Simple Microfluidics Research Articles

SMORES: a simple microfluidic operating room for the examination and surgery of Stentor coeruleus

Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.

Massively parallel single-cell sequencing of diverse microbial populations.

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.

(Invited) Optical Biosensors in Photonic Integrated Circuits for Smart System Integration

Photonic integrated circuits (PIC) are like electronic integrated circuits drivers of communication systems. Moreover, state-of-the-art technologies enable the fabrication of electronic-photonic integrated circuits (ePIC), which allows for example the cointegration of ultra-fast BiCMOS devices with high speed photo detectors. Besides that, this technology provides an attractive platform for biosensors. They open the door for label-free detection of various analytes with high specificity and in real-time. This gives perspective to highly scalable and cost effective technologies for smart system integration of biosensors and electronic readouts to realize a true lab-on-a-chip application.We present the integration of optical biosensors in a Si-based PIC-technology. This comprises a front-end of line with optical waveguides, an integrated Ge-photodiode and a complete back-end of line consisting of three thin and two thick aluminum metal layers. The optical biosensor is integrated by opening the optical waveguide from the backside of the chip. In particular, we released a Si-based slot waveguide, which provides an enhanced evanescent field to interact with the analyte and hence to increase the sensitivity. The slot waveguide is incorporated in a ring resonator and we analyzed the wafer-level performance using the full width at half maximum, which allows us to assess the optical losses. Thermal sensing in a wide temperature range is investigated. In addition, a simple and cost-effective microfluidic is realized and we show initial results of the surface functionalization. Our work demonstrates the compatibility of a photonic biosensor with a photonic integrated circuit technology without any restriction to the standard FEOL and BEOL processing on the wafer front-side, while it keeps the inherent high sensitivity of evanescent-field sensing and opens a route towards smart system integration. Figure 1

Simple Electroosmotic Pump and Active Microfluidics with Asymmetrically Coated Microelectrodes

Electroosmotic pumps can deliver liquid without moving parts, making them suitable for microfluidic and lab‐on‐chip systems. Previously, alternating current electroosmotic pumps were constructed using pairs of coplanar asymmetrical interdigitated microelectrodes on the same substrate. In this work, a simpler micropumping system is developed, separating the electrodes on two substrates and breaking the symmetry by half‐depositing electrodes with 3D microstructures. Numerical simulation models of the pumping system and experimental velocity profiles are used to explain the fluid motion mechanism and structure‐dependent pumping performance. In addition to its efficiency and simplicity, this new pumping system also allows for the creation of a microvortex device and an active microfluidics device. This scalable micropumping system provides a way to pump liquids at microscopic or macroscopical scale in complex microfluidics systems.

A digital microfluidic platform coupled with colorimetric loop-mediated isothermal amplification for on-site visual diagnosis of multiple diseases.

Traditional diagnosis of infectious diseases relies on advanced analyzers in central hospitals, which is not sufficient for rapid control of epidemics, especially in resource-limited areas, raising the importance of the development of diagnostic systems for point-of-care testing (POCT). Here, we developed a simple and cost-effective digital microfluidic (DMF) platform in combination with colorimetric loop-mediated isothermal amplification (LAMP) for simple on-site diagnosis of diseases by the naked eye. The DMF chip contained four parallel units for the simultaneous detection of multiple genes and samples at a time. After amplification, the results were visualized by endpoint detection with concentrated dry neutral red on the chip. The whole process could be completed within 45 min and the on-chip LAMP reaction was shortened to 20 min. The analytical performance of this platform was evaluated by the detection of Enterocytozoon hepatopenaei, infectious hypodermal and hematopoietic necrosis virus, and white spot syndrome virus genes of shrimp. The DMF-LAMP assay showed a detection limit of 101 copies per μl for each target, exhibiting comparable sensitivity to the conventional LAMP assay but was more efficient. The sensitivity was also competitive with that of microfluidic-based LAMP assays using other POCT devices such as centrifugal discs regarding the detection of the same targets. Moreover, the proposed device possessed a simple chip structure and high flexibility to integrate multiplex analysis, which was beneficial for further widespread use in POCT. The practicability of the DMF-LAMP assay was verified by the testing of field shrimp. The result of the DMF-LAMP assay indicated a good agreement with the qPCR method, with Cohen's kappa values ranging from 0.91 to 1.00 depending on different targets. For the first time, an image processing method was established based on RGB analysis under different lighting conditions, and a universally adaptable positive threshold was determined regardless of lighting conditions. Paired with a smartphone, the objective analytical method was facile to implement in the field. Moreover, the DMF-LAMP system is easy to extend for a broad range of bioassays, with the advantages of low-cost, rapid detection, ease of use, good sensitivity, and easy readout.

Simple droplet microfluidics platform for drug screening on cancer spheroids.

3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipment for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplet microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorogenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from a patient-derived-xenografts (PDX). As compared to microtiter plates methods, our system reduces the initial number of cells up to 10 times and opens new avenues towards primary tumors drug screening approaches.

Microfluidic encapsulation of soluble reagents with large-scale concentration gradients in a sequence of droplets for comparative analysis

Concentration gradient is important for comparative analysis in the fields of biomedicine and chemistry, especially with the in-depth research of material synthesis, enzyme kinetic analysis and cell biology. In this work, we reported a simple coaxial injection microfluidic device for encapsulation of soluble reagents with large-scale concentration gradients in a sequence of nanoliter-scale water-in-oil(W/O) droplets for comparative analysis. The concentration gradients can be adjusted straightforwardly by changing the flow rates of phases in two coaxial channels. The concentration gradients of the components can reach the order of 105. As proofs of this concept, acid-base reaction, L-Ascorbic acid redox reaction and synthesis of metal organic frameworks(MOFs) are conducted using this concentration gradient generator. Nanoliter-grade reagents preparation, reactions and analyses with higher concentration gradients and less sample consumption can be achieved without stopping experiments and changing solutions by using this simple microfluidics concentration gradient generator.

An ex vivo system for investigation of Plasmodium berghei invasion of the salivary gland of Anopheles stephensi mosquitoes

ABSTRACT Plasmodium sporozoites associated with the midgut and in the hemolymph of mosquitoes differ from sporozoites in the secretory cavities and ducts of the insects’ salivary glands in their transcriptome, proteome, motility, and infectivity. Using an ex vivo Anopheles stephensi salivary gland culture system incorporating simple microfluidics and transgenic Plasmodium berghei with the fluorescent protein gene mCherry under the transcriptional control of the Pbuis4 promoter whose expression served as a proxy for parasite maturation, we observed rapid parasite maturation in the absence of salivary gland invasion. While in vivo Pbuis4::mCherry expression was only detectable in sporozoites within the salivary glands (mature parasites) as expected, the simple exposure of P. berghei sporozoites to dissected salivary glands led to rapid parasite maturation as indicated by mCherry expression. These results suggest that previous efforts to develop ex vivo and in vitro systems for investigating sporozoite interactions with mosquito salivary glands have likely been unsuccessful in part because the maturation of sporozoites leads to a loss in the ability to invade salivary glands.

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today's availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

Emergent Behavior at the Calcite–Water Interface during Reactive Transport in a Simple Microfluidic Channel

Geochemical reactive transport processes in natural mineral–fluid systems may produce a wide array of emergent phenomena that are difficult to predict from basic principles and to reproduce in model systems. Here, we present experimental results obtained from a simple microfluidic system with which we explored the consequences of reacting the calcite (104) cleavage surface with an acidic Pb-bearing solution (pH = 3.5, [Pb]total = 5 mM) as a function of flow rate. This system is relevant to passive remediation systems for Pb-rich acid mine drainage. We observed periodic banding in the amounts of Pb sorption at flow velocities ≥926 μm s–1, where the band spacing was spatially correlated with the amount of calcite dissolution and the development of micropyramidal topography on the calcite (104) surface. The equivalent coverage of Pb deposited in these Pb-rich bands was at least several monolayers per unit cell, yet there was no evidence for precipitation of any secondary Pb phase implying incorporation of Pb within the near-surface calcite lattice. We also observed spatial variations in nucleation and growth of euhedral secondary Pb-carbonate minerals hydrocerusite and cerussite at flow rates ≤278 μm s–1. These findings demonstrate potential for exploiting the rich phenomenology afforded by the interplay among transport phenomena and chemical kinetics in experimental systems designed to yield deeper insights into geochemical self-organization.

Nanomotor-Derived Porous Biomedical Particles from Droplet Microfluidics.

Porous particles have found widespread applications in therapeutic diagnosis, drug delivery, and tissue engineering due to their typical properties of large surface area, extensive loading capacity, and hierarchical microstructures. Attempts in this aspect are focusing on the development of effective methods to generate functional porous particles. Herein, a simple droplet microfluidics for continuously and directly generating porous particles by introducing bubble‐propelled nanomotors into the system is presented. As the nanomotors can continuously generate gas bubbles in the unsolidified droplet templates, the desirable porous microparticles can be obtained after droplet polymerization. It is demonstrated that the generation process is highly controlled and the resultant microparticles show excellent porosity and monodispersity. In addition, the obtained porous microparticles can serve as microcarriers for 3D cell culture, because of their characteristic porous structures and favorable biocompatibility. Moreover, owing to the existence of oxygen in these microparticles, they can be used to improve the healing effects of wounds in the type I diabetes rat models. These remarkable features of the generation strategy and the porous microparticles point to their potential values in various biomedical fields.

Benign fabrication process of hierarchal porous polyurea microspheres with tunable pores and porosity: Their Pd immobilization and use for hexavalent chromium reduction

Developing new strategies for fabrication of porous materials with tunable morphology which have fewer environmental impacts is highly desired. Herein, conventional precipitation polymerization of polyurea (PU) synthesis is converted to interfacial polymerization via simple microfluidics and hierarchically porous and uniform PU microspheres (PPM) are prepared by the reaction of toluene diisocyanate and water, eliminating the use of toxic amines and organic solvents. The morphology and porosity of PPM are probed. The size of PPM is controlled by changing the diameter of flow channels of microfluidic device and their pore size and porosity are tuned by optimizing the polymerization conditions. Moreover, palladium (Pd) is immobilized on PPM by impregnation and subsequent reduction approach to get Pd@PPM. The hybrid composite, Pd@PPM, is used for catalytic reduction of toxic Cr6+ to benign Cr3+ by formic acid as a reductant along with sodium format as a promoter in aqueous system. Pd@PPM exhibited high catalytic activity and good stability for Cr6+ reduction and their large size with high uniformity eased their recovery to reuse them in subsequent Cr6+ reduction. Porous PU with controllable shape and morphology are rarely available. This work therefore provides new strategy to synthesis PPM with controllable porous morphology and their use as an effective support for catalysis.

Go with the capillary flow. Simple thread-based microfluidics

• Review of capillary flow enabled thread-based microfluidics development. • Review of threads’ capillarity and fluid flow control strategies. • Mainstream detection methods applied on thread-based microfluidics. • Applications on blood separation and analysis, immunoassays, and wearable sensing. • Trend in smartphone-compatible thread microfluidics for onsite applications. Studies on microfluidic thread-based analytical devices (μTADs) have been developed for a decade. Threads, made of natural cellulose or synthesized polymer fibres, are manufactured in the form which possesses a large number of capillary gaps for liquid transport, and show differences in structural morphologies and surface chemistry. Threads have been used to design simple thread-based microfluidics with different analytical functions. Due to their low-cost, portability, biocompatibility and ease of integration with other components, threads show a great application basis to fabricate point-of-care μTADs. To date, there are a limited number of published studies on μTADs in contrast to paper-based analytical devices. To facilitate further advances of μTADs, this review summarizes the development of these devices from the aspects of engineering control of capillary flow on threads and biological applications. Particularly, as liquid flow on threads affects the transport and detection efficiencies, strategies of controlling liquid flow on threads are firstly reviewed. This includes thread surface modification, proper thread type selection, and microfluidic channel design. Then various detection methods including colorimetry, fluorescence, electrochemistry and surface-enhanced Raman spectroscopy applied on μTADs are reviewed. The biological applications of μTADs in blood separation and analysis, immunoassay, wearable sensing, as well as compatibility with smartphones for biosensing are also systematically summarized. Finally, the future perspectives of μTADs regarding external collaboration to satisfy the “REASSURED” criteria are proposed. This review is expected to expand understandings of μTAD designs and applications, and to clarify the challenges and future development directions of μTADs.

Quantitative Nano-Platforms for Interrogation of Curvature Sensitive Proteins

Membrane curvature is a defining parameter which mediates many critical cellular processes like endocytosis and exocytosis. This parameter is mainly mediated by several types of curvature-sensing domains, including N-BAR, F-BAR, I-BAR, and ENTH domain. Previous studies of these curvature-sensing domains lacking precise quantification of curvature sensitivity while maintaining the ease of use the assay. Herein, we design a concise platform to quantitatively determine the curvature-sensing range for several curvature-sensing proteins, which implies the role of these proteins in several membrane-associated processes. The core of our technology consists of simple nanofabrication and microfluidics, integrated with quick lipid bilayer engineering and protein lysate preparation. This design provides us with the flexibility to switch lipid compositions, manipulate lysate components, and engineer complex curvatures, which serves as a powerful tool to investigate curvature-dependent cellular processes.

Biofabrication of Gelatin Tissue Scaffolds with Uniform Pore Size via Microbubble Assembly

AbstractThe control of pore size and uniform porosity remains as an important challenge in gelatin scaffolds. The precise control in building blocks of tissue scaffolds without any additional porogen is possible with costly equipment and techniques, though some pre‐requirements for polymeric material, such as photo‐polymerizability or sintering ability, may be needed prior to construction. Herein, a method for the fabrication of gelatin scaffolds with homogenous porosity using simple T‐junction microfluidics is described. The size of the microbubbles is precisely controlled with 5% deviation from the average. Porous gelatin scaffolds are obtained by building‐up the monodispersed microbubbles in dilute cross‐linker solutions. The effect of cross‐linker density on pore diameter is also investigated. After cross‐linking, pore size of the resultant five scaffold groups are precisely controlled as 135 ± 11, 193 ± 11, 216 ± 9, 231 ± 5, and 250 ± 12 µm. Porosity ratios above 65% are achieved in every sample group. According to the cell culture experiments, structures support high cell adhesion, viability, and migration through the porous network via interconnectivity. This study offers a practical and economical approach for the preparation of porous gelatin scaffolds with homogenous porosity which can be utilized in diverse tissue engineering applications.

Label-free purification of viable human T-lymphocyte cells from a mixture of viable and non-viable cells after transfection by electroporation

Recent developments in chimeric antigen receptor (CAR) T-cell therapy for cancer have shown promising results. CAR T-cells are engineered mainly by AC or DC electroporation to express CAR molecules on their surfaces. Electroporation has detrimental effects on the majority of T-cells and causes cell death via apoptosis or necrosis. These non-viable cells have potentially harmful effects on other healthy T-cells. Because there are no unique biomarkers that pinpoint the apoptosis and necrosis of T-cells, currently available cell separation methods are incapable of purifying healthy T-cells after electroporation. To address this critical issue, we have used dielectrophoretic cell isolation in a simple microfluidics chip. Our results indicate that it is possible to purify cell samples after electroporation and achieve ~100% purity and >90% target cell recovery. Therefore, this work presents a viable solution to a critical need in CAR T-cell manufacturing.

Conducting Polymeric Nanocomposites with a Three-Dimensional Co-flow Microfluidics Platform

The nanoprecipitation of polymers is of great interest in biological and medicinal applications. Many approaches are available, but few generalized methods can fabricate structurally different biocompatible polymers into nanosized particles with a narrow distribution in a high-throughput manner. We simply integrate a glass slide, capillary, and metal needle into a simple microfluidics device. Herein, a detailed protocol is provided for using the glass capillary and slides to fabricate the microfluidics devices used in this work. To demonstrate the generality of our nanoprecipitation approach and platform, four (semi)natural polymers—acetalated dextran (Ac-DEX), spermine acetalated dextran (Sp-Ac-DEX), poly(lactic-co-glycolic acid) (PLGA), and chitosan—were tested and benchmarked by the polymeric particle size and polydispersity. More importantly, the principal objective was to explore the influence of some key parameters on nanoparticle size due to its importance for a variety of applications. The polymer concentration, the solvent/non-solvent volume rate/ratio, and opening of the inner capillary were varied so as to obtain polymeric nanoparticles (NPs). Dynamic light scattering (DLS), transmission electron microscopy (TEM), and optical microscopy are the main techniques used to evaluate the nanoprecipitation output. It turns out that the concentration of polymer most strongly determines the particle size and distribution, followed by the solvent/non-solvent volume rate/ratio, whereas the opening of the inner capillary shows a minor effect. The obtained NPs were smooth spheres with adjustable particle diameters and polymer-dependent surface potentials, both negative and positive.

Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP.

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.

Simple and Inexpensive Microfluidic Thread Based Device for Teaching Microflow Injection Analysis and Electrochemistry

The miniaturization of flow injection analysis devices presents several improvements related to analytical procedures. However, a cost reduction and simplification are required to allow extensive use in undergraduate teaching laboratories. Here we present a low cost microfluidic electroanalytical device composed only of interlocking toy blocks, double sided tape, pencil leads, and hydrophilic cotton threads for performing microflow injection analysis (μFIA). With adequate analytical performance and a manufacturing cost of only $1.0/unit, the proposed device can be easily employed in undergraduate laboratories for teaching content related to μFIA and electrochemistry in a simple and inexpensive way.

Chemiluminescence detection with microfluidics for innovative in situ measurement of unbound cobalt ions in dynamic equilibrium with bound ions in binding study with polyethyleneimine and its functionalized nanoparticles

This work reports a novel method for in situ measurement of binding of cobalt ions to polyethyleneimine (PEI) and polyethyleneimine-functionalized poly (methyl methacrylate) nanoparticles (PEI-NPs) using simple microfluidics with a chemiluminescence detection system. The catalytic effect of free cobalt ion in solution on the luminol-hydrogen peroxide chemiluminescence was employed for the detection of unbound cobalt in dynamic equilibrium with cobalt bound to PEI or PEI-NPs. Many binding measurements lead to incorrect estimation of free metal ions due to insufficient separation of bound and free ions. The catalytic activity of only unbound cobalt ion on the luminol reaction was demonstrated by observing that PEI and PEI-NPs alone did not give chemiluminescence. Also, both Co-PEI and Co-PEI-NPs complexes gave no chemiluminescence when cobalt ion is fully bound with excess PEI or PEI-NPs. In addition diethylenetriamine (dien) as a model ligand to completely bind the cobalt ions was also employed as further confirmation. The chemiluminescence measurement employing microfluidics was then successfully applied for the measurement of binding cobalt ion to PEI and PEI-NPs. This in situ measurement of binding does not require filtration of the two species. As there is no perturbation of equilibrium, an accurate binding measurement can therefore be successfully performed. Experimental parameters, such as concentrations of polymers and cobalt ions, and equilibration time were investigated. Analysis of the experimental data employed the binding equation derived assuming independent and equivalent binding sites of the polymer for the metal ions. Also the binding constant of cobalt ions with PEI-NPs is first reported employing chemiluminescence detection. This work provides quantitative determination of the binding constant and total binding capacity of PEI and PEI-NPs with cobalt ions using chemiluminescence detection and microfluidics as an innovative in situ measurement of the unbound cobalt ions.