Microfluidic Chip Research Articles

Design and Application of Microfluidic Capture Device for Physical-Magnetic Isolation of MCF-7 Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a type of cancer cell that spreads from the main tumor to the bloodstream, and they are often the most important among the various entities that can be isolated from the blood. For the diagnosis of cancer, conventional biopsies are often invasive and unreliable, whereas a liquid biopsy, which isolates the affected item from blood or lymph fluid, is a less invasive and effective diagnostic technique. Microfluidic technologies offer a suitable channel for conducting liquid biopsies, and this technology is utilized to extract CTCs in a microfluidic chip by physical and bio-affinity-based techniques. This effort uses functionalized magnetic nanoparticles (MNPs) in a unique microfluidic chip to collect CTCs using a hybrid (physical and bio-affinity-based/guided magnetic) capturing approach with a high capture rate. Accordingly, folic acid-functionalized Fe3O4 nanoparticles have been used to capture MCF-7 (breast cancer) CTCs with capture efficiencies reaching up to 95% at a 10 µL/min flow rate. Moreover, studies have been conducted to support this claim, including simulation and biomimetic investigations.

Contribution of Osteoblast and Osteoclast Supernatants to Bone Formation: Determination Using a Novel Microfluidic Chip

We fabricated a microfluidic chip (osteoblast [OB]–osteoclast [OC] chip) that could regulate the mixture amounts of OB and OC supernatants to investigate the effect of different supernatant distributions on osteogenesis or osteoclastogenesis. Computer-aided design was used to produce an OB–OC chip from polydimethylsiloxane. A pressure controller was assembled and different blends of OB and OC supernatants were correctly determined. OB and OC supernatants were placed on the upper panels of the OB–OC chip after differentiation for an in vitro evaluation. We then tested the changes in osteogenesis using MC3T3-E1 cells in the middle chambers. We observed that a 75:25 distribution of OB and OC supernatants was the most potent in osteogenesis. We then primed the osteogenic differentiation of MC3T3-E1 cells using an OB–OC mixed supernatant or an OB supernatant alone (supernatant ratios of 75:25 or 100:0, respectively). These cells were placed on the calvarial defect sites of rats. Microcomputed tomography and histological analyses determined a significantly higher bone formation in the group exposed to the OB–OC supernatant at a ratio of 75:25. In this study, we demonstrate the applicability of an OB–OC chip to evaluate the effect of different supernatant distributions of OB and OC. We observed that the highest bone-forming potential was in MC3T3-E1 cells treated with conditioned media, specifically the OB–OC supernatant at a ratio of 75:25.

A Microfluidic Chip on a Robotic Manipulator for Loading and Reloading of Oocytes

Loading individual oocytes is a critical step for injecting RNA, expressing heterologous proteins, performing electrophysiological measurements, and so on. However, existing methods remain a challenge for automatically loading multiple single oocytes into different locations. Herein, a novel microfluidic chip on a robotic manipulator (chip‐on‐robot) with feedback control for flexible manipulation of multiple oocytes within a large spatial range is proposed. The manipulator automatically controls the microfluidic chip to reach different locations based on imaging feedback. The microfluidic chip then utilizes the hydrodynamic focusing effect of the main channel to separate oocytes for individual loading or reloading under capacitive sensor feedback. The separation distance reaches approximately 16 times the oocyte diameter. Moreover, capacitive signal feedback on the number of oocytes for flow direction control ensures the separation of all oocytes. For close‐loop control of the loading/reloading process, image‐based oocyte detection is combined using deep learning to calculate the target position of the oocyte. Finally, an automatic sequence is achieved to load multiple single oocytes into a well chip by using the chip‐on‐robot. As a demonstration, the oocytes are reloaded into a specified location based on the conditions. The proposed chip‐on‐robot with feedback control has significant advantages in the micromanipulation of oocytes.

Immune response to IL6 gradient in a diffusion-based microfluidic labchip

Cell migration plays a crucial role in uncovering many physiological phenomena and predicting several pathologies, such as immune cell trafficking, tissue-specific immunological responses, wound healing, cancer metastasis, etc. A controlled approach to cellular migration has been explored through microfluidic devices. In order to create multiple dilutions of interleukin 6, a symmetric microfluidic labchip is well-designed. Eight chambers for cell culture and two for collecting migrated T cells were considered. This research will focus on mimicking immune cell movement or chemotaxis in a diffusion-based microfluidic device with different chemokine concentration gradients. Immune cells co-cultured with fibroblasts migrate in response to different gradients of interleukin 6. Immune cell migration is spatially controlled for 48 h by monitoring immune cell migration time and dose dependency. The microfluidic chip developed in this research aims to study immune cell movement in response to the concentration gradient of chemokine in cancer studies and clinical applications.

Comprehensive analysis of resilience of human airway epithelial barrier against short-term PM2.5 inorganic dust exposure using invitro microfluidic chip and exvivo human airway models.

The updated World Health Organization (WHO) air quality guideline recommends an annual mean concentration of fine particulate matter (PM2.5) not exceeding 5 or 15 μg/m3 in the short-term (24 h) for no more than 3-4 days annually. However, more than 90% of the global population is currently exposed to daily concentrations surpassing these limits, especially during extreme weather conditions and due to transboundary dust transport influenced by climate change. Herein, the effect of respirable <PM2.5 inorganic silica particle exposures on epithelial barrier integrity was simultaneously evaluated within the biomimetic microfluidic platform-based airway epithelial barrier (AEB)-on-a-chip and human bronchoscopic exvivo airway tissue models, comparatively. Silica particles at an average size of 1 μm, referred to as <PM2.5, dose-dependently tested by MTT and LDH analyses. The elicited dose of 800 μg/mL was applied to human airway epithelial cells (Calu-3) seeded to the membrane at air-liquid interface in the AEB-on-a-chip platform, which is operated under static and dynamic conditions and to exvivo human bronchoscopy bronchial tissue slices for 72 h. For both models, healthy and exposed groups were comparatively investigated. Computational fluid dynamics simulations were performed to assess shear stress profiles under different flow conditions. Qualitative and quantitative analyses were carried out to evaluate the resilience of the epithelial barrier via cell survivability, morphology, barrier integrity, permeability, and inflammation. In the AEB-on-a-chip platform, short-term exposure to 800 μg/mL PM2.5 disrupted AEB integrity via increasing barrier permeability, decreasing cell adhesion-barrier markers such as ZO-1, Vinculin, ACE2, and CD31, impaired cell viability and increased the expression levels of proinflammatory markers; IFNs, IL-6, IL-1s, TNF-α, CD68, CD80, and Inos, mostly under dynamic conditions. Besides, decreased tissue viability, impaired tissue integrity via decreasing of Vinculin, ACE2, β-catenin, and E-cadherin, and also proinflammatory response with elevated CD68, IL-1α, IL-6, IFN-Ɣ, Inos, and CD80 markers, were observed after PM2.5 exposure in exvivo tissue. The duration and concentration of PM2.5 that can be exposed during extreme weather conditions and natural events aligns with our exposure model (0-800 μg/mL 72 h). At this level of exposure, the resilience of the epithelial barrier is demonstrated by both AEB-on-a-chip platform emulating dynamic forces in the body and exvivo bronchial biopsy slices. Lung-on-a-chip models will serve as reliable exposure models in this context.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT’s adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

SIMPA simplifies micropipette aspiration to measure multiple samples simultaneously

With a microfluidic chip fitted with sliding elements, SIMPA enables parallel measurements of mechanical properties of cells, vesicles, and tissue.

Nanobody-Based Microfluidic Immunosensor Chip Using Tetraphenylethylene-Derived Covalent Organic Frameworks as Aggregation-Induced Electrochemiluminescence Emitters for the Detection of Thymic Stromal Lymphopoietin.

In this letter, a sensitive microfluidic immunosensor chip was developed using tetrakis(4-aminophenyl)ethene (TPE)-derived covalent organic frameworks (T-COF) as aggregation-induced electrochemiluminescence (AIECL) emitters and nanobodies as efficient immune recognition units for the detection of thymic stromal lymphopoietin (TSLP), a novel target of asthma. The internal rotation and vibration of TPE molecules were constrained within the framework structure, forcing nonradiative relaxation to convert into pronounced radiative transitions. A camel-derived nanobody exhibited superior specificity, higher residual activity and epitope recognition postcuring compared to monoclonal antibodies. Benefiting from the affinity between silver ions (Ag+) and cytosine (C), a double-stranded DNA (dsDNA) embedded with Ag+ was modified onto the surface of TSLP. A positive correlation was obtained between the TSLP concentration (1.00 pg/mL to 4.00 ng/mL) and ECL intensity, as Ag+ was confirmed to be an excellent accelerator of the generation of free radical species. We propose that utilizing COF to constrain luminescent molecules and trigger the AIECL phenomenon is another promising method for preparing signal tags to detect low-abundance disease-related markers.

Advancing Tissue Culture with Light-Driven 3D-Printed Microfluidic Devices.

Three-dimensional (3D) printing presents a compelling alternative for fabricating microfluidic devices, circumventing certain limitations associated with traditional soft lithography methods. Microfluidics play a crucial role in the biomedical sciences, particularly in the creation of tissue spheroids and pharmaceutical research. Among the various 3D printing techniques, light-driven methods such as stereolithography (SLA), digital light processing (DLP), and photopolymer inkjet printing have gained prominence in microfluidics due to their rapid prototyping capabilities, high-resolution printing, and low processing temperatures. This review offers a comprehensive overview of light-driven 3D printing techniques used in the fabrication of advanced microfluidic devices. It explores biomedical applications for 3D-printed microfluidics and provides insights into their potential impact and functionality within the biomedical field. We further summarize three light-driven 3D printing strategies for producing biomedical microfluidic systems: direct construction of microfluidic devices for cell culture, PDMS-based microfluidic devices for tissue engineering, and a modular SLA-printed microfluidic chip to co-culture and monitor cells.

A microfluidic chip for immobilization and imaging of Ciona intestinalis larvae.

Sea squirts (Tunicata) are chordates and develop a swimming larva with a small and defined number of individually identifiable cells. This offers the prospect of connecting specific stimuli to behavioral output and characterizing the neural activity that links these together. Here, we describe the development of a microfluidic chip that allows live larvae of the sea squirt Ciona intestinalis to be immobilized and recorded. By generating transgenic larvae expressing GCaAMP6m in defined cells, we show that calcium ion levels can be recorded from immobilized larvae, while microfluidic control allows larvae to be exposed to specific waterborne stimuli. We trial this on sea water carrying increased levels of carbon dioxide, providing evidence that larvae can sense this gas.

Engineering free-standing electrospun PLLCL fibers on microfluidic platform for cell alignment

Here, a PLLCL-on-chip platform was developed by direct electrospinning of poly (L-lactide-co-ε-caprolactone) (PLLCL) on polymethyl methacrylate (PMMA) microfluidic chips. Designed microchip provides the electrospinning of free-standing aligned PLLCL fibers which eliminates limitations of conventional electrospinning. Besides, aligned fiber structure favors cell alignment through contactless manipulation. Average fiber diameter, and fiber alignment was evaluated by SEM analyses, then, leakage profile of microchip was investigated. 3D cell culture studies were conducted using HeLa and NIH-3T3 cells, and nearly 85% cell viability was observed in PLLCL-on-chip for 15 days, while cell viability of 2D control started to decrease after 7 days based on Live dead and Alamar Blue analyses. These findings emphasize biocompatibility of PLLCL-on-chip platform for 3D cell culture and its ability to mimic extracellular matrix (ECM). Immunostaining results prove that PLLCL-on-chip platform favors the secretion of ECM proteins compared to control groups, and cytoskeletons of cells were in aligned orientation in PLLCL-on-chip, while they were in random orientation in control groups. Overall, these results demonstrate that the developed platform is suitable for the formation of various 3D cell culture models and a potential candidate for cell alignment studies.Graphical

Combination of an Optically Induced Dielectrophoresis (ODEP) Mechanism and a Laminar Flow Pattern in a Microfluidic System for the Continuous Size-Based Sorting and Separation of Microparticles.

Optically induced dielectrophoresis (ODEP)-based microparticle sorting and separation is regarded as promising. However, current methods normally lack the downstream process for the transportation and collection of separated microparticles, which could limit its applications. To address this issue, an ODEP microfluidic chip encompassing three microchannels that join only at the central part of the microchannels (i.e., the working zone) was designed. During operation, three laminar flows were generated in the zone, where two dynamic light bar arrays were designed to sort and separate PS (polystyrene) microbeads of different sizes in a continuous manner. The separated PS microbeads were then continuously transported in laminar flows in a partition manner for the final collection. The results revealed that the method was capable of sorting and separating PS microbeads in a high-purity manner (e.g., the microbead purity values were 89.9 ± 3.7, 88.0 ± 2.5, and 92.8 ± 6.5% for the 5.8, 10.8, and 15.8 μm microbeads harvested, respectively). Overall, this study demonstrated the use of laminar flow and ODEP to achieve size-based sorting, separation, and collection of microparticles in a continuous and high-performance manner. Apart from the demonstration, this method can also be utilized for size-based sorting and the separation of other biological or nonbiological microparticles.

Triplex immunomagnetic separation combined with loop-mediated isothermal amplification on a microfluidic chip for simultaneous visual detection of Escherichia coli O157:H7, Salmonella enteritidis and Staphylococcus aureus in milk

Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus are widely present in food and can cause serious and large-scale cases of illness worldwide every year. Herein, we developed a triplex immunomagnetic separation (IMS) combined with loop-mediated isothermal amplification (LAMP) on a microfluidic chip (triplex IMS-mLAMP) for the simultaneous detection of these three important foodborne pathogenic bacteria. To optimize the triplex IMS method, the CEs of the three types of IMBs were determined for different particle sizes, amounts and incubation times. Under optimal reaction conditions, the CEs for the three bacteria were between 65.4% and 71.1% in PBS, and between 41.1% and 47.5% in milk, respectively. After triplex IMS, DNA from target bacteria captured by magnetic beads was extracted and subjected to a multiplexed LAMP reaction in a centrifugal microfluidic chip that allowed for the simultaneous detection of up to 8 individual milk samples. The results were judged directly by the color change. The method only detected the three target bacteria and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of the triplex IMS-mLAMP as low as 1.2 × 102 CFU/mL for E. coli O157:H7 and S. enteritidis, and 2.5 × 102 CFU/mL for S. aureus in milk samples. The overall testing time of the triplex IMS-mLAMP was just 80 min. This is the first report that IMS-mLAMP was used for simultaneous capture and detection of E. coli O157:H7, S. enteritidis, and S. aureus in milk. This cost-effective, easy-to-use, no need for expensive laboratory-based equipment, integrated on-chip colorimetric multi-target detection system opens a novel pathway for rapid diagnostic screening of pathogens in food.

A Microfluidic Chip-Based Automated System for Whole-Course Monitoring the Drug Responses of Organoids.

Tumor patients-derived organoids, as a promising preclinical prediction model, have been utilized to evaluate ex vivo drug responses for formulating optimal therapeutic strategies. Detecting adenosine triphosphate (ATP) has been widely used in existing organoid-based drug response tests. However, all commercial ATP detection kits containing the cell lysis procedure can only be applied for single time point ATP detection, resulting in the neglect of dynamic ATP variations in living cells. Meanwhile, due to the limited number of viable organoids from a single patient, it is impractical to exhaustively test all potential time points in search of optimal ones. In this work, a multifunctional microfluidic chip was developed to perform all procedures of organoid-based drug response tests, including establishment, culturing, drug treatment, and ATP monitoring of organoids. An ATP sensor was developed to facilitate the first successful attempt on whole-course monitoring the growth status of fragile organoids. To realize a clinically applicable automatic system for the drug testing of lung cancer, a microfluidic chip based automated system was developed to perform entire organoid-based drug response test, bridging the gap between laboratorial manipulation and clinical practices, as it outperformed previous methods by improving data repeatability, eliminating human error/sample loss, and more importantly, providing a more accurate and comprehensive evaluation of drug effects.

Microfluidic chips as drug screening platforms

Microfluidic chips as drug screening platforms

A microfluidic platform for automatic continuous flow layer‐by‐layer assembly of polymer multilayered nanofilms

AbstractCapillary flow layer‐by‐layer assembly is a microfluidic platform for high throughput preparation and screening of multilayered polymer nanofilms. However, it is time‐consuming and labor‐intensive, since the switching of polyelectrolyte solutions is done manually. As the emergence of flow chemistry and microdevices for artificial intelligence‐based lab automation, an automatic system that can switch polymer solutions and washing liquids is urgently needed for building functional LbL films inside microfluidic devices. Herein, we construct a continuous flow (ConF)‐layer‐by‐layer (LbL) device that can automatically fabricate polymer nanofilms inside a microfluidic device. By including a flow separator and a mixer (a model polyelectrolyte pair), chitosan and carboxymethyl cellulose can be deposited inside a microfluidic device using the ConF‐LbL system. Resulting nanofilms are studied by investigating thickness and surface roughness of the nanofilms and comparing with the other conventional LbL methods. The acid dissolution method was used to compare the differences in stability between nanofilms made from ConF‐LbL and static immersive method in similar conditions. Furthermore, by using one optimized film formulation for coating on the inside walls of a microfluidic chip, we successfully demonstrated the ability of this device to capture cancer cells in whole blood. This ConF‐LbL platform is expected to be a convenient tool to coat functional nanofilms inside microdevices for biomedical and sensor applications.

Reagent storage and delivery on integrated microfluidic chips for point-of-care diagnostics.

Microfluidic-based point-of-care diagnostics offer several unique advantages over existing bioanalytical solutions, such as automation, miniaturisation, and integration of sensors to rapidly detect on-site specific biomarkers. It is important to highlight that a microfluidic POC system needs to perform a number of steps, including sample preparation, nucleic acid extraction, amplification, and detection. Each of these stages involves mixing and elution to go from sample to result. To address these complex sample preparation procedures, a vast number of different approaches have been developed to solve the problem of reagent storage and delivery. However, to date, no universal method has been proposed that can be applied as a working solution for all cases. Herein, both current self-contained (stored within the chip) and off-chip (stored in a separate device and brought together at the point of use) are reviewed, and their merits and limitations are discussed. This review focuses on reagent storage devices that could be integrated with microfluidic devices, discussing further issues or merits of these storage solutions in two different sections: direct on-chip storage and external storage with their application devices. Furthermore, the different microvalves and micropumps are considered to provide guidelines for designing appropriate integrated microfluidic point-of-care devices.

Bimetal−organic framework-integrated electrochemical sensor for on-chip detection of H2S and H2O2 in cancer tissues

Studies on the interaction between hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) in redox signaling motivate the development of a sensitive sensing platform for their discriminatory and dynamic detection. Herein, we present a fully integrated microfluidic on-chip electrochemical sensor for the online and simultaneous monitoring of H2S and H2O2 secreted by different biological samples. The sensor utilizes a cicada-wing-like RuCu bimetal−organic framework with uniform nanorods architecture that grows on a flexible carbon fiber microelectrode. Owing to the optimized electronic structural merits and satisfactory electrocatalytic properties, the resultant microelectrode shows remarkable electrochemical sensing performance for sensitive and selective detection of H2S and H2O2 at the same time. The result exhibits low detection limits of 0.5 μM for H2S and 0.1 μM for H2O2, with high sensitivities of 61.93 μA cm−2 mM−1 for H2S, and 75.96 μA cm−2 mM−1 for H2O2. The integration of this biocompatible microelectrode into a custom wireless microfluidic chip enables the construction of a miniature intelligent system for in situ monitoring of H2S and H2O2 released from different living cells to differentiate between cancerous and normal cells. When applied for real-time tracking of H2S and H2O2 secreted by colorectal cancer tissues, it allows the evaluation of their chemotherapeutic efficacy. These findings hold paramount implications for disease diagnosis and therapy.

A PEDOT: PSS/GO fiber microelectrode fabricated by microfluidic spinning for dopamine detection in human serum and PC12 cells.

Rapid and accurate in situ determinationof dopamine is of great significance in the study of neurological diseases. In this work, poly (3,4-ethylenedioxythiophene): poly (styrenesulfonic acid) (PEDOT: PSS)/graphene oxide (GO) fibers were fabricated by an effective method based on microfluidic wet spinning technology. The composite microfibers with stratified and dense arrangement were continuously prepared by injecting PEDOT: PSS and GO dispersion solutions into a microfluidic chip. PEDOT: PSS/GO fiber microelectrodes with high electrochemical activity and enhanced electrochemical oxidation activity of dopamine were constructed by controlling the structure composition of the microfibers with varying flow rate. The fabricated fiber microelectrode had a low detection limit (4.56 nM) and wide detection range (0.01-8.0 µM) for dopamine detection with excellent stability, repeatability, and reproducibility. In addition, the PEDOT: PSS/GO fiber microelectrode prepared was successfully used for the detection of dopamine in human serum and PC12 cells. The strategy for the fabrication of multi-component fiber microelectrodes isa new and effective approach for monitoring the intercellular neurotransmitter dopamine and has highpotential as an implantable neural microelectrode.

Mechanism study on the influences of buffer osmotic pressure on microfluidic chip-based cell electrofusion.

Cell electrofusion is a key process in many research fields, such as genetics, immunology, and cross-breeding. The electrofusion efficiency is highly dependent on the buffer osmotic pressure properties. However, the mechanism by which the buffer osmotic pressure affects cell electrofusion has not been theoretically or numerically understood. In order to explore the mechanism, the microfluidic structure with paired arc micro-cavities was first evaluated based on the numerical analysis of the transmembrane potential and the electroporation induced on biological cells when the electrofusion was performed on this structure. Then, the numerical model was used to analyze the effect of three buffer osmotic pressures on the on-chip electrofusion in terms of membrane tension and cell size. Compared to hypertonic and isotonic buffers, hypotonic buffer not only increased the reversible electroporation area in the cell-cell contact zone by 1.7 times by inducing a higher membrane tension, but also significantly reduced the applied voltage required for cell electroporation by increasing the cell size. Finally, the microfluidic chip with arc micro-cavitieswas fabricated and tested for electrofusion of SP2/0 cells. The results showed that no cell fusion occurred in the hypertonic buffer. The fusion efficiency in the isotonic buffer was about 7%. In the hypotonic buffer, the fusion efficiency was about 60%, which was significantly higher compared to hypertonic and isotonic buffers. The experimental results were in good agreement with the numerical analysis results.