Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy.

Actin is an essential protein in almost all life forms. It mediates diverse biological functions, ranging from controlling the shape of cells and cell movements to cargo transport and the formation of synaptic connections. Multiple diseases are closely related to the dysfunction of actin or actin-related proteins. Despite the biological importance of actin, super-resolution imaging of it in tissue is still challenging, as it forms very dense networks in almost all cells inside the tissue. In this work, we demonstrate multiplexed super-resolution volumetric imaging of actin in both cultured cells and mouse brain slices via expansion microscopy (ExM). By introducing a simple labeling process, which enables the anchoring of an actin probe, phalloidin, to a swellable hydrogel, the multiplexed ExM imaging of actin filaments was achieved. We first showed that this technique could visualize the nanoscale details of actin filament organizations in cultured cells. Then, we applied this technique to mouse brain slices and visualized diverse actin organizations, such as the parallel actin filaments along the long axis of dendrites and dense actin structures in postsynaptic spines. We examined the postsynaptic spines in the mouse brain and showed that the organizations of actin filaments are highly diverse. This technique, which enables the high-throughput 60 nm resolution imaging of actin filaments and other proteins in cultured cells and thick tissue slices, would be a useful tool to study the organization of actin filaments in diverse biological circumstances and how they change under pathological conditions.

Live Cell Electron Microscopy Using Graphene Veils.

As a promising tool over the optical resolution limits, liquid electron microscopy is practically utilized to visualize the structural information on wet biological specimens, such as cells, proteins, and nucleic acids. However, the functionality of biomolecules during their observation is still controversial. Here we show the feasibility of live-cell electron microscopy using graphene veils. We demonstrate that the electron dose resistivity of live bacterial cells increases to 100-fold in graphene veils, and thus they maintain their structures and functions after electron microscopy experiments. Our results provide the guidelines and show possibilities for the electron microscopy imaging of live cells and functional biomolecules.

Recent Advances of Fluid Manipulation Technologies in Microfluidic Paper-Based Analytical Devices (µPADs) toward Multi-Step Assays.

Microfluidic paper-based analytical devices (µPADs) have been suggested as alternatives for developing countries with suboptimal medical conditions because of their low diagnostic cost, high portability, and disposable characteristics. Recently, paper-based diagnostic devices enabling multi-step assays have been drawing attention, as they allow complicated tests, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), which were previously only conducted in the laboratory, to be performed on-site. In addition, user convenience and price of paper-based diagnostic devices are other competitive points over other point-of-care testing (POCT) devices, which are more critical in developing countries. Fluid manipulation technologies in paper play a key role in realizing multi-step assays via µPADs, and the expansion of biochemical applications will provide developing countries with more medical benefits. Therefore, we herein aimed to investigate recent fluid manipulation technologies utilized in paper-based devices and to introduce various approaches adopting several principles to control fluids on papers. Fluid manipulation technologies are classified into passive and active methods. While passive valves are structurally simple and easy to fabricate, they are difficult to control in terms of flow at a specific spatiotemporal condition. On the contrary, active valves are more complicated and mostly require external systems, but they provide much freedom of fluid manipulation and programmable operation. Both technologies have been revolutionized in the way to compensate for their limitations, and their advances will lead to improved performance of µPADs, increasing the level of healthcare around the world.

Advection Flows-Enhanced Magnetic Separation for High-Throughput Bacteria Separation from Undiluted Whole Blood.

A major challenge to scale up a microfluidic magnetic separator for extracorporeal blood cleansing applications is to overcome low magnetic drag velocity caused by viscous blood components interfering with magnetophoresis. Therefore, there is an unmet need to develop an effective method to position magnetic particles to the area of augmented magnetic flux density gradients while retaining clinically applicable throughput. Here, a magnetophoretic cell separation device, integrated with slanted ridge-arrays in a microfluidic channel, is reported. The slanted ridges patterned in the microfluidic channels generate spiral flows along the microfluidic channel. The cells bound with magnetic particles follow trajectories of the spiral streamlines and are repeatedly transferred in a transverse direction toward the area adjacent to a ferromagnetic nickel structure, where they are exposed to a highly augmented magnetic force of 7.68 µN that is much greater than the force (0.35 pN) at the side of the channel furthest from the nickel structure. With this approach, 91.68% ± 2.18% of Escherichia coli (E. coli) bound with magnetic nanoparticles are successfully separated from undiluted whole blood at a flow rate of 0.6 mL h-1 in a single microfluidic channel, whereas only 23.98% ± 6.59% of E. coli are depleted in the conventional microfluidic device.

Pumpless Microflow Cytometry Enabled by Viscosity Modulation and Immunobead Labeling.

Major challenges of miniaturizing flow cytometry include obviating the need for bulky, expensive, and complex pump-based fluidic and laser-based optical systems while retaining the ability to detect target cells based on their unique surface receptors. We addressed these critical challenges by (i) using a viscous liquid additive to control flow rate passively, without external pumping equipment, and (ii) adopting an immunobead assay that can be quantified with a portable fluorescence cell counter based on a blue light-emitting diode. Such novel features enable pumpless microflow cytometry (pFC) analysis by simply dropping a sample solution onto the inlet reservoir of a disposable cell-counting chamber. With our pFC platform, we achieved reliable cell counting over a dynamic range of 9-298 cells/µL. We demonstrated the practical utility of the platform by identifying a type of cancer cell based on CD326, the epithelial cell adhesion molecule. This portable microflow cytometry platform can be applied generally to a range of cell types using immunobeads labeled with specific antibodies, thus making it valuable for cell-based and point-of-care diagnostics.

Rapid and background-free detection of avian influenza virus in opaque sample using NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay platform.

Rapid and sensitive on-site detection of avian influenza virus (AIV) is the key for achieving near real-time surveillance of AIV and reducing the risk of dissemination. However, unlike the laboratory-prepared transparent buffer solutions containing a single type of influenza virus, distinction between real- and false- positive outputs and detection of low concentrations of AIV in stool specimens or cloacal swabs are difficult. Here, we developed a rapid and background-free lateral flow immunoassay (LFA) platform that utilizes near-infrared (NIR)-to-NIR upconversion nanoparticles (UCNPs) to yield a sensor that detects AIV nucleoproteins (NPs) from clinical samples within 20 min. Ca2+ as a heterogeneous dopant ion in the shell enhanced the NIR-to-NIR upconversion photoluminescence (PL) emission without inducing significant changes in the morphology of the UCNPs. In a mixture of opaque stool samples and gold nanoparticles (GNPs), which are components of commercial AIV LFA, the background signal of the stool samples masked the absorption peak of GNPs. However, UCNPs dispersed in the stool samples still show strong emission centered at 800 nm when excited at 980 nm, which enables the NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay (NNLFA) platform to detect 10-times lower viral load than a commercial GNP-based AIV LFA. The detection limit of NNLFA for LPAI H5N2 and HPAI H5N6 viruses was 102 and 103.5 EID50/mL, respectively. Moreover, the viruses were successfully detected within dark brown-colored samples using the NNLFA but not the commercial AIV LFA. Therefore, the rapid and background-free NNLFA platform can be used for sensitive on-site detection of AIV.

Solenoid Driven Pressure Valve System: Toward Versatile Fluidic Control in Paper Microfluidics.

As paper-based diagnostics has become predominantly driven by more advanced microfluidic technology, many of the research efforts are still focused on developing reliable and versatile fluidic control devices, apart from improving sensitivity and reproducibility. In this work, we introduce a novel and robust paper fluidic control system enabling versatile fluidic control. The system comprises a linear push-pull solenoid and an Arduino Uno microcontroller. The precisely controlled pressure exerted on the paper stops the flow. We first determined the stroke distance of the solenoid to obtain a constant pressure while examining the fluidic time delay as a function of the pressure. Results showed that strips of grade 1 chromatography paper had superior reproducibility in fluid transport. Next, we characterized the reproducibility of the fluidic velocity which depends on the type and grade of paper used. As such, we were able to control the flow velocity on the paper and also achieve a complete stop of flow with a pressure over 2.0 MPa. Notably, after the actuation of the pressure driven valve (PDV), the previously pressed area regained its original flow properties. This means that, even on a previously pressed area, multiple valve operations can be successfully conducted. To the best of our knowledge, this is the first demonstration of an active and repetitive valve operation in paper microfluidics. As a proof of concept, we have chosen to perform a multistep detection system in the form of an enzyme-linked immunosorbent assay with mouse IgG as the target analyte.

Pseudaminobacter granuli sp. nov., isolated from granules used in a wastewater treatment plant.

A Gram negative, aerobic, non-motile and rod-shaped bacterial strain designated as Gr-2T was isolated from granules used in a wastewater treatment plant in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain Gr-2T grew at 18-37°C (optimum temperature, 30°C) and a pH of 6.0-8.0 (optimum pH, 7.0) on R2A agar medium. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Pseudaminobacter of the family Phyllobacteriaceae, and formed clusters with Pseudaminobacter defluvii THI 051T (98.9%) and Pseudaminobacter salicylatoxidans BN12T (98.7%). The G+C content of the genomic DNA was 63.6%. The predominant respiratory quinone was ubiquinone-10 (Q-10) and the major fatty acids were cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0. The overall polar lipid patterns of Gr-2T were similar to those determined for the other Pseudaminobacter species. DNA-DNA relatedness values between strain Gr-2T and its closest phylogenetically neighbors were below 18%. Strain Gr-2T could be differentiated genotypically and phenotypically from the recognized species of the genus Pseudaminobacter. The isolate therefore represents a novel species, for which the name Pseudaminobacter granuli sp. nov. is proposed with the type strain Gr-2T (=KACC 18877T =LMG 29567T).

Correction: A smart multi-pipette for hand-held operation of microfluidic devices.

Correction for 'A smart multi-pipette for hand-held operation of microfluidic devices' by Byeongyeon Kim et al., Analyst, 2016, 141, 5753-5758.

Rapid preparation and single-cell analysis of concentrated blood smears using a high-throughput blood cell separator and a microfabricated grid film.

Cytological examination of peripheral white blood cells inhomogeneously distributed on a blood smear is currently limited by the low abundance and random sampling of the target cells. To address the challenges, we present a new approach to prepare and analyze concentrated blood smears by rapidly enriching white blood cells up to 32-fold with 92% recovery on average at a high throughput (1mL/min) using a deterministic migration-based separator and by systematically analyzing a large number of the cells distributed over a blood slide using a microfabricated grid film. We anticipate that our approach will improve the clinical utility of blood smear tests, while offering the capability to detect rare cell populations.

A portable somatic cell counter based on a multi-functional counting chamber and a miniaturized fluorescence microscope.

A somatic cell count is the concentration or density of somatic cells in milk, and is an important indicator for monitoring mastitis incidence and milk quality in the dairy industry. Managing and controlling mastitis based on somatic cell counts can help ensure high milk quality and yield. A major challenge when translating existing cell counting methods to such application is that they require off-chip sample preparation and complicated sample and reagent delivery steps that cannot be easily performed in resource-limited settings such as dairy farms. Here, we describe an integrated cell counting platform that enables automatic sample delivery into a cell counting chamber and on-chip sample preparation without requiring any off-chip processes, and that simultaneously provides a miniaturized, hand-held fluorescence device for the identification of fluorescently-labelled somatic cells. Our platform thus allows simple, rapid and accurate enumeration of somatic cells in milk. We successfully demonstrated its capability of counting somatic cells in milk, which can be easily performed even by non-experts without additional instrumentation. The platform represents a promising tool for everyday milk-quality tracking and for controlling mastitis occurrence.

A smart multi-pipette for hand-held operation of microfluidic devices.

A smart multi-pipette for hand-held operation of microfluidic devices is presented and applied to cytotoxicity assays and micro-droplet generation. This method enables a continuous-flow and accurate pumping simply by pushing the plunger of the smart multi-pipette, thereby obviating the need for auxiliary equipment and special expertise in microfluidics. We applied the smart multi-pipette to a cytotoxicity assay using a gradient-generating device and water droplet generation using a T-junction device. In combination with general microfluidic devices, the smart multi-pipette enables the devices to successfully perform their own functions.

A Reconfigurable Microfluidics Platform for Microparticle Separation and Fluid Mixing.

Microfluidics is an engineering tool used to control and manipulate fluid flows, with practical applications for lab-on-a-chip, point-of-care testing, and biological/medical research. However, microfluidic platforms typically lack the ability to create a fluidic duct, having an arbitrary flow path, and to change the path as needed without additional design and fabrication processes. To address this challenge, we present a simple yet effective approach for facile, on-demand reconfiguration of microfluidic channels using flexible polymer tubing. The tubing provides both a well-defined, cross-sectional geometry to allow reliable fluidic operation and excellent flexibility to achieve a high degree of freedom for reconfiguration of flow pathways. We demonstrate that microparticle separation and fluid mixing can be successfully implemented by reconfiguring the shape of the tubing. The tubing is coiled around a 3D-printed barrel to make a spiral microchannel with a constant curvature for inertial separation of microparticles. Multiple knots are also made in the tubing to create a highly tortuous flow path, which induces transverse secondary flows, Dean flows, and, thus, enhances the mixing of fluids. The reconfigurable microfluidics approach, with advantages including low-cost, simplicity, and ease of use, can serve as a promising complement to conventional microfabrication methods, which require complex fabrication processes with expensive equipment and lack a degree of freedom for reconfiguration.

Magnetophoretic Sorting of Single Cell-Containing Microdroplets.

Droplet microfluidics is a promising tool for single-cell analysis since single cell can be comparted inside a tiny volume. However, droplet encapsulation of single cells still remains a challenging issue due to the low ratio of droplets containing single cells. Here, we introduce a simple and robust single cell sorting platform based on a magnetophoretic method using monodisperse magnetic nanoparticles (MNPs) and droplet microfluidics with >94% purity. There is an approximately equal amount of MNPs in the same-sized droplet, which has the same magnetic force under the magnetic field. However, the droplets containing single cells have a reduced number of MNPs, as much as the volume of the cell inside the droplet, resulting in a low magnetic force. Based on this simple principle, this platform enables the separation of single cell-encapsulated droplets from the droplets with no cells. Additionally, this device uses only a permanent magnet without any complex additional apparatus; hence, this new platform can be integrated into a single cell analysis system considering its effectiveness and convenience.

A centrifugally actuated point-of-care testing system for the surface acoustic wave immunosensing of cardiac troponin I.

A fully automated point-of-care testing (POCT) system with a surface acoustic wave (SAW) immunosensor was developed for rapid and sensitive detection of cardiac troponin I (cTnI) in body fluid (plasma and whole blood). The assay, based on gold nanoparticle sandwich immunoassay and subsequent gold staining, was performed on the SAW immunosensor packaged inside a disposable microfluidic cartridge. The entire fluidic process, including plasma separation, reagent transport, metering, and mixing, was carried out by controlling the centrifugal force acting on the rotating cartridge and laser-irradiated ferrowax microvalves. On investigation of sensor response to various cTnI concentrations, the system exhibited a high performance with a detection limit of 6.7 pg mL(-1), and the coefficient of variation was less than 10% over the entire test range (10 pg mL(-1) to 25 ng mL(-1)). On comparing this POCT system with a clinically utilized system in a physical laboratory (Centaur® XP; Siemens), a correlation coefficient of 0.998 was found, validating the diagnostic capability of the SAW immunosensor.

Label-free cell separation using a tunable magnetophoretic repulsion force.

This paper describes a new label-free cell separation method using a magnetic repulsion force resulting from the magnetic susceptibility difference between cells and a paramagnetic buffer solution in a microchannel. The difference in the magnetic forces acting on different-sized cells is enhanced by adjusting the magnetic susceptibility of the surrounding medium, which depends on the concentration of paramagnetic salts, such as biocompatible gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA), dissolved therein. As a proof-of-concept demonstration, Gd-DTPA solutions at concentrations of 0-80 mM were applied to separate U937 cells from red blood cells (RBCs) and to distinguish two different-sized polystyrene (PS) beads (8 and 10 µm in diameter). By increasing the Gd-DTPA concentration from 0 to 40 mM, the separation resolution of PS beads was increased from 0.08 to 0.91. Additionally, we successfully achieved label-free separation of U937 cells from RBCs with >90% purity and 1 × 10(5) cells/h throughput using a 40 mM Gd-DTPA solution.

Versatile immunoassays based on isomagnetophoresis.

We report an isomagnetophoretic immunoassay capable of detecting an attomolar level of proteins and adjusting the dynamic range of target analytes. Here, the magnetic nanoparticles are used as labels on microbeads in sandwich-type immunoassay, detecting the amount of bound analytes by isomagnetophoretic focusing the solid-support microbeads under the magnetic susceptibility gradient and magnetic field in a microchannel. For the practical purpose, this platform was applied to detect three types of breast cancer biomarkers.

Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel.

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.

Magnetophoretic immunoassay of allergen-specific IgE in an enhanced magnetic field gradient.

We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient ( approximately 10(4) T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum ( approximately 10 microL).