Computational Hyperspectral Microflow Cytometry.

Miniaturized flow cytometry has significant potential for portable applications, such as cell-based diagnostics and the monitoring of therapeutic cell manufacturing, however, the performance of current techniques is often limited by the inability to resolve spectrally-overlapping fluorescence labels. Here, the study presents a computational hyperspectral microflow cytometer (CHC) that enables accurate discrimination of spectrally-overlapping fluorophores labeling single cells. CHC employs a dispersive optical element and an optimization algorithm to detect the full fluorescence emission spectrum from flowing cells, with a high spectral resolution of ≈3 nm in the range from 450 to 650 nm. CHC also includes a dedicated microfluidic device that ensures in-focus imaging through viscoelastic sheathless focusing, thereby enhancing the accuracy and reliability of microflow cytometry analysis. The potential of CHC for analyzing T lymphocyte subpopulations and monitoring changes in cell composition during T cell expansion is demonstrated. Overall, CHC represents a major breakthrough in microflow cytometry and can facilitate its use for immune cell monitoring.

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.

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.

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.

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.

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.

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.

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.

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.

Smart Pipette and Microfluidic Pipette Tip for Blood Plasma Separation.

An integrated method for blood plasma separation is presented by combining a pneumatic device, which is referred to as a "smart pipette," and a hydrophoretic microchannel as a microfluidic pipette tip for whole-blood sample preparation. This method enables hemolysis-free, high-purity plasma separation through smart pipetting of whole blood, potentially providing the means for rapid, inexpensive blood sample preparation for point-of-care testing.

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.

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.

Smart microfluidic pipette tip enabled by flow-rate insensitive particle ordering.

Smart microfluidic pipette tip: A microfluidic method for removing cells and recovering liquid medium is presented and applied for blood cell rejection and cytotoxicity assay. This method enables continuous cell rejection by manual operation, potentially providing the means for rapid, inexpensive sample preparation for personalized diagnostics and mobile laboratory.

Automation of 3D digital rolling circle amplification using a 3D-printed liquid handler.

Automation of liquid handling is indispensable to improve throughput and reproducibility in biochemical assays. However, the incorporation of automated systems into laboratory workflows is often hindered by the high cost and complexity associated with building robotic liquid handlers. Here, we report a 3D-printed liquid handler based on a fluidic manifold, thereby obviating the need for complex robotic mechanisms. The fluidic manifold, termed a dispensing and aspirating (DA) device, comprises parallelized multi-pipette structures connected by distribution and aspiration channels, enabling the precise supply and removal of reagents, respectively. Leveraging the versatility of 3D printing, the DA device can be custom-designed and printed to fit specific applications. As a proof-of-principle, we engineered a 3D-printed liquid handler dedicated for 3D digital rolling circle amplification (4DRCA), an advanced biochemical assay involving multiple sample preparation steps such as antibody incubation, cell fixation, nucleic acid amplification, probe hybridization, and extensive washing. We demonstrate the efficacy of the 3D-printed liquid handler to automate the preparation of clinical samples for the simultaneous, in situ analysis of oncogenic protein and transcript markers in B-cell acute lymphoblastic leukemia cells using 4DRCA. This approach provides an effective and accessible solution for liquid handling automation, offering high throughput and reproducibility in biochemical assays.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

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

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

Field-free, sheathless cell focusing in exponentially expanding hydrophoretic channels for microflow cytometry.

Cell focusing into a narrow stream is an essential step prior to counting and sorting cells in microfluidic devices for flow cytometry and cell sorting applications. Hydrodynamic focusing techniques, however, rely on the need for large volumes of sheath liquid and complex mechanical setup for flow control, preventing miniaturization of the systems. Although microfluidic methods based on active or passive particle control offer sheathless and efficient focusing, they often accompany fabrication complexities or bulky external setups, and operate in a certain range of flow rates. We present here a microfluidic device to focus cells into a narrow stream. The device employs hydrophoresis to guide cells by locally patterned slanted grooves, and channel expansion to improve focusing efficiency and produce a narrow stream of cells. This device principle allows easy improvement of focusing efficiency by adding more expansion steps. Adjusting channel expansion also ensures successful cell focusing without defocusing by inertial effects even at high Reynolds numbers. Using this device, we successfully produced a narrow stream of cells having size variation of >11% in a coefficient of variation (CV), achieving a narrow cell stream with a focusing variation below CV of 3.0%.

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.