Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.

Revolutionizing sample preparation: a novel autonomous microfluidic platform for serial dilution.

Dilution is a standard fluid operation widely employed in the sample preparation process of many bio(chemical) assays. It serves multiple essential functions such as sample mixing with certain reagents at specific dilution ratios, reducing sample matrix effects, bringing target analytes within the linear assay detection range, among many others. Traditionally, sample processing is performed in laboratory settings through manual or automated pipetting. When working in resource-limited settings, however, neither trained personnel nor proper laboratory equipment are available limiting the accessibility to high-quality diagnostic tests. In this work, we present a novel standalone and fully automated microfluidic platform for the stepwise preparation of serial dilutions without the need for any active elements. Stepwise dilution is achieved using the coordinated burst action of hydrophobic burst valves to first isolate a precisely metered volume from an applied sample drop and subsequently merge it with a prefilled diluent liquid. Downstream, expansion chambers are used to mix both reagents into a homogeneous solution. The dilution module was characterized to generate accurate and reproducible (CV < 7%) dilutions for targeted dilution factors of 2, 5 and 10×, respectively. Three dilution modules were coupled in series to generate three-fold logarithmic (log5 or log10) dilutions, with excellent linearity (R2 > 0.99). Its compatibility with whole blood was furthermore illustrated, proving its applicability for automating and downscaling bioassays with complex biological matrices. Finally, autonomous on-chip serial dilution was demonstrated by incorporating the self-powered (i)SIMPLE technology as a passive driving source for liquid manipulation. We believe that the simplicity and modularity of the presented autonomous dilution platform are of interest to many point-of-care applications in which sample dilution and reagent mixing are of importance.

Microfluidic approach to correlate C. elegans neuronal functional aging and underlying changes of gene expression in mechanosensation.

The aging process has broad physiological impacts, including a significant decline in sensory function, which threatens both physical health and quality of life. One ideal model to study aging, neuronal function, and gene expression is the nematode Caenorhabditis elegans, which has a short lifespan and relatively simple, thoroughly mapped nervous system and genome. Previous works have identified that mechanosensory neuronal structure changes with age, but importantly, the actual age-related changes in the function and health of neurons, as well as the underlying genetic mechanisms responsible for these declines, are not fully understood. While advanced techniques such as single-cell RNA-sequencing have been developed to quantify gene expression, it is difficult to relate this information to functional changes in aging due to a lack of tools available. To address these limitations, we present a platform capable of measuring both physiological function and its associated gene expression throughout the aging process in individuals. Using our pipeline, we investigate the age-related changes in function of the mechanosensing ALM neuron in C. elegans, as well as some relevant gene expression patterns (mec-4 and mec-10). Using a series of devices for animals of different ages, we examined subtle changes in neuronal function and found that while the magnitude of neuronal response to a large stimulus declines with age, sensory capability does not significantly decline with age; further, gene expression is well maintained throughout aging. Additionally, we examine PVD, a harsh-touch mechanosensory neuron, and find that it exhibits a similar age-related decline in magnitude of neuronal response. Together, our data demonstrate that our strategy is useful for identifying genetic factors involved in the decline in neuronal health. We envision that this framework could be applied to other systems as a useful tool for discovering new biology.

In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy.

BACKGROUND: The analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. RESULTS: In this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. SIGNIFICANCE AND NOVELTY: By using the method of small flow perfusion (≤20 µL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

Automatic offline-capable smartphone paper-based microfluidic device for efficient biomarker detection of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a prevalent neurodegenerative disease with no effective treatment. Efficient and rapid detection plays a crucial role in mitigating and managing AD progression. Deep learning-assisted smartphone-based microfluidic paper analysis devices (µPADs) offer the advantages of low cost, good sensitivity, and rapid detection, providing a strategic pathway to address large-scale disease screening in resource-limited areas. However, existing smartphone-based detection platforms usually rely on large devices or cloud servers for data transfer and processing. Additionally, the implementation of automated colorimetric enzyme-linked immunoassay (c-ELISA) on µPADs can further facilitate the realization of smartphone µPADs platforms for efficient disease detection. RESULTS: This paper introduces a new deep learning-assisted offline smartphone platform for early AD screening, offering rapid disease detection in low-resource areas. The proposed platform features a simple mechanical rotating structure controlled by a smartphone, enabling fully automated c-ELISA on µPADs. Our platform successfully applied sandwich c-ELISA for detecting the ß-amyloid peptide 1-42 (Aß 1-42, a crucial AD biomarker) and demonstrated its efficacy in 38 artificial plasma samples (healthy: 19, unhealthy: 19, N = 6). Moreover, we employed the YOLOv5 deep learning model and achieved an impressive 97 % accuracy on a dataset of 1824 images, which is 10.16 % higher than the traditional method of curve-fitting results. The trained YOLOv5 model was seamlessly integrated into the smartphone using the NCNN (Tencent's Neural Network Inference Framework), enabling deep learning-assisted offline detection. A user-friendly smartphone application was developed to control the entire process, realizing a streamlined "samples in, answers out" approach. SIGNIFICANCE: This deep learning-assisted, low-cost, user-friendly, highly stable, and rapid-response automated offline smartphone-based detection platform represents a good advancement in point-of-care testing (POCT). Moreover, our platform provides a feasible approach for efficient AD detection by examining the level of Aß 1-42, particularly in areas with low resources and limited communication infrastructure.

Microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum.

BACKGROUND: Calcium and magnesium ions are highly abundant and important cations in human body. At the same time, both dyscalcemia and dysmagnesemia are frequently encountered in the clinical practice. As deficiency or excess of Ca(II) or Mg(II) can cause severe symptoms, determining these ions in serum is of great importance. Concentration of these ions in biological samples is typically assayed in clinical laboratories with the use of expensive and specialized equipment. Since those methods cannot be easily adapted for self-diagnosis purposes, there is a great need to develop a convenient tool for reliable determination of calcium and magnesium in serum at the point-of-care. RESULTS: The colorimetric methods employed for calcium and magnesium analysis were o-cresophtalein complexone assay and xylidyl blue assay, respectively. Analytical signal acquisition was accomplished using an ordinary flatbed scanner or smartphone and free software. For increased user-friendliness the device was optimized to perform simultaneous determination of calcium and magnesium ions in only 10 min. In the optimized conditions, the limit of detection for calcium ions was 0.09 mmol L-1, while for magnesium it was 0.04 mmol L-1. Determination of both ions requires only 4 µL of serum sample. The developed paper-based sensors were validated with control human serum samples and the obtained relative errors for majority of samples were below 20 %. SIGNIFICANCE: In this paper, a microfluidic paper-based analytical device for simultaneous determination of calcium and magnesium ions in human serum is reported for the first time. Additionally, this is also the first report on colorimetric determination in serum of any of these ions in paper-based format. Simultaneous detection of both ions allows for fast and user-friendly screening of disturbance in calcium and magnesium homeostasis.

Biosensor-Enhanced Organ-on-a-Chip Models for Investigating Glioblastoma Tumor Microenvironment Dynamics.

Glioblastoma, an aggressive primary brain tumor, poses a significant challenge owing to its dynamic and intricate tumor microenvironment. This review investigates the innovative integration of biosensor-enhanced organ-on-a-chip (OOC) models as a novel strategy for an in-depth exploration of glioblastoma tumor microenvironment dynamics. In recent years, the transformative approach of incorporating biosensors into OOC platforms has enabled real-time monitoring and analysis of cellular behaviors within a controlled microenvironment. Conventional in vitro and in vivo models exhibit inherent limitations in accurately replicating the complex nature of glioblastoma progression. This review addresses the existing research gap by pioneering the integration of biosensor-enhanced OOC models, providing a comprehensive platform for investigating glioblastoma tumor microenvironment dynamics. The applications of this combined approach in studying glioblastoma dynamics are critically scrutinized, emphasizing its potential to bridge the gap between simplistic models and the intricate in vivo conditions. Furthermore, the article discusses the implications of biosensor-enhanced OOC models in elucidating the dynamic features of the tumor microenvironment, encompassing cell migration, proliferation, and interactions. By furnishing real-time insights, these models significantly contribute to unraveling the complex biology of glioblastoma, thereby influencing the development of more accurate diagnostic and therapeutic strategies.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Photocell-Based Optofluidic Device for Clogging-Free Cell Transit Time Measurements.

Measuring the transit time of a cell forced through a bottleneck is one of the most widely used techniques for the study of cell deformability in flow. It in turn provides an accessible and rapid way of obtaining crucial information regarding cell physiology. Many techniques are currently being investigated to reliably retrieve this time, but their translation to diagnostic-oriented devices is often hampered by their complexity, lack of robustness, and the bulky external equipment required. Herein, we demonstrate the benefits of coupling microfluidics with an optical method, like photocells, to measure the transit time. We exploit the femtosecond laser irradiation followed by chemical etching (FLICE) fabrication technique to build a monolithic 3D device capable of detecting cells flowing through a 3D non-deformable constriction which is fully buried in a fused silica substrate. We validated our chip by measuring the transit times of pristine breast cancer cells (MCF-7) and MCF-7 cells treated with Latrunculin A, a drug typically used to increase their deformability. A difference in transit times can be assessed without the need for complex external instrumentation and/or demanding computational efforts. The high throughput (4000-10,000 cells/min), ease of use, and clogging-free operation of our device bring this approach much closer to real scenarios.

High-throughput viscoelastic characterization of cells in hyperbolic microchannels.

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress-strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress-strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, shows strain-thickening following a power law up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation times of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid-liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Young's moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with latrunculin B and nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.

Adaptively resettable microfluidic patch for sweat rate and electrolytes detection.

Skin-interfaced microfluidic patch has become a reliable device for sweat collection and analysis. However, the intractable problems of emptying the microchannel for reuse, and the channel's volumetric capacity limited by the size of the patch, directly hinder the practical application of sweat sensors. Herein, we report an adaptively resettable microfluidic sweat patch (Art-Sweat patch) capable of continuously monitoring both sweat rate (0.2-4.0 µL min-1) and total ionic charge concentration (10-200 mmol L-1). We develop a platform with a vertical and horizontal microchannel combined strategy, enabling repeatedly filling sweat and emptying the microchannel for autonomously resetting and detecting. The variation in the emptied volume is designed to be adaptively identified by the sensor, resulting in enhanced stability and an enlarged volumetric capacity of over 300 µL. By integrating with self-designed wireless transmission modules, the proposed Art-Sweat patch shows product-level wearability and high performance in monitoring variations in regional sweat rate and concentration for hydration status assessment.

Centrifugo-Pneumatic Reciprocating Flowing Coupled with a Spatial Confinement Strategy for an Ultrafast Multiplexed Immunoassay.

Immunoassays serve as powerful diagnostic tools for early disease screening, process monitoring, and precision treatment. However, the current methods are limited by high costs, prolonged processing times (>2 h), and operational complexities that hinder their widespread application in point-of-care testing. Here, we propose a novel centrifugo-pneumatic reciprocating flowing coupled with spatial confinement strategy, termed PRCM, for ultrafast multiplexed immunoassay of pathogens on a centrifugal microfluidic platform. Each chip consists of four replicated units; each unit allows simultaneous detection of three targets, thereby facilitating high-throughput parallel analysis of multiple targets. The PRCM platform enables sequential execution of critical steps such as solution mixing, reaction, and drainage by coordinating inherent parameters, including motor rotation speed, rotation direction, and acceleration/deceleration. By integrating centrifugal-mediated pneumatic reciprocating flow with spatial confinement strategies, we significantly reduce the duration of immune binding from 30 to 5 min, enabling completion of the entire testing process within 20 min. As proof of concept, we conducted a simultaneous comparative test on- and off-the-microfluidics using 12 negative and positive clinical samples. The outcomes yielded 100% accuracy in detecting the presence or absence of the SARS-CoV-2 virus, thus highlighting the potential of our PRCM system for multiplexed point-of-care immunoassays.

Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

A point-of-care testing platform for on-site identification of genetically modified crops.

Genetically modified (GM) food is still highly controversial nowadays. Due to the disparate policies and attitudes worldwide, demands for a rapid, cost-effective and user-friendly GM crop identification method are increasingly significant for import administration, market supervision, etc. However, as the most-recognized methods, nucleic acid-based identification approaches require bulky instruments, long turn-around times and trained personnel, which are only suitable in laboratories. To fulfil the urgent needs of on-site testing, we develop a point-of-care testing platform that is able to identify 12 types of GM crops in less than 40 minutes without using laboratory settings. Our system integrates sample pre-treatment modules in a microfluidic chip, performs DNA amplification via a battery-powered portable kit, and presents results via eye-recognized colorimetric change. A paraffin-based reflow method and a slip plate-based fluid switch are developed to encapsulate and release amplification primers in individual microwells on demand, thus enabling identification of varied targets simultaneously. Our system offers an efficient, affordable and convenient tool for GM crop identification, thus it will not only benefit customs and market administration bureaus, but also satisfy demands of numerous consumers.

Drug testing of monodisperse arrays of live microdissected tumors using a valved multiwell microfluidic platform.

Cancer drug testing in animals is an extremely poor predictor of the drug's safety and efficacy observed in humans. Hence there is a pressing need for functional testing platforms that better predict traditional and immunotherapy responses in human, live tumor tissue or tissue constructs, and at the same time are compatible with the use of mouse tumor tissue to facilitate building more accurate disease models. Since many cancer drug actions rely on mechanisms that depend on the tumor microenvironment (TME), such platforms should also retain as much of the native TME as possible. Additionally, platforms based on miniaturization technologies are desirable to reduce animal use and sensitivity to human tissue scarcity. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, microdissected tumors (µDTs) or "spheroids" that retain an intact TME have shown promising responses to immunomodulators acting on native immune cells. However, difficult tissue handling after microdissection has reduced the throughput of drug testing on µDTs, thereby constraining the inherent advantages of producing numerous TME-preserving units of tissue for drug testing. Here we demonstrate a microfluidic 96-well platform designed for drug treatment of hundreds of similarly-sized, cuboidal µDTs ("cuboids") produced from a single tumor sample. The platform organizes a monodisperse array of four cuboids per well in 384 hydrodynamic traps. The microfluidic device, entirely fabricated in thermoplastics, features 96 microvalves that fluidically isolate each well after the cuboid loading step for straightforward multi-drug testing. Since our platform makes the most of scarce tumor tissue, it can potentially be applied to human biopsies that preserve the human TME while minimizing animal testing.

Development of a microfluidic device to enrich and detect zearalenone in food using quantum dot-embedded molecularly imprinted polymers.

Mycotoxins are secondary metabolites of certain moulds, prevalent in 60-80% of food crops and many processed products but challenging to eliminate. Consuming mycotoxin-contaminated food and feed can lead to various adverse effects on humans and livestock. Therefore, testing mycotoxin residue levels is critical to ensure food safety. Gold standard analytical methods rely on liquid chromatography coupled with optical detectors or mass spectrometers, which are high-cost with limited capacity. This study reported the successful development of a microfluidic "lab-on-a-chip" device to enrich and detect zearalenone in food samples based on the fluorescence quenching effect of quantum dots and selective affinity of molecularly imprinted polymers (MIPs). The dummy template and functional polymer were synthesized and characterized, and the detailed microfluidic chip design and optimization of the flow conditions in the enrichment module were discussed. The device achieved an enrichment factor of 9.6 (±0.5) in 10 min to quantify zearalenone spiked in food with high recoveries (91-105%) at 1-10 mg kg-1, covering the concerned residue levels in the regulations. Each sample-to-answer test took only 20 min, involving 3 min of manual operation and no advanced equipment. This microfluidic device was mostly reusable, with a replaceable detection module compatible with fluorescence measurement using a handheld fluorometer. To our best knowledge, the reported device was the first application of an MIP-based microfluidic sensor for detecting mycotoxin in real food samples, providing a novel, rapid, portable, and cost-effective tool for monitoring mycotoxin contamination for food safety and security.

Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective.

Millions of people are subject to infertility worldwide and one in every six people, regardless of gender, experiences infertility at some period in their life, according to the World Health Organization. Assisted reproductive technologies are defined as a set of procedures that can address the infertility issue among couples, culminating in the alleviation of the condition. However, the costly conventional procedures of assisted reproduction and the inherent vagaries of the processes involved represent a setback for its successful implementation. Microfluidics, an emerging tool for processing low-volume samples, have recently started to play a role in infertility diagnosis and treatment. Given its host of benefits, including manipulating cells at the microscale, repeatability, automation, and superior biocompatibility, microfluidics have been adopted for various procedures in assisted reproduction, ranging from sperm sorting and analysis to more advanced processes such as IVF-on-a-chip. In this review, we try to adopt a more holistic approach and cover different uses of microfluidics for a variety of applications, specifically aimed at sperm separation and analysis. We present various sperm separation microfluidic techniques, categorized as natural and non-natural methods. A few of the recent developments in on-chip fertilization are also discussed.

Multiplex nanozymatic biosensing of Salmonella on a finger-actuated microfluidic chip.

A colorimetric biosensor was elaboratively designed for fast, sensitive and multiplex bacterial detection on a single microfluidic chip using immune magnetic nanobeads for specific bacterial separation, immune gold@platinum palladium nanoparticles for specific bacterial labeling, a finger-actuated mixer for efficient immunoreaction and two coaxial rotatable magnetic fields for magnetic nanobead capture (outer one) and magnet-actuated valve control (inner one). First, preloaded bacteria, nanobeads and nanozymes were mixed through a finger actuator to form nanobead-bacteria-nanozyme conjugates, which were captured by the outer magnetic field. After the inner magnetic field was rotated to successively wash the conjugates and push the H2O2-TMB substrate for resuspending these conjugates, colorless TMB was catalyzed into blue TMBox products, followed by color analysis using ImageJ software for bacterial determination. This simple biosensor enabled multiplex Salmonella detection as low as 9 CFU per sample in 45 min.

Screening for urothelial carcinoma cells in urine based on digital holographic flow cytometry through machine learning and deep learning methods.

The incidence of urothelial carcinoma continues to rise annually, particularly among the elderly. Prompt diagnosis and treatment can significantly enhance patient survival and quality of life. Urine cytology remains a widely-used early screening method for urothelial carcinoma, but it still has limitations including sensitivity, labor-intensive procedures, and elevated cost. In recent developments, microfluidic chip technology offers an effective and efficient approach for clinical urine specimen analysis. Digital holographic microscopy, a form of quantitative phase imaging technology, captures extensive data on the refractive index and thickness of cells. The combination of microfluidic chips and digital holographic microscopy facilitates high-throughput imaging of live cells without staining. In this study, digital holographic flow cytometry was employed to rapidly capture images of diverse cell types present in urine and to reconstruct high-precision quantitative phase images for each cell type. Then, various machine learning algorithms and deep learning models were applied to categorize these cell images, and remarkable accuracy in cancer cell identification was achieved. This research suggests that the integration of digital holographic flow cytometry with artificial intelligence algorithms offers a promising, precise, and convenient approach for early screening of urothelial carcinoma.

Microfluidic particle counter visualizing mucosal antibodies against SARS-CoV-2 in the upper respiratory tract for rapid evaluation of immune protection.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.