Microfluidic channel-integrated hanging drop array chip operated by pushbuttons for spheroid culture and analysis.

Although the hanging drop methods have a number of advantages for spheroid culture, they suffer from reagent exchange procedures that depend on tedious and accurate liquid handling by manual pipetting or robotic arms. To simplify these procedures, we developed a method for liquid handling in a hanging drop array (HDA) chip for spheroid culture and analysis by integrating microfluidic channels operated by pushbuttons. Six finger-actuated microfluidic pumping units connected to a 3 × 3 HDA can draw or replenish reagents in an HDA chip without any external equipment. The initial cell seeding, medium exchange, and staining for further analysis can be simply done by pushing the buttons in the programmed order. After the assessment of the reagent exchange ratio of the device, BT474 spheroids of various sizes were cultured in the device for 7 days by exchanging the medium once a day and stained on the same device by exchanging the medium with staining reagents for the LIVE/DEAD assay. Furthermore, the cultured spheroids were embedded into collagen by exchanging the medium with a collagen solution to mimic a cancer metastasis environment.

Colorimetric Detection of Escherichia coli O157:H7 with Signal Enhancement Using Size-Based Filtration on a Finger-Powered Microfluidic Device.

Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 102 CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.

Finger-Actuated Microfluidic Display for Smart Blood Typing.

Accurate blood typing is required before transfusion. A number of methods have been developed to improve blood typing, but these are not user-friendly. Here, we have developed a microfluidic smart blood-typing device operated by finger actuation. The blood-typing result is displayed by means of microfluidic channels with the letter and the symbol of the corresponding blood type. To facilitate the mixing of blood and reagents, the two sample inlets are connected to a single actuation chamber. According to the agglutination aspect in the mixture, the fluids are directed to both the microslit filter channels and bypass channels, or only to the bypass channels. The dimension of the microslit filter being clogged by the red blood cell aggregates was optimized to achieve reliable blood-typing results. The flow rate ratio between two channels in the absence of agglutination was subjected to numerical analysis. With this device, blood typing was successfully performed by seven button pushes using less than 10 µL of blood within 30 s.

Toxicity Assessment of Iron Oxide Nanoparticles Based on Cellular Magnetic Loading Using Magnetophoretic Sorting in a Trapezoidal Microchannel.

To accurately assess potential nanotoxicity on the basis of cellular iron content, the precise separation of cells into subpopulations according to their magnetic nanoparticle loading is of crucial importance. In this study, we developed a microfluidic magnetophoresis device consisting of a trapezoidal channel containing five side outlet branches and a narrow rectangular channel with three outlet branches. This unique structure enabled the sequential separation of cells loaded with tiny amounts of iron oxide and cells heavily labeled with iron oxide, in a single device. As a proof of concept, we attempted the sequential separation of Raw 264.7 cells with a large heterogeneity in uptake capabilities (1-50 pg of iron per cell). Consequently, we were able to differentiate the bulk cell population into seven subpopulations according to their mean iron oxide loading. We also evaluated potential nanotoxicity effects using the production of excess reactive oxygen species (ROS) and the inhibition of proliferation on the separated subpopulations, and we found that 46.6% of cells loaded with iron above the threshold value (16.4 pg) had higher ROS levels than the control group. Cells loaded with more than 3.7 pg of iron exhibited transiently inhibited cell-cycle progression. In particular, cells loaded with more than 35.4 pg of iron exerted a significant effect on cell proliferation. The proposed system could be useful in the investigation of nanotoxicity effects of iron oxide nanoparticle-induced cells, based on their iron oxide nanoparticle loading.

Visualization and label-free quantification of microfluidic mixing using quantitative phase imaging.

Microfluidic mixing plays a key role in various fields, including biomedicine and chemical engineering. To date, although various approaches for imaging microfluidic mixing have been proposed, they provide only quantitative imaging capability and require exogenous labeling agents. Quantitative phase imaging techniques, however, circumvent these problems and offer label-free quantitative information about concentration maps of microfluidic mixing. We present the quantitative phase imaging of microfluidic mixing in various types of polydimethylsiloxane microfluidic channels with different geometries; the feasibility of the present method was validated by comparing it with the results obtained by theoretical calculation based on Fick's law.

Functional Packaging of Lateral Flow Strip Allows Simple Delivery of Multiple Reagents for Multistep Assays.

This paper presents a rotary device designed for facile delivery of multiple reagents to a paper strip for multistep assays. Its purpose is to allow users to easily perform multistep assays and achieve sensitive detection. While the test strip remains stationary, rotating the top piece of the device aligns the reagent and absorbent pads to each end of the paper strip and initiates fluid flow. Further incremental rotation makes an adjacent pair of pads to align simultaneously, causing fluid flow of subsequent reagent that was preloaded in the reagent pad. In this work, various porous substrates were tested to observe their effect on overall flow rate of the system and multistep assays were performed to demonstrate its simple use. As a proof of concept, enzyme-linked immunosorbent assay was carried out to detect Escherichia coli O157:H7.

Pressed Paper-Based Dipstick for Detection of Foodborne Pathogens with Multistep Reactions.

This paper presents a pressed paper-based dipstick that enables detection of foodborne pathogens with multistep reactions by exploiting the delayed fluid flow and channel partition formation on nitrocellulose (NC) membrane. Fluid behaviors are easily modified by controlling the amount of pressure and the position of pressed region on the NC membrane. Detection region of the dipstick is optimized by controlling flow rate and delayed time based on Darcy's law. All the reagents required for assay are dried on the NC membrane and they are sequentially rehydrated at the prepartitioned regions when the device is dipped into sample solution. In this manner, multistep reactions can be facilitated by one-step dipping of the dipstick into the sample solution. As a proof of concept, we performed detection of two fatal foodborne pathogens (e.g., Escherichia coli O157:H7 and Salmonella typhimurium) with signal enhancement. In addition, we expanded the utilization of channel partitions by developing a pressed paper-based dipstick into dual detection format.

On-chip generation of monodisperse giant unilamellar lipid vesicles containing quantum dots.

Monodispersed lipid vesicles have been used as a drug delivery vehicle and a biochemical reactor. To generate monodispersed lipid vesicles in the nano- to micrometer size range, an extrusion step should be included in conventional hand-shaking method of lipid vesicle synthesis. In addition, lipid vesicles as a drug carrier still need to be improved to effectively encapsulate concentrated biomolecules such as cells, proteins, and target drugs. To overcome these limitations, this paper reports a new microfluidic platform for continuous synthesis of small-sized (∼10 µm) giant unilamellar vesicles (GUVs) containing quantum dots (QDs) as a nanosized model drug. To generate GUVs, we introduced an additional cross-flow to break vesicles into small size. 1,2 - dimyristoyl-sn-glycero - 3 - phosphocholine (DMPC) in an octanol-chloroform mixture was used in the construction of self-assembled membrane. Consequently, we have successfully demonstrated the fabrication of monodispersed GUVs with 7-12 µm diameter containing QDs. The proposed synthesis method of cell-sized GUVs would be highly desirable for applications such as multipurpose drug encapsulation and delivery.

Automated measurement of multiple cancer biomarkers using quantum-dot-based microfluidic immunohistochemistry.

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.

Bioprinted Multi-Composition Array Mimicking Tumor Microenvironments to Evaluate Drug Efficacy with Multivariable Analysis.

Current organ-on-a-chip technologies confront limitations in effectively recapitulating the intricate in vivo microenvironments and accommodating diverse experimental conditions on a single device. Here, a novel approach for constructing a multi-composition tumor array on a single microfluidic device, mimicking complex transport phenomena within tumor microenvironments (TMEs) and allowing for simultaneous evaluation of drug efficacy across 12 distinct conditions is presented. The TME array formed by bioprinting on a microfluidic substrate consists of 36 individual TME models, each characterized by one of three different compositions and tested under four varying drug concentrations. Notably, the TME model exhibits precise compartmentalization, fostering the development of self-organized vascular endothelial barriers surrounding breast cancer spheroids affecting substance transport. Multivariable screening and analysis of diverse conditions, including model complexity, replicates, and drug concentrations, within a single microfluidic platform, highlight the synergistic potential of integrating bioprinting with microfluidics to evaluate drug responses across diverse TME conditions comprehensively.

Evaluation of Fluid Behaviors in a Pushbutton-Activated Microfluidic Device for User-Independent Flow Control.

Although numerous studies have been conducted to realize ideal point-of-care testing (POCT), the development of a user-friendly and user-independent power-free microfluidic platform is still a challenge. Among various methods, the finger-actuation method shows a promising technique that provides a user-friendly and equipment-free way of delivering fluid in a designated manner. However, the design criteria and elaborate evaluation of the fluid behavior of a pushbutton-activated microfluidic device (PAMD) remain a critical bottleneck to be widely adopted in various applications. In this study, we have evaluated the fluid behavior of the PAMD based on various parameters, such as pressing velocity and depth assisted by a press machine. We have further developed a user-friendly and portable pressing block that reduces user variation in fluid behavior based on the evaluation.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

In situ analysis of heterogeneity in the lipid content of single green microalgae in alginate hydrogel microcapsules.

Microalgae, a group of microorganisms that grow using sunlight as the sole energy source and carbon dioxide as an only carbon source, have been considered as a feedstock of choice for the production of biofuels such as biodiesel. To explore the economic feasibility of such application, however, many technical hurdles must first be overcome; the selection and/or screening of competent species are some of the most important and yet challenging tasks. To greatly accelerate this rather slow and laborious step, we developed a droplet-based microfluidic system that uses alginate hydrogel microcapsules with a mean diameter of 26 µm, each of which is able to encapsulate a single microalgal cell. This novel device was successfully demonstrated using three microalgae species, namely, Chlorella vulgaris , Chlamydomonas sp., and Botryococcus braunii . In situ analysis of the lipid content of individual microalgal cells by nondestructive fluorescence staining using BODIPY (4,4-difluoro-1,3,5,7,-tetramethyl-4-bora-3a,4a-diaza-s-indacene) was possible. In all cases, we confirmed that the lipid content of microalgal species in alginate hydrogel microcapsules was comparable to that of free-living cells. Stochastic heterogeneity in the lipid content was verified under a highly viable physiological condition, implying that other analyses were possible after the determination of lipid content. Furthermore, the designed microwell arrays enabled us to distinguish the BODIPY fluorescence response of a single live alga within the microcapsules.

Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

Reduction in microparticle adsorption using a lateral interconnection method in a PDMS-based microfluidic device.

Microparticle adsorption on microchannel walls occurs frequently due to nonspecific interactions, decreasing operational performance in pressure-driven microfluidic systems. However, it is essential for delicate manipulation of microparticles or cells to maintain smooth fluid traffic. Here, we report a novel microparticle injection technique, which prevents particle loss, assisted by sample injection along the direction of fluid flow. Sample fluids, including microparticles, mammalian (U937), and green algae (Chlorella vulgaris) cells, were injected directly via a through hole drilled in the lateral direction, resulting in a significant reduction in microparticle attachment. For digital microfluidic application, the proposed regime achieved a twofold enhancement of single-cell encapsulation compared to the conventional encapsulation rate, based on a Poisson distribution, by reducing the number of empty droplets. This novel interconnection method can be straightforwardly integrated as a microparticle or cell injection component in integrated microfluidic systems.

Dielectrophoresis in a slanted microchannel for separation of microparticles and bacteria.

Dielectrophoresis (DEP) is an effective method to trap, manipulate and separate various dielectric particles. To generate a DEP force, a spatially nonuniform electrical field has been generated by an array of electrodes, while electrodeless DEP has been accomplished by placing an insulating material between two electrodes. Here, we describe a new DEP method for generating a nonuniform electrical field using a slanted microchannel. The electric field gradient is induced due to a slope in the channel and can be used to move and separate particles. Based on the gradual electric field induced by three dimensional structure of the microchannel, our method enables particles of different sizes to be separated solely by DEP force without flow. The slanted microchannel was easily fabricated by a replica molding technique using a commercial UV-cured photopolymer (NOA 63) and bonded as an insulating layer between two indium-tin-oxide films. By applying the electrical field, polystyrene beads of different sizes (6-45 microm in diameter) were trapped and separated depending on the applied electric strength and frequency. Using this method, the opportunistic pathogen Pseudomonas aeruginosa attached to antibody-conjugated microbeads was successfully separated from Escherichia coli in a slanted microchannel.

Fabrication of a self-assembled and vascularized tumor array via bioprinting on a microfluidic chip.

A tumor microenvironment (TME) is a complex system that comprises various components, including blood vessels that play a crucial role in supplying nutrients, oxygen, and growth factors, as well as delivering chemotherapy drugs to the tumor mass through the vascular endothelial barrier. To replicate the TME in vitro, several bioprinting and microfluidic organ-on-a-chip technologies have been developed. However, these technologies have not been fully exploited in terms of potential benefits of bioprinting and microfluidics, such as precise spatial control for biological samples, construction of multiple TMEs per microfluidic device, and the ability to adjust culture environments for better biological similarity. In addition, the complex transport phenomena within the vascular endothelial barrier and the aggregated tumor mass in the TME model should be considered before applying the model to drug treatment and screening. In this study, we describe a novel integrative technology that addresses these issues by introducing a self-organized TME array bioprinted on a microfluidic chip consisting of a vascular endothelial barrier surrounding breast cancer spheroids. To integrate the TME array onto the microfluidic platform, a microfluidic substrate for extrusion bioprinting was developed for a cell culture platform, which enables diffusivity control by microstructures and establishes a perfusion culture environment inside the culture channel. We also analyzed the cellular behaviors within the TME array to investigate the influence of the diffusivity on the self-organization process required to form the vascular endothelial barrier surrounding breast cancer spheroids.

Hybrid Biofabrication of Heterogeneous 3D Constructs Using Low-Viscosity Bioinks.

The application of cytocompatible hydrogels supporting extensive cellular activities to three-dimensional (3D) bioprinting is crucial for recreating complex physiological environments with high biomimicry. However, the poor printability and tunability of such natural hydrogels diminish the versatility and resolution of bioprinters. In this study, we propose a novel approach for the hybrid biofabrication of complex and heterogeneous 3D constructs using low-viscosity bioinks. Poly(lactic acid) (PLA) filament is extruded by fused deposition modeling on a micromesh to create PLA-framed micromesh substrates onto which fibrinogen is printed by microextrusion bioprinting. The micromesh supports the printed hydrogel with a capillary pinning effect to enable high-resolution bioprinting. Accordingly, the micromesh-bioink layers are aligned and stacked to form volumetric constructs. This approach, called the 3D micromesh-bioink overlaid structure and interlocked culture (3D MOSAIC) platform, enables the fabrication of complicated and multimaterial 3D structures, including overhangs and voids. Endothelial cells cultured under vasculogenic conditions in the platform self-organize within the biologically functional hydrogel to form vascular networks, and cancer cell migration can be observed across the layers. The multidisciplinary 3D MOSAIC platform is an important step toward the biofabrication of complex constructs with high biological and structural significance and functionality.

Characterization of PDMS Microchannels Using Horizontally or Vertically Formed 3D-Printed Molds by Digital Light Projection.

Three-dimensional (3D) printing is one of the promising technologies for the fabrication of microstructures due to its versatility, ease of fabrication, and low cost. However, the direct use of 3D-printed microstructure as a microchannel is still limited due to its surface property, biocompatibility, and transmittance. As an alternative, rapid prototyping of poly(dimethylsiloxane) (PDMS) from 3D-printed microstructures ensures both biocompatibility and efficient fabrication. We employed 3D-printed molds fabricated using horizontal and vertical arrangement methods with different slice thicknesses in a digital light projection (DLP)-based 3D printing process to replicate PDMS microchannels. The replicated PDMS structures were investigated to compare their optical transmittances and surface roughness. Interestingly, the optical transmittance of PDMS from the 3D-printed mold was significantly increased via bonding two single PDMS layers. To evaluate the applicability of the replicated PDMS devices from the 3D-printed mold, we performed droplet generation in the PDMS microchannels, comparing the same device from a conventional Si-wafer mold. This study provides a fundamental understanding of prototyping microstructures from the DLP-based 3D-printed mold.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis.

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.