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.

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.

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.

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.

Enhanced functionalities of immune cells separated by a microfluidic lattice: assessment based on holotomography.

The isolation of white blood cells (WBCs) from whole blood constitutes a pivotal process for immunological studies, diagnosis of hematologic disorders, and the facilitation of immunotherapy. Despite the ubiquity of density gradient centrifugation in WBC isolation, its influence on WBC functionality remains inadequately understood. This research employs holotomography to explore the effects of two distinct WBC separation techniques, namely conventional centrifugation and microfluidic separation, on the functionality of the isolated cells. We utilize three-dimensional refractive index distribution and time-lapse dynamics to analyze individual WBCs in-depth, focusing on their morphology, motility, and phagocytic capabilities. Our observations highlight that centrifugal processes negatively impact WBC motility and phagocytic capacity, whereas microfluidic separation yields a more favorable outcome in preserving WBC functionality. These findings emphasize the potential of microfluidic separation techniques as a viable alternative to traditional centrifugation for WBC isolation, potentially enabling more precise analyses in immunology research and improving the accuracy of hematologic disorder diagnoses.

Sessile droplet array for sensitive profiling of multiple extracellular vesicle immuno-subtypes.

The sensitive detection of the multiple immuno-subtypes of cancer-specific extracellular vesicles (EVs) has emerged as a promising method for multiclass cancer diagnosis; however, its limitations in sensitivity, accessibility, and multiple detection of EV subtypes have hindered its further implementation. Here, we present a platform for sensitive EV detection enabled by sessile droplet array (eSD) that exploits enhanced immuno-capture of EVs via evaporation-driven radial flows in a sessile droplet. Compared to a micro-well without internal flows, this platform demonstrates significantly enhanced EV capture and detection by detecting low levels of EVs with a detection limit of 384.7 EVs per microliter, which is undetectable in the micro-well. In addition, using a small sample consumption of ∼0.2 µL plasma per droplet, the platform detects EV immuno-subtypes against seven different antibodies in patient plasma samples of different cancer types (liver, colon, lung, breast and prostate cancers). Further, using the profiling data, the platform exhibits a sensitivity of 100% (95% confidence interval (CI): 83-100%) and a specificity of 100% (95% CI: 40-100%) for the diagnosis of cancer, and classified cancer types with an overall accuracy of 96% (95% CI: 86-100%) using a two-staged algorithm based on quadratic discriminant analysis technique for machine learning.

Real-time monitoring of mono- and dual-species biofilm formation and eradication using microfluidic platform.

In a human host, bacterial Staphylococcus aureus and fungal Candida albicans pathogens form a mixed biofilm that causes severe mortality and morbidity. However, research on the formation and eradication of mixed biofilms under dynamic conditions is lacking. Thus, this study employed a microfluidic technique to analyze the real-time formation of mono- and dual-species (S. aureus and C. albicans) biofilms and noninvasive optical treatment of the established mature biofilm using 405-nm laser light. A herringbone mixer thoroughly mixed both bacterial and fungal cells in the growth media before being injected into the observation channels on the microfluidic chip. At a flow rate of 1.0 µL/min of growth media for 24 h, the bacterial biofilm coverage was up to 15% higher than that of the fungal biofilm (50% for bacteria vs. 35% for fungus). On the other hand, the dual-species biofilm yielded the highest coverage of ~ 96.5% because of the collective interaction between S. aureus and C. albicans. The number of cell proliferation events in S. aureus was higher than that of C. albicans for 12 h, which indicates that the S. aureus biofilm was developed faster than C. albicans. The novel in situ test platform showed a significant bactericidal effect (80%) of the 405-nm laser light at 1080 J/cm2 towards the established S. aureus biofilm, whereas the same treatment removed approximately 69% of the mixed cells in the dual-species biofilm. This study revealed that the developed microfluidic platform could be utilized to monitor the formation of dual-species biofilms in real-time and laser-induced antimicrobial effects on dual-species biofilms.

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.

3D-Printed Capillary Circuits for Calibration-Free Viscosity Measurement of Newtonian and Non-Newtonian Fluids.

Measuring viscosity is important for the quality assurance of liquid products, as well as for monitoring the viscosity of clinical fluids as a potential hemodynamic biomarker. However, conventional viscometers and their microfluidic counterparts typically rely on bulky and expensive equipment, and lack the ability for rapid and field-deployable viscosity analysis. To address these challenges, we describe 3D-printed capillary circuits (3D-CCs) for equipment- and calibration-free viscosity measurement of Newtonian and non-Newtonian fluids. A syringe, modified with an air chamber serving as a pressure buffer, generates and maintains a set pressure to drive the pressure-driven flows of test fluids through the 3D-CCs. The graduated fluidic chambers of the 3D-CCs serve as a flow meter, enabling simple measurement of the flow rates of the test fluids flowing through the 3D-CCs, which is readable with the naked eye. The viscosities of the test fluids can be simply calculated from the measured flow rates under a set pressure condition without the need for peripheral equipment and calibration. We demonstrate the multiplexing capability of the 3D-CC platform by simultaneously measuring different Newtonian-fluid samples. Further, we demonstrate that the shear-rate dependence of the viscosity of a non-Newtonian fluid can be analyzed simultaneously under various shear-rate conditions with the 3D-CC platform.

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.

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.