Nanoporous electroporation needle for localized intracellular delivery in deep tissues.

The exogenous control of intracellular drug delivery has been shown to improve the overall efficacy of therapies by reducing nonspecific off-target toxicity. However, achieving a precise on-demand dosage of a drug in deep tissues with minimal damage is still a challenge. In this study, we report an electric-pulse-driven nanopore-electroporation (nEP) system for the localized intracellular delivery of a model agent in deep tissues. Compared with conventional bulk electroporation, in vitro nEP achieved better transfection efficiency (>60%) with a high cell recovery rate (>95%) under a nontoxic low electroporation condition (40 V). Furthermore, in vivo nEP using a nanopore needle electrode with a side drug-releasing compartment offered better control over the dosage release, time, and location of propidium iodide, which was used as a model agent for intracellular delivery. In a pilot study using experimental animals, the nEP system exhibited two times higher transfection efficiency of propidium iodide in the thigh muscle tissue, while minimizing tissue damage (<20%) compared to that of bulk electroporation. This tissue-penetrating nEP platform can provide localized, safe, and effective intracellular delivery of diverse therapeutics into deep tissues in a controlled manner.

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays.

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40 min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.

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.

On-site fabrication of injectable 131I-labeled microgels for local radiotherapy.

Nuclear medicine is a routine but essential clinical option for diagnostic imaging and disease treatment. Encapsulating radioisotopes in injectable biodegradable hydrogels is ideal for localizing radiation sources to target tissues or organs to achieve long-term, low-dose radiotherapy. However, difficulties in the on-site production of radioactive gels upon treatment and the unpredictable radiation level at the target region are major obstacles to their clinical use. In this study, we bypassed these limitations by developing locally injectable hydrogel microparticles based on 131I-labeled photo-crosslinkable hyaluronic acid (HA) and a microfluidic high-throughput droplet generator. This approach enabled rapid on-site production of injectable, radioactive, biodegradable (IRB) HA microgels, thus allowing their immediate therapeutic application with improved local retention and predictable radioactivity. We demonstrated the clinical utility of this comprehensive approach by preparing IRB HA microgels within 15 min and localizing them to the target tissue (rat muscle) with minimal off-target biodistribution and in vivo radioactivity that extended beyond 3 weeks.

One-Step Microfluidic Purification of White Blood Cells from Whole Blood for Immunophenotyping.

One-step purification of white blood cells (WBCs) is essential to automate blood sample preparation steps for WBC analysis, but conventional methods such as red blood cell (RBC) lysis and density-gradient centrifugation typically require harsh chemical or physical treatment, followed by repeated manual washing steps. Alternative microfluidic separation methods show limited separation performances due to the trade-off between purity and throughput. Herein, an integrated microfluidic device is developed to decouple the trade-off by synergistically combining a slant array ridge-based WBC enrichment unit as a throughput enhancer and a slant, asymmetric lattice-based WBC washing unit as a purity enhancer. The enrichment unit can maintain a high sample-infusion throughput while lowering the flow rate into the washing unit, thus enabling WBC-selective washing without significant influence by the overwhelming number of RBCs and inertial forces. The device delivers efficient separation performances by rejecting 99.9% of RBCs as well as 99.9% of blood plasma from canine and human whole blood in a single round of purification at a high throughput of 60 µL/min. The purified WBC population well preserves the composition of lymphocyte subpopulations, the major components of the adaptive immune system, thus providing the potential for the integrated device to be used as an essential sample-preparation tool for immunologic investigations.

Handheld Microflow Cytometer Based on a Motorized Smart Pipette, a Microfluidic Cell Concentrator, and a Miniaturized Fluorescence Microscope.

Miniaturizing flow cytometry requires a comprehensive approach to redesigning the conventional fluidic and optical systems to have a small footprint and simple usage and to enable rapid cell analysis. Microfluidic methods have addressed some challenges in limiting the realization of microflow cytometry, but most microfluidics-based flow cytometry techniques still rely on bulky equipment (e.g., high-precision syringe pumps and bench-top microscopes). Here, we describe a comprehensive approach that achieves high-throughput white blood cell (WBC) counting in a portable and handheld manner, thereby allowing the complete miniaturization of flow cytometry. Our approach integrates three major components: a motorized smart pipette for accurate volume metering and controllable liquid pumping, a microfluidic cell concentrator for target cell enrichment, and a miniaturized fluorescence microscope for portable flow cytometric analysis. We first validated the capability of each component by precisely metering various fluid samples and controlling flow rates in a range from 219.5 to 840.5 µL/min, achieving high sample-volume reduction via on-chip WBC enrichment, and successfully counting single WBCs flowing through a region of interrogation. We synergistically combined the three major components to create a handheld, integrated microflow cytometer and operated it with a simple protocol of drawing up a blood sample via pipetting and injecting the sample into the microfluidic concentrator by powering the motorized smart pipette. We then demonstrated the utility of the microflow cytometer as a quality control means for leukoreduced blood products, quantitatively analyzing residual WBCs (rWBCs) in blood samples present at concentrations as low as 0.1 rWBCs/µL. These portable, controllable, high-throughput, and quantitative microflow cytometric technologies provide promising ways of miniaturizing flow cytometry.

Integrated microfluidic pneumatic circuit for point-of-care molecular diagnostics.

Developing simple, portable, rapid, and easy-to-use diagnostic technologies is essential for point-of-care (POC) blood molecular testing. Integrated microfluidic devices that include the functionalities of blood separation, microfluidic pumping, and molecular detection are desirable for POC testing; however, current technologies still rely on off-chip sample processing or require bulky equipment. We report a fully-integrated microfluidic diagnostic device, i.e., an integrated pneumatic microfluidic circuit (iPC), that can autonomously pump whole blood, continuously sort blood plasma, and readily enable blood plasma proteomic analysis. The iPC contains vacuum pillars as a vacuum source and waste reservoir, as well as microchannels connecting the pillars as a plasma separator or a flow stabilizer. We combined the iPC and a miniaturized fluorescence microscope to create a portable diagnostic platform that enables fluorescence-based biomarker detection. First, we performed systematic parametric studies to establish design rules for determining the transport and distribution of fluid streams in the iPC. We then demonstrated the capability of the iPC-based diagnostic platform by successfully separating blood plasma from microliter quantities of whole blood while simultaneously quantifying thrombin in blood samples using an aptamer beacon within 5 min of sample injection. Our platform holds potential as a rapid, field-deployable, essentially universal diagnostic tool in POC settings.

An open-source programmable smart pipette for portable cell separation and counting.

Microfluidics offers great potential for biomedical applications, but the complexity, inconvenience, and low pumping equipment accessibility of operating microfluidic devices have limited their widespread use. Here we describe an open-source, programmable smart (OS) pipette as an easy-to-use, simple, handheld microfluidic pump that overcomes the major limitations of previous commercial- or research-level pumps for microfluidics. The OS pipette pumps fluid into a microfluidic device by precisely controlling the position of the plunger of a positive-displacement micropipette with stepper motor control. The intuitive pumping mechanism of the OS pipette enables the novel features of simple fabrication, straightforward device operation, and precise, predictable, and programmable flow-rate generation as an open-source pumping tool. Controlling the OS pipette using an open-source microcontroller board not only allows straightforward generation of constant flow rates with simple source code commands, but also permits varying flow rates to be programmed (including stepwise increase and decrease of the flow rate over time, and flow-rate pulse generation). We successfully validate the OS pipette's capabilities for portable microfluidic cell separation and counting. The OS pipette has promise as a rapidly evolving and potentially transformative pumping tool that freely allows unrestricted use, distribution, reproduction, and modification even by non-expert users, and further enables diverse usages, even beyond microfluidics.

Optofluidic Modular Blocks for On-Demand and Open-Source Prototyping of Microfluidic Systems.

Rapid prototyping of microfluidic devices has advanced greatly, along with the development of 3D printing and micromachining technologies. However, peripheral systems for microfluidics still rely on conventional equipment, such as bench-top microscopy and syringe pumps, which limit system modification and further improvements. Herein, optofluidic modular blocks are presented as discrete elements to modularize peripheral optical and fluidic systems and are used for on-demand and open-source prototyping of whole microfluidic systems. Each modular block is fabricated by embedding optical or fluidic devices into the corresponding 3D-printed housing. The self-interlocking structure of the modular blocks enables easy assembly and reconfiguration of the blocks in an intuitive manner, while also providing precise optical and fluidic alignment between the blocks. With the library of standardized modular blocks developed here, how the blocks can be easily assembled to build whole microfluidic systems for blood compatibility testing, droplet microfluidics, and cell migration assays is demonstrated. Based on the simplicity of assembling the optofluidic blocks, the prototyping platform can be easily used for open-source sharing of digital design files, assembly and operation instructions, and block specifications, thereby making it easy for nonexperts to implement microfluidic ideas as physical systems.

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.

A 3D-Printed Multichannel Viscometer for High-Throughput Analysis of Frying Oil Quality.

Viscosity as a sensitive measure of material changes is a potential quality-control parameter for simple and rapid assessment of frying oil quality. However, conventional viscometers require improvements in throughput, portability, cost-effectiveness and usability to be widely adopted for quality-control applications. Here we present a 3D-printed multichannel viscometer for simple, inexpensive and multiplexed viscosity measurement. The multichannel viscometer enables both parallel actuation of multiple fluid flows by pressing the plunger of the viscometer by hand and direct measurement of their relative volumes dispensed with naked eye. Thus, the unknown viscosities of test fluids can be simultaneously determined by the volume ratios between a reference fluid of known viscosity and the test fluids of unknown viscosity. With a 4-plex version of the multichannel viscometer, we demonstrated that the viscometer is effective for rapid examination of the degradation of a vegetable oil during deep frying of potato strips and the recovery of used frying oil after treatment with an adsorbent agent to remove frying by-products. The measurement results obtained by the multichannel viscometer were highly correlated with those obtained using a commercial oil tester. We also demonstrated the multiplexing capability of the viscometer, fabricating a 10-plex version of the viscometer and measuring the viscosities of ten test liquids at the same time. Collectively, these results indicate that the 3D-printed multichannel viscometer represents a valuable tool for high-throughput examination of frying oil quality in resource-limited settings.

On-Chip Cell Staining and Counting Platform for the Rapid Detection of Blood Cells in Cerebrospinal Fluid.

Counting blood cells in cerebrospinal fluid (CSF) is indispensable for diagnosing several pathological conditions in the central nervous system, such as meningitis, even though collecting CSF samples is invasive. Cell counting methods, such as hemocytometer chambers and flow cytometers, have been used for CSF cell counting, but they often lack the sensitivity to detect low blood cell numbers. They also depend on off-chip, manual sample preparation or require bulky, costly equipment, thereby limiting their clinical utility. Here, we present a portable cell counting platform for simple, rapid CSF cell counting that integrates a microfluidic cell counting chamber with a miniaturized microscope. The microfluidic chamber is designed not only to be a reagent container for on-chip cell staining but also to have a large control volume for accurate cell counting. The proposed microscope miniaturizes both bright-field and fluorescence microscopy with a simple optical setup and a custom cell-counting program, thereby allowing rapid and automated cell counting of nucleated white blood cells and non-nucleated red blood cells in fluorescence and bright-field images. Using these unique features, we successfully demonstrate the ability of our counting platform to measure low CSF cell counts without sample preparation.

Distributed Coordination for Optimal Energy Generation and Distribution in Cyber-Physical Energy Networks.

This paper proposes three coordination laws for optimal energy generation and distribution in energy network, which is composed of physical flow layer and cyber communication layer. The physical energy flows through the physical layer; but all the energies are coordinated to generate and flow by distributed coordination algorithms on the basis of communication information. First, distributed energy generation and energy distribution laws are proposed in a decoupled manner without considering the interactive characteristics between the energy generation and energy distribution. Second, a joint coordination law to treat the energy generation and energy distribution in a coupled manner taking account of the interactive characteristics is designed. Third, to handle over- or less-energy generation cases, an energy distribution law for networks with batteries is designed. The coordination laws proposed in this paper are fully distributed in the sense that they are decided optimally only using relative information among neighboring nodes. Through numerical simulations, the validity of the proposed distributed coordination laws is illustrated.

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.

Microfluidic Pipette Tip for High-Purity and High-Throughput Blood Plasma Separation from Whole Blood.

Blood plasma separation from whole blood is often limited by numerous blood cells which can compromise separation processes and thus deteriorate separation performance such as purity and throughput. To address this challenge, we present a microfluidic pipet tip composed of slant array ridges that enable autonomous blood cell focusing without significant deviation as well as facilitating a high degree of parallelization without compromising separation purity. With these advantages, we achieved high-purity (99.88%) and high-throughput (904.3 µL min-1) plasma separation from whole blood. In combination with a smart pipet, we successfully demonstrated rapid, inexpensive, and equipment-free blood plasma preparation for pretransfusion testing.

Intraoperative Templating in Lateral Meniscal Allograft Transplantation.

Recently, studies have emphasized the importance of anatomical placement of the lateral meniscal allograft to decrease postoperative extrusion. However, it is infeasible to identify the exact rotation of the allograft during transplantation. We present a patient who underwent a lateral meniscal transplantation using a wire for correct positioning of the allograft. The use of a wire intraoperatively shaped to resemble the contour of the lateral meniscal allograft will aid in more accurate and anatomical graft placement.

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.

Deterministic Migration-Based Separation of White Blood Cells.

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells.