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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Here, we report a portable microfluidic device to generate and dispense droplets simply operated by pushbutton for droplet digital polymerase chain reaction (ddPCR), which is named pushbutton-activated microfluidic dropenser (droplet dispenser) (PAMD). After loading the PCR mixtures and the droplet generation oil to PAMD, digitized PCR mixtures are prepared in PCR tubes after the actuation of a pushbutton. Multiple droplet generation units are simultaneously operated by a single pushbutton, and the size of droplets is controllable by adjusting the geometry of the droplet generation channel. To examine the performance of PAMD, digitized PCR mixtures containing genomic DNA of Escherichia coli (E. coli) O157:H7 prepared by PAMD were assessed by a fluorescence signal analyzer after PCR with a thermal cycler. As a result, PAMD can produce analytical droplets for ddPCR as much as a conventional droplet generator even though any external equipment is not required., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Molecular diagnostics can provide a powerful diagnostic tool since it can detect pathogens with high sensitivity, but complicated sample preparation procedures limit its widespread use as an on-site detection tool that relies on the skilled person and external equipment. To resolve these limitations, we report a solid-phase nucleic acid purification using a finger-actuated microfluidic device, which can control a set amount of flow regardless of differences in end-users. To increase the recovery rate, a finger-actuated reciprocator was newly developed and integrated into the microfluidic device that can efficiently react with silica microbeads and reagents. After verifying the finger-actuated microfluidic reciprocator, the effect of the reciprocating flow on the recovery rate was assessed to purify the standard DNA of the hepatitis B virus (HBV). The recovery rate was increased up to ∼50% and 955 to 955 000 IU mL -1 of HBV standard DNA was successfully purified and detected by a real-time polymerase chain reaction. Furthermore, the proposed microfluidic device was exploited to purify the HBV DNA from the patient's blood plasma samples.

Microfluidic technologies offer a number of advantages for sample preparation in point-of-care testing (POCT), but the requirement for complicated external pumping systems limits their wide use. To facilitate sample preparation in POCT, various methods have been developed to operate microfluidic devices without complicated external pumping systems. In this review, we introduce an overview of user-friendly microfluidic devices for practical sample preparation in POCT, including self- and hand-operated microfluidic devices. Self-operated microfluidic devices exploit capillary force, vacuum-driven pressure, or gas-generating chemical reactions to apply pressure into microchannels, and hand-operated microfluidic devices utilize human power sources using simple equipment, including a syringe, pipette, or simply by using finger actuation. Furthermore, this review provides future perspectives to realize user-friendly integrated microfluidic circuits for wider applications with the integration of simple microfluidic valves.

Here, we report an integrated operation of microfluidic pumps and valves only by finger actuation. As the working principle of the finger-actuated microfluidic pumps includes deflection of the poly(dimethylsiloxane) (PDMS) membrane, the pneumatic valves for controlling the flow direction can be easily integrated with the pumps. Using a single button, the flow path can be determined and flow generation can be achieved. We also verified the integrated operation of finger-actuated pumps and valves by demonstrating nucleic acid purification.

A blood cross-matching test should be carried out to prevent a hemolytic transfusion reaction as the final verification step. To simplify complicated procedures of a conventional blood cross-matching test requiring bulky systems and skilled people, we present a finger-actuated microfluidic device for the blood cross-matching test. Although finger actuation is a simple action that anyone can easily accomplish, there would be a variation in the individual finger actuation that may induce the user-dependent errors of the device. Therefore, the working principle of the finger-actuated microfluidic device is newly designed to reduce the user-dependent errors by indirectly controlling the pressure of fluidic channels. The constant volume was repeatedly dispensed by pushing and releasing a pressure chamber regardless of the different pushed depths of the pressure chamber, the pushing time interval, and the end-users. The dispensed volume was linearly increased according to the number of pushing times applied to the pressure chamber and determined by adjusting the diameter of an actuation chamber. In addition, multiple fluids can be dispensed with a desirable ratio by pushing and releasing the pressure chamber. Finally, a finger-actuated microfluidic device for the blood cross-matching test was developed, which can simultaneously actuate four fluidic channels. After loading 50 μL of whole blood samples from a donor and a recipient into two inlets of the device, the blood plasma from each individual was separated through the two plasma separation membranes. The blood cross-matching test results can be achieved by cross-reacting the donor's blood plasma with the recipient's whole blood as well as the donor's whole blood with the recipient's blood plasma by pushing and releasing only a single pressure chamber within 10 min.

Immunohistochemistry (IHC), which has been used to detect antigens in cells of a tissue section using an immunoreaction between an antibody and an antigen, is a practical tool for identifying the type and stage of diseases in cancer diagnosis and scientific research. However, conventional IHC requires long, laborious process times and high costs. Although microfluidic IHC platforms have been developed to overcome these limitations, the application of microfluidic IHC in real-world environments is still limited due to the additional equipment needed to operate the microfluidic systems. In addition, continuous flow in a microfluidic channel leads to a waste of unbound antibodies. In this study, we demonstrate a novel and easy-to-use microfluidic IHC platform operated only using a manual pipette that is commonly available in research laboratories or hospitals. No other device such as a pump or a controller is required to operate our system. Bidirectional flows of the antibody solution in a microfluidic device are induced by repetitive manual pipetting which facilitates the enhanced antigen-antibody reaction and enables the effective use of a limited amount of antibody. When breast cancer cell and tissue sections are reacted with antibodies using our platform, pipetting for less than 2 min is sufficient to obtain immunostaining results without damaging the sample. The staining intensity by our method is similar to that of the sample stained for 1 h by a conventional batch process. We believe that this pipetting-based approach to the operation of a microfluidic system allows end users to use microfluidic IHC more conveniently and easily in real-world environments.

Cell-containing hydrogel modules as cell-hydrogel microunits for creating a physiologically relevant 3D in vivo-like microenvironment with multiple cell types and unique extracellular matrix (ECM) compositions facilitate long-term cell maintenance and bioassays. To date, there have been many important advances in microfluidic bioassays, which incorporate hydrogel scaffolds into surface-accessible microchambers, driven by the strong demand for the application of spatiotemporally defined biochemical stimuli to construct in vivo-like conditions and perform real-time imaging of cell-matrix interactions. In keeping with the trend of fostering collaborations among biologists, clinicians, and microfluidic engineers, it is essential to create a simpler approach for coupling cell-containing hydrogel modules and an automated bioassay platform in a user-friendly format. In this article, we review recent progress in hydrogel-incorporated microfluidics for long-term cell maintenance and discuss some of the simpler and user-friendly 3D bioassay techniques combined with cell-containing hydrogel modules that can be applied to mutually beneficial collaborations with non-engineers. We anticipate that this modular and user-friendly format interfaced with existing laboratory infrastructure will help address several clinical questions in ways that extend well beyond the current 2D cell-culture systems.

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.

By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A quantitative, reproducible, fast and inexpensive multiplexed immunohistochemistry (IHC) system might play a locomotive role in drug screening and personalized medicine. Currently, fully automated IHC machines and sequential multiplexed IHC methods based upon multiple color reagents have been developed, with the evolution of such methods having revealed novel biological findings over the conventional IHC method, which is time consuming and labor intensive. We describe a novel parallel multiplexed IHC method using a microfluidic multiplexed immunohistochemistry (MMIHC) device for quantitative pathological diagnosis of breast cancer. The key factors for success of parallel multiplexed IHC are the fabrication of a robust microfluidic device, the interface between the device and a tissue slide, and an accurate fluidic control for multiple IHC reagents. In order to apply conventional thin-section tissues into on-chip systems without any additional modification process, a tissue slide-compatible assembler was developed for optimal compatibility of conventional IHC methods. With this approach, a perfect fluid control for various solutions was demonstrated without any leakage, bubble formation or cross-contamination. The results presented in this chapter indicate that the microfluidic IHC protocol developed can provide the possibility of tailored cancer treatments as well as precise histopathological diagnosis using numerous specific biomarkers.

Since most of the bioavailable drugs are impermeable through the blood-brain barrier (BBB), development of a rapid and reliable permeability assay system has been a challenge in drug discovery targeting central nervous system (CNS). Here, we designed a microfluidic device to monitor the drug permeability into the CNS. Human umbilical vein endothelial cells (HUVECs) were shortly (2 ~ 3 h) incubated with astrocyte-conditioned medium after being trapped on microholes in the microfluidic device and tested for chip-based permeability measurement of drugs. The measured permeability values were highly correlated with those measured by conventional in vitro methods and the brain uptake index representing the quantity of transported substances across the in vivo BBB of rats. Using the microfluidic device, we could easily monitor the effect of hydrogen peroxide on the trans-endothelial permeability, which are consistent with the finding that the same treatment disrupted the formation of tight junctions between endothelial cells. Considering relatively short period of time needed for endothelial cell culture and ability to monitor the BBB physiology continuously, we propose that this novel system can be used as an invaluable first-line tool for CNS-related drug development.

We constructed a mesh-grid integrated microwell array which enables easy trapping and consistent addition of droplets. The grid acts as a microchannel structure to guide droplets into the microwells underneath, and also provides open access for additional manipulation in a high-throughput manner. Each droplet in the array forms a stable environment of pico-litre volume to implement a single-cell-based assay.

This paper describes the active control of the capillary-driven flow in paper using a centrifugal device.

A novel active surface-enhanced Raman scattering (SERS) platform for dynamic on-demand generation of SERS active sites based on optoelectrofluidics is presented in this paper. When a laser source is projected into a sample solution containing metal nanoparticles in an optoelectrofluidic device and an alternating current (ac) electric field is applied, the metal nanoparticles are spontaneously concentrated and assembled within the laser spot, form SERS-active sites, and enhance the Raman signal significantly, allowing dynamic and more sensitive SERS detection. In this simple platform, in which a glass slide-like optoelectrofluidic device is integrated into a conventional SERS detection system, both dynamic concentration of metal nanoparticles and in situ detection of SERS signal are simultaneously possible with only a single laser source. This optoelectrofluidic SERS spectroscopy allows on-demand generation of 'hot spots' at specific regions of interest, and highly sensitive, reliable, and stable SERS measurements of the target molecules in a tiny volume (∼500 nL) of liquid sample without any fluidic components and complicated systems., (This journal is © The Royal Society of Chemistry 2011)

We report a contraction-expansion array (CEA) microchannel that allows inertial size separation by a force balance between inertial lift and Dean drag forces in fluid regimes in which inertial fluid effects become significant. An abrupt change of the cross-sectional area of the channel curves fluid streams and produces a similar effect compared to Dean flows in a curved microchannel of constant cross-section, thereby inducing Dean drag forces acting on particles. In addition, the particles are influenced by inertial lift forces throughout the contraction regions. These two forces act in opposite directions each other throughout the CEA microchannel, and their force balancing determines whether the particles cross the channel, following Dean flows. Here we describe the physics and design of the CEA microfluidic device, and demonstrate complete separation of microparticles (polystyrene beads of 4 and 10 μm in diameter) and efficient exchange of the carrier medium while retaining 10 μm beads., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Extraordinary advances in lab on a chip systems have been made on the basis of the development of micro/nanofluidics and its fusion with other technologies based on electrokinetics and optics. Optoelectrofluidic technology, which has been recently introduced as a new manipulation scheme, allows programmable manipulation of particles or fluids in microenvironments based on optically induced electrokinetics. Herein, the behaviour of particles or fluids can be controlled by inducing or perturbing electric fields on demand in an optical manner, which includes photochemical, photoconductive, and photothermal effects. This elegant scheme of the optoelectrofluidic platform has attracted attention in various fields of science and engineering. A lot of research on optoelectrofluidic manipulation technologies has been reported and the field has advanced rapidly, although some technical hurdles still remain. This review describes recent developments and future perspectives of optoelectrofluidic platforms for chemical and biological applications.

We report a contraction-expansion array (CEA) microchannel for rapidly and homogeneously mixing different types of fluids by multivortex induced in alternately formed rectangular structures of the channel. Rapid mixing can be achieved in a topologically simple and easily fabricated CEA microchannel, employing a synergetic combination of two kinds of vortices: (1) expansion-vortices being induced by flow separation due to an abrupt change of cross-sectional area of the channel in its expansion region, and (2) Dean-vortices being induced by centrifugal forces acting on a cornering fluid through the channel. We experimentally and numerically investigated expansion- and Dean-vortices, and demonstrated rapid mixing of an aqueous solution containing fluorescein or human red blood cells (RBCs) at different flow rates corresponding to Reynolds number (Re) ranging from 7.2 to 43.0. Over 90% mixing efficiency at a channel length of 14.4 and 19.5 mm was achieved at Re ≥ 28.6 (fluorescein solution and deionized water) and Re ≥ 21.5 (RBC suspension and phosphate buffered saline), respectively. The proposed CEA channel is expected to be useful for a wide range of applications where particles, cells and reagents must be rapidly and homogeneously mixed in microchannels.

Hepatocytes have been used for in vitro hepatotoxicity assays because of their ability to sustain intact liver-specific functions. Here, we demonstrate a hepatotoxicity assay system using primary human hepatocytes trapped in microholes of a microfluidic device, providing a microscale in vivo liver-like environment. We performed microfluidic hepatotoxicity assays of several drugs, including acetaminophen, verapamil, diclofenac, and benzopyrene, all of which are known to specifically affect hepatic function. The drug sensitivities in hepatocytes and HepG2 cells were measured by calculating the live cell fraction at various drug concentrations. The results indicated that hepatocytes were more sensitive to these drugs than HepG2 cells. The lethal concentration 50 values for all drugs tested were similar to those from the in vitro toxicity data with human hepatocytes obtained from the literature. Furthermore, we developed a mathematical hepatotoxicity model based on the time-dependent cell death profiles measured by our device. This novel assay system enabled us to analyze in vivo-like hepatotoxicity in a microfluidic device by exploiting microstructures to mimic the microenvironment of the liver.

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft-lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time-consuming manufacturing steps. We report a three-dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer-by-layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37 degrees C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on-demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites.

This Letter describes a quantitative phase microscopy for microfluidic devices using a simple self-referencing interferometry. Compared with the gross dimensions of the microfluidic device, the microchannel occupies only a small area of the device. Hence, the reference field can be generated by inverting the relative position of the specimen and background. Our system is realized using an extended depth-of-field optics in the form of Michelson interferometry, which allows quantitative phase measurement for an increased depth-of-field without moving objective lens or specimen. Furthermore, the system can be readily converted to a higher signal-to-noise ratio Hilbert phase microscopy thanks to the simultaneous acquisition of double interferograms. The performance of our system is verified using polymer beads, micropatterning poly(dimethylsiloxane) (PDMS), and embryo cells in the microchannels.

Simple and low-cost implementation of three-dimensional (3D) particle measurement is vital for designing and characterizing microfluidic devices that show spatiotemporally varying characteristics in three dimensions. However, the conventional 3D particle image velocimetry or particle streak velocimetry has proven difficult to address the needs, requiring complex and expensive equipment, precise alignment between optical components, and specialized image-processing algorithms. Here, we report mirror-embedded microchannels and a method of optical path-length (OPL) modulation for 3D particle measurement in the channels. The mirror, ideally at 45 degrees, reflects the side view of the channels and enables 3D positional information to be obtained easily from two different orthogonal-axis images with different optical paths. To offset the optical path difference between two image views, we utilized a cover glass as a medium of high refractive index and placed it in the light path through which the side-view image propagates, thereby prolonging the OPL of the side view and simultaneously shifting its depth of field (DOF) range. This modulation ensures imaging of in-focus side view as well as top view. This 3D imaging principle was verified by observing 3D positions of 6 mum-sized beads in the linear and grooved microchannels. The mirror-embedded scheme can be readily fabricated with existing microfluidic designs, and offer easy and simple implementation of 3D particle measurement.

We report a contraction-expansion array (CEA) microchannel that allows three-dimensional hydrodynamic focusing with a single sheath flow in a single-layer device. The CEA microchannel exploits centrifugal forces acting on fluids travelling along the contraction and expansion regions of the microchannel. Around an entrance of the contraction region, the centrifugal forces induce a secondary flow field where two counter-rotating vortices enable to envelop a sample flow with a sheath flow in three dimensions. We herein describe an underlying principle and a design of the CEA microchannel and demonstrate complete sheathing of a sample fluid (water and human red blood cells) in three dimensions. The focusing characteristics of the CEA microchannel are investigated in terms of the number of the rectangular structures, flow rate, and flow ratio between sample and sheath flows. This microfluidic channel for three-dimensional hydrodynamic focusing is easy to fabricate in a single-layer fabrication process and simple to operate with a single sheath flow.

This work demonstrates a novel microfluidic in vitro cultivation system for embryos that improves their development using a partially constricted channel that mimics peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. To investigate the effects of constriction geometry on embryonic development, different constriction widths of the channel were designed. Bovine embryos were loaded and incubated by simply placing them on a tilting machine to provide embryo movement via gravity. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. To confirm the quality of blastocysts in the microfluidic channel, double staining was performed and compared with bovine embryos cultivated by the conventional droplet method. The proportion of eight-cell development among total embryos in the constricted channel (56.7+/-13.7%; mean+/-SD) was superior to that in the straight channel (23.9+/-11.0%). This suggests that the effect of constriction is vital for the early development of bovine embryos in assisted-reproduction research.

This paper demonstrates a method for tuning elastomeric microchannels for hydrophoretic separation made in poly(dimethylsiloxane) (PDMS). Uniform compressive strain is imposed on the elastomeric microchannel between two acrylic substrates by fastening the bolts. The elastomeric microchannel can change its cross-section during compression, simultaneously tuning the criterion for hydrophoretic ordering. The change of the channel cross-section under compression is studied using a confocal microscope and finite element method (FEM). By pressing the channel for hydrophoretic separation, we achieved tuning of the separation criterion from 7 to 2.5 microm in particle diameter.

This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.

As orally administered drugs must be absorbed from the intestine into the blood circulation, permeability assays of drug candidates have been widely used in the early screening stages of drug discovery. In this study, a microfluidic device was developed for the drug permeability assay, considering the in vivo delivery path of drugs in humans. A microhole array for cell trapping was fabricated using the poly(dimethylsiloxane) (PDMS) molding technique by mimicking the intestinal epithelial cell membrane. On the basis of mathematical simulations, the configuration of the microfluidic device, including a microhole array and a mixing channel, was optimized to trap cells firmly in each microhole. At the flow rate under optimal conditions, cells were effectively trapped in a microhole array without cell damage. We measured the permeability of 10 drugs, including those with high and low permeability in microchannels, and compared the results with the reported values of permeability in the human and rat intestine. Most drugs had a high p value (p > 0.4), and only a few drugs had a low p value less than 0.05 by t test. Though their measured permeabilities are not the same as those in vivo human intestine, it shows that in vivo permeabilities in the human and rat intestine are highly correlated with those measured by the microfluidic device (R(2) = 0.9013 and R(2) = 0.8765, respectively). Also, the fraction of the dose absorbed in the human intestine (F(a)) indicated that the drug permeability measured using this device was significantly correlated (R(2) = 0.9641) with those in human subjects. As the microfluidic assay system is dependent on cells trapped inside a microhole array, it is a valuable tool in drug discovery as well as an alternative to animal testing.

Cell cycle studies for examining regulatory mechanisms and progression invariably require synchronization of cell cultures at a specific phase of the cell cycle. Current implementations to produce synchronous cell populations, however, tend to perturb normal cellular progression and metabolism and typically require complex, time-consuming preparations. Thus, it is challenging for the development of a simple, noninvasive, and effective means for cell cycle synchronization. We demonstrate the use of hydrophoretic size separation to sort cells in target phases of the cell cycle entirely based on a hydrodynamic principle. With this method, we found that there is a linear relationship between a cell's size and its position distribution in the hydrophoretic device. We also demonstrate the robustness of the hydrophoretic method for practical applications by sorting cells in the G(0)/G(1) and G(2)/M phases out of the original, asynchronous cells with a high level of synchrony of 95.5% and 85.2%, respectively. These results show that the hydrophoretic size separation can be used in order to collect cells at the same phase of the cell cycle in a gentle, noninvasive way.

A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator. As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment.

We demonstrate rapid manipulation and selective concentration of microparticles using AC electrokinetics such as dielectrophoresis (DEP) and AC electro-osmosis (ACEO) in an optoelectrofluidic platform based on a liquid crystal display (LCD). When 10 V bias at 10 kHz was applied to the optoelectrofluidic device, only the 1 microm-diameter polystyrene particles were concentrated into the projected LCD image patterns and closely packed, forming the crystalline structure by ACEO flow, while the 6 microm-diameter particles were repelled by negative DEP forces. We have characterized this frequency-dependency of the optoelectrofluidic particle behavior according to the particle diameter. On the basis of these results, we can rapidly concentrate the 1 microm-diameter particles and separate them from the 6 microm particles, by applying an AC signal of 10 kHz frequency. This novel technique can be applied to rapidly concentrate, separate and pattern micro-/nanoparticles in many biological and chemical applications.

We describe a hydrophoretic device that uses rotational flows induced by regularly patterned obstacles only on the top wall for preparing samples of biological particles, including micrometer and submicrometer particles, and DNA molecules. Many of the current continuous separation devices based on physical fields are limited to the separation of cells and micrometer-sized particles due to their dependence on a particle volume, and the purely hydrodynamic separation of macromolecules such as DNA or protein complexes remains a challenge. Hydrophoresis is entirely based on hydrodynamics using rotational flows induced by anisotropic obstacles. Different sizes of micrometer and submicrometer beads, as well as DNA molecules, were separated into distinct trajectories using two kinds of hindrance mechanisms. Continuous separation of these particles was achieved using the obstacles, demonstrating the potential of hydrophoresis for biological sample preparation on the micro- and nanoscales, with the advantages of continuous flow and sheathless passive operation.

A microfluidic platform to satisfy both 3D cell culture and cell-based assay is required for credible assay results and improved assay concept in drug discovery. In this article, we demonstrate a microvalve-assisted patterning (MAP) platform to provide a new method for investigating cellular dynamics by generating a linear concentration gradient of a drug as well as to realize 3D cell culture in a microenvironment. The MAP platform was fabricated by multilayer soft lithography and several microvalves made it possible to pattern a cell-matrix (scaffold) and to exchange media solutions without breaking cell-matrix structure in a microchannel. This approach provides not only exact fluids control, bubble removal, and stable solution exchange in a microchannel, but also reliable scaffold fabrication and 3D cell culture. In this study, hepatotoxicity tests with human hepatocellular liver carcinoma cells (HepG2) were also performed in real-time monitoring where cell morphologies exposed to different drug concentrations were observed at a time. Compared to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the MAP platform could be used to reduce drug amount and assay time for cell-based assays as much as 10 and 3 times, respectively.

This paper reports a new dielectrophoretic separation method of porcine oocytes for in vitro fertilization. Conventional manual selection of oocyte highly depends on the expert's experience and lacks universal standards for identifying the quality of oocyte. In this study, an electrode array chip with castellated shape was developed to evaluate dielectrophoretic velocities of oocytes, under applied bias conditions with an AC 3 V waveform at 1 MHz for 15 s. Based on different dielectrophoresis (DEP) response, the selected group of oocytes that moved showed a better developmental potential than the group of oocytes that stayed, representing a higher rate of blastocyst formation and a lower rate of polyspermic fertilization. In addition, the overall developmental potential of oocytes selected by the DEP device was comparable to that of oocytes selected by conventional manual method. These results demonstrate that the difference in dielectrophoretic velocity can be used to establish an objective criterion for the selection of oocytes. Consequently, this method will open the possibility to develop an automatic tool for oocyte selection, which would be helpful for assisted reproductive technologies such as transgenic and clonal animal production.

We present a novel microfluidic device with exponentially increasing obstacle arrays to enable sheathless particle focusing. The anisotropic fluidic resistance of slant obstacles generates transverse flows, along which particles are focused to one sidewall. In the successive channel with exponentially increasing widths, bent obstacles extended from the slant obstacles increase the focusing efficiency of the particles. With the device, we achieved the focusing efficiency of 76%, 94%, and 98% for 6, 10, and 15 microm beads, respectively. The focusing efficiency of the particles can be further improved in the devices with more extension steps. In addition, using the microfluidic devices with the symmetric structure of the slant and bent obstacles, we achieved complete focusing of biological cells to the centerline of a channel within 1.7% coefficient of variation. The results demonstrated the sheathless hydrophoretic focusing of microparticles and cells with the advantages of a sheathless method, passive operation, single channel, and flow rate independence.

A magnetophoretic continuous purification method is presented of single-walled carbon nanotubes (SWCNTs) from the superparamagnetic iron-catalyst impurities in a microfluidic device without any influence on inherent SWCNT properties. By employing microfluidics and a magnetic-field-induced saw-tooth nickel microstructure, a highly enhanced magnetic force in adjoining microchannels is exploited. The iron impurities of SWCNTs are attracted towards areas of higher magnetic-flux density in the microchannels where magnetic field was asymmetrically generated perpendicularly to the streamline. We obtained highly purified SWCNTs at a rate of 0.36 mg h(-1) and that are estimated to be about 99% purity.

A sensitive and rapid electrochemical microchip fabricated by assembling a surface-functionalized poly(dimethylsiloxane) (PDMS) microchannel with an interdigitated array (IDA) gold electrode was developed for the detection of human cardiac troponin I (cTnI) in the early diagnosis of acute myocardial infarction. Anti-cTnI was immobilized onto the internal surface of the PDMS channel on which protein G layer had been generated by silanization. To reduce electrode fouling, a PDMS channel was assembled with an IDA chip after surface treatment. The detection experiments were performed with successive injection of cTnI, alkaline phosphatase (AP)-labeled anti-cTnI, and p-aminophenylphosphate. Then, cyclic voltammograms were obtained by the oxidation peak current proportionally to the concentration of enzymatic product, p-aminophenol. The optimal packing density of anti-cTnI on the surface of the PDMS channel was determined at the anti-cTnI concentration of 30 microg/ml for the highest electrochemical signal. These demonstrate that the proper orientation and best packing density of antibody as well as no electrode fouling contributed to the low detection limit (148 pg/ml) of cTnI within 8 min.

We report a microfluidic separation and sizing method of microparticles with hydrophoresis--the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, we can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. Using such movements of particles, we completely separated polystyrene microbeads with 9 and 12 microm diameters. Also, we discriminated polystyrene microbeads with diameter differences of approximately 7.3%. Additionally, we measured the diameter of 10.4 microm beads with high coefficient of variation and compared the result with a conventional laser diffraction method. The slanted obstacle as a microfluidic control element in a microchannel is analogous to the electric, magnetic, optical, or acoustic counterparts in that their function is to generate a field gradient. Since our method is based on intrinsic pressure fields, we could eliminate the need for external potential fields to induce the movement of particles. Therefore, our hydrophoretic method will offer a new opportunity for power-free and biocompatible particle control within integrated microfluidic devices.

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.