High-resolution spiral microfluidic channel integrated electrochemical device for isolation and detection of extracellular vesicles without lipoprotein contamination.

Recent studies have indicated significant correlation between the concentration of immune checkpoint markers borne by extracellular vesicles (EVs) and the efficacy of immunotherapy. This study introduces a high-resolution spiral microfluidic channel-integrated electrochemical device (HiMEc), which is designed to isolate and detect EVs carrying the immune checkpoint markers programmed death ligand 1 (PD-L1) and programmed death protein 1 (PD-1), devoid of plasma-abundant lipoprotein contamination. Antigen-antibody reactions were applied to immobilize the lipoproteins on bead surfaces within the plasma, establishing a size differential with EVs. A plasma sample was then introduced into the spiral microfluidic channel, which facilitated the acquisition of nanometer-sized EVs and the elimination of micrometer-sized lipoprotein-bead complexes, along with the isolation and quantification of EVs using HiMEc. PD-L1 and PD-1 expression on EVs was evaluated in 30 plasma samples (10 from healthy donors, 20 from lung cancer patients) using HiMEc and compared to the results obtained from standard tissue-based PD-L1 testing, noting that HiMEc could be utilized to select further potential candidates. The obtained results are expected to contribute positively to the clinical assessment of potential immunotherapy beneficiaries.

Machine learning powered detection of biological toxins in association with confined lateral flow immunoassay (c-LFA).

Biological weapons, primarily dispersed as aerosols, can spread not only to the targeted area but also to adjacent regions following the movement of air driven by wind. Thus, there is a growing demand for toxin analysis because biological weapons are among the most influential and destructive. Specifically, such a technique should be hand-held, rapid, and easy to use because current methods require more time and well-trained personnel. Our study demonstrates the use of a novel lateral flow immunoassay, which has a confined structure like a double barbell in the detection area (so called c-LFA) for toxin detection such as staphylococcal enterotoxin B (SEB), ricinus communis (Ricin), and botulinum neurotoxin type A (BoNT-A). Additionally, we have explored the integration of machine learning (ML), specifically, a toxin chip boosting (TOCBoost) hybrid algorithm for improved sensitivity and specificity. Consequently, the ML powered c-LFA concurrently categorized three biological toxin types with an average accuracy as high as 95.5%. To our knowledge, the sensor proposed in this study is the first attempt to utilize ML for the assessment of toxins. The advent of the c-LFA orchestrated a paradigm shift by furnishing a versatile and robust platform for the rapid, on-site detection of various toxins, including SEB, Ricin, and BoNT-A. Our platform enables accessible and on-site toxin monitoring for non-experts and can potentially be applied to biosecurity.

Bone-on-a-chip simulating bone metastasis in osteoporosis.

Osteoporosis is the most common bone disorder, which is a highly dangerous condition that can promote bone metastases. As the current treatment for osteoporosis involves long-term medication therapy and a cure for bone metastasis is not known, ongoing efforts are required for drug development for osteoporosis. Animal experiments, traditionally used for drug development, raise ethical concerns and are expensive and time-consuming. Organ-on-a-chip technology is being developed as a tool to supplement such animal models. In this study, we developed a bone-on-a-chip by co-culturing osteoblasts, osteocytes, and osteoclasts in an extracellular matrix environment that can represent normal bone, osteopenia, and osteoporotic conditions. We then simulated bone metastases using breast cancer cells in three different bone conditions and observed that bone metastases were most active in osteoporotic conditions. Furthermore, it was revealed that the promotion of bone metastasis in osteoporotic conditions is due to increased vascular permeability. The bone-on-a-chip developed in this study can serve as a platform to complement animal models for drug development for osteoporosis and bone metastasis.

Capillary force-driven reverse-Tesla valve structure for microfluidic bioassays.

Biological assays involve the lysis of biological particles, enzyme reactions, and gene amplification, and require a certain amount of time for completion. Microfluidic chips are regarded as powerful devices for biological assays and in vitro diagnostics; however, they cannot achieve a high mixing efficiency, particularly in some time-consuming biological reactions. Herein, we introduce a microfluidic reverse-Tesla (reTesla) valve structure in which the fluid is affected by vortices and branch flow convergence, resulting in flow retardation and a high degree of mixing. The reTesla is passively operated by a microfluidic capillary force without any pumping facility. Compared with our previously developed micromixers, this innovative pumpless microfluidic chip exhibited high performance, with a mixing efficiency of more than 93%. The versatility of our reTesla chip will play a pivotal role in the study of various biological and chemical reactions.

Efficient separation of large particles and giant cancer cells using an isosceles trapezoidal spiral microchannel.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITSµC), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITSµC's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 µm) toward the inner wall, while directing smaller ones (avg. diameter: 23 µm) outward. Utilizing ITSµC, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITSµC suggests its potential in differentiating a wide range of heterogeneous cell populations.

Parathyroid-on-a-chip simulating parathyroid hormone secretion in response to calcium concentration.

The parathyroid gland is an endocrine organ that plays a crucial role in regulating calcium levels in blood serum through the secretion of parathyroid hormone (PTH). Hypoparathyroidism is a chronic disease that can occur due to parathyroid defects, but due to the difficulty of creating animal models of this disease or obtaining human normal parathyroid cells, the evaluation of parathyroid functionality for drug development is limited. Although parathyroid-like cells that secrete PTH have recently been reported, their functionality may be overestimated using traditional culture methods that lack in vivo similarities, particularly vascularization. To overcome these limitations, we obtained parathyroid organoids from tonsil-derived mesenchymal stem cells (TMSCs) and fabricated a parathyroid-on-a-chip, capable of simulating PTH secretion based on calcium concentration. This chip exhibited differences in PTH secretion according to calcium concentration and secreted PTH within the range of normal serum levels. In addition, branches of organoids, which are difficult to observe in animal models, were observed in this chip. This could serve as a guideline for successful engraftment in implantation therapies in the future.

Hand-held all-in-one (HAO) self-test kit for rapid and on-site detection of SARS-CoV-2 with colorimetric LAMP.

Throughout the COVID-19 pandemic, individuals potentially infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were forcibly recalled to local or central hospitals, where the diagnostic results were obtained a couple of days after the liquid biopsies were subjected to conventional polymerase chain reaction (PCR). This slow output of such a complex and time-consuming laboratory procedure hindered its widespread application. To overcome the limitations associated with such a centralized diagnostic system, we developed a hand-held and all-in-one type test kit in which the analytical results can be obtained in only 30 min. The test kit consists of three major steps for on-site SARS-CoV-2 RNA detection: 1) virus lysis by heat, 2) RNA enrichment by membrane, and 3) real-time detection by colorimetric loop-mediated isothermal amplification (c-LAMP). The proposed device operates in a sample-to-answer format, is fully automated, and reduces dependence on traditional laboratory settings, facilitating large-scale population screening.

Formation Pattern Analysis of Spheroids Formed by a Droplet-Based Microfluidic System for Predicting the Aggressiveness of Tumor Cells.

Examining tumor heterogeneity is essential for selecting an appropriate anticancer treatment for an individual. This study aimed to distinguish low- and high-aggressive tumor cells by analyzing the formation patterns of spheroids. The droplet-based microfluidic system was employed for the formation of each spheroid from four different subtypes of breast tumor cells. Additionally, heterotypic spheroids with T lymphocytes and cancer-associated fibroblasts (CAFs) were produced, and distinctions between low- and high-aggressive tumor cells were explored through the analysis of formation patterns using circularity, convexity, and cell distributions. In both homotypic spheroids and heterotypic spheroids with T lymphocytes, spheroids formed from low-aggressive tumor cells exhibited high circularity and convexity. On the other hand, spheroids formed from high-aggressive tumor cells had relatively low circularity and convexity. In the case of heterotypic spheroids with CAFs, circularity and convexity did not exhibit clear differences between low- and high-aggressive tumor cells, but distinct variations were observed in cell distributions. CAFs and low-aggressive tumor cells were evenly distributed, whereas the CAFs were predominantly located in the inner layer, and high-aggressive tumor cells were primarily located in the outer layer. This finding can offer valuable insights into predicting the aggressiveness of unknown tumor cells.

Enhanced enrichment of collected airborne coronavirus and influenza virus samples via a ConA-coated microfluidic chip for PCR detection.

The severe acute respiratory syndrome (SARS-CoV-2) outbreak triggered global concern and emphasized the importance of virus monitoring. During a seasonal influenza A outbreak, relatively low concentrations of 103-104 viral genome copies are available per 1 m3 of air, which makes detection and monitoring very challenging because the limit of detection of most polymerase chain reaction (PCR) devices is approximately 103 viral genome copies/mL. In response to the urgent need for the rapid detection of airborne coronaviruses and influenza viruses, an electrostatic aerosol-to-hydrosol (ATH) sampler was combined with a concanavalin A (ConA)-coated high-throughput microfluidic chip. The samples were then used for PCR detection. The results revealed that the enrichment capacity of the ATH sampler was 30,000-fold for both HCoV-229E and H1N1 influenza virus, whereas the enrichment capacities provided by the ConA-coated microfluidic chip were 8-fold and 16-fold for HCoV-229E and H1N1 virus, respectively. Thus, the total enrichment capacities of our combined ATH sampler and ConA-coated microfluidic chip were 2.4 × 105-fold and 4.8 × 105-fold for HCoV-229E and H1N1 virus, respectively. This methodology significantly improves PCR detection by providing a higher concentration of viable samples.

E2E-BPF microscope: extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution.

Several image-based biomedical diagnoses require high-resolution imaging capabilities at large spatial scales. However, conventional microscopes exhibit an inherent trade-off between depth-of-field (DoF) and spatial resolution, and thus require objects to be refocused at each lateral location, which is time consuming. Here, we present a computational imaging platform, termed E2E-BPF microscope, which enables large-area, high-resolution imaging of large-scale objects without serial refocusing. This method involves a physics-incorporated, deep-learned design of binary phase filter (BPF) and jointly optimized deconvolution neural network, which altogether produces high-resolution, high-contrast images over extended depth ranges. We demonstrate the method through numerical simulations and experiments with fluorescently labeled beads, cells and tissue section, and present high-resolution imaging capability over a 15.5-fold larger DoF than the conventional microscope. Our method provides highly effective and scalable strategy for DoF-extended optical imaging system, and is expected to find numerous applications in rapid image-based diagnosis, optical vision, and metrology.

An all-in-one platform to deplete pathogenic bacteria for rapid and safe enrichment of plant-derived extracellular vesicles.

Plant-derived extracellular vesicles (PDEVs) have exhibited several advantages, such as high biocompatibility, improvement of skin conditions, and the prevention of skin aging. However, traditional methods of extraction for plant substances, such as heating under reflux or solvent extraction, are complicated, time-consuming, and low in purity. Accordingly, a simple and efficient platform is necessary for purely isolating natural substances from plants. In this study, we report a newly designed platform for removing impurities to purify PDEVs. The proposed platform comprises three parts: (i) inflow of samples, (ii) depletion of impurities, and (iii) collection of PDEVs. The platform is designed to flow from top to bottom using gravity without the need for electric components. The platform allows the delimitation of impurities, such as the pathogenic bacteria in PDEVs, by capturing magnetic beads coated with Concanavalin A (Con A). We validate the practicality of our platform using extracellular vesicles derived from liquorice (LdEVs). Notably, the LdEVs purified using the Con A-coated magnetic beads provide better cell uptake and wound recovery than the commercialized extract LdEVs. This highlights the therapeutic potential of fresh LdEVs purified using our platform, particularly in preventing skin aging. The findings of this study hold significant practical implications for the cosmeceutical and therapeutic field, providing a promising approach for the extraction and purification of natural substances from plants to harness their benefits effectively.

Modularized dynamic cell culture platform for efficient production of extracellular vesicles and sequential analysis.

Extracellular vesicles (EVs) are nanometer-sized particles naturally secreted by cells for intercellular communication that encapsulate bioactive cargo, such as proteins and RNA, with a lipid bilayer. Tumor cell-derived EVs (tdEVs) are particularly promising biomarkers for cancer research because their contents reflect the cell of origin. In most studies, tdEVs have been obtained from cancer cells cultured under static conditions, thus lacking the ability to recapitulate the microenvironment of cells in vivo. Recent developments in perfusable cell culture systems have allowed oxygen and a nutrient gradient to mimic the physiological and cellular microenvironment. However, as these systems are perfused by circulating the culture medium within the unified structure, independently harvesting cells and EVs at each time point for analysis presents a limitation. In this study, a modularized cell culture system is designed for the perfusion and real-time collection of EVs. The system consists of three detachable chambers, one each for fresh medium, cell culture, and EV collection. The fresh medium flows from the medium chamber to the culture chamber at a flow rate controlled by the hydraulic pressure injected with a syringe pump. When the culture medium containing EVs exceeds a certain volume within the chamber, it overflows into the collection chamber to harvest EVs. The compact and modularized chambers are highly interoperable with conventional cell culture modalities used in the laboratory, thus enabling various EV-based assays.

Electrochemical detection and analysis of tumor-derived extracellular vesicles to evaluate malignancy of pancreatic cystic neoplasm using integrated microfluidic device.

Tumor-derived extracellular vesicles (tdEVs) are one of the most promising biomarkers for liquid biopsy-based cancer diagnostics, owing to the expression of specific membrane proteins of their cellular origin. The investigation of epithelial-to-mesenchymal transition (EMT) in cancer using tdEVs is an alternative way of evaluating the risk of malignancy transformation. An ultra-sensitive selection and detection methodology is an essential step in developing a tdEVs-based cancer diagnostic device. In this study, we developed an indium-tin-oxide (ITO) sensor integrated microfluidic device consisting of two main parts: 1) a multi-orifice flow-fractionation (MOFF) channel for extraction of pure EVs by removing blood cellular debris, and 2) an ITO sensor coupled with a geometrically activated surface interaction (GASI) channel for enrichment and quantification of tdEV. The microfluidic channel and the ITO sensors are assembled with a 3D printed magnetic housing to prevent sample leakage and to easily attach/detach the sensors to/from the microfluidic channel. The tdEVs were successfully captured on the specific antibody modified ITO surfaces in the integrated microfluidic channel. The integrated sensors showed an excellent linear response between 103 and 109 tdEVs/mL. Simultaneous evaluation of the epithelial and mesenchymal markers on the tdEV surfaces successfully revealed the EMT index of the corresponding pancreatic cancer cells. Our ITO sensor integrated microfluidic device showed excellent detection in the clinically relevant tdEVs-concentration range for patients with pancreatic cystic neoplasms. Hence, this system is expected to open a new avenue for liquid biopsy-based cancer prognostics and diagnostics.

Coil spring-powered pump with inertial microfluidic chip for size-based isolation and enrichment of biological cells.

Microfluidic chips have been widely used for in vitro diagnostics using pretreatment of biological samples; however, biologists and clinical researchers have difficulties using them in resource-limited settings. Sample injection systems for microfluidic chips are bulky, expensive, electricity-powered, and complex. A coiled spring-powered device, which can be used to isolate variously sized cells with high efficiency continuously and passively, was developed for portable, low-cost, electricity-free, and simple sample injection. The flow driving power was provided by releasing the compression spring in the mechanical syringe driver with a one-click action. In general, a syringe pump generates a stable passive flow rate. However, the syringe pumps are large in size and expensive because they have many functions such as infusion/withdrawal flow injection and the use of syringes of various sizes, allowing them to be applied in a variety of applications performed in the laboratory. In addition, it is not suitable for portable devices because of the considerable amount of electric power required. To overcome these drawbacks, we developed a device prototype that sorts different-sized particles and separates rare tumor cells or blood cells from blood with high efficiency. The performance of the coiled spring-powered device was evaluated and found to be comparable with that of syringe pump-powered devices. In situations where trained personnel cannot handle microfluidic chips for isolating circulating biomarkers (CTCs, WBCs, or plasma) from blood samples, the coiled spring-powered device can provide diagnostic tools, especially in resource-limited countries.

Simple ultrasensitive electrochemical detection of the DBP plasticizer for the risk assessment of South Korean river waters.

Rapid detection of contaminants for the purpose of sensitive and quantitative monitoring of environmental hazards is an essential first step in realizing the avoidance of human health risks. In this regard, we present a fast and simple electrochemical method of detecting di-n-butyl phthalate (DBP) from river water samples using a phthalic acid group specific aptamer modified on a gold nanoparticle (AuNP) functionalized graphene oxide nano-platelet (GO) and ionic liquid (IL) nanocomposite. Here, the IL/GO nanocomposite allows an enhanced interaction with phthalate esters, thereby increasing the sensitivity of the sensor surface. The proposed sensor showed a wide linear dynamic range from 0.14 pg mL-1 to 0.35 ng mL-1 and from 0.35 ng mL-1 to 7 ng mL-1 with a detection limit of ≤0.042 pg mL-1, which were evaluated using standard, analytical grade DBP; the limit of quantification was determined using different concentrations of DBP in DI water in comparison with gas chromatography-mass spectroscopy (GC/MS) values. The proposed sensor was used to monitor the DBP concentrations in river water samples collected from various locations across South Korea. The quantitative data from the measurements in comparison with standard GC/MS values were then used to ascertain the human health risk posed by the daily consumption of these river waters.

A modular microfluidic platform for serial enrichment and harvest of pure extracellular vesicles.

Extracellular vesicles (EVs) are recognized as promising biomarkers for several diseases. However, their conventional isolation methods have several drawbacks, such as poor yields, low purity, and time-consuming operations. Therefore, a simple, low-cost, and rapid microfluidic platform has been extensively developed to meet the requirement in biomedical applications. Herein, a modular microfluidic platform is demonstrated to isolate and enrich EVs directly from plasma, in a combination of continuous capture and purification of EVs. The EVs were selectively captured by target-specific antibody-coated beads in a horseshoe-shaped orifice micromixer (HOMM) chip within 2 min. A fish-trap-shaped microfilter unit was subsequently used to elute and purify the affinity-induced captured EVs from the microbeads. The ability of the modular chip to capture, enrich, and release EVs was demonstrated in 5 min (100 µL sample) at high throughput (100 µL min-1). The two chips can be modularized or individually operated, depending on the clinical applications such as diagnostics and therapeutics. For the diagnostic applications, the EVs on microbeads can be directly subjected to the molecular analysis whereas the pure EVs should be released from the microbeads for the therapeutic treatments. This study reveals that the fabricated modular chip can be appropriately employed as a platform for EV-related research tools.

Electrochemical biosensor for nucleic acid amplification-free and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA via CRISPR/Cas13a trans-cleavage reaction.

The outbreak of the COVID-19 pandemic has led to millions of fatalities worldwide. For preventing epidemic transmission, rapid and accurate virus detection methods to early identify infected people are urgently needed in the current situation. Therefore, an electrochemical biosensor based on the trans-cleavage activity of CRISPR/Cas13a was developed in this study for rapid, sensitive, and nucleic-acid-amplification-free detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, a redox probe conjugated with ssRNA is immobilized on the electrode surface modified with a nanocomposite (NC) and gold nanoflower (AuNF) for enhancing the sensing performance. The SARS-CoV-2 RNA is captured by the Cas13a-crRNA complex, which triggers the RNase function of Cas13a. The enzymatically activated Cas13a-crRNA complex is subsequently introduced to the reRNA-conjugated electrochemical sensor, and consequently cleaves the reRNA. A change in current occurs due to the release of the redox molecule labeled on the reRNA, which is trans-cleaved from the Cas13a-crRNA complex. The biosensor can detect as low as 4.4 × 10-2 fg/mL and 8.1 × 10-2 fg/mL of ORF and S genes, respectively, over a wide dynamic range (1.0 × 10-1 to 1.0 × 105 fg/mL). Moreover, the biosensor was evaluated by measuring SARS-CoV-2 RNA spiked in artificial saliva. The recovery of the developed sensor was found to be in an agreeable range of 96.54-101.21%. The designed biosensor lays the groundwork for pre-amplification-free detection of ultra-low concentrations of SARS-CoV-2 RNA and on-site and rapid diagnostic testing for COVID-19.

Multi-miRNA panel of tumor-derived extracellular vesicles as promising diagnostic biomarkers of early-stage breast cancer.

Extracellular vesicles (EV) have been emerging as potential biomarkers for disease monitoring. In particular, tumor-derived EV (TDE) are known to carry oncogenic miRNA, so they can be used for diagnosis of early cancer by analyzing the expression levels of EV-miRNA circulating in the blood. Here, using our novel microfluidic device, we rapidly and selectively isolate cancerous EV expressing breast cancer-derived surface markers CD49f and EpCAM within 2 minutes. Based on seven candidates of miRNA nominated from The Cancer Genome Atlas (TCGA) database, the expression levels of miRNA in TDE were validated in a total of 82 individuals, including 62 breast cancer patients and 20 healthy controls. Among seven candidates, four miRNAs (miR-9, miR-16, miR-21, and miR-429) from the EV were highly elevated in early-stage breast cancer patients compared with healthy donors. The combination of significant miRNAs from specific EV has high sensitivities of 0.90, 0.86, 0.88, and 0.84 of the area under the receiver operating characteristic curve (AUC) in each subtype (luminal A, luminal B, HER-2, and triple-negative) of early-stage breast cancer. Our results suggest that the combination of four miRNA signatures of specific EV could serve as a sensitive and specific biomarker and enable early diagnosis of breast cancer using liquid biopsy.

Circulating miR-122-5p and miR-375 as Potential Biomarkers for Bone Mass Recovery after Parathyroidectomy in Patients with Primary Hyperparathyroidism: A Proof-of-Concept Study.

Primary hyperparathyroidism (PHPT) is the leading cause of secondary osteoporosis. Although bone mineral density (BMD) tends to recover after parathyroidectomy in PHPT patients, the degree of recovery varies. Circulating microRNAs (miRNAs) profiles are known to be correlated with osteoporosis and fracture. We aimed to investigate whether osteoporotic fracture-related miRNAs are associated with postoperative BMD recovery in PHPT. Here, 16 previously identified osteoporotic fracture-related miRNAs were selected. We analyzed the association between the preoperative level of each miRNA and total hip (TH) BMD change. All 12 patients (among the 18 patients enrolled) were cured of PHPT after parathyroidectomy as parathyroid hormone (PTH) and calcium levels were restored to the normal range. Preoperative miR-19b-3p, miR-122-5p, and miR-375 showed a negative association with the percent changes in TH BMD from baseline. The association remained robust for miR-122-5p and miR-375 even after adjusting for sex, age, PTH, and procollagen type 1 N-terminal propeptide levels in a multivariable model. In conclusion, preoperative circulating miR-122-5p and miR-375 levels were negatively associated with TH BMD changes after parathyroidectomy in PHPT patients. miRNAs have the potential to serve as predictive biomarkers of treatment response in PHPT patients, which merits further investigation.

Microfluidic recapitulation of circulating tumor cell-neutrophil clusters via double spiral channel-induced deterministic encapsulation.

Circulating tumor cell (CTC)-neutrophil clusters are highly potent precursors of cancer metastasis. However, their rarity in patients' blood has restricted research thus far, and moreover, studies on in vitro methods for mimicking cell clusters have generally neglected in vivo conditions. Here, we introduce an inertial-force-assisted droplet microfluidic chip that allows the recapitulation of CTC-neutrophil clusters in terms of physical as well as biochemical features. The deterministic encapsulation of cells via double spiral channels facilitates the pairing of neutrophils and cancer cells with ratios of interest (from 1 : 1 to 1 : 3). The encapsulated cells are spontaneously associated to form clusters, achieving the physical emulation of CTC-neutrophil clusters. Furthermore, the molecular signatures of CTC-neutrophil clusters (e.g., their E-cadherin, VCAM-1, and mRNA expressions) were well defined. Our novel microfluidic platform for exploring CTC-neutrophil clusters can therefore play a promising role in cancer-metastasis studies.