybdenum Disulfide Nanoblisters.

Nanoblisters have attracted attention due to their ability to controllably modulate the properties of two-dimensional materials. The accurate measurement or estimation of their properties is nontrivial and largely based on Hencky's theory. However, these estimates require a priori knowledge of material properties and propagate large errors. Here we show, through a systematic atomic force microscopy study, several strategies that lead to vastly enhanced characterization of nanoblisters. First, we find that nanoblisters may contain both liquid and gas, resolving an ongoing debate in the literature. Second, we demonstrate how to definitively determine the membrane thickness of a nanoblister and show that Hencky's theory can only reliably predict membrane thicknesses for small aspect ratios and small membrane thicknesses. Third, we develop a novel technique to measure the internal pressures of nanoblisters, which quantitatively agrees with Hencky's theory but carries a 1 order smaller propagated error.

Inertial Microfluidic Purification of Floating Cancer Cells for Drug Screening and Three-Dimensional Tumor Models.

Floating cancer cells can survive the programmed death anoikis process after detaching from the extracellular matrix for the anchorage-dependent cells. Purification of viable floating cancer cells is essential for many biomedical studies, such as drug screening and cancer model development. However, the floating cancer cells are mixed with dead cells and debris in the medium supernatant. In this paper, we developed an inertial microfluidic device with sinusoidal microchannels to continuously remove dead cells and debris from viable cells. First, we characterized the differential inertial focusing properties of polystyrene beads in the devices. Then, we investigated the effects of flow rate on inertial focusing of floating MDA-MB-231 cells. At an optimal flow condition, purification of viable cells was performed and the purity of live cells was increased significantly from 19.9% to 76.6%, with a recovery rate of 69.7%. After separation, we studied and compared the floating and adherent MDA-MB-231 cells in terms of cell proliferation, protrusive cellular structure, and the expression of cyclooxygenase (Cox-2) which is related to epithelial-mesenchymal transition (EMT) changes. Meanwhile, drug screening of both floating and adherent cancer cells was conducted using a chemotherapeutic drug, doxorubicin (Dox). The results revealed that the floating cancer cells possess 30-fold acquired chemoresistance as compared to the adherent cancer cells. Furthermore, a three-dimensional (3D) double-cellular coculture model of human mammary fibroblasts (HMF) spheroid and cancer cells using the floating liquid marble technique was developed.

Fundamentals of Differential Particle Inertial Focusing in Symmetric Sinusoidal Microchannels.

Focusing and separation of particles such as cells at high throughput is extremely attractive for biomedical applications. Particle manipulation based on inertial effects requires a high flow speed and thus is well-suited to high-throughput applications. Recently, inertial focusing and separation using curvilinear microchannels has been attracting a great amount of interest because of the linear structure for parallelization, small device footprint, superior particle-focusing performance, and easy implementation of particle separation. However, the curvature directions of these microchannels alternate, leading to variations in both the magnitude and direction of the induced secondary flow. Accumulation of this variation along the channel causes unpredictable behaviors of particles. This paper systematically investigates the inertial-focusing phenomenon in low-aspect-ratio symmetric sinusoidal channels. First, we comprehensively studied the effects of parameters such as viscosity, flow conditions, particle size, and geometric dimensions of the microchannel on differential particle focusing. We found that particle inertial focusing is generally independent of fluid kinematic viscosity but highly dependent on particle size, flow conditions, and channel dimensions. Next, we derived an explicit scaling factor and included all four dimensionless parameters (particle-blockage ratio, curvature ratio, Dean number, and channel aspect ratio) in a single operational map to illustrate the particle-focusing patterns. Finally, we proposed a rational guideline to intuitively instruct the design of channel dimensions for separation of a given particle mixture.

Liquid marble coalescence via vertical collision.

The coalescence process of liquid marbles is vital to their promising roles as reactors or mixers in digital microfluidics. However, the underlying mechanisms and critical conditions of liquid marble coalescence are not well understood. This paper studies the coalescence process of two equally-sized liquid marbles via vertical collision aided by dielectrophoretic handling. A liquid marble was picked up using the dielectrophoretic force and then dropped vertically onto another liquid marble resting on a hydrophobic powder bed. The whole collision process was recorded by a high-speed camera and the recorded images were then analysed to derive the generalised conditions of liquid marble coalescence. By varying the marble volume, impact velocity and offset ratio in the experiments, we concluded that liquid marble coalescence may occur through the coating pore opening mechanism. We quantitatively measured the radius change versus time of the liquid neck formed between two coalescing marbles and estimated the maximum deformation of impacting marbles before rupture in rebound cases. We also qualitatively described the redistribution of coating particles at the impact area during coalescence as well as the consequent ejection of particles. Finally, we summarised the critical conditions for liquid marble coalescence, providing a frame for future applications involving liquid marbles as micromixers and microreactors.

Evaporation of Ethanol-Water Binary Mixture Sessile Liquid Marbles.

Liquid marble is a liquid droplet coated with particles. Recently, the evaporation process of a sessile liquid marble using geometric measurements has attracted great attention from the research community. However, the lack of gravimetric measurement limits further insights into the physical changes of a liquid marble during the evaporation process. Moreover, the evaporation process of a marble containing a liquid binary mixture has not been reported before. The present paper investigates the effective density and the effective surface tension of an evaporating liquid marble that contains aqueous ethanol at relatively low concentrations. The effective density of an evaporating liquid marble is determined from the concurrent measurement of instantaneous mass and volume. Density measurements combined with surface profile fitting provide the effective surface tension of the marble. We found that the density and surface tension of an evaporating marble are significantly affected by the particle coating.

Deformation of a floating liquid marble.

A rigid spherical particle floating on a liquid is a known problem with well-defined solutions. Under the combined effect of gravity and surface tension, the rigid particle deforms the liquid surface. However, in the case of a floating soft particle such as a liquid marble, not only the liquid surface but also the particle itself deforms. In this paper, we investigate the deformation of a floating liquid marble and characterise its height as well as aspect ratio. The experimental results show that theoretical models for a rigid spherical particle suit well for small liquid marbles. Larger marbles require an oblate liquid spheroid model. We will discuss the limitations of the two models and characterise the deformation of these marbles.

Calcium alginate elastic capsules for microalgal cultivation.

Calcium alginate elastic capsules with a core-shell structure are versatile spherical solid beads that can be produced in large quantities using various techniques. This type of capsule is a promising platform for cell culture applications, owing to its mechanical elasticity and transparency. This paper reports the production of calcium alginate capsules with high consistency, and for the first time, demonstrates the feasibility of the capsules for microalgal cultivation. Cell growth analysis reveals that the vibrationally-shaken calcium alginate elastic capsule platform yielded a higher maximum cell number (4.86 × 108 cells per mL) during the cultivation period than the control solution platforms. Aquafeed and food supplements for humans are the targeted applications of this novel platform.

Uniaxial Cyclic Cell Stretching Device for Accelerating Cellular Studies.

Cellular response to mechanical stimuli is a crucial factor for maintaining cell homeostasis. The interaction between the extracellular matrix and mechanical stress plays a significant role in organizing the cytoskeleton and aligning cells. Tools that apply mechanical forces to cells and tissues, as well as those capable of measuring the mechanical properties of biological cells, have greatly contributed to our understanding of fundamental mechanobiology. These tools have been extensively employed to unveil the substantial influence of mechanical cues on the development and progression of various diseases. In this report, we present an economical and high-performance uniaxial cell stretching device. This paper reports the detailed operation concept of the device, experimental design, and characterization. The device was tested with MDA-MB-231 breast cancer cells. The experimental results agree well with previously documented morphological changes resulting from stretching forces on cancer cells. Remarkably, our new device demonstrates comparable cellular changes within 30 min compared with the previous 2 h stretching duration. This third-generation device significantly improved the stretching capabilities compared with its previous counterparts, resulting in a remarkable reduction in stretching time and a substantial increase in overall efficiency. Moreover, the device design incorporates an open-source software interface, facilitating convenient parameter adjustments such as strain, stretching speed, frequency, and duration. Its versatility enables seamless integration with various optical microscopes, thereby yielding novel insights into the realm of mechanobiology.

Protein array processing software for automated semiquantitative analysis of serum antibody repertoires.

Effective immunotherapies activate natural antitumor immune responses in patients undergoing treatment. The ability to monitor immune activation in response to immunotherapy is critical in measuring treatment efficacy over time and across patient cohorts. Protein arrays are systematically arranged, large collections of annotated proteins on planar surfaces, which can be used for the characterization of disease-specific and treatment-induced antibody repertoires in individuals undergoing immunotherapy. However, the absence of appropriate image analysis and data processing software presents a substantial hurdle, limiting the uptake of this approach in immunotherapy research. We developed a first, automated semiquantitative open-source software package for the analysis of widely used protein macroarrays. The software allows accurate single array and inter-array comparative studies through the tackling of intra-array inconsistencies arising from experimental disparities. The innovative and automated image analysis process includes adaptive positioning, background identification and subtraction, removal of null signals, robust statistical analysis, and protein pair validation. The normalized values allow a convenient semiquantitative data analysis of different samples or timepoints. Enabling accurate characterization of sample series to identify disease-specific immune profiles or their relative changes in response to treatment may serve as a diagnostic or predictive tool of disease.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

Core-Shell Particles: From Fabrication Methods to Diverse Manipulation Techniques.

Core-shell particles are micro- or nanoparticles with solid, liquid, or gas cores encapsulated by protective solid shells. The unique composition of core and shell materials imparts smart properties on the particles. Core-shell particles are gaining increasing attention as tuneable and versatile carriers for pharmaceutical and biomedical applications including targeted drug delivery, controlled drug release, and biosensing. This review provides an overview of fabrication methods for core-shell particles followed by a brief discussion of their application and a detailed analysis of their manipulation including assembly, sorting, and triggered release. We compile current methodologies employed for manipulation of core-shell particles and demonstrate how existing methods of assembly and sorting micro/nanospheres can be adopted or modified for core-shell particles. Various triggered release approaches for diagnostics and drug delivery are also discussed in detail.

Controllable high-performance liquid marble micromixer.

A liquid marble is a liquid droplet coated with a shell of microparticles. Liquid marbles have served as a unique microreactor for chemical reactions and cell culture. Mixing is an essential task for liquid marbles as a microreactor. However, the potential of liquid marble-based microreactors is significantly limited due to the lack of effective mixing strategies. Most mixing strategies used manual and contact-based actuation schemes. This paper reports the development of a manipulation scheme that induces fluid motion into a liquid marble, leading to enhanced mixing. By inducing rotation on a horizontal axis, we significantly increased the mixing rate by 27.6 times compared to a non-actuated liquid marble and reduced the reaction time by more than 10 times. The proposed method provides a simple, continuous, precise, and controllable high-performance mixing strategy on a liquid marble platform.

Dynamic Behaviours of Monodisperse Double Emulsion Formation in a Tri-Axial Capillary Device.

We investigated experimentally, analytically, and numerically the formation process of double emulsion formations under a dripping regime in a tri-axial co-flow capillary device. The results show that mismatches of core and shell droplets under a given flow condition can be captured both experimentally and numerically. We propose a semi-analytical model using the match ratio between the pinch-off length of the shell droplet and the product of the core growth rate and its pinch-off time. The mismatch issue can be avoided if the match ratio is lower than unity. We considered a model with the wall effect to predict the size of the matched double emulsion. The model shows slight deviations with experimental data if the Reynolds number of the continuous phase is lower than 0.06 but asymptotically approaches good agreement if the Reynolds number increases from 0.06 to 0.14. The numerical simulation generally agrees with the experiments under various flow conditions.

Loop-Mediated Isothermal Amplification in a Core-Shell Bead Assay for the Detection of Tyrosine Kinase AXL Overexpression.

The upregulated expression of tyrosine kinase AXL has been reported in several hematologic and solid human tumors, including gastric, breast, colorectal, prostate and ovarian cancers. Thus, AXL can potentially serve as a diagnostic and prognostic biomarker for various cancers. This paper reports the first ever loop-mediated isothermal amplification (LAMP) in a core-shell bead assay for the detection of AXL gene overexpression. We demonstrated simple instrumentation toward a point-of-care device to perform LAMP. This paper also reports the first ever use of core-shell beads as a microreactor to perform LAMP as an attempt to promote environmentally-friendly laboratory practices.

Liquid marble-based digital microfluidics - fundamentals and applications.

Liquid marbles are droplets with volume typically on the order of microliters coated with hydrophobic powder. Their versatility, ease of use and low cost make liquid marbles an attractive platform for digital microfluidics. This paper provides the state of the art of discoveries in the physics of liquid marbles and their practical applications. The paper first discusses the fundamental properties of liquid marbles, followed by the summary of different techniques for the synthesis of liquid marbles. Next, manipulation techniques for handling liquid marbles are discussed. Applications of liquid marbles are categorised according to their use as chemical and biological reactors. The paper concludes with perspectives on the future development of liquid marble-based digital microfluidics.

Correction: Liquid marble-based digital microfluidics - fundamentals and applications.

Correction for 'Liquid marble-based digital microfluidics - fundamentals and applications' by Chin Hong Ooi et al., Lab Chip, 2021, DOI: .